The principal investigator's interest has been the subject of calcium and phosphate transport across the intestinal epithelium during maturation. Initially, the P.I. utilized in vivo perfusion and in vitro everted gut sacs to define the overall picture of transport. These studies were extended to the plasma membranes (brush border and basolateral membranes) and then to the subcellular organelles (mitochondria, golgi, endoplasmic reticulum). The role of vitamin D in these processes was defined. The current proposal will extend knowledge acquired during the tenure of the grant to explore the Na-phosphate transporter located at the brush border membrane of the intestine of the rat. Specifically, the intestinal Na-phosphate transporter will be expressed in Xenopus laevis oocytes by injecting poly(A)+ RNA into Xenopus laevis oocytes. This will be followed by size fractionation of poly(A)+ RNA to determine which size selected fraction encoding for a functional Na-phosphate transporter by screening for expression in Xenopus laevis oocytes. A cDNA library will be constructed from appropriate size selected mRNA fragments. A 32P labelled cDNA encoding for the transport protein will be synthesized and used to determine the amount and size of homologous transcripts in RNA isolated from intestinal and renal cortical homogenates by Northern blot analysis. The restriction maps of the gene as well as its assignment of its locus to the X-chromosome will be determined by analyzing genomic DNA from mice and humans. The protein encoding the Na-phosphate transporter cDNA will be incorporated into proteoliposomes and examined for the transport properties of this protein in reconstituted proteoliposomes. This extensive knowledge will then be applied for the investigation of the defect in Na-phosphate transport in the hypophosphatemic mouse which is a murine homologue of human X-linked hypophosphatemic rickets. These studies will first define the defect in phosphate transport in the intestine and kidney and its regulation by vitamin D and its active metabolite and second, will utilize our cDNA to measure and compare sequence homologies in RNA isolated from the hypophosphatemic mouse (Hyp/y) and its genetically age matched controls (+/y) by Northern blot analysis. The cloned probe will also be used to study genomic DNA from the (Hyp/y) by digesting their DNA with a variety of restriction endonucleases, performing Southern blots and hybridizing the blotted DNA to cloned cDNA probes. This will permit identification of gene deletions and genetic polymorphisms linked to the genes probed. Sequencing the affected genes will delineate the genetic defect in the hypophosphatemic mice. These studies will pioneer work on the Na-phosphate transporter and will shed light on the defect in phosphate transport in the (Hyp/y) mouse and eventually in man with hypophosphatemic rickets.
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