Announcements
-Solutions to problem set 3 and from the questions pertaining to last Fridays lecture have been posted on the course website.
-Problem set 4 (pertaining to last weeks lectures) is posted on the course website.
-A reading assignment on DNA hybridization has been posted on the course website.
Today:-Mutant analysis (screen vs. selection; reversion; suppression; mutation rate; mutagens).
-Repair of mutations
Mutant analysis (AKA Genetic Analysis)
The use of mutants to understand how a biological process normally works*
*See the Salvation of Doug article at the following site:http://bio.research.ucsc.edu/people/sullivan/savedoug.html
• Start with “unknown” system (e.g., metabolic pathway, embryonic development, behavior, etc.)
• Generate mutations that affect the “unknown” system (i.e., that “break” the “unknown” system)
• Study the mutant phenotypes to reveal the functions of the genes
• Map the genes
• Identify the genes (more on this later)
Wild-type yeast can survive on ammonia, a few vitamins, a few mineral salts, some trace elements and sugar…
They synthesize everything else they need, including adenine
What genes does yeast need to synthesize
adenine?
Case study: analyzing the adenine biosynthetic pathway by generating and studying “ade” mutants
Conducting a mutant analysis with yeast
-adenine plate
“complete” plate
sterile piece of velvet
Adenine-requiring colonies
(ade mutants)
m2
m1m3
Replica-plate
plate cells
Treat wt haploid cells with a mutagen:
Identifying yeast mutants that require adenine
This is an example of a “genetic screen”
Identifying interesting mutations—screen vs. selectionScreen Each member of the population is examined…
does it fit the phenotype criteria that have been set up?Individuals not meeting the criteria don’t survive (or are otherwise eliminated from the population)
Selection
Example 1: Looking for a translator
Example 2: Looking for wingless fly mutants
Primary selection or screen is often followed by secondary selection or screen
Russian EnglishScreen: read resumésSelection: advertise in Russian
Screen: Look at each fly… wings present?Selection: Open vial, let flies fly away
Reversion and Suppressors
Another approach - start with a mutant
Look for reversion to wild type (or less mutant)-sometimes called “reverse” genetics.
ww
look for red eyes
What kinds of mutations might you find?
Most genetic screens are “forward” screens - start with wild type organism and look for new phenotypes caused by mutation (e.g., screen for yeast ade mutants).
1)Mutations that restore function to the white gene (revertant).
w gene
X
XX
or
w gene
X
w gene
X
plus suppressor mutation in some other gene
What kinds of suppressor mutations might you imagine?
2) Mutations that bypass (or suppress) the need for a white gene.
Reversion and Suppressors
intermediate X
adenineADE2
YADE1
red pigment
Partial biosynthetic pathway for adenine in yeast
adenine
ade2
XX Y
ADE1
build up of
Revert ade- to Ade+… does RED revert to WHITE?
-Ade plate
Ade+ revertant
it’s white
Pretty good proof that one mutation (ade2) has two phenotypes
ade2 mutant
Treat wt haploid cells with a mutagen:
5’..AUG....UAC....UGA..3’
A working hypothesis… ade2 has reverted to ADE2
ADE2
ColorGrowth on -ade mRNA
sequence
ade2
revertant
(ade2-R)
+ white
- red 5’..AUG....UAG....UGA..3’
+ white 5’..AUG....UAC....UGA..3’
STOP!
Has the mutant gene changed back to WT?
A test of the hypothesis……do a cross: ADE
2ade2-Rx :)
If ade2-R is “true revertant”… :)
ADE2
ade2-R
ade2-R = ADE2
the diploid is homozygous WT meiosis
Spores from the diploid should be:
All Ade+, white
Most Ade+ revertants… like this. But some exceptions!
Some revertants behave differently…
ADE2
revertantx
meiosis
Most spores: Ade+, white
Some spores: ade-, red!
Ade+, white Ade+, white
DiploidsAde+, white
Interpretation?
In these revertants…
• ade2 is still in mutated form
• a new mutation somewhere else suppresses the ade2 phenotype!
The cross:
Summary of revertant types
ade revertants come in two varieties:
1. “True” revertants
2. Suppressors (aka “extragenic suppressors” or “second site suppressors”)
ade2 mutant allele ADE2 (wt)
A mutation in a different gene eliminates the ade2 mutant phenotype
Definition of suppressor?
