mutations

Biology 322 Lecture Nov 15, 2010
Anouncements: see course web site
Backtrack to a previous lecture: where do antibiotic resistance
genes and alleles come from?
Thinking about the nature of mutation & about mutation
frequency
Tuberculosis and Antibiotic Resistance
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How do they confer resistance?
Science 293: 1786 Sept. 7, 2001
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Where do antibiotic-resistance alleles/genes come from?
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Weapons of Microbial Drug Resistance Abound in Soil Flora
http://fire.biol.wwu.edu/trent/trent/microbialresistance.pdf
(+) = acquired from
an extraspecies source
What do we know about
the transfer
mechanisms?
What types of genetic
elements are involved?
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A = adaptive implying a darwinian process
What requirements for evolution by natural selection does the table on
the previous page illustrate?
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A recent article on antibiotic resistance is titled
The bacteria fight back
This is misleading: “adaptive” resistance happens
because mutation happens and the antibiotic is simply
selecting for preexisting mutations in the vast bacterial
populations
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rifs  rif r (rif = the antibiotic rifamycin = rifampin)
Target site alteration
Rifampin (rifamycin) is a major drug
used in the treatment of tuberculosis
infections, and increasing rifampin
resistance represents a worldwide
clinical problem. Resistance to
rifampin is caused by mutations in the
rpoB gene, encoding the beta-subunit
of RNA polymerase. The wildtype
Mycobacterium tuberculosis RNA
polymerase is 1000X more sensitive
to this antibiotic than the wildtype E.
coli RNA polymerase
Tough bug. Mycobacterium
tuberculosis (red) likes nothing better
than to be ingested by a macro- phage,
its usual home.
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Bacterial RNAP as an antibiotic target: Bacterial RNA polymerase (RNAP) is a
proven target for broad-spectrum antibacterial therapy and for antituberculosis therapy
RNAP is a suitable target for three reasons:
(1) RNAP is an essential enzyme (permits efficacy)
(2) bacterial RNAP-subunit sequences are highly conserved (permits broad-spectrum
activity)
(3) bacterial RNAP sequences and eukaryotic RNAP sequences are less highly conserved
(permits therapeutic selectivity).
• The rifamycin antibacterial agents–rifampin (also known as rifampicin) function by
binding to and inhibiting bacterial RNAP
• The rifamycins are in clinical use in treatment of Gram-positive and Gram-negative
bacterial infections, are first-line antituberculosis agents, and are among the few
antituberculosis agents that can kill nonreplicating tuberculosis bacteria.
• For all major bacterial pathogens, including the tuberculosis pathogen, strains
resistant to rifamycins have arisen
• Resistance to rifamycins involves substitution of residues within the rifamycin-binding
site on bacterial RNAP, i.e., substitutions that directly decrease rifamycin binding
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“ What we really need,” Rich adds, “is a good, cheap, point-of-service
test.” Wealthier countries have access to polymerase chain reaction
tests that monitor variable TB organism genes, signaling within 24
hours whether the strain is resistant to the first-line drugs isoniazid and
rifampin. “
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Follow-up on rifR mutants We will follow up on our rifR mutations by determining the location
and nature of mutatgenic changes that can confer resistance to the antibiotic without severely
compromising the ability of RNA polymerase to perform its cellular function
Figure 1. The Rif-Resistant Regions of the RNAP b Subunit
The bar on top schematically represents the E. coli b subunit primary sequence with amino acid numbering shown
directly above. Gray boxes indicate evolutionarily conserved regions among all prokaryotic, chloroplast,
archaebacterial, and eukaryotic sequences. indicate the four clusters where RifR mutations have been identified in E.
coli. Mutations that confer RifR in E. coli and M. tuberculosis are indicated directly above (for E. coli) or below (for M.
tuberculosis) as follows: D for deletions, V for insertions, and colored dots for amino acid substitutions (substitutions
at each position are indicated in single amino acid code in columns above or below the positions).
Color coding for the amino acid substitutions is as follows:
yellow, residues that interact directly with the bound Rif
green, residues that are too far away from the Rif for direct interaction
purple, three positions that are substituted with high frequency (noted as a % immediately below the substitutions) in
clinical isolates of RifR M. tuberculosis
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How common should spontaneous mutation to
antibiotic resistance be in a population not under
selection?
