Agrobacterium tumefaciens Ribonucleic Acid Synthesis in Tomato Cells and Crown Gall Induction

Purified Agrobacterium tumefaciens deoxyribonucleic acid (DNA) does not produce crown gall tumors in growing plants, conditioned by wounding, as the living bacteria do. Purified bacterial DNA migrates in the plant and replicates, but it is not transcribed in our experimental conditions. On the contrary, when DNA is released naturally from bacteria into plant cells, a bacterial ribonucleic acid (RNA) can be found in these cells. There seems to be a direct relation between the appearance of A. tumefaciens RNA in the plant cells and the induction of the tumor.     

Eight DNA insertion events of Agrobacterium tumefaciens Ti-plasmids in isogenic sunflower genomes are all distinct

We investigated whether the same or different T-DNA insertions occur every time Agrobacterium tumefaciens, the octopine type strain pTi 15955 strr, infects genetically identical sunflower plants. Eight newly established crown gall tissue culture lines were analyzed for their T-DNA content. Our data showed that all isogenic crown gall callus DNA produced distinct hybridization patterns. These eight patterns were also different from three standard lines included for comparison. In addition, all the tumor lines analyzed produced octopine, albeit in different quantities, and five produced agropine and mannopine. We concluded, that each A. tumefaciens crown gall tissue line derived from isogenic sunflower plants contained a distinct insertion pattern of T-DNA. Possible causes and reasons for this diversity will be discussed.     

Crown gall transformation of tobacco callus cells by cocultivation with Agrobacterium tumefaciens

Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype.     

Isoenzymes of Acid Phosphatase and Non-specific Esterases in Cultures of Neoplastic and Normal Tobacco Tissues

Axenic cultures of normal, habituated and crown gall teratoma tissues were grown under varying conditions to examine the effects of environment on the expression of neoplastic character. Acid phosphatase patterns on polyacrylamide gels did not vary greatly among tissues although there were differences in acid phosphatase activity between various strains of Agrobacterium tumefaciens , the bacteria which cause crown gall. Certain esterase isoenzymes were found only in tissues grown on specific media, while others were tissue-specific but independent of the nature of the medium. Comparisons of liquid and solid grown cultures revealed that culture conditions also influence esterase expression. Both sunflower and tobacco crown gall tissue contained an esterase not found in habituated or normal tissues, and similar in electrophoretic mobility to an esterase found in extracts of the bacteria that had induced the tumors. The basic difference between the three tissue types studied is the manner in which they respond to a given environment.     

Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA,and transmission of T-DNA to R1 progeny

Cloned DNA sequences encoding yeast alcohol dehydrogenase and a bacterial neomycin phosphotransferase have been inserted into the T-DNA of Agrobacterium tumefaciens plasmid pTiT37 at the “rooty” locus. Transformation of tobacco stem segments with the engineered bacterial strains produced attenuated crown gall tumors that were capable of regeneration into intact, normal tobacco plants. The yeast gene and entire transferred DNA (T-DNA) were present in the regenerated plants in multiple copies, and nopaline was found in all tissues. The plants were fertile, and seedlings resulting from self-pollination also contained intact and multiple copies of the engineered T-DNA. Expression of nopaline in the germinated seedlings derived from one regenerated plant was variable and did not correlate with the levels of T-DNA present in the seedlings. Preliminary evidence indicates that nopaline in progeny of other similarly engineered plants is more uniform. The disarming of pTiT37 by insertions at the “rooty” locus thus appears to produce a useful gene vector for higher plants.     

Deoxyribonucleic Acid of Marek''s Disease Virus in Virus-Induced Tumors

DNA was extracted from [(3)H]thymidine-labeled Marek's disease virus (MDV) and purified by two cycles of CsCl gradient centrifugation in a fixed-angle rotor. The DNA was transcribed in vitro into (32)P-labeled complementary RNA (cRNA). MDV cRNA did not hybridize with DNA from chicken embryo fibroblast cultures or from chicken spleen, but hybridized efficiently with DNA from MDV particles or MDV-infected cell cultures. Five Marek's disease tumors from different chickens and different organs (ovary, liver, testis) were all found to contain MDV DNA sequences. The relative amount of MDV DNA varied from tumor to tumor and was between 3 and 15 virus genome equivalents per cell. The content of virus DNA per cell in spleens from tumor-bearing chickens was much lower than in tumors from the same animals. MDV-infected cell cultures contained a large proportion (28-59%) of virus antigen-positive cells, as measured by immunofluorescence, but tumor cells were negative in this respect (<0.02% positive cells). These data indicate that MDV is present in a provirus form in tumor cells.     

