Introduction to the Workshop
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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.
Long range mapping and sequencing of the human X chromosome
D.L. Nelson, E.E. Eichler, B.A. Firulli, Y. Gu, G.B. Ferraro, B. Franco, A. Grillo, G. Borsani, M.C. Wapenaar, A. Ballabio, A.C. Chinault, E.J. Roth, H.Y. Zoghbi, F. Lu, M.A. Wentland, D.M. Muzny, J. Lu, R.L. Clingan, S. Richards, R.A. Gibbs
Department of Molecular and Human Genetics, 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. 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. The map is composed of nearly 500 YAC clones derived from five different YAC libraries, including the two CEPH libraries, which contributed the majority of the clones. YAC screening has been carried out through use of two methods: PCR sampling of YAC DNA pools and hybridization of pooled Alu PCR products. Contigs were assembled primarily by STS content analysis, with subsequent verification of YAC overlaps via extensive Southern blot studies of YAC DNAs. Verification of marker order was by use of numerous somatic cell hybrids retaining translocated and deleted chromosomes derived from patients whose breakpoints are well mapped. The map is highly annotated, with 85 breakpoints defining 53 deletion intervals, 175 STSs (20 of which are highly polymorphic), and l9 genes.
Cosmid binning. Two regions of the Xp physical map have been converted to cosmid based maps using the Lawrence Livermore National Laboratories flow sorted X chromosome-specific cosmid library. YAC probes have been used to identify cosmids from the library, and these have been sorted with the use of adjacent YACs and YAC fragments derived by rare-cutter digestion and pulsed-field gel isolation. One of these efforts was recently published (1). A systematic effort to identify cosmids for this region, and particularly for the genes contained in the region is ongoing. Additional cosmid contig construction in distal- Xq is described in the accompanying abstract (Parrish et al.).
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 1.2 Mb region between FMRl (the gene involved in fragile X syndrome) and IDS (iduronate sulfatase, Hunter syndrome) in Xq27.3-Xq28. This region is at the boundary of Xq27.3 and Xq28, and its sequence may provide interesting data regarding the nature of chromosome bands in addition to discovery of novel coding sequences (such as that involved in FRAXE related mental retardation). To date, the complete sequence of both the FMRl and IDS genes have been determined (62 and 40 kb, respectively), along with ~250 kb of the interval between. Shotgun sequencing using the sequence-mapped gap strategy in conjunction with standard ABI fluorescent chemistry is being employed. Additional sequence in Xq28 has been determined, including that of a cosmid containing the three genes, ALD, DXSl357E and a creatine transporter. Curiously, this cosmid exhibits strong homology with a region on 16p11.1-11.2 and clones derived from the Los Alamos chromosome 16 library have been isolated and are being characterized for extent of similarity. This duplication appears to have a very recent evolutionary origin.
(1) Wapenaar et al. Hum Mol Genet 3: 1155-1161.