PubMed Health⌕ Search

Biomedical subjects

M D Chilton

Publications and source records attributed to M D Chilton.

At least 37 records · Page 2Linked to original sources
PubMed1986

T-DNA and opine synthetic loci in tumors incited by Agrobacterium tumefaciens A281 on soybean and alfalfa plants.

We report here the molecular characterization of transferred DNA (T-DNA) in leguminous tumors incited by Agrobacterium tumefaciens A281 harboring the tumor-inducing plasmid pTiBo542. The T-DNA is composed of two regions named TL (left portion)-DNA and TR (right portion)-DNA, in accordance with the nomenclature for the octopine strains. TL-DNA is defined by several internal HindIII restriction fragments totaling 10.8 kilobase pairs (kbp) in uncloned soybean and alfalfa tumors. Alfalfa tumor DNA may contain one more HindIII fragment at the left end of TL-DNA than does soybean tumor DNA. TR-DNA has a 5.8-kbp BamHI-EcoRI internal fragment. All borders other than the left border of TL-DNA appear to be the same within the detection limits of Southern blot hybridization experiments. The two T-DNA regions are separated by 16 to 19 kbp of DNA not stably maintained in tumors. The distance from the left border of TL-DNA to the right border of TR-DNA is approximately 40 kbp. Loci for the mannityl opines are situated in TR-DNA, based on genetic and biochemical criteria.

Cloning, Molecular↗
PubMed1986

The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA.

We used a binary-vector strategy to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. We constructed plasmids (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs in trans with complementary regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results we suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.

Amino Acids↗
PubMed1985

Expression of the nopaline synthase gene in Escherichia coli.

The Agrobacterium tumor-inducing (Ti) plasmid pTiT37 encodes nopaline synthase (NOS) gene (nos) with eukaryotic promoter elements that is expressed in transformed plant cells but not in the bacterial host. We have fused the nos gene to the Escherichia coli trp promoter, and observed synthesis of NOS in E. coli. The nopaline produced by this enzyme is excreted into the culture medium. NOS is enzymatically active at 30 degrees C but not 37 degrees C, as based on nopaline production. NOS protein is produced at both temperatures, based on production in minicells.

Amino Acid Oxidoreductases↗
PubMed1984

Physical map of the Agrobacterium rhizogenes strain 8196 virulence plasmid.

Virulence of Agrobacterium rhizogenes, agent of hairy root disease, is conferred by large plasmids called Ri (root-inducing) plasmids. We have determined the BamHI fragment map of pRi8196, MW 143 Mda, principally by analysis of recombinant plasmids containing overlapping BamHI partial-digest fragments. Clones containing solitary BamHI inserts of remaining unmapped fragments were used to probe a series of Southern-blotted, pRi8196-derived EcoRI, PstI, HindIII, SalI, or SmaI digests. Continguous hybridized bands represented complements of EcoRI, PstI, HindIII, SalI, or SmaI fragments which bridged the unmapped BamHI fragments. We present, in addition, a detailed map of the core T-DNA region with respect to the restriction endonucleases SalI, EcoRI, HpaI, and HindIII.

Chromosome Mapping↗
PubMed1984

T-DNA fragments of hairy root plasmid pRi8196 are distantly related to octopine and nopaline Ti plasmid T-DNA.

Agrobacterium Ti (tumor-inducing) and Ri (root-inducing) plasmids transform dicot plant cells by insertion of a specific plasmid sector called T-DNA (transferred DNA) into host plant nuclear DNA. The mannopine -type Ri plasmid pRi8196 contains four BamHI fragments that encompass core T-DNA. We report Southern hybridization studies that show that these four fragments have no strong homology to octopine-, nopaline-, or agropine -type Ti plasmids. We detected and mapped very weak homology regions, most of which are assignable to opine synthase or opine catabolic functions on the Ti plasmid. We found no homology between Ri T-DNA and the region of Ti T-DNA that encodes tumor morphology functions.

Arginine↗
PubMed1984

Succinamopine: a new crown gall opine.

Agrobacterium tumefaciens strains can incite plant tumors consisting of transformed cells that synthesize novel metabolites called opines. The pattern of opine synthesis is dictated by plasmid-borne genes in the pathogen; additional plasmid genes confer on the pathogen the ability to catabolize the same pattern of opines synthesized. One group of A. tumefaciens strains, AT181, EU6, and T10/73, contains closely related tumor-inducing (Ti) plasmids that encode the ability to degrade the opine nopaline; but tumors incited by these strains do not synthesize nopaline. We demonstrated by Southern blot hybridization that AT181(pTi) has no DNA homologous to the nopaline synthase gene of pTi T37, a nopaline Ti plasmid that appears to be most closely related to this group based on fingerprint analysis. Tumors incited by these seemingly anomalous strains contain a new opine that we designate succinamopine. Its structure is analogous to that of nopaline, with asparagine replacing arginine. Evidence for the structure of succinamopine, as well as those of two related metabolites, succinamopine lactam and succinopine lactam, will be published elsewhere. Ability to catabolize succinamopine, succinamopine lactam, and succinopine lactam is encoded by pTi AT181, pTi EU6, and pTi T10/73, but not by any of 15 other Ti and root-inducing plasmids tested. Three avirulent strains tested did not catabolize succinamopine, succinamopine lactam, or succinopine lactam. We propose that pTi AT181, pTi EU6, and pTi T10/73 be designated the succinamopine Ti plasmids.

