Identification of the 30K protein of TMV by immunoprecipitation with antibodies directed against a synthetic peptide

Ooshika, Ikuo; Watanabe, Yoshihisa; Meshi, Tetsuo; Okada, Yoshimi; Igano, Ken'ichi; Inouye, Ken; Yoshida, Nobuo

doi:10.1016/0042-6822(84)90092-8

Cited by 34 publications

(5 citation statements)

References 37 publications

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“…This increase suggests that there are restrictions on sgRNA promoter sequences used for other functions (183-kDa coding sequence) besides transcription, because three of these substitutions led to alteration of the 183-kDa amino acid sequence. Alternatively, the MP sgRNA promoter might be negatively regulated, which is consistent with extremely low amounts of MP sgRNA relative to genomic RNA and CP sgRNA (Ooshika et al, 1984;Moser et al, 1988).…”

Section: Discussionmentioning

confidence: 67%

“…Although the MP and CP are expressed through sgRNAs, their expression patterns are markedly different. MP is produced maximally early in the infection (Watanabe et al, 1984;Lehto et al, 1990) and in low amounts (Ooshika et al, 1984), whereas CP is expressed late, reaching an extremely high level (Siegel et al, 1978). The time course of MP and CP expression is correlated with synthesis of the corresponding sgRNAs, suggesting transcriptional regulation (Ogawa and Sakai, 1984;Watanabe et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

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Mapping of the Tobacco Mosaic Virus Movement Protein and Coat Protein Subgenomic RNA Promoters in Vivo

et al. 2000

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The Tobacco mosaic virus movement protein (MP) and coat protein (CP) are expressed from 3'-coterminal subgenomic RNAs (sgRNAs). The transcription start site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T. Meshi, and Y. Okada (1984), FEBS Lett. 173, 247-250) and 4828 (K. Lehto, G. L. Grantham, and W. O. Dawson (1990), Virology 174, 145-157) for the TMV OM and U1 strains, respectively, has been reexamined and mapped to position 4838 for strain U1. Sequences of the MP and CP sgRNA promoters were delineated by deletion analysis. The boundaries for minimal and full MP sgRNA promoter activity were localized between -35 and +10 and -95 and +40, respectively, relative to the transcription start site. The minimal CP sgRNA promoter was mapped between -69 and +12, whereas the boundaries of the fully active promoter were between -157 and +54. Computer analysis predicted two stem-loop structures (SL1 and SL2) upstream of the MP sgRNA transcription start site. Deletion analysis and site-directed mutagenesis suggested that SL1 secondary structure, but not its sequence, was required for MP sgRNA promoter activity, whereas a 39-nt deletion removing most of the SL2 region increased MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem-loop structure. Deletion analysis suggested that the upper part of this stem-loop, located upstream of the transcription start site, was essential for transcription and that the lower part of the stem had an enhancing role.

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Section: Discussionmentioning

confidence: 67%

Section: Introductionmentioning

confidence: 99%

Mapping of the Tobacco Mosaic Virus Movement Protein and Coat Protein Subgenomic RNA Promoters in Vivo

et al. 2000

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“…The latter is synthesized by read-through over an amber termination codon of the 130K protein gene (5), incorporating tyrosine (6). The non-structural proteins are actually synthesized in vivo (7,8,9) but their functions are not yet fully resolved at the molecular level.…”

Section: Introductionmentioning

confidence: 99%

In vitromutagenesis of the putative replicase genes of tobacco mosaic virus

Ishikawa¹,

et al. 1986

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We have established an in vitro transcription system to produce infectious tobacco mosaic virus (TMV) RNA from a cloned cDNA copy. Using this system, several TMV mutants were transcribed in vitro from cDNA clones mutagenized at or near the leaky amber termination codon of the 130K protein gene, and their infectivity was assayed on tobacco plants.Three (two frame-shift and one non-sense) mutants with an intact 130K but a defective 180K protein gene were not infectious, while two mutants with a one-amino-acid insertion in the 180K protein gene were infectious. When the amber codon of the 130K protein gene was deleted, infectivity was lost. However, when the amber termination codon was replaced with ochre or tyrosine codon, infectivity was retained. Sequence analyses revealed that introduced mutations were retained in progeny viral sequences except in the progeny of the amber-to-tyrosine mutant, which was a mixture of the parental mutagenized virus and a pseudo-revertant with ochre codon.