Tumor Induction by Agrobacterium tumefaciens: Specific Transfer of Bacterial Deoxyribonucleic Acid to Plant Tissue

When Agrobacterium tumefaciens cells grown in the presence of tritiated thymidine to label specifically the bacterial deoxyribonucleic acid (DNA) are incubated with carrot root tissue for short periods of time, an appreciable fraction of the label becomes firmly associated with the root tissue. Such association is not observed in identical experiments when A. tumefaciens cell ribonucleic acid or protein are labeled. The extent of the retention of thymidine-derived label from bacterial cells by the root tissue in experiments with A. radiobacter and poorly tumorigenic strains of A. tumefaciens is significantly less than that afforded by tumorigenic strains of A. tumefaciens but greater than the level afforded by Escherichia coli. Transfer of DNA-specific label from A. tumefaciens to carrot root discs is not enhanced by treatments designed to provoke lysis of the bacterial cells, nor is it decreased by addition of deoxyribonuclease or excess unlabeled thymidine to the incubation medium. Bacterial cell-to-plant cell contact is necessary for transfer. Unlabeled A. radiobacter cells decrease in a competitive manner transfer of label when mixed with labeled A. tumefaciens cells. These findings suggest that transfer of DNA from A. tumefaciens to plant tissue after binding of the bacterial cells to specific plant tissue site(s) is a necessary feature of the mechanism by which A. tumefaciens provokes tumors in plants and provides an experimental technique of potentially great value in study of the early steps in the process of tumor induction by A. tumefaciens.     

Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis

Evidence is presented that crown gall tumors are caused by the incorporation of part of a virulence plasmid carried by the inciting bacterium, Agrobacterium tumefaciens. The rate of reassociation of labeled plasmid DNA was slightly accelerated in the presence of tobacco crown gall tumor DNA, but not normal tobacco DNA. Treatment of tumor DNA with DNAase abolished the acceleration. To determine whether all plasmid sequences are represented in tumor DNA, the labeled plasmid DNA was separated into specific fragments after digestion with restriction endonuclease Sma I. Renaturation rates for DNA from bands 1, 2, 7, 8, 9, 12 and 14 were not affected by tumor DNA. DNA from band 3 showed a slight rate increase in the presence of tumor DNA, indicating 21–27 copies of 14–18% of the DNA sequences in this (doublet) band. The band 3 doublet was separated by electrophoresis into bands 3a and 3b. Tumor DNA had little effect on the rate of reassociation of labeled band 3a DNA. Band 3b DNA renatured rapidly in the presence of tumor DNA, and its rate increase indicated that approximately 18 copies of 40% of band 3b DNA sequences are present per diploid tumor cell. This amounts to 3.7 × 10⁶ daltons of foreign genetic information and represents a contribution of 0.0011% to the DNA content of the tumor cell. The relationship between this plant tumor and virally induced animal tumor systems is discussed.     

Attempts to Detect Agrobacterium tumefaciens DNA in Crown-Gall Tumor Tissue

Primary and secondary crown gall tissue cultures were established from sunflower plants (Helianthus annuus, variety Mammoth Russian) wound-inoculated with Agrobacterium tumefaciens (Smith and Townsend) Conn strain B(6). Growth rates of tumor tissues and habituated healthy sunflower stem section tissues on basal medium lacking auxin and cytokinin were compared to those of healthy sunflower stem section tissue grown on the same medium with added phytohormones. No difference was detected in the thermal denaturation midpoints (74.8 C) and melting profiles in 25 mm sodium phosphate (pH 6.8), or the buoyant densities in cesium chloride equilibrium centrifugation (1.687 g cm(-3)), between deoxyribonucleic acids (DNAs) isolated from crude nuclear preparations of the four tissue types. No satellite DNA was observed in equilibrium centrifugation of unsheared plant DNAs.Heterologous DNA renaturation kinetic analyses were performed in 0.14 m sodium phosphate (pH 6.8) at 70 C. Thermal stability measurements of reassociated DNA revealed less than 1% of mismatched base pairs. Reannealing of sheared, denatured, radioactive A. tumefaciens B(6) DNA (molecular weight, 325,000 daltons) in the presence of a 5400-fold excess of sheared calf thymus, healthy tissue, or secondary sunflower crown gall DNA obeyed second order kinetics, with a Cot((1/2)) of 2.8, identical to that observed when B(6) DNA was reannealed in the absence of foreign DNA.Reannealing rates of B(6) DNA in the presence of 5400-fold excesses of DNA from two lines of primary sunflower crown gall were increased 2.24- or 1.47-fold. Digestion of the tumor DNA preparations with pancreatic deoxyribonuclease I until no detectable DNA remained, followed by restoration of solution viscosity by added calf thymus DNA, failed to remove the acceleration effect of the tumor DNA preparations. Reisolation of the reannealed nucleic acid formed in this experiment, and digestion with ribonuclease A or deoxyribonuclease I revealed that the double-stranded fraction was composed entirely of DNA-DNA duplexes, with no detectable DNA-RNA hybrids.The data indicate that tumor, but not healthy tissue DNA preparations contain some factor or factors (not DNA) which accelerate the reannealing of bacterial DNA. Sunflower tumor tissue DNAs, therefore, do not contain integrated A. tumefaciens DNA sequences in amounts greater than a random (1/5) of the bacterial genome per diploid amount of plant DNA, or a complete bacterial genome per five diploid plant cell DNA equivalents. Further, the possibility of the presence of many copies of a specific portion greater than 5% of the bacterial genome is excluded.     

