Vip Genes Research Articles

Transgenic cowpeas (Vigna unguiculata L. Walp) expressing Bacillus thuringiensis Vip3Ba protein are protected against the Maruca pod borer (Maruca vitrata)

Although cowpea is an important food security crop in Africa, its production is constrained by insect pests such as the legume pod borer (Maruca vitrata) (Lepidoptera). Potential control strategies have focussed on using insecticidal toxins such as the crystal Cry proteins and vegetative insecticidal proteins (Vips) encoded by the cry and vip genes, respectively, of the bacterium Bacillus thuringiensis (Bt). This study sought to identify vip genes encoding toxins active against Maruca Pod Borer (MPB), from Australian Bt isolates. A collection of 224 Bt isolates was screened with gene-specific primers to identify those containing target vip genes namely vip3Aa35, vip3Af1, vip3Ag, vip3Ca2 and vip3Ba1. The coding sequences of the vip3 genes were cloned and over-expressed in Escherichia coli to produce Vip3 protein. The proteins were incorporated into Maruca artificial diets for use in insect bioassays with MPB larvae to screen for toxicity. Of these, Vip3Ba1 protein was found to strongly inhibit larval growth and was selected as the candidate gene for cowpea transformation. A vip3Ba gene reconstructed for plant expression was used in transforming cowpea via Agrobacterium tumefaciens. Transgenic lines expressing Vip3Ba protein were used in insect feeding trials to assess protection against Maruca and were found to be completely protected from this pest. We propose that the vip-cowpea lines could be combined with existing cry-transgenic cowpea to introgress this additional resistance trait and thus avoid or greatly delay the development of resistance in Maruca.

Induction of out-of-season egg laying by artificial photoperiod in Yangzhou geese and the associated endocrine and molecular regulation mechanisms

This study was carried out to induce out-of-season breeding, in the summer, and to achieve high reproductive performance using artificial photoperiod manipulation in the long-day breeding Yangzhou goose. Young geese were subject to a two-phase short-to-long (group A) or a three-phase (long-short-long; group B) photoperiod program February through October. Egg-laying was induced to start similarly in both groups in May, increased to a peak level in July, and then decreased gradually through to October. The peak and post-peak laying rates were higher with the three-phase than with the two-phase program. Plasma progesterone concentrations changed similarly in the two groups, increasing from low levels during the pre-lay periods until the peak laying stage, then decreasing with decline in the egg-laying rate. Plasma T3 concentrations increased from the beginning of the experiment to form the first peak under a short photoperiod, declined to a trough at peak lay and then progressively increased to high levels towards the end of the experiment. Plasma T4 concentrations increased throughout the experiment, showing little response to changes in photoperiod. GnIH mRNA expression level in the hypothalamus steadily decreased from high levels under the short photoperiod to a nadir at peak of lay, but was abruptly up-regulated by over a thousand-fold thereafter. This mRNA expression pattern was also shared by GnIHR, VIPR, TRHR, TSH, and PRL genes in the pituitary gland, and to lesser extent, by GnRH, VIP, and TRH genes in the hypothalamus. Pituitary GnRHR mRNA expression levels changed in a similar manner to that of reproductive activities of geese in both groups. FSH beta subunits mRNA expression levels increased to high levels after day 11 of the long photoperiod, and were higher in group B than in group A at peak laying. LH beta gene expression level was similarly upregulated by photoperiod and was higher in group B than in group A when used the multivariable and two-way analyses of variance. Taken together, photoperiod, through regulation of expression of an array of genes in the hypothalamus and pituitary gland, synchronized stimulation and refractoriness of the reproductive system in Yangzhou geese. The higher out-of-season egg laying performance following the three-phase photo-program treatment was mediated by higher FSH beta and LH beta subunit mRNA expression levels.

Characterization of a highly toxic strain of Bacillus thuringiensis serovar kurstaki very similar to the HD-73 strain.

The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed a 6.4 and 9.5 increase in toxicity, against Manduca sexta and Trichoplusia ni, respectively, compared to HD-73. However, LBIT-1200 was still highly similar to HD-73, including the production of bipyramidal crystals containing only one protein of ∼130000 kDa, its flagellin gene sequence related to the kurstaki serotype, plasmid and RepPCR patterns similar to HD-73, no production of β-exotoxin and no presence of VIP genes. Sequencing of its cry gene showed the presence of a cry1Ac-type gene with four amino acid differences, including two amino acid replacements in domain III, compared to Cry1Ac1, which may explain its higher toxicity. In conclusion, the LBIT-1200 strain is a variant of the HD-73 strain but shows a much higher toxicity, which makes this new strain an important candidate to be developed as a bioinsecticide, once it passes other tests, throughout its biotechnological development.

