Monday. Ribonucleoproteins and RNA Processing (1261
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Monday. Ribonucleoproteins and RNA Processing (1261
Monday. Ribonucleoproteins and RNA Processing (1261-1266) 1261 1262 REEGION ON INFLUENCE OF 3'-UTR AND CODING HALF-LFEM OF BOVINE FFL. Schadacher, ant MCC. Neville)) Lab Pattanajitvfllai, Biology, Ohio Agric. Res. & Dev. Center, Ohio Sta Univ., Wooster, LACTOFERRIN mRNA. ((S. of Moll & Dev. Cob Health Sci Center, Denver, CO. Dept ofPbysolooy, Umv. R of 3e-3U the mNRNA destabiuaton comsess squences noted mammary mRNAm lactofern(n(bLf) ggeuedpostthairmcriptionalregulationofbLfbyNRNA degadation raten RNA half-ife). Consuuction of expressonvectors(pcDNA) with 5'comnplet cDNAs ( a lac hLn corone tarection allowed study of the effects of for bovine or aman andW m on RNA half-ife m of RNA segments (COS cells) ard potein exchange or deletion nse 1 ando e(ssion(COS(COMMA-(D) mammary cel). Exchange (KH) m ) RNAs, and deleton of haman growth hrmnone of the between bLf 33-UTRs Ohio and bovin e -l orpbmarybovin am! codingcregionsegments (t334-1021)ofbLf, alloweddetenninationofeffectsofbLfmRNA regions on mRNA half-life in tansfected celia and at 0, 6,12, 18, 24 hrs after inhibition of of the 3'-R of rnarcription by 5,6-dichlorobenzimidazdle-Rboside(DRB). Rplacement hG of bLf dramaticaly rdued the expression of H protein, decreased 33-UTR hGH with the 15.6+/ the hG mRNA, and shortenedtm of mH RNA from 31.3+/-7.9 Ins to the H 3'-UTR onto bLf increared bLf protein Susisltution of IG expession and the amount ofbLf mRNA in the cel but decreasedthe t,, of bLf mRNA from 31.3+/-14 his to 11.9+/-2.8 hb, niggefing the coding rgion to be criticaly sensitive to nalase degradaionn Deletion of the distal N-lobe coding region (nt 334-1021) from bLf Tof retducedthe t,, of the bLf 334-1021 ntivebLf or ofGH cDNA havg the 3'-eithero deletionmRNAs to 5.8+1-1.1 if with bLf 3'-UR and 6.25+/-1.8 his if with bOl 3'-UTR. m of and b Lf/C-lobe hLf RNAs were hLf chimeric N-lobe In corast, the half-ife an! 16.2+/-1.9 her, respectively. 33-UTR of bLf mRNA conrains strong destabilization The if mabdsted onto stable nRNAs (hOH) but is a stabilizer sn the signals as predicted of bLf nRNA itself, as is the distal N-Lobe coding egion of bLf mRNA, of the naucaw sensitive the C-Lobe coding region. Elevate bLf protein expression in mammay cells vemus native bLf cDNA ttarsfected with bLf cDNA lstiuted with thehGH3'3-U so ggests pnotrarucriptional regulation for Lf mRNA in mammary as for COS-1 cells. & Natnl. Res. Board. Dairy Prom Supported by the the 217a level of GH H 6.1 hrs as expected. his 13+1+03 context 1263 ANALYSIS OF CIS-REGUIATORY ELEMENTS DIRECTING ALTERNATIVE PRE-mRNA SPLICING OF THE MYOSIN HEAVY CHAIN GENE TRANSCRIPT IN DROSOPHILA. D.M. Standiford, M.B. Davis and C.P. Emerson, Jr. Dept. of Cell and Developmental Biology, University of Pennsylvania School ofMedicine, Philadelphia, PA 19104 Myosin heavy chain (MHC) protein isoform diversity in Drosophila is generated through alternative splicing of the pre-mRNA transcript from the single skeletalMhc gene. This process is highly regulated with specificMHC isoforms being expressed at certain developmental time and in specific muscle types. We are exploring the mechanisms that enforce this specificity by examining the cis-regulation of one alternative exon set, exon 11, in tranagenic flies. Based on sequence comparisons of exon 11 betweenD. melanogaster andD. virilis, we have constructed and are testing a model in which the conserved, non-consensus 5' splice donors and a large intronic conserved region direct splice specificity. Data will be presented to show that the 5' donor plays a role in regulating splicing for some but not all alternative exon lls. In addition intronic conserved region appears necessary for the correct specific regulation of exon lle. These data and others suggest that at least two seperable mechanisms function to regulate alternative exon 11 splicing. Data from a genetic screen designed to identify mutations in genes for the transregulation of alternative splicing will also be presented. the tissue A HETEROLOGOUS MODEL SYSTEM TO STUDY DEVELOPMENTAL SPLICING SWITCHES IN ERYTHROID PROTEIN 4.1 PRE-mRNA ((K. Aoyagi and J.G. Conboy)) Life Sciences Division, Lawrence Berkeley Laboratory, University of Clifrniia, Berkeley, CA 94720. distn Alterative pre-mRNA splicin regulates expression of functionally isoforrs of the stucturl protein 4.1 duing erythrid differentiation. Splicing of 4.1 pre-mRNA in mouse is developmentally regulated such exon 16 is compleely sipped in early erythroid precrsors. but is spliced in at the late eryhroblast stage. Because exon 16 encodes pait of the critical spiectrin-actin binding domain, only in late erithroid cells does 4.1 mechanically strengthen the erythroid membrane. This study was undertaken to explore regulated splicing of a model 4.1 pre-mRNA in simplified pre-mRNA into heterologous system. Microinjection of a synthetic three-exon 4.1 mRNA products, analysis of spliced Xenopru oocyte nuclei, followed by revealed accurate alterative splicing of 4.1 pre-mRNA into two mRNA species, srnmgly RT/PCR exon 16. Thus, the oocyte system recognizes the cis-acting sequences plus/minus which regulate spicing of mouse exon fuuncional aanaysis of 166 and should facilitate such sequences via site-directed mutagenesis. Phylogenetic clues as to regulatory sequenc were sough t by comparison of frog and mouse gene sequene arund exon 16. Isntrons of both have consensus 3' slice sites and putative brnch points, but the splice sites appear weak and may be rsponsible for exon 16 skipping. No evidence for other conserved cis-regulatory sequences was detected in the intrn. In contrast, exon 16 sequences of both species contaied a purine-rich 5' domain followed by a nt region of identity, which is also conserved in the human, dog, and chicken genes. This extraordia conservation suggests that exon sequences are umporannt splicing in model pre-mRNAs We regulation, as recenty described for alternative exonsotthr also found that the efficiency of exon 16 splicing was concentration-dependent, and could be altered by co-expression in oocytes of trans-acting splicing factors such as SF2/ASF. Webelieve this system pprovde wll important insights into the mechanism of alterative splicing which mediates a functionally important splicing switch duTing erythrdiddevelopmedL 5 42 1264 IN VITRO ANALYSIS OF A VHS-DEPENDENT RIBONUCLEASE ACTIVITY IN HERPES SIMPLEX VIRIONS. ((B.D. Zelus, R.S. Stewart and J. Ross)) McArdle Laboratory for Cancer Research, University of Wisconsin, Madison WI 53706. (Spon. by C.E. Somers) The infection of permissive cells by herpes simplex virus results in the rapid degradation of cellular mRNAs. Genetic evidence suggests that the virion host shutoff (vhs) protein, a 58 kDal polypeptide encoded by the UL41 ORF, is responsible for the observed destabilization of host cell mRNA. We have used soluble extracts of purified herpes virions and an in vitromRNA decay assay system to investigate the mechanism of vhs action. Extracts of wild-type virions accelerate the decay of both polysomal and in vitro synthesized mRNAs. Extracts from vhs-deficient mutant virions do not. Polyclonal antibodies raised against bacterially expressed vhs protein inhibit the ribonucleolytic activity of these extracts and can immunoprecepitate ribonuclease activity. Electrophoretic analysis of the decay products of in synthesized globin mRNA reveals specific decay vitro intermediates resulting from ribonuclease activity in the 3' portion of the mRNA. These results suggest that the vhs protein is a ribonuclease and provide a system with which to analyze its mechanism of action and reveal why mRNA, but not rRNA or tRNA, is targeted for destruction. 1265 1266 DISRUPTION OF THE MAJOR VAULT PROTEIN B GENE (mvpB) AND IDENTIFICATION OF A GROWTH DEFECT PHENOTYPE IN MVP NEGATIVE LINES. ((S.K. Vasu and L.H. Rome)) Departmt of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90024. (Spon. by L.H. Rome) VAULT RNA AND ITS ASSOCIATED PROTEINS. ((V.A. Kickhoefer, and L.H. Rome)) Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90024. (Spon. by V.A. Kickhoefer.) Vaults are large cytoplasmic ribonucleoprotein Vaults particles of unknown function. at Dictyostelium vault proteins migrate on SDS-PAGE gels as two bands, kDa (MvpA) and the other at 92 kDa (MvpB). An MvpB cDNA clone one 94 was isolated by immuno-screening Dictyos:elium vault Dicystelium expression library with an antipolyclonal antibody. The cDNA was identified as MvpB as it a shares 60% identity at the amino acid level with the previously cloned MvpA has been (Vasu et al., 1993, K 268, 15356-15360). The single mvpB disrupted in both wild-type and mvpA- genetic backgrounds. Although the mutant that show loss of MvpA and/or MvpB interferes with vault they viable, lines are fiuntion sufficiently to impede growth under conditions of nutritional stress. Ovoid structures have been isolated from single mvp- mutant lines and shown to gen represent what remains of vault structure in these cells. Ovoid structures have also been isolated from the mvpA- mvpB- line, M7, and characterized by electon microscopy and SDS-PAGE. A novel protein of MW 92 kDa (MvpC) copurifies with the ovoid structures. Antibodies directed against nornal vaults do not the mvpB reognize this protein suggesting that this protein might be induced in line. (Supported by USPHS grant GM 38097) are ubiquitous, evolutionarily conserved, cytoplasmic particles of unknown function. The 13-MDa ovoid ribonucleoprotein vauft has bifold and each half can open into a particle symmetry, flower-like structure, which contains eight petals surrounding a central ring. Vaults purified from rat liver consist of four protein species (210, 192, 104, and 56 kDa) and a 141-nt RNA polymerase lIl transcribed RNA (vRNA). The 104-kDa protein constitutes >70% of the total vault mass. We have characterized the distribution of the vRNA by fractionation of cellular extracts. Whereas all of the major vault protein is associated with the vault particle and pellets amount of with the microsomal fraction (100,000xg), a significant the vRNA is present in the fraction. These results demonstrate that not all of the vRNA is associated with the vault particle. We have determined by UV-crosslinking that the vRNA in the S100 fraction is associated with protein in two different RNP complexes. In label transfer experiments we have identified a 50 kDa protein present in one of the vRNA complexes. We are currently purifying the vRNA binding proteins. (Supported by USPHS Grant GM38097). S100 218a Stress Response Genes (1267-1272). Monday 1267 1268 VARIATIONS IN FERRITIN SUBUNIT SIZE CORRELATE WITH SURVIVABILITY OF YEAST TO HEAT AND OXIDATIVE STRESS. ((E.C. Thomborrow, M. Donovan, and A.A. Infante)) Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459-0175. CLONING OF STRESS-INDUCIBLE GENES OF S. porbe. ((M. Morimyo, E. Hongo, K Mita, H. Hama-Inaba, and I. Macbids)) National Institute of Radiological Sciences, Chiba 263, JAPAN S.cerevsuiae porbe which of To elucidate the repair mechanisms S. exhibits extreme resistance to radiation, stress-inducible genes were cloned. Firstly, cloning vector, pYMM5, was constructed by ligation of pBR322 vector with ars gene of S. porbe, URA3, and reporter gene lacZ carrying multi-cloning site on itt upstream site. Secondly, the fusion S. porbe DNA library was made by ligation of BarHI and CIAP treated pYMM5 vector with MboI digested and fractionated S. porbe DNA. Cells of a wild-type strain were transformed with these plasmids by Liacetate or electroporation treatment at a frequency of 106 transformants/g.g DNA. Thirdly, transformants exhibiting pale blue color on Xgal plates were obtained at about 1/200, and were replica-plated on Xgal plates. They were treated with various stresses such as UV, X-rays heat and oxygen radicals. Those colonies which showed darker blue color after stress treatment, were obtained at about 1/100. Plasmid DNA were extracted from 71 candidates and were classified into 26 groups by determining DNA sequences of S. porbe region flanking to multi-cloning site. Many of them were new genes and were induced by various stresses, indicating that they were involved in the later cascade of stress-induction. is a facultative anaerobe, capable of metabolism by both fermentation Weatem and reapiration. We report a protein in S.cerevisie that is recognized blots specifically by an anti-homse spleen ferritin antibody. The molecular weight of the putative ferritin subunit, however, appears to change depending on the mode of on yeast metabolism and during heat ahock. During normal fermentation the yeast ferritin migrates on SDS-PAGE at 42 kD. When the yeast are respiring, ferritin migrates at a poaition consistent with a 19 kD protein. Further, if fermenting cells are subjected protein is no longer observed to a one hour mild heat shock at 38 C, the at 42 kD; rather, its migration is shifted to 19 kD. Additional studies have shown ferritin that these transitions in ferritin migration rate correlate to the ability of the yeast to survive severe 52 C insult. Studies show that respiring cells-which contain the 19 kD ferritin-survive (as measured by colony forming units) 80-90% better than fermenting a 5 stress at 52 C. Prior to the 52 cells-which contain the 42 kD C stress, if fermenting cells are heat shocked at 38 C for one hour, causing a ferritin from 0-10% to 80-100%. Thus, shift from 42 kD to 19kD, their survivability increases a ferritin-foUowing min a close correlation between the presence of the 19 kD and the coils' ability to survive a 52 C heat stress. Similar results have been obtained using hydrogen peroxide as the stress. Compared to cells with the 42 kD ferritin, yeast possessing the 19 kD form of the protein have a greater survival rate when subjected to mM quantities of hydrogen peroxide. we have shown that there is ferritin subunit 1269 1270 CARDIAC MYOSIN LIGHT CHAIN GENE IS MODULATED ACTIVATION OF INVOLVE THE COILED-COIL STRUCTURE. and OF HUMAN HEAT SHOCK TRANSCsls?ON ABIIITY DNA-BINDING FROM TRI-TRANDED AN INTRAMOLECULAR TO AN TRANSMON DD adVRoelly)) of RIaU.milty R. Zo,. IL B.e,. 0. UUaiveyof Miami SdioolofMddciic Miam, 3311i. Bioby.i.o,yBipbysU. M.iacut b.hag. BY STRESS (EDUARDO MASCARENO) ANATOMY & CELL BIOLOGY DEPARTMENT, SUNY at Brooklyn, NY 11203 (spon. by M.A.Q.Slddlqui) The response ofanimals and humans to stress is reflected at the cellular level by an increment in heat-shock gene expression. Studies in our laboratory have indicated that the promoter of the MLC2 gene is, among others, the target for stress-.related transcription activator proteins, the heat shock factors (HFSs). We have observed that the heat shock responsive sequence elements (HSEs) located in the proximal promoter of MLC2 gene, binds the HSFs in primary cardiac cells in culture following stress induced heat shock recombinants MLC2 promoter/reporter treatment. transfected into primary cardiac cells, respond positively upon heat shock treatment but not when HSEs were deleted from the promoter. These results, therefore, strongly suggests a role for the HSFs in targeting stress responsive genes such as cardiac MLC2, indicating a new and novel molecular mechanism of stress-mediated modulation of gene Hat d mot (39-42C) ..y ds,q,a MAY FL ad of1 bum ofg.hs. .sock hYdrpbobic r_ (b,c Het FACTOR INTERR1LBCULAR aM'. I. zsppa or 12 be 1-3).La a cti_. and oci by bet Xc umd lumA..I HSFI baugb t.e -dhd genesa iza n mbiliz id by, .sracbo haS-o-tr5m bHSFIr (370C). HSFI a oeU ba binding bity. HHSFIexp x d Xapoocyso.n6 a_cripa ad HSE .,qis ass.., containing dee to bc pp ima in M_.5 & b.p70. 6..srcio. S6. (Hem E_at) DNA- oNA-DNAbiding l ias " .g.imi o. B.. dc. DNA-bading form upon expomm of oocyI. to bed ock (35-37C HSF DNA-bindg sivity l., ad .a&hHSFI anibody doe not f..ogaie 1.wp.s HSF. w. emplyed anIy.i of a yq d for m.,.ppag fgio tam requid for dke .ala...ac of dh .am aaeic sdo. hHS ins e of .asil deikdi bht -reg,atd DNA-bnding biity .ad trknerbti of hHSFI. lig a kg bHSFI Xeopw ooyt., duhe dierese, goa. contain" iZI-3, we identifd dat wm crit for of of LZ anaurm by amag6gdy. Xcg ..ive moomer atda on-beat hbock en aer .um (200C). Disrupt to a teric, .. hydr.pbobic rdue aboliabectd beti wid susitu of zpper reidu ek s ame tn..f.sien .A hydopob.c nos-hat owe u -der acdie an .eil. T1 iy involog vin eracbo. ll ma tedt d. and LZ 1.uve wh may Tl sadwia... oodd. shz* ve qpd-ryI_ige .6e g am HSF naata hckF amays widhub.. u ,i.ntA r ofbHSFIHSE DNA-badig .biity.W LZ12t dasg.kbdfor HSE DNA-bind ,guio bHSFI form r was * s. =.ad by baiied by csdid-coiL o f HSFI byf.iibatingcoop. tivce badagof_asIc T,ri.rtio emb DNA-bindingfbasi.o cead hHSFi DNA-bidcg wbs. with to thU HSE motif.to bindng boundto LcxA bid-" ia resultg cbbmr_ sithtUe DNA-bindag do.a of LcxA. b.seai tik d wi tU.iagrit of dbHSFILZ bert-reguladflab. Sagk cido coadiauivy oCigom.,, aWd OA-bidag.LteA bound_blyto DNAonly *die butast s_ i our DNA-bidig LZ of bHSFIm of Tbarfore. dU hydmopbobic intac D NA-bndingabiky. doai bat-mp,6d may expression. DNA- U.e doa. nots,.. great apte.. a am doma w ab.iti.a i a g rader.d y a a. tUe Uee confer oa b_..gA 1272 1271 HEAT SHOCK AND DEFLAGELLATION INDUCE A SHORT-LIVED mRNA IN CHLAMYDOMONAS. ((J.A.J. Burrows, P. Liggit, and E.J. Department of Biology, University of Nevada, Reno, NV 89557. HSP70 Baker)) Using 3' UTR probe specific for an hsp70 mRNA previously shown to be inducible by light as well as heat shock (Gromoff et al., Mol. Cell. Biol. 9: 3911, 1989), we find that this hsp7O mRNA is also inducible by flagellar amputation (deflagellation). Whereas induction of,the hsp70 mRNA in response to heat shock is not blocked in the presence of cydoheximide (a primary response), its induction in response to deflagellation is prevented, implying the existence of two different induction pathways. This finding allows us to study the posttranscriptional regulation of this mRNA in two different cytoplasmic environments. The hsp70 mRNA is short-lived under both conditions. Heatshock at 370C induces peak levels of hsp70 mRNA within 15 minutes. The mRNA decays rapidly back to low basal levels within 2 hours, exhibiting a half-life of 15-20 minutes. Unlike most short-lived mRNAs, inhibition of protein synthesis does not significantly stabilize the induced hsp7O mRNA. Hsp70 mRNA induced by deflagellation affains peak levels within 30 minutes, and also returns to basal levels within 2 hours with a half-life of 15-20 minutes. The relative instability of polysome-bound hsp70 mRNA is reproducible in extracts prepared from heat shocked or deflagellated cells. Analysis of poly(A) metabolism of the heatshock-induced hsp7O mRNA reveals that unusually rapid poly(A) tail shortening precedes its degradation, and this rapid shortening is independent of ongoing protein synthesis. Within 40 minutes of heat shock, >50% of the induced mRNA is poly(A)-deficient. The measurement of poly(A) shortening rates for deflagellation-induced hsp70 is in progress. Poly(A) tail shortening has recentlY been shown to play a key role in the post-transcriptional regulation of hsp7O mRNA in Drosophila (Dellavalle et al. Mol. Cell. Biol. 14: 3646, 1994), suggesting that regulated poly(A) metabolism may be a highly conserved CEREVISIAE 70 kDA MOLECULAR THE ROLE OF SACCHAROMYCES CHAPERONES IN PROTEIN TRANSLOCATION. ((Nancy M. Wang and William J. Chirico)) Department of Anatomy and Cell Biology, Science Center at Brooklyn, Brooklyn, NY 11203 SUNY-Health a property of this mRNA. 70 kDa heat shock proteins (Hsp7O) have been shown to act as molecular chaperones in many cellular activities including protein folding, One function is protein assembly, and the uptake of proteins into organelles. proteins into the to facilitate the post-translational translocation of which 70 kDa by mechanism the To reticulum explore (ER). endoplasmic heat shock proteins facilitate translocation, we chose to study their interactions (ppaf), a precursor of the a with prepro-a-factor in vitro. cerevisiae, can be post-translationally mating pheromone of translocated across the endoplasmic reticulum membrane in vitro. A biochemical dissection of this process would be greatly aided by the the entire coding availability of sufficient amounts of pure ppxf. Therefore, in the region of ppaf was cloned into an expression plasmid that resulted placement of six histidine codons at its 3' end. Histidine tags allow purification of proteins from E. coli in one step using nickel affinity chromatography. in an E. coli SecY resulting plasmid was The mutant to preserve the signal sequence of the overexpressed protein. The conditions. Upon histidine-tagged ppaf was purified under and dilution into containing yeast microsomes, it was translocated glycosylated. However, the amount of translocation decreased by 50%a when yeast Ssalp, If refolded histidine-tagged ppaf was used as a substrate. level of cytosolic Hsp7O, was present during the refolding reaction, the original Ssalp or Ydjlp, a 45 kDa heat shock translocation was restored. presecretory Prepro-a-factor Saccharomyces temperature-sensitive transformed denaturing reactions Furthermore, protein that modulates Ssalp, formed stable complexes with histidine-tagged ppaf. Together these results suggest that 70 kDa chaperones facilitate posttranslational translocation by binding presecretory proteins and maintaining them in a translocation-competent conformation. Monday. Stress Response Genes (1273-1274) 1273 EXPRESSION OF HUMAN HEAT SHOCK PROTEIN 27 kD IN BOVINE ENDOTHELIAL CELLS ALTERS CELL GROW1H ((RS Piotwicz', LE Weba&, E Hickey2, and EG Levin')) 'Dept. of Mol. and Exp. Medicine, The Scripps ResearchInstitue, La Jolla CA, 92121; 2The Univ. of Nevada, Reno, NV, 89557. Heat shock protein of molecular weight 27 kD (HSP27) exhibib ehancdexp and phosphoylation in endothelial celis exposed to activaing agents, ic stimuli el have bon or stessfl conditions. Bovine pulmonm y erial endo l tsuzsfbcted with either the complete human HSP27gum or a mutageniard gme which lacks key phosphoyLion sites (seine residues 15, 78 and 82). The enogeo bovin analog is sightly smaller (MW 25 kD), emsts as ree isofns ad fracionates witn . staining of the a hypotonic rleasate and an NP40 homog Immunofuoe bovine HSP25 show puctatio associated with plasma membme along with diffuse cytosolic staining. Both exogenous gene products co-localize wihi the HSP2S. The wivdtype human HSP27 is phosphosylated in respons to endogenous similar agonists which rsult in edogenuHSP25 phosphorylion, genati phospho-isoforms. Tritiated thymidine inporon into thiten clon exp wildtype human HSP27 was measured and compared to the meain obtained fom a panel of eleven vector control cloes. On average, the HSP27 exprsing clones in amomunt of triated thymidine inrpona-ed. of celis expressingthe mutgenied human HSP27 (which can not be phosphorylated) grw at significantly siower sates and exhibited masked decreases in thymide incoposation. These data suggest a causive relaionship betwee HSP27 expreson/phosphorylton ad the enhacd growth of endodheal cels in response to agents (immune andthrombogenic fctors) and even (wounding) which activate the a three-fold In contsast, culs 219a 1274 HEAT SHOCK PROTEIN (HSP27) IN MELANOCYTES. (T. Dizon, D.A. Reilly, M.D. ) Departments of Plastic Surgesy and Dermatology, University of California Davis Medical Center, Sacramento, CA 95817. (Spon. by R.R Isseroff.) Melanocytes found in the epidermiis are responsble for an individual's skin color. Because of their superficial location, these cells react to such How thes cells react to V light and heat. of our invetigaton. In particular we have protein expression after exposure to an environm ental streses as such sesses is the topic focused on heat shock environmental stress. Heat shock proteins are synthesized within human fibroblasts and keratnocytes after exposure to stress. It has been serve to protct the cel from hypotheszed that these protei environmn insults. Using indirect immunofluoresence, we determined the cytoplasmic presence and location of low molecular weight, 27Kdprotein (HSP27) in unstrssed cells. Using sodium arsenite as an oxidative stress inducer, HSP27 was then observed to translocate from the cytoplasm to the nucleus. In contrst, in the unteated control cells, HSP27 remained in the cytoplasn. Isoclectric focusing gel analysis was used to identify two different isofonns of HSP27. There is also an increase in the amount of HSP27 after stress. The precise role of heat shock proteins in meanocytes has yet to be documented, however given our initial characteization of these proteins we are one step closer to understanding this most remarkable cell the meanocyte. endoelium. Sperm and Spermatogenesis II(1275-1278) 12T5 12TS IMIJNOCYTOCHE3ICAL LOCALIZATION OF SPE-4, A PROTEIN REQUIRED FOR SPERMATOGENESIS IN C.ELEGANS.((P.Nichele Arduengo, O.K. Appleberry, and S.W. L'Hernault)) Dept of Biology, Emory University, Atlanta,GA 30322 IDENTIFICATION OF MULTIPLE PROTEASES IN GERM CELL-CONDMONED MEDIUM (GCCM) THAT AFFECT SERTOU CELI (SC) SECRETORY FUNCTION ((G. R. Arvinda, C. Pineam B. egon, C.W. Badin ad C. Yan aimn)) The Populaion Couil, 1230 York Avenue, New York, NY 10021. Germ odl (GC) developme in the testis ivelves exensve pwiapaton ad coordnaion of poteme ad prowe inimbito that allow the traocaton of GC from the bas to the adluminal crmps and the rease of m re spermatoa into the seminifeus tubul Spermatogenesis in C. elegans uses unusual organelles, called the fibrous body-membranous organelle cosplexes (FB-NO), to prepackage and deliver macromolecules to spermatids during their formation. The FB-NOs are segregated non-randomly into the spermatid during the second meiotic division while non-essential components are discarded in a structure called the residual body (RB). Mutations in the gene spe-4 (spermatogenesis defective) disrupt FB-HO morphogenesis and cytokinesis during spermatogenesis. Consequently, spe-4 mutants arrest spermatogenesis as terminal spermatocytes that contain four haploid nuclei; spermatids are never formed. The spe-4 gene is predicted to encode a novel 465 amino acid transmembrane protein. The ultrastructural phenotype of spe-4 cells suggests that spe-4 might reside in the FB-MO, but it could possibly be found in the plasma membrane also. To localize SPE-4, polyclonal antisera were generated against an 114 a.a. hydrophillic portion of SPE-4 fused to GST. Preliminary results reveal sperm specific punctate staining of wildtype sperm during all stages of spermatogenesis. This is consistant with FB-MO localization. Additionally, in wildtype sperm, signal is excluded from the residual body, as predicted for a protein located within the FB-NO. Future studies will look at SPE-4 localization in the six alleles currently available. lu1n _ile mntining the integrity of the bkod-1seis bwsier. Wehaveexmined if peimmy clture of GC roel _e any protes using ILSI-colagen substrate ins liquid-phse aay. When GCCM was prpeduo poedure involving trypization 26kDap---n tha ws purfied to appwent hemngncqity by sequential HPLC frm GCCM which yield a w a sdendnt inhibitio on Seatli el (SC) testin film secretion wa subequendy shown to trypsin to oine trypsin by GC and that typin at doses betw sewetion protease 10 pg ad I >g/ml indeed inhibited SC testin pdnmy cultures of SC, conclude that this aoigated from the typsn used for the GCCM prepuatoL To further examine in an is Witro bioasy system using whetdr other factonwe GC from ahult rat tese by ent we GCCM that afect SC echicsl procetur withdwo soeerery function, the we have ueof ay enzymatc prepued _tatments codition at 32 'C for 20 hi while nuntemning cdl viabiity of gter tha 90% at the end of the culture pesiod. When a 10-lite batch of GCCM was frcionated by prepswative anion-exchange HPLC, three peas of ptease activities were detcted togete with three peas of biocativity that modulate SC testin secretion. Two of these protease activities were co-eluted with fractions that modulated SC testin saretian whras the third out did not ovesap. One of thee proteaes designated GC-1 that cx stidmle SC testin sewemoa hs been puried to sptw hemnogeneity by sequentia BPLC using C8, C18, ad CCI8 reveed-ph solums which displayed appun Mr of 30,000 on SDS-polyacrylnide gd ude ucig conditions. In smmary, GCCM prepared by mechaoical without eazymatic teatme centais multiple biological factors affec SC testin eetion, seme of wich also edhibit prose acivity when daemined supported in pst by NIH gr aay. Tis wkw HD-13341. ad cultured seun-free a cedures vitro a 177 1278 EPrITlLIAL(E)- AD NEURAL(N)-CADHERIN PES SION IN THE DEVELOPING RAT TESTI AND OVARY. ((J-C Wul, L-FH Linl, MJ. Wheelock2, KR. Johnson2, and RM. DePhilipl)) lDeptment of Cell Biology, Neurobiology & Anstomy, Ohio State University, Columbus, OH 43210 and 2Depa1ment of Biology, University of Toledo, Toldo, OH 43606. GENETIC CONTROL OF GERM LINE RENEWAL IN THE DROSOPHILA TESTIS ((G. Hime, M. Fogarty, M.T: Fuller)) Deparm nt of Developmental Biology, Stanford University School of Cadherins have not yet been localized at contac stes betwee Seoli cells ublot analysis were scopy and ncen in the testis. Imnlh used to exploe the exWession of epitheial(E) and neural(N)-ca rin during the period of SaWfi cel (blood-tsts) barrier fomation. Throughout the frst four week of postnal developmet, E-cadherin was assocated win of the seninHers tubules, q il inaecular ree testis, and intstitl cells pre ed to be Leydig cells ESertoli between at conta of cells In cadhein was not detcted areas contrast, N-cadherin was llizedin de bal reon of seminiferous tubues of 3 and 4 week old esds, and rcatnin. N-cadherin a-, and the three catenins wae also locaHzed at contact sies between Serooli in tess and cells in culue. Cmpison between cadhn l i ovary reveald similarities that my eprs conmon featues of function. Germ cels in both orans expressed E-adherin, while supporting cels in both orgns (Serli cdls and ganuksa cells, resctvely) expressed Ncadhein Interstital cells in both gonads also expressed E-cadhrin. These results suggest roles for N-cadherin in junction fnation betwen Satoli vent and cells and granulosa cells and for E-cadherin in germ cell interstial cell egdon. with germ cells ces as were -, be through direct protlin sequencing (NH2-IVGGYTXAAN). Since falled confirm the synthesis of _mmunoprecipitation experments mig PS1S5-tn porcine Medicine, Stanford, CA 94305. Adult germ cells in Drosophila are constantly being replaced as they differentiate, by division of a small number of stem cells. In adult males the 5-7 germ line stem cells per testis are located in close physical association with the somatic hub cells at the apical tip of the testis. As the stem cells divide, one daughter remains associated with the hub, and maintains stem cell character, while the other daughter is displaced from the hub and differentiates into a spermatogonial cell. As the germ cells mature they move down the testis, such that cells at more advanced stages of spermatogenesis are located further away from the hub. Function of the shut off and 406 genes are required for continued renewal of the male germ line. In adult shat off males, cells at advanced stages of spermatogenesis including mature sperm are present, but cells at the early stages of spermatogenesis are missing. In newly eclosed 406 males, all testes are rudimentary in morphology, resembling agametic testes, but most contain a small number of sperm bundles and no early gonial cells. In wild type males, the hub cells are the only cells in the testis apical tip that express fasciclin III, a cell adhesion molecule. Using an antibody to fasciclin HI, we have detemined that the hub fasciclin Im staining dis as in shat off testes at about the same time that the germ line stops renewal, and in 406 mutants the hub becomes disorganized after the larval stage. This may indicate a role of the soma in stem cell maintenance. Sperm and SpermatogenesisII (1279-1284). Monday 220a 1279 LOCALIZATION AND REGULATION OF CONNEXIN43 IN THE RAT EPIDIDYMIS. ((D.G. Cyr8, D.W. Laird', andL. Hermo")) Maurice-LamontagneInstituta, MontJoli,QC,bDepartment of Anatomy and Call Biology, McGill University, Montreal, QC, Canada. Gap junctions are composed of complementary membrane proteins, connexins, which form a pore connecting the cytoplasm of adjacent cells. While gap junctions are present in the epididymal epithelium, little is known about the proteins which make up these junctions ortheir regulation in this tissue. The objectives of this study were to determine the presence and regulation of connexin43 (Cx43l inthe ratepididymis. Immunolocalization was done with antiCx43 antisera and either horseradish peroxidase or immunogold detection. Our resuits show that Cx43 was localized between principal and basal cells but not between adjacent principal cells where gap junctions have been characterized morphologically. At the electron microscope level, Cx43 was present at sites along the plasma membrane where basal cells interdigitate with principal cells. In order to determine if Cx43 was regulated by a testicular factor or specifically by testosterone, rats wereeither orchidectomized or orchidectomized and given a testosterone implant and sampled 7 and 14 days post-surgery. The testosterone implant (18.6 cm) raised serum concentrations of testosterone to levels present in the epididymis. Cx43 immunostaining between principal and basal cells in orchidectomized rats was stil detectable despite the absence of testes. Furthermore, in the initial segment and caputepididymidis, but not in the corpus or cauda epididymidis, staining was also present between adjacent principal cells. In epididymides from orchidectomized rats which received testosterone, Cx43 immunostaining was similar to controls. This is the first report that gap junctions are present between principal and basal cells. These results also suggest that the intracellular targeting of Cx43 to specific cell to cell interfaces is segment specific and androgen-dependent. Supported by MRC. 1281 AUTOPHOSPHORYLATION OF THE C-KIT RECEPTOR IN RAT TYPE A SPERMATOGONIA. ((M. Dym, M.C. Jia, G. Dirami, J.M. Price, S.J. Rabin, I. Mocchetti, and N. Ravindranath)) Department of Cell Biology, Georgetown University Medical Center, Washington, DC 20007. expression and activation of the c-ldt receptor in rattype A spermatogonia was from 9 day old rats and subjected to sequential enzymatic digestion. The mixture of testicular cell types was then separated by sedimentation velocity at unit gravity and the isolated type A spermatogonia were characterized by light and electron microscopy. They exhibited spherical nuclei containing several nucleoli and associated chromatin clumps and organelles generally in a perinuclear location. Syndtesis of the c-kit receptor by the spermatogonia was established by hybridization of total RNA with a specific cDNA for mouse c-kit receptor. Two mRNA transcripts migatng at 4.8 Kb and 12 Kb were observed. Localiion of the c-kit receptor in the isolated cells was determined by immunocytochemistry. Specific staining for c-kit receptor was observed in the cytoplasm of the isolated type A spermatogonia. Furthermore, the presence of the ckit receptor protein in the spermatogonia was confirmed by Western blot analysis using the same antibody. The antibody recognized the ckit receptor at -160 kDa. In an attempt to determine whether this receptor has a functional significance, we examined the effect ofkit ligand on the phosphorylation of the c-kit receptor. The c-it receptor appeared to be constitutively autophosphorylated on tyrosine at low basal levels and upon stimulation with kit ligand, the amount of phosphorylated protein increased significantly. These observations indicate that kit ligand induces autophosphorylation of the c-kit receptor which may lead to the activation of other celular target proteins responsible for spermatogonial proliferation and/or differentiation. The examined. Testes were obtained 1280 GERM Y IN THE ADULT RAT PROM SPERMAIOGONIA TO CELI -MORPHOMI SPERM((LR. IPm,M. S ,dbegand LD. Russell)) Depwqn tof Phbysolo of Stmcaral Biology, Souten lifioisn Univs, Carbondale, EL62901. LaboraIty One of the most amazing cell tran isdIaracsized bya prolifraavephase( occurs during a apeI oeeisTs process genetc rM division phase (melosis of spmuocyes), ad adiffean pe (spermatids). present stIdy forth to chaacteize the entie 7.5 week prces in ae usig quantitative exesion of cell mopolo coreatd with functona evens om se, cll surace volumes and surface mch that stuctr were areas zed to de for viualy al cell rat ouoldbe pm ua d rmaie cell bcelular paramet of adult rat germ cells, including sperm. Over three thousand electron were tab consuct monges from four nimasat min ofthethurees miropa ofthe ermogenkc cycle (Russell et aL, 1990). Viealy al germ cell components of tbeir dynamic properties associated with specific phaa For example the devw dsow surfae of smooth ndopasmic reticulum, expresd per increased subsamty (about x25) in gam cel development fom type A rmagonia diplosene It halved during the two meiotic divons, buticea slighty spermatids step 6 of md declined slowly 16-17 (is sta I-I). accelerated decra occurred in sbsequet step reuting in overll 52fold depki ofthis orlnele fm its peak at pachytee (930pm*e) its nair t step 19. in smooth endoplasmic temendous membrn and numae orpnelks occuring during late paled therise in lipid ineloogat spermatids, sugesting that lipid of spermatids is a reservoir for degraded smooth endoplamnic ticulum and other membanowuscompoen Membra processed to lipid reservoirs prior to permiation would not overwhelm th Setoli cell's ability o degrade membranous componts at the end ofsrmio also explains the pid cumulation in Setoli cells post spmmit. The nature ofthe methdolo fored ustoqutify germ cell subcellular components and necessitated that several structures, yet unnamed, be characteized and desnibed The data provide a srucura basis for physiological events relating to germ cell development Suppored, in part, by Brazilian Reseich Council (CNPq). ar an cel, sraOCY in young unl er ep An to reclum other rm It all 1282 IMMUNOCYTOCHEMICAL LOCALIZATION OF GERM CELL RNA-BINDING PROTEINS p48/p52 TO SPECIFIC STAGES OF MALE GERM CELL DEVELOPMENT AND THE CHROMATOID BODY. R. Okoa, R. Korley5, M. T. Murrayb, N. B. Hecht' and L. Hermoa, McGill University, Montreal, Canada&. Wayne State University, Detroit, MI 48202b. Tufts University, Medford, MA 02155c. Sequence-independent zaRNA-binding polypeptides, mRNP3 and URNP4, are associated with a pool of stable nontranslated poly(A)+mRNA in Xenopus oocytes. Proteins homologous to URNP3 and mRNPA (p48/52) have been identified in male mouse germ cells. Western and Northwestern blots of testes and isolated germ cells indicate that p48/52 are present during meiosis but reach highest levels post-meiotically at a tine when many mRNAs are stored [Dev. Biol. 158:90-l00 (1993)]. Here we analyze the distribution pattern of p48/p52 in the rat testis by LM and EM issunocytochemistry. p48/p52 izasunolabeling was found to be predominantly cytoplasmic and germ cell and stage specific. It began in early pachytene spermatocytes, progressively increased during meiotic development, reached a peak post-meiotically in round spermatids, and gradually declined in spermatids undergoing nuclear condensation and elongation. A proportionally high concentration of cytoplasmic was found in association with the lacunae of the granulofilamentous chromatoid body. The pattern of synthesis of these mRNA-binding proteins together with their presence in the chromatoid body suggests their role as germ cell specific mRNA stabilizing and/or storage proteins. Supported by MRC of Canada and NIH. isosunolabeling 1284 LEYDIG CELLS OF TESTIS BUT NOT IN GERM CELLS. S. Igdoura, THE L. Hermo and C.R. Morales, Anastasiades, Department of McGill Canada Montreal, Anatomy University, Biology, a-MANNOSIDASE IS II IN EXPRESSED SERTOLI AND RAT ADULT Cell and 2B2. a-Mannosidase processing a is II transmembrane modification of and Golgi enzyme involved in N-linked oligosaccharides. study, mannosidase II was immunolocalized in the epididymis using light and electron microscope Paraffin sections of the testis and immunocytochemistry. present and were epididymis staining specific. appeared against the catalytic light microscope, the Golgi antibodies In the II. cells was immunostained. The pattern of the Sertoli Golgi apparatus was stage At VII-VIII of the cycle the Golgi apparatus stages as a distributed not deeply the of microscope and Sertoli of the cycle, incubated with mannosidase of apparatus Leydig stained cell to anastomotic network the lumen. and extending from At other stages of the Electron supranuclear. imunocytochemistry revealed iunogold particles saccules of the Golgi apparatus. Mannosidase localized in germ cells. In the epididymis, a reaction was basal over over narrow cells of the initial caput, corpus and cauda regions. type specific expression of within the testis and epididymis suggesting mannosidase specialization in the processing of N-linked oligosaccharides. Supported by MRC of Canada. was segment and noted clear results thus II functional exclusively cells of the demonstrate cell BCL-2 AND BCL-X GENE EXPRESSION DURING CHICKEN SPERMATOGENESIS. ((X. Vilagrasa, C. Mesquita and J. Mesquita)) Molecular Genetics Research Group. Faculty of Medicine, University of Barcelona, Casanova, 143, 08036 Barcelona, Spain. The bcl-2 gene product has the ability to block apoptosis and may confer stress spermatogonia and Sertoli cells, which are unaffected by temperature elevation and other stresses. We have determined RNA levels of bcl-2 and bcl-x during spermatogenesis by Northern hybridization and PCR amplification using bcl-2 primers that amplify a region of 362 bp that comprises the C-terminal coding region (aa 190- 232) and 227 bp of the 3' region of the gene. The bcl-x primers amplify a region of 617 bp that corresponds to the whole coding region of the bcl-x gene (Boise et aL, 1993, Cell, 74:597). For Northern hybridization experiments the PCR products were used as probes. Bcl-2 and BcI-x transcripts are present in chick embryo testis and in 5-6 week-old chicken testis, where of the basal layer of cells, the spermatogonia, occurs. Later in spermatogenesis, when most of the cells are meiotic and postmeiotic, the bcl-2 and bcl-x mRNA's are no longer detectable. When the chick embryo was exposed to increased temperatures (42°C) for variable periods of time (5-24 hours) the expression of bcl-2 and bcl-x genes in the testis persists elevated. Bcl-2 and bcl-x are developmentally expressed in testis cells which are particularly resistant to hyperthermia and -resistance to certain testicular cells, such us niultiplication other stresses. Monday. Sperm and SpermatogenesisII (1285-1290) 221a 1286 1285 ZATIONOF RETINOIC AACID RECEPTOR-a TRANSCRIPTSIN THE RAT EPIDIDYMIS. ((K.M. Akmal and K.H. Kim)) Department of Genetics and Cell Biology, Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164. DIFFERENTIAL As spermatozoa traverse the epididymis, they undergo physiological, morphological, and biochemical changes which resuit In their maturation. The epkiddymal epithellum provides the appropriate microenvironment for these processes. Abnormalities in the epididymis have been reported in vitamin A-deficient animals. Although the mechanism of vitamin A and The development of spermatozoa in mice procedes normally at is severely inhibited by higher temperatures. To examine the effects of LOC heat stress action on the epididymis remains unknown, Itis postulated that the effects 3S-methionine, and the proteins extracted with 4% trichloroacetic acid (Hlt) 44°C 1288 LIGANDS FOR THE IGF-I1CATlON-INDEPENDENT MANNOSE 6-PHOSPHATE RECEPTOR MODULATE GENE EXPRESSION IN SPERMATOGONiA, PACHYTENESPERMATOCYTES AND ROUND SPERMATIDS. ((J.K Tsunra and DA. OBrien)) The Laboatories for Reprodudive Biology, Depts of Pediabis and Cell Biolgy & Anatonw, Univerity of North Carolna. Chapel HE, NC 27599 MANNOSE and round levels in both pachytene spem This elec is completely abolshed in the presenceof mM ManS-P, that this change in gene expression is mediated specifically by the IGFIU+I-MPR. Nolthem analysis and irnmunohistochemrstry have shown recently tha spermtogonia and eary spermatocytes contain markedly higher levels of ceels at ater stages of developnent K; VCIMPR mRNA than se Spermatogonia were treated wIth Scm, IGF-II and two IGF-II analogs to determine If spermatWds. indicag modubete IGFIVCI-PR expression marked, these celis. Spermatoonia dose-dependn inceaes l8S rRNA which were 3-fold highwr han changes observed in round spermatids and may reflect hb her cell-surface levels of the receptor in thesecelis. Quantitative RT-PCR wasused to determineretie leves of c-fos mRNA In spenratogonia after treatment levels the IGFNUC'I-MPR) or [g" IGF-ll (an analog that IGF-11, IGF-11 (an anabD VW binds to only the IGF-I receo). Both native IGF-11 and [Leu27] IGF-11 was not IGF-II mncreasTo-fos mRNA levels to sinilar lvls whle jArgS able to increase c-fs mRNA levels. Thus, MLP-bearing glycoproteins secreted by enic cels and IGF-11 Increases Sectoli cels aier gene expresslon in sperm of with [LuzY c-its mRNAspecificaly through the IGFIIICI-MPR. The secretion by Seroli cells may act as knportant cues for the reguiation of PR IGFIIIC spermatgenic cell gene expression durig spernatogenesis. Supported by NIH spermatid grant HD2"8 6-PHOSPHATE-BEARING GLYCOPROTEINS ARE ABUNDANT IN ((D.A. O'Brien', P.L. Magyar', D.E. SIeat2 and P. for Reproductive Biology, University of North Carolina, 'Laboratories LobeI2)) Chapel Hill, NC 27599. 