Mb Of DNA Research Articles

Dose – response for radiation-induced apoptosis, residual 53BP1 foci and DNA-loop relaxation in human lymphocytes

The purpose was to compare the radiation-induced apoptosis in human lymphocytes with DNA-loop relaxation and DNA damage as a function of radiation dose and time after exposure. Morphological changes were analysed by staining with fluorescent dyes and apoptotic fragmentation of DNA with conventional agarose gel electrophoresis, pulsed-field gel electrophoresis (PFGE) and alkaline comet assay. Viability was estimated by trypan blue assay. The levels of protein p53 (TP53) were determined with Western blot. Relaxation of DNA-loops was analysed by the method of anomalous viscosity time dependence (AVTD) and neutral comet assay. Induction and repair of double-strand breaks (DSB) was studied by PFGE and by immunostaining of the TP53 binding protein 1 (53BP1). At various time points of apoptosis, there was a linear dose dependence for all apoptotic end-points up to 1 – 2 Gy followed by a plateau at higher doses. Immediately after irradiation, relaxation of DNA-loops due to strand breaks was observed. This relaxation had a similar dose – response with saturation at 2 – 3 Gy. This dose induced approximately one single-strand break (SSB) per 2 Mb of DNA, a value close to the average size of DNA-loops in resting lymphocytes. Similar saturations in dose – responses for apoptosis and DNA-loop relaxation were also observed if cells were treated by camptothecin (CPT) or etoposide VP-16, drugs that relax DNA-loops by induction of SSB and DSB, respectively. The PFGE data showed that the vast majority of DSB were repaired within few hours after irradiation. However, approximately 1.4 foci/Gy/cell, that corresponded to around 3.5% of initial DSB, remained in cells even 24 h after irradiation as measured with immunostaining. The probability to produce one or more than one residual foci per cell was calculated. Radiation at 2 – 3 Gy induced at least one residual 53BP1 focus per cell. The dose – responses for DNA-loop relaxation, induction of at least one residual 53BP1 foci per cell and apoptosis saturated at 2 – 3 Gy. The correlation between dose – responses obtained suggested that the DSB in residual foci and relaxation of DNA-loops may be linked to induction of radiation-induced apoptosis in lymphocytes.

