Martin H. Drummond

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.

Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumours

Agrobacterium tumefaciens incites cancerous growths in dicotyledonous plants called crown gall tumours. Large plasmids in oncogenic Agrobacterium strains1 carry genetic information which is essential for tumour induction2,3. We recently reported that axenic crown gall tumour tissue contains multiple copies of a small part of the oncogenic (Ti) plasmid of the inciting bacterial strain4. The mechanism of induction of crown gall tumours thus seems to resemble that of some virally induced animal tumours: the eukaryotic cell is transformed by addition of new genetic information which is stably maintained through subsequent cell divisions5–7. The means by which foreign DNA brings about the tumorous growth pattern of crown gall cells is unknown. The first step in elucidating its mode of action is reported here: we have detected RNA transcripts of the foreign genetic information in the tumour cell.

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.

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.

The Incorporation and Expression of Agrobacterium Plasmid Genes in Crown Gall Tumors

Ever since Smith and Townsend proved in 1907 that a bacterium is the causative agent of crown gall tumors (1), the most intriguing question has been, how does the bacterium induce these modifications? The elegant experiments of Braun and his collaborators first convincingly demonstrated that the continued presence of viable bacteria is not required for tumor formation (2). If the bacteria are heat killed about 2 days after being applied to a wounded plant the tumor develops normally. Thus, attention has focused on the identification of a putative “tumor inducing principle” elaborated by the bacteria and transferred to the plant. This paper will present evidence that this principle is a fragment of a large plasmid which is transferred to the plant.