Long-range restriction map around 11p13 aniridia locus

Abstract

Using two random DNA markers, and pulsed field gel electrophoresis, a 1.5-Mb physical map surrounding the 11p13 aniridia locus (AN2) has been assembled. The map was constructed using a combination of single- and double-restriction digests on DNA from normal controls and a patient transmitting familial aniridia. The aniridia patient has a chromosome translocation and the two DNA markers flank the breakpoint. This 11p13 breakpoint lies no further than 100 kb from the DNA marker 1104 (D11S95), located on the centromeric side of the breakpoint. Two CpG islands, separated by 550 kb and flanking the translocation, suggest an upper limit to the size of the gene.

Somatic cell hybrid and long-range physical mapping of 11p13 microdissected genomic clones.

“…3 Upper. Patient cell lines that were critical to construction of the map included WT-21, a cultured Wilms tumor cell line, and two aniridia patients (11,20), DG-85 and SIMO; all three cell lines have 11p13 translocations, with the first two including deletions at the translocation breakpoints. …”

“…DAR15-14 is a human-mouse hybrid established from a patient who had Wilms tumor, but not aniridia, and a deletion of 11p12-p13 (31). DGL hybrids were established from a patient, DG-85, with familial aniridia and a cytologically balanced translocation between chromosomes 11 and 22 (32); the translocation involving chromosome 11 was located at llpl3 with an associated deletion of at least 600 kb (11,20). The patients, their clinical histories, and the cell hybrids have been described (11 (34), and filters were exposed to XAR-5 film (Kodak) at -70'C with an intensifying screen for 3-10 days.…”

“…Pulsed Field Gel Electrophoresis. Preparation of high molecular weight DNA from lymphocyte cells, digestion with rarecutting restriction enzymes, and electrophoresis through either transverse alternating, field inversion, or contourclamped homogeneous field electrophoresis have been described (20). After electrophoresis, gels were stained for 20-40 min with ethidium bromide, exposed to shortwave UV light (300 nm) for 5 min, transferred to nylon under alkaline conditions, and hybridized as described above.…”

“…The 7.5-Mb map extends from the centromere proximal side of CAT, through the WAGR region, to the distal side of FSHB. Locations for the Wilms tumor gene, as defined by the translocation/deletion breakpoint of cell line WT-21 (unpublished data), and the aniridia gene, as defined by the translocation of SIMO and the translocation/deletion of DG-85 (11,20), are indicated by the brackets. A detailed map of the aniridia region has been presented (20).…”

“…In the cases of Wilms tumor and aniridia, the translocation or deletion breakpoints of a few critical patients or tumors have been positioned on the physical map; consequently the aniridia gene has been localized to a region of <125 kilobases (kb) (19,20), and the Wilms tumor gene has been localized to 2.5 megabases (Mb) in one case (18), to 800 kb in another (17), and more recently to a region of <350 kb (21)(22)(23)(24). When the position of a gene is known to within a few hundred kilobases, strategies to identify additional clones directly at the presumptive locus can be devised, such as those undertaken to clone a candidate Wilms tumor gene (22,23).…”