Journal of Molecular Biology
Regular articleLarge-scale sequence comparisons reveal unusually high levels of variation in the HLA-DQB1 locus in the class II region of the human MHC1
Introduction
The human major histocompatibility complex (MHC) spans about 4 Mb on the short arm of chromosome 6 (6p21.3). Many of its gene products are highly polymorphic and have been shown to be associated with the immune system, and with susceptibility and resistance to various diseases Klein 1986, Trowsdale 1995. Because of this special interest, the MHC was targeted and subdivided for early sequencing at the last chromosome 6 workshop (Beck et al., 1997). We have been focusing on the MHC class II subregion, where we previously reported higher than average levels of variation in the non-coding sequences of two loci flanking HLA-Z1 and HLA-DOB (Beck et al., 1996). Furthermore, the availability of the genomic sequence has contributed towards the identification of 127 novel polymorphic markers within the class II region that were used to characterise the recombination pattern and several recombination hotspots in this region (Cullen et al., 1997). The identification of so-called single-nucleotide polymorphisms (SNPs), which are a specific type of sequence variation, is expected to contribute towards the understanding of complex diseases and disorders such as diabetes, asthma and others Collins et al 1997, Schafer and Hawkins 1998. To facilitate this, it is important that such data are made available in public databases. As part of the human chromosome 6 mapping and sequencing project, we have created such a database (6ace) in which we maintain and curate a variety of chromosome 6-associated data, including sequence variation (Theaker et al., 1997). The long-established association with diseases such as diabetes and narcolepsy have made the DQB1 locus a particularly competitive target for genomic sequencing Morel et al 1988, Mignot et al 1994. Although about 8 kb of the DQB1 gene were sequenced 15 years ago Larhammar et al 1983, Boss and Strominger 1984, the entire locus including the surrounding regions were sequenced only recently by us (this work) and by Ellis and co-workers during a study of predisposing factors to narcolepsy (Ellis et al., 1997). Variations between the early DQB1 sequences have been described (Radley et al., 1994). By analysing over 220 kb of overlapping sequences, we report here the level of genomic sequence variation in the MHC DQ locus and that of the non-MHC linked, spinocerebellar ataxia type 1 (SCA1) locus for comparison.
Section snippets
Results and discussion
Figure 1 shows the chromosomal locations of the DQB1 and SCA1 loci that have been analysed for sequence variation. Clones p797a11 and F1121, E1448, and 93N13 of the DQB1 locus (6p21.3) overlap by 86.2 kb and clones SGII and 467D16 of the SCA1 locus (6p23) overlap by 138.5 kb. In addition to providing a source for variation data, the availability of clone p797a11 (Ellis et al., 1997) indicated to us that the two gaps (between 93N13 and E1448 and E1448 and F1121) in our clone contig map had been
DNA sequencing and analysis
Cosmids F1121 and E1448 were isolated from the ICRF chromosome 6 library (RPETO1 cell line: Nizetic et al., 1994), whereas PACs 93N13 and 467D16 were isolated from the RPCI-1 PAC library (HSF7 cell line: Ioannou et al., 1994). The entire clones were randomly subcloned into M13mp18 and pUC18 (Bankier et al., 1987). Recombinant clones (80% M13 s, 20% pUCs) were picked, amplified and purified in 96-well microtitre plates (Beck and Alderton 1993, Mardis 1994; A. Smith & L. Baron, unpublished
Acknowledgements
We thank E. Mignot and D. Ruddy for providing sequence and associated data of clone p797a11 prior to publication; and S. Dear, I. Dunham, J. Kaufman and members of the chromosome 6 project group for comments and contributions (http://www.sanger.ac.uk/HGP/Chr6/). This work was funded by the Wellcome Trust.
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