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发信人: ghostneuron (synapse+IX), 信区: Biology
标 题: Re: MCBJC, cell division, Jan 20 2003, by Assasin
发信站: The unknown SPACE (Sat Jan 25 04:59:00 2003) WWW-POST
1. I learned the molecular concept of 'IN VIVO vs IN VITRO' from this paper,
because in my study, the in vivo means animal experiments, and i fully agree
this is the 'right'way to explore biological questions, at least in the
begining.
2. It's a tough paper for me, since I am not familiar with this field, but
good paper is always good, i did like it for the way they ask questions and
design exp. logically.
3. For the 2nd question of Assasin, it is not difficult if you can purify
seperase without destroying its bioactivity, and measure the cleavage after
mixed incubation.I don"t know in Figure 7, if they have used a blank control
without adding L or H after L, but with seperase itself, could it answer this
question? For the intramolecular hypothesis, can i use mutant methods to get
different seprases and try to cleave itself? The problem is I don't know where
to mutate, phosphorylation site or other modification site.
4. I want to know more on the tech of Mass Spectrometry, is there anyone could
suggest me some good readings?
【 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?
:
: 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?
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 195.143.]
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?
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 129.111.]
发信人: Marble (小石头哥哥), 信区: Biology
标 题: MCBJC, cell division, discussion
发信站: The unknown SPACE (Sun Jan 26 01:07:48 2003) WWW-POST
【 在 assasin (冷血---何时能被加热？) 的大作中提到: 】
: Hi, folks,
:
: please read and discuss. and make this first MCBJC work.
i do not think that everybody has read this paper. we need more enthuisasm and
effort to keep active discussion and make this MCBJC run. meanwhile, think
about the paper that you will pick up later.
: : 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?
these unknown proteins could interact with separase/securin and thus make the
result hard to interpret. an affinity purification might help, however, it
still
could not exclude contamination of tightly associated proteins. yet the major
goal for this part of work is to identify "inhibitory factors", particularly
phosphorylation of separase per se. i am glad that they indeed found the
inhibitory phosphorylation site by mass spec. and then later they showed that
expressed phosphomutants support their hypothesis that phosphorylation can
inhibit separase. i am wondering whether they use bacteria or mammalian
expression system. for mammalian system, my concern is that purified protein
might also associate with unknown polypeptides.
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 130.203.]
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:).
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 130.203.]
发信人: assasin (冷血---何时能被加热？), 信区: Biology
标 题: Re: MCBJC, cell division, Jan 20 2003, by Assasin
发信站: The unknown SPACE (Sun Jan 26 14:58:39 2003) WWW-POST
I think this is a good paper to discuss for that Xenopus egg extract system
enables researchers to combine biochemical, molecular, cell biological
approaches to address the mechanism of cell cycle control. The shortcoming of
this system is that most experiments were done in extract, i.e., in vitro. So
it’s always necessary to study other vertebrate system side by side. Another
shortcoming is that sometimes it’s very hard to clone frog genes or to
isolate protein factors. Frog is tetraploid. It has undergone extensive gene
duplication during evolution. So for one single biochemical function, there
could be several redundant factors, with few amino acid sequence differences,
involved.
In contrast, yeast S. cerevisea is very genetically characterized system. It
has haploid and diploid forms of life. People can do sophisticated gene
manipulations. But yeast is just yeast, it differs to vertebrates in a lot of
aspects of cell cycle control. Human cell lines (most are cancer cell lines)
are not amenable to biochemical assay. So assaying human proteins in frog egg
extract becomes an acceptable method, assuming that these proteins are
functionally conserved across vertebrates.
This paper used massspec to map the phosphorylation site of seperase. I think
this is a very powerful method. In fact, to characterize the protein factor(s)
you have purified from a biochemical system, you need to know the sequence of
that protein. You digest the protein with endo-peptidase, and you need to use
mass-spec to sequence the peptide fragments and finally know what the protein
is. To me, using mass-spec to map P-site is quite a new application. Imagine
how hard Tony Hunter was mapping the tyrosine receptor P-site. He was lucky
for there was few site(s) phosphorylated in tyrosine receptors. Actually a lot
of proteins have multiple P-sites, to quickly identify them, mass-spec
probably is the best way.
【 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?
:
: 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?
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 136.152.]
发信人: assasin (冷血---何时能被加热？), 信区: Biology
标 题: Re: MCBJC, cell division (discussion?)
发信站: The unknown SPACE (Sun Jan 26 15:07:00 2003) WWW-POST
【 在 Marble (小石头哥哥) 的大作中提到: 】
: come on, show time, folks. so should we all email assassin back and corrupt
: his mail box? i won't discuss all of the questions listed by assasin.
actually,
: to read papers on Cell is always headache, particularly manuscripts by
: biochemistry/cell biology people.
:
: : 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?
:
: Fig2A shows that purified separase can utilize in vitro translated(IVT)
hSCC1
: as well as endogenous hSCC1 as substrate in an in vitro cleavage assay. I
think
: the IVT sample (in RRL?) should be purified first (avoid incomplete
translation),
: there should be a mock w/o separase (don't like otherwise degradation
though),
: and include a control hSCC1 that could not be cleaved (i am not satisfied by
: the Cys/Ser mutation of separase). the left side is autoradiography,
sometimes
: hard to avoid showing those multiple bands; the right side is western, there
: are also some bands (maybe non-specific), or maybe intermediate cleavage
: products, however, i do not see a clear cleavage band on the top here
(compare
: lane 5 and lane 7). don't know the source of the antibody.
:
: btw, i have a question,. in Fig5A , how did they get the "separation%"?
: ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
I think they isolated human cell metaphase nuclei. And they break(?) the
nuclei membrane and isolate the chromosomes. They add the metaphase
(un-seperated) chromosomes into the frog extract and stain chromatid and
centromere. And count the seperated vs. unseperated chromatids. they got the
seperation %.
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 136.152.]
发信人: assasin (冷血---何时能被加热？), 信区: Biology
标 题: Re: MCBJC, cell division, Jan 20 2003, by Assasin
发信站: The unknown SPACE (Sun Jan 26 15:19:02 2003) WWW-POST
【 在 reer () 的大作中提到: 】
: 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 P-level reaches the highest just before metaphase-anaphase transition
(MAT) and goes down very quickly at the onset of annaphase. MAT is very quick.
I am wondering when sampling the rection, how do you know exactly at which
phase your CSF is. Oh, maybe you can sample every 5 minutes. Or we can add
some fluorescent cellular makers (green chromatid?) into the extract and
monitor the progression of cell cycle under microscope. .......?
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.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
good point, i agree.
: 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
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 136.152.]
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 18:38:34 2003) WWW-POST
A good CSF extract should be arrested @ metaphase.
After the addition of calcium or fertilization which causes a transient
increase in cytoplasmic calcium conc., the CSF will be inactivated, following
by APC activation, cyclin B destruction and mitotic exit. And this process is
roughly 30 min. It is easy to collect phosphorylated separase before the
addition of calcium. To collect the unphosphorylated one, like you say, you
can throw sperm chromatin into extract and monitor the progresstion by DAPI or
some other fluorescent dye of DNA.
Actually, can you guys answer me why they use human proteins in this study?
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
hard for me to understand.
For Q6, another regulation of separase which is introduced by phosphorylation
can be so-well fit into the network of cell cycle regulation( phosphorylation
counts for much relationship between proteins, even proteolysis is dependent
on phosphorylation although there may be some exception.) In Marc's paper "
the timing of events in mitosis", they proposed two extreme models of
regulation of the timing of events in each phase of the cell cycle, which are
"regulator-controlled model" and "substrate-controlled model" Their conclution
is that "during early mitosis the timing of biochemical events and
morphological events is at least partly controlled by the responses of the
substrates themselves to a common set of signals." Now they may add separase
as another substrate which can controll the downstream events.
【 在 assasin (冷血---何时能被加热？) 的大作中提到: 】
: 【 在 reer () 的大作中提到: 】
: : 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 P-level reaches the highest just before metaphase-anaphase transition
: (MAT) and goes down very quickly at the onset of annaphase. MAT is very
quick.
: I am wondering when sampling the rection, how do you know exactly at which
: phase your CSF is. Oh, maybe you can sample every 5 minutes. Or we can add
: some fluorescent cellular makers (green chromatid?) into the extract and
: monitor the progression of cell cycle under microscope. .......?
:
: 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.
: ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
: good point, i agree.
:
:
: : 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.
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 129.111.]
发信人: oil (brother), 信区: Biology
标 题: Re: MCBJC, cell division, Jan 20 2003, by Assasin
发信站: The unknown SPACE (Mon Jan 27 01:42:41 2003) WWW-POST
let me try to say something, see if I understand it right.
they claimed the 2 inhibitory functions on separase:
1. securin. no data on this part. assume it's well-known.
and this seems have no in vivo function as shown by mutations
(introduction) securin sequences are not conserved.
2. separase phosphorylation. then
2.1 what phosphorylate separase? ( did they show this clear?)
2.2 effect of the phosphorylation ( this should be convincing)
2.3 this happens in vivo (maybe convincing?)
I think the significance of this event is not fully revealed.
(I mean genetics)
3. separase self cleavage? I can't find real data on this point.
Please comment on these.
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 128.125.]
发信人: reer (), 信区: Biology
标 题: Re: MCBJC, cell division, Jan 20 2003, by Assasin
发信站: The unknown SPACE (Mon Jan 27 02:04:48 2003) WWW-POST
【 在 oil (brother) 的大作中提到: 】
: let me try to say something, see if I understand it right.
:
: they claimed the 2 inhibitory functions on separase:
: 1. securin. no data on this part. assume it's well-known.
: and this seems have no in vivo function as shown by mutations
: (introduction) securin sequences are not conserved.
:
: 2. separase phosphorylation. then
: 2.1 what phosphorylate separase? ( did they show this clear?)
CDC2/cycB or Erk2 or other kinases, but they do exclude some kinase like
polo,auroraA and CaMKII by in vitro kinase assay.
: 2.2 effect of the phosphorylation ( this should be convincing)
: 2.3 this happens in vivo (maybe convincing?)
:
: I think the significance of this event is not fully revealed.
: (I mean genetics)
:
: 3. separase self cleavage? I can't find real data on this point.
they do show some data in fig2A, they mutated the catalytic site of
separase and found that separase can not be cleaved. "As the active site lies
far from where cleavage occurs, this result implies that the cleavage of
separase is autocatalyzed" Not quite convincible.
If you are interested, you can go read "anaphase specific auto-cleavage of
separase" done by same group.
:
: Please comment on these.
:
:
--
※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 129.111.]
发信人: clio (我是谁), 信区: Biology
标 题: Re: MCBJC, cell division, Jan 20 2003, by Assasin
发信站: The unknown SPACE (Mon Jan 27 05:17:38 2003), 转信
For question 2: I guess maybe we can test hte kinetics of cleavage reaction.
We can tell whether it is intra or inter molecular cleavage from the
concentration dependence of hte reaction.
【 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?
: 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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※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 61.156.]
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