The purpose of this project is to utilize a combination of somatic cell genetic and recombinant DNA technologies to carry out fine structure gene mapping of two human chromosomes, numbers 5 and 12. These two chromosomes were chosen for study because genetic selective systems are available which make possible fine structure mapping of them using recombinant DNA probes. For each of these chromosomes there is available: a) a CHO-human hybrid cell line which retains, under selective pressure, the single human chromosome under study; b) a complete recombinant DNA library specific for each of the two chromosomes, and c) a selective system to isolate a large number of derivatives of the single human chromosome-containing hybrid which have deletions of different, well-defined parts of the chromosome. Low copy DNA fragments from the human chromosome-specific recombinant libraries will be identified and subcloned and will be used as probes for Southern blots of restriction endonuclease digested DNAs from the series of cell lines with deletions of various parts of the chromosome under study. One can then correlate the presence or absence of human specific DNA fragments that hybridize to the various probes tested with the regions of the chromosome that are deleted in the various cell lines. This approach to mapping the human genome provides an opportunity to construct very detailed fine structure maps of human chromosomes. DNA probes localized to specific regions if one of these two chromosomes will then be screened to identify those that detect common restriction fragment length polymorphisms (RFLP) in members of a large human kindred. The goal of this part of the project is to use common RFLP as genetic markets to examine, for a number of pairs of loci, the relationship between positions of, and physical distances between, genes to meiotic recombination frequencies. Studies on recombination for a number of different regions along a given chromosome may well define regions in which recombination is much more, or much less frequent than in other regions, which could potentially help lead to an understanding of the structural or sequence elements of DNA that affect recombination. A related part of the project is aimed at using three other recombinant DNA libraries, specific for human chromosomes, 3, 4, and 18, respectively, to identify DNA probes from each chromosome that detect common RFLP in humans, which will be very useful for examining linkage relationships in families with genetic disorders in which the chromosomal location of the altered gene is unknown.
