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. 1990 Jun;64(6):2599–2607. doi: 10.1128/jvi.64.6.2599-2607.1990

Characterization of a transpositionally active Ty3 element and identification of the Ty3 integrase protein.

L J Hansen 1, S B Sandmeyer 1
PMCID: PMC249438  PMID: 2159534

Abstract

Ty3 is a Saccharomyces cerevisiae retrotransposon associated with tRNA genes. Two Ty3 elements have been cloned and characterized. The complete nucleotide sequence for one element, Ty3-2, was reported previously (L. J. Hansen, D. L. Chalker, and S. B. Sandmeyer, Mol. Cell. Biol. 9:5245-5256, 1988). However, this element is incapable of autonomous transposition. The complete DNA sequence of a transpositionally competent Ty3 element, Ty3-1, is presented here. Its sequence translates into two overlapping open reading frames, TYA3-1 and TYB3-1, which encode proteins with homology to the proteins specified by the retroviral gag and pol genes, respectively. Comparison of the Ty3-1 nucleotide sequence to Ty3-2 suggests that the TYB3-2 open reading frame of Ty3-2 is truncated by the deletion of a single nucleotide, which causes a frameshift mutation. Restoration of the reading frame with insertion of a single adenine by site-directed mutagenesis converted Ty3-2 into a transpositionally active element, Ty3-2(+ A). Western blot analysis with antibodies made against synthetic peptides identified integrase (IN) proteins in viruslike particle preparations from cells expressing Ty3 elements. Cells expressing Ty3-1 and Ty3-2 (+A) produce antibody-reactive proteins with approximate molecular masses of 61 and 58 kilodaltons (kDa), while cells expressing Ty3-2 produce reactive proteins of approximately 52 and 49 kDa. Together, these data show that the 61- or 58-kDa protein, or both, provides the integrase function of Ty3.

