Department of Organismic and Evolutionary Biology, Harvard University, and U.S. Environmental Protection Agency, Atlantic Ecology Division, Narragansett, Rhode Island
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Abstract |
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Introduction |
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This approach makes use of the specific role of Mhc as a mediator of the adaptive immune response by way of antigen binding, taking advantage of some of the strongest signals of selection of any known genetic locus. Small human populations offer one of the strongest pictures of rapid Mhc change and selection in novel environments (Parham and Ohta 1996
). But although unusual allelic compositions in these cases are presumed to result from a combination of strong selection and drift, the actual selective forces are historical inferences at this point. Here, large wildlife populations, less subject to drift and currently experiencing known differences in strong selective forces, can be compared. Alarming environmental toxicant loads in an Environmental Protection Agency (EPA) Superfund site offer a "natural" experimental comparison with cleaner environments because the toxicant loads are well characterized and are frequently associated with altered vertebrate immune responses, in general (U.S. Environmental Protection Agency 2000, chapt. 4, p. 1
), and at this site are associated with unusual parasite loads and altered stressor responses (Nacci et al. 1999
; Bello et al. 2001
; Nacci et al. 2002b
).
The human Mhc class II molecule has been crystallized (Brown et al. 1993
; Stern et al. 1994
), and correlations between residue variation and antigen-binding ability have been elucidated using a variety of binding affinity assays (Davenport and Hill 1996
; Ou, Mitchell, and Tingle 1998
) and computer analysis (Sturniolo et al. 1999
). In the extra-membrane portion of Mhc class II molecules, an open cleft between alpha and beta subunits selectively binds antigens on the basis of the affinities of the particular amino acid residues lining the binding cleft (Stern et al. 1994
). A particular portion of the cleft, the B1 subunit, has been shown in a variety of vertebrates to contain variable regions with extremely high rates of amino acid substitution. In this region, high ratios of nonsynonymous substitution to synonymous substitution (Dn/Ds) appear to result from positive selection to recognize diverse antigens (Hughes, Ota, and Nei 1990
; Hughes, Yeager, and Carrington 1996
). Within the B1 subunit, particular residues have been identified as highly variable, and Dn/Ds comparisons between residues suggest that these highly variable sites are the specific residues driven by positive selection for diverse antigen recognition and binding. Thus, the relationship between residue variation within the Mhc antigen-binding region and ability to recognize and bind different antigen motifs is well established (Jardetzky 1996
). This relationship may be used to make population-level comparisons of functional immunogenetic repetoires. Populations that have experienced different suites of immune challenges might show different amino acid replacement patterns at antigen-binding sites on the basis of the different binding affinities of various pathogens. Rather than looking for specific allelic correlations with particular pathogens, by using this approach it is possible to assess the immunogenetic status of an entire population relative to other populations experiencing different suites of antigenic challenges.
In this study, the immunogenetic profiles of two fish populations that differ dramatically in exposure to environmental toxicants and parasite loads are compared. Fundulus heteroclitus is an extremely abundant resident estuarine teleost fish (Pisces: Cyprinodontidae) in salt marshes along the entire western North Atlantic coast (Bigelow and Schroeder 1953, p. 164
). These fish are tolerant to a wide range of environmental conditions, including salinity, temperature, and chemical contaminants. Fundulus heteroclitus is a well-established model system for studies of biochemical, physiological, ecotoxicological, and genetic adaptation to natural and artificial environmental variations (Mitton 1997, p. 44
; Powers and Schulte 1998
), including adaptation to an extreme accumulation of dioxin-like compounds (DLCs) in the EPA Superfund site at New Bedford Harbor, Mass. (Nacci et al. 1999
). Functional variation in immune system parameters has been measured in a variety of pollutant assays using F. heteroclitus (Rozell and Anderson 1996
; Armknecht, Kaattari, and Van Veld 1998
); however, immunogenetic Mhc variation has not been examined previously in this fish. DLCs cause a variety of immunological, endocrine, reproductive, tumorigenic, and other disorders as well as lethality in vertebrates, and exposures are linked to higher incidences of disease in mammals, birds, and fish (Luebke et al. 1997
; Monosson 2000
).
