Isolation of Mhc Class II DMA and DMB cDNA Sequences in a Marsupial: The Gray Short-Tailed Opossum (Monodelphis domestica)

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. Received: 2 March 1998 / Accepted: 2 June 1998     

Polymorphism and transcription of Mhc class I genes in a passerine bird, the great reed warbler

 The class I genes of the major histocompatibility complex (Mhc) are here investigated for the first time in a passerine bird. The great reed warbler is a rare species in Sweden with a few semi-isolated populations. Yet, we found extensive Mhc class I variation in the study population. The variable exon 3, corresponding to the α₂ domain, was amplified from genomic DNA with degenerated primers. Seven different genomic class I sequences were detected in a single individual. One of the sequences had a deletion leading to a shift in the reading frame, indicating that it was not a functional gene. A randomly selected clone was used as a probe for restriction fragment length polymorphism (RFLP) studies in combination with the restriction enzyme Pvu II. The RFLP pattern was complex with 21–25 RFLP fragments per individual and extensive variation. Forty-nine RFLP genotypes were detected in 55 tested individuals. To study the number of transcribed genes, we isolated 14 Mhc class I clones from a cDNA library from a single individual. We found eight different sequences of four different lengths (1.3–2.2 kilobases), suggesting there are at least four transcribed loci. The number of nonsynonymous substitutions (d _N ) in the peptide binding region of exon 3 were higher than the number of synonymous substitutions (d _S ), indicating balancing selection in this region. The number of transcribed genes and the numerous RFLP fragments found so far suggest that the great reed warbler does not have a "minimal essential Mhc" as has been suggested for the chicken. Received: 13 May 1998 / Revised: 18 August 1998     

Isolation and characterization of polymorphic microsatellite markers in the gray,short‐tailed opossum (Monodelphis domestica)

Twenty‐six polymorphic microsatellite markers were isolated from (AC)_n and (AG)_n microsatellite‐enhanced genomic libraries of the gray, short‐tailed opossum Monodelphis domestica. All 26 loci showed high allelic diversity, with allele numbers ranging from five to 11 in a subset of 35 animals. Normal Mendelian inheritance was confirmed for 24 loci by analysing allelic segregation in 10, two‐generation, families. Non‐amplifying (null) alleles were detected at two loci, which we recommend be used only if pedigree data are available. We conclude that all of these microsatellite markers would be useful for quantitative trait locus mapping and population genetic studies.     

Energetics of offspring production: a comparison of a marsupial (Monodelphis domestica) and a eutherian (Mesocricetus auratus)

This study compares the energetic cost of reproduction during gestation and lactation of a eutherian, the golden hamster (Mesocricetus auratus), and a similar-sized (60–120 g) marsupial, the gray short-tailed opossum (Monodelphis domestica). Food consumption was monitored in 20 reproductively active (RA) opossums and 16 RA hamsters from conception to weaning and at equivalent intervals in 19 non-reproductive (NR) opossums and 21 NR hamsters, all maintained within their zone of thermoneutrality (30 °C). Total energy assimilated from conception to weaning [opossums: 1261.3 ± 28.0 Kcal (1 Kcal = 4.1868 J) and hamsters: 1647.5 ± 60.6 Kcal] was positively correlated with litter size and mass per young in both species. Maternal mass-specific assimilated energy was significantly greater in hamsters than in opossums during gestation (P < 0.001), but not during lactation or from conception to weaning (P > 0.05). Efficiency of offspring production (energy stored in young/incremental energy in RA females) was higher in hamsters than in opossums and, in both species, it was higher during lactation than in gestation. The energetic cost of reproduction (per young per day) was higher in hamsters than in opossums. The marsupial mode of reproduction, as seen in opossums, yields young at lower cost but requires a longer reproductive period than is the case for a similar-sized eutherian. Accepted: 8 September 1998     

Partial nucleotide sequence of a bovine major histocompatibility class II DRβ-like gene

