Cloning of disease-causing genes for the syndrome with congenital heart malformations

我们对猫眼综合征(CES)心脏畸形的致病基因进行了探讨，获得了以下结果：(1)构建了CES染色体区域(CER；1.9Mb)的BAC/粘粒重叠群。我们使用了两个DNA文库，一个是来自CES细胞系CH91-157标记染色体的CES区域特异性粘粒文库(9,200个克隆；冗余，10个)，另一个是来自人总DNA的BAC文库(200,000个克隆，冗余，7.5个)，这两个文库都是我们自己建立的。利用一些已知的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，结果分离到1,740个粘粒和116个BAC克隆。应用载体聚合酶链式反应和指纹图谱技术构建了BAC/粘粒重叠群。到目前为止，已经构建了6个重叠群，包括33个BAC和43个粘粒克隆，覆盖了&gt；1.3Mb区域。(2)CER中4个BAC和7个粘粒克隆的基因组测序已基本完成，覆盖了约300kb。由于重复序列含量高，与其他染色体如14号染色体和其他含异染色质染色体的同源性很高，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。(3)DNA序列正在通过外显子预测程序(GENSCAN，GRAIL)和同源搜索工具(BLAST，FASTA)进行分析，然后分离相应的cDNA。

We have approached the disease-responsible genes for 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 33 BAC and 43 cosmid clones have been constructed to cover >1.3 Mb region.(2) Genomic sequencing of 4 BAC and 7 cosmid clones have almost been finished which covers 〜300 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.