A Three-Part Laboratory Exercise Using Flightless Fruit Flies

Transcription

This article reprinted from:
Chinnici, J. P. and R. Ketcham. 2008. A three-part laboratory exercise using flightless fruit
flies (Drosophila melanogaster) to study modes of inheritance. Pages 127-136, in
Tested Studies for Laboratory Teaching, Volume 29 (K.L. Clase, Editor). Proceedings
of the 29th Workshop/Conference of the Association for Biology Laboratory
Education (ABLE), 433 pages.
Compilation copyright © 2008 by the Association for Biology Laboratory Education (ABLE)
ISBN 1-890444-11-1
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or
transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise,
without the prior written permission of the copyright owner. Use solely at one’s own institution with no
intent for profit is excluded from the preceding copyright restriction, unless otherwise noted on the
copyright notice of the individual chapter in this volume. Proper credit to this publication must be
included in your laboratory outline for each use; a sample citation is given above. Upon obtaining
permission or with the “sole use at one’s own institution” exclusion, ABLE strongly encourages
individuals to use the exercises in this proceedings volume in their teaching program.
Although the laboratory exercises in this proceedings volume have been tested and due consideration has
been given to safety, individuals performing these exercises must assume all responsibilities for risk. The
Association for Biology Laboratory Education (ABLE) disclaims any liability with regards to safety in
connection with the use of the exercises in this volume.
The focus of ABLE is to improve the undergraduate
biology laboratory experience by promoting the
development and dissemination of interesting,
innovative, and reliable laboratory exercises.
Visit ABLE on the Web at:
http://www.ableweb.org
A Three-Part Laboratory Exercise Using Flightless
Fruit Flies (Drosophila melanogaster) to Study
Modes of Inheritance
Joseph P. Chinnici1 and Robert B. Ketcham2
1
Department of Biology
Box 842012
Virginia Commonwealth University
Richmond, VA 23284-2012
joechin@vcu.edu
2
Department of Biology
University of Delaware
Newark, DE 19716-2590
rketcham@UDel.Edu
Abstract: This is an inquiry-based investigation of genetic modes of inheritance using flightless
Drosophila as the experimental organism. We present the three-part laboratory writeup, suitable for
use as a student handout. We describe the flightless mutant strains and where they may be obtained,
how to use carbon dioxide as fly anesthesia, various molecular websites for students to research the
mutant genes, and helpful hints for setting up the laboratory. Practical benefits of using flightless
flies include convenience in handling the organisms by inexperienced students, and reduced
likelihood of flies escaping to invade other areas of the school building.
Association for Biology Laboratory Education (ABLE) 2007 Proceedings, Vol. 29:127-136
Modes of Inheritance Using Drosophila
127
Introduction
This objective of this exercise is for students to perform an inquiry-based investigation of
genetic modes of inheritance using flightless fruit flies as the experimental organism (see Chinnici et
al., 2005). We have used this exercise in introductory general education science college courses for
non-science majors, and many high school advanced biology courses also use this exercise. Students
learn how to anesthetize the flies, distinguish male and female fruit flies, identify unknown (to them)
mutant traits by comparing mutant and wild-type flies, and determine genetic modes of inheritance
for their mutant type by setting up parental, F1, and F2 generation crosses and observing the
offspring of these crosses. They analyze their F2 generation data using chi-square analyses. The
exercise consists of three 90-120 minute sessions each separated by two weeks (performed on days 1
[week 1], 15 [week 3], and 29 [week 5]) to allow the offspring of each generation to develop into
adults. Other lab exercises may be performed on days 8 [week 2] and 22 [week 4].
Materials
Here, we describe the flightless flies and where they may be purchased, how to use carbon
dioxide as an alternative to “Fly Nap” for fly anesthesia, various molecular websites for students to
research the different mutants, and helpful hints for setting up the laboratory.
Flightless Fruit Flies
Ten years ago, one of the authors (JPC) constructed the various mutant strains of flightless
flies used in this exercise. The available flightless strains of Drosophila are white eyes, whiteapricot eyes, yellow body, singed wings, and cut wings (all recessive X-linked traits); Bar eyes (a
dominant X-linked trait); dumpy wings, vestigial wings, scarlet eyes, sepia eyes, ebony body,
apterous wings, and eyeless eyes (all autosomal recessive traits). Each of these mutant strains has
the X-linked recessive trait miniature wings fixed in its genetic background. The “wildtype” or
normal strain used in exercises with these mutants is the miniature wing strain. Thus, miniature
wing is the genetic “standard” for all these strains. The practical benefit of using flightless flies is
convenience in handling flies by typically inexperienced students, and no likelihood of flies escaping
the laboratory to invade other areas of the school building.
Carolina Biological Supply Company carries the flightless fruit fly kits and individual
strains. Go to <carolina.com>, type in “flightless fruit flies” and select “teacher resources” for more
information about use of the flies. At the end of the article Using Flightless Fruit Flies in the
Genetics Teaching Lab, click on “Flightless Fruit Fly Kits” and, then, “Flightless Fruit Fly Mutants”
for more information.
Using CO2 As An Anesthetic
As an alternative to using “Fly-Nap” for fly anesthesia, one of us (RK) uses carbon dioxide.
Once a CO2 delivery system is constructed in a lab room, it provides a convenient, odor-free means
for students to anesthetize flies. Flies stay "out" as long as CO2 is supplied, but they recover within
minutes when removed from the CO2. Different CO2 delivery systems can vary a lot in details of
128 ABLE 2007 Proceedings Vol. 29
Chinnici and Ketcham
construction. Here, we describe the system we built into RK’s teaching labs at the University of
Delaware and add some comments on where the system at the University of Kentucky differs from
ours.
Tanks, regulators, control valves. We installed two CO2 tanks in each of our lab rooms. Each tank
has its own two-stage regulator (Fisher 10-572E) and the piping from the two tanks come together in
a T, where a 3-way selector valve (McMaster Carr 4373K51) allows the operator to choose which
tank is in use. This makes it possible for a lab instructor to restore the CO2 supply simply by
throwing the selector valve to the second tank if one tank is emptied during a class activity. Each
tank has a lever-style shut off valve (McMaster Carr 4726K72) installed between its regulator and
the selector valve.
Piping from tank to work stations. The University of Kentucky has an ideal set up for distributing
CO2 around the room. Their labs were built with gas cocks at each student seat and they converted
that system to CO2. At Delaware, we had to install our piping from scratch. An early version
