Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

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.     

Quantitative analysis of the fate of exogenous DNA in Nicotiana protoplasts

After a 5-hour incubation of protoplasts of Nicotiana tabacum L. ;Xanthi' with (3)H-DNA (7.26 mug/ml) from N. tabacum L. ;Xanthi nc' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE-dextran and poly-l-lysine to the incubation medium nearly doubled radioactivity adsorption. The absorption was inhibited by 2,4-dinitrophenol, KCN, and low temperature (0 C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cultivar that had been allowed to absorb DNA prepared from a resistant cultivar did not show transfer of the virus-resistant gene.A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli(3)H-DNA and Nicotiana glutinosa protoplasts. In 5 to 20 hours, about 10% of the (3)H-DNA entered the protoplasts. Competition experiments between the (3)H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded (3)H-DNA. Examination of intraprotoplast fractions revealed that 60 to 80% of the absorbed radioactivity resided in the "soluble" fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA had been depolymerized. Of the incorporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein, and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable (3)H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 x 10(-15)g donor DNA, or approximately half of the E. coli genome.     

Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp. Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the C31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of C31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.     

Genotype- and promoter-induced variability in transient β-glucuronidase expression in pea protoplasts

Summary Leaf mesophyll protoplasts isolated from pea (Pisum sativum L.) genotypes Century and PI244253 showed transient expression of -glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 F; and using 125 g of calf thymus carrier DNA and 75 of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expression.NRCC No. 30910     

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Factors affecting transient gene expression in protoplasts isolated from very slowly growing embryogenic callus cultures of wheat (Triticum aestivum L.)

Protoplasts isolated from embryogenic (Mustang and Chinese Spring) and non-embryogenic (Mit) calli of wheat (Triticum aestivum L.) genotypes transiently expressed -glucuronidase (GUS) activity when electroporated with a plasmid containing the GUS gene and driven by an enhanced 35S promoter and a TMV leader sequence. Conditions for the maximum expression of GUS activity were: electroporation of the freshly isolated protoplasts at 250 Vcm^-1 and 250 F for 2 s using 50 g/ml of plasmid DNA; incubation of the protoplasts with the plasmid before the pulse for 2 h; and a 15-min recovery period on ice after the pulse. In general, a higher GUS activity was obtained in protoplasts of non-embryogenic (NE) callus origin than in those of embryogenic (E) callus origin. Only GUS constructs containing a duplicate 35S promoter derivative resulted in a significant level of GUS expression. The presence of the TMV viral leader sequence in the pAGUS1-TN2 plasmid construct resulted in a significant increase of GUS activity in the electroporated protoplasts of both callus types. On the other hand, protoplasts electroporated with the Adh1 promoter and intron showed a threefold less GUS activity than those electroporated with pAGUS1-TN2. Optimized conditions for DNA uptake and expression were very similar for protoplasts of both callus types. The importance of these findings for the successful regeneration of transgenic and fertile wheat plants is discussed.     

Transformation of spheroplasts and protoplasts of Corynebacterium glutamicum

Summary Spheroplasts were prepared from Corynebacterium glutamicum ATCC13032 by growing cells in the presence of glycine followed by digestion with lysozyme. Using pUL330 a spheroplast transformation system was established routinely yielding 10³ to 10⁴ transformants per g of plasmid DNA. Spheroplasts were converted into protoplasts after incubation with the lytic enzyme achromopeptidase in the presence of additional lysozyme. Protoplasts prepared by this method regenerated at efficiencies of 10 to 30%. A protoplast transformation system was established routinely yielding 10⁵ to 10⁶ transformants per g of plasmid DNA. The Escherichia coli Brevibacterium lactofermentum shuttle vector pUL62 prepared from E. coli could be introduced into spheroplasts of C. glutamicum after heat treatment at 48 to 49°C for 10 min.     

Conjugal transfer of E. coli F''lac from Erwinia chrysanthemi to Pseudomonas syringae pv. glycinea and the apparent stable incorporation of the plasmid into the pv. glycinea chromosome

Summary The E. coli Flac plasmid was transferred from an Erwinia chrysanthemi Hfr8 donor to a multiply-auxotrophic, rifampicin-resistant Pseudomonas syringae pv. glycinea recipient. Transfer occurred at a frequency of approximately 10^-5/donor. Stable transconjugants which were able to utilize lactose as the sole carbon source after several transfers would not donate the Flac plasmid in detectable frequency to other pv. glycinea or E. coli recipients. The plasmid DNA was shown to be integrated into the pv. glycinea chromosome (Fig. 1).     

