发信人: reer (), 信区: Biology
标 题: Re: MCBJC, cell division, Jan 20 2003, by Assasin
发信站: The unknown SPACE (Sun Jan 26 00:39:22 2003) WWW-POST

If I am wrong, correct me.
For Q5, if they use CSF extract( a kind of Xenopus extract, which should be
natually mitotic extract, once you add Calcium, a good CSF extract will start
to cycle.), they can avoid the possible artifact. The problem is in their
paper they use all human proteins(separase, securin and cohesin.) If they use
CSF extract, the proteins they can analyze for MS are Xenopus proteins. What I
don't understand is why they use human proteins.

For Q6,
Nagao K, Yanagida M.
Regulating sister chromatid separation by separase phosphorylation.
Dev Cell. 2002 Jan;2(1):2-4. Review.
PMID: 11782307
I cannot get the full text paper. I believe this one is talking about Marc's
cell paper.

For me, this paper provides a new regulation point in mitosis. This mechanism
happens to be related to CDC2/cycB activity.


在 assasin (冷血---何时能被加热？) 的大作中提到: 】
: MCBJC, cell division, Jan 20 2003, by Assasin
:
: paper:
:
: Stemmann, O., Zou, H., Gerber, S.A., Gygi, S.P., and Kirschner, M.W. (2001)
: Dual inhibition of sister chromatid seperation at metaphase. Cell 107,
: 715-726.
:
: Questions:
:
: 1. Describe the differences between S. cerevisiae chromosome seperation
: pathway and that of metazoan cells.
:
: 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?

:
: 3. In Fig. 2 A, lane 5-8, they used in vitro translated (5,6) and purified
: endogenous human SCC1 as substrates for seperase. Why the gels shows
multiple
: bands besides the bands of cleaved and uncleaved hSCC1? Do you think this is
a
: neat experiment? How to solve this problem?
:
: 4. In Fig. 3 B & C, they tried to purify endogenous human securin/seperase
: complex. However, silver staining (Fig. 3 C) showed that there were other
: proteins in fraction 5 & 6. Do you think these unknown proteins would cause
: problems to the in vitro cohesin cleavage assay? How to make more pure
: securin/seperase preparatons?

It must be very hard. Go through more columns but you will lose a lot of
proteins and need more hela cells.
:
: 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?
:
: 6. In terms of studying the mechanism of chromosome seperation, What's novel
: about this paper compared to those published before it?
:
:


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