Role of DNase in recovery of plasmid DNA from Clostridium perfringens

Recovery of plasmid DNA from Clostridium perfringens 10543A and 3626B cleared lysates was significantly improved by the addition of 0.2% (vol/vol) diethylpyrocarbonate (DEP) before protoplast disruption in the cleared lysate protocol. Three previously undetected, large-molecular-mass plasmids (45.2, 51.9, and 68.2 megadaltons) were isolated from modified DEP-treated cleared lysates of C. perfringens 3626B. Two plasmids (9.4 and 30 megadaltons) were recovered from C. perfringens 10543A modified DEP-treated cleared lysates which previously required dye-buoyant density gradient centrifugation for visualization on agarose gels. Unsuccessful attempts to isolate plasmid DNA from Brij 58 cleared lysates of extracellular DNase-negative mutants of C. perfringens suggested the deleterious DNase activity was not extracellular. Cellular localization studies indicated that the cell wall-compartmentalized cell fraction contained 72.2% of the total DNase activity, whereas the extracellular and intracellular fractions demonstrated much less (26.8 and 1.0%, respectively). Cleared lysates prepared with DEP demonstrated much less DNase activity than cleared lysates prepared without DEP. The variable and irreproducible recovery of plasmid DNA from C. perfringens cleared lysates was attributed to cell wall-compartmentalized DNase.     

Role of DNase in recovery of plasmid DNA from Clostridium perfringens.

Recovery of plasmid DNA from Clostridium perfringens 10543A and 3626B cleared lysates was significantly improved by the addition of 0.2% (vol/vol) diethylpyrocarbonate (DEP) before protoplast disruption in the cleared lysate protocol. Three previously undetected, large-molecular-mass plasmids (45.2, 51.9, and 68.2 megadaltons) were isolated from modified DEP-treated cleared lysates of C. perfringens 3626B. Two plasmids (9.4 and 30 megadaltons) were recovered from C. perfringens 10543A modified DEP-treated cleared lysates which previously required dye-buoyant density gradient centrifugation for visualization on agarose gels. Unsuccessful attempts to isolate plasmid DNA from Brij 58 cleared lysates of extracellular DNase-negative mutants of C. perfringens suggested the deleterious DNase activity was not extracellular. Cellular localization studies indicated that the cell wall-compartmentalized cell fraction contained 72.2% of the total DNase activity, whereas the extracellular and intracellular fractions demonstrated much less (26.8 and 1.0%, respectively). Cleared lysates prepared with DEP demonstrated much less DNase activity than cleared lysates prepared without DEP. The variable and irreproducible recovery of plasmid DNA from C. perfringens cleared lysates was attributed to cell wall-compartmentalized DNase.     

Purification of mucin glycoproteins by density gradient centrifugation in cesium trifluoroacetate.

A procedure for the rapid isolation of mucin glycoprotein by density gradient centrifugation in cesium trifluoroacetate (CsTFA) is described. The separation of mixtures of rat tracheobronchial mucin, DNA, hyaluronic acid, and bovine serum albumin in CsTFA gradients was superior to that in cesium bromide gradients. Inclusion of guanidinium chloride or urea in the gradient had no influence on the separation obtained. The mucins isolated from sputum samples of cystic fibrosis patients by this procedure are largely free of nucleic acid, nonglycosylated proteins, and glycosaminoglycans. The results of the use of CsTFA gradient centrifugation for the isolation of mucin from extracts of bovine submaxillary gland are also presented. The CsTFA method is particularly suitable for the high-yield isolation of mucin from individual samples which are available in limited quantities.     

