Wise T L, Harris M
Department of Zoology, University of California, Berkeley 94720.
Somat Cell Mol Genet. 1988 Nov;14(6):567-81. doi: 10.1007/BF01535311.
Previous studies on V79 Chinese hamster cells have shown that bromodeoxyuridine (BrdU) -resistant variants deficient in thymidine kinase (TK) activity arise by a multistep process which is initiated by a random event and progresses gradually during serial culture in the presence of the drug. In order to determine the molecular basis for the loss of TK activity in these cells, the TK gene was isolated from a lambda phage library of genomic V79 DNA, using a fragment of the human TK gene as a probe. One phage isolated contained the entire TK gene in a 15-kb insert, as demonstrated by the ability of the phage DNA to transform Ltk- mouse cells to the TK+ phenotype. Five fragments spanning the entire gene were then subcloned into the plasmid pUC12 for DNA methylation studies. With these probes it was shown by hybridization analysis that the copy number of the TK gene in V79 cells is about four times the copy number in CHO cells and Chinese hamster liver cells. Southern hybridization analysis of the DNA from first-stage variants partially resistant to BrdU indicated that partial resistance was accompanied by deletion of a number of copies of the TK gene in V79 cells. However, the subsequent gradual transition to full BrdU resistance and full loss of TK activity was correlated with a gradual hypermethylation of sites in the 5' region of the TK gene, with no further change in gene copy number.
先前对V79中国仓鼠细胞的研究表明，缺乏胸苷激酶（TK）活性的抗溴脱氧尿苷（BrdU）变体是通过多步骤过程产生的，该过程由随机事件引发，并在药物存在下的连续培养过程中逐渐进展。为了确定这些细胞中TK活性丧失的分子基础，使用人TK基因的片段作为探针，从基因组V79 DNA的λ噬菌体文库中分离出TK基因。分离出的一个噬菌体在一个15kb的插入片段中包含了整个TK基因，这通过噬菌体DNA将Ltk-小鼠细胞转化为TK+表型的能力得以证明。然后将跨越整个基因的五个片段亚克隆到质粒pUC12中用于DNA甲基化研究。通过杂交分析表明，V79细胞中TK基因的拷贝数约为CHO细胞和中国仓鼠肝细胞中拷贝数的四倍。对部分抗BrdU的第一阶段变体的DNA进行Southern杂交分析表明，部分抗性伴随着V79细胞中TK基因多个拷贝的缺失。然而，随后向完全抗BrdU和TK活性完全丧失的逐渐转变与TK基因5'区域位点的逐渐超甲基化相关联，而基因拷贝数没有进一步变化。
