CONSTRUCTION AND CHARACTERIZATION OF ESCHERICHIA COLI PLASMIDS USEFUL IN THE MANIPULATION OF DNA
GAYLE, RICHARD BROWNLEY, III
Doctor of Philosophy
The restriction endonuclease MboII recognizes a nonpalindromic, pentanucleotide DNA sequence and cleaves both strands of the DNA eight bases downstream, leaving a single 3' protruding base. MboII is one of a group of restriction enzymes which has its site of cleavage removed from its recognition sequence. Insertions of DNA sequences into the MboII cleavage site will not disrupt the recognition site allowing further additions to be performed. Several synthetic oligonucleotides containing MboII sites in position for DNA manipulations. These oligonucleotides have been cloned into a plasmid vector which has no MboII sites. The plasmids constructed with the synthetic oligonucleotides allow a DNA sequence to be inserted into the MboII site, without disrupting the MboII recognition sequence. Following isolation of the recombinant plasmids, further insertions have been performed, thus juxtaposing DNA sequences without the need for compatible restriction enzyme sites. In addition, a small DNA fragment has been constructed. This sequence consists of the lac operator region, so that it may be easily screened, surrounded by two MboII recognition sequences. This DNA fragment has been randomly inserted into plasmid DNA using DNAase I. The MboII recognition sequences face outward and will cleave into surrounding DNA, removing up to 14 base pairs. This allows specific deletions to be performed, as well as insertion of DNA fragments into the cleaved DNA. Because of the mode of action of MboII, the entire sequence can be removed from the region of insertion. Some of the plasmids constructed in this thesis had unusual regions surrounding the origin of replication. This area was much smaller than had been previously reported necessary for stable replication. The stability of the plasmids in different strains was found to vary and they had a much higher copy number than pBR322 in exponentially growing cells, but did not amplify to the same extent as pBR322. The plasmids were found to be compatible with pBR322.