Solving Gene Expression

### Contents #### News Getting the Noise Out of Gene Arrays [Searching for the Genome's Second Code][1] A Fast and Furious Hunt for Gene Regulators #### Reviews and Viewpoints The ENCODE (ENCyclopedia Of DNA Elements) Project The ENCODE Project Consortium Systems Biology and New Technologies Enable Predictive and Preventative Medicine L. Hood, J. R. Heath, M. E. Phelps, B. Lin Gene Order and Dynamic Domains S. T. Kosak and M. Groudine Cis-Acting Regulatory Variation in the Human Genome T. Pastinen and T. J. Hudson See related [STKE and SAGE KE material][2], Editorial, [Perspective][3], papers by Liao et al. and [Stolc et al.][4], and the [Functional Genomics][5] Web site. W ords are static images on a page unless you know what they mean, how their meanings change depending on the context, and what the rules are for using them. Similarly, a complex regulatory code is buried within the genome, and researchers will need to decipher it to understand how genes are expressed, what their functions are, and how normal instructions are altered in disease. Throughout the magazine and online this week are features that describe different aspects of gene expression and its control. Kosak and Groudine (p. 644) describe how genomes may be organized (linearly and within three-dimensional space in the nucleus) for the regulation of gene expression. A News story by Pennisi (p. [632][1]) provides a clear introduction to the world of enhancers: regulatory elements that have been a pivotal force in evolution. Pastinen and Hudson (p. [647][6]) describe the pitfalls associated with analyses of cis-acting control mechanisms governing allele-specific differences in gene expression, some of which have been associated with disease susceptibility. At Science 's online Signal Transduction Knowledge Environment (STKE, [www.sciencemag.org/sciext/genome2004][2]) are features describing the dance of nuclear receptor complexes with DNA that lead to transcription (Fowler and Alaric) and approaches different organisms use to select genes within gene families for expression (Dalgaard and Vengrova). Aware of the magnitude of the challenge of developing a complete “parts list” for all of these activities, an international consortium of scientists has begun the ENCODE (ENCyclopedia Of DNA Elements) project (p. 636), whose goal is to identify all of the structural and functional elements of the human genome. In their pilot phase, researchers are comparing multiple approaches for detecting different elements on 30 Mb of DNA. Some of the medical applications of this information are tantalizing prospects, whereas others are already at our doorstep. Hood et al. (p. 640) present a sweeping view of how expression patterns will be combined with technological advances to further predictive medicine. However, it is not an entirely smooth path. For instance, as the number of gene array studies proliferates, some researchers are finding that they don't necessarily lead to quick diagnosis or prognosis (see News story by Marshall on p. 630). The Science Functional Genomics Web site ([www.sciencemag.org/feature/plus/sfg][5]) contains additional online discussion of whether microarrays have been oversold and how they can reach their full potential. The site also has updated links to other resources. At the Science of Aging Knowledge Environment (SAGE KE, [www.sciencemag.org/sciext/genome2004][2]) are articles describing how microarrays and other genome-scale technologies are being applied to aging research (Kaeberlein; Melov and Hubbard). The Editorial by Lord and Papoian (p. [575][7]) explores efforts to standardize microarray data so that regulatory agencies can use gene expression studies to evaluate drug safety. In the course of their analysis of RNA expression for protein-coding and non-protein-coding sequences during the Drosophila life cycle (p. 655), White and his group have come to see the task of assembling the functional parts of the genome as being like a Rubik's cube. Although ∼4.3 × 1019 different positions are possible, the cube can be resolved from any position by 29 or fewer manipulations. As we begin to understand biological systems through carefully designed experiments and analyses, the complexity we are seeing now may begin to resolve into simpler principles. [1]: /lookup/doi/10.1126/science.306.5696.632 [2]: http://www.sciencemag.org/sciext/genome2004 [3]: /lookup/doi/10.1126/science.1101104 [4]: /lookup/doi/10.1126/science.1101312 [5]: http://www.sciencemag.org/feature/plus/sfg [6]: /lookup/doi/10.1126/science.1101659 [7]: /lookup/doi/10.1126/science.1105854

Mutation Rate and Predicted Phenotypic Target Sizes in Ethylnitrosourea-Treated Mice

Chemical mutagenesis of the mouse is ongoing in several centers around the world, with varying estimates of mutation rate and number of sites mutable to phenotype. To address these questions, we sequenced approximately 9.6 Mb of DNA from G1 progeny of ethylnitrosourea-treated mice in a large, broad-spectrum screen. We identified 10 mutations at eight unique sites, including six nonsynonymous coding substitutions. This calibrates the nucleotide mutation rate for two mutagenesis centers, implies significance criteria for positional cloning efforts, and provides working estimates of effective genetic target sizes for selected phenotypes.

Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin.

The effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a reversible decondensation of dense chromatin regions and led to a more homogeneous distribution. These structural changes were quantified by image correlation spectroscopy and by spatially resolved scaling analysis. The image analysis revealed that a chromatin reorganization on a length scale from 200 nm to >1 microm was induced consistent with the opening of condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be assigned to the folding of chromatin during G1 phase by characterizing the effect of TSA on cell cycle progression and developing a protocol that allowed the identification of G1 phase cells on microscope coverslips. An analysis by flow cytometry showed that the addition of TSA led to a significant arrest of cells in S phase and induced apoptosis. The concentration dependence of both processes was studied.

Genome plasticity in Streptomyces: identification of 1 Mb TIRs in the S. coelicolor A3(2) chromosome.

The chromosomes of several widely used laboratory derivatives of Streptomyces coelicolor A3(2) were found to have 1.06 Mb inverted repeat sequences at their termini (i.e. long-terminal inverted repeats; L-TIRs), which are 50 times the length of the 22 kb TIRs of the sequenced S. coelicolor strain M145. The L-TIRs include 1005 annotated genes and increase the overall chromosome size to 9.7 Mb. The 1.06 Mb L-TIRs are the longest reported thus far for an actinomycete, and are proposed to represent the chromosomal state of the original soil isolate of S. coelicolor A3(2). S. coelicolor A3(2), M600 and J1501 possess L-TIRs, whereas approximately half the examined early mutants of A3(2) generated by ultraviolet (UV) or X-ray mutagenesis have truncated their TIRs to the 22 kb length. UV radiation was found to stimulate L-TIR truncation. Two copies of a transposase gene (SCO0020) flank 1.04 Mb of DNA in the right L-TIR, and recombination between them appears to generate strains containing short TIRs. This TIR reduction mechanism may represent a general strategy by which transposable elements can modulate the structure of chromosome ends. The presence of L-TIRs in certain S. coelicolor strains represents a major chromosomal alteration in strains previously thought to be genetically similar.

