An encapsidated,subgenomic messenger RNA encodes the coat protein of carnation mottle virus

The translation strategy of carnation mottle virus (CarMV) in vitro has been generally assumed to involve internal initiation events on full-length, genomic RNA (4.3 kb). We suggest that this is, at least in part, incorrect. Encapsidated RNA, fractionated on denaturing sucrose gradients, or total RNA from CarMV-infected leaves, fractionated under non-denaturing conditions, was translated in an mRNA-dependent rabbit reticulocyte cell-free system. Evidence for subgenomic RNAs which encode a polypeptide of M_r 38 000 was found. This product was shown to be related to authentic CarMV coat protein by partial proteolysis with -chymotrypsin and SDS/polyacrylamide-gel electrophoresis.     

Characterization of the full-length sequences of phospholipase A2 induced during flower development

The suppression subtractive hybridization (SSH) method was used to isolate developmentally regulated genes during carnation flower maturation. Carnation flower maturation-related clones obtained by the SSH were serially assigned as CFMI (carnation flower maturation-induced) clones. Northern blot analysis showed that several CFMI clones were differentially expressed during flower development. One of the clones, CFMI-3, showed similarity to various animal secretory phospholipases A2 (PLA2). Since little is known about PLA2 gene sequence in plant species, the CFMI-3 clone was selected for further characterization by sequence analysis. Full sequence analysis reveals that the CFMI-3 contains a Ca2+ binding domain, a PLA2 active site, and 12 conserved Cys residues, which is a distinct characteristic of PLA2. Amino acid sequence alignment of CFMI-3 to various putative plant PLA2 confirmed that the CFMI-3 cDNA is the full-length putative PLA2 cDNA identified in plant species.     

The elimination of four viruses from carnation and sweet william by meristem-tip culture

Carnation mottle virus (CarMV), carnation ringspot virus (CRSV), carnation vein mottle virus (CarVMV) and carnation latent virus (CarLV) were all eliminated from both carnation (Dianthus caryophyllus) and sweet william (D. barbatus) plants by meristem-tip culture. Carnation mottle virus was more readily eliminated from D. barbatus than from carnation. Carnation vein mottle and carnation latent viruses were more readily eliminated from carnation than from sweet william: they are rarely found in carnation but CarVMV is found frequently in sweet william. Carnation ringspot was eliminated equally readily from both hosts.     

Isolation of Carnation Ringspot Virus from a Canal Near a Sewage Plant: cDNA Hybridization Analysis, Serology and Cytopathology

A small water sample of only 60 ml from the Oker Aue canal near Braunschweig was ultracentrifuged. The resuspended pellet was rubbed on Chenopodium quinoa leaves which responded with the formation of almost 200 local lesions. The virus causing these lesions was identified as carnation ringspot virus (CarRSV) by means of dot blot hybridization using random-primed cDNAs to the viral nucleic acids and by means of serology. Northern blot analysis revealed that the two RNA species of the virus which consisted of c. 3.7 and 1.5 Kb, respectively, have little or no base sequence homology. In immunoelectrophoresis at pH 7.0 the virus migrated towards the cathode. The isometric particles were 33 nm in diameter, round to slightly angular in outline, and showed a distinct granular or knobly surface structure. Virus particles occurred in the cytoplasm, nuclei, and vacuoles of infected cells which in addition contained amorphous granules in the cytoplasm and/or proliferated endoplasmic reticulum. Heavily, affected cells were necrotized and contained large virus particle aggregates which sometimes were crystallized. CarRSV is the third carnation virus, after carnation mottle and carnation Italian ringspot viruses, which was identified in a natural water.     

昆明地区香石竹病毒病流行状况调查及脱病毒苗的制备

对昆明地区3种不同生产模式下的香石竹（Dianthus caryophyllus L.)进行了调查,采集样本146号,利用酶联免疫法和电镜检测法对样本感染香石竹病毒的情况进行检测,结果表明昆明地区主要流行的香石竹为香石竹斑驳病毒和香石竹坏死斑点病毒,以带香石竹斑驳病毒的香石竹品种“俏新朗”为实验材料,研究了直接剥茎尖法,高温处理结合剥茎尖法和病毒痤处理结合剥茎尖法3种方法在脱病毒效率和茎尖成苗率的差异,实验结果表明以加热处理结合剥茎尖法脱病毒效果最好,0.2mm茎尖脱病毒率可达77．78％,加5％病毒座处理对脱病毒有一定的影响,直接剥茎尖法脱病毒效果最差。     

Sequence and organization of barley yellow dwarf virus genomic RNA.

The nucleotide sequence of the genomic RNA of barley yellow dwarf virus, PAV serotype was determined, except for the 5'-terminal base, and its genome organization deduced. The 5,677 nucleotide genome contains five large open reading frames (ORFs). The genes for the coat protein (1) and the putative viral RNA-dependent RNA polymerase were identified. The latter shows a striking degree of similarity to that of carnation mottle virus (CarMV). By comparison with corona- and retrovirus RNAs, it is proposed that a translational frameshift is involved in expression of the polymerase. An ORF encoding an Mr 49,797 protein (50K ORF) may be translated by in-frame readthrough of the coat protein stop codon. The coat protein, an overlapping 17K ORF, and a 3'6.7K ORF are likely to be expressed via subgenomic mRNAs.     