consisting of 1/2" Tygon tubing running down each bench was workable but cumbersome. We
upgraded by installing 3/4" c-PVC tubing underneath each bench, with a branch point at each
student station. Each branch consists of a length of amber gas-line tubing (Fisher 14-178 2B), which
steps down to aquarium airline tubing.
Student stations. Our student stations use flexible silicone aquarium airline tubing (Penn Plax
STD25) and a two-valve aquarium airline gang valve (Penn Plax VN2). From the gang valve, one
line is attached to a 16G 3" hypodermic needle (Fischer 14-817-103) with the tip cut off; students
use this by inserting the needle into a vial or bottle to anesthetize the flies before dumping them onto
a working platform. The second line from the gang valve goes to the working platform, which
students use to inspect and sort flies under the dissecting microscope. Connections in the airline
tubing are made using Luer fittings (Value Plastics FTLL230-1 and MTLL230-1).
My choice of material for building student work platforms is floral foam. It has high
resistance to gas flow but disperses the gas very uniformly. I buy bricks (9" x 3" x 41/4") at a local
florist, and cut them to 1" x 3" x 23/4" blocks on a band saw (wearing a respirator is important - the
dust is irritating). A channel for the gas to enter the block is created by pushing the handle of a flysorting paint brush (Carolina 17-3094) down the center of the block, starting in the center of the
smallest face and extending about 7/8 of the length of the block. Each block is covered on the
bottom and four sides with card stock, as a gas barrier. A hole punched into the card stock at one
end accommodates a Luer fitting (FTLL230-1) that fits into the opening of the gas channel down the
center of the block. The top and four sides of the block are covered with Whatman #1 filter paper,
cut, folded, and taped to the bottom of the block to form a flat, smooth, gas-permeable working
surface for sorting flies.
Several other materials may make suitable substitutes for floral foam. I have used cellulose
sponges, Styrofoam (though most Styrofoam packaging is impervious to gas), and upholsterer's
foam. The University of Kentucky built working platforms using the bottom half of pipette-tip
boxes covered with Mylar fabric. The CO2 in their system is dispersed by an aquarium air stone in
the hollow base of the pipette-tip box.
129
Student Outline
We present the three-part laboratory exercise write-up, suitable for use as a student handout,
in APPENDIX A. MS-Word formatted files of these three exercises are available by emailing JPC
(joechin@vcu.edu).
Materials and Equipment
Initially, (week 1) students work in groups of two or three. Each group receives three
miniature wing cultures; a “wild-type” culture containing male and female flightless flies with the
miniature wing trait only; an “unknown” mutant culture with males and females possessing one
mutant trait; and, a culture containing only virgin wild-type females. Each group also receives an
empty culture vial (in which to place sleeping flies) and an anesthetizing chamber (an empty vial
with a foam stopped through which a wand from the “Fly-Nap” kit is placed) if “Fly-Nap” is used.
Each group also uses a dissecting microscope, index cards (on which sleeping flies are placed for
viewing and sorting), and either toothpicks or a small artists watercolor brush (for pushing the flies
around on the index cards). For the second part of the exercise (week 3), each student in the groups
receives a food vial without flies, in which to place some F1 flies to generate the F2 generation. For
the third part of the exercise (week 5), each student in the class will need a dissecting microscope in
order to collect data from his/her vial of flies set up in week 3, as well as an individual anesthetizing
chamber.
Notes for the Instructor
Culture Medium
We use Instant Drosophila food as the culture medium for maintaining our stocks and for the
experimental procedures: go to <www.carolina.com > and type in “17 3200” (for white medium) or
“17 3210” (for blue medium). To prepare student fly cultures, first, we add the dry Instant
Drosophila medium to a vial. Then, we sprinkle in some dry yeast (obtained from the supermarket).
When adding water, we use distilled water if available, or jugs of drinking water from the
supermarket. We avoid using tap water due to the risk of introducing mold into the cultures.
In addition, we add a small rectangle of white paper toweling impregnated with “Tegosept”
mold inhibitor to each vial. Tegosept may be ordered fro Carolina Biological Supply Co.:
<https://www2.carolina.com/webapp/wcs/stores/servlet/ProductDisplay?jdeAddressId=&catalogId=
10101&storeId=10151&productId=23738&langId=-1&parent_category_rn=&crumbs=n>. We mix
10 grams of Tegosept powder into 100 ml of 100% ethyl alcohol and soak full sheets of white paper
toweling, squeezing the excess fluid out, and then hanging the wet towels up on a clothesline until
the alcohol evaporates. Then, we cut the paper towels into small rectangles (1 x 4 inches = 2.5 x 10
cm). We then push the “tego-strip” into the surface of the fly-food in the vial with the handle of a
small artist’s watercolor brush. These “tego-strips” accomplish two purposes: protect against mold
infestation, and give the larvae more surface area for pupation.
Collecting “Virgin” Female Drosophila
In the parental generation, “virgin” (previously unmated) wild-type females are crossed with
mutant males. We collect the virgin females for the students to use, since the entire experiment will
be ruined if the parental generation females are not virgins, a trivial reason for students to have their
experiments fail. Virgin females are easy to collect, since female fruit flies cannot accept sperm
from males until they are at least four hours post-emergence from their pupal cases (it takes that time
for them to expel larval wastes from their seminal receptacles). So, a few days before the beginning
of the exercise, we clear all the wild-type culture vials of adults at 10:00AM, then return three hours
later (1:00PM) and collect any “new” females which have emerged, knowing that they are virgins.
We repeat this again three hours later (4:00PM) and collect more virgin females. If needed, we do
this again the following day. Then, the day before the exercise begins, we place 6-8 virgin females
each in fresh food vials and have students add mutant males to these vials to begin the parental
generation crosses.
Alternatively, one may ask the students to collect their own virgin females, but this would be
burdensome to the instructor who would have to clear the wild-type vials for each group of students
three hours before they collect the virgins. In addition, in very young flies, it is more likely for
inexperienced students to mis-sex the flies since the pigmentation is quite pale in newly emerged
flies, increasing the chance for error. Adding virgin males to the parental cross would ruin the
experiment.
On days 8 (week 2) and 22 (week 4), either the instructor or the students must remove the
adult flies from their vials, so that when the next generation of adults emerges several days later,
they will not intermingle and mate with their parents (thus ruining the experiment).
Molecular Websites for Researching the Mutant Genes
