hydroxyurea arrest

Namjin Chung nc1 at acpub.duke.edu
Wed Sep 11 15:33:21 EST 1996


Well, this was the exactly same question which I was going to ask when I 
read through messages in this newsgroup today.  Basically I had the same 
problem while I was adopting the experiment in a Science paper published 
early this year from S. Elledge lab.

My experimental procedure was:

1. Wild type lab strain cells were grown to saturation in YPD for overnight.

2. Cellw were, then, diluted to get OD(600)=~0.1, and regrown until the 
OD became 0.5-0.8 (log phase and more than 80% of cells had buds when 
observed under microscope).

3. Diluted cells with either 0.2M hyroxyurea (HU)/YPD or HU/water to get the 
cell density of around 2000/mL, and incubated cells in a roller drum at 
30C for 0, 2, 4, 6, 8 hours.

4. At specified time, 100uL was sampled and plated on YPD medium, and 
incubated for 2 days.


There were normal-sized big colonies and very small colonies, and I 
counted separately.  However, neither total number of colonies nor 
number of big colonies decreased over incubation time.  In the above 
mentioned reference, there was a large drop in the number of wild type 
colonies.

I did several times with triplicate each time.  Even used different 
batches of hydroxyurea (though both from Sigma).  I also tried cells from 
freshly streaked plate.  All didn't work with me.  

Does anyone out there with experiences in hydroxyurea effects on yeasts?  
Please give us some advice.  Thank you in advance.


Namjin Chung 
Duke University Medical Center


On Wed, 11 Sep 1996, Martin Stahl wrote:

> Subject: hydroxyurea arrest 
> 
> Hi everybody!
> Can anybody help me with hydroxyurea arrest? I used the amount of
> hydroxyurea as published (300 mM), but even the double amount did not
> arrest my cells (W303 background) in S-phase.
> Even after 5 hours of the arrest I found cells with large and small
> buds, as well as unbudded cells. The only effect I could see was that
> the fraction of cells with small buds has increased.
> Thank you in advance,
> 

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* Namjin Chung                             *                           *
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