Announcements -Solutions to problem set 3 and from the questions pertaining to last Fridays lecture have been posted on the course website. -Problem set

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


X-ray dose

% X-linkedrecessiveLethalmutations

Certain external agents (mutagens) can drastically increase mutation rates.

no X-raytreatment


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



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


C

3’ end base-paired extension rate high

3’ end NOT base-paired extension rate low

probability of excision high


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.




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


GACGTATATGCTGCATGTAC

repair is biased; tends to restore normal sequence


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’


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


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.)


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

Documents

Announcements -Solutions to problem set 3 and from the questions pertaining to last Fridays lecture have been posted on the course website. -Problem set