Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens

Cell wall regenerating protoplasts from soybean cells kept in suspension culture were cocultivated with bacteria which were derived from the nopaline strain C58 of Agrobacterium tumefaciens. When the bacteria carried an oncogenic Ti-plasmid, about 5% of the surviving protoplasts were able to form calli on hormone-free agar in contrast to controls, where bacteria without Ti-plasmid were applied, and where no calli were formed. After isolation of DNA from hormone-independently growing cells further evidence for transformation was obtained by hybridization to Ti-plasmid specific RNA and by rescue of a segment with a bacterial resistance gene which had been inserted before into the T-DNA. Transfer of T-DNA harboring a neomycin-resistance gene activated by the nos-promoter resulted in calli growing on kanamycin. Verification of segments located at the left and the right part of the T-DNA indicated the presence of its entire length in transformed soybean cells. Expression of T-DNA genes was measured by the assay of nopaline-synthase. Cells cultured on agar had a much higher level of nopaline-synthase than fast growing cells in suspension culture. Transferring them to agar or treatment with azacytidine strongly increased synthesis of nopaline-synthase indicating a reversible repression presumably via a methylation mechanism.     

Structure and expression of DNA transferred to tobacco via transformation of protoplasts with Ti-plasmid DNA: co-transfer of T-DNA and non T-DNA sequences

Summary The T-DNA structure and organization in tissues obtained via transformation of tobacco protoplasts with Ti-plasmid DNA was found to be completely different from the T-DNA introduced via Agrobacterium tumefaciens. It is often fragmented. Overlapping copies of T-DNA, having various sizes, as well as separated fragments of T-DNA were detected. The border sequences of 23 basepairs (bp), flanking the T-region in the Ti-plasmid as direct repeats are not used as preferred sequences for integration. Similar results were obtained with a T-region clone lacking one of the T_L-borders. This clone, which carried the cytokinin locus and only the right border sequence of T_L and the left border sequence of T_R, still had the capacity to transform protoplasts. Also the Vir-region of the Ti-plasmid is not required for integration of foreign DNA via DNA transformation. This is demonstrated by the results with the T-region clone mentioned and by the transforming capacity of a Ti-plasmid carrying a mutated Vir-region. Nevertheless, in a number of Ti-plasmid DNA transformants Vir-region fragments were found to be stably integrated. Furthermore, it has been established that co-transformation can occur with plant cells. Besides the detection of Ti-plasmid fragments from outside the T-region also DNA sequences originating from two DNA sources, which were both independently present in transformation experiments, have been found in some DNA transformants, e.g. calf thymus DNA, which was used as carrier DNA. No expression of the co-transferred DNA was observed. In total three phenotypical classes of DNA transformants were isolated. Although the T-DNA was often scrambled, polyA⁺ mRNA studies indicated that the different phenotypes studied can be explained by the presence of active T-DNA genes with known functions.     

Transfection and transformation of Agrobacterium tumefaciens.

Summary The freeze thaw transfection procedure of Dityatkin et al. (1972) was adapted for the transfection and transformation of A. tumefaciens. Transfection of the strains B6S3 and B6-6 with DNA of the temperate phage PS8cc186 yielded a maximum frequency of 2 10^-7 transfectants per total recipient population. In transformation of the strain GV3100 with the P type plasmid RP4 a maximum frequency of 3.5 10^-7 transformants per total recipient population was obtained. Agrobacterium Ti-plasmids were introduced in the strain GV3100 with a maximal efficiency of 4.5 10^-8. These experiments provide further evidence that the Ti-plasmid is responsible for the oncogenic properties of A tumefaciens and for its capacity to induce opine synthesis in Crown-gall plant cells.     

Studies on the nature and role of polyadenylated RNA in spore development of Bacillus subtilis

Summary cDNA probes synthesized on poly(A)RNAs isolated from sporulating cells of Bacillus subtilis were used for hybridization studies with RNAs derived from cells at different stages of growth and sporulation. It was shown that these cDNAs hybridized only to RNA from sporulating cells. No hybridization was observed if total RNA isolated from vegetative cells or from stationary phase cells of a zero stage asporogenic mutant was used. The hybridization studies also indicate that at each sporulation stage different poly(A)RNA species are synthesized. Furthermore, the hybridization kinetics have clearly demonstrated the existence of three distinct abundance classes of poly(A)RNA similar to those observed in eukaryotic cells. BamHI endonuclease restriction fragments of B. subtilis DNA that were found to hybridize to labeled poly(A)RNA were ligated to the pHV33 vector and hybrid clones that hybridized efficiently to poly(A)RNA were selected. Among these, three have been found to carry the spoOB gene.These results strongly suggest that the appearance of poly(A)RNA can be correlated to the expression of spore genes.     

