Summary
We have analysed functional properties of putative proteins encoded by the yeast transposable element, Ty1, by overexpression of TY genes. High-level expression was achieved by appropriate fusion of a Ty sequence, TY9C, to the yeast ADH1 promoter and transformation of yeast cells with this construction. As shown recently by others (Garfinkel et al. 1985; Mellor et al. 1985c) TY over-expression leads to an increase in particle-bound reverse transcriptase activity and to an intracellular accumulation of virus-like particles (Ty-VLPs). We have used a number of deletions in the second open reading frame (TYB) to identify functional domains required for processing and assembly of Ty proteins. Deletions in the TYB region with homology to acid proteases result in overproduction of an unprocessed form of the TYA protein (pro-TYA) which represents the major protein of Ty-VLPs. One particular mutant construction, TY9C-Δ36, led to the accumulation of a particle-bound, 160 kDa protein which cross-reacted with a mouse antiserum raised against purified pro-TYA protein. This supports the hypothesis that TYB is expressed as a TYA/TYB fusion protein which is processed by a TYB-encoded protease activity. Ty-VLPs are formed in the absence of protein processing and even when the TYB gene is not expressed. Thus, we assume that the assembly of Ty particles occurs prior to processing of Ty proteins.
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References
Ammerer G (1983) Expression of genes in yeast using the ADCI promoter. Methods Enzymol 101:192–201
Baltimore D (1970) Viral RNA-dependent DNA polymerase. Nature 226:1209–1211
Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 275:104–109
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Boeke JD, Garfinkel DJ, Styles CA, Fink GR (1985) Ty elements transpose through an RNA intermediate. Cell 40:491–500
Breilmann D, Gafner J, Ciriacy M (1985) Gene conversion and reciprocal exchange in a Ty-mediated translocation in yeast. Curr Genet 9:553–560
Clare J, Farabaugh P (1985) Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression. Proc Natl Acad Sci USA 82:2829–2833
Dickson C, Eisenmann R, Fan H, Hunter E, Teich N (1982) Protein biosynthesis and assembly. In: Weiss R, Teich N, Varmus H, Coffin J (eds) RNA Tumor Viruses, second edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 513–648
Dobson MJ, Mellor J, Fulton AM, Roberts NA, Bowen BA, Kingsman SM, Kingsman AJ (1984) The identification and high level expression of a protein encoded by the yeast Ty element. EMBO J 3:1115–1119
Elder RT, John TPS, Stinchcomb DT, Davis RW (1980) Studies on the transposable element Ty1 of yeast I. RNA homologous to Ty1. Cold Spring Harbor Symp Quant Biol 45:609–617
Elder RT, Loh EY, Davis RW (1983) RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA. Proc Natl Acad Sci USA 80:2432–2436
Garfinkel DJ, Boeke JD, Fink GR (1985) Ty element transposition: Reverse transcriptase and virus-like particles. Cell 42:507–517
Hauber J, Nelböck-Hochstetter P, Feldmann H (1985) Nucleotide sequence and characteristics of a Ty element from yeast. Nucleic Acids Res 13:2745–2758
Klebe RJ, Harriss JV, Sharp ZD, Douglas MG (1983) A general method for poly-ethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25:333–341
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Mellor J, Fulton AM, Dobson MJ, Roberts NA, Wilson W, Kingsman AJ, Kingsman SM (1985a) The Ty transposon of Saccharomyces cerevisiae determines the synthesis of at least three proteins. Nucleic Acids Res 13:6249–6263
Mellor J, Fulton SM, Dobson MJ, Wilson W, Kingsman SM, Kingsman AJ (1985b) A retrovirus-like strategy for expression of a fusion protein encoded by yeast transposon Ty1. Nature 313:243–246
Mellor J, Malim MH, Gull K, Tuite MF, McCready S, Dibbayawan T, Kingsman SM, Kingsman AJ (1985c) Reverse transcriptase activity and Ty RNA are associated with virus-like particles in yeast. Nature 318:583–586
Mount SM, Rubin GM (1985) Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins. Mol Cell Biol 5:1630–1638
Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039
Temin HM, Mizutani S (1970) RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226:1211–1213
Towbin H, Staelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Warmington JR, Waring RB, Newlon CS, Indge KJ, Oliver SG (1985) Nucleotide sequence characterisation of Ty1-17, a class II transposon from yeast. Nucleic Acids Res 13:6679–6693
Williamson VM, Young ET, Ciriacy M (1981) Transposable elements associated with constitutive expression of the yeast alcohol dehydrogenase II. Cell 23:605–614
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Communicated by C.P. Hollenberg
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Müller, F., Brühl, KH., Freidel, K. et al. Processing of TY1 proteins and formation of Ty1 virus-like particles in Saccharomyces cerevisiae . Mol Gen Genet 207, 421–429 (1987). https://doi.org/10.1007/BF00331610
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DOI: https://doi.org/10.1007/BF00331610