Yeast Genetics

Saccharomyces cerevisiae – Budding yeasts

• Ellipsoidal; about 5 µm diameter

Divides by budding
In certain characteristics - More similar to animal cells than to S. plombe

Schizosaccharomyces pombe – Fission Yeast

• Cylindrical; 3-4 µm diameter; 7-15 µm long

Divides by medial fission

In certain characteristics - More similar to animal cells than to S. cerevisiae

two very different types of yeast. They diverged over 1 billion years ago.
Life Cycle of Yeasts – Pyramidal Ascus

S. cerevisiae
Life Cycle of Yeasts –

S. pombe

Linear Ascus
Linear asci retain information regarding
the order of meiotic events.
Heterothallic haploid cells retain their mating types as they go through rounds of
mitosis. Cannot mate with each other within a single mating type
Homothallic cells frequently switch mating type. Thus the progeny of a single
homothallic spore colony can mate with each other to form diploids.
 Life Cycle of Yeasts

Life Cycle of Fission Yeasts Life Cycle of Budding Yeasts

Schizosaccharomyces pombe Saccharomyces cerevisiae
 Metabolism
 Both organisms grow fastest on glucose, which is
fermented into ethanol or oxidized to CO 2 and H2O
 S. cerevisiae prefers fermentation and will use it so long
as glucose is present, even in the presence of oxygen;
 S. pombe employs pure fermentation only in the absence
of oxygen
 S. cerevisiae shifts to aerobic growth on EtOH when the
supply of glucose runs out (diauxic shift)
 Genomes
 Both yeasts
o DNA ~60% A+T
o Normal core histones; no close relative to histone H1

 S. cerevisiae
o 16 chromosomes
o Entire genome sequenced (12 Mb; about 6,000 genes)
o Centromeres small (~120 bp), unique; three regions bind proteins,
allowing interaction with a single microtubule for segregation;
o Replication origins (ARS elements -Autonomously Replicating
Elements) 100-150 bp
 Genomes
 S. pombe
o 3 very large chromosomes (most markers on same
chromosome unlinked to each other)
o Entire genome sequenced (14 Mb; about 5,000
genes)
o Centromeres large (40-100 kb), mostly
repetitious; may bind more than a single
microtubule; genes within centromere are usually
silenced
o Replication origins (ARS elements) 500-1500 bp
 Extrachromosomal elements
 Mitochondria
o Budding yeast cells can grow aerobically or anaerobically
o mitochondrial respiratory function is not essential.
o mitochondrial DNA can be mutated at will, and mitochondrial genetics can be
readily studied.
o Mitochondrial mutants (S. cerevisiae) are recognized as mutants able to grow
(though poorly) on glucose (which can be fermented) but not on glycerol (which can
only be metabolized by aerobic respiration).
o Fission yeast mitochondrial DNA is essential for wild-type cells
 Plasmids
o S. cerevisiae
 2 µm circle
 6.3 kbp
 60-100 copies/cell
o S. pombe - no known naturally occurring plasmids

 Non-Mendelian determinants in S. cerevisiae

o Prion-like proteins
 Dominant
 Extranuclear
 Not associated with killer particles or mitochondria
 Self-seeding
o Killer particles
 Defective dsRNA virus encoding a lethal toxin - Polypeptide killing
 Genetic nomenclature
 S. cerevisiae
o Gene names are 3 letters followed by a number
o Dominant alleles are capitalized; recessives are lower case; usually but
not always the wild-type is dominant
o Gene names are italicized (YFG1)
o Wild type may, optionally, be denoted by a + sign (YFG1+)
o Mutant alleles are indicated by an allele-specific number (yfg1-1)
o Knockouts are indicated yfg1::URA3 (ura3 - orotidine-5′-phosphate decarboxylase)
o Proteins are partially lower case, not italicized: Yfg1 or Yfg1p

 S. pombe
o Gene names are 3 lower case letters followed by a number
o Wild type is denoted by a + sign (yfg1+)
o Mutants are denoted by an allele number (yfg1-1)
o Knockouts are designated yfg1::ura4 (ura4 - orotidine 5'-phosphate decarboxylase)
 Making mutants
 Ethyl methane sulfonate (safer; base transitions); nitrosoguanidine (more
effective; base transitions); UV (very safe, least effective, many different
types of mutation)
 5 x 10-4 to 1 x 10-2 per gene, with 20-80% killing
 Screen for desired phenotype
 Backcross desired mutants to wild-type
o 3 rounds recommended
 Tetrad analysis to confirm single mutant status
o Mate; sporulate; dissect asci; determine phenotypes of spore colonies
o Fig. 6 (results predicted if single gene)
o Can be used for mapping; see below
 Random spore analysis
(a poor or clumsy person's alternative to tetrad analysis)

