Molecular cloning,expression pattern and phylogenetic analysis of myosin light chain 2 gene from Antheraea pernyi: A potential marker for phylogenetic inference

Myosin light chain 2 (MLC-2) gene was isolated and characterized from Antheraea pernyi, a well-known wild silkmoth. The isolated cDNA sequence is 905 bp in length with an open reading frame of 612 bp encoding a polypeptide of 203 amino acids. Semi-quantitative RT-PCR analysis showed that the MLC-2 gene was transcribed during four developmental stages (egg, larva, pupa, and moth), and present in all tissues tested. Alignment analysis revealed that the deduced protein sequence has over 95% identity to myosin light chain 2 of lepidopteran species, and 57–88% identity to other insect species, suggesting that insect MLC-2 proteins are highly conserved throughout evolution. The protein sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate MLC-2 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of MLC-2 gene in phylogenetic study.     

Nucleotide sequence and molecular characterization of the structural glycoprotein gp41 gene homologue of <Emphasis Type="Italic">Spodoptera litura</Emphasis> nucleopolyhedrosis virus (SpltNPV-I)

The structural glycoprotein gene gp41 homologue of Spodoptera litura nucleopolyhedrosis virus (SpltNPV-I *) was identified in the 4.0 kb EcoRI-L fragment of the viral genome. The nucleotide sequence of 2063 bp of this fragment revealed an open reading frame of 1014 nucleotides to encode a polypeptide of 337 amino acids. Analysis of nucleotide and deduced amino acid sequences of the putative ORF indicated its identity with gp41 protein of other baculoviruses sharing maximum homology with that of Spodoptera frugiperda nucleopolyhedrosis virus (SfNPV). The coding sequence was preceded by an AT-rich region containing the consensus baculoviral late promoter motif RTAAG. The putative SpltNPV gp41 ORF was abundantly expressed as a 37 kDa apoprotein in E. coli and as a 50 kDa glycoprotein in Sf9 cells. The recombinant protein expressed in insect cells was glycosylated (20%) and has GlcNAc as the terminal sugar. The gene is conserved among baculoviruses and places SpltNPV-I close to Spodoptera frugiperda and Spodoptera exigua NPVs in phylogenetic tree.Assigned GenBank accession no. for the nucleotide sequence data is AF445192.abbreviated as SlNPV in earlier publications and GenBank     

Purification,properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of α-subunit (702 amino acid residues) and β-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX α- and β-subunits share as high as 87 and 95% sequence identity with the SNTX α- and β-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX β-subunit with the reported VTX β-subunit is 90%, being lower than that with the SNTX β-subunit.     

SfDronc,an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X.     

Molecular cloning of ribosomal protein L26 (RPL26) cDNA from Ailuropoda melanoleuca and its potential value in phylogenetic study

The cDNA fragment of ribosomal protein L26 (RPL26) was cloned from Ailuropoda melanoleuca using RT-PCR method. The cDNA fragment is composed of 475 bp, containing an open reading frame of 145 amino acids. Alignment analyses indicated that the nucleotide sequence and the deduced amino acid sequence showed high identity to other known RPL26 sequences from vertebrates and invertebrates. The cDNA sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate RPL26 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of RPL26 gene in phylogenetic analysis.     

Purification and characterisation of a protease inhibitor from Streptomyces chromofuscus 34-1 with an antiviral activity

The aim of this study was to purify and characterise a novel protease inhibitor (PISC-2002) isolated from culture supernatants of Streptomyces chromofuscus. PISC-2002 was purified by anion-exchange chromatography, and RP-HPLC analysis. PISC-2002 had a molecular mass of 11.2 kDa and a high content of hydrophobic amino acids and proline. N-terminal sequence gave two sequences differing by one residue. The main sequence is ASLPAVSALVLTV and the shorter sequence is SLPAVSALVLTV. This shows its homology to Streptomyces subtilisin inhibitor family. Besides its large spectrum of powerful inhibitory activities against various serine proteases, PISC-2002 displayed significant antiviral effect against influenza virus A/Rostock/34 (H7N7).     

