Organization /Function of Chromosomal Regions Required

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Despite of their importance for studying the chromosome organzation and for construction for Human Artificial Chromosomes (HACs), centromeric and telomeric regions remain poorly characterized. The main reason of that is that long stretches of repeated centromere and telomere-specific DNA sequences can not be cloned by standard YAC or BAC cloning techniques. During last year we developed a new recombinational cloning procedure that selects that includes positive and negative genetic selection and allows selective isolating large fragments of genomic DNA from heterochromatic chromosomal regions lacking ARS- like sequences. A new technique (modified TAR cloning) was successfully applied for cloning and physical characterization of centromeric sequences from five human chromosomes (11, 13, 15, 22 and Y). Different centromere DNA isolates were retofitted with a mammalian selectable marker and at present they are compared on their efficiency to form HACs during transfection into human cells. TAR cloning strategy has been also used for isolation of functional genomic copies of two human disease genes, the metastasis-suppressor gene for prostate cancer from chromosome 8p21-p12, and the human telomerase gene, hTERT. Entire copies of these genes were not previously found in large insert size BAC and YAC libraries. We have shown that an absence of these genes in BAC libraries is caused by toxicity of the sequences for E. coli cells. Based on our estimate, such toxic sequences represent approximately 6% of human genome and they are not present in published human genome draft sequence. To demonstrate utility of a modified TAR cloning protocol for completion of human genome sequence, two gaps of ~ 200kb in size containing new genes from human chromosome 5 were covered.