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“…This approach is similar to that used by Sutcliffe et al (20) for the putative protein R of Moloney leukemia virus. Very recently Ooshika et al (35) showed that the tobacco mosaic virus protein of molecular weight 30,000, which had been known from in vitro experiments for some years, could be found in infected cells by using an approach similar to that described here. This technique also could be applied to the geminiviruses whose genomes have been sequenced (36,37) and PV-B (Fig.…”

Section: Discussionmentioning

confidence: 99%

Detection in vivo of a new gene product (gene III ) of cauliflower mosaic virus

Xiong

Lebeurier

Hirth

1984

Proc. Natl. Acad. Sci. U.S.A.

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Cauliflower mosaic virus DNA contains six mauor open reading frames (ORFs). As only the mRNA corresponding to the transcription of gene VI and its translation product have been isolated, the identification in infected plants of products corresponding to the five other putative genes remains to be established. The present paper reports the detection of an ORF III product by means of antibodies raised against an NH2-terminal synthetic peptide of 19 amino acids corresponding to a sequence predicted from the nucleotide sequence of ORF HI. The detection of this gene product raises the question of the mechanism of its expression.Cauliflower mosaic virus (CaMV) contains circular relaxed double-stranded DNA as genetic material (for review, see refs. 1-3). Determination of the CaMV DNA sequence (4-6) revealed the presence of six large (I-VI; putative genes I-VI) and two small (VII and VIII; putative genes VII and VIII) open reading frames (ORFs). The functions of the ORF II (7-10) and the ORF IV (4, 11) have been deduced, but no direct demonstration of such functions has been given. Several RNA species (35S, 19S, and 8S) transcribed from the viral DNA have been detected in extracts from virus-infected turnip leaves (12)(13)(14). In an in vitro system only the 19S mRNA, transcript of the ORF VI, was translated to give a P66 protein, which corresponded to the main inclusion body protein (15)(16)(17). In 1980, Lebeurier et al. (18) demonstrated that the presence of the ORF III was necessary for viral multiplication, but up to the present time no mRNA corresponding to ORF III has been isolated and, thus, the presence of the corresponding protein in infected plants has remained speculative. However, since the DNA nucleotide sequence for gene III is known, it has been possible to use an alternative approach to detect the gene product. Synthetic peptides with the appropriate sequence can be used to elicit the formation of antibodies which also will react specifically with the native protein (19)(20)(21).In the work described here, we used this strategy to detect the protein product of ORF III in infected tissue. As far as we are aware, identification of an unknown gene product in plants has not been reported previously. Protein Blotting Procedure. Protein blotting was performed by a modification of the method described by Towbin et al. (27). Briefly, the semipurified inclusion bodies or nuclei were treated by boiling for 2 min in 62.5 mM Tris HCl, pH 6.8/2% NaDodSO4/2% 2 mercaptoethanol/10% glycerol, subjected to electrophoresis in a NaDodSO4/12.6% polyacrylamide slab gel, and then electrophoretically transferred to nitrocellulose sheets (0.45-,um pore size; Schleicher & Schull BA-85) prewetted with 150 mM NaCl/10 mM Tris HCl, pH 7.4 (NaCl/Tris). A voltage gradient of 7 V/cm was applied for 1.5 hr. The blot was either stained with amido black or used directly for immunological testing. MATERIALS AND METHODSPeptide Synthesis. Solid-phase peptide synthesis was carried out by the method of Merrifield (28,29).Purificati...

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Identification of the 30K protein of TMV by immunoprecipitation with antibodies directed against a synthetic peptide

Cited by 34 publications

References 37 publications

Mapping of the Tobacco Mosaic Virus Movement Protein and Coat Protein Subgenomic RNA Promoters in Vivo

Mapping of the Tobacco Mosaic Virus Movement Protein and Coat Protein Subgenomic RNA Promoters in Vivo

In vitromutagenesis of the putative replicase genes of tobacco mosaic virus

Detection in vivo of a new gene product (gene III ) of cauliflower mosaic virus

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