Exploitation of a high genomic mutation rate in Solenopsis invicta virus 1 to infer demographic information about its host, Solenopsis invicta

The RNA-dependent RNA polymerase (RdRp) region of Solenopsis invicta virus 1 (SINV-1) was sequenced from 47 infected colonies of S. invicta, S. richteri, S. geminata, and S. invicta/richteri hybrids collected from across the USA, northern Argentina, and northern Taiwan in an attempt to infer demographic information about the recent S. invicta introduction into Taiwan by phylogenetic analysis. Nucleotide sequences were calculated to exhibit an overall identity of >90% between geographically-separated samples. A total of 171 nucleotide variable sites (representing 22.4% of the region amplified) were mapped across the SINV-1 RdRp alignment and no insertions or deletions were detected. Phylogenetic analysis at the nucleotide level revealed clustering of Argentinean sequences, distinct from the USA sequences. Moreover, the SINV-1 RdRp sequences derived from recently introduced populations of S. invicta from northern Taiwan resided within the multiple USA groupings implicating the USA as the source for the recent introduction of S. invicta into Taiwan. Examination of the amino acid alignment for the RdRp revealed sequence identity >98% with only nine amino acid changes observed. Seven of these changes occurred in less than 4.3% of samples, while 2 (at positions 1266 and 1285) were featured prominently. Changes at positions 1266 and 1285 accounted for 36.2% and 34.0% of the samples, respectively. Two distinct groups were observed based on the amino acid residue at position 1266, Threonine or Serine. In cases where this amino acid was a Threonine, 90% of these sequences possessed a corresponding Valine at position 1285; only 10% of the Threonine¹²⁶⁶-containing sequences possessed an Isoleucine at the 1285 position. Among the Serine¹²⁶⁶ group, 76% possessed an Isoleucine at position 1285, while only 24% possessed a Valine. Thus, it appears that the Threonine¹²⁶⁶/Valine¹²⁸⁵ and Serine¹²⁶⁶/Isoleucine¹²⁸⁵ combinations are predominant phenotypes.     

Molecular characterization of a truncated antigen (Wb14) of SXP-1 of Wuchereria bancrofti from four endemic regions in India

Wb14 of Wuchereria bancrofti, an orthologue of Brugia malayiSXP-1 and W. bancrofti SXP-1, was amplified from genomic DNA of W. bancrofti microfilaria collected from four distant geographical locations in India viz., Vellore, Bhubaneshwar, Pondicherry and Sevagram. The gene was sub-cloned in a prokaryotic vector pRSET and expressed in Escherichia coli as a truncated protein (∼23 kDa). The nucleotide sequence of the gene is 98% similar to that of WbSXP-1 and is found to be intron-less. However, the analysis and comparison of the derived amino acid sequence with WbSXP-1 showed that Wb14 is truncated at amino acid position 153. The distribution of the two genes in the studied four geographical locations indicated that WbSXP-1 is prevalent only in parasite samples from Sevagram while Wb14 is present in parasites from all the other locations. Only a limited polymorphism was observed in both the genes among the parasites from different geographical locations.     

Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis)

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35 kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35 kDa protein was ~ 98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates.     

Mutanase from a Paenibacillus isolate: Nucleotide sequence of the gene and properties of recombinant enzymes

A mutanase (α-1,3-glucanase)-producing microorganism was isolated from a soil sample and was identified as a relative of Paenibacillus sp. The mutanase was purified to homogeneity from culture, and its molecular mass was around 57 kDa. The gene for the mutanase was cloned by PCR using primers based on the N-terminal amino acid sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of 3651-bp open reading frame that encoded a predicted 1217-amino acid polypeptide including a 43-amino acid signal peptide. The mature enzyme showed similarity to mutanases RM1 of Bacillus sp. strain RM1 and KA-304 of Bacillus circulans with 65.6% and 62.7% identity, respectively. The predicted molecular mass of the mutanase was 123 kDa. Thus, the enzyme purified from the isolate appears to be truncated by proteolysis. The genes for the full-length and truncated mutanases were expressed in Bacillus subtilis cells, and the corresponding recombinant enzymes were purified to homogeneity. The molecular masses of the two enzymes were 116 and 57 kDa, respectively. The specific activity was 10-fold higher for the full-length enzyme than for the truncated enzyme. The optimal pH and temperature for both recombinant enzymes was pH 6.4 in citrate buffer and 45 °C to 50 °C. Amongst several tested polysaccharides, the recombinant full-length enzyme specifically hydrolyzed mutan.     

