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M D Chilton

Publications and source records attributed to M D Chilton.

At least 55 records · Page 3Linked to original sources
PubMed1980

Agropine in "null-type" crown gall tumors: Evidence for generality of the opine concept.

Agrobacterium Ti (tumor-inducing) plasmids, the causative agents of crown gall disease, fall into four genetic groups based on the patterns of octopine and nopaline synthesis (by crown gall tumors) and catabolism (by Agrobacterium tumefaciens) for which they are responsible. Two classes of Ti plasmids induce tumors that synthesize neither octopine nor nopaline. The existence of these Ti plasmids challenged the view that opines such as octopine and nopaline play a central role in crown gall biology. We now report the occurrence of an opine in tumors induced by one of these classes of Ti plasmids, the "null-type" plasmids typified by pTi Bo542. The opine was purified by biological enrichment based on its utilization by bacteria containing pTi Bo542 but not by bacteria lacking a Ti plasmid. The mass spectrum and biological properties of this opine are identical to those of agropine, an opine recently discovered in octopine-type tumors. We propose that null-type Ti plasmids now be named for their signal opine, agropine-type Ti plasmids.

Journal Article↗
PubMed1980

T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells.

The crown gall teratoma tumor line BT37, incited by Agrobacterium tumefaciens strain T37, has been found to contain part of the tumor-inducing plasmid, pTi T37, of the inciting strain. This foreign DNA segment, called T-DNA, is maintained at several copies per diploid tumor cell. We have examined subcellular DNA fractions from this tumor line in an effort to determine whether T-DNA is in chloroplasts, mitochondria, or nuclei. Tumor cell chloroplast DNA exhibited EcoRI and Bst I endonuclease cleavage patterns identical to those of normal tobacco chloroplast DNA. Tumor cell mitochondrial DNA exhibited a complex Bst I cleavage pattern that did not differ detectably from that of normal tobacco mitochondrial DNA. Southern blots of tumor chloroplast and mitochondrial cleavage products did not hybridize with labeled pTi T37 DNA, whereas blots of tumor cell nuclear DNA cleavage products hybridized strongly. We conclude that T-DNA is located not in chloroplasts or mitochondria but rather in the nuclear fraction of this tumor line.

Journal Article↗
PubMed1977

Octopine and nopaline metabolism in Agrobacterium tumefaciens and crown gall tumor cells: role of plasmid genes.

Crown gall tumors produced octopine or nopaline or neither compound, depending on the bacterial strain that incited the tumor. The genes specifying production of octopine or nopaline by the tumor were transferred to recipient bacterial strains when the large plasmid associated with virulence was transferred by either conjugation or deoxyribonucleic acid-mediated transformation. Our results, which confirm the work of others (Bomhoff et al., 1976; Goldman et al., 1968; Petit et al., 1970), indicate that, in general, the strains that utilize octopine induce tumors that synthesize octopine, and those that utilize nopaline induce tumors that synthesize nopaline. However, there were several notable exceptions. One class utilized both octopine and nopaline, but the tumors induced by these strains produced only nopaline. Another class utilized nopaline, but their tumors synthesized neither nopaline nor octopine. Mutants were isolated from a number of either octopine- or nopaline-utilizing strains that no longer could utilize the relevant guanido amino acid. These strains, which were mutant in the gene specifying octopine or nopaline oxidase, still retained the permease for these amino acids as well as virulence. Tumors induced by these mutants still synthesized approximately the same levels of octopine and nopaline as tumors induced by their parents. These results suggest that the plasmid gene that determines production of octopine or nopaline by the tumor is distinct from the plasmid gene that determines their catabolism by the bacteria.

Arginine↗
PubMed1976

RP4 promotion of transfer of a large Agrobacterium plasmid which confers virulence.

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.

Arginine↗
PubMed1975

Attempts to detect Agrobacterium tumefaciens and bacteriophage PS8 DNA in crown gall tumors by DNA-DNA-filter hybridization.

A systematic study of the DNA-DNA-filter reaction is presented which measures its ability to detect small amounts of simple DNA (bacterial or bacteriophage) in model mixtures of DNA immobilized on filters. Saturation curves show qualitatively that significant binding occurs when there is 10% Agrobacterium tumefaciens DNA on the filter but not 1%. PS8 bacteriophage DNA is detectable at a level of 0.1%. True saturation is not attained in the bacterial DNA reaction : radioactivity bound represents only 3% of the theoretical saturation value. The bacteriophage DNA reactions attain 15-30% of the expected saturation value. When crown gall tumor DNA filters were tested for the presence of A. tumefaciens or PS8 bacteriophage DNA by saturation reactions, an apparently significant amount of binding was observed compared with usual background levels for heterologous DNA filters. However thermal dissociation profiles revealed that no well-matched duplexes were formed. Normal tobacco callus DNA filters exhibited the same type of binding of labeled DNA to a similar extent (50-100% as much as tumor DNA filters). Both types of DNA-filters bound Bacillus subtilis and bacteriophage T4 DNA as efficiently as A. tumefaciens and PS8 DNA. The high non-specific background binding of labeled DNA by filters containing DNA isolated from plant tissue culture materials is ascribed to low single strand molecular weight of the filterbound DNA. This study provides no evidence for foreign DNA in crown gall tumors, and raises objections to the interpretation of the data of earlier investigators (Quetier, F., Huguet, T. and Guille, E. (1969) Biochem, Biophys. Res, Commun. 34, 128-133 and Srivastava, B.I.S. (1970) Life Sci. 9, 889-892) who claimed to detect A. tumefaciens DNA in crown gall tumors by DNA-DNA-filter hybridization.

