RESUMO
Missing contact footprinting with formic acid as a modifying reagent was used to examine specific IE-1 binding contacts to double-stranded oligonucleotides that contained either a consensus hr repeat sequence or a sequence from the pe38 promoter, which is down regulated by IE-1. The hr repeat sequences contain two consensus IE-1 binding motifs (IBMs) flanking a central EcoRI site that are oriented in opposite directions with respect to each other. IE-1 was found to contact regions including both IBMs. The bases footprinted in the top strand included the left IBM (IBM-A), whereas bases in the bottom strand were footprinted in a region that included IBM-B and part of IBM-A. When substitution mutations were introduced into either IBM, bases on both strands of the remaining IBM were strongly footprinted. As with the hr IBM-mutant constructs, bases footprinted in the pe38 promoter construct included both strands of the single IBM.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Imediatamente Precoces/genética , Nucleopoliedrovírus/genética , Transativadores/genética , Animais , Sequência de Bases , Sítios de Ligação , Pegada de DNA , DNA Viral , Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Dados de Sequência Molecular , Mariposas/virologia , Mutagênese , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Transativadores/metabolismoRESUMO
LEF-3 and P143 are two of six proteins encoded by the Autographa californica multinucleocapsid nucleopolyhedrovirus genome which are required for DNA replication in transient replication assays. LEF-3 has the properties of an ssDNA-binding protein and P143 exhibits amino acid sequence homology to helicases. In this report, the interaction of LEF-3 and P143 was studied by yeast two-hybrid and immunoaffinity analyses. Using the yeast two-hybrid system, the interaction domain of LEF-3 (385 aa) was mapped to amino acids between positions 1 and 165. Deletion analysis of P143 failed to reveal an interaction domain, suggesting that there were either multiple interaction domains or that the deletions disrupted the secondary structures required for the interaction between LEF-3 and P143.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Nucleopoliedrovírus/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Sequência de Bases , DNA Helicases/química , DNA Helicases/genética , Primers do DNA/genética , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/genética , Nucleopoliedrovírus/genética , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Deleção de Sequência , Transativadores/química , Transativadores/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-1) is thought to play a central role in stimulating early viral transcription. IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulate the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2. Our results indicate that IE-1 negatively regulates the expression of certain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') near their mRNA start sites. The IE-1 binding motif was also found within the palindromic sequences of AcMNPV homologous repeat (hr) regions that have been shown to bind IE-1. The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by introducing mutations in these promoters in which the central 6 bp were replaced with BglII sites. GUS reporter constructs containing ie-2 and pe-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containing plasmid (pIe-1). Comparisons of GUS expression produced by the mutant and wild-type constructs demonstrated that the IE-1 binding motif mediated a significant decrease in expression from the ie-2 and pe-38 promoters in response to increasing pIe-1 concentrations. Electrophoretic mobility shift assays with pIe-1-transfected cell extracts and supershift assays with IE-1-specific antiserum demonstrated that IE-1 binds to promoter fragments containing the IE-1 binding motif but does not bind to promoter fragments lacking this motif.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Viral da Expressão Gênica , Proteínas Imediatamente Precoces/genética , Nucleopoliedrovírus/genética , Regiões Promotoras Genéticas , Transativadores/genética , Proteínas Virais , Animais , Sítios de Ligação , Linhagem Celular , Proteínas Imediatamente Precoces/metabolismo , Mariposas/virologia , Mutação , Spodoptera/citologia , Transativadores/metabolismo , TransfecçãoRESUMO
The Autographa californica multinucleocapsid nuclear polyhedrosis virus has six genes required and three genes stimulatory for transient DNA replication. We demonstrate that the products of two of these genes, LEF-1 and LEF-2, interact in both two-hybrid assays using Saccharomyces cerevisiae and glutathione S-transferase fusion affinity assays. Using yeast-two-hybrid assays, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Extensive deletion analyses of LEF-1 failed to reveal a delimited interaction domain, suggesting that there may be essential secondary structural elements that are inactivated by these deletions. All clones expressing LEF-1 and LEF-2 that were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction is required for DNA replication. Sequence analysis of LEF-1 revealed a primase-like motif, WVVDAD. When this motif was mutated to WVVQAD, LEF-1 no longer supported transient DNA replication.