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Selected References

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  1. Adams C. W., Hatfield G. W. Effects of promoter strengths and growth conditions on copy number of transcription-fusion vectors. J Biol Chem. 1984 Jun 25;259(12):7399–7403. [PubMed] [Google Scholar]
  2. Boeke J. D., Eichinger D., Castrillon D., Fink G. R. The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1. Mol Cell Biol. 1988 Apr;8(4):1432–1442. doi: 10.1128/mcb.8.4.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boeke J. D., Garfinkel D. J., Styles C. A., Fink G. R. Ty elements transpose through an RNA intermediate. Cell. 1985 Mar;40(3):491–500. doi: 10.1016/0092-8674(85)90197-7. [DOI] [PubMed] [Google Scholar]
  4. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  5. Bowerman B., Brown P. O., Bishop J. M., Varmus H. E. A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev. 1989 Apr;3(4):469–478. doi: 10.1101/gad.3.4.469. [DOI] [PubMed] [Google Scholar]
  6. Brodeur G. M., Sandmeyer S. B., Olson M. V. Consistent association between sigma elements and tRNA genes in yeast. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3292–3296. doi: 10.1073/pnas.80.11.3292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clare J., Farabaugh P. Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression. Proc Natl Acad Sci U S A. 1985 May;82(9):2829–2833. doi: 10.1073/pnas.82.9.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clark D. J., Bilanchone V. W., Haywood L. J., Dildine S. L., Sandmeyer S. B. A yeast sigma composite element, TY3, has properties of a retrotransposon. J Biol Chem. 1988 Jan 25;263(3):1413–1423. [PubMed] [Google Scholar]
  9. Copeland T. D., Grandgenett D. P., Oroszlan S. Amino acid sequence analysis of reverse transcriptase subunits from avian myeloblastosis virus. J Virol. 1980 Oct;36(1):115–119. doi: 10.1128/jvi.36.1.115-119.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donehower L. A. Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3' region of pol. J Virol. 1988 Nov;62(11):3958–3964. doi: 10.1128/jvi.62.11.3958-3964.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Donehower L. A., Varmus H. E. A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6461–6465. doi: 10.1073/pnas.81.20.6461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Doolittle R. F., Feng D. F., Johnson M. S., McClure M. A. Origins and evolutionary relationships of retroviruses. Q Rev Biol. 1989 Mar;64(1):1–30. doi: 10.1086/416128. [DOI] [PubMed] [Google Scholar]
  13. Duyk G., Leis J., Longiaru M., Skalka A. M. Selective cleavage in the avian retroviral long terminal repeat sequence by the endonuclease associated with the alpha beta form of avian reverse transcriptase. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6745–6749. doi: 10.1073/pnas.80.22.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eichinger D. J., Boeke J. D. The DNA intermediate in yeast Ty1 element transposition copurifies with virus-like particles: cell-free Ty1 transposition. Cell. 1988 Sep 23;54(7):955–966. doi: 10.1016/0092-8674(88)90110-9. [DOI] [PubMed] [Google Scholar]
  15. Emori Y., Shiba T., Kanaya S., Inouye S., Yuki S., Saigo K. The nucleotide sequences of copia and copia-related RNA in Drosophila virus-like particles. 1985 Jun 27-Jul 3Nature. 315(6022):773–776. doi: 10.1038/315773a0. [DOI] [PubMed] [Google Scholar]
  16. Garfinkel D. J., Boeke J. D., Fink G. R. Ty element transposition: reverse transcriptase and virus-like particles. Cell. 1985 Sep;42(2):507–517. doi: 10.1016/0092-8674(85)90108-4. [DOI] [PubMed] [Google Scholar]
  17. Gentry L. E., Rohrschneider L. R., Casnellie J. E., Krebs E. G. Antibodies to a defined region of pp60src neutralize the tyrosine-specific kinase activity. J Biol Chem. 1983 Sep 25;258(18):11219–11228. [PubMed] [Google Scholar]
  18. Gibson W., Verma I. M. Studies on the reverse transcriptase of RNA tumor viruses. Structural relatedness of two subunits of avian RNA tumor viruses. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4991–4994. doi: 10.1073/pnas.71.12.4991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goding J. W. Use of staphylococcal protein A as an immunological reagent. J Immunol Methods. 1978;20:241–253. doi: 10.1016/0022-1759(78)90259-4. [DOI] [PubMed] [Google Scholar]
  20. Grandbastien M. A., Spielmann A., Caboche M. Tnt1, a mobile retroviral-like transposable element of tobacco isolated by plant cell genetics. Nature. 1989 Jan 26;337(6205):376–380. doi: 10.1038/337376a0. [DOI] [PubMed] [Google Scholar]
  21. Haltiner M., Kempe T., Tjian R. A novel strategy for constructing clustered point mutations. Nucleic Acids Res. 1985 Feb 11;13(3):1015–1025. doi: 10.1093/nar/13.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hansen L. J., Chalker D. L., Sandmeyer S. B. Ty3, a yeast retrotransposon associated with tRNA genes, has homology to animal retroviruses. Mol Cell Biol. 1988 Dec;8(12):5245–5256. doi: 10.1128/mcb.8.12.5245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hauber J., Nelböck-Hochstetter P., Feldmann H. Nucleotide sequence and characteristics of a Ty element from yeast. Nucleic Acids Res. 1985 Apr 25;13(8):2745–2758. doi: 10.1093/nar/13.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  25. Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Inouye S., Yuki S., Saigo K. Complete nucleotide sequence and genome organization of a Drosophila transposable genetic element, 297. Eur J Biochem. 1986 Jan 15;154(2):417–425. doi: 10.1111/j.1432-1033.1986.tb09414.x. [DOI] [PubMed] [Google Scholar]
  27. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Johnson M. S., McClure M. A., Feng D. F., Gray J., Doolittle R. F. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7648–7652. doi: 10.1073/pnas.83.20.7648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kitagawa T., Aikawa T. Enzyme coupled immunoassay of insulin using a novel coupling reagent. J Biochem. 1976 Jan;79(1):233–236. doi: 10.1093/oxfordjournals.jbchem.a131053. [DOI] [PubMed] [Google Scholar]
