The gene responsible for Cystic Fibrosis (CF), the most common lethal inherited disorder of Caucasians, has recently been identified. However, little is known of the function of its protein product, the CF Transmembrane Conductance Regulator (CFTR) and its role in chloride secretion in epithelial tissue. The overall goal of this proposal is to extend our knowledge of the molecular basis of CF by pursuit of the following aims: 1) To determine functionally significant regions of the CFTR protein by identification of naturally-occurring deleterious mutations in the CF genes of patients from four ethnic groups (American Black and Caucasian, Irish and Israeli). For each mutation, rapid detection methods will be devised to facilitate screening of CF chromosomes. 2) To assess the effect of mutations by determining disease severity and CFTR function in patients of known genotype. CFTR protein function will be analyzed by patch clamping and 36Cl flux studies of epithelial cells and cell lines derived from patients of known genotype, as determined in specific aim 1. 3) To create mutations in the full-length CFTR cDNA using site-directed mutagenesis. The altered CFTR cDNA will be expressed by injection of in vitro transcribed RNA into Xenopus oocytes and by lipofection of rabbit kidney (MTAL) and immortalized tracheal CF cells with a mammalian vector containing the mutated CFTR cDNA (MTAL cells do not express the CFTR mRNA). 4) To complete the cloning and determine the function of two closely related human chloride channel genes (IC1 and IC2) by a) identifying the tissues expressing these genes using Northern blotting and in situ hybridization b) cloning the full-length cDNA for the second gene, IC2, by screening cDNA libraries created from tissues known to express IC2 c) determining the locations of these genes in the human genome using somatic cell hybrid panels d) expression of each gene by injection of in vitro transcribed RNA into Xenopus oocytes and by lipofection of rabbit kidney cells (MTAL) with an expression vector containing the full length cDNA for each gene.
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