The primary objective of this investigation is to determine the role of the BamHI-E fragment of HSV-2 DNA in oncogene activation associated with neoplastic transformation. Specifically, we will: (1) localize the minimal HSV DNA sequences responsible for transformation; (2) characterize the activated rat and fused HSV sequences transduced in extrachromosomal DNA isolated from transformed cells; (3) clone and characterize HSV-related cellular DNA sequences from untransformed cells and amplified DNA sequences from transformed cells and (4) determine whether DNA amplification is causal and relevant to HSV transformation.
The specific aims will be accomplished in the following steps: Individual restriction fragments of BamHI-E, subcloned in M13 vectors from enzyme digests (PstI, SalI, SacI) whose cleavages leave the transforming region intact, will be assayed for focus formation in Rat-2 cells in the presence of vector DNAs as controls. DNA sequence of the smallest overlapping subclones exhibiting transforming activity will be determined by Sanger dideoxy method. Activated rat DNA and fused HSV junctions of extrachromosomal DNA subcloned in puc-19 vector will be analyzed by the exonuclease III unidirectional sequencing technique Sequences will be analyzed by the Intelligenetics Computer Users Program. Phage library of normal genomic rat DNA will be screened with small HSV-2 BamHI-E subcloned probes and the smallest hybridizing cellular and viral fragments subcloned, sequenced and compared. Amplified sequences commonly detected in HSV-transformed cells will be cloned and characterized further by restriction enzyme mapping and DNA sequencing. Genomic DNA from primary HSV- transformants will be sheared and assayed for transforming activity in Rat-2 cells using neomycin coselection. Presence of transfected sequences in secondary HSV-transformants will be analyzed by Southern blot hybridizations using cloned amplified DNA and pSV-2 neo probes. The amplified DNA probe will be additionally used to detect occurrence of DNA amplification and enhanced expression of homologous RNA transcripts in cells transformed by various agents and in subclones of normal Rat-2 cells. This approach will allow us to define the second minimal transforming region of HSV-2, identify the oncogenic rat sequences activated in extrachromosomal DNA and determine the relevance of DNA amplification to HSV transformation.
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