DNA sequencing of the MHC class II region and the chromosome 6 sequencing effort at the Sanger Centre.

The human Major Histocompatibility Complex (MHC) is located on the short arm of chromosome 6 (6p21.3) and spans about 4 Mb. According to different gene families the MHC is subdivided into a class I, class II and class III region and many of its gene products are associated with the immune system and the susceptibility to various diseases. To date, we have sequenced about 40% (400 kb) of the class II region between HLA-DP and HLA-DQ and a coordinated effort to sequence the entire MHC is well underway. Analysis of the sequence revealed several novel genes and provides new insights into the molecular organisation and evolution of the MHC. All our data are publicly available via the MHC database (MHCDB) which allows rapid access, retrieval and display in the context of other MHC associated data. MHCDB is online available at (http:(/)/www.hgmp.mrc.ac.uk/) and, together with all our sequences also via anonymous ftp (ftp.icnet.uk/icrf-public).

Evolutionary dynamics of non-coding sequences within the class II region of the human MHC.

About 40% (350 kb) of the human MHC class II region has been sequenced and a coordinated effort to sequence the entire MHC is underway. In addition to the coding information (22 genes/pseudogenes), the non-coding sequences reveal novel information on the organisation and evolution of the MHC as demonstrated here by the example of a 200 kb contig that has been analysed for local and global features. In conjunction with cross-species comparisons, our results present new evidence on the structure of isochores, the evolutionary dynamics of repeat-mediated recombination and its effect on certain MHC encoded genes, and a higher than average degree of natural polymorphism that has implications for sequencing the human genome. We also report the finding of a class I-related pseudogene (HLA-ZI) in the middle of the class II region, which provides the first direct evidence for DNA exchange between these two related regions in man.

Cloning and characterization of human phosphatase inhibitor-2 (IPP-2) sequences.

cDNA clones similar to rabbit muscle phosphatase inhibitor-2 (IPP-2) were isolated from human libraries. On Northern blots two transcripts of approximately 2kbp and approximately 4kbp were detected in all tissues tested. Analysis of cDNA sequences showed that the longer transcripts were similar to the shorter clones but contained extended 3' ends. The human nucleotide sequence was highly homologous (94% identity) to the rabbit IPP-2 sequence and encoded a peptide of 205 amino acids. IPP-2 sequences were highly conserved throughout vertebrates. Southern hybridization results were consistent with the existence of a family of related IPP-2 sequences in the human genome. Most of these are likely to be pseudogenes, since all of the cDNA clones examined could have originated from a single gene. By in situ hybridization IPP-2 sequences were mapped to several different human chromosomes. We sequenced one gene located in the major histocompatibility complex (MHC) on Chromosome (Chr) 6 that contained the entire coding region of IPP-2.

Genomic organization of HLA-DMA and HLA-DMB. Comparison of the gene organization of all six class II families in the human major histocompatibility complex.

The genomic nucleotide sequences of HLA-DMA and HLA-DMB have been determined and their gene organizations have been compared with other human class II genes. The following features were found to be highly conserved throughout all human class II families. (i) All alpha genes are composed of 5 exons and all beta genes of 6 exons. (ii) The intron-exon boundary classes of exons 1-4 (alpha genes) and exons 1-5 (beta genes) are 100% conserved. Only the last boundary class which falls within the cytoplasmic domain appears to be variable. (ii) The size of exon 3 (membrane proximal domain) is also 100% conserved except for DMA (-1 codon) and DMB (+1 codon). The position and a possible functional implication of this deletion/insertion are discussed. Our findings confirm and extend the evidence based on sequence homology that DMA and DMB are different from typical class II genes suggesting that they may originate from a time prior to the divergence of the main class II genes. In addition we have identified various new repeat sequences within class II genes. Analysis of their classification and distribution reveal single and multiple repeat mediated recombination events. One of these events seems to have partially replaced exon 1 in DPA2. The possibility of this event causing DPA2 to become a pseudogene is discussed.

Automated DNA hybridization.

We have developed a system which fully automates the process of membrane-immobilized DNA hybridization. The system consists of three major parts: (i) a membrane-processing cassette which is thermo-controlled and which can hold multiple membranes simultaneously; (ii) a fluidic system which consists of seven independent input channels, a collecting waste container, and flow-controlling valves; and (iii) integrating software which controls the entire system. No pumps are required since the entire fluidic system is gravity driven. The hybridizer is compatible with nonradioactive detection such as chemiluminescence and has been tested on gridded template arrays as part of a large-scale DNA sequencing project.

A strategy for the amplification, purification, and selection of M13 templates for large-scale DNA sequencing.

A strategy addressing various aspects of generating and processing DNA sequencing templates is described. The strategy is designed for large-scale DNA sequencing projects and involves procedures for the amplification, purification, and selection of M13 DNA templates. The amplification is based on growth in liquid culture, the purification is based on a novel thermoextraction method which replaces phenol extraction and ethanol precipitation, and the template selection is based on hybridization of gridded template arrays on membranes with chemiluminescent probes. All steps are currently carried out manually but the use of standardized formats (96-well format) throughout the entire strategy provides for possible transition to partial or full automation in the future. The strategy has been developed and tested within the ongoing project of sequencing the class II region of the human major histocompatibility complex but it should be equally applicable to any large-scale DNA sequencing project.

DNA sequence analysis of 66 kb of the human MHC class II region encoding a cluster of genes for antigen processing.

The genomic sequence of a 66,109 bp long region within the human MHC has been determined by manual and automated DNA sequencing. From cDNA mapping and sequencing data it is known that this region contains a cluster of at least four genes that are believed to be involved in antigen processing. Here, we describe the genomic organization of these genes, which comprise two proteasome-related genes (LMP2 and LMP7), thought to be involved in the proteolytic degradation of cytoplasmic antigens and two ABC transporter genes (TAP1 and TAP2), thought to be involved in pumping of the degraded peptides across the endoplasmic reticulum membrane. Analysis of the sequence homology and the intron/exon structures of the corresponding genes suggests that one gene pair arose by duplication from the other. Comparison of the available sequence data from other organisms shows striking conservation (70 to 84%) of this gene cluster in human, mouse and rat. The presence of several potential interferon stimulated response elements (ISREs) is in agreement with the experimentally observed up-regulation of these genes with gamma-interferon.

Magnetic bead purification of M13 DNA sequencing templates.

A method for the preparation of multiple M13 DNA sequencing templates is described. No phenol extraction is required and the procedure can be carried out in Eppendorf tubes or 96-well microtiter plates. Starting with a phage supernatant, the entire procedure is carried out in the same reaction vessel and all separation steps are based on a novel application of magnetic bead separation. The design of a 96-well magnetic separator is presented and the application of the method for large-scale sequencing is discussed.