HELP!!! E-coli (XL1-Blue et al) point-mutating my CpG island???

Heidi Moss hmmoss at MAIL.MED.CORNELL.EDU
Mon Apr 14 16:22:34 EST 1997


Greetings netters!!

I am in desperate need of a good detective to help me figure out a 
strange occurrence:
		
     --	I cloned 3 identical 19kb genes from a human genomic library (lambda 
dashII expanded in XL1-Blue MRA P2) and subsequently subcloned fragments 
into pBluescript (ranging from 260bp->4.8kb in size, and numerous overlapping 
fragments).  

     -- The e-coli strains I used for subcloning varied from plain XL-1 
blue, XL1-blue MRF', to DH5alpha


    -- Upon sequence analysis of my gc-rich promoter, I found that 
several subclones had a C-->T subsitution within a CpG island. I know it is 
NOT a sequencing problem because:
	1) The region is in a high-quality sequence area
	2) The presence of the 'C' confers a SacII site--> ergo, some of 
mny clones cut with SacII, others do not!

   -- I have obtained a consensus for the ENTIRE 19kb, and the ONLY 
ambiguity lies in this region!! 

THE BIG QUESTION:  ?? Is it an effect of the seemingly identical 3 
genomic clones --  does one clone have a non-essential point mutation?? 
OR is it an e-coli artifact????)

	 -- Oddly, the database/genome project has 2 fragments that match my 
region: one was an EST and it contains the 'T', the other was a cosmid 
clone that contained the 'C' --- this made me totally confused

MY QUESTIONS ARE:

	1) What is the percent efficiency of these e-coli strains to 
handle such CpG islands??? My assumption was that the INITIAL expansion 
of the lambda clone in the XL-1 Blue MRA P2 would rid me of such a problem.
That is why I felt comfortable using more generic strains for further 
expansion. (And even those strains have state-of-the-art genetic 
modifications to handle CpG islands)

	2) Could it be that this is a REALLY HOT SPOT??? Thus explaining 
why even excellent strains have difficulty with it AND the fact that the 
genome project is ambiguous? 
(THE MUTATION IS RIGHT NEXT TO TANDEM STAT5 SITES IN THE PROMOTER,therefore, 
it is very important to know the 'real' story)

Where did this mutation happen??? During the subcloning??? In one of the 
original genomic clones?? I cannot figure out what is going on. Any imput is 
appreciated. Unfortunately, I cannot trace which strain I used for which 
clone (i do have a good notebook, but for most i just listed 
"transformation" and not the strain ): ), and when I was subcloning I pooled 
genecleans...


Thanks for your time!!!!!! Please respond personally via e-mail.

						Sincerely,

							Heidi M. Moss



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