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yeast strain for disr

Colin MacDiarmid macdiarm at sbsu1.auckland.ac.nz
Tue Feb 7 18:56:35 EST 1995

In article <108.7.585 at ghawk.com>, tod.lorenz at ghawk.com (Tod Lorenz) wrote:

>  -=> Quoting Colin Macdiarmid to All <=-
>  CM> Hi out there
>  CM> I need some help from experienced gene-disruptors.  I have two genes
>  CM> which I need to interupt, and want to do this by insertion of the URA3
>  CM> gene.  After that I want to be able to use 5-FOA to reselect for a
>  CM> strain which has excised the URA3 gene (but retained my disrupted
>  CM> version of the gene).  The genes were both from a library made from the
>  CM> S288C strain, and strains with this background are sensitive to low
>  CM> concentrations of 5-FOA on plates with proline as sole nitrogen source.
>  CM> So ideally I want a diploid yeast strain derived from s288C to do my
>  CM> disruptions.  Anyone have an idea which strain to use? Can I get it
>  CM> from the YGSC? Please post replies or send direct to me.
>        Is the gene(s) disruption lethal?  It should not matter what
>   the source of the clone bank is, just the background of the yeast
>   being transformed...try the Berkeley yeast stocks for other ura3
>   strains to use.  Why the need for proline as a nitrogen source
>   out of curiosity?  Why not just excise the URA3 gene out with a
>   restriction enzyme if the plasmid is mapped and get around all
>   the FOA problems?
>           /^\/^^^^\/^\     R. Todd Lorenz
>             | #  @ |      Ellicott City, MD
>              \ :: /      T.LORENZ at GENIE.GEIS.COM
>              =\||/=
>                #@

This helpful message was posted to me:

> See Fred Winston's paper in the Jan 95 issue of Yeast. The strains are 
> derived directly from S288C. They are available from the ATCC and they 
> are being used in the genome project.
> Preston Garrison                 garrisonp at uthscsa.edu
> Biochem. Dept.                   voice: 210-567-3702
> Univ Texas Health Sci Ctr        fax:   210-567-6595
> San Antonio, Tx 78284-7760

As for the proline question, and in case anyone else missed it;

AU  - McCusker JH
AU  - Davis RW
TI  - The use of proline as a nitrogen source causes hypersensitivity
      to, and allows more economical use of 5FOA in Saccharomyces
AD  - Department of Biochemistry, Stanford University School of
      Medicine, CA 94305.
AB  - The use of proline as a nitrogen source causes hypersensitivity
      to 5-fluoro-orotic acid (5FOA) and allows up to 40-fold less of
      this drug to be used to select for the loss of URA3 function in
      Saccharomyces cerevisiae. 5FOA hypersensitivity is presumably due
      to the absence of nitrogen catabolite repression when proline is
      substituted for (NH4)2SO4 as a nitrogen source. There are two
      constraints to the use of the proline-5FOA combination: (1) S288c
      genetic background strains are hypersensitive to 5FOA when grown
      in proline as a nitrogen source but at least one other genetic
      background is resistant to low levels of 5FOA under these
      conditions. (2) The addition of some nutritional supplements
      confers phenotypic resistance to the 5FOA-proline combination.
MH  - Culture Media
MH  - Mutation
MH  - Saccharomyces cerevisiae/GROWTH & DEVELOPMENT/GENETICS/
LA  - Eng
SO  - Yeast 1991 Aug-Sep;7(6):607-8

Hope this is useful.

Colin MacDiarmid
macdiarm at sbsu1.auckland.ac.nz

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