Targeting of Integration

Division Of Basic Sciences - Nci

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Abstract

To observe aberrant target site preference by mutagenized elements, we constructed a S. cerevisiae strain in which the URA3 gene was placed in the genome upstream of a preferred glycyl-tRNA (suf16) on chromosome III. The configuration of this construction is such that correct targeting of an element upstream of the suf16 disrupts the URA3 thereby permitting survival of cells in the presence of FOA. Using repair-deficient Escherichia coli, we randomly mutagenized a plasmid containing the Ty1 element under the control of the GAL1 promoter, and containing the retrotransposition indicator, his3AI. Approximately 10,000 mutagenized plasmids were screened for transposition by survival on media lacking histidine, for aberrant targeting by failure to survive on media containing FOA, and absence of chromosomal duplications by the ability to survive on plates containing canavanine, the toxic analogue of arginine permease. Candidates from this initial screen were further analyzed by PCR of extracted genomic DNA for Ty1 insertions upstream of the suf 16. This screen generated 31 mutants of sufficient interest to warrant sequencing. Sequencing results showed mutations dispersed throughout the element, falling within the frameshift site, protease, the N-terminal zinc binding domain and active site domain of integrase. However, several mutations were found within the C-terminal domain of integrase, a region of no known function. Also, several mutations fell within the N-terminal region of reverse transcriptase. Although it may be counterintuitive to hypothesize that the targeting determinant resides in reverse transcriptase rather than integrase, a number of recent observations that reverse transcriptase and integrase remain functionally, if not physically, associated after proteolytic cleavage to form mature proteins, leads to speculation that the targeting determinant may be comprised of multiple sites. These mutant strains have now been relocated to the University of Georgia.