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.

我们已经接近猫眼综合征（CES）的眼睛，肛门和心脏畸形的疾病基因，并获得了以下结果。（1）我们已经构建了CES染色体区域的BAC/COSMID重叠群（CER; CER; 1.9 MB的大小）。我们使用了两个DNA库，即来自CES细胞系CH91-157的CES分类标记染色体的CES特异性宇宙库（9,200个克隆；冗余，10），来自人类总DNA的CES细胞系CH91-157和BAC库（200,000克隆，7.5），它们都是由我们自己建立的。许多已知的DNA标记物，新成立的来自Cosmid克隆的STS，并使用这些DNA标记物分离的YAC克隆用于筛选BAC和CES CES宇宙库，因此隔离了1,740个宇宙和116个BAC克隆。矢量PCR方法和指纹已应用于构建BAC/cosmid重叠群。迄今为止，已经构建了6个由36个BAC和43个宇宙克隆组成的重叠群，覆盖> 1.4 MB区域。（2）几乎完成了4个BAC和4个宇宙克隆的基因组测序，其中涵盖了-500 kb的CER。我们遇到了很难构建此类序列的序列，并确定此类型号的序列，并确定了较高的等级序列，并且备受序列的重复序列和凸起的序列，并累积了较高的序列。作为染色体14和其他含异染色质染色质的染色体。（3）通过外显子预测程序（Genscan，Grail）和同源性搜索工具（BLAST，FASTA）对DNA序列进行分析，然后分离相应的CDNA。