Integration and organization of Ti plasmid sequences in crown gall tumors

Thomashow, Michael F.; Nutter, Robert C.; Montoya, Alice L.; Gordon, Milton P.; Nester, Eugene W.

doi:10.1016/s0092-8674(80)80049-3

Cited by 421 publications

(248 citation statements)

References 35 publications

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“…We investigated whether any sequences homologous to the IS66 sequence were present in Ti plasmid genomes. The octopine Ti plasmids contained at least three homologous sequences: two were found in the vir region and one was in the region where the right-hand border of TL-DNA and the left-hand border of TR-DNA were mapped (16). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, Barker et al (34) determined the entire sequence of the T region of the octopine Ti plasmid pTil5955, which was shown to be highly homologous to pTiB6806 (16). The analyses of the sequence showed that the T region is composed of three distinct domains, TL-DNA, TR-DNA, and the center region of (34), and that the insertion split the T region into two portions, TL-DNA and TR-DNA.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…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 contained the right portion (TR-DNA).…”

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confidence: 99%

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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.

Machida¹,

Sakurai²,

Kiyokawa³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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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.

Machida¹,

Sakurai²,

Kiyokawa³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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“…MA). The blots were analyzed by DNA gel blot hybridization according the protocol of Thomashow et al (1980), except that 0.2% sodium pyrophosphate was included in all solutions. Virus-and component-specific probes were randomly labeled using U-~'P-~ATP and the Klenow fragment (Sambrook et al, 1989).…”

Section: Replication Assaysmentioning

confidence: 99%

Geminivirus replication origins have a modular organization.

et al. 1994

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Tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) are closely related geminiviruses with bipartite genomes. The A and B DNA components of each virus have cis-acting sequences necessary for replication, and their A components encode trans-acting factors required for this process. We showed that virus-specific interactions between the cis-and trans-acting functions are required for TGMV and BGMV replication in tobacco protoplasts. We also demonstrated that, similar to the essential TGMV AL1 replication protein, BGMV AL1 binds specifically to its origin in vitro and that neither TGMV nor BGMV AL1 proteins bind to the heterologous origin. The in vitro AL1 binding specificities of the B components were exchanged by site-directed mutagenesis, but the resulting mutants were not replicated by either A component. These results showed that the high-affinity AL1 binding site is necessary but not sufficient for virus-specific origin activity in vivo. Geminivirus genomes also contain a stem-loop sequence that is required for origin function. A BGMV B mutant with the TGMV stem-loop sequence was replicated by BGMV A, indicating that BGMV AL1 does not discriminate between the two sequences. A BGMV B double mutant, with the TGMV AL1 binding site and stemloop sequences, was not replicated by either A component, indicating that an additional element in the TGMV origin is required for productive interaction with TGMV AL1. These results suggested that geminivirus replication origins are composed of at least three functional modules: (1) a putative stem-loop structure that is required for replication but does not contribute to virus-specific recognition of the origin, (2) a specific high-affinity binding site for the AL1 protein, and (3) at least one additional element that contributes to specific origin recognition by viral trans-acting factors.

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Gentechnik mit Pflanzen

1987

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Professor Heinz Harnisch zum 60. Geburtstag gewidmetIsolierung und Charakterisierung pflanzlicher Gene bereiten heute keine grundsatzlichen Schwierigkeiten mehr. Die Erkenntnisse iiber Struktur und Funktion der bisher sequenzierten Gene ermbglichen es bereits, die Genregulation in Pflanzen wenigstens in groBen Ziigen zu verstehen. In Verbindung mit den in letzter Zeit entwickelten Methoden des Gentransfers in pflanzliche Zellen ergibt sich der fur die Anwendung wichtigste Aspekt: die gentechnische Gewinnung von Pflanzen rnit neuen Eigenschaften. Da die zu ubertragenden Gene aus beliebigen Pflanzen, ja sogar aus beliebigen Organismen stammen konnen, werden hier vollig neue Moglichkeiten fur die Pflanzenzuchtung erbffnet.

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Integration and organization of Ti plasmid sequences in crown gall tumors

Cited by 421 publications

References 35 publications

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.

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.

Geminivirus replication origins have a modular organization.

Gentechnik mit Pflanzen

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