Toshihiko Iizuka

Analysis of genetic information of an insect picorna-like virus, infectious flacherie virus of silkworm: evidence for evolutionary relationships among insect, mammalian and plant picorna(-like) viruses*

SummaryWe synthesized the cDNAs of an insect picornavirus, infectious flacherie virus of silkworm (IFV), genomic RNA and inserted it into a bacterial plasmid (pUC119). The 9 650 nucleotides (nts) sequence except for the poly(A) tail was obtained from the cloned cDNAs, and the sequence integrity was confirmed by primer extension and direct RNA sequencing. The sequence has a large open reading frame (ORF) of 9 255 nts (3 085 codons) flanked by the short 5′ non-coding region (156 nts) and by the rather long 3′ non-coding (239 nts). The structural proteins VP3, 4, 1 and 2 were located at the N-terminus of the polyprotein in this order and were preceded by a tentative small peptide. Computer analysis identified the sequences similar to the consensus sequences of 2C (helicase?), 3C (protease), and 3D (RNA polymerase) conserved among mammalian and plant picorna(-like) viruses. In addition, the predicted genome organization of IFV was quite similar to those of picornaviruses. Further analyses of the characteristics of the genome structure and a tentative phylogenetic tree constructed on the basis of the amino acid sequence similarity emphasized the evolutionary relationships among the insect and plant viruses.

Two types of entomocidal toxins in the parasporal crystals of Bacillus thuringiensis kurstaki

Two types of entomocidal proteins of Bacillus thuringiensis kurstaki were isolated from the parasporal bodies (crystals), and their structures were compared with each other in relation to the toxic activity. When the crystals were dissociated in 2% 2-mercaptoethanol at pH 10, a protein of Mr = 135,000, called delta-endotoxin, was liberated. The crystals of a strain of B. thuringiensis kurstaki, the HD-1 strain, also released another protein in small quantities. This minor component of HD-1, which had been discovered and named mosquito factor by Yamamoto and McLaughlin (T. Yamamoto and R. E. McLaughlin (1981) Biochem. Biophys. Res. Commun. 103, 414-421) because of its toxicity to mosquito larvae, could be liberated selectively from the crystals by alkali treatment without any thiol reagent at pH 11. Electron microscopic observation suggested that the bipyramidal crystal is composed of a homogeneous component, presumably the delta-endotoxin, and the mosquito factor is not within the crystal matrix. The liberated toxins, including the mosquito factor, were purified by Sephacryl S-300 column chromatography and activated by proteinases obtained from gut juice of the cabbage looper (Trichoplusia ni). The activated toxins were characterized by peptide mapping using techniques of HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide mapping revealed that the mosquito factor is a protein distinctly different from the delta-endotoxin. Furthermore, a comparison between two strains of B. thuringiensis kurstaki indicated that minor differences in the structure of the delta-endotoxins, in particular the differences in their proteinase-resistant region, caused significant variations in their toxicity to susceptible insects.

A strain of Bacillus thuringiensis subsp. galleriae containing a novel cry8 gene highly toxic to Anomala cuprea (Coleoptera: Scarabaeidae)

Abstract A strain of Bacillus thuringiensis ( Bt ) subsp. galleriae highly toxic to the cupreous chafer, Anomala cuprea , was isolated from a soil sample collected in Japan. The strain, SDS-502, produces a crystal consisting of a 130-kDa protein. The gene encoding the protein was cloned in Escherichia coli using antiserum directed to the protein. The gene was expressed in E. coli , producing a 130-kDa protein toxic to A. cuprea . Sequencing of the cloned gene indicated a typical Bt cry gene with substantial homology to cry8 genes. Based on the peptide sequence comparison, the gene found in SDS-502 was designated as cry8Da (AB089299). The cry8Da gene was highly expressed in SDS-502 in a laboratory scale bioreactor. The fermentation product of SDS-502 was formulated for soil application and tested in a peanut field for chafer control along with a chemical insecticide reference, a fenthion organophosphate granular formulation. Judging from the amount of undamaged nuts harvested from plots of different treatments, plots treated with SDS-502 had significantly better insect control than the untreated plots. The chemical insecticide plots showed no significant difference in nut damage from the Bt -treated or control plots.

Analysis of the genetic information of a DNA segment of a new virus from silkworm.

SummaryIn 1983, a parvo-like virus (Yamanashi isolate) was newly isolated from silkworm. However, unlike parvovirus, two DNA molecules (VD1 and 2) were always extracted from purified virions. To investigate the structure and organization of the virus genomes, we determined the complete nucleotide sequence of VD2. The sequence consisted of 6031 nucleotides (nts) and contained a large open reading frame (ORF1) with 3513 nts. A smaller open reading frame (ORF2) with 702 nts was found in the complementary sequence. Computer analysis revealed that both ORFs did not code for the major structural proteins (VP1, 2, 3, and 4). These results suggest that VD2 has not enough information to produce progeny virions by itself. Further, the structural importance of the terminal sequence (CTS) common to both VD1 and VD2 was also predicted by a computer analysis.

Characterization of a cry2A Gene Cloned from an Isolate of Bacillus thuringiensis serovar sotto

Abstract. A cry2A-type gene, designated as cry2(SKW), was cloned from Bacillus thuringiensis serovar sotto SKW01-10.2-06, and some unique features of the gene were revealed. The cry2(SKW) gene encoded a polypeptide of 635 residues with a predicted molecular mass of 71,137 Da. Cry2(SKW) had 95.4% identity with Cry2Aa in amino acid sequence and was two residues longer than Cry2Aa. Two open reading frames (ORFs), designated as orf1 and orf2, were present upstream of the cry2(SKW) and showed high homology with the corresponding ORFs in the cry2Aa operon. The Orf2 from SKW01-10.2-06 contained a region of repeated sequences. However, unlike Cry2Aa, Cry2(SKW) formed the cuboidal crystalline inclusions when the cry2(SKW) gene was expressed in an acrystalliferous B. thuringiensis strain in the absence of the upstream ORFs. Furthermore, Cry2(SKW) was less toxic to a lepidopteran species, Bombyx mori, than Cry2Aa in spite of high homology between the two proteins.

