RESULTS

General: To simplify the interpretation, we identified nonfunctional intergenic regions for amplification. Presumably such regions evolve without the confounding influence of phenotypic selection. Our initial amplifications were made in the absence of genomic sequence data by using linker amplification to extend the sequence from known intergenic segments (Geraghtyet al. 1992; Leelayuwatet al. 1992) and by amplifying it into adjacent DNA segments. The subsequent sequencing of the TNF to HLA-B region (Mizukiet al. 1997; Guillaudeuxet al. 1998; Shiinaet al. 1998) simplified the task of identifying and choosing regions and of evaluating those already chosen.

Several duplications in the HLA class I region have been revealed by large-scale sequencing and they include some of the initially chosen segments for amplifications (notably, CL1, FGF, and DIST). Two factors enabled us to identify paralogous PCR amplification products when they occurred. First, where two or more different sequences were obtained from a putatively homozygous source, it is likely that paralogy is involved. Second, phylogenetic analysis revealed the clustering of paralogous segments into a clade separate from the targeted product. Since the genomic sequences are known in the Boleth haplotype (Mizukiet al. 1997), it was possible to assign definitive locations to the fragments amplified.

Much of the intergenic region is nearly identical in the fragments we sequenced and so the alignment is straightforward. There are many single substitutions, however, leading to a moderate degree of polymorphism. When an orthologous region was amplifiable, we used the chimpanzee and gorilla sequences to determine the likely direction of substitutions for each region. Figure 1 shows the approximate positions of the segments we amplified, as well as the positions of genes in the TNF to HLA-B interval. The number of nucleotides compared excluding gaps was 5664 bp, of which 263 sites are segregating. Of the segregating sites, 132 are phylogenetically informative and the remaining 131 are singletons. The description of the individual regions follows.

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Figure 1.

—Map of the TNF to HLA-B interval showing the approximate locations of the amplified fragments used in this study. Open boxes indicate the location of known genes. Thick lines indicate the location of PCR fragments.

TNF: The amplified segment of 665 bp is ∼1.5 kb telomeric from the stop codon of the TNF gene and some 200 kb distant from HLA-B. It contains part of an Alu element but shows no evidence of divergent duplicate copies. Single substitutions were found in EHM (A to T at site 284 and T to C at 461), in Renchr (T to A at 436), in EA (C to T at 440), and in DKB (A to G at 672). There are two published sequences encompassing this region. One of them (Guillaudeuxet al. 1998) is identical with our Boleth, Brip, DBB, and Madura sequences. The other (Iriset al. 1993, accession no. Z15026), reportedly from Boleth, shows several differences from the Boleth sequence reported here and the sequence of Guillaudeux et al. (1998). It differs from all sequences presented here by deletion of C at site 330, insertion of G at 386, and single substitutions at sites 373, 374, 375, 430, 433, 436, 448, and 453. None of the substitutions or indels were found in any of the sequences we examined, including Boleth. It was therefore excluded from our polymorphism estimates. In the set used, there are a total of five singleton polymorphic sites (Figure 2), giving a nucleotide diversity of 0.18% (SD 0.06%) at this locus.

CL1: This region was originally identified as part of a duplicated segment on a restriction enzyme fragment of 6 kb by Leelayuwat et al. (1992). It lies ∼75 kb distal from TNF and 130 kb proximal to HLA-B. Several primers were designed to amplify a segment of CL1 of up to 1.46 kb encompassing a single Alu element (sites 696–1034). None of a variety of PCR primers could amplify the CL1 segment from chimpanzee or gorilla, CL1 being presumably deleted in these two species. An Alu-less CL2 fragment could be obtained instead. Since CL1 and CL2 are related by duplication that occurred some 30 mya (Leelayuwatet al. 1992), the CL2 sequences can be used as outgroups to determine the substitutions that occurred in the CL1 sequences. Both CL2 and CL1 contain sequences related to the LTR8 repetitive element from chromosomes 7 and 14 (accession nos. AC002508 and AC005857).

