Liposome-Mediated transfer of bacterial RNA into carrot protoplasts

The uptake of liposome-encapsulated E. coli [³H]RNA by carrot (Daucus carota L.) protoplasts was examined. [³H]RNA extracted from protoplasts that had been incubated with [³H]RNA-containing, large, unilamellar lipid vesicles (liposomes) obtained by ether infusion, and examined by sucrose gradient centrifugation and formamide-polyacrylamide gel electrophoresis, appeared substantially degraded, with a total elimination of 23S RNA and a partial loss of 16S RNA. In contrast, no breakdown of the [³H]RNA was apparent in the liposomes after sequestration, even in the presence of externally added ribonuclease, or in the unfused liposomes remaining after incubation of protoplasts with liposomes. Thus, the degradation of the [³H]RNA extracted from the protoplasts must have occurred within the protoplasts and represents evidence for liposome-mediated RNA uptake. Naked RNA added to the protoplast culture was found to be totally degraded after incubation with the protoplasts. The uptake of liposome-sequestered RNA by protoplasts was demonstrated to be a function both of the lipid composition of the liposomal membrane and of the temperature of incubation of the liposomeprotoplast mixture. Furthermore, the mode of this uptake (fusion versus endocytosis) could be manipulated by adjusting the cholesterol content of the liposomal membrane. The implications of the ability to insert RNA into protoplasts without degradation by extracellular nucleases are discussed.     

Liposome-mediated delivery of tobacco mosaic virus RNA into petunia protoplast

Liposome-mediated delivery of TMV RNA into petunia protoplasts and resulting virus antigen production has been used as an assay for determining incubation conditions which favor increased uptake of vesicle contents by plant cells. Vesicle phospholipid composition, incubation buffer divalent metal ion concentration, the type and concentration of polyalcohol used to stimulate vesicle uptake and the RNA content of the liposome preparation were determined to be critical factors influencing the efficiency of delivery. Manipulation of these parameters resulted in a 50-fold improvement in virus antigen production over that obtained with conditions previously used for liposome-protoplast incubations (Proc Natl Acad Sci 79: 1859–1863, 1982). Virus antigen production could be detected following incubation of protoplasts with <0.5 ng of encapsulated TMV RNA, while at higher concentrations of added liposomes, >80% of the protoplasts could be infected. Comparisons with other techniques used to introduce nucleic acids into plant protoplasts indicated that liposome-mediated delivery was 10-to 1 000-fold more efficient than these other methods. The general use of liposomes to introduce RNA and DNA molecules into plant protoplasts is discussed.     

Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

Amplified expression of ribulose bisphosphate carboxylase/oxygenase in pBR322-transformants of Anacystis nidulans

Prior research suggested that the genes for large (L) and small (S) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) are amplified in ampicillin-resistant pBR322-transformants of Anacystis nidulans 6301. We now report that chromosomal DNA from either untransformed or transformed A. nidulans cells hybridizes with nick-translated [³²P]-pBR322 at moderately high stringency. Moreover, nick-translated [³²-P]-pCS75, which is a pUC9 derivative containing a PstI insert with L and S subunit genes (for RuBisCO) from A. nidulans, hybridizes at very high stringency with restriction fragments from chromosomal DNA of untransformed and transformed cells as does the ³²P-labeled PstI fragment itself. The hybridization patterns suggest the creation of two EcoRI sites in the transformant chromosome by recombination. In pBR322-transformants the RuBisCO activity is elevated 6- to 12-fold in comparison with that of untransformed cells. In spite of the difference in RuBisCO activity, pBR322-transformants grow in the presence of ampicillin at a similar initial rate to that for wild-type cells. Growth characteristics and RuBisCO content during culture in the presence or absence of ampicillin suggest that pBR322-transformants of A. nidulans 6301 are stable. The data also collectively suggest that a given plasmid in the transformed population replicates via a pathway involving recombination between the plasmid and the chromosome.     

