Identification and Mapping of Two Divergent, Unlinked Major Histocompatibility Complex Class II B Genes in Xiphophorus Fishes

Corresponding author: Thomas J. McConnell, Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353., mcconnellt{at}mail.ecu.edu (E-mail).

Communicating editor: C. KOZAK

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

We have isolated two major histocompatibility complex (MHC) class II B genes from the inbred fish strain Xiphophorus maculatus Jp 163 A. We mapped one of these genes, designated here as DXB, to linkage group III, linked to a malic enzyme locus, also syntenic with human and mouse MHC. Comparison of genomic and cDNA clones shows the gene consists of six exons and five introns. The encoded ß1 domain has three amino acids deleted and a cytoplasmic tail nine amino acids longer than in other teleost class II ß chains, more similar to HLA-DRB, clawed frog Xela-F3, and nurse shark Gici-B. Key residues for disulfide bonds, glycosylation, and interaction with chains are conserved. These same features are also present in a swordtail (Xiphophorus helleri) genomic DXB PCR clone. A second type of class II B clone was amplified by PCR from X. maculatus and found to be orthologous to class II genes identified in other fishes. This DAB-like gene is 63% identical to the X. maculatus DXB sequence in the conserved ß2-encoding exon and was mapped to new unassigned linkage group LG U24. The DXB gene, then, represents an unlinked duplicated locus not previously identified in teleosts.

MAJOR histocompatibility complex (MHC) class II ß heterodimers present peptides to T helper cells, providing a signal necessary for activation and subsequent initiation of a specific immune response to a foreign peptide or protein (GERMAIN 1994 ). Since the first isolation of an MHC class II B gene in a teleost (HASHIMOTO et al. 1990 ) and an elasmobranch (BARTL and WEISSMAN 1994 ), this gene has been identified in a number of teleosts (KLEIN et al. 1997 ). MHC and complement genes have been mapped in zebrafish (BINGULAC-POPOVIC et al. 1997 ) and medaka (KURODA et al. 1996 ), respectively. A teleost model system particularly suited to genomic mapping and linkage analysis is the assemblage of inbred strains of platyfish, Xiphophorus maculatus.

The platyfish and the swordtail, Xiphophorus helleri, (Teleostei: Poeciliidae) are livebearers native to streams of eastern Mexico and Central America. Hybrids of X. maculatus and X. helleri were originally noted to be tumor-susceptible by fish hobbyists, and further characterized as highly susceptible for development of malignant melanomas from pigment cells of the platyfish (BELLAMY 1922 ; GORDON 1927 ; KOSSWIG 1928 ; HAUSSLER 1928 ). To identify the genes responsible for the development of these tumors, inbreeding of platyfish was initiated in 1939. These platyfish strains were used in the first inbred fish histocompatibility studies (KALLMAN and GORDON 1957 ; KALLMAN 1958 ). These same strains, and others, were used in early attempts to estimate the number of histocompatibility loci in the fish X. maculatus (KALLMAN 1964 ). Inbred strains of platyfish have also been instrumental in the identification of melanoma-inducing loci in platyfish-swordtail hybrids (VIELKIND et al. 1989 ; WITTBRODT et al. 1989 ; ADAM et al. 1993 ; NAIRN et al. 1996 ). Availability of inbred strains of X. maculatus and the fertile hybrids of interspecific crosses have led to the development of extensive Xiphophorus gene maps (MORIZOT et al. 1991 ) that are presently unavailable in most other fish species. Members of this genus are, therefore, particularly suitable for determining genetic linkage relationships for study of evolution of the MHC in teleosts. In this study, we report the identification of a novel MHC class II B locus in Xiphophorus as well as the more commonly characterized II B locus, and the genetic linkage mapping of these two loci. The Xiphophorus gene maps allow prediction of ancestral vertebrate genome organization (MORIZOT 1990 , MORIZOT 1994 ) and could provide insight into evolution of vertebrate MHC gene complexes.

The genes we have cloned and mapped are designated as MhcXima-DXB*01 and MhcXihe-DXB*01, in general accordance with the guidelines of KLEIN et al. 1990 , where Xima and Xihe refer to X. maculatus and X. helleri, respectively, D to class II; X to a new and as yet uncharacterized family designation; B to the ß chain-encoding gene; and *01 to the allelic form of the gene shown. MhcXima-DAB*01 also represents a class II B gene, with the A referring to a relatively well-characterized family of teleost class II B genes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Fish:
Platyfish (X. maculatus), strain Jp 163 A (inbred since capture from Rio Jamapa, Veracruz, Mexico in 1939), and the swordtail (X. helleri), Sarabia strain pedigree 6243 originally from Rio Sarabia, Veracruz, were obtained from the Xiphophorus Genetic Stock Center at Southwest Texas State University, San Marcos, TX.