Analysis of Clinical Features of Williams-Beuren Syndrome Referred for Molecular Cytogenetic Study

Objective: Williams-Beuren syndrome (WBS) is a contiguous gene deletion disorder in which the commonly deleted region encompasses about 1.5~2.0 Mb of DNA at 7q11.23. Clinical features of WBS change with age and vary between patients. We attempted to establish useful indicators for clinical recognition of WBS in infancy and childhood. Materials and Methods: We analyzed the clinical characteristics of 25 patients referred to us to confirm a diagnosis of WBS. Those 25 patients were classified into 3 groups: group 1, younger than 3 years; group 2, between 3 and 5 years old; and group 3, older than 5 years. A diagnosis was made by fluorescence in situ hybridization (FISH) analysis. Results: Nineteen of the 25 suspected cases (76%) were found to have deletion of the elastin gene. Nine of the 14 patients in group 1 (64.29%) had the deletion as did 5 in 6 in group 2 (83.33%) and 5 in 5 (100%) in group 3. Seventy-two percent (18/25) of patients with deletion of the elastin gene had congenital cardiovascular defects and developmental delay, 68% (17/25) had dysmorphic features, 56% (14/25) had mental retardation, 40% (10/25) had a gregarious personality, 28% (7/25) had dental abnormalities in, 4% (1/25) had strabismus, 4% (1/25) had systemic hypertension. Most of the cases (19/25) with more than 2 phenotypic features were found to have deletion of the elastin gene, particularly those with cardiovascular defects. Conclusions: The clinical characteristics, including SVAS/PPS, mental retardation, developmental delay, a dysmorphic face, dental abnormalities, and a gregarious personality may be useful indicators of WBS. Although cardiovascular defects alone cannot serve as an absolute indicator, the combination of cardiovascular defects with 1 of the other phenotypic features can help make a diagnosis of WBS.

Analyzing Heterochromatin Formation Using Chromosome 4 of Drosophila melanogaster

organization within the nuclei of higher eukaryotes, the chromosome itself is further organized into multiple domains with distinct properties. This level of organization is visible in interphase nuclei as areas of condensed heterochromatin and regions of more dispersed euchromatin. Chromatin domains are biochemically distinguished by the types of histone modification and associated nonhistone chromosomal proteins. In most higher eukaryotes, domains of constitutive heterochromatin are normally restricted to pericentric and telomeric DNA. A remarkable property of heterochromatin in fungi, flies, and mammals is the ability to spread in cis, in response to loss of boundary constraints or to changes in dosage or activity of chromatin components; this results in silencing of euchromatic genes that are abnormally juxtaposed to heterochromatic domains by chromosome rearrangement or transposition, referred to as Position Effect Variegation (PEV) (for review, see Grewal and Elgin 2002). Recent studies in fungi and plants suggest that heterochromatin formation is targeted to repetitious elements through an RNAi mechanism, resulting in a domain of silenced chromatin (Volpe et al. 2002; Matzke et al. 2004; Schramke and Allshire 2004). The small fourth chromosome of Drosophila melanogaster exhibits a rather unusual chromatin organization compared to the other chromosome arms. The distal 20–25% of chromosome 4 is amplified in polytene nuclei to a similar extent as the other euchromatic arms, and contains 82 genes in 1.2 Mb of DNA (Flybase Consortium 2003), a gene density comparable to that found in other euchromatic regions. At the same time, chromosome 4 exhibits characteristics of heterochromatin throughout its length. Chromosome 4 is rich in dispersed repetitious sequences (Kaminker et al. 2002), shows no detectable meiotic recombination (Bridges 1935), and is late-replicating (Barigozzi et al. 1966), all well-established characteristics of heterochromatic regions. Further, immunofluorescent staining of polytene chromosomes shows that Heterochromatin Protein 1 (HP1), known to play a key role in heterochromatin-induced silencing, is localized both in the pericentric heterochromatin and across the whole of the fourth chromosome (Fig. 1A) (James et al. 1989; Eissenberg and Elgin 2000). Analyzing Heterochromatin Formation Using Chromosome 4 of Drosophila melanogaster

The comings and goings of a Y polymorphism

A deletion that removes 1.6 Mb of DNA and several genes from the Y chromosome increases its carriers' chance of infertility, yet is present in ∼2% of men. The Y chromosome can no longer be regarded as a neutral locus in evolutionary studies.

