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Literature DB >> 8127892

Expression and partial purification of enzymatically active recombinant Ty1 integrase in Saccharomyces cerevisiae.

Abstract

Integration of the Saccharomyces cerevisiae retrotransposon Ty1 into the genome requires Ty1 integrase (IN). Apparent functions of Ty1 IN are target-site determination, cleavage, and joining of donor strands. To further study the mechanism of Ty1 integration, an IN expression plasmid has been constructed for use in yeast. The recombinant IN coding sequence differs from mature Ty1 IN associated with Ty1 virus-like particles only in that it has several additional N-terminal amino acid codons. Inclusion of a polyhistidine tag facilitates purification of recombinant IN by metal chelate chromatography. Recombinant Ty1 IN is active in an in vitro assay with short double-stranded oligonucleotide substrates and has biochemical properties similar to those observed with Ty1 virus-like particles. The full-length Ty1 IN produced in yeast should be useful for further biochemical, genetic, and structural analyses of Ty1 integration and for comparative analyses with retroviral IN proteins.

Entities: Chemical Disease Species

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Year: 1994 PMID： 8127892 PMCID： PMC43260 DOI： 10.1073/pnas.91.5.1843

Source DB: PubMed Journal: Proc Natl Acad Sci U S A ISSN： 0027-8424 Impact factor: 11.205

35 in total

1. Reverse transcriptase activity and Ty RNA are associated with virus-like particles in yeast.

Authors: J Mellor; M H Malim; K Gull; M F Tuite; S McCready; T Dibbayawan; S M Kingsman; A J Kingsman
Journal: Nature Date: 1985 Dec 12-18 Impact factor: 49.962

2. Ty element transposition: reverse transcriptase and virus-like particles.

Authors: D J Garfinkel; J D Boeke; G R Fink
Journal: Cell Date: 1985-09 Impact factor: 41.582

3. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.

Authors: M M Bradford
Journal: Anal Biochem Date: 1976-05-07 Impact factor: 3.365

4. The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins.

Authors: S E Adams; J Mellor; K Gull; R B Sim; M F Tuite; S M Kingsman; A J Kingsman
Journal: Cell Date: 1987-04-10 Impact factor: 41.582

5. Functional organization of the retrotransposon Ty from Saccharomyces cerevisiae: Ty protease is required for transposition.

Authors: S D Youngren; J D Boeke; N J Sanders; D J Garfinkel
Journal: Mol Cell Biol Date: 1988-04 Impact factor: 4.272

5. The application of a homologous recombination assay revealed amino acid residues in an LTR-retrotransposon that were critical for integration.

Authors: A Atwood; J Choi; H L Levin
Journal: J Virol Date: 1998-02 Impact factor: 5.103

9. Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

Authors: S E Devine; J D Boeke
Journal: Nucleic Acids Res Date: 1994-09-11 Impact factor: 16.971

10. A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein.

Authors: G V Merkulov; K M Swiderek; C B Brachmann; J D Boeke
Journal: J Virol Date: 1996-08 Impact factor: 5.103

Expression and partial purification of enzymatically active recombinant Ty1 integrase in Saccharomyces cerevisiae.

1. Reverse transcriptase activity and Ty RNA are associated with virus-like particles in yeast.

2. Ty element transposition: reverse transcriptase and virus-like particles.

3. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.

4. The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins.

5. Functional organization of the retrotransposon Ty from Saccharomyces cerevisiae: Ty protease is required for transposition.

6. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction.

7. Purification and specificity of porcine enterokinase.

8. Compilation and comparison of the sequence context around the AUG startcodons in Saccharomyces cerevisiae mRNAs.

9. The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1.

10. Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli.

1. Correct integration of model substrates by Ty1 integrase.

2. Functional analysis of N-terminal residues of ty1 integrase.

3. Ty1 in vitro integration: effects of mutations in cis and in trans.

4. Ty3 integrase mutants defective in reverse transcription or 3'-end processing of extrachromosomal Ty3 DNA.

5. The application of a homologous recombination assay revealed amino acid residues in an LTR-retrotransposon that were critical for integration.

6. Increased length of long terminal repeats inhibits Ty1 transposition and leads to the formation of tandem multimers.

7. A Ty1 reverse transcriptase active-site aspartate mutation blocks transposition but not polymerization.

8. The Ty1 LTR-retrotransposon of budding yeast, Saccharomyces cerevisiae.

9. Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

10. A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein.