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Positional cloning of the genes related to malformations of eye, anus and heart

眼、肛门、心脏畸形相关基因的定位克隆

基本信息

批准号：
06670824
负责人：
MINOSHIMA Shinsei
金额：
$ 1.41万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670824/
关键词：
congenital malformations cat eye syndrome marker chromosome region specific cosmid library BAC library BAC cosmid contig genomic sequencing gene exon prediction コスミドライブラリー YACコンティグコスミドコンティグゲノミックシーケンシング猫目症候群 STS化エキソントラッピ

项目摘要

We have approached the disease-responsible genes for eye, anus and heart malformation in cat eye syndrome (CES) and obtained the following results.(1) We have constructed BAC/cosmid contigs of the CES chromosomal region (CER ; 1.9 Mb in size). We used two DNA libraries, a CES region-specific cosmid library (9,200 clones ; redundancy, 10) from the flow-sorted marker chromosomes of a CES cell line CH91-157 and a BAC library (200,000 clones ; redundancy, 7.5) from human total DNA,both of which were established by ourselves. A number of known DNA markers, newly established STS's from cosmid clones, and YAC clones isolated with these DNA markers were used to screen the BAC and CES cosmid libraries, and as a result 1,740 cosmids and 116 BAC clones were isolated. The vectorette PCR method and fingerprinting have been applied to construct BAC/cosmid contigs. To date, 6 contigs consisting of 36 BAC and 43 cosmid clones have been constructed to cover>1.4 Mb region.(2) Genomic sequencing of 4 BAC and 4 cosmid clones have almost been finished which covers -500 kb in CER.We have encountered a great difficulty to construct contigs and determine the sequence of this peri-centromeric region due to high contents of repetitive sequences and high homology with other chromosomes such as chromosome 14 and other heterochromatin-containing chromosomes.(3) The DNA sequence is being analyzed by exon-prediction programs (GENSCAN,GRAIL) and homology search tools (BLAST,FASTA) followed by isolation of corresponding cDNA's.

我们研究了猫眼综合征（CES）中眼睛、肛门和心脏畸形的疾病相关基因，并获得了以下结果。（1）我们构建了 CES 染色体区域的 BAC/粘粒重叠群（CER；大小为 1.9 Mb）。我们使用了两个DNA文库，一个来自CES细胞系CH91-157的流式分选标记染色体的CES区域特异性粘粒文库（9,200个克隆；冗余，10）和一个来自人类总DNA的BAC文库（200,000个克隆；冗余，7.5），这两个文库都是我们自己建立的。使用许多已知的DNA标记、从粘粒克隆中新建立的STS以及用这些DNA标记分离的YAC克隆来筛选BAC和CES粘粒文库，结果分离出1,740个粘粒和116个BAC克隆。小载体 PCR 方法和指纹分析已应用于构建 BAC/粘粒重叠群。迄今为止，已构建了由36个BAC和43个粘粒克隆组成的6个重叠群，覆盖>1.4 Mb区域。（2）4个BAC和4个粘粒克隆的基因组测序已基本完成，覆盖CER中的-500 kb。由于重复序列含量高且与其他染色体（例如，其他染色体）同源性高，我们在构建重叠群和确定该着丝粒周围区域的序列时遇到了很大的困难。如14号染色体和其他含有异染色质的染色体。(3)通过外显子预测程序(GENSCAN，GRAIL)和同源性搜索工具(BLAST，FASTA)分析DNA序列，然后分离相应的cDNA。