Preparation of genomic DNA:
Gill tissue of 10 specimens of X. maculatus Jp 163 A or X. helleri 6243 was removed, frozen in a dry ice/ethanol bath, ground into a powder in the presence of liquid nitrogen, and resuspended in lysis buffer [10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% sodium dodecyl sulfate (SDS), 1 mg/ml protease K], incubated overnight at 54°, phenol/chloroform extracted, ethanol precipitated and resuspended in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA.

Preparation of RNA:
Spleens of 10 specimens of X. maculatus Jp 163 A were dissected, minced, and resuspended in TRIzol reagent. RNA was isolated according to the manufacturer's protocol (Life Technologies, Gaithersburg, MD).

PCR amplification and cloning of Xima-DXB:
Genomic DNA of X. maculatus Jp 163 A and the oligonucleotide primers TM215 and TM216 (primers described in WALKER and MCCONNELL 1994 ) were used in the initial PCR amplification of a Xima class II B DNA fragment using previously described conditions (WALKER and MCCONNELL 1994 ). Briefly, primer TM215 corresponds to the amino acid sequence CSAYDFYP of a class II B gene in carp (HASHIMOTO et al. 1990 ) that includes the first cysteine of the ß2 domain. The antisense primer TM216 corresponds to the amino acid sequence CMVEHASL, including the second cysteine of the ß2 domain. The resultant 190-bp fragment was cloned into pCR I (Invitrogen, San Diego, CA) and sequenced. The initial database searches using NCBI BLASTN (ALTSCHUL et al. 1990 ) found the sequences to be similar to other vertebrate MHC class II genes. This DNA fragment was subsequently used for screening of a genomic library as described below.

The Xima-DXB sequence, as determined from clones isolated from a genomic library (see below), made possible the design of DXB-specific primers TM341 (5'-ATCTCTGTTGCCAATCTAAGA-3') and TM328 (5'-ATGTGTAAAAGGCTAAATGAT-3'). These primers were designed using Oligo Primer Analysis software (National Biosciences, Plymouth, MN) and used for amplification of Xihe-DXB genomic and Xima-DXB cDNA. For isolation of the Xima-DXB cDNA, the Capfinder protocol (CLONTECH, Palo Alto, CA) was used as directed by the manufacturer to transcribe cDNA from X. maculatus splenic RNA. Thirty cycles of amplification (94° for 30 sec, 58° for 30 sec, 68° for 4 min) were performed using 1 µl of the high-fidelity KlenTaq polymerase enzyme mixture, 5 µl 10x KlenTaq reaction buffer, 10 mM dNTP mix, 0.4 µg each of TM341 and TM328, and 2% of the amplified cDNA mix as per manufacturer's directions (CLONTECH). The resulting PCR mixture was then electrophoresed on a 1% agarose gel, the appropriate sized band excised, and DNA isolated from the agarose using the QIAquick protocol (QIAGEN, Chatsworth, CA). The DNA was then used as substrate for the +1 cycle of the PCR+1 reaction (BORIELLO and KRAUTER 1990 ; WALKER and MCCONNELL 1994 ; HARDEE et al. 1995 ) with primer TM342 (5'-GAGAAGCTTATCTCTGTTGCCAATCTAAGA-3') with a HindIII site (underlined), using Taq polymerase according to manufacturer's protocol (Life Technologies). The resultant 777-bp fragment was cloned into pGEM-T (Promega, Madison, WI) and sequenced. Plasmid restriction digests and DNA sequence data were used to confirm the identities of the Xima-DXB cDNA PCR+1 clone. Excision of the fragment from the vector with the primer HindIII site and the vector NotI site resulted in a 795-base pair (bp) fragment. PCR+1 amplification and cloning procedures for the Xihe-DXB genomic fragment were identical to that of the DXB cDNA clone, with 1 µg X. helleri 6243 genomic DNA as template and resulted in a 1759-bp genomic PCR+1 fragment.

PCR amplification and cloning of Xima-DAB:
Amplification of the Xima-DAB cDNA was performed as described for Xima-DXB cDNA, but using RNA isolated from intestinal tissue as template. Primers used were TM396 (5'-GCTGGGCTGGCTGCTGGTCAT-3') based on the leader sequence of the guppy (SATO et al. 1995 ), TM398 (5'-GAAGCAGGAGGAACCAGAACC-3') in the 3' untranslated region of the guppy (SATO et al. 1995 ), and TM399 (5'-AGAAAGCTTGCTGGGCTGGCTGCTGGTCAT-3') as the +1 primer with the underlined HindIII site. The program Oligo (National Biosciences) was used in the design of these primers.

