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PubMed2001

Life without a cell membrane: regeneration of protoplasts from disintegrated cells of the marine green alga Bryopsis plumosa.

When the multi-nucleate giant cells of the green alga Bryopsis plumosa (Huds.) Ag. are injured, the protoplasm is extruded from the cells and can generate spontaneously numerous new cells. The cell organelles aggregate rapidly in seawater and become covered with a gelatinous envelope within 15 minutes. A lipid cell membrane is formed inside the envelope within 9 to 12 hours and about 15% of the original cell membrane is recycled to make the membrane of new protoplasts. Cytochemical studies using Nile Red and various enzymes revealed that the primary envelope is initially composed of polysaccharides, and then transformed into a polysaccharide-lipid complex. Fluorescein diacetate staining showed that the primary envelope has some characteristics of a cell membrane including semi-permeability and selective transport of materials. The aggregation of cell organelles appears to be mediated by two kinds of materials, one present in vacuolar sap and the other on the surface of the cell organelles. About a thousand new cells were generated from a single disintegrated branch and 40% of them eventually developed into mature plants.

Cell Division↗
PubMed1987

Laser-induced fusion of mammalian cells and plant protoplasts.

An ultraviolet-laser microbeam was shown to be suitable for inducing fusion of individually selected plant protoplasts or of B-lymphocytes with myeloma cells. The fusion took place in normal culture medium and the fusogenic condition perturbed the cells only for a fraction of a millisecond. Without manipulating the cell culture except for exposing the cells to laser light, fusion products between preselected individual pairs may be produced.

Animals↗
PubMed1993

A mutant allele skt5 affecting protoplast regeneration and killer toxin resistance has double mutations in its wild-type structural gene in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains carrying the mutant allele skt5 are resistant to the killer toxin of Kluyveromyces lactis and are defective in protoplast regeneration. The DNA sequence analysis of the cloned mutant skt5 gene showed a nucleotide substitution (causing a glycine-to-glutamic acid substitution) and also a nucleotide insertion (causing a frameshift at the extreme carboxyterminal region) in the structural region from its wild-type gene.

Alleles↗
PubMed1995

Effects of degraded schizophyllans on regeneration of protoplast cells of Saccharomyces cerevisiae.

Schizophyllan was heated at 100 degrees C in 85% dimethyl sulfoxide (DMSO) containing 0.01 M H2SO4 for various times, and fractionated by gel-permeation chromatography. Molecular weights (M(r)) of the depolymerized products thus obtained were measured in water and DMSO by GPC-LALLS to estimate their conformations in water. The products with triple helical structure stimulated regeneration of Saccharomyces cerevisiae protoplast cells, while those of single chain conformation were totally inactive.

Hydrolysis↗
PubMed1997

Transient expression of goat growth hormone gene in poplar (Populus alba L.) protoplasts: a quick method for detection of foreign gene expression in mRNA level.

We developed a sensitive, accurate, and fast method to detect foreign gene expression using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) in poplar tree (Populus alba L.) protoplasts. Template total RNA was purified by removing the transfected foreign gene vector completely with DNase I treatment before the RT reaction. Expression of cDNA that encodes goat growth hormone was confirmed at the mRNA level 24 h after electroporation mediated DNA transfer.

Animals↗
PubMed1997

Mutational analysis of the potato virus Y 5' untranslated region for alteration in translational enhancement in tobacco protoplasts.

The 185 nucleotide 5' untranslated region (5'UTR) of potato virus Y ordinary strain (PVY-O) showed translation-enhancing activity on the beta-glucuronidase (GUS) gene in tobacco protoplasts. Mutational analysis of the 5'UTR was done to find sequence motifs necessary for the enhancement. Deletions within the 1-130 nucleotide region of 5'UTR stimulated the GUS expression in some cases, while the GUS activity declined with deletions in the 131-185 nucleotide region. The results indicated that the last 55 nucleotides of PVY-O 5'UTR might play the much important role in the translational enhancement in plant cells.

Amino Acid Sequence↗
PubMed2000

A simple and rapid extraction of high molecular weight chromosomal DNA from Bacillus subtilis protoplasts for cosmid cloning and interspecific transformation.