Assuntos
Nucleopoliedrovírus/metabolismo , Proteínas Virais/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , DNA Primase , Replicação do DNA , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Spodoptera/citologia , Proteínas Virais/genéticaRESUMO
The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) has high potential for development as a bio-insecticide for control of the beet armyworm (S. exigua). It is highly infectious for S. exigua larvae and its host range is very narrow. A prerequisite for such application is the possibility of growing this virus in large quantities, e.g. in insect cell lines. It was observed, however, that polyhedra of SeMNPV plaque-purified in Se-UCR1 cells did not cause larval mortality or morbidity when fed to S. exigua larvae. As this suggested a genetic alteration in in vitro produced SeMNPV, comparative restriction analysis of in vitro and in vivo produced SeMNPV DNA was performed. The restriction patterns of viral DNA from several different plaques always differed from that of the wild-type in the same way, suggesting that a large, single deletion had occurred in the in vitro produced viral genome. In order to localize this deletion more precisely a detailed physical map of the wild-type SeMNPV genome was constructed, using the restriction endonucleases XbaI, BamHI, Bg/II, PstI, SstI, HindIII and SpeI. In addition, the entire SeMNPV genome was cloned into a library containing five overlapping cosmids and a plasmid library. About 80 restriction sites were located and the orientation of the map was set according to the location of the polyhedrin and p10 genes. The approximate size of the viral genome was 134 kbp. Based on this map it could be established that mutant SeMNPV, obtained by passage in cell culture, contained a single deletion of approximately 25 kbp between map units 12.9 and 32.3.
Assuntos
Nucleopoliedrovírus/genética , Nucleopoliedrovírus/patogenicidade , Mapeamento por Restrição , Spodoptera/virologia , Animais , Linhagem Celular , DNA Viral/análise , Deleção de Genes , Genoma Viral , Nucleopoliedrovírus/crescimento & desenvolvimento , Spodoptera/citologia , VirulênciaRESUMO
Homologous regions (hrs), which are present at eight dispersed locations on the Autographa californica multinucleocapsid nuclear polyhedrosis virus genome, are composed of repeated imperfect palindromes within directly repeated sequences. Hrs act as transcriptional enhancers of RNA polymerase II-mediated transcription and as origins of DNA replication when incorporated into plasmids and tested in transient replication assays. To characterize the physical structure of these elements and to determine the role that mismatched nucleotides may play in hr function, oligonucleotides containing a consensus mismatched "imperfect" palindrome and a closely related "perfect" palindrome were synthesized. These sequences were cloned into individual plasmids and tested for their ability to form cruciform structures using nuclease P1 assays and two-dimensional (2-D) gel analyses of topoisomers. The perfect palindrome formed a cruciform structure and the energy requirement for its formation was predicted to occur under physiological conditions. In contrast, the construct containing an imperfect palindrome did not form a cruciform under these conditions. Both hr constructs were found to bind IE-1 in electrophoresis mobility shift assays and act as enhancers when cis-linked to the baculovirus 39K early gene promoter. However, a single oligonucleotide containing the palindrome sequence did not bind IE-1 when annealed under conditions conducive to hairpin formation.
Assuntos
Conformação de Ácido Nucleico , Nucleocapsídeo/química , Nucleopoliedrovírus/genética , Animais , Sequência de Bases , Linhagem Celular , DNA Viral , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos , Proteínas Imediatamente Precoces/metabolismo , Dados de Sequência Molecular , Mariposas/virologia , Nucleocapsídeo/fisiologia , Nucleopoliedrovírus/química , Oligonucleotídeos , Conformação Proteica , Sequências Repetitivas de Ácido Nucleico , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Spodoptera/citologia , Relação Estrutura-Atividade , Transativadores/metabolismoRESUMO
Plectreurys tristis cephalothorax mRNA was isolated and amplified by PCR using degenerate primers corresponding to reverse translated mature Plt-VI toxin. An oligonucleotide corresponding to a portion of the amplified product was then used to screen a P. tristis cDNA library. The cDNAs from 10 positive clones were sequenced. Eight of these cDNAs corresponded to Plt-VI toxin, one to Plt-XI toxin, and one was very similar to Plt-VIII toxin, with the exception of a single amino acid substitution. Analysis of these cDNAs indicated that these toxins are initially synthesized as prepro-forms which undergo signal cleavage followed by additional processing at both their N- and C-termini to produce the mature products.
