|Budget Amount *help
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1997: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1996: ¥3,500,000 (Direct Cost: ¥3,500,000)
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.