After conversion of Bacillus subtilis vegetative cells to protoplasts, a simple and rapid method for extracting high-molecular-weight chromosomal DNA was devised with the inclusion of bovine serum albumin and phenol-chloroform treatments. The DNA sample thus prepared was the size of 100-450 kb and could be used for cosmid cloning and interspecific transformation.

Animals↗
PubMed2000

Interspecific transformation of Bacillus subtilis competent cells by chromosomal DNA in lysates of protoplasts of Bacillus amyloliquefaciens.

Competent cells of Bacillus subtilis were transformed with chromosomal DNA in lysates of protoplasts of B. subtilis or B. amyloliquefaciens. The interspecific transformation frequency of B. subtilis by cysA in a conserved region was 3.1 x 10(4) transformants per microg DNA, 60 times higher than that for conventional transformation using purified DNA. Increased interspecific transformation frequencies of B. subtilis were also observed for arg-1, lys-1, leuB, aroG, thr-5, hisH, or metC markers outside the conserved region (3.1 x 10 approximately 5.2 x 10(2) transformants per microg DNA). An interspecific cotransformation ratio (33-50%) as high as an intraspecific one (46%) using purified DNA was also detected between cysA and rpsL markers, which are separated by 16 kb on the B. subtilis chromosome. Interspecific double transformation of the cysA-arg-1 or cysA-metC marker was observed, which have not been detected for conventional transformation. The involvement of mutS in the interspecific transformation was not significant.

Bacillus subtilis↗
PubMed2000

Hybridization and breeding of the benomyl resistant mutant, Trichoderma harziantum antagonized to phytopathogenic fungi by protoplast fusion.

A diploid strain obtained from heterokaryons of Trichoderma harzianum by protoplast fusion grew on minimal medium containing 100ppm benomyl. This strain inhibited the growth of the phytopathogenic fungus Fusarium oxysporum f. sp. raphani on paired cultures and also protected against radish yellows and a drop in germination induced by F. oxysporum f. sp. raphani.

Benomyl↗
PubMed2001

A simple and rapid method for intra- and interspecific transformation of Bacillus subtilis on solid media by DNA in protoplast lysates.

A simple method for intra- and interspecific transformation of Bacillus subtilis on solid media has been devised with DNA in protoplast lysates, 0.8% agar, glutamate, and yeast extract. The transformation frequency is 2.3 x 10(3) transformants per microg DNA, 10-20 times higher than that for conventional transformation on solid media. The method can be applicable to transformation in microtiter plates.

Bacillus subtilis↗
PubMed2005

Transient RNAi induction against endogenous genes in Arabidopsis protoplasts using in vitro-prepared double-stranded RNA.

RNA interference is a powerful technique for suppressing gene functions in many eukaryotes including plants. Here we show that introduction of double-stranded RNA into Arabidopsis protoplasts leads to marked silencing of endogenous genes, as observed previously for transgenes [Biosci. Biotechnol. Biochem., 67, 2674-2677 (2003)]. This simple system should be useful for functional analysis of genes involved in fundamental cellular processes.

Arabidopsis↗
PubMed2006

Gene transfer between different Trichoderma species and Aspergillus niger through intergeneric protoplast fusion to convert ground rice straw to citric acid and cellulases.

Single-stage direct bioconversion of cellulosic materials to citric acid using intergeneric hybrids obtained from three different Trichoderma species and Aspergillus niger was carried out. The recent results were obtained on the basis of either resistance or sensitivity to one or more of five metal ions, two catabolite repressors, and five antifungal agents, which were used in this study at different concentrations. Sixty-six fusants were isolated after using the three intergeneric protoplast fusion experiments, belonging to two types of intergeneric fusants. Fusants of the first type are heterokaryons (35 fusants). On the other hand, those of the second type are haploids (31 fusants), i.e., they were stable. The present study can be successfully applied in the construction of 14 new genetic fusants, which produced at least 100% more citric acid than the citric acid producer strain A. niger. Out of the fusants, three (1/18, 2/13 and 2/15) showed about a threefold increase of citric acid production in comparison with the parent A. niger strain. Furthermore, studies on DNA content showed that this finding may be submitted on the evidence that citric acid and cellulases production was not correlated with DNA content; however, the productivity depends on specific DNA content.

Antifungal Agents↗
PubMed1981

Phospholipid composition of chlorophyll-free mitochondria isolated via protoplasts from oat mesophyll cells.

