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At least 1,513 records · Page 84Linked to original sources
PubMed1993

Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems.

Nicotiana plumbaginifolia protoplasts were directly transformed by PEG treatment with a cloned 16S rRNA gene isolated from a double antibiotic-resistant Nicotiana tabacum plastid mutant. Putative plastid transformants were selected in cell culture by their spectinomycin resistance and identified by their unselected streptomycin resistance. Alternatively, cell lines were selected in the presence of both antibiotics. The cell line (and its regenerated plants) selected solely for spectinomycin resistance demonstrated an extensive segregation of streptomycin resistance in subsequent tests, while the double-selected line showed stable resistance for both antibiotics. The resistance markers were inherited maternally. In the putative plastid transformants the origin of the resistance mutations was identified by the absence of an AatII site, missing in the donor N. tabacum plastid gene (spectinomycin resistance site) but present in that of wild-type N. plumbaginifolia, and a sequence analysis of the particular nucleotide changes in both resistance sites. Restriction enzyme analysis of total plastid DNA (ptDNA), and the recloning and full sequencing of the fragment introduced, investigated in one of the plastid transformants, showed no DNA rearrangements accompanied with the integration process. Sequence analysis indicated a targeted, homologous integration of the DNA fragment introduced but an unexpectedly complete homology of the parental ptDNA sequences in this region prevented the location of borders. Although the frequency of plastid transformant colonies (2 x 10(-5)) should still be improved, this method for stable chloroplast DNA transformation is comparable with or more efficient than the particle bombardment techniques.

Base Sequence↗
PubMed1996

[Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. I. Construction of vector plasmids for transfer into plants and expression of a modified gene for beta-1,3-glucanase from Clostridium thermocellum in tobacco protoplasts].

We constructed two vectors, pC27-glc and pC29-glc, that allow expression of the beta-1,3-glucanase gene (glc) in plant cells. The glc gene was previously cloned from anaerobic thermophilous bacterium Clostridium thermocellum. To increase the efficiency of expression, the N-terminal fragment of the glc gene encoding bacterial transient peptide was deleted, and hybrid variants of lacZ-glc were obtained. Analysis of expression of the hybrid genes in Escherichia coli showed that deletion of the fragment corresponding to 31 amino acids (a.a.) of beta-glucanase affected neither activity nor thermostability of the enzyme. The modified gene was subcloned into two vectors, pC27 and pC29, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter from Arabidopsis, respectively. Each of the resulting plasmids, pC27-glc and pC29-glc, was transfected into protoplasts of Nicotiana plumbaginifolia. Both the plasmids were shown to allow a high level of activity of the thermostable beta-1,3-glucanase. We plan to use the vectors obtained for transformation of agrobacteria and construction of transgenic plants.

Base Sequence↗
PubMedDate not supplied

[Isolation of protoplasts from vegetable tissues using extracellular lytic enzymes from fusarium oxysporum f.sp. melonis].

Fusarium oxysporum f.sp. melonis, a pathogen of melon (Cucumis melo L.), was grown in shaken cultures at 26 degrees C in a mineral salts medium containing glucose, xylan and apple pectin as carbon sources. The extracellular enzymic complex obtained from these cultures showed lytic activity on plant tissues, causing maceration of melon fruits, potato tubers and carrot roots. Protoplasts were isolated from melon fruits when the maceration was carried out under appropriate osmotic conditions. This fact suggest a possible relationship between the enzymes produced by Fusarium oxysporum f.sp. melonis and their pathogenicity on melon plants.

Cell Wall↗
PubMed1996

Transformation of Pleurotus sapidus protoplasts by electroporation.

The total DNA of Pleurotus ostreatus was successfully transferred into the protoplasts of monokaryotic mycelia of Pleurotus sapidus by electroporation. The transformants were selected by their clamp connection of dikaryocyte, and the transformation frequency and transformation rate were 8.2 x 10(-5) and 3.6%, respectively. Analyzing the esterase isozyme, it was found that the transformants showed some isozyme bands of P. ostreatus and P. sapidus, and new non-parental bands. This proved that the recombination occurred between the DNAs of P. sapidus and P. ostreatus. The fruit bodies of the transformants showed morphological changes.

Electroporation↗
PubMed1995

Cytological events during the initiation of meristematic nodules in calli derived from eggplant protoplasts.

