Sen S, Siciliano M J, Johnston D A, Schwartz R J, Kuo T
J Biol Chem. 1985 Mar 10;260(5):3071-8.
Chicken glyceraldehyde-3-phosphate dehydrogenase gene (GAPD) and thymidine kinase gene (TK) were co-transfected into mouse LMTK- cells by the calcium phosphate precipitation technique. Four of the eight hypoxanthine/aminopterin/thymidine-containing medium-resistant, TK+ transfectants were shown to produce different amounts of chicken glyceraldehyde-3-phosphate dehydrogenase by zymogram analysis. Subcloning and further analysis revealed that the chicken GAPD was stably inherited and that its enzyme subunits randomly combined with mouse subunits in heterotetramers. Although the contribution of chicken enzyme varied from approximately 30 to approximately 90% of the total glyceraldehyde-3-phosphate dehydrogenase activity with a proportional increase in total activity in the different subclones, it did not appear to affect the expression of mouse endogenous glycolytic enzymes since there was no distinct change in the levels of either mouse glyceraldehyde-3-phosphate dehydrogenase mRNA nor mouse phosphoglycerate kinase enzyme activity. The levels of chicken GAPD copy number, mRNA, and enzyme apparently were generally correlated in the different subclones, suggesting that the chicken GAPD in the mouse cells were expressed constitutively. In situ hybridization revealed that the transfected genes were integrated into mouse chromosomes in one cluster, and the locations of these clusters were different in different clones. Chromatin structure analyses of the chicken GAPD in four different transfectants revealed three DNase I-hypersensitive sites located around 0.2, 2.0, and 3.4 kilobases upstream from the 5' side of the gene. These sites are also present in the same locations in chicken lymphoblastoid cells (Kuo, M. T., Iyer, B., and Schwartz, R. J. (1982) Nucleic Acids Res 10, 4565-4579), indicating the dominant transmission of DNase I-hypersensitive cleavage sites in the transfected gene.
通过磷酸钙沉淀技术将鸡甘油醛 - 3 - 磷酸脱氢酶基因（GAPD）和胸苷激酶基因（TK）共转染到小鼠LMTK - 细胞中。通过酶谱分析表明，八个含次黄嘌呤/氨基蝶呤/胸腺嘧啶核苷培养基抗性的TK + 转染子中有四个产生不同量的鸡甘油醛 - 3 - 磷酸脱氢酶。亚克隆和进一步分析显示，鸡GAPD稳定遗传，其酶亚基在异源四聚体中与小鼠亚基随机组合。尽管鸡酶的贡献在不同亚克隆中占总甘油醛 - 3 - 磷酸脱氢酶活性的约30％至约90％，且总活性成比例增加，但由于小鼠甘油醛 - 3 - 磷酸脱氢酶mRNA水平和小鼠磷酸甘油酸激酶酶活性均无明显变化，所以似乎不影响小鼠内源性糖酵解酶的表达。鸡GAPD拷贝数、mRNA和酶水平在不同亚克隆中通常呈大致相关，表明小鼠细胞中的鸡GAPD组成性表达。原位杂交显示，转染基因在一个簇中整合到小鼠染色体中，且这些簇在不同克隆中的位置不同。对四个不同转染子中鸡GAPD的染色质结构分析显示，在基因5' 端上游约0.2、2.0和3.4千碱基处有三个DNase I超敏位点。这些位点在鸡淋巴母细胞中也存在于相同位置（Kuo，M. T.，Iyer，B.，和Schwartz，R. J.（1982）Nucleic Acids Res 10，4565 - 4579），表明转染基因中DNase I超敏切割位点的优势传递。