Before we address this question in bacterial, let’s
indulge in thinking about us….
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• Achondroplasia is a completely
penetrant, autosomal dominant
disorder characterized by
disproportionate short stature -the arms and legs are short
compared with the head and
trunk.
• More than 80% of the people
who have achondroplasia are
born to parents of normal stature
and represent a new germline
mutation
Michael Dunn is best known for his recurring
role as the villain Dr. Miguelito Loveless on
The Wild Wild West (1965-68).
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Achondroplasia and the mutagenic male
An estimate of the mutation rate in FGFR is based upon the
accumulated data of newborn studies in four cities:
• In a total of 242,257 births, seven infants had achondroplasia
• From these numbers, the rate of mutation of the normal to the
achondroplasia allele is calculated to be
• 1.4 X 10-5 mutations in the achondroplasia gene per gamete =
• 1 mutation per 69,216 copies of the gene
** Who is at fault? **
• It has been established that fathers are the source of all achondroplasia
mutations
• The probability of having an affected offspring increases explonentially
with the father’s age
• 50% of children with achondroplasia are born to father older than 35 years
old.
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Sex, Errors and the Genome by Mark Ridley
Natural History (6/2001)
“At conception, human embryos average about 200
copying errors and about 50% of the embryos have a
botched number of chromosomes. “
WHO IS TO BLAME?
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When a thirty year old man breeds
with a 30 year old woman:
• his DNA (in his sperm cells) has been copied 430 times against her
33 cell division (in egg cells).
• with thirteen times as many errata in his DNA, about 185 of the 200
copying mistakes in each human conception may come from the
sperm.
• however, a woman’s eggs are more likely to carry serious errors in
chromosome numbers, and these errors increase with maternal age.
• The germline mutation rate in human males, especially older males,
is generally much higher than in females, presumably because in
males there are many more germ-cell divisions
• Why is number of cell divisions correlated with mutation rate?
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Every time a human cell divides it has to replicate 6 X 109
base pairs of DNA
Every time an E. coli divides it has to replicate 5 X 106
base pairs of DNA
Every time a base pair is copied, there is a very small but finite
probability that an error will be introduced at that site
Furthermore, if the DNA molecule has sustained damage that remains
unrepaired, then mistakes will occur during DNA replication of that
site by so-called error-prone bypass polymerases
What sort of damage occurs routinely to DNA?
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The Mutagenesis lab is a great illustration of the principle that
the power of bacterial genetics is the potential for studying rare events
We have looked at rare spontaneous [and EMS-induced] mutations:
Selection for
rifs  rif r (rif = the antibiotic rifamycin)
Screen for Forward mutation
lac+  lac - (lac = lactose)
Selection for Reverse mutation
lac-  lac +
How to explain the differences in mutation rates?
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MUTATION JARGON
GENE MUTATION = POINT MUTATION
(scales of mutation is small and is localized to a specific region,
a single nucleotide or a few adjacent base pairs)
↓
at the DNA level:
 single base pair substitutions: transitions & transversions
 single (or a few) base pair addition or deletion: indels
 gene mutation by transposon insertion
at the level of
gene expression:
at the protein
level:
promoter mutations
splicing mutations
regulatory mutations
nonsense
missense
[neutral]
silent
frameshift
at the level of gene function:
loss-of-function
gain-of-function
[neutral
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CHROMOSOME MUTATION
• involves segments of chromosomes or
whole chromosomes or whole genomes
• alterations in chromosome structure
and number
• deletion, duplications, translocations
and inversions
• CNVs: copy number variations
Decoding mutation jargon
“The (achondroplasia) mutations just discussed are single base
substitutions. The most striking is achondroplasia, in which 153 of
154 analysed cases are due to a glycine to arginine substitution at
codon 1,138. The mutations are in the transmembrane domain of the
fibroblast growth factor receptor 3 (FGFR3). Of the 153 mutations, 150
were guanine to adenine transitions and three were guanine to
cytosine transversions of the same nucleotide. This means that all
the cases of achondroplasia are due to changes in one nucleotide — a
nucleotide with the highest known mutation rate (about 10-5 per
generation). There are mutations at other sites in this gene, but the
phenotypes are different.”
The achondroplasia mutations are all at a CpG nucleotide pair, known to be a mutation hot-spot. What do
you know about CpG islands?
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MORE JARGON:
gain and loss-of-function
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