Diseases such as AIDS and leukemia caused by retroviruses have intensified the need to understand the mechanisms of retrovirus replication. Our primary objective is to understand how retroviral cDNAs are integrated into the genome of infected cells. Because of their similarities to retroviruses, long terminal repeat (LTR)-retrotransposons are important models for retrovirus replication. The retrotransposon under study in our laboratory is the Tf1 element of the fission yeast Schizosaccharomyces pombe. We are particularly interested in Tf1 because of its strong preference for integrating into pol II promoters. This choice of target sites is similar to the strong preferences human immunodeficiency virus 1 (HIV-1) and murine leukemina virus (MLV) have for integration into pol II transcription units. Very little is known about how these viruses recognize their target sites. We therefore study the integration of Tf1 as a model system from which we hope uncover mechanisms general to the selection of integration sites. Such an understanding of the mechanisms responsible for targeted integration may lead to new approaches for blocking the replication of HIV-1. ? ? A key goal of our research this year was to identify the mechanism that directs integration to regions upstream of ORFs. To study insertion patterns in specific genes, a target plasmid assay was developed. Integration into plasmids containing various genes occurred upstream of the ORFs in insertion windows of approximately 150 nt. Deletion analyses of the plasmids indicated that the target positions were the only sequence features required for integration. Separate plasmids containing smaller sequences demonstrated that the windows of integration themselves were sufficient to direct integration. The prominent insertion sites in fbp1 were just 30 and 40 bp downstream of where the transcription activator Atf1p bound. These data suggested the model that transcription factors bound at their promoters, mediate integration. This model was supported by the finding that a functional binding site for the transcription factor Aft1p played a critical role in the targeting of Tf1 integration to the two major insertion sites in the fbp1 promoter. In addition, we found that Atf1p is required for integration in the fbp1 promoter and that Atf1p interacts with integrase. These data provide strong support for the role of Atf1p in directing integration to target sites.? ? Another project conducted this year was to identify the function of the GPY domain present in the C-termini of some retrovirus and retrotransposon integrases. Based on sequence conservation, single amino acid substitutions were made in the GPY domain of Tf1 integrase. In vivo, these mutations greatly reduced transposition activity. Work with recombinant integrase revealed that mutating the conserved P365 greatly reduced catalytic activity. Gel filtration and cross-linking of a 71-amino acid fragment containing the GPY domain revealed this peptide formed dimers, trimers and tetramers. Mutations in the conserved amino acids of the GPY domain disrupted all multimerization. These results suggest that the GPY domain promotes the multimerization and catalytic activity of integrase. ? ? The integration of Tf1 occurs primarily into pol II promoters. Although we currently believe this preference is the result of a mechanism that actively targets Tf1, it is possible that the insertion bias is due to greater accessibility at the promoter sequences. We are currently testing this possibility by studying in S. pombe cells the integration pattern of hermes, a cut and paste transposon that was isolated from the house fly. Since hermes propagates in a host that is evolutionary distant from S. pombe, it is unlikely that a mechanism exists that would actively position insertion sites. Thus, any integration of hermes in S. pombe would likely occur at positions that were accessible to the transposase. In addition, unbiased activity of a transposon in S. pombe could be widely adapted as a tool for random mutagenesis. Since no methods currently exist for transposon mutagenesis of S. pombe, a method for insertional mutagenesis would be a significant contribution to the field. ? ? The transposase of hermes was expressed in S. pombe by fusing its gene to the promoter of nmt1. To measure transposition activity the cells that expressed the transposase also contained a plasmid encoded copy of neo flanked by the terminal inverted repeats (TIRs) of hermes. The ability of the transposase to cut out neo with the TIRs and insert this DNA into the pombe genome was tested. Twenty six independent strains that became G418 resistant were analyzed and each strain was found to have acquired a copy of hermes. Analysis of these inserted copies revealed that 54% of them disrupted ORFs. These results indicate that the insertion of hermes did not discriminate between coding and noncoding sequences. This is in strong contrast to the integration of Tf1 where virtually none of the inserts occur in ORFs. In pilot experiments cultures of mutagenized cells were screened for mutations in two specific genes, ade6, and ade7. Of 106,000 cells screened, seven produced red colonies, the phenotype expected for mutations in either ade7 or ade6. This number of ade- colonies was consistent with randomly distributed integration. Together, these data indicate that hermes can readily be used as a tool for the random disruption of pombe genes.
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