Determination of cellular carbohydrates in peanut fungal pathogens and baker's yeast by capillary electrophoresis and electrochromatography.

In this work, the quantitation of cellular carbohydrates, namely chitin and glucan, in peanut fungal pathogens and baker's yeast was carried out by capillary electrophoresis (CE) and capillary electrochromatography (CEC). The chitin and glucan of the fungi were hydrolyzed by the enzymes chitinase and glucanase, respectively, to their corresponding sugar monomers N-acetylglucosamine (GlcNAc) and glucose (Glc). These two monosaccharides were then tagged with 6-aminoquinoline (6-AQ) to allow their separation and detection in CE and CEC. The 6-AQ derivatives of GlcNAc and Glc formed the basis for the determination by CE and CEC of chitin and glucan in peanut fungi and baker's yeast. Several parameters affecting the separation of the 6-AQ derivatives of GlcNAc and Glc, including the separation voltage and the composition of the running electrolyte, were investigated. Under the optimized separation conditions, the contents of cellular carbohydrates including N-acetylglucosamine, chitin, glucose, and glucan in some fungi, such as Sclerotinia minor, Sclerotium rolfsii, and baker's yeast, were successfully determined. The method described here allowed the assessment of genetic differences in Sclerotium rolfsii isolates from various locations.

Characterization of the nucleic acid binding properties of tomato spotted wilt virus nucleocapsid protein.

Tomato spotted wilt tospovirus (TSWV) is the type member of the plant-infecting viruses of the genus Tospovirus in the family Bunyaviridae. The three TSWV RNAs are encapsidated with nucleocapsid (N) protein to form ribonucleoprotein (RNP) which serves as the template for viral transcription and replication. Regions of the open reading frame coding for the N protein on the small (S) RNA were subcloned into pET protein expression vectors and expressed in Escherichia coli BL21 (DE3) cells. Full-length N, N amino and carboxy halves, and two N carboxy-terminal regions were expressed and isolated by metal chelate affinity chromatography. The N protein, both of its halves and the extreme carboxy-terminal region, bound cooperatively and irrespective of sequence to radiolabeled single-stranded RNA produced by runoff transcription of clones of either TSWV S RNA or cowpea chlorotic mottle virus RNA3. N protein did not bind to radiolabeled double-stranded TSWV RNA. The density of the synthetic RNase-sensitive N protein-RNA complexes was 1.32 g/ml, similar to the density of authentic Bunyaviridae RNPs. These studies are the first to indicate differences in the nucleic acid binding abilities of Tospovirus and Hantavirus nucleocapsid proteins, the only characterized nucleocapsid proteins of the family Bunyaviridae.

Use of firefly luciferase for ATP measurement: other nucleotides enhance turnover.

Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (> 8 microM) or a fairly constant production of light with lower concentrations of ATP (< 1 microM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.

Comparison of the nucleotide sequence of the coat protein open reading frame of nine isolates of wheat streak mosaic rymovirus.

Wheat streak mosaic rymovirus (WSMV) is an important pathogen of wheat (Triticum aestivum L.). The coat protein region of nine isolates of WSMV were cloned by RT-PCR using primers that were inclusive of nucleotides 398-1825 (1) and sequence analysis indicated four regions of variability among the isolates.

Phylogenetic relationships reveal recombination among isolates of cauliflower mosaic virus.

Isolates of cauliflower mosaic virus (CaMV) differ in host range and symptomatology. Knowledge of their sequence relationships should assist in identifying nucleotide sequences responsible for isolate-specific characters. Complete nucleotide sequences of the DNAs of eight isolates of CaMV were aligned and the aligned sequences were used to analyze phylogenetic relationships by maximum likelihood, bootstrapped parsimony, and distance methods. Isolates found in North America clustered separately from those isolated from other parts of the world. Additional isolates, for which partial sequences were available, were incorporated into phylogenetic analysis of the sequences of genome segments corresponding to individual protein coding regions or the large intergenic region of CaMV DNA. The analysis revealed several instances where the position of an isolate on a tree for one coding region did not agree with the position of the isolate on the tree for the complete genome or with its position on trees for other coding regions. Examination of the distribution of shared residue types of phylogenetically informative positions in anomalous regions suggested that most of the anomalies were due to recombination events during the evolution of the isolates. Application of an algorithm that searches for segments of significant length that are identical between pairs of isolates or contain a significantly high concentration of polymorphisms suggested two additional recombination events between progenitors of the isolates studied and an event between the XinJing isolate and a CaMV not represented in the data set. An earlier phylogenetic origin for CaMV than for carnation etched ring virus, the caulimovirus used as outgroup in these analyses, was deduced from the position of the outgroup with North American isolates in some trees, but with non-North American isolates in other trees.

Effect of periodate-oxidized ATP and other nucleotides on firefly luciferase.

Addition of periodate-oxidized ATP (oATP) to firefly luciferase-containing reaction mixtures enhanced light production when the reaction mixture contained > approximately 8 microM ATP. The time course of light production was changed from a flash pattern to a constant light output during incubations of < approximately 10 min. During longer incubation, firefly luciferase was inactivated in a concentration-dependent fashion by oATP. Firefly luciferase has two different time courses of light production that depend on ATP concentration (DeLuca and McElroy, Biochem. Biophys. Res. Commun., 123, 764, 1984). The enhancement of light production occurred only when higher ATP concentrations (> 8 microM) were used. There is little effect of oATP on firefly luciferase activity at low ATP concentrations (< 2 microM) which gave steady production of light. ATP did not antagonize the inactivation of firefly luciferase by oATP. When the oATP was chemically reduced with sodium borohydride (giving or ATP), there was no inactivation of firefly luciferase on incubation. When or ATP was used in a short incubation the enhancement of light production and time course change were the same as those observed with oATP. The corresponding AMP and adenosine compounds (o and or) were slightly inhibitory to firefly luciferase activity. ADP was without effect but both oADP and or ADP enhanced light production. Of these periodate-oxidized ADP, AMP, and adenosine derivatives only oADP inactivated firefly luciferase. The activating effect can be explained by a change in the conformation of the enzyme-product complex so that the product is released faster. In addition there is an inactivation of the enzyme by certain periodate-oxidized nucleotides during longer incubations.

Patterns of nucleotide sequence variation among cauliflower mosaic virus isolates.

A consensus nucleotide sequence of the DNA of nine isolates of cauliflower mosaic virus (CaMV) was used to examine variation of nucleotide sequence in CaMV. Variability in coding regions was lowest in open reading frames (ORFs) 1, 2, 3 and 5 and higher in ORFs 4 and 6. Silent substitutions were not uniformly distributed among the ORFs. The large intergenic region was also variable, particularly in loops and bulges of a predicted secondary structure for this region of the 35S RNA transcript. A profile of frequencies of the substitution of consensus nucleotides with other nucleotides revealed a deficit of A to G transitions and an excess of transversions involving A. Most insertions/deletions could be accounted for by template misalignment during replication. The results suggest that the major source of variation in CaMV DNA sequences is associated with replication by reverse transcription.

The complete nucleotide sequence of cauliflower mosaic virus isolate BBC.

We report the complete nucleotide sequence of BBC, a new and unique isolate of cauliflower mosaic virus (CaMV). The organization of the BBC genome agrees with that of previously sequenced CaMV isolates.