Yeast artificial chromosomes (YACs), recently developed as cloning vectors may provide the basis for a new approach to systematic analysis of the contents of the human genome. Large YAC clones could bridge the gap between pedigree studies and cytogenetics, operating with 2-5 Mb resolution, and conventional recombinant DNA technology, operating at the 1 to 50,000 base pair level. Further preliminary work suggests that YACs containing inserts of up to at least 1 Mb of human DNA can be prepared and restriction mapped using a feature of the vector that permits specific end-labeling. We have prepared a pilot library containing the equivalent of 10% of the human genome and determined conditions to screen for probe sequences. Three test sequences have been found in YACs at frequencies expected from their representation in the total human genome (a sequence from one tip of the X chromosome, 55 rDNA, and a Type I HLA sequence). This Program proposes to develop, test, and exploit libraries of human DNA further and to continue to improve required mapping and associated technology. A Core facility would organize, maintain, and characterize a master library of YACs with 5-hit coverage of the entire human genome in overlapping clones (about 100,000 yeast colonies with an average insert size of 250 kb). The Core would also aid in collateral cloning project, including the subcloning of the YACs down to lambda-and plasmid-sized fragments. Sequencing and software development are being pursued in associated endeavors. The health relatedness of the Program, land the potential usefulness of the Core, are indicated by the six Projects that would interact with it: 1) to improve the mapping technology further, permitting progressive subcloning from total human DNA all the way to individual sequencing templates (""""""""vertical integration""""""""); 2) to clone and study tandem repeat families, exemplified by 18S-28S and 5S rDNA; 3) to analyze a complex locus (HLA); 4) to map and study a single cytogenetic band in detail (on chromosome 1, containing several complement receptors and other genes of interest); and 5- 6) to locate genes at sites of chromosomal abnormalities which are involved in certain tumors, but for which no specific hybridization probes are yet available.
| Haimovich, Gal; Zabezhinsky, Dmitry; Haas, Brian et al. (2016) Use of the MS2 aptamer and coat protein for RNA localization in yeast: A response to ""MS2 coat proteins bound to yeast mRNAs block 5' to 3' degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system"". RNA 22:660-6 |