Assuntos
Inseticidas , Peptídeos/genética , Aranhas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Aranhas/químicaRESUMO
A physical map of Anagrapha falcifera multinucleocapsid nuclear polyhedrosis virus (AfMNPV) DNA was constructed for restriction endonucleases EcoRI, HindIII, PstI and XhoI. The genome size was estimated to be 130 kbp. Ordering of the restriction fragments was accomplished by cross-blot hybridization, double digestion and DNA-DNA hybridization. The polyhedrin gene and homologous repeat (hr) regions were located by hybridization to the Autographa californica MNPV (AcMNPV) polyhedrin gene and hr4, respectively. Restriction pattern comparison and Southern blot analysis suggest that AfMNPV is closely related to AcMNPV.
Assuntos
Nucleopoliedrovírus/genética , Capsídeo , DNA Viral/análise , Hibridização de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
Hr1a consists of two 30-bp imperfect palindrome sequences separated by 58 bp and each palindrome contains a naturally occurring EcoRI site at its core. Plasmid subclones of the hr1a-containing AcMNPV HindIII-N fragment were examined for their ability to replicate in virus-infected (Spodoptera frugiperda) Sf9 cells, and to stimulate transcription when linked in cis with a 39K gene promoter-beta-glucuronidase fusion and cotransfected into cells along with a plasmid (ple-1) containing the gene encoding the trans-acting factor IE-1. Only those plasmids containing hr1a underwent infection-dependent replication and were able to stimulate transcription. Sequences mapping to the left of hr1a were required for maximal levels of replication. A single palindrome from hr1a was sufficient for supporting plasmid replication and for stimulating transcription, although both activities were more efficient when both palindromes were present. Plasmids containing only one-half of a palindrome or disruptions of the central EcoRI core either did not replicate, or replicated very poorly, and did not exhibit enhanced transcriptional activity from the 39K gene promoter. Gel retardation experiments showed that labeled hr1a-containing DNA fragments had retarded migration after incubation with extracts from cells transfected with ple-1. Supershift experiments using polyclonal antibodies to IE-1 indicated that IE-1 is a component of the protein complex bound to hr1a. Fragments containing disruptions of the EcoRI-core still bound IE-1, as shown by gel retardation assays, indicating that IE-1 binding alone is not sufficient to allow replication and transcriptional enhancement.
Assuntos
Replicação do DNA/genética , DNA Viral/genética , Elementos Facilitadores Genéticos/genética , Nucleopoliedrovírus/fisiologia , Replicação Viral/genética , Animais , Sequência de Bases , Extratos Celulares , Linhagem Celular , DNA Viral/metabolismo , Dados de Sequência Molecular , Nucleopoliedrovírus/genética , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência/fisiologia , Spodoptera , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica/genética , TransfecçãoRESUMO
The potency of venom from Bracon hebetor against lepidopterous larvae has been known for over 40 years, but previous attempts to purify and characterize individual protein toxins have been largely unsuccessful. Three protein toxins were purified from venom of this small parasitic wasp and the amino acid sequences of 22-31 consecutive residues at the amino-terminus were determined. These relatively large toxins (apparent molecular mass 73 kDa) were labile under many isolation techniques, but anion-exchange chromatography allowed purification with retention of biological activity. Two purified toxins were quite insecticidal (LD50 < 0.3 microgram/g) when injected into six species of lepidopterous larvae. On a molar basis, one toxin (Brh-I) has the highest known biocidal activity against Heliothis virescens (LD50 = 2 pmol/g).
Assuntos
Inseticidas/isolamento & purificação , Venenos de Vespas/química , Sequência de Aminoácidos , Animais , Larva , Dados de Sequência Molecular , Mariposas , Parasitos , VespasRESUMO
The rice glutelin Gt3 promoter was fused to a beta-glucuronidase (GUS) reporter gene and its expression evaluated in transgenic tobacco plants. Histochemical analysis revealed that the expression of the introduced Gt3 promoter/GUS (beta-glucuronidase) chimeric gene was confined to endosperm tissue of developing seeds. 5'-promoter deletion analysis revealed that two domains of the Gt3 promoter, -346 to -263 bp (domain I) and -945 to -726 bp (domain II) from the transcriptional start site, were essential for optimum expression of the GUS reporter gene. Removal of 5' sequences upstream of -726 resulted in a reduction in overall promoter activity and a shift in temporal expression from a maximum of 16-20 days after flowering to 24 days. Removal of DNA sequences from the 5' end to -346 yielded a promoter fragment that was still able to confer endosperm-specific expression, although a further deletion to -263 abolished promoter activity. These data suggest that at least two cis-regulatory elements are required for endosperm specificity and temporal regulation of glutelin Gt3 gene expression.
