ORGANIZATION AND EXPRESSION OF GENES IN VIRAL DNAS

腺病毒5的EIA和EIB转录区含有大约 3500个碱基对的DNA和编码的功能，调节表达的 其他病毒基因导致哺乳动物的致癌转化 细胞 该区域的DNA序列是已知的，其重叠的mRNA 定义并确定其编码的多肽。 实验 提出定义这些mRNA中的哪一个编码所需的多肽， 用于基因表达的调节或细胞转化。 此外，这些编码的多肽的功能结构域将是 确定并研究其作用机制。 调控 基因表达：I类宿主范围突变体定位基因产物 EIA内其他病毒基因表达所需的;以下 方案将确定需要哪些特定EIA编码函数。 真核转导载体将用于制备永久性猴 含有来自野生型和突变体的EIA和EIB区的细胞系 基因组 含有单个EIA全长拷贝的SV 40病毒 cDNA将被制备并用于补充突变体中的突变缺陷。 永久细胞系。 这将定义这些特定的基因功能 对调节基因表达至关重要。 这些功能域 多肽将通过比较许多人的EIA DNA序列来定义。 I类宿主范围突变体及其回复突变体，并通过直接改变 合适的cDNA的特异性区域。 的 基因表达调控的机制将通过以下方式进行研究： 过量生产EIA多肽并使用富集的提取物来研究 它们与DNA的结合以及它们对基因体外转录的影响。 转化：转导载体将用于插入全长 将来自Ad 5EIA和EIB的cDNA导入原代仓鼠细胞中，以确定 哪些基因产物诱导转化。 宿主范围突变体和体外 诱变将限定含有转化的结构域 功能协调发展的 此外，猴腺病毒7，这是有效的DNA促进 转化将用于补充这些Ad 5研究。 该数据 将作为实验的基础，研究分子机制， 哺乳动物细胞的致癌性转化。

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.