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Biomedical subjects

J Schell

Publications and source records attributed to J Schell.

At least 199 records · Page 11Linked to original sources
PubMed1986

A binary vector for transferring genomic libraries to plants.

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.

Cloning, Molecular↗
PubMed1986

Organization, structure and symbiotic function of Rhizobium meliloti nodulation genes determining host specificity for alfalfa.

In R. meliloti we have identified four nodulation genes determining plant host-range specificity and have designated them hsnABC and D. The genes code for 9.7, 41.7, 26.7, and 28.6 kd proteins, respectively, and are organized into two transcriptional units. Mutations in these genes affect nodulation of their natural plant hosts Medicago sativa and Melilotus albus to different extents and hsnD mutants have an altered host-range. These Nod- mutations are not complementable by nodulation genes of other Rhizobium species such as R. leguminosarum. The hsn genes determine plant-specific infection through root hairs: hsnD is required for host-specific root hair curling and nodule initiation while the hsnABC genes control infection thread growth from the root hairs.

Bacterial Proteins↗
PubMed1986

Primary structure of a proteinase inhibitor II gene from potato (Solanum tuberosum).

The isolation and characterization of a genomic clone encoding proteinase inhibitor II of potato (Solanum tuberosum) is described. The structure of this gene was determined by sequencing a genomic fragment of about 2 kb containing the entire RNA coding as well as about 900 nucleotides of the 5'-upstream and 250 nucleotides of the 3'-downstream region. The transcription start site was determined by RNase protection experiments. The comparison of the genomic sequence with cDNA sequences reveals the presence of one intron with a length of 117 nucleotides. The genomic clone contains an open reading frame of 462 nucleotides allowing for a protein of 154 amino acids. The proteinase inhibitor II gene displays typical features of eucaryotic genes. The sequence TATAAA is found 26 nucleotides upstream of the transcription initiation site and the sequence CAAAT at position--103. In the 3'-region the sequence AATAA is found 33 nucleotides in front of the poly-A addition site.

Amino Acid Sequence↗
PubMed1986

Transformed cell clones as a tool to study T-DNA integration mediated by Agrobacterium tumefaciens.

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.

Cells, Cultured↗
PubMed1986

Transposition of the maize controlling element "Activator" in tobacco.

Transposition of the maize autonomous controlling element Activator (Ac) and a nonautonomous derivative, Dissociation (Ds), was investigated in tobacco cells. Tobacco protoplasts were transformed with Ti-plasmid vectors that contained Ac or Ds flanked by short maize wx gene sequences. The structures of the elements and surrounding wx and T-DNA sequences were investigated in nine Ac and five Ds tobacco transformants by digestion with restriction enzymes, Southern blotting, and hybridization using specific probes. In four of the nine Ac transformed lines, Ac had excised from its original position in the T-DNA and inserted at new sites in the tobacco genome. Ds did not excise from its original T-DNA position in any of the transformants examined. Two Ac fragments and cellular flanking sequences were cloned from a line of tobacco in which Ac had transposed. Fragments, comprised of sequences flanking the newly integrated Ac elements, were used as hybridization probes to normal tobacco DNA and to the tobacco DNA from which they were isolated. The Ac copies were integrated into repetitive tobacco DNA sequences. Two tobacco fragments containing empty Wx donor sites were cloned from the DNA of the same Ac transformant and sequenced. Both sequences are among the types of excision products observed to result from Ac-catalyzed excision events in maize. Our results indicate that the maize controlling element Ac is capable of self-catalyzed transposition in tobacco.

Journal Article↗
PubMed1986

Expression of the nodulation gene nodA in Rhizobium meliloti and localization of the gene product in the cytosol.

The nodA gene of Rhizobium meliloti encodes a 21.8-kDa protein, which is conserved in several Rhizobium species. We overproduced the nodA protein as a fusion product with a portion of the lambda cI repressor in Escherichia coli. This fusion protein was purified from inclusion bodies by gel and hydroxyapatite chromatography in the presence of NaDodSO(4). Monospecific polyclonal antibodies against the hybrid protein were used to detect the nodA protein in the cytosol of E. coli and R. meliloti by immunoblotting. In contrast to experiments with antibodies against the R. meliloti nodC membrane protein, the alfalfa-R. meliloti nodulation was not affected by the addition of anti-nodA antibodies to medium and inoculum. This suggests that the nodA protein is located within the cell and is therefore not accessible to antibodies. The expression of the nodA gene is induced in R. meliloti by various compounds present in the exudate of leguminous plants, particularly by the flavone luteolin. We show that the plant hormone trigonelline also has some inducing activity. The nodC protein was further localized in the membrane fraction of R. meliloti. Our experiments demonstrate that the nodC transmembrane protein is not necessary for the uptake of the compounds inducing the synthesis of the nodA protein. The nodA and the nodC proteins were also detected in mature nodules. During nodule development, the nodC protein may be processed to a 34-kDa protein.

