RESUMO
BACKGROUND: Claspin and TopBP1 are checkpoint mediators that are required for the phosphorylation of Chk1 by ATR to maintain genomic stability. Here, we investigated the functions of Drosophila Claspin and mus101 (TopBP1 ortholog) during chorion (eggshell component) gene amplification, which occurs in follicle cells in the absence of global genomic DNA replication. RESULTS: Unlike Drosophila mei-41 (ATR ortholog) mutant embryos, Claspin and mus101 mutant embryos showed severe eggshell defects resulting from defects in chorion gene amplification. EdU (5-ethynyl-2'-deoxyuridine) incorporation assay during initiation and elongation stages revealed that Claspin and mus101 were required for initiation, while only Claspin had a major role in the efficient progression of the replication forks. Claspin proteins were enriched in the amplification foci both in the initiation and elongation stage-follicle cell nuclei in a mei-41-independent manner. The focal localization of ORC2, a component of the origin recognition complex, was not significantly affected in the Claspin mutant, whereas it was reduced in the mus101 mutant. CONCLUSIONS: Drosophila Claspin plays a major role in the initiation and elongation stages of chorion gene amplification by localizing to the amplification foci in a mei-41-independent manner. Drosophila mus101 is also involved in chorion gene amplification, mostly functioning in initiation, rather than elongation. Developmental Dynamics 246:466-474, 2016. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Córion , Proteínas de Drosophila/fisiologia , Amplificação de Genes , Animais , Proteínas de Ciclo Celular/genética , Replicação do DNA , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas MutantesRESUMO
A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin-cry1-5-polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin-Cry1-5-polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an â¼65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.
Assuntos
Baculoviridae/patogenicidade , Inseticidas/farmacologia , Lepidópteros/virologia , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Baculoviridae/genética , Endotoxinas/genética , Expressão Gênica , Proteínas Hemolisinas/genética , Larva/fisiologia , Larva/virologia , Lepidópteros/fisiologia , Organismos Geneticamente Modificados , Controle Biológico de Vetores/métodos , Recombinação Genética , Venenos de Escorpião/genética , Análise de SobrevidaRESUMO
Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures, and that these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with the Polh gene at the N-terminus, including a linker and enterokinase (EK) site between Polh and EGFP, was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells in three steps: cell harvest, sonication, and EK digestion. Through final enterokinase digestion, EGFP presented mainly in the supernatant, and this supernatant fraction also showed a pure EGFP band. These results suggest that a combined procedure of Polh fusion expression and enterokinase digestion can be used for rapid and easy purification of other proteins.
Assuntos
Baculoviridae/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/isolamento & purificação , Sequência de Bases , Linhagem Celular , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/químicaRESUMO
The flagellated vegetative cells of the Bacillus thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific factor sera but non-reactive for 3c and 3e factor sera. This creates a new serogroup with flagellar antigenic structure of 3a3b3d: B. thuringiensis serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipienspallens while no lepidopteran toxicity. It produced ovoidal parasporal inclusions (crystals) whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected.
Assuntos
Anopheles/microbiologia , Bacillus thuringiensis/fisiologia , Proteínas de Bactérias , Culex/microbiologia , Endotoxinas , Proteínas Hemolisinas , Inseticidas , Animais , Bacillus thuringiensis/classificação , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Controle de Mosquitos , SorotipagemRESUMO
To construct a new recombinant strain of Bacillus velezensis that has antifungal and insecticidal activity via the expression of the insecticidal Bacillus thuringiensis crystal protein, a B. thuringiensis expression vector (pHT1K-1Ac) was generated that contained the B. thuringiensis cry1Ac gene under the control of its endogenous promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K). This vector was introduced into a B. velezensis isolate that showed high antifungal activities against several plant diseases, including rice blast (Magnaporthe grisea), rice sheath blight (Rhizotonia solani), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans), and wheat leaf rust (Puccinia recondita), by electroporation. The recombinant B. velezensis strain was confirmed by PCR using cry1Ac-specific primers. Additionally, the recombinant strain produced a protein approximately 130 kDa in size and parasporal inclusion bodies similar to B. thuringiensis. The in vivo antifungal activity assay demonstrated that the activity of the recombinant B. velezensis strain was maintained at the same level as that of wild-type B. velezensis. Furthermore, it exhibited high insecticidal activity against a lepidopteran pest, Plutella xylostella, although its activity was lower than that of a recombinant B. thuringiensis strain, whereas wild-type B. velezensis strain did not show any insecticidal activity. These results suggest that this recombinant B. velezensis strain can be used to control harmful insect pests and fungal diseases simultaneously in one crop.
