elongated inviable vegetative cells with mitotic cell cycle arrest

[fypoadmin]

elongated inviable vegetative cells with mitotic cell cycle arrest parent to: elongated inviable vegetative cells with mitotic cell cycle arrest in S phase

Original comment by: Antonialock

[fypoadmin]

Re: elongated inviable vegetative cells with mitotic cell cycle arrest in S phase

doe this happen?

I didn't think you could detect elongated/ S-phase....

another Q!

Original comment by: ValWood

[fypoadmin]

This has only been used once by me for http://www.ncbi.nlm.nih.gov/pubmed?term=15388803 I'm going to check it now...most likely outcome will be rename

elongated inviable vegetative cells with mitotic cell cycle arrest in S phase to elongated inviable vegetative cells with mitotic cell cycle arrest

Original comment by: ValWood

[fypoadmin]

dpb4 is annotated to it..

Original comment by: Antonialock

[fypoadmin]

I get it now. Here https://sourceforge.net/p/pombase/fission-yeast-phenotype/341/ The conversation wnet

"has elongated vegetative cells which arrest with less than 2C DNA content so this could be mitotic cell cycle arrest in S-phase but I want to capture that they are elongated so could we have mitotic cell cycle arrest in S-phase with elongated cells "

but actually, just because the cells have less than 2C DNA content, doesn't mean they are still in S-phase does it? It just means they proceeded through the cell cycle without activating the DNA replication checkpoint....

so we could rename/ define this term to

elongated inviable vegetative cells with mitotic cell cycle arrest with less than 2C DNA content

Original comment by: ValWood

[fypoadmin]

Yeah I think it doesn't sound like they are necessarily in S-phase

Original comment by: Antonialock

[fypoadmin]

Hmmm, how are we defining S phase here? I would naively have thought that if the cells haven't finished replicating their DNA, they're still in S phase (unless they try to proceed and end up with mitosis going horribly wrong - but in that case, I think you'd see some cells with less than 1C DNA content).

Then again, maybe this just means it's a good thing I try to stay out of the GO cell cycle discussions ...

m

Original comment by: mah11

[fypoadmin]

I didn't look at the paper but couldn't they also have arrested in G1 without going into S-phase? i.e. 1C

Original comment by: Antonialock

[fypoadmin]

the FACS in the paper showed a lot between 1C and 2C ...

Original comment by: mah11

[fypoadmin]

You are right.

However, the classical elongated lethal phenotype I think is considered to be an interphase block so the cells have finished S-phase without completing DNA replication, and then activated the replication checkpoint to stop G2/M transition, so I think they are out of S-phase (without fully replicated DNA)....but are blocked at G2/M

The others were from a different experiment (its a promoter shut off), so for these they are inviable but some have got though G2/M, and some have 2 C DNA content (residual protein?) but failed cytokinesis

I still think you can get rid of "elongated inviable vegetative cells with mitotic cell cycle arrest in S phase" though...

Does that make sense? not sure I am getting confused

Original comment by: ValWood

[fypoadmin]

from http://www-bcf.usc.edu/~forsburg/main4.html

Aside: FACS analysis

People doing flow cytometry on exponentially growing S. pombe cells are puzzled that there is only one peak, corresponding to 2C DNA content. Fission yeast DOES have a G1 phase, but it is short (about 10% of the cell cycle, as noted above). And, the timing of cytokinesis and the timing of the nuclear division cycle are somewhat out of synch. This means that after nuclear division occurs, the nuclei go through G1 and enter S phase before the cells complete cytokinesis. As far as a flow cytometer is concerned, a binucleate cell with two 1C (G1) nuclei looks the same as a uninucleate cell with a single 2C (G2) nucleus. There are a couple of ways around this that can give you a true G1 peak. You can perform FACS on prepared nuclei (ghosts). Or, you can use 2-D analysis by comparing pulse area to width. This discriminates single nuclei from double nuclei. If you eliminate doublets by appropriate gating, you gain significant additional information. Both these methods were developed by Erik Boye's lab in Oslo.

This is one of the reasons I think we should remove the phase stuff from GO and put them in another ontology, and have all organism use them as "during" extensions otherwise it is totally impossible to get all the phases to integrate with cell cycle properly for all organisms.

One for the GO cell cycle meeting agenda....

Original comment by: ValWood

[fypoadmin]

I'm very confused.

One thing is straightforward: if, for whatever reason, you don't feel the need for the "in S phase" specificity, I can easily broaden FYPO:0001497 to just "with mitotic cell cycle arrest". Or I could make it "with mitotic cell cycle arrest with partially replicated DNA" if that's more useful. For either of those I wouldn't really need to worry about the rest of the "is it S phase" subtlety that we've been worrying about. Let me know which you prefer.

I gotta say, though, this comment really confused me:

"However, the classical elongated lethal phenotype I think is considered to be an interphase block so the cells have finished S-phase without completing DNA replication, and then activated the replication checkpoint to stop G2/M transition, so I think they are out of S-phase (without fully replicated DNA)....but are blocked at G2/M"

It brings me right back to the question I asked earlier: how is S phase defined?

In the old days, S phase was just the phase when DNA replication happened (and the PMID:15388803 authors say the cells in the Figure 4 FACS are in S phase). Under that interpretation, the bit about "have finished S-phase without completing DNA replication" makes no sense.

Now that we know a lot more, it's bound to be A Bit More Complicated Than That, but for the "finished S-phase" phrase to make sense, there must be some other way of defining S phase -- and then a given experiment may or may not actually check for the features that say "S phase is still on" or "cells have exited S phase even though they haven't completed replication". Although we can adjust FYPO:0001497 without pinning this down, I'm curious, and we may need the S phase definition for something else later.

