Songbird genomics: analysis of 45 kb upstream of a polymorphic Mhc class II gene in red-winged blackbirds (Agelaius phoeniceus)

Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative gamma-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.     

A 39-kb sequence around a blackbird Mhc class II gene: ghost of selection past and songbird genome architecture

To gain an understanding of the evolution and genomic context of avian major histocompatibility complex (Mhc) genes, we sequenced a 38.8-kb Mhc-bearing cosmid insert from a red-winged blackbird (Agelaius phoeniceus). The DNA sequence, the longest yet retrieved from a bird other than a chicken, provides a detailed view of the process of gene duplication, divergence, and degeneration ("birth and death") in the avian Mhc, as well as a glimpse into major noncoding features of a songbird genome. The peptide-binding region (PBR) of the single Mhc class II B gene in this region, Agph-DAB2, is almost devoid of polymorphism, and a still-segregating single-base-pair deletion and other features suggest that it is nonfunctional. Agph-DAB2 is estimated to have diverged about 40 MYA from a previously characterized and highly polymorphic blackbird Mhc gene, Aph-DAB1, and is therefore younger than most mammalian Mhc paralogs and arose relatively late in avian evolution. Despite its nonfunctionality, Agph-DAB2 shows very high levels of nonsynonymous divergence from Agph-DAB1 and from reconstructed ancestral sequences in antigen-binding PBR codons-a strong indication of a period of adaptive divergence preceding loss of function. We also found that the region sequenced contains very few other unambiguous genes, a partial Mhc- class II gene fragment, and a paucity of simple-sequence and other repeats. Thus, this sequence exhibits some of the genomic streamlining expected for avian as compared with mammalian genomes, but is not as densely packed with functional genes as is the chicken Mhc.     

Gene organization of the quail major histocompatibility complex (MhcCoja) class I gene region

 Class I genomic clones of the quail (Coturnix japonica) major histocompatibility complex (MhcCoja) were isolated and characterized. Two clusters spanning the 90.8 kilobase (kb) and 78.2 kb class I gene regions were defined by overlapping cosmid clones and found to contain at least twelve class I loci. However, unlike in the chicken Mhc, no evidence for the existence of any Coja class II gene was obtained in these two clusters. Based on comparative analysis of the genomic sequences with those of the cDNA clones, Coja-A, Coja-B, Coja-C, and Coja-D (Shiina et al. 1999), these twelve loci were assigned to represent one Coja-A gene, two Coja-B genes (Coja-B1 and -B2), four Coja-C genes (Coja-C1-C4), four Coja-D genes (Coja-D1-D4), and one new Coja-E gene. A class I gene-rich segment of 24.6 kb in which five of these genes (Coja-B1, -B2, -D1, -D2 and -E) are densely packed were sequenced by the shotgun strategy. All of these five class I genes are very compact in size [2089 base pairs (bp)–2732 bp] and contain no apparent genetic defect for functional expression. A transporter associated with the antigen processing (TAP) gene was identified in this class I gene-rich segment. These results suggest that the quail class I region is physically separated from the class II region and characterized by a large number of the expressible class I loci (at least seven) in contrast to the chicken Mhc, where the class I and class II regions are not clearly differentiated and only at most three expressed class I loci so far have been recognized. Received: 9 March 1998 / Revised: 12 October 1998     

Diversity of<Emphasis Type="Italic"> Mhc</Emphasis> class I and IIB genes in house sparrows (<Emphasis Type="Italic">Passer domesticus</Emphasis>)