Assuntos
Regulação da Expressão Gênica , Genes de Plantas/genética , Glutens/genética , Oryza/genética , Regiões Promotoras Genéticas/genética , Sequência de Bases , Análise Mutacional de DNA , Genes Reporter , Glucuronidase/análise , Glucuronidase/genética , Glutens/biossíntese , Histocitoquímica , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/biossíntese , Sementes/anatomia & histologia , Sementes/enzimologia , Distribuição Tecidual , Transformação GenéticaRESUMO
The replication of a series of plasmids, each containing one or more of 6 homologous regions (hrs) from the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) genome, was investigated in AcMNPV-infected Spodoptera frugiperda cells. Although a construct containing hr2 showed the most efficient replication, the sequences flanking the hr region appeared to influence the levels of replication. Plasmids with non-hr-containing AcMNPV inserts showed greatly reduced replication relative to hr-containing plasmids. A variety of features of hr-containing plasmid replication were characterized. These included the time course of plasmid replication and the influence of plasmid concentration and multiplicity of infection of the helper AcMNPV on replication efficiency. It was found that replicated plasmid DNA was detectable by 36 hr p.i. and plateaued by 60 hr p.i. Under our experimental conditions, plasmid levels of 0.5-2 micrograms/1.25 x 10(6) cells and multiplicities of infection of 0.1 to 10 produced optimal levels of plasmid replication. The transfection procedure was shown to delay viral DNA replication by about 12 hr. Experiments directed toward determining the form of the replicated hr-containing plasmid DNA were conducted using partial restriction enzyme digestion. The results indicated that the AcMNPV-replicated plasmids were composed of high molecular weight concatemers.
Assuntos
Baculoviridae/genética , Replicação do DNA , Mariposas/microbiologia , Plasmídeos/genética , Homologia de Sequência do Ácido Nucleico , Animais , Células Cultivadas , Plasmídeos/ultraestrutura , Mapeamento por Restrição , Fatores de TempoRESUMO
A chimeric gene consisting of the 5' flanking sequences of a rice glutelin gene (Gt3) linked to the chloramphenicol acetyltransferase (CAT) coding segment was introduced into tobacco via Agrobacterium tumefaciens-mediated transformation. CAT enzyme activity could be detected in extracts from seeds as early as 8 days after flowering and obtained a maximum level at 16 days after flowering, the onset of overall protein accumulation. Significant expression of CAT activity in non-seed tissues occurred in some, but not all plants, suggesting possible chromosome position effects on non-seed tissue expression. A positive correlation was observed between expression levels in seeds and gene copy numbers.
Assuntos
Genes de Plantas , Glutens/genética , Nicotiana/genética , Oryza/genética , Plantas Tóxicas , Regiões Promotoras Genéticas , Rhizobium/genética , Transformação Genética , Quimera , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Expressão Gênica , Cinética , Mapeamento por RestriçãoRESUMO
A 1.1-kb region of DNA containing the p26 gene of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was sequenced, transcriptionally mapped, and compared to the same region in the MNPV of Autographa californica (AcMNPV). The mRNA start site of the p26 gene occurs about 22 nucleotides downstream from an A/T-rich putative promoter sequence that is highly conserved between AcMNPV and OpMNPV. The p26 mRNA is transcribed through the p26 gene and coterminates with the p10 gene resulting in a mRNA containing copies of both genes. The reading frames of the OpMNPV and AcMNPV p26 genes showed 47% amino acid sequence homology which is clustered in six regions with over 65% amino acid homology. There was a distinct bias toward incorporation of G/C-rich codons in the OpMNPV p26 gene. No DNA homology was observed between the region upstream of the p26 gene in AcMNPV and OpMNPV. In AcMNPV, this region contains the homologous repeated (hr) sequence hr5. Hybridization of a plasmid containing an AcMNPV-repeated sequence (hr5) to Southern blots of the OpMNPV genome indicated that this repeated sequence is lacking in OpMNPV.
