The objectives of this research proposal are to exploit the novel way in which expression of the transformed phenotype is modulated in cells infected with the MuSVts110 murine sarcoma virus as a means to study the molecular basis of RNA splicing. MuSVts110 infected cells (6m2 cells) are transformed by the P85gag-mos, a virus-encoded gag-mos polypeptide synthesized from a spliced 3.5KB viral mRNA. The splice events which produce this RNA occur in infected cells only at growth temperatures of 33 degrees C (or lower). We plan to make advantage of the fact that although MuSVts110 viral transcripts (4kb) are efficiently synthesized at 39 degrees C, they are not spliced unless the growth temperature is lowered to at least 33 degrees C. Thus, we plan to dissect successive events in the mechanism by which this viral RNA is spliced in intact cells under physiologic conditions by shifting 6m2 cells from 39 degrees C to 33 degrees C for specified periods of time during the splicing process. By using S-1 nuclease protection analysis, primer extensive and nucleotide sequencing analyses with specific DNA reagents capable of measuring splice donor site cleavage, 'lariat' formation within the intron, events near the splice acceptor site, and ligation of the 5' and 3' exons, we plan to define splicing of this RNA in detail. Additionally, we plan to investigate the structural basis for and constraints on thermosensitive RNA splicing by altering the structure of sequences in and around the 'intron' in MuSVts110 DNA by, for example, site-directed mutagenesis, removal or additional of restriction fragments or digestion with processive enzymes such as BAL-31. The splicing properties of these altered viral DNAs will be studied in vitro after their introduction into, for example, one of the SP6 family of plasmid vectors and those that prove interesting will be transfected into NRK or 3T3 cells to study their splicing properties in vivo. Preliminary evidence indicates that in MuSVts110 revertant cells capable only of splice donor site cleavage that this is a thermosensitive process, suggesting that introducing alterations into the intron of viral DNA obtained from this cell line and then measuring donor site cleavage will provide a rapid assay for the effects of structural alterations on RNA splicing. If, as is our current expectation, we find that is the structure of the MuSVts110 intron is responsible for thermosensitive RNA splicing, we plan to construct an expression vector capable of the temperature-sensitive expression of cloned genes in euraryotic cells.