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At least 1,225 records · Page 68Linked to original sources
PubMed1988

Intergeneric Protoplast Fusion between Ruminococcus albus and an Anaerobic Recombinant, FE7.

Intergeneric protoplast fusion between Ruminococcus albus, a cellulolytic, gram-positive, anaerobic bacterium (Pc Sm Km), and an anaerobic recombinant, FE7 (Pc Sm Km), having lignin-related compound-degrading activities, was performed under strictly anaerobic conditions to introduce cellulase genes into strain FE7. The fusion frequency varied with different selected markers from 3.0 x 10 to 3.3 x 10. Two fusants, obtained from a synthetic medium with selective pressures of penicillin and streptomycin and with cellooli-gomer as the sole carbon source, were gram-negative rods. One of them, named FE7R2, showed 45 to 47% of the beta-glucosidase and cellobiosidase activities of its parent R. albus and still maintained a level of degradation activity against dehydrodivanillin, a lignin-related compound, of up to 87% of that of the parent strain FE7. To verify that the cellulolytic activities expressed in the fusant FE7R2 originated from R. albus cellulase genes, the beta-glucosidase gene of R. albus was cloned into Escherichia coli HB101 with plasmid pBR322. Cells bearing a recombinant plasmid, pRAII, produced high enzyme activities against both p-nitrophenyl-beta-d-glucoside and p-nitrophenyl-beta-d-cellobioside and could degrade cellobiose to glucose. Southern blot results showed that the cloned DNA fragment could hybridize with chromosomal DNAs of both R. albus and FE7R2, but did not with the chromosomal DNA of FE7, indicating that the beta-glucosidase gene fragment was introduced into the chromosome of FE7R2 from R. albus via the protoplast fusion. The fusant FE7R2 could utilize simultaneously both cellobiose and dehydrodivanillin. These results gave evidence that the fusion product FE7R2 is a recombinant strain between its parents R. albus and FE7. This recombinant has stably kept the above properties for about 2 years.

Journal Article↗
PubMed1970

Penicillin-Lysozyme Conversion of Clostridium botulinum Types A and E into Protoplasts and Their Stabilization as L-Form Cultures.

When logarithmically growing cultures of Clostridium botulinum types A and E are treated with penicillin in a liquid medium containing 8% polyethylene glycol, protoplast-like spherical bodies are formed. The penicillin effect shows a dose-response relationship; the largest yield of converted forms is obtained with 10,000 units/ml, but the treatment leaves many intact bacilli. Lower antibiotic concentrations produce smaller numbers of spherical bodies, but lysis of bacilli results in suspensions that are relatively free of rods. Cells grown under the same conditions and treated with 250 mug of lysozyme/ml do not form spherical bodies. However, a combination of 1,250 to 2,500 units of penicillin and 100 mug of lysozyme/ml yields suspensions which have sphere counts in excess of 1.0 x 10(8)/ml and only a few intact rods. The state of the culture at the time of addition of the antibiotic and enzyme is critical. Suspensions of these protoplasts can be adapted to grow as stable L-form cultures producing the same toxin type as the parent cultures.

Journal Article↗
PubMed1968

Structural and immunological studies on the protoplast membrane of the yeast Candida utilis.

Cell membranes of the yeast Candida utilis isolated by lysis of protoplasts have been shown to be lipoprotein in nature. Electron microscopy shows that Mg(++) is responsible for maintaining the integrity of the membrane. A close serological relationship was found between membranes and cell walls isolated from the yeast. This relationship was exhibited not only by membranes obtained by strepzyme treatment but also by those obtained from the action of helicase enzyme. No such relationship existed between membranes and whole cells. Related data have been obtained by treatment of yeasts with different digestive enzymes. All of the results suggest that the protoplast membrane possesses traces of structural cell wall material. This material is detectable by serological tests, but not by electron microscopy.

Agglutination Tests↗
PubMed1982

The detergent-resistant cytoskeleton of higher plant protoplasts contains nucleus-associated fibrillar bundles in addition to microtubules.