Establishment of pure neuronal and muscle precursor cell cultures fromDrosophila early gastrula stage embryos

Summary Primary cultures ofDrosophila gastrula stage embryonic cells will divide and terminally differentiate into morphologically recognizable neurons and muscles. The phenotypically mixed nature of this primary culture system has made it difficult to effectively analyze various parameters of cell growth and differentiation for individual cell types. We report here a simple and economic method to separate early embryonic precursors for different cell types, using a shallow linear reorienting Ficoll gradient at unit gravity. The separated cells were collected into fractions, cultured, and analyzed for their growth and differentiation patterns. The larger and denser cells of the first fractions differentiated to yield pure neuronal cultures, as judged by morphologic, immunologic, and biochemical criteria. Cells in the last fractions differentiated into a predominantly muscle-enriched cell population, which also contained a very small percentage of neurons morphologically distinct from those in the pure neuronal fractions. Approximately 35% of the early gastrula stage embryonic cells differentiate into neuronal cells, and 65% of the non-neuronal lineage cells later develop into predominantly muscle population. The method is highly reproducible, can process 3×10⁷ cells per procedure, and the recovery is >90% of the input cells. The separated cells are suitable for cell biological analyses as well as for biochemical and molecular studies of neuron and muscle precursors. Deceased.     

Simultaneous banding of rat embryo DNA, RNA, and protein in cesium trifluoroacetate gradients

A procedure for the simultaneous banding of cellular DNA, RNA, and protein by centrifugation in cesium trifluoroacetate (CsTFA) gradients is described. Starting with homogenates of Day 11 rat embryos, this procedure was used to separate total DNA, RNA, and protein. Under the conditions used DNA banded at a peak density of 1.63 g/ml, RNA at a peak density of 1.83 g/ml, and protein at a peak density of 1.40 g/ml. Nucleic acids isolated from CsTFA gradients were judged to be protein free. RNA isolated by this method is apparently free of DNA contamination; however, DNA isolated by this method does contain some RNA (less than 5% contamination).     

Isolation of DNA-free RNA, DNA, and proteins by cesium trifluoroacetate centrifugation

The ability to simultaneously isolate intact DNA-free RNA, genomic DNA, and proteins from a biological specimen can be useful in cloning genes and analyzing gene expression. Equilibrium density gradient centrifugation with CsCl is a useful tool for fractionating, quantitatively separating, and characterizing RNA, DNA, and the total quota of proteins, respectively, based on differences in their buoyant densities. In the present study we have reexamined the rarely used cesium salt, cesium trifluoroacetate, for the same purpose. A significant advantage of CsTFA lies in the fact that, unlike in CsCl, RNA can be recovered from a single, soluble fraction of the CsTFA gradient. Furthermore, unlike CsCl, CsTFA is freely soluble in ethanol so that co-precipitation of the salt in the recovered RNA upon alcohol precipitation does not take place. Hence, the RNA is recovered with minimum manipulations. The one-step separation of cellular macromolecule classes free of each other in small amount of starting materials provides a major advantage over other methods currently in use.     

A quantitative analysis of shotgun cloning in Bacillus subtilis protoplasts

Summary We used the Escherichia coli-Bacillus subtilis shuttle vector pHP13, which carries the replication functions of the cryptic B. subtilis plasmid pTA1060, to study the effects of BsuM restriction, plasmid size and DNA concentration on the efficiency of shotgun cloning of heterologous E. coli DNA in B. subtilis protoplasts. In a restriction-deficient strain, clones were obtained with low frequency (19% of the transformants contained a recombinant plasmid) and large inserts (>6 kb) were relatively rare (12% of the clones contained inserts in the range of 6–9 kb). The efficiency of shotgun cloning was severely reduced in restricting protoplasts: the class of large inserts (>6 kb) was under-represented in the clone bank (4% of the clones contained inserts in the range of 6–6.1 kb). Furthermore, BsuM restriction caused structural instability of some recombinant plasmids. Transformation of protoplasts with individual recombinant plasmids showed that plasmid size and transforming activity were negatively correlated. The size effect was most extreme with cut and religated plasmid DNA. The yield of clones was independent of the DNA concentration during transformation. It is therefore unlikely that clones were not detected because of simultaneous uptake of more than one plasmid. It is concluded that shotgun cloning in B. subtilis protoplasts is inferior to that in competent cells.     

Efficient Uptake of Cesium Ions by Rhodococcus Cells

Bacteria of the genus Rhodococcus were found to be able to accumulate cesium by means of active transport and nonspecific sorption on the cell surface structures. The maximum removal (up to 97%) of cesium from a medium supplemented with ammonium acetate was observed at 28°C, pH 7.8–8.6, and an equimolar content (0.2 mM) of potassium and cesium ions in the medium. The most active cesium-accumulating rhodococcal strains may be useful in biological treatment of industrial wastewaters contaminated with radionuclides.     

