D. L. Nelson, E.E. Eichler, B.A. Firulli, Y. Gu, J. Wu, E. Brundage, A.C. Chinault, M.Graves, A. Arenson, R. Smith, E.J. Roth, H.Y. Zoghbi, Y. Shen, M.A. Wentland, D.M. Muzny. J. Lu, K Timms, M. Metzger, and R.A. Gibbs
Department of Molecular and Human Genetics and Human Genome Center, Baylor College of Medicine, Houston, Texas
The human X chromosome is significant from both medical and evolutionary perspectives. It is the location of several hundred genes involved in human genetic disease, and has maintained synteny among mammals; both of these aspects are due to its role in sex determination and the haploid nature of the chromosome in males. We have addressed the mapping of this chromosome through a number of efforts, ranging from long-range YAC-based mapping to genomic sequence determination.
YAC mapping. The YAC-based map of the X is essentially complete. We have constructed a 40 Mb physical map of the Xp22.3-Xp21.3 region, spanning an interval from the pseudoautosomal boundary (PABX) to the Duchenne muscular dystrophy gene. This region is highly annotated. with 85 breakpoints defining 53 deletion intervals, 175 STSs (20 of which are highly polymorphic), and 19 genes.
Cosmid binning. The YAC-based physical is being used in a systematic effort to identify and sort cosmids prepared at LLNL from flow sorted X chromosomes into intervals Gene identification through use of a common database for cDNA pool hybridization data is continuing. Additional efforts in distal Xq are described in Parrish et al. Over 50 YACs have been utilized as probes to the gridded cosmic arrays. These have identified over 9000 cosmids from the 24,000 member library. An additional 4000 cosmids have been identified using a variety of probes, with the bulk corning from cDNA pool probes.
Cosmid contig construction. Creation of long-range continuity in cosmids proceeds from clones identified by the YAC-based binning experiments. Identification of STS carrying clones is carried out by a combined PCR/hybridization protocol, and adds to the specificity of the overlap data. Cosmids are grown and DNA is prepared by an Autogen robot. DNAs are digested and analyzed by the AB362 GeneScanner for collection of fingerprint data. The use of novel fluorescent dyes (BODIPY) in this application has increased signal strength markedly. End fragment detection is currently carried out with traditional Southern hybridization, however additional dyes will permit detection without hybridization in the GeneScanner protocol. Data are transferred to a Sybase database and analyzed with ODS (J. Arnold, U. Georgia) software for overlap. ODSoutput is ported to GRAM (LANL) for map construction. A fully automated approach has yet to be achieved, but this goal is increasingly in reach.
Sequencing. An independently funded project awarded to RAG seeks to develop long-range genomic sequence for ~2 Mb of the human X chromosome. In support of this project, cosmids have been constructed and isolated for the 1.6 Mb region between FRAXA and FRAXF in Xq27.3-Xq28. To date, the complete sequences of the regions surrounding the FMR1 and IDS genes have been determined (180 and 130 kb, respectively), along with an additional ~500 kb of the interval. This sequence has led to identification of the gene involved in FRAXE mental retardation. Additional sequence in Xq28 has been determined, including that of a cosmid containing the two genes, DXS1357E and a creatine transporter. This sequence has been duplicated to chromosome 16pl 1 in recent evolutionary history. Comparative sequence analysis reveals 94% sequence identity over 25 kb, and the presence of pentameric repeats which are likely to have mediated the duplication event. A number of technical advances in sequencing have been developed, including the use of BODIPY dyes in AB373 sequencing protocols, which has offered enhanced base calling due to reduced mobility shifting, improved single strand template protocols for much reduced cost, and streamlined informatics processes for assembly and annotation.
Supported by grants DE-FG05-92ER6l401 and DE-FG03-94ER61830 from the U.S. Department of Energy and a Center grant from the NCHGR of the NIH (NIH 5P30 HG00210) to DLN.
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