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location in the notes)
Consequences of loss of
function of a1
& a2
(usually leads to sterility)
STE
= wt genes
ste
= mutant
genes
1.
Loss of a1
results in sterility because of the lack of
expression of a-specific genes
2.
Loss of a2
allows expression of both a-specific & a-specific gene banks with result that antagonism
-> sterility
3.
Inactivation of both a1
and a2
renders a
cells a-like because a-specific
genes are off and a-specific genes are on.
(alf phenotype - for a-like
faker)
4. Loss of a1 makes no
difference as a-specific genes remain on
253
Switching rules in
homothallic strains (HO strains)
1.
Switch occurs in pairs of cells
2.
Only "experienced" mother*
cells switch
3.
"Experienced" cells switch
at least 50% of the time at minimum (usually
80-90% of time)**
*An "experienced"
cell is one that has been a mother
(previously produced a daughter)
** Switching occurs only at a
~ once/106 cell divisions/ho strains
270
Switching
rules suggest
1.
Switching is not random: rather cells
are directed to pick as a donor gene for the
mating-type switch gene of the opposite type
than the one already in the MAT locus.
2.
switching occurs prior to or at the
time of MAT DNA synthesis, and then the
genetic switch is replicated and passed to
both resulting cells (if occurred after DNA
synthesis then only one of the pair would be
switched)
271
Nature
of cassettes*
1.
Core region of unique,
transcribed information that defines each
mating type
Silenced by
ya
= 642 bp
}
idiomorphic sequences
Sir/Mar Repression
ya = 747 bp
}
2.
Two flanking regions**
x = 704 bp
z-1 = 239 bp
**
These are common to all three loci, HML,
HMR & MAT
**
Provide sequence homology which
facilitates the recombination that results
in exchange of the y regions
3.
MAT and HML share 2
additional loci
w & z-2 ~
723 & 88, respectively
*
both loci > 100 kbp away from centromere-linked MAT locus near
left & right telomeres
MAT <-> HML ~ 200
kb
MAT <-> HMR ~ 150
kb
274
Cell
cycle definition
The name given to the repeated
events that occur between the formation of a
daughter cell by division of its mother cell
and the time when the daughter itself
divides
In S.
cerevisiae
Time between daughter separating from
mother and daughter producing its own
daughter
Early leader in field – Dr.
Leland H. Hartwell see R.R. #14 + 15 for
example
280
Keys to early success
1.
Yeast cells of S.
cerevisiae were amenable to
synchronization
2.
So-called cell
division cycle (cdc)
mutants were relatively easily derived
3.
the insight that the yeast bud and
its growth could serve as a major landmark
of the progression of a eucaryotic cell
through its cell cycle
Initial goal: determine the
relationship between DNA synthesis –
nuclear division cylcle and the cell
growth/cell division cycle.
282
Types
of synchronization
1.
Induction - involves distortion of
cycle in way that all cells arrest in one
cell cycle stage, after which they are
released to divide in syncrony
2.
Selection - isolation of selected
population from among an asynchronously
dividing population, which is at same stage
of cell cycle, so when put in fresh medium
all cells divide in unison.
Bud formation and cell
division -> 2 easily scored landmarks
283
ts cdc mutants
for
functional analysis
Conditional “lethal”
strains that have altered alleles in their
genomes, which render them unable to
complete certain specific events of a normal
cell
division
cycle (cdc) at a restrictive condition,* but allow them to grow
normally at a permissive condition.**
*
usually 37oC
- 41oC
**
usually 25oC
* arrest in a single
"terminal phenotype"
* most not really lethal as
down shift shows cells often still alive –
just don’t grow reproductively to ->
turbid broth cultures or dense plate
colonies compared to wt
293
Initial events scored in
cdc
strains
1.
Terminal phenotypes at restrictive
temperature (37oC)
*
2.
Whether or not a 1st cycle
arrest mutant (arrested in 1st
cell cycle after shift to 37oC)
<100% increase in cell #.
3.
Mutation’s execution point
4.
What landmark(s)** inhibited
(particularly initial defect).
* selection based on little or
no growth at ~ 37->41oC
and production of a terminal phenotype
**
Bud emergence
}
DNA synthesis
}
Cell separation
}
change with deeper study
Nuclear migration
}
Etc.
}
study over when gene product
identified and function found
294
Use of cdc mutants for functional mapping
1.
Comparisons of the phenotypes of
single mutants
2.
Comparisons of the terminal
phenotypes of two single mutants with that
of the corresponding haploid double mutant
3.
Use in reciprocal shift experiments
300
Functional
sequencing with single mutants a
1.*
D1
La
Lb
Lb is not dependent on La
®/ ®/
®
2.*
D1 Lb La
La is not dependent on Lb
®
®
®
3.**
D1
LaLb
La
& Lb are interdependent
®
®
La
4.***
D1
®
La & Lb are independent events
®/
Lb
®/
L = landmark
D = initial defect.
