Cloning and characterization of class I Mhc genes of the zebrafish,Brachydanio rerio

The zebrafish (Brachydanio rerio) offers many advantages for immunological and immunogenetic research and has the potential for becoming one of the most important nonmammalian vertebrate research models. With this in mind, we initiated a systematic study of the zebrafish major histocompatibility complex (Mhc) genes. In this report, we describe the cloning and characteristics of the zebrafish class I A genes coding for the chains of the heterodimer and thus complete the identification of all four classes and subclasses of the Mhc in this species. We describe the full class I cDNA sequence as well as the exon-intron organization of the class I A genes, including intron sequences. We identify three families of class I A genes which we designate Bree-UAA,-UBA, and -UCA. The three families originated about the time of the divergence of cyprinid and salmonid fishes. All three families are members of an ancient lineage that diverged from another, older lineage also represented in cyprinid fishes before the radiation of teleost orders. The fish class I A genes therefore evolve differently from mammalian class I A genes, in which the establishment of lineages and families mostly postdates the divergence of orders.The nucleotide sequence data reported in this Papershave been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers Z46776–Z46779     

Cloning of the β2-microglobulin gene in the zebrafish

The ₂-microglobulin (₂m) is a protein found in the serum in a free form and on the cell surface in a form noncovalently associated with the chain of the class I major histocompatibility complex (Mhc) molecules. In mammals, the ₂m-encoding gene (B2m) is found on a chromosome different from the Mhc proper. We have isolated and characterized the B2m gene of the zebrafish, Brachydanio rerio, family Cyprinidae. We obtained both cDNA and genomic clones of the Brre-B2m gene. The cDNA clones contained the entire coding sequence, the entire 3 untranslated (UT) region, and at least part of the 5UT region. The genomic clone contained the entire Brre-B2m gene. The coding sequence specifies 97 amino acid residues of the mature protein so that the zebrafish ₂m is two residues shorter than human and one residue shorter than cattle, fowl, or turkey ₂m (codons at positions 85 and 86 have been deleted in the Brre-B2m. gene). The amino acid and nucleotide sequence similarities between zebrafish and human ₂m (B2m) are 45% and 59%, respectively. Approximately 24% of the positions are invariant and an additional 9% show only conservative substitutions in comparisons which include all known 2m sequences (fish, avian, and mammalian). Most of the conserved positions are in the strands (some 47% of the -strand positions are conserved in the three vertebrate classes). The Brre-B2m gene consists of four exons separated by three introns. All of the introns are considerably shorter than the corresponding introns in the mammalian B2m genes. The coding sequences of the cDNA and the genomic clones are almost identical but the sequences of the 3'UT regions differ at 1.7% of the sites, suggesting that the genes borne by these clones might have diverged at least 0.7 million years (my) ago. In contrast to the human B2m gene, the Brre-B2m gene shows no bias in the distribution of the CpG dinucleotides: the dinucleotides are distributed evenly along the entire available sequence. The haploid genome of the zebrafish contains only one copy of the B2m gene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L05383 (B2M) and L05384 (B2RG). Correspondence to: J. Klein.     

Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins

Olfaction in Drosophila is mediated by a large family of membrane-bound odorant receptor proteins (Ors). In heterologous cells, we investigated whether the structural features and signalling mechanisms of ligand-binding Drosophila Ors are consistent with them being G protein-coupled receptors (GPCRs). The detailed membrane topology of Or22a was determined by inserting epitope tags into the termini and predicted loop regions. Immunocytochemistry experiments in Drosophila S2 cells imply that Or22a has seven transmembrane domains but that its membrane topology is opposite to that of GPCRs, with a cytoplasmic N-terminus and extracellular C-terminus. To investigate Or signalling mechanisms, we expressed Or43b in Sf9 and HEK293 cells, and show that inhibitors of heterotrimeric G proteins (GDP-beta-S), adenylate cyclase (SQ22536), guanylyl cyclase (ODQ), cyclic nucleotide phosphodiesterases (IBMX) and phospholipase C (U73122) have negligible impact on Or43b responses. Whole cell patching of Or43b/Or83b-transfected HEK293 cells revealed the opening of plasma membrane cation channels on addition of ligand. The response was blocked by lanthanum and by 2-APB, but not by Ruthenium red or SKF96365. Based on these data, we conclude that Drosophila Ors comprise a novel family of seven transmembrane receptors that in HEK293 cells signal by opening cation channels, through a mechanism that is largely independent of G proteins.     

