An integrated metric physical map of human chromosome 19.

A metric physical map of human chromosome 19 has been generated. The foundation of the map is sets of overlapping cosmids (contigs) generated by automated fingerprinting spanning over 95% of the euchromatin, about 50 megabases (Mb). Distances between selected cosmid clones were estimated using fluorescence in situ hybridization in sperm pronuclei, providing both order and distance between contigs. An average inter-marker separation of 230 kb has been obtained across the non-centromeric portion of the chromosome. Various types of larger insert clones were used to span gaps between contigs. Currently, the map consists of 51 'islands' containing multiple clone types, whose size, order and relative distance are known. Over 450 genes, genetic markers, sequence tagged sites (STSs), anonymous cDNAs, and other markers have been localized. In addition, EcoRI restriction maps have been generated for > 41 Mb (approximately 83%) of the chromosome.

Isolation of chromosome 18-specific brain transcripts as positional candidates for bipolar disorder.

Several studies have proposed the existence of susceptibility loci for bipolar disorder on chromosome 18. To identify possible candidate genes for this disease, we isolated brain-expressed transcripts by direct cDNA selection on chromosome 18-specific biotinylated cosmid clones. Longer cognate cDNA clones of the selected cDNAs were isolated from a normalized infant brain cDNA library. Physical mapping by PCR on a panel of somatic cell hybrids was conducted by the use of primers derived from partial sequences on either the 5' or 3' ends of the clones. In our initial analysis, 48 cDNA clones were found to be chromosome 18-specific, mapping to different subchromosomal regions. Sequence redundancy among these clones yielded 30 unique transcripts, five of which were represented in previously known genes. Further sequencing of the remaining 25 unique cDNA clones confirmed the absence of significant homology to known genes, indicating that these transcripts represented novel genes. Mapping with the use of a radiation hybrid panel positioned the brain cDNAs to within = 100 to 1100 kb from reference sequence tag sites (STSs) and assembled them into six high resolution linkage groups. The majority of the transcripts were found to cluster to discrete locations on 18p and 18q, previously hypothesized as susceptibility regions for bipolar disorder, identifying them as positional candidate genes.

Generation and mapping of human chromosome 2 microdissection clone-derived STSs.

Twenty-nine new sequence-tagged sites (STSs) were derived from DNA sequences of clones from two human chromosome 2 microdissection libraries. The specificity of the STSs for human chromosome 2 was first demonstrated by PCR amplification of DNA from genomic human and hamster cells and a human chromosome 2-containing human x hamster hybrid cell line. The STSs were then mapped to chromosome 2 by two different approaches. In the first attempt, 12 of the STSs were shown to PCR amplify YAC clones associated with genetic markers on the chromosome. In the second approach, 27 of the STSs were localized to chromosome bands by FISH using cosmid or PAC clones encoding the STSs. The specific STSs mapped to chromosome 2 by these two approaches tie together the genetic and cytogenetic maps of the chromosome at the two termini. The distribution of these STSs further defines the region of the chromosome present in the two microdissection libraries.

Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries.

Three human chromosome 2-specific clone libraries were constructed and characterized. Chromosome 2-specific cosmid and fosmid clone libraries were constructed using flow-sorted DNA from the monochromosomal hybrid cell line GM10826. The cosmid and fosmid libraries consist of 38,496 and 26,400 arrayed clones, respectively, with an average size of 40 kb. Colony hybridization of a representative number of clones with both human and hamster genomic DNA probes demonstrates that between 58 and 66% of the clones in the flow-sorted libraries contain human inserts. Approximately 5% of the cosmid and fosmid clones are nonrecombinants. A chromosome 2-specific PAC library was also produced from the hybrid cell line GM10826. DNA from the hybrid cell line was cloned, and the human chromosome 2-specific clones were identified by colony hybridization. Approximately 5800 chromosome 2-specific PAC clones with an average insert size of approximately 85 kb were arrayed. Based on the size of the clones, the cosmid, fosmid, and PAC libraries are approximately 3.6x, approximately 2.5x, and approximately 1.9x, respectively in chromosomal coverage. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. The average number of clones identified for each STS in the library indicates the cosmid, fosmid, and PAC libraries to be approximately 3.2x, approximately 2.1x, and approximately 1.5x, respectively, in chromosome coverage. All except one of the 82 STSs were represented in the portions of the libraries screened.