Faculty Bibliography

Two related families of transposons were isolated from schizosaccharomyces pombe, an organism which has been the object of extensive genetic studies which had previously produced no evidence for the existence of such elements. These two classes of repeated DNAs, dubbed Tf1 (transposon of fission yeast 1) and Tf2 have many properties of retrotransposons. Tf1 and Tf2 both possess long terminal repeats and predicted protein sequences that resemble the protease, reverse transcriptase, and integrase domains of retroviruses. The chromosomal locations and total numbers of Tf1 and Tf2 differ greatly in various isolates of S. pombe. The Tf elements are expressed in the form of 4.5-kb mRNAs. The complete sequence of Tf1 was determined and suggests that a novel mechanism for regulating its gene expression may be used.

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.