A genetic locus of Helicobacter pylori inversely associated with gastric intestinal metaplasia

The genomic contents of Helicobacter pylori strain C1 from a patient with gastric cancer and strain 98587 from a patient with duodenal ulcer disease were compared using a rapid subtractive hybridisation approach. A total of 11 tester-specific sequences representing gene specificity, DNA rearrangement and sequence variation were identified. This included two novel sequences, clone P32 and clone F5, which have no significant homologue in the database. H. pylori strains positive for clone P32 were less prevalent in patients with gastric intestinal metaplasia (12.5%) than in duodenal ulcer (39.1%) (p=0.036), or chronic gastritis (38.1%) (p=0.036). The results suggest that H. pylori clone P32 is potentially a useful marker for distinguishing intestinal metaplasia associated strains from others.     

CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease

CARD15/NOD2 encodes a protein involved in bacterial recognition by monocytes. Mutations in CARD15 have recently been found in patients with Crohn disease (CD), a chronic inflammatory condition of the digestive tract. Here, we report the mutational analyses of CARD15 in 453 patients with CD, including 166 sporadic and 287 familial cases, 159 patients with ulcerative colitis (UC), and 103 healthy control subjects. Of 67 sequence variations identified, 9 had an allele frequency >5% in patients with CD. Six of them were considered to be polymorphisms, and three (R702W, G908R, and 1007fs) were confirmed to be independently associated with susceptibility to CD. Also considered as potential disease-causing mutations (DCMs) were 27 rare additional mutations. The three main variants (R702W, G908R, and 1007fs) represented 32%, 18%, and 31%, respectively, of the total CD mutations, whereas the total of the 27 rare mutations represented 19% of DCMs. Altogether, 93% of the mutations were located in the distal third of the gene. No mutations were found to be associated with UC. In contrast, 50% of patients with CD carried at least one DCM, including 17% who had a double mutation. This observation confirmed the gene-dosage effect in CD. The patients with double-dose mutations were characterized by a younger age at onset (16.9 years vs. 19.8 years; P=.01), a more frequent stricturing phenotype (53% vs. 28%; P=.00003; odds ratio 2.92), and a less frequent colonic involvement (43% vs. 62%; P=.003; odds ratio 0.44) than were seen in those patients who had no mutation. The severity of the disease and extraintestinal manifestations were not different for any of the CARD15 genotypes. The proportion of familial and sporadic cases and the proportion of patients with smoking habits were similar in the groups of patients with CD with or without mutation. These findings provide tools for a DNA-based test of susceptibility and for genetic counseling in inflammatory bowel disease.     

Ancestral Population Sizes and Species Divergence Times in the Primate Lineage on the Basis of Intron and BAC End Sequences

