| Summary: | 博士 === 國立陽明大學 === 臨床醫學研究所 === 87 === 英文摘要
Essential hypertension is an epidemic problem in the developed countries with a prevalence rate of about 15% or more. Epidemiological study reveals that essential hypertension is a polygenic disease. Though there are a number of drugs available for the control of hypertension. Knowledge of genetic factors involved in the pathogenesis of hypertension would lead to earlier detection, prevention and more efficient treatment of hypertension. Kidney has long been suspected as a major organ involved in the pathogenesis of hypertension. We therefore constructed a kidney cDNA library of spontaneously hypertensive rat (SHR), and performed differential hybridization with cDNA probe of SHR kidney and that of Wistar-Kyoto rat (WKY) kidney. We could not detect any clones expressing differentially between SHR kidney and WKY kidney after screening more than 100000 clones. In order to identify more genes expressing in the kidney to facilitate study of hypertension and other renal pathophysiology, a modified equalized kidney (MEK) cDNA library was constructed using Ko’s method with some modification. The cDNA of a SHR kidney was first sonicated to cDNA fragments of 100 to 200 basepairs (bp) in length and then equalized. All equalized cDNA fragments were inserted into λgt10 vectors instead of only the 3’ cDNA fragments inserted into plasmids in Ko’s method. Totally, 350 clones were randomly selected and their cDNA inserts amplified by PCR. The inserts were sequenced and 336 sequences were sent to GenBank for BLAST search. Three sequences were similar to bacteriophage DNA. Seven genes had redundant clones, one had 5 redundant clones and the other 6 each had two. All sequences could be grouped into 323 genes, including 77 known genes and 246 unknown genes. Compared with an expression profile of mouse kidney proximal tubule ordinary cDNA library, there were more proportion of genes for signal transduction and DNA/RNA binding proteins, and less proportion of ribosomal genes in this MEK cDNA library. Multiple tissue Northern blot analysis of eight genes revealed one kidney specific, one very rare and lung specific, and one relatively testis specific gene. cDNA ends of three genes were successfully amplified by PCR from Marathon-Ready cDNA of rat kidney. One cDNA was 938 bp in length and could be translated into a 182 amino acid novel protein. Analysis by Profile revealed this protein belonged to a metallo-phosphoesterase family. The second cDNA was 4.1 kilobases (kb) in length and could be translated into a 390 amino acid novel protein. Analysis by Prosite revealed this protein was a zinc-finger protein and contained 13 zinc-fingers. The third was 2.5 kb in length and could be translated into a 474 amino acid new protein of unknown function. In conclusion, a modified equalized kidney cDNA library was constructed and screened by direct sequencing of its cDNA clones. This special cDNA library had the following characteristics: 1. There were less redundant cDNA clones in the MEK cDNA library than in the ordinary cDNA library. 2. cDNA clones of genes expressing rarely could be identified more easily from this MEK cDNA library. 3. Every gene so far identified in this MEK cDNA library had only one corresponding DNA sequence. 4. Only 7.7% of cDNA sequences were identified as 3’ cDNA fragments. Because the cDNA sequences identified in a modified equalized cDNA library are probably more specific, a databases different from dbEST can be established after sequencing large number of cDNA clones from this special library. The sequences can also be used to prepare cDNA array for study of hypertension and other renal pathophysiology.
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