Prevalence of cry2-type genes in Bacillus thuringiensis isolates recovered from diverse habitats in India and isolation of a novel cry2Af2 gene toxic to Helicoverpa armigera (cotton boll worm).

Insecticidal cry and vip genes from Bacillus thuringiensis (Bt) have been used for control of lepidopteran insects in transgenic crops. However, novel genes are required for gene pyramiding to delay evolution of resistance to the currently deployed genes. Two PCR-based techniques were employed for screening of cry2-type genes in 129 Bt isolates from diverse habitats in India and 27 known Bt strains. cry2Ab-type genes were more prevalent than cry2Aa- and cry2Ac-type genes. Correlation between source of isolates and abundance of cry2-type genes was not observed. Full-length cry2A-type genes were amplified by PCR from 9 Bt isolates and 4 Bt strains. The genes from Bt isolates SK-758 from Sorghum grain dust and SK-793 from Chilli seeds warehouse, Andhra Pradesh, were cloned and sequenced. The gene from SK-758 (NCBI GenBank accession No. GQ866915) was novel, while that from SK-793 (NCBI GenBank accession No. GQ866914) was identical to the cry2Ab1 gene. The Bacillus thuringiensis Nomenclature Committee ( http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/toxins2.html ) named these genes cry2Af2 and cry2Ab16, respectively. The cry2Af2 gene was expressed in Escherichia coli and found to be toxic towards Helicoverpa armigera. The cry2Af2 gene will be useful for pyramiding in transgenic crops.

Virulence factors and resistance to antimicrobials in Listeria monocytogenes serotype 1/2c isolated from food.

The aim of this study was to assess the potential risk posed to the human population by the presence of Listeria monocytogenes serotype 1/2c in food based on the characterization of virulence factors of Listeria involved in the invasion of host cells and sensitivity to antimicrobial agents. In addition to sequencing of the inlA and inlB genes, the presence of genes lapB, aut, fbpA, ami, vip and llsX was tested. A premature stop codon (PMSC) in the inlA gene was detected in all tested strains of serotype 1/2c and, concurrently, two novel PMSC mutation types were identified. However, neither PMSC in the inlB gene nor deletion of the lapB, aut, fbpA, ami and vip genes were found in any of the strains. The presence of the llsX gene was not confirmed. Even though all L.monocytogenes strains showed sensitivity to the tested antimicrobials on the basis of their phenotype, sequencing revealed the presence of IS1542 insertion in the inlA gene, indicating the possibility of sharing of mobile genetic elements associated with antimicrobial resistance among strains. Other than the presence of PMSCs in the inlA gene, no PMSC in inlB or deletion of other factors linked to the invasiveness of listeria were detected. Tested strains showed sensitivity to antibiotics used in the therapy of listeriosis. Strains of L.monocytogenes serotype 1/2c typically carry a PMSC in the inlA gene, but these strains still represent a potential threat to public health. The possibility of transfer of IS1542, associated with resistance to vancomycin, between enterococci and Listeria spp. was revealed.

Molecular and insecticidal characterization of Vip3A protein producing Bacillus thuringiensis strains toxic against Helicoverpa armigera (Lepidoptera: Noctuidae).

Vegetative insecticidal proteins (Vip) represent the second generation of insecticidal proteins produced by Bacillus thuringiensis (Bt) during the vegetative growth stage of growth. Bt-based biopesticides are recognized as viable alternatives to chemical insecticides; the latter cause environmental pollution and lead to the emergence of pest resistance. To perform a systematic study of vip genes encoding toxic proteins, a total of 30 soil samples were collected from diverse locations of Kashmir valley, India, and characterized by molecular and analytical methods. Eighty-six colonies showing Bacillus-like morphology were selected. Scanning electron microscopy observations confirmed the presence of different crystal shapes, and PCR analysis of insecticidal genes revealed a predominance of the lepidopteran-specific vip3 (43.18%) gene followed by coleopteran-specific vip1 (22.72%) and vip2 (15.90%) genes in the isolates tested. Multi-alignment of the deduced amino acid sequences revealed that vip3 sequences were highly conserved, whereas vip1 and vip2 showed adequate differences in amino acid sequences compared with already reported sequences. Screening for toxicity against Helicoverpa armigera larvae was performed using partially purified soluble fractions containing Vip3A protein. The mortality levels observed ranged between 70% and 96.6% in the isolates. The LC50 values of 2 of the native isolates, JK37 and JK88, against H. armigera were found to be on par with that of Bt subsp. kurstaki HD1, suggesting that these isolates could be developed as effective biopesticides against H. armigera.

