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Introduction To The Cell Cycle: Promega Education Resources Unit 7
Introduction To The Cell Cycle: Promega Education Resources Unit 7
G1 S G2 M G1 S G2 M G1 S G2
Interphase: G1
• Events during G1
Cell growth
Preparation of chromosomes for replication
Duplication of cellular components
G1 checkpoint (or restriction point); cell commits to division or exits
from cell cycle
Interphase: S phase
• DNA replication
• Duplication of the centrosome
The centrosome is located near the nucleus of the cell and
contains the microtubule organizing center MTOC in animal cells. It
contains two centrioles that migrate to the poles before cell division
and serve to organize the spindle.
Interphase: G2
• Cell growth
• Checkpoint (restriction point) for entry into M phase
M phase
• Cell division (mitosis or meiosis for germ cells)
• Can be subdivided into four subphases:
Prophase
Metaphase
Anaphase
Telophase
• Factors that influence M phase entry
Cellular Mass
Growth Rate
Time (During early embryogenesis, divisions may proceed rapidly,
essentially alternating M and S phases, with little growth between
them.)
Completion of DNA Replication
Studying the Cell Cycle
• Much of what we know about the cell cycle comes from
the study of model experimental systems:
Genetics
• Yeast: Schizosaccharomyces pombe (fission yeast) and
Saccharomyces cerevisae (budding yeast)
• Short article from HHMI (http://www.hhmi.org/genesweshare/a300.html)
Biochemistry
• Frog eggs
• Mammalian cell culture
Control of the Cell Cycle
• Intrinsic Control: Cyclins
Proteins whose levels rise and fall during the various phases of the
cell cycle (primary regulators of the cyclin-dependent kinases)
Interact with the cyclin-dependent kinases (cdk)
Cdk levels are constant
Cdks must bind to cyclins to be activated
The complexes of cyclin+cdk act in concert. The cdks
phosphorylate proteins that initiate or regulate cell cycle activities.
Cyclins also may be involved in cdk target recognition.
Cdk activity is terminated by cyclin degradation (generally).
Cdk activity can be “fine tuned” by other mechanisms (i.e.,
inhibitory phosphorylation by Wee1 kinase, activation by cdc25
kinase. Cdk inhibitor proteins also can regulate the cyclin-cdk
complexes.
Cyclins
• Four classes
Defined by phase of the cell cycle in which they bind
their cdk
•G1/S phase cyclins-bind cdks at the end of G1,
commit cell to DNA replication (cyclin E)
•S phase cyclins- bind cdks during S phase, required
to initiate replication (cyclin A)
•M phase cyclins- bind cdks immediately before
M phase, initiate early mitotic (or meiotic) events
(cyclin B)
•G1 cyclins- involved in progression through the
checkpoint in late G1 (cyclin D)
M phase Promoting Factor (MPF)
• Cytoplasmic protein factor responsible for initiation of
meiotic and mitotic phases of cell cycles in eukaryotes
• First detected in unfertilized amphibian eggs
• Progesterone triggers oocyte maturation; MPF is
important for progression from meiosis I)
• MPF activity rises at beginning of meiosis II and before
mitotic divisions after fertilization
• MPF activation, oocyte activation and mitosis will not
occur without protein synthesis*
What is MPF?
• MPF is a complex of proteins
• In 1988, one of the proteins associated with MPF was
shown to be homologous to the cdc2 gene of S. pombe
• cdc2 gene encodes a protein kinase
• Could one of the other components be cyclins?
Xenopus oocyte maturation
• Progesterone triggers oocyte maturation
• Oocytes progress through two cycles of mieoisis, but the
second meiotic division is arrested at metaphase II until
fertilization
• Oocytes are released from metaphase II arrest when
exposed to sperm chromatin
• Scientists often prepare extracts from oocytes that have
been released from metaphase II arrest for their studies.
• Cytoplasm extracted from oocytes will “cycle” when
exposed to sperm chromatin
Key Experiment
• Murray and Kirschner (1989)
• Determine that cyclin is involved (isolated “cycling”
proteins from extract)
• Show cyclin synthesis is required to drive cell cycle
• Prepare extracts from unfertilized Xenopus eggs
Contain all materials required for multiple cell cycles
Can synthesize proteins from maternal mRNA in the cytoplasm
Can be mixed with chromatin from interphase sperm generates a
haploid nucleus with sperm DNA, which will replicate, and the
extract will go through “mitosis” (cycling extracts).
Experimental Details
• Treat cycling extracts with limited amount of RNase A
Degrades all of the maternal mRNAs
Does not degrade the tRNAs or rRNAs necessary for protein
synthesis (these RNAs are protected by proteins within the
protein/RNA complexes)
• Add RNasin Ribonuclease Inhibitor to inactivate RNase A
• Add back cyclin B mRNA
Extracts resume cycling activity
• Add cyclin B mRNA that encodes a protein with a
mutated protein-degradation signal to parallel extracts
Extracts start to cycle, but get “stuck” in early mitotic events