Progress in Molecular and Subcellular Biology by K. N. Timmis, S. N. Cohen, F. C. Cabello (auth.), Professor

By K. N. Timmis, S. N. Cohen, F. C. Cabello (auth.), Professor Fred E. Hahn Ph. D., H. Kersten, W. Kersten, W. Szybalski (eds.)

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These fragments could however be selected indirectZy by means of a specific probe that contains a selectable property, resistance to ampicillin (Ap), and that is incapable of self-replication. In vitro linkage of the probe fragment to a DNA fragment capable of self-replication leads to the formation of a replicon that can be selected in transformation. , 1978). , 1976). This was due to two features of the recombinant replicon: firstly, the inability of one replicon to function would not prevent perpetuation of the plasmid, and secondly, the two component replicons are dissimilar and the replication of each one could be individually inhibited (low levels of DNA polymerase I prevent replication of CoIE1; chloramphenicol treatment prevents the replication of pSC101).

For this purpose we used ColE1 for cloning EcoRI-generated DNA fragments and pML21 for cloning HindIII-generated fragments (see Table 2). Table 7 lists the DNA fragments that were cloned and their phenotypes. A specific advantage of the ColE1-based cloning vehicles is that when they are resident in a ~Ats host their replication activity can be switched off by a temperature shift. Thus one method to test for the presence of replication functions on a DNA fragment cloned on a ColE1type vector is to determine whether temperature-resistant replication of the hybrid molecule occurs in a polAts host.

Coli strains that require diamino pimelic acid and thymine and that can grow only under very stringent laboratory conditions have been developed for the biological containment of recombinant DNA molecules judged to be potentially biohazardous (Curtiss, 1976) . , 1977) and some Pseudomonas strains (Chakrabarty, 1976). D. Analysis of Plasmid Structure and Function Plasmids are autonomous ext~achromosomal elements that code for functions generally not essential for the survival of the host cell. They are very important to society in general and to molecular biologists in particular.

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