Since we live in a molecular age, students should become exposed to some of the molecular
aspects of fruit fly genetics. One way to incorporate some molecular biology into this exercise is to
have students submit a report on some molecular aspects of the particular mutant gene they are
following in their crosses. A good place for them to begin is at the “WWW Virtual Library:
Drosophila” website: <http://www.ceolas.org/fly/>. Then, the student might go to the “National
Center
for
Biotechnology
Information”
website
for
Drosophila
melanogaster
<http://www.ncbi.nlm.nih.gov/genome/guide/fly/>
and
finally
to
<http://www.ncbi.nlm.nih.gov/sites/entrez> where one selects “Gene” for Search and then types in
the mutant name (for instance, “white” ). APPENDIX B lists some results for various mutant genes.
An alternative molecular approach is to go to the FlyBase website, “A Database of Drosophila
Genes & Genomes” <http://www.flybase.org/>. Here, one can simply select “genes” for Data Class
(or “alleles” for “cut-6”) and then type in the mutant name (for instance, “miniature”). APPENDIX
C lists some results for various mutant genes or alleles.
Literature Cited
Chinnici, J. P., A. M. Farland, and J. W. Kent. 2005. An Inquiry-Based Investigation of Modes of
Inheritance Using “Flightless” Fruit Flies. The American Biology Teacher 67:38-44.
131
About the Authors
Dr. Joseph P. Chinnici received his A.B. in Biology from LaSalle University in 1965, and his Ph.D.
in Biology from the University of Virginia in 1970. He has been a faculty member in the Biology
Department at Virginia Commonwealth University in Richmond, VA since 1970. Currently, he is an
Emeritus Associate Professor of Biology and Life Sciences at VCU. In 2001, he was awarded the
Distinguished Teaching Award from the College of Humanities and Sciences at VCU. Dr. Chinnici
has published over 35 papers in a variety of research and teaching journals and has been a PI or coPI on several federal and state research grants totaling over five million dollars.
Dr. Robert B. Ketcham earned his undergraduate degree at Wesleyan University and his Ph.D.
degree at the University of Delaware. He currently works as Laboratory Coordinator in the
Department of Biology at the University of Delaware, managing the laboratory component of the
non-science majors' biology class.
©2007 Joseph P. Chinnici and Robert B. Ketcham
APPENDIX B. Molecular Websites of Interest for Drosophila Mutants
http://www.ceolas.org/fly/ [The WWW Virtual Library: Drosophila. This directory points to
internet resources for research on the fruit fly Drosophila melanogaster]
http://www.ncbi.nlm.nih.gov/ [National Center for Biotechnology Information]
http://www.ncbi.nlm.nih.gov/sites/entrez [type in Drosophila and mutant name (e.g., Drosophila
white)]. Some results are listed below. If one clicks on the gene symbol, a complete description
appears (the longer website at the end of each summary).
white [Drosophila melanogaster]
Other Aliases: Dmel_CG2759, BACN33B1.1, CG2759, DMWHITE, EG:BACN33B1.1, unnamed, w(AT)[[13]]
Other Designations: white CG2759-PA
Chromosome: X; Location: 3B6-3B6
Annotation: Chromosome X, NC_004354.3 (2684632..2690499, complement)
GeneID: 31271
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=31271&ordinalpos=9&ito
ol=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum
ebony [Drosophila melanogaster]
Other Aliases: Dmel_CG3331, CG3331
Other Designations: ebony CG3331-PA
Chromosome: 3R; Location: 93C7-93D1
GeneID: 42521
yellow [Drosophila melanogaster]
Other Aliases: Dmel_CG3757, CG3757, EG:125H10.2, T6
Other Designations: yellow CG3757-PA
Chromosome: X; Location: 1A5-1A5
Annotation: Chromosome X, NC_004354.3 (250542..255278)
GeneID: 30980
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=30980&ordinalpos=17&it
ool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum
vestigial [Drosophila melanogaster]
Other Aliases: Dmel_CG3830, CG3830, VG, vg21
Other Designations: vestigial CG3830-PA
Chromosome: 2R; Location: 49E1-49E1
GeneID: 36421
cut [Drosophila melanogaster]
Other Aliases: Dmel_CG11387, CG11387, Ct, Cut, kf
Other Designations: cut CG11387-PA, isoform A; cut CG11387-PB, isoform B
Chromosome: X; Location: 7B4-7B6
GeneID: 44540
133
apterous [Drosophila melanogaster]
Other Aliases: Dmel_CG8376, CG8376, LIM, S-2a, Xa, blt
Other Designations: apterous CG8376-PA, isoform A; apterous CG8376-PB, isoform B
Chromosome: 2R; Location: 41F8-41F8
GeneID: 35509
Bar [Drosophila melanogaster]
Other Aliases: FBgn0000154, BB, Bar eye, BarH1, InfraBar, Ultrabar, bar
Chromosome: 1; Location: 1-57.0
GeneID: 44798
This record was discontinued.
BarH1 [Drosophila melanogaster]
Other Aliases: Dmel_CG5529, BH1, Bar, Bar H1, Bar-H1, BarHI, CG5529, barH1
Other Designations: BarH1 CG5529-PA
GeneID: 32724
BarH2 [Drosophila melanogaster]
Other Aliases: Dmel_CG5488, B, BH2, Bar, Bar-H2, CG5488
Other Designations: BarH2 CG5488-PA
GeneID: 32723
dumpy [Drosophila melanogaster]
Other Aliases: Dmel_CG33196, CG15637, CG33196, CT35799, DP, SP460
Other Designations: dumpy CG33196-PB
Chromosome: 2L; Location: 24F4-25A1
GeneID: 318824
singed [Drosophila melanogaster]
Other Aliases: Dmel_CG32858, CG1536, CG32858, Sn, fs(1)K418, fs(1)M45
Other Designations: singed CG32858-PA, isoform A; singed CG32858-PB, isoform B; singed CG32858-PC, isoform C
Chromosome: X; Location: 7D1-7D2
GeneID: 31717
scarlet [Drosophila melanogaster]
Other Aliases: Dmel_CG4314, CG4314
Other Designations: scarlet CG4314-PA
Chromosome: 3L; Location: 73A3-73A3
GeneID: 39836
[sepia] CG6781 [Drosophila melanogaster]
Other Aliases: Dmel_CG6781
Other Designations: CG6781-PA
Chromosome: 3L; Location: 66D5-66D5
GeneID: 38973
brown [Drosophila melanogaster]
Other Aliases: Dmel_CG17632, CG17632, Pm, Su(w[coJ]), unnamed
Other Designations: brown CG17632-PA
Chromosome: 2R; Location: 59E2-59E3
GeneID: 37724
eyeless [Drosophila melanogaster]
Other Aliases: Dmel_CG1464, CG1464, DPax-6, EYEL, Ey, Ey/Pax6, Pax-6, Pax6, eye, l(4)33
Other Designations: eyeless CG1464-PA, isoform A; eyeless CG1464-PB, isoform B; eyeless CG1464-PC, isoform C;
eyeless CG1464-PD, isoform D
Chromosome: 4; Location: 102C2-102C2
Annotation: Chromosome 4, NC_004353.3 (718315..741787)
GeneID: 43812
135
APPENDIX C. More Molecular Websites of Interest for Drosophila Mutants
http://www.flybase.org/ [ FlyBase: A Database of Drosophila Genes & Genomes]
choose “genes”, type in “miniature”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=miniature&authors=&year=&alltext=&caller=quicksearch
select “m”:
http://www.flybase.org/reports/FBgn0002577.html
choose “genes”, type in “Bar”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=Bar&authors=&year=&alltext=&caller=quicksearch
select “B-H1”:
select “B-H2”:
choose “genes”, type in “white”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=white&authors=&year=&alltext=&caller=quicksearch
select “w”:
choose “genes”, type in “white apricot”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbal&context=white&authors=&year=&alltext=&caller=quicksearch
select “w^a”:
http://www.flybase.org/reports/FBal0018195.html
choose “genes”, type in “yellow”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=yellow&authors=&year=&alltext=&caller=quicksearch
select “y”:
choose “genes”, type in “cut”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=cut&authors=&year=&alltext=&caller=quicksearch
