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a strategy for gene replacements

Morris F. Manolson mm6y+ at andrew.cmu.edu
Mon Feb 7 10:50:53 EST 1994


>- a good general strategy for replacing a wt chromosomal gene by a cloned
>mutant allele without introducing markers or rearrangements into the
>locus would be:
> 
>1. Disrupt the gene of interest with URA3. If you are worried about
>sickness or inviability of the disruptant, do it in a diploid or in
>the presence of a plasmid-borne wt copy.


and if for any reason you can not use URA3, you can always use LYS2.  
You can select against LYS2 by growing the yeast on plates containing
DL-alpha-aminoadipate.  There are similar schemes for the CAN1 and CYH2
genes.   I would recommend Guthrie and Fink, Meth. in Enzymol., 
vol 194, (1991), chapter 19 by R. Rothstein, Targeting, Disruption,
replacement, and allele Rescue: Intergrative DNA transformation in
yeast.  and chapter 20 by Sikorski and Boeke:  In vitro mutagenesis and
plasmid shuffling: from cloned gene to mutant yeast. 
Both chapters have excellent protocols.   hope this helps, Morrie
******************************************************************************
Morris F. Manolson                     Tel:  416-813-6662  (office)
Division of Cell Biology                     416-813-5594  (lab)
Hospital for Sick Children                   416-813-5028  (FAX)
88 Elm St., McMaster building        email:  Morrie at resunix.ri.sickkids.on.ca
Toronto, Ontario, Canada
M5G 1X8





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