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At least 1,369 records · Page 76Linked to original sources
PubMed2001

ABI5 interacts with abscisic acid signaling effectors in rice protoplasts.

Abscisic acid (ABA) regulates seed maturation, germination, and adaptation of vegetative tissues to environmental stresses. The mechanisms of ABA action and the specificity conferred by signaling components in overlapping pathways are not completely understood. The ABI5 gene (ABA insensitive 5) of Arabidopsis encodes a basic leucine zipper factor required for ABA response in the seed and vegetative tissues. Using transient gene expression in rice protoplasts, we provide evidence for the functional interactions of ABI5 with ABA signaling effectors VP1 (viviparous 1) and ABI1 (ABA insensitive 1). Co-transformation experiments with ABI5 cDNA constructs resulted in specific transactivation of the ABA-inducible wheat Em, Arabidopsis AtEm6, bean beta-Phaseolin, and barley HVA1 and HVA22 promoters. Furthermore, ABI5 interacted synergistically with ABA and co-expressed VP1, indicating that ABI5 is involved in ABA-regulated transcription mediated by VP1. ABI5-mediated transactivation was inhibited by overexpression of abi1-1, the dominant-negative allele of the protein phosphatase ABI1, and by 1-butanol, a competitive inhibitor of phospholipase D involved in ABA signaling. Lanthanum, a trivalent ion that acts as an agonist of ABA signaling, potentiated ABI5 transactivation. These results demonstrate that ABI5 is a key target of a conserved ABA signaling pathway in plants.

Abscisic Acid↗
PubMed1981

Dose--effect relationships, r.b.e. and split-dose effects after gamma-ray and fast neutron irradiation of protoplasts from wild Nicotiana species.

Protoplasts from a diploid Nicotiana species were used to investigate the effects of gamma-rays and fast neutrons. The damage was assessed by scoring the relative plating efficiencies. The effects of single acute doses, as well as fractionated doses, are described for both qualities of radiation. The dose--r.b.e. relationship, expressing the effectiveness of fast neutrons relative to gamma-rays as a function of dose, appears similar to that of most animal systems, with higher values in the region of lower doses. The fractionation of gamma-ray doses, with sufficiently long time intervals between split doses, allowed a substantial recovery, as known also for X-rays. On the other hand, the fractionation of a fast neutron dose into low single fractions markedly increased the biological effectiveness of the total dose. This could not be explained by cell progression during resting time intervals and necessitated an additional hypothesis, possibly involving other mechanisms in the specific action of low doses of radiation.

Dose-Response Relationship, Radiation↗
PubMed1963

Studies on L forms and protoplasts of group A streptococci. II. Chemical and immunological properties of the cell membrane.

Intact bacterial membranes have been isolated from protoplasts prepared from Group A streptococci by a cell wall-dissolving enzyme. A membrane fraction with identical chemical and serological properties has been obtained by differential centrifugation of mechanically disrupted streptococci. The membrane is chemically distinct from the cell wall and is composed of 72 per cent protein, 26 per cent lipid, and 2 per cent carbohydrate. Capillary precipitin tests and analysis by microdiffusion have demonstrated that the membrane contains antigens distinct from those of the cell wall and from those of the cytoplasm which it envelops. Evidence is presented which demonstrates that this antigenic material is common to the membranes of Group A streptococci, and that it can be distinguished by immunodiffusion from related antigenic substances present in membranes of several other serological groups of hemolytic streptococci. This antigenic material does not cross-react with the membrane antigens of other Gram-positive cocci.

Antigens↗
PubMed1993

A ribozyme that enhances gene suppression in tobacco protoplasts.

Site-directed mutagenesis has been used to produce two hammerhead ribozyme molecules targeting the chloramphenicol acetyltransferase gene (CAT). One ribozyme has a single catalytic domain between two 12-nucleotide arms that can hybridize 5' and 3' of the GUC target site of the CAT RNA transcript. The second ribozyme is a full-length antisense RNA with four catalytic domains inserted along the length, each targeting a specific GUC site within the CAT mRNA. Our results show that both ribozymes can produce almost equivalent rates of cleavage of the CAT mRNA in vitro (T1/2 of 18 or 15 min, respectively). In tobacco protoplasts we show consistently greater gene suppression in the presence of the long ribozyme molecule, compared with the equivalent antisense (22% gene reduction for antisense compared with 44% with the long ribozyme). These results suggest that hammerhead ribozymes may be developed for the inactivation of gene activity in plant cells.

Binding Sites↗
PubMed1996

Noncomplementing diploids from Bacillus subtilis protoplast fusion: relationship between maintenance of chromosomal inactivation and segregation capacity.