2Center for Advanced Biotechnology and Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854. MOUSE TESTIS AND BRAIN. 6-phosphate receptor(lGFIICI-MPR) medates the uptake and targeting of mannose 6-phosphate beanngg copotems P-berg (MBP-gp) and triges second messenger cascades alter bind reported previously that Seytlcel condtd medium growthbtks or IGF-11.US (Scm) and MP-gppurfioed om Scm are able to elicit dose-dependent increases in of c-fos mRNA in spemaogenic celi (pedominantly steady-state spermatocstes and spemats). VWk nowreport that Scm ecits dose-dependent ls able to the treated with Scm demntae oigands andthe Melon Foundation. et 209:156) glycoproteins (M6P-glycoproteins) in paraffin sections of several mouse tissues using standard avidin-biotin immunoperoxidase methods. Binding specificity to M6P-glycoproteins was verified by inclusion of 5 mM M6P in the incubation buffer, which completely abolished sCI binding. M6P-glycoproteins were not detected in liver, pancreas, stomach, duodenum, large intestine, salivary glands, spleen, lung, kidney, thyroid, adrenal, pituitary, ovary, uterus, prostate, seminal vesicles, heart orskeletal muscle. However, M6P-glycoproteins were prominent in vesicular structures in the seminiferous epithelium and in large neurons in several regions of the brain and spinal cord. M6P-glycoproteins in the testis particularly abundant in Sertoli cells and in pachytene spermatocytes. Adjacent sections were immunostained with a monoclonal antibody to LAMP-1, lysosomal membrane protein. The distribution of M6P-glycoproteins and LAMP-1 was distinct in both brain and testis. Multiple M6P-glycoproteins were detected with 12Sl-labeled sCI on protein blots from these tissues. Our previous studies also indicate that Sertoli cells secrete several M6P-glycoproteins (O'Brien et al., 1993; Biol Reprod 49:1055). These results suggest that M6Pglycoproteins serve unique functions in the testis and brain, where they retain the M6P recognition marker and do not appear to be localized exclusively in and NSF DCB-9118681 (PL). lysosomes. Supported by NIH HD26485 were a (DAO) 1290 H IT PROMOTER. EVIDENCE FOR MULTIPLE CIS-ACTING TESTIS ELEMENTS. ((SA. Wolfe, J.M. vanWert, and S.R. Grimes)) Research Service (151), Overton Brooks Veterans Administration Medical Center, Shreveport, Louisiana 71101-4295; and Department of Biochemistry and Molecular Biology, Lousiana State University Medical Center, Shreveport, Louisiana 71130-3932. SUGGESTS proximal promoter of the testis specific histone Hlt gene from the human, monkey, mouse, and rat were compared for nucleotide homology. The HIt promoters are highly conserved from nucleotide -107 to nucleotide -24 upstream from the tracriptional initiation site of the sequence ((J.M. AN IMPORTANT ROLE IN Research Service (151), Center, Shreveport, Biochemistry and Overton Brooks Veterans 71101-4295, Louisiana Molecular Spi Analysis synthesized using the rat H It promoter sequence containing TE 1, TE2, and the centrally located SpI consensus (5'-GGGGCGGGG-3) does bind to SpI as determined by supershift assays using polrclonal anti-SpI was not antibodies, This workc was supported by the Veterans N.I.H. Grant ROIHD29381-01. Administraion and Grimes)) and Department Medical of LA 71130-3932. The testis-specific histone H lt is gene transribed spermatocytes during spermatogeness. cloned and sequenced and its mammalian species with regard unique to rat to sequence of the expression specific revealed the gene assays does not and TE2, Nuclear elements. sequence appear to codon, and promoter that contains to Veterans Administration sexually extracts the bind other two located proteins Within the from coding S I nuclease start ste Northern 66 blot nucleotides analysis of the indicated that histone H It mRNA is using nuclear to revealed 93% identity with the human trsnscription translation start of the H It binding GC-rich box. genes and 61% identity with the and accumulates only in testis from binding lt designated TE1 and human H shows protection analys upstream has been that of protein these conserved region, the primary It region compared H1/CCAAT the H to elements, promoter between the H 1/AC box and only in The conserved elements and promoter promoter contains two elements that bind to nuclear proteins from the testis, TEi and TE2. These two elements are spaced approxmately 10 nucleotides apart and flan the GC-rich element. Binding to the TEl element has been characterized using methylation interference. croshnling experiments indicate that a complex of testis-specific proteins that bind to this element have a molecular mass of approximately 180 kDa. The GC-rich element, common to all of the cell cycle dependent histone H 1 promoters and the H lt promoter, contains of a 39 bp oligonucleotide that S.R. and Administration Biology, LSU Medical Center, Shreveport testis bind specifically sequence. SAL Wolfe, H.R. Panek, P.S.Merchant, vanWert, rat nucleotide sequence, and they are essentially identical from the H1 /AC box through the TATAA box. In addition to the four conserved elements that are common to cell cycle dependent HI promoters, the H It consensus HIT PROMOTER ELEMENT TESTIS-SPECIFIC TRANSCRIPTION. A CONSERVED MAMMALIAN HISTONE binding activity. Studies of the WV- (sCI) A soluble form of the cation-independent mannose 6-phosphate receptor al., 1993; Anal Biochem isolated from fetal bovine serum (Valenzano was biotinylated and used to localize mannose 6-phosphate-bearing 1289 ANALYSIS OF A HIGHLY CONSERVED REGION WITHIN THE HISTONE The protein synthesis in various testicular cells, seminiferous on tubules from adult mice were cultured at 320C, 37°C or 440C, labeled with 1287 mannose PROTEIN ON Synthesis of the testis-specific analyzed by acid-urea PAGE. in pachytene primary spermatocytes was isoform of histone Hl slightly reduced by incubation at 37C, and sharply reduced by incubation at 440C. In contrast, 440C slightly reduced synthesis of transition proteins To determine whether heat stress 1 and 2 in elongated spermatids. inhibits translational initiation or elongation, the distributions of a variety of mRNAs in extracts of cultured tubules were analyzed in sucrose gradients and Northern blots. 440C greatly reduces the size of polysomes translating the Hlt mRNA, the lactate dehydrogenase C mRNA which is translated in pachytene spermatocytes and round spermatids, and the sulfated glycoprotein 2 mRNA which is translated in Sertoli cells. slightly reduces the size of polysomes translating the transition protein 2 and protamine 2 mRNAs in elongated spermatids. In combination, these results suggest that the initiation of translation in pachytene spermatocytes, round spermatids and Sertoli cells is more sensitive to heat stress than in elongated spermatids. grant HD25094). F-Iwcation-independent SHOCK HEAT were mediate vitamin A signal transduction in the epididymis, its expresslon pattem was Investigated using a rat RARa cDNA to perform Northem blot analyses and a rat RARa cRNA to carry out in situ hybridization analyses. Northem blots revealed two major RARa transcripts, 3.4 kb and 2.7 kb, expressedin the epididymis at approximately equal levels. Results from the in situ hybridization analyses demonstrate variation in expression of the RARa along the ingth of the epkidymal duct. RARa is expressed in the initial segment, then increases to its highest ievelsin the proximal caput. RARa expression then decreases to low levels in the corpus and caudal regions of the epkdidymis. These results suggest that RARa may play a role in spermatozoan maturation in the proximalepididymis The OF 32°C ofvitamin A may be mediated by two nuclear retinoid receptor families, retinoic acid receptors (RARa,p and y) and retinoid X receptors (RXRa, yi. These receptors may act as heterodimeric transcription factors P and in the regulation of gene expression. To determine whether RARa couid (supported by NIH EFFECTS ((L.M. SYNTHESIS IN VARIOUS TESTICULAR CELL TYPES. Cataldo and K.C. Kleene)) Dept. of Biology, University of Massachusetts, Boston, MA 02125. TE N.I.H. mice. Protein from various tissues show elements. site, This work Grant testis- These elements overlap SpI this element and mature but testis Spl supported by ROIHD29381-01 the 222a Sperm and Spermatogenesis II (1291-1296). Monday 1252 THE 5 FLANKING REGION OF THE MOUSE GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S (GAP" GENE CAN DIRECT SPERMATOGENIC CELLSPECIFIC GENE EXPRESSION. ((J.E. Welch, P.R. Brown, E.H. Gouldng, and E.M. Eddy)) Gamete BologW Secton, Labary of Reproductve and Devebpmerntal Toxicology, National Institue of Environmental Health Sdenoes, National InsiuAtes of Health, Research Trangle Park, NC 27709. 1291 ALTERATION IN HISTONE H4 PRaOTER GENE FUNCTION IN SPERNATOCYTE AFTER GOSSYPOL TREATIENT. ((C.S. Tang and N.Y. Yang)) Departmnt of Anatomy, Physiological Sciences and Radiology, North Carolina State University, Raleigh, NC 27606. This research project is to gain an understanding of the machanim of action of a male contraceptive drug - gossypol (C) at the genomic level in rat spermtogenic cells. After C treatment for various times (8 to 19 rk), the spermatogonial cells wera allowed to rest for 2 to 4 wk. The function of histone H4 promoter gene (H4PG) in the repopulating pachytene spermatocytes (RPS) was investigated. The sequances of the oligonucleotides for HIPG binding site I and II were synthesized by an ABI-392 DNA synthesizer. The complementary strands of oligonucleotide were annealed in Tris-saline containing EDTA, and than labeled with a-32p deoxy-GTP by filled-in reaction. RPS and the control pachytene spermatocytes (CPS) were obtained by centrifugal alutriation and subsequently they were used for the preparation of nuclear protein extracts (NPE). The NPE interaction with site I or II was studied by an electrophoresis nobility shift assay (EISA). EKSA of CPS-NPE revealed 5 mjor gel shift band group for sits I and II. After 2 to 4 wrk recovery from 8-, 12-, and 19-irk of G treatmnt, RPS-NPE failed to shift two bands (c and d) in site I. Results suggested that band c and d formed by site I represent binding activity involving transcription factors (e.g., H4TF-1). G treatmnt could affect the factors for interaction with the H4PG site I. No effect was dmonstrated by EKSA of RPS-NPE on H4PG site II (supported by Rockefeller Grant RF 90034). Many enzymes involved in spermfatogenc cel metabolsm are dIfferent from the metaboic enryfes preaent in somaft cels. This Is parduiarly true of enzymes wtich funcion In the hIgy conserved glycolyt pathway. The gene encoding one such s glcoy enzyme, gcerbyde 3-phosphae dehydrogenase (GspdLs), has been cacerzed In our labo y. Wib GAPD-S Is cbsely reated to the somatic GAPD enzyrme, Gapd-s gene exprion Is detectable only In spermatds, durIng the post-melotic phase of apermatogenesia. In orderto detefrrne what regions of the Gapd-s promoter are responsible for thls haploid gene expression, we inroduced DNA construct contairing the Gapd-s pnomoter and the E. co# Pgabclosdase gene into the mouse genome by drect pronuciear Injecion. Identification of the regions drecting haploid gene expression Involved a series of constructs (A,B,C,D) contairing progressive 5' deletions of the Gapd-s promoter. Haploid cell-spedcic expression of P-galaclosidase was detected in rnice Integting constructs wlth 1350 bp (A), 626 bp (B), or 336 bp (C) of Gapd-s promoter secquence 5' to the Initiation of transcrption, but no reporter gene expression was detected in the testes of two founders contairdng 162 bp (D) of Gapd-s pmoter. ThIs would Indicate that the 175 bp region present In the C construct, but rrissing from the D construct Is required to specify haploid cell-specIfic expression. Whie no sequence motifs reported to be involved in spermatogenic cel-sapecifc expression could be lderntfed in this region, possible binding sites for CCMT, AP-3, E2A, ets, and GAGA transcription factors are present. The presence of an ets binding site is inrguing since members of the ets gene fanrily are expressed at high levels In the testis (Rao et at., Science 244.66-70, 1989) and may serve to regulate Gapd-sgene expression. 1293 1294 WITH A RAPID TURNVER O?TESTEN uRaIM OF JUNClIONAL COMPLEX (JC) IN THE TESTIS (Josehne Onma, Prendu Mathw md C. Ya Cbhg) bem Population Council, 1230 York Avenue, New York. NY 10021. THE EXPRESSION the tests, the JC betwe Stoli cells (SC) and Satoh-gSm cels must be disrupted md In rgsneted in a prcidy coordinated famion to allow the pag of dveloping germ cells (GC) from the bsa to the adluminal cmparment while mintaining the integrity of tbe blood-testis barrier (BIB). Testinsha reody boen down to be a cotmp of the JC in the testis mad odbr tissues. Whe miniferous tibeles (SI) isolated from 60-daysd enlturd in vitro, ST e_psd epificandy les test mRNA compad to 10-dayold rats ratswere possibly becue of a extensive if GC have ay effec ae tisue on restuctuing in the tatis in the younge rats. To pesion. OC md GC-conditioned medium testmi mRNA (G0CM) dpreaeby mseadcal proarewidloomyynzaQatic tent_ weacn.sltured ormboad with SC. Both GC ad GCCM isduced virully so effect en the SC tesinamRNA levd indicain the age-related dages in tesis level observed in the ST mny be related not to ofustn is indeed craWed with qaid JC tumover, total RNAs md kideys of pre-. neo-. sd post-nal rats. Testes isoklaed from rat from 3- ad 20-days old rat expressd 3-8 fold testi adult rats. pattn of expemon ws dlar in the kiddwy whe pro md no-natd rats exprsed sigpificmdy maze treted with loidnne, Whben at testin post-natal md adult ic drug known to dirpt JC between SC, ad SC-OC, the testicular testin matiper mRNA levd iwesaed by umch 30-fold withn 24 hr. perked at day 6, mad declid by 15 days consie with the posuate tee the expresion of testin is corated with the rate of IC turnover. Since testis is a namber of the cathepin superfanily. without my apparen proteaw activity, we heve also ained the expession of multiple catepsin sNAs in tese same samples. Cathpsin B. H, L. md S showed respo to dte loidmine _ret wherei C md D showed a sigh incae se demea, raspectivdy, in thsir mRNA in the c tdasarId the uniquere of tein folowing JC following loaidsaine damnge We postulate thett depti of GC by1ondne induoss testi aeeo by SC in attempt to untoais the inteity of the BIB. In cedusica the expesson of tesdt in mad during GC devdopment. the testis is a senstive masker to imoitor the staten of the Tibis work was supported in pwt by NIH gmts HD- 13541 mad DK-0M13. GC. To exuaie whether the expression were a tes mere thm rats rat. s were an no n tstis In Mab Mous Gem Clls Cu-Zn Sup de Dsu i Und Both T p on and Tri Contol Ih ulil Tra from L Dofferertlal Two P e ((W. Ou, C. M Hmo, aid N.B. Heft')) 'Deatmt of Biolgy, Tuft U dsraY, MedIcd, MA 02155, Depminet of Anatofy, McGU UniersIy, Montreal, Cada. (Spon by N.C. Mibum) Cu-Zn supeadde dlsm (80-1) is a ewzme is widely sped in eukeryt-lc coa Nd perfrm a vIal role in protectIg cefll agaonat fe radical dwnage In mouse bt, tee dfeir sizes d OD-1 mFNAs of about 0.73, staes of 0.80 nd 0.93 kB are detected. The 0.73 Kb mRNA is omd In e mae germ cob and in go somatc tisues The mRNAs d 0.80 Kb and .93 kB are aekuIvely detacted in posmidc grm cals F4ie H dlgeilorm and norn blot aly reveal thatththree soD-I mRNAs e doved from two a po met bcts transsdt whIch dwerby abot 114 ucodds. RNae protction assap demonstate tW the addIonal ucleoodas prest in hFe post-"maltc mRNA ae soey In the 5! uriranslatad region (IT. Usn a probe dWerIfdfim the 5S UTR d the tip 8 SOD-1 mRNA, we have establd that the 0.93 kIS mRNA oignats from mnaornaive upetmr promoter contguouB wth the somatic SODI promoter. Polysomal gradIent sanlysIs d the re mouse test SOD-1 mRNAs res tha the 0.93 kB SODI mfRNA is prIwly nonpolysomal, while the 0.80 and 0.73 kB SOD-1 mRNAs are mostly polome associed A faster mIgratIg form of the 0.93 kB SOD-1 mRNA is pest on poloms as a result nl These data d NA tat male germ coa d par transcrbe two sIze clas of SOD-I mRNAs by UOINIng two fferent pmotrs post-mnltc SODI mNAs udWgo adyaton dc g and one of the postmelctIc SOD-1 tancrpts is hInIy stored s klIt is "t d at the end d an 1296 IN-SITU EXPRESSION 0F a-IN5IN IN HUMAN AZOOSPERMIC MALES W1TH SERTOLI-CL ONLY HISTOLOGY. ((CS. Ndrbg, DJ. Lamb,* S. Kim,* S. Maislos,* M Glinz,* S. Pursel S. Rizvi, LS. Ross, LILUpshultz,* of Urolo ad Y. Cho)) Depae and Genetcs, the University of linois at of Cell Biow and Urology, Chiag, Chicag, IL 60612, and *Dea Bayklr College of Medci, Hou. TX 77030 (Spon. by R. Bcaker) In situ hyWiizon present the powerul method to mechaoimS regulati S. and the abeant functon of mechanisms in human infertlty. Unti recenty, the fixatves and techuiques t commonly used to prpare teis issue for in situ hy sverely disoted a cellul demnstrate spe thedelicattstcula specfic gen have andthwarted it exopessed during peviously shon using ema ientify to of tiand cell- and stae diffn We ammble timage anlysis system in the an in Depawtment Pirofesionl gphicslanguagn an Amig 3000 platmn. an a-inhibin indin model in rat Serti cel cultur, that Zamboni's and pfixaes provd suprior siga deten of mnNA and slgul-to-nol ratio compred to Boin's, p adh+ %),hyde(l%), Art Carnoy's. and denstt Zambooi's human rded%) + ot-inhibis exprsion of whom dislayed Seol-cel only evaluationsfor eevated file stmlat in tess of histoy. infertlty t-Inhibibn We sough me, az to a Bght tstis biopss fm 6 males with normal horme (PSE) fixadve. buman high normal lwels wae hybrid ad moderat in situ y with a scdtions diqslyed expresson of inhibin in Serol cels, but the intesil exrson of a-inhibln varied beteoen patents with diffi PSH levds. In siu expression of ct-inhibin in hnman tests may prvide senivc of dinica subtp of infetlty. 227 base pa human a (Supported by American Fow pmbe All a- male bsdon for Urologic Disease Grant NI94 -04) 1296 EXPRESSION OF TiE I EDIATE EARLY GENE NUR77 IN-SlTU IN MURINE AND lNF T[LE HUMAN ISTIS. ((C.S. Ni erb , DJ. Lamb,* L Las, S. Kim.* S. PurwlL S. Rizvi, LS. Ross, LL Tpsliushz* and Y. Cho)) and Geneucs, the Uvrsity of Minois at Chicago, Depart s of Ur ag., IL 60612, and *Departlnts of Cdl Biology and Uroloy, Baylor College of Medicine, Houston. TX 77030 We invesdgaed a putative role for nur77 during murine and human spem ic difSe itio by probing for its expr n using in situ ld oeL Like othr imadiate eawly gunes, nw'77 ws Iadefied in thu search for gem rapidly exprssed as cells in G, re-ene the cdl cyce in response to growth factors. NurT7 cDNA hus boe shows t be bigMy ho to sequencs of membes of the sterdthyroid hmone recptor g superfaily, nd a human homeoog s identified in ests cDNA nd libraries. Using a sucrose buffrd peviousy proem parafomaldhyde fixative optmizd for both mRNA ra_nto and histogic prservation, and a user-r ammed ima anaysis system on a SUN SpaclO pladformusing theDLgraphics lguage. we dputed therelatvecell- ad stae spcfic expressin of murine =wr77 duig mmine mausis. We foud nurT7exreio to be rtiaced to the round smatd in sages IV tough IV imth low lves of Intbstitial expression. Eight dintg use estds bbopse fro 6 azoosPermck mals un ing evaluati for infility with nrmal high normal nd moderately devated folce stmuatn hormonme levels wer t probd in siu wth the humn nur77 qence Of note highly abundant ,sr77 expei was noted in the int tum of these paten who uniformly paten with displayed SerH-i-ol only histology. Expesn vari bet dfg clinical pfiles. Nur77 may ths be a adtia reulat i spermagnic differeti whose abant apaeon reults in hmn male Inft lity. (Supported by American Foundaton for Urologic Disease Grant N194-04) Monday. Sperm and Spermatogenesis II (1297) 223a 129T IDENTIFICATION OF NUCLEAR PROTEINS B1NDING TO THE C4MOS NEGATIVE REGULATORY ELEMENT (NRE) ((W. Xu and G. M. Cooper)) Dqeatent of Pathology, Harvard Medical School, DanaFarber Cancer Institute, Boston, MA 02115 The expression of the c-mos proto-oncogene is highly regted such that its eoxpssion is almost restricted to male and female germ cells, which appears consistent with its role beng a meiotic cell cycle regulator. Previous experiments in our laboratory indicated that an upstream negative regulatory element (NRE) was responsible for the suppression of c-mos transcripton in somatic cells. In order to find trnscription factors imvolved in interaction with the NRE, we used gel shift assay to identiIy nuclear proteins that bind to the NRE in a sequence-specific manner. One protein factor identified in seval somatic cell lines and tissues appeared to bind to a region (box 2) previously demonstrated to be required for NRE activity by site-directed mutageness. This box-2 binding protein was absent in nuclear aetracts of male germ cels, consistent with its role as a somatic cell repressor of c-mos. Sequence immediaely upstream of box 2 was similar to a negative regulatory sequence of germ cell specific phosphoglycerate kinas (PGK-2) gene. This sequence was required for repressng c-mos expression in NIH3T3 cells and for the binding of the protein to box 2. The box 24inding protein may thus be a general somatic cell repressor of the transcription of c-mos and other germ cell specific genes in somatic ces. Fertilization I: Gamete Recognition and Binding (1298-1301) 1299 STRUCIURE AND FUNCTION OF gp55 - AN mZP3-DERIVED GLYCOPEPTIDE THAT CONTAINS THE COMBINING-SITE FOR SPERM. ((E. S. Lischer and PM Wassarman)) Roche Institute of Molela Biology, Roche Research Center, Nutley, NJ 07110. During fertilization in mice, sperm fust bind to mZP3 (.83K Mr), one of 3 n undgo the oome th mabe-up the mouse egg zona peucida, and 1298 ACMVE mZP3 SYNTHESIS OF EXPRESSION ENCODING VECTORS ((S. Mortiio1, TRANSFERASE. 2DtpL. AND a1-3-GALACTOSYL- Litcb&l, D.H. Joziasse2, 1.J. Shspe3, E.S. Amstrdn, Medicinal Cem., Vrije Univ., Ceter, Jobns HpBoins mZP3 TRANSFECTED WrIH STABLY Mod Biol., Roche Rea. Center, Nutey, Waan1)) 1Roche InsL P.M. CV-I CELLS BY Me Netherands, ad NJ 07110, and 3ncology Sb., Balimo mZP3 (-83K Mr) is of 3 glycoproteins tat consattte the egg zx pelncida Dring ferlzatd, sperm to mZP3 0-linked migomcaides that have in a-lika wil t penuhimate sugr, at thek nonreducing end (1J2). EC ativEC-mZP3 iDts the medim saby rsfected wItb the mZP3 bhve been repnted forCV-l acel ansected with exjespfn rvecm (3). Similar Howev, (4). sequence cont ng the compite mZP3 coing since CV-1 cels not have functous al-3-pglacoytrnsfer (al-34T) ge (5), dte results suggst lHe, eatbisd stybly hfeed CVthat mZP3 may nolreqe a-al lines wit used previousy (3) ud cells tbe mZP3 pe CVmZP3 (-71K Mr) into medium. However, unlie mZ3 and EC-mZP3, CV-mZP3 in Med. MD 21205. one eacion mouse cels considerably do a for ac we y. con these secrete speem whether the binding and lack of acsosome tmiml a-al an CV-mZP3 acounts CV-mZP3 wenr c CV-1 ideosfifed wer mZP3/0;1) ino AR-inducing the Tbhs usro. m.73 e_gues Ithe pseneofa-at na itsO-liS O dcide. Ned. Aced. Sci. (1) Bleil, J.. Waarman, (1988). Wassman, P. (1990). Dcwlopswat 108,1. (3) Kinlocb, R. et el. P. 115, 655. (4) Beebe, S. et (1991). Proc. Nad. Aced. 264, Ccs el. Sa. USA Dev. Bfol. 88. ckones (CVad activity of 7401. (6) USA 85, 6778. (2) (1991). J. 48. A beaviy a specific size- s may be glycopeptide resuts rvea tat ies ci (5) GaiU, Swanso, K. Jozise, D. et et. (1989). J. Diol. 151, and bind to mZP3 (3); these o from compble al-3-GT binding tha the on orinlly suggested. was k do Proc. (1992). sem provide fiher cvidence resut ad lo sereterecombinantglycoprotei Purified CV-mZP3/GT exibited both modum. activity ceDls qxesming foud mZP3 both exocytosis (1.2). Sperm recogize mZP3 with papin and pifW by HPLC for furter anlys that (i) In addin to peventng speam fm biding to ovulated eggs, gpSS induces sperm to the AR in Removal of As- (N-) lned oligopclsaies viro. (ii) mdergo gpS55 (glycopeptdase F digestio) reduces its Mr by -35K ad signifantly increass its pI. However, gpSS lacking N-linked iigoach'ide retins both sperm binding and ARinducing activity. (iii) Cleavage of internal P-galactosidic linkages of gpSS odeswrin'_( _ _ _ ~~~digestio) ees its Mr by -15K, buthas no effect on its sperm binding and AR-inducing activity. (iv) Removal of sialc acid from gp55 (ne_xaminidse digestio) gnificantly incases its pL but has no effect o its sperm ke ionct mZP3, gpSS binding and AR-inducing activity. Tbes results suggest polypepdide ad 0-lnkd o uppboth sperm binding and AR-inductio (1) Wasuan, P. (1990). Dcvelopment 108, 1. (2) Wssarman, P. (1993). Semu,rs Dev. Biot. 4, 189. (3) Litcher, E., Wamnun, P. (1993). Trends Glycoci. Glycotech. To detrmine inacvity, for its expoessiovecoitrc enting the wih a1-3-GT coding sequence(6). drsdc_aractorlPsd, ad bovin reaction (AR)-induction assays. fom of of the inative in a smaller (-55K Mr), derived by poteolysis from the C-tminal half of the mZP3 polypeptide, binds to em d. ike intat mZP3, pivents the srm frm binding to ovulated egg in vitro (4). Tbee findings suggest that 0-linbed oh recogized by sperm are present on the -55K Mr mZP3 glycopeptide (gpS5); i.e., gp55 poesses the combining-site for sperm. Here, we extended our analysis of the rlationsip between isoled by digesion glycepepti gpSS ohigosacch-ides ad biolgical activity. seete reas cel (AR). class of SetIr- (O-) lind o._Igosachuid bind enI 5. 369. (4) Rosier 14290. T., Waainan, P. (1992). Dev. Biol. 154. 309. 1301 13W EVIDENCE FOR 5SkD SPERM RECEPTOR ACTIVITY OF A PELLUCIDA ZONA EXPRESSION SYSTEM. Depatmet of Cell Biobgy, EXPRESSED PROTEIN ((Wilkins, Baytor B., Prasad, College of RECOMBINANT RABBIT IN S.V., BACULOVIRUS A and Medicine, Houston, Dunbar, TX ELS.)) 77030. XENOPUS LAEVIS EGG JELLY CONSISTS OF A FIBROUS GLYCOPROTEIN MATRIX TO WHCH ARE BOUND GLOBULAR PROTEINS. ((B.S.Bonnell, D.Reinhart, and D.E.Chandler)) Department of Zoology, Arizona State University, Tempe, AZ. 85287. ZP proteins which ad as receptors for homoogous sperm have been described for species and those proteins have been shown to be important in the of lmmunocontraceptive vaccines.. However deviopment obtaining such proteins in quantity and of a purity suff8entto allow detailed charaderizatIon of bological a number of a and Immunolgical the activity Is frequently complicated by the extrme hetergeneity proteins due to extensive post translational m wo have expressed a cDNA encoding the rabbit 55kD ZP protein of native baukvr expression a as to near used to to system. The soluble, paetlally glyc tions. Therfore using a eukaryotic resuitant recombinant protein, BV5S, ated protein (BV5S) and has been is purified homogensity by lectin affinity chromatography. The purified protein was immunize guinea pigs or was biotinylated and tested for it's ability to bind sperm directly. Both the guinea pig antiseum (GP.a-BV5S ) and FAB purfied from the serum (FAn-a-BV55 ) were shon to recognize naive rabbit and recombinant rabbit and ELISA 55kD protein by one and two dimensional immunoblotting sperm in hi techniques. Blotinylated BV55 bound to capacitated rabbit binding exriments. The blotnylated protein localized to the anterior poxtIon head hI the acrosomal region. Occasional iabeHng of equatorial seen, and these sperm were presumed tobe acrosome readed. GPeiBVS5 ihibited rabbit sperm binding to rabbit eggs hi vbo In a dose dependent nnmre. However FAe-a-BV55 failed to block sperm binding, suggesting that the Inhibitory effect of thisserum was due to stearic hindrance, rtherthan biocking of a of the sperm segments wa igsnd specIfic sperm the devopme for puAtiv rabbit werit was spwm sugest that BV55 is a poeti immunogen vaccine. and that it's role as a ecepor waants fuher hIvestigation. by a gat to BSD lfom fte CONRAD pmgm. site. These data of an Wpoed Eggs from Xenopgg levi are imvested wih tree jelly layers klown as J1, J2, and J3. In this study, we have found, through rotary shadowing of whole egg jelly (WEJ) on ceaved mica chips, that these layers consist of a fibrous matix to which globular proteins are bound. In addition, WEJ, run on a SDS-PAGE gradient gel is shown to consist of- 700 kDa staining 450 kDa with Alcian blue, and prominent with PAS, a 630 90 and 100 kDa bands ain WEJ, sepated into J1, J2, and J3 and then by SDS-PAGE, showed that the PAS staining bands where found pred in J3, whereas the Alcian blue stained band was prominent in J2 and J1. Soubilized egg jelly was separated on a Sephacryl-500 column into fractions which wer analyzed by SDS-PAGE and rotary shadowing The 700, 630, and 450 kDa facons were found to be linear fibers whereas the fraction containing the 90 and 100 kDabands was found to be globulr in foxm Further SDS-PAGE analysis showed that eggs, incubated for 1 to 16 hours in buffer, released low molecular weight proteins inluding the 90 and the 100 kDa bands into the medium; in contras, the high molecular weight glycoproteins remained associated with the egg. These results suggest that egg jelly consists of a stable matix of high molcula weigt gyopoteins to which diffisble low moecular weight globular proteins are bound. Release of these proteins dring jelly hydration may either enhance or inhibit sperm passage and egg frlizability. staining with run sil Fertilization I: Gamete Recognition and Binding (1302-1307). Monday 224a 1302 1303 SIT TEROGENOUS DISTRIBUTION OF LECTIN BINDING XNOPUS LAEVIS EGG JELLY. ((N.M. Mozingo and J.L Molcular Davis, CA 95616. Secon of from the X anuran and Cellular Biology, Universty IN Hedrick)) of Califonia, X. evis d lization. successively tie th iso of 30 lec be essetial in elizat. -lein as with jelly coat layer was xamminedeEenyme-l Te reacvi lectn wer then led A). FITC- in conjunction with confocal micr These stdies of lectin binding sites among heteogenous di J stri within the thee jelly layers. 3 is a thick layer which has a flooclent appearane at its outer aspect. Several lectn bound well to the outer poronof including LcH, PSA, jacain and GS1. However, none of the lectis tested bound to the 3. isarelativel thin portion Jof J2 which non-uniformly stained with WGA. This lectin very intense, band of fluoresce at J1112 inefrface while the li y. Using ELLA we found that most of the remainder of stained showedme reactivity to al lecti howeve, two jelly lay,only bound lay lectins sowed jelly first, TKA, to J and slectivity. 13 while MAA bound only to JI and MAA produced inen fluorescencetaining pattem att heJ12 interac which extended wel into effect of jelly-lecn binding on ferdliztion is currentiy being J1. ininggrant HD0 7131. determined. NM was orted by NIHa labeledlectins ficttion can and as of Molecular microbicides tive ep ((11,. Hedrick, Secto CA 95616 tract C;ellular and by cros- prm c, STD uad -like matrix ae d reby entrappig the cel within a visous To test this hypothei we are quantify prevening; infection and coio the binding of frty to spem seminalplama, seum ant d an ELLA and detminig the viscosity ofloelctins on scretiom sperm wwe bound to 96 plates, fioowed pathogen oeci gell cervic ed usn by vaying concentratons we biotdnylae of m sem or lctin, thn alkaline stae y. revealed an ner the er an 1344 The rate of hydrolyis The maximal and reader. was determined m using a lctin binn kinetic mode plae con Seval lecins, suh as WGA, jaci ConA, and LcH emae sperm, reproductive trat secretions aN stronl whereas bound to male and sWGA and TKA bound Viscosity was detmined using a only the male trct sretions"perm conslate viscoseter at Hr rate. non-Newtonian viscosity. Semen vscosity increasd lneary with ConA concenn and a four fold increas in viscosity was obtained at Semen ezbibited 6.7 mg/mt ConA. As oberved cross-flinked pically, sminal Thee sperm wer plm glycptin aspport obsevtin i micides nd SI proposal that lectins can funcion glcoproin aelaeatemmtrix Sup entapping in par by a fom Lectin BioPharma Inc. to the Universy of California the icelsinacross-linked gffi by 1305 TOPOLOGY OF THE SEA URCHIN EGG RECEPTOR FOR SPERM. ((K.R. Foltz)) Department of Biological Sciences, Division of Cell, Molecular and Developmental Biology, University of California, Santa Barbar, The and Davi are may Some of AS Mah We propose that lectis srounded by a thick jelly cot which is essential during jely coat contains the distinct layer, added to the eg as it ignated Jl, J and 3, which are transits the oviduct. We xammined lectin bning propets of the g a s individ ual jelly coat layes as ep in identifyinjel ly which Egs ANDR LECTIANS ANDRISOBI MLI.ieduWit LJA. Biology, University of California CA 93116. egg receptor for INTERACTION OF THE SEA URCHIN EGG RECEPTOR FOR SPERM WITH THE ACTIN-BASED CYTOSKELETON. ((R. J. Belton Jr. and K RR. Foltz)) Department of Biological Sciences, Division of Cell, Molecular asa Developmental Biology, University of California, Santa Barbara, CA sperm has been purified, cloned and sequenced from Strongylocentrotus gpvWjurga [Foltz et al. (1993) Science 259, 1421-1425; Ohlendieck et al. (1993) J. Cell Biol. 122, 887-895]. Although it is known to be a transmembrane glycoprotein, the exact topology of the sperm receptor is unknown. Several topologies are possible; in addition to the presumed, single transmembrane domain (residues 1003-1019), there are two additional, slightly hydrophobic domains in the 258 C-terminal residues. These hydrophobic domains span residues 1043-1061 and residues 12251238. Because aknowledge of the topology of this protein is crucial to structure-function studies, we have used several approaches to determine the actual topology. First, antibodies directed against defined epitopes of the 931069 used in immunocytological studies of intact eggs. Antibodies directed against epitopes on the N-terminal side of the first hydrophobic domain labeled the membrane in permeabilized and nonpermeabilized eggs while antibodies directed against epitopes located on the C-terminal side labeled egg surfaces only when the eggs had been permeabilized. Thus, the C-terminal epitopes appear to be located on the intracellular face. Further, we used in vitrotranscription and translation of various receptor cDNA constructs in the presence and absence of microsomes followed by protease protection assays. The results indicate that the 258 C-terminal residues are intracellular. Therefore, based on the data of the two independent methods employed, we conclude that the sea urchin egg receptorfor sperm is a type I transmembrane receptor spanning the membrane at residues 1003-1019. Supported by an NIH Shannon Award ,and the California Cancer Research Coordinating Council. Fertilization of the sea urchin egg initiates an extensive reorganization of the egg cortical cytoskeleton. At the point of sperm entry, a fertilization cone is formed, consisting of actin microfilaments that extend out around the sperm, while cortical actin filaments elongatethroughout the egg microvilli. a transmembranous glycoprotein Yhe sea urchinfor egg receptor for sperm is interactions at fertilization. We responsible species-specific gamete that the for is spern associated with the actin-based egg receptor propose egg cytoskeleton and that it also may be involved in thereorganization of the egg cytoskeleton following fertilization. We have shown that the sperm receptor co-purifies with the actin based cytoskcleton, and that recombinant sperm receptor protein co-sediments with a crudefilamentous actin preparation. Using recombinant GST-sperm receptor fusion proteins, we are the actinattempting to determnine how the sperm receptor interactsbe with based cytoskeleton, and what other components may involved. Egg with cytoskeletal proteins were isolated, solubilized and incubated recombinant receptor protein attached to agarose beads. The interacting followed were eluted SDS-PAGE from the beads and by analyzed proteins Our preliminary data indicate that two proteins, of by silver staining. 79K and 32K, are candidates for mediating the caloulated molecular weights interaction. Currently, we are assessing the specificity receptor-cytoskeleton of the interactions using competition assays and are attempting to identify these proteins via antibody cross-reactivity and protein sequencing. Supported by an NIH Shannon Award and the California Cancer Research Coordchating Committee. 1306 1307 KINETICS OF MOUSE SPERM BINDING TO SOLUBILIZED ZONAE PELLUCIDAE. ((C.D. Thaler andRA. Cardullo)) University of California, Dept. of Biology, Riverside, CA 92521. CHARACTERIZATION AND LOCALIZATION OF FLUORESCENT ZONAE PELLUCIDAE ON MOUSE SPERM ((Q. Chen andRA. Cardullo)) Dept. of Biology, Univ. of Califonia, Riverside, CA 92521. sperm receptor were Mammalian sperm first bind to the egg at the zona pellucida (ZP), a glycoprotein matrix surrounding the egg. This species specific adhesion event additionally initiates a signal tranaduction cascade resulting in acrosomal exocytosis. Unlike many signalling systems, the biochemical interactions of the ligand (ZP3) and the sperm surfiace receptor have not yet been charcteized. In this study, we quantified binding of soluble rnI-ZP glycoproteins to mouse sperm and present data defining the biochemical interactions. Live, capacitated mouse sperm displayed a bimodal binding pattern with a transient binding event at -40s and a second binding peak at 10-20 min. Fixed sperm also bound ZPs, displayed similar overall binding as live sperm, and allowed us to quanty binding without iitiating exocytosis. Both capacited and '25I- non-cpadtae sperm bound similar amounts of 'I-ZPs at equilibrium, suggeting that n are required for eps other than spem uface change during cra binding to the ZPs. 12iI-ZP binding to fixed sperm was specific and saturable. Specific binding could be displaced by unlabeled ZPs and non-specific binding was les than 1%. Using apaed sperm that were fixed, equilibrum biding was attained within 60 min at 37°C, and had a t,, of 14 min. Initial shdies of saturation binding indicate a KI in the nanomolar range and a B of 14-20k fixed sites per sperm. These data will allow us to identify the ZP and complementary rcptors involved in primary and secondary binding. Funded by NI HD27244 (RAC) and a Lalor Fndn.Fellowship (CDT). total ZP binding components Theinitial adhesion and signalling events between mammaliangametes involve ligand (ZP3) within a rigid matrix (the zona pelhicida). animmobilid revealed that cAbe broken down events: (1) ligand-receptor adhesion, (2) acivation of effector moleules, and (3) opening of Ca2 chann. Undesanding theseeventa would be sided labeled and used toqantify the gready if ZP3andcould be fluorescently mobilit of the ZP3 receptor on the mouse sperm suface. We distriion have covalently labeled intact zonae pelucidae (ZP) with isothiocyanate (TRITC) at pH 9.0 for 2 hours. TRITC-ZPs were sohilized and separated on anHPLC size excluion column revealng 3 distinct peaks. i Biophysical modeing has this into 3 distinct The relative positions of the three peaks were identical to those using ZP2, and ZP3 indicaing that all 3 labeled ZPs corresponding to ZPI,labeled. were able to bind gycoproteins wre fluorecnt aed TRITC-ZPs sperm and initiate acrosomal exocyosis as determined using a TRITC-ZPs retain adhesion and signal properties similar to native ZPs. In addition, we have used unsduction video microscopy to show that solubilzed TRITC-ZPs are digitaly enhanced to the anterior head of the sperm overly" the aerosomal veside. localized Taken together, these data demonstrate that fluorescent zonae will be usefil tools for studying ZP3 loction and dynamics. Supported by HD27244 and the Whitaker Foundation. Coomassie Blue assay. Thus, roeeptor NIH Monday. Fertilization I: Gamete Recognition and Binding (1308-1311) 225a 1308 1309 A SPERM MEMBRANE PROTEIN THAT BINDS IN A SPECIES-SPECIFIC MANNER TO THE ZONA PELLUCIDA IS HOMOLOGOUS TO VON WiLLEBRAND FACTOR. ((D. M. Hardy and D. L. Garbers)) Dept. Pharmacology and Howard Hughes Medical Inst., Univ. of Texas Southwestem Med. Sch., Dallas, TX 75235. The molecular basis of species-specific adhesion of spefmatozoa to the egg extracellular matrix (zona pellucida, ZP) remains unclear. We previously Idenified sperm membrane proteins that bind in a species-specific manner to ZP. The Mr 105,000 and 45,000 subunits of one of these proteins (p105/45) were pufified from pig sperm membranes based on the ability to bind directly to ZP. Degenerate primers designed from tryptic peptide sequences were used to amplify a PCR product encoding part of the protein; this PCR product was subsequently used as a probe to clone overlapping cDNAs that represent the entire coding sequence of the p105/45 mRNA. The 7785 base composite sequence contained a 7431 bp open reading frame; the sequences of eight tryptic peptides from p105 and five tryptic peptides from p45 were present in the 2477 amino acid deduced sequence, confirming that the open reading frame encoded p105/45. The deduced sequence predicted a 2418 residue N-terminal extracellular region, a single transmembrane domain, and a 37 residue C-terminal intracellular segment. Dot matrix analysis identifed amino acid sequence similarities among four extracellular domains of approximately 400 residues each and a fifth 110 residue extracellular domain. These domains are homologous to the D-domains of von Willebrand factor. The putative extracellular sequence also contained a region of repetitive sequence rich in proline and threonine residues; these properties are typical of mucins. Northem blotting and in situ hybridization detected expression of the p105/45 message only within spermatids. Thus, p105/45 is a spem-specific ZP-binding protein that has structural characteristics similar to two different classes of molecules that participate in cellular interactions; these properties strongly imply a function for p105/45 in sperm adhesion to the zona pellucida. Supported by a grant from the USDA (37203-9024). TRANSCRIPTION AND TRANSLATION OF SP56, A MOUSE SPERM FERTILIZATION PROTEIN, IS RESTRICTED TO SPERMATOGENIC CELLS ((L.H. Bookbinder, A. Cheng, and J.D. Bleil)) Department of 1310 INTERSPECIFIC COMPARISION OF AN ABALONE FERTILIZATION PROTEIN. ((W.J. Swanson and V.D. Vacquier)) Center for Marine Biotechnology and Biomedicine, University of California, San Diego, La Jolla, CA 92093-0202. Abalone are large marine archeogastropod molluscs. There are seven species on the California coast. Fertilization is external and the species have overlapping breeding seasons and habitats. We have been studying the molecular mechanism of species specific fertilization in abalone. Here, we present an interspecific analysis of an abalone sperm acrosomal protein. Abalone sperm possess a large acrosomal granule containing two proteins. One protein, 16Kd lysin, dissolves the vitelline The second acrosomal protein, of 18Kd, coats the acrosomal process of the sperm, suggesting it is involved in fusion or egg activation. The 18Kd protein was purified from 2 species, and shown to have effect on dissolving the vitelline envelope The cDNA sequence of the 18Kd protein envelope. no was obtained from 5 species. The sequences are extremely divergent, ranging from 27%o 87% amino acid identity. Analysis of the nucleotide substitutions demonstrates that the divergence of the 18Kd has been promoted by positive Darwinian selection. In one comparison, the frequency of amino acid altering substitutions (Dn) is 4.7 times higher than the frequency of silent substitutions (Ds) (normalized for the excess number of amino acid altering sites so that Dn=Ds if divergence were neutral). One of the purified 18Kd proteins forms rod-shaped crystals. Molecular Biology, The Scripps Research Institute, 10666 N. Pines Rd., La Jolla, CA 92037. Torrey Sperm-egg recognition in mammals involves species-specific binding of sperm head plasma membrane to ZP3, one of three glycoproteins in the egg's extracellular matrix, or zona pellucida. We have identified a mouse sperm protein, sp56, which has many of the characteristics expected of the sperm protein responsible for recognition of ZP3. sp56 is a homomultimeric, peripheral membrane protein, confined to plasma membrane overlying the sperm acrosome. sp56 is the only mouse sperm protein having specific affinity for ZP3 and ZP3s functional domain oligosaccharide, the part of ZP3 recognized by sperm. cDNA encoding sp56 has been isolated. Primary sequence of sp56, derived from this cDNA, indicates that it is a member of a superfamily of protein receptors. Monoclonal antibodies and molecular probes developed from sp56 cDNA have been used to demonstrate that sp56 expression is restricted solely to spemiatogenic cells of the mouse. Both sp56 mRNA and polypeptide accumulate in round spermatids, which are early haploid cells of the spermatogenic pathway. These tools have also been used to demonstrate that presence or absence of sp56 on sperm from different species can account for species specificity of spermegg recognition. 