Histological and ultrastructural studies have been undertaken in order to identify the particularities and, if possible, some mechanisms involved in regeneration from protoplasts in the eggplant Solanum melongena. The most important result consists of a peculiar and concomitant association of meristematic and differentiated features only in cells from which regenerative processes originate.

Microscopy, Electron↗
PubMed1995

[Analysis of genetic properties of meiotic products from an interspecific triploid fusion-hybrid itself constructed by protoplast fusion in Saccharomvces].

A genetically stable interspecific triploid fusion-hybrid between a diploid Saccharomyces cerevisiae var. ellopsoideus and a haploid Saccharomyces diastaticus was constracted by protoplast fusion technique. Experimental results indicate that such hybrid can be induced to sporulate as a sexual hybrid. Genetic analysis of the complete and non-complete tetrads demonstrates that the prototrophic interspecific triploid fusion-hybrid HU-KDF-240 selected by nutritional complementation experienced segregation and exchange for the genetic markers, and showed parental ditype and recombination in the meiotic process of sporulation. The results of the tetrad analysis of the hybrid HU-KDF-240 showed a 1:2 segregation ratio for soluble starch fermenting and non-fermenting characters and a 1:1 segregation ratio for genetic markers of the auxotrophic character in 349 complete and non-complete tetrads from 186 asci of the hybrid.

Cloning, Molecular↗
PubMedDate not supplied

[Lysis of Micrococcus lysodeikticus protoplasts by gramicidin S derivatives].

Gramicidin S derivatives with the substituted amino groups of ornithines (carbamoylgramicidin and diacetylgramicidin) possess nearly the same level of the lytic activity when acting on Micrococcus lysodeikticus protoplasts in a 1 M sucrose solution as the original antibiotic. The lytic activity of gramicidin S and these derivatives considerably increases in a solution of sucrose in phosphate buffer. The dependence of the lytic activity of gramicidin on its concentration is of a complex character: two "peaks" of activity and a region of "nonlyzing" concentrations. The lytic activity of gramicidin derivatives with substituted amino groups directly depends on the concentration. When gramicidin S acts upon the intact cells of M. lysodeikticus, it impair with the permeability of their membranes and causes a loss of compounds absorbing at 260 nm; the derivatives with the substituted amino groups change the permeability of the cellular membranes only to a slight extent.

Adsorption↗
PubMed1996

Studies on the reaction model for the immobilized Streptomyces aureofaciens's protoplasts used to convert lincomycin.

A reaction kinetic model is proposed for immobilized S. aureofacien's protoplasts which can facilitate the conversion of lincomycin into clindamycin. The simulation result of the model has been given. Effects of various kinetic parameters of the reaction on the conversion rate are given in the formula. The formulas which can be used to calculate the maximum conversion rate and the optimal reaction time have been given. The results of model simulation are in good agreement with the experimental results.

Clindamycin↗
PubMed1984

The molecular basis for the differential translation of TMV RNA in tobacco protoplasts and wheat germ extracts.

Translation of tobacco mosaic virus (TMV) RNA in tobacco protoplasts yields the 17.5-K coat protein, a 126-K protein and a 183-K protein which is generated by an efficient readthrough over the UAG termination codon at the end of the 126-K cistron. In wheat germ extracts, however, only the 5'-proximal 126-K cistron is translated whereas the 183-K readthrough protein is not synthesized. Purification and sequence analysis of the endogenous tyrosine tRNAs revealed that the uninfected tobacco plant contains two tRNAs, both with GPsiA anticodons which stimulate the UAG readthrough in vitro and presumably in vivo. In contrast, 85% of the tRNA from wheat germ contains a QPsiA anticodon and 15% has a GPsiA anticodon. Otherwise the sequences of tRNAs from wheat germ and tobacco are identical. UAG readthrough and hence synthesis of the 183-K protein is only stimulated by tRNA(GPsiA) and not at all by tRNA(QPsiA). The tRNAs from wheat leaves were also sequenced. This revealed that adult wheat contains tRNA(GPsiA) only. This is very much in contrast to the situation in animals, where Q-containing tRNAs are characteristic for adult tissues whereas Q deficiency is typical for the neoplastic and embryonic state.