Inheritable variable sizes of DNA stretches in the human MHC: conserved extended haplotypes and their fragments or blocks.

The difference in sizes of conserved stretches of DNA sequence within the major histocompatibility complex (MHC) in human individuals constitutes an underappreciated genetic diversity that has many practical implications. We developed a model to describe the variable sizes of stretches of conserved DNA in the MHC using the known frequencies of four different kinds of small (< 0.2 Mb) blocks of relatively conserved DNA sequence: HLA-Cw/B; TNF; complotype; and HLA-DR/DQ. Each of these small blocks is composed of two or more alleles of closely linked loci inherited as one genetic unit. We updated the concept of the conserved extended haplotype (CEH) using HLA allele identification and TNF microsatellites to show that specific combinations of the four blocks form single genetic units (>/= 1.5 Mb) with a total haplotype frequency in the Caucasian population of 0.30. Some CEHs extend to the HLA-A and -DPB1 loci forming fixed genetic units of up to at least 3.2 Mb of DNA. Finally, intermediate fragments of CEHs also exist, which are, nevertheless, larger than any of the four small blocks. This complexity of genetic fixity at various levels should be taken into account in studies of genetic disease association, immune response control, and human diversity. This knowledge could also be used for matching CEHs and their fragments for patients undergoing allotransplantation.

Identification of a locus (LCA9) for Leber's congenital amaurosis on chromosome 1p36.

Leber's congenital amaurosis (LCA) is the most common cause of inherited childhood blindness and is characterised by severe retinal degeneration at or shortly after birth. We have identified a new locus, LCA9, on chromosome 1p36, at which the disease segregates in a single consanguineous Pakistani family. Following a whole genome linkage search, an autozygous region of 10 cM was identified between the markers D1S1612 and D1S228. Multipoint linkage analysis generated a lod score of 4.4, strongly supporting linkage to this region. The critical disease interval contains at least 5.7 Mb of DNA and around 50 distinct genes. One of these, retinoid binding protein 7 (RBP7), was screened for mutations in the family, but none was found.

Biallelic expression of HRAS and MUCDHL in human and mouse.

At least eight genes clustered in 1 Mb of DNA on human chromosome (Chr) 11p15.5 are subject to parental imprinting, with monoallelic expression in one or more tissues. Orthologues of these genes show conserved linkage and imprinting on distal Chr 7 of mice. The extended imprinted region has a bipartite structure, with at least two differentially methylated DNA elements (DMRs) controlling the imprinting of two sub-domains. We previously described three biallelically expressed genes ( MRPL23, 2G7 and TNNT3) in 100 kb of DNA immediately downstream of the imprinted H19 gene, suggesting that H19 marks one border of the imprinted region. Here we extend this analysis to two additional downstream genes, HRAS and MUCDHL (mu-protocadherin). We find that these genes are biallelically expressed in multiple fetal and adult tissues, both in humans and in mice. The mouse orthologue of a third gene, DUSP8, located between H19 and MUCDHL, is also expressed biallelically. The DMR immediately upstream of H19 frequently shows a net gain of methylation in Wilms tumors, either via Chr 11p15.5 loss of heterozygosity (LOH) or loss of imprinting (LOI), but changes in methylation in CpG-rich sequences upstream and within the MUCDHL gene are rare in these tumors and do not correlate with LOH or LOI. These findings are further evidence for a border of the imprinted region immediately downstream of H19, and the data allow the construction of an imprinting map that includes more than 20 genes, distributed over 3 Mb of DNA on Chr 11p15.5.

Resolution of fluorescence in-situ hybridization mapping on rice mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers.