1311 ANALYSIS OF CELL FUSION IN CHIAMYDOMONAS.((N. Wilson, G.Huang, G.Fletcher, W.Snell)) Department of Cell Biology and Neuroscience, UT Southwestern Medical Center, Dallas, TX 75235. We are interested in understanding the molecular mechanisms of cell fusion between gametes. In Chlamydomonas mt+ gametes, fusion occurs at a well-defined site, the tip of an actin-filled, microvillus-like fertilization tubule. Formation of this fusion organelle is triggered by signals induced by flagellar adhesion. To identify adhesion/fusion proteins we have begun to study fertilization tubules using both genetic techniques and cell fractionation. Screening of 11,000 insertional mutants yielded 7 fusion-defective clones that underwent normal flagellar adhesion, but did not fuse to form zygotes. Thin-section electron microscopy and fluorescence microscopy with phallacidin indicated that 2 mutants, 6D1 and 60A8, formed fertilization tubules that did not bind/fuse with the mating structures on mt- gametes, suggesting a defect in adhesion/fusion proteins. In a parallel approach we have employed cell fractionation to obtain fractionshighlyenriched in fertilization tubules as indicated by fluorescence microscopy and by immunoblottingwith anti-actin antibodies. SDS-PAGE combined with surface biotinylation has identified several proteins as candidates for fertilization tubule surface proteins. Studies are in progress to compare wild type and mutant tubule proteins and to generate monoclonal antibodies against tubule proteins that function in gametic fusion. (Supported by NSF EBM-9318708). Invertebrate Development (1312-1313) 1312 1313 AN EXPRESSED RNA HELICASE GENE IS EMBEDDED IN MANX, A GENE REQUIRED FOR DEVELOPMENT OF THE ASCIDIAN TAILED LARVA. ((E. L Pedersonl, B. J. Swalla2, M. L. Just 3, and W. R. Jefferyl,3)). iCell and Development Graduate Group, University of Califomia, Davis, CA 95616, 2Department of Biology, Vanderbilt University, Nashville, TN 37235, and 3Bodega Marine Laboratory, Bodega Bay, CA 94923. EXPRESSiON OFTHE hG4WGENE IS RKEURED FOR SPECIFYINGTHE LARVAL BODY PLAN IN ASCIDIANS. ((B. J. Swalls1 and W. R. Jeffery2)) 1Department of Biology, Vanderbiit University, Nashville, TN 37235, and 2Sectlon of Molecular and Cellullar Biology and Bodega Marine Laboratory, University of CaliHomia, Davis, Bodega Bay, CA 94923. The manx (uro-1 1) gene was identified by a subtractive screen for cDNA clones expressed during development of the tailed ascidian Molgula oculata but not the closely-related tailless species Molgula occulta. Manx encodes a zinc finger protein that is required for development of the tailed larva. The manx gene encodes 2.3 and 1.9 kb mRNAs, which differ only in the length of their 5' leader sequences. Several overlapping M. oculata genomic clones were isolated that encode the entire manx gene. The manx gene contais 9exons and spans about 6 kb of DNA. The first exon In the manx gene is separated from the downstream exons by a 2.7 kb intron. Surprisingly, this intron contains a single copy gene encoding a putative DEAD box-family RNA helicase with high sequence homology to the yeast and mammalian p68 genes. In contrast to the yeast and nmamallan p68 RNA helicases, the ascidian p68 gene lacks Introns. Northen blots indicate that manx and p68 have the same developmental expression patterns in M. oculata embryos, but both genes are down-regulated in M. occults embryos. However, manx but not p68 mRNA acccumulation is enhanced In hybrids produced by fertilizing M. eggs with M. . oculata sperm, suggesting the two genes can be regulated ind The unusual structure of the manxlp68 gene complex suggests that the p68 gene may have been retrovirally inserted before the divergence of M. oculata andM. occulta. occuvta The zinc finger gene manx (uro-1 1) was identified in a subtractive screen cDNA clones expressed during development of the tailed ascidian Molgula oculata but not the closely-related tailless species Molgula occulta. M. oculata embryos differentiate a brain sensory organ, a notochord, and tail muscle, whereas these tissues are lacking in M. occulta. The missing tissues are restored when M. occulta eggs are fertilized with M. oculata sperm, suggesting they are specIfied by zygotic genes that are inactive in the tailless species. The manx gene is expressed in presumptive notochord, neuroectoderm, muscle, and tail epidermis in M. oculata but not M. occulta, and manx mRNA accumulates In the same restored tissues In hybrid embryos, suggesting a role in specification of tailed larval features. Hybrid embryos were treated with manx antisense phosphorothioate oligodeoxynucleotides (ODNs) before first cleavage and their phenotypes were examined after hatching. Treatment with an antisense ODN corresponding to the +4/+21 region of the manx gene reduced manx mRNA to undetectable levels, but had no effect on muscle actin mRNA accumulation. The corresponding sense ODN did not affect manx mRNA levels. Antisense ODN-treated hybrids failed to develop the brain sensory organ, notochord, and secondary tall muscle cells, but these tissues developed normaily in sense ODN-treated hybrids. Similar results were obtained with antisense ODNa in other regions of the manx gene. The results indicate that manx expression Ia required for restoration of tailed larval features in hybrid embryos, suggesting that the manx gene plays a fundamental role in specfyng the larval body plan in ascdans. for 226a Invertebrate 1314 Development (1314-1319). Monday 1315 ASCIDDANS WWll EVOLU'rONARY CHANGES IN MUSCLE AC71N GENES IN ALTERNATE MODES OF DEVELOPMENT. ((T. Kusakabebl2, B. J. Swells3, N. 1 Bodega Marine Laboratory, University of Satoh2. and W. R. Jefferyll)). Califoria, Davia, Bodega Bay, CA 94923, 2Department of Zoology, Kyoto University, Kyoto 606-01, Japan, and 3Department of Biology, Vanderbilt University, Nashville, TN 37235. CELL CONTACT DIRECTED SPINDLE ORIENTATION IN EARLY DEVELOPMENT ((S. W. Wang', F. J. GrUn' & W. H. Cr,k Jr.15)) 'Bodeg Laboratory, MarIn Cdlornla, UniversIty of and Aquatic Scince DavI, Bodega Bay, CA 94023. estmet of Fisheries Uniesiy of Foidda, Genevl, Ft 32806. Sdyoh Ingendls During saly ceavages In eby mitoti c spindle orIeta er between blastomr and change a predictabe arnwe dll aat0angle succesive mitoels. From e through cevge, sp orent9 In Molgula occulta are closely-related ascidians with Molgula oculata and different modes of development. M. oculata embryos develop into tadpole (urodele) larvae with striated tail muscle cells, whereas M. occulta embryos develop into tailless (anural) larvae lacking differentiated muscle cells. The muscle actin genes MocuMA 1 and MocuMA2 were isolated from M. oculata genoric library. MocuMA has no introns and is expressed specifically In larval muscle cells, whereas MocuMA2 has four introns and resembles adult muscle actin genes in other ascidian 5' species. Despite lacking introns, MocuMA 1 is a functional gene because its flanking region is sufficient to drive in vivo expression of a microinjected lacZ fusion construct. Muscle actin mRNA was not detected in M. embryos, suggesting that the gene(s) corresponding to MocuMA is not expressed. Two different intron-less genes similar to MocuMA were isolated from an M. occulta genomic library. These genes have nucleotide substitutions and insertions that could make their products non-functional. Hybrid embryos, produced by fertilizing M. occulta eggs with M. oculata sperm, express MocuMA1 mRNA in vestigial muscle cells, suggesting that trans-acting factors regulating larval muscle actin genes have been retained in M. occulta. Muscle actin mRNA could not be detected in embryos of five other anural species, although transcripts were observed in closelyrelated urodele species. Analysis of structural changes in the larval 5' flanking regions, may elucidate the muscle actin genes, including their basis for lack of expression in M. occulta embryos. an occulta embryoswas pall which blatomere The e of Drosophila abdominal hypodermal muscles (AHM) are remodeled larval intersegmental muscles that survive the destruction of neighboring larval muscles during metasmorphosis. These larval muscles then lose their contractile apparati and Subsequently, they become strap-like bands of cytoplasm. regenerate into the muscles responsible for the emergence of pupal case and later for the inflation of the the adult from the wings. We have devised a selection scheme for the isolation of Drosophila mutants with defective AHN in order This procedure has been to study the regeneration process. applied to the isolation of mutants on the third chromosome. We have been successful in isolating several recessive mutants that cannot emerge from the pupal case as homozygotes unless the operculum is opened manually. These flies are also unable to inflate their wings after emergence. We are currently screening these mutants for defects in abdominal musculature by examination of abdominal "pelts" and have observed abnormal AHM. Isolation of ultimately permit genetic dissection regeneration process. such mutants will of this muscle tothe lay hI produced four paralle quartts. Then, ceavage respec 44'ange nrxmaldevWopnt to with orientatIon mitotic embryos. leave We bt rath s regions of ths pealle pairs hI to the substrate. the ret In embryos sugges where th ed spinl poles during Grant eauly deparKewlt but wIh (ceroom"es) cll-cd ontact during en Sea hI se of mitoic spndles nor age (Supported by NOAA as were not d tod orinwtation an property Itrrnc conunctIon thmes Furthr at a wer separated into lkiear halflike tiose observed for Intct In 4-cll lkiea liner emyos regresse hI arage sM ori reore Is mrtherelcel contact related. d nonmranulated cytopimic eeuthhg parll prepartion orietatI sugges of (3W emnbryonic cleavege) foor 5e eavage, spinles previou ceavage plan, rathe than parall In When 8-cl embryos, 8-cd lina Ue recbig the axle of the embryo, r a common pln which we a linea embryo wth the 16 the Is I specific res, vieated wea affected hI coy. embryos, subequnt spidle #NA36RG)7) early ACTIVE NUCLEI FROM ISOLATION ((R.OF A.TRANSCREAONAILY and K. Q. Tran)) Dept of Chemistry and Acey 90840, and ((J. C. Biochemistry, Cal.ofState Univ.,andLong Beach, CAWorcester Polytechnic Biotechnology, Bagshaw))Worcester, Dept MABiology 01609 Institute, the fators regulating gene expressin durng the Attempts at und ofandig mpered by theiabiltyto isolate eaiiy development acie Arma nuclei freehaveofbeen yolk pladets. We report here procedure for theisolation ofactive nuclei from both encysted embiyos and pH 7.5, 10 mM swimming naupl Shrimpare mgenized in 10mM MgQ2, 10 mM NaCl, 0.l%(vtv) NP40 andoentrifuged to obtain a crude buffer without pell The pellet is ruspended in homogenizato nuckl gradients.The nuclei band at the detergentandfiactionated on buffered Prcoll as of well asLDH is platet yolk Percoll/buff and SDH activity. The nuclei in at 32p UTP into RNA for up to 30 l sCi of 32pminutes at 30D C in the following buffersupplmented with 30 UTP: 30 mM Tris, pH 7.6, 75 mM KCG, 2.4 mM Mg(OAc)2, 20 mM 2mM 20 mM DTT, 200 &M 400isM CTP, 400 aM GTP, 100pM UTP, and 0.4 U placental ribonuclease inhibitor. Moreover, the ransciptioal activity of the isolate nuclei mimics the in vivo activity during i.e., nuclear actiiy peaks dwing the first 24 hrs of early development, then deases signifiany. Preinicubation of the nuclei with development and D activity while Actinmycin completdy abolishes their with a-amanitin causes a 30% decease in activity. Southern blot I RNA from nuclear nm-on assys indicates the pesene of analysis of the and tubulin gene Supported by NIH-M 08238 rRNA, sioy Tns, rliable free inter&fe.pTepeatio fiee (NH4)2S04, histone, 1318 orbtrton To detemin ble ing 4-cd tdbydesocl c wer hI a near erey. The enuing ceavage ARTEMIA. DROSOPHILA MUTANTS WITH DEFECTS IN REGENERATION OF ABDOMINAL MUSCLES. ((S.L. Tobin and A. Wilson Malaska)) Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104. the blasme. of and asked each spiNe pene that fome a 1317 Using G-50 Sephedex molecular exclusion and DEAE-Sephadex ion-exchange we reported the presen of a soluble, developmentally regulated,nlalothionein-lhe zinc bining poten (ZnBPII) in Artemia. We report here that ZnBPII isactually composed of fourindividualzinc binding activities Forty eight hr naupii were collected and homogizdin 50 mM Tris,pH 8.0, 0.1 mM DTT, 0.5 mM PMSF and 10pg/ml of SBTL The soluble proteins were obtained by highspeed centrifugton,incubated with on a G-50molecular exclusion column. The 109Cd, and thenfrac column eluate was monitored for 109Cd and endogenous Zn to locate metal binding activities. Peak fiacons peaning to ZnBPII wee pooled and applied to an FPLC Mono Q ion-exchange column.The column was eluted with alinear gradient of Tris, pH 8.0 (10 to 400 mM) Four endogenous zinc binding activities (ZnBPHab,c,d) are cleaiy resolved by this procedure and coelates dircty with the exogenous 109Cd binding activity. The individual aceamide andfa ated by SDS-PAGE pteinswere allated with i using a10-20% liear polyacrylamide gradient Each of the preparatin was judged tobe homogenous usng silver stain deectio Theindivdual poes all had a caculated relative molecular mass of 6700, cactistic of metallobhionein. Inteeting, itappearsthat the four proteins arediffeentially expressed during development Supported by NIH-GM08238 spinle of the par perdel to the spidle orintatwon 1316 chromatography, ne bastomer Thus, dcvage tuinsic propertes nhd udu st w soClatloMM how spindeb mic1 . confocal lawser ceseaseavage scumingbryoe and to the with respec MMTAL BINDING PURIFCATION OF METAILLOTONEIN-LIKE PRaOTINS FROM ARTEMIA. ((J. L. Brook, B. G. Harpham and R. A. of and Biochemistry, Cal. State Univ., Long Beach, Acey)) Dept Chemistry CA 90840 wh 7" ATP, MnCI2, transcripts. 1319 CLONING AND CHARACTERIZATION OF GASE GENES, A GROUP OF PUTATIVE SERINE PROTEASE GENES EXPRESSED IN THE MIDGUT OF DROSOPHILA MELANOGASTER. ((Q. Wu, C.M. Cheney, and J.E. Sadler.)) HHMI, Departments of Medicine & Genetics, The Jewish Hospital of St. Louis, Washington University School of Medicine, St. Louis, MO 63110. Serine proteases participate in a variety of essential biological events, such as food digestion, blood coagulation, complement activation, and To further explore the function of serine proteases in fertilization. four novel putative serine protease genes, Gasel-4 (for development, gastric serine protease), were cloned from a Drosophila embryo cDNA library. Gase cDNAs encoded a group of chymotrypsin-like or elastase-like enzymes that consisted of a signal peptide, a short activation peptide, and a domain. Expression of the Gase genes was carboxyl terminal protease The developmentally regulated. transcrpts of the Gase genes were abundant in late embryos (12-18 hr) as well as in larvae and adult flies, but completely absent in pupae. Northern analysis and in situ hybridization showed that in the gastric Gase mRNAs were localized in the anterior midgut, were hybridization to polytene chromosomes, Gase genes caeca. By 99C8, and to several 66C5, 65A1-2, 25B1-2, mapped Similar positions including 99F6-7. chromosomal locations were reported previously for the Jonah genes which also are expressed in the midgut, but the structrues of Jonah proteins have not been reported. Two of the original Jonah clones were analyzed and shown to contain typical serine protease gene sequences that were different from Gasel-4. These results indicate that Gase and Jonah genes belong to a multigene family of over 20 genes, and that the members of this gene family encode serine proteases that may function in the digestive system of Drosophila. especially Monday. Invertebrate Development (1320-1324) 1321 1320 SCREENING FOR GENES INVOLVED IN THE IMMUNE SYSTEM AND TUMOR FORMATION IN DROSOPHILA. ((D.A.Kimbrell, ARodrigue, C.Wu, X.Zhou, n)) Deparmn of E.Yonters,S.Meller.,Jhen, K.MaLtin. LMann, K.Hale, Biology. University of Houston, Houston, TX 77204, HHMI and Institute of Molecular Genetics, Baylor Colge Medicie Houston, TX 77030. H.BelW The Drosophilh immune effectively respond to boceril oo ponertsVt and c llular by the production of proteins i als orltical . Thas systemn to aggregation of hemocytes. We have hashumoral ifction swteib and the mobilizaonf hemocytes for tumor formatio, whichls hallmarkd by the the screend enhancer detectorattai to find two typesof genes relaed to e iriection, B procese": A -genes induced or decrease in expression by - genes exp immune system sse in tissues. i.e. hernocyts lymph glnds (Ih bacterial hematopoltc organ) and fat body (majorsite ofantibectrial proteinsyrhesis). For type A, we devised n a ew variation of enhancer detection in which slings of eachstrainareassayed _ r o wiIh folyforthelevelof response ogaiacoeidase Sains that show an increase or decrease inB and without inection. genes. 2,600 upon infecton are caKides forlines identifying knm e stocks were screened for type A and/orB, and 3 type A strains have been found. and the in Twosirains areki esd in Mcx_seexpresin both stages upon infection, and one strain is developmeally resticted to the laval hrval adult stage. For type B. embryos andVorlavae were screernd and 18 strains with hemocyte, lymph gland and/or fat body expression were identified. We characterized the reporter gene expression of many of these strains throughout development and are using these to study the developmental origins and relationships amongimmune systemtissues. Two Pelnemnts map to the third chromosome and the remaindermap to the second chromosome. P element mobilization of all strains tested far confirm that lethality resultsfrom the insertion. Geneticcomplementation toest and restriction mapping of pasmnid two of thesestains carryinrtions into the sane rescued DNA show that at ximately 100 base pais apart. These two enhancer detector locus and are so stmis are associed with foffation ((S.W. Chem, J. Hau, L Berg and GM. Weael)) Biology and Cell Biology & Bio. Department of Molecular eaist, Brown University, Providence RI THAT ALTER PATTERNS OF GENE EXPRESELEGANS. ((C. Xie, Y. Jia and F. Aamodt)) Department Biochemistry and Molecular Biology, Louisiana State University Medical SION IN C. Center-Shreveport, Shreveport, LA 71130-3932. developed a method for isolating mutations in Caenorhabdiis ecantibody or histochemical staming patterns. The basis for this method is a new procedure for making C. eLgans permeable that does not kill the eggs contained within the uterus of gravid adult hermaphrodites. We have used this procedure to isolate mutations in genes that affect the expression of and pag-S, will be a mec-71acZ fusion gene. Mutations in two genes, pag-i chromosome IIL described. pag-i is located between lon-i and dpp-17 Mutations in peg-i cause 8-16 fold higher expression of the mec-71ecZ the amount of and an not increase does wnc-861acZ gene. A pag-i mutation mec(B7lacZ mRNA but it does increase the amount of polyadenylated message we believe that the peg-i product is involved in deadenylation of mRNA in preventing polyadenylation. peg-S is on the right arm of the X-chromosome between the right end point of mnDfS and mnDf20, which lies between sne-S and wnc-7?. A mutation in peg-S results in expression of the touch neuron the specific genes mec-71ecZ, mec(B4lecZ and mec-7 in the BDU lineal sisters of the ALM touch neurons. The pag-S('lsO) mutation also causes a reverse kIinisr Uncoordinated phenotype and axonal guidance errors. The pag-S gene product may be involved in spedifying or interpreting spatial inforWe have that alter on gene so or neurons, . We are A CHAIN GENE Davalos', defects in hypodermal morphogenesis. differentiation. 1324 elegans A. DURING SEA Z. Rosenttal' URCHIN and of Hawaii. The spreading and rearrangement of epithelial cells is a widespread phenomenon development. Our laboratory is using digital time lapse, 04-dimensionalO, and confocal microscopy to characterize the morphogenesis of the embryonic hypodermis of C. elegans as a prelude to a genetic analysis of this model embryonic epithelium. The cells of the dorsal hypodermis, which initially comprise a strip two cells widenlnning the length of the anterior-posterior (A-P) axis, interalate to form a single row. Concurrent contralateral nuclearmigration also takes place in these cells. Based on analysis of unc-83 (e1408) embryos, in which nuclear migration is blocked, these processes are separable. The nuclear migration defects can be phenocopied by treatment with nocodazole without perturbing intercalation. Ventral hypodermal cells undergo epiboly, ultimately enclosing the embryo as they meet at the ventral midline. We have used 4-d microscopy and laser ablation to investigate the role specific hyp-6 and hyp-7 precursors play in enclosure. The bilateral pairs of cells derived from ABpraapp and ABplaapp seem tobe particularly important for the advance of the ventral margin. Ablation of the C blastomere or its progeny indicates that dorsal and ventral morphogenetic movements areregionally autonomous, at least within large sectors along the A-P axis. We are currently engaged in screening a bank of zygotic embryonic lethal mutations, as well as existing chromosomal deficiency strains, for MUTATIONS mation in C. LMUNfN Page', Biological Sciences, California State Hayward. 2Kewalo Marine Lab, University Laminin is a multi-domain basement membrane glycoprotein composed of three chains; Bi, 82 and A. A cDNA representing a region of laminin A chain was isolated from aS. purpuratus lambda ZAP cDNA library. Sequence analysis indicates this cDNA (WL 23)represents 1307 bp encoding a region of the globular or "G" domain at the carboxyl terminal region of laminin A chain. The G domain is composed of five homologous repeats (Gl-G5). WL 23 represents G2-G3. A homology search using the deduced amino acid sequence of the cDNA showed 30-40% sequence similarity with other laminin A chain genes in the carboxylterminal G domain. Other sequence features are more highly conserved including invariant cysteine residues which form the loops of the "fingers" in the G domain. Northern blot analysis indicated that the 11 kb mRNA was present in unfertilized eggs and remained at near steady state levels through development to the 72 hr pluteus stage embryo. Inhibition of zygotic transcription in 4-8 hr embryos by actinomycin D suggests steady state levels require continuing transcription. Northern blot analysis using RNA isolated from polysomes of embryos at 4 hours after fertilization indicates that there is active translation of the maternal mRNA at 4 hours post-fertilization. A polyclonal murine antibody was raised against a fusion protein and Used to invest- igate temporal and spatial accumulation of the laminin A chain protein. Developmentalimmunoblot analysis on total protein extracts indicates the presence of immunoreactive protein of 400 kD in eggs which increases with the formation of a basal lamina during blastulation. Immunofluorescence microscopy reveals reactivity confined to the basal lamina of blastula, gastrula and pluteus stage embryos. in animal a by transformation resu TE ((L. THE CELLULAR BASIS OF EPITHELIAL MORPHOGENESIS IN THE EMBRYONIC EPIDERMIS OF C. ELEGANS. ((J. Hardin, A. Malik, and E. Williams-Masson)) Dept. of Zoology and Program in Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706 The extracelular matrix (ECM) plays an important regulatory role during endoderm cell differentiation and gastrulationin the early sea urchin embryo. Most of the ECM molecules identified in the sea urchin embryo are stored in the egg within secretoryvesicles. Fertlization initiates series of regulated secretory events of these ECM vesicles that contribute to formation of the nascent basal lamina and the blastocoel matrix Herewe describe an ECM molecule of osregatus with a contrastng pattem of expresson that was identified by L1tec an expression cDNA screen using antibodies to the embryo ECM. We found that the ECM 18 protein is not detectable in eggs or early embryos eventhough its mrRNA of approximately 6kb is present matemnally and thoroughout early development. During gasnuabon, mRNA accumulatedseveral fold over matemal levels and was enriched approximately 3 fold in the endoderm. Using antibodies to reombinant ECM 18 proteins in either westem blot or immunofluoreseence assays, no protein was detectable in eggs or early embryos until just prior to gastrulation. Dunng gastrulation the protein accumulated to high lvels throughout the basallamina/blastocoel matrix. Partial characterzation of the ECM 18 DNA sequence shows a repeat motif withhigh cysteine content (10.5%). No sequences similar to ECM 18 were found within theGenbank. These results suggest that ECM 18 is anewly described ECM molecule whose expression is rgulated by translational control. Since this extracellular matrix protein is expressed just prior to gapulation and selectively by invagnating endoderm cells ECM 18 may participate gans OF 1323 0291. (Sponsred by AW. Colem ) in the regulation of endoderm EDCX8SSICN DEVELOPS(NT. en n'Det.5ept. BUniversity, of tumors. 1322 UNUSUAL EXPRESSION OF AN EXTRACELLULAR MATRIX MOLECULE DURING GASTRUIATION IN THE SEA URCHIIN EMBRYO. of 227a presently attempting to cone peg-I and pag-S 228a Cell Lineage (1325-1330). Monday 1325 1326 LIPOCORTIN-1 IS EXPRESSED IN MICROGLIA AND THEIR PROGENITOR CELLS IN CULTURES. ((S. Fedoroff, andJ.A. McKanna)) Department of Anatomy, University of Saskatchewan, Saskatoon, Canada and Depatment of Cell Biology, Vanderbilt University Medical School, Nashville, Tennessee. A MATHEMATICAL MODEL TO TEST HYPOTHESES ABOUT THE DEVELOPMENT OF IN CULTURES. ((JP. Novak S. 1800 mnonte Ste.-Julie, Varennes, Que., Canada J3XlSl Fedoroff)) and Department of Anatomy, University of Saskatchewan, Saskatoon, Sask, Canada S7N OWO. (Spon. by GD. Burkholder) In rat embryos lipocortin-l (LC-l) is expressed at E12.5 in primitive Disaggreged cells from newbon C3HJ armou nu llimonin cultur or microglia, depending culture astroglia,To oligodendrogliadevelopmental pathways of astrogia conditonns. we a mathemacal microglia quantitadve peimentaldataw Neopallial cells were planted atS 103 cells/cm2/l.5 medium (modified MEM, 5% horse serum, 10% medium condidoned by LM fibroblasts which contains CSF-1, and 5% mediumconditioed bySTO fibroblasts which contains IL-6). The resulting cell colonies were immunostained for OFAP (astroglia marker), and CR3(miicrogia markr), and cel nucki, were stained with Hoechst 33258.Th total cell number and numbers of varioustypes of colonies were counted after 1,3 days. A of a common astrocytemathematical model based on the assumptiontotal numbr of unbaeled, yielded good agreement microgiaprogenitor CR3+ and GFAP+ cells, total number of CR3+ andGFAP+ colonies and a glia in the floor plate of the hindbrain and spinal cord. LC1§ primitive glia give rise to LC-1l cells which have the characteristics of (McKanna, Res 491,1993). 36: We now report microglia. Neurosc. ttat LC-1+ cells appear to he pronitors of the microglia (Mac-I+) that are induced by CSF-l andLL-6 tissue cultures initiated from Po mouse neopallium We counted LC-1+ cells in 3, 5, and day neopalluaa cultures anddeermined the number of LC-l1 cellsthat were also Mac-lI or GFAP+. In 3 day cultures 67.8% of LC-l cells were LC-lI+Mac-l- and 31% were LC-l+IMac-l+. In 5 day culturs 50.7% were LC-l+/Mac-l- and 46.9% forming araphe 29.5% were LC-1+/Mac-1- and cells were also LC-1+.These results indicate that microglia precursor clls express LC-1 before they receptor d t CR3 immunoreacts with Mac-l antibody and is express the characteic to microglia. e observations that microgia can form from the primitive glia ofthe floor plate and thatin neopqalial cell culures the LCl+/Mac-l- progenitorcells can give rise to LC-l+/Mac-l+ mic glia,strongly support the notion that microglia are of neuroepithelial origin and may originate in more than one site in the CNS. This wok was supported by grant MT4235 from MRC Canada and by the anadi Network ofCentres of Excellence, NeuroScience Network LC-l+/Mac-l+. In 7 day cultures 66.5% were LC-l+/Mac-l+. All Mac-l+ were 1327 DORSAL AND VENTRAL CELLTYPES CAN ARISE FROM COMMON PROGENITORS IN THE CLOSING NEURAL TUBE((K.B. Artingerl, S.E. Fraser2 and M. Bronner-Fraser1)) lDevelopmental Biology Center, University of California, Irvine, CA 92717 and 2Division of Biology, California Institute of Technology To challenge the developmental potential of dorsal neural tube cells and test whethersingle neuroepithelial cells can give rise to the full range of neural tube derivatives, we grafted a notochord lateral to the dosing neural folds. This results in juxtaposition of dorsal and ventral cells types, by inducing floor plate cells and motor neurons dorsally. Clonal analysis with the vital dye lysinated rhodamine dextran (D) showed that both "dorsal" and"ventral" neural tube derivatives can arise from a single precursor. Cells as diverse as sensory ganglion cells, presumptive pigment cells, roof plate cells, motor neurons and floor plate cells were observed in the same done. The presence of such diversity within single dones indicates that the responses to dorsal and ventral signals are not mutually exdusive; even in the dosing neural tube, neuroepithelial cells are not restricted to form only dorsal or ventral neural tube derivatives. IREQ, MICROGLIA form determine the 1329 HOMEOBOX CONTAINING cDNA EXPRESSED IN ALVEOLAR TYPE II CELLS DURING LUNG DEVELOPMENT ((Hamdan, H., Metter, J., Horowitz, S., Dodds, S., deLenos, R., & Minoo, P.)) Dept. of Pediatrics, USC School of Medicine, Los Angeles, CA 90033 Immaturity of alveolar type II (AT2) cells which includes aberrant expression of SP-A, a protein comnponent of surfactant, has been described in premature neonates born in advance of complete in utero development and may involve absence (or inactivity) of AT2 cell-specific transcriptional factors. Cklning and analysis of a human SP-A genomic clone revealed the presence of helixturn-helix-like binding motif in the 5' upstream domain of this gene. Using degenerate oligonucleotides, and AT2 cell cDNA, we amplified and cloned a DNA fragment of approximately 150 nucleotides which contains a homeodomain. Screening 105 phage clones from a fetal lung cDNA library with this fgment yielded 8 cDNAs, suggesting high abundance of complementary sequences expressed in fetal lung. DNA sequence detmination of one clone, 12A2 revealed 98% homology to a thyroid transcription factor TTFl Analysis of mRNA complementary to 12A2 demonstrates: 1) expression of 12A2 occurs in AT2 cells and not adult lung fibroblasts; 2) 12A2 cDNA hybridizes to one major RNA band at 2.3 kb which is expressed during lung development and two minor, larger bands which appear to be developmtally regulated with the highest level of expression found in association with completion of fetal lung development. Priser extension/rapid amplification of 5' ends of the mRNA, RACE is underway to clone and characterz the developmentally regulated minor transcripts. We hypothesize that 12A2 may play a role in cell lineage and differentiation of AT2 cell during lung development. Supported by NHLBI 48298. and modeL x mi and 5 for number of mixed CR3 andGFAP+ colonies (typically within ±i15% with one exception of +28%). Differendal and cumulative distributions of the colonies according to specific cell counts and bivaiant distributions of the mixed colonies (CR3 plus GFAP+ and CR3 plus unlabeled cells) were well represented. A model assumingmonopotential progenitors yielded reasonable agreement for the total cell and colony counts and for colony distributions; however, the CR3+ andGFAP mixed colonies were underested by 70%. Overall compason withobservatons favors the hypothesis ofa common CR3+ and GFAP+ progenitor. Supported by grant MT4235 from MRC Canada. 1328 EMERGENCE OF SALIVARY GLAND CELL LINEAGE DIVERSITY SUGGESTS A ROLE FOR EGF RECEPTOR SIGNALING. ((E.M. Durban and P. G. University of Texas Houston, HSC, Dental Branch, Division of Oral Pathology, Houston, TX 77225. Nagpala)) Diversity of cell lineages within glandular is generated progenitor cells. not understood. Differentiative transitions of mouse submandibular salivary gland served as a model to assess the role of epidermal growth factor (EGF) receptor signaling in generation of cell lineage protection analyses revealed temporal fluctuations diversity. in EGF receptor mRNA levels coincident with crucial differentiative cell lineage transitions. EGF receptor mRNA levels were highest between Days 2-S when proacinar cells are maturing and striated duct cells emerge; all differentiating cells exhibited EGF receptorspecific immunoreactivity during this period. Following these differentiative events, EGF receptor mRNA levels declined sharply and immunoreactivity became confined to ductal cells. A second increase in receptor mRNA occurred between Days 11-16 coincident with emergence of granular convoluted cells which were strongly EGF receptor immunoreactive. Reductions in EGF receptor mRNA levels and intensity of immunoreactivity were observed at DAY 22 upon completion of GCT cell emergence. Thus, temporally distinct patterns of EGF receptor expression correlate with identifiable cell lineage transitions in the developing SSG suggesting a role for this signaling pathway in the emergence of cell lineage diversity in a organs by differentiation of committed postnatally Fundamental aspects of this process are (SSG) RNase (GCT) glandular A and organ. Supported by grant DE07766. 1330 PERMANENT CELL LINES FROM MOUSE VON EBNER GLAND. ((L.S. Chen and M.L. Snead)) The University of Southern California, Center for Craniofacial Molecular Biology, School of Dentistry, CSA 142, 2250 Alcaar Street, Los Angeles CA 90033 Yon Ebner's glands (VEG) are small tubulo-alveolar serous salivary glands whose secretory ducts open into the troughs at the base of the taste bud containing papilla. Morphological and biochemical data indicate that their anlages acquire functional acinar structures late after birh, at the when taste buds in the cicumvlae papillae divide and mature. Von Ebner's gland secretion is serous polypeptides such as acid phosphatase, amylase, lingual lipase, and members of the lipocalin superfamily of VEG-proteins. The anatomical organization, biochemical properties and neurological control of this gland strongly suggest that Von Ebner's secretion play a role in chemical discriminaton, perhaps similarly to the functions of define the role of VEG accessory glands of the olfactory epithelium. To cell products, we are developing permanent cell lines from the Von Ebner's mice bearing the MHC-promoter coupled to the glands of circumvallateexclusively timne including better transgenic transforming "1 antigen of polyoma virUS (Immortomice"). Using RT-PCR with amplimers unique to a VEG-specific messenger RNA, we demonstrate that isolated VEG.derived acinar cells grown in monolayer, maintain a dominant feature ofthe intact gland, namely, the capacity to transcribe a VEGcells have the potential to specific messenger RNA. These the execution of experimental strategies designed to understand VEG's functions difficult to execute due to the physical limitation of the gland in situ. VEG-derived pennit Monday. Cell ineage (1331) 229a 1331 MEGAKARYOCYTIC DIFFERENTIATION IN HUMAN LEUKEMIC CELL LINES: ALTERATIONS IN TRANSCRIPTION FACTOR ACTlVITY ((Karen M. Hudson and Michael A. Lieberman)) Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267. The mechanism of differentiation of a pluripotent bone marrow stem cell into a committed multinucleated megakaryocyte is poorly understood. To investigate the role of transcriptional regulation in megakaryocyte development, several experimental approaches were taken utilizing human kukemic cell lines as a model system. The human cell lines (CHRF-288-11, K562, HEL) all differentiate exclusively through the megakaryocytic lineage when treated with the appropriate concentration of phorbol ester. Studies with the CHRF-288-11 cell line demonstrated that a number of transcription factors were up-regulated in response to phorbol esters. Gel moblity shift assays and Northern analysis indicated that the transcription factors AP-1, NF-1, and ETS-1 were all up-regulated during differentiation, although the timing of the regulation differed between them. These changes were also observed with either the K562 or the HEL cell lines. GATA-1, a hematopoeitic specific transcription factor, does not change its level of expression or activity during CHRF-288-11 cell differentiation. Similarly, the level of expression of the transcription factors SCL, MYC, MAX, and SP-1 were unchanged as the cells differentiated. GATA-2 was found not be expressed in CHRF-288-11 cels as determined by RT-PCR. These data suggest that regulation of transcription during megakaryocyte differentiation may be occuring through changes in the levels and activity of the factors AP-1, NF-1, and ETS-1. The relationship of these factors to that previoudy identified (SKI, Oncogene 9:1407-1416,1994) is currently under investigation. This work was supported in part by grants from the NIH (HL-51555 and T32-ES07250) Pattern Formation (1332-1335) 1332 1333 E(PRESSION OF A HOMEOBOX GENE, msn 2, iS AFFECTED BY ABERRANT PaK 3 IN SPLOTCH MUTANT EMBRYOS. ((Y. Takahashi1, and J. A. Weston)) Institute of Neuroscience, University of Oregon, Eugene, Oregon, 97403. 1 Present address: Department of Bioscience, Kitazato University, Sagamihara, 228, Japan. MESODERM INDUCTION IN THE MAMMALIAN EMBRYO. ((C. A. Burdsall, and R. A. Pedersenl,2)) lLaboratory of Radiobiology and Environmental Health, 2Departments of Anatomy, Radiology and Obstetrics, Gynecology and Reproductive Sciences, University of Califomia, San Francisco, CA 94143. The Splotch (Sp) gene in mice codes for a transcription factor, Pax 3. Mouse embryos homozygous for the Sp mutation produce aberrantly spliced Par 3 mRNA and display spina bifida along with failure of the embryonic neural tube to clse. Alfthough Par 3 mRNA expression Is localized in the dorsal porton of the deveoping neural tube, it is not known what role Pax 3 plays in regulating neural tube development. We have observed that a homeobox gene, msx 2, is co-expressed with Pax 3 in the dorsal half of the neural tube. In order to study a possible regulatory relationship between Pax 3 and msx 2 expression during neural tube formation, we have examined max 2 expression pattems in embryos homozygous for the Sp mutation. In El 1.5 Sp embryos, Pax 3 mRNA is localized in the dorsolateral portion of the open neural tube, suggesting that the region-specific expression along the dorsoventral and mediolateral axes of the neural tube is maintained in mutant embryos. In contrast, msx 2 mRNA expression, which Is normally evident in the El 1.5 dorsal -hindbrain, is below the level of detection in the exencephalic hindbrain regions of Sp embryos. Likewise, the level of msx 2 mRNA expression in the spins bifida regions of Sp embryos Is markedly lower than in corresponding regions of the dorsal neural tube of wildtype embryos. These results suggest that, during development of the dorsal neural tube, Par 3 lies on an upstream regulatory pathway for msx 2 expression Supported by PHS Grant DE-04316. In amphibian embryos, peptide growth factors have been shown to specify mesodermal fate in embryonic tissue. To test the role of growth factors in mammalian mesodermal differentiation, we developed an in vitro system in which fragments of undifferentiated epiblast (embryonic ectoderm), obtained by manual dissection, were cultured in a serum-free, defined medium with addition of bFGF and TGF-01. Treated explants adopted a complex morphology (rather than a simple, flat epithelial morphology as in controls) and by immunofluorescence regions within these explants stained positively for vimentin, a marker of primitive streak mesoderm. In situ hybridizations demonstrated that in 2/3 of treated explants, small regions within the explants expressed brachyury, a marker of streak-stage mesoderm and later dorsal mesoderm. These results indicate that peptide growth factors may play a role in mesoderm induction in mammalian embryos. (Supported by NIH Grant No. HD26732, USDOE Contract# DE-AC03-76-SF01012 & and American Heart Association Postdoctoral Fellowship). 