Milbrandt J D, Azizkhan J C, Greisen K S, Hamlin J L
Mol Cell Biol. 1983 Jul;3(7):1266-73. doi: 10.1128/mcb.3.7.1266-1273.1983.
We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.
我们用黏粒载体pHC79构建了一个来自甲氨蝶呤抗性中国仓鼠卵巢细胞系（CHOC 400）的基因组DNA文库。利用小鼠二氢叶酸还原酶（DHFR）cDNA克隆，我们在通过菌落杂交筛选的11000个菌落中鉴定出了66个DHFR+克隆。为了分离出包含完整DHFR基因的重组黏粒，我们使用W. 沙夫纳开发的基因转移原生质球融合方法（《美国国家科学院院刊》77:2163 - 2167, 1980），测试了这些菌落拯救DHFR-中国仓鼠卵巢细胞系的能力。一个克隆（cH1）能够将DHFR-细胞转化为DHFR+表型，并且在杂交研究中显示其包含除3'非翻译区一小部分之外的所有基因。我们用多种限制酶对黏粒cH1和几个重叠黏粒进行了图谱绘制，并确定了DHFR基因内五个（可能六个）外显子的大致位置。结果表明，仓鼠细胞（24.5千碱基[kb]）和小鼠细胞（31.5 kb）中同源基因大小的差异主要存在于3'内含子的长度上，仓鼠基因的3'内含子长度为8 kb，小鼠基因的为16 kb。我们的研究证实了黏粒文库在分离大基因方面的实用性，正如R. 德·圣文森特等人之前所表明的（《细胞》27:267 - 277, 1981）。此外，一个包含功能性DHFR基因的黏粒将是用于其他克隆基因共扩增及随后过表达的有用载体。
