The cellular defect which appears to be responsible for cystic fibrosis is malfunction of a chloride channel in the apical surface of epithelial cells lining secretory ducts in a number of tissues. When mRNA from these epithelial cells, isolated from dog or normal human tracheas, is injected into Xenopus oocytes, apical- type chloride channels are synthesized and are detectable by virtue of increased rates of chloride uptake. Epithelial mRNA from CF patients does not induce such channels. This assays will be used to aid the identification and isolation of cloned DNAs for the CF gene. This will be accomplished by hybrid-arrest procedures. Then attempts will be made to isolate a full-length cDNA clone for the CF gene product, from which mRNA will be produced and used to complement the cloride channel deficiency in occytes injected with CF epithelial mRNA. To facilitate studies of the function and regulation of this apical chloride channel, similar procedures will be used to isolate cDNA clones for other channel components. The goal is to be able to produce active channels in oocytes by injection of purified mRNAs made in vitro from cDNA clones. This will allow detailed studies of the regulation of channel activity through cAMP, and possibly other second messengers. It will be determined whether any of the channel components is a target for phosphorylation by the cAMP-dependent protein kinase. In addition, the function of individual channel components and interactions between them will be investigated by mutagenesis of the cDNA clones. Ultimately, it is hoped to provide a complete description of apical chloride channel function, which will represent a paradigm for other ion channels and allow directed intervention in the treatment of cystic fibrosis.
| Urnes, M S; Carroll, D (1990) Amylase synthesis as a simple model system for translation and hybrid arrest in Xenopus oocytes. Gene 95:267-74 |
