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Cell biology > Lecture 14 > Flashcards

Lecture 14 Flashcards

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1
Q

What did lee Hartwell find whilst using selective temperatures to identify genes that control cell division?

A

Isolated ~400 different temperature sensitive mutants in S. cerevisiae

Ts mutants grow at 25 degrees (not higher than 35 degrees)

Complementation used to identify matured genes

Functional copy rescues cells at non-permissive temperatures

Wild type yeast digested and ligated to plasmids - cloning genes by complementation

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2
Q

Process of genetic complementation

A

cdc28ts cells grown at 25 degrees

Transform with plasmids of wild type S. cerevisiae DNA

Transformed cells grow at 35 degrees

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3
Q

What occurs in budding yeast cell cycle

A
  • Some Cdc28ts mutants arrest in G1
  • Cdc28 is a component of SPF (CDK)
  • Gene complementation to identify compensatory mechanisms identified G1 cyclins
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4
Q

How are G1 cyclins in S. cerevisiae identified?

A
  • Wild type cells showed colonies form between G1 and high affinity cdc28 at both 25 and 26 degrees
  • cdc28ts cells showed colonies form between Cdc28ts and G1 cyclin at 25 degrees, but Cdc28ts arrested in G1 at 36 degrees
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5
Q

When will SPF form between G1 cyclin and Cdc28ts at 36 degrees?

A

When G1 cyclin is in excess

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6
Q

Fission yeast cell cycle

A

G1 - Growth
S- DNA replication
G2 - Spindle pole body duplication
M - Chromosomes condense, spindle formation, Chromosome segregation, nuclear division
Cytokinesis

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7
Q

What was identified in fission yeast cell cycle of S. pombe

A
  • Cdc mutants in G2/M arrest
  • Complementation developed to identify cell cycle regulators
  • Complementation used a gene library
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8
Q

Colonies of S. pombe containing mutants at 25 vs 35 degrees

A

1 S. pombe cdc2ts -> No colonies at 35 degrees
2. S. pombe cdc2ts + cdc28 -> Colonies formed at 35 degrees

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9
Q

Are cdc28 homologues conserved in yeast?

A

Yes

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10
Q

what is structurally and functionally conserved in eukaryotes

A

Cdk1

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11
Q

How is Cdk1 structurally and functionally conserved?

A
  1. DNA coding region predicted by amino acid sequence
  2. Degenerate oligonucleotides synthesised corresponding to regions 1 and 2
  3. Oligonucleotides amplify DNA between regions 1 and 2 and then whole open reading frame from cDNA source
  4. Clone open reading frame into yeast expression plasmid
  5. Test conservation by complementation
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12
Q

How was it proven eukaryotes are universally conserved?

A

Eukaryote + CDK1 -> Colony formation

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13
Q

How do cdc different mutants affect G2/M transition

A

cdc2+ - Optimal cell division
Cdc2- - Unable to enter mitosis, cell growth continues
Cdc(D) - Enter mitosis inappropriately, Cell growth insufficient

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14
Q

How is phosphorylation aligned to the cell cycle?

A
  1. CDK are regulated by cyclin binding
  2. Mitotic CDK (Cyclin B-CDK1) activity regulated by activating and inhibitory phosphorylation in addition to cyclin binding
  3. Formation of MPF not sufficient to control mitosis
  4. Wee 1 kinase and Cdc25 phosphatase required
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15
Q

G2/M transition regulated by phosphorylation state of MPF

A
  1. Mitotic cyclin expression occurs in late S/G2
  2. MPF remains inactive due to inhibitory phosphorylation - Wee1
  3. Cdc2 requires phosphorylation at T161 for activity - CAK (CDK activating kinase)
  4. Inhibitory phosphorylation at Y15 must be removed for full activity
  5. Cdc25 removes inhibitory phosphorylation to induce mitosis at G2/M transition
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16
Q

What controls activation of MPF

A

Wee1 and Cdc25

Mitotic CDKs inactive in G2 due to inhibitory phosphorylation by Wee1

Removal of phosphorylation by Cdc25 enables rapid transition into mitosis

17
Q

Explain the process of temporal regulation

A
  1. Wee1 inhibits CDK via inhibitory phosphorylation (pY15)
  2. Cdc25 activates CDK
  3. CDK enhances Cdc25 activity
  4. CDK inactivates Wee1

Removal of inhibitory Y15 phosphorylation by Cdc25 phosphatases fully activates mitotic CDK

18
Q

Wee1 and Cdc25 phosphatase

A

Prevented mitosis - Wee1 excess, Deficit of Cdc25

Inappropriate mitosis - Wee1 deficit, Cdc25 excess

Regulated mitosis - Opposing and balanced CDK and Wee1 activities

Cell biology (22 decks)

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