The complete genomic sequence of 424,015 bp at the centromeric end of the HLA class I region: gene content and polymorphism

Abstract

We report here the genomic sequence of the centromeric portion of HLA class I, extending 424,015 bp from tumor necrosis factor alpha to a newly identified gene approximately 20 kb telomeric of Otf-3. As a source of DNA, we used cosmids centromeric of HLA-B that had been mapped previously with conventional restriction digestion and fingerprinting and previously characterized yeast artificial chromosomes subcloned into cosmids and mapped with multiple complete digest methodologies. The data presented provide a description of the gene content of centromeric HLA class I including new data on intron, promoter and flanking sequences of previously described genes, and a description of putative new genes that remain to be characterized beyond the structural information uncovered. A complete accounting of the repeat structure including abundant di-, tri-, and tetranucleotide microsatellite loci yielded access to precisely localized mapping tools for the major histocompatibility complex. Comparative analysis of a highly polymorphic region between HLA-B and -C was carried out by sequencing over 40 kb of overlapping sequence from two haplotypes. The levels of variation observed were much higher than those seen in other regions of the genome and indeed were higher than those observed between allelic HLA class I loci.

Figure 1

Schematic representation of the region sequenced in centromeric HLA class I. (A) The location of the region sequenced within HLA class I is identified and expanded beneath the HLA class I map. On the larger map, the locations of HLA class I genes are indicated by letters and of pseudogenes by numbers (20). In the expanded map, the locations of cosmids obtained from Spies et al. (24) and of YACs (19) and the cosmids generated from them are indicated beneath. Cosmids derived from YACs are named with the derivative YAC number followed by a designated cosmid number. (B) The positions and transcriptional orientations of known genes within the region are indicated to scale.

Figure 2

Multiple complete digest maps of YAC-derived cosmids. Restriction maps for the three indicated enzymes were constructed as described for YACs 3, 5, and 14 (19). The distance spanned is indicated at the top starting with the leftmost mapped restriction site. Only complete fragments verified by more than one cosmid subclone are included in the map, resulting in a portion of the sequences at the ends of the YACs to be excluded. YACs 3 and 14 were derived from the same chromosome, and YAC 5 was derived from the homologous chromosome in CGM1. The constructed maps verified this and identified a region between HLA-B and -C (indicated below) with substantial restriction site variability between YACs 5 and 3 (shaded areas). When comparing the maps derived for YACs 3 and 14, an apparent deletion in YAC 3 was detected as discussed in the text (bounded by vertical dotted lines).

Figure 3

Sequence comparison of allelic regions between HLA-B and -C. The sequence of cosmid Y5C028 was compared over the first 3 kb with cosmid O32A, which was derived from a non-CGM1 HLA haplotype (24) and over positions 4,000–42,000 with cosmid Y3C071, derived from YAC 3. cross_match analysis in sequential 1-kb blocks of Y5C028 against O32A or Y3C071 was used with the following parameters: minmatch 20, minscore 40, bandwidth 14, indexwordsize 10, masklevel 80. Y5C028 and Y3C071 span the region between HLA-B and -C identified by MCD mapping as highly variable between the two CGM1 chromosomes (shaded region in Fig. 2). Each bar represents the percentage difference over the 1-kb region compared. Included at the left of the bar graph is a similar comparison of the divergence between the HLA-B locus contained in O32A and a genomic HLA-B locus from the database. Below is a depiction of the genetic content including genome wide repeats and other homologies as discussed in the text, aligned to scale with the bar graph. Solid arrows indicate alu repeats, open arrows indicate MIR repeats, and shaded boxes indicate reverse transcriptase homologies. CHU, class I homology unit.

Figure 4

The gene content of centromeric HLA class I. The positions of genes, pseudogenes, and gene fragments identified by blastn and blastx–beauty analysis are indicated above and below the scale with the relative width of the bar indicating the extent of the gene. The given names of previously described genes (see ref. for references) are indicated above (transcriptional orientation from left to right) and below (transcriptional orientation from right to left) the bars, with the following abbreviations: Rvs, reverse transcriptase homology; HLA CLI fgt, homology to HLA-class I genomic fragment; FGF-R fgt, fibroblast growth factor receptor gene fragment. New putative genes identified as the result of this work are indicated immediately above the scale as Pg1–9 (putative gene) with homologies as described in Table 1.