Complete nucleotide sequence of a quinolone resistance gene (qnrS2) carrying plasmid of Aeromonas hydrophila isolated from fish

Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621 bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment.     

A DNA library from an individual Beta patellaris chromosome conferring nematode resistance obtained by microdissection of meiotic metaphase chromosomes

We have used a standard protocol established for human chromosomes to create a chromosome-specific plasmid library from a Beta patellaris chromosome conferring nematode resistance. A monosomic addition line was chosen carrying 18 sugar-beet (Beta vulgaris L.) and one wild-beet (B. patellaris) chromosome. The wild-beet chromosome can readily be identified as a univalent during metaphase I of meiosis. Highly synchronized meiotic material was used to excise the univalents from four pollen mother cells. The chromatin was lysed in a 1 nl collection drop, the DNA purified and restricted with Rsa I, ligated into a vector containing universal sequencing primers, and amplified by the polymerase chain reaction. The amplified DNA was inserted into a standard plasmid vector and cloned. Approximately 23 000 recombinant plasmids were obtained of which 15 800 could be utilized. Their insert sizes ranged from 80 to 700 bp with an average of 130 bp. 61 clones were tested in more detail by genomic Southern hybridization with sugar-beet and wild-beet DNA. Of these 32 plasmids (52%) contained single-copy inserts, 11 (18%) were specific for wild-beet DNA indicating that the DNA cloned originates in the univalent chromosome. The application of this technique for establishing high-density RFLP maps for discrete regions of plant genomes is discussed.     

Release of transforming plasmid and chromosomal DNA from two cultured soil bacteria

The release of chromosomal and plasmid DNA from Acinetobacter calcoaceticus and Bacillus subtilis cultivated in minimal medium and broth over a period of 50 h was monitored and related to growth phase, autolysis, DNase production and natural competence. The released DNAs were biologically active in natural transformation. In addition, the circular integrity of a released B. subtilis shuttle vector (pHV14) was demonstrated by artificial transformation of Escherichia coli. In cultures of both strains high molecular weight DNA accumulated, particularly during the stationary and death phase (up to 30 g ml^-1). Generally, despite the presence in culture fluids of DNase activity (and of an intracellular enzyme, catalase, indicating some cell lysis) there was high transforming activity of chromsomal and plasmid DNA even 40 h after the cultures reached the stationary phase. In cultures of B. subtilis in minimal medium a presumably active release of intact plasmids and chromsomal DNA occurred during the competence phase. The release of biologically functional DNA during essentially all growth phases of a gram-positive and a gram-negative member of soil bacteria might facilitate horizontal gene transfer by transformation in natural habitats.     

Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

Synthesis and turnover of cell body-agglutinin as a pool of flagellar surface-agglutinin inChlamydomonas reinhardii gamete

Chlamydomonas reinhardii cells were broken in a French press and the soluble fraction was tested for agglutination activity. Deflagellated cell bodies ofmt ⁺ andmt ^- gametes yielded soluble fractions that were able to isoagglutinate gametes of the opposite mating type. When the wild-type gametes of opposite mating types were mixed, the cell body-agglutinins were used up during flagellar agglutination and subsequent cell fusion. When thefus mt ⁺ andmt ^- gametes agglutinated without successive fusion, the amount of cell body-agglutinins sharply decreased, then increased and reached the premixing level: the recovery was blocked by cycloheximide. When cells were treated with EDTA or trypsin, the cell body-agglutinins as well as flagellar surface-agglutinins were completely lost without apparent loss of motility. The EDTA extract contained the same amount of agglutinins as observed in the cell bodies before extraction, and this amount was about 100 times higher than that in the EDTA extract of isolated flagella. By the addition of trypsin inhibitor, the trypsinized gametes resynthesized the cell body-agglutinins. The process was sensitive to cycloheximide in both mating type gametes and to tunicamycin inmt ⁺ gametes.Abbreviations mt ^+/- mating type plus or minus - CHI cycloheximide - TI trypsin inhibitor - TM tunicamycin     

Purification of plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata

We report a straightforward protocol for isolating plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata. Plastids were purified using differential centrifugation and 2-step sucrose density gradients. We found that 10% polyethylene glycol 4000 was essential for maintaining plastid integrity prior to lysis. Plastid DNA isolated directly from the purified rhodoplasts was sufficiently pure for restriction endonuclease fragment analyses. Database comparisons of sequences generated randomly from a shotgun genomic library indicated that plastid DNA was 89% pure following ultracentrifugation in isopycnic cesium chloride equilibrium gradients. The protocol yields 30–50 μg of plastid DNA per 100 g of fresh algal tissue.     

