1887

Abstract

Summary: Low copy number vector plasmid pCT571 was constructed to clone genomic fragments in . pCT571 confers Km, Tcand Cm in and Cm in . It has unique restriction sites within the Km and Tcmarkers to allow screening for recombinant plasmids by insertional inactivation of these genes. It contains the pSC101 replicon and replicates normally at six to eight copies per chromosome equivalent in . It also contains , which when supplied with the product of the gene of RK2 in , allows pCT571 to replicate at 35−40 copies per chromosome equivalent. A gene bank was created by cloning partially 3A-digested and size-fractionated fragments of chromosomal DNA into the HI site of pCT571. DNA from 1097 KmTctransformants was extracted and analysed electrophoretically as supercoiled DNA and after digesting with RI or RI and I. Approximately 1000 hybrid plasmids were found with reasonably sized fragments. The mean size of the inserts in pCT571 is 8 kb, ranging from 4 to 20 kb in different plasmids. The gene bank covers most of the chromosome, as demonstrated by the results of screening the gene bank for selectable nutritional markers in and . Hybrid plasmids which complement mutants for , , , , , and markers were identified from the gene bank. In the presence of , , , , , , , , , , , , and was established by transformation experiments. The effects of copy number on cloning and long-term maintenance in the bacterial strains were also investigated. At high copy number some hybrid plasmids cannot be maintained at all, while others show an increased rate of structural deletions and rearrangements.

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1986-07-01
2021-10-21
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