select “ct”:
choose “alleles”, type in “cut”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbal&context=cut&authors=&year=&alltext=&caller=quicksearch
select “ct^6”:
http://www.flybase.org/reports/FBal0001934.html
choose “genes”, type in “dumpy”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=dumpy&authors=&year=&alltext=&caller=quicksearch
select “dp”:
choose “genes”, type in “vestigial”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=vestigial&authors=&year=&alltext=&caller=quicksearch
select “vg”:
choose “genes”, type in “scarlet”:
choose “genes”, type in “sepia”:
choose “genes”, type in “brown”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=brown&authors=&year=&alltext=&caller=quicksearch
select “bw”:
choose “genes”, type in “ebony”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=ebony&authors=&year=&alltext=&caller=quicksearch
select “e”:
choose “genes”, type in “eyeless”:
http://www.flybase.org/cgibin/uniq.html?species=Dmel&field=SYN&db=fbgn&context=eyeless&authors=&year=&alltext=&caller=quicksearch
select :ey”:
Exercise 1.
1-1
© Joseph P. Chinnici
Exercise 1
Using the Scientific Method:
Fruit Fly Studies
A.
B.
C.
Introduction; Fruit Fly Life Cycle
Setting up a Fruit Fly Study
Determining Inheritance Patterns
A.
Introduction.
The Scientific Method consists of observation, hypothesis, experimentation, and
drawing conclusions based on the results of the experiments performed. In this
exercise, we will use elements of the Scientific Method to gather information about
certain variable physical traits in fruit flies. From the observations, you will be asked to
form hypotheses about the inheritance of these traits and devise experiments that will
test the validity of your hypotheses.
We will use fruit flies in our studies because these organisms are readily
available. Fruit flies have been important insect organisms for genetic research since
the early 1900s; today, more is known about the genetic control of fruit fly development,
behavior, and evolution than is known about humans.
In order for you to better understand the experiments you will perform, please
read the following sections to gain some basic knowledge of the life cycles of fruit flies.
Fruit fly life cycle.
Fruit flies (Drosophila melanogaster) continue to be extremely useful organisms
for the study of genetics. There are various reasons for this: they are small, being easy
and economical to culture in the laboratory; they breed prolifically - each female is
capable of laying several hundred eggs; they have a short generation time - from egg
to adult in about 10 days at 25ΕC.; they have a large number of easy to see (under low
magnification) external characteristics that show genetic variation; and they have a well
understood life cycle, including descriptive embryology that is now being understood at
the molecular level.
Fruit flies are typical insects whose life cycle shows complete metamorphosis
(the young stages do not resemble the adult stage; Fig. 1-1). Mature male and female
flies mate and produce fertilized eggs each of which are oval in shape and about 0.5
mm in length. Within 24 hours, each egg hatches into a tiny nearly transparent wormlike larva, highly adapted to burrowing into and eating fleshy fruit. As each larva grows,
it sheds ("molts") its external skin ("exoskeleton") twice, becoming whiter and larger
after each molt. Thus, the larval period consists of three stages (called "instars"); the
1-2
Exercise 1.
third instar larva reaches a length of 4.5 mm. The larvae are such intensely active and
voracious feeders that the culture medium in which they are crawling becomes heavily
channeled and furrowed, a sure sign that the mating was successful.
When the third instar larvae are mature (about 5 days after the eggs were laid),
they will crawl up the sides of the container and adhere to some relatively dry surface
such as the side of the bottle or the paper toweling which has been inserted in the food.
The larvae then pupate, during which their outer skin hardens and darkens and internal
changes occur leading to the formation of the adult body from buds of tissue within the
puparium. This is similar to what occurs when caterpillars metamorphose into
butterflies.
About 5 days after puparium formation in Drosophila, each puparium splits open
at the anterior end and either a young male or female adult emerges, pale, wet, and
with soft wings folded up like a parachute on its back (dorsal surface). Within 4 hours of
emergence, each adult has produced its body pigmentation, has elongated and
stiffened its wings, and has sexually matured. Matings between males and females
then may occur, leading to another generation of offspring.
Figure 1-1. Life cycle of fruit flies (Drosophila melanogaster).
Exercise 1.
1-3
B.
Setting up a fruit fly study.
1.
Objectives.
You will be given several vials each containing a small colony of fruit flies
homozygous (pure breeding) for some physical trait known to the lab instructor. One
vial contains normal flies, another contains mutants. The objectives of working with
these flies are:
2.
A.
To describe in as much detail as possible, by observation of the flies in
these vials, the mutant trait.
B.
To set up appropriate matings to determine the mode of inheritance of the
mutant trait, namely:
1.
Is the mutant trait dominant or recessive?
2.
Is the gene for the mutant trait located on a sex chromosome (the X
chromosome) or on a non-sex chromosome (one of the
autosomes)?
Fruit fly morphology (Fig. 1-2)
A.
B.
Prominent features. Fruit flies have a typical insect body arrangement of
head, thorax (chest), and abdomen.
1.
Head. Prominent features on the head are the antennae, the
mouth parts (called the proboscis), the large compound eyes (each
consisting of about 800 individual facets), a triangular arrangement
of three simple eye spots (called ocelli), and bristles.
2.
Thorax. Prominent features are three pairs of walking legs, one
pair of wings for flying, a pair of globular structures called halteres
(analogous to the second pair of wings found in most other insects),
a triangular region on the posterior dorsal surface called the
scutellum, and many bristles.
3.
Abdomen. Consists of a number of segments; the external
genitalia and anal region are located at the posterior tip.
Distinguishing males from females (Figure 1.2). Male and female
Drosophila may be distinguished from each other very easily by a number
of clear-cut differences.
1.
Sex combs. Males possess sex-combs, a group of about 10 stout,
black bristles towards the front of the first pair of legs. Females
lack sex-combs.
1-4
Exercise 1.
Figure 1-2: Fruit fly morphology.
2.
External Genitalia. The external genitalia of males and females
have obvious differences. Males have a series of brown to black
Exercise 1.
1-5
external structures related to sperm transfer, but females do not.
This is a striking difference even in extremely young flies.
3.
3.
Number of Abdominal Segments. Females have 7-8 visible
abdominal segments whereas males have 5-6. Except in very
young flies, the tip of the abdomen is elongated in the female and
somewhat more rounded in the male. As females age, their
abdomens become swollen with maturing eggs.
4.
Abdominal Pigmentation. In normal flies (but not all mutant stocks),
the abdominal pigment pattern is sufficiently distinct in the two
