| Project/Area Number |
10470338
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Urology
|
|---|
| Research Institution | KYORIN UNIVERSITY |
|---|
Principal Investigator |
SHIMIZU Yoshiko Kyorin University, Health Sciences, Professor, 保健学部, 教授 (50255410)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
YAMAKI Akiko Kyorin University, Health Sciences, Res.Associate, 保健学部, 助手 (40296546)
TAMURA Takashi Kyorin University, Health Sciences, Assist.Prof., 保健学部, 講師
HIGASHIBARA Eiji Kyorin University, Medicine, Professor, 医学部, 教授 (00092312)
|
|---|
| Project Period (FY) |
1998 – 1999
|
| Project Status |
Completed (Fiscal Year 1999)
|
| Budget Amount *help |
¥12,900,000 (Direct Cost: ¥12,900,000)
Fiscal Year 1999: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 1998: ¥8,000,000 (Direct Cost: ¥8,000,000)
|
|---|
| Keywords | polycystic kidney disease / DNA diagnosis / PKDI mutation / human genome BAC library / cloning of kidney cyst-related genes / FISH mapping / differential display method / デイファレンシャルデイスプレイ法 / ヒトゲノムBACライブラリースクリーニング |
|---|
| Research Abstract |
1. We have established the method of DNA diagnosis of polycystic kidney disease1(PKD1). Polycystic kidney disease is one of the most frequent monogenic diseases. Since theregion from exon 34 to exon 46 of PKD1 gene has unique sequnce, we carried out ordinary PCR-SSCP analysis to detect mutations in this region. One nonsense mutation from CAG to TAG in exon45 (Gln4124stop) and deletion of 20bp in intron 43 were found. This deletion caused the splicing mutation because of the size of the intron is small. Three polymorphisms were found : five alleles of different size in intron42,10 among 123 patients mutated from GAC to GAT in exon 46(Asp4234Asp) and 7 among 107 patientsmutated from GCC to GCA in exon 43(Ala 3910Ala). Since the similar sequence to those from exon 1 to 33 has been found to be present in at least three other loci elsewhere on the same chromosome, we carried out the long range PCR and RT-PCR using the anchored PKD1-specific primers. We are analysing the PCR products using WAVE DNA fragment analysis system.
2. To clone new genes related to the formation of kidney cyst, wetooe two techniques. (1) We have screened human genomic BAC library using PKD1 cDNA as probes. We have mapped 26 clones by FISH analysis. Seven clones were mapped on 16p13 and nineteen clones on chromosomes other than chromosome 16. After digesting with restriction enzymes we seperated with pulse-field gel electrophoresis. By Southern blot hybridization analysis we have isolated DNA fragments as positive bands. We are subcloning and sequencing them. (2) RNA was isolated from kidney tissues of PKD patient and normal individual and cDNA was synthesized. We carried out Differential Display technique. PCR were performed using 24 upstream primers and 9 down stream primers and the products were analyzed with PAGE.By comparing the patterns we found 40 bands only in normal kidney and 25 bands only in polycystic kidney. We are sequensing these DNA fragments and confirming the different expression.
|
