Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials

cDNAs encoding IgM heavy chain constant region (Cμ) were isolated from two metatherians (marsupials) — the Australian common brushtail possum (Trichosurus vulpecula) and the South American grey short-tailed opossum (Monodelphis domestica). Analysis of the sequences suggested that they correspond to the secreted form of Cμ in both species. The domain size and structure of the marsupial Cμ sequences were compared with other Cμ sequences and a high degree of conservation throughout vertebrate evolution was observed. Amino acid sequence comparisons revealed a marked level of sequence similarity between the two marsupial sequences (79%), relatively high similarity between the marsupials and eutherians (63%), and lower similarities between marsupials and birds (45%), marsupials and amphibians (47%), marsupials and reptiles (45%) and marsupials and fish (37%). These data allow the incorporation of metatherians into the study of mammalian IgM evolution.     

Cloning and structural analysis of IgM (μ chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica

To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (γ chain), IgE ( chain) and IgA (α chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (μ chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.     

Molecular cloning of the brushtail possum (Trichosurus vulpecula) immunglobulin E heavy chain constant region

The immunobiology of marsupial IgE is poorly understood. As a first step towards the development of immunological reagents for marsupials and to obtain a further understanding of immunoglobulin evolution, a brushtail possum (Trichosurus vulpecula) mesenteric lymph node cDNA library was screened for the heavy chain constant region of IgE (Cε), using a partial Cε probe from the American marsupial, Monodelphis domestica. The cDNA sequence for T. vulpecula Cε was determined and found to be most similar to the M. domestica Cε sequence [(76%) at the amino acid level]. T. vulpecula Cε has amino acid sequence similarities ranging from 43–52% with various eutherian Cε sequences. The secondary structure of T. vulpecula Cε, based on loops formed by internal disulfide bonds, more closely resembles rodent Cε than the American marsupial sequence.     

Comparison of the body wall myosin heavy chain sequences from Onchocerca volvulus and Brugia malayi

The complete coding sequence of Onchocerca volvulus myosin heavy chain has been determined from a series of overlapping cDNAs. The protein sequences from the 2 filarids, one responsible for subcutaneous filariasis, the other for lymphatic filariasis, show 92% identity, and are 1957 amino acids long. Each protein sequence is also equally related, with 75% identity, to MHC-B, the protein encoded by the unc-54 gene of the free-living nematode C.elegans. Such analysis is useful in phylogenetic studies among nematodes, as well as in structure-function relationships among myosin isolates.     

Buffalo (Bubalus bubalis) interleukin‐2: sequence analysis reveals high nucleotide and amino acid identity with interleukin‐2 of cattle and other ruminants

A 4400‐bp genomic sequence and a 332‐bp truncated cDNA sequence of the interleukin‐2 (IL‐2) gene of Indian water buffalo (Bubalus bubalis) were amplified by polymerase chain reaction and cloned. The coding sequence of the buffalo IL‐2 gene was assembled from the 5′ end of the genomic clone and the truncated cDNA clone. This sequence had 98.5% nucleotide identity and 98% amino acid identity with cattle IL‐2. Three amino acid substitutions were observed at positions 63, 124 and 135. Comparison of the predicted protein structure of buffalo IL‐2 with that of human and cattle IL‐2 did not reveal significant differences. The putative amino acids responsible for IL‐2 receptor binding were conserved in buffalo, cattle and human IL‐2. The amino acid sequence of buffalo IL‐2 also showed very high identity with that of other ruminants, indicating functional cross‐reactivity.     

Nucleotide sequences and characterization of haemolysin genes from Aeromonas hydrophila and Aeromonas sobria

Extracellular haemolysin is thought to be one of the important virulence factors in Aeromonas infection. Two extracellular haemolysin genes (AHH3 and AHH4) from Aeromonas hydrophila strain 28SA, one (AHH5) from A. hydrophila strain AH-1 and one (ASA1) from Aeromonas sobria strain 33 were cloned into cosmid and plasmid vector DNA in Escherichia coli. The nucleotide sequences of the open reading frames of AHH3 and AHH4 are both 1476 basepairs (bp), whereas AHH5 and ASA1 are 1455 and 1467 bp in length, respectively. The deduced amino acid sequences of AHH3, AHH4, AHH5 and the previously reported aerolysin from A. hydrophila showed a significant degree of sequence homology of over 90% each. The amino acid identity of the ASA1 haemolysin and those from A. hydrophila and Aeromonas trota aerolysins ranged from 58–68%. From DNA hybridization analysis using our cloned haemolysin genes as probes, we found that the AHH5 and ASA1 DNA probes hybridized with about 31 and 75% strains of motile Aeromonas species, respectively. The activity of haemolysins of cloned genes were different in medium agar containing various erythrocytes.     