*good for 1 & 2 if a & b far
apart in timing
**poor for #3
*** very good for #4
Often was done by time lapsing
on agar medium.
301
EXAMPLES
D1 = DNA synthesis
La
= mitosis
La1
= bud emergence
Lb
= cytokinesis
D1
La
Lb
“dependent”
®/
®/
®/
cytokinesis depends on
mitosis, which
depends on DNA synthesis
La1
D1
®/
mitosis dependent upon DNA
®/
La
synthesis, but bud emergence
®/
is independent of DNA synthesis.
302
IN double cdc
mutants*
Mutant
|
Initial
Defect
|
Terminal
phenotype
|
|
cdc
24
|
BE
|
|
|
cdc
8
|
DNA
syn
|
|
|
cdc
24,8
|
|
|
** unique phenotype
Important to remember that
these are haploid double mutants as
heterozygous diploids would be like wt
because of complementation - however, could
construct
cdc
24
cdc
8
cdc
24
cdc 8
"strains homozygous
recessive diploids --> same results"
306
Landmark events in the cell cycle
earlier, broader definition:
events
which can be monitored by an available assay
that provides information about the position
of cells within the cell cycle.
more recent, narrower definition:
events
that actually duplicate and segregate cell
constituents and produce daughter cells.
RHO-type GTP-Binding proteins, such as Cdc42p
1. Carry prenylation sequences and
the addition of lipid moieties to these
domains modulates their binding to membranes
and may therefore affect their activity
2. Like Ras, all bind guanine
guanine nucleotides, being active when bound
to GTP and inactive when bound to GDP;
3. All are able to hydrolyze GTP,
for which they require a GAP, which acts as
a negative modulator;
4. Involve a GEF in their
activation, closing the GTPase cycle;
5. Are thought to stimulate actin
reorganization in vivo.
A unified theory of cell cycle control
this
prevailing theory invokes two central
coordinative mechanisms so that cell cycle
events occur in the proper order with
repsect to each other: e.g. chromosome
segregation follows dna replication.
1.
a cell cycle clock based on a set of highly conserved serine threonine
protein kinases (cdks; cyclin-dependent-kinases)
2.
checkpoint controls, which involve regulatory pathways that monitor the
progress of key cell cycle events and delay
progression, if those events have not been
satisfactorily completed.
The cell cycle clock
the
ticking of the clock is manifested as
cyclical changes in cdk kinase activities*
these
phosphorylations regulate many processes,
including even the synthesis, activation
levels, and proteolysis of cdk regulators
that contribute to the oscillations of cdk
activities themselves.
*main cdk
is cdc28p,** although there are others
**all cdk
are inactive as monomers
*require
association with positive regulatory
proteins, called cyclins for activity.
**cdc28p
levels do not fluctuate, and are
produced in excess
Cyclins
diverse
family of kinases, all of which have a
distinctive "cyclin box" required
for binding and activation of cdks.
most, but
not all, exhibit periodic accumulation.
main
cyclins*
Cln1p-3p
cell cycle control: G1
Clb1p-6p
cell cycle control S, G2 M
*all these activated Cdc28p,
leading to posttranslational regulation of
Cdc28p activity.
The checkpoint controls
Early studies showed that certain
events are linked in "dependent
pathways" dna replication
ŕ no mitosis
Later studies support the idea of
central cell cycle clock based on cyclin/cdk
regulators
ŕparadox*
*How can cells maintain dependency
relationships if events are triggered
independently by an autonomous clock?
Paradox resolution
Possibility #1:
events are mechanistically linked:
completion of event #1 produces substrate
for event #2.
Possibility #2:
events are mechanistically unlinked,
but a regulatory pathway ensures that the
later event does not begin until earlier one
has been completed*
*Finding of mutations
and drugs that uncoupled the dependent
events and permitted the second event to
occur, even when the first event was
blocked, provided needed evidence for
possibility #2.
Looking back
cdc genes and cdc mutant
groups
1.
cdc genes important to the ticking of the clock: cdc28, cdc4,
cdc34, cdc53, cdc16, etc.
2.
cdc genes important for processes monitored by checkpoint controls:
cdc2, cdc6,
cdc7, cdc8, cdc9,
etc.
3.
cdc genes that, if mutated, cause cells to arrest in g1 in ways that mimic
effects of extracellular signals: (e.g. cdc70
[gpa1,
scg1] and cdc72 [nmt]
pheromone response) and cdc35
[cyr1]
nutritional-deprivation response.
4. cdc genes involved directory in
morpho-genesis: cdc24,
cdc42,
cdc3, cdc10; with these,
the uniform terminal morphology reflects the
role of the gene products in bud formation
and cytokinesis and not actual cell cycle
arrest.
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