Protoplasts isolated from cell suspensions of albino Petunia hybrida were grown for 10 d at the interface between aqueous culture medium and oxygenated perfluorodecalin. Protoplasts synthesised new cell walls and divided normally at the fluorocarbon/culture medium interface, the mean division frequency of protoplasts being increased by 37% (P < 0.05) in this system when the perfluorodecalin was saturated with oxygen prior to use. The mean division frequency of protoplasts was further increased to a maximum of 52% above control (P < 0.01) when the medium overlaying the oxygenated perfluorodecalin was supplemented with 0.01% (w/v) of the co-polymer surfactant, Pluronic F-68. These results demonstrate a beneficial and synergistic effect of supplementing protoplast culture systems with oxygenated perfluorodecalin and Pluronic F-68.

Cell Division↗

PubMed1994

Transformation of Solanum dulcamara protoplasts and regeneration of transgenic plants.

We have achieved successful transformation of Solanum dulcamara protoplasts by direct DNA uptake and regeneration of transgenic plants. The plasmids pDW2 carrying CAT gene and pCaMVNEO carrying NPTII gene were used. The electroporation voltage was 1500 V, which gave a field strength of 1500 V/cm with a time decay constant of 59.4 sec. The concentration of plasmid was 20 micrograms/2 x 10(6) protoplasts. Under these conditions, a very high transformation efficiency was obtained, with relative transformation frequency being up to 12.4% and absolute transformation frequency 2.4 x 10(-4). The activity of NPTII was detected in 75% of the kanamycin resistant calli and all of the plants regenerated from resistant calli. Southern blot analysis showed that the DNA sequence of NPTII gene derived from pCaMVNEO plasmid existed in the transformed plants of S. dulcamara.

Blotting, Southern↗

PubMed1994

Equipment for the large-scale electromanipulation of plant protoplasts.

Electric fields are now used extensively for the genetic manipulation of plant cells through protoplast fusion and direct gene uptake. The cost of commercially available electrofusion and electroporation equipment remains prohibitive for many laboratories. This paper describes an electronic apparatus, suitable for the large-scale electrofusion and electroporation of plant protoplasts that is compatible in both function and cost with commercially available equipment.

Biomedical Engineering↗

PubMed1992

Quantification and comparison of chloramphenicol acetyltransferase activity in transformed plant protoplasts using high-performance liquid chromatography- and radioisotope-based assays.

Rice and petunia leaf and cell suspension protoplasts were transformed by electroporation in the presence of pDW2. This plasmid contains a chloramphenicol acetyltransferase (CAT) coding region under the control of a promoter constructed from sequences of the cauliflower mosaic virus genome. We have compared two different approaches to measuring CAT activity in this system, namely high-performance liquid chromatography (HPLC) and a radioisotope-based method. Our results show that both techniques have a similar detection limit of 10 mU CAT and (with an activity greater than 10 mU CAT) the standard error for measuring known amounts of CAT activity was less than +/- 12% for both assays. The HPLC technique, however, has a greater linear response range of 10-600 mU CAT than the radioisotope method, which has a range of 10-400 mU CAT. The HPLC assay also requires a shorter assay time. As a result of this work we believe that HPLC is a viable alternative to the radioisotope-based assay described.

Cells, Cultured↗

PubMed1991

Design and use of synthetic oligonucleotide probes in the cloning of delta-endotoxin genes from Bacillus thuringiensis.

A detailed protocol is described for the design and use of synthetic oligonucleotide probes for screening DNA libraries from Bacillus thuringiensis var. kurstaki (strain HD191) for copies of the gene (tox) encoding the insecticidal delta-endotoxin. Two homologous tox genes were identified in this organism; one of these was located on a 75-kb plasmid and the other on a second large plasmid or the bacterial chromosome. A tox gene was isolated as a 6.5-kb HindIII fragment of B. thuringiensis plasmid DNA.

Bacillus thuringiensis↗

PubMed1991

Surface exposure of O1 serotype lipopolysaccharide in Klebsiella pneumoniae strains expressing different K antigens.

Surface exposure of the O1 serotype lipopolysaccharide in encapsulated Klebsiella pneumoniae strains belonging to different serotypes was examined by using the O1 antigen-specific bacteriophages FC3-1 and FC3-2 in conjunction with immunogold electron microscopy and enzyme immunoassays with specific antisera. Despite the presence of the capsular polysaccharide, the O1 antigen was exposed at the cell surface in strains producing K2, K7, K8, K12, K19, K21, K22, K34, K35, K42, K45, K55, K57, K62, K66, K69, and K70 capsular polysaccharides. However, in strains producing K1, K10, and K16 capsular polysaccharides, the O1 antigen was masked by the K antigen. These results suggest that, since the O1 antigen is surface exposed in many different strains of K. pneumoniae with different capsular serotypes and is also able to immunoprotect, its potential as a useful vaccine component should not be overlooked.

Animals↗

PubMed1990

Introduction of a yeast artificial chromosome vector into Saccharomyces cerevisiae cells by electroporation.

Explore the source record for details and available documents.

Chromosomes, Fungal↗

PubMed1990

Conservative approach to the management of pelvic inflammatory disease.