Fluorescence in-situ hybridization (FISH) is a quick and affordable approach to map DNA sequences to specific chromosomal regions. Although FISH is one of the most important physical mapping techniques, research on the resolution of FISH on different cytological targets is scarce in plants. In this study, we report the resolution of FISH mapping on mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers in rice. A majority of the FISH signals derived from bacterial artificial chromosome (BAC) clones separated by approximately 1 Mb of DNA cannot be resolved on mitotic prometaphase chromosomes. In contrast, the relative positions of closely linked or even partially overlapping BAC clones can be resolved on a euchromatic region of rice chromosome 10 at the early pachytene stage. The resolution of pachytene FISH is dependent on early or late pachytene stages and also on the location of the DNA probes in the euchromatic or heterochromatic regions. We calibrated the fiber-FISH technique in rice using seven sequenced BAC clones. The average DNA extension was 3.21 kb/microm among the seven BAC clones. Fiber-FISH results derived from a BAC contig that spanned 1 Mb DNA matched remarkably to the sequencing data, demonstrating the high resolution of this technique in cytological mapping.

Mapping and identification of essential gene functions on the X chromosome of Drosophila.

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.

Association between divergence and interspersed repeats in mammalian noncoding genomic DNA

The amount of noncoding genomic DNA sequence that aligns between human and mouse varies substantially in different regions of their genomes, and the amount of repetitive DNA also varies. In this report, we show that divergence in noncoding nonrepetitive DNA is strongly correlated with the amount of repetitive DNA in a region. We investigated aligned DNA in four large genomic regions with finished human sequence and almost or completely finished mouse sequence. These regions, totaling 5.89 Mb of DNA, are on different chromosomes and vary in their base composition. An analysis based on sliding windows of 10 kb shows that the fraction of aligned noncoding nonrepetitive DNA and the fraction of repetitive DNA are negatively correlated, both at the level of an entire region and locally within it. This conclusion is strongly supported by a randomization study, in which repetitive elements are removed and randomly relocated along the sequences. Thus, regions of noncoding genomic DNA that accumulated fewer point mutations since the primate-rodent divergence also suffered fewer retrotransposition events. These results indicate that some regions of the genome are more "flexible" over the time scale of mammalian evolution, being able to accommodate many point mutations and insertions, whereas other regions are more "rigid" and accumulate fewer changes. Stronger conservation is generally interpreted as indicating more extensive or more important function. The evidence presented here of correlated variation in the rates of different evolutionary processes across noncoding DNA must be considered in assessing such conservation for evidence of selection.

Identification and Sequencing the Juvenile Spermatogonial Depletion Critical Interval on Mouse Chromosome 1 Reveals the Presence of Eight Candidate Genes

In mice, the recessive, non-pleiotropic, juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis, followed by failure of type A spermatogonial stem cells to differentiate, rendering adult males sterile. As part of an effort to identify the gene underlying this mutation, we report here the construction of a high-resolution genetic map involving more than 1000 meioses and 24 polymorphic loci. Our data define a critical jsd interval of approximately 0.4 cM at 49 cM on mouse chromosome 1, between D1Mit215 and 257SP6. We have constructed a physical map spanning the region comprising 24 overlapping BACs. Eighteen of these BACs have been fully sequenced, or are in draft form, allowing us to annotate approximately 2.5 Mb of DNA surrounding the jsd locus. The critical 0.4 cM jsd interval corresponds to a physical distance of ∼1.5 Mb. Eight genes have been identified in this interval, two of which appear to be possible candidates for the jsd mutation.

Sequence analysis and transcript identification within 1.5 MB of DNA deleted together with the NDP and MAO genes in atypical Norrie disease patients presenting with a profound phenotype.

Mutations at the Norrie disease gene locus, NDP, manifest in a broad range of defects. These range from a relatively mild, late-onset, exudative vitreoretinopathy to congenital blindness and sensorineural deafness combined, in some cases, with mental retardation. In addition, extensive deletions involving the NDP locus, located at Xp11.3, the adjacent monoamine oxidadase genes MAOA and MAOB, and additional material, result in a more severe pattern of symptoms. The phenotypes include all or some of the following; mental retardation, involuntary movements, hypertensive crises and hypogonadism. We extended an existing YAC contig to embrace the boundaries of three of the largest deletions and converted this into four PAC contigs. Computer analysis and experimental data have resulted in the identification of several putative loci, including a phosphatase inhibitor 2-like gene (dJ154.1) and a 250-bp sequence which resembles a homeobox domain (dA113.3), 1.2 Mb and 400 kb respectively from the MAO/NDP cluster. The pattern of expression of dJ154.1 suggests that it may represent an important factor contributing to the complex phenotypes of these deletion patients. Hum Mutat 17:523, 2001.