Fusions of Bacillus subtilis protoplasts from two genetically marked strains produce noncomplementing heterodiploid bacteria. These noncomplementing diploids (Ncds) carry both parental chromosomes, but only one is expressed. Fusion products of strains polymorphic for NotI restriction sites provide new physical evidence to support the conclusion that Ncds are not an artifact of cross feeding or cell adhesion. We show that reversible chromosomal inactivation can only account for the biparental trait of unstable Ncds. Two types of cells were recovered from the late progeny of unstable Ncds: Ncds with irreversible chromosome silencing (stable Ncds) and secondary recombinants that displayed a genomic mosaic NotI profile. Segregants from an unstable Ncd population gave rise to two viable haploid cell types. By contrast, stable Ncds segregated into a population of viable and inviable haploid cells. We propose that the latter are derived from irreversible chromosome silencing. Our results indicate that clonal populations of stable Ncds are heterogenous and suggest that segregation and inactivation are independent parameters.

Bacillus subtilis↗
PubMed1976

Some properties of tobacco protoplast chromatin.

Chromatin was prepared from tobacco-leaf protoplasts. Its solubility in increasing molarities of NaCl was studied and the structure of the soluble fraction observed by electron microscopy. We demonstrate that in plants, the DNA and histones are associated in beaded structures similar to those called omicron-bodies or nucleosomes in animal chromatin. The nucleosomes were associated with DNA in either compact or extended forms. The compact arrangement was predominant in the fraction solubilized between 0.1 and 0.4 M NaCl. The extended form, present at 0.5 and 0.6 M NaCl. showed DNA filaments of various lengths interspacing the nucleosomes. At these ionic strengths ring structures were present, associated with the DNA. At 0.7 M NaCl and above, only DNA filaments were present, occasionally associated with big rings, and nucleosomes were compoetely dissociated. Free DNA molecules were present at all ionic strengths used. The possible origin and significance of the rings are discussed.

Cell Nucleus↗
PubMed1992

Replication of grapevine fanleaf virus satellite RNA transcripts in Chenopodium quinoa protoplasts.

A set of full-length cDNA clones of the satellite RNA of grapevine fanleaf nepovirus isolate F13 (GFLV-F13) was constructed with a variable number of additional, non-viral nucleotides at the 5' and 3' ends. The biological activity of the RNAs transcribed from these constructs was tested in Chenopodium quinoa protoplasts using a helper virus. When inoculated with arabis mosaic virus S (ArMV-S) RNA as helper, transcripts with 33 non-viral nucleotides at the 5' end (tr45p4) did not replicate, whereas transcripts with only one non-viral nucleotide at the 5' end (tr3S and tr3M) did replicate. Capping of the transcripts enhanced their replication. On the other hand, the presence of extra nucleotides at the 3' end had little influence on the biological activity of the in vitro transcripts. In contrast with ArMV-S, GFLV isolate 24 was not a helper for tr3M transcripts, indicating a specific interaction between the helper strain and the satellite RNA.

Base Sequence↗
PubMed2001

Umbravirus-encoded movement protein induces tubule formation on the surface of protoplasts and binds RNA incompletely and non-cooperatively.

Various functions of the cell-to-cell movement protein (MP) of Groundnut rosette virus (GRV) were analysed. The GRV ORF4-encoded protein was shown by immunofluorescence microscopy to generate tubular structures that protrude from the surface of the protoplast. The protein encoded by ORF4 was assessed also for RNA-binding properties. This protein was tagged at its C terminus with six histidine residues, produced in Escherichia coli using an expression vector and purified by affinity chromatography. Gel retardation analysis demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-cooperatively to viral ssRNA and formed complexes of low protein:RNA ratios. Competition binding experiments showed that the ORF4-encoded protein bound to both ssRNA and ssDNA without sequence specificity, but did not bind to dsDNA. UV cross-linking and nitrocellulose membrane-retention assays confirmed that both the GRV and the CMV MPs formed complexes with ssRNA and that these complexes showed similar stability in NaCl. Probing the MP-RNA complexes by atomic force microscopy demonstrated that the ORF4-encoded protein bound RNA incompletely, leaving protein-free RNA segments of varying length, while the CMV 3a protein formed highly packed complexes. The significance of the two properties of limited RNA binding and tubule formation of the umbraviral MP is discussed.

Arachis↗
PubMed1982

Effect of tunicamycin on exo-1,3-beta-D-glucanase synthesis and secretion by cells and protoplasts of Saccharomyces cerevisiae.

