Biomedical subjects
Publications and source records attributed to D Inzé.
A new class of binary vectors has been constructed, containing the origin of replication of the W-type plasmid Sa. These vectors are designed to express foreign genes in plants under control of the TR gene 2' promoter or the promoter of a light-inducible ribulose-1,5-bisphosphate carboxylase small subunit gene of Arabidopsis thaliana.
Two lines of transgenic Nicotiana tabacum transformed to kanamycin resistance by means of a binary Agrobacterium vector containing a nos-npt gene were investigated over three generations. Southern hybridization and crossing analyses revealed that a single copy of T-DNA had integrated in each line and that the kanamycin resistance was regularly transmitted to the progeny as a monogenic dominant trait. Homozygous transgenic plants were fully fertile, morphologically normal and did not significantly differ from wild-type plants in the quantitative characters examined (plant height, flowering time). The two lines showed very low, but significantly different levels of meiotic instability: kanamycin-sensitive plants occurred among backcross progeny from homozygous transgenic plants with frequencies of 6/45,000 and 25/45,000, respectively. The sensitive plants arose independently of each other and thus resulted from meiotic rather than mitotic events. These findings demonstrate for the first time that integrated foreign genes can be transmitted to progeny with the high degree of meiotic stability required for commercial varieties of crop plants. They emphasize the importance of non-homologous integration and of avoiding co-integration of inactive gene copies for achieving meiotically stable transformants.
Phenotypic alterations induced by the cytokinin 6-benzylaminopurine in cell suspensions of Nicotiana plumbaginifolia were studied at the level of the NH(2)-terminal sequence of the constituent proteins. Total protein extracts were separated by classical two-dimensional PAGE, and the proteins were recovered by electroblotting onto support materials allowing direct gas-phase sequence analysis of the immobilized proteins. The systems used consist of an efficient electrotransfer buffer (50 mM Tris borate, pH 8.3) in combination with either glass-fiber sheets to which poly(4-vinyl-N-methylpyridinium iodide) is adsorbed or with membranes of polyvinylidene difluoride. The former is an improved version of our previously reported Polybrene-coated glass-fiber sheets and was found to be at least twice as efficient as the polyvinylidene difluoride blots. Thirteen proteins were selected for analysis. They were either induced, repressed, or independent of cytokinin. Ten proteins yielded a sequence, ranging from 10 to 38 residues. Three of the studied Nicotiana proteins show a degree of homology higher than 85% with the amino acid sequences of other eukaryotic proteins-triose-phosphate isomerase, Mn superoxide dismutase, and (1,3)-beta-glucanase. The latter enzyme was repressed by the plant hormone. This study demonstrates that proteins associated with phenotypic variations in cells can now be sequenced by a straightforward procedure involving two-dimensional gel separation of total cellular proteins, recovery by electroblotting, and gas-phase sequence analysis of the immobilized proteins.
The transformation of mutant plants with a complete recombinant library derived from wild-type DNA followed by assay of transformed plants for complementation of the mutant phenotype is a promising method for the isolation of plant genes. The small genome of Arabidopsis thaliana is a good candidate for attempting this so-called shotgun transformation. We present the properties of an A. thaliana genomic library cloned in a binary vector, pC22. This vector, designed to introduce genomic libraries into plants, contains the oriV of the Ri plasmid pRiHR1 by which it replicates perfectly stably in Agrobacterium. Upon transfer of the library from E. coli to A. tumefaciens large differences in transfer efficiencies of individual recombinant clones were observed. There is a direct relation between transfer efficiency and stability of the recombinant clones both in E. coli and A. tumefaciens. The stability is independent of the insert size, but seems to be related to the nature of the insert DNA. The feasibility of shotgun transformation and problems of statistical sampling are discussed.
A large number of tobacco SR1 cell clones transformed by the wild-type Agrobacterium C58 have been analysed for the presence of screenable markers such as tumour morphology, opine synthesis and hormone dependence. Distinct phenotypic classes were observed depending upon whether the cell clones were isolated from primary tumours or were obtained via cocultivation of protoplasts. These classes of tobacco SR1-C58 transformants appear to arise from errors in the Ti plasmid (T-DNA) transfer and integration mechanism itself rather than from subsequent T-DNA rearrangements, since 900 subclones, obtained by recloning a wild-type SR1-C58-transformed cell clone, yielded no variation in the phenotypes. A detailed genomic T-DNA analysis showed the presence of characteristic, abnormally short T-DNAs in the teratoma-forming, Acs- class and also in the Nos- class. The abnormal right border in two Nos- clones ends close to a sequence that resembles the normal T-DNA terminus and lies adjacent to the nos promoter, suggesting that this sequence could have functioned as a recognition site directing these particular T-DNA transfers. On the basis of the phenotypic and genomic blotting data it is clear that the short T-DNAs are characteristic of the cocultivation method. Other phenomena causing phenotypic variation, such as the loss of the T-DNA, and the gradual repression of T-DNA gene expression by methylation, are the main causes of aberrations in primary tumours. Moreover, the physical data suggest that early in the transformation cycle of Agrobacterium a replication step of a preselected T-DNA occurs before integration into the plant genome.