Although only twice the size of the TNF segment, CL1 contains 50 polymorphic sites (Figure 2) compared with 5 found at TNF. The estimated nucleotide diversity of 1.24% (SD 0.19%) is more than six times greater than that estimated for TNF. Nineteen of the substitutions at the locus are informative. To determine the extent of incompatibility among the informative nucleotide sites in this region, an incompatibility matrix was produced (Takahata and Satta 1998b). Of the 1225 pairs of informative sites, 103 pairs in two phylogenetically distinct blocks were incompatible. In the first 700 bp, substitutions were scattered and no pattern of relationships between sequences emerged. By contrast, at the 3′ end of the CL1 fragment, the Brip and Madura sequences shared six sites and most sites were compatible (Figure 2).

CL2: The CL1 region and some 10 kb of its flanks were duplicated in an event estimated to have occurred some 30 mya (Leelayuwatet al. 1992), giving rise to a related segment called CL2. This segment lies 150 kb distal to TNF and ∼50 kb proximal to HLA-B. The amplified fragment is 449 bp in length and is homologous to part of the CL1 region. By positioning one primer across the site of the Alu insertion in CL1, we were able to preferentially amplify CL2, which does not contain the Alu element. A total of 15 polymorphic sites were found in humans, giving a nucleotide diversity of 1.52% (SD 0.27%). Of these, two sites occurred in single haplotypes and the remaining 13 were shared by at least two haplotypes (Figure 2). At the 5′ end (∼290 bp), the sequences form at least two distinct groups on phylogenetic trees, with EHM, EA, Madura, and Boleth falling in one group (with a total nucleotide diversity of 0.48%; SD 0.25%). Three other sequences (DBB, DKB and Brip) are identical and share 10 substitutions that distinguish them from the former group. However, at the 3′ end of the segment, the Boleth sequence clusters with Brip, DKB, and DBB, suggesting at least one recombination breakpoint in the region.

Despite the higher diversity at CL2 compared to CL1, no human sequence is more closely related to either the gorilla or the chimpanzee sequences than to other human sequences; in other words, we found no evidence for trans-species evolution of CL2. The high level of nucleotide diversity (1.5%) could indicate that the comparisons made are of a paralogous type. However, database searches could not identify any paralogs of CL2.

FGF: The FGF region is adjacent to a fibroblast growth factor-like pseudogene. We amplified a segment of 888 bp that lies 175 kb distal from TNF and 31 kb proximal to HLA-B. The segment was obtained as follows: a primer based on the sequence of Leelayuwat et al. (1996) was used in combination with a primer specific for a linker added to a restriction fragment that contained the Leelayuwat et al. (1996) sequence. The amplification product was sequenced and primers were then designed for the amplification of the FGF segment on the basis of this sequence. The primers were, however, not specific for this region; they also amplified at least one paralogous segment 50 kb proximal to the site described by Leelayuwat et al. (1996). The paralogous segment, labeled D83543-P in Figure 2, could be identified on the basis of sequences from Shiina et al. (1998). A second paralogous segment occurred at an unidentified position and only this segment could be obtained from the DBB cell line (Figure 2). The fixed sequence differences enabled us, however, to distinguish both paralogous amplification products, which group in distinct clades on phylogenetic trees. By these means we could identify the sequences derived specifically from the FGF region. After the exclusion of the DBB paralog, nine polymorphic sites were found in this segment, eight of which were singletons. There was no incompatibility among the substitutions. The nucleotide diversity of the seven orthologous sequences was 0.32% (SD 0.07%).

DHFR: The DHFR region lies adjacent to a DHFR pseudogene identified by Mizuki et al. (1997) and is 12 kb proximal to HLA-B. The amplified segment is 583 bp in length. In the sequences we analyzed, 15 polymorphic sites were found, giving a nucleotide diversity of 1.02% (SD 0.15%) for the eight human DNAs. Nine sites were phylogenetically informative and all sites are mutually compatible.