Location of glucose oxidase during production by Aspergillus niger

The production of the enzyme glucose oxidase by Aspergillus niger is well documented. However, its distribution within the fungal culture is less well defined. Since the enzyme location impacts significantly on enzyme recovery, this study quantifies the enzyme distribution between the extracellular fluid, cell wall, cytoplasm and slime mucilage fractions in an A. niger NRRL-3. The culture was separated into the individual fractions and the glucose oxidase activity was determined in each. The extracellular fluid contained 38% of the total activity. The remaining 62% was associated with the mycelia and was distributed between the cell wall, cytoplasm and slime mucilage in the proportions of 34, 12 and 16%, respectively. Intracellular cytoplasmic and cell wall sites were confirmed using immunocytochemical labelling of the mycelia. In the non-viable cell, the mycelial-associated enzyme was distributed between these sites, whereas in the viable cell, it was predominantly associated with the cell wall. The distribution of the enzyme activity indicates that recovery from the solids would result in a 38% loss, whereas recovery from the extracellular fluid would result in a 62% loss. The results also suggest, however, that this 62% loss could be reduced to around 34% by disintegrating the solids prior to separation due to the contribution of the enzyme in the cytoplasm and slime mucilage. This was confirmed by independently establishing the percentage activity in the liquid and solid portions of a disintegrated culture as 62 and 38%, respectively.     

Biologically Active Oligosaccharides from Pectins of Pisum sativum L. Seedlings Affecting Root Generation

Two physiologically active oligosaccharide fractions were isolated from pectin of Pisum sativum L. cell wall after its partial acid hydrolysis. These fractions displayed stimulating and inhibiting effects on root formation in thin-layer explants. The subsequent separation of these fractions by gel permeation and anion-exchange chromatography resulted in fractions with effective concentrations two orders of magnitude lower than the concentrations of the initial fractions. The resulting oligosaccharides displayed their effect on the earliest stage of the rhizogenesis associated with formation of root primordias. The rhizogenesis-inhibiting fraction suppressed cell division by 30-50%. The stimulating fraction mainly contained fragments of xyloglucan and galactan, and the inhibiting fraction contained fragments of xyloglucan, galactan, and arabinan. The polymerization degrees of the stimulating and of the inhibiting oligosaccharides were 10-11 and 5-6, respectively.     

一株感染铜绿假单胞菌的双链RNA噬菌体PaP6的分离和鉴定

【目的】鉴定一株新分离的铜绿假单胞菌噬菌体PaP6的生物学特性。【方法】利用铜绿假单胞菌临床分离株PA038为宿主,从西南医院污水中分离得到一株裂解性噬菌体PaP6,观察其噬斑特点;氯化铯密度梯度离心纯化噬菌体颗粒后,用透射电子显微镜观察噬菌体形态;提取PaP6基因组,通过DNA酶和RNA酶酶切,做基因组酶切图谱分析;按照感染复数(MOI)分别为10、1、0.1、0.01、0.001和0.000 1加入噬菌体和宿主菌,裂解细菌后,测定噬菌体滴度;以MOI=10的比例加入噬菌体和宿主菌,绘制一步生长曲线;用112株铜绿假单胞菌临床分离株检测PaP6宿主谱。【结果】PaP6的噬斑直径约2 mm-4 mm,圆形透明,边缘清晰;PaP6噬菌体呈多面体立体对称的头部,直径约45 nm;酶切图谱表明PaP6基因组对DNase不敏感,对RNase敏感,未酶切基因组具有3节段双链RNA(dsRNA),长度分别约为9.0、4.5、3.5 kb,共约17 kb;当MOI为0.1时PaP6感染其宿主菌产生的子代噬菌体滴度最高,达到3.4×109 PFU/m L;用一步生长曲线描绘了其生长特性;PaP6可以感染40.1%的临床分离株,是一株比较广谱的噬菌体。【结论】首次报道了一株铜绿假单胞菌的ds RNA分节段噬菌体,分类学上属于囊病毒科,该噬菌体具有较广的宿主谱,在噬菌体治疗领域具有应用前景。     

