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Biomedical subjects

E W Nester

Publications and source records attributed to E W Nester.

At least 109 records · Page 6Linked to original sources
PubMed1983

Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA.

Insertion of the transposon Tn5 into the T-region of the octopine Ti plasmid of Agrobacterium tumefaciens gives rise to crown gall tumors having altered morphology. Three loci within the T-DNA that control tumor morphology have been detected [Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., Gordon, M. P. & Nester, E. W. (1981) Cell 27, 143-153]. They influence tumor size (tml), production of roots (tmr), or production of shoots (tms). Cytokinin and auxin levels in such mutant tumors were examined by HPLC/radioimmunoassay and HPLC/fluorescence assay, respectively. Free indoleacetic acid levels (in pmol/g) were: uninfected tobacco stem tissues, 128; wild-type A348 tumors, 295; tml mutant tumors, 307; tmr mutant tumors, 129; and tms mutant tumors, 70. Average trans-ribosylzeatin levels were correspondingly: 0.97, 48, 40, 0.54, and 1,400 pmol/g. trans-Ribosylzeatin/indoleacetic acid ratios were as high as 24 in shoot-producing tumors and as low as 0.003 in root-producing tumors. The evidence strongly suggests that tumor phytohormone levels are determined by genes in the T-DNA.

Journal Article↗
PubMed1983

Multiple mutations in the T region of the Agrobacterium tumefaciens tumor-inducing plasmid.

Three genetic loci affecting tumor morphology lie within pTiA6NC T-DNA: tms, tmr, and tml. Using deletions and multiple transposon insertions, we constructed tumor-inducing (Ti) plasmids representing every possible double and triple mutant combination. tms tmr and tms tmr tml mutants did not incite tumors on most plants and produced a very weak response on a few other hosts but tms tml and tmr tml mutants were virulent. Thus, either tms+ or tmr+ alone can promote significant tumor growth but tml+ by itself is not sufficient. On hosts where tms mutants induce tumors accompanied by shoot proliferation, addition of a tml mutation reduces or eliminates shoot proliferation, suggesting that tml+ promotes shoot development. The small calli incited by tms tmr and tms tmr tml mutants contain agropine, an indication that these plant cells incorporate T-DNA in the absence of substantial tumor growth.

DNA Transposable Elements↗
PubMed1983

In vivo packaging of cosmids in transposon-mediated mutagenesis.

A technique was developed that permits the analysis of large regions of DNA by transposition mutagenesis. Large fragments of the pTiA6NC plasmid were cloned into the broad host range cosmid pHK17 and subjected to transposition mutagenesis by Tn3. Cosmids containing Tn3 insertions were selected by in vivo packaging by lambda cI857 and transduction to a new host. The insertions were localized by DNA restriction endonuclease analysis and transferred to the Ti-plasmid by marker exchange.

Bacteriophage lambda↗
PubMed1983

Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid.

Forty-nine Tn3 and Tn5 transposition insertion mutations were introduced into the virulence region of the pTiA6NC plasmid of Agrobacterium tumefaciens. Five Tn5 transposition mutations from an earlier study (D. Garfinkel and E. Nester, J. Bacteriol. 144:732-743, 1980) were also mapped more accurately. These mutations defined five separate loci within the virulence region. Two Tn3 insertions into one of these loci, virA, result in a strain which is only weakly virulent; however, a Tn5 insertion into this locus eliminates virulence. One Tn5 insertion into another locus, virC, results in a strain which is weakly virulent. Two additional Tn5 insertions into this locus eliminate virulence. Insertions into the remaining three loci eliminate virulence entirely.

DNA Transposable Elements↗
PubMed1983

Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines.

Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homology. Fingerprints of nopaline Ti plasmids from strains of a given biotype resemble each other but not fingerprints of Ti plasmids from strains of the other two biotypes. The wide host range octopine Ti plasmid from the biotype 3 strain Ag86 shares more DNA homology with narrow host range Ti plasmids, nopaline Ti plasmids, and octopine catabolism plasmids than with the wide host range octopine Ti plasmid from biotype 1 strain 20/1. pTiAg86 does share homology with the portion of pTi20/1 integrated and expressed in plant tumor cells. Since all wide host range Ti plasmids studied contain these sequences, we suggest that natural selection for a wide host range resulted in the presence of the common sequences in distantly related plasmids. The lack of homology between this "common DNA" and limited host range Ti plasmids shows that the DNA sequences per se are not required for tumorigenesis.