1335 1334 ALTERNATIVE SPLICING YIELDS TRANSCRIPTS FOR BNP-l, NANNALIAN TOLLOID HONOLOGUE *TId, AND A NETALLOPROIEASE CONTAIsN/ A UNIQUE HISTIDINf-RICH DONAIN. ((Y. Takahara G.E. Lyons and D.S. Greenspan )) Departments of Pathology and Anatomy, University of Wisconsin Nedical School, Nadison, WI 53706 Bone morphogenetic protein-I (EBP-1) is a metalloprotease found in extracts capable of inducing ectopic bone formation. In humans, it has a domain structure similar to that of Drosophila dorsal-ventral patterning protein tolloid (Tld), but is shorter. We have found that, in humans and mice, alternatively spliced transcripts encode both BNP-1 and longer protein which we have designated mammalian tolloid (oTld) due to a domain structure identical to that of Tld. A third alternatively spliced product, in which a novel domin is inserted near the BNP-1 C-terminus, was also found. Low levels of RNA transcripts for *Tld were found in all adult human tissues surveyed, while BNP-1 transcripts were found in all adult tissues except brain. This difference in distribution of expression in adult tissues is mirrored in embryonic mouse tissues where in situ hybridization found high levels of *Tld transcripts but no detectable BNP-l transcripts in floor plate of the neural tube of the developing CNS. The third alternatively spliced form was not found in adult human tissues. Northern blots showed high levels of all three forms in human placenta. In situ hybridizations found punctate signal for all three forms, in 17.5 day mouse placenta, localized to trophoblast giant cells throughout the labyrinth, with higher levels of expression, especially for BNP-1, at the Junctional zone near the aternal interface. , a , WITHDRAWN 230a Pattern Formation (1336-1341). Monday 1336 1337 EXPRESSION OF RETINOIC ACID BINDING PROTEIN I IN THE DEVELOPING CHICK OTOCYST. ((J.-L. Sanne and D. K. Wu)) NIDCD, 5 Research Court, Rockville, MD 20850. A NEWLY ISOLATED QUAIL ZINC FINGER GENE EXPRESSED IN THE DORSAL NEURAL TUBE. Barembaum and Bronner-Fraser)) Department of Developmental and Cell Biology, University of Irvine, CA 92717. The inner ear, derived from the otic placode, undergoes tremendous morphological changes during development. We retinoic acid (RA) may piay in this developmental are interested in the role Exogenous RA has process. been shown to induce supemumerary hair cella in organ of Corti cultures from mice (Develop. 119:104-1053, 1993). RA also inhibited proliferation and induced differentiation of chick otocysts in vitro (Develop. 110:1081-1090, 1990). RA is thought to act through nuclear RA receptors (RAR; RXR), which belong to the steroid/thyroid hormone receptor family. However, the amount of RA reaching the nucleus is thought to be modulated by a mechanism involving cytoplasmic binding protein for retinol (CRBP) and RA (CRABP I-il). Whole- mount in situ hybridization was performed to study the distribution of mRNA ear (stage 11 -18). CRABP I encoding for CRABP I in developing chick inner 0 0-11). When the otic placode mRNA was not found in the otic placode (stage invaginated to form the otic pit, CRABP I was expressed mostiy in the dorsal and anterior region of the otic pit. After complete invagination of the otic pit to form the otocyst (stage 18-19), CRABP mRNA was found in the entire otocyst except the most ventral portion. At embryonic day 2.5 (stage 18), positive hybridization signals were also found in neural tube, neural crests, branchial arches and limb buds as reported by others. The expression of other retinoic acid receptors and binding proteins, as well as the effects of RA in the developing otocyst are currently under investigation. ((M. California, Genes containing zinc finger motifs constitute a family of DNA binding eWe proteins, some of which have been found to be important are interested in determining whether zinc finger genes are involved the a cDNA of neural tube and neural crest. We have sceened hlbrary development made from quail neural crest culture RNA with a degenerate oligonudeotide probe corresponding to the H/C-link region of C2-H2 class of zinc fingergenes. Among the 60 positive plaques picced, we have studied one in detai. This cDNA contains 14 zinc finger domains and aKRAB domain. Several other cDNA clones with regions of high sequence identity were also found, indicating that this gene belongs to a cosely related group within the C2-H2 zinc finger family. Whole mount in situ hybridization shows that the gene is expressed in the dorsl neural tube as early as the four somite stage and at least as late as stage 25 with the strongest expression located in caudal neural tube. Signal can also be detcted in the ecoderm of stage 14 embryos but not older embryos. The limbe also express the RNA as early as stage 16, and later the message is localized distally. Other places where signal is found in older (stage 19-25) embryos are the branchial arches and the tail. This gene is an interesting candidate for involvement in dorsoventral patteming during neural tube development, and perhaps in cell proliferation in other regions of the in in in molecule embryo. 1338 1339 PHOTORECEPTOR PATTERNING IN EMBRYONIC AND ADULT GOLDFISHRETINA. ((D.L. Stenkamp and P.A. Raymond)) Dept. of Anat. and Celi Biology, Univ. of Michigan School of Medicine, Ann Arbor,MI 48109. The photoreceptor layer of the teleost retina consists of an array of repeating mosaic units, each a precisearrangement ofspecific cone photoreceptor typea. This mosaic may develop as a consequence of lateral interactions among quentially committed cones,with an early-differentiating cone type acting as a "founder, as in Drosophilaretina Using in situ hybridization to identifyspecific P-CATENIN LOCALIZATION DURING XENOPUS EMBRYOGENESIS: ACCUMULATION AT TISSUE BOUNDARIES. ((F.Fagotto and B.M.Gumbiner)) Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 photoreceptortypes bytheir opsn mRNA, have studied rod and cone cell patare detectable first, in a nasally-positioned patch of retina in stage 24 (85 h) embryos. A wave of rod differentiation then proceeds centrally along the choroid fissure, and cieumferentially around the eye. By stage 26 (120 h), scattered cells throughout the eye also expr rod opsin. A slightly later wave of conediffentiation also begins in a nasal patch and progresses similarly, with red conesfirst, at earlystage 25(95 h), followed by blue and green (100-105 h). Regular spacing ofblue cones in rows is evident even at the earliest times. We have also studied the order of cone commitment in peripheral retina of adult goldfish, where new retina (including cones) continues to differentiate. The distance between the proliferative germinal zone (BrdU-labeled) and the nearest cone labeled with one of the opsin probes represents time between cell birth and cell commitmnent; this distance would be smallest for the cone type which iffeentiateS first. Our results indicate that the blue conemay be first to differentiate at the retinal margin. The observations suggest that mosic formation in embyonic goldfish retina may include cellular mechanisms distinct from those which continue to generate cone mosaic in the adult. we terning in the embryonic goldfish retina. Rods 1340 OVEREXPRESSION OF P-CATENIN CAUSES THE FORMATION OF A SECONDARY AXIS IN XENOPUS LAEVIS EMBRYOS. ((Kadtleen A. Guger and Barry Gumbiner)) Departmnt of Cellular Biochemsitry and Biophysics, Memorial Sloan Kettering Cancer Center, New York, NY, 10021. p-catenin is a protein known to associate with the cytoplasmic tails of members of the cadherin family of cell adhesion molecules. Recently, it has been shown that injection of Fab fragments directed against pcatenin can cause the formation of a duplicate axis in Xenopus lwvis (McCrae et al. 1993, JCB 123:473-484). To further explore the role played by P-cnin inXenopus development, P-catenin was overexpressed in early Xenopus embryos by mRNA injection. Injection of mRNA into the vegetal-ventral blastomere of 8 or 32 cell embryos was found to cause duplication of the dorso-anterior axis and was also found to rescue axis formation in UV-ventralized embryos. Lineage tacing experiments revealed that cells receiving D-cat mRNA do not contribute to axial sucues. In this respect, overexpression of 0-caienin is simlar to the ectopic expression of certain members of the Wnt gene family (Sokol et al. 1991. Cell 67:741-752., Smith and Harland. 1991. Cell 67:753-765.). Like Wnts (Olson et al. 1991. Science 252:1173-1176.), overexpression of P-cauenin was also found to increase gap junctional communication in cells of the ventral animal cap. These findings ae intriguing, particularly in light of the fact that P-cat shaes homology with armadillo (McCreaetal. 1991. Science 254:1359-1361.), a protein which is Inown to act downstresm of a Wnt-signaling event during patterning in the epidermis of the fly (Pfeiferet al. 1991. Development 111:1029-1043.). Data from this that a similar signaling pathway nmy play a role in the specification of the dorsal-ventral axis in Xenopus. study suggests p -catenin is a cytoplasmic protein associated with cadherir adhesion molecules, and has beenimplicatedin axis formationin Xenopus (McCrea, Brieher and Gumbiner, J. Cell Biol. 127, 1993, 447). We have studied Its distribution in Xenopus embryos by on frozen sections. Consistent with its immunofluorescence function cell adhesion, it is present in every cell. However, variable amounts have been found in various regions and different tissues of the embryo. No simple correlation appears to exist between the levels of P-catenin with the expected strength of adhesion. High levels of p-catenin were found in regions undergoing active morphogenesis, such as the marginal zone of blastulae and gastrulae. This suggests that high expression of pcatenin could be involved in dynamic adhesion events. Surprisingly, p-catenin also accumulates on plasma membranes that probably do not establish direct or strong contacts with other cells. In particular, high amounts of P-catenin are found transiently at boundaries between tissue anlagen and at the intersomitic boundaries. This unexpected pattern of P-catenin that this molecule participates in expression raises the developmental processes, perhaps independently of its classical role in cell-cell adhesion. in possibility 131 EXPRESSION OF ECTOPIC XWNT8 OR TREATMENT WTH LICL IN ALTERED INVOLUTION OF THE DORSOANTERIOR RESULTS MESODERM IN XENOPUS. ((J.R. Fredieu, D. Maier, M. Danilchik, and L. Christian)) Dept of Cell Biology and Anatomy, Oregon Health Sciences University, Pordand, OR 97201 The centl nervous system (CNS) of Xenopus is derived from an layer of cells which have been provided cues as to their relatve ectodennal position along the anterior-posterior axis by underlying mesodermal cells. mesoderm is positioned beneath the neural ectodem during the flse morphogenetc movements of gastulation. Misexpression of the protoin the dol anterior meoderm of early Xenopus oncogene gastrulac causes this tissue to adopt a more ventml fate followed by aloss ofof the most anteior portions of the CNS. In similar manner, the embryos with LiCl just before gsrulaon reslts in the loss of forebrain. of staged control, plasmid Xwnt8-injected, and Midsagital optical sections wee examined by confocal microscopy to delineate embryos LiCI-teated the extent of dorsal mesodermal involution during gatulation.In these misexpion of Xwnt-8 in the dorsal mesoderm of studies, we fnd thatasthewell as the exposure of eary gastulse to LiCl retards gastnlae, Xenopus the involution of the dorsal anteior mesoderm, thus preventing it from its normal position beneath the most anterior neural ectoderm. in reaching addition, the extent of meodensa involution correlates dicty with the extent of anterior CNS dfentainsuggesting that normal mesodermal involution is critical for the tansmission of neural pattening signals. We are embryos as a model system to using these characterie the nature of signals involved in neural paterning in the Xwnt-8 a currendy embryonic frog. comct teratogenized treatment Monday. Pattern Formation (1342-1347) 231a 1342 1343 REGULATION OF HOXA13 EXPRESSION DURING LIMB REGENERATION IN THE AXOLOTL. ((D.M. Gardiner, B. Blumberg*, Y. Komine, and S.V. Bryant)) Developmental Biology Center and Department of Developmental and Cell Biology. UCI, Irvine, CA 92717 and *The Salk Institute for Biological Studies, La Jolla, CA 92037. important in the regulation of outgrowth and pattern formation during development of the vertebrate limb. Although leas ia known about the expreaaion of homeobox genea during limb regeneration, it is likely that they play an equally important role aa limbs regrow and pattern is reformed. We have iaolated and identified axolotl homologs of 17 different homeoboxcontalning genes expressed by cells of regenerating limbs. Nearly half of the clones represent genes belonging to the HoxA complex, which are thought to be involved in pattern formation along the proximal-diatal limb axis. We have analyzed expression of the 5' most member of this complex, HoxA13. This gene ia expressed in developing limb buda and ia reexpressed in the diatal-moat celia of regenerating limbs. Its expression pattern is consistent with the hypothesis that is functions in the specification of distal limb structures as has been suggested for developing mouse and chick limbs. HoxA 13 reexpression is induced within 24-48 hours after amputation and is an early molecular marker for dedifferentiating limb stump cells. Retinoic acid acts to down regulate HoxA13 expression, coincident with the change in positional information of blastema cells from distal to proximal. expression is more distally restricted in anterior regions and extends more proximally in posterior regions, and thus is asymmetric with respect to the anterior-posterior axis as has been observed in developing limbs. Homeobox are genea HoxA13 1344 HOX D EXPRESSION IN DEVELOPING AND REGENERATING AXOLOTL LIMBS ((M.A. Torok, D.M. Gardiner, S. V. Bryant )) Department of Developmental and Cell Biology, University of California, Irvine, CA 92717 Hox complex consists of an evolutionarily duplicated group of homeobox-containig genes that are expressed along the body and limb axes in overlappig regions. Hox genes are expressed durig and thought to play role in the pattern formmtion of developing limbs. Urodeles have longbeen studied for their abilty to regenerate lost tissues, wicluding ims bs their limbs. Amputation of axolotl, results in a blastema from which theregenerate is formed. Blastema issue was used to make a cDNA librry from which seventeen homeobox genes were isolated. (Gardiner and Bryant, 1994, in press). Three of the genes identified by the library screen show significant nucleotide sequence identity to theHoxD complex genes HoxDll, D10, and D8. in pettening of the HoxD genes are believed to be involved anterior/posterior axis of the limb. This work details the temporal and spatial localization in the axolotl limb of these three genes. HoxDll, D10 and D8 have three, one and two transcripts, respectively, as detected by Norther analysis. Al tascripts studied to date appear exclusively in then meschyme and are developmentally regulated. Retinoic acid, which has been implicated altenrg the patter of gene expression in regeneratng and developing vertebrate limbs, was analyzed and was found, insome cases, to alter the expression patter of the HoxD genes. One of the HoxDll transcripts is expressed only in response to retinic acid treatment and one of the D8 trancripts is upregulated by such treatment. The implications of these results for pattern formation will be explored. 1345 DLX-3 LS IVOLVED IN VERTEBRATE LIMB DEVELOPMENT AND REGENERATION. ((L. M. Mullen, M.A. Torok, S.V. Bryant and D.M. Gardiner,)) Department of Developmental and Cell Biology, University of California, Irvine, CA 92717 TME DRO6OPHIL TER 18-WHEELE ENCODES A LEUCIE ((X William, RECETR-LIKE RICS 1. Departmet of Biological Blslen2, ofEldon1)) X University Dusman, Notre Dana, Notre Dana, 46556. sciences, (219) 631-4161. 2. Howard Hughes nstitute, Baylor of 77030. Medicine, Houston, Colleg REPEAT MOLECULE. t. IN Medical Tx Distalless (Dli) is a homeobox containing gene expressed in the developing head andlimbs of Drosophila embryos. Six vertebrate genes related to DlU have been isolated in mouse, frog, zebrafish, newt and human, and comprise the Dlx family. We have isolated an axolotl homolog of Dlx3 from a regeneratinglimb cDNAlibrary and studied the expression ofthis gene throughlimb development and regeneration. In other species, the Dlx3 homolog has been shown to be expressed in embryonic ectoderm andlimb primordia (mouse) and in the otic vesicles (developing zebrafish). By whole mount is situ hybridization analysis, we find Dlx3 is expressed in the epidermis of developinglimb buds as well as in that of regenerating limb blastemas. Dlx3 expression during limb regeneration is markedly upregulated approximately 6-8 days following amputation (medium to late bud stage) and is seen as an anterior-posterior band in the terminal epidermis. Immunohistochemical analysis shows the protein to be in cells of the limb epidermis as well as in differentiated limb muscle cells, a result supported by RT-PCR from musde fractions. 18-whe.ler(1lw) UREA ia expressed unique 9w aRK& expression patterns (detected through whole mount in situ hybridization with digoxygeninUTP-labeled19w cDOA fragment) were observed in ebryos mutant for the segmnt polarity genes wlnglesa(vg), patched(ptc), naked(rkd), engralled(en), or azmadlllo(arm). Escaping show bypcorphic high frequency leg, antenna, and wing deformities, presumaly resulting frcm improper aversion of appendage imaginal discs. 19w is exprossed in all larval imginal discs. Expression of 18W protein in nonadhesive Schneider-2 cells prmotes cell aggregation to a degree in similar experimnts with To}L and other data indicate that the 18w receptor-ligand molecules. Theseand/or protein functions as cell adhesion molecule, receptor ccmunication during various mediating intercellularincluding pattern formation and imaginal events, developosntal cell determination. Recent experimnts have shown that 1lw be involved in the tranalocation of Dorsal-related isnity factor(Dif) protein into the nuclei of larval fat body. Dif is a cytoplamic protein found in larval fat body that quickly accumulates in the nucleus upon injury or with lipopolysaccharide (1p, 1993). The potential injection role of 19w in the wg->en and/or the*n->wg signalling pathways, imaginAl cell determination, central nervous systm developmnt, and the larval ine response is currently under investigation. 13" 134T DEVELOPMEL CONPOCAL Hughes BlUrERPLY WINGS: A STUDY BY LASER SCANNING Institute, apposed layers of epithelium, arranged in precise linear arrays (1). and is Each develops from two wing butterfly Laboratory 1525 Wisconsin, Paddock, J. Gates and S.B. Carroll)) Howard of Molecular Biology, Bock Building, Linden Drive, Madison, WI 53706, USA. ((SW. MICROSCOPY. Medical University The ON thousands of scales overall wing pattern, which usually surface of the wings of most butterfly species. wing in the imaginal discs the butterfly processes pattern have described, and are remarkably conserved between recently and flies (2). As a first step to an understanding of the which by specific colours are assigned to individual scales, we have undertaken structural study of scale cell development in the pupal the butterfly, Erseia..xsenn wing epithelium using laser scanning confocal microscopy. Tne scale forming cells are first detected as large polyploid between the pupal epithelial cells, and eventually interspersed The scales precise rows in the pupal wing epithelium. small subsequently develop and grow out from the scale cells; first single colour to the scale diffen on upper lower and that been a of protrusion, which shape. preparations protrusions thickens This where and is and best bundles eventually eventually flattens to form the charactersitic viewed in fluorescent phalloidin-stained polymerised parallel arrays of form actin are detected the scales flatten as in out. the It of the mature wing in appear much laser on in the wing disc field cells in the imnainal some scales but only a subset of them lateral pupal epithelium, perhap inhibition. using a mechanism akin (1) Nijhout (1991) The Development And Evolution Of Butterfly Pater. Smithonian Press. (2) Carroll S. et al. (1994) Science 265:109-114. first wherea scales imaginal discs, might sugest that all discs. kDowledge of their future identities to note that the sigs pattern have to H.P. Wing in pattern. segmntal a Abnormal a adult flies of a seen a ny HOW DO SEA URCHINS GASTRULATE? A BIOMECHANICAL STUDY. ((L. Davkio&', R. KelO2, M. A R. Kohi, and G. F. Oer2)) 'Gra Group in Biophysics, Cell Biolgy, A Bioogy, 31ntegraive and Uniesity of Calfrni, Berkeley, CA 94720. Formabon of th early rchen-on mechanical deformation of th in th epithl as shoe urchin involv a comprising th vegea plabt. What the m tat dri this invagiratn? We hav developed a meail mode which can simulate four m of re gene , and which allows us to define what nmati mechanc propertes the cabt and of ft fte nee to be in order to reproduce observed mnbryonic is of extraelular th The four models are: (i) apicl (N) apical ctotrng of a -ubpopulat1on of vegeta geometriss. plat clls, of vegea pate (ib) swellg of Poneogaycan gel apical by plae cals, and (iv) anular conrbacbton within a subpoplation of cells, a secred veget out differnt range of vege pat cells. We hawhichmapped each mec can operate. For mateil stiffneseswiftin and col tractoring meaim requlire stiff sweFng instac, etraellular nmtr la with a high deomabe ler, whie i gel c apical constriction and cas an hav mtal specify which matea measse in orde deteri conctionb the properte to distngis what drive grnvt 92-20525. anular requlre tness. and cel shp t Thes th matr simublions changes to between these mechanism, and to pnmary gantration. Supported by NSF Neural Development I (1348-1353). Monday 232a 1348 1349 NEK1, A MEMBER OF THE ELK CLASS OF RECEPTOR TYROSN KINASES IS EXPRESSED IN HENSEN'S NODE. ((D. Kenny, M. Bronner-Fraser, and C. Marcelle)) Developmental Biology Center, University of Calffornia, Irvine, CA 92717 PLATELET-DERIVED GROWTH FACTOR RECEPTORS OF MOUSE CENTRAL NERVOUS SYSTEM CELLS IN VITRO To explore a possible role for receptor tyrosine kinases (RTK) in early avian embryonic development, we examined the distribution of two genes that belong to the ELK class of RTKs. Expression of one of these genes, NEK1, was determined using in situ hybridization. During early embryogenesis Nekl mRNA expression was confined to cells of Hensen's node where it can be seen from stage 4 until the completion of gastrulation. Cells expressing NEK1 are within Hensen's node as it regresses and also in the newly laid down notochord. Later in development, expression expands to cells of the presumptive neural plate and floor plate. After several days of development NEK1 is expressed in other tissues that include neural, placodal and mesenchymal cells. Cells within the node differentiate into diverse cell types that are critical for neurogenesis and somitogenesis. Presence of NEK1 mRNA in cells of the node suggests a possible role incell differentiation, and expression in the node and floor plate may indicate a function in inductive interactions necessary for Receptors for platelet-derived growth factor (PDGF) are likely to play a key role in the differentiation of cells in the central nervous system. PDGF patterning of the ((JiB. The limbic e prtein sysem-asociated memba (LAMP) is a 64-68 kDa neuronl surface suboirtcal regions of theIhmbic systm. LAMP byhbmophic bindi and me inked to the naeon membrae by a Rat-LAMP wu oned fm glycosyl-phospbatdylinosis (GPI) cDNA (Ckotec)usin degenera DNA prbes deduced from the LAMPN I clond by PCR mino acid sequence. Human-LAMP of human cDNA Both encode a 338 (Clontec) anchored by oligo prime from the at cDNA seqse 99%identity. Consevedpais ofcystines amino acid polypepad withseque a was sharn idenify the off* mai_rpas three stuccme of the Ig-domains reveals LI, FascicIn andother adhesion OBCAM with new identy in the homology proteins. The stafaunly. with speifi g-fDs on Analsis of TAG-i, N-CAM, highst homology is between LAMP and fim two domains. Te coem prusin ha eight pumtive N- NorQth sites. bota nysis linkedgycosyt dies and elev pose phoal LAMP RNAprobesiniatd the pmsec of two mRNA transcripts in the aInt rat ung bra whih arenot cqx sed in noel tiss. Twot scripts ofidaical wealso detected inhum brain mRNA blot. In situ hy t analysis shows reticted i zed with LAMPimm exrssion of LAMP tscripts, parculy The trecmbist prtein, expressd on the surface oftrsected wihiDn limbic brin is reonzed by at-LAMPmonooala CHO-KI ad is reased body by Pl-PLC da indicates that LAMP is a new member of the immeoglobulin trament. col men with hypot that by NIMHgl GPt-linkage LAP ad resated ptcie in limbic ntMH4SS07. anatomical exprsso, conistent with sysem our devdopmt and fencton. SuWported is type 1 (NFM) is one of the most freuety vonRecinghausens neuob inherited autosomal dominant disorders in humans. Although the symptoms and severity of the dies me variable, the mnjority oftissus affected inMFI me of neural is 1 (aNFI) 432-bp cDNA which ve have crest origin. The avian neuroo cloned is 82% identical to the human NFI gene at the nucleic acid level and 93% the to preicted protidn identica sqce. We have used this probe to eblsh the normal exprsion patern of aNFI in eary chick embryos Northe bbt anslysi aNFI traript expressed as ealy Hamburger & Hailton shows a large (12.6kb) stage 11 (40-45 hours in oo) that pesis into adulthood. IA situ hybridization aNFI is ubiquitously in the early embryo while experiments show exprssd a a prtin, as delineated by antibody saining. is neual crest cells t stages 13 (2 days inovo) nd more highly expressed in 20 (3 days i ovo) dneua suboetof crest derivatives at stage 29 (6 days in ovo). NFI is a tumor suppressor gen which has been shown to regule p2lras in vitro. Since p2lras ha been implicd in cell differentiation, it is likely that NFI is also important to the process of coll neumn differntiaion. We me using approache so Fs, we have produced seveal investigate the role of NFl in neuronal diff crest src-transfeomed quail newal crest cell lines by infecting primary quail cultures with the PAIOI temprature sensitive mutant of the Rous Sarcoma Second, we me genataig mouse neural ct cell lines from mmortomice differentiation, spcfically neuWal virus. mice which am transgenic for an inducible-promoter conouct of SV40 re T angs. We the can manipulate tese sytems to differntia into nesrons in culte, d changes in NFI levels and isoforms during in vitro differentiation. To pinpoint the. ion, our third approch is to invesipte she involvement of NFI in neuronal d ES cells. diffrutsin capacity of cells defcet in NFI using NFI nve"at Medical 1351 HOMOPHILIC BINDING BETWEEN RECOMBINANT AND NATIVE LIMBIC SYSTEM-ASSOCIATED MEMBRANE PROTEIN SELECTIVELY REGULATES NEURITE OUTGROWTH. ((P.Levitt, V.A.Zhukareva, and A.F.Pimenta)). Dept. of Neuroscience and Cell UMDNJ-Robert Wood Johnson Medical School, Piscataway Biology, 08854. NJ We have recently shown that the limbic system associated membrane protein (LAMP) can facilitate substrate adhesion, via homophilic binding, and neurite outgrowth of neurons from areas of rat embryonic brain. rsults provide direct evidence that LAMP expression supports The presentadhesiveness of primary brain cells on LAMPincreased cells. CHO-Kiandcellsgrowth were transfected with full-length LAMP transfected cDNA. Stable transfectants were obtained by calcium phosphate and cloned by limiting dilution. Recombinant LAMP is precipitation on thesurface of CHO cells and susceptible to PI-PLCtreatment expressed has been shown previously that LAMP binds homophilically;rapid fluorescent Covaspheres. Here, aggregation occurs between LAMP-coatedLAMP from the rat bran, bind to Covaspheres, coatedwith CHO cellstransfected with LAMP cDNA, forming aggregates of 3 5 beads on the cell surface. Monolayers oftransfected cells were used as a culture substrate for embryonic neurons from limbic (perirhinal cortex) and non limbic (olfactory bulbs) areas of rat brain. LAMP-transfected cells showed a markedly enhance ability to promote neurite outgrowth of perirhinal neurons, known to express LAMP on the cell surface, compared with olfactory bulb neurons that are LAMP-negative. These results support the as cell adhesion molecule through hypothesis that LAMPandfunctions regulate neuronal outgrowth and homophilic interaction, can possiblytarget recognition. Supported by NDMH grant MH 45507. specific It affimty-purified - selectively 1353 1352 THE ROLE OF NEUROFIBROMATOSIS-1 (NFl) IN THE DEVELOPI NERVOUS SYSTEM ANDNEURALCREST. ((AL Kavka &K F. Barl) Depwmnt ofAnatomy and Cdl Biology, University of Michiga Medical School. Ann Arbor, MI 48109-0616. neurofibromin Mississippi cells and Neuobogy, Medical College ofPensylvaa, Philadelphia, PA 19129. Inc., Proein Chenistry, S. Sm Fracsco, CA 94080. molecu intect of . tech, glycopotein expressed in cordcal and Anatomy, University are found on central nervous system cells isolated from fetal or newborn mice and maintained in titro The presence of PDGF receptors has been shown with colloidal gold-labeled immunocytochemical markers at the electron microscopic level. PDGF receptors are sparsely distributed over the surface of type 1 astrocytes, apparent type 2 astrocytes, and neurons. Receptors appear to be preferentially associated with filopodia-like extensions of the cell membrane. The existence of functional receptors was confirmed using the impermeant, water-soluble affinity cross-lsnking agent bis(sulfosuccinimidyl)suberate to covalently link radiolabeled PDGF to its receptor. The PDGF/receptor complexes could also be immunoprecipitated with the same antibody as was used in immunocytochemical experiments. The improved resolution of these techniques allows definitive identification of PDGF receptors on cultured mammalian central nervous other than oligodendrocytes. These data expand the range of system possible roles of PDGF during nervous system development. 1350 MOLECULAR ANALYSIS OF THE LIMBIC SYSTEM-ASSOCIATED MEMBRANE PROTEIN (LAMP): A NEW MEMBER OFTHE IMUN40GLOBULIN SUPERFAMILY HIGHLY CONSERVED IN HUMAN AND RAT. ((A. F. Pimente, V. Zh A. skeva, B. S. Fisher ad P. Levitt)). Depl. Neuocin Reinoo. C. Grimey, W. Helzel d Cell Biology, UMDNJ-Robert Wood Jobhon Medical School Piscatawy, NJ 08854; Dept Anatomy of receptors system. nervous Hutchins)) Dept. Center. AND FUNCTION OF ANNEXIN II IN DEVELOPING RAT LOCALIZATION NEURONS IN VITRO. ((W.J. Zats and P.C. Bridgman)) SYMPATHETIC and Anatomy and NeurobIology, WashIngton University Medicine Departmerls d School d Medicine, St. Louis MO 63110. Annexln II is a Ca2+-and Is protein whose and PC-12 cel dllferentaton. To knvstIgate annexn neuronl durhg enhanced role nourke outgrowth, we have examned Ka ddatbion and ihbted 1ts plays fnctIon devebping rat yIXthC nerons cuture. Immunoluorecence revead that both annehdn II and ks p11 lght chain were datrbied exression I a in in in microscopy in puntae pattem throgu them c1 at the body staining a neurke and growth oone. In the cel membra for coqswrd to body, annrodn Their p11 grater plama Anneidn and p11 appeaed paslaly coocake growth cone. thickened ceotral doain, but also extended staining was greatest thedomain. the neumns were m'croijed wth a throuot the perlheral1 artbody, When both annexin 11 and p11 wer perthly antiannexdn poWcbnal eded patches, together with the sequestered bho plama alffted, antbody. abl for at bast the and th remained sen. neurons soma wer nwmplogyd 4.6 days clture. Folowng a 3 hr recovery perId, newIte outgrowth from or neurn hrs. was measured for up to 6 Alhough antbody- coutrorneded dNlferert for the firt 90 mh, arnbody-injed ceb outgrowth was not statistcally and by 280 min had grown only 28 % d the dItance d the thrter, grew poorly The mean outgrowth veocilty 252 min for autuiodycornrol (P < 0.001). wa -0.0012 t 0.024 snvrmin (n . 55) 0.147 t O.2 pWmmn (n . ijected oe 42) for control (P < 0.001). Foynne percent d the aw0body-irjcted coe we this time 17 % d the controla (P < 0.001). Those corilnu to ratracthg grow had grwth cone morphlogs. These data idcate that annexi II. In to in jcted Annesdn VI darbutlon not no gro nes in in at n at 1 -. or p11 necessae andior ocalzatlon may be functon in cultured sympathetic nerons. for normal outgrowth Monday. Neural Development I (1354-1359) 1354 233a 1355 OFANTI-N-CADHERIN ANTIBODIES ONTH E XEN OPUS RETINA HISTOGENESIS AND IFFERENTIA ON OF THE RV VIVO ((TD. Folsom, J. Leonad, and D.S. Sakaguchi)) Signal Transduction Training Group, the Neurascience Program, and the Deptof Zoology and Genetics, Iowa Stat University, Ames, IA. In the preset study, we have investigaed the role of N-Cadhrin during the dif of the Xenopus retina. Functio normal histgenesis and blkxing antibodies directed aginst N-cadherin injected into optic vesicles of stage 23 embryos. Embryos were allowed to develop for 36 hours. imm unoist using development of the retia was thn examined irected against the snptictrminal retd pteins sy d antibodies (p65) and Rab 3A In control eyes, the anibodies labeled the outer andinner plexiform layers and the optic fiber layerof the retimarveahing its distinct laminatio. Compared to thecontrol eyes, those injed with the anti-Ncadhern antibodies appearedimnmaure, exhibiting unusual lamination patterns or no lamination of the In addition, the eyes often lacked a prominent lens and other morphological abnormahites were obsered including unusual normal eyes. invaginations of the eye cup and small retnotectal project was also examined in antibodyinjected embryos at 24 hours following injections. Horseradish peroxidas was approximately use d to label the visua projecton fron the injected eyes. Many optic fibers a relavely normal cous from the antibody injected eyes followed the optic the antibody does rot block axon outgrowth. However, tectunm, rvealing the projection from injected eyes tended be delayed their growth when compared to nonimmune andbody injectedcontrols. We conclude that Ncadherin plays an essentia role durig the normal histogenesis and of the vertebrate retina. Supported by grants from the NSF, the differenttion ISU Biotechnology Council and the Carvvr Tns EFFECFS wer reti to I IDENTIFIC O OF A NEURONAL COLLAGEN RECEPTOR: EXPRESSION OF a2l WEGRIN IN THE DEVELOPIN FUNCTIONAL ANALYSIS AND DiB. Gervin, MdNsgy, and D.O. COegg)) BDBdow,U Unvaity ofCalfornia SBaBatw, Santa Rnate CA9310. (Span. Bradshaw.) nrvons the all exo migon,; amo uo to guide aniao ofcell RETINA. ((AD. NeosciencResech Barba K-& by In the doelophig exe of mic AD. matrix smm md en cc doc edr by data- Qt2 im aY highe at the n day 1. culs d The the npt With res Reta pngo ntlre ioe ydeecne-i_ d a expressed epsiOf02I Leves atemiryomic day 4. As develepinesa roceeds, litl orso by postal ideiid neotic 1 colsgenreceptr t2l n2lxsein mid mRNA were rn. significast Although cull a inea m edim p We have ideiedinigr q2 developing avin is bavebeem of colge ewie onogw samonderzkod. tens~O ofdevelopingemtryos. prntigacvke dfor endiyo nic retid cullS, eik in di povides apotentia uoba fainly Of fao, SW O oc gow he Colla synae n ate exu onesaprese ehihemaric comp that lite if a2 is inlogrin Pr-culls. By employing function exprasedcm unifeentd retinal n amias a collagen reeptor, blocking asalbdies we have foted thatA iesser mediatin bath cull adhesio andp nr cms outgrowth by embryonic retinal cull on fioaction bath collgen type I mid type IV. of -n regulatd with down dhis' nsgrin in amociatd widh thedifsetto data mid wtol aWm appeas to be activity of p in p a rtnald cdellsbose sggesta dk die expession pater w forecaol in the whdy role events avim retina ovelpm ontof the to 1356 INTEGRIN v41 AND VCAM-1 AREE(PRESSED IN THE DEVELOPING P45D (PNS0AROM) IN TIHE PRIMARY CULTURES ASTROC-YME BY PCR AND DAL,UNOCYTOCflsrY ((ainat I-L Zwam Cheeg) Poipaltoa Casicil. 1230 York Avasie, New York, NY 1002 1. NSODAROM is the enzyme that catalysi the conversion of androgen to estrogen in the brain, cesrgen is required to promote netwona growt asd regulate the sexual behavor minimal. Rcasen studie also sugges that estroge agaLni Aluheimeors may he invevedin the protectio IDENTIFICATION OF AROMATASE RETINA AND MEDIATE PROCESS OUTGROWTH MOUSE Bradshaw and ((G.M. Can, A.D. Ceg.)) Neunroence ResearchInsttue, Utvray of Barbar Ca. 93106. OF RAT D.O. ClMorrisa Sa mid C. Yasi teractns are o hi the spelatn DurIng rel devebpmet, ce l of relina pwnpe, aXonal paSthldrt and and the are rlefsti of the segregatnio of alemalt cea wd Me lyers e and homoplic eel h wIe p of ure vertbrate r adhesion nolecule are btly medae these ert ad delition of col adheson nmlcules expre_d hI the deveopI reina dsod alow a better Ial deveopnt In o the moeculrm anme Invovd In undrstadI n inmt inherant edIated VLA-4 (a41) and countr receptorthe vascterl adhesion by molculwe- (VCAM-1).Prouslywwreported theeprssIo of mRNA encoing syqsogepnes, diseas cneste aunil at- deopIn cick reI hi usng are nigran We tods ertaigt sowble VCAM-1suppots pocess reconant hi cAr odes spnet the protein, acidic resub sugos betwee arole that this enzyme Fl2ID ME (1: 1, of P450AROM II re-amplified then above to detec in astrcytes the 2nd PCR reaction a using P450AROM also studied was dreurmining by yield mRNA that the with the action of 1358 oqp IL-li5 conclusion: dose- a estadiol to peared he spetiric since its action astrcytes sine the brai in by are a new to cedh role for prdoduc in prtctn ee was blocked IS ALTERED N-CADHERN MOLECULAR BY EXPRESSION DUWUNG FUNCTION AND Fenre-Cornwell, REINAL DEVELOPMENT ((iC. Grnnwald)) D _atmnt of Antomy, Pathoogy, Univrty, Philadel, PA 19107. NEURAL GENETIC RJ. and Buoo Jeknon CeU Biola, Tboms ad G.B. TIHE eatAsto mid expres IN PROTEN, IS UP-REGULATED IN GLOSIS AND GUOMA. ((D.M. Jaworaid, Yale GlIt N-cadberin molcue is cadberin fmildy of to mediae ceii-cu d been N-cadherin Optia fincton depends f the a shown which de v ia its aic methods for tunatd N-cnherin domais pr_tin mnauker i (8-a). promoter to in ly e_enI both homophilic the cytos icet dsevlin ina embmyoni seon or potion desiwgn encodi retin mRNA of th via molecul (2) xracblular e hitochemially Intt enbyonic day 7 chick enxyo no"ul pTBI, which uilizes the eprI-o of 8-gal, Nd pSG5NCAD(+) or (-), _dsense N-cadherin mRNA, respetvely, dnven Rwus wbich ct by exprem truncutd N-csdherin, or SV4O promote of the pSG5 plasmd kxprm vector. Folowin thee days in oran Whole mount emimbion reveaed ta tui, ssa were fixed d staid for wer to be incorporftd icaed by 8-gal expr on retn cel Whik blg noues could be detectd asmog cut ranelced with pTBlI Uad/or pSGS, no neurites were obsrved amnwg markd culls N-caherin essing antsn Aberrant nturile growth observed along marked cul tncae Niad peubation of N-cadherin retsin These dat suggt that pa cadhei the constru in mRNK wa neurie lOPM expssn amog retinal NRSAEY06470 to MC2FC and ROIEY06658 to nuron UIBO. Supported by NIH Hockfield)) contribute to the cel Section of Neurobiology, Medicine, New Haven CT 06510. vnmet of the CNS. for omyoliation While rqepre sitocytes a more fucinlyheterogenonus populstion of cells. Severalextracellularmatrix curimuponent areexprsussd by aitocytes (HA) and the HA incuding hyalur Previosly, we repoted the cloning and restricted tothe CNS (JCB regulationof BEAB,a Abinding with gl 125:495) that is tmpoally and spataly regulated in para enesis. drepto fCYlEr alyze dBEHAB exre hRA hypCtopy C.dconditions promoascyte We I now blot analym of priay tRocyten or with the wh plsmid drive on during of wegd respo sible adesion gowth etinal deveopment by mRNA ideufy successlly transected by vir directed at hioduced N-cadhri ase mima and (3) wer murite with di teactio of N-cdn which coud (1) be of eher retin smrcoma the role ng veco m domain e-peimea Th pasmid _ep drive the exPri dtect o extracd its cl calcium GJMZ Kelly and S. University School Prviu sdies have underping remodeling. growth is adult rats invaion. mfpare breast danag BEtAB could prmmy g and is l (l cultu and hyperplasisY BEHAB Is abundt Northern expressed by Assues wa was might play a role in glin performed following a cortical verified .BEHAB in the vicinity eHA hasbeenrelported toplay expession is brain. hot is to normal colon dt HA shown s Glioss ignifcndy o-egultd gliomawasexamined beca indicates wheher BEHAB To in oro stab wound cutue of the stab. BEHAB expression in cpificandyup-reguled ntmdetectbl in anileintu in non-neurkl glioblastoma tumors, inclxudng resonse to CNS lug Incrad BEHAB expreion have sigios implications for the role ofHA ad HA ind nd CNS posifaintinsue remodeling dPiin.g tmogednbysiat.