Characterization of a transpositionally active Ty3 element and identification of the Ty3 integrase protein.
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.
Hansen
L J
LJ
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
Sandmeyer
S B
SB
D000595:Amino Acid Sequence; D001483:Base Sequence; D002678:Chimera; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification; D004251:DNA Transposable Elements; D015684:Gene Products, pol / Q000235:genetics; D005800:Genes, Fungal; D019426:Integrases; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D012343:RNA, Transfer / Q000235:genetics; D015183:Restriction Mapping; D012190:Retroviridae / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D012808:Sigma Factor / Q000235:genetics
D004251:DNA Transposable Elements; D015684:Gene Products, pol; D015345:Oligonucleotide Probes; D012808:Sigma Factor; D012343:RNA, Transfer; D004254:DNA Nucleotidyltransferases; D019426:Integrases