Cytoskeletons prepared from carrot protoplasts often retain the nucleus, which is associated with bundles of 7 nm fibrils. These were also seen in cytoskeletons from oil palm, maize and Italian rye grass cells. By examining such cytoskeletons by immunofluorescence, negative staining and stereo scanning electron microscopy it appears that the bundles could be part of a more delicate system of fibrils distal to the nucleus. Optical diffraction of the bundles, staining with heavy meromyosin, drug treatments and phalloidin-staining all confirm that the bundles are not composed of actin. Bundles could not be isolated from such cytoskeletons in quantities sufficient for analysis, but by disrupting protoplasts prepared from stationary-phase cells it was possible to purify fibrillar bundles that appeared in the lysate. These were similar in size and properties to the nucleus-associated bundles. The former were shown by sodium dodecyl sulphate/polyacrylamide gel electrophoresis to be composed of several proteins and were sensitive to reducing agents. Since they are not composed of actin, the possibility was explored that these fibrils are related to the P-proteins of phloem sieve tube and accessory cells.

Cell Adhesion↗
PubMed1994

Oryzacystatin exogenously introduced into protoplasts and regeneration of transgenic rice.

Oryzacystatin (OC) is a proteinaceous cysteine proteinase inhibitor involved in the biodefense of rice seeds. To create transgenic rice plants with increased OC activity, we introduced an OC expressing vector into rice protoplasts and obtained transformed calli. The expression vector contained a bacterial inaA DNA fragment in the 3'-noncoding region as a tag to distinguish the introduced DNA from the intrinsic OC gene. The OC vector and a selection marker gene conferring hygromycin resistance were used together to transfect into rice protoplasts. A number of hygromycin-resistant calli were obtained and studied by polymerase chain reaction and genomic Southern blotting to find if the exogenous OC gene had been integrated. The calli were studied by northern blotting as well to examine mRNA expression. The results showed that integration and expression of the introduced OC gene occurred in 51% and 27%, respectively, of 156 subcultures from 15 hygromycin-resistant calli. As a final step, transgenic rice plants were regenerated from the calli expressing OC. Leaves and seeds from the plants had higher OC activities than those from nontransgenic plants.

Base Sequence↗
PubMed2002

Assignment of UVB-responsive cis-element and protoplastization-(dilution-) and elicitor-responsive ones in the promoter region of a carrot phenylalanine ammonia-lyase gene (gDcPAL1).

Expression of a carrot phenylalanine ammonia-lyase (PAL) gene (gDcPAL1) in suspension-cultured carrot cells is induced by dilution of the culture or by application of a fungal elicitor, as well as by ultraviolet B (UVB) irradiation. We demonstrated that among its upstream cis-elements (Takeda et al. [1997] Photochem. Photobiol. 66, 464-470), L4 is UVB responsive, and L1 is protoplastization- (dilution-) and elicitor responsive, from studies with transiently transformed mutated or truncated g-DcPAL1 promoter-luc constructs. This conclusion is consistent with our observation that PAL activities induced by UVB and by protoplastization (dilution) or elicitor are additive.

Daucus carota↗
PubMed1991

[The study of Bacillus sphaericus Ts-1 protoplast-plasmid transformation of electroporation].

This report gave the best conditions of Bacillus sphaericus Ts-1 protoplast-plasmid pHV33 electroporation. The highest transformation frequency and transformation efficiency induced by three pulse of 21 KV/cm and 10 microseconds duration applied at an interval of one sec., was 2.44 x 10(2) transformants/micrograms DNA and 3.16 x 10(-6) respectively. The saturated concentration of DNA absorbed by the protoplast was 5 micrograms DNA/10(9) cells/ml. By means of this method, pJB417, a recombinant mosquito larvicide clone, was introduced into B. subtilis 168M and B. sphaericus Ts-1. The transformants of B. subtilis 168M with biocide activity were obtained, but the toxicity of B. sphaericus Ts-1 was not increased.

Animals↗
PubMed1991

Anthracycline antibiotic 2-hydroxyaclacinomycins. I. 2-Hydroxyaclacinomycin-producing recombinant obtained from aclacinomycin-blocked mutants of Streptomyces galilaeus by a technique of protoplast fusion.