A mutation in a second gene that eliminates the mutant phenotype of a mutation in the first gene.
What is this suppressor?
Linkage analysis… mapped to chr XV
SUP3 codes for tRNATyr
huh?
To recap…
ADE2
ade2
sup3 SUP3
Ade+
white!
5’..AUG....UAG....UGA..3’
Explanation?
TyrSTOP mutation
WT
How does SUP3 suppress ade2?
WT tRNA (sup3)
AUG
Tyr
AUC
Tyr
Mutant tRNA (SUP3)
TyrSTOP5’..AUG....UAG....UGA..3’
ade2 mRNA ade2 mRNA
5’..AUG....UAG....UGA..3’
The mutant tRNA suppresses the nonsense codon! --full-length ADE2 protein is made--Ade+, white colony!
Doesn’t the suppressor tRNA cause problems for cells?
• Yeast has 8 tRNA-TYR genes• Only one of them has the suppressor mutation.
What reads the normal TYR codons, UAC?
What about genes that normally end in UAG?
• Not all ORFs end with UAG.• For those that do, there’s still a competition between the suppressor tRNA and termination factor.
Even so, a cell with a SUP mutation can be quite sick.
Another kind of suppression (unrelated to ade2)
WT protein 1 WT protein 2
Mutant protein 1 WT protein 2
Mutant protein 1 Mutant protein 2
Restoration of function!
Red/White and Ade+/ade-
One gene or two closely linked ones?
1. Isolate new red mutants: do they also require adenine?
2. If the adenine mutation is reverted to wild type (ADE) does the red color also revert to white?
yes
yes
3. If red is reverted to white, does ade- revert to Ade+?
AdenineYXade2 ADE1
red pigmen
t
gene R
Can we get redwhite revertants that are still ade-?
Some ideas
WADE3ade3
In an ade2 strain (red)… LOF mutation of either gene R or ADE3 white colonies, but still ade-.
ade2 mutant revert phenotype to white
Using complete media . . .
Some white colonies could be true revertants.Some mutations could be suppressors.Some could be in other genes of the pathway?
Making redwhite revertants
Treat with mutagen
rev#1
rev#2
Summary…
revert ade2 ADE2
color?
grow without adenine?
mutate ADE3 to LOFmutate gene R to LOF
white yes
white no
white no
from
Growth without adenine
distinguishes
AdenineYXade2 ADE1
red pigmen
t
gene R
WADE3
In ade2 strain:
How many mutants are like ade3?
How many are like “gene R”?
adenineADE2 ADE1
gene R
• 10 complementation groups are ade- and white
• lots and lots, but “gene R” has never been identified!
red
• Respiration defective cells can’t make red pigment. Respiration mutations are epistatic to red pigment!
*** use up 1 ATP, non-reversible step
• 2 are ade- and red.
Final tally…
Quiz Section this week:
Genetic Analysis in Caenorhabditis elegans
An introduction to C. elegans. . .
A bit of background on Caenorhabditis elegans
• 1 mm long nematode worm.
• 3.5 day generation time.
• Predominantly internally selfing hermaphrodite (make sperm and oocytes).
• Rare males arise spontaneously and can cross with hermaphrodite (male sperm fertilize hermaphrodite oocyte).
• Moves by wriggling (like a snake).
C. elegans hermaphrodite
head tail
C. elegans generates bends using dorsal and ventral muscle strips.
worm movie
Inbreeding is important for model organism genetics
• Outbred (wild) populations are genetically heterogeneous.
•Highly inbred strain has little or no genetic variability.
With each generation, ½ of the previously heterozygous alleles become homozygous.
Inbreeding makes strains homozygous for everything
QuickTime™ and aNone decompressor
are needed to see this picture.
X X XX
XX X X
Inbreeding is important for model organism genetics
• Outbred (wild) populations are genetically heterogeneous.
•Highly inbred strain has little or no genetic variability.
• Mutant alleles behave simply - only change present in cross.
• E. coli, yeast, fruit fly, C. elegans, zebrafish, mouse are highly inbred.
To conduct a mutant analysis begin with inbred WT strain, then treat with a mutagen to generate a large population of mutagenized animals
Why mutagenize?
FREQUENCY!!
Spontaneous mutations are VERY RARE.
