Yeast Molecular Biology I

Question 1

Why is yeast a popular in tool in molecular biology?

Answer 1

Transformation with recombinant DNA is efficient
Targeted gene replacement is straightforward
Gene expression and regulation systems are well characterised
Genetic map is very detailed and complete sequences of the genome is known

Question 2

What methods can be used for yeast transformation?

Answer 2

Use of: sphaeroplasts, lithium acetate, electroporation

Question 3

What’s in a yeast plasmid?

Answer 3

Replication origin (unless the plasmid is integrative)
Selectable marker for transformation of yeast
Sequences for selection and replication of E. coli (yeast vectors are shuttle plasmids)
Additional sequences for cloning and/or expression in yeast

Question 4

What type of selectable marker should be used for genetically defined strains? (usually haploid)

Answer 4

Selectable markers that complement an auxotrophic requirement are often used

Question 5

What type of selectable marker should be used for industrial or WT strains (usually polyploid)?

Answer 5

Dominant selectable markers

Question 6

What are 5 examples of yeast plasmids?

Answer 6

YIp, YRp, YCp, YEp, YAC

Question 7

What are the properties of YIps?

Answer 7

Yeast integrative plasmid, its transformation frequency is very low but stability is high

Question 8

What are the properties of YRps?

Answer 8

Yeast replicating plasmid, contains ARS, transformation frequency very high, not stable

Question 9

What are the properties of YCps?

Answer 9

Yeast centromeric plasmid. Same as YRps but with addition of a chromosomal centromere to increase its stability

Question 10

What are the properties of YEps?

Answer 10

Yeast episomal plasmids. Contain all or part of the yeast 2µm circle. Transformation frequency high, copy number can be very high (>100 copies)

Question 11

What are the properties of YACs?

Answer 11

Yeast artificial chromosomes. Used to clone very large DNA fragments - stable

Question 12

What can YAC cloning be used for?

Answer 12

Preparations of higher eukaryote genomic libraries (positional gene cloning)
Gene therapy (allowing preparation and testing on mammalian artificial chromosomes)
Synthetic biology
Metabolic pathway engineering

Question 13

What type of plasmid would you use for functional cloning?

Answer 13

YCp

Question 14

What type of plasmid would you use if you wanted high stability?

Answer 14

YIp

Question 15

What type of plasmid would you use if you wanted higher expression levels of a gene

Answer 15

YEp (due to high copy number)

Question 16

What is the plasmid eviction experiment used for?

Answer 16

To check the complementation phenotype is due to the plasmid

Question 17

How can you determine if you’ve cloned the correct gene?

Answer 17

Delete cloned gene in WT yeast
Mate to original mutant
Sporulate resulting diploid
Examine progeny following tetrad analysis

Question 18

What is the structure of a yeast gene?

Answer 18

D R A W

Question 19

What are some gene deletion/disruption methods?

Answer 19

Traditional cloning (with recombinant DNA)
PCR - short flanking homology (SFH) or long flanking homology (LFH)

Question 20

If a gene is non-essential what is the ratio of Yfg+ to Yfg- after sporulation?

Answer 20

2:2 (4 viable progeny)

Question 21

If a gene is essential what is the ratio of Yfg+ to Yfg- after sporulation?

Answer 21

2:0 (2 viable progeny)

Question 22

What is used if you want to delete more than one gene in the same yeast species?

Answer 22

Bacteriophage P1 Cre recombinase

Question 23

What is a shuttle vector?

Answer 23

A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species [1]. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types. The main advantage of these vectors is they can be manipulated in E. coli, then used in a system which is more difficult or slower to use (e.g. yeast)

Question 24

Outline an example of plasmid eviction experiment

Answer 24

URA3 plasmid eviction experiment with 5-FOA
Ura3+ yeast (containing plasmid with URA3 gene) are grown on a medium containing 5-FOA and a small amount of uracil
5-FOA is decarboxylate due to toxic 5-fluorouracil by the product of the URA3 gene
Yeast cells that survive FOA treatment are (usually) ura3 mutants i.e. have lost the plasmid
Check the phenotype of cells - are they are the original mutant?
The screen for the mutant allele can be time consuming if the mutant does not display an obvious phenotype