Download lecture_10(LP)

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*
• 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)
*See the Salvation of Doug article at the following site:
http://bio.research.ucsc.edu/people/sullivan/savedoug.html
Conducting a mutant analysis with yeast
Case study: analyzing the adenine biosynthetic
pathway by generating and studying “ade” mutants
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?
Identifying yeast mutants that require adenine
Treat wt haploid
cells with a
mutagen:
Adeninerequiring
colonies
(ade
mutants)
plate
cells
This is an example of a “genetic
-adenine
plate
m3
“complete”
plate
screen”
Replica-plate
m2
m1
sterile
piece of
velvet
Identifying interesting mutations—screen vs. selection
Screen
Each member of the population is examined… does it fit
the phenotype criteria that have been set up?
Selection
Individuals not meeting the criteria don’t survive (or are
otherwise eliminated from the population)
Example 1: Looking for a translator Russian
Screen: read resumés
Selection: advertise in Russian
English
Example 2: Looking for wingless fly mutants
Screen: Look at each fly… wings present?
Selection: Open vial, let flies fly away
Primary selection or screen is often followed by
secondary selection or screen
Reversion and Suppressors
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).
Another approach - start with a mutant
Look for reversion to wild type (or less mutant)-sometimes
called “reverse” genetics.
look for red eyes
ww
What kinds of mutations might you find?
Reversion and Suppressors
1) Mutations that restore function to the white gene (revertant).
X
w gene
or
X
X
2) Mutations that bypass (or suppress) the need for a white
gene.
X
w gene
X
w gene
plus suppressor mutation in some other gene
What kinds of suppressor mutations might you imagine?
Partial biosynthetic pathway for adenine in yeast
ADE2
Y
intermediate X
build up of
X
X
red
pigment
ADE1
adenine
ADE1
ade2
Y
adenine
Revert ade- to Ade+… does RED revert to WHITE?
Ade+ revertant
it’s white
Treat wt haploid
cells with a
mutagen:
ade2 mutant
-Ade plate
Pretty good proof that one mutation (ade2) has two
phenotypes
A working hypothesis… ade2 has reverted to ADE2
ADE2
Growth
on -ade
Color
mRNA sequence
+
white
5’..AUG....UAC....UGA..3’
STOP!
ade2
-
red
5’..AUG....UAG....UGA..3’
revertant
(ade2-R)
+
white
5’..AUG....UAC....UGA..3’
Has the mutant gene changed back to WT?
A test of the hypothesis…
…do a cross:
ADE2
x
ade2-R
:)
ADE2
the diploid is homozygous WT
ade2-R
:)
If ade2-R is “true
revertant”…
ade2-R = ADE2
meiosis
Spores from the diploid should be: All Ade+, white
Most Ade+ revertants… like this. But some exceptions!
Some revertants behave differently…
The cross:
ADE2
Ade+, white
x
revertant
Ade+, white
Diploids Ade+, white
meiosis
Most spores: Ade+, white
Some spores: ade-, red!
Interpretation?
In these revertants…
• ade2 is still in mutated form
• a new mutation somewhere else
suppresses the ade2 phenotype!
Summary of revertant types
ade revertants come in two varieties:
1. “True” revertants
ade2 mutant allele  ADE2 (wt)
2. Suppressors (aka “extragenic suppressors” or
“second site suppressors”)
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
sup3
ade2
SUP3
Ade+
white!
5’..AUG....UAG....UGA..3’
WT
Explanation?
TyrSTOP
mutation
How does SUP3 suppress ade2?
WT tRNA (sup3)
Tyr
Mutant tRNA (SUP3)
Tyr
AUG
AUC
5’..AUG....UAG....UGA..3’
5’..AUG....UAG....UGA..3’
TyrSTOP
ade2 mRNA
ade2 mRNA
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?
What reads the normal TYR codons, UAC?
• Yeast has 8 tRNA-TYR genes
• Only one of them has the suppressor mutation.
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
Mutant protein 1
WT protein 2
Mutant protein 2
Restoration of function!
Red/White and Ade+/adeOne gene or two closely linked ones?
1. Isolate new red mutants: do they also require adenine?
yes
2. If the adenine mutation is reverted to wild type (ADE)
does the red color also revert to white?
yes
3. If red is reverted to white, does ade- revert to Ade+?
Can we get redwhite revertants that are still ade-?
Some ideas
W
ade3
ADE3
X
ade2
Y
ADE1
Adenine
gene R
red
pigment
In an ade2 strain (red)… LOF mutation of either gene R
or ADE3  white colonies, but still ade-.
