Construction of a 750-kb bacterial clone contig and restriction map in the region of human chromosome 21 containing the progressive myoclonus epilepsy gene.

Abstract

The gene responsible for progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPMI) is located on human chromosome 21q22.3 in a region defined by recombination breakpoints and linkage disequilibrium. As part of an effort to clone the EPM1 gene on the basis of its chromosomal location, we have constructed a 753-kb bacterial clone contig that encompasses the region containing the gene. Because DNA markers from the region did not identify intact yeast artificial chromosome (YAC) clones after screening several libraries, we built the contig from cosmid clones and used bacterial artificial chromosome (BAC) and bacteriophage PI clones to fill gaps. In addition to constructing the clone contig, we determined the locations of the E¢oRI, Sadl, EaBI, and Nod restriction sites in the clones, resulting in a high-resolution restriction map of the region. Most of the contig is represented by a level of redundancy that allows the orders of most restriction sites to be determined, provides multiple data points supporting the clone orders and orientations, and allows a set of clones with a minimum degree of overlap to be chosen for efficient additional analysis. The clone and restriction maps are in excellent agreement with maps generated of the region by other methods. These ordered bacterial clones and the mapping information obtained from them provide valuable reagents for isolating candidate genes for EPMI, as well as for determining the nucleotide sequence of a 750 kb region of the human genome.Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is inherited as an autosomal recessive disorder and is characterized by severe stimulus-sensitive myoclonus, tonicclonic seizures, and progressive neural degeneration. The age of onset is typically between 6 and 15 years. This condition is one of a group of five different subtypes of progressive myoclonus epilepsy, all of which show progressive neurodegeneration and variable degrees of severity. Unlike the other progressive myoclonus epilepsies, EPM1 is not characterized by biochemical markSCorresponding author. E-MAIL myers@shgc.stanford.edu; FAX (415) 725-9689.

Linkage disequilibrium mapping in isolated populations: The example of Finland revisited

“…Adjacent to but outside and centromeric of the presumed critical region was a marker (D21S141) that showed a relatively high degree of linkage disequilibrium (p excess ϭ 0.66; for a definition of this parameter of linkage disequilibrium, see below). This information and data on recombinations prompted physical mapping efforts to be targeted in the centromeric direction of the YAC-unclonable region (73). Fig.…”

Multi‐transgenic pigs for xenotransplantation

“…Transgenic pigs were healthy and showed strong β-cell-specific transgene expression. (Shizuya et al, 1992;Stone et al, 1996) and PAC vectors are capable of carrying inserts between 100-300 kb (Coren and Sternberg, 2001). The initial development of BAC and PAC libraries was largely driven by the Human Genome Project to allow construction of genomic libraries and physical maps for genome sequence analysis.…”

High-Resolution Physical and Transcriptional Mapping of the Autoimmune Polyendocrinopathy–Candidiasis–Ectodermal Dystrophy Locus on Chromosome 21q22.3 by FISH

“…Cloned cDNAs from a cDNA selection experiment were hybridized with PAC 92C23 and also with the cosmid contig overlapping with PAC 92C23 (Stone et al 1996). The selected cDNA F9 (size 1.0 kb) hybridized with PAC 92C23 and the cosmids 46C7, 1B4, and 5E4.…”

“…Parts of the APECED region have so far been cloned only into cosmids. Both Lafreniere et al (1995) and Stone et al (1996) have constructed restriction maps of cosmids including the region between markers D21S25 and D21S154.…”