Transfer of Liposome-Sequestering Plasmid DNA into Daucus carota Protoplasts

Reverse-phase evaporation lipid vesicles (REV) liposomes, consisting of phosphatidyl choline and stearylamine in 1:3 molar ratio, encapsulated approximately 30% of exogenously supplied recombinant DNA vector, pBR322. The DNA sequestered in REV liposomes was highly tolerant to DNase.     

A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

Three methods of plasmid delivery to mesophyll protoplasts ofNicotiana tabacum cv. Xanthi have been evaluated. Specifically;a) chemically stimulated uptake of isolated plasmid, b) deliveryof plasmid encapsulated in liposomes, and c) fusion of plasmid-containingspheroplasts, were combined with divalent cation (Ca²⁺ and Mg²⁺)or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol(PVA)] treatments. The quantity and quality of plasmid associatedwith intact protoplasts, was assessed by DNA-DNA blot hybridisationanalysis, following stringent washing to separate intact protoplastsfrom non-viable protoplasts and debris. Treatments which increasedassociation of plasmid with protoplasts decreased protoplastviability. Optimum association of plasmid with protoplasts,in the context of acceptable loss of viability, was achievedwhen protoplasts were interacted with either naked plasmid orliposomeencapsulated DNA in the presence of 15% w/v PEG 6000,or with Escherichia coli spheroplasts containing chloramphenicol-amplifiedplasmid in the presence of 25% w/v PEG 6000. Divalent cationsdid not stimulate significant plasmid delivery without unacceptableloss of protoplast viability. Strategies to further increasethe efficiency of plasmid delivery are discussed. (Received June 21, 1984; Accepted August 20, 1984)     

Transformation of Streptococcus lactis Protoplasts by Plasmid DNA

Polyethylene glycol-treated protoplasts prepared from Streptococcus lactis LM3302, a lactose-negative (Lac⁻) derivative of S. lactis ML3, were transformed to lactose-fermenting ability by a transductionally shortened plasmid (pLM2103) coding for lactose utilization.     

Polyethylene Glycol-induced Uptake of Bacteria into Yeast Protoplasts

Cells of the nitrogen-fixing bacterium Azotobacter vinelandii and the unicellular cyanobacterium Anacystis nidulans were introduced into protoplasts of Saccharomyces cerevisiae by the polyethylene glycol (PEG) method. Factors influencing the uptake frequency were examined, and experimental conditions were established for maximizing the uptake frequency. Under optimal conditions, each protoplast took-up a few bacterial cells. Electron-microscopic studies showed the localization of integrated bacterial cells in membrane-bound vesicles of the cytoplasm or large vacuoles. The protoplasts at the intermediate stages of uptake revealed two major mechanisms of uptake: (a) “endocytosis” by a single protoplast and (b) “cell fusion” between two or more protoplasts. Some bacterial cells disintegrated during the subsequent incubation period through a heterophagy-like process.     

Transformation of Bacillus Protoplasts by Plasmid pTP4 DNA

Polyethylene glycol (PEG)-induced protoplast transformation by plasmid pTP4 DNA encoded chloramphenicol resistance determinant was developed for Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus. Protoplasts were formed by treatment of cells with lysozyme and the transformation frequencies (transformants per regenerants) were in the range of 1.3 × 10^?2 to 7.1 × 10^?1. Reisolated plasmid DNA prepared from transformants exhibited covalently closed and open circular forms similar to those of the donor DNA. These results indicate that PEG-induced protoplast transformation is an adequate method for plasmid transformation and pTP4 is a useful plasmid as a cloning vector in a wide range of varieties of the genus Bacillus.     

Rapid Delivery of Foreign Genes into Plants by Direct Rub-Inoculation with Intact Plasmid DNA of a Tomato Bushy Stunt Virus Gene Vector

Tomato bushy stunt virus (TBSV) cDNA, positioned between a modified cauliflower mosaic virus 35S promoter and the hepatitis delta virus antigenomic ribozyme with a downstream nopaline synthase gene polyadenylation signal, established infections upon rub-inoculation of plants with intact plasmids. Application of this methodology produced a TBSV DNA-based gene vector which yielded readily detectable levels of localized foreign gene expression in inoculated leaves. This is the first demonstration of an infectious DNA from a member of the Tombusviridae which permits rapid TBSV-mediated foreign-gene expression upon direct rub-inoculation of miniprep DNA onto a variety of plant species.     

Binding of ColE1-kan Plasmid DNA by Tobacco Protoplasts: Nonexpression of Plasmid Gene

Protoplasts prepared from cultured tobacco cells were treated with ColE1-kan plasmid DNA, a hybrid of ColE1 and pSC105 plasmids bearing a gene for kanamycin resistance. The conditions employed permitted the uptake or irreversible binding of 2.9% of the added DNA in acid-insoluble form. Upon commencement of division, the treated cells were plated in agar medium containing kanamycin and differentiating hormones. Plantlets or shoots obtained as presumptive transformants were further tested on kanamycin medium by subculturing small leaf pieces. No evidence was obtained for expression of the kanamycin resistance gene of ColE1-kan in tobacco tissue.     

Yeast Spheroplasts Formed by Cell Wall-Degrading Enzymes from Oerskovia sp

Oerskovia sp. produces inducible extracellular enzymes which degrade the walls of various yeasts. Yeast spheroplasts are formed from both log- and stationary-phase cells.     