Polyethyleneglycol-induced transformation of Bacillus subtilis protoplasts by bacterial chromosomal DNA

Summary Bacillus subtilis protoplasts, which in the presence of polyethyleneglycol (PEG) are transformed by plasmid DNA (Chang and Cohen 1979) can also be transformed under these conditions by chromosomal DNA. Transformation in this case occurs at a much lower frequency, not fully accounted for by the heterogeneity of this DNA. Another unexpected feature of the transformation studied, which may explain why it previously went unnoticed, is that DNA concentrations higher than 1–2 g/ml decrease the yield of transformants, without showing signs of general toxicity.PEG-induced protoplasts (PIP) transformation for chromosomal markers operates normally with protoplasts prepared from a non-transformable bacterial mutant. The evidence indicates that both native linear and plasmid DNAs must somehow be forced into the cells as a result of PEG action. Denatured chromosomal DNA however is almost inactive in PIP transformation. No competition between chromosomal and plasmid DNAs could be detected, when the DNA tested as inhibitor was in tenfold excess.     

Investigation of novel oligoelectrolyte polymer carriers for their capacity of DNA delivery into plant cells

Development of modern agriculture and biotechnology is closely connected with the use of novel and effective genetic engineering methods. Presently, non-viral nanoparticle-mediated plant transformation methods gain more attention because of their stability, safety, and convenience of performance. In this work, new polymeric dimethylaminoethyl metacrylate (DMAEM)-based polymers were synthesized and investigated for their properties in gene delivery. Formation of stable complexes between TN 83/6, TN 84/5, DLM-9-DM and LM-8-DM polymers and plasmid DNA, as well as the DNA protection by the PDMAEM polymers against nuclease degradation were confirmed by electrophoresis in agarose gel. In addition, model organisms Allium cepa and Nicotiana tabacum L. were studied to evaluate cytotoxic effect of the PDMAEM carriers. The created PDMAEM-based carriers were effective in delivery of plasmid DNA into moss and tabacco protoplasts (obtaining stable transformants of Ceratodon purpureus moss, as well as in transient expression of the reporter yfp gene product in N. tabacum protoplasts). Thus, novel PDMAEM-based polymers were shown to be promising carriers for delivery of DNA into plant cells, and carriers possess high potential for further applications in this field.     

Transformation ofStreptomyces avermitilis by plasmid DNA

Summary Polyethylene glycol (PEG) efficiently mediated the transformation ofStreptomyces avermitilis protoplasts by plasmid DNA to yield 10⁷ transformants per g of plasmid DNA. Under conditios in which the maximum transformation frequency was observed, the cotransformation frequency exceeded 10%. The number of transformants increased linearly with the amount of DNA and number ofS. avermitilis protoplasts. Relaxed and supercoiled, but not linear DNA transformed protoplasts efficiently. Dimethyl sulfoxide (DMSO)-mediated transformation of protoplasts was 1000-fold less efficient. PEG and, less efficiently, DMSO also mediated the transformation of whole cells ofS. avermitilis by DNA.     

RNase-sensitive DNA polymerase activity in cell fractions and mutants of Neurospora crassa

RNase-sensitive DNA polymerase activity (RSDP) was tested in different cell fractions of Neurospora crassa cell types and its morphological mutants. This RSDP was found localized in the microsomal pellet fraction and absent in the purified nuclear pellets isolated from different N. crassa cell types: conidia, germinated conidia, and mycelia. This enzyme is capable of synthesizing a DNA product only in the presence of all four deoxyribonucleoside-5-triphosphates and Mg²⁺. Removal of RNA from the pellet fraction by RNase strongly inhibited the DNA synthesis. The endogenous synthesis of DNA in the microsomal pellet fraction was associated with the formation of an RNA:DNA hybrid as analyzed by Cs₂SO₄ equilibrium density gradient centrifugation. The DNA product after alkali hydrolysis hybridizes with the RNA isolated from the same pellet fraction, as analyzed by elution from hydroxylapatite column at 60 C. This DNA product did not hybridize with poly(A). A few mutants tested showed this RNase-sensitive DNA polymerase activity.This work was supported in part by a contract with the U.S. Department of Energy and a grant from the U.S. Naval Research.     

