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POSITIONAL CLONING OF HEREDITARY CATARACTS IN MICE

小鼠遗传性白内障的定位克隆

基本信息

批准号：
13480281
负责人：
HIRAI Hiroshi
金额：
$ 6.66万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

We performed positional cloning of the genes responsible for two mouse hereditary cataracts found in Japan. These genes are supposedly involved in organogenesis and maintenance of transparency of the lens. According to the results of fine mapping of Rupture of lens cataract (RLC), we sequenced a number of the cDNA clones at the map positions obtained from the mouse full-length cDNA encyclopedia developed by RIKEN. One of cDNA clones, rlcl, was found to have deletion of 27 bp, when compared with the normal cDNA sequence. This deletion was specific to RLC mutant. Rabbit antiserum to the C-terminus peptide of rlcl product detected a 110 kDa polypeptide in the lens. Immunohistochemistry with this antiseum revealed unique distribution in lens epithelium, nervous system and other tissues. Mechanism of cataractgenesis is now under investigation. On the other hand, the same positional approach in Nakano cataract (NCT) demonstrated a 13 bp deletion in nctl cDNA. This deletion was conserved in NK11, an established cell line of lens epithelium provided from NEL Careful examination of the lenses in N2 cross to MSM revealed two distinct forms of lens opacity in respect to distribution of cloudiness and latent period. We could map two modifier loci determining the subtypes of NCT cataract on Chr. 3 and 10.To further confirm that these deletions are indeed causative mutation, rescue with normal full size cDNA and targeting experiments are now in progress in this laboratory.

我们对在日本发现的两种小鼠遗传性白内障的基因进行了定位克隆。这些基因被认为与器官发生和保持透镜的透明度有关。根据透镜性白内障破裂（RLC）的精细定位结果，我们对RIKEN开发的小鼠全长cDNA百科全书中的一些cDNA克隆进行了测序。其中一个cDNA克隆rlcl与正常的cDNA序列相比有27 bp的缺失。这种缺失是RLC突变体所特有的。rlcl产物C-末端肽的兔抗血清在透镜中检测到110 kDa多肽。免疫组化结果显示，该抗血清在透镜上皮、神经系统及其他组织中有独特的分布。白内障的发生机制正在研究中。另一方面，在中野白内障（NCT）中的相同位置方法证实了nctl cDNA中的13 bp缺失。这种缺失在NK 11中是保守的，NK 11是NEL提供的一种已建立的透镜上皮细胞系。对N2与MSM杂交的晶状体进行仔细检查，发现在浑浊和潜伏期的分布方面存在两种不同形式的透镜浑浊。本实验室在Chr. 3和10上定位了两个决定NCT白内障亚型的修饰位点。为了进一步证实这些缺失确实是致病突变，本实验室正在进行正常全长cDNA的拯救和靶向实验。