Studies on superoxide dismutase activities in virulent and avirulent strains of Agrobacterium tumefaciens and also in normal and crown gall tumor cells of Bryophyllum calycinum

Superoxide dismutase activity in virulent strains of Agrobacterium tumefaciens was found to be higher than that in avirulent strains. Polyacrylamide gel electrophoresis revealed two isoenzymes in both these strains. These isoenzymes are suggested to be iron and manganese containing superoxide dismutases. Crown gall tumor cells of the plant Bryophyllum calycinum were found to have higher superoxide dismutase activity than the normal plant cells. Polyacrylamide gel electrophoresis revealed two isoenzymes in both normal and crown gall tumor cells. Advantages of the higher superoxide dismutase activities in respect of the survival of virulent strains of A. tumefaciens and crown gall tumor growth have been discussed.     

Transgenic Arabidopsis plants expressing Agrobacterium tumefaciens VirD2 protein are less susceptible to Agrobacterium transformation

Agrobacterium tumefaciens causes crown gall disease on many plant species and can result in considerable economic losses. Here we report a new strategy to control crown gall disease by over-expressing Agrobacterium tumefaciens VirD2 protein in plants. Transgenic Arabidopsis plants over-expressing virD2 from constitutive or wound-inducible promoters are less susceptible to Agrobacterium -mediated transformation. Additionally, the transient introduction of an A. tumefaciens virD2 gene in tobacco BY-2 cells reduces subsequent Agrobacterium -mediated transformation.     

Evidence for the Presence of Bacteria-specific Proteins in Sterile Crown Gall Tumor Tissue

Cross-reacting antigens were found in bacteria-free crown gall tumor tissue tested with serum prepared against Agrobacterium tumefaciens (Smith and Towns.) Conn., but no such antigens were detected in callus tissue. Soluble proteins from tumor tissue, callus tissue, and the crown gall bacteria were fractionated on a DEAE-Sephadex (A-50) column. The diethylaminoethyl-Sephadex elution profile for tumor tissue showed three protein fractions that were not detected in the callus tissue. Two of these protein fractions were shown to be exclusively bacteria specific. Besides these qualitative differences between the two tissues, significant quantitative differences in the amount of protein fractions were also observed. The diethylaminoethyl-Sephadex column fractions from tumorigenic strain of A. tumefaciens corresponding in position to the three additional peaks in the tumor tissue also showed cross-reacting antigens when tested with serum prepared against sterile tumor tissue. It is suggested that tumor formation by A. tumefaciens involves integration of the bacterial genome into the host-cell genome.     

RNA from callus cultures and leaves of Nicotiana tabacum

MAK column chromatography has been used to analyse RNA from normal and crown gall callus cultures and leaves of Nicotiana tabacum. To determine the elution behaviour of well-defined DNA-like RNAs with different GC content, complementary RNAs (c-RNA) synthesized on Agrobacterium tumefaciens DNA and crown gall DNA were used. The elution profile of the RNA from all three tissues followed a similar pattern. By salt gradient elution the RNA in the tRNA region showed a remarkably high CMP content which was significantly higher for the normal tissues than for crown gall tissue. RNA from the callus cultures contained more DNA-like RNA (D-RNA) with a higher turnover rate than RNA from leaves. Because of its relatively low poly A content, measured as RNase A + T₁ resistance, as well as its high turnover rate, the salt-eluted D-RNA is thought to be heterogeneous nuclear RNA (Hn-RNA) and not mRNA. RNA molecules that might represent the mRNA population, having intramolecular poly A tracts, were subsequently eluted by a salt gradient, a low salt buffer and with the chaotropic agent guanidine thiocyanate, which removed tenaciously bound (TB-RNA) in two fractions, α and β. Crown gall RNA showed both a different labelling behaviour and a higher poly A content in the α and β fractions compared to the normal tissues. c-RNAs may be eluted at different salt concentrations because of their different GC content. They give rise to a considerable fraction of TB-RNA which in the presence of tobacco leaf RNA was split into fractions similar to α and β. No fraction was found amongst these RNAs which did have intramolecular poly A tracts.     

