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At least 883 records · Page 49Linked to original sources
PubMed1999

Two-dimensional analysis of proteins secreted by Saccharomyces cerevisiae regenerating protoplasts: a novel approach to study the cell wall.

Protoplasts of Saccharomyces cerevisiae incubated in regenerating conditions secrete cell wall components in order to allow the biosynthesis of this structure. During the first hours of incubation, many of these are not retained in the forming cell wall but remain in the medium. We have developed a method for collecting the secreted proteins and have analysed these by two-dimensional electrophoresis to obtain a reference map of putative cell wall proteins. Several proteins were identified by microsequencing or immunoblotting; namely, cell wall hydrolytic enzymes, heat shock proteins, glycolytic enzymes and others. Some beta-1,3- and beta-1, 6-glucosylation was detected in the proteins secreted by regenerating protoplasts.

Amino Acid Sequence↗
PubMed1987

Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts.

A method to obtain a high metaphase index and thereafter a plant chromosome suspension is described for Petunia hybrida (2n = 14). Mesophyll protoplast cultures have been used, giving easily disrupted cell walls and a high percentage of dividing cells after 42 h. On 2.5 mM colchicine-treated cells, metaphase indexes reaching 10% were routinely obtained. The lysis medium in which the protoplast-derived cells were disrupted was a simplified culture medium. After chromosome release, samples were stained with Hoechst 33342 dye and analysed by flow cytofluorometry. The histogram of fluorescence intensities included three peaks of metaphase chromosomes and a duplication of this flow karyotype provoked by "monochromatid chromosome." This interpretation was established after flow sorting; micronuclei could also be observed and sorted. Of the 7 chromosomes, only the largest formed a distinct peak while the others were incompletely resolved, due to the similar DNA content of various chromosomes. Model distributions of Petunia hybrida chromosomes have been computed according to the relative chromosome length. The theoretical histograms indicated that low variability is indispensable for resolving distinctive chromosome peaks. The experimental flow karyotype was consistent with one of the models having CV of 2.5%.

Benzimidazoles↗
PubMed1988

Preparation by two-dimensional electrophoresis of proteins for antibody production: antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts.

Using phenol extraction from tobacco callus, we have prepared extracts with a high protein content. These proteins were separated in cylindrical non-equilibrium pH gradient gels and visualized by dipping in sodium dodecyl sulfate (SDS)-containing solution. Three gel sections, each containing proteins previously detected as abundantly synthesized in tobacco mesophyll protoplasts and whose synthesis is reduced by auxin application, were excised from each gel and collected. These proteins were further separated on slab SDS gels and protein bands were excised after Coomassie Brilliant Blue R-250 staining and used to inject three rabbits. After one booster, highly specific antibodies were detected in their sera by ELISA and immunoblotting. Using these sera we have confirmed that the corresponding proteins are identical in callus and mesophyll protoplast and demonstrated that they are abundantly accumulated in tobacco roots but are undetectable in aerial organs and seeds.

Antibody Formation↗
PubMed1982

The localization of SPO1 phage resistance in the genome of Bacillus subtilis as revealed by fusion of protoplasts.

We localized the gene for resistance to phage SPO1 relatively to the markers pur B 34 and ura by means of the polyethylene-glycol induced fusion of bacterial protoplasts of three-fold auxotrophic Bacillus subtilis strains S3 and S13. By this same method, the site of some auxotrophic markers was tentatively determined. The application of the protoplast fusion technique to exact genetic analysis will not be possible until the exo- and endogenous factors influencing cell wall regeneration are standardized. Fluctuations of this kind are very significant for the determination of genetic segregation.

Adsorption↗
PubMed1984

Gene expression after transformation of Streptomyces lividans protoplasts with plasmid pIJ2 DNA.

A new method has been developed for the selection of antibiotic-resistant clones after transformation of Streptomyces protoplasts with plasmid DNA. This method is based on establishing a spatial concentration gradient for the antibiotic, the resistance to which is encoded by the transforming plasmid. By this method, the resistance development of regenerating protoplasts can be followed. The results suggest that antibiotic resistance is inducible. In addition, we were able to show that resident plasmids incompatible with the incoming ones are eliminated when this direct selection principle is used. Moreover, this method, which may facilitate the application of gene technology in Streptomyces, works even though the transformation procedure gives variable results.

DNA, Bacterial↗
PubMed1998

Intra- and interspecies virus transfer in Aspergilli via protoplast fusion.

Intra- and interspecies transfer of dsRNA viruses between black Aspergilli and Aspergillus nidulans strains has been investigated using protoplast fusion. We found interspecies transfer of virus in all combinations of black Aspergillus and A. nidulans strains and vice versa. Using the same conditions, intraspecies virus transfer among heterokaryon incompatible strains was also tested. Whereas such transfer was always found among A. nidulans strains, transfer among black Aspergilli was frequently unsuccessful. The lack of virus transfer between black Aspergillus isolates was further investigated by using a mitochondrial oligomycin resistance marker as a positive control for cytoplasmic exchange. These experiments showed independent transfer of the oligomycin resistance and dsRNA viruses during protoplast fusion of heterokaryon incompatible black Aspergilli. The inefficient transfer of dsRNA viruses between black Aspergilli is not caused by absolute resistance to viruses but may be related to heterokaryon incompatibility reactions that operate intraspecifically. Consequences for the dynamics of mycoviruses in populations of black Aspergilli are discussed.

Anti-Bacterial Agents↗
PubMed1993

Transcripts of a maize chlorotic mottle virus cDNA clone replicate in maize protoplasts and infect maize plants.

A full-length cDNA clone (pMCM41) was constructed to contain the exact 5' end of MCMV behind a T7 RNA polymerase promoter and a Smal site at the 3' end. Uncapped RNA synthesized from pMCM41 has the exact 3' end of viral RNA (vRNA) but is missing the cap found on vRNA. This RNA was infectious in protoplasts from black Mexican sweet (BMS) maize (Zea mays) suspension cultures. Uncapped transcripts were also infectious when inoculated onto maize plants and produced an infection indistinguishable from vRNA-inoculated plants. Capped pMCM41 transcripts which initiated at position +2 of the cDNA clone, as well as capped or uncapped RNA synthesized from a clone containing an extra G between the T7 promoter and the 5' end of MCMV sequence (pMCM721), were less infectious than uncapped pMCM41 transcripts in BMS protoplasts. The transcripts one nucleotide longer or shorter than uncapped pMCM41 transcripts were not able to infect maize plants.

Base Sequence↗
PubMed1994

Replication of barley yellow dwarf virus satellite RNA transcripts in oat protoplasts.

A small RNA associated with an isolate of barley yellow dwarf virus (BYDV) has been described which has the physical properties of a satellite RNA (Miller et al., Virology 183, 711-720, 1991). Here we demonstrate that this RNA has the biological properties of a satellite RNA: it requires the presence of helper virus (BYDV genomic) RNA for replication and the helper RNA does not require the satellite. To obtain pure satellite RNA, a permuted dimeric clone was constructed from which infectious satellite RNA could be transcribed in vitro. The dimeric transcript self-cleaved to produce monomeric satellite RNA. When this RNA was coelectroporated with BYDV genomic RNA into oat protoplasts, replication of both RNAs was detected by Northern hybridization. The RPV, but not the PAV, serotype of BYDV supported satellite RNA replication. The presence of discrete oligomeric forms of (+) and (-) sense satellite RNA in infected protoplasts suggests that both strands replicate by a rolling circle mechanism.

Base Sequence↗
PubMed2003

Identification of a minimal sequence required for activation of the tomato golden mosaic virus coat protein promoter in protoplasts.

Transient expression studies using a Nicotiana benthamiana suspension cell-derived protoplast system have identified a minimal sequence that is necessary and sufficient for activation of the tomato golden mosaic virus coat protein (CP) promoter by the viral TrAP protein (also called AL2). The sequence has a bipartite arrangement in which elements located between -125 to -107 and -96 to -60 from the transcription start site are both required for TrAP-mediated activation. One of the sequences (-96 to -60) also appears to interact with a repressor, as its deletion increases basal promoter activity in the absence of TrAP. That competition experiments using the -107 to -60 sequence to titrate the repressor also resulted in increased basal transcription is consistent with this idea. Thus, in a protoplast system which models mesophyll, regulation of the minimal CP promoter involves both activation and derepression by TrAP.

Capsid Proteins↗
PubMed1991

Stress responses in alfalfa (Medicago sativa L.). 8. Cis-elements and trans-acting factors for the quantitative expression of a bean chalcone synthase gene promoter in electroporated alfalfa protoplasts.