Addition of tunicamycin to the culture medium of growing Saccharomyces cerevisiae protoplasts or cells resulted in the formation of a modified exo-1,3-beta-D-glucanase which was detectable in both extracellular and intracellular fractions. This modified enzyme had a lower molecular weight than the native form and did not bind to concanavalin A. The activation energy and Km values of both enzyme forms were identical. Antibodies raised against the native protein readily precipitated the exo-1,3-beta-D-glucanase produced after tunicamycin treatment. The latter enzyme was comparable, in terms of molecular size and lack of affinity for concanavalin A, to the beta-D-glucanase obtained by treatment of the native form with endoglycosidase H; both lacked the carbohydrate moiety present in the native enzyme. The exo-1,3-beta-D-glucanase obtained in the presence of the antibiotic was more sensitive to variations in temperature and pH than both endoglycosidase H-treated and non-treated enzymes. Our results suggest that the carbohydrate moiety, if not necessary for exo-1,3-beta-D-glucanase secretion, may play a role in the conformation of the protein and in stabilizing the enzymic activity.

Amino Acids↗
PubMed1983

Aspects of genetic interaction in hybrids of Aspergillus nidulans and Aspergillus rugulosus obtained by protoplast fusion.

Hybrids were produced by protoplast fusion between strains of Aspergillus rugulosus and mitotic master strains of Aspergillus nidulans with a genetic marker on each linkage group. Analysis of segregants induced by growth on benomyl revealed recombination between every pair of unlinked markers. Parental combinations of markers were often recovered at significantly higher frequencies than expected. This aberrant segregation was not correlated with any particular pair of linkage-groups and was attributed to inter-species incompatibility. The segregation of genetic markers of A. rugulosus from the hybrids suggested that A. nidulans and A. rugulosus may differ in haploid chromosome number and chromosome size. In sexual crosses between A. nidulans and strains containing chromosomes of mixed parental origin recombinants were recovered. The results support the classification of A. nidulans and A. rugulosus as separate species.

Aspergillus↗
PubMed1984

Further studies on protoplast fusion and interspecific hybridization within the Aspergillus nidulans group.

Hybridization of eight species of the Aspergillus nidulans group was attempted using auxotrophic mutants and protoplast fusion methods. Viable fusion products were obtained from eight crosses. Allodiploid hybrids were recovered from crosses involving A. nidulans with A. rugulosus, A. quadrilineatus, A. nidulans var. echinulatus and A. violaceus, although some mutants only gave heterokaryons. Crosses involving these latter species also gave heterokaryons. Crosses between A. nidulans and A. unguis, A. stellatus and A. heterothallicus were unsuccessful. Fusions involving three parents gave heterokaryons made up of only two of them.

Aspergillus nidulans↗
PubMed1985

Protoplast fusion as a tool for genetic analysis in Cephalosporium acremonium.

Protoplasts of nutritionally complementary strains of Cephalosporium acremonium were fused and plated onto media which supressed the growth of both parents. The regenerating colonies were used for genetic analysis and were found to be of two types, stable haploid recombinants and unstable heterozygotes (aneuploids and/or diploids). Analysis of these colonies provided evidence for eight linkage groups and a relatively high rate of mitotic crossing-over. The gene order for three of the markers on one linkage group was also determined.

Acremonium↗
PubMed1985

Transfection of Corynebacterium lilium protoplasts.

A protoplast transfection system has been developed for a lysine-producing bacterium, Corynebacterium lilium, using the DNA of phage CL31. Phage CL31 is lytic and specific to C. lilium and has a genome of approximately 48 kb. The transfection procedure involves a polyethylene-glycol-mediated introduction of the DNA into lysozyme-treated cells and has a maximum efficiency of 3 X 10(4) transfectants per microgram DNA.

Bacteriophages↗
PubMed1986

An efficient method for the introduction of viral DNA into Brevibacterium lactofermentum protoplasts.

A method for the introduction of a bacteriophage DNA into Brevibacterium lactofermentum protoplasts is described. Frequencies of 10(5) infective centres per micrograms DNA were easily achieved, the relationship between the number of infective centres and the amount of DNA being linear up to 5 micrograms DNA per assay. This method can be used to introduce foreign DNA into these bacteria.

Bacteriophages↗
PubMed1989

Heat shock at an elevated temperature improves transformation efficiency of protoplasts from Podospora anserina.

We have developed an improved transformation procedure for the filamentous fungus Podospora anserina. This procedure is based on the observation that a heat shock at an elevated temperature (48 degrees C) improves the competence of P. anserina protoplasts for transformation 5- to 10-fold. This is observable only if the heat shock is applied before the addition of transforming DNA. An increase in competence is observed immediately after the heat shock, and heat-shocked cells are still competent after 20-30 min. The mechanism by which heat shock improves competence remains unclear. The modified transformation procedure gives as many as 200-500 stable transformants per microgram of plasmid DNA containing the P. anserina ura5 gene. This should allow direct cloning of P. anserina genes from a cosmid library.

Ascomycota↗