Mutagenesis can increase frequency by about 10,000 fold.
Mutant Analysis: generating mutants
X-Rays (H. J. Muller’s X-linked “ClB” system in Drosophila)
C
l
B
How frequently do new mutations appear on this X-chromosome?
Estimation of mutation rate: X-ray-induced mutations
rossover suppressor = X-chromosome with inversions… no recombination
lethal (l) = recessive lethal (XlY males are dead)
Bar (B) = bar-shaped eyes; bar shape is DOMINANT
X-rays
x Bar-eyed femaleB l
x
How frequently do new mutations appear on this X? x Bar-eyed
female
x wt
look just at sons
If new lethal mutation…
dead
dead!
no sons!
B l
Pick Bar-eyed female progeny B l
1 female/cross; repeat many times
dead
viable
If no new mutations…
B l B l
Estimation of mutation rate: X-ray-induced mutations
X-ray dose
% X-linkedrecessiveLethalmutations
Certain external agents (mutagens) can drastically increase mutation rates.
no X-raytreatment
Estimation of mutation rate: X-ray-induced mutations
Spontaneous mutation rate (2/1000 X-chromosomes)
Spontaneous mutation rates
Measurement of spontaneous mutation rates:
2 mutations per 1000 X-chromosomes
2 mutations per 1,000,000 genes
Mutation rate = 2 x 10-6/gene/generation
Very similar rate calculated for humans!
(from assumption of 1000 genes on X)
Rough calculation:
If 35,000 genes in human genome…
2 x 10-6 x 35000 = ~ 0.07 mutations per generation
or 1 mutation (somewhere in the genome) per 14 gametes…
i.e., you would only get ~2 mutants/1,000,000 animals analyzed from spontaneous mutations - using a mutagen can increase this rate to ~2 mutants/100 animals
Radiation
- X-rays, -rays: ionizing radiation
cause breaks in DNA chromosomal rearrangements!
- Ultraviolet light: non-ionizing radiationthymine dimers
impede DNA polymerase
Some mutagens (electromagnetic radiation)
C
Chemical mutagens
- Alkylating agents, e.g., ethylmethane sulfonate (EMS)
G
EMS
O6-ethyl-G
T
- intercalating agents, e.g., acridine orangeQuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
cause frame shift mutations
base substitutions
Some mutagens (chemical mutagens)
Transposons: jumping genomic segments of DNA
Transposase gene
Small pieces of DNA (a few hundred to a few kbp in length) that can move from one site in the genome to another.
•ALL organisms have them (~45% of our genome: transposon remnants!)•Jumping genes, Selfish DNA•Mechanism for rapid evolutionary change
Transposons can also cause mutations if they hop into or near genes
Allele R
Allele r
Transposon insertion
The wrinkled pea trait that Mendel studied was caused by a transposon insertion that inactivated a gene
What are the sources of spontaneous mutations?
How are mutations repaired?
Mutation; repair of mutations
mutation!
Spontaneous mutations
- Base alteration or loss
- Replication errors
CA C G
CA A C GT T G C A T G
AT T G C A T G
new
old
corrected?
yes T A C
no CA A C GT T G C A T G
A C
CA A C GT T G C G T G
A C
replication
probably exceeds 50,000/cell/day
Damage control
Experimentally observed mutation rate in E. coli (inside the cell):
Expected error rate of E. coli DNA polymerases (from physical/chemical properties of the bases:
Experimentally observed error rate of E. coli DNA polymerases (in the test tube):
Conclusions:
-DNA polymerases must possess a “proofreading” ability.-There must be yet another backup error detection system in the cell.
1 mutation/1010 bases polymerized
1 mutation/105 bases polymerized
1 mutation/107 bases polymerized
Proof-reading by DNA polymerase
A CA C GT T G C A T G
A A C GT T G C A T G
new
oldTcorrection
DNA polymerase has 3 activities:
- can add bases to 3’ end
- the end must be base-paired
- template must be available
- can excise (remove) bases from 3’ end
Normally, addition rate >> excision rate
- can remove bases from 5’ end (involved in DNA replication and some forms of repair)
(for optimal activity)
(not covered in this course)
Damage control
Proof-reading (cont’d)
A A CT T G C A T G
TA A C GT T G C A T G
A A C GT T G C A T G
C
3’ end base-paired extension rate high
3’ end NOT base-paired extension rate low
probability of excision high
A A C GT T G C A T G
C
3’ end base-paired again!