Activity of vegetative insecticidal proteins Vip3Aa58 and Vip3Aa59 of Bacillus thuringiensis against lepidopteran pests

Vegetative insecticidal proteins (Vips) secreted by some isolates of Bacillus thuringiensis show activity against insects and are regarded as insecticides against pests. A number of B. thuringiensis strains harbouring vip3A genes were isolated from different sources and identified by using a PCR based approach. The isolates with the highest insecticidal activity were indicated in screening tests, and their vip genes were cloned and sequenced. The analysis revealed two polymorphic Vip protein forms, which were classified as Vip3Aa58 and Vip3Aa59. After expression of the vip genes, the proteins were isolated and characterized. The activity of both toxins was estimated against economically important lepidopteran pests of woodlands (Dendrolimus pini), orchards (Cydia pomonella) and field crops (Spodoptera exigua). Vip3Aa58 and Vip3Aa59 were highly toxic and their potency surpassed those of many Cry proteins used in commercial bioinsecticides. Vip3Aa59 revealed similar larvicidal activity as Vip3Aa58 against S. exigua and C. pomonella. Despite 98% similarity of amino acid sequences of both proteins, Vip3Aa59 was significantly more active against D. pini. Additionally the effect of proteolytic activation of Vip58Aa and Vip3Aa59 on toxicity of D. pini and S. exigua was studied. Both Vip3Aa proteins did not show any activity against Tenebrio molitor (Coleoptera) larvae. The results suggest that the Vip3Aa58 and Vip3Aa59 toxins might be useful for controlling populations of insect pests of crops and forests.

Synergism and antagonism between Bacillus thuringiensis Vip3A and Cry1 proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda.

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.

Genomic sequencing identifies novel Bacillus thuringiensis Vip1/Vip2 binary and Cry8 toxins that have high toxicity to Scarabaeoidea larvae.

The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), which has previously been shown to encode the cry8Ga toxin gene, is active against both Holotrichia oblita and Holotrichia parallela. Recombinant Cry8Ga however is only weakly toxic to these insect pests suggesting the involvement of additional toxins in the native strain. We report that through the use of Illumina sequencing three additional, and novel, genes, namely vip1Ad1, vip2Ag1, and cry8-like, were identified in this strain. Although no protein corresponding to these genes could be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the HBF-18 proteome, reverse transcription (RT)-PCR indicated that all three genes were transcribed in the native strain. The two vip genes were cloned and expressed and, as with other Vip1/2 toxins, appeared to function as a binary toxin and showed strong activity against H. oblita, H. parallela and Anomala corpulenta. This is the first report to demonstrate that the Vip1/Vip2 binary toxin is active against these Scarabaeoidea larvae. The cry8-like gene appeared to be a C-terminally truncated form of a typical cry8 gene and was not expressed in our usual recombinant Bt expression system. When however the missing C-terminal region was replaced with the corresponding sequence from cry8Ea, the resulting hybrid expressed well and the toxin was active against the three test insects.

Diversity of <i>Bacillus thuringiensis</i> strains isolated from coffee plantations infested with the coffee berry borer <i>Hypothenemus hampei</i>

The coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) was first reported infecting Costa Rican coffee plantations in the year 2000. Due to the impact that this plague has in the economy of the country, we were interested in seeking new alternatives for the biological control of H. hampei, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 202 B. thuringiensis isolates obtained from Costa Rican coffee plantations infested with H. hampei were analyzed through crystal morphology of the crystal inclusions and SDS-PAGE of 6-endotoxins, while 105 strains were further evaluated by PCR for the presence cry, cyt and vip genes. Most of the Bt strains showed diverse crystal morphologies: pleomorphic (35%), oval (37%), bipyramidal (3%), bipyramidal and oval (12%), bipyramidal, oval and pleomorphic (10%) and bipyramidal, oval and cubic (3%). The SDS-PAGE analyses of the crystal preparations showed five strains with delta-endotoxin from 20 to 40 kDa, six from 40 to 50 kDa, seven from 50 to 60 kDa, 19 from 60 to 70 kDa, 29 from 70 to 100 kDa and 39 from 100-145 kDa. PCR analyses demonstrated that the collection showed diverse cry genes profiles having several genes per strain: 78 strains contained the vip3 gene, 82 the cry2 gene, 45 the cry1 and 29 strains harbored cry3-cry7 genes. A total of 13 strains did not amplified with any of the cry primers used: cry1, cry2, cry3-7, cry5, cry11, cry12 and cry14. Forty-three different genetic profiles were found, mainly due to the combination of cry1A genes with other cry and vip genes. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against H. hampei and other insect pests of agricultural importance.

BIOLOGICAL AND MOLECULAR STUDIES ON THE TOXIC EFFECT OF VEGETATIVE INSECTICIDAL PROTEIN (VIPs) OF Bacillus thuringiensis EGYPTIAN ISOLATES AGAINST WHITEFLIES

Whitefly Bemisia tabaci (Gennadius) transmitted geminiviruses cause epidemics in vegetable and fiber crops. It can infect more than 600 species of host plants. It also considered as the major vector transmitting various types of geminiviruses such as tomato yellow leaf curl virus (TYLCV) which cause great damage to tomatoes crop. Previous studies have reported that the vegetative insecticidal proteins (VIPs) of Bacillus thuringiensis (Bt) revealed insecticidal activity against different insect species. In this study, two local isolates of B. thuringiensis named BtC-18 and BtDI-29 were screened for the insecticidal activity of VIPs against whitefly population. Analysis of median lethal concentration (LC50) revealed that B. thuringiensis strain BtC-18 is more potent and toxic than BtDI-29 strain against whiteflies with an estimated LC50 of 90 ppm and 160 ppm for BtC-18 and BtDI-29, respectively. However, the median lethal time (LT50) value did not show significant difference between both isolates. PCR analysis of vip genes confirmed the presence of vip1, vip2 and vip3 genes on BtC-18 genome. Proteins extract from the BtC-18 culture pellet were further purified by 80% saturation of ammonium sulfate precipitation. The purified protein showed a clear band at 88 KDa corresponding to Vip3A protein as previously demonstrated. The LC50 of the purified band at 88KDa showed insecticidal activity against white fly with an estimated LC50 of 898 ppm.

Characterization of a Bacillus thuringiensis strain collection isolated from diverse Costa Rican natural ecosystems

Costa Rican natural ecosystems are among the most diverse in the world. For this reason, we isolated strains of the entomopathogenic bacteria Bacillus thuringiensis (Bt) to determine their diversity, distribution and abundance. A total of 146 Bt strains were obtained from environmental samples collected from diverse natural ecosystems and life zones of Costa Rica. We recovered Bt strains from 71%, 63%, 61% and 54% of soil samples, fresh leaves, other substrates and leaf litter respectively. Bt was isolated in 65% of the samples collected in the humid tropical forest in national parks (Braulio Carrillo, Gandoca Manzanillo, Sierpe, Hitoy Cerere, and Cahuita), and in 59% of the samples collected in the dry tropical forest (Parque Nacional Marino las Baulas, Palo Verde and Santa Rosa). In the very humid tropical forest (Tortuguero) Bt was isolated in 75% of the samples and in the very humid tropical forest transition perhumid (Carara) it was found in 69% of the samples. The strains exhibit a diverse number, size and morphology of parasporal inclusion bodies: irregular (47%), oval (20%), bipyramidal (3%), bipyramidal and cubic (1%), bipyramidal, oval and irregular (5%) and bipyramidal, oval and cubic crystals (2%). Strains isolated from Braulio Carrillo, Tortuguero and Cahuita, presented predominantly irregular crystals. On the other hand, more than 60% of the isolates from Térraba-Sierpe and Hitoy-Cerere had medium oval crystals. Strains from Gandoca-Manzanillo, Palo Verde and Carara presented mainly combinations of oval and irregular crystals. Nevertheless, the greatest diversity in crystal morphology was observed in those from Santa Rosa, Llanos del Rio Medio Queso and Parque Marino las Baulas. Protein analyses of the crystal-spore preparations showed delta-endotoxin with diverse electrophoretic patterns, with molecular weights in the range of 20 to 160 kDa. Fifty six percent of the strains amplified with the cry2 primer, 54% with vip3, 20% with cry1, 9% with cry3-cry7 and 8% with cry8. The cry11 and cyt genes were found in 8% and 7% of the strains, respectively. When analyzed with specific primers for the cryl subfamily, 13 different genetic profiles were obtained. In addition, twenty-four strains did not amplify with any of the primers used, suggesting they contain novel cry genes. The diversity of Bt genes found in this collection indicates it could have great potential for the control of different species of insect pests. The toxicological characterization of the strains by bioassays against important insect pests will provide useful information about their potential use for the formulation of biological insecticides and their respective cry and vip genes for the transformation of crops to confer resistance to insects.