Screening of genomic library and subcloning of positive plaques for Xima-DXB:
A platyfish genomic library in lambda FIX II vector, prepared from X. maculatus Jp 163 A adult males, was obtained from Stratagene (La Jolla, CA). The complexity of the original library was 2 x 10⁶ plaque-forming units (PFU); the titer of the amplified library used for screening was 2.0 x 10¹⁰ PFU/ml. Fifty nanograms of the MHC class II DXB gene fragment described above was radiolabeled with [-³²P]dCTP with the RadPrime DNA Labeling System (Life Technologies) according to the manufacturer's protocol. Replicate nylon filters (MSI, Westboro, MA) containing DNA of approximately 6 x 10⁵ genomic clones were screened with the radiolabeled platyfish probe after a 2-hr prehybridization in 5x Denhardt's, 6x standard sodium citrate (SSC), 0.5% SDS, and 50 µg/ml calf thymus DNA at 42° in a Hybridizer 600 oven (Stratagene). Hybridization was in the identical solution with 10⁶ cpm/ml radiolabeled probe added, and incubated at 65° for 16 hr. Filters were washed two times at 25° in 5x SSC, 0.5% SDS for 15 min; two times at 37° in 1x SSC, 0.5% SDS at 37°; two times at 37° in 0.1x SSC, 1% SDS; and three times at 65° in 0.1x SSC, 1% SDS. The filters were then used to expose X-ray film (Fuji Photo Film Co., Ltd., Japan) with DuPont Cronex intensifying screens for 1–3 days at -70°. Fourteen primary plaques were positive, five of which remained positive through secondary and tertiary screenings. Two of the plaques were analyzed by restriction enzyme mapping. One plaque yielded a 6.5-kb HindIII-NotI DNA single hybridizing fragment that was subcloned into the HindIII and NotI sites of pCR II (Invitrogen, Carlsbad, CA). Primers designed from the sequenced 190-bp ß2-encoding fragment were used to begin DNA sequence analysis of this 6.5-kb cloned genomic DNA. As this fragment was sequenced, new primers were designed until the complete sequence was determined.

DNA sequencing and analysis:
Sequencing was performed using universal Forward and Reverse primers (DNA International, Lake Oswego, OR) on PCR-derived fragments, and with gene-specific primers designed with Oligo. Both strands were sequenced by the dideoxy chain termination method (SANGER et al. 1977 ) using the fluorescence-based PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer's protocol. The results were analyzed on the Applied Biosystems Model 373A DNA Sequencing System. Exon-intron splicing at the only ambiguous sites (intron 5, due to the nonconsensus nature of the splice signals) is described as predicted by PROCRUSTES (GELFAND et al. 1996 ).

DNA sequence alignments and construction of dendrograms:
DNA sequence analysis, fragment assembly, homologous sequence overlays, and amino acid translations were generated using the DNAsis (Hitachi Software Engineering Co., Limited, 1991) sequence analysis as well as the Genetics Computer Group (GCG) (DEVEREUX et al. 1984 ) programs. Searches for sequences similar to Xiphophorus class II B genes were performed and preliminarily aligned using the NCBI Blast E-mail server (ALTSCHUL et al. 1990 ). The final nucleotide sequence alignments were performed using the PILEUP and PRETTY programs of the GCG. Pairwise distances were calculated for exon 3 (class II ß2-domain encoding) using the p-distance algorithm and the neighbor-joining method of SAITOU and NEI 1987 . Calculations and dendrogram construction were completed using the Molecular Evolutionary Genetics Analysis (MEGA) programs (KUMAR et al. 1993 ).

Gene mapping:
Interspecific hybrids used to produce backcrosses were made by artificial insemination (CLARK 1950 ), using Xiphophorus clemenciae from the Rio Sarabia, Oaxaca, Mexico, maintained in closed colony since capture in 1968; Xiphophorus milleri, collected in tributaries to Lago Catemaco, Veracruz, Mexico in 1982; X. helleri, collected in the Rio Sarabia in 1963; and X. maculatus strain Jp 163 A, inbred by brother-sister matings for 89 generations since collection in the Rio Jamapa, Veracruz, Mexico in 1939. Backcrosses were produced by KLAUS KALLMAN at the New York Aquarium Osborn Laboratories of Marine Sciences, Brooklyn, NY and at the Xiphophorus Genetic Stock Center at Southwest Texas State University, San Marcos, TX.