Assuntos
Bacillus/genética , Engenharia Genética , Controle Biológico de Vetores/métodos , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Animais , Antibiose , Bacillus/química , Bacillus/fisiologia , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Endotoxinas/genética , Endotoxinas/metabolismo , Fungos/fisiologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Mariposas/microbiologia , Mariposas/fisiologiaRESUMO
To identify novel crystal proteins, Bacillus thuringiensis 2385-1 was isolated from Korean soil samples and characterized. The H-serotype of 2385-1 was identical to that of subsp. kenyae (H4a4c), and its crystal toxin was bipyramidal-shaped. However, 2385-1 showed a much higher toxicity towards Plutella xylostella and Spodoptera exigua larvae than subsp. kenyae. In addition, the crystal protein profile and plasmid DNA pattern of 2385-1 differed from those of subsp. kenyae. To verify the crystal protein gene types of 2385-1, a PCR-RFLP analysis was performed, and the results revealed that 2385-1 contained two novel cry1-type crystal protein genes, cry1-5 and cry1-12, in addition to the cry1Ja1 gene. The deduced amino acid sequences of cry1-5 and cry1-12 showed a 97.9% and 75.7% sequence similarity with the CrylAb and Cry1Ja crystal proteins, respectively. Among the novel crystal proteins, Cry1-5 showed a high toxicity towards P. xylostella and S. exigua larvae. In conclusion, B. thuringiensis 2385-1 is a new isolate in terms of its gene types, and should be a promising source for an insecticide to control lepidopteran larvae.
Assuntos
Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Animais , Toxinas de Bacillus thuringiensis , Endotoxinas/farmacologia , Endotoxinas/toxicidade , Genes Bacterianos , Inseticidas/toxicidade , Lepidópteros/efeitos dos fármacos , Lepidópteros/microbiologia , Spodoptera/efeitos dos fármacos , Spodoptera/microbiologiaRESUMO
To clone novel cry1-type genes from the Bacillus thuringiensis K1 isolate, about 2.4-kb-long PCR fragments were amplified with two primer sets of ATG1-F/N400-R and 1BeATG1-F/N400-R. Using PCR-RFLP, three novel cry1-type genes, cry1-1, cry1-7, and cry1-44, were obtained from B. thuringiensis K1 and the complete coding sequences of these novel genes were analyzed. The Cry1-1, Cry1-7, and Cry1-44 proteins showed maximum similarities of about 78.0%, 99.7%, and 91.0% with the Cry1Ha1, Cry1Be1, and Cry1Ac2 proteins, respectively. These novel cry1-type genes were expressed using a baculovirus expression vector system and their insecticidal activities were investigated. Whereas all three novel genes were toxic to Plutella xylostella larvae, only Cry1-1 showed insecticidal activity against Spodoptera exigua larvae.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Endotoxinas/genética , Genes Bacterianos , Proteínas Hemolisinas/genética , Animais , Bacillus thuringiensis/isolamento & purificação , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Sequência de Bases , Bioensaio , Primers do DNA/genética , DNA Bacteriano/genética , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Insetos/efeitos dos fármacos , Coreia (Geográfico) , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
Ionizing radiation (IR) treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs) generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis.
