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首页> 外文期刊>Molecular and Cellular Biology >Transfection of mouse fibroblast cells with a promoterless herpes simplex virus thymidine kinase gene: number of integrated gene copies and structure of single and amplified gene sequences.
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Transfection of mouse fibroblast cells with a promoterless herpes simplex virus thymidine kinase gene: number of integrated gene copies and structure of single and amplified gene sequences.

机译：用无启动子单纯疱疹病毒胸苷激酶基因转染小鼠成纤维细胞：整合的基因拷贝数以及单个和扩增基因序列的结构。

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Plasmids carrying the herpes simplex virus thymidine kinase (tk) gene were used to transfect thymidine kinase-deficient cells of the mouse fibroblast cell line LM(tk-). Individual cell clones were cultivated in selective hypoxanthine-aminopterin-thymidine medium to determine the number of integrated plasmid copies which was almost always in the range of one to three copies per genome. In contrast, cells transfected with plasmids carrying a promoterless "truncated" tk gene typically contained between 10 and 25 copies per genome. Surprisingly, when the truncated tk gene was transfected together with a simian virus 40 DNA segment, including its transcriptional enhancer, the number of integrated tk gene copies was always low, between one and three copies per genome. We have analyzed the genomic organization of integrated truncated tk genes by blot hybridization of restricted cellular DNA and concluded that integrated units of plasmid DNA molecules are arranged in tandem arrays which remain stable in most cases for many cell generations. In only 1 of ca. 20 cell clones did we observe a retraction and expansion of the number of integrated promoterless tk genes as a response to the removal or readdition of selective pressure. Surprisingly, the thymidine kinase activity determined in extracts from cells growing in selective hypoxanthine-aminopterin-thymidine medium (high numbers of integrated tk gene copies) was nearly the same as the enzymatic activity in cells growing in nonselective medium (low copy numbers). Moreover, Northern blots of polyadenylated RNA, extracted from cells growing under selective and nonselective conditions, showed that, in both cases, the major species of tk-specific transcripts was ca. 1.5 kilobases in size, as expected for a tk-specific mRNA containing the entire coding region of the gene. Thus, disproportionate DNA replication appeared not to be essential for an active tk gene expression in these cells. We discuss possible pathways leading to the formation of tandem arrays of integrated truncated tk genes and the conditions required for disproportionate DNA replication in the unique case in which we found a retraction and expansion of tk gene copy numbers as a response to selective growth conditions.
机译：携带单纯疱疹病毒胸苷激酶（tk）基因的质粒用于转染小鼠成纤维细胞系LM（tk-）的缺乏胸苷激酶的细胞。在选择性次黄嘌呤-氨基蝶呤-胸腺嘧啶核苷培养基中培养单个细胞克隆，以确定整合的质粒拷贝数，每个基因组几乎总是在1-3个拷贝的范围内。相反，用携带无启动子“截短的” tk基因的质粒转染的细胞每个基因组通常包含10至25个拷贝。出乎意料的是，当将截短的tk基因与猿病毒40 DNA片段（包括其转录增强子）一起转染时，整合的tk基因拷贝数总是很低，每个基因组介于1-3个拷贝之间。我们已经通过限制性细胞DNA的印迹杂交分析了整合的tk基因的基因组组织，并得出结论，质粒DNA分子的整合单位以串联排列排列，在大多数情况下，在许多细胞世代中都保持稳定。大约只有1个。我们观察到20个细胞克隆，观察到整合的无启动子tk基因数目的回缩和扩展是对选择性压力的去除或重新分配的反应。令人惊讶地，在选择性次黄嘌呤-氨基蝶呤-胸腺嘧啶核苷培养基（大量整合的tk基因拷贝）中生长的细胞的提取物中测定的胸苷激酶活性几乎与在非选择性培养基（低拷贝数）中生长的细胞的酶活性相同。此外，从在选择性和非选择性条件下生长的细胞中提取的聚腺苷酸化RNA的Northern印迹显示，在两种情况下，tk特异性转录本的主要种类均为ca。如含有该基因整个编码区的tk特异性mRNA所预期的那样，大小为1.5千个碱基。因此，不成比例的DNA复制对于这些细胞中活跃的tk基因表达似乎不是必需的。我们讨论了导致整合的tk基因截短串联阵列形成的可能途径，以及在独特的情况下不成比例的DNA复制所需的条件，在这种情况下，我们发现tk基因拷贝数的回缩和扩展是对选择性生长条件的反应。

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