Brain and eye, skeletal muscle, testis, and liver tissues were prepared and used for starch gel electrophoresis and histochemical staining following methods of MORIZOT and SCHMIDT 1990 . Genomic DNA was extracted from gill, testis, spleen, and/or kidney tissues using protocols of HARLESS et al. 1990 . Preliminary digestions with a variety of restriction endonucleases identified interspecific polymorphisms at DXB cut with BamHI and at DAB digested with PstI. Genomic DNA from appropriate backcross individuals was digested according to manufacturers' directions, electrophoresed through 0.8% agarose gels, and blotted onto hybridization membranes by methods of HARLESS et al. 1990 , HARLESS et al. 1991 . Probes for DXB (190-bp fragment in the ß2-encoding domain of X. maculatus cDNA) and DAB (full-length 795-bp X. maculatus cDNA) were ³²P-radiolabeled by nick translation and/or random priming and hybridized to membranes, washed at high stringency, and autoradiographed to visualize hybridizing fragments (WALTER et al. 1993 ). Genotypes of each backcross hybrid individual were scored, usually based upon codominant inheritance models, for all allozyme, DNA RFLP, and arbitrarily primed PCR (AP-PCR; KAZIANIS et al. 1996 ) polymorphisms.

Each polymorphic locus was assessed for agreement with the expected 1 homozygote:1 heterozygote backcross segregation; loci significantly (P 0.05) deviating from Mendelian expectations were excluded from linkage analyses. Pairwise tests for deviation from 1 parental:1 recombinant independent assortment expectations were performed using MAPMAKER software (LANDER and GREEN 1987 ) with LOD > 3.0 or ²_{1 d.f.} > 13.8 (P < 0.001) used as the criterion for presumption of genetic linkage. Map positions were compared to existing gene map assignments summarized in MORIZOT et al. 1991 , MORIZOT et al. 1993 , MORIZOT et al. 1998 and KAZIANIS et al. 1996 , which references also should be consulted for gene nomenclature and mapping methodology.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Nucleotide sequence of Xima and Xihe DXB genes:
The X. maculatus, strain Jp 163 A, genomic library was screened with the PCR-derived ß2-encoding fragment as described in MATERIALS AND METHODS. A 6.5-kb HindIII-NotI fragment from a positive plaque was subcloned and sequenced with gene-specific primers and found to contain the complete coding region of a Xima class II B gene. The sequence (Figure 1) includes the 5' untranslated region (UTR), six exons, five introns, and the 3' UTR to the polyadenylation signal. The exon-intron boundaries were determined from comparisons with a cDNA clone (PCR-amplified from X. maculatus RNA with primers designed to the 5' and 3' ends of the genomic sequence) that was identical in coding regions. Proposed transcription start sites (underlined in Figure 1 in the region upstream of the START codon and listed in order) of the 5' UTR including S box, pyrimidine-rich region, X box, X2 box, Y box, and CCAAT box are marked (BENOIST and MATHIS 1990 ; PRESTRIDGE 1991 ; GLIMCHER and KARA 1992 ; SINGAL and QIU 1995 ). Exon 1 encodes the first 20 amino acids of the leader peptide followed by intron 1, which is 137 bp long (data of introns not shown). Exon 2 encodes 2 additional amino acids of the leader and 86 of the ß1 domain. Intron 2 is 116 bp and exon 3 encodes the complete ß2 domain (94 amino acids). A third intron of 369 bp follows. The 9 amino acids of the connecting peptide, the 23 amino acids of the transmembrane region and the first 5 amino acids of the cytoplasmic tail are encoded in exon 4. Intron 4 is 133 bp. Exons 5 and 6, together encoding the remaining 19 amino acids of the cytoplasmic tail, are separated by a fifth intron. Exon 6 proceeds to the TAG stop codon, followed by 3' UTR. This exon-intron structure of the DXB locus is more similar to the class II B genes found in amphibian (KOBARI et al. 1995 ), chicken (XU et al. 1989 ), and human (KAPPES et al. 1984 ) than to the class II B genes characterized in zebrafish (SULTMANN et al. 1994 ) and cichlid (ONO et al. 1993 ). This exon-intron organization, in combination with the longer cytoplasmic tail encoded by DXB (see below), raises the interesting question of whether a DXB-like locus originally led to the tetrapod lineage of MHC class II B genes.

View larger version (82K): In this window In a new window Download PPT slide   Figure 1. Genomic nucleotide sequences of platyfish MhcXima-DXB*01 and swordtail MhcXihe-DXB*01. Dashes indicate identity of Xihe with Xima. Dots represent intron sequences. Asterisks denote the position of the initial primers used to amplify swordtail genomic DNA and also platyfish cDNA to obtain the coding regions. The proposed positions of the S box, pyrimidine-rich region, X box with X2 box, Y box, and CAAT box (in order from 5' to 3') are underlined; protein domains, exons, and introns are in bold; and the deduced amino acid sequence is printed above the codons. The Xima DXB*01 and Xihe-DXB*01 genomic sequences (complete with introns) have been submitted to the GenBank database and assigned accession nos. AF040762 and AF040763, respectively.