  31. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Liu F. T., Zinnecker M., Hamaoka T., Katz D. H. New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. Biochemistry. 1979 Feb 20;18(4):690–693. doi: 10.1021/bi00571a022. [DOI] [PubMed] [Google Scholar]
  35. Marlor R. L., Parkhurst S. M., Corces V. G. The Drosophila melanogaster gypsy transposable element encodes putative gene products homologous to retroviral proteins. Mol Cell Biol. 1986 Apr;6(4):1129–1134. doi: 10.1128/mcb.6.4.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Marschalek R., Brechner T., Amon-Böhm E., Dingermann T. Transfer RNA genes: landmarks for integration of mobile genetic elements in Dictyostelium discoideum. Science. 1989 Jun 23;244(4911):1493–1496. doi: 10.1126/science.2567533. [DOI] [PubMed] [Google Scholar]
  37. Mellor J., Malim M. H., Gull K., Tuite M. F., McCready S., Dibbayawan T., Kingsman S. M., Kingsman A. J. Reverse transcriptase activity and Ty RNA are associated with virus-like particles in yeast. Nature. 1985 Dec 12;318(6046):583–586. doi: 10.1038/318583a0. [DOI] [PubMed] [Google Scholar]
  38. Mount S. M., Rubin G. M. Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins. Mol Cell Biol. 1985 Jul;5(7):1630–1638. doi: 10.1128/mcb.5.7.1630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Müller F., Brühl K. H., Freidel K., Kowallik K. V., Ciriacy M. Processing of TY1 proteins and formation of Ty1 virus-like particles in Saccharomyces cerevisiae. Mol Gen Genet. 1987 May;207(2-3):421–429. doi: 10.1007/BF00331610. [DOI] [PubMed] [Google Scholar]
  40. Panganiban A. T., Temin H. M. The retrovirus pol gene encodes a product required for DNA integration: identification of a retrovirus int locus. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7885–7889. doi: 10.1073/pnas.81.24.7885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Robinson H. L., Gagnon G. C. Patterns of proviral insertion and deletion in avian leukosis virus-induced lymphomas. J Virol. 1986 Jan;57(1):28–36. doi: 10.1128/jvi.57.1.28-36.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Saigo K., Kugimiya W., Matsuo Y., Inouye S., Yoshioka K., Yuki S. Identification of the coding sequence for a reverse transcriptase-like enzyme in a transposable genetic element in Drosophila melanogaster. Nature. 1984 Dec 13;312(5995):659–661. doi: 10.1038/312659a0. [DOI] [PubMed] [Google Scholar]
  43. Sandmeyer S. B., Bilanchone V. W., Clark D. J., Morcos P., Carle G. F., Brodeur G. M. Sigma elements are position-specific for many different yeast tRNA genes. Nucleic Acids Res. 1988 Feb 25;16(4):1499–1515. doi: 10.1093/nar/16.4.1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sandmeyer S. B., Olson M. V. Insertion of a repetitive element at the same position in the 5'-flanking regions of two dissimilar yeast tRNA genes. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7674–7678. doi: 10.1073/pnas.79.24.7674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schwartzberg P., Colicelli J., Goff S. P. Construction and analysis of deletion mutations in the pol gene of Moloney murine leukemia virus: a new viral function required for productive infection. Cell. 1984 Jul;37(3):1043–1052. doi: 10.1016/0092-8674(84)90439-2. [DOI] [PubMed] [Google Scholar]
  47. Shehee W. R., Chao S. F., Loeb D. D., Comer M. B., Hutchison C. A., 3rd, Edgell M. H. Determination of a functional ancestral sequence and definition of the 5' end of A-type mouse L1 elements. J Mol Biol. 1987 Aug 20;196(4):757–767. doi: 10.1016/0022-2836(87)90402-5. [DOI] [PubMed] [Google Scholar]
  48. Shiba T., Saigo K. Retrovirus-like particles containing RNA homologous to the transposable element copia in Drosophila melanogaster. Nature. 1983 Mar 10;302(5904):119–124. doi: 10.1038/302119a0. [DOI] [PubMed] [Google Scholar]
  49. Shih C. C., Stoye J. P., Coffin J. M. Highly preferred targets for retrovirus integration. Cell. 1988 May 20;53(4):531–537. doi: 10.1016/0092-8674(88)90569-7. [DOI] [PubMed] [Google Scholar]
  50. Stucka R., Lochmüller H., Feldmann H. Ty4, a novel low-copy number element in Saccharomyces cerevisiae: one copy is located in a cluster of Ty elements and tRNA genes. Nucleic Acids Res. 1989 Jul 11;17(13):4993–5001. doi: 10.1093/nar/17.13.4993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Toh H., Kikuno R., Hayashida H., Miyata T., Kugimiya W., Inouye S., Yuki S., Saigo K. Close structural resemblance between putative polymerase of a Drosophila transposable genetic element 17.6 and pol gene product of Moloney murine leukaemia virus. EMBO J. 1985 May;4(5):1267–1272. doi: 10.1002/j.1460-2075.1985.tb03771.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vijaya S., Steffen D. L., Robinson H. L. Acceptor sites for retroviral integrations map near DNase I-hypersensitive sites in chromatin. J Virol. 1986 Nov;60(2):683–692. doi: 10.1128/jvi.60.2.683-692.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Voytas D. F., Ausubel F. M. A copia-like transposable element family in Arabidopsis thaliana. Nature. 1988 Nov 17;336(6196):242–244. doi: 10.1038/336242a0. [DOI] [PubMed] [Google Scholar]
  54. Warmington J. R., Waring R. B., Newlon C. S., Indge K. J., Oliver S. G. Nucleotide sequence characterization of Ty 1-17, a class II transposon from yeast. Nucleic Acids Res. 1985 Sep 25;13(18):6679–6693. doi: 10.1093/nar/13.18.6679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yuki S., Inouye S., Ishimaru S., Saigo K. Nucleotide sequence characterization of a Drosophila retrotransposon, 412. Eur J Biochem. 1986 Jul 15;158(2):403–410. doi: 10.1111/j.1432-1033.1986.tb09767.x. [DOI] [PubMed] [Google Scholar]

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