Partial purification and serological relationship of three strains of bean yellow mosaic virus

Three biologically distinct strains of bean yellow mosaic virus (BYMV^S, BYMV¹ PV-2) were partially purified by centrifugation at relatively low g forces. Serologically these strains appeared to be distinct from each other but were related.     

Partial purification and characterization of the ribonucleotide reductase induced by herpes simplex virus infection of mammalian cells.

In this report we confirm and further characterize the induction of a novel ribonucleotide reductase after herpes simplex virus infection of mammalian cells. Induction of the enzyme was observed at a multiplicity of infection of 1 PFU/cell or greater and was found to be maximal (three- to sixfold the activity in mock-infected controls at 6 to 8 h postinfection at a multiplicity of infection of 10 PFU/cell. Partial purification and subsequent characterization of the reductase activity from infected cells demonstrated the existence of two enzymes which could be separated by precipitation with ammonium sulfate. One of the activities precipitated at between 35 and 55% salt saturation, as did the enzyme from control cells, whereas the novel activity precipitated at 0 to 35% saturation. This latter enzyme was similar to the herpes simplex virus-induced reductase described by others in its lack of requirement for Mg2 and its resistance to inhibition by dTTP and dATP; in addition, we found that it was inhibited by ATP, whereas the enzyme from control cells displayed an absolute requirement for the nucleotide. Both enzymes were equally inhibited by pyridoxal phosphate and showed similar cold and heat stability. The enzyme induced by herpes simplex virus infection, however, was much more labile than the control enzyme upon purification.     

Nodule infection by bean yellow mosaic virus in Phaseolus vulgaris.

Infection of root nodules of beans, Phaseolus vulgaris L., by bean yellow mosaic virus (BYMV) and the effect of the disease on the specific activity of the nodule are reported. Infectivity and serological microprecipitin assays with two sources of BYMV antiserum demonstrated that nodules from bean plants whose leaves had been inoculated with BYMV contain BYMV antigen. The disease reduced the fresh weights of tops, roots, and root nodules and induced premature nodule decay and/or nodule drop. The disease also reduced leghemoglobin content, on a plant weight basis, and N2 fixation rate, on an individual plant basis, as measured by the acetylene reduction assay. The increased leghemoglobin content per gram-nodule in BYMV-infected nodules relative to healthy nodules might be associated with multiplication of the virus in the nodule and/or unknown cellular effects derived from the BYMV-Rhizobium interaction.     

Soluble pathogenesis-related proteins ofCapsicum annuum leaves induced by cucumber mosaic virus infection

Eight major pathogenesis related (PR) proteins were found in a soluble extract of cucumber mosaic virus (CMV) infectedCapsicum annuum leaves. None of them was present in the soluble extract of sham-inoculated controls. The proteins were detected by two-dimensional polyacrylamide gel electrophoresis using (2D-PAGE)PhastSystem, native-PAGE in the first dimension and SDS-PAGE in the second. Two major acidic PR-proteins were identified on the basis of their relative molecular mass (M_r), PR1 of 15.3 kD and PR2 of 29 kD. Six proteins were basic and were identified as PRla of 15.9 kD, PRlb of 15.0 kD, PRlc of 15.9 kD, PR2 of 27.0 kD, PR3 of 36.3 kD and PR4 of 48.8 kD.     

Metachromasia of basic dyes induced by mercuric chloride. I

Summary Interactions of the cationic dye methylene blue with mercuric chloride have been studied conductometrically, analytically and spectrophotometrically. Methylene blue produces red colored precipitate with mercuric chloride; in presence of large excess of mercuric chloride a strong metachromasia is induced in the dye. Metachromasia induced by mercuric chloride is more hypsochromic as well as hypochromic than that induced by chromotopes like heparin. The complexes formed between methylene blue and mercuric chloride have variable compositions, the complex responsible for the red metachromatic color of the dye has the composition 2 dye: 1 HgCl₂. A model has been proposed for the metachromatic complex consisting hexa-coordinated mercury, dye is coordinated to the mercury by donating the lone pair electrons of terminal nitrogen. The non-metachromatic dye capri blue also interacts with mercuric chloride but without any change in the visible spectrum. Potassium iodide also gives metachromatic reddish blue colored precipitate with methylene blue.University Research Scholar.     

An inhibitor in faba bean sap suppressing infection of Chenopodium quinoa by bean yellow mosaic virus

An inhibitor in faba bean sap decreased the infectivity of bean yellow mosaic virus in undiluted sap to 0.2% of its potential infectivity calculated from diluted preparations.     