The effective sizes of ancestral populations and species divergence times of six primate species (humans, chimpanzees, gorillas, orangutans, and representatives of Old World monkeys and New World monkeys) are estimated by applying the two-species maximum likelihood (ML) method to intron sequences of 20 different loci. Examination of rate heterogeneity of nucleotide substitutions and intragenic recombination identifies five outrageous loci (ODC1, GHR, HBE, INS, and HBG). The estimated ancestral polymorphism ranges from 0.21 to 0.96% at major divergences in primate evolution. One exceptionally low polymorphism occurs when African and Asian apes diverged. However, taking into consideration the possible short generation times in primate ancestors, it is concluded that the ancestral population size in the primate lineage was no smaller than that of extant humans. Furthermore, under the assumption of 6 million years (myr) divergence between humans and chimpanzees, the divergence time of humans from gorillas, orangutans, Old World monkeys, and New World monkeys is estimated as 7.2, 18, 34, and 65 myr ago, respectively, which are generally older than traditional estimates. Beside the intron sequences, three other data sets of orthologous sequences are used between the human and the chimpanzee comparison. The ML application to these data sets including 58,156 random BAC end sequences (BES) shows that the nucleotide substitution rate is as low as 0.6–0.8 × 10^–9 per site per year and the extent of ancestral polymorphism is 0.33–0.51%. With such a low substitution rate and short generation time, the relatively high extent of polymorphism suggests a fairly large effective population size in the ancestral lineage common to humans and chimpanzees.[Reviewing Editor: Dr. Magnus Nordborg]     

Biglycan-Like Extracellular Matrix Genes of Agnathans and Teleosts

Biglycan and decorin are two members of a family of small extracellular matrix proteoglycans characterized by the presence of 10 leucine-rich repeats and one or two attachment sites for glucosaminoglycans. Both have thus far been described only from tetrapod species, mainly mammals. Because the extracellular matrix has played an important part in the evolution of Metazoa, the phylogeny of its components is of considerable interest. In this study, biglycan-like (BGL) cDNA sequences have been obtained from two teleost (Oreochromis cichlid and zebrafish) and two lamprey species. The analysis of the sequences suggests that, like tetrapods, the lampreys possess two types of proteoglycans, both of which are biglycan-like; decorin-like proteoglycans could not be identified in these species. The genes specifying these two types apparently arose by duplication in the lamprey lineage after its divergence from gnathostomes. The two teleost species possess a BGL proteoglycan and a bona fide decorin. The BGL proteoglycan is highly divergent from the tetrapod biglycan and related to the BGL proteoglycans of the lamprey. Hence, although the duplication generating the ancestors of biglycan and decorin genes occurred after the divergence of agnathans but before the emergence of teleosts, only decorin acquired its characteristic properties in the bony fishes. The BGL gene presumably turned into a typical biglycan only in the tetrapod lineages. The presumed acquisitions of new functions appear to have been accompanied by changes in the evolutionary rate. Received: 13 April 2000 / Accepted: 4 July 2000     

Expression of the Dentin Matrix Protein 1 Gene in Birds

The emergence of jawed vertebrates was predicated on the appearance of several innovations, including tooth formation. The development of teeth requires the participation of several specialized genes, in particular, those necessary for the formation of hard tissues—dentin, enamel, and cementum. Some vertebrates, most conspicuously birds, secondarily lost the tooth-forming ability. To determine the fate of some of the tooth-forming genes in the birds, we tested a domestic fowl cDNA library for the expression of the dentin matrix protein 1 (DMP1) gene. The library was prepared from the poly(A⁺) RNA isolated from the jaws of 11- to 13-day-old embryos and the testing was carried out by the polymerase chain reaction with degenerate primers designed on the basis of the available mammalian and reptile sequences. A chicken homologue of the DMP1 gene identified by this approach was shown to be expressed in the jaws and long bones, the same two tissues as in mammals. The chicken DMP1 gene has an exon/intron organization similar to that of its mammalian and reptile counterparts. The chicken gene contains three short highly conserved segments, the rest of the gene being poorly alignable or not alignable with its mammalian or reptilian homologues. The distribution of similarities and dissimilarities along the gene is indicative of a mode of evolution in which only short segments are kept constant, while the rest of the gene is relatively free to vary as long as the proportion of certain amino acid residues is retained in the encoded polypeptide. The DMP1 gene may have been retained in birds because of its involvement in bone formation. Received: 5 April 1999 / Accepted: 9 August 1999