Reddish Yeast in 2-Hy

Tod Lorenz tod.lorenz at ghawk.com
Tue Jan 31 20:50:00 EST 1995


 -=> Quoting Heidi Moss to All <=-
 HM> Using the Brent 2-Hybrid System I have so far successfully cloned
 HM> by bait into the DNAbd plasmid, transfected EGY48 w/ pSH18-34, checked
 HM> for self reactivity, done a western to detect presence of the fusion
 HM> protein, proceeded to transfect lib.plasmid containing a Hela cell
 HM> lib,  made glycerol stocks and proceeded to screen for protein
 HM> interaction by  plating onto Gal/Raf -His, -Ura, -Trp, -Leu plates.
 HM> For curiosity I also  plated the same cells onto Gal/Raf -H,-U,-T and
 HM> Glu -H,-U,-T.  The yeast  colonies that grew the next day were reddish
 HM> in color, where as the yeast  on the Glu -H, -U,-T were white and
 HM> present in high density as expected.   Most interesting was the Gal/Raf
 HM> -H,-U,-T plate contained a lawn of white  colonies as well as a smaller
 HM> number of large reddish colonies.  In all  the plates the number of
 HM> reddish colonies was the same so I assumed that  these were in fact
 HM> yeast that were growing on -Leu plates because of some  relevant
 HM> protein interaction.  Subsequently I have tested for Gal  dependence
 HM> and no color change was observed.  What is going on?  Can  anyone help
	  Are you using Saccharomyces?   Red mutants can be
  expressed with some adenine mutations such as ade2, these will
  typically form normal size colonies.  Also some heme/porphyrin
  mutations can cause a red phenotype, but these would grow slow or
  not at all without supplementation.
     Could you explain what the Brent 2-hybrid system is for those
  that are not familiar with what it is and what it is used for?
  More details and particulars may help us solve the mystery....

	  /^\/^^^^\/^\     R. Todd Lorenz
	    | #  @ |      Ellicott City, MD
	     \ :: /      T.LORENZ at GENIE.GEIS.COM
	     =\||/=
	       #@

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