Journal Article↗
PubMed1986

Fate of selectable marker DNA integrated into the genome of Nicotiana tabacum.

To compare the effects of different transformation methods on the integration behavior and structural stability of integrated foreign genes in plant cells, tobacco protoplasts were transformed with Escherichia coli plasmid pLGV2103neo DNA using the Ca phosphate DNA coprecipitation technique. Parallel transformations were done by cocultivation with Agrobacterium tumefaciens harboring the Ti plasmid derivatives pGV3850::2103neo or pGV3850::1103neo. A comparison of the fine structure of the integrated donor DNA obtained by direct gene transfer and by cocultivation indicates that the donor DNA in cells transformed by the former technique undergoes structural changes and concatemerizations, while the DNA integrated by the latter procedure is often unaltered. The cotransformed nopaline synthase gene, which is present in the donor Ti plasmid DNA, was inactivated in two out of nine cases. Once integrated, the arrays of selectable marker DNA appear to be structurally stable under different cell culture and selection conditions, as well as after genetic transmission.

Amino Acid Oxidoreductases↗
PubMed1985

Selection-expression plasmid vectors for use in genetic transformation of higher plants.

Plasmid vectors containing both a selectable marker for plant transformation (kanamycin resistance) and a second, directly adjacent, divergent promoter for the transcription of inserted DNA fragments have been constructed. These vectors make use of a small (479 bp) dual-promoter DNA fragment, originally isolated from the T-DNA of Agrobacterium tumefaciens, fused to the neomycin phosphotransferase gene of Tn5. Several unique restriction enzyme cleavage sites, as well as a polyadenylation signal sequence, have been introduced downstream of the open promoter, allowing simple insertional cloning of DNA fragments to be expressed in plants. To test the vectors, the coding region for the chloramphenicol acetyltransferase gene (CAT) from Tn9 was inserted, and the resulting plasmids introduced into tobacco cells. Transformed calli, selected only for Km resistance, contained, in every case tested, both NPTII and CAT activities.

Cell Line↗
PubMed1985

Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants.

A two-component cloning system to transfer foreign DNA into plants was derived from the octopine Ti plasmid pTiB6S3. pGV2260 is a non-oncogenic Ti plasmid from which the T-region is deleted and substituted by pBR322. pGV831 is a streptomycin-resistant pBR325 derivative that contains a kanamycin resistance marker gene for plant cells and a site for cloning foreign genes between the 25-bp border sequences of the octopine T-region. Conjugative transfer of pGV831 derivatives to Agrobacterium and cointegration by homologous recombination between the pBR322 sequences present on pGV831 and pGV2260, can be obtained in a single step. Strains carrying the resulting cointegrated plasmids transfer and integrate T-DNA into the genome of tobacco protoplasts, and transformed tobacco calli are readily selected as resistant to kanamycin. Intact plants containing the entire DNA region between the T-DNA borders have been regenerated from such clones. In view of these properties we present pGV831 and its derivatives as vectors for efficient integration of foreign genes into plants.

Arginine↗
PubMed1985

The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts.

The light-inducible nuclear gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco), produces a precursor protein with an amino-terminal transit peptide which is transported into the plastids and cleaved by a specific proteinase. To test whether the promoter and transit peptide-coding sequences of the small subunit gene can be used to direct the light-inducible synthesis and transport of a foreign protein into chloroplasts, a chimeric gene was constructed consisting of the promoter, first exon and intron as well as part of the second exon of the small subunit Rubisco gene fused to the amino-terminal end of the neomycin phosphotransferase II gene (nptII) of Tn5. Tobacco tissue, as well as whole plants, into which this chimaeric gene was introduced, were resistant to kanamycin. The transcription of the chimaeric gene as well as the NPTII activity of the resulting fusion protein were shown to be light inducible. The fusion protein is processed and located within the chloroplasts of the transformed plants.

Artificial Gene Fusion↗
PubMed1985

Chloroplast transformation by Agrobacterium tumefaciens.

A chimeric gene consisting of the promoter region of the nopaline synthase gene (Pnos) fused to the coding sequence of the chloramphenicol acetyltransferase gene (cat gene) of Tn9 was introduced by co-cultivation in tobacco protoplasts followed by selection with 10 mug/ml chloramphenicol. The chloramphenicol-resistant plants derived from these selected calli were unable to transmit the Cm phenotype through pollen. A typically maternal inheritance pattern was observed. Southern blot analysis showed that the chimeric Pnos-cat gene was present in the chloroplasts of these resistant plants. Furthermore, the chloramphenicol acetyltransferase activity was shown to be associated with the chloroplast fraction. These observations are the first proof that the Agrobacterium Ti-plasmid vectors can be used to introduce genes in chloroplasts.