In order to understand the expression and evolution of host resistance to pathogens, we need to examine the links between genetic variability at the major histocompatibility complex (Mhc), phenotypic expression of the immune response and parasite resistance in natural populations. To do so, we characterized the Mhc class I and IIB genes of house sparrows with the goal of designing a PCR-based genotyping method for the Mhc genes using denaturing gradient gel electrophoresis (DGGE). The incredible success of house sparrows in colonizing habitats worldwide allows us to assess the importance of the variability of Mhc genes in the face of various pathogenic pressures. Isolation and sequencing of Mhc class I and IIB alleles revealed that house sparrows have fewer loci and fewer alleles than great reed warblers. In addition, the Mhc class I genes divided in two distinct lineages with different levels of polymorphism, possibly indicating different functional roles for each gene family. This organization is reminiscent of the chicken B complex and Rfp-Y system. The house sparrow Mhc hence appears to be intermediate between the great reed warbler and the chicken Mhc, both in terms of numbers of alleles and existence of within-class lineages. We specifically amplified one Mhc class I gene family and ran the PCR products on DGGE gels. The individuals screened displayed between one and ten DGGE bands, indicating that this method can be used in future studies to explore the ecological impacts of Mhc diversity.     

Linkage of a new member of the lectin supergene family to chicken Mhc genes

With the use of tissue-specific cDNA probes, several genes, which do not correspond to the class I (B-F), class II (B-L), or class IV (B-G) genes, were detected within the cosmid clusters containing the chicken major histocompatibility genes. We isolated cDNA clones with a probe corresponding to one of them, the 17.5 gene, located between two class I genes. The 17.5.3 cDNA, isolated from a chicken spleen cDNA library, encodes a 257-residue-long protein. This sequence shows significant similarity with several members of the C-type animal lectin superfamily and is probably a type II transmembrane protein. Analysis of several cDNA clones, together with Southern blot experiments, strongly suggest that this gene belongs to a multigene family, with at least some of its members being polymorphic. Several arguments lend support to the possibility that, together with the linked Mhc genes, the 17.5 gene is part of the recently described Rfp-Y system.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M88072.     

Functional characterization of a chicken major histocompatibility complex class II B gene promoter

 A 0.7 kilobase (kb) DNA fragment from the 5′ flanking region of a chicken major histocompatibility complex (MHC) class II B gene was cloned into chloramphenicol acetyltransferase (CAT) reporter vectors and was transfected into a chicken macrophage cell line that expresses a low level of MHC class II antigens. Positive orientation-dependent promoter activity of the chicken DNA was evident in a reporter construct containing an SV40 enhancer. Deletion analysis of this 0.7 kb DNA fragment revealed a short fragment in the 3′ end that was crucial for the promoter function and negative regulatory elements (NRE) located further upstream. The conserved MHC class II X and Y boxes did not have a significant effect on promoter activity. Sequence analysis of the 0.7 kb class II B gene upstream region suggests possible involvement of interferon (IFN), E twenty-six specific (ETS)-related proteins, and other factors in regulating this promoter. A chicken T-cell line culture supernatant increased surface expression of MHC class II antigens, as well as class II promoter activity, in this macrophage cell line. This first functional characterization of a chicken MHC class II B gene promoter will aid in understanding the regulatory mechanisms that control the expression of these genes. Received: 9 July 1996 / Revised: 7 October 1996     

Characterization of Mhc genes in a multigenerational family of ring-necked pheasants

Little is known about the major histocompatibility (Mhc) genes of birds in different taxonomic groups or about how Mhc genes may be organized in avian species divergent by evolution or habitat. Yet it seems likely that much might be learned from birds about the evolution, organization, and function of this intricate complex of polymorphic genes. In this study a close relative of the chicken, the ring-necked pheasant (Phasianus colchicus), was examined for the presence and organization of Mhc B-G genes. The patterns of restriction fragments revealed by chicken B-G probes in Southern hybridizations and the patterns of pheasant erythrocyte polypeptides revealed in immunoblots by antisera raised against chicken B-G polypeptides provide genetic, molecular, and biochemical data confirming earlier serological evidence for the presence of B-G genes in the pheasant, and hence, the presence of a family of B-G genes in at least a second species of birds. The high polymorphism exhibited by the pheasant B-G gene family allowed genetic differences among individuals within the small experimental population in this study to be detected easily by restriction fragment patterns. Further evidence was found for the organization of the pheasant Mhc class I and class II genes into genetically independent clusters. Whether these gene clusters are fully comparable to the B and Rfp-Y systems in the chicken or whether yet another organization of Mhc genes has been encountered in the pheasant remains to be determined.     