Uptake of liposome-encapsulating plasmid DNA by plant protoplasts and molecular fate of foreign DNA

Summary Conditions faborable for the uptake of artificial lipid vesicles (liposomes) encapsulating plasmid DNA byDaucus carota protoplasts were investigated. Incubation period necessary for the maximum uptake of liposome-DNA was in the neighborhood of 10 minutes under the circumstances where liposomes (approximately 1.28 moles lecithin/ml) were mixed with 5 × 10⁶ protoplasts/ml. Results obtained by Southern hybridization indicated that the recombinant DNA vector, pBR325 was found in protoplasts after 20 hours incubation in the open circular, linear and some complexed forms. There were some variations in DNA-uptake among protoplasts from different plant species. The gradual disappearance of plasmid molecules was confirmed after 1 week culture, suggesting instability of pBR325 in prolonged cell culture.     

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure

Summary. We have analysed the incorporation of [³H]sucrose and [³H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [³H]sucrose or [³H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2–3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [³H]Sucrose uptake was twice that of [³H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [³H]sucrose and [³H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [³H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [³H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [³H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum. Correspondence and reprints: Unidad de Fisiología Vegetal, Facultad de Biología, Universidad de Barcelona, Avenida Diagonal 645, 08028 Barcelona, Spain.     

Thymidine Metabolism and the Measurement of the Rate of DNA Synthesis in Carrot Suspension Cultures: EVIDENCE FOR A DEGRADATION PATHWAY FOR THYMIDINE

The kinetics of [³H]thymidine incorporation into the DNA of carrot suspension cultures were investigated. At a thymidine concentration of 0.1 micromolar, incorporation into DNA is not quantitative but ceases after only 14% of the thymidine has been incorporated. Thymidine incorporation into DNA is resumed following addition of a second aliquot of thymidine, which is consistent with substrate depletion. In vivo tracer experiments indicate that this may be due to a catabolic route for converting thymidine to β-aminoisobutyric acid. Bearing these observations in mind, conditions for determining the rate of DNA synthesis using [³H]thymidine incorporation have been investigated. It is concluded that by increasing the thymidine concentration to 10 micromolar the assay period may be increased, by reducing the influence of the degradative pathway, and that cell density and incubation time are critical factors in establishing a valid measure of the rate of DNA synthesis using this method.     

Separation and analysis of cell types involved in early stages of carrot somatic embryogenesis

The occurrence of the polarized synthesis of DNA in embryogenic cell clusters of carrot on the third and fourth days after transfer to an embryogenesis-inducing medium was observed by labeling with [³H]thymidine and autoradiography. The cells that were actively synthesizing DNA were separated from cells that were not synthesizing DNA by maceration of cell clusters into individual protoplasts and centrifugation in a Percoll density gradient. [³⁵S]Methionine-labeled proteins extracted from the two types of cell were analyzed by SDS-PAGE and fluorography. Three polypeptides (of 69, 98 and 108 kD, respectively) were found only in cells that were actively synthesizing DNA and could be candidates for markers of the polarity of DNA synthesis that is specific to embryogenesis.     

Targeting of large liposomes with lectins increases their binding to plant protoplasts

Soybean agglutinin, peanut agglutinin, and concanavalin A were covalently bound by condensation reaction to gangliosides and ceramides incorporated within the bilayer of multilamellar and unilamellar liposomes. These modified liposomes had a much higher affinity for carrot and tobacco protoplasts except when concanavalin A was used.

In addition, soybean agglutinin and concanavalin A were attached by ligand-specific binding to liposomes containing cholesterol molecules derivatized with each lectin-specific sugar. This procedure allowed efficient crosslinking of liposomes to protoplasts. The same effect was achieved with soybean agglutinin and peanut agglutinin when derivatized cholesterol was replaced by gangliosides. The implications of these findings for the liposome-mediated nucleic acid transfer into protoplasts are discussed.

     

Osmotic stress inhibits thymidine incorporation into soybean protoplast DNA

DNA synthesis in protoplasts isolated from soybean cell suspension cultures has been investigated by [³H] thymidine uptake and incorporation kinetics. Initial rates of incorporation in exponential and 5-fluorodeoxyuridine synchronized protoplasts are inhibited by increased osmolarities of the medium. The inhibition was not readily reversible during 3 h culture in low osmotic medium. Velocity sedimentation analyses of replicating DNA from such protoplasts shows a complex pattern of inhibition. The inhibition probably effects replicon initiation as well as strand elongation and ligation of replication intermediates.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.     

