A truely Rec minus host?

nigel at codon1.berkeley.edu nigel at codon1.berkeley.edu
Thu Oct 31 03:25:48 EST 1991


Howdy Nettsters,

Im in need of a truely rec minus strain of E.coli strain...does anyone out therehave any suggestions?

Purhaps a few details are in order:
1) I'm trying to clone an autonomous transposon from maize.
2) Two other labs have reported cloning the gene I'm after, but their sequences
show deletions when compared to each other.
3) I seem to be able to clone this element in Lambda, but it appears to   
rearrange before I can get it pure enough to subclone.

The following ref. might make this request a little clearer:

->Ishiura M; Hazumi N; Koide T; Uchida T; Okada Y.
->A recB recC sbcB recJ host prevents recA-independent deletions in
->recombinant cosmid DNA propagated in Escherichia coli.
->Journal of Bacteriology, 1989 Feb, 171(2):1068-74.
->
->Abstract: Segments of DNA are deleted from recombinant cosmid DNAs with high
->frequency during propagation in standard recA Escherichia coli hosts. An
->attempt has been made to derive an appropriate strain of E. coli, suitable
->for cosmid cloning, in which such deletions do not occur. We examined the
->effects of a series of host recombinational mutations on the deletion
->process, using six independent recombinant cosmids that carry inserts of
->mouse, Chinese hamster, or human DNA. Various E. coli host cells carrying
->the recombinant cosmids were cultured serially in liquid medium, and the
->recombinant cosmid DNAs were extracted from the host cells and analyzed by
->agarose gel electrophoresis and by gene transfer of the DNAs into cultured
->mammalian cells. Of the mutations examined, only a recB recC sbcB recJ (or
->recN) quadruple combination of host mutations prevented the deletion of DNA
->segments. The recombinant cosmid DNAs propagated in E. coli hosts that
->carried this combination of mutations were functionally as well as
->structurally intact. We propose that the recJ (and/or recN) gene is
->involved in some aspect of the events that lead to deletions of cosmid DNA
->in a recB recC sbcB genetic background.

I intend writing to the authors to ask for some of their magic bugs, but I
was hopping one of you might have an even better solution.

caio

-nigel
.



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