Thelander M, Thelander L
Department of Medical Biochemistry and Biophysics, University of Umeå, Sweden.
EMBO J. 1989 Sep;8(9):2475-9. doi: 10.1002/j.1460-2075.1989.tb08383.x.
Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. M2-related DNA sequences are present on mouse chromosomes 4, 7, 12 and 13. However, M2-overproducing mouse cells show amplification of a chromosome 12-specific, single 13 kb HindIII fragment, which probably represents the active gene. We have isolated this fragment from parental mouse cell DNA and used it to clone and characterize the functional M2 gene. The 5770 bp transcribed M2 sequence contains ten exons separated by nine 95-917 bp introns. The 501 bp of 5' flanking DNA is G + C rich and contains TTTAAA and CCAAT sequences as well as potential Sp1 binding sites. The M2-related sequence on chromosome 13, which contains only the last six exons and several internal rearrangements, is a pseudogene. Transfection of BALB/3T3 cells with the M2 gene resulted in stable transformants with a 10-fold reduction in sensitivity to hydroxyurea, compared to control cells. This confirmed that the cloned M2 genomic DNA represents the functional gene and conclusively establishes the link between hydroxyurea resistance and M2 expression in mammalian cells. M2 genomic DNA should be a valuable dominant, selectable marker for identifying and isolating stable co-transformants.
哺乳动物核糖核苷酸还原酶由两个不同的亚基，即M1和M2蛋白组成。与M2相关的DNA序列存在于小鼠的4号、7号、12号和13号染色体上。然而，过量产生M2的小鼠细胞显示出12号染色体特异性的一个13 kb HindIII单一片段的扩增，该片段可能代表活性基因。我们已从亲代小鼠细胞DNA中分离出该片段，并用于克隆和鉴定功能性M2基因。转录的5770 bp M2序列包含十个外显子，由九个95 - 917 bp的内含子分隔。5'侧翼DNA的501 bp富含G + C，包含TTTAAA和CCAAT序列以及潜在的Sp1结合位点。13号染色体上与M2相关的序列仅包含最后六个外显子和一些内部重排，是一个假基因。用M2基因转染BALB/3T3细胞产生了稳定的转化体，与对照细胞相比，对羟基脲的敏感性降低了10倍。这证实了克隆的M2基因组DNA代表功能性基因，并最终确立了哺乳动物细胞中羟基脲抗性与M2表达之间的联系。M2基因组DNA应该是用于鉴定和分离稳定共转化体的有价值的显性选择标记。
