The Cleavage of ɸX174 DNA with Restriction Endonucleases

Citation

Lee, Amy So-Ming Shiu (1975) The Cleavage of ɸX174 DNA with Restriction Endonucleases. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/3qag-c766. https://resolver.caltech.edu/CaltechTHESIS:03252022-213533588

Abstract

Restriction endonucleases from Hemophilus influenzae (Hin), H. parainfluenzae (Hpa) and H. aegyptius (Hae) have been prepared and purified from contaminating exonuclease activities. Using ɸX174 RFI as substrate, Hin produces 13 specific fragments, Hpa produces 8 and Hae produces 11 fragments. These have been analyzed by polyacrylamide gel electrophoresis and their molecular size determined to be within the range of 1600 to 70 base pairs. The 5' end group of the fragments produced by the action of Hin on ɸX RF have been identified to be adenine (50-57%), and guanine (30-40%), whereas in the case of Hpa, preliminary results show that a majority of the fragments terminate at the 5' end with cytosine.

By analyzing partial digestion products, as well as overlapping sets of fragments produced by two different restriction enzymes, the physical order of the three sets of DNA fragments produced by hydrolysis of ɸX RF by the Hin, Hpa and Hae restriction endonucleases have been determined. This analysis has been facilitated by the adaptation of a continuous electro-elution device to separate and recover individual DNA restriction enzyme fragments.

The physical cleavage map has been in turn related to the ɸX genetic map by the fact that many ɸX174 mutations are available and that methods have been developed by which restriction enzyme fragments may be assayed to determine which genetic loci they contain.

With the availability of the ɸX174 cleavage map, it is possible to locate the unique, methylated cytosine of the ɸX genome. Bacteriophage ɸX DNA is labeled in vivo with [³H-methyl]methionine and used as template for in vitro synthesis of the complementary strand. The resultant RF DNA is then cleaved in separate experiments with the Hin and Hae enzymes. The DNA fragments are analyzed by polyacrylamide gel electrophoresis. It is concluded that the single methyl group in the viral DNA is located at a specific region of the ɸXl74 genome, very likely in gene H.

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