MOLECULAR APPROACH TO THE NA-PHOSPHATE TRANSPORTER

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.