By W.A. Scott (Eds.)
ISBN-10: 0126342504
ISBN-13: 9780126342505
ISBN-10: 0323158722
ISBN-13: 9780323158725
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Extra info for Molecular of Cloning of Recombinant DNA
Sample text
Y. A. Although :he earliest DNA cloning experiments were reported little more than three years ago, molecular cloning of recombinant DNA has since become an important tool for the study of proka::yotic and eukaryotic biology. The initial experiments (1) involved linkage of EcoRI restriction endonuclease-generated DNA fragments to a bacterial plasmid replicon, and subsequent introduction of the composite molecule into Escherichia coli by transformation. Using this general method, genes from a wide variety of diverse sources have been propagated in bacteria using plasmid cloning vehicles such as pSClOl and ColEl and their derivatives, or bacteriophage λ cloning vectors (for reviews, see refs.
Purpuratus and pSClOl appear to be quite stable, but one containing L. pictus histone genes inserted at the same EcoRI cleavage site of pSClOl seems to undergo recombinational events. The basis for these differences isn't clear to us. C. WEISSMANN: Are there any data on the actual preservation of the sequences inserted over many generations of replication? N. COHEN: We have not done DNA sequence analysis of cloned fragments of DNA that have been propagated in E. coli for varying periods of time.
Acad. M. Casadaban, J. M o l . , 104 (1976) 557-566. 56 S. Ν. COHEN et al. Y. A. Α. N. Cohen, Cell, 6 (1975) 231-244. C. L. W. Boyer, submitted publication. for DISCUSSION L. KODITSCHECK: I believe you said that in some cases there was more than one replicating site in a plasmid, is that right? Does that have any relation to the number of copies of the plasmid per cell? N. COHEN: No, it does not appear to. However, the pSC134 plasmid is a composite of pSClOl plus ColEl. Ken Timmis, and Felipe Cabello in my laboratory show that this plasmid ordinarily used only its ColEl replication system, and its copy number is maintained at the copy number of ColEl.
Molecular of Cloning of Recombinant DNA by W.A. Scott (Eds.)
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