Remember that we will start the morning

1
Remember that we will start the morning by voting
on the identify of mystery strains A, B, and C
from the experiment on Wed.
Please analyze the results before class and bring
your yeast plate with you!
2
How do you determine number of genes?
Mutant 1 (a strain) crossed to Mutant 2 (a
strain)
If diploid has mutant phenotype
If diploid has wild-type phenotype
3
Complementation Analysis
a strain
A
B
C
D
allele

-


E
-



F
a strain
-
-


G




H
Is the screen saturated??
4
Exceptions to the rules (complementation group
gene)
Intragenic complementation
Non-allelic, non-complementation
5
How can we verify if our interpretation is
correct?
If diploid is wild type
If diploid is mutant
6
Cloning by functional complementation...
Galactose
Glucose
GAL
gal
gal
gal
7
Yeast plasmids come in several flavors
adapted from http//saturn.roswellpark.org/huberma
n/BIR572/Fig13YeastGenetics.html
8
Is the gene we have cloned essential?
YFG
KANR
YFG
9
What kinds of mutations provide useful genetic
tools?
Null (or knockout)
Hypomorphic
Conditional
Separation of function
Gain-of-function
Dominant-negative
10
How is genetics used to explore protein function?
Epistasis
Suppressor Analysis
Synthetic lethality
11
Epistasis
Ade2
Ade3
AIR
Red
RNA
DNA
ATP
cell growth
A
B
C
12
Activating (gain-of-function) mutations aid
epistasis analysis
13
Suppressor Analysis Identification of
interacting genes
Interacting genes
Suppressor
Permissive temperature
Restrictive temperature
14
Overexpression Suppression
15
Overexpression Suppression
Advantages
Disadvantages
16
What about suppression in the absence of
overexpression?
tRNA suppressor
Revertant or intragenic suppressor
Extragenic suppressor
How do you distinguish these classes of
suppressors?
17
Suppressor Analysis
ats-sup x A
Revertant or intragenic suppressor
ats bsup x A B
Extragenic suppressor
18
How can we interpret extragenic suppression?
Allele-specific suppressor
Non-allele specific suppressor
Bypass suppressor
19
An example of allele-specific suppression...
EST1 CDC13
est1-62 CDC13
est1-60 CDC13
est1-62 cdc13-9
est1-60 cdc13-2
20
Synthetic Lethality Another type of genetic
interaction
A
1) Redundant genes
growth
2) Parallel pathways
B
3) Interacting proteins
21
How do we screen for a mutation that results in
lethality?
Ade2p
Ade3p
AIR
Red
RNA
DNA
ATP
Colony color?
ADE2 ADE3
ade2 ADE3
ade2 ade3
22
Screen for Synthetic Lethality
Colony color?
ade2 ade3
yfg1
ADE3
YFG1
ade2 ade3
yfg1
ade2 ade3
yfg1
23
Screen for Synthetic Lethality
ade2 ade3
yfg1
SLY1
ade2 ade3
yfg1
sly1
24
How do you clone sly1?
ade2 ade3
yfg1
sly1
25
Yeast has been a leader in development of genomic
resources
Complete sequence
Transcription arrays
Gene knock-out collection
Genome-wide synthetic lethal screens
Genome-wide 2-hybrid screens
Efforts to epitope tag every gene
26
Resource for Saccharomyces information
Saccharomyces Genome Database
http//genome-www.stanford.edu/