H. De Greve

Genetic identification of functions of TL-DNA transcripts in octopine crown galls

The TL‐DNA in octopine crown galls encodes seven transcripts most, if not all, expressed from individual promoters. Site‐specific deletions and substitutions in the T‐region of the octopine plasmid pTiB6S3 indicate some of the functions of the TL‐DNA transcripts. Two of the seven genes are sufficient to allow tumorous growth. T‐DNA transfer and oncogenicity are controlled by different and independently acting functions. None of the transcripts of TL‐DNA appear to be essential for T‐DNA transfer. Four, possibly five, of the TL‐DNA transcripts act by suppressing organ development. Shoot and root formation are suppressed by the action of different transcripts.

The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5

We have determined the complete primary structure (13 637 bp) of the TL‐region of Agrobacterium tumefaciens octopine plasmid pTiAch5 . This sequence comprises two small direct repeats which flank the TL‐region at each extremity and are involved in the transfer and/or integration of this DNA segment in plants. TL‐DNA specifies eight open‐reading frames corresponding to experimentally identified transcripts in crown gall tumor tissue. The eight coding regions are not interrupted by intervening sequences and are separated from each other by AT‐rich regions. Potential transcriptional control signals upstream of the 5′ and 3′ ends of all the transcribed regions resemble typical eukaryotic signals: (i) transcriptional initiation signals (‘TATA’ or Goldberg‐ Hogness box) are present upstream to the presumed translational start codons; (ii) ‘ CCAAT ‘ sequences are present upstream of the proposed ‘TATA’ box; (iii) polyadenylation signals are present in the 3′‐untranslated regions. Furthermore, no Shine‐Dalgarno sequences are present upstream of the presumed translational start codons.

Restoration of virulence of Vir region mutants of Agrobacterium tumefaciens strain B6S3 by coinfection with normal and mutant Agrobacterium strains

SummaryThree avirulent Tn7 insertion mutants mapping in the vir E region of the Agrobacterium tumefaciens plasmid pTiB6S3 regain virulence by co-infection with several wildtype strains and with a number of strains carrying mutations in other regions of the Ti plasmid. This finding indicates that during tumour induction normal Agrobacterium strains produce a diffusable factor required for transformation and might allow the isolation of such a factor.

The functional organization of the octopine Agrobacterium tumefaciens plasmid pTiB6S3

We have used the transposon Tn7 to isolate insertion and deletion mutations in the octopine Ti-plasmid pTiB6S3. Mutations that inactivate most of the known Ti phenotypes have been located on the physical map. Most importantly, we have positioned several regions involved in the determination of oncogenicity. They correspond to homology regions between octopine and nopaline plasmids. One of these regions, the T region, is part of the Ti-plasmid DNA present in transformed plant cells. Some Tn7 insertions in this region are weakly virulent and the tumor tissue, incited by these mutant Ti plasmids, readily produce proliferating shoots.

The pathogenesis of edema disease in pigs. A review

Edema disease is known to cause important losses in the period shortly after weaning. Although the disease is known for many decades, intensive studies with bacterial lysates of pathogenic E. coli, followed by biotechnological research the last ten years, has led to a better understanding of its pathogenesis. Especially the impact of the toxin is clearly established. Evidence also exists that adhesion factors play a crucial role in the pathogenesis of edema disease.

Identification of sequences involved in the polyadenylation of higher plant nuclear transcripts using Agrobacterium T-DNA genes as models.