Plant crown gall tumor cells result from the insertion and expression of a defined DNA sequence, called T-DNA, which is derived from the Ti plasmid, harbored by Agrobacterium tumefaciens strains. To study the function of the genes of the T-DNA of the nopaline Ti plasmid, pTiC58, a collection of mutants was isolated so that T-DNA genes are inactivated either separately or in various combinations. It was found that no single T-DNA gene or T-region border is absolutely essential for stable tumor formation. We have identified the gene responsible for synthesis in transformed cells of the phosphorylated sugar, agrocinopine, and at least three additional genes controlling the morphology of plant tumors. Two of these latter genes work together to inhibit shoot formation and ensure efficient tumorous growth. Inactivation of these genes can be suppressed by the addition of auxins. The third gene inhibits root formation and appears to play a role in the cytokinin-independent growth of transformed cells. Mutants missing all three genes do not induce tumors, nor shoot or root formation, although the mutant T-DNA sequence is transferred to plant cells.
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The structure of the cointegrate plasmids formed by fusion of RP4 and the tumour-inducing plasmid (pTi) of Agrobacterium tumefaciens was analyzed. In all of the nine independently isolated pTi::RP4 cointegrates, the integration occurred at the same site on the RP4 genome. Moreover, a 1.2 Md (1750 bp) RP4 sequence (IS8) was directly repeated at both junction sites of the two replicons. The insertion of RP4 generated deletions, starting from the IS8 sequence and extending into the Ti part of the cointegrate. Dissociation of the cointegrates resulted in wild-type RP4 and Ti-plasmids with the IS8 sequence inserted at the original RP4 insertion site. The processes of integration and dissociation and the genetic properties of the cointegrates indicate that the IS8 sequence has unique characteristics defining a new insertion sequence.
The mRNAs encoding orthodiphenol-O-methyltransferases (OMTs; EC 2.1.1.6), which are involved in the biosynthesis of lignin precursors, are highly induced in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus (TMV). OMT messengers were fractionated on a sucrose gradient and translated in vitro. Protein A-Sepharose columns adsorbed with specific antisera raised against purified OMTs were used to select translation products, and the translatable activity of OMT mRNA was measured at different stages of infection. Oligonucleotides derived from peptide sequences of purified OMT I were used to prime polymerase chain reactions; total RNA was used as template to allow the isolation of an OMT I clone. RNA blots, hybridized with the OMT I probe, revealed a unique messenger of 1.7 kb. The kinetics of accumulation of OMT I mRNAs during the hypersensitive reaction to TMV parallels the kinetics of translation and suggests that an increase in mRNA controls the increase in the rate of enzyme synthesis. In healthy plants, RNA blot hybridization showed that the steady-state level of OMT I mRNA is very high in vascular tissue compared to the level measured in leaves.
We have isolated a cDNA encoding a catalase (Cat2St) by differential screening of a cDNA library constructed from potato roots infected with the cyst nematode Globodera pallida. Expression analysis confirmed the local induction of Cat2St and showed that it was highest at the adult stage of the parasite. It also revealed that Cat2St was induced in uninfected roots, stems, and leaves of infected plants. Localized and systemic induction of Cat2St was also observed upon root-knot nematode (Meloidogyne incognita) and root bacteria (Erwinia carotovora, Corynebacterium sepedonicum) infections. Based on sequence and expression analysis, Cat2St was found to belong to the recently described class II of dicotyledonous catalases, suggesting that these catalase isoforms could also be pathogen induced. Plant-parasitic nematodes are known to induce, in the roots of their hosts, highly metabolic feeding cells that function as nutritional sinks. Whereas the local induction of Cat2St is probably a consequence of an oxidative stress of metabolic nature, the systemic induction of Cat2St shows striking similarities with the induction of systemic acquired resistance (SAR) genes. The possible role of catalase in compatible plant-pathogen interactions is discussed.