Base Sequence↗
PubMed1982

Units of genetic expression in the virulence region of a plant tumor-inducing plasmid of Agrobacterium tumefaciens.

The effect of a large number of Tn3 insertions in the vir region of the Ti plasmid pTiA6NC on the virulence of Agrobacterium was determined. The Vir- insertions were mapped in three of the five loci that have been defined previously. Merodiploid Rec- strains carrying one insertion mutation on the Ti plasmid and another insertion mutation (or the homologous wild-type region) on a compatible plasmid were constructed and used in complementation tests for virulence in test plants. This analysis has revealed that there are ten units of gene expression, presumably transcription units in the vir region. Mutation in one of these units is confirmed to be dominant while those in all others are recessive. Co-infection of test plants with pairs of insertion mutants did not restore virulence.

DNA Restriction Enzymes↗
PubMed1982

T-DNA organization in homogeneous and heterogeneous octopine-type crown gall tissues of Nicotiana tabacum.

Octopine-type tumor tissue was obtained both by infection of plants or isolated protoplasts with Agrobacterium tumefaciens and by somatic hybridization of normal and crown gall tobacco cells. Analysis of T-DNA by Southern blotting of clones and uncloned tissue reveals that, whereas tumors induced on plants are heterogeneous mixtures of cells differing in T-DNA organization, each tissue derived from transformed protoplasts or from somatic hybridization is homogeneous. Detailed analysis of T-DNA organization showed that TL- or "core" T-DNA was always present at one or two copies per diploid genome. However, sometimes it was present in a modified form, either deleted, extended, tandemly duplicated or probably methylated. TR-DNA was not detected. The observed variation in the organization of T-DNA in octopine crown gall tissue did not appear to be a characteristic of the way the tissue was derived.

Arginine↗
PubMed1982

DNA from the A6S/2 crown gall tumor contains scrambled Ti-plasmid sequences near its junctions with plant DNA.

The A6S/2 tumor incited on tobacco by Agrobacterium tumefaciens harboring the octopine-type A6 Ti plasmid contains one insert of Ti-plasmid sequences (the T DNA). This 13 kb insert is derived from a colinear sequence in the Ti plasmid (the T region) and becomes attached to plant DNA in the nucleus of the host cell. We have determined the DNA sequence encompassing the left end of the T region of the A6 Ti plasmid and the corresponding portion of the A6S/2 T DNA. The two sequences are identical for at least 806 bp. To the left of the divergence point, the tumor contains five partially overlapping sequences that are direct or inverted repeats of sequences to the right of the divergence point. The Ti plasmid contains only the right member of each of these repeats. We have also performed heteroduplex studies that indicate that this T DNA has a 520 bp inverted repeat of an internal sequence at the right end near its junction with plant DNA. The repeated sequences near the ends of the T DNA resemble the repeats of adenovirus type 12 sequences found near its junction with host DNA. We discuss data suggesting that the 23 bp to the immediate right of the divergence point of the A6 left junction form a site important in some step in the transfer of T-region DNA from the bacteria to the plant.

Base Sequence↗
PubMed1982

Sizes and map positions of several plasmid-DNA-encoded transcripts in octopine-type crown gall tumors.

The sizes and relative map positions of RNA molecules encoded by the plasmid DNA of several octopine-type tobacco crown gall tumors were determined by blot hybridization. At least six discrete polyadenylylated RNAs (sizes 0.73-1.75 kilobases) were detected in octopine-producing tumors (lines A6S/2, E9, and 15955/01). In one tumor (line 15955/1) in which octopine could not be detected, one of the RNA species was missing and another was diminished in abundance. Evidence is presented suggesting that the transcripts are encoded entirely by the plasmid DNA and do not originate from the surrounding plant DNA sequences.

Arginine↗
PubMed1982

Tumor induction by Agrobacterium rhizogenes involves the transfer of plasmid DNA to the plant genome.

