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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
DEVELOPMENT AND TESTING OF A METHOD FOR THE PURIFICATION OF EVOLUTIONARILY CONSERVED SEQUENCES FROM CLONED HUMAN DNA
[1,3]Lisa Stubbs, Karen Glantz, and Elbert Branscomb
Mammalian Genetics and Development Section, Biology Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37831-8077; Human Genome Center, Lawrence Livermore National Laboratory, P.O. Box 808, L-452, Livermore, CA 94551; Corresponding author.
As a corollary to our groups' efforts to define genomic homologies between human chromosome 19 and related murine regions, we are interested in devising means through which these genomic relationships can be exploited for the identification of new human genes. This project has been designed to capitalize upon LLNL's collection of contiguous cosmid, YAC and other clones which span the length of human chromosome 19. A large number of cloned chromosome 19 genes and DNA markers have already been localized to specific clone contigs, but a vastly larger number of new genes remains undiscovered. In order to address the pressing problem of large-scale gene identification, we have developed a novel method to permit rapid and efficient identification of genes and other functionally-significant DNA sequences from cloned human DNA.
Our approach relies upon the fact that DNA sequences with important biological functions are most likely to be conserved throughout evolution. Because of this fact, the genes of mouse and man are, on the whole, very similar in DNA sequence; nongene regions, by contrast, will generally vary greatly between two such highly divergent species. Our goal is thus to selectively clone the sequences which are most similar between mouse and human DNA. The approach focuses upon using conserved sequences in murine YAC or P1-based clones to "trap" similar sequences from human cosmids spanning homologous genomic regions, and is loosely based upon cDNA selection methods (such as those developed by M. Lovett and colleagues). To test and troubleshoot our 'conserved element mapping' strategy, we have worked with a pair of homologous cosmids carrying the mouse and human XRCC1 gene sequences. Since both mouse and human cosmids have been completely sequenced (by J. Lamerdin and colleagues, LLNL), the exact size, position, and degree of similarity of conserved DNA sequences are known. Initial results with this cosmid model system have demonstrated that XRCC1 exons, which occupy less than 5% of cosmid sequences, can be efficiently purified using this approach; significant contamination is derived only from Alu repeats, which are most likely binding to related B1 repeats of mouse. Experiments now in progress are aimed at improving the purity and yield of our products in the cosmid model system, and at expanding the length of scanned regions to include large human contigs and mouse P1-based contigs and YAC clones. Ultimately, we intend to apply these methods to the identification of genes and conserved regulatory elements along the length of human chromosome 19.
This work was supported by USDOE under contract DE-AC05840R21400 with Martin Marietta Energy Systems, Inc., and contract W-7405-ENG-48 with the Lawrence Livermore National Laboratory.