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1.
Insect Mol Biol ; 11(6): 577-84, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12421415

ABSTRACT

Wolbachia is an intracellular symbiont that causes reproductive disorders in many insects. Its presence in the leafhoppers Hishimonoides sellatiformis and Hishimonus sellatus, vectors of mulberry dwarf-Phytoplasma, was confirmed by the PCR analysis of 16S rDNA, ftsZ and wsp. Sequencing of cloned PCR products revealed that two Wolbachia strains coexist in both leafhoppers. The phylogenetic analysis of wsp revealed that these strains belong in novel positions in the B-group of Wolbachia. These strains were detected by PCR and/or PCR-RFLP in all of the tested non-genital organs including salivary glands, as well as in the tested genital organs of Hishimonoides sellatiformis. In addition, Wolbachia-like organisms were observed by electron microscopy in all PCR-positive organs. We discuss the possible horizontal transmission of Wolbachia via mulberry trees.


Subject(s)
Insecta/microbiology , Wolbachia/classification , Animals , DNA Primers , DNA, Ribosomal/genetics , Female , Fruit/parasitology , Ovary/microbiology , Ovum/microbiology , Phylogeny , Polymerase Chain Reaction , RNA, Ribosomal, 16S/genetics , Salivary Glands/microbiology , Trees/parasitology , Wolbachia/genetics , Wolbachia/isolation & purification
3.
J Gen Virol ; 82(Pt 2): 307-312, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11161267

ABSTRACT

Non-occluded viruses (NOVs) of Bombyx mori nucleopolyhedrovirus (BmNPV) are poorly infectious to silkworm larvae when administered by peroral inoculation, although they are highly infectious when injected into the insect haemocoel. In the present study, it is demonstrated that NOVs of BmNPV became highly infectious even through peroral inoculation when administered with spindles (proteinaceous structures) of Anomala cuprea entomopoxvirus (AcEPV). Marked enhancement of peroral infectivity of NOVs by AcEPV spindles (nearly 1000-fold higher in the strongest case) was observed in all growth stages of silkworm larvae tested (2nd to 5th instar). Similarly, peroral infectivity of polyhedrin-negative recombinants of BmNPV, which do not produce polyhedra, was also enhanced remarkably by AcEPV spindles. In contrast, spheroids (proteinaceous structures containing AcEPV virions) did not enhance the peroral infectivity of either NOVs or the recombinant BmNPV in silkworm larvae.


Subject(s)
Baculoviridae/physiology , Bombyx/virology , Entomopoxvirinae/chemistry , Larva/virology , Nucleopolyhedroviruses/physiology , Viral Proteins/physiology , Animals , Baculoviridae/genetics , Bombyx/growth & development , Entomopoxvirinae/physiology , Gene Deletion , Larva/growth & development , Lethal Dose 50 , Occlusion Body Matrix Proteins , Viral Proteins/genetics , Viral Structural Proteins
4.
Arch Virol ; 145(7): 1465-71, 2000.
Article in English | MEDLINE | ID: mdl-10963350

ABSTRACT

The biological function of entomopoxvirus (EPV) spindles (inclusion bodies that lack virions), has not been elucidated. We characterized the function of EPV spindles in the cupreous chafer, Anomala cuprea (Coleoptera: Scarabaeidae). Spheroids or spheroids mixed with spindles of Anomala cuprea EPV were administered per os to the A. cuprea larvae. A significant increase in infectivity was induced by the addition of spindles to the spheroids, strongly suggesting that the spindles play an important role in the infection of the host insect.


Subject(s)
Coleoptera/virology , Entomopoxvirinae/physiology , Entomopoxvirinae/ultrastructure , Inclusion Bodies, Viral/physiology , Animals , Inclusion Bodies, Viral/ultrastructure , Virulence
5.
Biosci Biotechnol Biochem ; 64(3): 660-4, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10803977

ABSTRACT

We report here the nucleotide sequence of a full-length cDNA encoding ent-kaurene synthase that was isolated by a reverse-transcription polymerase chain reaction from Gibberella fujikuroi (Gcps/ks). This cDNA encodes 952 amino acid residues with a relative molecular mass of 107 kDa. The sequence similarity between Gcps/ks and ent-kaurene synthase of the gibberellin A1-producing fungus, Phaeosphaeria sp. L487, is very high, suggesting that Gcps/ks is also a bifunctional diterpene cyclase. Its recombinant protein expressed in Escherichia coli converted geranylgeranyl diphosphate to copalyl diphosphate and ent-kaurene.