To test this population-level approach to immunogenetic variation, a highly variable Mhc class II B locus was isolated from F. heteroclitus using degenerate PCR methods. Patterns of variability at this locus were characterized to determine (1) if this fish locus shows similar variability and positive selection in comparison with other vertebrate Mhc class II loci responding to pathogen pressure, and (2) if patterns of amino acid substitution at the antigen-binding locus vary between populations experiencing strongly differing environmental stresses. This information is used to determine if populations that differ strongly in immune challenges also show differences in immunogenetic profiles at a highly variable Mhc locus. Such differences found by looking at composite patterns of unique amino acid substitutions in the antigen-binding region may provide a useful technique for evaluating population adaptation to extreme environmental stress. To control for Mhc differences that may reflect a signal of geographic isolation and drift rather than selection, complimentary information from the hypervariable region I of the mitochondrial control region, a noncoding locus thought to reflect nonselective evolutionary forces, was obtained for the same populations.
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Materials and Methods |
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Samples
Fish were collected from field sites with minnow traps or seines and fatally anesthetized with MS-222 (Sigma). Hot Spot, NBH, Mass., and Beaufort, N.C., fish were dissected before vouchering to confirm general patterns of parasitism. Hot Spot fish used in this study all showed two unusual and elevated levels of internal digenean trematode infestation as encountered in previous sampling (M. Huber and R. Overstreet, personal communication), relative to reference populations in West Island and Gloucester. All Hot Spot fish showed evidence of swim bladder infestation by an adult stage hemiurid digenean tentatively identified as Stomachicola rubea (an unusual organ location and life-history stage for this organism to appear in F. heteroclitus) as well as high incidences of heterophyid digenean metacercaria heart parasites (identified as Ascocotyle tenuicollis by R. Overstreet) in comparison with reference populations (M. Huber, R. Overstreet, and S. Cohen, unpublished data). Beaufort, N.C., fish used in this study showed no evidence of heart, swim bladder, or any other parasitic infestation in a gross examination of the internal and external anatomy, excluding gills. All sampled fish were either frozen as a whole at -80 or a posterior section including a portion of the muscle and tail was removed and stored in 95% ethanol.
Molecular Methods
DNA was extracted from the muscle tissue by standard phenol-chloroform methods (Duda and Palumbi 1999
) or by using Nucleospin tissue columns (Clontech). For MHC sampling, degenerate PCR primers were designed from an alignment of guppy, topminnow, cichlid, and bass Mhc class II B sequences found in GenBank. Primers were designed using coding sequences from exons 1 and 2 as well as noncoding sequence from cichlid intron 1. This region was chosen for amplification because it includes nearly all of exon 2 in the B1 unit, where the highly variable portion of the peptide-binding region (PBR) is located (as well as the preceeding intron and a small portion of the leader peptide). Using these primers, the B1 subunit lacks 14 bases at the 3' end of the exon because these nucleotides are located in the reverse primer. This portion of the exon includes all the inferred peptide-binding codons for this region (24 residues) and 92% (60/65 residues) of the nonpeptide-binding codons. PCR primers used were forward XIS (sense, 3' exon 1 and 5' intron 1) CASCSTCHRCWCWGCAGGT and reverse MRS (antisense, 3' exon 2) RCTYACCTGATTTAKMCAGA. The PCR reaction followed the manufacturer's recommended conditions using TaKaRa Ex Taq (Promega) with the modification of one-half of the lowest recommended Taq concentration in a 25-µl hot-started reaction. PCR cycling conditions were usually one cycle at 94°C for 2 min; 35 cycles at 94°C for 30 s, 5053°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 05 min. PCR conditions were varied to search for additional related loci. Different concentrations of magnesium, Taq, primers, templates, and dNTPs were tested; none produced additional Mhc alleles beyond those found with the standard conditions. Annealing temperatures and extension times were varied as well and also did not produce additional Mhc alleles. Extension times below 15 s sometimes produced additional artifactual alleles by recombination during PCR of incompletely extended products. These recombinant alleles were not found when the reaction was repeated with longer extension times.