A genome-wide integrative study of microRNAs in human liver

BackgroundRecent studies have illuminated the diversity of roles for microRNAs in cellular, developmental, and pathophysiological processes. The study of microRNAs in human liver tissue promises to clarify the therapeutic and diagnostic value of this important regulatory mechanism of gene expression.ResultsWe conducted genome-wide profiling of microRNA expression in liver and performed an integrative analysis with previously collected genotype and transcriptome data. We report here that the Very Important Pharmacogenes (VIP Genes), comprising of genes of particular relevance for pharmacogenomics, are under substantial microRNA regulatory effect in the liver. We set out to elucidate the genetic basis of microRNA expression variation in liver and mapped microRNA expression to genomic loci as microRNA expression quantitative trait loci (miR-eQTLs). We identified common variants that attain genome-wide significant association (p < 10-10) with microRNA expression. We also found that the miR-eQTLs are significantly more likely to predict mRNA levels at a range of p-value thresholds than a random set of allele frequency matched SNPs, showing the functional effect of these loci on the transcriptome. Finally, we show that a large number of miR-eQTLs overlap with SNPs reproducibly associated with complex traits from the NHGRI repository of published genome-wide association studies as well as variants from a comprehensive catalog of manually curated pharmacogenetic associations.ConclusionOur study provides important insights into the genomic architecture of gene regulation in a vital human organ, with important implications for our understanding of disease pathogenesis, therapeutic outcome, and other complex human phenotypes.

Screening of vip genes from a Spanish Bacillus thuringiensis collection and characterization of two Vip3 proteins highly toxic to five lepidopteran crop pests

A Spanish Bacillus thuringiensis strain collection was screened for the presence of vip genes. One hundred strains from a Canary Island collection were screened for vip1 and vip2 genes and 7% contained potentially novel vip1 and vip2-like genes, as indicated by the low degree of similarity with previously known vip1 and vip2 genes. Four hundred strains from a collection originating from the Spanish mainland were screened for vip3 genes and 14.5% of them contained potentially novel vip3-like genes. Reconstruction of the full-length vip sequences could only be achieved for two vip3 gene variants encoding 789 and 787 amino acid proteins that were designated as Vip3Aa45 and Vip3Ag4, respectively. These proteins showed 82% pairwise identity between them and differed from Vip3Aa1 in the putative signal peptide, two specific proteolytic processing sites and the 66-kDa insecticidal fragment. The purified proteins were tested against nine lepidopteran pest species and displayed toxicity, expressed as mean lethal concentration, for five of them. The two toxins were highly toxic for Lobesia botrana (∼1–2μg/ml) and Spodoptera littoralis (∼20ng/cm2), moderately toxic for Spodoptera exigua (∼100–300ng/cm2), and varied greatly in their toxicity for Mamestra brassicae or Chrysodeixis chalcites, with high toxicity for Vip3Aa45 in M. brassicae (∼40ng/cm2) and for Vip3Ag4 in C. chalcites (∼45ng/cm2).