<Emphasis Type="Italic">Clonorchis sinensis</Emphasis>: molecular cloning and functional expression of a novel cytosolic glutathione transferase

Glutathione transferases (GSTs) represent a large family of enzymes. In the high throughput sequencing of the cDNA library constructed from the adult stage of Clonorchis sinensis (Cs), we isolate another cDNA clone encoding a novel cytosolic GST enzyme. To discriminate with our former reported CsGST, we designated this GST as CsGST1. This new cDNA contains 744 bp with a putative open reading frame of 213 amino acids. The deduced amino acid sequence exhibits 88% identity to Opisthorchis viverrini 28GST (Ov28GST), 60 and 52% identity to C. sinensis cytosolic 28-kDa GST (Cs28GST) and CsGST, respectively. The CsGST1 was expressed in Escherichia coli BL21(DE3) as a His-tag fusion protein and was purified by Ni-NTA agarose. The recombinant CsGST1 showed moderate GST activity of 0.79 U mg⁻¹. The average K _m of the CsGST1 for 1-chloro-2, 4-dinitrobenzene is 150 μM. Cibacron blue F3GA and albendazole inhibited the CsGST1 activity with average IC₅₀ values of 9.1 and 265.4 μM, respectively. The nucleotide sequence reported in this paper was submitted to the GenBank Database with accession number DQ342327.     

Cloning, characterization, and expression of a novel secretory lipase-like gene from Clonorchis sinensis

A secretory lipase-like gene was isolated from total cDNA of adult Clonorchis sinensis. The gene has an open reading frame of 1,218 bp long and encodes for a protein of 406 amino acids including a putative signal peptide of 20 amino acids. The deduced amino acid sequence including signal peptide has 42–45% identity with lipase of other species and two typical enzymic active sites that contain consensus sequence (Gly-X-Ser-X-Gly) of lipase. The cDNA encoding this protein was subcloned into pET-28a (+) expression vector and expressed in Escherichia coli. The expressed fusion protein has a molecular mass of about 45 kDa. Prediction of signal peptide and Western blot analysis indicated that the secretory lipase-like protein is an excretory–secretory product of C. sinensis. Immunostaining revealed that the secretory lipase-like protein was localized in the tegument of the adult worm and metacercaria. These results provide basis for further studies on the nutrition taking and invasion of C. sinensis mediated by the secretory lipase-like protein.     

Full‐length sequence and expression analysis of a myeloid differentiation factor 88 (MyD88) in half‐smooth tongue sole Cynoglossus semilaevis

Myeloid differentiation factor 88 (MyD88) is a universal and crucial adaptor protein, which plays an essential role in the intracellular signalling elicited by IL‐1R/TLR superfamily. In the present study, we report the full‐length sequence of MyD88 gene in half‐smooth tongue sole (Cynoglossus semilaevis). In the 2855 bp genomic sequence, five exons and four introns were identified. The cloned cDNA exhibited 110 bp of 5′ UTR, 576 bp of 3′ UTR and 858 bp of the entire open‐reading frame encoding a polypeptide of 285 amino acids. The protein sequence included a typical conserved cytosolic Toll/interleukin‐1 receptor (TIR) domain, an intermediate domain (ID) and a death domain (DD), and shared greater than 70% identity with Japanese flounder Paralichthys olivaceu ortholog. Real‐time polymerase chain reaction (RT‐PCR) analysis indicated a broad expression of csMyD88, especially in ovary and spleen. Quantitative RT‐PCR analysis indicated that the csMyD88 mRNA levels were significantly increased in the spleen and head kidney after inactive Vibrio anguillarum challenge and the expression of csMyD88 appeared to be developmentally regulated during C. semilaevis ontogeny. Although, species‐specific differences were present, the similarity between mammalian and piscine MyD88s suggested that the main function of MyD88 might be conserved across vertebrates.     

DNA sequence analysis of an allelic variant of the Actinobacillus pleuropneumoniae-RTX-toxin I (ApxIA) from serotype 10

The structural gene encoding Actinobacillus pleuropneumoniae-RTX toxin I (ApxIA), one of the major hemolytic and cytolytic toxins of the organism, was cloned from serotype 10. The nucleotide sequence data showed that the gene from serotype 10 was 98% identical to that from serotype 1 at both DNA and amino acid levels. The sequence difference was found to localize at the 3′ terminal region of the gene. We then analyzed the 3′ terminal region of the apxIA gene from other serotypes, 5a, 5b, 9 and 11, using polymerase chain reaction-amplified DNA fragments. Results of DNA sequence indicated that apxIA gene can be divided into the original form including serotypes 1, 9 and 11, and the allelic variant including serotypes 5a, 5b and 10. These gene products, ApxIA proteins, appear to have different second structures at the carboxyl terminal proximal region.     

Isolation and sequence of a cDNA coding for the heavy chain constant region of IgG from the Australian brushtail possum, Trichosurus vulpecula.

A brushtail possum (Trichosurus vulpecula) mesenteric lymph node cDNA library was screened with a South American short-tailed opossum (Monodlelphis domestica) immunoglobulin gamma heavy chain constant region (Cgamma) probe, resulting in the isolation of a 1518 nucleotide cDNA clone. The sequence corresponds to exons 1-3 of Cgamma. The Australian marsupial (T. vulpeculla) sequence is 70% identical at the amino acid level with the American marsupial (M. domestica) sequence, but less similar to the eutherian mammals (45-50%). These data provide the opportunity to compare the evolution of IgG between orders of marsupials separated by at least 75 million years and confirm the appearance of IgG prior to the metatherian/eutherian divergence.