Yeast Flashcards
What is different between S. Cerevisiae and S.pombe that can be used in research?
The first is budding yeast, the second is fission yeast. S.pombe more closely resembles higher eukaryotes at aspects like entry into mitosis, centromere structure, presence of RNAi pathway
How do fission yeast reproduce?
Usually proliferate as haploids (whereas budding yeast diploids). Two mating types, M and P, conjugate to form diploid cell. Diploid immediately undergoes meiosis (unlike budding yeast fission yeast are very unstable as diploids) to give four haploid spores
How is forward genetics used in genetic screening?
How are dominance relationships investigated once you have your haploid mutants?
What needs to be considered?
Start with a phenotype, mutate cells, identify mutants and replicate plate to grow colonies of the mutants, examine mutants and their phenotypes
Perform the screens on haploids - recessive mutations can show a phenotype ie removes dominance relationships from interfering. Helps when studying essential genes to use conditional mutations so the mutants can still be investigated without killing the organism by mutating
oss mutated haploid to wild type haploid to give a diploid that is heterozygous for the mutated locus - if the diploid is mutated then the mutation is dominant, if not it is recessive
Can investigate dominance relationships, location in genome, how many genes identified by mutants and if/how mutations interact
Are dominant or recessive mutations more dominant and what characterises them?
Recessive tend to be loss of function and are more common
Dominant tend to be:
Gene product is no longer regulated
Haploinsufficiency- one copy of gene is not enough to provide wild type function
For gene products that form a dimer, dimer between a wild type and a mutated protein not functional
Following a genetic screen, what tests can be used to analyse the mutants?
Cross wild type and mutated haploids to investigate dominance
Complementation tests to determine how many genes the mutations define
What is the basis for complementation?
What is a drawback of this method?
Cross two mutant haploids to give a diploid
If mutations are in different genes, for each mutation a wild type copy will be present on the other gene so will cancel out mutation (complement) or give wild type phenotype. If you have a set of mutants, perform all possible pair-wise combinations and sort into complementation groups (group of genes that don’t complement = one group)
Requires mutations to be recessive otherwise only one copy of mutation would cause mutated phenotype even if wt gene present. It is also possible to get two mutations in the same gene complementing one another (intravenous complementation) identification of this requires further analysis
What is intragenic complementation and when might it arise?
What is required in this case in order to study/identify?
What two mutations in the same gene complement one another, eg if encoded protein is made up of two domains that function independently
Genetic mapping or molecular characterisation
What is a back cross?
What is a prototroph?
Crossing a wild type to a mutant
An organism that can synthesise its own nutrients from inorganic material
What is tetrad analysis?
Uses the fact that all four spores from meiosis remain together in an ascus - separate single spores from ascus using a micromanipulator and enzyme to digest the ascus wall. Pick each spore with needle and I’ve to defined place on Petri dish. Incubate and you get colony from each of the spores. Investigate the phenotype of the colonies.
Can use to analyse two unlinked genes by crossing a mutant with two mutations, each in a different gene, with a wild type, giving a diploid that is heterozygous for each locus. Diploid undergoes meiosis to give tetrad, get parental ditype or non-parental ditype in the tetrad depending on how the mutations separated at first meiosis. As the segregation of homologous chromosomes at first meiosis is random, both cases are equally likely
Can use to investigate linked genes, do the same but the mutant has two mutations on the same chromosome in linked genes, still get a diploid heterozygous at each locus). Only one way in which homologous chromosomes can separate at first division. As genes can’t be separated at either division, get mainly parental ditypes - shows gene linkage
If genes are less closely linked allowing recombination to occur - still only one option for first division but multiple options for second when recombination occurs.
What are the different results of recombination in tetrad analysis and what to they result in?
Single crossover results in tetratype (AB,ab,Ab,aB)
Double crossover between four chromatids results in NPD
Other possibilities with two crossovers
What can follow tetrad analysis if genes are linked?
Genetic mapping - the genetic distance between two linked genes is the frequency of recombination in centimorgans (see formula)
What can tetrad analysis be used for?
Generate genetic maps, investigate linkage and characterise mutations, to create double mutants
See last page of lecture 1
How do budding yeast reproduce sexually?
What determines what sex type they are?
Haploid cells exist in one of two mating states, a or alpha, two different types conjugate to give a diploid a/alpha cell
The diploid cell may undergo meiosis to give four haploid cells in an ascus, two a and two alpha which can germinate and continue haploid growth, it the diploid can continue diploid by budding
Mating type is determined by a single locus MAT ( MATa or MATalpha depending on cell type)