Assuntos
Genes Virais , Vírus de Insetos/genética , Sequência de Aminoácidos , Sequência de Bases , Vírus de DNA/genética , Dados de Sequência Molecular , Transcrição GênicaRESUMO
A 32P-labeled cloned DNA fragment (AcMNPV HindIII-Q) containing one of the repeated sequences and a portion of the p10 gene from Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was used to probe Southern blots containing restriction endonuclease digests of Orgyia pseudotsugata muticapsid nuclear polyhedrosis virus (OpMNPV) DNA. A single 3.6-kb fragment, OpMNPV HindIII-Q, was hybridized. The OpMNPV HindIII-Q fragment was cloned into pUC-18, mapped with restriction endonucleases, and reprobed with the AcMNPV HindIII-Q fragment. A small region of ca. 700 bp, near the left end of the cloned fragment, was cross-hybridized. DNA sequencing in this region revealed an open reading frame of 279 bp which shares detectable homology with the p10 gene of AcMNPV. The sequences downstream from the p10 gene in both viruses also contain long open reading frames which share homology. Northern blot analysis of RNA from OpMNPV infected O. leucostigma cells was used to define the temporal and spatial organization of transcripts from this region. S1 analysis of both termini of the major p10 mRNA indicates nontranslated regions of 52-53 bases at the 5' end and 175 bases at the 3' end. The 5'-mRNA start site was located within a 12-nucleotide sequence which is conserved in all late hyperexpressed baculovirus genes.
Assuntos
Genes Virais , Vírus de Insetos/genética , Lepidópteros/microbiologia , Mariposas/microbiologia , Transcrição Gênica , Animais , Sequência de Bases , DNA Viral/análise , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Two overlapping restriction fragments containing the Pieris brassicae granulosis virus (GV) granulin gene were cloned into plasmids. The regions containing the coding region and the 5' and 3' flanking regions were subcloned into M13 and sequenced. The nucleotide sequence data were compared to those for the granulin gene from the Trichoplusia ni GV and the polyhedrin gene from the Autographa californica nuclear polyhedrosis virus (AcMNPV). The amino acid sequences derived from these DNA sequences indicated that the two GV proteins are more closely related to each other (77% amino acid homology) than either is to the AcMNPV (about 53% amino acid homology for either GV). The N-terminal region shows the greatest degree of variation between these proteins. Highly conserved amino acid sequences were identified between the two GVs and were also found between NPVs. Certain of these conserved regions are shared between GVs and NPVs while others are not.
Assuntos
Borboletas/microbiologia , Genes Virais , Vírus de Insetos/genética , Lepidópteros/microbiologia , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Matriz de Corpos de Inclusão , Especificidade da Espécie , Proteínas da Matriz Viral , Proteínas Estruturais ViraisRESUMO
The genome of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata was mapped by examining overlapping HindIII fragments from cosmid clones which had been constructed from partial HindIII digests of viral DNA. Five OpMNPV cosmid clones containing fragments encompassing the entire OpMNPV genome were hydridized to blots of DNA from the multicapsid nuclear polyhedrosis virus of Autographa californica. The hybridization pattern indicated that the genomes of these viruses are similarly organized.
Assuntos
Genes Virais , Vírus de Insetos/genética , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , DNA Recombinante/metabolismo , Hibridização de Ácido Nucleico , PlasmídeosRESUMO
Polyadenylated RNA was isolated from Orgyia pseudotsugata larvae 8-10 days postinfection with the multicapsid nuclear polyhedrosis virus. This RNA was centrifuged through a sucrose gradient and fractions enriched for polyhedrin mRNA were identified by in vitro translation. Complementary DNA made to this RNA hybridized predominantly to a 5-kb fragment of XhoI-digested viral DNA. This fragment was cloned into the plasmid pACYC177 and mapped with restriction endonucleases. A SalI subclone with a 2.5-kb insert derived from the cloned XhoI fragment was found to select by hybridization only polyhedrin mRNA as determined by the size of the in vitro translation product and its precipitation by anti-polyhedrin antibodies. The orientation of the polyhedrin gene and the region of the insert encoding the N terminus of the polyhedrin protein were determined by DNA sequencing. R-Loop mapping indicated polyhedrin mRNA is 980 +/- 75 bases long and contains about 250 nucleotides not represented in the final protein. The polyhedrin gene had no observable intervening sequences.