The significant difference in organization of the DXB locus from the DAB organization of other advanced teleost fishes leads to some additional observations. ONO et al. 1993 originally described the presence of an additional intron that splits the coding region of the ß2 domain for DAB-like gene of cichlids (Percomorpha). A subsequent survey among teleost fishes by FIGUEROA et al. 1995 also found this intron in several other percomorph species and in Melanotaenia trifasciata of the Atherinomorpha, sister clade to the Percomorpha (NELSON 1994 ), but the intron was absent in more basal teleost clades (i.e., Ostariophysi and Protacanthopterygii). Our data clearly indicate that this intron is absent in the DXB gene of Xiphophorus (Poeciliidae), also a member of the Atherinomorpha. Thus the exon-intron structure of the DXB gene is more similar to the DAB of primitive teleosts, than to the DAB gene of advanced teleosts, including other atherinomorph fishes. This leads to two alternative evolutionary hypotheses. In the first, the DXB and DAB genes were produced by a gene duplication event after the acquisition of the sixth intron, but this intron was subsequently lost in the DXB gene of Xiphophorus. Alternatively, the gene duplication event giving rise to the DXB gene occurred before the evolution of the sixth intron, very early in the diversification of teleost fishes. The latter hypothesis is supported by the phylogenetic analysis of nucleotide changes for the class II B genes (see below). If so, the investigation of evolution of the DXB gene adds an additional independent line of inquiry to the important insights already provided by the study of MHC genes in elucidating phylogenetic patterns (KLEIN et al. 1997 ).

The genomic sequence of Xima-DXB*01 has conserved features with other teleost class II B genes previously studied (ONO et al. 1993 ; SULTMANN et al. 1994 ; VAN ERP et al. 1996 ). Compared to human MHC class II introns (KLEIN 1986 ), those of teleosts are relatively short. All but one exon-intron boundary has canonical splice signals (SHAPIRO and SENEPATHY 1987 ; SENEPATHY et al. 1990 ). Intron 5 in Xiphophorus, splitting exons 5 and 6 encoding the cytoplasmic tail region, has not been reported in other teleosts. Intron 5 in Xiphophorus has nonconsensus splice signals both at the 5' donor and 3' acceptor site. Even though these nonconsensus splice sites appear to be rare, they nevertheless have been reported in 0.7% of 7500 mammalian splice sites examined by SENEPATHY et al. 1990 . VAN ERP et al. 1996 reported a nonconsensus splice signal in the Cyca-DAB3*01 and Cyca-DAB4*01 genomic sequences, as did DIXON et al. 1996 in Barbus intermedius intermedius and Barbus bocagei. It has been suggested that nonconsensus splice sites may be important in slowing the upregulation of the expressed gene (HAVILAND et al. 1991 ). Incomplete splicing may compromise the stability of the mRNA, thus providing a means of gene regulation (GLIMCHER and KARA 1992 ).

To test for the presence of the DXB locus in a species closely related to X. maculatus, swordtail (X. helleri) genomic DNA was isolated and a Xihe-DXB PCR fragment was amplified. The resulting fragment was cloned and sequenced, revealing a complete MHC class II B gene including start and stop codons (Figure 1). The two sequences are very similar, differing in only 25 nucleotides throughout the compared sequence, plus an insertion of 4 nucleotides in intron 2 (data not shown). Eleven of the nucleotide differences between the two DXB sequences are clustered in exon 2, the ß1 encoding domain. Nine of these nucleotide changes result in nonsynonymous amino acid substitutions indicative of a functionally encoded peptide binding region (HUGHES and HUGHES 1995 ), though these are not two sequences from the same species. In contrast, only three nucleotide differences occur in exon 3, resulting in two amino acid changes. Xihe-DXB*01 has an identical intron 5 with the same noncanonical exon-intron splice sites as the platyfish.

The promoter region at the 5' end of the Xima-DXB*01 sequence shows similar features to those previously identified (reviewed in BENOIST and MATHIS 1990 ; GLIMCHER and KARA 1992 ; MACH et al. 1996 ). Considerable allelic polymorphism in this region has been reported in the literature and may account for differences in gene expression (SINGAL and QIU 1995 ). The CCAAT box is present, but a typical TATA box cannot be found in the expected region of the promoter (-180 to +10). SULTMANN et al. 1994 also reported the lack of a TATA box in the zebrafish Dare-DAB and Dare-DEB genes. It has been suggested that the TATA box may not be needed for proper transcription initiation in those genes that are tissue-specific, where other transcription factor binding sites such as X box, X2 box and Y box are involved (GLIMCHER and KARA 1992 ). A sequence suggestive of an octamer motif (GATTTGTT) is immediately adjacent to the Y box (CATTGGTG). Initially described in immunoglobulin genes, this element appears to be involved in gene expression (GLIMCHER and KARA 1992 ).