Partial purification and characterization of nuclear triiodothyronine binding proteins

Nuclear high affinity triiodothyronine binding proteins from rat liver were purified about 500-fold over Triton-washed nuclei by a three-step procedure using gel filtration, DEAE-Sephadex and DNA-Sepharose chromatography. Although unstable, they retain most of the properties exhibited in crude nuclear extract: affinity and stereospecificity for T₃, thermolability, molecular size. Furthermore, their isolation from several chromatin constituents under reducing conditions allows their study with decreased aggregation risks.     

Protective action of salicylic acid against bean yellow mosaic virus infection in Vicia faba leaves

In this study, morphological, ultrastructural and physiological modifications of faba bean (Vicia faba cv Giza 461) leaves in response to bean yellow mosaic virus (BYMV) infection and salicylic acid (SA) treatments were examined. Under BYMV stress, leaves showed symptoms including severe mosaic, mottling, crinkling, size reduction and deformations. Three weeks after virus inoculation, photosynthetic rate, pigment contents and transpiration rate were significantly reduced in response to BYMV infection.

Ultrastructural investigations of BYMV-infected leaves demonstrated that most chloroplasts with increased stromal area became spherical in shape and some lost their envelopes, either partially or totally. The internal structures of chloroplast, grana and thylakoids were dilated. Two kinds of inclusions were detected in BYMV-infected leaves: straight or slightly curved bands sometimes coiled or looped at the end, and electron opaque crystals with varied shapes. BYMV-infected cells showed lower chloroplast number in comparison to the control.

Spraying of SA on faba bean leaves helped to reduce or prevent the harmful effects produced after virus infection. Application of 100 μM SA three days before inoculation restored the metabolism of infected leaves to the levels of healthy controls. SA treatment improved plant health by increasing the photosynthesis rates, pigment contents and levels of other parameters studied similar to control values.

Moreover, SA treatment increased plant resistance against BYMV. This was observed through induction of chloroplast number, reduction in percentage of infected plants, decrease in disease severity and virus concentration of plants treated with SA prior to BYMV inoculation. Cells of SA-treated samples showed well-developed chloroplasts with many starch grains and well-organized cell organelles.

The present results provide an overview of the negative effects on faba bean leaves due to BYMV infection from physiological and subcellular perspectives. Also, a role of SA involved in induction of resistance against BYMV infection in bean plants is discussed.     

Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.     

Accumulation of alfalfa mosaic virus RNAs 1 and 2 requires the encoded proteins in cis.

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (A1MV) encode the replicase proteins P1 and P2, respectively. P1 expressed in transgenic plants (P1 plants) can be used in trans to support replication of A1MV RNAs 2 and 3, and P2 expressed in transgenic plants (P2 plants) can be used in trans to support replication of A1MV RNAs 1 and 3. Wild-type RNA 1 was able to coreplicate with RNAs 2 and 3 in P1 plants, but this ability was abolished by frameshifts or deletions in the P1 gene of RNA 1. Similarly, wild-type RNA 2 coreplicated with RNAs 1 and 3 in P2 plants, but frameshifts or deletions in the P2 gene of RNA 2 interfered with this replication. Apparently, the P1 and P2 genes are required in cis for the accumulation of RNAs 1 and 2, respectively. Point mutations in the GDD motif of the P2 gene in RNA 2 interfered with accumulation of RNA 2 in P2 plants, indicating that replication of RNA 2 is linked to its translation into a functional protein. Plants transformed with both the P1 and P2 genes (P12 plants) accumulate replicase activity that is able to replicate RNA 3 in trans. An analysis of the time course of the accumulation of RNAs 1, 2, and 3 in protoplasts of P12 plants supported the conclusion that translation and replication are tightly coupled for A1MV RNAs 1 and 2 but not for RNA 3.     

Dimerization of papain induced by mercuric chloride and a bifunctional organic mercurial.

The bifunctional mercurial meso-1,4-bis(acetatomercuri)-2,3-diethoxybutane and mercuric chloride are capable of dimerizing papain, by the attachment of the thiol group of two molecules of papain to each molecule of reagent. This is evident from the titration data, gel filtration and sedimentation equilibrium. The conformational change of papain necessary for this reaction is discussed.     

Partial purification and characterization of CTP:cholinephosphate cytidylyltransferase from castor bean endosperm

CTP: cholinephosphate cytidylyltransferase (EC 2.7.7.15) has been purified approximately 600-fold from postgermination endosperm of castor bean. The enzyme was solubilized with n-octyl beta-D-glucopyranoside and then subjected to ion exchange and gel filtration chromatography. The Km's of the purified enzymatic activity were 0.37 and 1.1 mM for CTP and choline phosphate, respectively. Magnesium was required for activity. The purified cytidylyltransferase activity was inhibited by both phosphate and ATP. The extent of ATP inhibition was dependent on preincubation time, temperature, and Mg2+ and Ca2+ concentrations. The possible regulation of cytidylyltransferase in castor bean endosperm by protein phosphorylation is discussed.     

