High-resolution transcript map of the region spanning D12S1629 and D12S312 at chromosome 12q13: triple A syndrome-linked region

Abstract

For those searching for human disease-causing genes, information on the position of genes with respect to genetic markers is essential. The physical map composed of ESTs and genetic markers provides the positional information of these markers as well as the starting point of gene identification in the form of genomic clones containing exons. To facilitate the effort of identification of genes in the region spanning D12S1629 and D12S312, we constructed a high-resolution transcript map with PAC/BAC/cosmid clones. The strategy for the construction of such a map involved utilization of STSs for the screening of the large insert bacterial chromosome libraries and a chromosome 12-specific cosmid library by hybridization. The contig was constructed based on the STS contents of the clones. The resulting high-resolution transcript map of the region between P273P14/SP6 and D12S312 spans 4.4 cM from 66.8 to 71.2 cM of the Généthon genetic map and represents approximately 2.4 Mb. It was composed of 81 BAC, 45 PAC, and 91 cosmid clones with a minimal tiling path consisting of 16 BAC and 4 PAC clones. These clones are being used to sequence this part of chromosome 12. We determined the order of 135 STSs including 74 genes and ESTs in the map. Among these, 115 STSs were unambiguously ordered, resulting in one ordered marker per 21 kb. The order of keratin type II locus genes was determined. This map would greatly enhance the positional cloning effort of the responsible genes for those diseases that are linked to this region, including male germ cell tumor as well as palmoplantar keratoderma, Bothnian-type, and triple A syndrome. This transcript map was localized at human chromosome 12q13.

Figure 1

An STS content map for the region spanning D12S1629 and D12S312. The line extending across the top represents the 12q13 region. The centromere is to the left and the telomere is to the right. Genetic distances in centimorgans (Généthon Genetic map) are indicated below the line. (A) A YAC map of the region. The map consists of 34 YAC clones and contains 58 STSs. Clones are from Center d'Etude du Polymorphisme Humain (CEPH) II YAC library. (B) A high-resolution transcript map. The map is composed of 217 BAC, PAC, and cosmid clones with 135 STSs. The order of most STSs was determined unequivocally, although that of a few STSs was not (depicted by brackets). The boxed clones represent minimum number of clones to represent the whole contig (minimal tiling path). Lines represent the clones and their names correspond to the plate addresses. Clones with B prefix; RPCI 11 BAC library, DB prefix; CIT-HSP BAC library, P prefix; RPCI 1 PAC library, C prefix; chromosome 12-specific cosmid library LL12NCO1. The STS content of each clone is indicated by symbols: black circle, genetic marker; blue square, random genomic marker; square with red border, STS derived from the clone-end sequence; red triangle pointing upward, gene; triangle pointing downward, ESTs; unfilled symbol, not confirmed by PCR.

Figure 2

Determination of clone insert sizes by pulsed-field gel electrophoresis. (A) Ethidium bromide-stained BAC DNA analyzed by pulsed-field gel electrophoresis. PAC or BAC DNA was digested with NotI. The released inserts were separated on a BioRad CHEF Mapper as described in Methods. (Lane 1) B830P21; (lane 2) B680A11; (lane 3) B774I22; (lane 4) B1044N1; (lane 5) P385I2; (lane 6) B658B23; (lane 7) B657H18. (B) Sizes of the clone inserts that constitute a minimal tiling path. Size was determined based on the standard curve determined from λ DNA multimer.