Assuntos
Apoptose/efeitos da radiação , Drosophila melanogaster/efeitos da radiação , Embrião não Mamífero/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Pontos de Checagem da Fase S do Ciclo Celular/efeitos da radiação , Animais , Padronização Corporal/efeitos da radiação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero/anormalidades , Feminino , Expressão Gênica , Meiose/efeitos da radiação , Mitose/efeitos da radiação , Oogênese/genética , Oogênese/efeitos da radiação , Radiação IonizanteRESUMO
BACKGROUND: To evaluate the location of tumor relapse and imaging modality for detection according to the breast cancer subtype: luminal A, luminal B, HER2 positive luminal B, nonluminal HER2 positive, and triple negative. MATERIALS AND METHODS: A total of 1244 patients with breast cancer with known estrogen receptor (ER), progesterone receptor (PR), Ki-67 and human epidermal growth factor receptor 2 (HER2), who underwent breast surgery from 2009 to 2012 were analyzed. Patients were classified into the following categories: luminal A (n=458), luminal B (n=241), HER2 positive luminal B (n=227), nonluminal HER2 positive (n=145) and triple negative (n=173). A total of 105 cases of relapse were detected in 102 patients: locoregional recurrence (n=46), recurrence in the contralateral breast (n=28) and distant metastasis (n=31). Comparison of proportions was used to determine the difference between subtypes. RESULTS: Relapse rates by subtypes are as follows: luminal A 23 of 458 (5.02%), luminal B 19 of 241 (7.88%), HER2 positive luminal B 15 of 227 (6.61%), nonluminal HER2 postive 19 of 145 (13.10%) and triple negative 29 of 173 (16.76%). Luminal A tumors had the lowest rate of recurrence and had significantly lower recurrence rate in comparison with nonluminal HER2 postive (p=0.0017) and triple negative subtypes (p<0.0001). Compared with all other subtypes except nonluminal HER2 positive, triple negative tumors had the highest rate of tumor recurrence (p<0.01). Triple negatives were most likely to develop contralateral recurrence against all subtypes (p<0.05). Detection rate of locoregional and contralateral tumor recurrence were 28.3% on mammography (n=17/60). CONCLUSIONS: Luminal A tumors are associated with a low risk of recurrence while triple negative lesions have a high risk. In case of triple negative tumors, the contralateral breast has much more recurrence as compared with all other subtype. In terms of detection rates, breast USG was the best modality for detecting tumor recurrence, compared with other modalities (p<0.05). Subtyping of breast tumors using a molecular gene expression panel can identify patients who have increased risk of recurrence and allow prediction of locations of tumor recurrence for each subtype.
Assuntos
Metástase Linfática/patologia , Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/patologia , Neoplasias de Mama Triplo Negativas/patologia , Adulto , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Antígeno Ki-67/metabolismo , Radiografia , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/cirurgiaRESUMO
ATR and Chk1 are protein kinases that perform major roles in the DNA replication checkpoint that delays entry into mitosis in response to DNA replication stress by hydroxyurea (HU) treatment. They are also activated by ionizing radiation (IR) that induces DNA double-strand breaks. Studies in human tissue culture and Xenopus egg extracts identified Claspin as a mediator that increased the activity of ATR toward Chk1. Because the in vivo functions of Claspin are not known, we generated Drosophila lines that each contained a mutated Claspin gene. Similar to the Drosophila mei-41/ATR and grp/Chk1 mutants, embryos of the Claspin mutant showed defects in checkpoint activation, which normally occurs in early embryogenesis in response to incomplete DNA replication. Additionally, Claspin mutant larvae were defective in G2 arrest after HU treatment; however, the defects were less severe than those of the mei-41/ATR and grp/Chk1 mutants. In contrast, IR-induced G2 arrest, which was severely defective in mei-41/ATR and grp/Chk1 mutants, occurred normally in the Claspin mutant. We also found that Claspin was phosphorylated in response to HU and IR treatment and a hyperphosphorylated form of Claspin was generated only after HU treatment in mei-41/ATR-dependent and tefu/ATM-independent way. In summary, our data suggest that Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks, and this difference is probably due to distinct phosphorylation statuses.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Proteínas de Drosophila/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem , DNA/efeitos da radiação , Replicação do DNA/efeitos dos fármacos , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Raios gama , Hidroxiureia/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Proteínas de XenopusRESUMO
The supernatant of Beauveria bassiana SFB-205 reduced the population of cotton aphid, Aphis gossypii Glover, with a dosage-dependent manner, which allowed a quality control (QC) factor to be determined for the evaluation of the supernatant as the first step of a development. Enzymes were assumed as possible QC factors based on 1) the comparable aphicidal activity of the supernatant protein pellet to the raw supernatant, 2) the supernatant-induced degradation of the insect cuticles, observed by transmission electron microscopy, and 3) the confirmation of enzymes related to the fungal penetration - chitinase, and the Pr1- and Pr2 proteases - in the supernatant. Finally, from the bioassay with the enzyme-inhibited supernatants processed by substrate inhibition one by one, decreased aphicidal activities were observed for all three enzyme-inhibited treatments. This phenomenon, furthermore, was more remarkable in the chitinase-inhibited supernatant. This finding provides that those enzymes (and most particularly the chitinase) in the supernatant were strongly involved in the aphicidal activity. Consequently, the amount of the chitinase may be used as one of the QC factors to determine the insecticidal activity of the supernatant of B. bassiana SFB-205 in the optimization of mass production.
