The retrovirus HIV is the major disease causing member of the long terminal repeat (LTR)-containing retroelements. The development of new antiviral strategies requires an increased understanding of retrovirus biology. To understand the mechanisms that retroviruses use to propagate, we study LTR-retrotransposons, a closely related variety of retroelement that exists in model organisms such as yeast. The LTR-retrotransposon we study is the Tf1 element of the fission yeast, Schizosaccharomyces pombe. The focus of our efforts is to understand the molecular details of mechanisms such as reverse transcription, transport of Tf1 into the nucleus, and the integration of Tf1 cDNA. The process of reverse transcription is a complex set of reactions that requires two specialized primers and the transfer of DNA intermediates to specific segments of the transposon template. Although the biochemical properties of reverse transcriptase (RT) have been studied, little is known about which domains of RT recognize the primers or mediate the transfer events. To this end, we used assays of yeast genetics to identify residues of RT that are required for late steps in the production of cDNA. Ten of the mutations clustered within a small region of RNase H, the domain of RT that degrades RNA in RNA:DNA duplexes. Interestingly, most of these mutations occur in residues that in X-ray crystal studies form direct contacts with critical nucleotides of the primers. Since the process of integration has the potential to compromise the fitness of the host, we wish to understand the balance between the ability of the transposon to insert into the host genome verses the efforts of the host to maintain its viability. The recently completed sequence of the S. pombe genome allowed us to ask whether Tf1 integration results in the disruption of host genes. Surprisingly, the only transposons found in the genome were related to the Tf1/Tf2 family of LTR retrotransposons. A phylogenetic analysis indicated that these two families were probably the most recent to expand within the genome. Examination of all the 202 transposon sequences revealed that each element was located within intergenic regions of sequence. Since 60.2% of the genome of S. pombe is coding sequence, the positions of the LTRs was strongly biased. On a larger scale, the LTRs were found to be widely distributed throughout each of the three chromosomes of S. pombe. However, it was particularly surprising that the concentration of LTRs on chromosome 3 was twice that of the other two chromosomes. A genome-wide study of Tf1 transposition in S. pombe revealed that the events occurred within intergenic regions. This result demonstrates that the location of the preexisting transposon sequences was due to selection during integration. The systematic insertion of Tf1 into intergenic regions represents a novel method for protecting host genes from damage due to integration. Perhaps the most significant result of this study was the surprising observation that Tf1 integration had a significant preference for chromosome 3. Per unit length of DNA, chromosome 3 received approximately twice the number of inserts that occurred in chromosome 1 or 2. This preference was found not to be due to differences in the distribution or composition of intergenic sequences within the three chromosomes. Our results demonstrate that chromosome 3 was in some physiological aspect, distinct from the other chromosomes.
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