In vitro integration of retrotransposon Ty1: A direct physical assay

Lelita T. Braiterman, Jef D. Boeke

Research output: Contribution to journalArticle

Abstract

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half- reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.

Original languageEnglish (US)
Pages (from-to)5719-5730
Number of pages12
JournalMolecular and Cellular Biology
Volume14
Issue number9
StatePublished - Sep 1994

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Integrases
Retroelements
Virion
Genetic Selection
DNA
Retroviridae
Autoradiography
Genetic Recombination
Saccharomyces cerevisiae
Electrophoresis
Plasmids
Complementary DNA
Yeasts
Gels
Genome
In Vitro Techniques

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

In vitro integration of retrotransposon Ty1 : A direct physical assay. / Braiterman, Lelita T.; Boeke, Jef D.

In: Molecular and Cellular Biology, Vol. 14, No. 9, 09.1994, p. 5719-5730.

Research output: Contribution to journalArticle

Braiterman, LT & Boeke, JD 1994, 'In vitro integration of retrotransposon Ty1: A direct physical assay', Molecular and Cellular Biology, vol. 14, no. 9, pp. 5719-5730.
Braiterman, Lelita T. ; Boeke, Jef D. / In vitro integration of retrotransposon Ty1 : A direct physical assay. In: Molecular and Cellular Biology. 1994 ; Vol. 14, No. 9. pp. 5719-5730.
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