Amino Acids↗
PubMed1984

Mannityl opine analogs allow isolation of catabolic pathway regulatory mutants.

Five virulent Agrobacterium spp. strains that can catabolize the mannityl opines mannopine (MOP), mannopinic acid ( MOA ), and agropinic acid (AGA) were tested for their ability to grow on analogs of these compounds. Analogs containing alternative amino acids replacing glutamic acid or glutamine were generally refused by these bacteria, but mutants were obtained that catabolized the entire family of analogs. In the case of strain C58C1 (pRi 8196), we demonstrated that typical mutants were constitutive for MOP uptake, whereas the wild-type parent was inducible by MOP. Analogs of MOA prepared from a variety of sugars instead of mannose were generally refused, except for a strain carrying pTi B6-806, which grew well on all such analogs. The analogs allowed selection of mutants of all strains. Although most wild-type strains were inducible for AGA uptake, typical mutants selected from strain C58C1 (pRi 8196) were found to be constitutive for uptake of AGA, as was the wild-type strain carrying pTi B6-806. Such constitutive mutants grew on all sugar analogs of MOP, MOA , and AGA tested. The pTi B6-806-containing strain was tested for growth on a more extended series of analogs, including tetrose , triose, diose , and disaccharide analogs, all of which were accepted. Only ketose analogs were refused. Selection of promiscuous regulatory mutants by the two types of opine analogs suggests that the repressor proteins of MOP and AGA permease/ catabolase systems are chiefly responsible for the specificity of the pathways.

Bacterial Proteins↗
PubMed1983

Structure and transcription of the nopaline synthase gene region of T-DNA.

We present the DNA sequence and plant-tumor transcription pattern of some 2400 base pairs from the right border region of pTi T37 DNA from the virulent Agrobacterium tumefaciens strain T37. This region includes the entire transcription unit encompassing the nopaline synthase gene, together with parts of other transcription units. The strategy used to determine the sequence also produced two opposing series of defined, asymmetric deletions across the target DNA region, some of which may serve future purposes in the exploitation of this sequence, which is known to be expressed in a wide variety of host plant tissues.

Amino Acid Oxidoreductases↗
PubMed1983

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.

Alcohol Oxidoreductases↗
PubMed1983

Structure of T-DNA in roots transformed by Agrobacterium rhizogenes.

DNA isolated from hairy roots incited on Daucus carota by Agrobacterium strains harboring the Ri plasmid was probed with Ri plasmid fragments to identify plasmid sequences transferred and integrated into the plant genome. One hairy root line was incited by an A. rhizogenes natural isolate strain harboring two large plasmids in addition to the Ri plasmid. DNA from hairy roots incited by this strain contained a total of five nonidentical copies of T-DNA which together comprised a T-DNA complement representing a 34-42 kb segment of the Ri plasmid. A second hairy root line was incited by a Ti plasmidless A. tumefaciens strain into which the Ri plasmid had been conjugated. Hairy root DNA incited by this strain contained four identical T-DNA copies 17-18 kb in length and one truncated copy. T-DNA borders occurred at preferential sites of the Ri plasmid. T-DNA structure in these hairy root lines is similar to T-DNA structure in tumors incited by Ti plasmids of A. tumefaciens.

DNA↗
PubMed1982

Short direct repeats flank the T-DNA on a nopaline Ti plasmid.

Crown gall disease results from the insertion of a segment of the Agrobacterium Ti plasmid, called T-DNA, into host plant nuclear DNA. We have subjected to sequence analysis the border regions of pTi T37 (ends of T-DNA) and one left T-DNA/plant DNA border fragment isolated from BT37 tobacco teratoma by molecular cloning. These sequence studies, taken together with published sequence of a right T-DNA/plant DNA border fragment, allowed us to identify the positions of left and right borders at the DNA sequence level. Comparison of left and right border regions of the Ti plasmid revealed a "core" direct repeat of 13 of 14 bases (12 contiguous) precisely at the borders of T-DNA. An extended repeat of 21 of 25 bases overlaps this core repeat. T-DNA on the Ti plasmid exhibits no longer direct or inverted repeats in the border regions, based on Southern hybridization studies. The physical structure of T-DNA differs from that of known prokaryotic and eukaryotic transposable elements but bears a structural resemblance to the prophage of bacteriophage lambda.

