Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to N-ethyl-N-nitrosourea.

We have determined the mutational spectrum of N-ethyl-N-nitrosourea (ENU) in exon 3 of the hypoxanthine (guanine) phosphoribosyltransferase gene (Hprt) in splenic T cells following in vivo exposure of male B6C3F1 mice (5-7 weeks old) to ENU. Hprt- mutants were isolated by culturing splenic T cells in microtiter dishes containing medium supplemented with interleukin 2, concanavalin A, and 6-thioguanine. DNA was extracted from 6-thioguanine-resistant colonies and amplified by the polymerase chain reaction (PCR) using primers flanking Hprt exon 3. Identification of mutant sequences and purification of mutant DNA from contaminating wild-type Hprt DNA was accomplished by denaturing-gradient gel electrophoresis. Purified mutant DNA was then sequenced. Treatment of mice with ENU at 40 mg/kg of body weight produced a Hprt- mutant frequency of 7.3 x 10(-5) in splenic T cells, approximately 35-fold above background levels. Sixty-nine of the 521 Hprt- mutants analyzed contained mutations in exon 3 (13%). Transversions and transitions at A.T base pairs dominated the spectrum; 62 of the 69 exon 3 mutations were at A.T base pairs (14 different sites). Thirteen of 14 thymine bases undergoing mutation (61 of 62 mutations at A.T bases) were located on the nontranscribed strand of exon 3. The majority of the remaining mutations (6 of 69) were transitions at a single G.C base pair. These results suggest the importance of thymidine alkylation in ENU-induced mutagenesis in vivo. The mouse Hprt- T-cell cloning/sequencing assay described here may represent a useful system for studying the molecular mechanism of chemically induced mutation occurring in vivo in an endogenous gene.

本研究明确了5~7周龄雄性B6C3F1小鼠经体内暴露于N-乙基-N-亚硝基脲（N-ethyl-N-nitrosourea, ENU）后，其脾脏T细胞中次黄嘌呤（鸟嘌呤）磷酸核糖基转移酶基因（hypoxanthine (guanine) phosphoribosyltransferase gene, Hprt）第3外显子的突变谱。本研究通过将脾脏T细胞接种于添加了白细胞介素2、刀豆蛋白A与6-硫代鸟嘌呤的培养基的微量培养板中进行培养，分离得到Hprt缺陷型（Hprt⁻）突变体。从6-硫代鸟嘌呤抗性克隆中提取DNA，并以侧翼覆盖Hprt第3外显子的引物通过聚合酶链式反应（polymerase chain reaction, PCR）进行扩增。通过变性梯度凝胶电泳完成突变序列的鉴定，以及从混杂的野生型Hprt DNA中纯化突变体DNA。随后对纯化后的突变体DNA进行测序。以40 mg/kg体重的剂量对小鼠进行ENU染毒后，脾脏T细胞中的Hprt⁻突变频率为7.3×10⁻⁵，约为本底水平的35倍。在分析的521株Hprt⁻突变体中，有69株的第3外显子携带突变（占比13%）。突变谱以A-T碱基对的颠换与转换为主；69个第3外显子突变中有62个发生于A-T碱基对（共14个不同位点）。在发生突变的14个胸腺嘧啶碱基中，有13个（对应A-T碱基对突变中的61个）位于第3外显子的非转录链上。剩余的绝大多数突变（69例中的6例）为单一G-C碱基对的转换突变。上述结果表明，胸腺嘧啶烷基化在ENU体内诱导诱变的过程中发挥关键作用。本研究构建的小鼠Hprt⁻ T细胞克隆/测序检测体系，可作为研究内源基因体内化学诱导突变分子机制的实用模型。