Transcript Review of Lecture 1 - University of Kentucky

Gene Nomenclature
Budding Yeast:
CDC28 = wild type gene
cdc28 = recessive mutant allele
Cdc28 or Cdc28p = CDC28 protein
Fission Yeast:
cdc2 = the wild type gene
cdc2- = recessive mutant allele
p34cdc2 = the protein
Non-genetic systems:
not standard
use either yeast, or use unique name with yeast name superscript
or whatever
Regulation of G1 to S
Role in G1 for cdc2/cdc28 at START
But cdc2/cdc28 regulators acted at G2 to M ONLY
cdc2D
cdc13/B-type Cyclins
cdc25 and wee1
CKIs
wee1
?????
G1?????
Cyclins
cdc25
cdc2
G2
G1
Mitosis
S
phase
Cyclins Nomenclature
All contain “cyclin box” domain
Cyclin A
Cyclin B’s
Vertebrate Cyclin B1, B2
S. pombe cdc13...
S. cerevisiae CLB1,2,3,4,5,6
cdc2 binding domain
A and B’s closely related
-destruction box
- required for S and M
Differ in expression pattern
- “A” accumulates earlier,
- degraded earlier
G1 Cyclins
G1’s more similar to each other
S. cerevisiae CLN1, 2, 3
- PEST domains
Mammalian Cyclin D, E
- required for exit from G1
What do Cyclins do?
1. Positive Regulator of CDK catalytic subunit
2. Potentially impart CDK substrate specificity
3. Potentially impart CKD inhibitor specificity
4. Potentially determine specific CDK localization
Cyclins can be grouped by expression patterns
- CLN1 and CLN2 and CLB5 and CLB6 - CLB3 and CLB4 - CLB 1 and CLB2
CLN1,2,CLB5,6
CLB1,2
CLB3,4
CLN3
G1
Start
Metaphase
Yeast Cyclin Genes: CLNs and CLBs
Figure 13-22
Quadruple clb1,2,3,4 Mutant
S phase Cyclins
CLB5, CLB6
NO PHENOTYPE
- double mutants
-gain of function
-????????????
G1 Cyclins:
CLN1
CLN2
CLN3
Triple cln1,2,3 mutant
G2 Cyclins
CLB1
CLB2
CLB3
CLB4
Dominant CLN Mutations
Role of Cyclins in G1 to S????
Cdc28/Cln3
“Start-specific” Transcription
Cdc28/Cln1,2
Budding
SPB dup
Sic1
Cdc28/Clb5,6
DNA
Rep
Regulation of G1 to S
Starting point of this study:
- Specific role of CLNs in regulation of G1 to S not clear
- triple mutant doesn’t bud, dup SPB, or rep DNA
- CDC34, CDC53, and CDC4 Required specifically
for DNA replication
- high Cdc28/Cln activity
- Cdc34 = UBC
- CLB1,2,3 and 4 not required for S Phase
- What about CLB5 and CLB6, and what’s this to do with
CDC34 et al ?
Clb5/Cdc28 kinase accumulates near/after Start
Protein Levels
Clb/Cdc28 Activity
by Histone H1 Kinase Assay
Clb5,6/Cdc28 Necessary and Sufficient for
DNA Replication
clb1,clb3,clb4,clb5,clb6
clb2(ts)
GAL::CLB5
Grows at 23 deg on
Galactose medium
Dies at 37 degrees on
Glucose medium
Cdc34 involved in
degradation of a
Cdc28/Clb inhibitor??
CLBs redundant for S
Phase, with 5 and 6
most important
Inhibition of Clb/Cdc28 by Sic1 in vitro
Cdc28/Clb specific
inhibitor accumulates
in cdc34 et al mutants
Inhibitor depends on
SIC1 function
Sic1 is a Cdc28
inhibitor
In vitro
demonstration of Clbspecific inhibition and
Sic1/Cdc28/Clb
complex formation
Implications and Predictions...