Analysis of zebrafish Mhc using BAC clones

 Although major histocompatibility complex (Mhc) genes have been identified in a number of species, little is yet known about their organization in species other than human and mouse. The zebrafish, Danio rerio, is a good candidate for full elucidation of the organization of its Mhc. As a step toward achieving this goal, a commercially available zebrafish BAC library was screened with probes specific for previously identified zebrafish class I and class II genes, as well as for genes controlling the proteasome subunits LMP7 and LMP2. Restriction maps of the individual positive clones were prepared and the Mhc (LMP7) genes localized to specific fragments. The total length of genomic DNA fragments with Mhc genes was approximately 1700 kilobases (kb) (200 kb of fragments bearing class I loci and 1500 kb of fragments bearing class II loci). One of the two class I loci (Dare-UCA) is closely associated with the LMP7 locus; the second class I locus (Dare-UAA) is more than 50 kb distant from the UCA locus and has no LMP genes associated with it. None of the class II genes are linked to the class I or the LMP genes. All six of the previously identified class II B genes and one of the three class II A genes were found to be present in the BAC clones; no new Mhc loci could be identified in the library. Each of the six previously identified class II B loci was found to be borne by a separate group of BAC clones. The Dare-DAB and -DAA loci were found on the same clone, approximately 15 kb apart from each other. An expansion of DCB and DDB loci was detected: the zebrafish genome may contain at least five closely related DCB and two closely related DDB loci which are presumably the products of relatively recent tandem duplication. These results are consistent with linkage studies and indicate that in the zebrafish, the class I and class II loci are on different chromosomes, and the class II loci are in three different regions, at least two of which are on different chromosomes. Received: 14 August 1997 / Revised: 16 September 1997     

Mapping and characterization of the DQ subregion of the ovine MHC

A map of the ovine MHC class II DQ subregion has been constructed from overlapping cosmid clones. This region consists of two loci linked on a linear tract of 130 kb DNA. Each locus consists of a DQA and a DQB gene in a tail-to-tail orientation. The genes in each locus are transcribed but only those designated DQ1 express class II molecules at the surface of mouse L cells following DNA-mediated gene transfection. The DQA1 and DQB1 genes are separated by 11kb while the DQA2 and B2 genes are 25 kb apart. The loci are separated by 22 kb.     

Short-range linkage relationships of the valyl-tRNA synthetase gene in Fugu rubripes

 The class III region of the human major histocompatibility complex (MHC) is gene-dense, averaging one gene every 10–20 kilobases (kb). Its gene order has been compared with other organisms. To extend this analysis further in another non-mammalian vertebrate, the compact genome of Fugu rubripes was investigated for the existence of orthologues of these class III genes and their relative arrangements. Orthologues of the M _r 70000 heat shock protein (HSP70) and valyl-tRNA synthetase genes have been isolated. They do not seem to be closely physically linked as compared with mammals (supported by longer-range analysis using pulsed field gel electrophoresis). Random shotgun sequencing of the two Fugu cosmids containing the gene encoding valyl-tRNA synthetase revealed sequences resembling genes encoding tenascin-X, the nuclear antigen A/Ro of Sjogren’s syndrome, and the Landsteiner-Wiener blood group glycoprotein. These linkage relationships recapitulate some mammalian data, albeit imperfectly. Tenascin-X has been located both in the human and mouse Mhc class III regions. Three copies of a sequence found in the gene encoding Sjogren’s syndrome nuclear antigen A/Ro have been identified in the human Mhc class I region; the mouse Mhc class I region contains one copy. It is postulated that a fragmented gene pattern had existed prior to convergence in the ancestral mammalian immune response-related Mhc region, and that some of these genes had belonged to the same linkage group. Received: 17 February 1997 / Revised: 25 March 1997     