(Suppored by EYo6511 and injury and EY06451.) diring factoirs in PiooAROM supporting theunwons. ad EXPRESSION OF BEHAB, A CNS SPECIFIC HYALURONAN BI 1 by anti-ILli involved in synptic reorgaiization xt iu id provding biological otllutadd am 1358 OF 5'- mid growth, the ability of astrocytes vito suggeting PERTURBATION prinmer, antzmsepMm derdcribed idntci eas in estmadol production by astrocytes. Intedeasidn-li (IL-18). a kownP50AR?oMA nKxduatorin gtatadt mid extragoninla non-neurml cells, induced adoedependent isdbitionof estsdiol prdodction by astrocytes. The cultures. Testosterone induced fornerm MORPHOLOGY a the further bp product. Aromaitization by production by astroyte 168 herd of was was performed (sense) and 5'This productwa [y-32pJ-radiolabeled a a llary using PCR of TrGGAGAGTTCATGAGAGTCTGA-3- (sme), ftoethe hiegi ffrs mpicainstepa. The greate fri localixd masrcytes in the mechaiical Was study was 5'-ATACCA0GTCC`rGGCTAC`rG-3' IUI rAAATATGATGCC-3' (aniseose) gScnnate a 272 bp product specific primers two cal cnditio altered in two unsocytochemical Im shown presenc hot-nested RT-PC3 onmirnied by using dirthshowtu man owth fommouse and chick ld by mownmonal inhibitory antibody. nou P450AROM nasrcytes. fthe developing retnal cels. NEURITE detamined. yet bans in astroytes mid the isolated from the cerebral cels w masker of astrocytes. a agnat rat outrwth Is Wopct dth and chik beta-I sburit Thes and VCAM-1 hI meiadIag interactons rkrtgrin and that mous not aittred in sera n-suppkanensed camid cortx, pathoogicsl Nixed estrasici. tats by 1-day-old of prodhuce cells to chan growthcones the cerebral growth located neurons degemesate this vlv) reacton (Cann, GM. and Cego, D.041991)J. Ce# ib. sud we report the detection of VCAM-1 integhi ata4 eipessIon dneveping mouse reIVCAM-I eNesin is especaly stn I the optc ber layer and opti head, at Ines when retinal ganglon co polynm 115:11Oa). In where mediui. for a day. w then sepwar from oligodentAocyo- by cottaud aiderserum-4reecconditions for 24 n "mioig Thpuity of masocytes 90% stto allng using anasiibody aginst judg by m kgln neuronal areas: forebrain. the P450AROM ability of hiegn the ick moduates hormone basa disorder. However, the soaurc e(s) of esuogen in crebralt cortex hat Studies were petformed to investigat the expresion of to bynqhoposisis, since this hippoesnpcms and hara Neural Development I (1360-1365). Monday 234a 1360 1361 BINDING BETWEEN THE NEURAL CELL ADHESION MOLECULES AXONIN-1 AND NR-CAMIBRAVO IS INVOLVED IN NEURON GLIA INTERACTION. ((D.M. Suter, G.E. Pollerberg, A. Buchstafler, and P. Sonderegger))Institute of Biochemistry. University of Zurich, CH-8057 *Max-Panck-instItute for Developmental Biology, Zurich, Switzeriard. Department of Biochemistry, D-72076 TObngen, EFFECTS OF L-SERINE ON THE GROWTH PATTERN OF NEURONS IN VITRO. ((1. Savoca, U. Ziegler, and P. Sonderegger)) Institute of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland. molecues comprising both immunoglobulinImmunoglobulin superfamily type and IIke domains have been found to be Inoled In a l te f5bronectin and heterophilk of ktracthant rv ovide the molcular variety homophilic basis for many different cell-cell and cell-matrix contacts during system. Recently we have shown that the morphogenesis of ternervous mo gii nural cal adheslon kmoles axornn-I and Ng-CAM (neuron chicken stituting the culture medium. We have examined the influence of non-essential amino acids on embryonic chick dorsal root ganglia (DRG) neurons invitro. DRG-neurons were grown for 16 h in a serum-free, chemically defined medium containing minimal essential medium, nerve growth factor and N3 (insulin, triiodothyronine, corticosterone, transferrin, putrescine and Na-selenite). If ine was supplemented at physiological concentrations (10-200 AM), the neurites were up to 100 percent longer and developed a more complex branching pattern. The growth cones were smaller than in control cultures without serine. Gernmny. c Interaction, which IsIrnolved a adhesion molue) undergo hhterophilic also interacts wih Nr-CAM in neurite outgrowth. Here we report that of (Ng-CAM related cell adhesion molecule). Firs evidence for an other than was found when fluorescent axonin-1 withmolecels Ng-CAM coupled axonin-1 atthe surface beads (Covaspheres) covailely carrying (DRG) in an bound to glial cells of cultured chicken dorsal root Interactin that could not be perturbed wlth anti-Ng-CAM Fab fragments. Preincubatlon of these cultures wih aritbodies against potentlal receptor candidates revealed that only antibodles against Nr-CAM inhIbit this that I these DRG in mmunostainIn andsi interaction. hybriaion revealed glial cells indeed express Nr-CAM at their suriace. The bindIng between axonin-I and Nr-CAM was confimred with Isolated molecules using a cultured in the Covasphere aggregation assay. dissociated DRG cell ensheathment presence of anti-axonin-I or anti-Nr-CAM Fab fragments, of neuritas by the gIlal cab was markedly perturbed. We conclude that the membrane and Nr-CAM of the gial binding between axonIn-I ofneurite tth early phase of axon cells may have a role during cell axonin-I kiteractiDn ganglna were tth morphology of neuronal processes originating from depend on the substratum as well as on a variety of components The length and the in culture neurons con- ser- The described effects of Lserine were concentration dependent, stereospecific and found on several different substrata, such as laminin, Ng-CAM and axonin1. Simllar observations were made with neurons of the central nervous system such as embryonic retinal explants. Addition of other non- essential amino acids to DRG-neurons had no effect. We conclude that serine, althongh it belongs to the group of non-essential amino acids and is not known as a neurotransmitter, is an important factor modulating neurite outgrowth and branching of the central and peripheral nervous system neurons in vitro. ensheathmenl. 1362 RAPID REGULATION OF NEURITE EXTENSION AND RETRACTIONBY ARACHIDONIC ACID METABOLITES. ((N.R. Smalheiser)) Dept. of Pediatrics, MC 5058, University of Chicago, Chicago,IL 60637. Recent studies have indicated a role for arachidonic acid (AA) metabolites in regulating F-actin levels in non-neural cell types. Since rapid-onset neurite extension on laminincoated substrata and acute lysophosphatidic acid-induced retraction in NG108-15 cells both F-actin dependent responses, the possible regulatory roles of AA and its cyclooxygenase (COX) and lipoxygenase (LOX)metabolites were examined in these two assays (methods in DeveL Brain Res. 62: 81-89 and J. Neurochem. 61: 340343). Outgrowth measured at 1.5 hr post-plating was stimulated by AA (1 micro M) and by the COX inhibitor indomethacin, but was inhibited by phospholipase A2 AA inhibitors (BPB, ONO-RS-082, quincrine)and by the 5-LOX inhibitor Acute increased neurite lengths with little effect on probability of neurite inhibitors, A2 AA, and was inhibited by PL was also by retraction enhanced neurite by AA861, and by the FLAP inhibitor MK-886. The DAG lipase inhibitor RHC80267 had no effect in either assay. Cells exposed to high doses of PL A2 or LOX inhibitors exhibited decreased contractility, in that many neurites failed to retract when detached mechanically from the substratum. These results indicate that phospholipase A2-derived formation of AA, and its metabolism via 5-LOX, are not only neceary for acute neuritechanges in either direction, forward or backward, but that activity in this pathway can actively regulate responses of neurites to other stimuli. It is possible that some of the effects of AA and its metabolites on synaptic plasticity may be mediated directly at the level of the neurite. Supported by NIH HD 09402, NS 26055 and the Brain Research Foundation, Inc. are AA861. initiation. 1363 CRITICAL PERIODS OF OPIOID SENSITIVITY IN A PHARMACOLOGICALLY SUBPOPULATION OF ASTROGLIA DURING DEVELOPMENT. Ryan and C.S. Turbek)) Department of Anatomy ((KF. Hauser, Gurwell, KY 40536. School of & University of DISTINCT JA. S.E. Medicine, Lexington, Kentucky Neurobiology, glial In free Inltrellularcalium (KCC2li) development, oplod-Irduced chanrge messednIn prmary mixed glial cultures from 1-day-old mouse cerbra. Prior To explore the ontogeny of esponsiveness during CNS to opbids wer opid [Ca82]i in Irocytes, can affect growth. Opioid effects w reexprd ch s [Ca2 immunoreacwe, acidic protein measured by using days 1-10, 14 and 21 Invitro. Changes lC821i ic1 opiold analis of ratlometrlc dye fura-2. The computer-sidedImage caused signifIcant Ises InlCa21 U69,593 In100 nM agonist, in some astcytes. Equimolar nentrIonsof the opioid antagonist, naoxone, or the selective K antagonist, nor-binaltorphimine,bocked the effects of U69,593. In and aonts can vence that asocyt (GFAP) gIal flbury flat In (tp 1) astrocytes on wer the entrtion, and 5[D-Pen2,D3orpaD-Ala2,MePhe4, Gly(o051enkephalin) agonistsfoploid UW9,593 (Ca2+i. t ype of In In a subpopulation I ncrases [Ca&'li signilIcant Moreover, ICa2+i Increases were Induced by but not i and6,agonists. When thep cntae of type astrocytes owing signIfIcant Incaes in [Ca24liwas examined at dferet times in vltfO, the propordion of type astrocytes responding to opioods changed dramatically. Fewerthan 20% of astrcytes responded before few responded. day 4in culture. At days In vitr in confluent cultures responded. This suggests that transient, develpmentally regulated periods of oploid responsivenes occur In a pharmacologIcaly disnct populatlon of astocytes. Critical pedods of opioid sensitivity during develpment may be, part, defined by regionsl and temporal differences in the functional expression ofK opiold receptors by astrogla. Supported by NIH grant DA 06204. Selective Pen5penkephalin) receptor tvse (100 nM) did not effect caused I ascytes. xi, I I 44 60-90% astrocytes However, In 1364 1365 DN VITRO EFFECT OF OKADAIC ACID ON NEURITE OUTGROWTH IN RETINAL EXPLANTS. ((M.E. Vaxqucx and R. Chiesa)), Department of Ophthalmology, Columbia University, New York, NY 10032. NEURiTE OUTGROWTH OFPC12 CELLS IS INHlIBiED BY rANUSAND BOTULUM-A, -B and -C NEUROTOXINS ((G. Bi, and R. A. Steinhardt)) Departent ofMolea and Cell Biology, University of Califonia at Berkeley, 391 ISA, Several reports indicate that okadaic acid (OA), a potent and specific inhibitor of serine/thronine protein phosphatases 1 and 2A, can indwe neuronal cytoskeleton disrption. This effect is due to changes in the phosphorylation state of the cytoskleletal proteins, primarily, the microtubule associated proteins MAP and Tau. Since the neuonal cytoskeleton plays a pivotal roll in neuritogenesis and neurnal degeneration, an study to delineate the effects of OA on neurite outgrowth Rednal explants taken from six daysin refinal explants was uner old chick donor embryos (white Leghorn), cultured on collagen gels, covered with nutrient medium (BME eniched with optic lobe extract, 5OOg/ml) and incubated for three days. After 24 hr post explantation, the cultures were treated with nanomolar concentations of OA (0.5-10 nM) for 48 h. Neurite number (NN), length (NL) and growth index (NGI) were determined at the end of the expeim in an inverted microscope coupled to an image analyzer. We have found that exposure to OA (0.5 to 2.5 nM) induce a dramatic dose dependent reduction (50-80 0%) of the rate of NN and NL, with an overall decrease of NGI. Viability of the explants was unaffected in this range of exposure, 10 nM induce a complete inhibition of neuritogenesis. In conclusion, OA at concentations low as 0.5 nM, ificantly inhibits the outgrowth and affect the morphology of neurtes without impairment ofthe viability of the cultures. as Berkeley, CA94720-3200 To investogate the role ofexooytosis in neurite outgro , we used the well-chaacterized bacterial neurotoxin targeting sion complex to disrupt vesicle do proteins in synaptic NGF c in PC12 rat pheoebromo vesidle exocytosis primed PC12 cells were plated on matrigel oated glass with coveraip for a few hours before being microisjected control buffer or one of the four neurotoxins: tetanus toxin, s. (cleaving synaptobrevin), botulinum-B (cleaving and synaptobrevin), botulinum-A (cleaving SNAP-25),length was betulinum-CI toxin (cleaving syntaxin). Neurite outgroth in all of 40 hours after injection. Neurite measured by injected PC12 eafs four compared to that in control buffer injected cel These results suggest that membrane addition by exocytosis mediated by similar do ion complex (snaptobrevin/SAPis partially responsible for neuite outgrowth 25/syntaxin) cells. PC12 the toxins is inhibited 30-50% a in Monday. Neural Development I (1366) 235a 1366 ULTRASTRUCITURAL AND MORPHOMETRIC MANIFESTATIONS OF CHRONIC ETHANOL TREATMENT ON RAT SUPRAOPTIC NEURONS. ((J. Joshua, T. Thrower, A. Andrews and W. H. Woods)) Department of Biology, Philander Smith College, Little Rock, AR 72202. (Spon. by B. Lyn-Cook) The synthetic activity and/or release of posterior pituitary hormones (oxytocin and vasopressin) which are produced in part by the hypothalamic supraoptic nucleus (SON) are strongly inhibited by ethanol. The present study was designed to determine the effect of chronic ethanol administration on the ultrastructure of SON neurons. One week old Sprague-Dawley rats were given daily intragastric 3.0 g/kg doses of ethanol until 4 wks, 3-4 mo or 6-7 mo old. Brains were removed and hypothalamic regions containing the SON were processed using standard EM procedures. Electron micrographs were studied for ultrastructural changes, and cytoplasmic, nuclear, nucleolar cross-sectional areas were determined from the micrographs using a Cal Comp digitizing pad connected to a computerized image analysis system. Total counts of organelles which appeared altered (mitochondria, neurosecretory granules (NSO) and lysosomes/dense bodies) were made for each cell. The most significant changes in soma, cytoplasmic and nuclear areas of SON neurons were the smaller size of the 4 wk controls and experimentals as compared with the older control and experimental groups (3-4 mo and 6-7 mo). Likewise, there was a significantly smaller number of NSGs per 100 pm2 in 4 wk controls and experimentals and between this and the 3-4 mo and 6-7 mo control and experimental animals. Lysosomes and mitochondrial number also increased per 100 pm2 in 3-4 mo and 6-7 mo control and experimental groups over that of the 4 wk. However, the experimental groups in each case showed a significant increase in the number of highly vacuolated and generally enlarged mitochondria over that of controls. Synaptogenesis and Synaptic Plasticity (1367-1370) 1367 KINETICS OF COMPARTMENTALIZATION AND DIFFERENTIATION OF THE GOLGI APPARATUS IN MUSCLE FIBERS DURING DEVELOPMENT OF THE MOUSE DIAPHRAGM. ((C. Antony1, B. J. Jasmin2, M. Huchet3, J.P. Changeux3 and J. Institut Jacques Monod, Paris, Francel; University of Ottawa, Canada2; Institut Pasteur, Paris, France3. Cartaudl)). Shortly after the onset of synaptogenesis (El3-14), compartmentalization of the expression of the genes encoding the acetylcholine receptor subunits occurs (Piette et al., Dev. BioL 157: 205-209, 1993), suggesting a regional specializaion of the machinery responsible for the targeting of the receptor to the postsynaptic membrane. Indeed, in adult chicken or rat muscles, the Golgi apparatus (GA) appears both to be restricted to the subneural domain of the endplate and to be biochemically differentiated (Jasmin et aL, PNAS, 86: 7218-7222, 1989; and in preparation, 1994). In the present work we analyzed the kinetics of relocation of the GA in the developing diaphragm at various times after innervation using a panel of antibodies mapping to different subcompartments along the secretory pathway. We show that i) compartnentalization of most of the GA markers occurs at E16, i. e. 2-3 days after innervation, ii) some GA markers are no longer expressed even in the subsynaptic Golgi, iii) by contrast, the rough endoplasmic reticulum or the intermediate compartment, labelled with rabl, do not display compartmentalized patterns of staining, iv) upon denervation, a burst of reexpression of all Golgi markers is observed. Taken together, these data show that in innervated muscle fibers only part of the secretory pathway, i e. the GA, is compartnenslized and placed under nerve controL 1368 PRESYNAPTIC LOCAUZATION, MEMBRANE ASSOCIATION, AND AXONAL TRANSPORT OF GUINEA PIG TYPE I BRAIN HEXOKINASE ((JA Gamer, KD Unse, and RK Polk)) Cell and Neurobiology, USC School of Med., Los Angeles, CA 90033; Beckman Instruments, Fullerton, CA 92634. The identification of central nervous system presynaptic terminal proteins has been of increasing recent interest Previously, we reported unique characteristics of a 118 kD protein (pp118) axonally transported to the presynaptic terminals of retinal ganglion cells as part of slow component b (SCb), the proteins hought to compose the cytoplasmic matrix Afthough one of many proteins in SCb, pp118 uniquely co-isolated with the synaptic junctional complex upon biochemical hacionation of the radiolabeled synaptic regions. Also, pp118 was found to exhibit a marked increase in hydrophobicity, and thus potential relbtonship with membranes, after entry into the presynaptic environment. Purification of pp118 from synapses and amino acid sequencing of proteolytfc digest fragments revealed that the protein was a guinea pig isoform of the glycolytic enzyme Type I (brain) hexobnase. Further biochemical treatments support the identity of the protein. Polyclonal antibodies made to pp118 reveal an expected restiction of the protein to CNS neurons and an enrichment of the antigen in presynaptic terminal areas of neurons. One question in the study of neuronal hexokinase has been the fidelity of its known reversible association with mitochondrial membranes. While it is clear that hexokinase does associate with mitochondrial membranes, it is not known if that association is restricted to mitochondria, or might also represent association with other membrane systems. The preferential enrchment of putative hexokinase during synaptic junctional complex puification was unexpected, and could resuft from its sbtng association with membranes of the presynaptic portion of the synapse. This enrichment was not mimicked by similar biochemical enrichment of two intrinsic mitochondrial enzymes. Under the conditions used, few mitochondria appear to be identifiable in the fractions highly enriched for ppl 18, though synaptic 'junctional structures are visible (Matus and Taff-Jones, 1978). 1369 1370 PICCOLO, EXPRESSION OF AGRIN ISOFORMS IN DEVELOPING RAT: AN IN 'Dept SITU HYBRIDIZATION STUDY ((D.M.Stone and K.Nikolics)), Department of Neuroscience, Genentech, Inc., South San Francisco, CA. 94080. (Spon. by J. Mather) A 420 KDA SYNAPTIC JUNCTIONAL PROTEIN LOCALIZED TO THE ACTIVE ZONES OF SYNAPSES IN THE RAT CNS ((C. Cases-Langhoff, K. Takei*, P. De Camilli*, C. C. Garner)) of Pharmacology, Yale University, New Haven, CN 06536; NRC, University of Alabama at Birmingham, Birmingham, AL 35294. We have utilized a molecular and immunological approach to isolate and characterize novel structural components of synaptic junctions from the CNS of the rat. This has lead to the identification of cDNA clones encoding a 420 kDa brain specific protein called Piccolo. Antibodies generated against short segsnents coding sequence of have been used to determine the and subcellular distribution of Piccolo in rat brain. At level, Piccolo show a similar with the vesicle associated the of dendrites and cell bodies. This that Piccolo is associated with pattern spatial light microscopic punctate expression pattern synaptic protein synaptophysin, outlining profiles punctate suggesting synapses was confirmwd by EM analysis. Using the ABC method peroxidase reaction product has been shown to be concentrated in the presynaptic nerve terninals at the active zones of synapses. EM-immunogold labeling of cellular fragments of cerebelum show gold particles decorating the cytoskeletal matrix in between synaptic vesicles of synaptsones. These data suggest that Piccole is part of the cytskeeal network ankoring synaptic vesides to the active zone of these synaptic junctions. This hypothesis is supported by biochemical studies revealing that Piccolo is tightly assoicated with synaptic junctional preparation. Piccoio appears to be preforming a central role in the formation of synaptic juctions since it appears in synapses, both in vivo and in vitro, as soon as they are formed. Agrin, a large multi-domain protein localized to the extracellular matrix at the neuromuscular junction (NMJ) and derived from spinal cord (SC) thought to play a key role in synapse formation at the NMJ and possibly in the brain. Several different alternatively-spliced isoforms of rat agrin have been identified, which differ by the inclusion or exclusion of small inserts at three sites in the C-terminal half of the molecule, and by nicotinic acetylcholine receptor (AChR)-aggregating activity on skeletal muscle fibers (Ferns et al., Neuron 11, 491, 1993). We used in situ hybridization histochemistry to examine differential isoform expression in developing rat. Six 36-mer oligonucleotide probes were designed to distinguish between known mRNA isoforms at both the Y site (Exon 28: contains either a 0- or a 4-amino acid insert) and the Z site (Exons 31-34: contains either a 0-, 8-, 11-, or 19-anino acid insert). From developmental age E I S-PO, Y4ZO was the major isoform expressed in the nervous system: in mitotic ventricular zones, dorsal root ganglia, and diffusely throughout the brain. YOZO was not found in nervous tissue, but motor neurons, is specifically labelled microvessels within the brain and SC; expression was not detected in peripheral blood vessels, however, suggesting a potential role for YOZO in blood-brain barrer function. Y4Zl9 and Y4Z8, the forms most active in AChR aggregation, were expressed specifically in SC motoneurons; Z19 expression declined from E15-Pi, whereas Z8 expression increased slightly from E15-adult ZI1 could not be detected. These data are consistent with results from PCR analysis (Hoch et. al., Neuron 11, 479, 1993), and suggest that different agrin isoforms may serve different functional roles. Synaptogenesis and Synaptic Plasticity (1371-1376). Monday 236a 1371 THE EFFECr OF ACRIN ISOlORMS ON CULTURED XENOPUS MYOTOMAL MUSCLE CELLS. ((D.F. Daggettt CellBiol. and NK Haaelll D. Stnel, J.R. and Cur. in NurobioL., Genentech, Inc., S. San , Anat olicA and IKB. UNC, Chapel Frandsco, CA Hill, and of Ophthalmolo, Univ. of Pittsburgh, Pittsburgh, PA 15233. Deept. Agrr n has long been imiplcted as a molecu involved in postsyaptic of theneuromuscular juncin. As a atep towards furte differentiati which agrin induces the aggregatin of have begun to characterize its activity in Xenopus muscle cell 14 hr incubation with full-length souble recombnant chick dusters on the ventral agrin isoforms led to the formation of numerous small aurface of cells with differing efficacea A.B.>A,B,,=A,B. with being dependent as the inffective. This activity appeared to be tyrone aboished the effect After kinase inhibitor tyrphostin RG50864 (80 tM) completely 30 all agrin isofom could be labeled with an agnn incubetion at 4-C for min antibody in an evenly distributed, punctete manner on the muscle cell surface. After a 22C Untreatedcells ahowed negligilesating withthis incubation, the activeisoforms induced ventral AChRclustering, and agrin was to the edg of theaell and at the few dorsal enriched at ventral dusters showing agrin redistribution. However, dusters, in agreement with previousstudies in addition to the dustered agrin, with al isoforma there remained a punctste distnrbution across thesurface of the cell, suggesting thatit was notall redistbuted to sites ofcdustered AChRs Agrin bindingto the myotomal cell surface and induction of AChRclustering were Ca'-dependent, inhibited by Ca'-freemedium have with 1 mM EGTA, as previously shown in other systems. Asp been implicated in synaptognesis, the distnrbution of perlecan,the major myotomal undersatnding the nehn ( AChR acetylcholine receptors by we AChRR A,, , antibody. 14hr, coae heparan sulfate and chondrodtin sulfate were studied in reference to agrin binding sites. The even, punctateagr labing, the punctate, fibrousperlecan labeling and the sparse, punctate CS labeling showed distinct distributions, yetshared some areas of enrichment and others of oioal overlap. The relevance of prtoglycan to agrin's activity is being investigated. (Supported by NIH, Muscular Dystrophy Association andGenentech, Inc.) 1373 SPONTANEOUS CLUSTERING OF ACETYLCHOLINE RECEPTORS IN C2 MUSCLE CELLS IS ASSOCIATED WITH CHONDROITIN SULFATE. ((I. Mook-Jung, Colge of MedIIe, Universy of ArIzona, and H. Gordon)) Departmnt of Anm Tucson, AZ 85724. Proteoglycans have beenImpllcated In thecustering of acetylcholine receptors (AChRs) on cultured myobubes and at the neuronuscular junction. We have prevbusly descrbed theIsolatIon of genetic variants in protoglyn bIosynthesis from the C2 mouse musclecoell e andhave caacteedhI del the phenotype of one of those variants, S27.S27 Is defectivein the biosynthesis of glycosaminoglycans (GA(is), fails to form spontaneous clusters of AChRs, does formnerveassociated AChR clusters, doe not bind laminin to the surface ofmyotbes, and responds only weakly to agrln (J.Neurosci.,13Z5 595; Neuron,11:491-502). Two othe oftheorihl variant,S1i and S26, formnmobes but fal to sponanously cluster AChRs (Abstract 410, ASCB 1993). The three variat show dIferentboadand are especially deflclrthi thesynthesis of spectrum defectshi GAGbI chondroltn sulate (CS) chains thatelute at high salt hI ion-exchange chromatography.Heterokaryona formed in pair-wise fusbns of the variants sponnouwly clustered AChRs and recovered syrnhesIs of GAGs, espedally of CSe tln at high As lt is highly urlikely that defects hi AChR salt Inbn-exchange chr clustering would have arisen through a chance association with three different defecs hI GAGbosynihesis, we conclude that there is a requirement for poper GAG bbsynthesishi AChR clustering. We were also able to manipulate GAG biosynthesis In wild type C2 cells wlth severalp adim and to examIe the effects on AChR clustering. Chiorate was found to hbi both GAG synthesis and the clustefing of AChRshi a dose-dependent manner. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures ofwild ype C2myotubes, both the frequency of spontanos AChRclusters and the level of cl layer-assolated CS were Increased. Culture of wNld type C2nmoyubes in the presence of chonasklte ABC eliminated cel layerassocated CS and prevented the ornation of AChR clusters. Cell layer hoparan suNate was unaffected. Treatment wlth chondroktinase ABC only prevented AChR clustern I begun prior to the fomaton of spontneou clusters. This Uggests that CSis required In the initiation but not themairtenance of AChRcusters. 1375 DEVELOPMENTAL REGULATION OF A PROTEIN KINASE C ISOFORM LOCALIZED IN THE NEUROMUSCULAR JUNCTION. ((L Hllgenberg and K. Miles)) Department of Anatomy and Ceil Biology, SUNY H"eth Science Center at Brooklyn, Brooklyn, NY 11203. - Protein kinase C (PKC) has been suggested to play a role in synapse formation and signal transduction at the neuromuscular junction. The specifi PKC lsotorm(s) Involved In these various proesses has not been Identfled. Skeletal muscle has been shown to express high levels of mRNA encoding for cPKCa and nPKCe. To examine the protein distribution of nPKCe we raised a antisera against nPKCe. The affinity purified antisra specifically rcognIzed an 80 kDa protein highly enriched In skeletal muscle compared to other tissues and long term phorbol ester treatment of myotube cultures resulted in the down-regulation of this 80 kDa protein. A comparison of the subcellular distribution of nPKCe and cPKCa In rat skeletal musce reveaisd that nPKCe was enriched In the membrane traction whereas cPKCa was more abundant In the cytosol fmraction. Furthermore, nPKCe was found to be postnatally developmentally regulited wth a 4-told increase in expression occurring during days 4 to 21. Expresson of nPKCe In rat spleen, another tissue expresing detectable levels of this isoform, was not developmentally reguiated during this time. Only a moderate Increase In cPKCa expression In skeletal musce was obeved during postnatal days 4 to 21. Immunocytochemical staining for nPKCe hI skeletal muscis was associated wlth the sarcoismma and appeared to be predominantly located at the nouromuscular junction. This lmmunoreactivtty persisted after denervation suggesting that it was aiso localized at that the stain Is posaynaptic. Staining for cPKCa motor endplate but the moat lntehra staining patterns were extralunctlonal. that nPKCO is highly and spciftically The results of our experiments demonstrat expreaed In the membrane fraction of skelel muacle. The developmental increas In nPKC5 exprmoon during the time of synapse maturation and the distribution of nPKCe in nouromuscular junctions support the hypothesis that the nPKCe isoform pisys an Imporant role In the neuromuecular synapse. peptidh-spcilftc revzesd 1372 POSTSYNAPTIC CLUSTERING OF GLUTAMATE AND GABA RECEPTORS IN CULTURED HIPPOCAMPAL NEURONS. ((A.M. Craigt and G. Banker2)) 'Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801 and 2Department of Neuroscience, University of Virginia School of Medicine, Charlottesville, VA 22908. Several immunocytochemical and physiological studies have demonstrated of receptors at postsynaptic sites on neurons, a concentration but neurotransmitter has not been clear whether receptor clusters are selectively localized opposite terminals that release the corresponding neurotransmltter. Using antibodies against the excitatory AMPA-selective glutamatereceptor subunit and the inhibitory GABAA receptor 12/3 subunits, we found that these different receptor types cluster at distinct sites on cultured rat subunits clustered on cell bodies hippocampal neurons. The and dendritc shafts opposite GABAergic terminals, while GluR1 mainly on spines and was associated with glutamatergic synapses. The metabotropic glutamate receptor mGluRl a, which is not endogenously at detectable levels in cuitured pyramidalcells, was expressed in expressed the neurons from a defective herpesvirus vector. The expressed mGluRl a, like was restricted to the domain and formed postsynaptic clusters on spines. Furthermore, mGluRl a and formed clusters at many of the same postsynaptic sites, suggesting that similar mechanisms may regulate their localization. Chronic blockade of evoked release did not block receptor clustering at sites. These resuits suggest that complex mechanisms invoMng nerve GluRl GABAARI2/3 postsynapfic dustered dendritic GluRi, dendritfc somatodendrltc GluR1 postsynaptfc transmitter terminal-specific signals are required to generate such a postsynaptfc receptor mosaic. Supported by NIH NS17112, NS09248 and NS33184. 1374 CHICK ARIA IS CONCENTRATED AT EMBRYONIC NEUROMUSCULAR Sandrock A.,Yee A.& Flschbach G.)) JUNCTIONS Neurobiology Dept., Harvard Medical School, 220 Longwood Ave, Boston MA 02115 ((A.Goodearl, Chick ARIA (acetylcholine receptor inducing activity) was purified and cloned bymeasuring increased AChR expression on the surface of cultured primary muscle cells (Falls et al. 1993 Cell 22 801). It is a member of a diverse family of growth factors including GGF, NDF and theheregulins, encoded by asingle gene that generates many isoforms through alternative splicing. As motor neurons express ARIAmRNA during embryonic development, we propose that ARIA acts in vivo as a trophic factor secreted bymotor neurons that regulates focal neurotransmitter receptor expression (and probably other synaptic specialization) on its target muscle. We have developed a panel of polyclonal antibodies raised against sequences from in order to investigate this hypothesis. An antiserum proARIAl, aP1 isoform, (HM22) specific for the N-terminus of proARIAl gives strong staining of neuromuscular junctions (nmjs) in slow (anterior latissimus dorsi, ALD) and fast (posterior latissimus dorsi, PLD) muscles in late (E18) embryonic chicks. Although no ARIA staining is seen in motor neuron cell bodies, nerves immediately proximal to the nmjs are lightly stained, in a pattern that appears to be extra-axonal. Thbus ARIA may be rapidly transported from the spinal cord and accumulate at nerve terminals. Preliminary studies using electron microscopy suggest that ARIA at nmjs is concentrated between nerve and muscle cells in the synaptic cleft. HM22 is first observed at E16, some days after initial synaptic contact, suggesting that this isoform may play a role in thematuration and maintainance of synaptic may play in the receptor levels. The role that other initial formation of synaptic AChR clusters is under investigation using other antisera. immuno- imnmnoractivity currentlyisofonrns neurotransmitter 1376 ARE THE NEUROTOXIC EFFECTS OF METHYL MERCURY (MeHg) MEDIATED THROUGH THE ACTIVITIES OF PROTEIN KINASE A (PKA), PROTEIN KINASE C AND/OR ACETYLCHOLINESTERASE? ((M. Davis, M. Darville and 0. Turner)) Department of Biology, Morgan State University, Baltimore, Maryland. 21239. (Spon. by T. J. Robinson) In our attempt to clarify the neurotoxic basis of MeHg at the enzyme level in synaptosomes from the brain of rats, we investigated the effects of PKC and activity in the bmin of MeHg on the working hypothesis was that the effects of Sprague Dawley rats.areOur mediated through the phosphorylation of brain MeHg as well as by the inactivation of the proteins by PKC and enzyme. In this study, the animals were treated with for 5 from the forebrain, (3 MeHg mg/kg, i.p.) days. The midbrain, and hindbrain of these animals were prepared according to standard biochemical techniques. The PKA and PKC activities were determined using the Protein Kinase Assay System Reaction Kits (Sigma (Upstate Biotechnology, Inc.) and the Chemical Co., St. Louis, Missouri) was used to assay for activity. The results of our investigatiDons showed that a 1.43, 9.4, and 1.1 fold increases in PKA MeHg treatment producedand 1.6 fold. increases in PKC activity for 5.5, activity; and, 1.92, isolated from the forebrain, midbrain and hindbrain, synaptosomes PKA, acetylcholinesterase neurotoxicity PKA acetylcholinesterase synaptosomes Non-Radioisotopic Ellman acetylcholinesterase gespectively. Additionally, an inhibition of acetylcholinestemwa activity observed in synaptosones of all of the brain regions following MeHg These increases in PIKA and P3KC actiivity observed, suggest treatment. that MeHg produces its neurotoxic effects by intercting synergistically with both the cAMP and the inositol pathway and by inhibiting acetylcholinestemse. (Supported by Grant #SRC-7 (20) 2 T34 OMO 7977-11 from NIH). was 237a Monday. Synaptogenesis and Synaptic Plasticity (1377) 1377 F1/GAP-43 LEVELS ARE ALTERED AFTER THE INDUCTION OF HIPPOCAMPAL MOSSY FIBER LONG-TERM POTENWIATION ((D.T. Rivera, M.L Escobar, E Barea-Rodriguez, B.E Derrick and J.L Martinez, Jr.)) Departments of Psychology and Cell and Molecular Biology, University of California, Berkeley, CA 94720. Hippocampal long-term potentiation (LTP) is a long lasting process of synaptic plasticity that currently is considered the best model of memosy storage in the mammalian brain. Thus far, two forms of hippocampal LTP have been characterized: NMDA receptor-dependent and opioid receptor-dependent mossy fiber (MF) LTP, which is associated with bilateral synaptogenesis even in the absence of seizures (Escobar et al., Soc. Neurosci. AbsL in press). To explore further our previous findings, we measured the levels of F1/GAP-43 after the induction of MF LTP. Fl/GAP-43 is a neuronal, PKC substrate that has been associated with synaptic plasticity. Using Western blot analysis, we measured the protein levels of F1/GAP-43 four and seven days after the induction of LTP at the MF-CA3 synapse of an anesthetized rat Both sham and seizure control animals were used. We observed that F1/GAP-43 levels are decrased in the side where the stimulus was applied, but increased in the contralateral hippocampus at four days. At seven days following LTP induction, F1/GAP-43 levels were reduced in both hippocampi. Since F1/GAP-43 is associated with growth, its increase in expression in the contralateral side after 4 days suggests a possible regulatory role in the development of new synapses following MF LTP. Supported by DA01495 (JLM), NSF-DIR 9101951 (DTR), CONACYT MEX (MLE). Endoplasmic Reticulum (1378-1381) 1378 A mRNA SEQUENCE RESEMBLING A -1 TRANSLATION FRAMESHIFT INVOLVED IN SRP RECEPTOR MEMBRANE ASSEMBLY. ((J.C. Young and D.W. Andrews)) Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada. The site of signal recognition particle (SRP) mediated membrane targeting of nascent secretory proteins is the SRP receptor. Using deletion mutagenesis and a cell free assay system, we have now mapped the region of SRa required for membrane assembly. Examination of the translational properties of mRNA encoding SRa revealed a sequence necessary and sufficient to cause ribosomes to pause during translation. RNase protection and primer extension assays (toeprinting) demonstrated that the pause sequence is positioned within the SRat mRNA such that the SRa membrane assembly domain exits from the ribosome just prior to pausing. The primary sequence and predicted secondary structure of the mRNA at the ribosome pause site closely resembles a -1 frameshifling sequence. Moreover, it was possible to demonstrate a small amount of -1 frameshifting at this site in vilm. By separating membrane bound polysomes from cytoplasmic ones it was also possible to demonstrate that membrane assembly correlates with ribosome pausing. Thus, ribosomes translating SRa mRNA sequences 5' of the pause site are either cytoplasmic or tethered to ER membranes by the 3' end of a previously targeted mRNA. Ribosomes translating sequences beyond the pause site are membrane associated. Experiments with puromycin confirmed this result and indicated that at steady state translation, the 3' end of the SRa mRNA is attached to the ER membrane via the interactions of the nascent SRca polypeptide. 1380 RECONSTITUTION INTO PROTEOLIPOSOMES AND PARTIAL PURIFCATION OF TIE ENDOPLASMIC RETICULUM ATP TRANSPORTER. ((E. Guillen, and C.B. Hirschberg)) Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA 01655. Recent studies in vitro demonstrated an ATP transpr in the membrane of the endoplasmic reticulum (ER) of mammals and yeast This transport allows ATP, which is synthesized primarily in mitochondria, to be available in the lumen of the ER for reactions requirng energy durng folding and polymerization of proteins as well as phosphorylation. Purification and charcteion of this tansporter is important to (a) determine whether it is regulated and thereby affects the amount of ATP available in the lumen of the ER for the above descsibed reactions, (b) understand the mechanism of ATP translocation and (c) detemine the transporter arrangement within the ER mmbrane and its reltionship with other cellular ATP transps. Towards bne proteins were solubilized with detergent and this goaL ER inc ated into unillar phosphatdylcholhne liposomes. The resulting proteollposomes transported ATP in a satrable manner with an apparent Km of 0.5 pM, a value very similar to that of intact ER-derived vesicles. Transport of AT? into proteoliposomes was inhibited by DIDS and atractyloside in a manner similar to intact ER-derived vesicles. The above proteoliposomes were highly specific towards solute transport: nucleotide sugars and nucleoside phosphates which do not enter the lumen of the ER were not transport Supported by NIH grant GM34396. 1379 RER-65, a memnbrane protein of the Rough Endoplasmic Reticulum is related to p63, a protein of an ER-Golgi intermediate compartment. By Y-J., Chen, A.Stieber,N.K.Gonatas,University of Pennsylvania School of Medicine, Phiadelphia, PA 19104, and W.S.Lane, The Biological Laboratories, Harvard University, Boston, MA 02138. RER-65, identified by monoclonal antibody 2H1, is a protein of the rough endoplasmic reticulum (RER) of several rat cells including neurons, hepatocytes and pheochromocytoma (PC1 2), (Chen et al. J.Histochem.Cytochem. 1991, 39,635).We report here that RER-65 and p63, a protein of an ER-GoIgi intermediate compartment of primates, are closely related (Schweizer et al. J.Cell Sci. 1993,104,685). By immunoelectron microscopy, RER-65 is localized in the RER and nuclear envelope of brain neurons; by Westem blotting, mAb2H1 reacts with a 65 kDa band of liver rough microsomes. Partitioning in Triton X114, extractions in carbonate, and tryptic digests of membrane fractions from PC1 2 cells, show that RER-65 is an intrinsic membrane protein with a 10-15 kDa cytoplasmic segment. RER-65 does not display intrachain disulfide bonds but it may form interchain disulfide bridges and homomultimers. Metabolic babeling of PC1 2 cells in the presence of tunicamycin, digestions with 0-glycanase and chromatography of PC1 2 membrane fractions on a concanavalin-A column show that RER-65 is not glysosylated. Amino acid sequences of nine peptides, 132 residues, derived from tryptic digests of RER-65 purified from PC12 cells, reveal 75% to 100% identities with p63, while two peptides,35 residues, of RER-65 show no identities with p63. The biochenmical and sequence data strongly suggest that p63 and RER-65 are cosely related. Supported by N IH grant NS 05572. 1381 CHARACTISATION IN THE OF YEAST MUTANTS DOLIC55DL-LIliXKD PATHWAY. ((J. Roos, N. Dean, J. Eu, and W. J. Lennarz)) Department of Biochemistry and Cell Biology, State University of New York at stony Brook, Stony Brook, NY 11794, OLIOOS CCIARIDE N-linked glycoproteina are the product of two bioaynthetic processes in the endoplassic reticulumi the assembly of the and and the translation oligosaccharide lipid-linked In the yeast glycosylation of nascent polypeptide chains. Baccharoayces corevisaae, the enzyme that transfers the oligosaccharide chain from the lipid carrier to nascent We have polypeptide chains is still poorly understood. simple, yet highly snsitive assay to screen for devloped This in defective assay peptide glycosylation. yeast mutants utilizes endogenous oligosaccharylpyrophosphoryldolichol, the end product ?A, the dolichol phosphate synthetic pathway, and an for N-linked substrate I-labeled peptide exogenous glycosylation. Characterization of mutants identified by this screen allowed us to group the mutants into three classesi those defective ln (1) dollchol phosphate synthesis, (2) lLpid-linked ynthesis, or (3) the transfer of the oligosaccharide A putative temperatureoligosaccharide chain to proteln. sensitive Class 3 mutant (JRY163) has been identLfied and two genes that affect glycosylation in thl mutant have ben cloned. MNG is an essentLal gene that suppresses the temperature sensitivity of JRY163, but not the peptide glycosylation dfefct. NUGi effects the oligosaccharyl transferase mutant, wbpl-1, ln The socond gene, O083, rescues both the the same fashion. growth and peptide glycosylation defect of JRY1Y63, yet has no a effect efforts when are introduced into focused the on the f wbpl-1 fect of mutant 8I50 on strain. other Current mutants in the glycosylatLon pathway and the subcellular locatLon of Neglp. In addition, the role of 0873 in N-linked glycosylation is boing studied. (Supported by NIB grants GK33184 and 01133185 to 13L). 238a Endoplasmic Reticulum (1382-1387). Monday 1382 1383 ISOLATION OF MUTANTS DEFECTIVE IN KARMELLAE MEMBRANE BIOGENESIS IN S. CEREVISIAE ((A. Koning, S. Nygaard, K. Johnsen, G. Hedden, and R. Wright)) Dept. of Zoology, University of Washington, Seattle, WA 98195 GENETIC CHARACTERIZATION OF S. CEREVISIE MUTANTS DEFICEENT IN ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION (ERAD). ((.E.Ernaga, E.D.Werner, I.V.Karpichev, and A.A.McCracken)) Department of Biology, University of Nevada, Reno, Reno, NV 89557 Proliferation of a specific type of ER membranes called karmellac can be induced by a ten-fold increase in the levels of the integral ER protein, HMGCoA reductase (HMGR). This response allows us to investigate the mechanisms that underlie cellular membrane biogenesis. Using a vital membrane dye, DiOC6, we have screened mutagenized yeast cells for defects in karmellae biogenesis and identified several non-conditional mutants and many temperature-sensitive mutants that still have high levels of HMGR. Several of the non-conditional mutants, AK1, 2, and 4, do not generate karmellae with elevated levels of HMGR from a multicopy HMGR plasmid, but do generate karmellae with higher levels of HMGR from a galactose-inducible HMGR plasmid. In contrast, AK3 generates low amounts of karmellae with both the multicopy and galactose-inducible HMG-R constructs. A temperature-sensitive mutant, AK5, does not generate karmellae at the permissive growth temperature, but does at the restrictive growth temperature. One mutant,SN6, has been identified that does not produce karmellae at any level of HMGR in the cell. Interestingly, karmellae deficiency inSN6 co-segregates with improved growth on media containing galactose and raffinose as the carbon sources, and reduced growth on media containing only galactose. This phenotype has been used to identify genomic clones that complemnent the reduced growth phenotype on galactose, and that allow the mutant to generate karmellae. A 12 kb genomic clone that complementsSN6 is currently being analyzed. 