Mitochondria were isolated from oat primary leaves via mesophyll protoplasts and subjected to phospholipid analysis. In mesophyll cells mitochondria account for only small proportions of cellular phospholipids (in the order of 5%) and proteins (in the order of 2%). Contamination by lipids from other membranes was insignificant as indicated by the absence or very low levels of chlorophyll, galactolipids and steryl glycosides. The absence of 3-trans-hexadecenoic acid in phosphatidylglycerol from mitochondria of green cells serves an an additional criterion of purity. The phospholipid mixture extracted from these mitochondria resembles phospholipids in mitochondria from non-green tissues regarding composition as well as fatty acid profiles. Therefore, mitochondria maintain a rather constant lipid profile and in contrast to plastids do not respond at this level to differences in the physiological status of their housing cell. Palmitic acid in mitochondrial phosphatidylcholine and phosphatidylethanolamine is primarily localized at the C-1 position of the glycerol moiety. Two enzymatic activities so far not described in mitochondria, formation of acylgalactosyl diacylglycerol and hydrolysis of acyl-CoA, were found in the purified mitochondrial fraction.

Chlorophyll↗
PubMedDate not supplied

[The influence of protoplast media on the tumor induction on kalanchoe leaves].

Experiments concerning tumor transformation of plant cells or protoplasts need complete knowledge of the effects of media components. Here inhibiting effects of cellulase Onozuka, glucose, sucrose and mannitol (up to 0.3M), of tissue culture media, and of the antibiotic aureomycin on the tumor initiation process in Kalanchoe leaves are reported. Also the influence of these substances on the growth of the tumor inducing Agrobacterium tumefaciens is studied. The results lead to the design of conditions which should be favourable to in vitro transformation.

Bacteria↗
PubMedDate not supplied

Fusiogenic activity of natural amphiphiles, 5-n-alkylresorcinols in a yeast protoplast system.

Two homologues of cereal grain resorcinolic lipids, 5-n-heptadecylresorcinol and 5-n-heptadecenylresorcinol studied in the system employing yeast cell protoplasts showed marked fusiogenic activity. The frequency of hybrid formation induced by studied amphiphiles was significantly higher than that obtained with the use of 40% (w/v) polyethylene glycol 4000. The resorcinolic lipids as fusion-inducing agents did not affect regeneration of the cellular wall. The fusiogenic activity of resorcinolic lipids lost when calcium ions were absent in the medium. Fusiogenic activity of studied amphiphiles is related to their ability to induce non-bilayer structures within the cellular membranes.

Alkylation↗
PubMed1987

Protoplasts fusion hybrids from Candida albicans morphological mutants.

Three pairs of strains, derived from Candida albicans 1001 (wild type) and carrying appropriate selection markers, were crossed by means of protoplast fusion. The corresponding fusion products were regenerated in selection medium and hybrids of rapid growth and more defined characteristics were eventually isolated and analyzed for relevant properties. Hybrids from a cross of two auxotrophic strains with no morphological alteration had a normal morphology but gave rise to rough-filamentous segregants of mostly pseudomycelial appearance, probably through DNA content reduction. The observation was consistent with a previous suggestion that C. albicans 1001 carries recessive mutant alleles that can be unmasked. A permanently filamentous strain (Y- phenotype) when crossed with a mycelium deficient (F-) strain gave rise to wild-type hybrids with a normal capacity for mycelium formation. Finally, two Y- strains, with similar phenotypes consisting of rough colony formation and mostly pseudomycelial morphology of cells under all conditions, also complemented since they gave rise to wild-type hybrids. It is concluded that the determinants of the mutant phenotype must be different in these two strains.

Candida albicans↗
PubMed1983

Staining air dried protoplasts for study of plant chromosomes.

The air drying technique used in mammalian cytology was applied to isolated plant protoplasts for study of chromosomes. For cultured celery cells, this technique resulted in good spreads of metaphase chromosomes with high resolution. Mitotic chromosomes of Brassica species are relatively small, poor stained by common stains, and difficult to spread by the squash technique. In this study, however, the chromosomes of B. carinata in callus culture were spread well and stained clearly with Giemsa staining solution. The chromosome preparations by the present techniques should also be amenable to chromosome banding studies in plants.

Azure Stains↗
PubMed1984

Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts.

Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set.

Cell Fusion↗