Clinical criteria and treatment protocols are outlined for 663 cases of mild, moderate and severe pelvic inflammatory disease (PID). Data on 176 patients requiring admission to hospital and who were treated conservatively with antibiotics are analysed. Criteria for operative intervention are outlined; only 1 patient required surgical intervention. The study suggests that, provided strict criteria are adhered to, conservative management of PID is both safe and effective and offers outlying hospitals and rural practitioners a plan of management that can be safely followed before resorting to referral to major centres.

Amoxicillin↗

PubMed1989

Direct DNA transfer to plant cells.

A range of somatic cell and molecular techniques are now available to supplement conventional plant breeding. The introduction and expression of foreign DNA has been used to modify basic aspects of physiology and development, to introduce commercially important characteristics such as herbicide and insect resistance into plants and to insert genes suitable as dominant selectable markers for somatic hybridisation. Several techniques for direct DNA delivery are available, ranging from uptake of DNA into isolated protoplasts mediated by chemical procedures or electroporation, to injection and the use of high-velocity particles to introduce DNA into intact tissues. Direct DNA uptake is applicable to both stable and transient gene expression studies and utilises a range of vectors, including those employed for gene cloning. Although the frequency of stable transformation is low, direct DNA uptake is applicable to those plants not amenable to Agrobacterium transformation, particularly monocotyledons.

DNA↗

PubMed1987

Labour after caesarean section--the problem of scar dehiscence.

Conventional criteria for the prediction and diagnosis of dehiscence of a caesarean section scar during labour were prospectively evaluated. Of 70 patients selected to undergo trial of labour, scar dehiscence occurred in 2 of 35 mothers delivered vaginally and in 4 delivered by caesarean section. Conventional predictive and diagnostic criteria correlated poorly with the occurrence of scar dehiscence. These limitations should be recognised and, during trial of labour after caesarean section, emphasis should be placed on careful monitoring of maternal and fetal condition.

Adult↗

PubMed1986

Transfer of antibiotic resistance genes between yeast and mammalian cells under conditions favoring cell fusion.

Antibiotic resistance to G418 has been transferred into Chinese hamster cell lines via a plasmid vector. The same plasmid, which also contained the Leu2 gene, has been used to transform Leu2- yeast (strain MC16) to leucine prototrophy. Subsequent fusion between transformed yeast and untransformed hamster cells demonstrated that plasmid DNA could be transferred and its genes expressed within the mammalian cell genome. The fusion of transformed hamster cells with untransformed MC16 yeast cells demonstrated that DNA integrated within the mammalian cell genome could be transferred to correct the Leu2 deficiency and also confer G418 resistance on some yeast colonies.

Animals↗

PubMed1986

Transformation of the genomic expression of plant cells.

Agrobacterium-induced transformation of plant cells results from integration of T-DNA of the Ti or Ri plasmids into the genome of susceptible plants. Expression of T-DNA genes induces physiological changes in transformed cells which modify normal plant development to produce proliferations characteristic of crown gall and hairy root diseases. Understanding of the molecular basis of the transformation events associated with these examples of naturally occurring genetic engineering of plant cells, has stimulated efforts to construct vectors for transferring specific genes into plants. Vector construction has progressed from the use of wild-type Ti plasmids, giving phenotypically abnormal regenerated plants, to non-oncogenic plasmids. The range of vectors now available should enable useful foreign genes to be inserted into a range of dicotyledons and monocotyledons without impairing normal plant development.

Arginine↗

PubMed1982

Ultrastructure of the cell envelope layers and surface details of Legionella pneumophila.

Ten strains representing six serogroups of Legionella pneumophila were examined by electron microscopy using freeze-etching, thin-sectioning and negative-staining techniques. In addition, selected strains were examined further as shadowed and freeze-dried preparations and by scanning electron microscopy. The cell envelope consisted of two membranes, evident in fractured specimens as four short ridges. The major fracture plane was through the inner membrane, and therefore the protoplasmic and extracellular fracture faces of this membrane were predominant. With the exception of one strain (Togus 1), the particle arrangement on these fracture faces was random. A peptidoglycan-like cell wall layer was revealed only in sections of partially plasmolysed cells. Membrane-bounded poly-beta-hydroxybutyrate-like granules were evident in the cytoplasm and these frequently showed plastic deformation due to fracturing. Although appendages were present, the surfaces of organisms and of isolated cell membranes showed no regular arrays of particles.

Cell Wall↗

PubMed1981

Effect of inoculation of Zea mays with Azospirillum brasilense strains under temperate conditions.

Seven strains of Azospirillum brasilense were compared for their effect on the growth of Zea mays grown under temperate conditions in sand--vermiculite pot cultures. Inoculation with all seven strains tested, including Fix(-) mutant strains, increased dry weight and total nitrogen content of shoots, but nitrogen concentrations were unaffected. Low and variable rates of acetylene reduction activity were observed from excised roots of inoculated plants without preincubation. Estimates of N2-fixing A. brasilense associated with inoculated roots showed differences between strains in establishing themselves in the rhizosphere and endorhizosphere. In some strains enrichment in the endorhizosphere of roots occurred following inoculation, but the relative numbers and location of the strains did not appear to affect the yield response.

Acetylene↗