Bacteriophages↗
PubMed1975

On the question of the integration of exogenous bacterial DNA into plant DNA.

Extensive studies with pea, tomato, and barley failed to confirm the evidence presented by previous investigators for integration or replication of exogenously applied bacterial DNA in these plants. Labeled DNA of buoyant density in CsCl intermediate between that of high density donor bacterial DNA and of plant DNA was never observed with axenic plants. Intermediate peaks, similar to those used as evidence for recombination by earlier investigators, were observed only when the plants were contaminated with bacteria. Plant DNA prepared by a published procedure [Ledoux, L. & Huart, R. (1969) J. Mol. Biol. 43, 243-262] was found to be contaminated with unidentified impurities. Such DNA was partially protected from the action of DNase and produced aberrant banding patterns in CsCl after shearing. Much of the published evidence for integration of foreign DNA in plants is based upon experiments with plant DNA prepared by this procedure. We conclude that contamination is the likely explanation for what has been interpreted as evidence for integration.

Centrifugation, Density Gradient↗
PubMed1975

Plasmid required for virulence of Agrobacterium tumefaciens.

The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37 C is shown to be due to loss of a large plasmid (1.2 X 10-8 daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6 is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 X A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

Amino Acids↗
PubMed1975

On the question of integration of Agrobacterium tumefaciens deoxyribonucleic acid by tomato plants.

Treatment of tomato plants with Agrobacterium tumefaciens causes subsequently administered [3H]thymidine to be preferentially incorporated into a satellite deoxyribonucleic acid (DNA) whose buoyant density is between that of bacterial DNA (rho = 1.718 g/cm3) and plant main band DNA (rho = 1.692 g/cm3). Satellite DNA upon shearing or sonic treatment releases fragments of higher and lower buoyant density, as reported by earlier investigators. The satellite has no significant base sequence homology with A. tumefaciens DNA, for its rate of reassociation is not accelerated by the addition of high concentrations of the latter. Tomato DNA isolated from shoots or from leaf nuclei accelerates renaturation of labeled satellite DNA. We conclude that the intermediate density labeled DNA is a plant satellite and not the product of covalent joining of bacterial and plant DNA as suggested by earlier investigators.

Centrifugation, Density Gradient↗
PubMed1974

Detection and quantitation of octopine in normal plant tissue and in crown gall tumors.

Octopine has been detected in normal tobacco leaf and stem tissue, normal sunflower stem tissue, pinto bean leaves, and normal tobacco callus tissue in culture. Octopine was identified in extracts by means of electrophoresis and chromatography in several solvent systems. Tobacco and sunflower tumor lines induced by various strains of Agrobacterium tumefaciens were found to contain from 1 to 240 times as much octopine as the normal plant tissues examined. Several strains of A. tumefaciens produce undifferentiated tobacco tumors containing high levels of octopine, but produce undifferentiated sunflower tumors containing normal levels of octopine and high levels of arginine. Further, strain CGIC of A. tumefaciens produces in tobacco an undifferentiated tumor which contains high levels of octopine and a teratoma which contains normal levels of octopine. This evidence shows that there is no consistent relationship between the causative strain of A. tumefaciens and the octopine content of the resulting crown gall tumor.

Journal Article↗
PubMed1974

Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors.

Renaturation kinetics of labeled Agrobacterium tumefaciens DNA are not influenced by addition of 10(4)-fold excess of crown gall tumor DNA. Reconstruction experiments demonstrated that 0.01% added bacterial DNA produces a detectable increase in rate of renaturation of labeled DNA. Crown gall tumor DNA therefore cannot contain as much as 0.01% A. tumefaciens DNA (one entire bacterial genome per three diploid tumor cells). By the same technique, PS8 bacteriophage DNA is not detected in crown gall tumor DNA under conditions that allow detection of 0.0007-0.001% added phage DNA. Crown gall tumor DNA cannot contain as much as one entire phage genome per diploid tumor cell. The presence of a small fraction of either genome (less than 5%), even if present as multiple copies, would escape detection by this method.

Bacteriophages↗
PubMed1974

Attempts to detect deoxyribonucleic acid from Agrobacterium tumefaciens and bacteriophage PS8 in crown gall tumors by complementary ribonucleic acid-deoxyribonucleic acid-filter hybridization.

Labeled ribonucleic acid (RNA) complementary to Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA (cRNA) were used in a systematic study of the sensitivity of cRNA/deoxyribonucleic acid (DNA)-filter hybridization for detection of small amounts of phage or bacterial DNA immobilized on filters. A. tumefaciens cRNA of specific activity 10(6) to 2 x 10(6) counts per min per mug reacted to a significant extent when the DNA-filter contained 1% A. tumefaciens DNA in a salmon DNA background, but 0.1% A. tumefaciens DNA was not detectable. PS8 phage cRNA of the same specific activity reacted to a significant extent when the DNA-filter contained as little as 0.01% PS8 DNA in a salmon DNA background. Both kinds of cRNA were found to bind to tobacco crown gall tumor DNA-filters. Similar reaction was found with control normal callus DNA-filters but not with tobacco seedling DNA-filters. The "hybrids" formed by cRNA with normal callus and tumor DNA-filters had low thermal stability. Attempts to purify the tumor and normal callus DNA prior to immobilization on the filter resulted in elimination of this spurious binding. No evidence was found for bacterial or phage DNA in crown gall tumor DNA.

Bacteriophages↗