Calyx fluid proteins of two Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) biotypes in Kenya: implications to biological control of the stem borer Busseola fusca (Fuller) (Lepidoptera: Noctuidae)

Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) is an indigenous larval endoparasitoid of Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in sub-Saharan Africa. In Kenya, reports suggest that C. sesamiae occurs as two biotypes. Biotype avirulent to B. fusca gets encapsulated by haemocytes in this host and is unable to complete development. Biotype virulent to B. fusca is able to overcome immune defences. Factors present in the calyx fluid such as the PolyDNAviruses (PDV), venom and calyx fluid proteins have been implicated in the variation of C. sesamiae virulence against B. fusca. In the present study, calyx fluid proteins of the two C. sesamiae biotypes were compared using 2-D gel electrophoresis. More protein spots were observed in the virulent parasitoid calyx fluid, but some proteins were specifically observed in the avirulent parasitoid calyx fluid while others were observed in both. To study changes in proteins due to parasitism of B. fusca larvae by the two strains, SDS-PAGE gel were performed on fat body tissues and the haemolymph at three time points. Differences between the two strains were observed in both the fat body and haemolymph tissues. Parasitism-specific protein bands were detectable in fat body tissues of B. fusca larvae parasitized by the two C. sesamiae strains. These proteins were absent in unparasitized larvae. Implications for using C. sesamiae as a biocontrol agent of B. fusca in Africa are discussed.     

Differential expression of two small Hsps during diapause in the corn stalk borer Sesamia nonagrioides (Lef.)

We isolated and characterized two members of the α-crystallin/sHsp family, SnoHsp19.5 and SnoHsp20.8 from Sesamia nonagrioides (Lepidoptera: Noctuidae). The cDNAs encoded proteins of 174 and 185 amino acids, with calculated molecular weights of 19.5 and 20.8 kDa, respectively. The deduced amino acid sequences of SnoHsp19.5 and SnoHsp20.8 showed highest homology to Hsp19.7 of Mamestra brassicae and to Bombyx mori Hsp20.4, respectively. Expression patterns of SnoHsp19.5 and SnoHsp20.8 in non-diapausing individuals under different environmental conditions (heat or cold) showed different accumulation profiles for the two genes after heat and cold treatment. SnoHsp19.5 was consistently expressed, while SnoHsp20.8 gene was down-regulated in deep diapause and was up-regulated at the termination of diapause. Our results suggest that these two genes play distinctive roles in the regulation of diapause.     

Effects of cadmium chloride and nonylphenol on the expression of StAR-related lipid transfer domain containing protein (START1) gene in aquatic midge, Chironomus riparius

We identified and characterized a partial cDNA of StAR-related lipid transfer domain containing protein gene from Chironomus riparius (CrSTART1) having homology with human MLN64 and Drosophila melanogaster START1 (DmSTART1) and evaluated the effects of cadmium chloride (Cd) and nonylphenol (NP) on its expression. Pfam analysis identified the presence of two StAR-related lipid transfer (START) domains in CrSTART1 having several conserved amino acid residues, characteristic of the MLN64 and DmSTART1. The mRNA expression of CrSTART1 was observed in all developmental stages. The modulation in the mRNA expression of CrSTART1 was investigated after exposure to different concentrations Cd (0, 2, 10, and 20 mg/L) and NP (0, 10, 50, and 100 μg/L) for different time intervals in fourth instar larvae of C. riparius. Significant downregulation of CrSTART1 mRNA was observed after exposure to 2, 10 and 20 mg/L of Cd for 24, 48 and 72 h. Significant upregulation of CrSTART1 was observed after exposure to 10 and 50 μg/L of NP for 24, and 48 h period. At 100 μg/L of NP significant upregulation of CrSTART1 was observed after 12 h and downregulated after 24, 48 and 72 h.     

Construction of two vectors for gene expression in Trichoderma reesei

We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei. The two newly constructed vectors can be efficiently transformed into T. reesei with Agrobacterium-mediated transformation. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. As a result, pWEF32 easily undergoes homologous recombination. On the other hand, pWEF31 undergoes random recombination. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30. Additionally, we measured the exo-1,4-β-glucanase activity of the recombinant cells. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. reesei. It also provides a method for recombination at a specific chromosomal location. Finally, both vectors will be useful for the large-scale gene expression industry.     

Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus, Photorhabdus luminescens W14 P. luminescens TTO1, and Yersinia pestis CO92. The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity.     