sexes to permit their identification on this basis without use of a
microscope. The most posterior 3-4 segments in males are solid
black, whereas each segment in the female bears a transverse
narrow black band. In newly emerged adults, however, the
abdominal pigmentation has not yet darkened.
Experimental Procedures.
A.
B.
Sorting out males and females.
1.
Obtain a vial of normal flies ("wild type", vial labeled "+") and an
empty vial. Anesthetize the flies using "Flynap" solution, following
directions given by your lab instructor.
2.
When the flies are no longer moving, place them onto a white plate
and view them under a dissecting microscope.
3.
Using a toothpick, push each fly into one of two groups, forming a
group of females on the left side of the plate, and a group of males
on the right side. When finished, have the student sitting next to
you verify the accuracy of your sorting. If you are in doubt about
your accuracy, ask the lab instructor for help.
4.
When finished, leave about 10 males and 10 females on the plate
and discard the other flies by dumping them into the jar of motor oil
(the "fly morgue") on each table.
Determining and describing the abnormal phenotype in the mutant fly
colony.
1.
Anesthetize the flies from the vial marked "M__", and place the
sleeping flies onto the white plate. Be careful not to overdose the
flies, since dead flies are not useful for mating purposes.
2.
As you did for the normal flies, sort the mutant flies into male and
female groups.
3.
Compare the mutant males with normal males, and the normal
1-6
Exercise 1.
females with the mutant females. Determine which physical trait
differs consistently between normal and mutant flies. In as much
detail as possible (including drawings if you desire), describe the
mutant trait in the space below.
FULL DESCRIPTION OF MUTANT TRAIT FROM VIAL "M_____"
How does it differ from the normal (+) flies?
C.
Setting up a fly mating.
1.
After determining and describing the mutant trait, place 10 of the
healthiest looking mutant males in a clean vial.
2.
Place these males into the vial marked "VIRGIN + FEMALES."
Your lab instructor will demonstrate the proper procedure for this.
This vial contains 5 or 6 normal females collected shortly after they
emerged from their pupal cases before they became sexually
mature. Thus, these females have never mated previously. Be
very careful not to let the virgin females escape!! Fill in the M___
designation on the vial label.
3.
Turn the vial upside down and gently tap the vial until the sleeping
males fall onto the stopper. Then, lay the vial on its side until the
males revive, after which you should stand the vial upright.
4.
Then, discard all remaining normal and mutant flies remaining on
the plate by dumping them into the fly morgue.
Exercise 1.
1-7
5.
Write your name on the labels of the following vials:
- The "+" vial, containing eggs from normal flies.
- The "M___" vial, containing eggs from mutant flies.
- The "VIRGIN + FEMALE x M___" vial, containing adult normal
virgin females and the mutant males you just put in.
Now, Place a rubber band around the three vials and put them into
the constant temperature incubator.
6.
C.
Check your vials next week to see the larvae and pupal cases.
Also, you must remove any adults from these vials next week to
prevent the offspring that will emerge from comingling with the
parents. In two weeks time, enough offspring (the F1 generation)
will have emerged to permit the next stage of this experiment.
Determining Inheritance Patterns.
The purpose of the fruit fly mating is for you to scientifically determine two
important aspects of the inheritance of the mutant trait, namely whether it is a dominant
or recessive trait and whether it is associated with the chromosomes that determine
gender (the X and Y chromosomes) or the chromosomes that are common to both male
and female flies (the autosomes). There are four realistic possibilities: autosomal
dominant, autosomal recessive, X-linked dominant, and X-linked recessive (why
couldn't the mutant gene be located in the Y chromosome?) Use the information on the
following pages to predict the outcomes of your mating for each of these possibilities.
1.
1.
Autosomal dominant inheritance.
2.
Autosomal recessive inheritance.
3.
X-linked dominant inheritance.
4.
X-linked recessive inheritance.
Autosomal dominant inheritance. If the mutant trait shows autosomal
dominant inheritance, the mutant gene is designated "A", and all homozygotes
(AA) and heterozygous (Aa) flies would display the mutant trait. Only the
homozygous recessive (aa) flies would be normal. Fill in the following chart,
predicting the phenotypes and ratios of flies in the F1 and F2 generations.
P generation: (all parents are homozygotes)
1-8
Exercise 1.
phenotypes:
normal females
x
mutant males
genotypes:
______________
9
____________
phenotypes:
______ females
x
_______ males
genotypes:
______________
9
_____________
F1 generation:
F2 generation:
(Use a Punnett square to determine the expected results)
sperm
eggs
F2 FEMALES
PHENOTYPES
GENOTYPES
F2 MALES
RATIOS
PHENOTYPES
GENOTYPES
RATIOS
Exercise 1.
1-9
2.
Autosomal recessive inheritance. If the mutant trait shows autosomal
recessive inheritance, the normal gene is designated "B", and all homozygotes (BB) and
heterozygous (Bb) flies would display the normal trait. Only the homozygous recessive
(bb) flies would be mutant. Fill in the following chart, predicting the phenotypes and
ratios of flies in the F1 and F2 generations.
phenotypes:
normal females
x
mutant males
genotypes:
______________
9
____________
phenotypes:
______ females
x
_______ males
genotypes:
______________
9
_____________
F1 generation:
F2 generation:
sperm
eggs
F2 FEMALES
PHENOTYPES
3.
GENOTYPES
F2 MALES
RATIOS
PHENOTYPES
GENOTYPES
RATIOS
X-linked dominant inheritance. If the mutant trait shows X-linked
1-10
Exercise 1.
dominant inheritance, the mutant gene is designated "XA", and all flies that are
XAXA and XAXa females and XAY males would display the mutant trait. Only XaXa
females and XaY males would be normal. Fill in the following chart, predicting
the phenotypes and ratios of flies in the F1 and F2 generations.
phenotypes:
normal females
x
mutant males
genotypes:
______________
9
____________
phenotypes:
______ females
x
_______ males
genotypes:
______________
9
_____________
F1 generation:
F2 generation:
sperm
eggs
F2 FEMALES
PHENOTYPES
4.
GENOTYPES
F2 MALES
RATIOS
PHENOTYPES
GENOTYPES
RATIOS
X-linked recessive inheritance. If the mutant trait shows X-linked
recessive inheritance, the normal gene is designated "XB", and all flies that are
Exercise 1.
1-11
XBXB and XBXb females and XBY males would display the normal trait. Only XbXb
females and XbY males would be mutant. Fill in the following chart, predicting
the phenotypes and ratios of flies in the F1 and F2 generations.
phenotypes:
normal females
x
mutant males
genotypes:
______________
9
____________
phenotypes:
______ females
x
_______ males
genotypes:
______________
9
_____________
F1 generation:
F2 generation:
sperm
eggs
F2 FEMALES
PHENOTYPES
GENOTYPES
F2 MALES
RATIOS
PHENOTYPES
GENOTYPES
RATIOS
Exercise 2.
2-1
Exercise 2
Fruit Flies:
Collecting and Analyzing Data
A.
B.
Interpreting Data: the Chi-Square Test
Collecting Fruit Fly Data:
1.
Eliminate Hypotheses
2.
Setting up the F2 Generation