Effects of beta-lactam antibotics on thymidine incorporation of bacteria

Five strains each ofEscherichia coli, Proteus mirabilis, andStaphylococcus aureus were grown in Trypticase soy broth (TSB) with or without penicillin, oxacillin, ampicillin, amoxicillin, or cefamandole. [³H]Thymidine incorporation and the optical density of the cultures were determined at an hourly interval for the duration of incubation. All strains ofP. mirabilis showed after 2 and 3 h of incubation of 5-to 16-fold increase in the specific activity of [³H]thymidine incorporated in the presence of MIC of the antibiotics as compared to controls grown without the drugs.E. coli andS. aureus showed smaller increases in thymidine incorporation than didP. mirabilis. After 4–5 h the antibiotics produced an inhibition of [³H]thymidine incorporation. At the MAC, the responses were of a smaller magnitude. Regardless of whether these changes are the result of specific interference or just lack of [³H]thymidine incorporation, they are directly related to the antibiotic activity of agents known not to affect DNA synthesis. The monitoring of [³H]thymidine incorporation detects early antibiotic activity probably earlier than other current systems which are used for this purpose.     

The recF-dependent endonuclease from Escherichia coli K12. Formation and resolution of pBR322 DNA multimers

Summary The instability of supercoiled pBR322 DNA obtained from different cells has been investigated. Partially purified plasmid DNA species from rec ⁺, recA and recBC sbcB cells are converted in vitro first to relaxed and then to linear molecules. The recA and recBC sbcB cells produce the best conditions for the monomerization of the pBR322 DNA and the stable maintenance of plasmids. The supercoiled pBR322 DNA from the recBC sbcB recF144 cells has been isolated preferentially in multimeric from (circular oligomers). These DNA forms are not converted to plasmid monomers and are converted to linear molecules three-fold slower than the monomer linearization in the case of the recBC sbcB cells.On the other hand, incubation of the pure pBR322 DNA with the recF-dependent protein Z (Krivonogov and Novitskaja 1982) results in the ATP-independent conversion of supercoiled plasmid DNA to relaxed and linear molecules. These results demonstrate an endonuclease activity of the recF-controlled protein Z, which may be involved in general recA-dependent recombination and formation of the pBR322 monomers in the cell.The results also show that the recF144 mutation in recBC sbcB recF and recF cells leads to the absence of detectable amounts of a 49,000 molecular weight protein.     

PEG-induced fusion of phosphatidylcholine-liposomes with protoplasts and post-fusion evaluation of plating efficiency and enrichment in plasmamembrane phosphatidylcholine of protoplasts in Datura innoxia Mill

Liposomes entrapping fluorescein diacetate were fused with protoplasts of Datura innoxia Mill by employing polyethylene glycol (PEG) as the fusogen. Factors that influence liposome-protoplast fusion were optimized as a function of PEG-concentration and incubation duration, liposome composition and surface charge and liposome:protoplast ratio. Phosphatidylcholine-liposomes were found ideal for the objectives of the study. Fusion index based on per cent fluorescing protoplasts varied among the protoplast types. PEG-incubation duration in the fusion assay and growth ability of protoplasts to form microcalli subsequent to liposome-protoplast fusion was determined based on protoplast plating-efficiency. Plating efficiency of post-fusion protoplasts increased due to incorporation of liposome-phosphatidylcholine in the plasmamembrane of protoplasts. Results are discussed in relation to the application of liposome-protoplast fusion system in selective modification of plasmamembrane phospholipids of protoplasts.     

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

Summary In anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Ap^r). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.     