Journal Article↗
PubMed1990

Mapping of the glucoamylase gene of Trichosporon adeninovorans by mitotic haploidization using hybrids from protoplast fusions.

In two strains of Trichosporon adeninovorans genes of unknown location were assigned to linkage groups. Diploid strains were produced by protoplast fusion and partially haploidized by growth on benomyl causing mitotic haploidization. We show that both strains synthesize an extracellular glucoamylase with different properties. These differences could be used to map the corresponding gene in the segregants. We could map the glucoamylase gene from one of these yeast strains on linkage group II, one of the three linkage groups identified so far.

Chromosome Mapping↗
PubMed1980

Light microscopic investigations on lysozyme- and penicillin-induced morphological changes in Erysipelothrix rhusiopathiae and on propagation of its protoplast type L-form.

Although lysozyme and penicillin are different in their molecular action on cell wall murein they produce similar morphological changes in Erysipelothrix rhusiopathiae grown on agar media. 2,000--5,000 micrograms/ml lysozyme and 0.1--2 IU/ml penicillin induce filament formation. Filaments are able to divide in rods, which shows that only cross wall formation and separation are inhibited. Higher doses of lysozyme (10,000 micrograms/ml) and penicillin (less than 1 IU/ml) inhibit cell wall synthesis and induce L-form growth. The propagation of this protoplast type L-form was investigated by microphotographic series in phase contrast microscope during L-form induction and in the stable L-form state. In both cases L-form cells propagate by formation and growth of small granular elements of about 0.2--0.6 micrometers in diameter, which spread in different directions in the agar medium. The multiplication process may be explained by the plasticity and flexibility of the L-form cell and its cytoplasmic membrane and by the structural and functional interaction between the "folded chromosome" and the surrounding cytoplasm.

Erysipelothrix↗
PubMed1997

The satellite RNA of barley yellow dwarf virus-RPV is supported by beet western yellows virus in dicotyledonous protoplasts and plants.

The subgroup II luteovirus barley yellow dwarf virus-RPV (BYDV-RPV) acts as a helper virus for a satellite RNA (satRPV RNA). The subgroup II luteovirus beet western yellows virus (BWYV) and the ST9-associated RNA (ST9a RNA), a BWYV-associated RNA that encodes a polymerase similar to those of subgroup I luteoviruses, were assayed for their ability to support replication of satRPV RNA. SatRPV RNA was replicated in tobacco protoplasts in the presence of BWYV RNA or a mixture of BWYV plus the ST9a RNA, but not in the presence of ST9a RNA alone. ST9a RNA stimulated BWYV RNA accumulation which, in turn, increased the accumulation of satRPV RNA. SatRPV RNA was encapsidated in BWYV capsids primarily as circular monomers, which differs from the linear monomers found in BYDV (RPV + PAV) particles. SatRPV RNA was transmitted to Capsella bursa-pastoris plants by aphids only in the presence of BWYV and ST9a RNA. SatRPV RNA reduced accumulation of both BWYV helper and ST9a nonhelper RNAs in plants but did not affect symptoms. The replication of satRPV RNA only in the presence of subgroup II luteoviral RNAs but not in the presence of RNAs with subgroup I-like polymerase genes, in both monocotyledonous and dicotyledonous hosts, suggests that the specificity determinants of satRPV RNA replication are contained within the polymerase genes of supporting viruses rather than in structural genes or host plants.

Animals↗
PubMed1992

A cdc2 gene of Petunia hybrida is differentially expressed in leaves, protoplasts and during various cell cycle phases.

Analysis of p34cdc2 kinase in higher eukaryotes has demonstrated that p34cdc2 function is conserved in all eukaryotic cells. The p34cdc2 kinase (the product of the cdc2 gene) is required during the G1 cell cycle phase at the initiation of DNA replication and also in G2-M phases for entry into mitosis. In this paper we report the isolation and characterization of a cdc2 Petunia hybrida PCR fragment (cdc2Pet). Using a DNA probe based on this fragment and a p34cdc2-specific antibody, cdc2Pet transcript and p34 protein levels were found to be constant both in 2C nuclei of highly proliferating mesophyll 2C cells derived from protoplasts and in 2C nuclei isolated directly from expanded petunia leaves. Both the cdc2Pet transcript and p34cdc2 protein levels were found to be higher in nuclei at 4C than in those at 2C, even when these 4C nuclei were from non-proliferating tissue. Thus cdc2Pet mRNA and protein levels measured in different tissues should not be interpreted to reflect exclusively the proliferative state of the tissue but also the frequency of G2 cells including those in the differentiated state.