A flower senescence-related mRNA from carnation encodes a novel protein related to enzymes involved in phosphonate biosynthesis

We have isolated a cDNA clone (pSR132) representing a mRNA which accumulates in senescing carnation flower petals in response to ethylene. In vitro translation of RNA selected by hybridization with pSR132 indicated the mRNA encoded a polypeptide of approximately 36 kDa. This was confirmed by DNA sequence analysis, which predicted a peptide composed of 318 amino acids with a calculated molecular weight of 34.1 kDa. Comparison of the predicted peptide sequence of pSR132 with other proteins compiled in the NBRF data base revealed significant homology with carboxyphosphonoenolpyruvate mutase and phosphoenolpyruvate mutase from Streptomyces hygroscopicus and Tetrahymena pyriformis, respectively. These enzymes are involved in the formation of C-P bonds in the biosynthesis of phosphonates. C-P bonds are found in a wide range of organisms, but their presence or formation in higher plants has not been investigated.     

Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA.

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5' noncoding region of the viral RNA. The following results were obtained. The binding of cDNA fragments to the first 41 nucleotides, to the poly(C) tract (between nucleotides 149 and 263), and to the sequence between nucleotides 309 and 338 did not affect translation of the viral RNA; the binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; and the binding of fragments to eight different sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused high degrees of inhibition. The results suggest that the first part of the 5' untranslated region, at least to nucleotide 338, may not be required for encephalomyocarditis viral RNA translation; however, the region near nucleotide 450 is important for translation of the viral RNA. The possibility that initiation occurs at an internal site is discussed.     

Insertion and topology of a plant viral movement protein in the endoplasmic reticulum membrane

Virus-encoded movement proteins (MPs) mediate cell-to-cell spread of viral RNA through plant membranous intercellular connections, the plasmodesmata. The molecular pathway by which MPs interact with viral genomes and target plasmodesmata channels is largely unknown. The 9-kDa MP from carnation mottle carmovirus (CarMV) contains two potential transmembrane domains. To explore the possibility that this protein is in fact an intrinsic membrane protein, we have investigated its insertion into the endoplasmic reticulum membrane. By using in vitro translation in the presence of dog pancreas microsomes, we demonstrate that CarMV p9 inserts into the endoplasmic reticulum without the aid of any additional viral or plant host components. We further show that the membrane topology of CarMV p9 is N(cyt)-C(cyt) (N and C termini of the protein facing the cytoplasm) by in vitro translation of a series of truncated and full-length constructs with engineered glycosylation sites. Based on these results, we propose a topological model in which CarMV p9 is anchored in the membrane with its N- and C-terminal tail segments interacting with its soluble, RNA-bound partner CarMV p7, to accomplish the viral cell-to-cell movement function.     

Molecular cloning of cDNA for rat liver catalase

For the studies on the induction of peroxisomal enzymes by hypolipidemic agents, we have tried to isolate a cDNA clone for rat liver catalase. A recombinant clone, pMJ501, was isolated, of which cDNA insert specifically hybridized to catalase mRNA in hybridization-selected translation. On RNA blot hybridization, it hybridized to 2.4-kilobases RNA which was increased about 1.5-fold by the administration of di-(2-ethylhexyl)phthalate to the rats. The nucleotide sequence of the cDNA contains a reading frame for 109 amino acid residues which match the reported amino acid sequence of bovine liver catalase at the carboxyl end with 82% homology. It is concluded that pMJ501 contains a cDNA sequence for rat liver catalase.     

The complete nucleotide sequence of the maize chlorotic mottle virus genome.

The complete nucleotide sequence of the maize chlorotic mottle virus (MCMV) genome has been determined to be 4437 nucleotides. The viral genome has four long open reading frames (ORFs) which could encode polypeptides of 31.6, 50, 8.9 and 25.1 kd. If the termination codons, for the polypeptides encoded by the 50 and 8.9 kd ORFs are suppressed, readthrough products of 111 and 32.7 kd result. The 31.6 and 50 kd ORFs overlap for nearly the entire length of the 31.6 kd ORF. Striking amino acid homology has been observed between two potential polypeptides encoded by MCMV and polypeptides encoded by carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 25.1 kd ORF most likely encodes the capsid protein. The similar genome organization and amino acid sequence homology of MCMV with CarMV and TCV suggest an evolutionary relationship with these members of the carmovirus group.     

Cloning of a cDNA encoding EIN3-like protein (DC-EIL1) and decrease in its mRNA level during senescence in carnation flower tissues

A cDNA clone encoding a putative EIN3-like protein (DC-EIL1) was obtained from total RNA isolated from senescing carnation (Dianthus caryophyllus L.) petals using RT-PCR and RACE techniques. The cDNA (2382 bp) contained an open reading frame of 1986 bp corresponding to 662 amino acids. The amino acid sequence of the N-terminal half of the protein, ranging from 80-300 amino acid residues, had 84% identity with that of the corresponding regions of Arabidopsis EIN3 and tobacco TEIL, although the overall identity was 49% and 52%, respectively. Northern blot analysis revealed that the amount of mRNA corresponding to DC-EIL1 decreased in flower tissues, especially in petals, during natural senescence and senescence induced by exogenously applied ethylene or ABA.     

Influenza C virus RNA 7 codes for a nonstructural protein.

The complete nucleotide sequence of RNA segment 7 of influenza C/California/78 virus was determined by using cloned cDNA derived from viral RNA. The gene is 934 nucleotides long and possesses a long open reading frame which can code for a protein of 286 amino acids. Hybrid arrest translation experiments with the cloned cDNA fragment and poly(A)-containing RNA isolated from virus-infected cells showed that a 28,500-molecular-weight protein is coded for by RNA 7. Comparison of the proteins induced in the cell-free system and in virus-infected cells with those found in purified virus suggests that the 28,500-molecular-weight protein is a nonstructural protein.