Yeast

Question 1

temperature sensitive mutant screen

Answer 1

1. mutagenize haploid yeast using EMS
2. plate on YPD media @ 25 degrees Celsius
3. replica plate on YPD media @ 36 degrees
4. select mutants that don’t grow @ 36 degrees but 25 degrees
   -allows you to study function of essential genes in haploid yeast

Question 2

what are the two types of suppressors?

Answer 2

1. intragenic- same gene, which is true revertant and second site mutation
2. extragenic- different gene

Question 3

interaction suppressor

Answer 3

-allele specific
-does not rescue the null mutation

Question 4

yeast nomenclature

Answer 4

-YFG1 = WT
-yfg1 = mutant
-yfg1 with a triangle = deletion

Question 5

bypass suppressor

Answer 5

-rescues a null because the suppressor gains a function and allows the cell to no longer need the protein that has been mutated
-can provide novel function

Question 6

epistatic suppressor

Answer 6

-a type of bypass suppressor
-rescues a null since it no longer needs either component of a pathway and the pathway can continue forward as it would without proteins

Question 7

dosage suppressor

Answer 7

-restores balance in a system
-does not rescue a null
-may be allele-specific since the allele may help restore balance

Question 8

mass action suppressor

Answer 8

-type of dosage suppressor
-can stabilize mutant by interaction
-does not rescue a null
-may also be allele-specific

Question 9

screen

Answer 9

-distinguish individual organisms by phenotype
-BOTH WT and mutants grow
Ex. can’t lose plasmid experiment and temperature sensitive mutants generated by EMS

Question 10

suppressor screen

Answer 10

take mutant cells –> mutagenize them –> plate on rich media and look for mutations that in combination with original mutation allow the cells to grow
-may miss duplicated genes, essential genes, and small genes
-send the suppressor strains for sequencing to see common mutations in them and you’ll find gene where suppressor mutation is located

Question 11

selection

Answer 11

allows for the specific enrichment/growth of mutants of interest
Ex. isolate suppressors of a temperature sensitive allele

Question 12

“can’t lose plasmid” yeast screen

Answer 12

1. start with plasmid that has TUB2 and URA3
2. transform tub2-1 yeast with the plasmid
3. mutagenize with EMS
4. plate on non-selective medium
5. replica plate onto 5-FOA medium
6. sequence the cells that grow on non-selective media but not 5-FOA
   -synthetic lethal mutant will grow on non-selective medium and not on 5-FOA since it must retain plasmid carrying the URA3 gene with TUB2

Question 13

synthetic genetic array (SGA)

Answer 13

-genome-wide scale in unbiased way to ID genetic interactions and gene functions
-helps you to find genes with similar or same functions to gene of interest- turns out when you do this that genes with same or similar functions have the same pattern of genetic interactions with other genes and you’ll see groups of them forming
-genes showing strong positive interactions often code for subunits of a protein complex
-can’t do this with essential genes since they will die with mutations

Question 14

SGA analysis

Answer 14

1. using haploid yeast deletion collection, construct all possible double mutants
2. quantitatively assess growth phenotype of single and double mutants by measuring diameter of colonies and assigning growth scores
3. ID positive or negative interactions
   -use multiplicative model: deletion of gene 1 * deletion of gene 2 = expected

Question 15

positive interaction

Answer 15

grows better than expected and indicates same pathway

Question 16

negative interaction

Answer 16

grows worse than expected

Question 17

dead interaction

Answer 17

synthetic lethal