Transgenic Rice Plants Produced by PEG-Mediated Plasmid Uptake into Protoplasts

Embryogenic cell suspension cultures were obtained from calli developed from mature rice seeds of a Japonica type Itahan cultivar, Roncarolo. Protoplasts were isolated and transformed by PEG-treated method with plasmid pHP23 carrying the NPT Ⅱ gene which encodes resistance to antibiotic G-418. Protoplast-derived colonies were selected in presence of the inhibitor. Plants were regenerated and transplanted into soil in the green house. The presence of foreign gene in the regenerated plants was verified by PCR and Southern analysis.     

Transformation of Heat-Treated Clostridium acetobutylicum Protoplasts with pUB110 Plasmid DNA

Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55°C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44°C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55°C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55°C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44°C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 55°C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heatsensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycin-resistant (Km^r) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Km^r transformants.     

Surface Structure of Intact Cells and Spheroplasts of Pseudomonas aeruginosa

This report describes the ultrastructural features of Pseudomonas aeruginosa after freeze-etching of intact cells and enzymatically prepared spheroplasts. Freeze-etching of intact cells revealed two convex layers of the cell wall and particles within the hydrophobic interior of the cell membrane. Areas of the membrane free of particles were sometimes elevated in the form of rather large dome-shaped structures. Spheroplasts were formed from intact cells by the addition of trypsin to a reaction mixture of lysozyme and ethylenediaminetetraacetic acid. Spheroplasts contained the outer lipoid layer of the cell wall. It was possible to observe this cell wall layer in freeze-etch preparations of spheroplasts. The spheroplast membrane like that of intact cells was cleaved along a central plane to expose particles and particle-free areas.     

Transfer of Isolated Nuclei into Protoplasts of Trichoderma harzianum

Protoplasts released from young hyphae of Trichoderma harzianum contained 0 to 10 nuclei per protoplast, and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 μm in diameter. Nuclei were isolated from protoplasts of an auxotrophic mutant of T. harzianum and transferred into protoplasts obtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerous progeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from a wild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of a second strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when the regenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating. Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-type nuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern and colony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine 10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of this nuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.     

Cultivation of Rice Protoplasts and Their Transformation Mediated by Agrobacterium Spheroplasts

Improvement of the cultivation of rice (Oryza saliva L.) protoplastsisolated from suspension cultures led to their division at afrequency of 5 to 10%. Rapidly growing colonies were obtainedon a hormone-free medium when Agrobacterium tumefaciens spheroplastswere introduced into the protoplasts by polyethylene glycoltreatment. Opines corresponding to the strains of A. tumefaciensused for the spheroplast treatments were detected in some ofthese colonies at a frequency of about 10^–4. Using radioactiveprecursors, [¹⁴C]--ketoglutaric acid and [³H]-arginine, activitiesof nopaline synthase, a marker enzyme of nopaline-type crowngall, were also detected in some of these clones. These resultsshow that the rice cells were transformed by Ti plasmid introducedby the spheroplast method. (Received September 6, 1985; Accepted January 24, 1986)     

Surface Structure of Yeast Protoplasts

The fine structure of the yeast cell wall during protoplast formation was studied by means of phase-contrast microscopy and the freeze-etching technique. The freeze-etching results indicated that at least in some cases the entire wall substance was not removed from the surface of the protoplasts. After a treatment of 30 min to 3 hr with 2% snail enzymes, an innermost thin wall layer as well as remnants of the fibrillar middle layer sometimes could be demonstrated.     

用酚-氯仿直接从克隆中提取质粒DNA

目的:对用酚-氯仿直接从克隆中提取质粒DNA的方法进行了研究。方法:采用不同次数的酚-氯仿抽提,在不同RNaseA作用温度及作用时间下提取了三种质粒。结果:用酚-氯仿抽提2次,RNase A45℃作用8min,质粒纯度(OD260/OD280)为1.85左右、产量大于0.8μg/克隆。结论:本法不但快速,且所提质粒均能满足后续实验如酶切鉴定、测序等方面的需要。对于大批量重组克隆的筛选尤为适合。     

Exploitation of Plasmid pMRC01 To Direct Transfer of Mobilizable Plasmids into Commercial Lactococcal Starter Strains

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 × 10⁻⁴ transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01. All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the oriT-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that oriT of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.     

Introduction of Functional RNA into Plant Protoplasts by Electroporation

A simple apparatus was constructed for producing electric dischargeof varying intensities between two electrodes in a spectrophotometercuvette. This apparatus was used to study the conditions forefficient introduction of functional RNA into plant protoplastsusing RNAs of tobacco mosaic virus (TMV) and cucumber mosaicvirus (CMV). Electroporation under optimal conditions resultedin the production of TMV and CMV in 80% of the protoplasts fromtobacco cell line BY2. Infection by TMVRNA occurred in 60% ofVinca rosea suspension culture protoplasts and in 40% of tobaccomesophyll protoplasts. Electroporation in the presence of TMVand CMV particles also resulted in infection, suggesting thatpores larger than 30 nm are formed. The advantages and usesof the electrical method for introduction of functional RNAare discussed. (Received December 21, 1985; Accepted March 1, 1986)     

植物线粒体DNA质粒研究进展

本文列出了已发现的高等植物中的线粒体DNA质粒,按分子形状分为线粒体环状DNA质粒和线粒体线状DNA质粒,环状线粒体DNA质粒的特征是分子较小, 序列中有正向/反向重复序列,ORF一般较小。线状线粒体DNA质粒的特征是分子较大,末端有重复序列,5'端与蛋白质共价结合,有较长的ORF。还分别介绍了它们的复制机制、转录和起源。质粒间及质粒与核基因组、线粒体基因组、叶绿体基因组的同源性也作了介绍。最后,综述了植物线粒体DNA质粒与植物的细胞质雄性不育（CMS）之间的关系。