RESUMEN
The gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A was cloned and expressed in E. coli plasmid pUC8. Plasmid (pNC-1A.14.8) encoded M.CviBIII methylates adenine in TCGA sequences both in vivo in E. coli and in vitro. Transposon Tn5 mutagenesis localized the M.CviBIII functional domain to a 1.5 kbp region of pNC-1A.14.8 and also indicated that a virus promoter directs transcription of the gene in E. coli. The 2.1 kbp insert containing the M.CviBIII gene was sequenced and a single open reading frame of 1131 bp was identified within the domain determined by Tn5 mutagenesis. When the M.CviBIII gene was fused in-frame with the 19 amino-terminal codons of lacZ a 45 kD polypeptide was identified in maxicells as predicted by the DNA sequence. The M.CviBIII gene was not essential for virus replication since a virus M.CviBIII deletion mutant also replicated in Chlorella.
Asunto(s)
Chlorella/genética , Clonación Molecular , ADN (Citosina-5-)-Metiltransferasas/genética , Genes , Secuencia de Aminoácidos , Secuencia de Bases , Chlorella/enzimología , Clonación Molecular/métodos , ADN (Citosina-5-)-Metiltransferasas/aislamiento & purificación , Análisis Mutacional de ADN , Escherichia coli/genética , Operón Lac , Datos de Secuencia Molecular , Virus de Plantas/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Fagos T/genéticaRESUMEN
Nineteen plaque-forming viruses of the unicellular, eukaryotic Chlorella-like green alga, strain NC64A, were isolated from various geographic regions in the United States and characterized. Like the previously described virus, PBCV-1, all of the new viruses were large polyhedrons, sensitive to chloroform, and contained large dsDNA genomes of ca. 300 kbp. All of the viral DNAs contained 5-methyldeoxycytidine which varied from 0.1 to 47% of the deoxycytidine. In addition, 10 of the viral DNAs contained N6-methyldeoxyadenosine which varied from 8.1 to 37% of the deoxyadenosine. These viruses, along with 11 previously described viruses which replicate in the same Chlorella host, were grouped into 11 classes based on at least one of the following properties: plaque size, reaction with PBCV-1 antiserum, or the nature and abundance of methylated bases in their genomic DNA.
Asunto(s)
Chlorophyta , Virus de Plantas/genética , Composición de Base , Enzimas de Restricción del ADN/metabolismo , ADN Viral/genética , Metilación , Homología de Secuencia de Ácido Nucleico , Ensayo de Placa ViralRESUMEN
PBCV-1, a large dsDNA-containing virus which replicates in a Chlorella-like green alga, is composed of approximately 64% protein, 25% DNA, and 5-10% lipid on a weight basis. Polyacrylamide gel electrophoresis of the dissociated virus particle resolves 50 to 60 proteins which range in apparent molecular weight from 10,000 to 135,000. Two of these proteins are glycoproteins and at least four are located on the viral surface. The major lipids are phosphatidyl choline, phosphatidyl ethanolamine, and an unidentified component. The effect of organic solvents, surfactants, and chelating and reducing agents on viral infectivity and ultrastructure are reported. Inhibitor studies established that PBCV-1 protein synthesis occurs on cytoplasmic ribosomes.
Asunto(s)
Chlorella/genética , Replicación del ADN , Virus ADN/crecimiento & desarrollo , Lípidos/análisis , Proteínas Virales/análisis , Chlorophyta/genética , Virus ADN/genética , Virus ADN/ultraestructura , Microscopía Electrónica , Peso Molecular , Proteínas Virales/genética , Replicación ViralRESUMEN
A chemical assay for P-700 was developed using 0.36 mM potassium ferricyanide as oxidant and 1.6 mM sodium ascorbate as reductant. The major difference from other chemical assays for P-700 is procedural. The method is designed to take advantage of the availability of microprocessor-linked spectrophotometers to obtain greater accuracy by minimizing the spectral changes due to irreversible oxidized antenna chlorophyll molecules. The value measured for the P-700 concentration in a sample of chloroplasts was not changed by the presence of EDTA, Mg2+ or sucrose in the assayed solution. Similarly, half of the detergents tested (Triton X-100, Nonidet P-40, digitonin, Deriphat 160, Miranol S2M-SF and Miranol M2M) did not alter the value when added to the chloroplasts. The remainder of the detergents examined caused a significant decrease or increase in the value for P-700 content. Sodium dodecyl sufate, of particular interest due to its widespread use, caused a doubling in the amount of apparent P-700. This effect may be due to this detergent and some others enabling an additional long wavelength form of chlorophyll, possibly an intermediary electron acceptor in Photosystem I, to be chemically oxidized and reduced under the assay conditions.