E.coli host cells (rec deficient)

Douglas Brutlag brutlag at cmgm.Stanford.EDU
Thu Jun 13 00:19:29 EST 1991


Borries,

	You may be out of luck.  I ran into this same problem
attempting to clone tandemly repeated satellite DNAs into bacterial
plasmids.  We examined many recombination deficient strains all to no
avail.  The only solution we found that stabilized the repeats was to
have the total length of the tandemly repeated region shorter than an
average okazaki fragment.  We felt that the recombination was caused
by unequal exchange during replication.  Here are two references and
their abstracts:

Brutlag, D., Fry, K., Nelson, T. and  Hung, P. (1977).  Synthesis of
hybrid bacterial plasmids containing highly repeated satellite DNA..
Cell, 10(3), 509-19.

Hybrid plasmid molecules containing tandemly repeated Drosophila
satellite DNA were constructed using a modification of the (dA)-(dT)
homopolymer procedure of Lobban and Kaiser (1973). Recombinant
plasmids recovered after transformation of recA bacteria contained 10%
of the amount of satellite DNA present in the transforming molecules.
The cloned plasmids were not homogenous in size. Recombinant plasmids
isolated from a single colony contained populations of circular
molecules which varied both in the length of the satellite region and
in the poly(dA)-(dt) regions linking satellite and vector. While
subcloning reduced the heterogeneity of these plasmid populations,
continued cell growth caused further variations in the size of the
repeated regions. Two different simple sequence satellites of
Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both
recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose
that this recA-independent instability of tandemly repeated sequences
is due to unequal intramolecular recombination events in replicating
DNA molecules, a mechanism analogous to sister chromatid exchange in
eucaryotes. 

Lohe, A. R. and  Brutlag, D. L. (1986).  Multiplicity of satellite DNA
sequences in Drosophila melanogaster..  Proc Natl Acad Sci U S A,
83(3), 696-700.

Three Drosophila melanogaster satellite DNAs (1.672, 1.686, and 1.705
g/ml in CsCl), each containing a simple sequence repeated in tandem,
were cloned in pBR322 as small fragments about 500 base pairs long.
This precaution minimized deletions, since inserts of the same size as
the fragments used for cloning were recovered in a stable form. A
homogeneous tandem array of one sequence type usually extended the
length of the insert. Eleven distinct repeat sequences were
discovered, but only one sequence was predominant in each satellite
preparation. The remaining classes were minor in amount. The repeat
unit lengths were restricted to 5, 7, or 10 base pairs, with sequences
closely related. Each sequence conforms to the expression (RRN)m(RN)n,
where R is A or G. The multiplicity of simple repeated sequences
revealed despite the small sample size suggests that numerous repeat
sequences reside in heterochromatin and that particular rules apply to
the structure of the repeating sequence.

	We also tried host mutants that altered the size of Okazaki
fragments and these had deleterious effects on the stability of the
entire plasmid.  Sorry.

Doug Brutlag





More information about the Methods mailing list