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Candidate gene approach in corneal degenerations

角膜变性的候选基因方法

基本信息

批准号：
05454477
负责人：
KANAI Atsushi
金额：
$ 2.62万
依托单位：
Juntendo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

1. Candidate gene screening in corneal dystrophiesWe analyzed the prealbumin gene using SSCP (single strand conformation polymorphism), in 8 unrelated Japanese corneal dystrophies, gelatinous drop-like, lattice, granular, and macular corneal dystrophy patients. The DNA fragments for each exon were amplified by PCR (polymerase chain reaction) and were tested by SSCP method. No mutation was detected in the prealbumin gene.2. Cloning of the rabbit corneal endothelial cDNAThe rabbit corneal endothelial cDNA library (Yamaguchi et.al., J.Biol.Chem., 1989) was employed. Plus minus screening was performed in 1000 clones usiong corneal and iris total RNA of rabbit as probes. Twenty clones which were positive to the corneal probe and negative to the iris probe were obtained. Regions of inserts of clones were amplified by PCR and directly sequenced. Sequenced data were analyzed using Gene Works and EMBL.There were several clones that showed high homology with each knowen cDNA ; B22 subunit of bovine mitochondrial NADH-ubiquinone oxidoreductase, human and cattle FK-506/rapamycin binding protein 25 (FKBP25), human thrombospondin 2 and rabbit ferritin. 5 unknown clones that showed no homology with any previous reported cDNA were also isolated. The rabbit FKBP25 cDNA clone has the entire coding sequence except an initiation codon and 3' untranslated sequence until poly A.RT-PCR analysis showed high expression of FKBP25 mRNA in cornea. Since the calcium release channel of endoplasmic reticulum is modulated by FKBP and Ca^<2+> plays a important role in pump function of corneal endothelium. FKBP may play an great role in the pomp function in corneal endothelium. Although further study is required, these clones may become the candidate gene for the corneal dystrophies. Furthermore, some of them seem to be important for corneal research.

1. 角膜营养不良候选基因筛选我们使用SSCP（单链构象多态性）分析了8例不相关的日本角膜营养不良、凝胶滴状、晶格状、颗粒状和黄斑状角膜营养不良患者的白蛋白前基因。每个外显子的DNA片段用PCR（聚合酶链反应）扩增，并用SSCP法检测。白蛋白前基因未见突变2。兔角膜内皮cDNA文库（Yamaguchi et.al.）。, J.Biol.Chem。（1989）被雇用。以兔角膜和虹膜总RNA为探针，对1000个克隆进行正负筛选。获得角膜探针检测阳性、虹膜探针检测阴性的克隆20个。用PCR扩增克隆插入片段，并直接测序。测序数据使用Gene Works和EMBL进行分析。有几个克隆与每个已知cDNA具有高度同源性；牛线粒体nadh -泛醌氧化还原酶B22亚基，人和牛FK-506/雷帕霉素结合蛋白25 (FKBP25)，人血栓反应蛋白2和兔铁蛋白。还分离到5个未知克隆，与以往报道的cDNA无同源性。兔FKBP25 cDNA克隆具有除起始密码子和3'未翻译序列外的全部编码序列，直到poly a.t - pcr分析显示FKBP25 mRNA在角膜中高表达。由于内质网钙释放通道受FKBP调节，Ca^<2+>在角膜内皮泵功能中起重要作用。FKBP可能在角膜内皮的充血功能中起重要作用。虽然需要进一步的研究，但这些克隆可能成为角膜营养不良的候选基因。此外，其中一些似乎对角膜研究很重要。