Alice L. Montoya

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

Abstract 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 × 10 6 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.

Fingerprints of Agrobacterium Ti plasmids

Abstract Many crown gall-inducing Agrobacterium tumefaciens strains have multiple plasmids, only one of which, the tumor-inducing (Ti) plasmid, is essential for oncogenicity. For comparison of Ti plasmids, single-plasmid-containing transconjugant or transformant derivatives were used as sources of pure Ti-plasmid DNA. Fingerprinting was undertaken using the restriction endonuclease Sma I because it produced a relatively simple cleavage pattern. Three groups of Ti plasmids are discernible based upon both their genetic characteristics and their Sma I fingerprints: (1) Octopine-type Ti plasmids, which confer oncogenicity and octopine utilization on the bacterium. Tumors incited produce octopine. This group of plasmids is highly conservative; fingerprints of all members were identical except for two minor variations. (2) Nopaline-type Ti plasmids, which confer oncogenicity and nopaline utilization on the bacterium. Tumors incited may or may not produce nopaline; these plasmids have fingerprints that suggest variable degrees of relationship, including one that appears unrelated to the rest (3) Null-type plasmids, which confer oncogenicity but neither utilization trait on the bacterium. Tumors incited by this class of strains produce neither octopine nor nopaline. Only one member of this group has been examined thus far. Fingerprints of plasmids from several nononcogenic strains examined bore no resemblance to fingerprints of any of the Ti plasmids.

Molecular and genetic analysis of factors controlling host range in Agrobacterium tumefaciens

SummaryWe have investigated the factors which contribute to the host specificity of a tumor inducing plasmid of Agrobacterium, pTiAg162, which confers a narrow host range. Determinants both within the T-DNA and virulence regions contribute to host specificity. Within the T-DNA a defective cytokinin biosynthetic gene limits host range. Nucleotide sequence analysis revealed a large deletion in the 5′ coding region of this gene when compared with the homologous gene from the wide host range tumor inducing plasmid, pTiA6. Introduction of the wide host range cytokinin biosynthesis gene into the T-DNA of the limited host range strain expanded the host range and suppressed the rooty morphology of tumors incited by the limited host range strain. Two genes from the virulence region of the wide host range plasmid, designated virA and virC, must also be introduced into the limited host range strain in order to restore a wide host range phenotype. The wide host range strain is avirulent on some cultivars of Vitis plants on which the limited host range strain induces tumors. This avirulence is apparently due to a hypersensitive response in which infected plant cells are killed at the site of inoculation. Mutations within the virC locus of the wide host range plasmid prevented the hypersensitive response and allowed the formation of tumors by the wide host range strain.

Foreign DNA sequences in crown gall teratomas and their fate during the loss of the tumorous traits

SummaryA cloned tobacco crown gall teratoma induced by A. tumefaciens strain T37 was found to contain part of the Ti plasmid (T-DNA) of the causative bacterium. The tumor line contained transcripts of the ends of the T-DNA; transcripts of the intervening T-DNA were not detected.When grafted to a healthy tobacco plant, the cloned tumor line produced a plant that flowered and set seed. Tissues from leaves and flower petals when placed in tissue culture resumed a malignant growth pattern and grew in the absence of phytohormones. Both leaf and flower petal tissues were found to contain T-DNA, but in smaller quantity than observed in the parental line. Haploid tissue derived from anther and tissue from F1 progeny plants, which morphologically appear to be completely normal and require phytohormones for growth in vitro, were found to be free from T-DNA. Thus, meiosis acts either to cause or select for loss of the foreign DNA. The correlation between the presence of foreign DNA and the tumor phenotype indicates that the continued presence of T-DNA is required for the maintenance of the tumorous state.

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.

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

SummaryThe 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 pTi-B6-806. 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).

Restriction endonuclease mapping of a plasmid that confers oncogenicity upon Agrobacterium tumefaciens strain B6-806.

Abstract The 26 SmaI digest fragments of pTi-B6-806 plasmid have a total molecular weight (121 × 106) which accounts for the size of the plasmid as determined by contour length measurements. We have determined the physical arrangement of all SmaI digest fragments with reference to HpaI digest fragments. Hybridization of individual labeled SmaI digest fragments to HpaI digest fragments (cellulose nitrate transfers) allowed the latter to be ordered and located the SmaI boundary fragments. Recleavage of isolated HpaI fragments with SmaI revealed the SmaI fragments located within each HpaI fragment. The order of these internal SmaI fragments within a given HpaI fragment was determined by partial digestion of the latter with SmaI and hybridization of the resulting fragments with SmaI boundary fragments. From the sizes of partial digest fragments containing each boundary, the order of occurrence of SmaI fragments from each end was deduced. The complete map of the SmaI digest fragments is presented. The map of the HpaI digest fragments is presented with the following ambiguity: The order of fragments 12, 15, and 16, which map within SmaI fragment 1, was not determined. The SmaI digest fragments that contain DNA sequences transferred to plant cells during tumor induction, fragments 3b and 10c, were found to be contiguous on the physical map.

Plant tumor reversal associated with the loss of foreign DNA

SummaryTransformation of plant tissues into crown gall tumors has been associated with the transfer of a portion of a tumor-inducing plasmid (Ti-plasmid) into plant DNA. Various laboratories have regenerated normal-appearing plants from a number of crown gall tumors. This study investigates the fate of the foreign DNA in a series of tissues derived from various parts of a plant regenerated from the tumor BT-37 by Braun and his coworkers. It was found that all the foreign DNA sequences were lost from tissues that had lost all their tumorous traits; whereas the plasmid DNA sequences were still present in tissues that appeared normal but still exhibited tumorous traits when returned to tissue culture media. From these studies it would appear that the presence of the Ti-plasmid sequences in the plant DNA is required for the maintenance of the transformed state.

Cross pathway regulation of tyrosine and histidine synthesis in Bacillus subtilis. Biochemical, genetic, and transfer RNA studies.

Abstract A large number of mutants resistant to the histidine analog, 1,2,4-triazole-3-alanine or the tyrosine analogs, d -tyrosine and fluorotyrosine were isolated in the wild type or bradytrophic strains of Bacillus subtilis . Most of these mutants had elevated levels of enzymes in both the tyrosine and histidine biosynthetic pathways. These mutants could be divided into six different groups based on the levels of these enzymes in cells grown on minimal medium and on medium supplemented with tyrosine or histidine. The gene or genes conferring resistance to these analogs mapped near the purB locus by DNA-mediated transformation. The activating enzymes and chromatographic patterns of tRNA for histidine and tyrosine were compared in preparations from wild type cells and several mutant classes, but there were no detectable differences in these components. The possibility that the common element in the control of enzyme synthesis in the tyrosine and histidine pathways is an aporepressor is discussed.

The molecular basis of plant cell transformation by Agrobacterium tumefaciens.

Crown gall is an uncontrolled proliferation of tumor cells incited by Agrobacterium tumefaciens on most dicotyledonous plants. To accomplish this, the bacterium transfers and integrates a portion (the T-region) of its tumor inducing (Ti) plasmid into plant nuclear DNA (1,2,3,4). The transferred DNA (T-DNA) is expressed in the transformed cell and encodes enzymes involved in the synthesis of amino acid condensation products called opines (5,6,7,8,9,10). This paper will report data which suggest that T-DNA alos encodes genes which specify the synthesis... also encode the synthesis of the phytohormones, Indole acetic acid, Isopentenyl adenosine, Isopentenyl adenosine 5 monophosphate, and Isopentenyl adenine.