Autonomous replication in Drosophila melanogaster tissue culture cells

This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and tested them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA fragments were able to replicate autonomously, as were plasmids containing random human and Escherichia coli genomic DNA fragments. Most of the plasmids were detectable 18 days after transfection in the absence of selection, suggesting that transfected DNA is maintained in Drosophila cells without rapid loss or degradation. The finding that all plasmids containing Drosophila, human, or bacterial DNA replicate autonomously in Drosophila cells suggests that the signals that direct autonomous replication in Drosophila contain a low degree of sequence specificity. A two-dimensional gel analysis of initiation on one of the plasmids was consistent with many dispersed initiation sites. Low sequence specificity and dispersed initiation sites also characterize autonomous replication in human cells and Senopus eggs and may be general properties of autonomous replication in animal cells.     

Specific detection of RNA molecules by fluorescent in situ hybridization in living cells

The antisense therapeutic strategy makes the assumption that sequence-specific hybridization of an oligonucleotide to its target can take place in living cells. The present work provides a new method for the detection of intracellular RNA molecules using in situ hybridization on living cells. The first step consisted in designing nonperturbant conditions for cell permeabilization using streptolysin O. In a second step, intracellular hybridization specificity was evaluated by incorporating various types of fluorescently labeled nucleic acid probes (plasmids, oligonucleotides). Due to its high expression level, the 28S ribosomal RNA was retained as a model. Results showed that: (1) no significant cell death was observed after permeabilization; (2) on living cells, 28S RNA specific probes provided bright nucleoli and low cytoplasmic signal; (3) control probes did not lead to significant fluorescent staining; and (4) comparison of signals obtained on living and fixed cells showed a colocalization of observed fluorescence. These results indicate the feasibility of specific hybridization of labeled nucleic acid probes under living conditions, after a simple and efficient permeabilization step. This new detection method is of interest for investigating the dynamics of distribution of various gene products in living cells, under normal or pathological conditions.Abbreviations PI propidium iodide - SLO streptolysin O     

Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation

Summary The 200 kb Agrobacterium Ti-plasmid pTiT37 carries a 25 kb segment of T-DNA which it transfers to plant cells during crown-gall tumorigenesis. We have previously engineered into this T-DNA a pBR322-derived cloning vector which enabled us to rescue-clone full length T-DNA from the Ti-plasmid into a 36 kb MINI-Ti plasmid. We report here the deletion of oncogenes from MINI-Ti to produce Micro-Ti containing the nopaline synthase gene and the ampicillin resistance gene and origin of replication of pBR322, flanked by left and right T-DNA borders. Micro-Ti was recloned into the wide host range plasmid pRK290 and transformed into an A. tumefaciens strain carrying a helper plasmid that could supply Virulence (VIR) genes in trans. Using the octopine Ti-plasmid pTiB6-806 as a helper, transformed tobacco cells were obtained which produced both nopaline and octopine. Two cloned cell lines producing both opines were found to be hormone dependent and to produce fertile tobacco plants. We selfed one of these plants and found that the two opine markers segregated in the F1 progeny in a Mendelian fashion. This showed that the T-DNAs were not linked in the transformed plant genome. Southern blot analysis of the genomic DNA from the regenerated plant showed that only part of the (oncogenic) octopine T-DNA was present indicating that it had suffered a deletion in the auxin producing locus (tms region). Presence of the cytokinin autonomy locus presumably accounts for the abnormal rooting behavior of the F1 progeny seedlings containing this T-DNA.Abbreviations NAA Naphtalene acetic acid - IAA Indole-3-acetic acid - BA 6-benzylaminopurine - pCPA para-chlorophenoxyacetic acid Part of this work was presented for her doctoral thesis by A. JdF at the National Institute of Agronomy of Paris-Grignon, January 1983     

Hybridization of Rous Sarcoma Virus Deoxyribonucleic Acid Polymerase Product and Ribonucleic Acids from Chicken and Rat Cells Infected with Rous Sarcoma Virus

Rous sarcoma virus (RSV)-specific ribonucleic acid (RNA) in virus-producing chicken cells and non-virus-producing rat cells infected with RSV was studied by hybridization with the endogenous deoxyribonucleic acid (DNA) product of the RSV virion DNA polymerase system. By hybridizing the total DNA product with excess virion RNA, the product DNA was separated into hybridized (“minus”) and nonhybridized (“plus”) DNA. The “minus” DNA was complementary to at least 20% of the RNA from RSV which remained of high molecular weight after denaturation. A maximum of approximately 65% hybridization was observed between “minus” DNA and RSV RNA or RSV-infected chicken cell RNA. A maximum of about 60% hybridization was observed between “minus” DNA and RSV-infected rat cell RNA. RSV-infected chicken cells contained RSV-specific RNA equivalent to about 6,000 virions per cell. RSV-infected rat cells contained RSV-specific RNA equivalent to approximately 400 virions per cell. Neither cell type contained detectable RNA complementary to virion RNA. The RSV-specific RNA in RSV-infected rat cells did not appear to be qualitatively different from that in RSV-infected chicken cells.     

