This proposal has two primary aims, both bearing directly on the objective of the human genome initiative. Firstly, to generate a high resolution physical map of the fission yeast genome consisting of a series of approximately 500 overlapping cosmid clones. Secondly, fission yeast is to be used to develop improved methods for cloning large fragments of mammalian DNA for the purpose of chromosome mapping. A map of the 14 megabase genome of S. pombe will be obtained at the """"""""six-cutter"""""""" (BamHI or EcoRI) level of resolution. The mapping strategy is a top down approach that takes advantage of the existing NotI restriction amp of the whole genome. The various stages of mapping will include the following. 1. Insertion of new NotI sites in areas such as chromosome 3 from which they are absent. 2. Preparing cosmid libraries from each NotI restriction fragment. 3. Preliminary alignment of overlapping """"""""contigs"""""""" on the basis of hybridization """"""""multiplexing"""""""". 4. Formal contig alignment by means of cosmid """"""""fingerprinting"""""""". 5. Annealing of contigs by hybridization walking. Regions that appear to be unclonable in cosmids will be spanned with yeast YAC vector cloning. 6. Contigs of each NotI region will be annealed by hybridization walking, using a cosmid library prepared with an enzyme other than NotI. 7. The classical and physical maps will be aligned by preparing oligonucleotides to known genes and locating the corresponding cosmid clone for each gene in a PCR (polymerase chain reaction) multiplex analysis. 8. In addition to the well established but time consuming mapping methods described above, effort will be devoted to development of innovative mapping strategies. These will be based on the use of the PCR reaction to detect short overlaps between cosmid clones. Finally, fission yeast will be investigated as a host organism for cloning large fragments of mammalian DNA. Improvements will be sought over existing yeast artificial chromosome (YAC) methods.
| Zhang, M Q; Marr, T G (1993) Genome mapping by nonrandom anchoring: a discrete theoretical analysis. Proc Natl Acad Sci U S A 90:600-4 |