The Human Contactin-Associated Protein-like 2 Gene (CNTNAP2) Spans over 2 Mb of DNA at Chromosome 7q35

Contactin-associated genes are members of the neurexin superfamily that encode a group of transmembrane proteins that mediate cell–cell interactions in the nervous system. To study the human contactin-associated protein-like 2 gene (CNTNAP2), we have determined its complete DNA sequence and its genomic organization to comprise 25 exons spanning greater than 2.0 Mb of DNA at 7q35. Our results indicate that CNTNAP2 encompasses almost 1.5% of chromosome 7 and is one of the largest genes in the human genome.

Genome sequence of E. coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 is the agent responsible for several outbreaks of bloody diarrhea in the USA, UK, Japan and many other countries. Recently Perna and collaborators 1xGenome sequence of enterohaemorrhagic Escherichia coli O157:H7. Perna, N.T. Nature. 2001; 409: 529–533Crossref | PubMed | Scopus (1329)See all References1 reported the genome sequence of this pathogen and compared it with the already known sequence of the non-pathogenic E. coli K-12. The availability of this genome sequence will help the identification of genes responsible for pathogenesis, help develop better methods for detection of this strain and also enlighten our knowledge of E. coli evolution. These authors observed that E. coli O157:H7 has 1.34 Mb of DNA that is absent in K-12, and K-12 has 0.53 Mb of DNA that is absent in O157:H7. These strains share a common ‘backbone’ sequence of about 4.1 Mb that is co-linear except for one 422 kb inversion spanning the replication terminus. The ‘backbone’ is punctuated by hundreds of islands of apparently introgressed DNA designated ‘K-islands’ for the DNA segments present only in E. coli K-12 and ‘O-islands’ for DNA segments present only in E. coli O157:H7. These results suggest that lateral gene transfer is far more extensive than previously anticipated. Of the O-islands, only 40% could be assigned a function and many classifiable proteins are related to known virulence-associated proteins from other E. coli strains or related enterobacteria. Together, these findings reveal a great level of diversity between two members of the E. coli species. Most differences are attributable to horizontal transfer and offer a wealth of candidate virulence genes.

The pericentromeric region of human chromosome 11: evidence for a chromosome-specific duplication

We have identified a chromosome duplication in the pericentromeric region of human chromosome 11 located in 11p11 and 11q14. A detailed physical map of each duplicated region was generated to describe the nature of the duplication, the involvement at the centromere and to resolve the correct maps. All clones were evaluated to ensure they were representative of their genetic origin. The order of clones, based on their marker content, as well as the distance covered was determined by SEGMAP. Each duplication encompasses more than 1 Mb of DNA and appears to be chromosome 11 specific. Ten STS markers were mapped within each duplication. Comparative sequence analysis along the duplication identified 35 nucleotide changes in 2,036 bp between the two copies, suggesting the duplication occurred over 14 million years ago. A suggested organization of the pericentromeric region, including the duplications and alpha-related repetitive sequences, is presented.

Genetic organization of the human MHC class III region.

The human major histocompatibility complex (MHC), or human leukocyte antigen (HLA) region, encompasses over 4 Mb of DNA on the short arm of chromosome 6 and is traditionally divided into the class I, class II and class III regions. The MHC has now been entirely sequenced and ~220 genes have been defined of which ~62 are in the class III region. It is becoming clear that many of the latter encode proteins that are likely to be involved in the immune and inflammatory responses. The MHC is known to contribute to a large number of immune-related disorders including insulin dependent diabetes mellitus, rheumatoid arthritis, common variable immunodeficiency and IgA deficiency and there is growing evidence that genes within the class III region are important in determining susceptibility to many of these complex conditions. Genes in the class III region have also been implicated in a number of non-immune-related diseases such as congenital adrenal hyperplasia and sialidosis. Now that the full gene content of the class III region is known the stage is set for the identification and characterisation of candidate disease genes, which will allow greater understanding of the causes of many MHC-linked diseases and thus aid the development of improved treatments.