Romac S, Leong P, Sockett H, Hutchinson F
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
J Mol Biol. 1989 Sep 20;209(2):195-204. doi: 10.1016/0022-2836(89)90272-6.
The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Escherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt- colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR), and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes.
通过紫外线诱变在培养的中国仓鼠卵巢（CHO）细胞染色体上的一个基因中诱导产生的DNA碱基序列变化已被确定。该基因是大肠杆菌的gpt基因，其单拷贝稳定整合并在CHO细胞基因组中表达。用紫外线照射细胞，并通过对6-硫鸟嘌呤的抗性选择gpt-菌落。利用聚合酶链反应（PCR）从染色体DNA中扩增gpt基因，并通过双脱氧法直接对扩增的DNA进行测序。在58个独立起源的测序突变体中，53个是碱基变化突变。41个碱基替换是单碱基变化，10个有两个相邻（或串联）碱基变化，1个有两个被单个碱基对隔开的碱基变化。只有1个突变体有两个或更多间隔良好的碱基变化的多碱基变化突变。相比之下，在灵长类细胞中穿梭载体上的基因进行紫外线诱变时，报道的此类突变水平要高得多。观察到两个单碱基对的缺失和三个6到37个碱基对的缺失。gpt基因中的突变谱与原核生物中几个基因的紫外线突变谱有相似之处，这表明原核生物和真核生物在突变机制上有相似之处。