Proof-reading corrects 99% of incorporation errors!
DNA pol
Experimentally observed mutation rate in E. coli (inside the cell):
Expected error rate of E. coli DNA polymerases (from physical/chemical properties of the bases:
Experimentally observed error rate of E. coli DNA polymerases (in the test tube):
Conclusions:
-DNA polymerases must possess a “proofreading” ability.-There must be yet another backup error detection system in the cell.
1 mutation/1010 bases polymerized
1 mutation/105 bases polymerized
1 mutation/107 bases polymerized
mismatch repair system
Damage control
“mismatched” base
Mismatch repair
Proofreading catches many errors but some still slip by; how are they detected and repaired?
GACGTACATGCTGCATGTAC
GACGTACATGCTGCATGTAC
GACGTATATGCTGCATGTAC
repair is biased; tends to restore normal sequence
GACGTACATGCTGCATGTAC
GACGTATATGCTGCATATAC
repaired unrepaired
Best understood in bacteria
1. Identify mismatched bases in DNA
2. Recognize the template strand
3. Correct the OTHER strand
mutS protein in E. coli
use methylation state of DNA to identify template strand
TGATCAACTAGT
TGATCAACTAGT
CH3
CH3
deoxyadenosine
methylase (DAM)
Mismatch repair
DNA replication
DNA replication
transiently hemimethylated template strand can be distinguished from newly synthesized strandtransiently hemimethylated template strand can be distinguished from newly synthesized strand
Mismatch repair
mutL
mutHmutS
Mismatch repair—the mutSHL system
5'-CACGTTACAAGGTCATGTTTCCGATCTA-3’3'-GTGCAATGTTCCAGGACAAAGGCTAGAT-5'
CH3
mutS protein recognizes mismatch
mutH protein recognizes parental strand
mutL protein promotes mutH activity (make cut in new strand)
TTACAAGGTCCTGTTT
TTACAAGGTCATGTTTexcise mismatch region
5'-CACG CCGATCTA-3’3'-GTGCAATGTTCCAGGACAAAGGCTAGAT-5'
CH3re-synthesize DNA
mismatch
250nm
0
10
100
% surviving
cells
0 6 12Minutes of UV irradiation
phruvrAuvrBuvrCuvrD
Repair of UV light induced DNA damageWhat genes are involved?
E. coli
WT cellsMutants defective in UV repair
2 mechanisms of UV damage repair: light-dependent and light-independent.
250nm
# pyrimidine dimers/kb
DNA
time
Blue light (300-
500nm)
in the dark
UV light pulse
5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
Phr=photolyase (+ blue light)
Pyrimidine dimers in the genome converted to small ss DNA fragments
pyrimidine dimers ‘disappear’
Repair of UV light induced DNA damage
Phr=photolyase (+ blue light)
Light-dependent UV repair mechanism
uvrD
uvrBuvrC uvrA
5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
excise damaged region
5’-CACGT TTTCCGATCT-3’3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
TACAAGGTCCTG
pyrimidine dimer
Repair of UV light induced DNA damage (cont’d)Light-independent mechanism
uvrBuvrC uvrA
This repair system also corrects alkylation damage induced by chemical mutagens (e.g. EMS, MMS, etc.)
5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
TACAAGGTCCTG
5’-CACGT TTTCCGATCT-3’3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
TACAAGGTCCTG3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
replace damaged region
5’-CACGT TTTCCGATCT-3’
Light-independent mechanism
Repair of UV light induced DNA damage (cont’d)
mut genes found in humans also… mutations in mut genes associated with colon cancer.
Mechanisms of DNA damage repair are conserved
Xeroderma pigmentosa—defective UV repair system… mutations affect genes that resemble uvrA-D (not clear if there is a phr counterpart in humans).
Testing for mutagens… the Ames test
Premise:
- start with his- bacteria (Salmonella)
- spot test compound on plate
- if the compound causes mutations… sometimes his- will mutate to his+
Interpretation:
Compound #1 = non-mutagenic
#2 = mildly mutagenic
#3 = strongly mutagenicQuestion: some compounds that are known to be mutagenic in mammals only yield positive results if pre-incubated with a liver extract; why?
1
2 3
test compounds