Original comment by: mah11

[fypoadmin]

I am going to transfer the discussion about "s-phase" definintion to the GO cell cycle meeting

Original comment by: ValWood

[fypoadmin]

Good. If they/you can get a definition pinned down, it should be a lot clearer what to do with phenotype terms.

Original comment by: mah11

[fypoadmin]

One more comment so I get this straight. Isn't part of the problem that here we are looking at mutants, so we are not looking at normal processes? S-phase is the phase when DNA replication normally takes place, but in some mutants replication does is not complete. Doe this affect the S-phase definition? S-phase is still the period when DNA replication takes place regardless of whether it completed or not...(the definition doesn't say anything about completion)

Original comment by: ValWood

[fypoadmin]

Well, one of the shortcomings of the definition is that it ought to make this more explicit, but I've always interpreted it as implying that S phase starts and ends along with nuclear DNA replication. So if DNA replication isn't completed, the implication is that S phase hasn't ended. That's perhaps the most important thing for the meeting to pin down -- especially if I've been wrong all this time ;)

in some mutants replication does not complete. Does this affect the S-phase definition?

For this, I'd say no, it's the other way around. The definition of normal S phase affects how we describe our mutants and define their phenotypes.

Original comment by: mah11

[fypoadmin]

Let me check I have this right. I am having a block.

If S-phase is not complete until DNA replication has ended, then the mutants which have not completed replication, would be considered to be still in S-phase, whatever. However, if the definition changes so that completion of replication is not a requirement, then a mutant can be described as going in G2 (or M phase) even if replication is incomplete? Have I got it?

Original comment by: ValWood

[fypoadmin]

Although we already know that some mutants can proceed to mitosis with incompletely replicated DNA if the checkpoint is compromised.

human www.ncbi.nlm.nih.gov/pubmed/9927185 sogenic p53-null fibroblasts do enter mitosis with incompletely replicated DNA

mcb.asm.org/content/25/2/563?cited-by=yes&legid=mcb;25/... We show here that cells lacking Chk1, but not those lacking Chk2, enter mitosis with incompletely replicated DNA

nar.oxfordjournals.org/content/27/2/462 rfc2Δ cells proceed into mitosis with incompletely replicated DNA

pombe www.molbiolcell.org/content/10/12/3991.long In the fission yeastSchizosaccharomyces pombe, a number of genes have been ..... because the cell would enter mitosis with incompletely replicated DNA.

So we know that some mutants are out of S-phase but haven't finished replication.

But I don't find this inconsitent with

A cell cycle process comprising the steps by which a cell progresses through S phase, the part of the cell cycle during which DNA synthesis takes place.

because i) DNA synthesis is no longer taking place ii) the definition does not say that replication needs to be complete...

It just says that if replication is occuring the cell is in S-phase

Original comment by: ValWood

[fypoadmin]

So we know that some mutants are out of S-phase but haven't finished replication.

No, a few comments ago this was the big question at hand: whether they're "out of S phase" depends critically upon how S phase is defined.

i) DNA synthesis is no longer taking place

Do we actually know it has stopped? (Obviously this depends on the experiment.) The fact that the cell attempts mitosis in and of itself doesn't say whether DNA synthesis is still going on or not.

ii) the definition does not say that replication needs to be complete...

But we've already noted that the definition may not be optimal. You can't have it both ways -- if the definition isn't precise enough, then any argument built on it in its present form is similarly unreliable.

And until everyone is confident that the S phase definition is as good as possible it's premature to say we know which phase a mutant is in.

For the cell cycle meeting, just try to get the definitions for all the normal cell cycle phases really nailed down -- starts, ends, distinguishing features, necessary & sufficient criteria, etc.

Then we'll have something much more precise and reliable to use to figure out how phenotypes should be defined. In other words, once we have a better, more detailed understanding of what "normal" is, we'll be in a better position to define how this or that mutant deviates from normal. We already know that some mutants don't have S or M phases taking place as in normal cells ...

Original comment by: mah11

[fypoadmin]

what happens with the term? :p

Original comment by: Antonialock

[fypoadmin]

LOL, good question!

For FYPO:0001497, you & Val decide whether you want to keep any phase specificity at all. If you don't need the specificity, I edit the existing term; otherwise I add a new term.

For any term that mentions a cell cycle phase (whether FYPO:0001497 remains one of them or not) I see what the GO cell cycle meeting comes up with, and change logical definitions if necessary.

Original comment by: mah11

[fypoadmin]

watch this GO SF item for phase definitions: https://sourceforge.net/tracker/?func=detail&atid=440764&aid=3610881&group_id=36855

Original comment by: mah11

[fypoadmin]

I think I'm ok with elongated inviable vegetative cell - I don't think I need the mitotic cell cycle arrest tacked on.

1. I suppose the 'elongated' bit makes it implicit that the cells are not lysed and
2. the fact that they are vegetative means they are mitotic and not meiotic, so I suppose specifying 'mitotic cell cycle arrest' doesn't really add anything to the term.

54de4db66c7dfe2e and 82e75444a0553c25

Original comment by: Antonialock

[fypoadmin]

Val just said today that elongated is interpreted to mean cell cycle arrest (or at least delay) in interphase in pombe, so maybe the easiest thing is just to use 'elongated inviable vegetative cell' (FYPO:000190), and merge the 'S phase' term into it (could keep name as a narrow synonym.

There aren't any annotations to FYPO:0001497 anyway ...it was added for the ticket now known as [fission-yeast-phenotype:#341]

Original comment by: mah11

[fypoadmin]

• labels: --> other ontology change
• status: open --> closed-out-of-date
• assigned_to: Midori Harris

Original comment by: mah11

[fypoadmin]

renamed FYPO:0001497 - see ticket 1539 (linked)

closing at last - yay!

Original comment by: mah11