Analysis of proteins encoded in the bipartite genome of a new type of parvo-like virus isolated from silkworm — structural protein with DNA polymerase motif

Bombyx mori densonucleosis virus type 2 (BmDNV-2) is a small, spherical virus containing two complementary single-stranded linear DNA molecules (VD1, VD2). BmDNV-2 is a new type of virus with a unique, yet unspecified replication mechanism which is different from that of parvoviruses (Bando, H., Choi, H., Ito, Y., Nakagaki, M. , Kawase, S., 1992. Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence, Arch. Virol. 124, 187-193; Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M. , Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Hayakawa, T., Asano, S., Sahara, K., Iizuka, T., Bando, H., 1997. Detection of replicative intermediate with closed terminus of Bombyx densonucleosis virus. Arch. Virol. 142, 1-7). Recent analyses on the genomic information of BmDNV-2 identified open reading frames which code for three tentative nonstructural proteins and four (VP1 to 4) of the six known structural proteins (Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M., Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Nakagaki et al., in preparation). In this report we demonstrate that the two largest ORFs, VD1-ORF1 and VD2-ORF1, code for the two remaining structural proteins. In addition, computer-assisted analysis revealed that the structural protein encoded in VD1-ORF1 contains sequences conserved among various DNA polymerases, and showed an evolutionary relationship with the DNA polymerases involved in protein-primed replication.

Restriction endonuclease analysis and mapping of the genomes of granulosis viruses isolated from Xestia c-nigrum and five other noctuid species

Restriction endonuclease analysis was performed on the genomic DNA of granulosis viruses isolated from noctuid species of six genera: Xestia c-nigrum, Autographa gamma, Hydraecia amurensis, Celaena leucostigma, Aletia pallens and Pseudaletia separata. All of the isolates gave very similar restriction endonuclease profiles with only minor variations. An isolate obtained from X. c-nigrum was chosen as the reference genotype, and a genomic library was constructed for this isolate using plasmid vectors. The genome was mapped using EcoRI, BamHI and BglII, and Southern hybridization; the size of the genome was estimated to be 179 kbp. Hybridization of labelled clones to fragments of other isolates revealed that genotypic variation among isolates resulted from changes in restriction sites, and from deletion or insertion of DNA. Comparative restriction mapping revealed that all of the isolates were variants of one virus, even though they originated from different host species.

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis ☆

Three crystalliferous (Cry+) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry-) mutants, either induced or spontaneously derived from a single Cry+ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry+ and Cry- variants were analyzed by both a cleared lysate- and a modified Eckhardt lysate-electrophoresis technique. All of the Cry- mutants derived from the Cry+ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry- variants. All three Cry+ strains, including the parent of the Cry- strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry+ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry- derivatives differed in other respects from the pattern for their parent strain. The various Cry+ and Cry- strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.

Insecticidal Activity of The Protein Encoded by The cryV Gene of Bacillus thuringiensis Kurstaki INA-02

Abstract. A new host specificity was discovered with the insecticidal protein encoded by the cryV gene. The cryV gene was cloned from the Bacillus thuringiensis kurstaki INA-02 strain, which was selected among a number of B. thuringiensis isolates because of its high activity against Spodoptera litura. Analyses by polymerase chain reaction (PCR) revealed that INA-02 contained the cryIA(a) and cryV genes. Since no Spodoptera activity was observed with B. thuringiensis sotto, which contained only cryIA(a), insecticidal activity of the protein encoded by the cryV gene was investigated with several insect species including S. litura. For bioassay, the cryV gene was highly expressed in an acrystalliferous B. thuringiensis strain, BT51. The CryV protein from BT51 was assayed against larvae of three lepidopteran species, Bombyx mori, S. litura, and Plutella xylostella. The protein was highly active against S. litura and P. xylostella, suggestive that the protein contributes to the unique activity of INA-02.

Detection of replicative intermediate with closed terminus of Bombyx densonucleosis virus

SummaryTwo kinds of Bombyx densonucleosis virus (BmDNV), BmDNV-1 and 2, have been isolated from sericultural farms in Japan or China. These viruses are classified into the family Parvoviridae because of the small spherical virus particle containing a single-stranded linear DNA genome. Recent studies on the genome structure of these viruses suggested that BmDNV-2 was a new type of virus with unique replication mechanism, though that of BmDNV-1 was similar to parvoviruses. However, details about the replication mechanism of BmDNVs have not been reported so far. Here, in order to elucidate the difference on replication mechanism between BmDNVs and parvoviruses, we analyzed the structure of the replicative intermediate (RI) of BmDNV DNAs by CR using specific primers designed for detection of RI with closed terminal structure (RI-CT) which is expected to be formed by replication with self-priming mechanism. PCR using the DNA from the cells infected with BmDNV-1 could detect the expected DNA fragment, showing the existence of RI-CT. On the other hand, no fragment could be amplified from the virion DNA of the BmDNVs and the DNA extracted from BmDNV-2-infected cells, respectively. These observations strongly suggested that the BmDNV-1 replicates with the “self-priming and hairpin-transfer” mechanism similar to the human parvoviruses, while BmDNV-2 does not.