Romani M, De Ambrosis A, Alhadeff B, Purrello M, Gluzman Y, Siniscalco M
Laboratory of Molecular Biology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
Gene. 1990 Nov 15;95(2):231-41. doi: 10.1016/0378-1119(90)90366-y.
Human fibroblasts transformed with an adenovirus-5/simian virus 40 recombinant construct (Ad5/SV40) were analyzed to determine the chromosomal site(s) of virus integration. This was firstly done by in situ hybridization using metaphase and prometaphase chromosomes and 125I-labeled Ad5 DNA. Out of seven transformed cell lines (six of clonal origin and one uncloned), six were proven to have integrated the viral genome at the short- or the long-subtelomeric regions of autosome 1, two regions known to include chromosomal modification sites induced by acute infection with Ad12. Characterization of the integration sites was carried out by restriction analysis. Transformed cell lines with the same major chromosomal integration site were found to have the viral genome inserted in restriction fragments of different size, indicating that viral integration has occurred at different sites within a relatively small chromosomal region. Molecular studies carried out on one of the transformed cell lines (H13.1) gave an independent confirmation of the viral integration at the subterminal region of autosome 1 short arm. Nucleotide sequencing at this cellular-viral junction has shown that the virus has integrated within tandemly repeated Alu-like elements and that the cellular flanking sequences have several homologies with variable number of tandem repeats core sequences. Many possible open reading frames were identified in the DNA segment adjacent to the Alu-like elements.
对用腺病毒5型/猴病毒40型重组构建体（Ad5/SV40）转化的人成纤维细胞进行分析，以确定病毒整合的染色体位点。首先通过使用中期和早中期染色体以及125I标记的Ad5 DNA进行原位杂交来完成。在七个转化细胞系（六个克隆起源和一个未克隆）中，六个被证明在常染色体1的短或长亚端粒区域整合了病毒基因组，这两个区域已知包含由Ad12急性感染诱导的染色体修饰位点。通过限制性分析对整合位点进行表征。发现具有相同主要染色体整合位点的转化细胞系将病毒基因组插入不同大小的限制性片段中，这表明病毒整合发生在相对较小的染色体区域内的不同位点。对其中一个转化细胞系（H13.1）进行的分子研究独立证实了病毒在常染色体1短臂亚末端区域的整合。在这个细胞-病毒连接处的核苷酸测序表明，病毒已整合到串联重复的Alu样元件中，并且细胞侧翼序列与可变数量的串联重复核心序列有几个同源性。在与Alu样元件相邻的DNA片段中鉴定出许多可能的开放阅读框。