 Mass isolation of spores from asci followed by characterization

of phenotypes of the resulting spore colonies
 permits much larger numbers of spores to be analyzed
without the difficulties of micromanipulation,
 ascus information is lost
 If mutation is single gene, then half of spore colonies (from cross
of mutant x wt) will show mutant phenotype
 If none of the phenotypes is lethal, then analysis of random spores
can also provide recombination frequencies, which can be used for
mapping
 S. cerevisiae
o Diploids of desired genotype are induced to sporulate on starvation
medium
o Potential problem due to incomplete sporulation; some vegetative diploid
cells inevitably remain
o One approach is to use a can1-r allele in one of the haploid parent strains
o Diploid cells (CAN1-S+/can1-r) and half the spores (CAN1-S+) will be
killed by canavanine; the survivors will all be spore colonies
o This procedure works generally, except for the rare cases in which one of
the genes of interest is linked to the CAN1 gene
o After 2 days on starvation medium, incubate briefly with Glusulase (an
enzyme prep from snails that hydrolyzes yeast cell walls) and shake with
glass beads to remove the ascus wall and free the spores
o Plate onto appropriate medium containing canavanine (and other
supplements, as appropriate)
 S. pombe
o Generation of free spores is facilitated by tendency of diploids to
spontaneously sporulate and by fragility of asci and vegetative cells
o Make diploids from desired haploids by crossing on ME (malt extract;
carbohydrate only; no nitrogen) plates. Incubate 2 days at 25°-30°.
 Absence of nitrogen leads to G1 arrest and promotes mating
competence. Like S. cerevisiae cells, S. pombe cells mate while in G1.
o Collect some of mating mixture (which will contain asci due to
spontaneous sporulation of diploids) and incubate briefly with Glusulase.
 Opens asci, liberates spores, kills haploid and diploid vegetative cells
o Plate on appropriate medium to generate spore colonies
o Check phenotypes of spore colonies
 Complementation
 Used to test whether two mutations producing the same phenotype are
in the same or different "complementation groups”
 Procedure in S. cerevisiae

Those colonies displaying the mutant

phenotype (for example, the colony
in the upper left, because it is
homozygous for m1) have mutant
genes in the same complementation
group.

Colonies displaying wild type

phenotypes have mutant genes in
different complementation groups.
Procedure in Schizo. pombe
 Mapping by tetrad analysis
 Possible outcomes of 2-factor cross (A/a and B/b)

PD:NPD:T = 6:6:24 = 1:1:4.

When both genes are close to their centromeres,

frequency of crossovers between both genes
and their centromeres is reduced,
frequency of Ts is reduced,
ratio PD:NPD:T = 1:1:<4

Genes on different chromosomes

 6 X6 Table showing PD:NPD:T = 6:6:24 = 1:1:4.

  AAaa AaAa AaaA aAAa aAaA aaAA

BBbb AB AB ab ab AB aB Ab ab AB aB ab Ab aB AB Ab ab aB AB ab Ab aB aB Ab Ab
PD T T T T NPD

BbBb AB Ab aB ab AB ab AB ab AB ab aB Ab aB Ab AB ab aB Ab aB Ab aB ab AB Ab
T PD T T NPD T

BbbB AAaa AaAa AaaA aAAa aAaA aaAA

           
bBBb AAaa AaAa AaaA aAAa aAaA aaAA
           
bBbB AAaa AaAa AaaA aAAa aAaA aaAA
           
bbBB AAaa AaAa AaaA aAAa aAaA aaAA
           
For most sensitive detection of centromere linkage
of an unknown gene (A)
a cross should be made with a gene known to be so close to its
centromere that the probability of recombination between it and
its centromere is essentially 0.

TRP1 on chromosome IV of S. cerevisiae is a good example of

such a tightly centromere-linked gene.

Thus the extent to which A is centromere-linked can be

determined by tetrad analysis of the cross (A TRP1) x (a trp1)
or (A trp1) x (a TRP1).
Note:
Genes close to their centromeres display reduced SDS
SDS gives rise to tetratypes (T), so the frequency of SDS is
the frequency of T.
 Genes on the same chromosome
 If there are no crossovers between the genes, then PDs are produced.

 Single crossovers produce Ts

The four possible single crossovers between two genes on the same chromosome. 
  1/4 of double crossovers produce NPDs
 In a recombinant ascus (T or NPD), 2 (half) of the strands
recombine if there is a single crossover and 4 strands
recombine if there is a double crossover.

The four possible double crossovers between two genes on the same chromosome.
Thus, even if all of the strands do not participate in the double crossover,
other strands participate more than once so that the total number of
participating strands is 4, which is equal to the number of spores in the
ascus.

 If genes are close, so that map distance is less than or equal to 40

centimorgans (cM), then the following equations can be used to calculate
recombination frequency and map distance. These equations do not
account for more than two crossovers in an interval or for chromatid
interference (local deviations from this formula).
 Recombination frequency = total recombination events/total spores
 = total recombination events/4(total asci)
 = (2[single crossovers] + 4[double crossovers])/4(total asci)
 = (1/2[single crossovers] + double crossovers)/total asci
 = (1/2[T-2NPD] + 4NPD)/(PD + NPD + T)
 = (T + 6NPD)/2(PD + NPD + T)
 Note: the frequency of all double crossovers = 4NPD. Some of the Ts are
also due to double crossovers. The frequency of Ts due to double
crossovers is 2NPD (Fig. 12). Thus T-2NPD is the frequency of single
crossovers.
 Multiply by 100 to get map distance in centimorgans
Modern mapping procedures

 Determine partial sequence of protein or gene of interest

 Compare with S. cerevisiae or S. pombe genome sequence
 http://www.ncbi.nlm.nih.gov/BLAST/
 http://genome-www.stanford.edu/Saccharomyces/
 http://www.sanger.ac.uk/Projects/S_pombe/
 YI-YR vectors

Other modern techniques

(just a few examples from many possibilities)

 Introducing DNA on plasmidsw

o Integrating vectors
o ARS vectors
o CEN vectors (not needed for S. pombe)
 Gene Replacement

One Step Gene Replacement

 Useful for gene knockouts

 Can be used to insert linear PCR
products, if they contain selectable
markers and terminal regions of
homology to the yeast genome
 Gene Replacement

o Two-step Gene Replacement

 Useful for introducing any

mutation into the chromosome at
any location