Gene products which express an altered structure or function provide a rational approach to defining the genetic defect in cystic fibrosis (CF). This is an alternate approach to that of random recombinant DNA analysis without a gene product and has the advantage, when successful, of describing the basic defect. Information obtained in a number of laboratories describing abnormal glycoproteins in CF provide a strong rationale for using selected enzymes of glycoprotein metabolism as the starting point for gene probes and genetic linkage studies. One of these enzymes will be isolated, characterized and used to provide the potentially abnormal gene product. Phase I describes the experiments designed to examine certain enzymes of glycoprotein synthesis in the CF and control fibroblasts. Available information suggests that differences exist in the fucosylation of membrane glycoproteins. Therefore, the fucosyltransferases are potential candidates for an altered gene product. The activity of these transferases will be studied with synthetic and endogenous substrates, including glycopeptides with defined structures. The goal is to purify one of the fucosyltransferases and to determine the partial amino acid sequence by Edman degradation. Phase II describes experiments to use the partial amino acid sequence data of the purified fucosyltransferase to design and then synthesize oligonucleotides which will be used as molecular DNA probes. It is anticipated that these probes will directly detect the defect or show a linkage between the inheritance of the gene and CF. These experiments will provide direct information concerning the glycosylation alterations observed in CF. Moreover, information on a molecular level about an enzyme of glycoprotein biosynthesis will be obtained.
