The long-term objectives of the research outlined by this proposal are (1) to clone and sequence cDNAs and genes encoding the polypeptide core (apomucin) of pig trachea mucin glycoproteins, (2) to determine the mechanisms regulating transcription/translation of the apomucin genes and mRNAs, (3) to use expression vectors to synthesize sufficient apomucin(s) for chemical and physical characterizations, (4) to use transfected cell lines and primary cell cultures to evaluate gene regulation by various pharmacological and topical agents, and (5) to use the pig trachea cDNA and genomic clones to cross-hybridize with human RNA and DNA to isolate comparable cDNAs and genomic clones from human trachea or from cell cultures of human trachea epithelium. There is widespread acceptance that mucus glycoproteins, or mucins, are correlated with the pathogeneis of pulmonary diseases. However, relatively little is known about the chemistry and control of synthesis and secretion of these heteromacromolecules. A central characteristic in cystic fibrosis, for example, is the abnormal behavior of the mucus secretion: They are thick, giving rise to obstruction of organ passages and as a consequence to almost every clinical and pathological feature of bronchial infection and obstruction. The oligosaccharides have been removed from pig tracheal mucin(s) and polyclonal and monoclonal antibodies to the carbohydrate- free polypeptide chain are available. Recent success with the isolation and translation of pig trachea RNAs now make it possible to characterize the apomucin mRNA(s) and to clone the respective cDNA(s) The tools and procedures of recombinant DNA technology and molecular biology which have proved so successful in our laboratory in the determination of the structure, function and regulation of the tissue-specific inducible multigene families encoding the proline-rich proteins will be applied to the understanding of tracheal mucin biosynthesis and properties. General methods to be used include cloning and other recombinant DNA procedures, nucleotide and peptide sequencing, cell transfections, primary cell cultures gene induction and activation analyses, and analysis of peptides, proteins and oligosaccharides by NMR, CD, GC/MS, HPLC and GC.
