Genetics of cell cycle control

Budding yeast – Saccharomyces cerevisiae

• Cell cycle order tightly linked to morphology:

  • G1 (no bud)→ bud emergence

  • S phase (small bud) → DNA replication (bud grows)

  • G2/M (large bud)→ nuclear division into bud

  • Cytokinesis → daughter separates

• Key feature:

  • START checkpoint (G1/S) = major control point

• arrest phenotype: bud morphology

Fission yeast – Schizosaccharomyces pombe

• Rod-shaped cells grow by elongation

• Cell cycle:

  • G1 (short)

  • S phase

  • Long G2 phase

  • Mitosis → division in the middle

• Key feature:

  • Major control at G2/M transition

  • G2 arrest → elongated cells

  • Premature mitosis → small (“wee”) cells

• arrest morphology: cell length

Cloning by genetic complementation

Concept to Identify a mutated gene by restoring function using a wild-type copy

Hartwell’s experiment (temperature-sensitive mutants)

• Generated ~400 cdc mutants

• Temperature-sensitive (ts) mutants:

  • 25°C → normal growth (permissive)

  • 35°C → defective protein → arrest (non-permissive)

Complementation workflow

• Mutant strain (e.g., cdc28ts) cannot grow at 35°C

• Transform with plasmid library containing wild-type yeast DNA

• Plate at non-permissive temperature (35°C)

• Only cells that receive the correct gene survive

• Isolate plasmid → identify gene (e.g., CDC28)

Key idea:

• Wild-type gene rescues mutant phenotype

• Allows cloning of genes based on function, not sequence

cloning by genetic complementation extensions

• Dosage suppression:

  • Overexpression of related genes (e.g., G1 cyclins) can rescue defects

• Helped identify:

  • Cyclins

  • CDKs

Experiment observations showing G2/M control is governed by phosphorylation

(Nurse, fission yeast)

• cdc2 mutants:

  • Loss of function → cells grow but don’t enter mitosis

  • Gain of function → premature mitosis (“wee” phenotype)

Suggested regulation is not just gene expression, but activity contro

Experiment critical findings showing G2/M control is governed by phosphorylation

1. Cyclin B accumulates before mitosis

2. But MPF (Cyclin B–CDK1) is initially inactive

3. Activation requires:

   • Removal of inhibitory phosphorylation

Conclusion

• Entry into mitosis is controlled by:

  • Post-translational modification (phosphorylation)

  • NOT just protein abundance

Mechanisms regulating G2/M transition

• Core complex - CDK1 (Cdc2/Cdc28) + Cyclin B (Cdc13) = MPF

• Inhibition (prevents premature mitosis)

• Activation (triggers mitosis)

• Activating phosphorylation

• Feedback loops (switch-like behavior)

Inhibition (prevents premature mitosis)

Wee1 kinase

• Adds inhibitory phosphate to Y15 (and T14)

• Keeps CDK inactive

• increase Wee1 - Delayed mitosis → elongated cells

Activation (triggers mitosis)

Cdc25 phosphatase

• Removes inhibitory phosphate

• Activates CDK → mitosis

• increased cdc25 - Premature mitosis → small cells

Activating phosphorylation

CAK (CDK-activating kinase)

• Phosphorylates T161

• Required for full activity

Feedback loops (switch-like behavior)

• CDK:

  • Activates Cdc25

  • Inhibits Wee1

Creates rapid, irreversible transition into mitosis

Experiments defining SPF and phase-specific CDK complexes

Hartwell’s work (budding yeast)

Discovery of SPF (Start Promoting Factor)

Phase:

• G1 = G1 cyclins + CDK

• S = S-phase cyclins + CDK

• G2/M = Cyclin B + CDK1

key insight:

CDK activity depends on:

• Cyclin binding

• Cyclins are phase-specific

Major conclusion:

• Eukaryotic cell cycle uses:

  • Multiple cyclins

  • One or few CDKs

• Timing controlled by:

  • Cyclin expression

  • CDK regulation

Hartwell’s work (budding yeast)

• Identified cdc28 mutants:

  • Arrest in G1

• Showed:

  • Cdc28 = central regulator at START

Discovery of SPF (Start Promoting Factor)

• Cdc28 + G1 cyclins

• Drives G1 → S transition

Evidence for conservation of Cdk1

• Cross-species complementation

• Molecular cloning approach

Key findings:

• Cdc28 = Cdc2 = CDK1

• Highly conserved: Sequence, Structure, Function

Conclusion:

Cell cycle control is:

• Universal in eukaryotes

• From yeast → humans