Blastocatella fastidiosa gen. nov., sp. nov., isolated from semiarid savanna soil – The first described species of Acidobacteria subdivision 4

Acidobacteria represent abundant members of soil microbial communities but only few representatives could be isolated and validly described so far. Currently, eighteen species of subdivision 1, one species of subdivision 3, three species of subdivision 8, and one species of subdivision 10 are recognized. In contrast, Acidobacteria of subdivision 4 have largely escaped cultivation although they belong to the most abundant and diverse acidobacterial groups in soils. A member of subdivision 4, strain A2-16^T, was isolated from a semiarid savanna soil. Cells were motile spheres to rods with a tendency to form chains and larger aggregates. Cultures were orange to pink colored, neutrophilic mesophiles, and showed aerobic chemoorganoheterotrophic growth on very few complex substrates and protocatechuate, and weak growth on chitin, cellulose and starch. While protein substrates such as casamino acids or peptone were utilized, individual amino acids did not promote growth. Also, growth on alternative electron acceptors or fermentative growth could not be observed. Major fatty acids were summed features 1 (15:1 iso H/13:0 3-OH) and 3 (16:1ω7c/15:0 iso 2-OH). The major quinone was MK-8. The DNA G+C content was 46.5 mol%. Phylogenetic analysis placed A2-16^T amidst uncultured members of Acidobacteria subdivision 4. The most closely related environmental 16S rRNA gene sequences (96–97% nucleotide identity) were several clone sequences from terrestrial environments. Based on these characteristics, the isolated strain is proposed as a new species of a novel genus, Blastocatella fastidiosa gen. nov., sp. nov. The type strain of B. fastidiosa is A2-16^T (=DSM 25172^T = LMG26944^T).     

Characterization of emY162 encoding an immunogenic protein cloned from an adult worm-specific cDNA library of Echinococcus multilocularis

A cDNA library based on mRNA from adult worms of Echinococcus multilocularis was constructed. One cDNA clone, emY162, was isolated from this cDNA library. The putative protein from emY162 cDNA consists of 153 amino acids and has a predicted molecular weight of 17.0 kDa. The amino acid sequences of EMY162 are predicted to be a hydrophobic N-terminus conserving a secretory signal, and a hydrophobic C-terminus encoding a transmembrane domain or glycosyl-phosphatylinositol membrane anchor, and to have single fibronectin type III-like domain. In addition, it was shown that the emY162 gene (1738 bp) in the E. multilocularis genome DNA consists of three exons and two introns, and that emY162 is expressed in all four stages (protoscoleces, cultured metacestodes, immature adult worms and mature adult worms). Moreover, immunity to recombinant EMY162, which comprises the fibronectin type III-like domain on the EMY162 protein, was examined. Immune responses to the recombinant EMY162 were studied by using serum from dogs infected with E. multilocularis. Strong IgG immune responses were detected in Western blots.     

Morphological and molecular evidence of three species of pikes Esox spp. (Actinopterygii,Esocidae) in France,including the description of a new species

This integrative taxonomy study of French pikes compares morphological characters and molecular sequence data (mitochondrial COI and nuclear Plagl2 genes). In addition to the expected E. lucius, DNA sequences and morphology both support a new species in France, E. aquitanicus sp. nov. from the Charente to the Adour drainages. It is characterized by a color pattern of sides with narrow 1–1.5-scale-wide oblique vertical bands, conferring it a marbled coat, a snout only 0.9 times larger than the postorbital length, an anal fin basis 1.1–1.2 times larger than the caudal peduncle length, 101 to 121 lateral scales, 53 to 57 vertebrae, as well as 24 diagnostic sites in the COI gene and 3 in the Plagl2 gene. Partial COI sequences (131 bp) from modern and historical specimens indicate also the presence of E. cisalpinus and E. lucius during the 19th century in Lake Geneva. Morphological and molecular data points to a possible hybridization between E. lucius with both other local pike species, representing a risk for them. Their endangerment status should be evaluated rapidly in order to take conservation measures.     

cDNA,genomic sequence and overexpression of crystallin alpha-B Gene (CRYAB) of the Giant Panda

αB-crystallin, a small heat-shock protein, has been shown to prevent the aggregation of other proteins under various stress conditions. Here we have cloned the cDNA and the genomic sequence of CRYAB gene from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned contains an open reading frame of 528bp encoding 175 amino acids and the length of the genomic sequence is 3189bp, containing three exons and two introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species studied, including Homo sapiens, Mus musculus, Rattus norvegicus and Bos taurus. The homologies for nucleotide sequences of Giant Panda CRYAB to that of these species are 93.9%, 91.5%, 91.5% and 95.3%, respectively, and the homologies for amino acid sequences are 98.3%, 97.1%,97.7% and 99.4%, respectively. Topology prediction shows that there are only four Casein kinase II phosphorylation sites in the CRYAB protein of the Giant Panda. The cDNA of CRYAB was transfected into E. coli, and the CRYAB fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 24KDa polypeptide, which accorded with the predicted protein. The expression product obtained could be used for purification and study of its function further.