The DNA gyrase of Escherichia coli participates in the formation of a spontaneous deletion by recA-independent recombination in vivo

Summary A system for detecting a spontaneous deletion in Escherichia coli was developed and the role of DNA gyrase in deletion formation was studied. A derivative of plac5, AM36, was isolated in which whole pBR322 DNA was inserted in the lacZ gene and 227 by of the lac gene duplicated at both sides of the pBR322 DNA. E. coli lac ^–strains lysogenized by AM36 had a Lac^– phenotype and segregated Lac^– revertants. Sequence analyses showed that the revertant was formed by a deletion that eliminated the inserted pBR322 DNA and one copy of the duplicated segments. The frequency of lac ^– revertant formation was independent of recA function, was increased by oxolinic acid, an inhibitor of DNA gyrase, but was not increased in a lysogen of a nalidixic acid-resistant derivative. The reversion frequencies of temperature sensitive mutants of gyrA gene are 10 to 100 times lower than that of the wild-type strain. These results indicate that the DNA gyrase of E. coli participated in the in vivo deletion formation resulting from the direct repeats.     

Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.     

Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli

Summary Monomeric pBR322 DNA that had been linearized at its unique SalI site transformed wild-type Escherichia coli with 10² to 10³ times less efficiency than CCC plasmid DNA. Dose-response experiments indicated that a single linear plasmid molecule was sufficient to produce a transformant. Transformation with linearized pBR322 DNA was reduced 10 to 40 fold in recA ^–, recBC ^– or recF ^– backgrounds. In contrast, transformation with CCC DNA was unaffected by the rec status of the host. Transformation with linear pBR322 DNA was increased 3-fold in a DNA ligase-overproducing (lop11) mutant and decreased to a similar degree by transient inactivation of ligase in a ligts7 mutant.A proportion (ranging from about 9% in the wild-type to 42% in a recBC, lop11 mutant) of the transformants obtained with SalI-linearized pBR322 monomeric DNA contained deleted plasmids. Deletion rates were generally higher in rec ^– strains. Dephosphorylation of the termini on linear DNA or the creation of blunt-ended pBR322 molecules (by end-filling the SalI 5 protrusions or by cleavage with PvuII) decreased the transformation frequencywhilst increasing the deletion rate.Linear pBR322 dimeric DNA gave transformation frequencies in recA ⁺ and recA ^– strains that were reduced only 3 to 7 fold respectively relative to frequencies obtained with dimeric CCC DNA. Furthermore, in contrast to transformation with linear monomeric DNA, deletions were not observed.We propose that the majority of transformants arise, not by simple intracellular reannealing and ligation of the two cohesive SelI-termini of a linear molecule, but by intramolecular recombination. Deleted plasmids could be generated therefore during recyclization caused by recombination between short directly repeated sequences within a pBR322 monomer. We suggest that perfectly recircularized monomeric pBR322 molecules, which are found in the majority of transformants, arise primarily by intramolecular recombinational resolution of head-to-tail linear pBR322 dimers. Such linear oligomeric forms are created during preparation of linearized plasmid DNA by annealing of the SalI cohesive termini and constitute a variable proportion of the total molecules present.     

Monitoring the expression of green fluorescent protein in carrot

Green fluorescent protein (GFP) was successfully used as a visual reporter at various stages of carrot (Daucus carota L.) transformation. GFP-fluorescence was non-invasively observed in protoplasts, callus and plants after the delivery of mgfp5-er gene using two transformation methods: direct DNA transfer into polyethylene glycol (PEG) -treated protoplasts and inoculation of root discs with Agrobacterium rhizogenes. Transient GFP-expression was detected in the treated protoplasts and monitored during the first week of the cell culture until the stable level of expression was observed. It was useful for the comparison of protoplast susceptibility to DNA uptake and the transgene expression as the fluorescence declined with various rates depending on the used carrot genotype and PEG-concentration. GFP-monitoring in callus enabled the selection of stably expressing lines. It also allowed verification of the homogeneous tissue composition with regard to the expression of the transgene. In plants, GFP-performance depended on the assayed tissue and organ despite of the constitutive 35S promoter. The expression was visually detected in both vegetative and generative parts, but particularly strong fluorescence was observed in leaf marginal meristems, petioles, stems, and styles. Those tissues can be convenient for examination of the transgenic plants during their growth. The results encourage that GFP is a valuable reporter and can be routinely used for optimization of transformation protocol, selection of transformants and monitoring transgenic carrot.