The technique of protoplast fusion which optimized prototrophic recombination in aclacinomycin-producing Streptomyces galilaeus was studied and applied to the construction of new anthracycline analog-producing recombinant upon genetic cross of two specific mutants blocked in aclacinomycin biosynthesis. Thus, 2-hydroxyaclacinomycin-producing recombinant was obtained by the protoplast fusion.

Aclarubicin↗
PubMed1978

Diploid formation of Candida tropicalis via protoplast fusion.

Haploid auxotrophic mutants were produced from Candida tropicalis, and protoplast usion was induced by polyethylene glycol. The resulting nutritional complementation was due to heterokaryon formation and, at a much lower frequenty, to spontaneous diploidization. During cultivation, heterokaryotic clones regularly gave rise to heterozygous diploids from which, in turn, haploids could be isolated. The technique of protoplast fusion gives an opportunity for genetic analysis of this and similarly asexual fungal species.

Candida↗
PubMed1980

Characterization of conidiation mutants in Trichoderma viride by hyphal anastomosis and protoplast fusion.

Conidiation mutants were isolated from the fungus Trichoderma viride, and tested for complementation by both anastomosis and protoplast fusion. They could be grouped into three classes: (1) colour mutants; (2) mutants with reduced conidiation; and (3) nonconidiating mutants. Several mutants of classes (2) and (3) were incapable of anastomosis, but via protoplast fusion they produced heterokaryons suitable for complementation tests.

Genetic Complementation Test↗
PubMed1997

Expression of a bacterial endo beta-1,3-glucanase gene under the control of CAMV 35S promoter in Nicotiana plumbaguinifolia protoplasts.

A gene which codes for a thermostable endo beta-1,3-glucanase (EC 3.2.1.39) from a Gram positive anaerobic thermophilic bacteria Clostridium thermocellum F7, was fused to 35S promoter and polyadenylation signal of Cauliflower Mosaic Virus (CaMV) strain Cabb B-D. This chimaeric gene fusion was introduced into Nicotiana plumbaguinifolia protoplasts using PEG-mediated DNA transfer method of transformation. Transient expression of the thermostable endo beta-1,3-glucanase was carried out in the protoplasts and was assayed at 70 degrees C pH 8.0, suggesting that the chimaeric gene fusion: (i) is correctly transcribed and translated in plant cells; (ii) the product of translation (the thermostable endo beta-1,3-glucanase protein) is easy to assay since most of the plants' enzymes have their optimal reaction temperature at 40-60 degrees C and at neutral or weak-acidic condition which is a characteristic of plant cells; (iii) can be used as a model for studying and understanding some of the mechanisms of plant defence systems at the enzyme protein level, in case of stress conditions.

Caulimovirus↗
PubMed2001

Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts.

BACKGROUND: Quantifying plant gene expression by flow cytometry (FCM) would allow multidimensional cell-parameter analysis on a per-cell basis, thereby providing insight into the cellular mechanisms of plant gene regulation. Here we sought to establish quantitation by FCM of plant hormone (abscisic acid, ABA)-inducible green fluorescent protein (GFP) expression and to compare the method directly with traditional reporter enzyme assays. MATERIALS AND METHODS: GFP, beta-glucuronidase, and luciferase reporter genes driven by ABA-inducible or constitutive promoter constructs were expressed in transiently cotransformed rice protoplasts and reporter activities quantified by FCM (for GFP) or traditional enzyme assays. Treatments included cotransformations with specific ABA signaling effector cDNA constructs (encoding VIVIPAROUS-1, an ABA transcription factor, and ABA-INSENSITIVE1-1, a dominant-negative protein phosphatase regulator) and the ABA agonist lanthanum chloride. Dual-color FCM was also performed on GFP-expressing cells immunodecorated with an mAb recognizing a rice cell surface epitope. RESULTS: Quantitative analysis of ABA-inducible gene expression by FCM using GFP as reporter gave comparable results to traditional reporter enzyme assays, although the signal-to-noise ratio was less for FCM, which can be a limitation of the method at low promoter strengths. Multiparameter-correlated analysis of ABA-inducible GFP expression with a plasma membrane marker showed no apparent correlation between ABA sensitivity, marked by GFP, and presence of a cell surface arabinogalactan glycoprotein. CONCLUSIONS: Quantitative FCM of GFP-expressing plant cells is a rapid, robust, reproducible, and value-added method relative to traditional enzymatic reporter gene assays.

