Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

A variable number of tandem repeats locus within the human complement C2 gene is associated with a retroposon derived from a human endogenous retrovirus.

We have previously described multiallelic restriction fragment length polymorphisms of the C2 gene, suggesting the presence of a variable number of tandem repeats (VNTR) locus. We report here the cloning and sequencing of the polymorphic fragments from the two most common alleles of the gene, a and b. The results confirm the presence of a VNTR locus consisting of a nucleotide sequence, 41 bp in average length, repeated tandemly 23 and 17 times in alleles a and b, respectively. The difference in the number of repeats between the two alleles is due to the deletion/insertion of two noncontiguous segments, 143 and 118 bp long, of allele a, and of a 40-bp segment of allele b. The VNTR region is associated with a SINE (short interspersed sequence)-type retroposon, SINE-R.C2, located within the third intron of the C2 gene. SINE-R.C2 is a member of a previously described large retroposon family of the human genome, apparently derived from the human endogenous retrovirus, (HERV) K10, which is homologous to the mouse mammary tumor virus.

Genetic control of an insect neuronal network.

Motor activity responsible for the calling song of crickets is generated by a small neuronal network whose output is genetically determined. Genes controlling certain output features are located on the X chromosome. The genetic system involved is polygenic and multichromosomal. In some patterns, genetically derived information is adequate to specify the difference of a single impulse in the output of homologous neurons from different genotypes.

Postembryonic development of adult motor patterns in crickets: a neural analysis.

Adult crickets have stereotyped patterns of motor output which are generated by the central nervous system, and which serve as a standard against which emerging nymphal patterns can be measured. The neural circuits generating these patterns are not functional at hatching. The pattern elements appear in an ordered sequence over the course of the last four molts. The circuits are completely functional before the final molt. Circuits which might be prematurely activated are suppressed in the nymph by descending inhibition from the brain.

Analysis of vertebrate SCL loci identifies conserved enhancers.

The SCL gene encodes a highly conserved bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis. We have sequenced and analyzed 320 kb of genomic DNA composing the SCL loci from human, mouse, and chicken. Long-range sequence comparisons demonstrated multiple peaks of human/mouse homology, a subset of which corresponded precisely with known SCL enhancers. Comparisons between mammalian and chicken sequences identified some, but not all, SCL enhancers. Moreover, one peak of human/mouse homology (+23 region), which did not correspond to a known enhancer, showed significant homology to an analogous region of the chicken SCL locus. A transgenic Xenopus reporter assay was established and demonstrated that the +23 region contained a new neural enhancer. This combination of long-range comparative sequence analysis with a high-throughput transgenic bioassay provides a powerful strategy for identifying and characterizing developmentally important enhancers.

Chromosome 20 deletions in myeloid malignancies: reduction of the common deleted region, generation of a PAC/BAC contig and identification of candidate genes. UK Cancer Cytogenetics Group (UKCCG).

Deletion of the long arm of chromosome 20 represents the most common chromosomal abnormality associated with the myeloproliferative disorders (MPDs) and is also found in other myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Previous studies have identified a common deleted region (CDR) spanning approximately 8 Mb. We have now used G-banding, FISH or microsatellite PCR to analyse 113 patients with a 20q deletion associated with a myeloid malignancy. Our results define a new MPD CDR of 2.7 Mb, an MDS/AML CDR of 2.6 Mb and a combined 'myeloid' CDR of 1.7 Mb. We have also constructed the most detailed physical map of this region to date--a bacterial clone map spanning 5 Mb of the chromosome which contains 456 bacterial clones and 202 DNA markers. Fifty-one expressed sequences were localized within this contig of which 37 lie within the MPD CDR and 20 within the MDS/AML CDR. Of the 16 expressed sequences (six genes and 10 unique ESTs) within the 'myeloid' CDR, five were expressed in both normal bone marrow and purified CD34 positive cells. These data identify a set of genes which are both positional and expression candidates for the target gene(s) on 20q.

Characterisation of a novel murine intestinal serine protease, DISP.

A putative novel murine serine protease, DISP, was identified by cDNA indexing and shown to be expressed primarily in distal gut. FISH analysis showed it to be localised to mouse chromosome 17A3. A possible human homologue for DISP has been identified. DISP is a novel member of clan SA/family S1 of the serine proteases, at present of unknown function.

C2 and factor B: structure and genetics.

