Analysis of NotI linking clones isolated from human chromosome 3 specific libraries.

We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences

A group of NotI jumping and linking clones cover 2.5 Mb in the 3p21-p22 region suspected to contain a tumor suppressor gene.

The chromosomal region 3p21.2-p22 has been shown to be involved in the development of several forms of solid tumors. Such deletions, translocations, and rearrangements presumably result in the disturbance or loss of a critical gene function. Pulsed-field gel electrophoresis (PFGE), using NotI linking clones as a probe represent a powerful tool for analyzing such rearrangements. A NotI linking clone, AP20 (D3S1641), was localized by in situ hybridization to 3p21.3-p22. Two NotI jumping clones adjacent to this clone were isolated, clone J32-612 covering 0.5 Mb and clone J31-611 covering approximately 1 Mb. Clone J31-611 crosses the border of the deletion present in hybrid cell line MCH939.2, which contains a deleted 3p21 region. For these jumping clones, corresponding NotI linking clones, NLJ3 (D3S1642) and NL3-003, were isolated. Altogether, linking and jumping clones from the AP20 locus hybridize to NotI fragments totaling 2.5 Mb in length. These NotI-containing clones detect expressed sequences in several human tissues. Clone NLJ3 possesses homology to the human platelet-derived endothelial cell growth factor gene and may represent a new member of this gene family. Another clone (AP20) revealed 66% sequence similarity to rat skeletal muscle voltage-sensitive sodium channel subtype 2. Therefore, this group of clones will be useful not only for analyzing rearrangements in tumors, but also for the isolation of new genes from the 3p21.3-p22 region.