PCR products were ligated into TOPO or TA (Invitrogen), or pGEM (Promega) vectors using TOPO or TA kits. Clones were screened in PCR reactions with the cloning primers T7 and M13R. PCR products for clones of appropriate sizes were cleaned using a shrimp alkaline phosphataseexonuclease reaction or with Qiaquick PCR purification columns (Qiagen) before cycle sequencing with Big Dye for analysis on an ABI 377 or ABI 310 sequencer. Clones of many sizes were sequenced to look for additional alleles and loci; clones between 500 bp and 1 kb were routinely screened, and clones outside this size range (both smaller and larger) were occasionally sequenced. All sequences were submitted to BLAST searches against the GenBank database. All alleles shown in this study were confirmed by sequencing a minimum of two, but in many cases more independent clones (frequently 35, occasionally up to 10 or more). As an additional means of searching for additional loci and alleles, interior degenerate and nondegenerate primers were designed using the Fundulus sequence obtained in this study, in alignment with other GenBank sequences. Interior primers provided the same alleles as those obtained with the standard outer primers named above. DNA sequences were aligned in Sequencher. Inferred amino acids and Dn/Ds values were calculated with MEGA (Kumar, Tamura, and Nei 1993
) 1.02 using Jukes-Cantor distance measures. A phylogenetic neighbor-joining distance tree was constructed using all alleles in the program using PAUP* (Swofford 2000
) version 4.0b3a with Jukes-Cantor distances.
Other summary statistics were obtained using PAUP* and DnaSP (Rozas J. and Rozas R. 1999
), version 3.14.3.
Mitochondrial Hypervariable Control Region Sequences
Genomic extracts of F. heteroclitus were used in 25-µl PCR reactions using Perkin-Elmer PCR buffer and Amplitaq (Applied BioSystems) with the following conditions: one cycle at 94°C for 2 min and 35 cycles at 94°C for 1 min, 51°C for 1 min, 72°C for 1.5 min. The following PCR primers were used to obtain the sequence for hypervariable region I of the mitochondrial control region: forward primer Pro-5' (CTA CCC CTA ACT CCC AAA GC) (Palumbi 1996
) and reverse primer M-3b (AGA AAT AGG AAC CAG ATG CCA G) (courtesy, C. Reeb). PCR products were cleaned with a shrimp alkaline phosphataseexonuclease reaction and cycle sequenced with Big Dye for analysis on an ABI 3100 sequencer. Sequences were aligned in Sequencher, and Fst values between populations were estimated with the program HEAP (courtesy, S. Palumbi).
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Results |
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Mhc Sequences and Polymorphism
A 400- to 500-bp PCR product (including PCR but not cloning primers and vectors) from all populations (Beaufort, N.C. [n = 3], Gloucester, Mass. [n = 10], Hot Spot, New Bedford Harbor, Mass. [n = 17], West Island, Mass. [n = 5]) showed similarity to GenBank Mhc sequences in BLAST searches. The Mhc matching clones were inferred to include all of intron 1 and most of exon 2 (i.e., the entire exon minus the final 14 bases at the 3' end, which are included in the reverse primer) on the basis of primer design and matches with various fish class 2 DB1 sequences and cichlid class 2 B group 4 sequences in GenBank. The number of clones sequenced per individual ranged from 2 to greater than 10. In one case where an unusual motif (ERW NDP) could be identified by an RFLP assay, additional clones were screened by PCR and RFLP. No more than two functional alleles were found in any individual. An inferred pseudogene (containing a stop codon) was found in a single individual from Gloucester. The pseudogene sequence was excluded from further analysis. No indels were found in the coding sequence.
For a subset of individuals, the initial PCR and cloning was repeated, and identical alleles for that individual were always obtained. Also, interior primers only located alleles identical to the sequences obtained using the external primers that amplified the entire intron-exon piece. Thus, the alleles found in this study are inferred to represent a single functional DB locus in F. heteroclitus. One or more additional class II B loci have been found in a variety of fish (McConnell, Godwin, and Cuthbertson 1998
), sometimes in high numbers, e.g., cichlids (Malaga-Trillo et al. 1998
). But other species, including those most closely related to Fundulus and with characterized Mhc class II, show evidence of only a single locus in assays using cDNA, Southern blotting, or family genotype comparisons, e.g., topminnows (Hedrick and Parker 1998
) and guppies (Sato et al. 1995
), and McConnell, Godwin, and Cuthbertson (1998)
provide evidence for a second highly divergent DXB locus. Alternatively, the primers used in this study may be sampling only a limited subset of the existing alleles at a single locus, thus providing a limited sample of the actual amino acid replacements occurring in these populations at this locus. There is a low frequency of nulls (4%, 2/48 individuals subjected to PCR, one each from West Island and Gloucester). But in either case, comparison of amino acid replacement profiles is appropriate because populations were consistently sampled with the same primer set.
