The overall objectives of the proposed research are to: 1) isolate and characterize the human genes for the GTP-binding proteins--Ns and transducin, and 2) determine the molecular lesions in the human Ns gene that result in the heritable disease, pseudohypoparathyroidism type Ia. We shall use molecular and genetic methods to achieve these objectives. The homologous family of GTP-binding regulatory proteins are involved in the transduction of hormonal, neural and other regulatory signals across cell membranes. The analogous subunits of these proteins are structurally similar and in some instances, can functionally substitute for those of another. One of these proteins, transducin, is abundant in the retina. Using polyclonal antibodies prepared against the subunits of transducin, we have screened a bovine retinal cDNA library in the expression vector gt11 and isolated partial cDNA clones encoding two of the three subunits and a putative cDNA clone encoding the third subunit. These partial cDNA clones will be used to isolate: 1) full-length cDNA clones encoding the bovine transducin subunits by screening a bacteriophage library containing large, bovine retinal cDNA inserts (The identity of these clones will be verified by comparing the deduced amino acid sequence to actual amino acid sequences.) and 2) cDNA clones for the human transducin subunits by screening a cDNA library prepared from human retinas. (The identities of these clones will be verified by Northern blot analysis.) Next, we shall isolate the human genomic and cDNA clones of the homologous Ns protein genes by using the human transducin cDNA as a hybridization probe of a bacteriophage library containing human genomic DNA or appropriate cDNA under reduced stringency hybridization conditions. The identity of these clones will be verified by Northern blot analysis of poly(A)+RNA isolated from human cultured cells or tissue and by DNA sequence analysis. Analysis of these cDNA's in expression vectors will be necessary to correctly identify the Ns clones. Our second objective is to determine the molecular lesions that result in the reduced Ns bioactivity found in patients with pseudohypoparathyroidism type Ia. To this end, we shall use the cDNA or genomic clones for normal Ns to: 1) probe Southern restriction blots of genomic DNA from PHP-Ia-affected patients to identify partial or full-length gene deletions and gene polymorphisms, and 2) isolate and sequence the mutant cDNA or genomic DNA isolated from affected patients. To verify that base sequence alterations we find in the Ns gene are causative for the PHP-Ia phenotype, we shall construct mutant-wild-type-chimeric-genes in expression vectors to test for altered bioactivity.