Sequences in the 3′‐untranslated region of two different octopine T‐DNA genes were analyzed with regard to their significance in polyadenylation. Poly(A) addition sites were localized precisely by S1 nuclease mapping with T‐DNA‐derived mRNAs isolated from tobacco. The gene encoding transcript 7’ contains two AATAAA hexanucleotides, respectively 119 bp and 170 bp downstream of the TAA stop codon. A single poly(A) site was mapped 24‐25 bp downstream of the first AATAAA. Further, we show that a mutant octopine synthase gene, which has lost part of its 3′‐untranslated region by deletion, is still active. This mutant gene terminates 19 bp upstream from the major wild‐type polyadenylation site. The deletion also removes the AATAAT signal preceding this site. The mutant octopine synthase gene contains a minimum of four different poly(A) sites. The most prominent of these sites is identical to the minor poly(A) site of the wild‐type gene, and is preceded by a sequence AATGAATATA. Three other sites are located within the adjacent plant DNA, giving rise to hybrid T‐DNA/plant DNA transcripts. The two most distal sites are probably dependent on a motif AATAAATAAA, found 29 bp away from the T‐DNA/plant DNA junction.

An analysis of the boundaries of the octopine TL-DNA in tumors induced by Agrobacterium tumefaciens

SummaryThe octopine Ti plasmid of Agrobacterium tumefaciens strain Ach5 contains a 13.5 kb TL-region and a 6 kb TR-region which can independently be transferred to plant nuclear DNA. A direct repeat of 25 bp flanks the TL-region, and is related to the direct repeat flanking the nopaline T-region (Zambryski et al. 1982; Yadav et al. 1982). Two right TL-DNA borders, recloned from transformed plant cells, are located in or very near to the right copy of the direct repeat. One left TL-DNA endpoint lies within the left repeat copy, another one is located 57 bp inside of this sequence. These observations are analogous to and generalize the ones made with the nopaline system. A further analogy is the observation that one tumor clone contained a tandem junction of two TL-DNA copies. The junction sequence of 389 bp contains, to the left, a 15 bp sequence representing a direct repeat of a sequence in the right end of TL. The rest of the junction consists mainly of a unit of 40 bp of plant origin directly repeated 6 times. This structure indicates that the generation of the tandem repeat of two TL-DNA copies in this particular tumor line took place during or after the insertion of an original copy in the plant genome.

Broad-host-range cloning vectors derived from the W-plasmid Sa.

Four nonconjugative broad-host-range cloning vectors were derived from the W-plasmid Sa. They are small (Mr 5.6−7.2 × 106), carry several drug-resistance markers, and allow constructing and screening for recombinant plasmids generated by the restriction enzymes EcoRI, PstI, BglII, HindIII, BamHI and SalI,

Mutagenesis by insertion of the drug resistance transposon Tn7 applied to the Ti plasmid of Agrobacterium tumefaciens

Insertions of the linked trimethoprim and streptomycin resistance transposon Tn7 were isolated in a cointegrated plasmid formed between the Ti-plasmid of Agrobacterium tumefaciens strain B6S3 and the broad host range resistance plasmid RP4. By using the spontaneous dissociation of this cointegrate, we could demonstrate that these insertions had occurred into the Ti as well as into the RP4 part of the cointegrated plasmid. Among the insertions in the Ti part, mutants affected in different Ti plasmid-determined phenotypes, including non-oncogenic mutants, were detected. These mutants are useful for the physical mapping of these plasmid markers.

The opine synthase genes carried by Ti plasmids contain all signals necessary for expression in plants.

Signals necessary for in vivo expression of Ti plasmid T‐DNA‐encoded octopine and nopaline synthase genes were studied in crown gall tumors by constructing mutated genes carrying various lengths of sequences upstream of the 5′ initiation site of their mRNAs. Deletions upstream of position ‐294 did not interfere with expression of the octopine synthase gene while those extending upstream of position ‐170 greatly reduced the gene expression. The estimated size of the octopine synthase promoter is therefore 295 bp. The maximal length of 5′ upstream sequences involved in the in vivo expression of the nopaline synthase gene is 261 bp. Our results also demonstrated that Ti plasmid‐derived sequences contain all signals essential for expression of opine synthase genes in plants. Expression of these genes, therefore, is independent of the direct vicinity of the plant DNA sequences and is not activated by formation of plant DNA and T‐DNA border junction.