Complement components C2 and factor B are novel types of serine protease that are encoded by single loci in the major histocompatibility complex on human chromosome 6. The two proteins share 39% homology, or 50% taking into account conservative amino acid replacements. The catalytic chains, C2a (509 residues) and Bb (505 residues) show homology in their C-terminal domains to the catalytic polypeptides of other serine proteases. The non-catalytic chains, C2b (223 residues) and Ba (234 residues) both contain three tandem repeats of approx. 60 amino acids each, which are homologous to the repeats in C4b-binding protein and factor H, and also the repeats in the non-complement protein beta 2-glycoprotein I. Molecular mapping and DNA sequence analysis has shown that the factor B gene is 6 kb in length and contains 18 exons, while the C2 gene is 18 kb in length; 425 bp separates the 3' end of the C2 gene from the 5' end of the factor B gene. C2 and factor B are polymorphic and structural variants have been detected at the protein level by differences in charge. The degree of polymorphism at the factor B locus has been defined by DNA sequence analysis of the two common alleles F and S. In addition restriction fragment length polymorphisms have been detected in the C2 gene. These DNA polymorphisms subdivide the common allelic variant of C2 (C2C) and reveal that there is much greater variability at the C2 locus than that detected by protein typing.

Improved method for detecting differentially expressed genes using cDNA indexing.

In cDNA indexing, differentially expressed genes are identified by the display of specific, corresponding subsets of cDNA. Subdivision of the cDNA population is achieved by the sequence-specific ligation of adapters to the overhangs created by class IIS restriction enzymes. However, inadequate specificity of ligation leads to redundancy between different adapter subsets. We evaluate the incidence of mismatches between adapters and class IIS restriction fragments during ligation and describe a modified set of conditions that improves ligation specificity. The improved protocol reduces redundancy between amplified cDNA subsets, which leads to a lower number of bands per lane of the differential display gel, and therefore simplifies analysis. We confirm the validity of this revised protocol by identifying five differentially expressed genes in mouse duodenum and ileum.

The use of DNA amplification for genetic counselling related diagnosis in haemophilia B.

A single base pair variation in the coding sequence of coagulation factor IX produces a protein polymorphism detectable with monoclonal antibodies and a restriction fragment length polymorphism (RFLP). This allows carrier and prenatal diagnoses in 48% of Caucasian families segregating for haemophilia B. However, this RFLP cannot be detected by standard Southern blotting, while the antibody assay may give equivocal results in some females and can only allow prenatal diagnoses on second trimester fetal blood samples. We show that, using the polymerase chain reaction, the polymorphic DNA segment can be amplified and directly tested for the presence of the alternative sequences by a non-radioactive procedure that has the advantage of speed (1-2 days), partial automation and applicability to first trimester diagnoses. We also show that the method gives results on a single drop of dried blood.

Genetics and molecular biology of haemophilias A and B.

The development of rapid procedures for the characterization of mutations is advancing the knowledge of the molecular biology of the haemophilias and transforming the strategies for the diagnoses required for genetic counselling. In haemophilia B more than 300 mutants have been fully characterized. These comprise complete and partial deletions, rare insertions, and 'point' mutations. The latter may impair transcription (promoter mutations), RNA processing (splicing mutations) and translation (frameshifts and stop codons) or cause single amino acid (aa) changes. Eighty-four residues are involved in the 105 presumed detrimental aa substitutions reported so far and these are usually conserved in the factor IX homologues (factors VII, X and protein C) and/or the factor IX of different mammalian species. There are clear correlations between the mutation and clinical features. In addition mutations causing gross physical or functional loss of coding information appear to predispose to the development of antibodies against therapeutic factor IX. Hotspots of mutations have been identified and are usually associated with CpG sequences. In haemophilia A the size and complexity of the factor VIII gene has hindered the analysis of mutants. Most of the studies published so far have analysed only a small fraction of the essential region of the factor VIII gene and this led to the repeated observation of specific types of mutation. The recent development of a rapid method to analyse RNA splicing and the whole coding region of the factor VIII gene should unblock this situation. With regard to genetic counselling, the direct detection of gene defects has increased the proportion of haemophilia B families that can be helped from 60% to virtually 100% and similar expectations may now be formulated for haemophilia A. In the UK a national database of haemophilia B mutations is being constructed to optimize genetic counselling. This should offer a model for a similar development in haemophilia A.

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.

Decoding the human genome sequence.