Abscisic Acid↗
PubMed1984

Recombination efficiency is a quadratic function of the length of homology during plasmid transformation of Bacillus subtilis protoplasts and Escherichia coli competent cells.

Recombination efficiency between transforming and resident plasmids was 100 times higher in Bacillus subtilis protoplasts than in Escherichia coli competent cells. In both systems it varied with the square of the length of the sequences common to two plasmids within the studied range 0.96-8.8 kb in B. subtilis, 0.72-3.9 kb in E. coli. The observed exponential dependence may be characteristic of recombination occurring within a relatively short interval bordered by relatively long heterologous regions.

Bacillus subtilis↗
PubMed1985

Stabilized non-complementing diploids (Ncd) from fused protoplast products of B. subtilis.

Non-complementing diploids (Ncd) displaying the parental phenotype can be selected from polyethylene glycol (PEG)-treated fused polyauxotrophic protoplasts of Bacillus subtilis. These bacteria carry the two parental genomes, but only one of them is phenotypically expressed, the other being replicated but not expressed. Cellular cloning and DNA-DNA in situ hybridization led to the discovery of non-complementing diploid cells which at first sight could have been considered as parental haploids. The new class of stabilized Ncd (10(-7) segregants) can be obtained either directly after the primary fusion event or from segregating Ncd after further growth. The totally inactive chromosome of a stable Ncd can be activated after PEG-induced self fusion. DNA-mediated transformation studies using crude stable Ncd lysates as DNA donors show low frequencies for the genetic markers from the 'silent' chromosome. Contrary to the unstable Ncd situation, however, these frequencies remain low even with purified donor DNA. The differences in the transformation properties of the non-expressed markers are correlated to Ncd clone stability. These facts suggest that chromosome inactivation in PEG-induced fusion involves at least a two-stage process. The first would be reversible and the second irreversible, thus preserving the inactive chromosome state.

Bacillus subtilis↗
PubMed1982

Fatty acid composition of lipids of Escherichia coli W 1655 F+ and its stable protoplast type L-form.

The comparative fatty acid analysis of extractable and non-extractable lipids of Escherichia coli W 1655 F+ and its stable protoplast type L-form shows quantitative as well as qualitative differences. From 10 different fatty acids obtained 16:0, 17:0 and 18:0 are present at about the same quantities in the lipid fractions of the bacterial and L-form. The absence of larger amounts of 12:0, 14:0, and 14:beta OH fatty acids in non-extractable L-form lipids reflects the loss of the cell wall in L-form cells. 16:1 fatty acid was found in L-form lipids only. This qualitative difference and the 2-3 times higher content of 18:1 in L-form lipids and the 7 times lower content of cyc 19:0 in extractable lipids of the L-form may be interpreted as alterations characteristic for the changed composition of the cytoplasmic membrane in L-form cells.

Chromatography, Gas↗
PubMed2004

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts.

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.

Amino Acid Sequence↗
PubMed1993

The nonradioactive chloramphenicol acetyltransferase-enzyme-linked immunosorbent assay test is suited for promoter activity studies in plant protoplasts.

Four different promoters--the 35S, a double 35S promoter, and two different promoter fragments of the ribulose-bisphosphate carboxylase small subunit (rbcS)--were fused to the reporter gene chloramphenicol acetyltransferase (CAT) and transient expression studies were performed in potato protoplasts. Promoter strength was evaluated by using a nonradioactive CAT immunoassay not previously tested with plant cells. The activities of the rbcS promoters were 10-fold less than those of the 35S promoters. Unspecific reactions due to the immunoassay adopted were very low, and the sensitivity was in the range of that of other radioactive and nonradioactive reporter gene assays. The CAT-enzyme-linked immunosorbent assay test is a suitable nonradioactive alternative to test relatively weak promoters in plant systems.

Chloramphenicol O-Acetyltransferase↗