Naegleria fowleri: nfa1 gene knock-down by double-stranded RNAs

Nfa1 protein expressed by the nfa1 gene that was cloned recently from pathogenic Naegleria fowleri was found in pseudopodia, especially food-cups, and concerned with a mechanism of pathogenicity of N. fowleri. In the present study, N. fowleri nfa1 gene was knocked down using double-stranded RNAs, and the expression of Nfa1 protein was observed. Using synthetic double-stranded RNA of the nfa1 gene in vitro, the nfa1 gene and Nfa1 protein were knocked down about 50.4+/-3.1% and 52+/-2%, respectively. These results suggest that RNA interference (RNAi) may be an effective technique for gene knock-down in N. fowleri trophozoites.     

Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae)

Two full-length genes encoding different acetylcholinesterases (AChEs), designated as Ch-ace1 and Ch-ace2, were cloned from strains of the rice stem borer (Chilo suppressalis) susceptible and resistant to the organophosphate insecticide triazophos. Sequence analysis found an amino acid mutation A314S in Ch-ace1 (corresponding to A201 in Torpedo californica AChE) that was consistently associated with the occurrence of resistance. This mutation removed an MspA1 I restriction site from the wild type allele. An assay based on restriction fragment length polymorphism (RFLP) analysis was developed to diagnose A314S genotypes in field populations. Results showed a strong correlation between frequencies of the mutation and phenotypic levels of resistance to triazophos. The assay offers a prospect for rapid monitoring of resistance and assisting with the appropriate choice of insecticide for combating damage caused by C. suppressalis.     

Comparison between allicin and fluconazole in Candida albicans biofilm inhibition and in suppression of HWP1 gene expression

Candida albicans is an opportunistic human pathogen with the ability to differentiate and grow in filamentous forms and exist as biofilms. The biofilms are a barrier to treatment as they are often resistant to the antifungal drugs. In this study, we investigated the antifungal activity of allicin, an active compound of garlic on various isolates of C. albicans. The effect of allicin on biofilm production in C. albicans as compared to fluconazole, an antifungal drug, was investigated using the tetrazolium (XTT) reduction-dependent growth and crystal violet assays as well as scanning electron microscopy (SEM). Allicin-treated cells exhibited significant reduction in biofilm growth (p<0.05) compared to fluconazole-treated and also growth control cells. Moreover, observation by SEM of allicin and fluconazole-treated cells confirmed a dose-dependent membrane disruption and decreased production of organisms. Finally, the expression of selected genes involved in biofilm formation such as HWP1 was evaluated by semi-quantitative RT-PCR and relative real time RT-PCR. Allicin was shown to down-regulate the expression of HWP1.     

鲤TYK2基因的克隆鉴定及组织表达分析

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Identification of cis-regulatory elements from the C. elegans Hox gene lin-39 required for embryonic expression and for regulation by the transcription factors LIN-1, LIN-31 and LIN-39

Expression of the Caenorhabditis elegans Hox gene lin-39 begins in the embryo and continues in multiple larval cells, including the P cell lineages that generate ventral cord neurons (VCNs) and vulval precursor cells (VPCs). lin-39 is regulated by several factors and by Wnt and Ras signaling pathways; however, no cis-acting sites mediating lin-39 regulation have been identified. Here, we describe three elements controlling lin-39 expression: a 338-bp upstream fragment that directs embryonic expression in P5-P8 and their descendants in the larva, a 247-bp intronic region sufficient for VCN expression, and a 1.3-kb upstream cis-regulatory module that drives expression in the VPC P6.p in a Ras-dependent manner. Three trans-acting factors regulate expression via the 1.3-kb element. A single binding site for the ETS factor LIN-1 mediates repression in VPCs other than P6.p; however, loss of LIN-1 decreases expression in P6.p. Therefore, LIN-1 acts both negatively and positively on lin-39 in different VPCs. The Forkhead domain protein LIN-31 also acts positively on lin-39 in P6.p via this module. Finally, LIN-39 itself binds to this element, suggesting that LIN-39 autoregulates its expression in P6.p. Therefore, we have begun to unravel the cis-acting sites regulating lin-39 Hox gene expression and have shown that lin-39 is a direct target of the Ras pathway acting via LIN-1 and LIN-31.     

