The yeast artificial chromosome (YAC) technology has been utilized in order to establish the physical map of portions of the chromosomal region Xq24-qter. The main effort has been directed towards mapping the Xq27 band. Work in this region has been quite successful, since it has been possible to assemble 246 YACs in a single contig spanning approximately 12 Megabases (Mb) including the whole Xq27 band. This region joins a contig across Xq26 at the centromeric side and is merged with the IDS contig in the proximal portion of Xq28 at the telomeric side. The YAC contigs from FRAX (A) to U6.2 in Xq27.3-q28.1 have been consolidated and extended and currently an effort is being made to try to close the last gap. The remainder of the effort has been focussed in the lesser mapped Xq24-25 region. As a result of extensive YAC library screening, over 500 YACs have been identified which map to this region, some of which have been assembled into contigs of up to 3 Mb.
Sequence tagged sites (STSs) from the Xq 24-Xqter region of the X chromosome will be generated and YAC clones isolated from our resource in order to assign sequence landmarks to the map of the region that is now being constructed, and to order YAC clones relative to one another with reference to such landmarks. The location of potential CpG islands will be identified by the presence of multiple rare cutting restriction enzyme sites (eg EagI, SacII, NotI, etc). STSs will be generated from these regions and, hence, presumably near to genes.
Such STSs will be generated either by sequencing from the EagI site of the EagI-coRI human DNA inserts of lambda clones obtained from the rodent x human hybrid (X 3000.11) which contains the Xq24-Xqter segment of the X chromosome, or by examining human DNA-ontaining cosmids prepared from the same hybrid. Other STSs will be generated by Alu-CR of radiation hybrids containing small regions of the human X chromosome as the only human component. Such STSs should be close to polymorphic sites usually present in Alu repeats.
Primers for PCR reactions will be obtained from each STS, and YAC clones will be isolated by PCR screening. Additional YACs will be isolated by hybridization screening using the human DNA cosmids prepared from the X 3000.11 hybrid as the probe or as starting material for probes. Overlaps between YACs will be detected by virtue of the fact that they share some of the STSs defined above, but probes obtained from the ends of YACs and YAC fingerprinting will also be used. Extension of contigs with YAC end probes will also be performed. In addition the STSs will be located in existing map contigs when available and will contribute to an STS database.
Funding SchemeCSC - Cost-sharing contracts
SE1 9RT London