ORGANIZATION AND EXPRESSION OF GENES IN VIRAL DNAS

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The EIA and EIB transcription regions of Adenovirus 5 contain approximately 3500 base pairs of DNA and encode functions that regulate the expression of other viral genes as result in the oncogenic transformation of mammalian cells. The DNA sequence of this region is known, its overlapping mRNAs defined and their encoded polypeptides determined. Experiments are proposed to define which of these mRNAs encode the polypeptides required for either the regulation of gene expression or cellular transformation. In addition the functional domains of these encoded polypeptides will be determined and their mechananism of action investigated. Regulation of Gene Expression: The class I host range mutants locate the gene products required for the expression of other viral genes within EIA; the following scheme will determine which specific EIA encoded function(s) are required. Eukaryotic transducing vectors will be used to prepare permanent monkey cell lines containing the EIA and EIB regions from wild type and mutant genomes. SV40 viruses containing full length copies of the individual EIA cDNAs will be prepared and used to complement the mutant defects in the permanent cell lines. This will define these specific gene functions essential for regulating gene expression. The functional domains of these polypeptides will be defined by comparing the EIA DNA sequences of numerous class I host range mutants and their revertants and by direct alteration of specific regions of the appropriate cDNAs by in vitro mutagenesis. The mechanisms by which gene expression is regulated will be studied by overproducing the EIA polypeptides and using enriched extracts to study their binding to DNA and their effect on in vitro transcription of genes. Transformation: Transducing vectors will be used to insert full length cDNAs from the Ad5 EIA and EIB into primary hamster cells to determine which gene products induce transformation. Host range mutants and in vitro mutagenesis will define the domain(s) containing the transformation functions. Also, simian Adenovirus 7 which is efficient in DNA promoted transformation will be used to complement these Ad5 studies. This data will serve as a basis for experiments to study the molecular mechanism of the oncogenic transformation of mammalian cells.