Analysis of the site-specific integration function of the streptomycete bacteriophage phiC31
Abstract
A 2.1 kb segment of DNA from the streptomycete bacteriophage $\phi$C31 was found to be sufficient to direct site-specific integration of plasmid vectors in Streptomyces ambofaciens and Streptomyces fradiae (E. T. Seno, personal communication) in the absence of any streptomycete origin of replication. Sequencing and analysis of phage, chromosomal, and junction attachment sites of Streptomyces ambofaciens and Streptomyces fradiae revealed that recombination is conservative and that crossover takes place between phage and host within a three base-pair region of homology. Deletion analysis, sequencing, and site-directed mutagenesis of the $\phi$C31 DNA revealed a large open reading frame (orf 613) whose expression was necessary for integration. This orf begins near the point of crossover and is oriented away from the attachment site. A comparison of the predicted amino acid sequence of orf 613 with twenty known or predicted integrases, twelve resolvases and invertases, and three putative streptomycete transposases failed to reveal any substantial similarities. Genetic analysis of the amino terminal region of orf 613 suggested that translation could initiate at an ATG and at two GTGs. Amino-terminal sequencing of an orf 613-lacZ fusion protein expressed in Escherichia coli showed that translation initiated at both the ATG and the second GTG. Primer extension experiments showed that transcription initiated at a T and a C only 4 and 5 bases (respectively) from the site of crossover. This analysis suggested that the act of integration would separate orf 613 from its promoter.
Degree
Ph.D.
Advisors
Somerville, Purdue University.
Subject Area
Biochemistry|Molecular biology|Microbiology
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