S. Cerevisiae cross Flashcards
Why use S. Cerevisiae?
1) Easy to grow and cross, produces many progeny
2) Lots of mutant strains available, possible to demonstrate several genetic phenomena in 1 experiment
3) Has lots of life cycle as haploid organism so no need to worry about dominance relationships
What is S. Cerevisiae?
A unicellular eukaryotic budding yeast
How does it get energy?
Using fermentation to degrade glucose and other organic nutrients
Draw life cycle and describe
(see notes for diagram)
Cells of opposite type are a and alpha and they fuse to make diploid cells that continue to proliferate by mitotic division
Diploid cells undergoing carbon/ nitrogen starvation undergo meiosis and sporulation to give rise to 4 haploid progeny enclosed in an ascus
Once spores are released they can germinate and proliferate again
How can S. Cerevisiae be genetically modified?
Targeted mutagenesis
Homologous recombination has a high efficiency so targeted insertion of exogeneous DNA fragments into the genome can be used to delete/tag ORFs (ie. RNA ORF never gets made)
(NB: ORF is also in DNA, it is the bit that gets made into the RNA ORF)
What are steps of making a master plate?
1) spread spores from the cross on a petri dish with rich medium
2) Incubate the petri dishes at 23 degrees for a week
Species will germinate and give rise to different colonies
3) Use template (see notes for diagram) to create a master plate of 48 regularly arranged colonies that can be plated onto different media to detect mutant phenotypes
Why were all resulting colonies white after making the master plate?
(bear in mind ade2 mutants red in limiting adenine)
The plate contained a large excess of adenine preventing red pigment accumulation even in ade2 mutants
Why are large numbers of progeny required?
To detect rare recombinants
What are the p1 and p2 bits on the master plate?
Controls allowing you to check phenotypes (not included in final count)
How do you create a random sample?
1) Pick randomly ie. don’t target any big/small colonies
2) Don’t pick colonies that are touching other colonies as there is a risk of mixing genotypes
3) use the flat end of a sterile toothpick and transfer it as a small streak of less than 0.5 cm onto the master plate
4) Inoculate the p1 and p2 spots with the parental strains (checks that you inoculated the samples correctly)
What did our experiment show about how Ras1 and Ras2 delta interact?
Ras 1 delta and ras2 delta are colethal and disrupt the PKA pathway that is essential in S. Cerevisiae
Do ras2 delta mutants grow on the URA plate?
No
Draw out Ras1 delta/+ +/Ras2 delta cross
(see notes for diagram)
What is the equation for recombination frequency?
Recombination frequency = (Recombination/total progeny)
What does it mean if a) RF=50%? b) RF is under 50% ?
a) genes segregate independently
b) genes are linked
