发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: MCBJC, cell division (more discussion, 我又来乐)
发信站: The unknown SPACE (Sun Jan 26 02:16:06 2003) WWW-POST

: 2. If seperase can cleave itself, as suggested by the authors. I can imagine
: two ways how this self-cleavage works. One is that a seperase molecule
: recognizes and cleaves another seperase molecule (intermolecular). The other
: is, although unlikely, that the seperase molecule cleaves itself, in a way
: similar to RNA self-splicing (intramolecular). Can you design an experiment
to
: test theser two possibilities?
i think it requires knowledge about the structure of separase. if
intermolecular, there should be protein-protein interaction, and mutations
that inhibit the interaction should abolish the cleavage, otherwise, not. i do
not know how those genius found out the intramolecular cleavage in ribozymes,
yet i think that sequence-specific mutations help to distinguish "inter" from
"intra" activation. for "intra", the problem is to find how the cleavage
occurs, there should be significant conformational change upon activation.
btw, if it is "intra", then addition of extra cleavage product that is also
active should essentially increase the cleavage rate.

:
: 5. The high phosphorylation level of seperase on Ser1126 by a cryptic
ser/thr
: kinase(s) seems to be true in the in vitro frog extract. However, the
authors
: used a very high concentration of non-degradable cyclin B (delt 90). In that
: way, they increase the CDC2 kinase activity unusally high. This high CDC2
: activity may not occur during normal metaphase. Althogh they did measure the
: percentage of phosphorylated seperase in synchronized cells and showed a
high
: ratio of P-seperase. This still could be an artifact of unnatural increase
of
: CDC2 kinase activity caused by nocodazole arrest. How would you measure
: unarrested, normally progressing metaphase cells' seperase phosphorylation
: ratio? And why this measurement is necessary?

to address the endogenous phosphorylation level of separase, i would like to
separate cells by FACS (does this work, without synchronization using some
sort of drugs?) and then compare the P-level. btw, a CDC2 mutant cell line
should tell how much phosphorylation CDC2 contributes to separase.

see, it is very easy to get "publication" here, particulary when i am BM:).






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