E2F1 regulation of the human myo-inositol 1-phosphate synthase (ISYNA1) gene promoter

Human myo-inositol 1-phosphate synthase (IP synthase; E.C. 5.5.1.4), encoded by ISYNA1, catalyzes the de novo synthesis of inositol 1-phosphate from glucose 6-phosphate. It is a potential target for mood-stabilizing drugs such as lithium and valproate. But, very little is known about the regulation of human IP synthase. Here, we have characterized the minimal promoter of ISYNA1 and show that it is upregulated by E2F1. Upregulation occurs in a dose-dependent fashion and can be suppressed by ectopic expression of Rb. EMSA and antibody supershift analysis identified a functional E2F binding motif at -117. Complex formation at this site was competed by an excess of unlabeled Sp1 oligo consistent with the -117 E2F site overlapping an Sp1 motif. Because the -117 E2F motif is not a high-affinity binding site, we propose that the upregulation of ISYNA1 occurs through the cooperative interaction of several low-affinity E2F binding motifs present in the minimal promoter.     

Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. In addition, it has been shown that HP1 is also involved in telomere capping in Drosophila. Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila. By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes. Because the puffs are the cytological phenotype of intense gene activity, we did a detailed analysis of the heat shock-induced expression of the HSP70 encoding gene in larvae with different doses of HP1 and found that HP1 is positively involved in Hsp70 gene activity. These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.     

Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden Takin (Budorcas taxicolor Bedfordi)

A bacterial strain with high cellulase activity was isolated of feces sample of Golden Takin (Budorcas taxicolor Bedfordi). The bacterium was classified and designated Bacillus subtilis LN by morphological and 16SrDNA gene sequence analysis. Two putative cellulase genes, CelL15 and CelL73, were simultaneously cloned from the isolated strain by PCR. The putative gene CelL15 consisted of an open reading frame (ORF) of 1470 nucleotides and encoded a protein of 490 amino acids with a molecular weight of 54 kDa. The CelL73 gene consisted of an open reading frame (ORF) of 741 nucleotides and encoded a protein of 247 amino acids with a molecular weight of 27 kDa. Both genes were purified and cloned into pET-28a for expression in Escherichia coli BL21 (DE3). The ability of E. coli to degrade cellulose was enhanced when the two recombinants were cultured together.     

Involvement of HMG-12 and CAR-1 in the cdc-48.1 expression of Caenorhabditis elegans

Caenorhabditis elegans possesses two p97/VCP/Cdc48p homologues, named CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8), and their expression patterns and levels are differently regulated. To clarify the regulatory mechanisms of differential expression of two p97 proteins of C. elegans, we performed detailed deletion analysis of their promoter regions. We found that the promoter of cdc-48.1 contains two regions necessary for embryonic and for post-embryonic expression, while the promoter of cdc-48.2 contains the single region necessary for embryonic expression. In particular, two elements (Element A and Element B) and three conserved boxes (Box a, Box b and Box c) were essential for cdc-48.1 expression in embryos and at post-embryonic stages, respectively. By using South-Western blotting and MALDI-TOF MS analysis, we identified HMG-12 and CAR-1 as proteins that bind to Element A and Element B, respectively, from the embryonic nuclear extract. Importantly, we found the decreased expression of p97 in embryos prepared from hmg-12(RNAi) or car-1(RNAi) worms. These results indicate that both HMG-12 and CAR-1 play important roles in embryonic expression of cdc-48.1.     

Deletion of the Na/K-ATPase alpha1-subunit gene (Atp1a1) does not prevent cavitation of the preimplantation mouse embryo

Increases in Na/K-ATPase activity occur concurrently with the onset of cavitation and are associated with increases in Na(+)-pump subunit mRNA and protein expression. We have hypothesized that the alpha1-isozyme of the Na/K-ATPase is required to mediate blastocyst formation. We have tested this hypothesis by characterizing preimplantation development in mice with a targeted disruption of the Na/K-ATPase alpha1-subunit (Atp1a1) using embryos acquired from matings between Atp1a1 heterozygous mice. Mouse embryos homozygous for a null mutation in the Na/K-ATPase alpha1-subunit gene are able to undergo compaction and cavitation. These findings demonstrate that trophectoderm transport mechanisms are maintained in the absence of the predominant isozyme of the Na(+)-pump that has previously been localized to the basolateral membranes of mammalian trophectoderm cells. The presence of multiple isoforms of Na/K-ATPase alpha- and beta-subunits at the time of cavitation suggests that there may be a degree of genetic redundancy amongst isoforms of the catalytic alpha-subunit that allows blastocyst formation to progress in the absence of the alpha1-subunit.