Holger Sültmann

Nonlinkage of major histocompatibility complex class I and class II loci in bony fishes.

Abstractu2002In tetrapods, the functional (classical) classu2009I and classu2009II B loci of the major histocompatibility complex (Mhc) are tightly linked in a single chromosomal region. In an earlier study, we demonstrated that in the zebrafish, Danio rerio, order Cypriniformes, the two classes are present on different chromosomes. Here, we show that the situation is similar in the stickleback, Gasterosteus aculeatus, order Gasterosteiformes, the common guppy, Poecilia reticulata, order Cyprinodontiformes, and the cichlid fish Oreochromis niloticus, order Perciformes. These data, together with unpublished results from other laboratories suggest that in all Euteleostei, the classical classu2009I and classu2009II B loci are in separate linkage groups, and that in at least some of these taxa, the classu2009II loci are in two different groups. Since Euteleostei are at least as numerous as tetrapods, in approximately one-half of jawed vertebrates, the classu2009I and classu2009II regions are not linked.

Zebrafish Mhc class II α chain-encoding genes: polymorphism, expression, and function

Its small size and short generation time renders the zebrafish (Brachydanio rerio) an ideal vertebrate for immunological research involving large populations. A prerequisite for this is the identification of the molecules critical for an immune response in this species. In earlier studies, we cloned the zebrafish genes coding for the β chains of the class I and class II major histocompatibility complex (MHc) molecules. Here. we describe the cloning of the zebrafish α chain-encoding class II gene, which represents the first identification of a class II A gene in teleost fishes. The gene, which is less than 3 kilobases (kb) distant from one of the β chain-encoding genes, is approximately 1.2 kb long and consist of four exons interrupted by very short (<200 base pairs) introns. Its organization is similar to that of the mammalian class II A genes, but its sequence differs greatly from the sequence of the latter (36% sequence similarity). Among the most conserved parts is the promoter region, which contains X, Y, and TATA boxes with high sequence similarity to the corresponding mammalian boxes. The observed striking conservation of the promoter region suggests that the regulatory system of the class II genes was established more than 400 million years ago and has, principally, remained the same ever since. Like the DMA, but unlike all other mammalian class II A genes, the zebrafish gene codes for two cysteine residues which might potentially be involved in the formation of a disulfide bond in the α1 domain. The primary transcript of the gene is 1196 nucleotides long and contains 708 bucleotides of coding sequence. The gene is expressed in tissues with a high content of lymphoid/myeloid cells (spleen, pronephros, hepatopancreas, and intestine). The analyzed genomic and cDNA sequences are probably derived from different loci (their overall sequence similarity in the coding region is 73% and their 3′ untranslated regions are highly divergent form each other). The genes are apparently functional. Comparison of genes from different zebrafish populations reveals high exon 2 variability concentrated in positions coding for the putative peptide-binding region. Phylogenetic analysis suggests that the zebrafish class II A genes stem form a different ancestor than the mammalian class II A genes and the recently cloned shark class II gene.

A Contig Map of the Mhc Class I Genomic Region in the Zebrafish Reveals Ancient Synteny

In contrast to the human and mouse Mhc, in which the clusters of class I and class II loci reside in close vicinity to one another, in the zebrafish, Danio rerio, they are found in different linkage groups. Chromosome walking using BAC (bacterial artificial chromosome) and PAC (P1 artificial chromosome) clones reveals the zebrafish class I region to occupy a segment of ∼450 kb and to encompass at least 19 loci. These include three class I (Dare-UDA, -UEA, -UFA), five proteasome subunit β (PSMB8, -9A, -9C, -11, -12), two TAPs (TAP2A, TAP2B), and one TAP binding protein (TAPBP). This arrangement contrasts with the arrangements found in human and mouse Mhc, in which the orthologues of the PSMB, TAP, and TAPBP loci reside within the class II region. In addition to this main zebrafish class I contig, a shorter contig of about 150 kb contains two additional class I (UBA, UCA) and at least five other loci. It probably represents a different haplotype of part of the class I region. The previously identified UAA gene shares an identical 5′ part with UEA, but the two genes differ in their 3′ parts. One of them is probably the result of an unequal crossing over. The described organization has implications for the persistence of syntenic relationships, coevolution of loci, and interpretation of the origin of the human/mouse Mhc organization.

Mhc class II B gene evolution in East African cichlid fishes.

Abstractu2002A distinctive feature of essential major histocompatibility complex (Mhc) loci is their polymorphism characterized by large genetic distances between alleles and long persistence times of allelic lineages. Since the lineages often span several successive speciations, we investigated the behavior of the Mhc alleles during or close to the speciation phase. We sequenced exon 2 of the classu2009IIu2009B locus 4 from 232 East African cichlid fishes representing 32 related species. The divergence times of the (sub)species ranged from 6000 to 8.4 million years. Two types of evolutionary analysis were used to elucidate the pattern of exon 2 sequence divergence. First, phylogenetic methods were applied to reconstruct the most likely evolutionary pathways leading from the last common ancestor of the set to the extant sequences, and to assess the probable mechanisms involved in allelic diversification. Second, pairwise comparisons of sequences were carried out to detect differences seemingly incompatible with origin by nonparallel point mutations. The analysis revealed point mutations to be the most important mechanism behind allelic divergences, with recombination playing only an auxiliary part. Comparison of sequences from related species revealed evidence of random allelic (lineage) losses apparently associated with speciation. Sharing of identical alleles could be demonstrated between species that diverged 2 million years ago. The phylogeny of the exon was incongruent with that of the flanking introns, indicating either a high degree of convergent evolution at the peptide-binding region-encoding sites, or intron homogenization.