Making redwhite revertants
Using complete media . . .
rev#1
Treat with
mutagen
ade2 mutant
rev#2
revert phenotype to white
Some white colonies could be true revertants.
Some mutations could be suppressors.
Some could be in other genes of the pathway?
Summary…
W
ADE3
X
ade2
Y
ADE1
Adenine
gene R
red
pigment
color?
grow without
adenine?
white
yes
white
no
white
no
Growth without adenine
distinguishes In ade2 strain:
revert ade2  ADE2
from
mutate ADE3 to LOF
mutate gene R to LOF
Final tally…
How many mutants are like ade3?
• 10 complementation groups are ade- and white
• 2 are ade- and red.
ADE2
ADE1
*
gene R
red
How many are like “gene R”?
adenine
*
* use up 1 ATP, nonreversible step
• lots and lots, but “gene R” has never been identified!
• Respiration defective cells can’t make red pigment.
Respiration mutations are epistatic to red pigment!
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.
Inbreeding makes strains homozygous for everything
XX
X
X
X
X
X
X
QuickTime™ and a
None decompressor
are needed to see this picture.
With each generation, ½ of the previously
heterozygous alleles become homozygous.
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.
Mutant Analysis: generating mutants
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.
Estimation of mutation rate: X-ray-induced mutations
X-Rays (H. J. Muller’s X-linked “ClB” system in Drosophila)
C rossover suppressor = X-chromosome with inversions… no recombination
l ethal (l) = recessive lethal (XlY males are dead)
B
B ar (B) = bar-shaped eyes; bar shape is DOMINANT
l
X-rays
x
x
How frequently do new mutations
appear on this X-chromosome?
B
l
Bar-eyed
female
Estimation of mutation rate: X-ray-induced mutations
How frequently do new
mutations appear on this X?
x
B
1 female/cross; repeat many times
wt
x
B
l
look just at sons
Pick Bar-eyed
female progeny
If no new mutations…
dead
Bar-eyed
l female
B
l
If new lethal mutation…
B
l
dead
dead!
viable
no sons!
Estimation of mutation rate: X-ray-induced mutations
% X-linked
recessive
Lethal
mutations
Spontaneous
mutation rate
(2/1000 Xchromosomes)
X-ray dose
no X-ray
treatment
Certain external agents (mutagens) can drastically increase
mutation rates.
Spontaneous mutation rates
Measurement of spontaneous mutation rates:
2 mutations per 1000 X-chromosomes
 2 mutations per 1,000,000 genes (from assumption of 1000 genes on X)
 Mutation rate = 2 x 10-6/gene/generation
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
Very similar rate calculated for humans!
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…
Some mutagens (electromagnetic radiation)
Radiation
- X-rays, -rays: ionizing radiation
cause breaks in DNA
 chromosomal rearrangements!
- Ultraviolet light: non-ionizing radiation
thymine dimers
impede DNA polymerase
Some mutagens (chemical mutagens)
Chemical mutagens
- Alkylating agents, e.g., ethylmethane sulfonate (EMS)
C
T
EMS
G
O6-ethyl-G
 base substitutions
- intercalating agents, e.g., acridine orange
cause frame shift mutations
QuickTime™ and a TIFF (Uncomp resse d) de com press or are nee ded to s ee this picture.
Transposons: jumping genomic segments of DNA
Small pieces of DNA (a few
hundred to a few kbp in length)
Transposon insertion
that can
move
Allele
R from one site in the genome to another.
Allelethem
r (~45% of our genome:
•ALL organisms have
transposon remnants!)
•Jumping genes, Selfish DNA
The wrinkled
•Mechanism for rapid evolutionary
change
pea trait that
Mendel studied
caused by
Transposasewas
gene
a transposon
insertion that
inactivated a
gene
Transposons can also cause mutations if they hop
into or near genes
Mutation; repair of mutations
What are the sources of spontaneous mutations?
How are mutations repaired?
Spontaneous mutations
- Base alteration or loss probably exceeds 50,000/cell/day
- Replication errors
yes
new
old
AACG C
TT GC A TG
AACG C
TAC
TT GC A TG
corrected?
no
AA CG CAC
TT GC A TG
replication
mutation!
AA CG CAC
TT GC GTG
Damage control
Experimentally observed mutation rate in E. coli (inside the
cell):
1 mutation/1010 bases polymerized
Expected error rate of E. coli DNA polymerases (from
physical/chemical properties of the bases:
1 mutation/105 bases polymerized
Experimentally observed error rate of E. coli DNA polymerases
(in the test tube):
1 mutation/107 bases polymerized
Conclusions:
-DNA polymerases must possess a “proofreading” ability.
