- Volume 69, Issue 9, 1988
Volume 69, Issue 9, 1988
- Plant
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Molecular Characterization of Sorghum Chlorotic Spot Virus, a Proposed Furovirus
More LessSummaryA virus morphologically and physicochemically similar to the type member of the furovirus group, wheat soil-borne mosaic virus (WSBMV), has been isolated from Sorghum bicolor and partially characterized. The virus, sorghum chlorotic spot virus (SCSV), causes symptoms which include distinct elongated chlorotic spots and ring spots as well as yellowing on systemically infected leaves of sorghum and inbred Zea mays lines. SCSV is mechanically transmissible to and produces symptoms on the inoculated leaves of Nicotiana clevelandii, Chenopodium amaranticolor and C. quinoa. The virus is bipartite with two distinct rods, 20 nm in diameter and 260 and 140 nm in length. Virions are composed of a single 20·5K capsid protein and two non-homologous, non-polyadenylated, genomic RNAs of approx. 6·2 kb (M r 2·2 × 106) for RNA-1 and 3·5 kb (M r 1·2 × 106) for RNA-2. SCSV and WSBMV capsid proteins are serologically related as determined by Western blot and immunogold cytochemical analysis. Northern blot hybridizations indicated that there is no homology between SCSV RNA and WSBMV RNA under high stringency conditions. Unfractionated SCSV RNAs direct the synthesis of 180K, 170K, 110K, 50K, 42K, 25K and 20.5K polypeptides in vitro. The 110K, 25K and 20.5K products are immunoprecipitated by antiserum raised against SCSV capsid protein. Comparative in vitro translation analysis with WSBMV suggests that the SCSV capsid protein cistron resides on the 5′ terminus of RNA-2. A 1·8 kb cDNA clone was synthesized using SCSV RNA-2. T7 transcripts from this clone directed the synthesis of several polypeptides, none of which was immunoprecipitated by antiserum to the capsid protein. SCSV is similar to, but distinct from, WSBMV and is proposed to be a new member of the furovirus group.
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Biologically Active Transcripts of Beet Necrotic Yellow Vein Virus RNA-3 and RNA-4
More LessSummarySynthetic transcripts of beet necrotic yellow vein virus (BNYVV) RNA-3 and RNA-4 were prepared from cloned cDNA in a bacteriophage T7 in vitro run-off transcription system. The RNA-3 transcripts tested had 5′ non-viral extensions of 1, 23 or 64 nucleotides and identical 3′ non-viral extensions of 12 nucleotides. An RNA-4 transcript with a 12 nucleotide 5′ non-viral extension and a 28 nucleotide 3′ non-viral extension was also synthesized. All of the transcripts were biologically active when coinoculated to Chenopodium quinoa with BNYVV isolates deficient in RNA-3 and/or RNA-4 but the presence of a long 5′ non-viral sequence on the RNA-3 transcripts was found to diminish their specific activities considerably.
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In vitro Translation of Natural and Synthetic Beet Necrotic Yellow Vein Virus RNA-1
More LessSummaryThe in vitro translation of beet necrotic yellow vein virus RNA-1 was investigated in rabbit reticulocyte lysate and in wheatgerm extract using as messenger either RNA extracted from virions or a synthetic RNA-1 produced by in vitro transcription of full-length cDNA. In wheatgerm extract, RNA-1 directed the synthesis of approximately equivalent amounts of two long polypeptides of approximate M r 220000 (220K) and 240K. The size of the translation products of 3′-truncated RNA-1 transcripts suggested that synthesis of the 240K translation product was initiated at an AUG near the 5′ terminus, probably AUG(154), the first initiation codon in the RNA-1 sequence. The 220K polypeptide is initiated internally, at AUG(496). In rabbit reticulocyte lysate only the internal initiation codon, AUG(496), was used for initiation on full-length RNA-1 although AUG(154) was accessible on short 3′-truncated transcripts.
