Ⅰ
The first clear link between
cyclin and MPF
was demonstrated by Joan
Ruderman and her colleagues at the
Woods Hole Marine Biological
Laboratore
• .In these studies,an mRNA encoding
cyclin A was transcribed in vitro from a
cloned DNA fragment that contained the
entire cyclin A coding sequence.
• The identity of this mRNA was verified by
translating it in vitro and finding that it
encoded authentic clam cyclin A.
.When the
synthetic cyclin
mRNA was
injected into
Xenopus
oocytes,the cells
underwent
germinal vesicle
breakdown and
chromosome
compaction over
a time course not
unlike that
induced by
progesterone
treatment(Figure
5).
• These results suggested that the
rise in cyclin A,which occurs
normally during meiosis and
mitosis, has a direct role in
promoting entry into M phase.
•
(The amount of cyclin A normally drops rapidly and must be resynthesized
prior to the next division or the cells cannot reenter M phase.)
But what is the relationship
between cyclins and MPF?
Ⅱ
• One of the difficulties in answering this
question was the use of different organisms.
• (MPF had been studied primarily in
amphibians,and cyclins in sea urchins and clams.)
• Evidence indicated that frog oocytes contain a
pool of inactive preMPF molecules,which are
converted to active MPFs during meiosis Ⅰ.
• Cyclin,on the other hand,is totally absent from
clam oocytes but appears soon after fertilization.
• Ruderman considered the
possibility that cyclin A is an
activator of MPF.
Ⅲ
• Meanwhile,another line of
research was initiated to purify
and characterize the substance
responsible for MPF activity.
• (In 1980,Michael Wu and John Gerhart of the University
of California,Berkeley,accomplished a 20-to 30-fold
purification of MPF by precipiating the protein in
ammonium sulfate and subjecting the redissolved material
to column chromatography.)
• .In addition to stimulating oocyte
maturation,injections of the partially purified MPF
stimulated the incorporation of 32P into protins of
the amphibian oocyte.
• .When partially purified MPF perparations were
incubated with [32P]ATP in vitro,proteins present
within the sample became
phosphorylated,suggesting that MPF induced
maturation by acting as a protein
kinase.
Ⅳ
• 1.MPF was finally purified in 1988 by a series
of six successive chromatographic steps.
• 2.MPF activity in these purified preparations was
consistently associated with two polypeptides,one
having a molecular mass of 32kDa and the other
of 45 kDa.
• 3.The purified MPF preparation possessed a high
level of protein kinase activity,as determined by
the incorporation of radioactivity from [32P]ATP
into proteins.
• 4.When the purified preparation was incubated in
the presence of [32P]ATP,the 45-kDa polypeptide
became labeled.
Ⅴ
• .By
the end of the 1980s,the efforts to uncover the
role of cyclins and MPF had begun to merge with
another line of research that had been conducted on fission
yeast by Paul Nurse and his colleagues at the University of Oxford.
• .It had been shown that yeast produced a protein
kinase with a molecular weight of 34kDa whose
activity was required for these cells to enter M
phase .
•The yeast protein was called p34cdc2 or simply cdc2.
• The first evidence of a link between cdc
and MPF came as the result of a
collaboration between yeast and
amphibian research groups.
Antibodies formed against cdc2 from
fission yeast were shown to react
specifically with the 32-kDa component of
MPF isolated from Xenopus eggs.
•These
findings indicate that this
component of MPF is a homologue
of the 34-kDa yeast kinase
and,therefore,that the machinery
controlling the cell cycle in yeast
and vertebrates contains
evolutionarily conserved
components.
Ⅵ
• A similar study using antibodies against
yeast cdc2 showed that the homologous
protein in vertebrates does not fluctuate
during the cell cycle.
• .This supports the proposal that the 32kDa protein kinase in vertebrate cells
depends on another protein.
.In all of these cases,it was shown that the active
MPF present in M-phase animal cells is a complex
consisting of two types of subunits:(1)a 32-kDa
subunit that contains the protein kinase active site
and is homologous to the yeast cdc2 protein
kinase,and (2) a larger subunit (45 kDa) indentified
as a cyclin whose presence is required for kinase
activity.
The studies described in this Experimental Pathway provided a unified
view of the regulation of the cell cycle in all eukaryotic organisms.
.
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