-There must be yet another backup error detection system in
the cell.
Damage control
Proof-reading by DNA polymerase
new
old
AA CG C
TT GC A TG
correction
AACG T
TT GC A TG
DNA polymerase has 3 activities:
- can add bases to 3’ end
- the end must be base-paired (for optimal activity)
- 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) (not covered in this course)
Proof-reading (cont’d)
3’ end base-paired  extension rate high
AA C
TT GC A TG
AACG T
TT GC A TG
AA CG
TT GC A TG
3’ end NOT base-paired 
extension rate low
probability of excision high
AACG
TT GC A TG
3’ end basepaired
again!
Proof-reading corrects 99% of incorporation errors!
Damage control
Experimentally observed mutation rate in E. coli (inside the
cell):
1 mutation/1010 bases polymerized
Expected error rate of E. coli DNA polymerases (from
physical/chemical properties of the bases:
1 mutation/105 bases polymerized
Experimentally observed error rate of E. coli DNA polymerases
(in the test tube):
1 mutation/107 bases polymerized
Conclusions:
-DNA polymerases must possess a “proofreading” ability.
-There must be yet another backup error detection system in
the cell.
mismatch repair system
Mismatch repair
Proofreading catches many errors but some still slip by; how
are they detected and repaired?
GACGTACATG
CTGCATGTAC
GACGTACATG
CTGCATGTAC
“mismatched”
base
GACGTATATG
CTGCATGTAC
repair is biased; tends to
restore normal sequence
GACGTACATG
CTGCATGTAC
repaired
GACGTATATG
CTGCATATAC
unrepaired
Mismatch repair
Best understood in bacteria
1. Identify mismatched bases in DNA
mutS protein in E. coli
2. Recognize the template strand
use methylation state of DNA to identify template strand
CH3
TGATCA
ACTAGT
deoxyadenosine
methylase (DAM)
TGATCA
ACTAGT
CH3
3. Correct the OTHER strand
Mismatch repair
transiently
hemimethylated
template strand can
be distinguished
from newly
synthesized strand
transiently
hemimethylated
template strand can
be distinguished
from newly
synthesized strand
DNA replication
DNA replication
Mismatch repair—the mutSHL system
mutS protein recognizes mismatch
mutH protein recognizes parental strand
mutL protein promotes mutH activity (make cut in new strand)
mismatch
mutS
mutH
5'-CACGTTACAAGGTCATGTTTCCGATCTA-3’
3'-GTGCAATGTTCCAGGACAAAGGCTAGAT-5'
CH3
mutL
excise mismatch region
TTACAAGGTCATGTTT
5'-CACGTTACAAGGTCCTGTTTCCGATCTA-3’
3'-GTGCAATGTTCCAGGACAAAGGCTAGAT-5'
CH3
re-synthesize DNA
Repair of UV light induced DNA damage
What genes are involved?
250nm
E. coli
100
WT cells
% surviving
10
cells
0
Mutants
defective in
UV repair
0
6
12
Minutes of UV irradiation
phr
uvrA
uvrB
uvrC
uvrD
Repair of UV light induced DNA damage
2 mechanisms of UV damage repair: light-dependent and
light-independent.
Pyrimidine dimers in the
genome converted to
small ss DNA fragments
in the dark
# pyrimidine
dimers/kb
DNA
250nm
Blue light
(300-500nm)
UV light pulse
time
pyrimidine dimers
‘disappear’
5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’
3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
Phr=photolyase (+ blue light)
5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’
3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
Light-dependent UV repair mechanism
Phr=photolyase (+ blue light)
Repair of UV light induced DNA damage (cont’d)
Light-independent mechanism
pyrimidine dimer
uvrC
uvrA uvrB
5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’
3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
excise damaged region
uvrD
5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’
3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
Repair of UV light induced DNA damage (cont’d)
Light-independent mechanism
uvrC
uvrA uvrB
5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’
3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
TACAAGGTCCTG
5’-CACGT
TTTCCGATCT-3’
3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
replace damaged region
5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’
3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’
This repair system also corrects alkylation damage induced by
chemical mutagens (e.g. EMS, MMS, etc.)
Mechanisms of DNA damage repair are conserved
mut genes found in humans also… mutations in mut
genes associated with colon cancer.
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 hiswill mutate to his+
test compounds
1
2
Interpretation:
Compound #1 = non-mutagenic
3
#2 = mildly mutagenic
#3 = strongly mutagenic
Question: some compounds that are known to be mutagenic in mammals
only yield positive results if pre-incubated with a liver extract; why?