EXPRESSION OF CYSTATHIONINE SYNTHASE AND HUMAN DISEASE

该提案的主要目标是确定如何水平

The major goal of this proposal is to determine how levels of expression of the--cystathionine 8-synthase (CBS) gene may play a role in three human disease states: homocystinuria; peripheral and cerebral occlusive arterial disease; and Down syndrome. CBS, a central enzyme in eukaryotic sulfur metabolism, is a tetramer of 63 kDa subunit It is encoded in humans on chromosome 21; its inherited deficiency causes the most common form of homocystinuria. Further, 28% of patients with occlusive are trial disease reportedly are heterozygous for CBS deficiency. Conversely, elevated levels of CBS has been documented in trisomy 21 and may contribute to the generation of the Down syndrome phenotype. We have recently isolated cDNA clones encoding CBS. Interestingly, three types (I, II and III) of apparently full-length cDNAs were found, which differ significantly in both their translated and untranslated regions; they code for a 63, 39 and a 61.5 kDa polypeptide, respectively. Our specific aims are 1) to clone the entire gene for CBS and to determine its restriction map and intron/exon structure; 2) to determine, whether the three synthase mRNAs found in rat liver reflect alternative splicing of the, synthase transcript; 3) to ascertain whether these mRNAs are responsible for difference, in tissue-specific synthase expression; 4) to achieve expression of the three different cloned cDNAs in transfected cultured rodent cells and, thereby, address the mechanism < synthase interaction with S-adenosylmethionine and its activation by specific proteoly-sis which yields 48 kDa subunits with increased catalytic activity; 5) to provide evidence at a molecular level that some patients with premature arterial disease are CI heterozygotes; and 6) to establish transgenic mice carrying a rat CBS transgene as a model for CBS dosage in Down syndrome. Biochemical, cell biological, and molecular biological approaches will be employed, including: screening of genomic libraries with (32P) labeled DNA and RNA probes; DNA sequencing by Sanger's dideoxy technique; DNA amplification by polymerase chain reaction; DNA, RNA, and protein blotting with detection by (32p)labeled probes or by anti-CBS antibodies and (125I) protein G; DNA transfec-tion into cultured cells and oocyte microinjection. The results of this work will complement our studies of normal and mutant CBS genes and will elucidate the significance of their products in human disease.