Research was carried out in order to create large contiguous arrays of genomic clones on chromosome 22. A strategy for the generation f ordered arrays of overlapping clones covering the chromosome 22 long arm was developed based on the use of a common set of chromosome 22 specific cosmids, a panel of somatic cell hybrids which distinguishes 30 different subregions on the long arm of chromosome 22 and a large number of single copy probes. In this process, genes involved in chromosome alterations associated with human pathologies were identified by positional cloning.
The study involved the following:
mapping loci and expansion into cosmids4 (single copy, small insert clones were obtained from several sources and, 55 clones from the proximal third of the long arm of chromosome 22 were mapped by somatic cell hybrid analysis);
some probes detected more than one locus on physical mapping (the map spans 5 megabase pairs of genomic deoxyribonucleic acid (DNA) and includes 26 previously isolated markers and genes including VIIIF2, Ewing's sarcoma translocation breakpoint, the neurofilament heavy chain gene (NFH), the leukaemia inhibiting factor (LIF), the oncostatin M gene (ONM) and SCH/NF2 genes);
genomic clone walking (9 cloned loci that had been identified in the region flanked by D22S1 and D22S15 were progressively expanded by the recurrent isolation of overlapping cosmids from the gridded libraries);
gene identification the large 1.5 megabase contig constructed in the D22S1/D22S15 region was shown to contain 3 genes previously known to reside on chromosome 22, NFH, LIF and ONM and 2 new genes that are relevant to human pathology were identified in this region, EWS and SCH.
The study has enabled the isolation of 2 genes of major importance, the EWS gene which is systematically altered in the family of Ewing tumours and in malignant melanoma of soft parts and the SCH/NF2 gene which when altered caused neurofibromatosis.
The work has also provided reagents of diagnostic interests which have been distributed to many research and diagnostic laboratories.
Chromosome 22 is the second smallest autosome. Its long arm amounts to approximately 1% of the whole human genome (35 Mbp). Possibly because of its high content of R-bands which are known to be rich in coding sequences, this chromosome segment is involved in many pathologies. Inherited diseases with no known cytogenetic anomalies include enzymopathies and predisposition to neuronal tumours.
Diseases associated with chromosome 22 alterations are frequent and comprise both constitutional anomalies and aberrations acquired by neoplastic cells during tumorigenesis. Thus a physical map of this part of the genome will provide not only a contribution to the knowledge of the structure of a whole chromosome arm, but is expected to lead rapidly to the isolation of a number of presently unknown genes involved in human pathologies.
A strategy for the generation of ordered arrays of overlapping clones covering the entire chromosome 22 long arm will be developed in three European laboratories. It is based on the use of a common set of chromosome 22 specific cosmids, a panel of somatic cell hybrids which distinguishes close to 30 different subregions on the long arm and on a large number of probes (over 400) that are already or will soon be mapped to specific subregions of chromosome 22.
The three collaborating teams will each contribute to the mapping of a third of the long arm (approximately 12 Mbp each). The work will be performed stepwise by screening the library with all available probes from defined subregions and extending the primary contigs from each subregion by short range chromosome walking. This method, which has already provided contigs over 300Kbp in size, will enable construction of regional maps which will be progressively extended so as to include the whole chromosome. Every cosmid belonging to an identified contig will be characterized by its restriction fragment length pattern (using normalized procedures) that will be systematically transferred to hybridization membranes.
It is expected that approximately 150 contigs covering most of the chromosome will thus be generated. The final organization will be achieved by the use of YAC and P1 libraries, by analysis of hybridization patterns of pulsed field electrophoresis gels, by cosegregation studies in reference families or by any new technique that may be available at that time. During the construction of the map, membranes will be used when needed to solve any inconsistencies that might appear.
In contrast to mapping approaches by random picking of clones, this targeted strategy will rapidly integrate in the map identified loci of biological interest. A computerized database specific to chromosome 22 will be set up. It will include all the data obtained during the generation of the contigs, will be associated with a bibliographic database devoted to chromosome 22 and will be interconnected with the main DNA sequence databases. It will be accessible to the scientific community through electronic networks.
Funding SchemeCSC - Cost-sharing contracts