Journal Article↗
PubMed1985

Expression of the nodulation gene nod C of Rhizobium meliloti in Escherichia coli: role of the nod C gene product in nodulation.

The nod C gene of Rhizobium meliloti encodes a protein of mol. wt. 44 000 which is highly conserved in at least three Rhizobium species. In order to overproduce this protein, a gene fusion of lambda cI repressor sequences to a large fragment of nod C was constructed. The fusion was placed under control of the tac promoter on plasmid pEA305 to yield pJS1035. IPTG-induced Escherichia coli cells harbouring pJS1035 accumulated the cI-nod C hybrid protein up to 19% of total cellular protein. The synthesis of the hybrid protein drastically inhibits the growth rate of the bacterium. The fusion protein was purified by gel and hydroxyapatite chromatography in the presence of SDS. Antibodies raised against the purified fusion protein precipitated the mol. wt. 44 000 nod C proteins of R. meliloti and of the broad-host range Rhizobium strain NGR234, which were both expressed in E. coli mini-cells. The hybrid protein is associated with the outer membrane of E. coli cells, and the cI-nod C fusion protein appears to be an integral membrane protein. Nodulation of alfalfa by R. meliloti and of clover by R. trifolii was markedly inhibited (approximately 50%) by the addition of antibodies against the hybrid protein to plant growth medium and inoculum.

Bacterial Proteins↗
PubMed1985

Light-inducible and tissue-specific expression of a chimaeric gene under control of the 5'-flanking sequence of a pea chlorophyll a/b-binding protein gene.

We have investigated the regulatory functions of the 5'-flanking sequences of a chlorophyll a/b-binding protein gene from Pisum sativum, using the neomycin phosphotransferase (II) activity from Tn5 as an enzymatic reporter. We show that 0.4 kb of the upstream flanking sequences of this gene are sufficient for both organ-specific and light-regulated expression of our chimaeric constructs in transformed tobacco plants. In addition, we show that sequences farther upstream have a significant influence on the level of transcription of these constructions.

Journal Article↗
PubMed1985

Genetic analysis of integration mediated by single T-DNA borders.

Transformation of plant cells by the T-DNA of the Ti plasmid of Agrobacterium tumefaciens depends in part upon a sequence adjacent to the right T-DNA end. When this sequence is absent, the T-DNA is almost avirulent; when it is present, DNA between it and the left T-DNA border region becomes integrated in plants. To investigate further this process of DNA transfer and integration, we introduced the right border region and the nopaline synthase (nos) gene of plasmid pTiC58 into a variety of new positions around Ti plasmids. The border region functioned when separated from the remainder of the T-DNA by almost 50 kilobases. It also worked when placed outside of the T-DNA region where there were no known left-border sequences with which to interact. Indeed, the nos gene could be transferred to plants even when no other Ti plasmid sequences were present on the same plasmid. These results may indicate that the sequence requirements for the left borders are not as stringent as those for the right borders. In addition, mutants with an extra copy of the right border region within their T-DNA were found to transfer or integrate only parts of the bacterial T-DNA region. It is possible that abnormally placed T-DNA borders interfere with the normal process of DNA transfer, integration, or both.

Base Sequence↗
PubMed1984

An Agrobacterium-transformed cell culture from the monocot Asparagus officinalis.

Cultured stem fragments from the monocotyledonous plant Asparagus officinalis infected by the oncogenic bacterium Agrobacterium tumefaciens developed tumorous proliferations. This tissue was propagated in vitro on hormone-free culture medium. The T-DNA-encoded markers nopaline and agrocinopine were unambiguously detected in these tissues. The data demonstrate that stable T-DNA transfer as well as expression of T-DNA genes is possible in at least some monocotyledonous plants. This opens new possibilities for plant genetic engineering using the Ti plasmid as a gene vector.

Journal Article↗
PubMed1984

Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens.

The two most abundant transcripts derived from TR-DNA within plant cells transformed by an octopine strain of Agrobacterium tumefaciens arise from divergent transcription, both originating within an 500 bp section of the T-DNA. Using a combination of subcloning and exonuclease digestion, a 479-bp DNA fragment, directly flanked by the initiation codons for the two adjacent open reading frames, was isolated. The resulting DNA fragment was fused, in both orientations, to the neomycin phosphotransferase (NPT II) gene of the transposon Tn5 prior to introduction into Nicotiana tabacum cells via the Ti plasmid. The intergenic fragment was found to initiate expression of the NPT II gene in either orientation as assayed by kanamycin resistance of the transformed plant tissue as well as by enzymatic assay of the NPT II gene product. The plasmids described here are potential selection-expression vectors for plant systems.

Journal Article↗