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 to the subcellular organelles (mitochondria, golgi, endoplasmic reticulum). The role of vitamin D in these processes was defined. The competing renewal which was submitted on March 1, 1989, deals with identification of the intestinal phosphate transporter across the brush border membrane utilizing molecular biology tools. The strategy will be to express the Na-phosphate transporter in Xenopus laevis oocytes. This has been already accomplished by injection of poly(A)+ RNA into vegetal pole of the Xenopus laevis oocytes. This will be followed by size fractionation of poly(A)+ RNA to determine which size selected fraction encode for a functional phosphate transporter by screening for expression Xenopus laevis oocytes. A cDNA library will be constructed from appropriate size selected mRNA fragments. A radiolabelled cDNA encoding for the transport protein will be synthesized and used to measure and compare sequence homologies in RNA isolated from mouse intestinal and renal cortical homogenates by Northern blot analysis. Mapping of the gene encoding for the phosphate transporters will be done utilizing a genomic library. The P.I. will need the technical expertise in construction of cDNA libraries, Northern blots, Southern blots to carry the proposed studies effectively. These proposed studies will pioneer work on the phosphate transporter in the intestine and kidney and shed light on the possible molecular defect in phosphate transporter in the hypophosphatemic mouse and man with vitamin D resistance hypophosphatemic rickets.
| Acra, S A; Ghishan, F K (1991) Methods of investigating intestinal transport. JPEN J Parenter Enteral Nutr 15:93S-98S |
