Fried M, Griffiths M, Davies B, Bjursell G, La Mantia G, Lania L
Proc Natl Acad Sci U S A. 1983 Apr;80(8):2117-21. doi: 10.1073/pnas.80.8.2117.
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.
我们采用了一种策略来分离和鉴定细胞控制（表达）序列，该策略依赖于这些序列赋予一个没有自身表达序列的可选择基因表达的能力。多瘤病毒（Py）的Hae II - BamHI DNA片段，它包含完整病毒DNA的84%，含有Py转化区但缺乏Py 5'表达序列，其对大鼠细胞的转化能力显著降低。将经Hae II切割的小鼠细胞DNA与Py Hae II - BamHI片段连接。将连接后的DNA转染到大鼠细胞上后分离得到的一个转化菌落（H1）被发现含有多个Py DNA插入片段，其中大多数在生物学上无活性。用H1总DNA转染大鼠细胞后分离得到的一个转化菌落（H2）被发现含有单个Py DNA插入片段。H2细胞具有高度致瘤性并能合成三种Py肿瘤抗原。H2细胞中Py早期mRNA的转录起始位点与完整Py DNA中的相同Py核苷酸处。从H2细胞DNA中克隆出病毒及相邻的细胞DNA序列。克隆的Py转化区和相邻的H2细胞DNA的转化效率是含有Py表达序列的病毒DNA的20 - 40%。通过BAL - 31缺失图谱分析观察到，H2细胞DNA的前58个碱基对足以使Py转化区表达。测定了H2细胞DNA前149个碱基对的序列，未发现其与许多病毒和宿主结构基因的上游5'序列有任何显著相似性。讨论了H2细胞序列的特征。
