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Isolation and characterization of plasmids carrying genes of bacteriophage T7

University of British Columbia

The restriction endonuclease Hpa I cleaves wild type T7 DNA into nineteen fragments. These Hpa I fragments were inserted into the ampicillin resistance gene of the plasmid pBR322. The poly A - poly T method of construction was chosen in order to insure single T7 DNA inserts. Two hundred fifty clones were identified as carrying T7 DNA segments by colony hybridization with ³²P T7⁺ DNA. From these, clones carrying specific areas of the T7 genome were identified by various methods described in this thesis. Clones carrying Hpa I fragments E and G were identified by hybridization with Southern filters of electrophoressed Hpa I digested T7 DNA. Clones carrying most of gene 5 and parts of gene 4 were identified by hybridization with a purified restriction fragment carrying those genes (Hpa I fragment D). Clones containing parts of genes 10 and 11 were identified by marker rescue tests. Marker rescue "spot tests" were developed as a fast screening procedure for T7 genes and rely on generalized recombination between the T7 segment carried by the plasmid and the infecting phage DNA.

Text

2010-03-03

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http://hdl.handle.net/2429/21468

Microbiology and Immunology

University of British Columbia

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