Graduate Thesis Or Dissertation

 

Bacteriophage T4-coded dihydrofolate reductase : cloning and structural analysis of its gene and organization of flanking regions Public Deposited

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  • The dihydrofolate reductase gene of bacteriophate T4 has been investigated at the structural level to elucide the unusual regulation of expression of this gene and possible evolutionary relationships between the ohage and drug resistance factors-encoded dihydrofolate reductases (DHFR). A 1.1-kilobase-pair restriction fragment of the T4 multiple mutant dec8 containing frd, the structural gene for DHRF, has been cloned in E. coli by using pBR322 as the vector. It has been demonstrated, both by restriction and biochemical analysis, that the recombinant plasmid pSP19 carrying this fragment contains the entire frd gene, and the enzyme synthesized by these transformants is indistinguishable from T4 DHFR. Overproduction of this enzyme has been achieved by inserting this fragment into the hyper-expression vector pGW7. The length of the cloned fragment, as determined by the base modification technique of Maxam and Gilbert, is 1073 base pairs. Two of the four open reading frames identified are the structural genes for DHFR and part of thymidylate synthase (TS). The frd gene is of 579 base pairs and is translated into 193 amino acid residues. frd and td genes show a four-base-pair overlap with each other. Positions of these genes were identified by correlating the predicted and experimentally determined sequences of 20 N-terminal amino acid residues of DHFR (T. Bailey) and TS (162). Two additional open reading frames are located upstream to frd. The first open reading frame (ORF), from the 5' end, when translated, shows sequence homology to the conserved active site region of all known DHFRs. The second reading frame, a small leader sequence, flanks the 5' end of frd. At least four complementary inverted repeats have been identified in the segment that includes the leader sequence and 20 nucleotides from the 5' region of frd. Also, the termination codons of the ORF and the leader sequence overlap with the Shine and Dalgarno sequence of the leader and frd, respectively. This fragment also contains at least five sequences which show homology to the consensus sequence of E. coli promoters; however, no conventional transcriptional termination sequence is present. Comparison of the deduced amino acid sequence of both T4 and R-plasmid enzymes does not show homology more than what is observed among other DHFRs, suggesting that these enzymes are not unusually closely related.
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