Bromodeoxyuridine combined with UV light and gamma irradiation promotes the production of asymmetric somatic hybrid calli

Summary The degree of gamma- or X-ray-induced donor chromosome elimination in asymmetric somatic hybrids is highly variable. Here the beneficial use of bromodeoxyuridine and UV light as additional chromosome destabilizing agents is described. Protoplasts of Nicotiana tabacum were fused with protoplasts of Nicotiana plumbaginifolia (Np) that carried the kanamycin-resistance and glucuronidase (GUS) genes on separate chromosomes. Prior to fusion, the Np donor protoplasts were pretreated with bromodeoxyuridine and then were inactivated by treatment with iodoacetate ± UV light ± 200 Gy gamma irradiation. Hybrids were selected on medium containing kanamycin. The elimination of Np DNA was assessed by scoring of the fraction of hybrid calli that expressed GUS and by dot-blot analysis using a Np-specific probe. Gamma irradiation alone resulted in elimination of 50% of Np DNA. Pretreatment with bromodeoxyuridine (10 M) followed by 2.5 to 5 min UV light resulted in the elimination of 35–45% of the donor genome, but incorporation of bromodeoxyuridine (10 M) followed by 2.5 to 5 min UV light and 200 Gy gamma irradiation resulted in 85 to 90% elimination of Np DNA.Abbreviations BrdU bromodeoxyuridine (5-bromo-2-deoxyuridine) - GUS glucuronidase - HPT hygromycin B phosphotransferase - KmR kanamycin resistant - Np Nicotiana plumbaginifolia - NPT neomycin phosphotransferase II - Nt Nicotiana tabacum - UV ultraviolet     

Characterization of chromosome and plasmid transformation in Bacillus subtilis using gently lysed protoplasts

Competent cells of Bacillus subtilis were transformed with DNA from gently lysed protoplasts. Significant linkages among markers separated by distances of approximately 2.3% of the total chromosome were found, which have not been detected for conventional transformation. In comparison to previous reports, enhanced plasmid transformation was observed [4.0×10⁷ transformants per g DNA (one transformant per 5×10⁴ molecules added)], when competent cells were transformed with DNA from lysed protoplasts harboring pUB110.     

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis: further characterization of unstable association between donor and recipient DNA and the involvement of the cellular membrane

Summary Although heterospecific transformation is extremely inefficient and very little heterologous donor DNA integrates into the recipient chromosome in a stable way, we have previously shown that B. pumilus DNA entering competent B. subtilis efficiently associates with the recipient chromosome in an unstable way. This association can be stabilized by photocrosslinking in the presence of 4,5,8-trimethylpsoralen; it depends on the recombination proficiency of the recipient strain and on strand-separation of the recipient chromosome (te Riele and Venema 1982b). The present study provides further evidence that the heterologous donor DNA and the recipient DNA are associated by regions of base-pairing. Based on the high sensitivity of the donor moiety in the complex to nuclease S1 (90%) and the high sensitivity of the complex to moderate denaturing conditions (Tm=48°C), we presume that donor and recipient DNA are associated either by several short sequences of 15–25 fairly well matched base pairs or by a region of base-pairing of about 200 bases, which contains 25% of mismatches. During incubation, the unstable complex disappears, probably due to nucleolytic degradation.The unstable heterologous donor-recipient complex (DRC) was found to be membrane-bound. However, in contrast to homologous DRC, the unstable heterologous DRC remains membrane bound during incubation. Apparently, the predominantly single-stranded character of the heterologous DRC prevents release of the complex from the membrane.Abbreviations DRC donor-recipient complex - TMP 4,5,8-trimethyl-psoralen - DNAase I deoxyribonuclease 1 - TCA trichloroacetic acid     

Effect of repeated DNA sequences on direct gene transfer in protoplasts of Nicotiana plumbaginifolia

Summary Highly repeated nuclear DNA sequences from leaves of Nicotiana plumbaginifolia were cloned in pBR322 and tested for their effect on direct gene transfer in protoplasts of the same organism. Protoplasts were prepared from suspension cultures and were incubated in the presence of the plasmid pHP23 carrying the kanamycin resistance gene APH(3)II and in the presence of the plasmids carrying the cloned sequence. DNA uptake was induced by a polyethyleneglycol (PEG) treatment. Out of the 22 tested clones, 3 significantly stimulated the frequency of appearance of transformed colonies. DNA was extracted from some of the kanamycin-resistant calli obtained by co-transformations. Dot-blots have shown that the stimulatory effect on transformation frequency is often accompanied by a consistent increase in integrated genes sequences.