The objectives are to establish overlapping cosmid and YAC clone libraries of the end of the long arm of the human X chromosome (the region between the position of the fragile site associated with the FraX mutation and the telomere) and to correlate these clone maps with the physical and genetic map of this area.
As a first step towards the construction of ordered clone libraries in the Xq28 region of the human X chromosome and the identification of genes located in this area, a cosmid library has been constructed of 1 million clones from Q1Z, a cell hybrid containing the Xq28 region in a hamster background.
All cosmid clones in the library hybridizing to human deoxyribonucleic acid (DNA) have been identified by one or more other cosmid clones or single copy probes and this has, in combination with the yeast artificial chromosome (YAC) insert hybridizations, allowed the establishment of 39 cosmid contigs with sizes up to 800 kb throughout the Xq28 region. Out of the 39 contigs, 34 cosmid contigs could be physically assigned according to the pulsed field gradient (PFG) map. In the last phase of the contig building process, remaining gaps are being closed by hybridizing cosmids from the ends of existing contigs to high density filter grids of cosmid libraries constructed from human X chromosomes (isolated by fluorescence activated chromosome sorts) as well as to high density filter grids carrying clones from a P1 library containing the entire genome and libraries constructed from subcloning of YACs. The cloned material has been used to establish new technology for the isolation of transcribed and conserved sequences. These techniques have been used to establish transcription maps, resulting in the isolation of over 40 new genes. As a further result of the systematic molecular analysis of Xq27.3-qter the gene for X-chromosomal adrenoleukodystrophy has been cloned. By molecular analysis of patients with MASA syndrome, a deletion has been identified in a large family and point mutations in 2 further families were located in the L1CAM gene. In addition the material established in the research has been helpful in the identification of a number of deletions in fragile X patients. Finally a new polymorphic microsatellite marker has been isolated and used to delineate the myotubular myopathy gene.
This region of approx. 8.5 megabases has the highest density of mutations associated with human genetic diseases and has therefore been analyzed intensely in a number of laboratories, using genetic, cytogenetic and physical mapping techniques. In addition to its inherent interest due to the large number of genetic diseases located there, it can therefore serve as a prototype for the further analysis of larger regions of the human genome.
As an essential step in the analysis of this region cosmid, linking and jumping clone libraries have been established using cell lines which contain the Xq27.3 to Xqter region as the only human component in a hamster background. Libraries have been picked into microtitre plates and used to construct the high density filter replicas, using a robotic device at the ICRF. .SP 1 Preliminary work in deriving ordered subsets of clones by the use of oligonucleotide fingerprinting as well as by sequence fingerprinting using other types of probes has been started, and has already resulted in the characterization of a number of (as yet unchecked) clone-contigs. This work has been, and will be, complemented by the isolation of yeast artificial chromosome clones from a library constructed from a cell line with 4 X chromosomes currently consisting of 20 000 clones with an average insert size of 600 kb (approx. 4 fold coverage of the human genome, 8 fold coverage of the X chromosome), which similarly has been spotted to generate high density robot filters.
These libraries will be used to establish complete contigs of YAC clones, indexed to contigs of cosmid and linking clones, and related to the available physical map. These clones will be analyzed to identify genes and will be used to test databases able to integrate the large varying types of information established on this region.
Funding SchemeCSC - Cost-sharing contracts
WC2A 3PX London
OX3 9DU Oxford