Agrobacterium-mediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants

Summary Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification of NPT II and GUS gene fragments from genomic DNA of transgenic plants with polymerase chain reaction (PCR) and integration of these genes into nuclear genome of transgenic plants was confirmed by genomic Southern hybridization analysis. Histochemical assay revealed the expression of GUS gene in leaf, stem and root tissues of transgenic plants, further confirming the integration and expression of T-DNA in these plants. This protocol allows effective transformation and regeneration of quaking aspen using greenhouse-grown materials as an explant source. Whole plant regeneration from cuttings of fieldgrown mature quaking aspen and hybrid poplar (P. alba x P. grandidentata) was also readily achieved by using this protocol, which represents a potential system for producing transgenic quaking aspen and hybrid poplar of valuable genotypes.Abbreviations AMV RNA4 Alfalfa mosaic virus RNA4 - BA 6-benzyladenine - CaMV cauliflower mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraacetic acid - FAA formalin-acetic acid-alcohol - GUS ß-glucuronidase - NAA 1-naphthylacetic acid - NPT II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - TE Tris-Cl/EDTA - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl-urea (thidiazuron) - WPM woody plant medium (Lloyd and McCown 1980) - X-GLUC 5-bromo-4-chloro-3-indolyl-ß-glucuronic acid     

Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts

Summary Genetic transformation of Nicotiana tabacum protoplasts was achieved by incubation of protoplasts with a plasmid DNA-calcium phosphate coprecipitate, followed by fusion of the protoplasts in the presence of polyvinyl alcohol and subsequent exposure to high pH. A derivative of the plasmid pBR322 containing a chimaeric gene, consisting of the nopaline synthase promoter, the coding region of the aminoglycoside phosphotransferase gene of Tn5 and the polyadenylation signal region of the octopine synthase gene, was used for these transformation experiments. This chimaeric gene confers resistance of transformed plant cells to kanamycin. This novel transformation procedure reproducibly yielded transformants at frequencies of approximately 0.01%. Aminoglycoside phosphotransferase II activity was detected in both transformed calli and in regenerated plants. DNA from some of the transformed clones was analyzed by Southern blot hybridization. The input DNA appears to be integrated into high molecular weight cellular DNA. Genetic analysis of one of the kanamycin resistant plants shows that the chimaeric gene is transmitted to the progeny as a single dominant trait in a Mendelian fashion. As a comparison the input DNA was also introduced into tobacco protoplasts using Agrobacterium tumefaciens and Ti-plasmid derived gene vectors.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

The activity of two heat shock loci of Drosophila hydei in tissue culture cells and salivary gland cells as analyzed by in situ hybridization of complementary DNA

Complementary DNA was made to poly A⁺ nuclear or polysomal RNA isolated from heat shock tissue culture cells of Drosophila hydei. A number of loci other than the four major heat shock loci are labelled after in situ hybridization of these cDNA preparations, while solution hybridization indicated that only about 10% of the cDNA was specific for heat shocked cells. Removal of the fraction of cDNA which could react with 25° C RNA and subsequent in situ hybridization of heat shock specific cDNA indicated that locus 4–81 B, a major salivary gland heat shock locus, is also active at 25° C in tissue culture cells, while locus 4–85 B is specifically activated by heat shock in tissue culture cells. This latter locus is not seen to be clearly puffed in salivary glands, but was shown to be active in that tissue both by direct autoradiography of salivary gland chromosomes after 3H-uridine labeling and by hybridization of cDNA to chromosomal RNA.     

Polybrene-mediated transfection of cultured lepidopteran cells: Induction of aDrosophila heat shock promoter

Summary We have introduced hsp-cat plasmid DNA intoSpodoptera frugiperda (Lepidoptera: Noctuidae) cells by transfection with purified DNA (1 to 48 μg/ml) mixed with the polycation polybrene (100 μg/ml) in serum-free Grace's medium. The hsp-cat construct contains a gene coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT), whose expression is controlled by a promoter derived from aDrosophila heat shock protein (hsp) gene. Expression of CAT activity in transfectedSpodoptera cells was induced by a 2-h heat shock at 43°C. The temperature of the heat shock was based on conditions that maximized the expression of endogenous heat shock protein genes in these cells. CAT activity was maximal in cells that were exposed to the heat shock 2 d after transfection; by 4 d, activity was diminished, and little activity was detectable after 6 d. Transfection frequencies, which varied with DNA concentration and ranged as high as 6000 per million cells, were determined using a histochemical staining procedure. This work was supported by grant 88-37263-4020 from the United States Department of Agriculture, Washington, DC, and by the University of Minnesota Experiment Station. This is contribution 17,543 from the University of Minnesota Experiment Station, St. Paul, MN.