Partial purification and characterization of two Nicotiana tabacum leaf ribonucleases

Two enzymes with similar properties that degrade RNA but not DNA have been partially purified from tobacco leaves. They differ in sub-cellular localization and in ability to hydrolyse ribonucleoside 2′,3′-cyclic phosphates.     

Melon chlorotic leaf curl virus: characterization and differential reassortment with closest relatives reveal adaptive virulence in the squash leaf curl virus clade and host shifting by the host-restricted bean calico mosaic virus

The genome components of the Melon chlorotic leaf curl virus (MCLCuV) were cloned from symptomatic cantaloupe leaves collected in Guatemala during 2002. The MCLCuV DNA-A and DNA-B components shared their closest nucleotide identities among begomoviruses, at ~90 and 81%, respectively, with a papaya isolate of MCLCuV from Costa Rica. The closest relatives at the species level were other members of the Squash leaf curl virus (SLCV) clade, which is endemic in the southwestern United States and Mexico. Biolistic inoculation of cantaloupe seedlings with the MCLCuV DNA-A and -B components resulted in the development of characteristic disease symptoms, providing definitive evidence of causality. MCLCuV experimentally infected species within the Cucurbitaceae, Fabaceae, and Solanaceae. The potential for interspecific reassortment was examined for MCLCuV and its closest relatives, including the bean-restricted Bean calico mosaic virus (BCaMV), and three other cucurbit-infecting species, Cucurbit leaf crumple virus (CuLCrV), SLCV, and SMLCV. The cucurbit viruses have distinct but overlapping host ranges. All possible reassortants were established using heterologous combinations of the DNA-A or DNA-B components. Surprisingly, only certain reassortants arising from MCLCuV and BCaMV, or MCLCuV and CuLCrV, were viable in bean, even though it is a host of all of the “wild-type” (parent) viruses. The bean-restricted BCaMV was differentially assisted in systemically infecting the cucurbit test species by the components of the four cucurbit-adapted begomoviruses. In certain heterologous combinations, the BCaMV DNA-A or -B component was able to infect one or more cucurbit species. Generally, the reassortants were less virulent in the test hosts than the respective wild-type (parent) viruses, strongly implicating adaptive modulation of virulence. This is the first illustration of reassortment resulting in the host range expansion of a host-restricted begomovirus.     

Partial purification and characterization of a ribonucleic acid N2-guanine methyltransferase associated with avian myeloblastosis virus.

A nucleic acid methylase, N2-guanine ribonucleic acid (RNA) methyltransferase, which is associated with type C RNA tumor viruses, has been purified from avian myeloblastosis virions by gel filtration on Sephadex G-200, followed by chromatography on hydroxylapatite. The molecular weight estimated by gel filtration is 220 000, and the methylase activity has a pH optimum of 7.6--7.9. Magnesium and ammonium ions both stimulate activity 1.5-fold at 9.5 mM and 0.36 M, respectively, but apparently neither is essential for activity. Both daunomycin and adriamycin, antineoplastic drugs, also increase activity 1.5-fold at 1 mM. The enzyme was purified 120-fold from the virions and the activity is partially stabilized by dithiothretiol, but large losses were sustained during 24-h dialysis. The purified enzyme retains 75% of its activity on storage at -25 degrees C for 2 months in buffer containing 50% glycerol. Escherichia coli tRNAPhe and tRNAVal are preferred substrates with methylation occurring at position 10 of E. coli tRNAPhe.     

Partial purification of mitochondrial ribosomes from broad bean and identification of proteins encoded by the mitochondrial genome

An approach towards the identification at the protein level of the ribosomal proteins encoded by the mitochondrial genome of broad bean (Vicia faba) has been developed. After Triton X-100 treatment of isolated mitochondria, a fraction enriched in mitochondrial ribosomes was obtained by successive centrifugation, first onto a sucrose cushion, and then in a sucrose gradient. Mitochondrial translation products were labelled in isolated mitochondria with [³⁵S]methionine and added to the enriched mitochondrial ribosomal proteins before separation by two-dimensional gel electrophoresis. Six spots, identified both by Coomassie blue staining and autoradiography, were analysed by protein micro-sequencing. Two of these were shown to correspond to ribosomal proteins S10 and S12. We conclude that these two proteins are encoded by the mitochondrial genome of broad bean and that the method described here can be used to identify other proteins encoded by the mitochondrial genome. Received: 4 September 1996 / Accepted: 30 November 1996