The B1 exon alignment of 254 nucleotides consisted of 41 unique alleles found across all populations (GenBank accession numbers AF529585AF529625, alignment included in supplemental material and submitted to GenBank). A few alleles were found more than once in different individuals, including a total of three duplicated alleles from the Hot Spot location (alleles H150c4, H6c5, and H6c4 all had identical matches within the population), two duplicated alleles in Gloucester (G10c4 had an identical match), and one allele shared between Hot Spot and West Island (an allele identical in nucleotide sequence to W5c2 was also found in Hot Spot). The DNA sequences were translated using MEGA (Kumar, Tamura, and Nei 1993
) into the 84 residues shown in figure 1
. All inferred amino acid alleles were encoded by only a single DNA sequence, i.e., there were no differing DNA alleles redundant for the same amino acid allele.
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Mhc Variation in Amino Acid Replacement Patterns Related to Structure
The PBR codons were inferred from the human model of the Mhc class 2 DRB1 locus (Brown et al. 1993
) (fig. 1
). Comparison with zebrafish (Ono et al. 1992
) and salmonids (Dixon et al. 1995
; Miller, Withler, and Beacham 1997
) (truncated coding region) shows similar patterns of generally higher variability at inferred antigen-binding codons.
Population-specific replacements for the Gloucester fish are concentrated at residue 11 located in the first strand of the B-pleated sheet. Although fish from the Hot Spot do show variation at this residue, few of the replacements are population specific, i.e., unique to Hot Spot in this study. Instead, Hot Spot population-specific replacements predominantly occur in the a-helix. The southern Mhc alleles found in this study include only a single population-specific amino acid replacement (at residue 11). Residue 11 is among the most variable in other organisms including primates and other teleosts (Klein and O'hUigin 1995
).
Phylogenetic analysis of DNA alleles using a neighbor-joining algorithm and Jukes-Cantor distances places Beaufort alleles separated from each other within clusters containing other localities (fig. 2
). Indeed, as predicted for highly variable Mhc PBRs under balancing selection and undergoing recombination (Jakobsen, Wilson, and Easteal 1998
; Martinsohn et al. 1999
; Edwards, Nusser, and Gasper 2000
), there is no phylogeographic signal to this exon either in analyses of the entire region or of the a-helix and B-pleated sheet sections analyzed separately under the supposition that they may be experiencing separate evolutionary histories (Bergstrom et al. 1998
).
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Discussion |
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The Superfund site population (Hot Spot) shows a significantly elevated rate of nonsynonymous substitutions in the PBR. A similar pattern of elevated Dn/Ds in other populations indicates that the inferred antigen-binding sites are under strong positive selection outside the contaminated site as well as is expected for this Mhc locus in vertebrates in general. This is consistent with a response by each population to local selection, affecting the antigen-binding region. Nonsynonymous values in the PBR are elevated in comparison with synonymous PBR sites, showing that silent changes occur significantly less frequently than residue-changing substitutions at the antigen-binding residues in each of these particular environments. Non-PBR sites show similar levels of substitution between synonymous and nonsynonymous sites, thereby indicating a lack of positive selection at these residues, and non-PBR rates of change match the PBR synonymous rates. Thus, the signal of positive selection is detectable at inferred peptide-binding codons, as predicted by the human Mhc functional model. More limited sampling of salmonids (Miller, Withler, and Beacham 1997
; Binz et al. 2001
) has also shown a positive signal of selection, again suggesting that structural inferences from the human model to Fundulus are appropriate. Thus, differences in Dn/Ds ratios between PBR and non-PBR sites and differences at antigen-binding sites in amino acid replacement patterns between populations may be related to differences in pathogen pressures between environments.