Amino Acid Sequence↗
PubMed1982

Study of the phenomenon of marker rescue in plasmid transformation and transduction of intact cells, protoplasts and different rec mutants of Bacillus subtilis.

Marker rescue in plasmid transformation of competent cells of different rec mutants of B. subtilis was studied. In most cases the value of marker rescue decreased proportionally to reduction of plasmid transformation efficiency (although there were certain exceptions). Marker rescue was not observed either in plasmid transformation of protoplasts or in plasmid transduction of intact cells.

Bacillus subtilis↗
PubMed1982

Isolation and characterization of the nucleoid of non-complementing diploids from protoplast fusion in Bacillus subtilis.

Nucleoids of non-complementing diploids (Ncd) from protoplast fusion of B. subtilis were isolated. Their purified DNA banded in neutral CsCl gradient as a single unimodal peak of buoyant density 1.711 g/cm3, a value which is similar to that of the DNA purified from the original parental strains, suggesting that methylation of bases is not a significant factor in chromosome inactivation. Nucleoids released from a Ncd clone give two peaks in a sucrose gradient with a characteristic S value for each nucleoid. That is in contrast to nucleoids from the haploid parents whose sedimental patterns show only one peak. Both nucleoid preparations from Ncd strains assayed for transformation activity show the fast sedimenting nucleoid devoid of transformation activity while the slow nucleoid was active in transformation for the alleles carried by the genome which is expressed in vivo. Both nucleoids of the Ncd strains are transcribed in vivo. The RNA associated with the inactive chromosome is synthesized by the RNA polymerase of the active one. This study provides evidence that inactivation of one parental genome in the Ncd strain may be related with the tertiary organization of its DNA.

Bacillus subtilis↗
PubMed1979

Induced segregation in interspecific hybrids of Aspergillus nidulans and Aspergillus rugulosus obtained by protoplast fusion.

Interspecific hybrids produced by polyethylene glycol induced fusion of protoplasts from auxotrophic mutants of Aspergillus nidulans and Aspergillus rugulosus were grown in the presence of the recombinogens benomyl and chloral hydrate to stimulate segregation. The A. nidulans parental strains used had a known genetic marker in each linkage group. Hybrids grown on complete medium containing benomyl yielded more segregants. Analysis of the segregants showed that the distribution of A. nidulans linkage groups was random. No specific linkage group appeared in all the segregants. The two parents are closely related taxonomically and the findings from these experiments suggest that a high degree of chromosomal homology may exist between them.

Aspergillus↗
PubMed1980

Differentiation of mycoplasmatales from bacterial protoplast L-forms by assay for penicillin binding proteins.

Membrane proteins with the specific ability for binding penicillin with high affinity (penicillin binding proteins) were found to be present in two strains of the cell wall-less protoplast L-form of P. Mirabilis and were absent from different species of Mycoplasma and from Acholeplasma laidlawii. Thus, the assay for penicillin binding proteins appeared to be suitable for the differentiation of the cell wall-less procaryotes. The absence of penicillin binding proteins from the mycoplasmatales further confirmed the unrelatedness of this group to the bacteria.

Acholeplasma laidlawii↗
PubMed1981

Protein synthesis in cucumber mosaic virus-infected tobacco leaves, pre-infected cells and protoplasts.

Polypeptide synthesis has been investigated in cucumber mosaic virus-infected tobacco by radiolabelling and polyacrylamide gel electrophoresis. In addition to the coat protein polypeptide of molecular weight 27,000, others of higher molecular weight ca. 120,000 to greater than or equal to 160,000 (probably two) and possibly one of ca. 32,000--34,000 were detected in the particulate fractions only. A polypeptide of ca. 53,000--57,000 was particularly evident in leaves systemically infected using a differential temperature treatment. Two polypeptides of low molecular weight, ca. 23,000--24,000 and 21,000, were only observed in pre-infected cells and protoplasts and probably represent coat protein degradation products.

Cells, Cultured↗