Mendelsohn E, Baran N, Neer A, Manor H
J Virol. 1982 Jan;41(1):192-209. doi: 10.1128/JVI.41.1.192-209.1982.
The structure of the polyoma virus (Py) integration site in the inducible LPT line of Py-transformed rat cells was determined by biochemical methods of gene mapping. LPT cell DNA was digested with various restriction enzymes. The digestion products were electrophoresed in agarose gels and transferred onto nitrocellulose sheets by Southern blotting. Fragments containing viral or cell DNA sequences, or both, were identified by hybridization with Py DNA or with a cloned flanking cell DNA probe. Cleavage of LPT DNA with enzymes that restrict the Py genome once generated linear Py DNA molecules and two fragments containing both cell and viral DNA sequences. Cleavage of LPT DNA with enzymes which do not restrict Py DNA generated series of fragments whose lengths were found to differ by increments of a whole Py genome; the smallest fragment in each series was found to be longer than the viral genome. These data indicate that LPT cultures contain Py insertions of various lengths integrated into the same chromosomal site in all the cells. The length heterogeneity of the viral insertions is due to the presence of 0, 1, 2, 3. . . Py genomes arranged in a direct tandem repeat within invariable sequences of viral DNA. Double-digestion experiments were also carried out with the above enzymes and with enzymes that cleave the Py genome at multiple sites. The data obtained in these experiments were used to construct a physical map of the integration site. This map showed that the early region of the virus remained intact even in the smallest insertion (which contains no whole duplicated genomes), whereas the late region was partially duplicated and split during integration. The smallest insertion is colinear with the Py physical map over a region including the entire Py genome and at least a part of the duplicated segment. This structure could give rise to nondefective circular viral DNA molecules by single homologous recombination events. Similar recombination events may occur at a higher frequency in the longer insertions, which include longer regions of homology, and may yield many more free viral genomes. The presence of these insertions in LPT cells could thus be one of the factors which account for the high inducibility of the LPT line.
通过基因定位的生化方法确定了多瘤病毒（Py）在Py转化的大鼠细胞的诱导型LPT系中的整合位点结构。用各种限制性内切酶消化LPT细胞DNA。消化产物在琼脂糖凝胶中进行电泳，然后通过Southern印迹转移到硝酸纤维素膜上。通过与Py DNA或克隆的侧翼细胞DNA探针杂交来鉴定含有病毒或细胞DNA序列或两者的片段。用只切割一次Py基因组的酶切割LPT DNA，产生线性Py DNA分子和两个同时含有细胞和病毒DNA序列的片段。用不切割Py DNA的酶切割LPT DNA，产生一系列片段，其长度相差一个完整的Py基因组；每个系列中最小的片段比病毒基因组长。这些数据表明，LPT培养物中含有各种长度的Py插入片段，它们整合到所有细胞的同一染色体位点。病毒插入片段的长度异质性是由于在病毒DNA的不变序列内存在0、1、2、3……个以直接串联重复排列的Py基因组。还用上述酶以及在多个位点切割Py基因组的酶进行了双酶切实验。这些实验中获得的数据用于构建整合位点的物理图谱。该图谱显示，即使在最小的插入片段（不包含完整的重复基因组）中，病毒的早期区域仍保持完整，而晚期区域在整合过程中部分重复并断裂。最小的插入片段在包括整个Py基因组和至少一部分重复片段的区域与Py物理图谱共线。这种结构可能通过单次同源重组事件产生无缺陷的环状病毒DNA分子。类似的重组事件在较长的插入片段中可能以更高的频率发生，这些插入片段包含更长的同源区域，可能产生更多的游离病毒基因组。因此，LPT细胞中这些插入片段的存在可能是LPT系高诱导性的原因之一。