Distribution of &lt;i&gt;vip&lt;/i&gt; Genes, Protein Profiling and Determination of Entomopathogenic Potential of Local Isolates of &lt;i&gt;Bacillus thuringiensis&lt;/i&gt;

The aim of this study was to screen the Bacillus thuringiensis ( Bt ) strains isolated from local ecological niche for the presence of vegetative insecticidal protein ( vip ) genes and its characterization. PCR screening was carried out to determine different vip genes viz., vip1 / vip2 and vip3 genes, harboured by native Bt isolates using four gene specific primers. The PCR results depicted the presence of all three vip -type genes in these isolates. Strains containing vip3 -type genes were the most abundant (12.5%) in local isolates, followed by vip1 / vip2 -type genes (10%). Insect bioassays for these proteins were conducted against Helicoverpa armigera ( H. armigera ). Four Bt isolates PDKV-08 (161.80), PDKV-21 (173.62), NCIM-5110 (170.57) and NCIM-5132 (178.61) depict lower LC 50 values against H. armigera than the standard HD-1 (190.42). The present foundation work can be used for the production of more effective Bt based biopesticide and the identified genes can be explored for the production of next generation insect resistant transgenic plants.

Screening, Diversity and Partial Sequence Comparison of Vegetative Insecticidal Protein (vip3A) Genes in the Local Isolates of Bacillus thuringiensis Berliner

Characterization, direct sequencing of the PCR amplicon and phylogenetic relationship was done to discover a novel Vip protein genes of the Bt isolates, to improve the prospects for insect control, more Vip proteins should be sought out and researched to predict their insecticidal activity. Characterization was based on direct sequencing of PCR amplicon using primers specific to vip3A gene was presented here. 12 out of 18 isolates screened were positive for vip gene-specific primers. Homology search for the partial sequences using BLAST showed that 11 isolates had high similarity to vip3Aa gene and only one fragment with vip3Ae gene (25-100% at nucleotide and amino acid level). Phylogenetic analysis showed that the gene sequences were responsible for geographic separation for divergence within vip genes, consistent with the evaluation of distinct bacterial population. Despite the geographical distances, strains harbouring vip genes have originated from common ancestors may significantly contribute to control resistant insect pests. Some strains have evolved to be quite distinct and others remain as members of closely related groups. The reported method is a powerful tool to find novel Vip3A proteins from large-scale Bt strains which is effective in terms of time and cost. Further the Vip proteins produced by different strains of B. thuringiensis are unique in terms of the sequence divergence and hence may also differ in their insecticidal activities.

Characterization of Bacillus thuringiensis isolates from soil and small mammals that harbour vip3A gene homologues

The insecticidal and psychrotropic potential of 132 new isolates of Bacillus thuringiensis from northeastern Poland (74 from animals and 58 from soil) were determined by screening these for vip and cry genes encoding, respectively, vegetative insecticidal proteins (Vip) and Cry proteins, and cspA that encoded the CspA cold shock protein that confers psychrotropy in Bacillus species. The vip3A gene, encoding Vip3A toxic to lepidopterans, was found in ~5% of the isolates from animals and ~17% the isolates from soil, whereas coleopteran-specific vip1 and vip2 genes were present in 8% of the isolates from soil. Nucleotide sequences of vip3A-specific amplicons were highly conserved, with only a few containing minor differences from vip3A. Despite the high level of vip3A conservation, isolates harbouring the gene demonstrated a high level of heterogeneity based on whole-cell genomic DNA RFLP analysis with pulsed-field gel electrophoresis (PFGE) and plasmid profiling. Eight isolates positive for vip3A contained cry1 and six also harboured the cry2 gene, which encodes an endotoxin toxic to lepidopteran insects. However, none of these isolates contained cry genes coding for proteins toxic to coleopteran or dipteran insects. Due to the known potential for synergistic interactions between Vip and Cry proteins, the isolates positive for vip3A and cry genes may be used in resistance management strategies directed against lepidopteran larvae. Finally, all of the B. thuringiensis vip3A-positive isolates harboured the cspA gene, but only two were confirmed to be psychrotrophs.

Characterization of Vegetative Insecticidal Protein vip Genes of Bacillus thuringiensis from Sichuan Basin in China

Vegetative insecticidal proteins (Vip), the second generation of insecticides, are produced during the vegetative growth stage of Bacillus thuringiensis (Bt). To perform a systematic study of vip genes in Bt strains from different ecological regions of Sichuan Basin, 1,789 soil samples were collected from this basin, which is situated in the western region of China. The basin has a complicated geomorphology and contains mountains, forests, highlands, hursts, and plains. A total of 2,134 Bt strains have been screened from the 1,789 soil samples. According to the results, three vip-type genes were found in this basin, namely the vip1, vip2, and vip3-type genes. Strains containing vip3-type genes were the most abundant in our collection (67.4%), followed by vip2-type genes (14.6%) and vip1-type genes (8.1%). The three types of vip genes were distributed in most of the regions, but E Mei Mountain and the Ba Lang Mountains only contained vip3 genes in environments with high elevation, low temperature, insufficient oxygen, and abundant snow. Moreover, five novel vip3 genes were found, and these Vip proteins were toxic for Chilo suppressalis. All the results mentioned above suggest that Sichuan Basin is a rich resource for vip genes.

