Gasser C S, Schimke R T
J Biol Chem. 1986 May 25;261(15):6938-46.
We have used the technique of DNA-mediated gene transfer to introduce dihydrofolate reductase genes into dihydrofolate reductase-deficient Chinese hamster ovary cells. The transferred sequences include: dihydrofolate reductase minigenes, dihydrofolate reductase cDNA clones, and genomic DNA from mouse cells with highly amplified dihydrofolate reductase genes. The hamster cells were capable of utilizing the murine transcription initiation sites, splice junctions, and polyadenylation sites in complete murine dihydrofolate reductase genes. Only a short region of 5'-flanking sequence (160 base pairs (bp] was sufficient for proper initiation of dihydrofolate reductase transcription. In contrast only those clones with all of the dihydrofolate reductase introns and extensive (greater than 6.5 kilobase pairs) 3'-flanking regions utilized the dihydrofolate reductase polyadenylylation sites. Four of nine clones tested regulate the transfected genes normally, synthesizing dihydrofolate reductase preferentially at the onset of S phase. Regulated genes include one which entirely lacks intervening sequences, two with fewer than 420 bp of 5'-flanking sequence, and two in which polyadenylylation occurs predominantly in the adjacent hamster sequences. Two of the five cell lines which do not exhibit normal regulation contain genes which appear to be transcribed primarily from promoters present in the flanking hamster DNA. The other genes which fail to regulate do not appear to differ significantly in the structure of their transcripts or protein products from genes which regulate normally. We conclude that only the coding sequences, a region of less than 340 bp of sequence 5' of the translation initiation codon, and a short region of 3'-flanking sequence are required for regulated production of dihydrofolate reductase. The regulation can be abolished, however, by unknown properties of the site of insertion.
我们运用了DNA介导的基因转移技术，将二氢叶酸还原酶基因导入缺乏二氢叶酸还原酶的中国仓鼠卵巢细胞。转移的序列包括：二氢叶酸还原酶微型基因、二氢叶酸还原酶cDNA克隆，以及来自具有高度扩增二氢叶酸还原酶基因的小鼠细胞的基因组DNA。仓鼠细胞能够利用完整小鼠二氢叶酸还原酶基因中的鼠类转录起始位点、剪接位点和聚腺苷酸化位点。仅5'侧翼序列的一个短区域（160个碱基对）就足以实现二氢叶酸还原酶转录的正确起始。相比之下，只有那些含有所有二氢叶酸还原酶内含子和广泛的（大于6.5千碱基对）3'侧翼区域的克隆才利用二氢叶酸还原酶聚腺苷酸化位点。所测试的9个克隆中有4个正常调节转染基因，在S期开始时优先合成二氢叶酸还原酶。受调节的基因包括一个完全缺乏间隔序列的基因、两个5'侧翼序列少于420 bp的基因，以及两个聚腺苷酸化主要发生在相邻仓鼠序列中的基因。5个未表现出正常调节的细胞系中有两个含有似乎主要从侧翼仓鼠DNA中存在的启动子转录的基因。其他未能调节的基因在转录本或蛋白质产物的结构上与正常调节的基因相比似乎没有显著差异。我们得出结论，二氢叶酸还原酶的受调节产生仅需要编码序列、翻译起始密码子5'端小于340 bp的序列区域以及3'侧翼序列的一个短区域。然而，调节可能会因插入位点的未知特性而被消除。
