Title: ? The first clear link between cyclin and MPF was demonstrated by Joan Ruderman and her colleagues at the Woods Hole Marine Biological Laboratore
1?The first clear link between cyclin and MPF
was demonstrated by Joan Ruderman and her
colleagues at the Woods Hole Marine Biological
Laboratore
2- .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.
3.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).
4- 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.)
5- But what is the relationship between cyclins and
MPF?
6?
- 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 ?. - Cyclin,on the other hand,is totally absent from
clam oocytes but appears soon after fertilization.
7- Ruderman considered the possibility that cyclin A
is an activator of MPF.
8?
- 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.)
9- .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 32PATP in vitro,proteins present
within the sample became phosphorylated,suggesting
that MPF induced maturation by acting as a
protein kinase.
10?
- 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 32PATP
into proteins. - 4.When the purified preparation was incubated in
the presence of 32PATP,the 45-kDa polypeptide
became labeled.
11?
- .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.
12 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.
13These 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.
14?
- 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 32-kDa
protein kinase in vertebrate cells depends on
another protein.
15.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. .
16Thanks!!