Assuntos
Afídeos/crescimento & desenvolvimento , Beauveria/enzimologia , Quitinases/farmacologia , Meios de Cultivo Condicionados/farmacologia , Gossypium/parasitologia , Controle Biológico de Vetores , Animais , Afídeos/efeitos dos fármacos , Beauveria/crescimento & desenvolvimento , Quitinases/metabolismo , Meios de Cultivo Condicionados/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Doenças das Plantas/parasitologiaRESUMO
Plasmid capture system (PCS) facilitates cloning and manipulation of circular double-stranded DNA. We recently developed an improved PCS (PCS-LZ) to clone relatively large DNA molecules of 30-150 kb. The PCS-LZ donor consists of a mini-F replicon and a kanamycin resistance marker between Tn7 left and Tn7 right ends. Both the replicon and marker gene of the PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition, followed by replication in E. coli. Colonies are tested for lacZ expression by blue/white screening. Circular DNAs were obtained from plasmids of Bacillus thuringiensis, genome segments of Cotesia glomerata bracovirus and polymorphic genomes of Autographa californica nucleopolyhedrovirus. PCS-LZ is a powerful tool for use in genomic analysis and mutagenesis in microorganisms including invertebrate pathogens.
Assuntos
Clonagem Molecular/métodos , DNA Circular/genética , DNA Viral/genética , Plasmídeos/genética , Bacillus thuringiensis/genética , Escherichia coli/genética , Modelos Biológicos , Nucleopoliedrovírus/genéticaRESUMO
Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki Kl which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pKlS-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pKlS-1, ORF2 (MobKl) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8-25.2%) with the Rep protein coded by RCR plasmids, however. The putative double-strand origin of replication (dso) and single-strand origin of replication (sso) of pKlS-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pKlS-1, seven subclones were contructed in the B. thuringiensis ori-negative pHTIK vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (ReplK), dso and ORF4, exhibited replication ability. These findings identified pKlS-1 as a new RCR group VII plasmid, and determined its replication region.
Assuntos
Bacillus thuringiensis/genética , Clonagem Molecular , Replicação do DNA , Plasmídeos/genética , Origem de Replicação , Sequência de Bases , Dados de Sequência Molecular , Fases de Leitura Aberta , Alinhamento de SequênciaRESUMO
The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and non-recombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.
Assuntos
Baculoviridae/genética , Polydnaviridae/genética , Regiões Promotoras Genéticas/genética , Ribonucleases/metabolismo , Vespas/virologia , Animais , Proteínas de Bactérias , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleases/genética , Spodoptera , TransfecçãoRESUMO
Cotesia plutellae polydnaviruses (CpBV) has a segmented genome consisting of multiple circular double stranded DNAs. Recently, we have developed an easy, simple, and convenient system based on Tn7 transposition in order to clone genomic segments of CpBV in Escherichia coli cell and designated plasmid capture system (PCS). The PCS donor-S transferred a pUC19 origin of replication and an ampicillin resistance marker into CpBV genomic DNA by in vitro transposition. Through PCS system, we were able to clone 53 genomic clones ranging from 0.1 to 25.5 kb and further they were classified into 29 segments by their sizes and restriction endonuclease patterns. Among them, a complete nucleotide sequence of CpBV-S28 segment was determined and 10 putative genes were predicted from this segment. Interestingly, 9 of 10 putative ORFs had high level of similarities with catalytic domain of protein tyrosine phosphatase. Also, ORF2807 showed similarity with EP1-like proteins of C. congregata polydnavirus.