A chimeric gene consisting of a bean (Phaseolus vulgaris L.) chalcone synthase (CHS) promoter fused to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed, and further induced by fungal elicitor, when electroporated into alfalfa (Medicago sativa L.) suspension cell protoplasts. Functional analysis of 5' deletions of the CHS promoter-CAT construct in these protoplasts indicated that the region between -326 and -130 contained both activator and silencer elements. Co-electroporation experiments confirmed that these cis-acting elements were binding sites for functionally active trans factors. In vitro DNase I footprinting revealed four potential binding sites for alfalfa suspension cell nuclear proteins between positions -326 and -130 of the CHS promoter. These sites mapped to regions shown to contain functional cis-acting elements on the basis of the deletion analysis. Three of these sites mapped to previously identified binding sites for bean nuclear proteins. Competition gel retardation analysis using oligonucleotide probes containing binding site sequences revealed sequence-specific binding of alfalfa nuclear proteins to an AT-rich element and a putative GT-1 factor consensus binding sequence. Our results define cis elements and their cognate trans factors functionally active in determining the quantitative expression of a defense response gene in a heterologous transient expression system.

Acyltransferases↗
PubMed1992

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts.

The effects of methylation on plant viral DNA replication have been studied in Nicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m5C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m5C residues were substituted for cytosine residues in vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequences in vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.

Blotting, Southern↗
PubMed1992

Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplasts.

Expression cassettes containing a duplicated cauliflower mosaic virus (CaMV) 35S promoter fused to a polylinker preceded by the CCACCATGG and AACAATGG sequences were constructed. These two sequences correspond to the consensus sequences around the translation start codons in vertebrates and plants respectively. Translational fusions were made with the beta-glucuronidase-coding sequence and transient expression was recorded in tobacco mesophyll protoplasts. Approximately three times more GUS activity was found in protoplasts incubated with the constructs harbouring translational fusions as compared to a control harbouring a transcriptional fusion. No significant difference was observed between GUS activities obtained with the two consensus sequences.

Base Sequence↗
PubMed1993

The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants.

A Bacillus thuringiensis (B.t.) cryIIIA delta-endotoxin gene was designed for optimal expression in plants. The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t. cry genes. We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs. For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs. We compared modified cryIIIA gene with native gene expression by electroporation of dicot (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene. Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250,000 CPB larvae were placed on replicate clones of 56 transgenic potatoes. No CPB larvae developed past the second instar on any of these plants. Plants expressing high levels of delta-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation between insect control and the levels of delta-endotoxin RNA and protein.

Amino Acid Sequence↗
PubMed1992

Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts.

A hammerhead ribozyme designed against the mRNA coding for the Escherichia coli beta-glucuronidase (GUS) reporter enzyme was constructed. The synthetic ribozyme appeared able to correctly cleave in vitro the target RNA. This catalytic molecule was then assayed for in vivo activity in plant protoplasts. Plasmids coding either for the ribozyme or for the GUS target gene were cotransfected into the cells by the PEG-calcium procedure and GUS gene expression monitored following transient expression by measuring the intracellular GUS enzymatic activity. Expression of the ribozyme to high molar excess over the GUS transcript did not lead to any significant decrease of GUS activity in the transfected protoplasts. Insertion of the ribozyme sequence in the 3'-untranslated region of the GUS mRNA also had no detectable effect on GUS reporter gene expression whereas the corresponding RNA appeared able to self-cleave in vitro. These results indicate that the ability of ribozymes to perform catalytic cleavage of their substrate mRNA in vitro is essential but clearly not sufficient to ensure that efficient inhibition of the corresponding target gene will occur upon endogenous expression of this catalytic RNA in the plant cell.

Arabidopsis↗
PubMed1994

Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination.

Whole leaves and guard-cell protoplasts of the C3 plant Vicia faba L. (broad bean) were separately extracted following a period of illumination or following a period of darkness. Kinetic parameters of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), Vmax and Km(PEP.Mg), were determined as a function of assay pH (7.0 or 8.1), the presence of 5 mM glucose-6-Pfree (Glc-6-P, an activator), and the presence of 5 mM malatefree (an inhibitor). On the basis of these parameters, guard-cell PEPC was distinguished from that of whole leaf, indicating either that guard cells contain a unique isoenzyme of PEPC or a different complement of isoenzymes or--and less likely--that the obligatorily different methodologies for the leaf (intact organ) and the guard-cell (protoplast) enzymes altered them specifically. The values of Vmax were relatively unchanged, regardless of assay conditions or tissue pretreatment. The values obtained for whole-leaf PEPC Vmax were restricted to a small range (52.4 +/- 5.9 (SD) to 64.4 +/- 4.8 (SD) mumol.g fresh mass-1.h-1; the high value coincided with the presence of Glc-6-P, and the low value was obtained in the presence of malate. Guard-cell PEPC Vmax was also restricted to a small range: 7.48 +/- 0.89 (SD) pmol.guard-cell pair-1.h-1 (pH 8.1, light, +Glc-6-P) to 5.79 +/- 0.60 (SD) pmol.guard-cell pair-1.h-1 (pH 7.0, dark, +malate). Depending on effectors, and particularly pH, large changes in Km(PEP.Mg) were calculated (whole-leaf PEPC: 0.03 to 3.84 mM; guard-cell PEPC: 0.06 to 3.43 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Fabaceae↗
PubMed1995

