HUMAN GENOME CLONING AND ISOLATION OF SPECIFIC DNAS IN YEAST

工作总结：人类基因组计划已经完成

Summary of Work: The Human Genome Project has made great strides in the decade since its inception including the cloning of most of the chromosomal DNA, the identification of a unique sequence (STS) approximately every 100 kb and the sequencing of short regions of nearly all expressed genes (EST's). Critical in the characterization of the human genome has been the cloning of large chromosomal fragments, which has traditionally involved the isolation of random DNA fragments into vectors followed by bacteria or yeast. Previously we described a novel nonenzymatic method for cloning large fragments of human DNA into yeast based on Transformation- Associated Recombination (TAR cloning) that we applied to the direct isolation of specific DNAs. TAR cloning has now been applied to the selective isolation as linear and/or circular yeast artificial chromosomes (YACs) of i) human DNA from a radiation hybrid rodent cell line, ii) specific isolation of genes and regions directly from total human DNA including rDNA genes, and single copy genes BRCA1, BRCA2, and HPRT. It is being applied to the isolation from mouse cells of an element that is responsive to environmental agents. We have established that TAR cloned genes can be fully functional based on the isolation of HPRT and transfer back to an hprt- cell line. Thus, TAR cloning and opportunities to modify the isolated material enables genetic modification and correction. In addition we have established that a gene region can be isolated with only sequence information from one end of the gene, as one TAR "hook" and a common repeat as the other hook, thereby increasing the opportunities to examine regions around a gene. Current directions also include assessment of minimum size of sequence needed for TAR cloning and refinement of clone detection. Since only a few weeks are required, TAR cloning provides many opportunities for investigating genes and chromosomal regions directly from individuals. It can be used for studying human polymorphisms, clinical diagnosis, gene therapy and the filling-in of gaps in sequenced regions.