Sequence comparison of Xima-DXB and -DAB:
The nucleotide and amino acid sequences of Xima-DXB*01 and -DAB*01 are compared in Figure 2. The two sequences are 43 and 63% identical in the ß1- and ß2-coding regions, respectively, and 27 and 55% identical in the ß1 and ß2 amino acid sequences. Thus ß1 sequence identity between these two genes is low, but more informative is the low level of 55% identity for ß2. This is at the lower limit of the 54–85% identity found between the protein chains coded by known class II loci, excepting DM (CHO et al. 1991 ; KELLY et al. 1991 ), which is 31–39% identical to the other respective class II ß2 domains. Neither Xima gene demonstrated sequence patterns indicative of a DM-like locus, although similar functions could be carried out without sequence homology. The longer cytoplasmic tail of Xima-DXB could be involved in either specific trafficking or in MHC class II signaling to the interior of the cell. The three codon deletions in the ß1-encoding region and the one codon deletion in the CP-encoding region of Xima-DXB relative to Xima-DAB also indicate DXB is a significantly divergent locus. The nucleotide and amino acid sequence variability coding for the ß1 and ß2 domains is readily apparent in Figure 3. Surprisingly, the longest string of consecutive identical nucleotides in the cDNAs of these two loci in Xima is 16. The extensive diversity between these two genes may also affect preferential pairing of the encoded ß chains with as yet uncharacterized chains from different class II A loci in Xiphophorus. Differences in glycosylation sites and the length of the cytoplasmic tail are discussed below.

View larger version (72K): In this window In a new window Download PPT slide   Figure 2. Nucleotide and putative amino acid sequence comparisons of Xima-DXB*01 and Xima-DAB*01 cDNA clones. The Xima-DXB*01 and Xima-DAB*01 cDNA sequences have been submitted to the GenBank database and assigned accession nos. AF040761 and AF040760, respectively.

View larger version (57K): In this window In a new window Download PPT slide   Figure 3. Comparison of deduced amino acid sequences of the platyfish MHC class II ß chains with other teleost species. Cyca-DAB4*01 (VAN ERP et al. 1996 ), Dare-DAB4*01 (ONO et al. 1992 ), Onmy-DAB*01 (GLAMANN 1995 ), Sasa-C-157 (HORDVIK et al. 1993 ), Auha-M-231 and Cyfr-T-141 (ONO et al. 1993 ), MosaC-1 (WALKER and MCCONNELL 1994 ), Pore-DB-4-28 (SATO et al. 1995 ), and Icpu-DAB*01 (GODWIN et al. 1997 ) were chosen to represent a range of teleost species. Dashes indicate identity with the consensus (simple majority). Dots were inserted for optimal alignment of the sequences. The glycosylation site is underlined, and conserved cysteines involved in disulfide linkages are bold. The numbering system defines the putative mature protein of Xima-DXB*01 as determined by SignalP (NIELSEN et al. 1997 ). The proposed domains of the ß chain are labeled above the sequence.

Sequence alignment of selected teleost class II ß chains:
An amino acid (aa) sequence alignment of Xima-DXB*01 and Xima-DAB*01 with those of other representative teleost class II B chains found as related with an NCBI BLAST search, is shown in Figure 3. The Xima-DXB*01 leader sequence/ß1 boundary was determined with the SignalP computer program (NIELSEN et al. 1997 ). The hydrophobicity of the leader peptide, variability between the two Xiphophorus species in the ß1 domain, overall consensus residues of the ß2 domain, connecting peptide, transmembrane and cytoplasmic region are all similar to those of the previously identified organisms. Key residues such as those involved in disulfide bonds, glycosylation, and interaction with the chain are also seen in the Xima-DXB sequence. Differences in DXB compared to the other class II chains shown include 3-aa deletions (positions 65, 80, and 81) in ß1 and a 1-aa deletion (position 186) in the connecting peptide. Atlantic salmon cDNA clones 144 and 22 (HORDVIK et al. 1993 ) and zebrafish pseudogene genomic sequences DCB and DBB (SULTMANN et al. 1994 ) show codon deletions in the ß1 domain that differ from those of DXB.

The teleost MHC class II ß sequences shown all have a glycosylation signal sequence at positions 12–14 or 36–38 (Figure 3), including Xima-DXB, with the single exception of the guppy sequence Pore-DB-4-28, which lacks any such site. The Xima-DAB sequence, clearly related to the guppy sequence at 92% identity, does have a glycosylation signal sequence, at consensus positions 12–14.

The 22-aa cytoplasmic tail of Xima-DXB is surprisingly long, but similar in length to the human HLA-DRB (TIEBER et al. 1986 ), the clawed frog Xela-F3 (SATO et al. 1993 ), and the nurse shark Gici-ß (BARTL and WEISSMAN 1994 ). This could have functional significance as discussed above.

