Browsing School, Graduate by Author "Wu, Chung-hsiun Herbert"
Mechanism of gene amplification of genes 17 and 18 in bacteriophage T4Wu, Chung-hsiun Herbert; Black, Lindsay W. (1992)Bacteriophage T4 gene amplification mutants (Hp17s) carrying two to more than six copies of genes 17 to 18 in tandem were isolated by plating gene 17 amber mutant on an ochre suppressor containing host. Almost all (190/191) of the independently isolated Hp mutants arise from rearrangement in a 5 bp sequence, GCTCA, in two G-C rich regions of 24 bp partial homology (4 mismatches) located within genes 16 and 19. Analysis of several dispensable mutants showed that phage T4 topoisomerase (39, 52 and 60), T4 DNA primase (61), and T4 uvsX gene product are required for gene amplification, whereas the denV and the host recA gene products are not. The absence of the internal protein Alt is essential to isolate gene amplification mutants (Hp17) because more DNA can be packaged into the alt head which compensates the selection pressure against gene duplication. However, similar gene amplification mutants can not be found in genes 16, 18, 23, 32, 37, and 43 from the alt mutant. Site-directed mutations in the 24 bp recombination box in the phage T4 genome either reduced or eliminated the gene amplification mutants in T4 phage. This evidence suggests that sequence specificity rather than homology is important for the gene amplification event. DNAs isolated from T4D wild-type phage, plasmid pCBR2 which contains genes 16-67, and Hp17 phage had recombined in the GCTCA sequence in the 24 bp box as shown by the polymerase chain reaction (PCR) and direct sequencing of the PCR products. Mutations in E. coli genes recA, recB, recC, recE, recF, and recN did not eliminate the 200 bp recombination PCR signal in the plasmid pCBR2 DNA preparations indicating that their gene products are not required for the initial rearrangement event. Two E. coli proteins, of 69 kd and 30 kd, which bind a DNA fragment containing the recombination box in gene 16, were co-purified through DEAE-cellulose, MonoQ, and single-stranded DNA cellulose columns. However, protein preparations containing these two proteins did not exhibit sequence specific DNA binding activity in a competition assay. Although T4 late gene in the plasmid may not express well with the host RNA polymerase, removal of the N-terminal region of gene 16 from the plasmid eliminated the recombination signal suggesting that gp16 is involved in catalyzing the initial recombination event. Possible mechanisms of the gene amplification in mutant Hp17s are discussed.