Faculty Bibliography

Genetic screening of a yeast genomic library in a high-copy-number vector identified the normally single copy tRNA(CCU Arg) gene as one of the genes and reduces Ty1 transposition frequency when overexpressed. Immunoblot analyses of Ty1-encoded proteins indicate an inverse correlation between the copy number of the tRNA gene and the production of the TYB protein. Thus, Ty1 transposition frequency is apparently regulated by the level of tRNA(CCU Arg) in yeast cells.

In order to identify and characterize sequences within Ty1 elements which are required in cis for transposition, a series of mini-Ty1 plasmids were constructed and tested for transposition. Mini-Ty1s are deletion mutants of the Ty1-H3 element; Ty1 gene products required for transposition are supplied in trans from a helper Ty1 which has intact open reading frames but lacks a 3' long terminal repeat (LTR) and therefore cannot transpose itself. Up to 5 kilobase pairs of internal sequences of the 6-kilobase-pair-long Ty1 element can be deleted without a significant effect on transposition. The smallest mini-Ty1 element capable of transposition contains the 3' LTR and the transcribed portion of the 5' LTR, 285 base pairs (bp) of internal sequence 3' to the 5' LTR, and 23 bp of internal sequence 5' to the 3' LTR. We conclude that Ty1-encoded proteins can act in trans and that cis-acting sequences in Ty1-H3 are all within or near the LTRs. Further deletion of the 285-bp internal sequence adjacent to the 5' LTR significantly reduced transposition frequency, and the mini-Ty1 RNA produced failed to be packaged into the viruslike particles efficiently. Surprisingly, several nonhomologous cellular mRNAs were also associated with viruslike particles.

Yeast retrotransposon Ty1 directs the synthesis of virus-like particles (VLPs) consisting of Ty1-encoded proteins, RNA, and reverse transcripts. Ty1 reverse transcripts, tagged with a selectable marker and found within VLPs, are capable of transposing into naked target DNA in vitro. Cassettes consisting of a Ty long terminal repeat (LTR), or delta, marked with supF, and flanked by appropriate restriction sites were constructed. These artificial substrates, whose termini resemble those of linear, full-length Ty1 reverse transcripts, can be coincubated with VLPs (containing unmarked reverse transcripts), resulting in the very efficient integration of the artificial substrate. The results suggest that Ty DNA is limiting for transposition in vivo, suggesting that inefficient reverse transcription regulates Ty1 transposition. Analysis of the transposition of these model substrates, which resemble in vivo Ty1 transposition intermediates or differ from them in subtle ways, shows that Ty transposition proceeds by the linkage of the 3' hydroxyl residue of the reverse transcript to target DNA.

The tandemly arrayed miniexon genes of the trypanosomatid Crithidia fasciculata are interrupted at specific sites by multiple copies of an inserted element. The element, termed Crithidia retrotransposable element 1 (CRE1), is flanked by 29-base-pair target site duplications and contains a long 3'-terminal poly(dA) stretch. A single 1,140-codon reading frame is similar in sequence to the integrase and reverse transcriptase regions of retroviral pol polyproteins. Cloned lines derived from a stock of C. fasciculata have unique arrangements of CRE1s. In different cloned lines, CRE1s, in association with miniexon genes, are located on multiple chromosomes. By examining the arrangement of CRE1s in subclones, we estimate that the element rearranges at a rate of ca. 1% per generation. These results indicate that the C. fasciculata miniexon locus is the target for a novel retrotransposon.

To learn more about the variety of Ty elements capable of activating gene expression, we characterized 206 spontaneous Ty transpositions that activate the promoterless gene his3 delta 4. Most of the Ty elements appear to be full-length, although a few deleted elements were recovered. Over 95% of the insertions belong to the Ty1 family, and the rest are Ty2 elements. The excessive number of Ty1 transpositions was unexpected because there are only 2-fold more Ty1 than Ty2 elements in the yeast strains used in the selection. However, there is 20-fold more Ty1 than Ty2 RNA present in these yeast strains. This difference in RNA level explains the greater number of Ty1 verses Ty2 transpositions at his3 delta 4, because Ty elements transpose through an RNA intermediate. A similar association between the Ty transcript level and transpositional activation of his3 delta 4 is obtained in cells expressing GAL1-promoted Ty2-H556 or Ty2-917 elements, but only if the element does not contain a marker. Genetically marked Ty2-H556NEO and -917NEO elements transpose into and activate his3 delta 4 with the same efficiency as the previously characterized Ty1-H3NEO element, but are underrepresented relative to the levels of TyNEO transcript. We also found that chromosomal Ty transcripts are even more abundant than previously estimated and comprise about 1% of total cellular RNA.