Molecular analyses of UV^s syndrome and development of simple diagnostic methods for inherited photosensitive diseases

UV综合征的分子分析和遗传性光敏性疾病简单诊断方法的开发

• 批准号: 09670887
• 负责人: ITOH Toshiki
• 金额: $ 2.18万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670887/
• 关键词: UV^S syndrome; Xeroderma pigmentosum; Cockayne syndrome; UV^s症候群

I investigated the following experiments using this grant.1) DEVELOPMENT OF SIMPLE DIAGNOSTIC METHODS FOR INHERITED PHOTO-SENSITIVE DISEASES1. To obtain the candidates of photosensitive diseases, such as xeroderma pigmentosum group A through G (except for E) and Cockayne syndrome group A and B, I isolated the cDNAs using colony-hybridization or PCR method.2. cDNAs were subcloned into eukaryotic expression vectors (pcDNA1, pcDNA3, pTarget, pcDM8).3. I established the method of classification of photosensitive diseases using three DNA repair markers (UDS (unschduled DNA synthesis), RRS (recovery of RNAS synthesis), and RDS (recovery of replicative DNA synthesis)).4. Following the classification, we establised the definitive diagnostic method using micro-injection or transfection methods.2) MOLECULAR CLOING OF UV^S SYNDROME1. I established two independent immrotal UV^SS clones (Kps3SVY and Kps3SVI3) using SV40 T antigen.2. To confirm that two immrotal clones were derived from parental primary cells, we performed VNTR (variable number of tandem repeat) analyses.3. I constructed EB virus based cDNA library using HeLa S3 mRNA (size fractination (+)).4. I constructed immortal UV^SS clones expressed EBNA antigen.5. I am now screening the candidate gene complemented the UV^SS defect.

我利用这项研究进行了以下实验：1)开发遗传性光敏性DISEASE的简单诊断方法。为了获得光敏性疾病的候选基因，如色素性干皮病A组到G组(E组除外)和Cockayne综合征组A组和B组，我用菌落杂交或聚合酶链式反应的方法分离了cDNA。亚克隆入真核表达载体(pcDNA1、pcDNA3、pTarget、pcDM8)。建立了利用三种DNA修复标记(UDS、RRS、RDS)对光敏性疾病进行分类的方法。在分类的基础上，我们建立了采用显微注射或转染法的明确诊断方法。2)紫外光S综合征的分子闭合1。我利用SV40T抗原建立了两个独立的免疫UV、SS克隆(Kps3SVY和Kps3SVI3)。为了证实两个免疫克隆来自亲代原代细胞，我们进行了VNTR(可变数目串联重复)分析。利用HeLa S3mRNA(Size Frtination(+))构建了EB病毒DNA文库。构建了表达EBNA抗原的不朽UV、SS克隆。我现在正在筛选补充UV？SS缺陷的候选基因。