HLA-B: This is the locus under balancing selection that may be driving the polymorphism of adjacent regions. The HLA-B cDNA sequences for alleles found in seven of the eight cell lines have been characterized (Prasad and Yang 1996) and the HLA-B intron sequence is available for Boleth (Mizukiet al. 1997), DBB, and Madura (Cerebet al. 1996). To supplement the available data, we sequenced a segment encompassing exon 2 to exon 3 from each of the eight human sources. This collection enabled us to compare both the intronic and synonymous variation. In the intron 2 segments, the nucleotide diversity reaches 2.10% (SD 0.40%) for the haplotypes surveyed here. The variation at synonymous sites is higher at 3.36% (SD 0.36%) in the same haplotypes.

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Figure 2.

—Variant sites found in the complete alignments of six amplified segments adjacent to HLA-B. The label in boldface type gives the segment name. Paralogous human sequences (indicated by the accession code and the extension -P), as well as sequences of chimpanzee (Patr) and gorilla (Gogo), are shown where available. A consensus for each segment was determined by simple majority. A dash indicates identity with the consensus; an asterisk indicates a deletion. When available, known paralogs are shown immediately below the consensus sequences and other human sequences are grouped according to their similarities.

DIST: This segment lies 11 kb distal from HLA-B and 80 kb proximal to HLA-C. The primers we used were not specific for this site and amplified at least one other segment that presumably arose with the duplication of the ancestral HLA-B and HLA-C loci. The amplified fragment was 596 bp long and contained 41 variant sites (Figure 2). Only paralogous fragments could be obtained from the Renchr and DBB haplotypes. After the exclusion of the paralogous segments, the estimated nucleotide diversity in the remaining six haplotypes was 1.56% (SD 0.38%).

Other loci: In addition to the data we have obtained for these six loci from the eight human cell lines, other surveys have been conducted of coding sequences in the HLA-B to TNF interval. Most prominent among these is the search for polymorphism at the class I-related MICA and MICB loci (Bahramet al. 1994; Fodilet al. 1996; Andoet al. 1997; Pelletet al. 1997; Visseret al. 1998; Yaoet al. 1999). These surveys have concentrated on determining whether the sites of polymorphisms can be related to molecular function. However, the patterns found are quite unlike those of class I genes in which polymorphism is concentrated on sites specifying the peptide-binding region (PBR). In both MICA and MICB, little variation is found at putative PBR-specifying sites; it is instead concentrated in the fourth exon (Yaoet al. 1999), which is not variable in classical class I genes. The estimated nucleotide diversities of 20 near-complete MICA and 13 MICB coding sequences are 1.02 and 0.23%, respectively, but comparison with other diversity estimates is difficult since the haplotypes have not been systematically selected. The patterns of variability in MIC genes are consistent with the hypothesis that these genes represent nonclassical class I genes. There is evidence that MICA may be involved in induction of antitumor NK and T cell responses (Baueret al. 1999). On the other hand, some MICA alleles contain in-frame stop codons, suggesting that functionality of this locus may not be critical. Rates of synonymous and nonsynonymous substitution per site have been approximately equal since the divergence of MICA and MICB (∼7.5% synonymous substitutions per site vs. 5.5% nonsynonymous substitutions). In contrast to HLA-B, MIC polymorphisms are not trans-specific between apes and humans (Cattleyet al. 1999). There is evidence of a linkage disequilibrium between the MIC and HLA-B genes (Yaoet al. 1999) and thus their polymorphism fits a picture of being generated, as in noncoding regions, by virtue of their linkage to HLA-B. In this view the MIC genes may be functional and polymorphic, but selection at MIC loci does not produce polymorphism in the manner found for HLA-B.

Table 2 illustrates the extent of nucleotide diversity found for the regions we sequenced. The lowest levels of diversity are found in segments most distant from HLA-B, close to the TNF locus. The diversity found at TNF is nevertheless approximately double the average levels found by Li and Sadler (1991) for unselected sites of human gene sequences. The nucleotide diversity increases with increasing proximity to HLA-B, although the increase is not monotonic. It is unclear what effect any functional restrictions on MICA and MICB may have on the polymorphism in regions adjacent to those loci and whether these loci may be a cause of the nonmonotonic relationship. The absence of a simple relationship between diversity and distance from a locus under selection is also seen in the analysis of two haplotypes in the class I region (Satta and Takahata 2000). The highest nucleotide diversity is found at HLA-B itself, whether measured at the synonymous sites or in introns.

