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)
temperature sensitive mutants are typically?
thought to be caused by typically point mutations prone to mis-folding into an inactive form at restrictive temperatures
Permissible temperature - e.g. 25oC you’ve got a functional protein, 37oC the protein misfolds and you see the phenotype of the mutant
Remember - the mutation is present at both temperatures, it’s impact/phenotype is only visible at a certain temperature
why was the secretion study relevant?
The proteins involved in secretion are conserved across eukaryotic organisms - so this study was very relevant in wider biology and medicine. Secretion is essential for delivery of membrane proteins, neurons, biotechnology (want cells producing your drug to pump it out) etc…
chromosome instability (CIN) -
what is it?
caused by?
what does it result in?
Chromosome instability = hallmark of cancer
Its a change in chromosome structure/number causing chromosome gain/loss
Caused by - failures in mitotic chromosome transmission (a failure to divide chromosomes correctly in mitosis) OR defects in the mitotic spindle checkpoints
Unbalanced division of chromosomes in mitosis leads to aneuploidy offspring (as in cells) - leading to cell death OR cancer progression
CIN exacerbates tumorigenesis - accumulative large-scale genome rearrangements increases chances of disrupting expression/function of oncogene or tumour suppressor gene
explain what is meant by variants of uncertain significance
Cancer cells accumulate LOTS of mutations as cell cycle checkpoints etc… are not functioning
So they have lots abnormal cDNAs - in sequence and expression level
Finding out the functional consequence of these variants is a problem - the variants are being identified faster than their function can be figured out. We need to know which variants/changes in genes are driving cancer progression
how do you identify which is the driver mutation amongst passenger mutations?
name a few methods used to distinguish passenger and driver mutations (VUS classification)
biochemical functional assays
integrated algorithms
personal and family history of cancer of the carrier
severity of protein modification
loss of heterozygosity
what’s the idea behind the CIN model in yeast (paper 1)?
29% of all ‘essential’ genes in yeast were known to give a CIN phenotype when mutated
They asked can the human copy of the gene - orthologue - do the job in a yeast cell for a yeast gene that has been knocked out - this is cross species complementation
define orthologue and paralogue
orthologue = genes inherited from a common ancestor performing the same role in different organisms
Paralogue = genes which resulted from gene duplication events within a genome
what did this paper - ‘Paper 1 - using yeast as a platform to study human genetic variants (Hamza, Akil et al)’ - do?
(with 322 genes)
Diploid yeast cell heterozygous for a mutation in an essential gene (one mutant one functional) → add a a WT functional copy of the human orthologue via a plasmid
NOTE - they chose the human orthologue, going by one with the closest sequence to the yeast version
Allow sporulation - division into four haploid cells
Possibilities -
The two cells with the mutant gene die
The other two survive thanks to the functional yeast gene
OR
All four spores survive because complementation occurred, and in the cells with the mutant yeast copy of the gene, the human homologue compensated
Hamza, Akil et al did pool by pool screens as well - what were these and why?
Used ‘pools’ of yeast of heterozygous mutants for 622 essential yeast genes
Used a mixture of plasmids with a total of 1010 human cDNAs to transform the yeast
why - Unbiased strategy
To identify unknown orthologues, OR complementation between non-orthologous genes i.e. see if there were any surprises
Found -
65 human cDNAs that complement 58 yeast null mutants, 20 of which were newly discovered pairs
Looked for patterns - didn’t really know, said they were cytoplasmic proteins unlikely to have regulatory roles. But the researchers did not give up…
