DNA sequence and analysis of human chromosome 9.

Chromosome 9 is highly structurally polymorphic. It contains the largest autosomal block of heterochromatin, which is heteromorphic in 6-8% of humans, whereas pericentric inversions occur in more than 1% of the population. The finished euchromatic sequence of chromosome 9 comprises 109,044,351 base pairs and represents >99.6% of the region. Analysis of the sequence reveals many intra- and interchromosomal duplications, including segmental duplications adjacent to both the centromere and the large heterochromatic block. We have annotated 1,149 genes, including genes implicated in male-to-female sex reversal, cancer and neurodegenerative disease, and 426 pseudogenes. The chromosome contains the largest interferon gene cluster in the human genome. There is also a region of exceptionally high gene and G + C content including genes paralogous to those in the major histocompatibility complex. We have also detected recently duplicated genes that exhibit different rates of sequence divergence, presumably reflecting natural selection.

A physical map of the human genome.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X.

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.

A 6.9-Mb high-resolution BAC/PAC contig of human 4p15.3-p16.1, a candidate region for bipolar affective disorder.

Bipolar affective disorder (BPAD) is a complex disease with a significant genetic component and a population lifetime risk of 1%. Our previous work identified a region of human chromosome 4p that showed significant linkage to BPAD in a large pedigree. Here, we report the construction of an accurate, high-resolution physical map of 6.9 Mb of human chromosome 4p15.3-p16.1, which includes an 11-cM (5.8 Mb) critical region for BPAD. The map consists of 460 PAC and BAC clones ordered by a combination of STS content analysis and restriction fragment fingerprinting, with a single approximately 300-kb gap remaining. A total of 289 new and existing markers from a wide range of sources have been localized on the contig, giving an average marker resolution of 1 marker/23 kb. The STSs include 57 ESTs, 9 of which represent known genes. This contig is an essential preliminary to the identification of candidate genes that predispose to bipolar affective disorder, to the completion of the sequence of the region, and to the development of a high-density SNP map.

Physical map of human 6p21.2-6p21.3: region flanking the centromeric end of the major histocompatibility complex.

We have physically mapped and cloned a 2.5-Mb chromosomal segment flanking the centromeric end of the major histocompatibility complex (MHC). We characterized in detail 27 YACs, 144 cosmids, 51 PACs, and 5 BACs, which will facilitate the complete genomic sequencing of this region of chromosome 6. The contig contains the genes encoding CSBP, p21, HSU09564 serine kinase, ZNF76, TCP-11, RPS10, HMGI(Y), BAK, and the human homolog of Tctex-7 (HSET). The GLO1 gene was mapped further centromeric in the 6p21.2-6p21.1 region toward TCTE-1. The gene order of the GLO1-HMGI(Y) segment in respect to the centromere is similar to the gene order in the mouse t-chromosome distal inversion, indicating that there is conservation in gene content but not gene order between humans and mice in this region. The close linkage of the BAK and CSBP genes to the MHC is of interest because of their possible involvement in autoimmune disease.

A detailed physical and transcriptional map of the region of chromosome 20 that is deleted in myeloproliferative disorders and refinement of the common deleted region.

Acquired deletions of the long arm of chromosome 20 are the most common chromosomal abnormality seen in polycythemia vera and are also associated with other myeloid malignancies. Such deletions are believed to mark the site of one or more tumor suppressor genes, loss of which perturbs normal hematopoiesis. A common deleted region (CDR) has previously been identified on 20q. We have now constructed the most detailed physical map of this region to date--a YAC contig that encompasses the entire CDR and spans 23 cM (11 Mb). This contig contains 140 DNA markers and 65 unique expressed sequences. Our data represent a first step toward a complete transcriptional map of the CDR. The high marker density within the physical map permitted two complementary approaches to reducing the size of the CDR. Microsatellite PCR refined the centromeric boundary of the CDR to D20S465 and was used to search for homozygous deletions in 28 patients using 32 markers. No such deletions were detected. Genetic changes on the remaining chromosome 20 may therefore be too small to be detected or may occur in a subpopulation of cells.

Genomic analysis of the Tapasin gene, located close to the TAP loci in the MHC.

The Tapasin molecule is a member of the immunoglobulin (Ig) superfamily required for the association of TAP transporters and MHC class I heterodimers in the endoplasmic reticulum. In this study, the Tapasin gene was precisely mapped in relation to the MHC. The gene was centromeric of the HLA-DP locus between the HSET and HKE1.5 genes and within 500 kbp of the TAP1 and TAP2 genes. A homologous mouse EST was mapped to a syntenic position on chromosome 17, centromeric of the H-2 K locus. Similarly, the rat Tapasin gene was shown to be in an equivalent location with respect to the RT1.A locus. The localization of Tapasin, TAP, LMP and class I genes within such a short distance of each other on the chromosome implies some regulatory or functional significance. We determined the Tapasin gene sequence for comparison of its structure to that of other Ig superfamily members, such as MHC class I genes. The IgC domain was encoded by a separate exon. However, the positions of the other introns were not characteristic of other Ig superfamily genes, indicating that Tapasin has a distinct phylogeny.

From long range mapping to sequence-ready contigs on human chromosome 6.

Our aim is to construct physical clone maps covering those regions of chromosome 6 that are not currently extensively mapped, and use these to determine the DNA sequence of the whole chromosome. The strategy we are following involves establishing a high density framework map of the order of 15 markers per Megabase using radiation hybrid (RH) mapping. The markers are then used to identify large-insert genomic bacterial clones covering the chromosome, which are assembled into sequence-ready contigs by restriction enzyme fingerprinting and sequence tagged site (STS) content analysis. Contig gap closure is performed by walking experiments using STSs developed from the end sequences of the clone inserts.

An integrated YAC map of the human X chromosome.

The human X chromosome is associated with a large number of disease phenotypes, principally because of its unique mode of inheritance that tends to reveal all recessive disorders in males. With the longer term goal of identifying and characterizing most of these genes, we have adopted a chromosome-wide strategy to establish a YAC contig map. We have performed > 3250 inter Alu-PCR product hybridizations to identify overlaps between YAC clones. Positional information associated with many of these YAC clones has been derived from our Reference Library Database and a variety of other public sources. We have constructed a YAC contig map of the X chromosome covering 125 Mb of DNA in 25 contigs and containing 906 YAC clones. These contigs have been verified extensively by FISH and by gel and hybridization fingerprinting techniques. This independently derived map exceeds the coverage of recently reported X chromosome maps built as part of whole-genome YAC maps.

Physical mapping of chromosome 6: a strategy for the rapid generation of sequence-ready contigs.

The development of radiation hybrid (RH) mapping (Cox et al., 1990) and the availability of large numbers of STS markers, together with extensive bacterial clone resources provided a means to accelerate the process of mapping a human chromosome and preparing bacterial clone contigs ready to sequence. Our aim is to construct physical clone maps covering those regions of chromosome 6 that are not currently extensively mapped, and use these to determine the DNA sequence of the whole chromosome. We report here a strategy which initially involves establishing a high density framework map using RH mapping. The framework markers are then used for the identification of bacterial genomic clones covering the chromosome. The bacterial clones are analysed by restriction enzyme fingerprinting and STS-content analysis to identify sequence-ready contigs. Contig gap closure will also be performed by clone walking.