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

我们对猫眼综合征（CES）中眼睛、肛门和心脏畸形的致病基因进行了研究，并获得了以下结果。(1)我们已经构建了BAC/粘粒重叠群的CES染色体区域（CER ; 1.9 Mb的大小）。我们使用了两个DNA文库，一个来自CES细胞系CH 91 -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;冗余度，10）和一个来自人总DNA的BAC文库（200，000个克隆;冗余度，7.5），这两个文库都是我们自己建立的。用许多已知的DNA标记、新建立的来自粘粒克隆的STS和用这些DNA标记分离的YAC克隆筛选BAC和CES粘粒文库，结果分离出1，740个粘粒和116个BAC克隆。载体PCR方法和指纹图谱已被应用于构建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，以覆盖> 1.4Mb的区域。(2)4个BAC克隆和4个粘粒克隆的基因组测序工作已基本完成，其中约500 kb的片段在CER中，由于重复序列含量高，与14号染色体和其它含异染色质的染色体同源性高，给构建重叠群和确定其序列带来很大困难。(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。

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.