The year 2000 is marked by the production of the sequence of the human genome. A 'working draft' of high quality sequence covering 90% of the genome has been determined and a quarter is in finished form, including the first two completed chromosomes. All sequence data from the project is made freely available to the community via the Internet, for further analysis and exploitation. The challenge which lies ahead is to decipher the information. Knowledge of the human genome sequence will enable us to understand how the genetic information determines the development, structure and function of the human body. We will be able to explore how variations within our DNA sequence cause disease, how they affect our interaction with our environment and ultimately to develop new and effective ways to improve human health.

Structural superfamilies of the complement system.

The complete primary structures of nearly all the components, regulatory proteins and membrane-associated receptors of the complement system have now been determined by a combination of protein and cDNA sequence analysis. Based on homologies at the amino acid sequence level, three distinct classes of protein domain are evident. They are the C3, C4, C5 family; the serine proteases, and the short consensus repeat family, which is characterized by the widespread occurrence of a novel 60 amino acid repeat structure. A particular feature of the repeat structures in this last family is that they form the structural and functional domains of a range of complement proteins of distinct function, all of which interact with C3b and/or C4b. Detailed analysis of the gene structures of some of these proteins has been carried out. The results have shown that the intron/exon organization of the serine protease domains of factor B and C2 are related to the classical serine proteases at the DNA level. Each short consensus repeat in factor B, C2, C4b-binding protein, murine factor H and also the functionally unrelated interleukin-2 receptor in all except one case is encoded exactly by a single exon, which defines this novel structural unit at the DNA level.

The Human Genome Project--an overview.

The human genome sequence will underpin human biology and medicine in the next century, providing a single, essential reference to all genetic information. The international program to determine the complete DNA sequence (3,000 million bases) is well underway. As of January 2000, 50% of the sequence is available in the public domain. A comprehensive working draft is expected this year, and the entire sequence is projected to be finished in 2003. DNA sequencing is carried out on mapped, overlapping bacterial clones of 150-200 kb. The working draft comprises assembled unfinished sequence and is released immediately in the public domain. The draft sequence of each clone is then completed, by closing any remaining gaps and resolving any ambiguities, before the entire sequence is checked, annotated, and submitted to the public databases. The sequence of each clone is finished to an accuracy of >99.99%. The availability of a reference sequence of the genome provides the basis for studying the nature of sequence variation, particularly single nucleotide polymorphisms (SNPs), in human populations. SNP typing is a powerful tool for genetic analysis, and will enable us to uncover the association of loci at specific sites in the genome with many disease traits. SNPs occur at a frequency of approximately 1 SNP/kb throughout the genome when the sequence of any two individuals is compared. Programs to detect and map SNPs in the human genome are underway with the aim of establishing a SNP map of the genome during the next two years. The human genome sequence will provide a complete description of all the genes. Annotation of the sequence with the gene structures is achieved by a combination of computational analysis (predictive and homology-based) and experimental confirmation by cDNA sequencing. Detecting homologies between newly defined gene products and proteins of known function helps to postulate biochemical functions for them, which can then be tested. Establishing the association of specific genes with disease phenotypes by mutation screening, particularly for monogenic disorders, provides further assistance in defining the functions of some gene products, as well as helping to establish the cause of the disease. As our knowledge of gene sequences and sequence variation in populations increases, we will pinpoint more and more of the genes and proteins that are important in common, complex diseases. A more detailed understanding of the function of the human genome will be achieved as we identify sequences that control gene expression. Given the availability of gene sequences, the expression status of genes in particular tissues can be monitored in parallel. By comparing corresponding genomic sequences in different species (for example: man, mouse, chicken, and zebrafish), regions that have been highly conserved during evolution can be identified, many of which reflect conserved functions such as gene regulation. These approaches promise to greatly accelerate our interpretation of the human genome sequence.

Primary structure of human complement component C2. Homology to two unrelated protein families.

The primary structure of the second component of human complement (C2) was determined by cDNA cloning and sequence analysis. C2 has 39% identity with the functionally analogous protein Factor B. The C-terminal half of C2a is homologous to the catalytic domains of other serine proteinases. C2b contains three direct repeats of approx. 60 amino acid residues. They are homologous to repeats in Factor B, C4b-binding protein and Factor H, suggesting a functional significance of the repeat in C4b and C3b binding. The repeats are also found in the non-complement proteins beta 2-glycoprotein I and interleukin-2 receptor, and this repeat family may be widespread.