Fwd: Common integration loci for yeast?

Eric Meltzer via methods%40net.bio.net (by eric.meltzer from gmail.com)
Wed Feb 4 05:42:03 EST 2009


Hi Jennifer,

Thanks very much for the info!  I hunted down some of those resources,
and I think I am fairly clear on what I want to do.  I figured I'd run
this plan by the list in case anyone has experience with this type of
screen, so I can avoid whatever pitfalls there might be.

I have two constructs, both fairly small (800bp and 500bp roughly)
that I want to integrate into URA3 and LYS2 loci by ends-out
integration (i.e. deletion.)  I want to PCR my fragments with homology
tails for those two loci, then transform into URA3 and LYS2 strains.
After transformation, I figure I can do 5FOA and aAA screens for the
integrants.  Doing both of the screens as deletion screens seems to be
kind of uncommon, so I wanted to make sure I wasn't missing anything .  I'm
wondering whether using an 800bp construct to delete a roughly 4kbp gene is
viable?  It seems that at some point if your deletion construct is too
small, you would be likely to get single crossover events leading to
duplication over deletion.

Thanks!



-Eric





On Wed, Jan 28, 2009 at 1:36 PM, Chang, Jennifer
<Jennifer.Chang from nyumc.org> wrote:
>
> Hi, Eric,
>
> I hope I can be of some use here.
>
> When I worked in a yeast lab, we always used metabolic loci (HIS3, URA3,
LEU2, TRP1, ADE2). Once we knew had a integration, it was easy to select fo=
r
that clone on media lacking the metabolite (screening through the different
clones to find the right one is another matter). If you plan on doing
something else later with these yeast cells, such as mating to a different
strain or transformation with a plasmid, then you might want to consider
which metabolic loci you use. Just be aware that you want your nutritional
markers to be compatible at all times. From my experience, HIS is the most
common one used so having the integration at a different loci might be
better.
>
> I don't know exactly what you're using your constructs for but if you wan=
t
to control expression levels, perhaps integrating into the locus of an
inducible (GAL1) or high-expressing gene (ADH1) is desirable?
>
> I can't remember the specific references but try searching for R.
Rothstein. The Rothstein lab constructed many of important strains in use
now and I know they must have published something on one-step or two-step
gene replacement in yeast. Fred Sherman's Getting Started with Yeast
(published 1993 but I'm sure there's newer versions now) was really handy
when I worked in the yeast lab.
>
> Regards,
> Jennifer Chang, PhD
> Post Doctoral Fellow, Cell Biology
> Medical Sciences Building, Room 698
> New York University School of Medicine
> 550 First Avenue
> New York, NY 10016
> Tel 212=96263-5316
> Fax 212-263-8561
>
>
>
> -----Original Message-----
> From: methods-bounces from oat.bio.indiana.edu on behalf of Eric Meltzer
> Sent: Wed 1/28/2009 9:05 AM
> To: methods from magpie.bio.indiana.edu
> Subject: Common integration loci for yeast?
>
> I'm looking to integrate a series of reasonably large constructs into a
> yeast strain.  I know that people commonly integrate into HO, and of
course
> various metabolic loci like HIS or URA.  In trying to figure out the best
> place to integrate, I've been trying to see if there are some other commo=
n
> loci that don't affect growth much (or only affect growth on selective
media
> like HIS etc.).  If anyone can point me in the direction of a good paper,
or
> just a few good loci, I'd appreciate it!
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