Loren Hauser, Mark Shannon, Melissa York, Lisa Stubbs, and Richard Mural
Biology Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37831-8080
One of the most efficient ways of finding genes on cosmid or P1 clones is to shotgun sequence random fragments and analyze the data for potential coding regions using computer programs such as GRAIL. In gene rich regions of the mouse or human genome, a cosmid sized clone (about 40kb) may contain 3 to 5 genes. In such regions about 1 in 5 to 1 in 10 random 1kb subclones should contain protein coding exons and over 90% of these will be recognized by GRAIL analysis. A joint comparative human-mouse mapping project between Lawrence Livermore National Laboratory and Oak Ridge National Laboratory has previously identified a conserved cluster a Kruppel-type zinc-finger (ZNF) genes distal to XRCC1. Mapping data using mouse KRAB and zinc-finger probes placed one ZNF gene about every 25 kb in a tandem arrangement on the human cosmid contig. Therefore, there should be 1-2 ZNF genes per cosmid. A probe from a unique region of a cDNA mapped to this cluster was used to identify a cosmid from both a mouse and human library. Shotgun libraries from each cosmid were prepared in M13 and sequenced. The data was analyzed directly using BatchGRAIL. BatchGRAIL is designed to search for potential coding regions and if the coding potential exceeds a threshold its translation is searched against Swissprot. Consistent with expectation, approximately 1 in 10 clones are positive for ZNF coding regions. In addition, a LINE was identified via its weak homology to DNA polymerases. Comparative sequence of the mouse and human cosmids will be presented. The structure of both human and mouse cosmids containing orthologous genes from this cluster will provide useful information on the evolution and possible function of the cluster.
This Research was supported by the Office of Health and Environmental Research, United states Department of Energy, under Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.