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.

In a newborn female, an abnormal karyotype, 46,XX/47,XX,+mar/47,XX,+9, was found associated with several malformations. The marker chromosome was present in 70% of peripheral blood lymphocytes, and its size appeared to be less than half of the smallest chromosomes. Several differential staining methods provided no indication as to its origin.Chromosomes isolated from EBV-immortalized lymphocytes of the patient were fractionated on a FACS-440. The marker was resolved as a sharp peak in the region close to the chromosomal debris: its DNA content seemed to be close to 40% of chromosomes 21 and 22.About 580000 minichromosomes were sorted. In order to optimize cloning conditions, a pilot cloning experiment was performed on a pool of sorted chromosomes 9, 10, 11 and 12.

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NotI cleavage sites are frequently associated with CpG islands that identify the 5' regulatory sites of functional genes in the genome. Therefore we analyzed a sample of 22 NotI linking clones prepared from mouse brain DNA, to determine whether these mouse NotI site associated clones could be used for comparative analysis of mouse and human genomes by cross-reaction with both mouse and human genomic DNA and RNA in Southern and Northern hybridization. We further examined whether we could establish the identity of these clones with known genes by comparing the nucleotide sequences surrounding the NotI site with the GenBank database. We observed that 70% of the clones cross-hybridized with human DNA and that 4 of 11 tested clones (36%) detected a transcript in human HeLa cells RNA whereas 73% clones (8/11) detected transcripts in mouse RNAs from one or more organs. Single pass sequence analysis was successful on 16 of 19 clones. The GC content in these sequence was very high (48.8% to 73.8%) suggesting that 12 of 16 sequenced clones contained a CpG island. Three out of 19 clones showed significant similarity with previously analyzed mouse gene sequences in GenBank, including the mouse rRNA gene family, cathepsin and the scip POU-domain genes. In addition, two sequences showed significant similarity to the human and rabbit protein phosphatase 2A-beta subunit and the human transforming growth factor-beta. Thus, 5 of 16 clones showed homology with identified genes. These results and the recent work of using RLGS methods for genetic mapping indicate that NotI linking clones can be used to efficiently cross reference a comparative analysis of the mouse and human genomic maps.