1384 ER-ASSOCIATED PROTEIN DEGRADATION IN YEAST. ((E.D.Wernerand A.A.McCracken)) Departent of Biology, University of Nevada, Reno, Reno, NV 89557 Secretory proteins that are incorrectly processed and assembled acclate in the ER and are selectively degraded by a quality control mechanism associated with the ER (ERAD). This proteolytic process is thought to occur of the ER utilizing novel proteases that are not within an earlyco associated with previously reported intracellular protein degradation systems (McCrackenand Kruse, 1993, Mol.Biol.Cell 4:729). To study this process, we monitored the degradation of the mutant form of human .1-anti-trypsin (AlPiZ) expressed in various yeast strains. Our results showed that this ERAD substate was not stabilized ina strain deficient in vacuolar protease ng enzyme UBC6. activity, nor ina strain deficient in the ubiquitinco However, when the reporter substrat was monitored by ELISA and pulsechase protein radiolabelling in strains with mutations which alter the "chymotrypsin-like' activity of the yeast proteosome complex, the rapid degradation of AlPiZ was restricted. For example, the half-life of A1PiZ in the double mutant Prel-1 Pre2-2 (Heinmyer, et al., 1991, EMBO 10:555) was increased to 80 mintes compared to the half-life in the parent strain of 52 minutes. These results suggest that ERAD may depend, at least partially, on a proteolytic activity of the yeast proteosome complex. In another set of experiments, we studied the effects of ALLN and PMSF, two membrane permeable protease inhibitors often used to characterize proteolytic activity. In the presence of either inhibitor, strains expssg AlPiZ show a 4-5 fold increase in substrate. Onq possible inpretation of these results is that multiple proteolytic activities are involved in ERAD. (Supported by grants to A.M. from the American Cancer Society -MV551 and the Cystic Fibrosis Foundation #G789). Yeast mutants have been identified that lack the ability to degrade certain ERAD reporter proteins, including an aberrant form of al-anti-trypsin (A1PiZ). These mutants have been shown to stabilize this protein ina preGolgicompament, most likely the ER. Pulse-chase protein radiolabelling experiments showed that the half-life of AlPiZin various mutant strains ranged from 50-120 minutes compared to 30-38 mimutesin the wild type strains. The genetic analysis of these mutants was carried out tofacilitate the identification of the gene(s) involved in ERAD. Tetrad analysis ofthese mutants was performed to determine the number of genes involved in the mutant phenotype. About 40% of the mutants characterized conformed to simple Mendelian genetics, i.e. 2:2 segregation of phenotype. Further genetic testing revealed that the phenotypes of all but one of the mutants was expressed in a manner recessive to the wild type gene. Also, the phenotype of at least one of the mutants was determined to be controlled by two genes. Complementation analysis of these mutants identified at least seven complementaion groups. Experiments examining the stability of the mutant phenotypes were conducted to identify mutants competent for molecular cloning by complementation of the gene(s) responsible for ERAD. (Supported by grants to A.M. from the American Cancer Society -MV551 and the Cystic Fibrosis Foundation #G789). 1385 DEGRADATION OFAN ER-RETAINED FORM OF VSV GLYCOPROTEIN LEADS TO CLASS II PRESENTATION ((S. M. Bartido, S. Diment, and C. S. Reiss)) Departments of Biology and Pathology and the Kaplan Comprehensive Cancer Center, Naw York University, New York NY 10003-6888. A20 lymphoma clls infected by a vaccinia recombinant incorporating the 'poison tail' construct of the vesicular stomatitis virus glycoprotein IVSV GI and Bergmann, Cell 34: 513) express a mutated form of the protein that is retained in theendoplasmic reticulum (ER). Pulse-chase studies show degradation of the protein (TT -4h). Immunofluoresence studies as wel as ENDO sensitivities show the protein to be limited to the ER. This degradation was found unaffected by monensin or by leupeptin or NH4CI. These results also indicate that autophagy is an unlikely mechanism of protein degradation. A neutral, non-reducing environment, which is characteristic of the ER, is required. Pulse-chase experiments utilizing the proton ionophore CCCP, which equilibrates the pH in the intracellular compartments with that of the outside milieu, indicate optimal conditions occuring at pH 6-7 and not at pHI 8. The use of diamide, a thioloxidizing reagent, resulted in inhibition of protein degradation suggesting the possible involvement of serine or cysteine proteases. An inhibitor of cysteine protesses such as E-64 did not block the degradation. We have found that presentation of epitopes derived from VSV G in this system is affected by NH4C, emetine, and Brefeldin A (Reiss et al Cal Immunol. 139: 229; Together our resuits indicate that proteolytic fragments of the protein generated in the ER o lumen may traffic with the nascent class 11 complex to the compartment of peptide acquisition. Subcellular fractionation studies are being carried out to identify the site of peptide binding. Supported by NIH Grant All 8083 to CSR. (Rose D/H 1992). presently 1386 138T DEGRADATION OF P-SUBUNITS OF GONADOTROPINS RETAINED IN THE ENDOPLASMIC RETICULUM OF HYPERSTIMULATED GONADOTROPHS. ((P. Rosa, M. Bassetti, and D. Lodi Pasini)) CNR IN VIVO PHOSPHORYLATION OF CALNEXIN OCCURS ON THE CYTOPLASMIC DOMAIN A REGION REQUIRED FOR COPRECIPITATION WITM CLASS MHC MOLECULES((G. G. Capps, S. L Moseley, and M. C. Department of B University of Califomia, Santa Cruz, A Calnexin is a major calium-binding protein of the endoplasmic retiulum (ER) which is implicated in the biosynthesis of class Major Histocompatibiliy Compblx (MHC) molecules and other cell-surfaceif glycoproteins. Calnexin is phosphorlated in vitro, but it is unclear vWo phospho ation of calnexin oocurs on lumenal or on cytopasmic resides. This issue is of particular importance for understanding calnexin's chaperone functions because previous studies in this laboratory showed that not al class I MHC molecules with phosphorylated calnexin and that association with calnexin Cytopharmacology, Department of Medical Pharmacology, University of Milan, 1-20129 Milan, Italy. (Spon. by P. Rosa.) Center of the fate of the gonadoopin -subunits, accumulated in the endoplasmic reticulum (ER) of gonadotrophs after gonadectomy, using markers for the ER, the lysoms and the sectory granules on antenor pituitay ultra-thin fozen sections. After castration, the intacllular evels of the subunits were found to increase more than that of the a-subunit. Both gonadotropin subunits co-localized in secretoy granules with secretogrnnin I, a protein present in secretory granules of many specis. Conversely, the psubunits, but not the a-subunit and secrtogranin II, were localized in the dilated cisternae of the ER and the ransidonal elements as well as in irregularly-shaped vacuoles with one thick membrane. Using makers for the ER (PDI), the prelysosomal compartet and lysosomes (cathepsin D and lgpl20), we found that these vacuoles correspond to a degrdative ates, which may represent different compartment with two types of in stages in the pathway of gonadotropin -subunits degradation. These vacuoles do not appear to derive from autophagy, since vesicles rounded by a double or multilamellar membrane containing profiles of dstnae of the ER together with small amounts of the cytoplasm were never detected Moreover, they do not correspond to cnnophagic bodies, since the lakmr contained psubunits as well as a-subunit and SgI. Taken together these results show that rpha are degraded by a snits reined in the ER of gon gonadoin diffent from the clsscal auophagy andis not dmilarto the non-autophagic pathway for the diversion of the inracisternal ganules accumulated in the ER to lysosomes, recently described in hyperstimulated thyrotoph (Noda andFarquhar, J. Cell BioL 119, 85, 1992). We invesdgated Zuhga)) logy, in rylated correlates with slower acquisition of Endo H We now show thatpoorylated calnexin is phosphorylated exclusively on its cytop domain in vivo. Proteinase K treatment of vesicbs rom cells labeled with [5S] methionine and [s1P104 th H2Ld and from calnexin. Surprisingly, this tr alo resuits in the loss of coprecipitated calnexin in immunopreipitates of H-2Ld and in the oss of coprecipitated H-2Ld in immunopcipiates of calnexin. is required cytop Thus, the cainexin for its nc domain coprecipitationofwith class I MHC molecules. This is acalnexin surpsing resuit in Eght the observation that phosphorylated efficlently coprecipitates with H-2Ld molecules which lck a domain. Thus, is unlikely thatdomain domain the cytopasmic cvtopbasmkc of the cla ozf calnexin associates I with the cytopbsmb that the of MHC molecule. These findings sugest assoclation calnexin with class I MHC molecules is influenced in Dart by the conformation of the cainexin molecule. (Supported by NSF grants DCB-9196051 and DCB-9096241) removes rmoves Monday. Endoplasmic Reiiculum (1388-1391) 239a 1388 1389 RETINOL-MODULATED PROTEIN DISULFIDE ISOMERASE ACTIVITY OF RAT LIVER TRANSMONAL ENDOPLASMIC RETICULUM. ((E. Jacobs, R. de Cabo, D. J. Morrd and D. M. Morr6)) Department of Foods and Nutrition, and Department of Medicinal Chemistry, Purdue Univesity, W. Lafayette, IN 47907. ALL-TRANS RETINOL INHBITS BINDING OF GTP-y-S TO A 55 KD PROTEIN OF TRANSITIONAL ENDOPLASMIC RETICULUM OF RAT LIVER. ((J. Stevenson and D. M. Morrd)) Department of Foods and Nutrition, Purdue University, W. Lafayette, IN 47907 The formation and fusion of transition vesicles (TV) from transitional endoplasmic reticulum (TER) to cis Golgi apparatus (GA) was reconstituted in our laboratory in a cell-free system from rat liver (Nowack et al., BBA, 1051:250, 1990). At an optimum conc. of 1 gg/ml, the rate and amount of transfer was approx. doubled over 1-2 h incubation and was found to be the result of an effect on TV formation. A protein disulfide isomerase (PDI) activity of TER, stimulated by retinol with reduced and denatured RNase as substrate correlates with retinol-stimulated budding with TER incubated under the complete conditions required to induced vesicle budding in a cell-free transfer assay (ATP plus ATP-regenerating system and cytosol). Oxidized glutathione or a mixture of reduced and oxidized glutathione also supported the retinol-stimulated activity. With oxidized and denatured RNase, retinol, as well as NADH, stimulated reactiviation in the complete system. However, in the presence of NADH, retinol at all concentrafions tested, inhibited, rather than stimulated. The results are interpreted as evidence for a and retinolstimulated PDI activity of rat liver TER that may play a role in vesicle budding or membrane trafficking. Supported by USDA 9301253 and Phi Beta Psi Sorority. Nowack et al. (Biochim. Biophys. Acta, 1051: 250-8, 1990) previously described a role for retinol in stimulation of formation of transition vesicles from transitional endoplasmic reticulum (TER) of rat liver and suggested that this stimulation was due to retinol sparing cytosolic GTP. Zhao et al. (Biochim. Biophys. Acta, 1055:230, 1990) described a retinol-inhibited GTPase activity of rat liver TER. This activity was monitored and isolated by solubilization of isolated TER membranes with C12E8 detergent and by DEAE cellulose and gel filtration chromatography. Active fractions with retinolinhibited GTPase activities also had retinol-inhibited ADPase activity. Western blots of active fractions incubated with 5'-p-fluorosulfonyl benzoyladenosine (FSBA) (an adenosine analog) with detection using antiFSBA sera (gift of R. Geahlen, Purdue Univesity) with and without GTP or ADP revealed a band at 55 kD that was competed by the nucleotide. Blotted fractions incubated with [35S]GTP-y-S revealed a radiolabeled band at 55 kD with intensity that correlated with the retinol-inhibited GTPase activity. Binding of GTP-y- S to TER was greatly reduced with the addition of retinol. The findings suggest that the retinol-inhibited GTPase has a monomer Mr of 55 kD on denaturing gels. Supported in part by USDA 9301253 and Phi Beta Psi Sorority. 1390 ANALYSIS OF THE ENDOPLASMIC RETICULUM PROTEIN TRAFFIC RESPONSE ELEMENT (ERPTRE) OF ERP72, ALUMINAL ER STRESS PROTEIN ((N. Marcus and M. Green)) Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104 ERp72, a resident protein of the endoplasmic reticulum (ER) is both a stress protein and a member of the protein disulfide isomerase family of proteins. The promoter region of the murine ERp72 gene contains several potential transcriptional control elements, including multiple CCAAT elements and Sp1 and AP-2 consensus sequences. Functional analysis of the ERp72 promoter identified an 82 bp segment that is sufficient to mediate the response of the ERp72 gene to such stresses as exposure to the Ca"+ ionophore A23187 or accumulation of incompletely assembled immunoglobulin p heavy chain in the ER. This 82 bp sequence contains two CCAAT elements and a putative AP-2 site and has some limited additional homology to protein traffic responsive sequences of other members of the ER stress protein family. Mobility shift analysis using the 82 bp fragment showed no significant difference in the pattern of bands produced by extracts derived from control and stressed cells. Competition experiments using a set of overlapping synthetic oligonucleotides spanning the 82 bp segment demonstrated that a 30-mer containing one of the CCAAT sites could abolish the binding to the 82 bp sequence. This CCAAT site is a good candidate for interaction with CP-1. These studies should allow us to elucidate the target of the intracellular signaling pathway initiated by protein traffic in the ER. Supported by: NSF Grant MCB-9317238. 1391 CROSS-LINKING OF BETA-LACTAMASE TO A HIGH MOLECULAR WEIGHT PROTEIN COMPLEX IN THE LUMEN OF THE ROUGH ENDOPLASMIC RETICULUM. ((H. Pestana, R. MooreBatchelder, S. Alcala, P. Tamirisa, and J.B. Denny)) Division of Life Sciences, University of Texas at San Antonio, San Antonio, TX 78249. We have added the membrane-permeable cross-linking reagent BSOCOES to material sedimented from cellfree translation mixtures synthesizing beta-lactamase. The sedimented material included rough microsomes. Cross-linked material containing processed beta-lactamase appeared at the top of a 4% and a 12% polyacrylamide gel, and was still observed if RM were treated with proteinase K following crosslinking, indicating that the material was located in the RM lumen. The high molecular weight complexes were readily apparent in sedimented RM, but could not be immunoprecipitated using antibodies to beta-lactamase, even though uncross-linked betalactamase was immunoprecipitated. We have shown that the epitopes recognized by the antibodies are not destroyed by cross-linking and that free betalactamase that has reacted with one end of BSOCOES The results therecan still be immunoprecipitated. fore indicate that the lumenal RER proteins have bound to beta-lactamase in a way that prevents Beta-lactamase may access of antibody molecules. thus enter a complex of RER lumenal proteins. Golgi Complex (1392-1393) 1392 EMP47P. A YEAST HOMOLOGUE OF THE VIP36/ERGIC53-FAMILY OF POTENTIAL INTRACELLULAR LECTINS, REQUIRES ITS C-TERMINAL KXKXX,MOTIF FOR LOCAUSATION TO A POST-ER COMPARTMENT. ((S. Schr6der, F. Sc&hmmOler, B. SingerOger# and H. RJezman)) Blozertmrum, University of Basel, Switzerland; #present address: EMBL, Heldeberg, We have determined the primary sdtucture of the Integral 1393 membrane protein Emp47p of Sacehwomyces cerevisiae (orignaly termed p44; Singer-KrOger et at., 1993, JBC 268, 14376-14386). The mature type I transmbrane protein has a moecuar weight of 47-103K. Emp47p has a weak but satistically sgnificant hology o ERGIC53 (Sehindler et at, 1993, Eur. J. Cel Biol 61, 19). The homobgy extends Into the short cytopassc tail with an ER-Iocalsatlon signal of the KKXX-(ERGIC 53) and KXKXX-(EMP 47) type, respectvely. The homology to Vlp36 Is restricted to the N-terminal -280 residues, which constitute a domain odginayekntfed In VipW as being homologous to plnt lecdns (Fiedler and Slmons, 1994, Cell 77, 625-626). Deletion disruption of the EMP47-locus does not elcit any readily observable growth detect. By Immunofiuorescence Emp47p is locaized to punctate structures with no apparent spatal affinity br ER, vacuoles or plsma membrane, reminiscent of Golgi-staining. In fact Enp47p colocalizes in immunofluorescence wfth the presumed Golgi-protein Pmr1p (Antebi and FirK 1992, MBC 3, 633454) and fractionates on sucrose gradents ike several Golgi-markers. In addition, Ermp47p with an engineered N-inked glcosyLon site, displaying a wild type lmnwnoflurescence pattem, receives a1.6. and a1.3-mannose-modificaions. We show that dhngfg a sgle lysine in the K)XKXX-otif leads to the escape of the mutant protein to the vacuole. Enmp47p may be the 1irst exmpe of a protein with a fucton KXiOKX-mO and a G"oi steady state dibuln WITEIDRAWN 240a Golgi Complex (1394-1399). Monday 1394 1395 PROTEIN RETENTION IN THE GOLGI APPARATUS ((T. Nilsson and G. Warren)) Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Field, London WC2A 3PX, England IN VTRO REASSEMBLY OF A POLARISED GOLGI STACK FROM M[TOTIC GOLGI FRAGMENTS ((C. Rabouille and G. Warren)) Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Field, London WC2A 3PX, UK We and others recendy demonstrated the importance of the membrane spanning domain in retention of resident Golgi cnzymcL To explain etntion, we have posulated that Golgi enzymes ptcipae in the formaion of heteo-oligoms. Thcse oligomers would conslat of tousand or more molecules of related ("kin") nature which would be large enough to ensure exclusion from budding transot vesicles. We have shown that medial Golgi enzymes have the capacity to form hetero oligomers and are prsendy invesdgating their role in organclle architecture. The pesence of resident medial enzymes is a prerequisite for maintaining the cisternal structure and subte changes in their primary structure result in drstic alterations of the Golgi sucue. We propose dtat the role of hetero oligomers is to maintain the flattened apecarance of the Golgi cisterna and to prvent fencstration of the membrae. a On the onset of mitosis, the Golgi apparatus undergoes dramatic morphological changes: The stack disassbles into small fragments which form the Golgi clusters (Lucocq and Warren, 1989, JCB,109:463). Exiting mitosis (at telophase), the Golgi reassemble into the characteristic polarised stack as the transport resumes (Souter et al, 1993, JCB,122:533). To monitor the reassembly of these mitotic Golgi fragments, we have developped an in vitro assay using similar approaches than Misteli and Warren (1993, JCB, 125:269) for the disassbly of the stack. Preliminary studies showed that the Golgi can reassemble into a polarised stack after it has been fragmented under mitotic conditions. 1396 1397 IDENTIFICATION OF FACTORS REQUIRED FOR THE DISASSEMBLY OF THE GOLGI COMPLE DURING MiTOlS. ((PA. Barr, T. Mistelli, and G. Warren)) Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX. (Spon. by G. Warren.) GOLGI ANKYRIN: ASSOCIATION OF AN WITH THE GOLGI COMPLEX ((K. A. Beck, Nelson)) Dept. Molecular and Cellular University School of Medkice, Stamford, CA complex disassbles into clusters of throughout the cytoplasm. After cell reassemble to gve a functional copy of the Golgi complex in each of the daughter cells. Immunodepletion of coatomer from mitotic cytosol prevents this disassembly process, indicating that ARF and coatomer dependant vesicle formation is required for mitotic Golgi breakdown. Further investigation indicates that Golgi disassembly is a two stage process: the first step requiring only ARF and coatomer and giving rise to Golgi stacks with shorter cisternae (core-Golgi stack), and the second step bein the unstacking and fragmentation of these core-Golgi stacks m an ARF and catomer indeendant fashion. We are now isolating components from mitotic cytosol which are required for the second step in Golgi disassembly, the unstacking and fragmentation process. During mitosis Golgi small vesicles dispersed division these vesicles 1398 CELL-CYCLE DEPENDENT PHOSPHORYLATION OF GLNTIN, A CYIOSKELETAL-LIKE GOLGI MEMBRANE PROTEIN. ((A.D. Linstedt and H.-P. Hauri)) Dept. of Pharmacology, Biocenter, Basel, Switzerland CH-4056. The Golgi complex vesicularizes at mitosis and then reassembles in each daughter cell as a centrally localized, subcompartmentalized, and stacked membrane network. We have identified a protein, giantin, that may be involved in regulating Golgi structure. Subcellular fractionation and EM studies indicated that giantin is localized to cis and medial Golgi cisternae. Sequence analysis and expression of truncation mutants showed that a C-terminal membrane anchor and adjacent amino acids are required for proper localization of giantin. The cytoplasmic domain of giantin, comprising over 3000 amino acids, is largely composed of heptad repeats. Giantin's migration on velocity gradients and gel filtration columns suggested that the cytoplasmic domain forms a -250 nm coiled-coil rod. Glantin from non-synchronized cell cultures was resistant to extraction with non-ionic detergents, and bound to microtubules in an in vitro assay. Thus giantin is unique in being a cytoskeletal-like integral membrane protein. Two dramatic changes were observed in cells blocked at mitosis: giantin became readily extractable,.and the incorporation of phosphate into its cytoplasmic domain was increased -20 fold. Giantin was also phosphorylated in vitro by p34cdc2. These findings suggest that phosphorylation of giantin may regulate cell-cycle dependent changes in Golgi structure, and that giantin may link the Golgi to the cytoskeleton. ANKYRIN ISOFORM J. Buchama and W. J. Physiology, Stamford 94305-5424 The Golgi complex has a sructual organization composed of sequentially armnged comprments. While much is Inown about how this structural organization relates to Golgi function, little is known about how it is maintaine We have previously demonstrated that an isoform of spectin immunologically related to m i ey oyte spectrin (£1) resides at the level of the Golgi apparatus (Beck et al., (1994) J. Cell Biol. in press), suggesting that a spectrin-based membrane skeleton plays a role in Golgi organization and function. To the examine nature of the int ion of this spectrin isoform with the Golgi complex we sought to identify an isoform of ankyrin that associates with this organelle. Two lines of evidence in support of a Golgi associated ankyrin are presented. First, by indirect immunofluorescence we show that an antibody raised against purified canine erythrocyte ankyrin stains the Golgi complex in a variety of cell types including MDCK, NRK and 293 cells. In addition, we have prepared an HA-epitope tagged, recombinant human erythrid ankynn cDNA composed of an 384 amino acid portion of the 90-kDa band 3 binding domain. We find that upon transient expression in human 293 cells, this construct localizes to the Golgi apparatus; plasma membrane staining is also observed. Te presence of spectrin and ankyrin isoforms at the Golgi appars suggests that these two proteins exist in the form of a macromolecular complex analogous to the spectrin membrane cytoskeleton associated with the plasma membrane. We are currently examining the association of these proteins with the Golgi complex at the ultrastcal lewVl by immuno-electron microscopy. 1399 EFFECTS OF WINIBTORS OF SPHINGOLIPID SYNTHESIS ON LOCALIZATION OF GOLGI PROTEINS. ((MCW.Maceyka and C Machamer)) Deparmet of Cell Biology and Anatomy, The Johns Hopkins Universty School of Medicine, Balime, Maryland 21205 has boen shown that ftansmembrane domains contain information for the correct locaization of many Golgi memnane proteins. Because diffnin Lipid conios'on through the sectoy pathway are to thought exst, proin locaization may be drectly related to membrane co Son. M glycpra from the avian v infcdous It neessary co (IBV)is targeted to the cis Golgi although indirect evidence suggests that its localiration may be maintained by retrieval from later ments her by tight retention. first encountaphn Be potins in the in the cim Golgi, we asked wheter ongoin phingolid synthess is necessary for correct llin of IBV M pro The inhibitor l-phenyl-2-(decanoylaino)-3secret pway p morpollno-l-propanol (PDMP) blo ucosyceamide and Treatment of BHR-21 cels with PDM1P: 1) the steady-stat ditibuon of IBV M frm the Golgi to the ERas assessed by i2di)c _ . V dows the movement of seps the secretoy patway as assed by pulseitnemtproteinp atll chase experiment with VSV-0, and 3) does not chage aning pattern of sphingoyelinsytei appearsto shift IL, which is thought to be stably retained rathr than aypiments, p-chloroalanine, an to roEdihibute the IBV M protein to the ineneateco ent, while there is no effect on mannosi iI locaizaionL Tese data suggest tha when aphingolipid synthesis is blocked, the change in steady state disribution oFIBVMmay be due to a decrease in anterograde taffc and/or an increae in retrograde traffic. dyna lly retrieved. In inhibitor of ceramide synthesis, appears Monday. Golgi Complex (1400-1405) 241a 1401 1400 ANALYSIS OF THE GOLGI COMPLEX. ((E.B. Cluett and C.E. Machamer)) Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, MD 21205. LIPID lipids in the structure and function of the Golgi complex is unknown. Evidence indicates that compartmentalization of lipids may occur in the Golgi complex. Both sphingomyelin and cholesterol are preferentially concentrated in the trans region of the organelle, but the significance of this is not understood In the case of cholesterol this is particularly intriguing, since the pathways and mechanism by which cholesterol is transported within the cell are unclear. The transport of newly-synthesized cholesterol appears to be unaffected by BFA treatmet or 2 a 0°C temperature block, suggesting a Golgiindependent transport route. Cholesterol is also presumed to influence the flotation of intracellular membanes during subcellular fractionation. As a first step in investigating the role of lipids in the structure andfunction of the Golgi complex, we have isolated a highly-enriched population of intact Golgi stacks from rat liver. Lipids were extracted and analyzed byHPTLC and scanning densitometry with results comparable to previous studies. Furthermore, two populations of Golgi complexes could be isolated which were virtually identicalin protein profile, enzymatic marker activity, andlipid composition, including cholesterol. Only the amount of cholesterol esters differed in each fraction. Interestingly, a heavier fraction, which appeared to be smooth ER/Golgi-like membrane, was devoid of galactosyl transferase activity but contained an unexpectedly significant amount of cholesterol. These studies are currently being extended toMDCK and WIF B cultured celllines. The role of TWO RELATED GOLGI-RESIDENT CHIMIRIC PROTEINS ARE RETAINED BY DIFFERENT MECHANISMS. ((O.A. Weisz, C.S. Sevier, and C.E. Machamer)) Dept. ofCell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD 21205. We have been investigatng the mechanisms by which membrane proteins are retained in the Golgi complex. Previously, we showed that the first membrane spanning domain (ml) of the M glycoproten of infectious bronchitis virus (formerly caUled IBV El) contains cis Golgi retention information. When the membrne spanning domain of a protein that is delivered to the plasma membrane (VSV G) is replaced with ml, efficiently the resulting chimera (Gml) is retained in the Golgi. We recently demonstrated that Gml forms large oligom ers which are insoluble in Triton X-100 and SDS (Weisz, 0. A., A. M. Swift and C. E. Machamer (1993) J. Cell 1185-1196). Point mutants of Gml which disrupt the Golgi The Biol.22, spanning domain do not form that the oligomers. Experiments using truncated Gml constructsofsuggest tail a in the role plays tight Golgi retention Gml. When the cytoplasmic Gml tail is replaced with theIBV M tail, the resulting chimera (GmlM) is retentioninformationin the membrane retained in the Golgi, although at high expression levels, some of the protein reaches the plasma membrane. Surprisingly, internal deletions of the GmlMI tail result in rapid delivery of the chimeras to the cell surface. This suggests that Gml and GmlMK are retained in the Golgi complex by different mechanisms. We are currendy investigating whether these mechanisms involve the dirt retention of ml-conaining proteins or the retrieval of these proteins from later compartments. 1402 1403 CHOLESTIEROL-NDEPENDENT TARGETING OF GOLGI MEMBRANE PROTEINS. ((M.M. Rolls*, M.T. Marquardt+, M. Kielian+, C. E. Machame_o)) *Departsent of Cell Biology and Anatomy, IDENTIFICATION OF 4 NOVEL PROTEINS OF THE GOLGI COMPLEX USING HUMAN AUTOANTIBODY PROBES. ((Marvin J. Fritzler, Kevin J. Griffith, Edward Chan)) University of Calgry, Calgary, Alberta, Canada T2N 4NI and 'T Scripps K.L. Research Institute, La Jolla, CA 92037. Bronx, NY 10461. Autoantibodies directed agist the Golgi complex have been reportedi the sea of patients with certain autoimmme, infectious and neuro-degenative diseases. The ofdiffcrent cellularmembranes may provide physical basis for specifictargeting of some membrane proteins. In that cholesterol could play an essential been it has suggested particular, role intranamembrane domain-mediated loclization of proteins to the Golgi complex (Bretscher, M.S. and S. Munro,1993, Science 261: 1280- Human auanb directed agaist the Golgi complex wrae used to screen complex. cDNA exprsson lbraes. Four groups ofcDNA clones, designated golgin 95, golgm 160, study. These cDNAs wer ch_id CDF20, ACJF 5.21 were selected for fuher reacvities of the recombinant by resrictbon mapping and DNA seq analysis antibodies were eamid by Westeni nobti using proteins with Johns Hopkins University School of Medicine, Baltimore, and+Department of Cell Biology, Albert Einstein College MD 21205, of Medicine, Distinct lipid compositions a 1281). To determine whether cholesterol is required for thetargeing of Golgimembrane proteins, we exanined the localzation of several Golgi resident proteins in cholesterol-deplted cultured cells.Insect cells cannot synthesize cholesterol and canbe depleted of sterol by growthin delipidated senum We used the C6/36 cell line derived from Aedes albopicts (mosquito), which after four passgesin delipidatsd serum containedundetectable levels of cholesteol (Marquardt,MIT. et al, 1993, J. Cell Biol. 123:57-65). Because no probes are available for mosquito Golgi proteins, we expressed two well-cha ized mam malian Golgi enymes using a Semliki Forest virus-based vetor. In non-depleted C6/36 cells, both p1,4-galactosyltransferase andawere localized to the Golgi complex by indirect mannosidaseI immunofluoresence microscopy. When the same proteins were expressed in cholesterol-depleted C6/36 cells the staining pattern was indistinguishable from that soen in non-depleted cells. Thus, at leastin insect cells, cholestrol does not appear to be required for targetng of several proteins to the Golgi complex. objective of our research is to understand the nature of argets in the Golgi and human recombinant protein produced in E. coi and by pcipitation using in vitro translation products. Antibodies affinity purified by adsorpn to the recombinant phage The binding specificity Golgi staining HEp-2 Golgi complex was confirmed byd ting that the reactivity cokocalized to the same subcellular organelle bound by wheatgeSm agglutiin and antibodies to the coatomer protein,PCOP. InWvtro translation prodcts usingthese cDNA templates produced 40 5.21), 65 (golgin 160) and -95 (golgin 95, CDF20) kDa proteins Sequence analysis (ACJF of all four cDNAs indicated that they encode unique proteins, 3 of which (golgin 95, golgin 160, CDF20) had 20-2Woh sequence identity to the heavy chain of myosin and kineis. Analysis for secondary stmcturedemonstated that golgin 95 had coiled-coil domain (golgin 95) and CDF20 has a predicted alpha helical structurethroughout the majority of protein reprodued on to the cells. a the protein. sequences. None of the sequenc had potential HEp-2 cells, the autoantgma tranamembranc domains After befeldin A treuent of or wer sign redistrbuted 5 min, absent at 10-15 min and then reconstituted 120 minutes after eptnishing with fresh media. Insummary, we have cloned and identifed 4 novel components of the but stuctul analysis suggests that complex. The function ofthese proteins is not they are part of myosin-like family of proteins assoced with the Goigi complx. after Golgi known a 1404 1405 PERMEABILITY CHANGES IN LIPID BILAYERS INDUCED BY ILIMAQUINONE, A GOLGI DISRUPTIVE METABOLITE. ((IS. Fisher, S. Sohraby, F.G. Grillo)) Dept. Nephrology, WRAMI, Washington, DC 20307; Dept. Physiopathologie, Univ. Libre Bruxelles, Brussels, Belgium. DIARYLSULFONYLUREA LY181984 ((S. Moya-Camarena, ANTITUMOR D. M. Morrd, M. W. L.-Y. Wu and D.J. Morrd)) Recently, Takizawa et aL (Cell 73:1079, 1993) demonsrated that 25 pM ilimaquinone (IQ), a metabolite from sea sponges, reversibly disrupts Golgi membranes in several cell culture lines. Its mechanism of action is unknown. We eamined the effects of IQ on the properties of lipid bilayers. Bilayers were "painted" on a 250 p diameter aperture and consisted of 15 mg/ml 1,2Diphytanoyl-sn-Clcero-3-Phosphocholine in decane. Membrane ca ce be (60 Hz triangle wave) was used to estimate bilayer area. Specific conductance (G) averaged .04 pS/cm2 for untreated bilayers (150 NaCl, 10 MES, pH=7; AV= ±100 mV, DC). Addition of 1.0 pM IQ (trans) to the ce were bilayer inased G about 2-fold. Bilayer noise and ca unchanged by IQ. Addition of 10 pM IQ inreased conductance 4-fold within 2 min. G continued to increase over the next 40 min to values 14-fold greater than untreated bilayers. I-V plots were linear. The response was independent of pH (6 to 9) and was not selective for Na+ or Cl-. After IQ (2 to 14 p, a step change of bilayer voltage caused an additional exponential component in the decay of the current response with a time constant of about 10 s. We conclude that similar permeability changes of Golgi cisternae after IQ may occur which could be important in undrstanding the mechanisn(s) leading to membrane breakdown and loss of Golgi function. We thank Dr. Vv;ek Malhotra, Dept. Biol. UCSD, for the kind gift of IQ. A RESPONSE OF HELA CELL GOLGI APPARATUS TO THE MacKellar, Paulik, Department of Foods and Nutrition and Department of Medicinal Chemistry, Purdue University, W. Lafayette, Indianapolis, IN and Eli Lilly Research Laboratories, IN Growth of HeLa cells is markedly inhibited by the antitumor N-(4-methylphenyisulfonyl)-N'-(4-chlorophenyl)urea diarylsulfonylurea In (LY181984). parallel studies, the drug was shown to block an ameliorideinsensitive proton export by HeLa cells (E. Sun et al., Purdue University, Results Unpublished). Evidence was sought for a similar inhibition of proton transport at the level of the Golgi apparatus. If cells were pretated 1 to 3 h subsequently challanged with monensin, the resultant with drug and the monensin-induced vacuoles were smaller and less abundant The total volume of the monensin-sensitive trans Golgi apparatus compartment was reduced by more than treatment with LY181984. The swelling response was unaffected by sulfonylurea in cells (e.g. MDCK or PC-12 cells). A stuctully similar but inactive analog N4methylphenyisulfonyl)-N-(4-phenyl)urea (LY181985) was without effect on Golgi apparatus morphology following monensin treatment. The results active antitumor suggest a response of the trans Golgi apparatus to the trans Golgi cells 70%/o by sulfonylurea that resulted cssternae. Experiments sulfonylurea-unresponsive in reduced acidification of the to identify an antitumor apparats sulfonylurea-responsive enzymatic activity as a marker to guide isolation of Golgi membranes from HeLa homogenates are underway. appartus Golgi Complex (1406-1410). Monday 242a 140? 1406 EFFECT OF TEMPERATURE BLOCKADE ON THE PROCESSING OF PROTRH. ((E.A. Nilini, Perez de la Cruz and D. Jackson)) Division of Endocrinology, Brown University, Rhode Island Hospital, Providence, RI I.M 02903. AtT20 cells with a cDNA encoding preproTRH we have Using transfected previously reported that the processing of proTRH (26 kDa) begins in a subcompartment of Golgi Complex (GC) with proteolytic cleavage at two 15 kDa positions in the center of the molecule, generating an intrmediate peptide and a carboxyl-terminal 16.5 kDa intermediate peptide. In this study, we investigated the effect oftempeature blockade on the initial and AtT2O cell line, cells were pulsed subsequent processing of proTRH. In the with 3H-leucine for 4 hours at 37 OC followed by a 90, 120 and 180 minutes chase at 20 OC, in order to accumulate peptides in the trans-Golgi network, and compared to a chase at 370C as control. The release of peptides was further measured by increasing the discharge in response to PMA (phorbol 12myristate ester). Peptides were immunoprecipitated followed by analysis on SDS gel elotoihoresis. Endoproteolytic cleavage of proTRH to generate the 15 kDa and 16.5 kDa intermediates was not blocked by incubating the cells at 20 OC; however, generation of smaller intcmediates and cryptic peptides was seen only at 37 OC. After 120 minutes chase, the 15 kDa peptide at 20 OC accumulated five fold over the level at 37 OC. In contrast, the 16.5 kDa intermediate did not accumulate and was further processed with the level only 50% of the amount at amino-trmminal kDa in the GC while that of the 15 kDa intrmediate occurs in the This data 370C. suggests that further processing of the 16.5 to smaller forms occurs secretory granules (SG) after leaving the GC. We conclude that processing of proTRH begins prior to packing into SG and that N- and C-terminal intermediates are subjected to differential processing. Caifomria, hIssitute, A novel method was developed to introduce fluorescent probes selectively into the compartment of trans-Golgi in living cells. Normal human skin fibroblasts microinjected with a suspension of uniform-sized liposomes (70 ± 1 nm 101 diameter) containing fluid-phase fluorescent indicators, such as (SR). Liposomes fused with trana-Golgi and delivered their aqueous-phase contents with a half-time of min 15 at 37 'C. mfocal microscopy showed Dolocazation of SR Effcient liposome with the lipid-phase trans-Golgi-specific fusion in vito required 37 'C temperature, ATP, and precise liposome size. Fusion was also sensitive to NEM but not to GTP-y-S. To measure pH in the trans-Golgi aqueous were sulforhodamine mlarker NBD-ceramidde. compartment, two membrane-impermeant fluorophores were encapsulated in liposomes: fluorescein-sulfonate (pH-sensitive, pKa=6.3) and sulforhodamine 101 (pH-insensitive). These compounds were chosen for their self-quenching when present at high concentrations in liposomes, bright fluorescence when diluted in After trans-Golgi, non-overlapping spectra, low membrane permeability, and fusion of microinjected liposomes, confocal cellimages were collected by a cooled (oil x100, N.A. 1.4). Fluorescein-to-sulforhodamine signal CCD-camera objective: ratios (F/SR) werecalculated from background-subtracted pixel intensities integrated over the area of the trans-Golgi. In normul human skin fibroblasts, F/SR was .230 ± 0.009 (SE,n=89). The relationship between F/SR andtrans-Golgi pH was calibrated in vivo using solutions titrated to various pH containing bafilomycin, the ionophores monensin and CCCP, and highK. Simiar pKa values (-63) were determined in vitro and in vivo. Trns-Golgi pH was caklculated from F/SR and the calibration curve to be 6.20 + 0.05. pH was sensitive to bafilomycin, weak bases, solution anion pKa. content, and cAMP stimulation. These results provide the first direct masurment of trans-Golgi pH in living cells and introduce an effective methodto deliver fluid-phase indicators into the secretorycom t. 1410 Acw=RMcnzOP',EBNWtKShUCR REGMOANLAX APPARAVS (GA) WIDIINIDOPIISOFIHCAMALSNBUROM IN CULTUR.L ((E. Tonr, 0. Seward)). DepL of Nuauckence. Univbsity of Visginia (2IarIousville, VA 22908 an ervecells, N mogouly RNAsla mRNAs eemd eua dkl dihlbgeof vh eaode som so beuuu usglyosyled, orguseles IDdieRE td ieGAmetbePotip evadasd de; pme of at low aIC. musdg g or m dug dads Thepsn op mere es dh E tGA comapex in enluned hi;wocundlemrnsby syeiiwd wy _me0 or3H-plecomewhm by ham betwen wee-scir-.eat-wi aesiadwid BfA.due r amk gbpnkA"p1 labdidg ih3Hn slrm onnsthent w*bvocked BDrid A (BfA).When orb am syndheized xm meaoftcmdy heimcelbarmeaainisied at 2DIC.exit Of fas ike GA is bbcke& ds way. spouable so dede tde saim ofa Il e eGA). cen (14 days in ad _o gadwor(m di (s deER) glyooproisd horntheER vitro) won pruiacu d (S ta l) heGA lb loked so W wilh BEA (5 pghal, orat 15 ,20 3PC (cotl 3H eannoe Uced pilac (200 p3eCAa for h ud chaad in medisontining naulbeed 30 asi or s N21 mdaenah we glon. or3 lkllaetwawefinedsdjIorpouAdfor ag= for me waepnlxed atW won ep do GA hd bee bloked ees-ceoflocalg laim_ca only _o ee W hsropod do thttias _ea dmd 6e,lbe t s uesenoof W dandritc labeing neimos warepuls4abel gwujxeuep ovearcafbodies nd do bkbm of iD yd of ldin wit pbk ispm no nomism&a tdeIR GA dotno Wadatg Alp mle atvties widhIn forlacsoe Mr Xo _t-ofreceidy 1yntyhuiaSigly avail dhe t edwh fom dyonpaot d_oft wads norq. Inomc ddcoe When forMAP-2Imiddfy 3,,eR bodibesoawnndeimswereheavily lddee When cells 20l at at2-P DB A. bedingwinreuicidtocei bodies and Ilbue.ofxo.1bbe sdku Xtdseeofrexetly syndtzed dseIb dno-i. at l Fuseliaa, Santarone*, Girolamo.