Isolation of cellular DNA sequences that allow expression of adjacent genes.

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RESUMO

We have employed a strategy for the isolation and identification of cellular control (expression) sequences dependent on their ability to confer expression on a selectable gene devoid of its own expression sequences. The polyoma virus (Py) Hae II-BamHI DNA fragment, which comprises 84% of the intact viral DNA and contains the Py transforming region but lacks Py 5' expression sequences, was decreased markedly in its transformation of rat cells. Hae II-cleaved mouse cellular DNA was ligated to the Py Hae II-BamHI fragment. A transformed colony (H1) isolated after transfection of the ligated DNA onto rat cells was found to contain multiple inserts of Py DNA, most of which were biologically inactive. A transformed colony (H2) isolated after transfection of rat cells with total H1 DNA was found to contain a single insert of Py DNA. The H2 cells are highly tumorigenic and synthesize the three Py tumor antigens. Initiation of transcription of the Py early mRNAs in H2 cells occurs at the same Py nucleotides as in complete Py DNA. The viral and adjacent cellular DNA sequences were cloned from H2 cellular DNA. The transforming efficiency of the cloned Py transforming region and adjacent H2 cellular DNA was 20-40% of that of the viral DNA containing Py expression sequences. By BAL-31 deletion mapping it was observed that the first 58 base pairs of H2 cellular DNA were sufficient for the expression of the Py-transforming region. The sequence of the first 149 base pairs of the H2 cellular DNA was determined and does not show any striking similarities to upstream 5' sequences of a number of viral and host structural genes. Features of the H2 cellular sequence are discussed.

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