An analysis of the genome of actinophage phi EC.
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Biomedical subjects
Publications and source records attributed to K Denniston-Thompson.
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The mapping of the sites of cleavage of nine restriction endonucleases (EcoRI, HindIII, BamHI, SalI, KpnI, SstI, BglII, XhoI, and XbaI) on 21 Charon phage vectors is described. Maps of individual subsections were obtained and then combined to assemble the complete vector maps. Calculations of maximum and minimum sizes of inserts which may be carried by the vectors using different restriction endonucleases or pairs of restriction endonucleases are presented. The regions mapped include several parts of phi 80 that had not been mapped previously.
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Shotgun collections of Charon 3A bacteriophages containing Eco RI fragments of human and mouse DNA were constructed with the use of in vitro packaging. Plaques were screened by hybridization, and globin-specific clones were isolated from both human (Charon 3AHs51.1) and mouse (Charon 3AMm30.5). The fragments cloned were detected in unfractionated genomic DNA by the Southern method of hybridization.
Two globin-related clones isolated from collections of bacteriophages containing unfractionated Eco RI fragments of human and mouse DNA were characterized. Charon3AHs51.1Hbgamma includes 2.7 kilobase pairs of human DNA containing a large part of a fetal gamma globin chain structural gene; Charon 3AMm30.5 includes 4.7 kilobase pairs of mouse DNA related to alpha globin. The human fetal gamma globin gene has within its coding region two intervening sequences of noncoding DNA, IVS 1 and IVS 2, of approximately 1-0 and 900 base pairs. Sequence IVS 1 is located at the position of one of the two intervening sequences occurring in adult globin genes; IVS 2 is located at the position of the other.
Segments of the replication control region of bacteriophage lambda (lambda) and lambda mutants defective in replication were attached in vitro to the phi80 phage vector Charon 3 and to the plasmid vector mini Col El (pVH51). The chimeric phages and plasmids have been used to localize the origin of lambda DNA replication and to facilitate a structural analysis of the lambda replicator.
The nucleotide sequence of part of the replication region of wild-type bacteriophage lambda and of four mutants defective in the origin of DNA replication (ori-) has been determined. Three of the ori- mutations are small deletions, and one is a transversion. The sequence of the origin region, defined by these mutations, contains a number of unusual features.
The Charon lambda bacteriophages have been developed as vectors for cloning. Their construction incorporates mutations that make them simple to use and also greatly increases their safety for the biological containment of cloned recombinant DNA. Three of the Charon vector phages, 3A, 4A, and 16A, have been certified for use as EK2 vector-host systems, when propagated in bulk in a special bacterial host, DP50SupF. We present here some of the data on which the safety of these systems was evaluated. DNA fragments ranging in size from 0 to 2.2 X 10(4) base pairs can be cloned in these EK2 Charon phages.
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