MCB 252 Lecture 38

The parallel between a biochemical and genetic pathway was demonstrated in:

Phage T4 assembly

What chemical secreted by follicle cells surrounding the oocyte triggers maturation?

Progesterone

Advantages for studying yeast:

Rapid growth (90 minute cell cycle), both haploids and diploids undergo mitosis, isolate recessive mutants in haploids, cell morphology correlates with position in cell cycle

What implications did repeating the 1/100,000 volume cytoplasm transfer for one more step have?

1. Factor other than progesterone is involved = MPF 2. MPF may be or regulate an enxyme 3. MPF may be autocatalytic

How was the Maturation Promoting Factor (MPF) first discovered?

1/100,000 volume of the cytoplasm from an MII arrested egg drives interphase oocytes into meiosis as though they had been treated with progesterone

Starving diploid yeast cells of N makes them undergo meiosis to produce:

2 alpha haploids and 2 a haploids

Cyclin protein increases in abundance until crashing in what phase of the cell cycle?

Anaphase

Advantages of studying the embryonic cell cycle:

Cell division cycles are synchronous, early cycles are rapid, good source of materials for biochemistry (Xenopus egg 1 mm in diameter = huge)

What must occur in order for cells to exit from mitosis?

Cyclin degradation

G1 cell fused to an S cell causes:

DNA replication induced in G1 cell due to factor in S cell

Cell Fusion Experiments

Fuse cells together, monitor effects

Xenopus oocytes first arrest at 8 months when in the ____ phase.

G2

MPF drives (or regulates) which transition in the cell cycle? A. G2 > M B. S > G2 C. M > G1 D. G1 > S

G2 > M

What transition does progesterone trigger?

G2 > M

G2 cell fused to an S cell:

G2 cells do not respond to the factor in S cells that causes replication; block to replication so that is occurs once and only once

What experiment showed that MPF is only present and active in M phase?

Injections to assay MPF activity: take cytoplasm from cells in different stages to see which trigger G2 > M transition

Fusing a mitotic cell with an interphase cell causes:

Interphase nuclei to go into mitosis (nuclear envelope breaks down and chromosomes condense), suggesting that M phase cells have a factor that drives Interphase cells into M phase

What region on the cyclin protein marks it for polyubiquitination?

Last 90 N-terminal amino acids

Xenopus embryo cells arrest in ___ prior to fertilization.

MII

Cyclin protein levels correlate with ____ activity.

MPF

Cytoplasm from arrested mammalian cells also contains MPF activity, suggesting that:

MPF is highly conserved

What experiment led to the identification of Cyclin?

Study of translation in sea urchin embryos: take fertilized eggs, add 35-S-Met, take samples every 10 minutes, SDS-PAGE, autoradiography

How was a Xenopus egg extract prepared to go through the cell cycle in vitro?

Tightly packed cells in mineral oil, eggs crushed and separated from oil by centrifugation, added sperm nucleus with membrane removed, reassembly of nucleus, DNA replication, mitosis

How were egg extracts used and treated to reveal the role of Cyclin B?

Treat extracts with low dose RNAse to destroy mRNAs, Inactivate RNAase by removing Ca2+, Add back sea urchin Cyclin B mRNA transcribed in vitro

Background: In lecture we talked about a Xenopus egg extract that can undergo cell cycles in vitro. We saw that addition of an RNAase resulted in degradation of the mRNAs in this extract and blocked the ability of the extract to undergo cell cycles in vitro. True or False? Inhibition of the RNAase followed by addition of the mRNA for cyclin restored cell cycling. As a result we can conclude that cyclin is required for progression through the cell cycle and that cyclin is the only protein that needs to be translated in these extract to get cycling to occur.

True

True or False? Cyclin protein is both necessary and sufficient for (and therefore must be a regulator of) the cell cycle.

True

True or false? MPF function is conserved from frogs to humans.

True

Background: In lecture we talked about a Xenopus egg extract that can undergo cell cycles in vitro. We saw that addition of an RNAase resulted in degradation of the mRNAs in this extract and blocked the ability of the extract to undergo cell cycles in vitro. After inhibiting the RNAase in the previous question one can add back different versions of the cyclin mRNA. As described above, addition of the wild-type version results in multiple rounds of cycling. Addition of no mRNA results in replication of the DNA but the extract does not enter mitosis. You next add back a version of the cyclin mRNA that lack the region that codes for the N-terminal 90 amino acids of cyclin. This mRNA codes for a version of the cyclin protein that does not get degraded. You find that addition of this mRNA to your extract allows your extract to enter M-phase but not exit M-phase. Do these results support the conclusion that cyclin synthesis is required for entry into M-phase and that cyclin degradation is required for exit from M-phase?

Yes