A polymorphic system related to but genetically independent of the chicken major histocompatibility complex

Analyses of the major histocompatibility complex (Mhc) in chickens have shown inconsistencies between serologically defined haplotypes and haplotypes defined by the restriction fragment patterns of Mhc class I and class II genes in Southern hybridizations. Often more than one pattern of restriction fragments for Mhc class I and/or class II genes has been found among DNA samples collected from birds homozygous for a single serologically defined B haplotype. Such findings have been interpreted as evidence for variability within the Mhc haplotypes of chickens not detected previously with serological methods. In this study of a fully pedigreed family over three generations, the heterogeneity observed in restriction fragment patterns was found to be the result of the presence of a second, independently segregating polymorphic Mhc-like locus, designated Rfp-Y. Three alleles (haplotypes) are identified in this new system.     

Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity

Summary Three nodulation-deficient (nod) mutants of Rhizobium leguminosarum were isolated following insertion of the transposon Tn5 into pRL1JI, the R. leguminosarum plasmid known to carry the nodulation genes. DNA adjacent to the nod: Tn5 alleles was subcloned and used to probe a cosmid clone bank containing DNA from a Rhizobium strain carrying pRL1JI. Two cosmid clones which showed homology with the probe contained about 10 kb of DNA in common. The R. leguminosarum host-range determinants were found to be present within this 10 kb common region since either of the cosmid clones could enable a cured R. phaseoli strain to nodulate peas instead of Phaseolus beans, its normal host. Electron microscopy of nodules induced by Rhizobium strains cured of their normal symbiotic plasmid but containing either of the two cosmid clones showed bacteroid-forms surrounded by a peri-bacteroid membrane, indicating that normal infection had occurred. Thus it is clear that this 10 kb region of nodDNA carries the genes that determine host range and that relatively few bacterial genes may be involved in nodule and bacteroid development.     

In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).     

A class I jumping clone places the HLA-G gene approximately 100 kilobases from HLA-H within the HLA-A subregion of the human MHC

By the combination of cosmid cloning, chromosomal jumping, and pulsed-field gel electrophoresis (PFGE), we have fine-mapped the HLA-A subregion of the human major histocompatibility complex (MHC). Through the isolation of a class I jumping clone, the Qa-like HLA-G class I gene has been placed within 100 kb of HLA-H. The tight physical linkage of these class I genes has been further supported by hybridizing PFGE blots with locus-specific probes. It has been found that both of the above class I genes are linked to HLA-A, with HLA-H residing no more than 200 kb from the HLA-A gene. These data support the possible existence of a Qa-like subregion composed of nonclassical HLA class I genes within the human MHC linked telomerically to the HLA-A locus.     

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti)

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB088371–AB088374, AB089199, AB154393–AB154399, and AB162144.     

Cloning and characterization of hydrogenase genes from Azotobacter chroococcum

Summary Genomic DNA from Azotobacter chroococcum was shown by DNA hybridization to contain sequences homologous to Rhizobium japonicum H₂-uptake (hup) hydrogenase genes carried on the plasmid pHU1. Two recombinant cosmid clones, pACD101 and pACD102, were isolated from a gene library of A. chroococcum by colony hybridization and physically mapped. Each contained approximately 42 kb of insert DNA with approximately 27 kb of overlapping DNA. Further hybridization studies using three fragments from pHU1 (6 kb HindIII, 6.4 kb BglII and 5 kb EcoRI) showed that the hup-specific regions of R. japonicum and A. chroococcum are probably highly conserved. Weak homology to the hydrogenase structural genes from Desulfovibrio vulgaris (Hildenborough) was also observed. A 24 kb BamHI fragment from pACD102 subcloned into a broad host-range vector restored hydrogenase activity to several Hup^- mutants of A. chroococcum.     