+, Corda+, We have previously shown that brefeldin A (BFA), stimulates the mono-ADPribosylation of two cytosolic proteins of 38 and 50 KDa (De Matteis et al., PNAS 91:1114). The reaction is blocked by known inhibitors of mono ADP-ribosylation such as nicotinamide, aminobenzamide and novobiocin. We now show that, in addition to a coumarinic ring, such as dicumarol and novobiocin, other compounds coumermycin A1 can inhibit the BFA-stimulatnd ADP-ribosyltaraferase. Dicumarol in known to block the NAD binding site of DT dinphorase and of the mitochondrial NADH oxidase complex quinone binding site sugesting that 1. Both these enzymes contain amight bind quinones. The effects of also the BFA-dependent several natural and synthetic quinones were thus examined. They were all found to inhibit the reaction with varying degrees of potency. In order to corrlate these in vitro results the complex and with in vivo events, the effects of agents on Gogig were studied in rat basophilic leukemia (RBL) cells by immunofluorescence and electron with the (labelled microscopy. In the presence of these compounds the Golgi complex antibody) lost II FITC-conjugated lectn from Helix pomatia and anti-mannosidase sensitivity to BFA and appeared as spot-like seacture located in the perinuclear area At the ultrastnuctural level this spot was seen to corispond to a tightly packed ring of Golgi the above mentoned inhibitors suppressed cinternae around the centrosome. Finally, constittive glycosaminoglycan port from the cis-Colgi trto the ans Golgi network an enzyme (TON) but not from TON to the plasma membrane. Our results suggest conuaining NAD and quinone binding sites in involved in the regulation of constitutive secretion and in the mechanism of action of BFA. work was partialy supported by the Agenzia per la Promoznine Acknowledgments. Sud). Sviluppo del Mezzogiomo (PR-2 and PR-3) and by CNR (Convenzione CNR-Mario Negri containing ADP-ribosyltransferase coumarsnic quinone tara This e lo 1409 1406 TRANS-GOLGI pH MEASURED IN FIBROBLASTS BY MICROINJECTION OF LIPOSOME-ENCAPSULATED FLUOROPHORES ((0. Seksek, J. Biwersi, and A.S. University of San Francisco Verkman)) Cardiovascular Research In CELLULAR EFFECTS OF INHIBITORS OF BFA-STIMULATED ADPRIBOSYLTRANSFERASE. ((0. Santini', M.G. Sciulli', A. Colanzi', A. Mironov M.A De D. S. M. Di G Innamorati', A. Matteis.* and A. Luini)) *Unit of Physiopatology of Secretion, +Laboratory of Molecular and Callular Endocrinology and 'Laboratory of Molecular Neurobiology, di Ricerche Farmacologiche Mario Negri Snd, 66030 Maria Imbaro, Chieti, Istituto Italy (Spon. by A. Luini) sume was so e demndriesocaee indiamndlud milie elyestiesuanihevechxis ofdh ER-A co.mpbm Sppmwd by MS1233 to OS. orgrni tionc berac erisfic 115 IS A GENERAL VESICULARTRANSPORT FACTOR RELATED TO THE YEAST ER-GOLGI TRANSPORT FACTOR USOlp ((S.K. Sapperstein, D.M. Walter, A.R. Grosvenor, J.E. Heuser, and M.G. Waters)) Department of Molecular Biology, Princeton University, Princeton, WJ 08544. p vesictuar A recently discovered transport factor, termed p11, is required along with NSF and SNAPs for in vitro Golgi transport 15 is a peripheral membrane protein found predominantly on the Golgi Biochemical and electronmicroscopic analyses indicate that p1 15 is an elongated homo-dimer with to globular "heads" and an exlended "tail" reminiscent of myosin N. We have clonedandsequenced cDNAsfor bovine and rat 1 The predicted translation products are 90% identical, and each can be divided into three bovine protein consists of an aminodomains. The predicted 108 terminal 73 kDo globular domain follwod by a 29 kDa coied-coil acdic domain of dimerization domain, aminker segment of 4 kDa and ahighly ER 3 kDa. p1 5 is p,a protein requedfor toGolgi p has a Usol vesicular transport in the yeast Saccharomyces cerevisiae. 15 and similar"head-coll-acid" domain structure. The head domains of pland 728aa, Usol p, which are about 25% identical, are simiar in size (651 respectively), and they possess two highly homiogous regionsThe first is 62%identical and 74% similar over 34 residues and the second is 60% identical and 77% similar over 53 residues. There is a third region of homology (50%identity over 28 residues) between the coEded-cil and acidic domains. The acidic domains at the extreme C-termini of p1 and Usol p do Since pl15 is not appear to be conserved at the primary sequence required for intra Golgi transport, and Usol p is required for ER to Golgi transport, we propose that pl15 may be a general transport factor. pl pl 5. kDa homologoustoUsol level. 15 Monday. Blood VesselsI (1411-1416) 243a 1411 LDL INCREASES ENDOTHEUAL CELL VULNERABIUTY TO SHEAR STRESS. ((M.S.F. Clake, KA Pitda Jr., KS. Mdowand P.L MCNs1)) Bly bw dansiy lpopictsi Elead plma nical -tharoad as paru lion of lechan lciy-In ro s. ucd EC repot he expour t of factor- for th rela 0.005) th reveabd of LDL-4reatd toxs andlor mor nu at of *vu. badsc fbrolast o f (sudfered lees Flw uravvd EC cytofluorometly she had Sheared membrane ous We The wa ureated EC. LDL-tread EC releasd 24old mor (p< 0.02) bFGF tn sheard conrol EC. LDL treamt of EC resubAd in an 0% incae above km em dcol rol, and an binrase in evekls oftl ontrol cholesterol phospholpid rao from 0.6 to 1.3. Stedys"ta - otrop threscence mebane th reveaed fluktpy (PLF) LDLWreabd EC sigdflctiy lower hn that of ctol EC. When PMF of LDWItreatd EC ws raisd usin the membane fluldzimg shear alty agen AaC, we obsved a sgnd cut reducion In EC th whereas reducion In PMF, usion sti ste ant PFP8, had th opposte affw Our dat LWI-induced hincea in ECvwu abilitytovmechani s*essmaypromotesendothellal th do o.sceosI. t ijry i of wa a poo , inlllation 1413 tunmor a celUs from the circulatoy seconday tissue loca the mignte monolayer of or lymphatc systems, endodelil cells (EC) lining fiboneci mroorouscel culture is and E-STIM Endothelial Cel Culture Medium promotes the rapid formation of confluent EC monolayers withbarrier fncto i vuiro. These EC monolayers were used to examine the migrato of human prmasytumors and in wwo measatic potentials, including meanomas, sacomas, tumor cell lines of differing carcinomas of various origins. For each used tumor type,cor ng normal cells or and less controls. Cells placedintoiserts contmag EC and allowed to bind to and invade the endothelium for up to 24hours, then fixed invasive cell lines monolayers were as we and processed for microscopy (phase or SEM) or for quantitation of invasion (by microscopy or y scintillation counting of ICr-labeled tumor cells). In each case, tumor cells that were highly invasive inWio showed rapid invasion of the endothelium inWitro, migratingthrough to the underside of the insert. In hse ara of invasion, complete scattering of the EC monolayer was observed, while adjacentregions of the monolayer which were free of tumor cells remaiedintact. Control cells weremuchkss invasive, with most cells remaining only wealdy atached to the apical surface of the monolayer. Invasion of the endotheliumby highly invasive cells couldbe decreased or inhibitedcither by pro-treatment of EC with antibodies against certain CAMs (e.g., P-sdectin), or by treatment of the tumor cels with anti-tumor compounds such as taxol, vincristin, or vinblstine. These results demonstra th t the BioCoat Transendothea Tumor Migration Environmentserves as physiologically relvant a insitro model oftumor cell extravasation during metastasis, which can used to study the extravastion process, to assess the metastatc poential of varioustumor cells, and potentially to examine the efficacy of candidate anti-tumor be dtrapmstics. 1414 WING ENDOTHELIAL PERMSELECTIVITY. ((R.N. Feinberg and E. Cafasso)) Department of Anatomy, Cell Biology and Injury Sciences, New Jersey Medical School, Newark, NJ 07103 CHICK Normal vertebrate limb development requires the formation of characteristic vasculature. Vascular patterns of embryonic but little is known limbs have been extensively studied, about the permeability characteristics of the developing The microcirculation of the chick wing was circulation. examined by in vivo confocal microscopy following the systemic injection of a graded series of fluorescein isothiocyanate (FITC) labeled dextrans. Chick embryos at stages 21-23 were injected with FITC-DX-150, 70, 40 in order to establish how selective the endothelial lining of microvessels are to macromolecules of various molecular weights. A confocal image of the perfused wing bud was obtained by image analysis software. Videodensitometry, over a gray scale range of 0255, was used to quantitate the amount of tracer found within the interstitium. Images were acquired at 2, 7, and 12 min postinjection. FITC-DX-150 and 70 did not extravasate from the endothelium of limb microvessels for all three stages. the surrounding FITCDI--40 was found to extravasate into Thus, differences in all three stages. interstitium for microvascular extravasation were attributed to the size of the molecular tracer rather than the stage of the embryo. These data indicate that embryonic wing microvessels demonstrate efflux across to macromolecular the permselectivity provide a basis endothelium. The present results for additional permeability studies and challenge our concepts about the role of diffusible morphogens in limb development. a 1415 A NEUROPHIL SECRETORY PRODUCT INCREASES ENDOTHELAL CEI CALCIUM AND MONOLAYER PERMEABILITY. ((AJ. Huag, K.R. Haner, T.T. Chen, and S.C. Siverstin)) epmmeasts of Medicine an Physiolg, Cohsmbia Universy, Colege of Physics & Sugo, New York, NY 10032. P ucs barrier by reguates (PMN) migrate betwe crawl PMN tanst 120:1371-1380). woes EC. EC acrn an EC molcule by mandodelbal ceU (EC) coolic fre acrs com eteoftUa is ty (Ca calcium at al. 1993. monolayr (Hu PMN bindnto and mirtion by tie ligation of EC arhe pmti J.GCeZ BIoL acmnied on Ie PMN Wteraction of PMN sereoy wth EC. To determine whoher PMN-indced increases in EC 1Ca++]. and EC monolyer pemabiliy, ectrical rance, e inited by tie liaon a meassred by transendot of EC urfac PMN product, examined effict of cross lining EC srface m bcules and of cell fee a e derived from to evets. PMN Cell frec t IMLPlsmulated PMN increased EC [Ca++], ad EC monolayer pernebility to sons in a na _riodependent fahkon MAb 60.3, dircted againstP2 istgrn onthe PMN srface, inbibed 85 4% PMN adhesion to and migration acws EC but did not prevent PMN-inucd bxnr EC [CaL in response to fMLP. PMN tplasts, which lack grals and respiraory burs acivity, and by t products molels or a tie on migrt wit tstatic the lumenal surfaces of the vessels. To model this prcess oftumor cell extravasaton, we nwtro eanvironmnst, the BioCoate Transendothel hawv contcted Tumor Migrtion Environment. Previous workfiom our laboartories has shown that a combinaton of BioCoat an ECto 240 mgdchoLestsrUdL vess coitrol pm t of mle* cCl nmogen smoot ilasVW severity d shear-d*ed injry h IL-realsd EC Vth sured mechnIcal smherng 20%; p vsl injry ooaun fbr te m vascular a Duringinvasion into cdM (EC) andothala (WI) rsk Is men alheaogmnesis In m*o ad a* groAh facto (bFGF), poter- la Colg Mdical Anaomy, & Augus, GA 30912. (proo R. Cadwel) Gaorge, meha Caul ment 1412 CONSTRUCTON OF AN ENVIRONMENTFOR MODELLING TRANSENDOTHELIAL M IGRATION OF TUMOR CELLS. ((BJ. Del Buono)) Becton Dickinson Collabotive Biomedical Products, Bedford, MA 01730. om and increase tat PMN. molecues- with provide evio Crs muibodie pernme- bility of EC ICAM I a as com vral oier EC EC [Ca-1. bctinbfaled tD hmatrctastbnulatd PMN ad product which snc,reases BC [Ca4L and EC monolayer per-me akility. OVEREXPRESSION OF A MUTANT MYOSIN UGHT CHAIN (MLC) IN ENDOTHEUAL CELLS ATTENUATES THE INCREASE IN ENDOTHELIAL PERMEABILITY ((P.G. Nagpala, H. Lum, G. Nikevics, G. Nowak, S. Ono, D. Lodme', P. de LaneoW, and A.B. Malk)) Rush Medick Univeriy lEnois School Medcie Chicago, IL60612. of Colege, and of enkhl_ Contra_ion of cal (EC) in resporne to infammaty mecatDr resut in incread EC permeabit. We hypothesed tt ph MLC and by MLC kins aclivng the cadin-mnyosin motor endothelalo coracon. The role of MLC phoephorlallon on EC contrcion we inveigated by stdablbrodon of inmortefied human umb5csi vein EC kw EAhyb 928 wIt vector confinin mutant rat aoric smooth muscebMLC cDNA (muMLC). The muMLC wa gerae by conve thr18 and serB aleS and The muMLC DNA ws paced under the contol of the promoter region in th retrovira 5' lng bnrini repet and wa teggd at the end with an 11 amino saod myc epipe for idekaln. The transfedad cle showed no appernt moo difrence fom th control cel. Co-immunopeii wth confirmed the presencedof ogenous muMLC in the cel. The muMLC trarnfectante showed decreasd MLC phohoya Both the and muMLC b showd imlr basal taneendotellal pneabit to values 0.340.0B and 0.41±0.07 pifin, Human repecval-y). (1 laM) caused 26-fod increase over basene in _ b po.. of whereas muMLC transfetente w1rg O memat an a a al1a9. to myoin _trasfeted _ansfeate responded with 1.7-fod increase a MLCK- dependet irease 'ni-albunin ceabnceof '5i_. m-hronbin '1tunmin; (trarew'd endothelyl a beselne. These findngs indicate that MLC ser19 andfor thrl8 in pert mndate fe over hophomlon of pennabit in response to pro-inllmmatoiry meditore. Since mulSon of mer9 thri8 did not inhibit resporns, ph MLC by protein ins C may alo conribte to the response. (Supported by HL45638, HL27016, and Amnecn Heart Aawo ation of Chicago). the erir of the 1416 CHEMOATTRACTANT-STIMULATED NEUTROPIILS SIGNAL PHOSPHORYLATION OF MYOSIN REGULATORY UGHT CHAINS IN ENDOTHEUAL CELLS. (tEA. Hiuxebeghl, Z.M. Goeckaler2, A.J. HUan3, R.B. WyaoImersl4, ad S.C. Silverstein'l) Depts of Physiology' end Medicine3, Colunbia Uniesity, New York, NY 10032, and Depts of and Anesthesiology4, Saint Louis University, Saint Louis, MO 63104. Pathdogy24 Chemotaxing neutrophils ondothelial cel (EC) monolayers by crawling between neighboring ECs. ECs iceas their intracelllar C2 in response to chemo attractant-st umdted neutrophils. Agent that block this C jnctions and transendothelial rise inhibit opening of neutrophil migration (L Cal Bio. 135:355 119931). Inrease in EC Ca2+ promote phoephoryltion of reulatory myosin light chains and EC retraction (PNAS 87:16 119901). To determin whether neutrophils promote myosin light chain phosphorylation, ECs were prelabebd with 32P4 or u5methionine, incubated with neutrophils in the presence or absence of chemoattractant, and myosin was immunoprecipitated and subjected to one- or two-dimensional gel analysis. Using both methods, phosphorylation of EC myosin regulatory light chains increased 130-240% within 1-2 minutes afteraddition of chemoatractant-stimuated, but not unstimulated neutrophils. However, in interleukin-l-treated EC monolayers, addition of unstimulated neutrophils signaled a 1 10150% increase in phosphorylation of EC myosin regulatory light chains. Two-dimensional gelanalysis showed stimulated nurophils icreased both mono- and di-phosphorylation of EC tave intor-EC a lght urfce myosin regulatory sudies 19 and threonine 18 activity. Thes chai, and tryptic peptide mapping revealed serine phshrltion, results suggest that indicatin myosin light chain kinas phortion of the EC myosin regulatory light chains plays a role in neutrophil migration across endothelia. Blood VesselsI 244a 1417 TRANSENDOTHELIAL MIGRATION OF HUMAN MONOCYTES IS ATATENUATED BY THE STABLE PROSTACYCLIN ANALOGUE CICAPROST ((A. Griitzkau, S. Weliershoff and H. Graf)) Schering Research Laboratories, 13342 Berlin, Germany prostacyclin (PGI2) and Recent data indicated additional properties of exceeding the well known antiaggregatory activity on platelets, its analogues such as enh of endothelial barrier function, inhibition of neutrophil adherence to vascular endothelium and inhibitory effects on leukocyte chemotaxis. The aim of the present study was to investigate PGI2-dependent mechanisms responsible for the regulation of monocyte extravasation during inflammatory conditions. In this respect the influence of the stable PGI2analogue Cicaprost (ZK 96480) on the tansendothelial migration of human monocytes in response to various chemotcic compounds was investigated in an in-vitro model for the vascular endothelium. For this purpose endothelial cells isolated from bovine pulmonary artery were cultured on porous poycarbonate-membranes separating a two compartment system. The upper compartment contained monocytes prepared from fresh human blood by of 2h mocounterflow centrifugation elutriation. During an incubation nocytes migrated across the endotihealized membranes into the lower decreased migration in a ttrnsendothelial dose-depenpartment. Cicaprost dent manner mainly on the level of the endothelium. Rolipram (ZK 62711), IV specific phosphodiesterase, potentiated the inhibian ihibitor of the tion evoked by Cicaprost, whereas the cAMP analogue dibutyryl cAMP only caused an additive effect These studies suggest that prostacyclin and its analogues are capable of modulating the extravasation of monocytes during inflammatory processes via a cAMP-dependent mechanism. ent tme co- (1417-1422). Monday 1418 EXPRESSION OF THE GROWTH FACTOR-INDUCIBLE GENE PS4 IN BALLOON INJURED RAT ARTERIES AND IN HUMAN ATHEREC- TOMY SPECIMENS. ((R Mora, C. Haudenschild5, and G. Liau)) Department of Molecular, Biology, and Department of Experimental, Pathology*, Holland LAboratory, American Red Cross, Rockville, Maryland 20855. We recently identified a novel serum and growth factor- inducible gene called PS4 in rabbit vascular smooth muscle cells Sequence analysis of a full-length PS4 cDNA revealed an open reading frame encoding a polypeptide of 276 amino acids. The deduced protein sequence exhibits a 94% identity with human TSG-6; a specific TNF and IL-1 inducible hyaluronan binding protein synthesized by human fibroblasts. However, unlike human fibroblasts, expression of PS4/TSG-6 in vascular SMCs is regulated by both cytokines (IL-1) and growth fsctors (FGF-1). To determine if PS4 (SMCs). has a role in the development of vascular lesions, we examined the expression of this protein in a rat vascular injury model as wel as in human atheretomy specimens An intense immunostaining for PS4 was detected in the rat whereas in the medial layer and in non-ballooned carotid arteries was not expressed. This confinement to the neointima suggests that PS4 may be involved in vascular remodeling neointima PS4 also highly expressed in human atherectomy specimens. The highest level of PS4 immunolocalized in was found in the PS4 was PS4 immunoetaining macrophage-rich areas. the macrophage cytoplasm as well as diffusey in the matrix immediately surrounding the lipid-laden macrophages. In regions which were macrophags-negative and rich in SMCs, PS4 expression, when observed, was predominantly intracelluar. We conclude PS4 expression that the in localization of is consistent with its regulation by growth factors vvoi and cytokines in vitro and increased of PS4 is a marker of phenotypically and associated with activated macrophages. (Supported by NIH Grant SMCs is a of a Research Career Development Award HL-02449). recipient HH-37510,G.L. altered 1419 1420 ADHESIONMOLECULE ENDOTHEUN-1 DOES NOT STIMMLATE CELL EXPRESSION IN HUMAN UMBILCAL VEIN ENDOTHELIAL CELLS. ((R. Zaragoza,G.P. Budzik and J.R. Wu-Wong)). (Spon.G. P. Budzik) Abbott Laboratories, Pharmaceutical Products Division, Abbott Park, IL 60064 STABLE TRANSFER OF GENES INTO ENDOTHELUAL CELLS USING UPOSOME MEDIATED TRANSFECTION AND RETROVIRAL TRANSDUCTION.((P.G. Nagpolet H. Coag/ Rush Lum, and A.B. Melik)) Departnent of Pharmacology, Rush Presbytorin-St Luo 's Medical Canter, Chicago, IL60 12. ISpon. by EA. Everitt) Endothelin (ET-1), a 21 amino acid peptide, is potent endothelium- a derived vasoconstrictor. Previously it has been reported that ET-1 stimulates the expression of intercellular adhesion molecule-1 (ICAM- 1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial cell adhesion molecule (ELAM) in human brain microvascular endothelial cells (McCarron et al., 1993). In this study we investigated theeffectB of ET-1 on CAM expression in human umbilical veinendothelial cells (HUVEC). ET-1 up to1 LsM had no effecton the expression ofthese CAMs in HUVEC, although TNFa at 10 nglml stimulated the expression ELAM,ICAM and VCAM by 5.2, 4.0 and 8.3 fold, respectively. Bindingstudies show that HUVEC exhibits few ET binding sites (0.41 ±0.06 fmolmg). We further treated HUVEC with phosphoramidon to up-regulate the number of ET receptors and then examined the effect of ET-1 on CAM expression. After phosphoramidon treatment at 0, 0.1, 1, 2 and 6 mM for 48 h, the binding of 1251 ET-1 to HUVEC increased from a basal level of 0.41±0.06 to 1.8±0.2, 4.1±0.2, 6.2±0.6 and 11.9±0.9 fmoVmg protein, respectively. In HUVEC pretreated with phosphoramidon (2 mM) for 48 h, TNFa (10 nglml, 4 h incubation) was again shown to induce the expression of ELAM, FiM) still failed to exert any ICAM, and VCAM. However, ET-1 (0.01-1 effecton the expression of these CAMs. These data suggest that ET-1 in HUVEC does not stimulate the expression of receptor to its binding ICAM-1, VCAM-1 and ELAM. Medkcl EndotIhat coe (EC)wae mown to be refractoryto transtectionusingcalcum phosphate, DEAE dextrn, and electoporaon. Problems in obtainingoffcient gene traer and stable eprsionlimitthe appctn oftr nfection in EC. We compard two methods: (RMT) and lipofectemine- medatedtrao n (LMT) retrovirsl-mdiabd protocolsthat resultd in persitt eox on ofvarouscDNAs in EC. Wedoned cDNAs into retrovirel vectora pLNCXand pLXSN which containing theimmedate ealy promoter of ofth human cytomeglovu and the retrovirsl tong terminl repeatforthe tran tle marker,the voctos Wo hadthe insertedcDNA, (NPT) As aThe" constucte used troduce w genes io two EC gne. phoephotwasferese vein EC drivtve) nd HMEC(human dermal lins: EA hyb92t(a human pariles we generated by microvscur EC). For RMT, replication incompetent plsrrid conabucts into amphotopic retovrs packaging colls, itoducing EC. In LMT, pA317. The viruscon rng cndioned medium was then used to tbanfeci noomycin redpectively. umbllcl rerovlra EC were tOnuc inubated with a suspension of lipofcteineo and the DNA. The bfton Tho DNA efficincy wa about 1004-od grear in RMT than LMTfor both cei f1hgment with NPT and theinsorted cDNAintgratinginto the EC chromosome was lines RMT. However, ths was notthec definod, andthus both genes wero wacoexpresed was necesy to etash intdegration of random, and hence for LMT in wtich DNA donl survMng coels in LMT made lecion ofcwhich clonal populations. The low denit ater in populaions possible immediately screning HMEC,but not in EA hyb92t Both methods oftransfection yielded stabletwb exrssingthe eventuallyanddiod. inrted gene for at least up to 2 monts poet traection confirmed the mar by Wetewn bkL. Theme resut show that stable long term expression of exogenous DNA in EC is poible using RMT and LMT,but th RMT is the prefrred metod itgive of the marker andtheinsrted higher transfectlon effency and cornient andcontegraon Heart Association of In It th ectnts gene. (Supported by HL45638, HL27016, Americm Chicago.) 1421 1422 IL-1 INDUCES PHENOTYPIC TRANSDIFFERENTIATION OF SKIN MICROVASCULAR ENDOTHELIAL CELLS INTO SPINDLE-SHAPED CELLS: RELEVANCE TO KAPOSI'S SARCOMA. ((LI. Romero, D.N. Zhang, and M.A. Karasek)) Department of Dermatology, Stanford University School of Medicine, Stanford, CA 94305. DEVELOPMENT OF THE AORTIC VESSEL WALL AS DEFINED BY VASCULAR MUSCLE AND EXTRACELLULAR MATRIX MARKERS W.S.Argraves C.D.L)) * ('JEHungerford, Cha,otesvile, VA 22908. +Biochemisty Labt,A UniVersity VI Red Cross, Rockvle, MD 20655. Cardivaseular Developmental Bbgy Center, Kaposi's sarcoma is a neoplasm of vascular origen, histologically characterized by an abnormal proliferation and perivascular infiltration of spindle-shaped cells. The purpose of this investigation was to deter-mine the effect of IL-1 and bFGF, two cytokines known to be secreted and to stimulate growth of Kaposi's sarcoma cells in itro, on transdifferentiation of normal microvascular endothelial cells. IL-1 A and IL-1ba induced morphologic transdifferentiation of normal epithelioid endothelial cells into spindle-shaped cells in 24 to 72 hrs. Changes in morphology of factor VIII were accompanied by immunostaining. IL- a decrease effects or were complete absence reversible after 3 days of stimulation and became irreversible after 7-10 days. Pretreatment of endothelial cells with cycloheximide (5gg/ml) did not inhibit transdifferentiation. The effect of IL-1 was more marked in the presence of bFGF (10 U/ml). In the absence of IL-1, bFGF slightly stimulated growth without changes in cell morphology; in the presence of IL-i, bFGF potentiated spindle cells growth dramatically. These data indicate that IL-1 can induce permanent transdifferentiation of endothelial cells into mesenchymal like cells and that these cells become more susceptible to stimulation by bFGF. These observations may begin to explain the inital steps in the pathogenesis of Kaposi's sarcoma. SMOOTH Medical Universit of South Carolna, Charlsto, SC 29425. The bul"d ot vessel wallfrom s cellularand ltraelllarmatrix(ECM) and maturaon of th cardovascular S aacuical event in tde events ta occur aftere iniil system. However, very Ut es known abo d vasmur nstwot anasent ndolu, esabshed. Usiqn quail the VWdlls X roe ntand as amodel (VSMC) thoracc aorta mot o devep gveselwall has edby usig St 12-22 nr luor qun been _ oed and indces tatth de monoconl enlbodes M bs) VSMC differetan ofdebryonic VSMCS vntral to . The are on the vental sufe ote fusigW cab first dosal p a ent t thee ndo lium, one er side dt midine. In orr to aortae, dorsal addresse rtd ttissue speicwy markers to VSMCs in em n tissue, weNhave gnad MAbs om e n sselwa anIens. 1E12, which iske muscle sourMAb, vs study Criticaltoths MAbs tob wnVSMC markers (suchs smoo muscle is b Imm seen wi h a SMA h t osa ms msst, patemd vascuar muscle caD of b bsto foud now and problms n td lwaldel alha adh) ianoo pattn #aWs tD Mrdiawnd vernrJ uno heel cl'ril spcrnin 1E12hlg withth we show t kmuolxuor MAb, 8-12 hourspoynonal labrin d abo ob 1 showsa m d on siarto he beigwkiha th of the VSMCmars Fin- lbngsufounii the VSMCe before estin pecurson are prsntwit the d vesor wVaLSWeMtwCo.bdevelbui-I expesslon to bean early marker for dIW MVMs Monday. Blood Vessels I (1423-1427) 245a 1423 PAF-INDUCED ALTERATION OF MORPHOLOGY, CYTOSKELETAL ORGANIZATION AND lCAM-1 RECEPTOR EXPRESSION IN MICROVASCULAR ENDOTHELIAL CELL MONOLAYERS. S.S. Kantak C.L. Jones, B. Singh, D.E. Bossung and J.M. Onoda, Department Radiation Oncology, Wayne State University Schoold Medicine, Detroit, Ml 48201. We characterized the effects of short-term exposure of nanomolar concentrations of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) on those endothera cell reaponses (to PAF) which characterize PAF-kiduced kig pathologies (e.g., edema). We examined PAF effects on edothelial cel morphlogy using phae cornrast mncocopy and mirofiamerit nt and luminal surface expression of adhesion receptors (i.e., ICAM-1) using immunofluorescence microscopy. Exposure of confluent, contact-inhibited munrne pulmonary mnicrwasculature endothelial cell monolayers to 10 nM PAF results in significant celular retraction ocring < 30 minutes post PAF exposue. in Inasnuch as F-acin micfaments have been shown to play a significant the regulation of endothelal mophology, we examined PAF effects on F-actin organization and observed PAF-induced F-actin depolynmrization conomitant with ceHular retraction. We observed that PAF-induced endothelial cell retmction is a reversible event. Reorganization of F-actin fibers occurs > 8 hours post PAF exposure, concomitant with restoration of monolayer morphology. Our results indicate a direct causal relationhip between PAF exposure and the induction of endtelial cell retraction and F-actin organization which are determinants in the pathology of inflammation. The induced expression of endothelial adhesion receptors is also causal for the inflammatory response. Therefore, we also characterized the effects of PAF on apical surface expression of ICAM-1, an adhesion receptor which is at east partialy responsible for maintenance and amplification of the intlammatory response. We observed that 10 nM PAF induces significant lCAM-1 expresion. These studies were supported in part by CAS0465 and Gers on Radiation Oncology Center, Detroit, Ml 48201 rle 1424 APPROACHING THE ROLE OF CD34 IN VASCULAR DEVELOPMENT AND HEMATOPOIESIS: LOCALIZATION, HEMATOPOIETIC POTENTIAL, AND PROTEIN-PROTEIN INTERACTIONS. ((P.E. Young, C. Fennie, H. Avraham, J. Groopman, & LA. Lasky)) Dept. Immunology, Genentech, Inc., South San Francisco, CA 94080. CD34 is a cell-surface protein expressed on vascular endothelial cells and hematopoietic progenitor cells. Our lab demonstrated that CD34 presents carbohydrate ligands to L-selectin in the high endothelial venules of the lymph node and potentially throughout the vasculature. However, an understanding of the function of CD34 in hematopdiesis and of its relative importance to cell survival and/or differentiation at vascular locations remains elusive. Towards this end, we completed an immunofluorescent confocal analysis of the distribution of CD34 in developing murine embryos throughout development. Our studies localized CD34 throughout the vasculature, to endothelial processes and filopodia associated with angiogenesis, and to hematopoletic progenitor cells within the embryonic yolk fetal liver, and other intraembryonic sites of hematopoiesis. Our results identified CD34 as the first known useful marker for the analysis of mammalian vascular development. In addition, we used anti-CD34 affinitypurified antibodies to isolate hematopoietic progenitor cells from embryonic sources. These studies demonstrated that CD34+ progenitors can give rise to a variety of hematopoletic lineages In vitro, when cultured with appropriate cytokines and/or growth factors. Finally, we isolated seven distinct proteins via the yeast two-hybrid system that interact with the intracellular domain of CD34 and may therefore be involved in signalling and/or CD34 function. Most proteins isolated thus far show no significant homology with the database. Interestinly, two proteins are 7096 identical to each other, and may represent a novel family of unknown function. sac, the 1425 1426 ENDOTHELIAL CELL CD36 EXPRESSION CORRELATES WITH PARENCHYMAL TISSUE FATTY ACID UTILIZATION. (D. E. Greenwalt*, S. Scheck§, and T. Rhinehart-Jones*)*Holland Laboratory, American Red Cross, Rockville, MD, U.S.A. and §Loyola Narymount University, DISIRIBUBlON OF PAF RECEPOR IN THE VASCUIATURE ((D. Los Angeles, CA, U.S.A. CD36 has been described as a receptor/binding protein for thrombospondin, collagen, oxidized low density lipoproteins, and long chain fatty acids. We have exaained CD36 tissue distribution and expression in a mouse model. Nurine capillary endothelial cell (CEC) CD36 expression was most prominent in adipose and maary epithelial tissues which take up fatty acids and store and/or secrete triacylglycerol and highly oxidative tissues such as skeletal and cardiac muscle which use fatty acids for energy production. CEC of the brain, which does not utilize fatty acids as an energy source, did not express CD36. We then hypothesized that increased levels of plasma triglyceride (PT) would lead to increased expression of CEC CD36. Both diabetic mice and mice fed diets high in fat have elevated PT levels and were examined for cardiac muscle CEC expression. NOD (nonobese diabetic) mice had PT levels 3fold higher than normal mice and expressed cardiac muscle CD36 at a level 7-fold higher than normal mice as quantified by densitometry of immunoblots. Female mice fed ad l1bltua a diet containing 40% fat (% caloric intake) for 5 weeks, expressed CD36 at a level 4-fold greater than mice fed normal diets (9% fat). In both diabetic and high fat diet mice, brain CEC remained CD36-negative. These data suggest cardiac muscle CEC can regulate CD36 expression in response to increases in PT. 142 DEVELOPMENT OF A HUMAN BRAIN MICROVASCULAR ENDOIIHELIAL CELL LINE WITH BLOOD BRAIN BARRIER CHARACTERISTICS: TRANSFECInON WnI SV40-LARGE T. ((M.F.Stins, P.V. Neman.i, KS.Kim)). Div. Infectious Diseases, Chlldrens Hospital LA, 4650 Sunset Blvd, Boc 51, Los Angeles, CA, 90027. Studieswith human brain microvascular endothelial cells (HBEC) have been limited because these cells are difficult to obtain and culture in vitro. Normal HBMEC usually showed signs of senescence at early passage. We trnsfcted HBMEC with a pBR322 based plasmid containing simian virus 40 large T antigen. The transfected endothelal cells showed a somewhat spindly morphology, were positive for fictor VIm carbonic anhydrase IV, took up fluorescently labeled acetylated low density lipoprotein, and expessed gamma glutamyl transpeptidase, demonstrating both their endothelial and brain origin. We have cultured these cells past passage 15. The HBMEC were used as an in vitro model of the blood brain barrier in minmg the pathogenesis of E.gl meningitis Le. the role of S-Fimbriae and OmpA in binding and invasion of HBMEC. Binding to and invasion of HBMEC was significantly greater for S-fimbriated and OmpA+ E..cli. These differenceswere not observed with human systemic arterial or venous endothelial cells. In additon, atvation of HBMEC by TNF reslted in a time dependent increase in VCAM expre. In conclusion, our tanfecd HBMEC's are the first reported human brain endothelial cells that retain morphologic, phenotypic and functional chracteristics of normal HBMEC for an extended period of time. Predescu, S. Predescu, L Ihida and G.E Palade)) Division of Cellular and Molecular Medicine, School of Medicine, University of Califomia, San Diego, CA 92093. Although the platelet activating factor (PAF) is one of the most active inflammatory mediators known to date, little is known about the cellular and subcellular distribution of its receptor(s). The latter has already been identified as a membrane protein of 39 kD. In order to better understand the signaling mechanisms responsible for PAFs unusually high potency, the first question that we decided to answer concerned the tissue distribution of the PAF receptor(s) (PAF-R). In our laboratory, we raised monoclonal antibodies agnat synthetic peptides reproducing sbort segments (12 and 14 as) at the N and C terminal parts of PAF-R. With the anti N terminal monoclonal antibody, we localized PAF-R by immunofluorescence on semithin frozen sections of lung, heart, diaphragm, kidney and brain specimens. With the exception of brain, the signal was restricted in all cases, primarily, to the vascular endothelium. Using a preembedding Immunogold procedure, we localized the PAF-R In small clusters on endothelial surfaces, without preferential localization to any differentiated microdomain. A morphometric analysis revealed, however, a greater signal density at the level of venoles than at the level of any other segment of the microvasculature. With the same antibody, we immunoprecipitated PAF-R from whole homogenates of the same tissues, and with an ELISA we obtained quantitative data. The purified antibody detected PAF-R at a dilution of 1:10,000 in the lung and at a dilution of 1:150 in the case of brain, which correspond to calculated values of 322 pmol/mg tissue protein and 26,8 pmol/mg tissue protein, respectively. Supported by NIH Grant HL 17080 to GEP and by Bugher Fellowship 001 to DP. - 246a Platelets (1428-1432). Monday I 1428 REDISTRIBUTION OF PLATELET-BOUND FIBRINOGEN MODULATES PLATELET AGGREGATION. ((J.D. Wencel-Drake)) Department of Medical Laboratory Sciences, University of Illinois, Chicago, IL 60612. ammsis converted fron *inactive' agonist-stimulated platelets, the integrin 'active fibrinosen (FBG) receptor, thereby mediating platelet aggregation. In to an an FBG an,sthat agP With time after agonist addition, at least two events occur, beocmes irreversibly bound to the platelet, and in the absence of stirring, platelets lose the is ability to aggregate. Since we previously identified a mobile pool of actively internalized in platelets, we explored the possibility that both of these events might result from the internalization of FBG bound to 'active' As Under conditions of irreversible FBG binding, fluorescence microscopy revealed that bound FBG is rpidly redistributed by activated platelets to a surface inaccesible, intracellular pool. To investigate the relationship between the rapid internalization of bound FBG and the loss of aggregation response, unstirred, washed platelets augmented with FITC-FBG were stimulated with thrombin receptor peptide agonist (TRP) for a various times, and the samples were rapidly brought to 4'C. FACS analyses revealed a time-dependent increase in the binding of FITC-FBG to unstirred, TRP activated platelets. Rabbit anti-fluorescein was subsequently added to quench surface-associated fluorescence and the samples were re-analyzed. A plot of the percent surface-associated FITC-FBG revealed a paaraleled a loss of aggregation loss of FITC-FBG from the platelet surface that response. Thus, the removal of FBG from the activated platelet surface via internalization appears to contribute not only to the irreversible phase of FBG binding, but also to the downregulation of platelet adhesiveness. To examine the effect of inhibition of irreversible FBG binding on the loss of aggreption response and the cellular localization of bound FBG, studies were repeated in the presence of ZnCl2. Incubation of platelets with ZnCl5 resulted in the retention of aggregation response in TRP desensitized platelets and inhibited internalization of biotinylated FBG previously observed by immunofluorescence microscopy. Taken colectively, these data sugest that internalization bound FBG represents a fundamental regulatory mechanism that modulates platelet function. HL-60 cells, a human promyelocytic stem cell line, have been used as model leukocytes in studies investigating platelet-leukocyte binding. Platelets activated bya-thrombin and related agonists bind to neutrophils and monocytes. Several groups have strong evidence that P-Selectin receptors on platelets and an unidentified ligand on leukocytes mediate this binding. It has also been reported that GPIIbEIa receptors are involved in platelet-leukocyte binding. Our laboratory has been interested in the functional interaction of these cells. We, and others, have demonstrted that coincident with binding, activated platelets cause an enhanced production of superoxide anion by neutrophils. Recently, we have also shown that activated platelets can modulate superoxide anionin DMS0differentiated HL-60 cells in a similar manner. The purpose of the present study was to investigate the relative roles of P-Selectin and GPIIbIIa receptors in the functional inteacion of these cells. This was accomplished by the use of monoclonal blocking antibodies against the respective receptors and RGDS peptides in platelet:HL-60 while measuring superoxide anion. (HL-40615) coincubations 1432 PHOSPHORYLATION OF CYTOSOLIC PHOSPHOLIPASE A2 IN HUMAN PLATELETS. ((Y. P. CWte and M. B. Feinstein)) Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06030-6125. Phospholipase A2 (PLA2) catalyscs the hydrolysis of the sn-2 fatty acyl phospholipids to liberate free fatty acids and lysophospholipids. Recently a Ca2-dependent high molecular weight cytosolic PLA2 (cPLA2) that is specific for arachidonic acid (AA) has been described. cloned and sequenced This enzyme has been identified and catezed in a variety of cells and has been prposed to be involved in the release of AA. More recently its activation by phosphorylation has been bond of demonstrated in human platelets and it has been proposed that it plays a mobilization of AA in human platelets. The goal of this determine the signal transductions involved in the phosphylation of cPLA2 in human pltelets. More specifically we have studied the phosphoylation of cPLA2 in human platelets stimulated with a-throbn, throbin receptor pbptide, A23187, tasiggin and phorbol12-myritate-I3-acetate (PMA). We have shown that (1) Although the release of the intracellular Ca2+ pool by thapsigagin or A23187, or the activation of PKC by PMA, rsult in increed by phosphorylaton of key IL-2 is required, but not sufficient, for the conal proiration of T lymphoThe activation of ymphocytes ruires concurrent occupancy of the T cellantigen receptor. IL-2 is known to induce the phosphorylation and activation of PI-3-K in lymphocytes, but does not affect intracellular calcium levels. In the patelet system, IL-2 presented alone does not result in pldelet aggregation, however, it does enhance the platelet response to suboptimal levels of collagen or thrombin. This study focused on the effect of EL-2 on patelet Platelets exposed to IL-2 were lyses and immunoPI-3-K. a precipitated withPI-3-K specific antibody. The immunoprecipitates were subjected to Western blot analysis with an antibody specific for phosphoof tyrosine contaning proteins. The phosphorylationPI-3-K (85 kDa subunit) peaked at 5-10 seconds. This is much more rapid than that noted in the the PI-3-K In lymphocyte system which peaks at 15 minutaes. adddtion, cytes. activity was directly meaured. Plateets incubated with IL-2 for varying times lysed and imunnoprecipitated with anti-PI-3-K. The incorporation of was from y`P-ATP into phosphatidylinositol substrate PI-3-K by y`2pd;2 determined by thin layer chromatograhy. The kinase activity was maximal within 1a0 second incubation of platelets with IL-2 These studies indicate that IL-2 modulatessignal transduconin platelets and may havesignificant effects on platelet function in conditions whereIL-2 levels are elevated. were 1431 1430 PLATELE;T MODUIALION OP E0O CELL PRODUCTION OF SUPEROXIDEANION:INHIBIONOOFP-SEECINANDGPIbMia RECEPTORS. ((D.G. Moon, P.S. Chin, T.B. Nguyen, S.M. Branley, S.R. Braemer, J.W. Fenton,II and G.A. Wetmore )) Departments of Biology and Pharmacy Prkcice, Albany College of Pharmacy, Albany, New York 12208 the was to role in work 1429 INTERLEUKIN-2 (IL-2) INDUCES PHOSPHORYLATION OF PHOSPHATIDYLINOSITOL-34KNASE (PI-3-K) IN HUMAN PLATELETS.((M.L. Aiken)) Univ. ofTexas Health Center at Tyler, Dept. of Biochem Tyler, TX 75710. cPLA2, (2) phosphorylation of cPLA2 induced by a-thrombin is extracellular Ca2+, is inhibited by prostacyclin, not GDPpS (in permesbii and not affected by GTPS by platelets). These results demonstrate that thrombin can stimulate the mchanism a phosphorylaton of cPLA2 by independent of a Gem protein and its downstramsignals, and may therefore be mediated by Py subunits. independent of mediated PKC or PIATELET ACIVAnON BY SODIUM INHIBMON OF HUMAN N AS VIEWED BY VIDEO-ENHANCED DIC IROPRUSSIDE Loftus, M.L Harlow and SJ Smith)) Department of Molecular and Cellular Physiology and Division of Hematology. Stanford University School of Medicine, Stanford, CA 94305. MICROSCOPY. ((DJ. We have used the technique of video-enhanced differential contrast (DIC) microscpy to examine the effect interference of a nitric oxide donor, sodium nitroprusside (SNP), on the morphological transformation that takes place when platelets become activated. In the absence of inhibitors, activating agents platelets atreatedW vth thrombin or other undergo dramatic changein shape with extension of numerousiflopodia. Plateletfllopodia exhibit extension, retraction, waving, bending and other complex movements. Near completeinhibition offllopodial formation and movement is seen in the presence of 100 gM SNP. Platelet adhesion and spreading on glass surfaces, however, which occursin the absence of activating agents, appears unaffected by SNP. These results indicate that nitric oxide specifically inhibits the platelet cytoskeletal transformation that is associated with platelet activation. Monday. Imaging Technology (1433-1438) 1434 1433* 3-D AND 4-D IMAGING OF LIVING CELLS WITH DIC AND POLARIZATION Inoue)) Marine Biological Lab., Woods MICROSCOPY. ((S. lnou6 and T.D. Hole, MA 02543, and Universal Imaging Corp., West Chester, PA 19380. We report here 247a a practical method for rapidly acquiring and generating high-resolution 3-D and 4-D images of live cells and developing embryos non-confocal, DIC and polarization (pol) microscopy. Current biological confocal microscopes do not allow confocal imaging in DIC orpol, and low-noise confocal fluorescence images require several seconds to acquire. Some confocal microscopes do permit video-rate image acquisition, but their utility for the current application is limited by photon statistics. Our method entails: Al acquire background-subtracted, lownoise, through-focal, high-numerical-aperture DIC orpoI video images (the z-stack at rates of 30 optical sections per second, repeatable every few seconds(Inoue et al., 1991, Biol. Bull. 181: 336-337); B) remove haze from 16-bit gray scale images using filtered nearest-neighbor optical sections; C) reconstruct volumetric 3-D projections with gray values of intermediate planes interpolated as required; D) sharpen and adjust contrast of the projections; E) play back as stereo movies from computer memory or from a video-rate laser disk recorder. The high-rate image acquisition (A), stack processing(B-D), and 3-D and 4-D playback (E) are controlled by a personal computer equipped with newly developed programs. 