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Sharon, G., Lab. of Eukaryotic Gene Expression, NCI-Frederick Cancer R. and D. Ctr., ABL-Basic Research Program, Frederick, MD 21702-1201, United States, Volcani Center, Inst. of Field and Garden Crops, Bet-Dagan 50280, Israel
Burkett, T.J., Lab. of Eukaryotic Gene Expression, NCI-Frederick Cancer R. and D. Ctr., ABL-Basic Research Program, Frederick, MD 21702-1201, United States, Proteinix Company, Rockville, MD 20852, United States
Garfinkel, D.J., Lab. of Eukaryotic Gene Expression, NCI-Frederick Cancer R. and D. Ctr., ABL-Basic Research Program, Frederick, MD 21702-1201, United States, NCI-Frederick Cancer R. and D. Ctr., ABL-Basic Research Program, Building 539, P.O. Box B, Frederick, MD 21702-1201, United States
Integration of the yeast retrotransposon Ty1 into the genome requires the self-encoded integrase (IN) protein and specific terminal nucleotides present on full-length Ty1 cDNA. Ty1 mutants with defects in IN, the conserved termini of Ty1 cDNA, or priming plus-strand DNA synthesis, however, were still able to efficiently insert into the genome when the elements were expressed from the GAL1 promoter present on a multicopy plasmid. As with normal transposition, formation of the exceptional insertions required an RNA intermediate, Ty1 reverse transcriptase, and Ty1 protease. In contrast to Ty1 transposition, at least 70% of the chromosomal insertions consisted of complex multimeric Ty1 elements. Ty1 cDNA was transferred to the inducing plasmid as well as to the genome, and transfer required the recombination and repair gene RAD52. Furthermore, multimeric insertions occurred without altering the levels of total Ty1 RNA, virus-like particle-associated RNA or cDNA, Ty1 capsid proteins, or IN. These results suggest that Ty1 cDNA is utilized much more efficiently for homologous recombination when IN-mediated integration is blocked.
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Efficient homologous recombination of Ty1 element cDNA when integration is blocked
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Sharon, G., Lab. of Eukaryotic Gene Expression, NCI-Frederick Cancer R. and D. Ctr., ABL-Basic Research Program, Frederick, MD 21702-1201, United States, Volcani Center, Inst. of Field and Garden Crops, Bet-Dagan 50280, Israel
Burkett, T.J., Lab. of Eukaryotic Gene Expression, NCI-Frederick Cancer R. and D. Ctr., ABL-Basic Research Program, Frederick, MD 21702-1201, United States, Proteinix Company, Rockville, MD 20852, United States
Garfinkel, D.J., Lab. of Eukaryotic Gene Expression, NCI-Frederick Cancer R. and D. Ctr., ABL-Basic Research Program, Frederick, MD 21702-1201, United States, NCI-Frederick Cancer R. and D. Ctr., ABL-Basic Research Program, Building 539, P.O. Box B, Frederick, MD 21702-1201, United States
Efficient homologous recombination of Ty1 element cDNA when integration is blocked
Integration of the yeast retrotransposon Ty1 into the genome requires the self-encoded integrase (IN) protein and specific terminal nucleotides present on full-length Ty1 cDNA. Ty1 mutants with defects in IN, the conserved termini of Ty1 cDNA, or priming plus-strand DNA synthesis, however, were still able to efficiently insert into the genome when the elements were expressed from the GAL1 promoter present on a multicopy plasmid. As with normal transposition, formation of the exceptional insertions required an RNA intermediate, Ty1 reverse transcriptase, and Ty1 protease. In contrast to Ty1 transposition, at least 70% of the chromosomal insertions consisted of complex multimeric Ty1 elements. Ty1 cDNA was transferred to the inducing plasmid as well as to the genome, and transfer required the recombination and repair gene RAD52. Furthermore, multimeric insertions occurred without altering the levels of total Ty1 RNA, virus-like particle-associated RNA or cDNA, Ty1 capsid proteins, or IN. These results suggest that Ty1 cDNA is utilized much more efficiently for homologous recombination when IN-mediated integration is blocked.
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