Class I Mhc genes of cichlid fishes: identification, expression, and polymorphism

Abstractu2003Cichlid fishes of the East African Rift Valley lakes constitute an important model of adaptive radiation. Explosive speciation in the Great Lakes, in some cases as recently as 12u200a400 years ago, generated large species flocks that have been the focus of evolutionary studies for some time. The studies have, however, been hampered by the paucity of biochemical markers for phylogenetic reconstruction. Here, we describe a set of markers which should help to alleviate this problem. They are the class I genes of the major histocompatibility complex. We provide evidence for the existence of at least 17 class I loci in cichlid fishes, and for extensive polymorphism of three of these loci. Since the polymorphism has a trans-species character, it will be possible to use it in investigating the founding events of the individual species. The sequences of the cichlid class I fishes support the monophyly of actinopterygian fish on the one hand, and of tetrapods on the other.

Major histocompatibility complex class II A genes in cichlid fishes: identification, expression, linkage relationships, and haplotype variation.

Abstractu2002Two cichlid species, the haplochromine Aulonocara hansbaenschi and the tilapiine Oreochromis niloticus, were used to study the major histocompatibility complex (Mhc) classu2009II A variation within this group. Multiple classu2009II A sequences were recovered from A. hansbaenschi and O. niloticus cDNA libraries and three sequence families, DAA, DBA, and DCA, were identified. Sets of O. niloticus haploid embryo families were used to determine the linkage relationships of these genes. Two independently assorting linkage groups were detected, DAA and DBA/DCA, neither of which is linked to the previously described Mhc classu2009I gene cluster. Three DCA genes and up to four DBA genes were found to segregate in different haplotypes, whereas DAA occurred as a single locus. Four DBA haplotypes, DBA*H1-H4, were identified and shown to co-segregate with the previously described classu2009II B haplotypes. Four DCA haplotypes, DCA*H1-H4, were found at a distance of 37u2009cM from the DBA/classu2009II B cluster; in one DCA haplotype, DCA*H5, the genes were tightly linked to the DBA/classu2009II B clusters. Transcripts of DAA and DBA genes were found in O. niloticus hepatopancreas and spleen; transcripts of DCA genes were detected in the A. hansbaenschi cDNA library, but not in O. niloticus. These findings provide a basis for using classu2009II haplotypes as markers in the study of adaptive radiation in the cichlid species flocks of the East African Great Lakes.

Conservation of Mhc Class III Region Synteny Between Zebrafish and Human as Determined by Radiation Hybrid Mapping

In the HLA, H2, and other mammalian Mhc, the class I and II loci are separated by the so-called class III region comprised of ∼60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the Mhc 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-Mhc that existed before the class I/II genes arose. The pre-Mhc may not have contained the complement and other class III genes involved in immune response.

ISOLATION OF MHC CLASS II DMA AND DMB CDNA SEQUENCES IN A MARSUPIAL : THE GRAY SHORT-TAILED OPOSSUM (MONODELPHIS DOMESTICA)

Abstract. We report the cDNA sequences for the DMA and DMB family of Mhc genes of the gray short-tailed opossum. Until now DM sequences were available only in eutherian mammals. The marsupial sequences indicate that both members of the family are old and probably diverged from other classical class II families about the time of the radiation of jawed vertebrates some 450 million years ago. We examine the evolutionary rates of equivalent sets of classical and nonclassical genes to check for rate heterogeneity. We find the α-1 domain of the DR genes to be untypically conservative in its evolutionary mode. The DM genes appear to evolve at rates typical of other class II genes, indicating that their placement at the root of class II gene evolutionary trees may be justified.

CHAPTER 4 – Reconstruction of Cichlid Fish Phylogeny Using Nuclear DNA Markers

This chapter introduces the reconstruction of cichlid fish phylogeny from the family of Cichlidae that constitutes a monophyletic group in the order Perciformes. Various methods used for reconstructing cichlid phylogeny are highlighted in this chapter.. It describes and discusses the application of two PCR-based methods. The Random Amplification of Polymorphic DNA (RAPD) procedure, originally developed as a method for fingerprinting genomes, has been applied to the discovery of genetic markers for mapping studies. Allele frequency distributions for 10 Haplochromis species from the lakes Victoria, Kayugi, Kayania, and Nabugabo at the microsatellite locus DXTUCA15 are demonstrated with the summaries. From several microsatellite loci typed, this chapter illustrates the example of the DXTUCA15 locus, which was amplified from haplochromine genomic DNA with the primers MS16 and MS17 by PCR for various time slots and temperatures. The reaction was completed by a final primer extension step for 10 minutes at 72 degrees Celsius. In general, RAPD and the microsatellite approach were both able to detect polymorphism between closely related taxonomic groups. With respect to cichlid phylogeny, RAPD can be primarily applied to genera under comparison.

Linkage relationships and haplotype variation of the major histocompatibility complex class I A genes in the cichlid fish Oreochromis niloticus.

Abstract: The haplochromine cichlid species flocks of the East African Great Lakes are one of the best examples of adaptive radiation. Analysis of genetic variation among these species provides valuable information on species relationships and timing of speciation events. Although the haplochromine cichlids generally display little genetic variation, the major histocompatibility complex (Mhc) genes have been found to be highly variable. A study of the linkage relationships of the Mhc class I A genes in the cichlid fish Oreochromis niloticus was therefore undertaken. Class I loci were identified, and their segregation in seven mothers and their haploid embryos was determined. In total, 56 class I A sequences were found among the seven families. A strong concordance of segregation was observed in five haplotypes among the embryos, indicating a close linkage of all loci. The number of loci per haplotype varied from 11 to 17, while the total number of distinct loci found among all families was 22. These findings show that all class I A loci are linked in a single genetic cluster in O. niloticus.