An alternative explanation for population-level differences in replacement patterns, introgression of southern Fundulus genotypes or more generally phylogeographic sorting, was excluded by sampling mitochondrial genotypes. The Mhc PBR substitution patterns found in this study might have reflected genetic differences between the two clades or subspecies of F. heteroclitus, i.e., southern and northern fish (Bernardi, Sordino, and Powers 1993
; Powers and Schulte 1998
). Such a finding would represent an unusually far northern population of southern fish because the geographic break for these two clades is located in New Jersey. In any case, the control region mitochondrial data show that fish sampled from Massachusetts for this study all represent the northern form of F. heteroclitus. Thus, differences between Hot Spot and Gloucester populations cannot be attributed to an anomalous occurrence of southern genotypes at either location.
Allele sharing among distant populations and allelic lineage sharing across species boundaries are characteristic features of some Mhc loci (Klein et al. 1998
) and are thought to result from balancing selection (Hughes, Yeager, and Carrington 1996
). MHC evolution may occur over both very long (transspecific) and much shorter time scales (e.g., local variation in protective alleles for severe malaria in Africa) depending on population genetic parameters (Parham and Ohta 1996
; McNicholl et al. 2000
). An overwhelming phylogeographic Mhc signal is not predicted for species such as Fundulus with high levels of genetic variability and large population sizes, where positive selection for antigen recognition acts on Mhc genotypes. Instead, Mhc variation may reflect the pattern of antigenic stressors in the local environment (Edwards and Hedrick 1998
; Hughes 1999, p. 63
; Hedrick and Kim 2000, p. 205
).
Other population-level Mhc studies on fish (Miller, Withler, and Beacham 1997
) have found a phylogeographic signal related to patterns of diversification at other loci; however, this work involved relatively limited Mhc (and other genetic) variation in comparison with that in Fundulus. For example, in a comparison between two populations, the three salmonid class II DB exon 2 alleles showed a mean pairwise nucleotide difference of 3% (Miller, Withler, and Beacham 1997
) in contrast to mean differences of 15%18% in interpopulation comparisons of Fundulus alleles. The limited variation found in salmonids is most likely a result of population bottlenecking (Miller, Withler, and Beacham 1997
) that would affect Mhc as well as other loci if population numbers are constricted sufficiently. In contrast, Fundulus population numbers are large, often with 10,000 or more individuals per estuary (Abraham 1985
, 23 p.). Lack of historical bottlenecks in Fundulus is evident in the high genetic diversity at Mhc and other loci both in numbers of alleles per population and in levels of genetic divergence between Mhc alleles within a population. Indeed, levels of genetic variation at other Fundulus loci are high along the East Coast (Ropson, Brown, and Powers 1990
) including New Bedford Harbor (McMillan et al. 2000
; Roark, Guttman, and Nacci 2000
; McMillan et al. 2001
). In addition, Fundulus populations are not affected by the harvesting and aquacultural practices common to many salmonid populations that may reduce genetic diversity in commercial fish populations.
Despite much motif or epitope sharing between populations, e.g., HATAF, HIWN, LQQM/LQSM, NER in the a-helix area (fig. 1
), unique patterns of substitution between the two populations are outstanding (figs. 1
, 4
, and 5
). Differences found in this study between a-helix and B-pleated sheet substitutions relate to the location of pockets in the Mhc-binding cleft, where antigen anchor residues are thought to bind (Brown et al. 1993
; Stern et al. 1994
). In the Gloucester population, unique replacements occur mostly at position 11, an important residue in binding pocket 6. In contrast, in Hot Spot, replacements are concentrated around pocket 7, which includes the area associated with residues 61, 47, 67, 28, and 71 in the human class II structural model. Although antigen-binding pockets may shift somewhat between species and alleles, the functionally significant pattern of amino acid replacement differences between Gloucester and Hot Spot populations is clear statistically (fig. 4
) and has a functional difference between strand 1 of the B-pleated sheet (pocket 6) and the a-helix (pocket 7).