Recombination frequency: The most likely factor driving the high levels of polymorphism in the six adjacent regions reported here is their linkage to HLA-B. Since the degree of polymorphism can be related to recombination frequency, it is of some interest to determine what recombination frequency can account for the observed degree of polymorphism. By using the population genetics models of Takahata and Satta (1998a) and Satta and Takahata (2000), we can attempt to fit a recombination rate to the observed data (Figure 3). The least c-squares estimate gives a rate of 0.15% per megabase (Figure 3), which is one-sixth of the average rate found over the entire human genome (in humans estimated at 1% per megabase per generation: see Vogel and Motulsky 1997; Takahata and Satta 1998a). The maximum and minimum diversity values found can be encompassed by recombination frequencies of 0.02 and 0.54%, respectively. The curve of the estimate is monotonic and does not account for variations found in the vicinity of the MIC loci. However, these variations affect primarily the shape of the recombination curve rather than its magnitude. The present estimate agrees with another estimate for this region from Satta and Takahata (2000) that is based on a comparison of the sequences of Guillaudeux et al. (1998) and Shiina et al. (1998) extending throughout the HLA-B and HLA-C regions. It therefore appears that recombination frequency in proximity to HLA-B is not elevated compared to other genomic regions, but rather reduced.

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Figure 3.

—The relationship between the percentage of nucleotide diversity (ordinate) found for six amplified intergenic segments, as well as exons 1–7 of HLA-B in the same haplotype, plotted as a function of distance from HLA-B. The nucleotide diversity found in a range of MICA and MICB haplotypes is also shown. The distances are given in kilobases with negative and positive numbers used for locations proximal to and distal from HLA-B, respectively. Lines show estimates of expected diversity at particular recombination frequencies following the model of Takahata and Satta (1998a). The solid lines show the leastsquares estimate of recombination rate (0.15% per megabase per generation) and the dashed lines show the rates required to incorporate the maxima (0.02% per megabase) and minima (0.54% per megabase). The mutation rate is assumed to be 10^–9 per site per year and the selection intensity is 1%.

To examine the extent of incompatibility in this region, an incompatibility matrix was constructed (Figure 4). Among all the pairs of informative sites (9870 pairs), including those in HLA-B, 3769 pairs are incompatible. From the diagram, there appear to be at least five phylogenetically distinct blocks (Figure 4). Block 1 is composed of the 5′-most segment (5′ region of CL1, the entirety of CL2, and exon 2 of HLA-B). These three regions are mutually compatible and support a single phylogeny. Block 2 contains the only the 3′ part of CL1. Block 3 is composed of FGF and DHFR. Block 4 is formed by exon 1 as well as exons 3–7 of HLA-B and block 5 by DIST. We reconstructed a phylogeny for each block. Although the number of informative sites for each block is not large, most clusters in the phylogenies are statistically supported. Then we counted the minimum number of branch switching by a modified method of Robinson and Foulds (1981) for all blocks to support a single phylogeny (not shown). The minimum number was 18. Taking this number as a minimum number of recombination events in the 200-kb region during the past 17 million years (this date is based on the average synonymous divergence of eight sampled HLA-B exons, 0.034/site, and the synonymous substitution rate of 10^–9 per site per year), the recombination frequency is calculated as 1.1/200 kb/million years. Assuming 20 years per generation in humans, the recombination rate per megabase per generation is 0.01%. This possible minimum rate, although roughly estimated, is not much different from the lowest limit of the least-squares estimate.

The polymorphisms at the HLA-B locus are thought to be trans-specific between humans and chimpanzees, having arisen at least 5 million years before present (∼250,000 generations ago, taking 20 years as generation time). For TNF, CL2, and DIST, orthologous segments from the chimpanzee and gorilla were obtained. In every case the nucleotide diversities found in humans were smaller than the nucleotide divergences between humans and apes. This observation suggests that, unlike at HLA-B, the polymorphisms at the adjacent linked loci have not evolved trans-specifically between humans and apes.