The DNA from tumors of Nicotiana glauca initiated by strains of Agrobacterium rhizogenes was shown to contain sequences that are homologous to the root-inducing (Ri) plasmid of the bacterium. Two independently established tumor lines contained a similar portion of the Ri-plasmid. The Ri-plasmid also hybridized to DNA fragments from uninfected N. glauca. A cosmid clone of the Ri-plasmid encompassing the region containing the Ri-plasmid sequences that are stably transferred to the plant also hybridized to the Ri-plasmid-related fragments found in uninfected plants. Five of six tumor lines tested produced a tumor-specific compound that is similar to agropine.

Journal Article↗
PubMed1982

Complementation analysis of Agrobacterium tumefaciens Ti plasmid mutations affecting oncogenicity.

A wide host range cosmid vector has been constructed by insertion of the lambda cos site into the plasmid pRK2501. This cosmid, which is maintained in Agrobacterium tumefaciens and is compatible with the Ti plasmid, has been used to make a clone bank of the A. tumefaciens pTiA6 plasmid. Several pTiA6 cosmids have been used to complement Tn5-induced Ti plasmid mutations. Five avirulent mutations which map outside of the region of the plasmid maintained in plant tumours (T-DNA) could be complemented in a trans orientation. Two mutations which are located on a single HpaI restriction fragment outside of the T-DNA, as well as three mutations which map within the T-DNA region, could not be complemented in a trans orientation in a REC- host.

Cloning, Molecular↗
PubMed1982

Agrobacterium tumefaciens mutants affected in attachment to plant cells.

An analysis of Agrobacterium tumefaciens mutants with Tn5 insertions in chromosomal DNA showed that the chromosome of A. tumefaciens codes for a specific ability of this bacterium to attach to plant cells. This ability is associated with tumorigenesis by A. tumefaciens, the ability of avirulent A. tumefaciens to inhibit tumorigenesis, and the ability to adsorb certain phages. A second class of chromosomal mutations affects tumorigenesis without altering the ability to attach to plant cells. The attachment of A. tumefaciens to plant cells was assayed by mixing radiolabeled bacteria with suspensions of tobacco tissue culture cells or freshly isolated Zinnia leaf mesophyll cells. Under the conditions of this assay, an avirulent Ti plasmid-cured strain attached to the same extent as the same strain containing pTiB6806. Six of eight avirulent mutants with Tn5 insertions in chromosomal DNA showed defective attachment, whereas two retained wild-type attachment ability. In contrast to the strains showing wild-type attachment, the attachment-defective mutants failed to inhibit tumorigenesis when inoculated onto Jerusalem artichoke slices before inoculation of a virulent strain and also showed a loss of sensitivity to two Agrobacterium phages. The loss of phage sensitivity appeared to be due to a loss of ability to adsorb the phages. Staining with Calcofluor indicated that the mutants retained the ability to synthesize cellulose fibrils, which have been implicated in the attachment process. Southern filter hybridizations demonstrated that each mutant contained a single Tn5 insertion, and genetic linkage between the Tn5 insertion in one mutant and the attachment phenotype has also been demonstrated.

Adsorption↗
PubMed1981

Relationship between the limited and wide host range octopine-type Ti plasmids of Agrobacterium tumefaciens.

The relationship between the limited host range octopine Ti plasmids and the wide host range octopine Ti plasmids pTiB6806 and pTiA6 was studied. The limited host range Ti plasmids shared extensive deoxyribonucleic acid homology; pTiAg63 and pTiAg162 were essentially completely homologous with pTiAg158 while pTiAg57 shared approximately 64% homology with pTiAg158. In contrast, the limited host range octopine Ti plasmids only shared 6 to 15% homology with the wide host range octopine Ti plasmid pTiB6806. Thus, limited and wide host range octopine Ti plasmids comprise distinct families of plasmids. The deoxyribonucleic acid homology shared between the limited host range Ti plasmids and pTiB6806, however, was distributed over some 50% of pTiB6806, suggesting that both families of plasmids evolved from a common progenitor plasmid. The limited host range Ti plasmids showed relatively strong homology with pTiB6806 HpaI fragment 7, a region which codes for octopine utilization by the bacterium, but showed only weak homology with pTiB6806 HpaI fragment 12, a region required for virulence. In addition, homology between the limited host range octopine Ti plasmids and the "common deoxyribonucleic acid," sequences shown to have a central role in plant cell transformation, was barely detectable when stringent hybridization conditions were used. We therefore conclude that a highly conserved version of the common deoxyribonucleic acid is not required for crown gall tumorigenesis on all plant species.

Arginine↗