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Microdissection and microcloning of banded human metaphase chromosomes have been used to construct a genomic library of 20,000 clones that is highly enriched for chromosome llpl3 DNA sequences. Clones from this library have been mapped on a panel of human-rodent somatic cell hybrids that divides the region from distal p12 to proximal p14 into seven physical intervals. A total of 1500 clones has been isolated, 250 clones have been characterized, and 58 clones have been mapped. Six of the clones were used to complete a long-range physical map of 7.5 megabases through the region. Two of the clones are localized to the Wilms tumor (WT) region, three are localized to the aniridia (AN2) region, and two are localized to the region between WT and AN2. The library represents DNA sequences spanning a distance of -13 x 106 base pairs, with an average density of one clone per 37,000 base pairs.

We have cloned and sequenced the murine homologue of the human EXT1 gene. At the protein level, these genes show almost complete identity as divergence is limited to only 5 amino acid positions that are scattered about the whole sequence. In addition, similarity searches identified a protein from chromosome III of C. elegans that shows significant similarity to the human and murine EXT/Ext genes. Using high resolution backcross mapping, the murine Ext1 was mapped at 26.55 cM between D15Mit143 and D15Mit153 on mouse chromosome 15. Therefore, Ext1 is part of an evolutionarily conserved linkage group including SDC2/Hspgl, TKHR/Trhr, EXT1/Extl, MYC/Myc, and TG/Tgn.

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Vertebrate DNA contains a small fraction of unmethylated CpG-rich DNA sequences, many of which include the 5' end of a gene. This fraction can be detected by its cleavage to tiny fragments with the methylation-sensitive restriction enzyme Hpall. Thus, the Isolation of Hpall tiny fragments (HTFs) from a specific chromosome region may be a useful approach for making an Inventory of the genes contained in it. Using microdissection, we have Isolated DNA from human chromosome band 8q24.1.The DNA was digested with Hpall, Iigated to a Clol-cut pUC plasmid, and amplified with Toq DNA polymerase and the standard M13/pUC forward and reverse sequencing primers. The amplification products were used to construct an HTF library, which is enriched for CpG-rich single-copy clones.