Subject(s)
Alkyl and Aryl Transferases/genetics , Gibberella/enzymology , Alkyl and Aryl Transferases/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Complementary , Molecular Sequence Data , Molecular Structure , Sequence Homology, Amino Acid
6.
Phytopathology ; 90(8): 909-14, 2000 Aug.
Article in English | MEDLINE | ID: mdl-18944513

ABSTRACT

ABSTRACT The presence of mulberry dwarf (MD) phytoplasmas in organs of the inoculative vector insects Hishimonoides sellatiformis and Hishimonus sellatus was determined by means of electron microscopy (EM) and polymerase chain reaction (PCR) assays. Many MD phytoplasmas were detected in genital organs as well as in the intestines, salivary glands, brains, fat bodies, and thoracic ganglia of Hishimonoides sellatiformis, but only in the intestine and salivary glands of Hishimonus sellatus. Many phytoplasmas with characteristic morphology were observed via EM in ovaries, seminal receptacles, and testes, and they were further identified by PCR assays with group I-specific primers. In addition, the organisms were detected by direct or nested PCR assays in eggs (head pigmentation stage of embryos) laid on mulberry shoots by inoculative leafhoppers and in the newly hatched nymphs from these eggs. These findings indicate that transovarial transmission of MD phytoplasmas occurs in Hishimonoides sellatiformis.

7.
Plant Physiol ; 118(4): 1517-23, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9847128

ABSTRACT

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA1, the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3beta-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3beta-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3beta-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action.


Subject(s)
Gibberellins/biosynthesis , Lactuca/drug effects , Lactuca/metabolism , Phytochrome/pharmacology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , DNA, Plant/genetics , Gene Expression Regulation, Developmental/radiation effects , Gene Expression Regulation, Plant/radiation effects , Genes, Plant , Lactuca/genetics , Light , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Models, Biological , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Seeds/drug effects , Seeds/growth & development , Seeds/metabolism , Sequence Homology, Amino Acid
8.
Virus Res ; 55(1): 61-9, 1998 May.
Article in English | MEDLINE | ID: mdl-9712512

ABSTRACT

The complete nucleotide sequence of the spheroidin gene of Anomala cuprea entomopoxvirus (AcEPV) was determined. The sequence was compared with those of spheroidin genes of EPVs such as Melolontha melolontha EPV (MmEPV, Genus A), Choristoneura fumiferana EPV (CfEPV, Genus B) and Amsacta moorei EPV (AmEPV, Genus B). The gene harbored an open reading frame (ORF) of 2826 nt, with the same size as that of MmEPV belonging to the same genus, capable of coding for a polypeptide of 109.0 kDa. The predicted amino acid (aa) sequence showed a greater or moderate similarity to the corresponding sequence of the other EPVs, showing a 94, 40 and 41% aa identity with MmEPV, CfEPV and AmEPV, respectively; the identity was 89, 53 and 54% at the nucleotide level. The hydropathy plots also showed a greater similarity in organization to MmEPV and moderate similarity to the viruses of Genus B. In the polypeptide, 44 cysteine residues, which are likely to be involved in paracrystal formation and seven potential N-glycosylation sites were detected. The number of cysteine residues and N-glycosylation sites also depended on the difference in genera (A or B). Thus, the spheroidin gene of EPVs was well conserved within the same genus.


Subject(s)
Entomopoxvirinae/genetics , Viral Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cysteine , Glycosylation , Inclusion Bodies , Molecular Sequence Data , Molecular Weight , Open Reading Frames/genetics , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Viral Structural Proteins
10.
Insect Biochem Mol Biol ; 27(10): 869-76, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9474783

ABSTRACT

The fusolin gene of Anomala cuprea entomopoxvirus (AcEPV) was cloned and sequenced. The sequence was compared with that of fusolin genes of EPVs from other insects such as Melolontha melolontha (MmEPV), Choristoneura biennis (CbEPV) and Heliothis armigera (HaEPV) previously reported. The gene harbored a single open reading frame (ORF) of 1119 nt capable of coding for a protein of 43.3 kDa. The microsequencing of the protein showed the cleavage of a signal peptide of 16 amino acid (aa) residues. The predicted aa sequence revealed significant homologies with the corresponding sequence in other EPVs, showing a 53, 51 and 52% aa-identity with MmEPV, CbEPV and HaEPV, respectively. In the ORF, five 'conserved regions' described by Vialard et al. (1990, Journal of Virology 64, 5804-5811) like other EPVs were detected. Eleven cysteine residues, presumably involved in paracrystal formation, were also detected. On the other hand, AcEPV, belonging to Genus A, harbored a potential N-glycosylation site in the ORF like CbEPV and HaEPV belonging to Genus B which was not, however, detected in MmEPV belonging to the same Genus as AcEPV. Furthermore, the last 330 bp region of the ORF revealed a low homology with that of MmEPV.


