yeast and mutant screens Flashcards
useful properties of yeast for genetics?
- Can exist stably as haploids (n) or diploids (2n)
- Are single-celled eukaryotes
- Forms compact colonies on plates
- Grown in large quantities in liquid medium
- Rapid life cycle (90 minutes)
- Small, compact genome (12 Mb)
- Efficient homologous recombination (handy for making knockouts)
- Excellent genomic resources https://yeastgenome.org/
CON - cant be used to study multi-cellular processes involving cell-cell signalling
explain the yeast life cycle
Two different haploid mating types, a and α. Determined by MATa and MATα
Two haploids of the different mating types can fuse to form a diploid…
Can enter mitosis and produce more diploids
Can undergo meiosis, forming an ‘ascus’. forms four haploid spores, two will be ‘a’, two will be α
***Haploids can also just undergo mitosis (no fusion first) and continue producing more haploids (not four tho obvi as its not mitosis)
how can the haploid/diploid aspect of yeast be useful?
You can immediately see the impact of a recessive mutation in a haploid (only one mutant copy needed)
Can look at lethal mutations in diploid heterozygotes, when this would kill haploids immediately
what was the goal of the Saccharomyces Genome Deletion Project?
generate a complete set (as far as possible) of yeast deletion strains with the overall goal of assigning function to the ORFs through phenotypic analysis of the mutant
how were mutants created in the Saccharomyces Genome Deletion Project?
Used a PCR-based deletion strategy
had a normal yeast genome…
- Designed linear DNA to perform knockout:
Ends = 45 bp primers - homologous to target gene to ensure the knockout occurs in the right place
- Use of an upstream and downstream tag to later map where the knockout occurred/which gene was knocked out
- Kanamycin resistance cassette replaced the yeast ORF to prevent replication/knock out the gene in question, and allow for selection of successful integration by homologous recombination (by applying the antibiotic kanamycin)
what mutant collections did the Sac. genome deletion project make?
Haploid (n) mating type a
Haploid (n) mating type α
Diploid (2n) homozygous for knockout (for non-essential genes)
Diploid (2n) heterozygous - necessary for knocking out essential genes (will still be able to live as it should have one functional copy)
give an overview of a forward genetic screen
Finding the gene responsible for the phenotype you are interested in
Amass mutants that can’t do what you are interested in
Cross with WT individuals to see if offspring show ratios of WT:mutant phenotype to see if it shows single-gene inheritance (has to be this way, you want to focus on one gene at a time)
Deduce gene function at molecular level
Deduce how the gene interacts with other genes to produce the trait you are interested in
Identify and test homologues in other species (yeast is a good starting point, but not entirely applicable to humans, tho high conservation between yeast and humans = likely to be a homologue)
in a forward genetic screen - what choice would you make for recessive mutations?
mutations in genes for essential processes?
recessive - immediately see the phenotype in a haploid
essential - use a conditional mutant (e.g. temperature sensitive) in order to keep your cells alive - turn up the heat - view the mutation is action
how are temperature sensitive mutants (or condition sensitive ones) found?
- Add mutagen to yeast to create point mutations
- Collect lots of colonies
- Use velveteen ‘stamp’ to copy colonies from one plate to another
- You can then grow identical colonies in two different conditions (for us it would be temperature) and play spot the difference, e.g. one of the colonies on your plate growing only at the low temperature and not at the more restrictive temperature
These are mutations across the genome, so not all secretion obviously, just all lethal (if its growing vs not growing at all) and temperature sensitive
why do you need temperature sensitive mutants when looking for genes involved in secretion?
mutations in secretion genes would be lethal
so for haploids it would have to be temperature sensitive to allow them to survive at all (at the permissive temperature)
for diploids you could use heterozygotes but then how do you see the result of the mutation ALONE?
once a load of temperature sensitive lethal mutants had been identified, how did they find out which were mutants in the secretion pathway?
secretion assay - looked for mutants unable to uptake or secrete assayable molecules
Focused on -
- Acid phosphatase and invertase (converts sugars from one to another), two enzymes usually secreted by yeast
- Chromate (lethal IF uptaken)
- Sulphate permease - transport protein that allows chromate into the cell
A WT cell - the surrounding media should have acid phosphatase and invertase present (you can assay for their activity)
In a mutant - these enzymes would be stuck inside the cell
- Grow mutant in permissive (low) temperature and then shift to non-permissive temperature
- Assay the growth medium for enzyme (acid phosphatase) activity at various time points across the experiment
Select mutants that fail to secrete acid phosphatase at non-permissive temperatures - the graph shows the shift to the restrictive temperature and the SEC mutants no longer secreting acid phosphatase (less of its activity seen in the assay)
what was seen in the first sec mutants viewed?
How was this information used to perform a more efficient, second screen for secretion mutants?
Loads of vesicles - cus it cannot secrete them
Smaller - cannot grow as cannot deliver stuff to the membranes
Darker - dense as it is producing materials to grow but cannot
As the mutants appeared darker - he used a sucrose density gradient to isolate all the different mutants easily
Grows mutants at permissive temperature
Puts them at the restrictive temperature
Allows to grow
Separates on density gradient - to get all the ones with mutations affecting secretion
Allows him to screen more and more quickly
surprise karaoke! Sing your favourite song
what is the purpose of complementation tests?
what are they?
Purpose - to identify if these were mutations in different genes, or just a different mutations in the same gene
Cross two recessive mutations
Check whether the resultant F1 diploid individual(s) have wild-type or mutant phenotype…
If the F1 progeny display the mutant phenotype, then the two mutations must be recessive alleles of the same gene (in a diploid a recessive mutant only shows if both alleles are mutated)
If wild-type, then the two mutants are said to have complemented (been in different genes). This must be the case because their has to be a functional copy of each gene
what was identified in this screen for mutations of the secretion pathway?
sec1 - was for a SNAP….
The density gradient allowed him to find another 23 sec mutants
Group 1 - Ten genes that when mutated accumulate secretory vesicles (like sec1) look like an aero
2. Group 2 - Nine genes that when mutated accumulate ER-like structures (ER to golgi)
3. Group 3 - Two genes that when mutated caused strange golgi-like organelle to form (golgi to vesicle)
