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.

最近发现了囊性纤维化（CF）基因，即囊性纤维化基因， 纤维化跨膜传导调节因子（CFTR）基因（Riordan JR et 例如，Science 1989;245：1066-73），揭示了该基因属于一个家族， 包括多药耐药性的膜转运分子 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（Δ F508）。该项目的重点是确定新的突变， 该基因包含剩余的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 用聚合酶链反应扩增了几个编码外显子。这些 已检查了110名患者的包含127条染色体的区域 没有常见的CF突变（Δ F508）。八个额外的突变 在这个组中已经确定，总共有四个不同的外显子。 大多数突变最初都是使用单一的检测方法鉴定的， 链构象多态性所有突变随后 其特征在于对扩增的DNA进行直接测序， 通过限制酶消化或等位基因特异性寡核苷酸 杂交方法 突变分为两类：（1）一个或两个核苷酸插入 和将终止密码子引入基因的缺失， 预测导致严重截短的蛋白质产物，和（2）点 在假定的跨膜结构域中的突变， 具有非极性残基的氨基酸。跨膜序列测定 从几个物种的区域表明，该地区是高度 在物种间是保守的。