Date of Award
Doctor of Philosophy (PhD)
Expression of the ten genes on the small filamentous phage f1 genome must be closely regulated during the infection cycle, since this nonlytic coliphage lives in delicate balance with its host. The sequence and context requirements of specific regions involved in f1 mRNA metabolism were investigated by: 1) relocation of an intact transcription unit on the phage genome; 2) construction of a hybrid E. coli-f1 transcription unit which could be studied in a phage or plasmid context; 3) identification and characterization of a rho-dependent transcription termination signal in f1 by cloning it between the promoter and gene of an E. coli transcription unit, and by studies of its function in its usual phage context. Expression of the f1 major coat (gene VIII) protein was studied in vivo in cells infected with variant f1 phage whose genomes had been restructured in vitro. This gene with its most proximal promoter and terminator was found to behave as an independent transcription unit. Coat protein gene expression was unaffected by transposition of the gene to the phage's large intergenic region, and was independent of orientation, but was dependent upon its own intact promoter. Gene VIII was placed under control of the E. coli lac promoter and the RNA from the new transcription unit was characterized. Coat protein was expressed from a hybrid mRNA initiated at the lac promoter. This mRNA, which contained the 5' 36 nucleotides of the lac operon mRNA at its 5'end, attached to the entire gene VIII mRNA sequence, was unstable (half life -1.5 min), as is the E. coli lac operon mRNA. In contrast, authentic f1 gene VIII mRNA is extremely stable (half life -10 min). The f1 large intergenic region was shown to encode a rho-dependent transcription termination signal. The minimal sequence required for terminator function in a heterologous plasmid system encompasses about 100 nucleotides. Like most known rho-dependent terminators, the signal contains a region of dyad symmetry. It differs from previously characterized rho-dependent terminators, in that the sequence at the termination site is G-C rather than A-T rich. In a rho mutant host, f1 tranvii scripts pass through the normal termination site, and stop downstream within a region of high potential secondary structure near the f1 origin of DNA replication. A method is described for the efficient construction in vitro of recombinant DNA molecules from fragments produced by cleavage with the restriction endonuclease HgaI. The method relies upon the unique properties of HgaI and is applicable to any viral or plasmid DNA that contains several HgaI recognition sites. Using f1 DNA, it is shown that only HgaI fragments that were originally adjacent on the genome can anneal, that infectious molecules are reassembled with high efficiency from a mixture of fragments, and that recombinant genomes can be easily constructed from parental DNAs containing genetic markers which map in different HgaI fragments.
Moses, Phyllis Barbara, "Structure-Function Relationships of Elements of the Bacteriophage f1 Genome" (1983). Student Theses and Dissertations. 456.