Comparison of the Xima sequences to zebrafish (Dare-DAB4*01), among others, is shown in Figure 3. The genes coding for the zebrafish class II ß sequences have been particularly well characterized at the genomic level (SULTMANN et al. 1994 ). Within the Dare-DAB1 family of related loci, coded amino acid sequence identity of DAB2*01, DAB3*01, and DAB4*01 to DAB1*01 ß2 domain is 84–89%, though evidence indicated only one locus was transcribed. Comparison of Dare-DAB ß2 sequence to the presumed pseudogenes of distantly related loci Dare-DBB, -DCB, -DDB, and -DEB revealed 63–71% identity. This contrasts with the more distantly related Xima-DXB vs. -DAB at 55% identity in ß2, particularly when considering that both the Xima loci are transcribed with all features necessary for expression.

Mapping of MHC class II B loci:
Linkage analyses for DXB and DAB are presented in Table 1 and Table 2, respectively. Unfortunately, polymorphisms for both genes were not detected in the same cross type, so that direct linkage tests could not be performed. However, each locus was assigned to a multipoint linkage group with high confidence.

Assignment of DXB to Xiphophorus linkage group (LG) III: Table 1 documents linkage of DXB and GAPD1 with ~18% recombination. Unfortunately, crosses involving X. clemenciae were made years before our incorporation of many DNA RFLP and AP-PCR polymorphisms as markers; thus, many LG III markers could not be analyzed in the present study. Because GAPD1 is near the end of LG III in the current Xiphophorus map comprising >20 markers (MORIZOT et al. 1993 , MORIZOT et al. 1998 ), it is likely that DXB lies toward the center of the linkage group near GUK2. LG III also contains a malic enzyme locus, probably coding for a cytosolic isozyme, which is also the case in human chromosome 6, carrying both malic enzyme loci and the major histocompatibility complex.

Assignment of DAB to Xiphophorus newly designated LG U24: DAB was found to be polymorphic in the most extensively mapped Xiphophorus cross type, with >250 informative markers located in 25 multipoint linkage groups, one more than the 24 acrocentric or telocentric chromosome pairs. DAB was found to be linked to two AP-PCR markers, XD0226 and XD0154; these three markers here are assigned to the newly designated LG U24. The gene order is uncertain because of small sample sizes, but an order of XD0154–12%–XD0226–9.8%–DAB minimizes multiple crossovers. It is impossible to determine with certainty that DAB and DXB reside on different chromosomes, particularly since DAB yields recombination estimates of <40% with some LG III markers. Additional mapping data will coalesce multi-point linkage groups into 24 chromosomal linkage groups in time, but at present it can be stated with reasonable certainty based upon recombination estimates with all LG III loci (Table 2) that DXB and DAB are not tightly linked and assuredly are not members of a gene cluster. BINGULAC-POPOVIC et al. 1997 performed linkage analysis of MHC class I and class II, and ß2m genes in zebrafish. These investigators found that MHC class I genes were not linked to class II. They also found Dare-DAB and DDB (a pseudogene) class II B genes to be tightly linked, and DFB (a pseudogene) not to be linked to the expressed gene DAB. The Xima-DXB and DAB, however, represent two very different class II B genes of different clusters, that are both expressed. The data presented here thus support the possibility that tight clustering of MHC loci in mammals may represent a relatively recent evolutionary arrangement.

It is uncertain whether more than one copy of DXB and DAB exists in the Xiphophorus genome. DXB exhibits only one strongly hybridizing fragment on Southern blots with the 190-bp cDNA probe used (Figure 4A), and likely is present as a single copy. DAB, on the other hand, exhibits several fragments in addition to three obviously polymorphic fragments (Figure 4B). Whether the additional fragments represent pieces of DAB, which is quite possible as a full-length cDNA was used for probe, or a gene duplicate cannot be determined at present. Cloning of the genomic DAB sequence and location of restriction sites will help to resolve this issue. SATO et al. 1995 present evidence suggesting that the MHC class II B sequences found in guppy are encoded at a single locus, the DAB-like locus. Guppy may also possess the DXB locus in addition to the DAB locus characterized by SATO et al. 1995 . Exon 3 in Xima DXB*01, encoding the more conserved ß2 domain, is 61% similar to exon 3 in the guppy (Pore-DB-4-28), a surprisingly low degree of identity between two very similar species until considering that DXB and the guppy DAB-like gene are two very different loci. The detection of one predominant set of bands in our Southern blots supports the detection of a single DXB locus without cross-hybridization to DAB.

