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Novel molecular approaches towards a high resolution genetic and physical map of chromosome 11

Objective


A high density radiation hybrid map was constructed consisting of 506 sequence tagged site (STS) markers spanning human chromosome 11. The map includes 143 unique map positions that could be ordered with respect to each other. Based on the estimated length of 144 megabases (Mb) for the chromosome, this provides a framework map with a resolution of approximately 1 Mb. Overall, there are 299 unique map positions with an average resolution of 1 position for 480 kb. The data generated lead to the conclusion that most distal loci are close to the telomeres. The pericentric region is extremely well defined in the radiation hybrid map.
The STSs included 256 polymorphic microsatellite markers and 56 new microsatellite were included in the map. Genetic linkage data were available for 98 of the microsatellite markers which allowed detailed comparison of physical and genetic maps across a whole chromosome.
In all 151 genes were mapped representing the vast majority of genes known to reside on chromosome 11. Since these are integrated with genetic markers, it is possible to obtain candidate genes rapidly from a region of interest that has been defined by linkage studies.

A yeast artificial chromosome (YAC) contig spanning 2.8 Mb from the 11q12 to q13 region was also constructed. The YAC contig contained 15 STSs which were ordered physically; the order could be confirmed in the radiation hybrid map. 7 genes were placed in the contig map, including the high affinity immunoglobulin E receptor which is thought to harbor variants implicated in atopy. A second contig was constructed in the region of chromosome 11q22 to 23 which contains a gene responsible for ataxia telangectasia. These results have led to the possibility of identifying a complete set of YACs spanning the whole of chromosome 11 based on STS information from the radiation hybrid map.
Several approaches will be taken to defining both a high resolution genetic map and a physical map for regions of human chromosome 11. The strategy chosen will integrate the work of four groups to develop detailed maps of this region. Radiation-reduced hybrids containing chromosome 11 will be used to map a series of markers and produce a detailed series of ordered probes along the chromosome. 177 hybrids containing human chromosome 11 will be used to screen RFLP probes, cloned genes and PCR loci generated by AluPCR, microsatellites or simple STSs.

Polymorphism is not a requirement for this mapping strategy and it should permit a high resolution map to evolve quickly for much of this chromosome. A major source of new length polymorphisms throughout mammalian genomes is microsatellite repeat sequences such as (CA)n. Using this new technology we will attempt to resolve such short polymorphisms on Southern blots. Should this prove feasible it will greatly improve the efficiency by which new repeats can be screened for polymorphism and subsequently be characterized in large numbers of individuals.

This approach for generating multiple new microsatellites will be tested by generating multiple markers on chromosome 11, utilizing cosmids and YACs defined by the other collaborators. Cosmids will be obtained from two chromosome 11 specific libraries and YACs by PCR screening of two libraries. Markers from the radiation hybrids will also be used to identify localized cosmids or YACs for this study.

The area 11qcen-1q13 will be mapped physically using YAC clones to generate a YAC contig of approximately 30 cM. This will provide a well defined physical map for this region and will provide valuable reagents for the generation of a genetic map in the region. New and established markers will be localized on metaphase or interphase nuclei using recently described in situ techniques.

These in situ studies will be used to assist in identifying low copy number hybrids and to verify the location of such hybrids to specific regions of chromosome 11. They will also be used for excluding coligation events in YAC clones and for mapping both dinucleotide repeat based markers and cosmid clones. These studies will form an integrated collaboration between CEPH and Oxford University and will provide the opportunity for considerable interchange of techniques and reagents.

Coordinator

FONDATION JEAN DAUSSET - CENTRE D'ETUDE DU POLYMORPHISME HUMAIN
Address
Rue Juliette Dodu 27
75010 Paris
France

Participants (2)

Institut National de la Santé et de la Recherche Médicale (INSERM)
France
Address
27 Rue Juliette Dodu
75010 Paris
The Chancellor, Masters and Scholars of the University of Oxford
United Kingdom
Address
John Radcliffe Hospital Headington
OX3 9DU Oxford