MUTATIONAL ANALYSIS OF THE CYSTIC FIBROSIS GENE

囊性纤维化基因的突变分析

• 批准号: 3874794
• 负责人: STEPHEN J O'BRIEN
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3874794
• 关键词: biochemical evolution; cystic fibrosis; gene deletion mutation; gene mutation; genetic polymorphism; genetic terminator element; membrane permeability; membrane proteins; molecular pathology; nucleic acid hybridization; point mutation; polymerase chain reaction; protein transport; restriction mapping; transport proteins

The recent identification of the cystic fibrosis (CF) gene, the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Riordan JR et al., Science 1989;245:1066-73), revealed that the gene belongs to a family of membrane transport molecules that includes the multi-drug resistance genes. Approximately 70% of CF chromosomes contain a deletion of 3-base pairs resulting in the loss of a phenylalanine codon at amino acid position 508 (delta F508). The focus of this project is to identify new mutations in this gene that comprise the remaining 30% of CFTR gene mutations. Using oligonucleotide primers based on the sequence of the CFTR gene, we have used the polymerase chain reaction to amplify several coding exons. These regions have been examined from 110 patients that contain 127 chromosomes without the common CF mutation (delta F508). Eight additional mutations have been identified in this group, in a total of four different exons. Most of the mutations were initially identified using an assay for single-- stranded conformation polymorphisms. All mutations were subsequently characterized by direct sequencing of the amplified DNA and can be assayed by restriction enzyme digestion or allele-specific oligonucleotide hybridization. The mutations fall into two classes: (1) one or two nucleotide insertions and deletions that introduce termination codons into the gene and are predicted to result in severely truncated protein products, and (2) point mutations in the putative membrane-spanning domains that replace charged amino acids with nonpolar residues. Sequencing of the membrane-spanning region from several species demonstrates that this region is highly conserved across species.

囊性纤维化（CF）基因的最新鉴定 纤维化跨膜电导调节因子 (CFTR) 基因 (Riordan JR et al., Science 1989;245:1066-73)，揭示该基因属于一个家族 膜转运分子，包括多药耐药性 基因。大约 70% 的 CF 染色体含有 3 个碱基的缺失 导致氨基酸位置上苯丙氨酸密码子丢失的配对 508（德尔塔 F508）。该项目的重点是识别新的突变 该基因包含剩余 30% 的 CFTR 基因突变。使用 基于 CFTR 基因序列的寡核苷酸引物，我们有 使用聚合酶链式反应来扩增几个编码外显子。这些 已对 110 名患者的包含 127 条染色体的区域进行了检查 没有常见的 CF 突变 (delta F508)。八个额外的突变 已在该组中鉴定出总共四个不同的外显子。 大多数突变最初是通过单一检测来识别的—— 链构象多态性。所有突变均随后 通过对扩增的 DNA 进行直接测序来表征并可以进行测定 通过限制酶消化或等位基因特异性寡核苷酸 杂交。 突变分为两类：(1) 一个或两个核苷酸插入 以及将终止密码子引入基因的缺失 预计会导致蛋白质产物严重截短，并且 (2) 点 假定的跨膜结构域发生突变，取代了带电结构域 具有非极性残基的氨基酸。跨膜测序 多个物种的区域表明该区域高度 跨物种保守。