MECHANISM OF MUTATION INDUCED IN MAMMALIAN GENE

哺乳动物基因突变的机制

• 批准号: 3178660
• 负责人: DEZIDER GRUNBERGER
• 金额: $ 18.74万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3178660
• 关键词: DNA footprinting; RNA splicing; acridines; active sites; adduct; benzanthracenes; benzopyrenediol epoxide; benzopyrenes; chemical carcinogen; clone cells; conformation; dihydrofolate reductase; gene expression; gene mutation; hamsters; molecular cloning; nucleic acid sequence; phenanthrene; point mutation; site directed mutagenesis; southern blotting; stereochemistry

The proposal describes approaches to define the spectrum of mutations induced two model chemical carcinogens in an endogenous mammalian gene at the DNA sequence level. The carcinogens we used were N-acetoxy-N-2-acetylaminofluorene (AAAF) and (plus/minus)-r- 7,t-8-dihydroxy-t-9, lO-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE). The target for mutation was the dominant autosomal dihydrofolate reductase (dhfr) gene of Chinese hamster ovary cells. DHFR- mutants are triple auxotrophs for glycine, thymidine, and hypoxanthine and were selected starting with a hemizygote cell line (dhfr+/delta). Characterization of the induced mutants by Southern blot analysis showed that in the case of AAAF, a significant number (28%) carried gene rearrangements (deletion, translocation, large insertion, or inversion). In the case of BPDE, the dhfr- mutants virtually all carried small lesions (base substitution, + or -1 frameshift mutation). We also examined the expression phenotype of the induced mutants by Northern analysis and RNA heteroduplex mapping. About 50% of the putative point mutants showed markedly reduced steady-state levels of poly(A)+ dhfr mRNA. By cloning and sequencing, we determined that two mutants displaying a low mRNA phenotype carried chain termination codons at different positions in the dhfr coding sequence. Our proposal describes plans for the further study of these mutants. We describe methods to facilitate the DNA sequence analysis of the putative point mutants. We intend to isolate dhfr- mutants induced with a related aromatic amine, N- hydroxy-AF in order to determine if the mutational spectra of AAAF is associated with the conformational changes in DNA structure induced by the dG-adduct. In collaboration, we will determine if chromosomal location influences the frequency and spectra of induced mutations. Finally, we will attempt to suppress the nonsense mutations by transfection of Su+ tRNA and selection for a DHFR+ phenotype. We will then evaluate the dhfr mRNA levels in these transfected cells.

该提案描述了定义频谱的方法 突变诱导内源性的两种模型化学致癌物 DNA序列水平的哺乳动物基因。 我们使用的致癌物 是N-乙酰氧基-N-2-乙酰氨基氨基氟烯（AAAF）和（加/减）-r- 7，T-8-二羟基T-9，Lo-Epoxy-7,8,9,10-Attrahydrobenzo（a）pyrene （bpde）。 突变的靶标是主要常染色体 中国仓鼠卵巢细胞的二氢叶酸还原酶（DHFR）基因。 DHFR突变体是甘氨酸，胸苷和甘氨酸和 低黄嘌呤，并从半合子细胞系开始选择 （DHFR+/delta）。 通过南部对诱导的突变体的表征 印迹分析表明，在AAAF的情况下，大量 （28％）携带基因重排（删除，易位，大型 插入或反转）。 在BPDE的情况下，DHFR突变体 几乎全部携带小病变（基本替代， +或-1 移码突变）。 我们还检查了表达表型 通过北方分析和RNA异形电子诱导的突变体的诱导突变体 映射。 大约50％的推定点突变体明显显示出 降低的聚（A）+ DHFR mRNA的稳态水平降低。 通过克隆和 测序，我们确定两个表现出低mRNA的突变体 表型在不同位置携带链终止密码子 在DHFR编码序列中。 我们的建议描述了 进一步研究这些突变体。 我们描述了促进的方法 推定点突变体的DNA序列分析。 我们打算 分离用相关的芳香胺诱导的DHFR突变体，n- 羟基-AF，以确定AAAF的突变光谱是否 与DNA结构的构象变化有关 由DG-ADDUCT诱导。 在合作中，我们将确定是否 染色体位置影响 诱导的突变。 最后，我们将尝试压制 通过转染SU+ TRNA和选择 DHFR+表型。 然后，我们将评估DHFR mRNA水平 这些转染的细胞。