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Gene, 18 (1982) 289-296 Elsevier Biomedical Press

Summary: Gene, 18 (1982) 289-296
Elsevier Biomedical Press
Construction of a broad host range cosmid cloning vector and its use in the genetic analysis
of Rhizobium mutants
(Recombinant DNA; Rhizobium meliloti; clone bank; X COS)
Alan M. Friedman *, Sharon R. Long *, Susan E. Brown, William J. Buikema and
Frederick M. Ausubel **
Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA 02138 (U.S.A.)
(Received January 2Oth, 1982)
(Accepted March 5th, 1982)
We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector
pRK290 (Ditta et al., 1980) by inserting a 1.6kb Bg/II fragment containing X cos into the unique BglII site
in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique
EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We
constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean
insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R.
meliloti auxotrophic mutants, tetracycline-resistant (Tc') transconjugants were obtained at frequencies
ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from


Source: Ausubel, Frederick M. - Department of Genetics, Harvard University


Collections: Biology and Medicine