Nucleotide sequence of the insertion sequence found in the T-DNA region of mutant Ti plasmid pTiA66 and distribution of its homologues in octopine Ti plasmid.

Abstract

The octopine tumor-inducing (Ti) plasmid pTiA66 has an insertion mutation in its T region (the DNA region incorporated into the plant genome) that results in the slow growth of crown gall tumors. These tumors exhibit hormonal autonomy different from that of the crown gall tumors caused by wild-type Ti plasmids. In the present study, the nucleotide sequences of both the DNA segment inserted into pTiA66 and its target site have been determined. The inserted segment is 2548 base pairs long and has 20-base-pair terminal inverted repeats. An 8-base-pair sequence at the target site is duplicated at both integration junctions. These structural features of the insert suggest that it is a bacterial insertion sequence (IS) element, which we have named IS66. Blot-hybridization analyses using IS66 probes revealed that genomes of octopine Ti plasmids contain at least three sequences homologous to IS66: two homologues are located in the virulence region and one is located between the left-hand (TL-DNA) and right-hand (TR-DNA) portions of T-DNA. The chromosome of Agrobacterium tumefaciens A66 also contains two sequences highly homologous to IS66. These results suggest that the mutant pTiA66 plasmid was generated by translocation of one of the sequences showing homology with IS66 into the T region. The fact that a sequence homologous to IS66 is present between TL-DNA and TR-DNA also suggests that the octopine T region was split into two portions, TL-DNA and TR-DNA, by translocation of IS66 or its relatives. Thus, IS66 may cause genetic and structural variations of the T region and the vir region of the octopine Ti plasmids.Tumor-inducing (Ti) plasmids harbored by oncogenic strains of Agrobacterium tumefaciens cause formation of tumors on dicotyledonous plants, called crown galls (1). Cells isolated from the crown gall tumors are characterized by their unlimited proliferation in medium lacking phytohormones, such as cytokinins and auxins, that are required for the culture of normal plant cells (2). It has been shown that a specific DNA region of the Ti plasmid, called the T-DNA region (Fig. 1), causes tumor formation when transferred into plant chromosomes and that the T-DNA persists in the chromosomes of the tumor cells (3-6). The virulence (vir) region of the Ti plasmid (Fig. 1) is located outside the T region and is also required for tumorigenesis by the Ti plasmid. However, this region is probably not stably maintained in the tumor cells (7-10).The Ti plasmids are classified on the basis of the novel amino acid metabolites, such as octopine or nopaline, whose syntheses are directed by T-DNA genes in the tumor cells (11-15). The octopine T region is composed of twQ DNA segments near each other in the Ti plasmid (16) (Fig. 1), whereas the nopaline T region consists of one contiguous DNA segment (17, 18). The chromosomes of the tumor cells caused by the octopine strain contain either one or two portions of the T region (16). All tumor cells that have been analyzed contained the left portion of T-DNA (TL-DNA) but not all...

Structural and Functional Characterization of IS 679 and IS 66 -Family Elements

“…(for a review, see reference 16). Unlike IS679, many have more than three ORFs; for example, the early isolates IS66 (15) and IS866 (2) have, respectively, four and five ORFs. No study so far, however, has addressed the transposition capability and requirement of ORFs in IS66 family elements.…”

Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors

“…As far as gram-negative bacteria are concerned, most IS elements have been isolated from enteric bacteria (mainly E. coli; for reviews and lists of isolated elements, see references 14 and 20). However, there have been a substantial number of reports on IS elements identified in other gram-negative bacterial species, such as Agrobacterium tumefaciens (3,15,24,42), Xanthomonas campestris (22, 23), and various Rhizobium (9,28,32) and Pseudomonas (6,13,35,41,45) strains.In general, these elements have been found more or less by chance because of the capacity to inactivate or activate particular genes. However, there have been successful attempts to screen for transposable elements by means of direct selection procedures (8,15,30,35).…”

“…As far as gram-negative bacteria are concerned, most IS elements have been isolated from enteric bacteria (mainly E. coli; for reviews and lists of isolated elements, see references 14 and 20). However, there have been a substantial number of reports on IS elements identified in other gram-negative bacterial species, such as Agrobacterium tumefaciens (3,15,24,42), Xanthomonas campestris (22,23), and various Rhizobium (9,28,32) and Pseudomonas (6,13,35,41,45) strains.…”

IS292: a novel insertion element from Agrobacterium

“…However, no known sequence was found related to ORF4. IS272 is readily distinguishable from IS426 and IS427 isolated from the Agrobacteritrm nopaline-type strain "37 (Vanderleyden et al, 1986;De Meirsman e t al., 1989, 1990, from IS7 737 isolated from the nopaline-type strain PO22 (Wabiko, 1992), from IS866 and IS867 isolated from the octopine/vitopine-type strain Tm4 (Bonnard e t al., 1989;Paulus et al, 1989bPaulus et al, , 1991a and from IS66 isolated from the octopine-type strain A66 (Machida et al, 1984). However, we found similarity between terminal inverted repeats of IS272, IS66, IS866 and IS7737 from Agrobacteritrm, ISRl7 from R. legzlminosarzim and ISRm2 from R. meliloti, but limited to a six nucleotide consensus sequence (Fig.…”