Acquired lactose intolerance is a common deficiency found in certain human populations; it results from an absence of the disaccharide lactase in the brush border of the small intestine. Congenital sucrase and lactase deficiencies also exist but are more rare. The appearance of these disaccharidases is well regulated both developmentally and during maturation of intestinal cells. We propose to apply the techniques of modern biochemical genetics to study the molecular basis for the regulation during development and cell maturation as well as the inactivity in disaccharide intolerance syndromes. The enzyme complexes lactase-phlorizin hydrolase (L-P) and sucrase-isomaltase (S-I) will be isolated from rabbit intestine and antibodies prepared against them. The mRNAs for these polypeptides will be cloned as cDNA plasmids, using hybrid-selected in vitro translation as a screening procedure. Libraries of rabbit and human DNA will be screened by hybridization with the cDNA plasmids to isolate the genes for S-I and L-P. We will determine whether or not the S-I and L-P are synthesized as a large precursor containing both subunits, and what the structure of the putative hybrid gene is. The nucleotide sequence of the cDNA plasmids and portions of the genes will be determined. The regulated steps in disaccharidase gene expression will be determined by using the gene- and protein-specific probes to measure the concentrations of all intermediates in expression in neonatal versus adult intestinal cells and in crypt versus villus cells. These same probes will also be used to identify which step is blocked in disaccharidase deficiency, i.e., gene deletion, transcription, RNA processing and transport, translation or posttranslational processing.