3-D and 4-D examples displayed at the ASCB Poster Session will include: birefringent chromosomal spindle fibers in mitosis with enhanced visibility; dynamic positioning of mitotic spindle in cell division; through-focal, high-resolution 3-D images of developing sea urchin plutei. (Supported in parts by NIH grant R-37 GM31617 and NSF grant MCB890816 awarded to S.I.) in 1435 Theory, Application And Comparison Of Real-time, Fluorescence Polarization Image Microscopy Using Polarized And Natural-Light Excitation. ((Yuling Yan-Marriott & Gerard Marriott), Max Planck Inst. for Biochemistry, 82152, Martinsried, Germany) polarization techniques are widely used in the measurement of orientational distributions, rotational diffusion of molecules in solution or in interactions, and membrane fluidity in artificial molecular suspension, liposomes and cells.The microscope measurement enables an analogous study on microvolumes of cells, especially for samples of highlyordered orientations, such as fluorophores aligned along membrane phospholipids. These probes are easily imaged and the orientations canbe calculated from the polarization values on a pixel by pixel base. In this study, we present a derivation of fluorescence polarization using natural and polarized excitations in the microscope, followed by some biological applications. A multi-view fluorescence image microscope is employed for simultaneous capture of two polarized emission images is employed in the microscope measurements. In the microscope measurement, the numerical aperture effect on the limiting polarization value of samples is well known. However, so far, only the depolarization factor from emission cone effect has been theoretically established. Here, we consider the depolarization from both excitation and emission cone effects and as an example, a derivation for the limitng polarization value for samples with 2-D isotropical frozen dipoles is given. We also study the fluorescence polarization image microscopy with natural-light excitation to observe the orientation and dynamics of ordered molecules or artificial fluorescent lipids and fluorescent proteins encapsulated within liposomes, and phalloidin labeled actin molecules in Factin intecting with myosin. We show that with natural-light excitation, the polarization of randomly oriented dipoles is theoretically zero, which makes it convenient to distinguish ordered dipoles from randomly oriented ones. Fluorescence 4-Dimensional Digital Imaging and Display System for Dynamic Processes. ((C.F.Thomas, P.J.DeVries, V.E.Centonze, J.D.Hardin* and J.G.White)) Integrated Microscopy Resource and *Deept Zoology, Univ. of Wisconsin, Madison, WI 53706. We have developed a 4D digital imaging system to study intracellular and multicellular dynamics. Images are with a Sierra Scientific video camera mounted on a Nikon Diaphot equipped with high extinction DIC is used to preprocess the video signal optics. A DSP-2000 image before digitization by the Scion LG-3 framegrabber card. Images are stored to the hard drive. The operating software is based on digitally computer NIH-Image, a public domain Macintosh-based image processing package run on a Quadra 950. Using the extensive macro language, the frameMac-1000 stage drive were integrated into a grabber card and the mouse-driven 4D acquisition operating system. We have incorporated the following features for flexible data acquisition: user-definable region of interest, choice of acquisition modes (time-lapse of single focal plane or zsetup, and variable series), image annotation, graphic menu for delay between time points. The data are subsequently organized into one large Apple QuickTime movie where images from each focal plane are appended together and compressed using the QuickTime JPEG compressor. The dataset is then made into a navigable movie using Video Navigator software from Radiant Interactive. This utility creates links between the timeframes at different focal planes. The resulting dataset is viewed using our custom-designed 4D Navigator program. This program allows the user to move up and down in focus and forward and backward in time, go to specific frames, set bookmarks etc. It also displays the current frame coordinates. This 4D data display package can be applied to any optical confocal, and 2sectioningmicroscopy technique such as Photon excitation. (Supported by NIH Grant RR-00570) collected processor Ludl z-series Nomarski-DIC, 1436 THE AUTOMATED INTERACTIVE MICROSCOPE: MEASUREMENT AND OF CHEMICAL AND MOLECULAR DYNAMICS IN LIVING CELLS, F. Lanni, D.L. Farkas, S. D.W. Deerfield and ((A.H. Gough, L.D. Taylor)) Center. for Light Microscope Imaging and Biotechnology, Camegie D.L. Mellon University, Pittsburgh, PA 15213 MANIPULATION Harris, Fahiman, goal of this project is to develop an Automated Interactive Light Microscope (AIM) thatwill be capable of real-time acquisition, processing, analysis and display of N-dimensional data sets from living cells, tissues and organisms. An advanced will be possible when researchers can generation of biological measure and manipulate biological processes during the time of the events. information fromliving Todays workstations can obtain complex event such samples minutes to days after a linear experiment, when the as been completed. The development and use of the division, hasWorkstation of has demonstrated value the hamessing (MMLMW) Ught Microscope from different modes of the distinct chemical and structural informatlon into a machine-vision when semi-automated, system. light microscopy integrated Together with the growing list of specific reagents designed to measure and the allowed has manipulate cell chemical and molecular dynamics, the MMLMW of cell and developmental functions. However, there exploration of are fundamental limitations of the present generation workstations that must be the maximum information. The major limitations include: overcome in order to of focusing devices; (3) (1) performance of computers; (2) speed and and of speed flexibility wavelength modulation for multispectral imaging; (4) resolution and read out rate of solid-state cameras; (5) axial resolution of motion fluorescence microscopy; (6) intelligence and power of feature extraction, and (7) integration and speed of switching and event detection tracking between key modes of microscopy. Solutions to these limitations are leading to the design and implementation of AIM. Recent advances in these areas will be presented with examples of biological applications. This project is supported by NSF grants: BIR-8920118 & BIR-9217091 The experiments temporal-spatial biological Multimode cell available mechanisms gain precision algorithms 1437 1438 NEAR-FEELE SCANING OPTICAL MICROSCOPY AS A PROBE OF FLTORSCENTF-LABEIED M 0BRANES. ((JeeseongHwng*,Eic Bezig, Robert CQiirster# and Michael Edidin*)) *Dqprten of Biology, The Johns OBSERVATION OF FLAGELLA BY CONTACT MODE AFM AND TAPPING MODE AFM(Kyoko Yoshioka, Hideo Komizu and Hiroshi Miyamoto, National Inst. Biosci. & Human-Technol. Tsukuba Ibaraki 305 JAPAN) Isopkirs Univesity, Balimore, MD 21218 USA, #AT&T Bell Laboraiores, 07974 USA. Mkmay Hill, While convetional otical miacscopy has a diffraction limited resolution of at best (where A is the wavelength of the light source), the Ner-Field Scaing Optical Nfiscopy (NSOf etinely gaenrates imagesw ith resolution as good as SOnm InourNSOMa fluorescently-labeled sample is illsuinated by a taered fiber oic probe with an aperture diamter of 80nm and the probe is raster-scaned over the sample at a distance of lOnm from the srface. The 2-D imwity map of fleence light from those smal areas constructs an image 0.5 hly srlius of the probe apeture. We used api pobe the distribution of lipids and proteins in plasma nnbranes of fibrlasts. Tei distnbution of fluoresctt lipid analogs in model sy was also imvestigoted and consared with the cell dta. Cell ya whose resolution is e NSOM to hianm skin n mnmbranes we labeled with the flhorescerst lipid analog, BODIPY-PC well as with uI_ted anti-HLA IgG. Patchy distribution of the fluorescest lipid analogs idicative of lipid domains was sem in all cells. These patches w"e not spatially correlated with patches of labeled HLA molecules. In conhast, the distribution of fluorescence was uniform, not plasma as oatd into single phase, DPPC, model patched, when BCODIPY-PC was in nI n.ce, the patches of BODiPY-PC seen in cell nmemranes are , domains, in thse nmemranes. dmo likely to reflect BACTERLAL Bacterial flagellum is an assembled structure of its protein subunit, flagellin. By using contact mode AFM and tapping mode AFM, we observed flagella of E. coli, S. typhimurium and its mutant SJW46 whose flagellin has deletion at central portion in amino acid sequence. Each flagellum is mounted on the surface of glass substrate and observed under ambient atmosphere. Flagella of wild type E. coli and S. typhimurium showed normal helical wave length of 2.7 u m, however that of mutant strain showed little deformed wave form. Since flagella of mutant SJW46 showed quite nornal structure in water from dark field light microscope observation, deformation after drying process suggests that flagellum of mutant SJW46 is fragile compare to that of wild type. This is also ascertained from height measurement of each flagellum filament. According to height of each filament, these flagella are deformed during AFM observation more or less and order of deformation is *Author(s) and/or presenter(s) have noted that there is potential conflict of SJW46>SJWllO03(wild interest. type)>E.coli(wild type). 248a Imaging Technology (1439-1444). Monday 1440 REQUIREMENTS FOR AND LIMITATIONS OF CONFOCAL IMAGING WITH RED AND FAR-RED LIGHT. ((Christopher Cullander)) Departments of Pharmacy and Pharmaceutical Chemistry, School of Pharmacy, University of Califoria, San Francisco, 94143 1439 SEQUENTIAL FORCE IMAGING OF CELLS USING THE ATOMIC FORCE MICROSCOPE (AFM). (S. R Marchese-Ragona and M. Wortge) TopoMetrix, 5403. Betsy Ross Drive, Santa Clara, CA. 95054. (408)-982-9700. tight will penetrate further (and with less scattering) into a specimen excitationwavelengths will, and the emitted fluorescent photons will exit the are *f long-wavelength fluorophores specimen in a similar fashion. Consequently, within a used, ls possible to obtain high quality confocal images from deeper thick specimen. While resolution is somewhat decreased longer wavelengths, with biological material is much less autofluorescent when excited with red tight. The acquisition of confocal images with red and far-red light thus has both advantages and limitations, and an understanding of the latter is necessary both to acquire high quality images and to avoid the misinterpretation of experimental data. A dual set was designed for the Bio-Rad MRC-600 for imaging the fluorescent tiiter probes cyanine 3.18 (CY3) and cyanine 5.18 (CY5). Visualization of CY5 with this Red than shorter instrument measuring Mkroscope is an extremely senisiive force The Atomic Force whichis routinefy used to measure the force exerted on the AFM tip by the sample, with a routine force resolution inpiconwton the to nanorewton range. By plotting to tp, one can tth tip-sample separation(positive or negative) vs. force produce what is traditionally known as a force distance' carve. Analysis of the dis tance curve allows one to extract information on micro elastict and force as magnitude of any tip-sample binding that plasticity of cell surfaces as welltth al., may occur. Recently several groups (e.g. Lee e1 1994, langmuir, 10, 354-357) the force-distance curve to investigate the binding (unbinding) strength of have used utility of the force-distance curve the streptavidin-blotin interaction. Toincrease the for cell blological studies we have develoed a sottware-dnven data acquisition program that will perform a force-distancecurve for every pixel of a topographic image. For a 200 x 200pixel image, 40,000 force-distance curves are generated. In order to analyze this vast quantity of information in a convenient and meaningful image isviewed in sequence, and can beviewed as 'movie', a way, each force eachframe being animage taken at a particular force. Asthe force on the sample increases (thetip pressing onthe sample) hard incompressible material appears bright, while soft compressible matenal appears dark. We have used this technique to imagefibroblasts in PBS solution. By analyzing the force movie one can determine(1) the optimum force for image contrasting, and (2) differences in compressibility of the cell surfaces. This technique represents a new form of microscopy, where differencs in compressability are used as the contrast mechanism for the image. app6edtth CY5 fiter set is neariy ideal, since is excited almost stits absorption maximum by the 647 nm laser line, and the collection fiher can be centered on its emisslon In the 568 nm laser line happens to be at the emission spectrum. contrast, maximum of CY3, which thus limits the collectable CY3 fluorescence. The poor quantum efficiency of photomultiplier tubes in the red region mandates a high optical transfer efficiency for the system, and light throughput was maximized by the use of special high transmission optical fitters with sharp bandpass cutoffs. The transmittance of objective decreases gradually as wavelength increases, and the axial and lateral chromatic aberration of a given lens are also since far-red not focus in the same plane as lower important, photons may confocal lightpath mirror efficiency rapidly degrades above wavelengths. 700 nm, the loss of this portion of the CY5 emission spectrum was acceptable since (a) the fluorophore is very bright and (b) these very long wavelengths will probably introduce aberration. Imaging with long-wavelength fluorophores thus requires the use of high quality optical components, bright dyes, and attention to lenses While the selection of the objective lens. 1441 CONFOCALMICROSCOPY AND CALCIUM IMAGES IN HUMAN EPIDERMAL KERATINOCYTES EXPOSED TO SULFUR MUSTARD ((RJ. Werrlein, J. Madren-Wsalley, S.D. Kirby and M.AE. Mol)) Pharmacology Division, United States Army Medical ResearchInstitute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425. Human epidermal keratinocytes (HEK) were grown on glass coverslips in a mixture ofHam's F-12 and Dulbecco's MEM. From day 7, culture medium contained 0.15 mM calcium. Experiments were performed on days 10-14. Cultures, preloaded with Indo-lAM, were excited with a 355 nm laser-line and subjected to image analyses using a Meridian ACAS-570 confocal laser cytometer. From control populations, the emissions at 485nm (Ca2e-free Indo-1) and 405nm (Ca2"-bound Indo-1) produced evidence of selective cell loading. Epidermal cells at the periphery of seed colonies were relatively large, morphologically distinct and brightly fluorescent. Smaller cells at each colony core were not fluorescent. Five-minute exposures to 400 sulfur mustard in buffer containing 0.15 mM calcium did not disrupt calcium images. They did produce significant (p<.05) increases in ratios of Ca2e-bound Indo-l emissions. Populations acutely Indo-l, without increasing Ca2+-bound exposed to 400 (SMsulfur mustard and to 1.02 mM calcium showed an early post-exposure efflux of Indo-l, and a corresponding loss of calcium image/cell definition. Control populations acutely exposed to 1.02 mM calcium, without mustard, showed no efflux of Indo-l. Results indicate that acute, sinmltaneous exposure tosulfur mustard and to increased calcium concentrations may effect changes in the plasma membrane and selective permeability of basal-type HEK. were IuM :Ca2+-free Indo-l 1443 (Tllll), a T111. side a a a of Guinea Pig amnion and chorion. 1444 KINETICAL STUDY OF DNA SYNTHESIS AND MORPHOLOGICAL ESTABLISHMENT OF APPROACH IN LIVING CULTURE CELL ((L. Lehong)) China Academy of Traditional Chinese Medicine, Beijing, China CHANGES In this document, an unique approach has been developed by which time lapse in vivo observation of dynamic behavior of DNA synthesis were successively observed during 12 hours and the paralleled morphological changes in the same cell Living culture cells (N1H3T3) were efficiently obtained. were stained by Hoechst33342 at the possible limited concentration and DNA changes was measured by cytofluorometry. Phase-contrast photomicrographs were recorded in the same cells in which DNA was examined. Morphologic features (cell areas, nuclear areas and shape constant) and the paralleled DNA dynamic synthesis were processed through image analyzer controlled by computer. In order to evaluate the method, the DNA synthesis time obtained by this approach was compared to the one come from AH-thymidine ('H-tdR) autoradiography. In addition, by using the established method, some tumor cell lines such as A431 and HL-60 cell were also observed and the effect of some anti-tumor drugs derived from Traditional Chinese Medicinal herbs were examined. It 1442 CELL VIABILITY AND EPITHELIAL IMPERMIABILITY CAN BE TESTED SIMULTANEOUSLY WITH THE STYRYL DYE RH414 WHEN VIEWED WITH A CONFOCAL MICROSCOPE. ((DAVID CARTER)) Department of Zoology and Robarts Research Institute, University of Western Ontario, London, ON, Canada. N6A 5B7. The styryl dye RH414 (Molecular Probes Tllll) has significant advantages over Propidium Iodide and Fluorescein Diacetate for distinguishing living from dead cells. At the same time, it provides information about plasma membrane distribution and the permiability of epithelial layers, when viewed by confocal microscopy. This water-soluble dye becomes fluorescent when it interchalates into the exposed face of lipid bilayer, but is not otherwise fluorescent. It has excellent photostability, low toxicity, adn can be washed out of living cells, once their viability has been established. Although designed as an indicator of membrane potentials, signal differences are too low (<5%) to interfere with its use as a vital stain. Living cells are revealed as a bright outline when their If dye solution is presented to one side plasma membranes accumulate of an impermiable epithelium, only the exposed of each cell is labelled, the dye being blocked at the level of the tight junctions. In fully permiable cell layers, both sides of the cell are labelled. When a cell ruptures, the membranes of all its organelles are exposed, resulting in highly fluorescent cytoplasm surrounding characteristically unstained nucleus. T111 has successfully been used to demonstrate cell viability and plasma membrane distribution in range of insect tissues. It has been used to demonstrate the absence of intracellular movement of solutes across insect epidermis and to show the membrane integrity was concluded that the established approach may effectively provide reliable dynamic information for vital cell and can be applied to the research on the mechanisms of anti-tumor drugs. IMPROVED PHOTOELECTRON IMAGING OF HUMAN BREAST CARCINOMA CELLS USING MULTIPLE WAVELENGTH EXCITATION. ((D.L. Habliston, K.K. Hedberg, G.B. Birrell and O.H. Griffith)) Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403 Photoelectron microscopy (PEM) provides a sensitive low-damage method imaging cell surfaces. The specimen is exposed to UV light, and the photoejected electrons are accelerated and imaged. PEM serves as the electron optical analogue of fluorescence microscopy, and provides both a topographical map of unlabeled cell surfaces and the distribution of gold-labeled surface antigens. A UV light source of 254 nm is used to generate maximal photoemission (image brightness). However, the use of this excitation wavelength can lead to charging in thicker specimens, and therefore PEM of cultured cells has been generally used for only thin, well-spread cell types. We now find that combining the short wavelength UV excitation with a longer wavelength UV source (310-325 nm) greatly extends the range of cell thickness which can be imaged. While the shorter UV illumination is primarily responsible for the photoelectron emission from the biological surface, the longer wavelength of evidently serves to increase photoconductivity and reduce charging. The use of excitation sources has the additional advantage of allowing the immunogold label brightness to be varied relative to cell surface brightness, generating the unique capability of tunable label contrast. The effects of varying the excitation wavelength is illustrated here with PEM images of cultured MCF-7 human breast carcinoma cells, both alone and in co-culture with thinner, normal cell types. two Supported by PBS grant # CA 11695 from the National Cancer Institute. Monday. Imaging Technology (1445-1448) 1445 NOVEL APPLICATIONS OF TOTAL INTERNAL REFLECTION MICROFLUORIMETRY (fiRM) IN UVING CELLS; MEASUREMENT OF CELL VOLUME AND CHARACTERIZATION OF MEMBRANE-ADJACENT CYTOPLASM. ((S.E. Bicknese, J. Farinas, HIP. Kao and AS. Verkman)) CVRI, U.C.S.F. TIRM pemrmts the selective excitation of cell-entrapped fluorophores that are very (within -100-250 nrn) the plasma numbrane. The effective excitation 'depth" is determined by illumnination angle and refractive index. A TIRM microscope was constructed to image cells nounted in a perfusion chamber and illuminated by Ar near and/or He-Cd laser beams directed at specified incident angles. To permit rapid selection of incident beam angles, the microscope contained a plane mirror mounted on a software-controlled microstepper motor, an off-axis ellipsoidal mirror, and a hemicylindrical sapphire prism. Fluorescence was detected by a cooled CCD camera or photomultiplier. Several biological applications of TIR were developed. Cell volume. Cells grown on a coverglass were loaded with the fluid-phase fluorescent indicator calcein. The time course of relative cell volunre was measured directly from the TIR fluorescence signal. Applications to cell volume regulation and water permeability were developed. 2. Solute mobility in membrane-adjacent cytoplasm. Fluid-phase fluorescent indicators (BCECF and FITC-dextrans) near the plasma membrane were excited selectively to measure rotational diffusion by frequency-domain fluorimetry (Bicknese et al, Biophys. J. 65:1272-82,1993) and translational diffusion by microsecond photobleaching recovery. 3. Calcium signaling near the plasma membrane. Agonist-induced calcium activity in bulk and membrane-adjacent cytoplasm was measured continuously by alternate TIR and trans-illumination of 3T3 fibroblasts loaded with the indicator Calcium-green. 4. Submicroscopic distance determination. From tieoretical modeling, Laplace transform of multi-angle TIR images should provide information on spatial distribution of intracellular fluorophores. This approach is being applied to image fluorescently 249a 1446 QUANTITATIVE ASPECTS OF DIGITAL MICROSCOPY APPLIED TO CELLULAR LOCALIZATION OF HEPARIN IN SMOOTH MUSCLE CELLS. (( Johnston(l) D. Hanzel(l), B. Brandley(2) and J. Castellot(3))) 1: Molecular Dynamics, Sunnyvale, CA. 2: Glycomed, Alameda, CA. 3: Tufts University School of Medicine, Boston, MA. High Resolution digital acquisition allows a great deal of flexibility in the types of questions that can be directed to microscopic samples. To eliminate subjective bias and provide quantitative results we have approached microscopy with an automated digital format. This mode can return quantitative data at high resolution over large fields. The digital format makes accessible data including multispectral colocalization, seeding and connectivity, particle size and shape distribution and population analysis. We have begun a program to investigate this approach using the confocal microscope. Scanning larger fields-of-view at lower spatial resolutions (e.g., low magnification objective) defines large maps that allow alignment of high spatial resolution (defraction limited) sampling. The selection of the field-of-view with low spatial resolution reduces the subjective nature of the selection of a "typical staining pattern". High resolution digital scanning in three d! imensions contribute both to the objective" nature of the analysis and allow for quantitation of characteristics not historically available/accessible The complex carbohydrate heparin is implicated in tumor growth and wound healing by affecting angiogenesis, cell proliferation and motility. The internal localization of heparin within vascular cells appears to be a good predictor of the sensitivity of those cells to the action of heparin. Cells resistant to the antiproliferative action of heparin are able to sequester the heparin in large vacuoles whereas those cells sensitive to the carbohydrate do not exhibit these structures. We have applied our approach to QUANTITATIVE DIGITAL MICROSCOPY to the analysis of intracellular heparin distribution. labeled endosomes and skeletal components near the cell plasmn membrane. 1447 EVALUATION OF THE TOMCITY OF THE CANCER TREATMENT DRUG CISPLATIN AT SINGLE CELL RESOLUTION BY ION MICROSCOPY ((I. Gay, S. Chandra, and G.H. Morrison)) Department of Chemistry, Cornell University, Ithaca, NY 14853 (Spon. by R.Wayne.) 1448 CHARACTER RECOGNrON OF RETINAL OUTGROWTH BASED ON FAST FOURIER TRANSFORM (FF) OF DIITAL IMAGES. (N.G.Carri and E. GaNego Uuesam). IMBICE and ClOp, Box 403,1900 La Plata, Argentina. Cisplatin is used as a therapeutic drug for the treatment of various types of cancers. One of the main problems with its use, however, has been the evaluation of the drug toxicity. Ion microscopy provides a powerful technique for the evaluation of the health status of cells. It is an imaging image from a spatial context into an spatial frequency context. In the Fourier spectrum of the images the explanted retina will appear as low spatial frequencies while the fibres will be represented by high spatial frequencies. An easily recognizeble image can be obtained from the FFT spectrum. In others words what we have been working on is the study of FFT of retinal images. The images were the outgrowth of chick refina E6 technique based on secondary ion mass spectrometry. It is capable of localizing elements or molecules at a spatial resolution of approximately 0.5 im with a sensitivity reaching into the ppm range. It is possible to evaluate toxicity by imaging the cellular K, Na, and Ca composition with ion microscopy. LLC-PKi porcine kidney epithelial cells were treated with various doses of cisplatin and then cryogenically prepared prior to ion microscopic analysis. Injured cells characteristically showed high levels of sodium and calcium and low levels of potassium, dependent on the cisplatin dose given. Additionally, dead cells revealed higher levels of calcium in their nuclei. We have also been able to detect the toxicity response in correlation to various stages of the cell cycle by using bromodeoxyuridine (BrdU) as a cell cycle marker which can be imaged independently and in addition to K, Na, and Ca from the same cell. While other toxicity tests only discriminate live versus dead cells, ion microscopy provides a powerful approach for the evaluation of drug toxicity at the single cell level. Supported by the Department of Energy and the National Institutes of Health. The use of FFT in digi explanted in short-term images give assay. us the possibility to translate an Retina were then cultured four days on collagn with the addition of trophic factors (TF) in the media. Retina cultured under the effect of trophic factors gave dense outgrowth elongating from the explant to the substrata. Controls show a discrete outgrowth and easy to measure the length of fibers. However, it is impossible to evaluate the pattem of the fibre network. This FFT pattern gives as the possibility to evaluate the efficiency of TF. In this work we have used digital images (phase contrast micoscopy) to characterize the outgrowth in Fourier transformation. In the outgrowth network appears as high spatial frequencies located in the periphery of the spectrum while the explants are represented by of lower frequencies located centrally in the spectrum. Our job is, by far, more simple, quicker and more accurate than other techniques used for pattern recognition. The Optica Winner Spectra will complete a more subtantial evaluation of the efficiency of dosimetry on different trophics. N.G. CA: E-ll CwdiMMK-MAI d Fax: 4(21)253320 (IMBC) mi 1115 (COOp) CONICET, Grwoe S143141i411 P04D A(149-15 Pre-College Science Education (1449-1450) 1449 1450 Precollege Science Education (CAPSE), University of Southern Califoria, Los Angeles, CA 90033. (Spon. by H.C. Slavkin.) ESTABLISHING Foused, sategic, long-term commitment to profesional devlopment that integrates science content, pedagogy and intuctional matrials results in a coordinated, inquiry-basd schoolwide science intuctional program at twentyfour innerity elementary schools in Los Angeles. PRAXIS has worked with twenty-four school teams of ree teachers and their principal for three years to prepare the teams with sffiient scientific and leadership knowledge and skills to make significant gains in siec education by: 1) Articlating K-6 science education at eah school; 2) Providing on-site peer expert, resources and trining; 3) Etlishing school-based instuctonal materials systems; 4) Maximizing student exposure to hands-on sience; 5) Stimulating parent involvement; and 6) Becoming models for otl schools School tams are rained in life, phyical and earth sciences, and provded with a frmwwork for schoolwide change in summer institutes and follow-up sessions. They acquire in group dynamics, presentation statgies, leadership, lning styles and resource acquisition in winter institute Sumn Institutes are cted by 'Teaching Cadres' conssting of scientiss, secondary science teachers and elementary currilum specialists who are oriented to the Califnia Science Framework, St-adopted instrctiona mateials and e ional reform philosophy and techniques. School teams receive salary poit credit for attending insitutes, stipends for carrying out school-based change objectives, and are igible to apply for grants to enhace thi ools' science programs with a skills community matching inservicing and in school l resouc matels a dedicated to teacher tin. planning, Beging its third and final year, schools in the PRAXIS powct compledng the three projet objectives of deveoping and implting a curriculum Content Matrix, organizing an ins tional mateials man mt center and establishing an After-School Stuet Science Club. Major funding for PRAXS is derived from the National Science Foumdation, with added support from copate and family foundations. are AN NRY SCIENCE AND MATH EDUCATION PARTNER/MENTOR. PROGRA USING STUDENT AND FACULTY VOLUNTERS. ((R. L. DeHaan and F. H. Dabney)) Department of Anatomy and Cell Biology, Emory University, Atlanta, GA 30322. The Emory Elementary Science Education Partnership (ESEP) Program provides elementary teachers with Emory faculty mentors and student partners to help with hands-on teaching of science. In the spring of 1994, we asked the principals and teachers of four neighboring schools how the Emory science community could be of help to them. Teachers asked for contact with a working scientist and weekly in-classroom help with active learning exercises from an undergraduate science student. In response to questionnaires that we sent to the Emory community and the schools, 47 faculty members and 97 students volunteered to partner with 40 teachers. To help define the goals and strategies of the program, we established a steering committee that includes scientists, educators, teachers, and school administrators, and a 14member student council. We have instituted a one semester course that begins with three 3-hour training sessions to familiarize partners and mentors with guided-discovery teaching strategies and activities, and to sensitize them to diverse learning modes. During the remainder of the semester students devote 6 hours per week to in-school classroom teamteaching activities with their assigned teacher. Scientist mentoring partners share information, donate spare equipment, offer lectures and laboratory tours. Student journals, statements, and pre-post testing are used for assessment. 250a Pre-College Science Education (1451-1454). Monday 1451 1452 PRE-COLLEGE SaENCE MENTOR PROGRAM - AN EVOLVING SaENCE EDUCATION PARTNERSHIP. ((S.H. Mayrand)) Science Educadon Program, Worcester Foundation for ExperImental Biology, Shrewsbury, MA 01545. PRE-COLLEGE SCIENCE EDUCATION: RESEARCH AS AN EDUCATIONAL TOOL ((M.C. Fields, 21st Century Biology Class)) The Sidwell Friends School, 3825 Wisconsin Ave., NW, Washington D.C. 20016 Five years ago, In cooperadon with the iocal high school science teachers and administrators, the Worcester Foundadon launched the Science Mentor Program, which gives students the opportunity to meet two evenings a month with sciendsts who engage them In hands-on projects In an Informial setting. These encounters with scientists, and with the idnd of thIn1dng that fuels the research process, helps students to appreciate the Importance of standard laboratory bask sciendfic principles In a way that textboox exercises and lectures do not. Science Mentors, recruited from the Worcester Foundation, local blotechnology companies, insttutions of higher education and govenment agencies, bring sinple experiments that allow students to acdvely explore topics ranging from human genedcs to the environment. Through these acMtides students begin to see the relevance of science to their daily lives and, for some, to consider sclendfkc careers. Additionally, Informal mentor/student Interactions often spawn In-depth discussions focusing on Issues InvoMngsclence, ethics and soclety. The program has enjoyed much success as evidenced by the duplication of our model by other Massachusetts school systems, favorable attention by the local media, and growing Institudonal and corporate support , 1453 DEMONSTRATION OF BACIERIAL CELL TRANSFORMATION BY ELEC[ROPORATION FOR THE HIGH SCHOOL SCIENCE CURRICULUM ((R. A. Acey)) Dept. of Chenistry and Biochemistry, Cal. State Univ., Long Beach, CA 90840, ((M. Ostresh)) BTX/Genetronics, Inc., San Diego, CA 92121 and ((T.F. Unger)) BioLogics, Inc., Irvine, CA 92718 The last sevemal years has seen a dramatic evolution in recombinant DNA technology and its impact on society. A major goal of educators has been to implement this technology int the high school and cofl_ee un aduate science curiculunt We here on a safe and elibe nl taches that the tan of proceduem bacterial cell pnnciples on and n to in provides students with hands on experience using ele the tasfioratio Students are supplied with a kit containing known amounts of s, and pe rd agar plates for pre-aliquoted cells andplasmid, cell of handlng. The HBlOl strain with ol by electr ation using a IhnPnor Plus (BXGneonics). Int n of plasmid DNA imparts antibiotic restance to the transfomed cells. Aiquot of untsunsrmed cells are plaed on agar with without anpcillin to show that fte oranisms are viable, yet sensitive to the antibiotic. Plabng of the ee pwe cells on agar cotiIng antibiotic reslts in cell growth, donsaing the sucessl 'p-e of the plasmid by the bacteria. Detrmn the number of drug resistant bace colonies allows students to calulte ta ii (transfiornants4pg DNA). Moreover, the peCnage of oells tmafored is eaily calcuatd by compaison with an equal number of cells not having undee procedure can be coupleted in three consctie on l ab periods without the need of ease or I sohistidi equipment. (Sponsored by R. D. Fields) Motivating teen-agers while exposing them to the concepts and process of scientific research is a challenging task. Our current system of lecture, discussion and reading tends to present the teacher as the expert and often fails to develop the creativity of students. The stanrd laboratory investigations designed to fit into a ninety minute period while reiterating facts do not encourage innovative thinking. Seniors at The Sidwell Friends School have participated in an experimental course that discards the usual method of teaching and replaces it with a research approach. Students undertake a three-month laboratory investigation of neuron specific enolase (NSE) in mouse neurons. NSE is localized by immunocytochemistry, the protein is identified by Western blot, and RNA is extracted and reverse transcribed into cDNA for PCR amplification. This exposes students to modern research techniques while promoting critical thinking and problem solving that is student centered and teacher facilitated. As a result, students gain an appreciation of scientific research while they master key concepts from the core of cell biology, including immunology, neuroscience, cytology, transcription, translation and DNA replication. Students are no longer the passive recipients of knowledge, but acquire it actively and through collaborative effort to complete the research and produce a scientific poster. This poster presents the work completed by students this past fall. 1454 "MEDICINES: THE INSIDE STORY" EDUCATION PROGRAM. AN INTERDISCIPLINARY RESOURCE FOR HIGH SCHOOL SCIENCE TEACHERS. (( T.M. Horn, J. Bennett, J. Berryman, L.A. Pierce, and C. Raphael.)) Life Science and Biotechnology Laboratory, Thomas Jefferson High School for Science and Technology, Alexandria, VA 22312. (Spon. by R.A. Bloodgood.) People all over the world use medicines to help restore maintain health. Cellular molecules are the ultimate targets of medicines. Thus, the concepts and methods of inquiry of cell biology are invaluable to understanding the actions of medications. This resource will enable high school science teachers of biology, chemistry and health professions, and their students, to follow in the footsteps of the people who, through hard work, persistence, luck, ingenuity, and prepared minds, discovered modern medicines. The resource will contain labs, lessons, interviews, images and cultural and historical documents. Teachers will be able to access a variety of ways to model techniques and investioative approaches used by scientists. Information available will include origin, identification, structure, and modes of action and side effects of medicines familiar to high school students. These classroom materials are being developed for use as a CD-ROM. The education program is one facet of Medicines: The Inside Story, a traveling museum exhibition, symposium, and planetarium show sponsored by Glaxo Inc. or Symposium VI: Molecular Machinery of the Secretory Pathway (1455-1456). Tuesday 1455 MOLECULAR MECHANISMS OF INTRACELLULAR PROTEIN TRANSPORT. ((Jaaes E. Rothaan)) Cellular Biocheaistry and Biophysics Program orial SloanKettering Cancer Center, 1275 York Avenue, New York, New York. Reconstitution of intracellular protein transport in cell-free systems has led to insights into the mechanisms responsible for vectorial transport by vesicles. Transport between Golgi cisterna is iediated by COP-coated vesicles whose principal subunits are complex of Coatomers and a small GTPbinding protein, ADP-Ribosylation Factor (ARF). Coatomer is composed of seven distinct subunits. The concomitant budding of the transport vesicle occurs In a series of steps. GTP-dependent binding of ARF to the membrane in a nucleotide exchange reaction is followed by binding of coatomer and assembly into buds. Fatty-acyl-CoA is required for the buds to pinch off. Coated vesicles then uncoat when the ARF-bound CTP is hydrolyzed. Uncoating exposes a vesicle-born targetting molecule (termed v-SNARE) that docks the vesicle as it pairs with its cognate t-SNARE at the target membrane. The general fusion machinery, involving NSF and SNAP proteins, then assembles on the SNARE complex and fusion is initiated upon ATP hydrolysis by NSF. This mechanism is of great generality, as yeast temperature-sensitive mutants defective In transport and fusion encode homologues of costomer and fusion components discovered in animal cells, and similar components operate in neurons at synapses. carrier 1456 COMMUNICATION BETWEEN THE ER AND THE CELL NUCLEUS: MONITORING PROTEIN FOLDING. ((M.-J. Gethingl.2. K. Moni2.4, W. Ma2, and J.F. Sambrookl.3)) lDepartment of Biochemistry, 2Howard Hughes Medical Institute, and 3McDermott Center, UT Southwestern Medical Center, Dallas, TX 75235, and 4HSP Research Institute, Kyoto 600, Japan. a BiP, the endoplasmic reticulum (ER)-located member of the hsp7O family of molecular chaperones, is required both for translocation of nascent proteins across the ER membrane and for their subsequent folding and assembly in the ER lumen. Transcription of the BiP gene is increased many fold when mutant or unfolded proteins accumulate within the ER. This unfolded protein response appears to be initiated by the decrease in the concentration of free BiP that occurs when complexes are formed between BiP and unfolded proteins. In the yeast Saccharomyces cerevisiae, BiP is encoded by the essential nuclear K4R2 gene. Thus a novel intracellular sensing system must exist that monitors events in the lumen of the ER and transduces signals across the ER membrane and to the nucleus. To identify proteins involved in this signaling pathway, we devised a genetic screen to isolate yeast mutants that are unable to activate the BiP gene when unfolded proteins are present in the ER. Several of these mutants lie in a gene (ERNIV) encoding a 11 15 amino acid polypeptide with an N-terminal signal sequence and a single-transmembrane spanning domain approximately halfway along its length. Ernlp is a type I integral membrane protein with its glycosylated N-terminal domain in the lumen of the ER and its C-terminal domain in the cytosol. Part of the cytosolic domain is homologous to the catalytic domains of serine/threonine-specific protein kinases. Phosphorylation by the Ernlp kinase could therefore be the first step in transcriptional activation of KAR2 and other genes which contain UPR elements and encode proteins resident in the ER that are required for protein folding.