Our goal is to understand better the relationship between polyoma virus genome structure and gene expression. Our particular interests are in the late leader unit and in the late promoter. Polyoma late mRNA molecules have multiple, tandem copies of a 57-base, untranslated sequence the """"""""late leader"""""""" at their 5'-ends. The function of the leader in late gene expression is somewhat unclear. We shall study its role in RNA synthesis, processing and translation. Our recent work has shown that the length of the leader, but not its sequence, is important for both late RNA splicing and stability. We shall extend these studies in order to elucidate the molecular events involved in late leader splicing. We shall use deletion and point mutants to test the role of leader length and of the 3' splice site at its 5' boundary in splicing and RNA stability. We shall insert the ALM leader region into other transcripts, upstream or downstream of other exons, to determine whether it can confer instability on other pre-mRNAs. We shall use chimeric double-genome plasmids and DNA transfections to study the order of exon splicing in polyoma late transcripts and we shall examine the possibility of trans splicing of polyoma late RNAs. We shall establish in vitro splicing of late viral pre- mRNAs in HeLa cell extracts and use this system to test leader region mutants for splicing, RNA stability, and splicing complex formation. In order to investigate the significance of leader multiplicity we shall study a mutant genome with the 3'-end processing/polyadenylation region of SV40 inserted into the 3'-end region of polyoma. This virus should only generate single leader units on all late messages. We shall test the leader region for a role in translation. We shall use reticulocyte lysates for in vitro translation of VP1 or VP2 from capped, T7 transcripts. We shall translate RNAs individually or use them in competition experiments with rabbit beta-globin. Experiments will be done with RNAs containing deleted, substituted or reiterated leader units. In in vivo experiments, we shall use S1 analysis to determine whether larger polysomes form infected cells contain viral mRNAs with more leader units. Another of our goals is to define cis-acting regulatory sequences that are involved in the initiation and control of transcription from the late promoter. Toward this end we shall use CAT assays, nuclear run-off experiments and S1 analysis to measure and compare early and late promoter activity in a large number of mutants, many made by site-directed oliqonucleotide deletion mutagenesis. We shall perform these experiments in the presence or absence of large T antigen or DNA replication inhibitors. Nuclear run-off experiments will be done as a function of time after wild type or mutant transfection/infection in order to determine the time course of late promoter activation and its relationship to large T, DNA replication and specific cis-acting DNA sequences.
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