Hormonal control of tobacco crown gall tumor morphology

The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus.     

Initiation of Infection by Deoxyribonucleic Acid From Bacteriophage λ

Kinetic studies conducted on the early stages of infection of Escherichia coli K-12 by deoxyribonucleic acid (DNA) isolated from bacteriophage lambda indicate a rapid adsorption of the phage DNA to receptor sites at the bacterial surface prior to deoxyribonuclease-insensitive incorporation. A direct relationship found between the number of DNA molecules adsorbed per bacterium and the multiplicity of helper phage infection indicates a requirement for helper function during the attachment process. An apparent lack of attachment specificity with regard to the source of the DNA preparation, to the size of the inhibiting fragment, to the base ratio of the inhibiting DNA molecule, and to "cohesive" ends suggests a nonspecific interaction between the infectious DNA and the sites of helper phage attachment.     

Cytokinins in tobacco crown gall tumors

Bacteria-free tobacco ($\text{Nicotiana tabacum}$ cv. Wisconsin #38) crown gall strains incited by $\text{Agrobacterium tumefaciens}$ C58, 27, B6, CGIC, and AT4 have been analyzed for cytokinin content with the tobacco callus bioassay. All tumor strains contained high total levels of cytokinins ranging from 4–810 kinetin equivalents per kg fresh weight compared to 0.5 kinetin equivalents per kg for normal callus growing on medium with 0.1 μM N6-benzyladenine. Fractionation on a column of Sephadex LH-20 separated cytokinin activity from B6 tumors into a number of components among which ribosyl-$\text{trans}$-zeatin has been purified and characterized based on uv spectrum, biological activity and mass spectrum.     

Polyamines in the Synthesis of Bacteriophage Deoxyribonucleic Acid. I. Lack of Dependence of Polyamine Synthesis on Bacteriophage Deoxyribonucleic Acid Synthesis

To determine whether polyamine synthesis is dependent on deoxyribonucleic acid (DNA) synthesis, polyamine levels were estimated after infection of bacterial cells with ultraviolet-irradiated T4 or T4 am N 122, a DNA-negative mutant. Although phage DNA accumulation was restricted to various degrees in comparison to cells infected with T4D, nearly commensurate levels of putrescine and spermidine synthesis were observed after infection, regardless of the rate of phage DNA synthesis. We conclude from these data that polyamine synthesis after infection is independent of phage DNA synthesis.     

日本樱花根癌病病原菌的鉴定及其防治

从浙江省的慈溪、奉化、嵊州等地的日本樱花苗圃内,采集到具有典型症状的日本樱花根癌病植株。经分离纯化及在指示植物番茄、向日葵幼苗的致病性测定,共分离到致病性病原菌株１１株,经形态学、生理生化学特征鉴定及菌体可溶性蛋白ＳＤＳ－聚丙烯酰胺凝胶电泳,确定引起日本樱花根癌病的病原细菌为根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓ）生物型１,经平皿拮抗和盆栽试验表明,生防菌Ｋ８４能够明显抑制致病菌株的致癌能力。     

Amino Acid Control over Deoxyribonucleic Acid Synthesis in Escherichia coli Infected With T-Even Bacteriophage

Starvation for a required amino acid of normal or RC(str)Escherichia coli infected with T-even phages arrests further synthesis of phage deoxyribonucleic acid (DNA). This amino acid control over phage DNA synthesis does not occur in RC(rel)E. coli mutants. Heat inactivation of a temperature-sensitive aminoacyl-transfer ribonucleic acid (RNA) synthetase similarly causes an arrest of phage DNA synthesis in infected cells of RC(str) phenotype but not in cells of RC(rel) phenotype. Inhibition of phage DNA synthesis in amino acid-starved RC(str) host cells can be reversed by addition of chloramphenicol to the culture. Thus, the general features of amino acid control over T-even phage DNA synthesis are entirely analogous to those known for amino acid control over net RNA synthesis of uninfected bacteria. This analogy shows that the bacterial rel locus controls a wider range of macromolecular syntheses than had been previously thought.     

Serological Relatedness of Bacterial Deoxyribonucleic Acid Polymerases

A number of bacterial species have been surveyed for serological activities with antiserum to Escherichia coli B deoxyribonucleic acid (DNA) polymerase I (EC 2.7.7.7.). The degree of serological cross-reaction is taken as a measure of relatedness of both the enzyme molecules from various species and the bacterial species themselves. Extracts were assayed by complement fixation only after treatment with deoxyribonuclease, since DNA bound to DNA polymerase alters the serological activity of the enzyme. Antiserum to E. coli DNA polymerase I did not react with either purified E. coli DNA polymerase II or the phage T4-induced DNA polymerase.