Subject(s)
Coleoptera/virology , Entomopoxvirinae/genetics , Genes, Viral , Viral Proteins/genetics , Animals , Base Sequence , Cloning, Molecular , Molecular Sequence Data , Sequence Homology, Amino Acid
11.
Plant Physiol ; 104(2): 401-407, 1994 Feb.
Article in English | MEDLINE | ID: mdl-12232091

ABSTRACT

An activity stain was used after native polyacrylamide gel electrophoresis, and at least 17 different endopeptidase activities were detected in maize (Zea mays L.) endosperm extracts prepared during the first 6 d after imbibition. The enzymes detected were classified into four groups based on their time of appearance and on their mobility in polyacrylamide gels. The first group, which included two enzymes present in dry endosperms, disappeared soon after imbibition. The second group, comprising five activity bands, appeared during the first 2 to 3 d after imbibition and then disappeared. The third set of enzymes increased continuously throughout the experimental period. The fourth group appeared after d 3 and remained at a constant level after that time. The endopeptidase activities were characterized by the effect of specific inhibitors on their activities. The two enzymes of the first group are metalloendopeptidases based on their sensitivity to ethylenediaminetetracetate (EDTA). Enzymes of the second, third, and fourth groups are sulfhydryl-endopeptidases as judged by their sensitivity to antipain, chymostatin, leupeptin, and E-64 and by their requirement for 2-mercaptoethanol. Pepstatin, phenylmethylsulfonyl fluoride, or EDTA had no effect on these enzymes. Many of the second, third, and fourth group enzymes cleaved [alpha]-zein-rich proteins as well as such easily obtained proteins as gelatin (used in our standard assay) and hemoglobin. The second group had a high affinity for [gamma]-zein, whereas none of the bands in the fourth group of enzymes cleaved this type of zein. The two metalloenzymes of the first group cleaved neither [alpha]- nor [gamma]-zeins.

12.
Plant Physiol ; 100(4): 2100-5, 1992 Dec.
Article in English | MEDLINE | ID: mdl-16653246

ABSTRACT

The catabolism of nuclear-encoded stromal proteins was investigated in intact chloroplasts isolated mechanically from pea (Pisum sativum) leaves. Glutamine synthetase, phosphoribulokinase, and nitrite reductase (quantified by immunoblotting) were more rapidly degraded in the light than in the dark. Furthermore, the degradation rates depended on exogenously supplied metabolites. For example, 2-oxoglutarate accelerated the catabolism of all three enzymes in chloroplasts incubated in the light, whereas oxaloacetate stabilized glutamine synthetase and at the same time destabilized the other two enzymes.

14.
Plant Physiol ; 89(1): 274-9, 1989 Jan.
Article in English | MEDLINE | ID: mdl-16666526

ABSTRACT

A sulfhydryl-endopeptidase was purified as a 33 kilodalton (kD) mass polypeptide from cotyledons of Vigna mungo seedlings. Immunoblot analysis with antiserum made against the purified enzyme showed that the sulfhydryl-endopeptidase was synthesized only in the cotyledons during germination and that the amount of the enzyme increased until 4 days after imbibition and decreased thereafter. Next, an RNA fraction was prepared from cotyledons of 3 day old seedlings and translated in a wheat germ system. The synthesis of a 45 kD polypeptide was shown by the analysis of its translation products by immunoprecipitation with the antiserum to the endopeptidase and gel electrophoresis. When the RNA fraction was translated in the presence of canine microsomal membranes, a smaller polypeptide, having a 43 kD molecular mass, was detected as the translation product. When membrane-bound polysomes, but not free polysomes, prepared from cotyledons were used for translation in the wheat germ system, both the 43 and 45 kD polypeptides were synthesized. By incubation of a crude enzyme extract from cotyledons at 5 +/- 1 degrees C at neutral pH, the 43 kD polypeptide was sequentially cleaved to the 33 kD polypeptide via 39 and 36 kD intermediate polypeptides. The endopeptidase was activated simultaneously with the processing. Two-dimensional polyacrylamide gel electrophoresis showed that the 33 kD polypeptide was the fully activated form of the enzyme, whereas little or no activity was detected in other forms. From the present results, we postulate that the sulfhydryl-endopeptidase is first synthesized as the 45 kD precursor with a 2 kD signal peptide being cleaved, and that the 43 kD polypeptide is further cleaved to give the 33kD mature enzyme.

15.
Plant Physiol ; 80(3): 628-34, 1986 Mar.
Article in English | MEDLINE | ID: mdl-16664675

ABSTRACT

Two major endopeptidases were present in cotyledons of germinating Vigna mungo seeds, as detected by the zymogram after polyacrylamide gel electrophoresis. They were not detectable in cotyledons of dry seeds, but their intensities on the zymogram increased during germination. During incubation of detached cotyledons, however, the activities showed only a slight increase for 5 days. These two endopeptidases could be separated by Sephacryl S-200 column chromatography. One of them was found to be a serine-endopeptidase as judged by phenylmethylsulfonylfluoride and diisopropyl fluorophosphate inhibition. The other was a sulfhydryl-endopeptidase because of its dependency on 2-mercaptoethanol and inhibition by leupeptin, chymostatin, and antipain. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis indicatd that the two endopeptidases digested the Vigna mungo seed globulin subunits at different rates. The serine enzyme digested the 56 kilodalton subunit at first, but the sulfhydryl enzyme digested the 54 kilodalton peptide more efficiently than the 56 kilodalton peptide. The pattern of digestion of globulin by the combination of the serine- and sulfhydryl-endopeptidases was similar to that using crude enzyme extracts.

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