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Comparison of the Nucleotide Sequences of Viroid-like Satellite RNA of the Canadian and Australasian Strains of Lucerne Transient Streak Virus
More LessSummaryA complete cDNA copy of the viroid-like satellite RNA of the Canadian strain of lucerne transient streak virus (LTSV) has been cloned and the nucleotide sequence of the RNA has been determined using this and other clones. The sequence comprises 322 residues and shares 80% homology with satellite RNAs (stRNAs) of the Australasian isolates of LTSV. A proposed secondary structure for the RNA is highly base-paired and thermodynamically stable, and consists of a rod-like structure interspersed with single-stranded loops, an arrangement similar to that proposed for circular stRNA of other strains of LTSV and other sobemoviruses as well as for viroids. Whereas certain regions of the sequence are very similar or identical to those of stRNA from Australasian LTSV, including the putative self-cleavage sites on both the positive and negative sense forms of the RNA, other regions of the sequence are quite dissimilar between the Canadian and Australasian strains.
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Molecular Properties of Bari 1, a Mild Strain of Cauliflower Mosaic Virus
More LessSummaryWe studied aspects of the structure and expression of the genome of Bari 1, a mild strain of cauliflower mosaic virus. Differences were observed between gene products of Bari 1 detected in inclusion body preparations and those of the more typically severe strain, Cabb B-JI. The most striking difference was the gel mobility of the Bari 1 gene VI polypeptide (apparent M r 70K) which contrasted with that of Cabb B-JI (M r 62K). This difference was also observed between products of in vitro translation of viral mRNA suggesting that it was not due to post-translational modification. The open reading frame in the nucleotide sequence of the Bari 1 gene VI region was very similar in size to that of other CaMV strains but corresponded to an amino acid sequence with a much lower overall homology and diverged greatly in a 40 base pair sequence in the 3′ region compared to gene VI sequences of other strains. The level of the Bari 1 aphid transmission polypeptide P18, the product of gene II, was much lower than that of Cabb B-JI. Some of the possible subcellular consequences resulting from the molecular properties of Bari 1 were examined by electron microscopy. Differences were observed in the composition and intactness of Bari 1 cytoplasmic inclusion bodies compared with those of a severe strain, and the presence of nuclear inclusions.
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Ultrastructural Location of Non-structural Protein 3A of Cucumber Mosaic Virus in Infected Tissue Using Monoclonal Antibodies to a Cloned Chimeric Fusion Protein
More LessSummaryCloned DNA representing the sequence coding for the non-structural protein 3A from cucumber mosaic virus (CMV) was inserted in an expression vector containing a truncated portion of the Protein A gene from Staphylococcus aureus. Expressed fusion protein was purified from Escherichia coli cell extracts by affinity chromatography on rabbit gamma globulin-conjugated Sepharose and used as an immunogen for the production of monoclonal antibodies (MAbs). Five MAbs that reacted with pXCM3A13 fusion protein in solid phase ELISA were able to immunoprecipitate specifically protein 3A from mixtures of in vitro translation products of CMV RNA. None of these antibodies reacted with the analogous protein in translation products of brome mosaic virus RNA, but all of them reacted with the 3A protein from tomato aspermy virus. Immunogold labelling of ultrathin sections of CMV-infected tobacco tissue with MAb 3H12 demonstrated that the 3A protein accumulated within the nucleoli of these cells. This location differs from that of the analogous 3A protein of alfalfa mosaic virus which is associated with the middle lamella of the cell walls of infected tobacco cells.
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Molecular Cloning of Single-stranded RNAs of Potato Leafroll Virus and Beet Western Yellows Virus
More LessSummaryComplementary DNA libraries were synthesized using the genomic RNAs of potato leafroll virus (PLRV) and beet western yellows virus (BWYV), by random as well as oligo(dT) priming of polyadenylated RNA, and cloned into pBR322. Two restriction endonuclease maps were constructed, extending to 6·1 kbp and 5·5 kbp for the PLRV and BWYV genomes respectively. The 3′ ends of genomic RNAs were verified by sequence analysis of oligo(dT)-primed clones; 5′-terminal sequences of PLRV and BWYV were not detected among the cDNA fragments mapped. Selected clones were used to demonstrate specific virus detection in total nucleic acid preparations of PLRV- or BWYV-infected Physalis floridana Rydb. One PLRV-derived clone showed cross-hybridization with purified BWYV RNA.
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