Population differences in amino acid replacement patterns seen in the comparison of Gloucester and Hot Spot populations suggest that the inferred antigen-binding region is responding to habitat-specific selective pressures in Gloucester and in New Bedford Harbor. Apparently the selective regime affecting antigen-binding abilities at these two geographic locations is altering the allelic composition at different functional locations within the molecule. This suggests that the different geographic locations have different antigens or suites of antigens that are interacting with Mhc receptors or that Mhc receptors are functioning differently in the two environments. Environmental differences between the New Bedford Harbor Superfund site and a relatively healthy marsh environment are extreme. Selective pressures at the New Bedford site are related to the harsh chemical environment and unusual suites of pathogens found in association with the Superfund site. Indeed, antigen differences between the two sites are clear on a gross level because New Bedford Harbor Hot Spot fish have a number of unusual helminth parasites not found in Gloucester or other Fundulus populations at all, or in some cases, at the high frequencies seen in Hot Spot (see Materials and Methods for further parasite description). Although the ecological basis for these local abnormal patterns of parasitism is unknown, in experimental infections in the laboratory with a ubiquitous bacterial pathogen Vibrio carchariae, Fundulus from New Bedford Harbor show survival rates equal to or greater than those for fish from clean reference populations (Nacci et al. 2001
).
Genetic adaptation to DLCs has been demonstrated in Fundulus from New Bedford Harbor and other toxic waste sites in comparison with control sites using assays of embryonic tolerance to PCB laboratory challenges that have shown strong heritable differences in survival rates (Nacci et al. 1999
, 2002a
) (and see Study Sites in Materials and Methods). Genetic adaptation to DLCs contaminating New Bedford Harbor since the 1940s supports the likelihood of accompanying immunogenetic adaptation to severe environmental stress. Thus, the extraordinary DLC concentrations in the Superfund area may be having selective effects on the Mhc allelic repetoires directly as well as through indirect pathways related to differential parasite loads.
Analysis of primate and other Mhc class 2 DB data has led to vigorous discussion on the possibility that the B-pleated sheet and a-helix experience different kinds of evolutionary change, with the B-pleated sheet accumulating changes in a conventional manner by single mutations, whereas the a-helix may be subject to substantial levels of gene conversion, moving blocks of amino acids between alleles, e.g., mice (Wakeland et al. 1990
) and primates (Gaur and Nepom 1996
; Takahata and Satta 1998
). If this is true for the Fundulus populations studied in this work, then interpopulation differences in the locations of unique replacements may also reflect population differences in the mechanism of evolutionary change occurring at this locus. Different intensities of selection as well as differences in specific selective agents in the two environments may actually be responsible for the production of changes in different parts of the molecule. The geographic location experiencing a more recent shift in selective pressure, the Superfund site, has unique amino acid substitutions concentrated in the a-helix section, where recombinational mechanisms may permit particularly rapid generation of new alleles.
Functional analysis, somewhat similar to the approach used in this study, has been applied to specific human pathologies and to patterns of amino acid replacement in humans as a whole by cataloging functional motif changes in Mhc (Ou, Mitchell, and Tingle 1998
). Allelic correlations with particular diseases may often involve more than a single amino acid replacement at any particular residue, making detection of allelic linkages, as opposed to linkages by functional categories, statistically challenging (Apple and Erlich 1996
). The strategy used in this study has an advantage over standard allelic frequency methods in its ability to detect a pattern of disease association involving more than one or a few alleles by grouping functionally similar changes together in analysis.
As demonstrated in this study, Mhc offers an additional opportunity to relate functional analysis at the genetic level to environmental stresses and allelic composition at the population level in wildlife. Each population experiencing a different suite of antigenic stressors may show novel patterns of amino acid replacement in the antigen-binding cleft. The Mhc sequence data provide the information to calculate Dn/Ds ratios, a measure of the relative strength of positive selection, and specific patterns of replacement may be used to make structural comparisons on the basis of the human crystallographic Mhc models. Further studies are underway with Fundulus to compare immunogenetic profiles in field populations and laboratory challenges to test whether particular stressors affect wildlife immune systems in diagnostic ways. By taking full advantage of Dn/Ds measures and positional information on amino acid replacement patterns in different environments, it may be possible to understand how this enigmatic polymorphic genetic recognition system maintains such extraordinary levels of diversity in wild populations.
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Acknowledgements |
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Footnotes |
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Abbreviations: DLC, dioxin-like compound; PBR, peptide-binding region; LC20, modeled estimate of the concentration killing 20% more than the control.
Keywords: Mhc
major histocompatibility complex
positive selection
Fundulus heteroclitus
chemical stress
Address for correspondence and reprints: Sarah Cohen, BioLabs, 16 Divinity Avenue, Harvard University, Cambridge, Massachusetts 02138. scohen{at}oeb.harvard.edu
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References |
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