A.
Interpreting data: the Chi-Square test.
Scientists make use of statistical analyses to determine which of several
competing explanations is better supported by the actual data obtained from an
experiment. One especially useful statistical tool for analyzing the results of genetic
crosses is called Chi-Square Analysis (X2). It is based on some basic principles of
probability (the chance that something will happen under certain circumstances) and the
fact that expected results do not occur exactly as predicted due to chance whenever
real experiments are performed.
Theoretical ratios are based on hypotheses. For example, if two heterozygotes
for a dominant trait mate, we expect a 3:1 ratio of phenotypes among the offspring, or, if
one tosses a coin into the air 1000 times, we theoretically expect the coin to land headsup 500 times and tails-up 500 times. Observations will be very close to the expected
hypothesized ratios if large samples are taken and the hypothesis is correct. In smaller
samples, chance variations may occur that give a ratio apparently much different than
the expected one. For example, if one tosses a coin into the air only 10 times, there is a
good chance of it landing heads-up 8 times and tails-up twice. The observed ratio of
4:1 apparently is considerably different from the expected ratio of 1:1.
In such cases, one must decide whether this difference is caused by accidental
chance alone or whether the hypothesis is wrong (maybe the coin is "loaded" so that
one side is heavier than the other). Likewise, it is important for a geneticist to know the
degree to which actual results may differ from expected results due to chance rather
than due to a wrong hypothesis or some error in the way the experiment was
performed. Knowing this, the scientist can decide whether or not the results support the
hypothesis.
Coin Tossing. When tossing coins, we expect that the coin will land heads-up 50%
of the time. This seldom happens exactly unless we toss the coin a very large number
of times, which would eliminate chance fluctuations (called "sampling error"). Since
actual experiments involve a limited number of observations, some sampling error is
expected and the results will not be exactly as expected. We could propose a "null
hypothesis" stating that nothing (gravity or the way the coins are tossed) has an effect
Exercise 2.
2-2
on their landing heads or tails. If so, then how much deviation from the expected 1:1
ratio can occur before the null hypothesis is rejected (something is making the coins fall
heads-up more than tails-up, for instance)? In most biological experiments, the null
hypothesis is rejected when the deviation is so large that it could be accounted for by
chance less than 5 percent of the time (a "significant deviation"). Statistics can never
provide absolute proof of the validity of a hypothesis, but may set limits to our
uncertainty of its correctness.
If the coin tossing experiment is based on small numbers, large deviations from
expected ratios occur quite often due to chance alone. But as the sample size
increases, the deviation should become smaller so that if the sample was infinite in size,
we would obtain an exact 1:1 ratio with no deviation at all.
Degrees of Freedom. We toss a coin into the air. If it does not land heads-up, it
must land tails-up. Although there are two sides to a coin, it has only one "choice" as to
which side is up. Or, when we put our shoes on, if we put one shoe on the right foot
first, then we must then put the other shoe on the left foot. Thus, given two possibilities,
there is only one free choice or "degree of freedom." In general, we have n-1 degrees
of freedom (df) in assigning numbers at random to n classes within an experiment.
Thus, if there are four possible phenotypic combinations possible (say, in a 9:3:3:1
dihybrid ratio situation), there are three degrees of freedom in assigning an organism to
one of these (if we choose not to place it in the first, second, or third group, we must
place it in the fourth category). For most genetics situations, the number of degrees of
freedom will be one less than the number of phenotypic classes.
Chi-Square Test (X2). The chi-square test enables an experimenter to convert the
amount of deviation from expected values into the probability of such differences
occurring by chance. This test takes into account the size of the sample tested and the
number of variables (degrees of freedom). The question we try to answer with the X2
test is "How small can the deviations be to be probably attributed to chance alone?"
The formula for X2 is:
chi-square =
Σ (O - E)
2
/E
where Σ = the grand total of the squared deviations (observed number minus
expected number, O-E)2 divided by the expected number (E) for each class. The
value of chi-square may then be converted into the probability (P) that the
deviation is due to chance by using the table below for the proper number of
degrees of freedom.
Exercise 2.
2-3
Chi-Square Distribution Table.
__________________________________________________________
Probability that
deviation is due
Numbers of Degrees of Freedom
to chance alone
1
2
3
4
5
__________________________________________________________
these values 0.95 (95%)
0.004 0.10
0.35
0.71
1.15
support the
0.70 (70%)
0.15
0.71
1.42
2.20
3.00
hypothesis
0.50 (50%)
0.46
1.39
2.37
3.36
4.35
under
0.30 (30%)
1.07
2.41
3.66
4.88
6.06
consideration 0.10 (10%)
2.71
4.60
6.25
7.78
9.24
__________________________________________________________
don't support 0.05 ( 5%) **
3.84
5.99
7.82
9.49
11.07
hypothesis
0.01 ( 1%) **
6.64
9.21
11.34 13.28 15.09
(P= or <.05)
0.001(0.1%) **
10.83 13.82 16.27 18.47 20.52
__________________________________________________________
** Observed results are significantly different from the expected results.
The chi-square test has two important limitations. First, it must be used only for
the numerical data itself, never on any percentages or ratios derived from the data.
Second, it cannot be used for experiments where the expected number in any
phenotypic class is less than 5.
Examples of using Chi-Square analysis.
Suppose you observe that, from a mating, 9 fruit fly offspring are males and 3 are
females. Can we say with scientific confidence that the data display a 3:1 ratio and not
a 1:1 ratio? Let's see if a Chi-Square analysis will reject the hypothesis that the results
support a 1:1 ratio.
Phenotype
Classes
observed
number=O
expected
number=E
deviation
(O - E)
deviation2
(O - E)2
(O - E)2
E
Males
9
2 x 12=6
(9-6)= 3
[3]2= 9
9/6= 1.5
Females
3
2 x 12=6
(3-6)=-3
[3]2= 9
9/6= 1.5
12
12
0
totals →
18
X2 = 3.0
How many degrees of freedom do we have? _______
What is the probability that this deviation from the expected 1:1 ratio is due to
chance? ______
Is the 1:1 ratio hypothesis supported or should we reject it? Explain.
Here, we see the effect of a too-small sample size leading to an inability to
Exercise 2.
2-4
statistically determine whether a 3:1 ratio or a 1:1 ratio is the "true" result.
Now, suppose you observe that 90 flies are males and 30 flies are females. With
this larger sample size, can we say with scientific confidence that the data display a 3:1
ratio and not a 1:1 ratio? Let's see if another Chi-Square analysis will reject the
hypothesis that the results support a 1:1 ratio. Fill in the blanks to complete the
