There are 30-35 copies of the retrotransposon Ty in the haploid genome of most laboratory strains of Saccharomyces cerevisiae. Ty elements are 5.9 kb long, have LTRs of 340 bp and produce a genomic RNA that is 5.7 kb in length, through which it transposes. They contain two ORFs; the TYA gene of the class I element Tyl-15 is 1,319 nucleotides long whilst its TYB gene, which possesses the consensus sequences of retroviral acid proteases, reverse transcriptases and integrases, is 3,984 nucleotides long. TYB overlaps TYA by 38 nucleotides at its 5' end and is expressed as a TYA;TYB fusion protein. In an attempt to asign some in vivo functions to the Ty encoded proteins, the whole transcriptional unit of Tyl-15 was overexpressed in yeast. Electron micrographs of the overexpressing cells revealed an abundance of cytoplasmic, ~60 nm virus-like particles (Ty-VLPs). These contain the 5.7 kb Ty RNA and have an associated reverse transcriptase activity. Their major protein is p2 (48 kD), which is a proteolytic derivative of the primary translation product of TYA, pi (50 kD), a protein that is also assembled into particles. A truncated pi, encoded by a TYA gene shortened by 60 codons at its 3' end, still aggregates into particles. Furthermore, the addition of α2-IFN (20 kD) onto its carboxy terminus, to produce a pl-IFN fusion protein of ~68 kD, does not interfere with its particle forming properties. The hybrid VLPs are easy to purify and elicit an antibody response in rabbits to the Ty and IFN epitopes. A series of fragments spanning Tyl-15 were inserted into the packaging analysis vector, pMA924. The association of the resulting PGK-Ty-IFN hybrid RNAs with Ty-VLPs, in the presence of abundant Ty encoded proteins, was analysed by Northern blotting and used to assess the packaging capabilities of Ty RNA. Unexpectedly, RNA sequences that, direct specific packaging are produced from regions throughout Tyl-15. The signals corresponding to TYA are probably located within two regions of the genomic RNA, 5' to nucleotide 236 and 3' to nucleotide 737; those corresponding to TYB are in at least two, currently undefined, areas. The TYB gene of the class II element Tyl-17 overlaps TYA by 44 nucleotides. The fusion of α2-IFN cDNA fragments into all three reading phases of TYB followed by a Western blot analysis demonstrated that, like the TYB gene of Tyl-15, it too is expressed as a TYA:TYB fusion protein.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:382694 |
| Date | January 1987 |
| Creators | Malim, Michael H. |
| Contributors | Kingsman, A. J. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:d02fc435-ceb2-443a-b650-dd0d216b2c7c |
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