Download Genetics 200A Monday, September 28, 2009 Day 5: Yeast Lecture

Genetics 200A
Monday, September 28, 2009
Day 5: Yeast Lecture #1 (Hiten Madhani)
[email protected]
Peter Walter’s Yeast Genetic Mystery
mmm1 -> sick
mmm1 smm1 -> healthy
goal: find smm1
sequence genome: identical genomes
cross to yeast KO collection to map: Not linked to any gene
What’s going on?
Fungi
Fungi are eukaryotes
There are many identified species (>70,000)
Many are saprophytes (eat decaying plant matter)
Some are human pathogens (e.g. Candida albicans, Aspergillus fumigatus)
Are interesting for many biotechnology applications
Sacchromyces cerevisiae
a.k.a. “budding yeast” (but wt can form hyphae also!!!)
Used as brewing yeast
Can be grown on minimal media (carbon source, nitrogen source, some
vitamins, salt, trace elements)
Useful resource: www.yeastgenome.org
S. cerevisiae genome
12.5 MB, ~6000 genes
Linear chromosomes (range in size from 240 kB to 1 MB)
Can run out the whole genome on a single gel (“pulse field gel”)
Useful for identifying differences in chromosome copy number
(aneuploidy)
S. cerevisiae mitochrondial genome
Is separate from the nuclear genome
Mostly encodes hydrophobic components of the mitochondria
(Some mitochondrial components are also encoded in the nuclear genome)
Genome can be defective/absent (lacks genes for respiration)
 “petite” yeast
BUT MUST STILL HAVE MITOCHONDRIA!
S. cerevisiae L-A virus genome
L-A virus is a cytoplasmic parasite of yeast
Has a small dsRNA genome
There are also some viruses that can parasitize L-A as well
S. cerevisiae prions
Most famous is [Psi+]
Most are amyloid (-sheet aggregation)
S. cerevisiae cell cycle
Mitosis (asymmetric division: budding)
G1 (growth phase, no division)
S (DNA synthesis)
Start (Checkpoint: enough nutrients --> proceed)
G2 (Yeast don’t really do this, unless they get caught at Start)
S. cerevisiae budding (cell division)
Budding location is nonrandom (i.e., regulated)
Two patterns:
Axial budding: next bud near the last site
Bipolar budding: next bud at opposite side of bud
Pattern is cell-type dependent
S. cerevisiae DNA replication (S)
REQUIRES
1x CEN: Centromere
2x ARS: Origins of replication (1x ARS for circular plasmid)
***CEN-ARS plasmids are self-replicated in yeast***
Replicated DNA (“sister chromatids”) held together by cohesin
S. cerevisiae Mitosis (M)
Metaphase
Each chromosome captures a single microtuble (from each
centrosome) in yeast
Centrosome is the microtuble organizing center
Also called spindle pole body in yeast
Are attached the nuclear envelope in yeast
Must have 2 centrosomes at opposite ends of cell
Anaphase
Loss of sister chromatid cohesion
Sister chromatids separate to opposite cell poles
S. cerevisiae sex
Two cell types: a and 
a secretes a factor,  secretes  factor
a has  factor receptor,  has a factor receptor
When cells detect pheromone (a,  factor), they “shmoo” (or conjugate)
Cells arrest in G1 (stop dividing)
Grow toward mating partner (chemotropism)
Cell-cell attachment (agglutination)
Cell fusion
Karyogamy (nuclei approaching)
Nuclear fusion
After conjugation, cells are diploid a/
N-starved cells form pseudohyphae
C- and N-starved cells undergo sporulation
Includes meiosis
Produces 4 haploid spores (2x a, 2x )
Cell types are specified at MAT (mating type) locus
MAT is on chromosome III
MATa has two genes: a1, a2
MAT has two genes: 1, 2
In a cell:
a1, a2 don’t do anything
a is default state without MAT locus
In  cell:
1 up-regulates -specific genes (sgs)
2 down-regulates a-specific genes (asgs)
In diploids:
a1/2 down-regulates haploid-specific genes (hsgs)
a1/2 down-regulates 1 too
a1/2 down-regulates RME1, a repressor of meiosis
S. cerevisiae meiosis
Requires specific mechanisms to solve problems not encountered during
mitosis
One round of pre-meiotic S including sister cohesion
Homolog pairing (e.g. both copies of chromosome 3 come together)
Attachment of homologs via homologous recombination*
*This mechanism is not universal and not required in all organsims
Ex. Male flies don’t allow any recombination during meiosis
Sister chromatid cohesion holds together homologs after recombination and
during meiosis I
Meiosis I involves monopolar spindle attachment, i.e., microtubules attach to
one spindle pole body and not both
S. cerevisiae meiosis (cont.)
First meiotic division
(Metaphase I)
Loss of arm cohesion
Maintenance of centromeric/peri-centromeric cohesion
Leads to anaphase I (separation of homologs)
Second meiotic division
(No S phase)
Metaphase II (as normal)
Anaphase II (separation of sister chromatids)
Four gametes (spores) == TETRAD!!
S. cerevisiae nomenclature and markers
Every gene is three letters and a number
Gene names are italicized
Wild type and dominant alleles are capitalized (e.g., HIS3)
Recessive alleles are lowercase and the allele is designated by a second
number (e.g., his3-1)
Yeast markers are generally auxotrophies (nutrient requirements)
“Drop-out” media lacks a nutrient
Media is –His (histidine drop-out media) if it lacks histidine
Phenotype is His- if it fails to grow (as opposed to His+ if it can)
Replica plating allows preservation of colonies grown under permissive
conditions (rich media), assayed under non-permissive conditions (drop-out media)
Ben Schiller
Email questions/corrections to [email protected] or any TAs/instructors