View larger version (95K): In this window In a new window Download PPT slide   Figure 4. (A) Representative Southern blot of the DXB cross-hybridizing fragments using the 190-bp probe described in the text. Lane 1, X. clemenciae; lane 2, X. milleri; lanes 3–6, backcross hybrids from X. clemenciae x (X. milleri x X. clemenciae) crosses. Lanes 3 and 5 are homozygotes for X. clemenciae alleles, while lanes 4 and 6 are heterozygotes. (B) Representative Southern blot of the DAB cross-hybridizing fragments using a full-length cDNA probe. Lanes 1–7, backcross hybrids from X. helleri x (X. maculatus x X. helleri) crosses. Lanes 1–4 and 6 are heterozygotes, while lanes 5 and 7 are homozygotes for X. helleri alleles.

Phylogenetic analysis:
To examine the relationship of Xima class II B sequences with those of other fishes, a dendrogram was constructed employing the neighbor-joining method (SAITOU and NEI 1987 ) on distances based on the p-distance algorithm as calculated by MEGA (KUMAR et al. 1993 ). The phylogenetic tree shown in Figure 5, based on exon 3 sequences, demonstrates the wide disparity between the Xiphophorus DXB sequences and the DAB sequences. An analysis using the Jukes-Cantor (JUKES and CANTOR 1969 ) and Tajima-Nei (TAJIMA and NEI 1984 ) methods of correction for multiple substitutions led to a dendrogram with identical topology as shown in Figure 5. Sequences of a class II B locus in carp, and of closely related loci in zebrafish, cluster together, while Xiphophorus DXB genes and the closely related guppy Poecilia reticulata DB-4-28 (SATO et al. 1995 ) (both species are in the Family Poeciliidae, Subfamily Poeciliinae, Tribe Poeciliini; NELSON 1994 ) are shown as unrelated in Figure 5. Xiphophorus-DAB and the guppy sequence DB-4-28 are, however, shown as closely related. The dendrogram also places the Xiphophorus-DXB sequences as ancestral to those of the class II sequences of representative members of the orders Salmoniformes and Perciformes, with the very low bootstrap value of 38. In fact, this placement of the Xiphophorus DXB genes is the only grossly inconsistent feature of this dendrogram, which otherwise fits with presumed phylogenetic relationships based on a cladistic analysis of morphological characters (NELSON 1994 ). The basal placement of DXB on the teleost dendrogram implies a very early duplication event with much subsequent independent evolution of the DXB and DAB genes. This timing is consistent with intron-exon structure that we discussed earlier. Our findings raise an important caution for investigators using MHC genes to make phylogenetic inferences: proper assignment of alleles to the correct homologous gene will be critical when tracing MHC evolution. Future experiments will test for the presence DXB (and DAB, if necessary) in other species of fish to determine the evolutionary history of these two loci relative to one another and relative to MHC class II B loci in tetrapods. The "X" family designation of the DXB locus will need to be reassigned as more is learned about the evolutionary relationship of this gene to other class II B genes in other species. Also, the possibility of different functions for these two loci will be investigated. The levels of polymorphism of DXB versus DAB, tissue expression patterns, and cellular trafficking patterns will lead to a more detailed understanding of the function of the products of the DXB locus.

View larger version (28K): In this window In a new window Download PPT slide   Figure 5. A phylogenetic tree based on the genetic distances between exon 3 nucleotide sequences from different teleosts. The tree was constructed as described under MATERIALS AND METHODS. Numbers on nodes indicate frequency with which this node was recovered per 100 bootstrap replications in a total of 500 replications. References correspond with those given in Figure 2, with additional references: Auha-M-195a, Auha-M-195b, Auha-M-231a, Auha-M-231b, Cyfr-T-141a, Cyfr-T-141b, Cyfr-T-141c, Cyfr-T-141d (ONO et al. 1993 ); Onmy-DAB*02, Onmy-DAB-522 (GLAMANN 1995 ); Dare-DAB1*01, Dare-DAB2*01, Dare-DAB3*01 (ONO et al. 1992 ); Dare-DBB, Dare-DCB, Dare-DDB, Dare-DEB (SULTMANN et al. 1994 ); Cyca-DAB1*01, Cyca-DAB2*01, Cyca-DAB3*01 (VAN ERP et al. 1996 ); Bain-DAB3-A02 (DIXON et al. 1996 ); and Gici-11 (BARTL and WEISSMAN 1994 ).

	ACKNOWLEDGMENTS

We thank BRENDA B. MCENTIRE, LUIS DELLA COLETTA, and BARBARA SANTI for excellent technical assistance. This research was supported in part by U.S. Public Health Service grants CA55245 and CA09480, and by National Institute of Environmental Health Sciences center grant ES07784.

Manuscript received December 22, 1997; Accepted for publication May 11, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

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