Summary. A genomic clone containing a bovine DRβ-like gene, BoDRβ II , was isolated from a bovine genomic library and characterized by restriction enzyme mapping and nucleotide sequencing of exon regions. Alignment of this sequence with the human DRβ cDNA sequence allowed identification of exon/intron boundaries. The clone contains a 13.3-kilobase (kb) insert, and includes 1.3kb 5' of the β₁ exon and 6.7kb 3' of the transmembrane (TM) exon. Open reading frames were present in the BoDRβ exons sequenced. Nucleotide identities of the bovine β₁, β₂ and TM exons with the corresponding human DRβ exons were 73, 91 and 83%, respectively. Nucleotide identities of these exons with those of a previously described bovine DRβ-like pseudogene, BoDRβ I , were 69, 95 and 81%, respectively. Although a limited amount of sequence data was obtained for the intron regions, a 71% identity was found within a 514-nucleotide region immediately 3' to the β₂ exons in BoDRβ I and BoDRβ II . A series of GT residues followed by a longer series of GA residues began about 35 nucleotides 3' of the β₁ exon in both BoDRβ I and BoDRβ II .     

Molecular analysis of an MHC class II deficiency patient reveals a novel mutation in the RFX5 gene

 Patients suffering from major histocompatibility complex (MHC) class II deficiency, a rare primary immunodeficiency, are characterized by a lack of MHC class II expression which is the result of defects in trans-acting factors. At least four complementation groups, A, B, C, and D, can be discerned. The gene affected in group C patients is known to be RFX5 and encodes one of the subunits of the multimeric phosphoprotein complex, RFX. In the present study we fused fibroblasts of a recently identified MHC class II deficiency patient, OSE, with fibroblasts derived from patients representative of each of the four complementation groups. Transient heterokaryon analysis indicated that OSE belonged to complementation group C. Furthermore, transfection of wild-type RFX5 cDNA into OSE fibroblasts resulted in restoration of the defect. Mutation analysis revealed that the RFX5 mRNA lacked four nucleotides and that this deletion was the consequence of a G to A transition in a splice acceptor site. Genomic oligotyping demonstrated that OSE was homozygous for the splice site mutation. Received: 15 July 1998 / Revised: 3 September 1998     

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus)

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5′UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered 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 AB301478, AB301944–AB301950, AB302087–AB302090, AB302190–AB302192, AB302843, AB302844, and AB303942–AB303945.     

Regulation of MHC class I and II gene transcription: differences and similarities

Major histocompatibility complex (MHC) molecules serve as peptide receptors. These peptides are derived from processed cellular or extra-cellular antigens. The MHC gene complex encodes two major classes of molecules, MHC class I and class II, whose function is to present peptides to CD8⁺ (cytotoxic) and CD4⁺ (helper) T cells, respectively. The genes encoding both classes of MHC molecules seem to originate from a common ancestral gene. One of the hallmarks of the MHC is its extensive polymorphism which displays locus and allele-specific characteristics among the various MHC class I and class II genes. Because of its central role in immunosurveillance and in various disease states, the MHC is one of the best studied genetic systems. This review addresses several aspects of MHC class I and class II gene regulation in human and in particular, the contribution to the constitutive and cytokine-induced expression of MHC class I and II genes of MHC class-specific regulatory elements and regulatory elements which apparently are shared by the promoters of MHC class I and class II genes. Received: 12 January 1998     

Identification of the Tapasin gene in the chicken major histocompatibility complex

