Jef Seurinck

Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana.

The multigene family encoding the small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase/oxygenase in the crucifer Arabidopsis thaliana has been isolated and the organization and structure of the individual members determined. The family consists of four genes which have been divided into two subfamilies on the basis of linkage and DNA and amino acid sequence similarities. Three of the genes, designated ats1B, ats2B, and ats3B, reside in tandem on an 8 kb stretch of the chromosome. These genes share greater than 95% similarity in DNA sequence and encode polypeptides identical in length and 96.7% similar in amino acid sequence. The fourth gene, ats1A, is at least 10 kb removed from, or completely unlinked to the B subfamily. The B subfamily genes are more similar to each other than to ats1A in nucleotide and amino acid sequence. All four genes are interupted by two introns whose placement within the coding region of the genes is conserved. The introns of the B subfamily genes are similar in length and nucleotide sequence, but show no similarity to the introns of ats1A. Comparison of the DNA sequences within the immediate 5′ and 3′ flanking sequences among the genes revealed only limited regions of homology. S1 analysis shows that all four genes are expressed.

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.

Differential expression of five Arabidopsis genes encoding glycine-rich proteins.

Five cDNA clones coding for glycine-rich proteins in Arabidopsis thaliana were isolated. The corresponding genes are present in the genome as single copies. The derived protein sequences contain highly repetitive glycine-rich motifs. There is, however, little homology among them, nor with previously described glycine-rich proteins from other species. All five genes are expressed in leaves and stems of 6-week-old plants but show different patterns of expression in other organ systems. Analysis of the effect of different external stimuli on the expression pattern showed that, in most cases, the transcript levels were moderately but selectively affected. With flooding stress, the accumulated level of the transcript from one of the genes was remarkably increased.

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called ‘opines’, e. g. octopine (N-α-(D-l-carboxyethyl)-L-arginine) and nopaline (N-α-(l, 3-dicarboxypropyl)-L-arginine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a ‘genetic colonization’ of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.

Developmental expression of tobacco pistil-specific genes encoding novel extensin-like proteins.

We have sought to identify pistil-specific genes that can be used as molecular markers to study pistil development. For this purpose, a cDNA library was constructed from poly(A)+ RNA extracted from tobacco stigmas and styles at different developmental stages. Differential screening of this library led to the isolation of cDNA clones that correspond to genes preferentially or specifically expressed in the pistil. Seven of these cDNA clones encode proteins containing repetitions of the pentapeptide Ser-Pro4, which is a typical motif found in extensins. Unlike extensin genes, the extensin-like genes described here are not induced under stress conditions. RNA gel blot hybridizations demonstrated the organ-specific expression of the extensin-like genes and their temporal regulation during pistil development. After pollination, the transcript levels of the pistil-specific extensin-like genes change relative to levels in unpollinated pistils. In situ hybridization experiments showed that at least one of these pistil-specific genes is specifically expressed in cells of the transmitting tissue. The possible roles of the extensin-like proteins in pistils are discussed.

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.

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.

cry IA(b) transcript formation in tobacco is inefficient.

Chimaeric PCaMV35Scry genes direct in tobacco mesophyll protoplasts mRNA levels of less than one transcript per cell. We provide evidence that this low cytoplasmic cry IA(b) mRNA level is not due to a rapid turnover but rather results from a marginal import flow of cry messenger into the cytoplasm. Run-on assays indicate that the frequency of transcription initiation is not limiting. However, the cry precursor mRNA carries at least three regions that are recognized as introns. The absence of high cytoplasmic levels of spliced cry mRNAs suggests that these mRNAs are unstable and/or not efficiently made. Point mutations in the 5′ splice site of the most distal intron allows high accumulation levels of the full-length mRNA. This implies that the inefficient formation of full-size mRNA is a major cause of the low expression level of chimaeric cry IA(b) genes in tobacco.

Isolation of the LEMMI9 gene and promoter analysis during a compatible plant-nematode interaction

Plant-endoparasitic root-knot nematodes feed on specialized giant cells that they induce in the vascular cylinder of susceptible plants. Although it has been established that a number of plant genes change their expression pattern during giant cell differentiation, virtually no data are available about the mechanisms involved in that change. One possibility is differential promoter recognition by the transcription factor(s) responsible for the expression of specific genes. We have isolated and characterized a genomic clone from tomato containing the promoter region of LEMMI9, one of the few plant genes that have been reported to be highly expressed in galls (predominantly in giant cells). The analysis of transgenic potato plants carrying a LEMMI9 promoter-beta glucuronidase (GUS) fusion has demonstrated that the tomato promoter was activated in Meloidogyne incognita-induced galls in a heterologous system. We have located putative regulatory sequences in the promoter and have found that nuclear proteins from the galls formed specific DNA-protein complexes with the proximal region of the LEMMI9 promoter. The nuclear protein-binding sequence mapped to a region of 111 bp immediately upstream from the TATA box. This region contains a 12-bp repeat possibly involved in the formation of DNA-protein complexes, which might be related to the LEMMI9 transcriptional activation in the giant cells.

Nucleotide sequence of gene cryIIID encoding a novel coleopteran-active crystal protein from strain BTI109P of Bacillus thuringiensis subsp. kurstaki.

The nucleotide sequence of a novel insecticidal crystal protein(Cry)-encoding gene from a Bacillus thuringiensis serotype kurstaki isolate is described. The gene is related to the known coleopteran-active cryIII genes and encodes a CryIIID that is much more active against Colorado potato beetle than other CryIII.