analysis.
Phenotype
Classes
observed
number=O
Green leaf
90
White leaf
30
totals →
120
expected
number=E
deviation
(O - E)
deviation2
(O - E)2
(O - E)2
E
120
X2 =
How many degrees of freedom do we have? _______
What is the probability that this deviation from the expected 1:1 ratio is due to
chance? ______
Is the 1:1 ratio hypothesis supported or should we reject it? Explain.
B.
Collecting fruit fly data.
Introduction.
Two weeks ago, you set up a mating involving virgin normal ("wild type") females
and mutant males. Last week, you removed the parental flies from the vial so that they
would not mate with the F1 flies when they emerge. Today, you will examine the F1 flies
that have emerged, set up a mating between the F1 males and F1 females, and
eliminate any hypotheses about the inheritance of the mutant trait that no longer are
supported by your observations.
As part of lab Exercise 2, you turned in a description of your mutant flies. Your
lab instructor has by now returned Part B of Lab Report 2, commenting on the accuracy
of your description of the mutant trait in your flies. If your description of the trait was
accurate, simply copy the description below. If your description was innacurate,
reexamine the mutant flies and give a new description of the trait below, for your lab
Exercise 2.
2-5
instructor to check again.
Below, briefly redescribe the mutant trait (M_____; refer back to Exercise 1, or look at
the flies in your mutant male x mutant female vial:
Examining the F1 generation flies.
Retrieve your group of 3 vials from the constant temperature incubator. Perform
the following only after your lab instructor has demonstrated the correct procedure for
transferring flies for observation:
1.
Remove about half the flies from the vial marked "Virgin + Females x Mutant
males" by quickly transferring them into a clean vial stoppered with a foam plug
containing a small brush containing a small amount of "Flynap" anesthesia.
2.
Do not overexpose the flies to Flynap since dead flies are of little further use.
When the flies have stopped moving about, remove the stopper and let the flies
fall onto a white plate.
3.
Place the plate with flies onto the stage of a dissecting microscope and, using a
toothpick, sort the flies into a group of females on the left and a group of males to
the right.
4.
Examine each group of flies, noting the presence or absence of the mutant trait,
and whether any change has occurred in the appearance of the mutant trait
between the P and F1 flies. Fill in the table on the next page.
SEX OF THE FLIES
PHENOTYPE:
NORMAL OR MUTANT
COMMENTS ABOUT THE
MUTANT TRAIT
Exercise 2.
2-6
FEMALES
MALES
Setting up the F1 x F1 crosses.
1.
Obtain two fresh food vials from your lab instructor.
2.
Label each vial as follows: F1 female x F1 male (M
date on each label.
3.
Insert the anesthetized flies you just examined into one vial, stopper the vial, and
lay it on its side until the flies awaken.
4.
Transfer the remaining flies from the Virgin + Females x Mutant males vial into
the second fresh vial, following the instructions of your lab instructor.
You now have two vials of flies of the identical F1 x F1 cross. In two weeks time,
you will be able to collect a considerable amount of data from the F2 generation
produced by these matings.
5.
Next week, check your vials to see the larvae and pupal cases. Also, you must
remove any adults from these vials next week to prevent the offspring that will
emerge from co-mingling with the parents. In two weeks time, enough offspring
(the F2 generation) will have emerged to permit definite determination of the
mode of inheritance of the mutant trait and statistical analysis of the results.
) and write your name and
Interpretation of the F1 generation data.
Refer back to Exercise 1, which describes the four possible inheritance patterns
for mutant traits in fruit flies. Based on the F1 data from your cross, is the mutant trait in
Exercise 2.
2-7
your flies inherited as a dominant or as a recessive? Explain.
Can you rule out either that the mutant trait is X-linked or autosomal in
chromosomal location? Elaborate.
So, cross out from the list below any mode of inheritance that no longer applies
for your mutant trait:
AUTOSOMAL DOMINANT
AUTOSOMAL RECESSIVE
X-LINKED DOMINANT
X-LINKED RECESSIVE
Note below anything unusual about the inheritance of your mutant trait:
2-8
Exercise 2.
Exercise 3
3-1
Exercise 3
Fruit Flies:
Collecting and Analyzing Data
A.
B.
C.
E.
Introduction
Collecting Fruit Fly Data
Chi Square Analysis of Fruit
Fly Data
Eliminating or Supporting
Hypotheses
Writing a Lab Report
A.
Introduction.
D.
During Exercise 1, you were given a vial of fruit flies displaying a mutant
phenotype. By comparing various physical characteristics of normal flies with those of
the mutants, you wrote a description of the mutant phenotype, which your lab instructor
checked for accuracy [If the description was inaccurate, you re-described the trait during
Exercise 2.] Also during Exercise 1, you set up a P generation mating involving normal
females and mutant males. A week later, you removed the parents.
During Exercise 2, you observed the phenotypes of the F1 generation flies and
noted whether your observations supported dominant or recessive inheritance and
autosomal or X-linked inheritance. Also, during Exercise 2, you set up two vials of the
F1 x F1 mating. Last week, you removed the F1 flies.
Today, you will observe the F2 generation flies from the two vials set up during
Exercise 2, analyze the data using the Chi-Square test, and determine or verify the
exact mode of inheritance for your mutant fly trait. Also, your lab instructor will give you
some guidance in writing a scientific lab report of the entire fruit fly experiment.
B.
Collecting fruit fly data: the F2 generation.
Retrieve your vials from the constant temperature incubator. Perform the
following only after your lab instructor has demonstrated the correct procedure for
transferring flies for observation:
1.
Remove all adult flies from one of the F1 x F1 vials by quickly transferring them
Exercise 3.
3-2
into a clean vial stoppered with a foam plug to which a small wand dipped in
"Flynap" anesthesia is attached.
2.
You may overexpose the flies to Flynap since you no longer need the flies for
mating purposes, and dead flies will not awaken and fly off. After the flies have
been thoroughly anesthetized, remove the stopper and let the flies fall onto a
white plate or index card.
3.
Place the plate with flies onto the stage of a dissecting microscope and, using a
toothpick, sort the flies into a group of females on the left and a group of males to
the right.
4.
Examine each group of flies, and further subdivide the flies (if necessary) into
groups of normal females, mutant females, normal males, and mutant males.
Count the number of flies in each group and record these data in the table at the
top of the next page.
5.
Repeat steps 1-4 for the second F1 x F1 vial.
C.
Chi Square analysis of fruit fly data.
To statistically support the likelihood that the data you collected represents either
a 3:1 ratio or a 1:1 ratio, perform the following Chi-Square analyses:
1.
Refer to the table on the next page. Compare the total number of females to the
total number of males [use the data from the TOTALS (B) row]. What would you