 The Tapasin molecule plays a role in the assembly of major histocompatibility complex (Mhc) class I molecules in the endoplasmic reticulum, by mediating the interaction of class I-β₂-microglobulin dimers with TAP. We report here the identification of the Tapasin gene in the chicken Mhc (B complex). This gene is located at the centromeric end of the complex, between the class II B-LBI and B-LBII genes. Like its human counterpart it comprises 8 exons, but features a significantly reduced intron size as compared to the human gene. Chicken Tapasin codes for a transmembrane protein with a probable endoplasmic reticulum retention signal. Exons IV and V, and possibly exon III, code for separate domains that are related to the immunoglobulin (Ig) superfamily (this relationship was so far unrecognized for human Tapasin domain IV which has lost its two cysteines). Two different cDNAs corresponding to the Tapasin gene were isolated, possibly related to alternative splicing events; the Ig-like domain encoded by exon IV is missing in one of the cDNAs, suggesting either that this domain is not necessary for the protein to perform its function, or that the two alternatively spliced cDNAs are translated into two functionally different forms of the protein. Received: 8 July 1998 / Revised: 5 October 1998     

家蝇抗菌肽Attacin-2基因的克隆、序列分析和诱导表达

攻击素(attacin)作为昆虫抗菌肽之一, 在昆虫的先天免疫中起着重要作用。本研究通过家蝇Musca domestica EST序列筛选并结合RACE技术克隆了家蝇的Attacin-2基因(Mdatta2) cDNA序列。其全长819 bp, 包含一个726 bp的完整开放阅读框(open reading frame, ORF), 以及42 bp的5′末端非翻译区(5′-UTR)和51 bp的3′末端非翻译区(3′-UTR), 编码241个氨基酸残基, 推导的多肽N端22个氨基酸残基为信号肽序列。同源性分析表明, 其氨基酸序列与嗜凤梨果蝇Drosophila ananassae的Attacin一致性最高(46%)。以邻接法(Neighbor-Joining, NJ)构建的系统关系表明, 家蝇的Attacin-2与其他双翅目昆虫的Attacin起源于共同的祖先, 属于Attain_C超家族。应用实时荧光定量PCR的方法研究家蝇幼虫在受到外源细菌刺激时Mdatta2基因的表达, 结果显示, 在大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus分别刺激后3 h和6 h, 家蝇幼虫Mdatta2表达量出现显著上调。Mdatta2基因在家蝇幼虫体内呈诱导型表达, 表达水平随诱导时间的不同而变化, 提示Mdatta2基因可能在家蝇免疫防御过程中起着重要作用。     

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     

家蝇防御素基因的cDNA克隆及序列分析

Defensin is a kind of cationic.inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms.with their broad antimicrobial spectrum and strong pharmaceutical effects.antimicrobial peptides,including defensins,represent a source of novel antibiotic agents.A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDnA library of houseflies(Musca domestica) that had been challenged by E.coli and staphylococcus taincd an NH2-terminal signal sequence(1-22)followed by a propeptide and the mature peptide(53-92),The sequence identity with other insect defensin is between 51% and 73%.The mature peptide,with a predicted molecular weight of 4.0kDa,and pI of 8.69,has 1 negative charged amino acid and 4 positice ones,the putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds,Cys1-Cys4,Cys2-Cys5 and Cys3-Cys6,These results suggest that the novel full-length cDNA of the defensin gene.Denominated Mdde,has been successfully cloned from houseflies.     

Partial nucleotide sequence of a novel bovine major histocompatibility complex class II β-chain gene, BoLA-DIB

A bovine genomic clone that hybridized to HLA-DQ beta cDNA was isolated and fragments containing the beta 1, beta 2 and transmembrane (TM) exons subcloned. The nucleotide sequences of the exons and flanking intron regions were determined. Comparisons of these exon nucleotide sequences and derived amino acid sequences to human class II beta-chain sequences showed that this gene is only 77% identical to HLA-DQ beta and about 75% identical to bovine DQ beta-like genes. The exon sequences were more divergent from other class II beta-chain genes. However, structural features such as conserved cysteines and regions of amino acids strongly suggest this to be a class II beta-chain gene. When exon-containing fragments were used as hybridization probes on Southern blots of bovine genomic DNA digested with Eco RI or Pvu II, each exon hybridized to a single band. Based on these results we have referred to this gene as a novel bovine class II beta-chain gene, BoLA-DIB.