New feature: pathways and important genes from PharmGKB

In this issue of Pharmacogenetics and Pharmacogenomics, we are pleased to introduce a regular new feature, provided in collaboration with the Pharmacogenomics Knowledgebase (PharmGKB, http://www.pharmgkb.org/). Each issue of our journal will highlight two elements from the PharmGKB site. First, we will present an important pathway of drug metabolism (usually affecting the pharmacokinetics of a drug) or an important pathway of drug action (describing its targets and mechanism of action or pharmacodynamics). The PharmGKB has a staff of PhD and MS level biologists who constantly survey the literature and create these summaries for users of the database. In addition, members of the NIH-support Pharmacogenetics Research Network and others often coauthor those pathways that figure significantly in their own work. PharmGKB currently offers 57 such pathways, all provided with a downloadable graphic, an extended caption, a set of links into the database describing more about the genes and drugs, and an excel file with pertinent literature references. In each issue, we will feature one of these pathways and provide a useful summary of the significance and scientific understanding of these pathways, along with links back to the PharmGKB for more information. We hope that these summaries will serve both as a starting point for new scientists getting interest in pharmacogenomics, as well as a way for established researchers to expand their understanding of current challenges in the field. Second, we will present detailed gene summaries for PharmGKB ‘Very Important Pharmacogenes’ (VIP genes) which are, in many ways, the field’s greatest hits – genes that have been shown to be important for modulating the response to one or more drugs, and whose variants have been shown to impact drug response phenotypes. The PharmGKB curators and/or collaborators (from Pharmacogenetics Research Network and elsewhere) create these summaries along with specific mention of important variants. The summaries not only provide a textual summary of the gene’s significance, but also provide extensive links to the literature, as well as lists of pertinent drugs, diseases, and side effects. At present, PharmGKB offers 39 VIP gene summaries. We hope these summaries will serve both as a tutorial and continuing education function for the field. PharmGKB tracks its usage patterns carefully and the pathways and VIP gene summaries are two of the most popular features of the site. We are happy to collaborate in bringing them to the readership of the journal to increase their impact and utility, and encourage readers to learn more on the PharmGKB site. We understand that in rapidly changing field such as pharmacogenomics, knowledge can become outdated. For this reason, we encourage our readers to provide feedback and commentary about the presented information. Each of the pathway diagrams and VIP gene summaries are time stamped, and are rereviewed on a regular basis, however experts in the field may become aware of new developments sooner than our staff. Therefore, the PharmGKB curatorial staff would be happy to interact with the readership to improve these summaries in an ongoing manner, and we hope that they will continue to be an important resource for our discipline.

Screening and identification ofvipgenes inBacillus thuringiensisstrains

To identify known vip genes and to detect potentially novel vip genes in a collection of 507 strains of Bacillus thuringiensis. Following a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy, four restriction patterns were found within the vip1 family: vip1Aa1, vip1Ba1/vip1Ba2 and vip1Ca. In the screening of vip2 genes, patterns similar to those of vip2Aa1, vip2Ba1/vip2Ba2 and vip2Ac1 genes were observed. Patterns for vip3Aa1, vip3Ae2 and vip3Af1 were found among vip3 genes. Two new patterns revealed novel vip1 and vip3A genes. The observed frequency of genes belonging to vip1 and vip2 families was around 10%, whereas 48.9% of the strains showed amplification of vip3 genes. A tendency of vip and cry genes to occur together has been observed in this collection of B. thuringiensis strains. Ten different patterns of vip genes belonging to the three vip families and two novel vip genes have been identified in this study. This is the first time that vip1 and vip2 genes have been identified by PCR-RFLP. Furthermore, the results show that the strategy used in this study can lead to the classification of known vip genes as well as the identification of novel vip genes.