expect the sex ratio to be from this mating? Explain your answer.
Data from F2 generation:
Exercise 3
PHENOTYPES
3-3
FEMALES
vial 1 +
vial 2 = total
MALES
vial 1 +
vial 2 = total
NORMAL
+
=
+
=
MUTANT
+
=
+
=
TOTALS (B)
NOTE ANY DIFFERENCES IN
THE MUTANT PHENOTYPES
OF FEMALES AND MALES
TOTALS
(A)
Exercise 3.
3-4
Perform the Chi Square analysis of the sex ratio data (from TOTALS (B) row). A ChiSquare distribution table is given below for your convenience.
Chi-Square Distribution Table.
__________________________________________________________
Probability that
deviation is due
Numbers of Degrees of Freedom
to chance alone
1
2
3
4
5
__________________________________________________________
these values 0.95 (95%)
0.004 0.10
0.35
0.71
1.15
support the
0.70 (70%)
0.15
0.71
1.42
2.20
3.00
hypothesis
0.50 (50%)
0.46
1.39
2.37
3.36
4.35
under
0.30 (30%)
1.07
2.41
3.66
4.88
6.06
consideration 0.10 (10%)
2.71
4.60
6.25
7.78
9.24
__________________________________________________________
don't support 0.05 ( 5%) **
3.84
5.99
7.82
9.49
11.07
hypothesis
0.01 ( 1%) **
6.64
9.21
11.34 13.28 15.09
(P= or <.05)
0.001(0.1%) **
10.83 13.82 16.27 18.47 20.52
__________________________________________________________
** Observed results are significantly different from the expected results.
Chi-Square analysis of the sex ratio data:
Phenotype
Classes
observed
number=O
expected
number=E
deviation
(O - E)
deviation2
(O - E)2
(O - E)2
E
Females
Males
totals →
Degrees of Freedom: ______; Probability that deviation is due to chance:
______
Do your data support your hypothesis regarding sex ratio? If there is a
disagreement, how might this be explained?
2.
Compare the total number of normal flies to the total number of mutant flies [use
Exercise 3
3-5
the data from the TOTALS (A) column].
What would you expect the normal : mutant ratio to be from this mating? Explain
your answer.
Perform, in the table below, the Chi Square analysis of the normal : mutant ratio data.
Chi-Square analysis of the normal : mutant ratio:
Phenotype
Classes
observed
number=O
expected
number=E
deviation
(O - E)
deviation2
(O - E)2
(O - E)2
E
Normal
Mutant
totals →
______
Do your data support your hypothesis regarding the normal:mutant ratio? If there
is a disagreement, how might this be explained?
3.
Compare the total number of normal female flies, normal male flies, mutant
female flies, and mutant male flies [use the data from the table].
Exercise 3.
3-6
What would you expect the ratios of the four categories of flies to be to be from
this mating? Explain your answer.
Perform, in the table below, the Chi Square analysis of the data.
Phenotype
Classes
observed
number=O
expected
number=E
deviation
(O - E)
deviation2
(O - E)2
(O - E)2
E
Normal
females
Normal
males
Mutant
females
Mutant
males
totals →
______
Do your data support your hypothesis regarding the normal:mutant ratio? If there
is a disagreement, how might this be explained?
D.
Eliminating or supporting hypotheses.
Exercise 3
3-7
From the data you collect for the F2 generation, you should be able to eliminate
all but one of the four original hypotheses about the mode of inheritance for your mutant
trait. Recall from Exercise 1 that the four hypotheses are: autosomal dominant,
autosomal recessive, X-linked dominant, and X-linked recessive. Based on all your
data (F1 and F2 generations), give the reasons you are able to either reject or support
each of the following modes of inheritance:
Autosomal Dominant:
Autosomal Recessive:
X-linked Dominant:
X-linked Recessive:
E.
Writing a Lab Report.
Exercise 3.
3-8
One important method of communication used by scientists to tell others about
the research they do is the journal article or research paper. Many scientific journals
are published on a monthly or quarterly schedule, and each contains a number of
research papers. In these papers, the scientists involved in doing the research write
about their studies using a standard format of Introduction, Methods and Materials,
Results, Discussion, and Conclusion:
1. First, the authors introduce the topic they are researching by talking about previous
studies that have been published and the objectives of their study (i.e., why their studies
are important).
2. Then, they describe the experiments they have performed, giving the methods and
materials used in enough detail that some other scientist could repeat the study.
3. Next, the results of the experiment are fully presented, along with appropriate
statistical analyses.
4. These results then are discussed as to their significance and meaning, including how
the results might suggest additional experiments that might be done.
5. Finally, the authors reach conclusions based on the results of their study and the
discussion of their results.
Most research papers also include a references of bibliography section in which
previous research studies relevant to the present study are cited as to journal volume,
page numbers, and year of publication.
Your Report.
As part of Exercise 3, you are to write up the results of your fruit fly study based loosely
on the research paper style just described. Follow the directions given below, and any
additional information given by your lab instructor. Be sure to use a computerized word
processing program to prepare your report. Your research report must contain the
following sections:
1.
Title page: Your name, your partner’s name(s), mutant designation (M_____),
lab section, lab instructor's name, title of your research.
2.
Objectives: the objectives of the research project. What were you, as an
amateur scientist, trying to find out?
3.
Methods and Materials: the organisms used, what was done (the techniques),
when each step was performed, etc.
4.
Results: It is best to present the data and statistical analyses in the form of
tables similar or identical to the ones in this manual. You are encouraged to
photocopy the data and statistical tables from Exercises 1, 2, or 3 for this section.
Exercise 3
3-9
Include statistical analyses of F2 generation data (chi-square analyses of sex
ratio and phenotypic ratio data).
5.
Discussion: Discuss whether the results either support or do not support each of
the four potential modes of inheritance.
6.
Conclusions: Give what conclusions you reached about the correct mode of
inheritance, and what further studies you would suggest that might lead to
additional information about the mutant trait.
3-10
Exercise 3.

A Three-Part Laboratory Exercise Using Flightless Fruit Flies

Transcription

Similar documents

Indoor Flies and Their Control - Insects in the City!

Mary Lyon hypothesis: Inactivation of all but one X chromosome

flying insect management

- Journal of Clinical Investigation

Drosophila - Virtual Courseware

to view our Pest Control Product line

Warner, R. R. (1984).

Chapitre 1 - thèse - Université Toulouse III

Bioreactors and transgenic animals

The Central Dogma of Molecular Biology

Suppression of neuropil aggregates and neurological symptoms by

this PDF file - E-Journal Faculty of Medicine Universitas

this PDF file - Journals

to view the full text as an Adobe PDF.

15 FREE ready-to-use worksheets to use with almost any book.

The Role of Arabidopsis thaliana P80 in Inositol Signaling

Gene Cloning and DNA Analysis: An Introduction, Sixth Edition

мссмв`07 - Moscow conference in computational molecular biology

Hamilton, D. 14