We would like to construct a set of yeast artificial chromosomes (abbreviated as YACs) each carrying up to 500 kb of Dictyostelium DNA flanked by telomeres. By arranging several hundred such chromosomes relative to each other on the basis of ordered restriction sites, it will be possible to establish a set of overlapping clones carried in yeast strains that covers all or almost all of the Dictyostelium genome. The Dictyostelium genome is small relative to that in other eukaryotes consisting of 50,000 kb + - 10,000 kb of DNA and so could be covered by 500 contiguous clones averaging 200 kb each with about 50% overlap. Evidence suggests that there are less than 10,000 genes in Dictyostelium so we would expect each artificial chromosome to carry only about 20 genes. These could be isolated from the appropriate yeast strains by digesting the artificial chromosome with restriction enzymes followed by electrophoresis on agarose. The fragment of interest could then be recognized on Southern blots of the fragments and eluted. Subcloning of the specific fragment in bacterial plasmids should then be relatively straightforward. This project has recently become feasible as the result of advances in electrophoretic techniques for separation of very large (up to 1 mb) fragments of DNA and the development of yeast vectors able to carry such large pieces. This project will have several immediate benefits: it will facilitate and coordinate gene isolation projects; overcome several problems encountered over the last few years in the maintenance of complete genomic banks of Dictyostelium DNA; and definitely map genes along the chromosomes. Moreover, it will be an essential step toward the goal of determining the complete sequence of nucleotides in the total genome once automated sequencing techniques are effective. Comparison of total genomic sequences of several distantly related organisms will be necessary to gain a complete understanding of the function and evolution of eukaryotic genomes.
| Titus, M A; Kuspa, A; Loomis, W F (1994) Discovery of myosin genes by physical mapping in Dictyostelium. Proc Natl Acad Sci U S A 91:9446-50 |
| Kuspa, A; Loomis, W F (1994) REMI-RFLP mapping in the Dictyostelium genome. Genetics 138:665-74 |
| Kuspa, A; Maghakian, D; Bergesch, P et al. (1992) Physical mapping of genes to specific chromosomes in Dictyostelium discoideum. Genomics 13:49-61 |