Journal Article↗
PubMed1982

T-DNA of the Agrobacterium Ti and Ri plasmids.

The study of T-DNA transmission from Agrobacterium Ti or Ri plasmid into the genomes of higher plant cells has revealed much about the consequences of transformation. It is now clear that the transformed phenotype is caused by hormonal changes produced directly or indirectly by T-DNA genes. The opine synthases are enzymes encoded in T-DNA that function in the plant cell. Our level of understanding of T-DNA-encoded functions is already sufficient to reveal clear and feasible ways to exploit T-DNA as a gene vector. What remains to challenge the crown gall investigator are many questions of fundamental importance: What is the mechanism of the seemingly illegitimate recombination between T-DNA and plant DNA, and is this process catalyzed by bacterial or host plant enzymes, or both? Do T-DNA genes encode enzymes that catalyze biosynthesis of auxin- and cytokinin-active substances? What gene in T-DNA confers immunity to A. tumefaciens, and what is its mode of action? Does T-DNA insert into random or specific sites in the host plant genome? Did T-DNA derive from plant genetic information or has prokaryotic DNA arrived at functional eukaryotic gene structure by convergent evolution? Although there is keen interest in T-DNA as a vector for genetic engineering, it holds equal interest as a unique interface between the biology of prokaryotes and eukaryotes.

Arginine↗
PubMed1982

Multiple transcripts of T-DNA detected in nopaline crown gall tumors.

Crown gall plant tumors contain neoplastic cells transformed by incorporation of a foreign DNA element, T-DNA, derived from a large tumor-inducing plasmid in the inciting Agrobacterium strain. T-DNA is covalently joined to the nuclear DNA of the tumor cell, and RNA transcripts from T-DNA are present in polyadenylated form on polysomes. This paper presents a detailed analysis of those parts of T-DNA transcribed in a nopaline-type tobacco teratoma, BT37, whose T-DNA has been mapped and cloned. Northern blots of polyA+ RNA were probed with 21 different nick-translated T-DNA fragments, and at least 13 well-defined transcripts were visualized.

Arginine↗
PubMed1981

Site-specific insertion of genes into T-DNA of the Agrobacterium tumor-inducing plasmid: an approach to genetic engineering of higher plant cells.

This paper presents a method for insertion of genetic material into a specific site in T-DNA, the portion of Agrobacterium tumor-inducing (Ti) plasmid that becomes incorporated into the nuclear DNA of transformed plant cells when crown gall tumors are incited by this plant pathogen. The three stages of our procedure are as follows: 1. A T-DNA subfragment cloned in pBR322 is cleaved by a restriction endonuclease at a unique central site and target DNA (a kanamycin resistance marker) is ligated into this site. 2. The resulting recombinant plasmid is purified and cleaved with EcoRI; the resulting fragment bearing the kanamycin resistance marker is ligated into the unique EcoRI site of pRK290, a wide-host-range plasmid. 3. The pRK290 recombinant plasmid is transformed into an agrobacterium tumefaciens strain containing a wild type Ti plasmid. Double recombination between the altered T-DNA fragment of the clone and its wild-type counterpart in the Ti plasmid is selected for by introduction of R751-pMG2, a plasmid incompatible with pRK290. The approach described here can be adapted for introducing genes into higher plant cells with the Ti plasmid as vector. It can likewise be used for site- or fragment-specific mutagenesis of the Ti plasmid as a means of detailed functional analysis.

DNA↗
PubMed1980

The boundaries and copy numbers of Ti plasmid T-DNA vary in crown gall tumors.

The Ti plasmid DNA maintained in octopine-type crown gall tumor lines is variable, but always includes at least part of the Ti plasmid that maps over the region of Hind III fragment 1 of pTiB6-806. The right-hand boundary of transferred DNA (T-DNA) varies considerably among the three independent tumor lines examined; the left boundary was not located definitively. The T-DNA of two sibling clones of the same tumor line, E1 and E9, appears identical. The copy number of T-DNA in E9 tumor DNA appears higher for the right end (about 30 copies) than for the left end (approximately 1 copy).

Cell Line↗
PubMed1980

Recombination between higher plant DNA and the Ti plasmid of Agrobacterium tumefaciens.

The Ti plasmid sequences (T-DNA) from the octopine-producing crown gall tumor A6S/2 were isolated by molecular cloning, using the bacteriophage lambda vector Charon 4A. Analysis of the cloned DNA segments indicates that the Ti plasmid sequences are covalently joined to plant nuclear DNA. These data demonstrate that genetic recombination between a eukaryote and a prokaryote can occur as a natural phenomenon.

Journal Article↗