Dynamics of Mhc evolution in birds and crocodilians: amplification of class II genes with degenerate primers

Genes of the major histocompatibility complex (Mhc) are the most polymorphic functional loci in mammalian populations, but little is known of Mhc variability in natural populations of nonmammalian vertebrates. To help extend such studies to birds and relatives, we present a pair of degenerate primers that amplify polymorphic segments of one chain (the β chain) of the class II genes from the major histocompatibility complex (Mhc) of archosaurs (birds + crocodilians). The primers target two conserved regions lying within portions of the antigen-binding site (ABS) encoded by the second exon and amplify multiple genes from both genomic DNA and cDNA. The pattern of nucleotide substitution in ABS codons of 51 sequences amplified and cloned from five species of passerine birds and an alligator (Alligator mississippiensis) indicates that archosaurian class II β genes are subject to selective forces similar to those operating in mammalian populations. Hybridization of a genomic clone generated by the primers revealed highly polymorphic bands in a sample of Florida scrub jays (Aphelocoma coerulescens coerulescens). Because the primers amplify only part of the ABS from multiple class II genes, they will be useful primarily for generating species specific clones, thereby providing a critical inroad to more detailed structural and evolutionary studies.     

Major histocompatibility complex class II A gene polymorphism in the striped bass

Adaptions of the polymerase chain reaction were used to isolate cDNA sequences encoding the Major histocompatibility complex(Mhc) class II A gene(s) of the striped bass (Morone saxatilis). Four complete Mhc class II A genes were cloned and sequenced from a specimen originating in the Roanoke River, North Carolina, and another three A genes from a specimen originating from the Santee-Cooper Reservoir, South Carolina, identifying a total of seven unique sequences. The sequence suggests the presence of at least two Mhc class II A loci. The extensive sequence variability observed between the seven different Mhc class II clones was concentrated in the 1 encoding domain. The encoded 2, transmembrane, and cytoplasmic regions of all seven striped genes correlated well with those of known vertebrate Mhc class II proteins. Overall, the striped bass sequences showed greatest similarity to the Mhc class II A genes of the zebrafish. Southern blot analysis demonstrated extensive polymorphism in the Mhc class II A genes in members of a Roanoke river-caught population of striped bass versus a lesser degree of polymorphism in an aquacultured Santee-Cooper population of striped bass.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers (Mosa-A-S5) L35062, (Mosa-A-S8) L35066, (Mosa-A-R7) L35067, and (Mosa-A-S7) L35072 L35066, (Mossa-A-R7) L35067, and (Mosa-A-S7) L35072     

Mapping of Mhc class I and class II regions to different linkage groups in the zebrafish, Danio rerio

 The mammalian major histocompatibility complex (Mhc) consists of three closely linked regions, I, II, and III, occupying a single chromosomal segment. The class I loci in region I and the class II loci in region II are related in their structure, function, and evolution. Region III, which is intercalated between regions I and II, contains loci unrelated to the class I and II loci, and to one another. There are indications that a similar Mhc organization exists in birds and amphibians. Here, we demonstrate that in the zebrafish (Danio rerio), a representative of the teleost fishes, the class II loci are divided between two linkage groups which are distinct from the linkage group containing the class I loci. The β₂-microglobulin-encoding gene is loosely linked to one of the class II loci. The gene coding for complement factor B, which is one of the region III genes in mammals, is linked neither to the class I nor to the class II loci in the zebrafish. These results, combined with preliminary data suggesting that the class I and class II regions in another order of teleost fish are also in different linkage groups, indicate that close linkage of the two regions is not necessary either for regulation of expression or for co-evolution of the class I and class II loci. They also raise the question of whether linkage of the class I and class II loci in tetrapods is a primitive or derived character. Received: 16 December 1996 / Revised: 6 February 1997