Clb5,6 expressed in late G1
Clb5,6 major role in S phase induction after Start
Clb5,6/Cdc28 and other Clb/Cdc28s held inactive by Sic1
Sic1 function is opposed by cdc34,4,53 after Start…
Sic1 should be regulated - on before start, off after start
Sic1 regulation dependent on cdc34,4,53
Phenotype of cdc34 mutants should be dependent on Sic1
sic1 mutants should be sensitive to CLB5 over expression
Cdc34 dependent Regulation of Sic1 Accumulation
Sic1 phosphorylated by
Cdc28/Cln in vitro
Sic1 destroyed in a CDC34dependent manner
Phosphorylation correlates
with time of destruction
Sic1 is required to prevent S phase in cdc34
G2/M arrest?
Regulation of G1/S
Cdc28/Cln3
cln1,cln2,cln3 dead
cln1,cln2,cln3,sic1 alive
“Start-specific” Transcription
only essential
function of CLNs!
Cdc28/Cln1,2
Budding
SPB dup
Sic1
Cdc28/Clb5,6
DNA
Rep
Mammalian regulation of G1 to S
PICK 1
CDK/CKI
Pair
Compare and Contrast Roles
of G1 CDKs and CKIs in G1 to S in
budding yeast and mammalian cells
Ubiquitin Dependent Proteolysis in Cell
Cycle Regulation
Cell Free Cell Cycling Extracts
Visualizing Mitotic Spindle Formation and
Chromosome Segregation in vitro
Cyclin is destroyed at the time of sister chromatid
separation by ubiquitin-dependent proteolysis
Ubiquitin-dependent proteolysis of other mitotic
proteins control separation of sister chromatids
Identification of the Anaphase Promoting Complex
(aka Cyclosome or APC/C)
Cell Free Cell Cycling Extracts
Cyclin mRNA is
necessary and sufficient
to drive cell oscillations
90 = nondegradable
NH2-terminus or D-box sufficient for
mitotic-specific, ubiquitin-dependent, proteolysis
Role of Cyclin B destruction in progression
through Mitosis
Cdc2/Cyclin B (MPF) activity peaks at metaphase
Cyclin destruction correlates with Cdc2/Cyclin B inactivation
Regulation of metaphase to anaphase by Cyclin B Proteoysis
half right...
“Cell Free Mitosis”
Reversible Arrest at Metaphase
Visualize MTs and DNA
Synchronous release from Metaphase-Anaphase-G1
+ EGTA (minus calcium)
extract arrests in “mitosis” = “CSF extract
analogous to mature egg
haploid genome
+ sperm nuclei, + rhodamine-labelled tubulin
+ calcium, enter G1, S
-sister chromatids
+ “CSF extract”, enter and arrest at metaphase
+ initiate anaphase by adding calcium again
Metaphase to Anaphase in vitro
Extract dependent on exogenous Cyclin B mRNA - WT Cyclin B
TIME AFTER SECOND CALCIUM ADDITION
Extract dependent on exogenous Cyclin B mRNA = 90 Cyclin B
TIME AFTER SECOND CALCIUM ADDITION
Extract contains endogenous Cyclin B mRNA PLUS D-box peptide
Interpretation...
Data:
Preventing Cyclin B destruction blocks after anaphase
Adding competitive inhibitor of Cyclin B destruction
blocks at metaphase
Interpretation...
Cell Cycle Regulation of Ubiquitin
Dependent Proteolysis
SCF
- Skp1, Cdc53, F-box (Cdc4)
- Sic1, Cln’s...
APC/C - Anaphase Promoting Complex or Cyclosome
- Securing (anaphase inhibitor), mitotic Cyclins,...
Exam 1 - will write in after Tuesday’s lecture
questions on cell cycle analysis using mutants,
synchronous cultures
- interpret data
- design experiment
your comparison contrast of G1 to S regulation by
Cdk/Cyclin/CKI in mammalian cells and budding yeast
review article is sufficient for mammalian cdk info
no wrong answers
I don’t care how long/short - just thoughtful
can bring the answer to class with you
questions on cell cycle regulated, ubiquitin-dependent
proteolysis
- interpret data from in vivo (genetic) and in vitro
experiments