Applicability of the co-inoculation technique using Agrobacterium tumefaciens shooty-tumour strain 82.139 in silver birch

In the co-inoculation technique, genetic transformation is performed using a mixture of Agrobacterium strains – shoot regeneration is induced by the wild-type strain 82.139, while the transferable genes are provided in a binary plasmid by another, non-oncogenic Agrobacterium strain. The aim of the present work was to study the applicability of co-inoculation under both in vitro and greenhouse conditions for in planta transformation in silver birch (Betula pendula Roth). In addition to the original method, several modifications of the technique including an genetically engineered 82.139 strain harbouring the binary pGUSINT were tested. The co-inoculations resulted in a gall formation frequency of 52–94% with greenhouse seedlings, and 4–63% with tissue-cultured plantlets, the shoot induction percentage varying by 0–13 in the greenhouse and 42–75 in vitro. PCR analysis verified that the majority of the regenerated material was non-transgenic, with a few individuals showing integration of the oncogenic T-DNA. According to the histochemical tests, however, some of the numerous differentiating buds and small shoots on gall tissues were transgenic, and contained the GUS reporter gene. The results show that it would have been necessary to apply selection pressure during differentiation in order to recover shoots transformed with the desired genes from the binary plasmid. The morphology and growth of all the regenerated plantlets was normal, suggesting that the oncogenic T-DNA was not expressed even though it was present. In conclusion, it was possible to obtain transgenic silver birch plantlets using the A. tumefaciens strain 82.139, but the co-inoculation method is not directly applicable as in planta transformation protocol.     

Plant regeneration from transformed embryogenic callus of an elite indica rice via Agrobacterium

The present study describes a simple and efficient protocol for plant regeneration from scutellar-derived embryogenic calli of an elite basmati indica rice (Oryza sativa L., cv Pusa Basmati 1) transformed with Agrobacterium. A supervirulent plasmid pTOK233 as well as a non-supervirulent plasmid pJB90GI containing -glucuronidase (gus) and hygromycin phosphotransferase (hpt) chimeric genes were used to assess transformation and regeneration efficiency. The effects of some factors like the bacterial density and inclusion of sorbitol in the medium on the co-culture and transformation have been evaluated; the procedure for selection and regeneration from transformed calli was found to be critical. Furthermore, co-culture and selection on regeneration medium was found to be better than callus medium and led to minimal media manipulations. Regeneration medium supplemented with 3% maltose was found to be better for regeneration as compared to 3% sucrose. The transformed calli were subjected to three cycles of regeneration, thus converting a higher number of transformation events into regenerants. The selected calli as well as leaf sections and roots of the transformants were GUS positive. The stable integration of the transgene was confirmed by polymerase chain reaction and Southern blot analysis of the transformants. Interestingly, the presence of three additional vir genes in supervirulent plasmid pTOK233 was not required for transformation as transformation was successful with non-supervirulent plasmid pJB90GI, although the transformation and regeneration frequency was higher with the former. This effective protocol for regeneration from transformed calli resulted in a relatively high transformation frequency.     

Rapid extraction of plasmid pGT5 from the hyperthermophilic archaeonPyrococcus abyssi

Hyperthermophilic archaea, specificallyPyrococcus spp., are the target of current efforts in developing heterologous expression systems. However, the published plasmid purification and plasmid screening protocols are long and tedious. We describe a fast, simple protocol for plasmid purification fromPyrococcus spp. developed while extracting the plasmid pGT5 fromPyrococcus abyssi cells. The protocol is modified from the procedures for commercial plasmid minipreps and is completed in about 20 min. The DNA is easily digested by restriction enzymes and can be used in sequencing reactions without additional purification.