Kinetic analysis of two simultaneously activated K+ currents in root cell protoplasts of Plantago media L.

Two different, simultaneously activated outward rectifying K+ currents were analyzed in the plasmalemma of root cortex protoplasts of Plantago media. Their gating is dependent on the diffusion potential for K+(EK). The threshold potential was more negative than EK allowing small inward currents at potentials below EK thereby keeping cells with little pump activity in the K state (Vogelzang & Prins, 1994). Time and voltage dependence of the outward rectifying K+ currents have been analyzed with Hodgkin-Huxley-like (HH) models. Dynamic responses of whole cell currents to pulse potentials were analyzed with two voltage dependent functions, the Boltzmann distribution for open probability per gate and the transition rate towards the open state (alpha). The transition rate in the opposite direction (beta), was calculated from alpha and the Boltzmann distribution. These functions were used for an integral analysis of activation and deactivation currents measured over a range of pulse potentials. Both whole cell and single channel data were used for the determination of the number of closed and open states. The effects of single channel flickering on time response and amplitude of tail currents were added to the model. The dominant K+ channel present in the plasmalemma of P. media has a characteristic nonlinear single channel I-V curve reducing the amplitude of whole cell currents at positive potentials. To compensate for this nonlinearity, a four state translocator model was added to the whole cell open probability model. The analysis presented here provides a general basis for the study and comparison of K+ channel kinetics in plant protoplasts.

Ion Transport↗
PubMed1994

Ion channels in the plasma membrane of protoplasts from the halophytic angiosperm Zostera muelleri.

Patch clamp studies show that there may be as many as seven different channel types in the plasma membrane of protoplasts derived from young leaves of the halphytic angiosperm Zostera muelleri. In wholecell preparations, both outward and inward rectifying currents that activate in a time- and voltage-dependent manner are observed as the membrane is either depolarized or hyperpolarized. Current voltage plots of the tail currents indicate that both currents are carried by K+. The channels responsible for the outward currents have a unit conductance of approximately 70 pS and are five times more permeable to K+ than to Na+. In outside-out patches we have identified a stretch-activated channel with a conductance of 100 pS and a channel that inwardly rectifies with a conductance of 6 pS. The reversal potentials of these channels indicate a significant permeability to K+. In addition, the plasma membrane contains a much larger K+ channel with a conductance of 300 pS. Single channel recordings also indicate the existence of two Cl- channels, with conductances of 20 and 80 pS with distinct substates. The membrane potential difference of perfused protoplasts showed rapid action potentials of up to 50 mV from the resting level. The frequency of these action potentials increased as the external osmolarity was decreased. The action potentials disappeared with the addition of Gd3+, an effect that is reversible upon washout.

Action Potentials↗
PubMed1990

Branched respiratory chain in aerobically grown Staphylococcus aureus--oxidation of ethanol by cells and protoplasts.

Addition of ethanol and some other primary alcohols, except methanol, to cells and protoplasts (but not membrane particles) considerably stimulated the rate of oxygen consumption. This additional respiration was strongly inhibited by 0.1 mM KCN. The cyanide inhibition curve of endogenous substrate oxidation was slightly biphasic while in the presence of ethanol it became clearly biphasic having Ki values of approx. 0.1 and 0.5 mM. Based on the steady-state cytochrome spectra in the presence of 0.1 mM KCN, we attributed the lower Ki to cytochrome a602. Proteolysis of protoplasts external membrane proteins did not change the rate of endogeneous substrate oxidation but prevented the inhibition of this respiration by low concentrations of KCN and stimulation of oxygen consumption by ethanol. The activity of NAD(+)-dependent ethanol dehydrogenase in the cytoplasm was found to be 520 nmol NADH- x min-1 x mg-1 protein. Proteolysis of external membrane proteins apparently inhibits the operation of the cytochrome a602-containing electron transport branch inducing the suppression of electron flow from NADH to oxygen.

Aerobiosis↗