Yeast Genetics

Question 1

What can yeast mutants be used for?

Answer 1

To work out metabolic pathways, dissect molecular interactions, identify proteins involved in particular biological processes (e.g. mitochondrial biogenesis)

Question 2

What does URA3 mean? (nomenclature)

Answer 2

A dominant allele with Ura+ phenotype

Question 3

What does ura3 mean? (nomenclature)

Answer 3

A recessive allele with Ura- phenotype

Question 4

What does ura3-1 mean? (nomenclature)

Answer 4

A specific recessive allele

Question 5

What does ura3-∆4 mean? (nomenclature)

Answer 5

A specific deletion, either full or partial

Question 6

What does leu2-3,112 mean? (nomenclature)

Answer 6

Specific point mutations in LEU2 gene

Question 7

Explain each element of YAR050w (nomenclature)

Answer 7

Y = yeast; A = no. of chromosome; R = side of centromere; w = strand of coding sequence

Question 8

What does cyc1::URA3 mean? (nomenclature)

Answer 8

Insertion of the URA3 allele at the CYC1 locus

Question 9

What does cyc1∆1::URA3 mean? (nomenclature)

Answer 9

Insertion of the URA3 allele at the CYC1 locus with deletion of part/all of CYC1

Question 10

What does ρ+ mean? (nomenclature)

Answer 10

Respiratory-competent (yeast possess functional mitochondria)

Question 11

What does ρ- mean? (nomenclature)

Answer 11

Respiratory-defective (mitDNA defective due to mutation giving rise to the petite phenotype)

Question 12

What does ρ0 mean? (nomenclature)

Answer 12

Respiratory-deficient (mitDNA absent but there are mitochondria!)

Question 13

What does cir+ mean? (nomenclature)

Answer 13

Yeast contains 2µm plasmid

Question 14

What does circ0 mean? (nomenclature)

Answer 14

Yeast lacks 2µm plasmid

Question 15

What yeast should be used in mutagenesis and why?

Answer 15

Haploid yeast to allow expression of mutant phenotype

Question 16

How is mutagenesis achieved?

Answer 16

Use chemicals (e.g. ethyl methanesulphonate); UV light; targeted recombination

Question 17

What are the steps of mutagenesis?

Answer 17

1. Treat population of cells with mutagen (usually mutate isogenic a and α strains?)
2. Plate cells onto a permissive medium (i.e. one on which all cells will grow into colonies)
3. Select or screen for mutants e.g. by replica plating onto selective medium

Question 18

What are the different types of mutants?

Answer 18

Loss of function (null mutants) - full or partial
Conditional lethal mutants e.g. temperature sensitve (ts) or cold sensitive (cs)
Gain of function
Suppressor mutation (a second mutation that alleviates or reverts the phenotypic effects of an already existing mutation)

Question 19

How can yeast mutants be identified? What are the differences between them?

Answer 19

Select or screen (select is first choice).
Positive selection: only mutants grow
Negative selection: mutants can’t grow
Screen: everything grows but mutants look different

Question 20

What is an example of positive selection?

Answer 20

Cadmium resistance. Plate mutagenised cells onto plate containing toxic concentration of cadmium, pick colonies that grow

Question 21

How can yeast mutants be screened?

Answer 21

ts mutants: grow cells at permissive temperature (23ºC) before replica plating onto fresh medium and incubate and restrictive temperature (37ºC)
Auxotrophic mutants, eg Leu: grow on medium + leucine then replica plate onto medium lacking leucine (leu mutants cannot grow on minimal media lacking leucine)

Question 22

What are the steps in replica plating?

Answer 22

Mutagenised yeast on a permissive medium are transferred using sterile velvet onto an appropriate medium and then incubated. The mutants on the test medium can be identified

Question 23

Explain the colony sector assay

Answer 23

It can be used to distinguish between particular markers. For instance, ade1, ade2, and ade3, involved in the adenine biosynthetic pathway, are adenine auxotrophs. ade1 and ade2 will accumulate a red pigmented precursor but the ade3 mutant cannot take up this precursor so will appear white as usual. This is because the enzyme encoded by ADE3 is before ADE1 and ADE2 in the adenine pathway. White sectors are where cells have lost the ADE3 carrying plasmid to reveal the Ade3- phenotype, and the solid red colonies never lose the plasmid. Therefore, it is essentially a measure of the plasmid’s stability in yeast.

Question 24

What is the advantage of using colour markers over typical markers?

Answer 24

Colour markers can be assayed in a single step, whereas typical selectable markers are usually assayed by replica plating to media lacking a particular metabolite

Question 25

How can splice-site mutants be isolated?

Answer 25

Construct a reporter gene dependent on splicing, insert an exon and intron upstream of the reporter. The WT can splice intron so Lac+; mutant cannot splice intron so Lac-

Question 26

What are the two kinds of suppressor mutations?

Answer 26

Intra-genic and extra-genic (very useful in identifying genes/proteins that interact)

Question 27

How can mutants be characterised?

Answer 27

Through phenotype testing (e.g. viability and/or sensitivity testing - drugs etc; biochemical tests - enzyme assays, protein interactions or “pull down” assays) and genetic interaction (complementation, suppressors, co-lethality)

Question 28

What is an example of sensitivity test and how does it work?

Answer 28

Spot test. Used with any toxic compound e.g. cadmium. Used to compare mutants and is a low-tech screen

Question 29

What three genetic tests are used to characterise mutants?

Answer 29

Tetrad analysis, mating to WT, and complementation analysis

Question 30

What is tetrad analysis used for?

Answer 30

To see if the phenotype is caused by a single mutation

Question 31

What is mating to WT used for?

Answer 31

To see if mutations are dominant or recessive

Question 32

What is complementation analysis used for?

Answer 32

To see how many genes are responsible for your phenotype

Question 33

Explain the process of mating to WT

Answer 33

Mate mutant to opposite mating type WT and check the phenotype of the diploid
If diploid is phenotypically WT, the mutation is recessive; if phenotypically mutant, mutation is dominant (most mutations are recessive)

Question 34

Explain process of complementation analysis

Answer 34

Mate all recessive MATa mutants to all recessive MATα mutants and screen phenotypes.
If progeny is mutant, no complementation so mutation is on the same gene
If progeny is WT, complementation so mutation on different gene

Question 35

Explain process of tetrad analysis

Answer 35

Mate mutant and WT haploids, the resulting diploid will be phenotypically WT if mutation is recessive. After meiosis, a tetrad is produced. If mutation is in single gene there will be a 2:2 ratio of inheritance - 2 WT and 2 mutant spores

Question 36

How is the haploid progeny recovered (from the tetrad)?

Answer 36

Briefly treat with zymolyase or lyticase and separate spores onto agar plate using a micromanipulator

Question 37

What does something other than a 2:2 ratio mean?

Answer 37

There is more than 1 mutation responsible for phenotype

Question 38

What does a 4:0 ratio mean?

Answer 38

(i.e. all progeny have the same phenotype) suggests a non-nuclear determinant

Question 39

What are some non-nuclear determinants?

Answer 39

mitDNA; plasmids (2µm, killer plasmids); prions (Ure3, psi)

Question 40

What else can tetrad analysis be used for?

Answer 40

Mapping yeast genes. Now done by cloning and sequencing, or hybridisation

Question 41

What is meant by the petite phenotype?

Answer 41

A mutation found first in yeast where “petite” yeast cannot grow on media containing non-fermentable carbon sources and grow only small colonies on media containing fermentable carbon sources. This is due to a defect (mutation) in the respiratory chain