Download “The cell cycle and other schmoos”

Michelle Attner, Dept. of Biology, Koch Institute for integrative cancer research
“The cell cycle and other schmoos”
Lecture Notes (below) courtesy of Mary Brunson
1)
Why yeast?
a)
Nontoxic organism
b)
Simple, eukaryotic cell
c)
Compact (sequenced genome), very few introns
d)
Can grow in plates and culture
e)
Short generation time (90 min)
f)
Live as haploids and diploids (as a haploid, whatever allele they have will
show)
g)
Easy to manipulate genes (swap promoters, delete genes in a matter of days)
h)
Many yeast genes have homologs to humans
2)
Life cycle of budding yeast
a)
2 mating types – a and alpha haploids (visibly indistinguishable - can mate
with known to tell)
i)
Both can divide mitotically and live happily
ii)
A and alpha can mate and produce a/alpha diploids
iii) a/alpha diploid can divide asexually or go through sporulation to make 4
haploid gametes (called spores)
iv) In the wild, they prefer to be diploid
b)
You can determine if an allele is dominant or recessive by mating it with
another spore with a different allele
c)
Yeast mating is a fusion event
i)
Signal for mating
(1) A produce a factor and alpha produce alpha factor (factors are 12-20
amino acids long)
(2) Alpha cells have receptors for a factor and vice versa
(3) What determines the type of the cell?
(a) Locus on chromosome 3 encoding mating type (matA or matAlpha)
(b) They can switch mating type in the wild
ii)
Factor binds to receptors, activating a MAP kinase pathway, arresting cell
cycle in G1 (so DNA is not replicated) and forming a shmoo (a mating
projection that necessary for cell fusion)
iii) What if there are no alpha cells around?
(1) BAR1 is a secreted protease that destroys alpha factor (made by a cells)
(2) If a lot of time has passed, causing all a cells to be arrested in G1
(3) If there are no alpha cells around, the a cells will just be paused in G1
(4) BAR1 destroys alpha factor so they can continue reproducing
(5) BAR1 knockouts – keep them in cell arrest with a shmoo
iv) In the lab, they use alpha factor to arrest cells (a factor is not easily
synthesized in the lab)
d)
How do yeast bud?
i)
Build a tiny bud in G1 and gets bigger in S and G2
ii)
During M, the nucleus migrates to the intersection and completes mitosis
iii) Bud size can give you an indication of where in cell cycle the cell is
iv) Cytoskeleton role
(1) Actin forms cables and patches, pulling at bud membrane as it grows
(2) Organelles are shifting into the bud
(3) Tubulin in anaphase (spindle – very few produced in yeast)
v)
Closed mitosis – nuclear envelope is never broken down and MTOC are
embedded in the nuclear envelope
vi) What genes control cell cycle progression?
(1) Discovered through genetic screens
(a) Large scale investigation of mutants possessing a phenotype of
interest
(b) Phenotype – arrested at a specific point in cell cycle based on bud
morphology
(c) Apply a chemical mutagen to the yeast to induce mutations
(d) If cell is in cell cycle arrest, it’s dead (lethal phenotype)
(e) Try growing them in different temperatures – sometimes making the
newly mutated protein more or less functional (alive at room
temperature, dead at high temperature)
(f)
Found cells that don’t bud at all (G1 – cdc28, cyclin dependent
kinase), small buds (late G1 – cdc53), anaphase (roughly equal size bud –
cdc14)
(2) How do you find the gene?
(a) Rescue the phenotype by adding plasmids (with WT genes)
(b) Isolate the plasmid out of the rescued cell and figure out what gene
it is
e)
Yeast meiosis
i)
Starvation induces sporulation
(1) DNA replicates
(2) Tetrads form and crossing over occurs
(3) Homologues segregate
(4) Sister chromatids segregate
ii)
Can manipulate spore formation to put certain chromosomes in specific
spores
f)
CDC14
i)
Arrests cell in telophase
ii)
E3 ubiquitin ligase
iii) Promotes ubiquitination and disposal of cyclins
iv) CDC14 mutant – cyclins stick around, can’t enter next G1
v)
CDC14 sequestered in nucleolus and released into nucleus and cytoplasm
during anaphase (only activated when bud is really big and ready to segregate)
3)
Can use budding yeast to study lots of other things – secretory pathways,
gametogenesis, etc