SELECTION FOR ANIMAL-CELLS THAT EXPRESS THE ESCHERICHIA-COLI GENE CODING FOR XANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE
SELECTION FOR ANIMAL-CELLS THAT EXPRESS THE ESCHERICHIA-COLI GENE CODING FOR XANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE
复制标题
DOI：
10.1073/pnas.78.4.2072
复制
发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
BERG, P
中科院分区：
文献类型：
--
作者：
MULLIGAN, RC;BERG, P
用携带相应细菌基因 Ecogpt 的 DNA 载体转染后，培养的猴 (TC7) 和小鼠 (3T6) 细胞合成大肠杆菌酶，黄嘌呤-鸟嘌呤磷酸核糖基转移酶 (XGPRT；5-磷酸-.α.-D-核糖-1-二磷酸:黄嘌呤磷酸核糖基转移酶，EC 2.4.2.22)。与不能有效利用黄嘌呤合成嘌呤核苷酸的哺乳动物细胞相反，产生大肠杆菌 XGPRT 的细胞可以通过 XMP 从黄嘌呤合成 GMP。用载体-Ecogpt DNA 转染后，产生 XGPRT 的存活细胞可以在含有阻断嘌呤核苷酸从头合成的抑制剂（氨基蝶呤和霉酚酸）的培养基中选择性生长，黄嘌呤作为鸟嘌呤核苷酸形成的唯一前体。转化 Ecogpt 的细胞出现频率为 10-4 至 10-5；它们似乎在遗传上是稳定的，因为在非选择性培养基中繁殖后，XGPRT 形成没有明显减少，也没有在选择性培养基中生长能力丧失。尽管一些载体-gpt DNA 可以在猴和小鼠细胞中复制，但没有一个转化体含有自主复制的载体-gpt DNA。 gpt 转化体含有 1-5 个与细胞 DNA 序列相关的转染 DNA 拷贝，并且很可能整合到细胞 DNA 序列中。在一些转化体中，还合成了未经选择的载体编码基因产物。含有 Ecogpt 作为选择性标记的重组 DNA 可用于非选择性基因的共转化。
Cultured monkey (TC7) and mouse (3T6) cells synthesize an E. coli enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT; 5-phospho-.alpha.-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase, EC 2.4.2.22), after transfection with DNA vectors carrying the corresponding bacterial gene, Ecogpt. In contrast to mammalian cells, which do not efficiently use xanthine for purine nucleotide synthesis, cells that produce E. coli XGPRT can synthesize GMP from xanthine via XMP. After transfection with vector-Ecogpt DNA, surviving cells producing XGPRT can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purine nucleotide synthesis. Cells transformed for Ecogpt arise with a frequency of 10-4 to 10-5; they appear to be genetically stable because there is no discernible decrease in XGPRT formation or loss in their ability to grow in selective medium after propagation in nonselective medium. Although several of the vector-gpt DNA can replicate in monkey and mouse cells, none of the transformants contain autonomously replicating vector-gpt DNA. The gpt transformants contain 1-5 copies of the transfecting DNA associated with, and most probably integrated into, cellular DNA sequences. In several transformants, vector-coded gene products for which there was no selection are also synthesized. Recombinant DNA containing Ecogpt as a selective marker may be useful for cotransformation of nonselectable genes.