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J Schell

Publications and source records attributed to J Schell.

At least 235 records · Page 13Linked to original sources
PubMed1982

Crown gall: tumor as a result of oncogenic DNA transfer.

It has been well documented that some viruses can cause neoplasmic transformations in animal cells because particular segments of the virus genome integrated in the animal cell DNA alter the differentiated state of these transformed cells. Therefore, the DNA of these viruses is oncogenic. Recent studies on so-called crown gall tumors in plants have shown that oncogenic DNA's need not always be of viral origin. Indeed, it was shown that these plant tumors result from the integration, maintenance, and expression in the plant cell nucleus of a specific DNA segment introduced in the plant cells by some pathogenic bacteria. Thus crown gall neoplastic transformation is a natural example of "genetic engineering" because it was shown that the growth of the tumor-inducing bacteria is stimulated by organic compounds (so-called "opines") excreted by the transformed plant cells. The bacterial DNA transferred to and expressed in the plant cells is directly responsible for the synthesis of the opines and for the tumorous character of the transformed plant cells. The bacterial oncogenic DNA (T-DNA) is a segment of an extrachromosomal element (Ti-plasmid) carried by the tumor-inducing bacteria. A complicated set of genes on the Ti-plasmid control the various steps involved in the tumorous transformation and are thus responsible for the transfer of the oncogenic T-DNA. A molecular study of the products of the T-DNA will allow us to have a better understanding of the control of growth and differentiation in eukaryotic cells. Furthermore, the Ti-plasmid can be used as a gene vector and thus allow the transfer of selected genes into plants.

Cell Transformation, Viral↗
PubMed1982

Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene.

We have determined the complete nucleotide sequence of the gene for the crown gall enzyme, octopine synthase. The sequence was derived from cloned fragments of the Agrobacterium tumefaciens Ti plasmid Ach5. It displayed a continuous open reading frame encoding a polypeptide chain of 358 amino acids. The nucleotide positions corresponding to the 5' end and poly(A) addition site of the mature octopine synthase mRNA from a tobacco tumor cell line were determined by S1 nuclease mapping. Two sequences closely resembling transcriptional control regions found in eukaryotic genes transcribed by RNA polymerase II were identified in the flanking genomic DNA: a sequence 5'-TATTTAAA-3' was located 32 base pairs upstream from the initiation site of transcription, and a hexanucleotide 5'-AATAAT-3' occurred 17 base pairs in front of the poly(A) addition site. No Shine-Dalgarno sequence was present in the untranslated 5' leader sequence. The observations indicate that this DNA sequence, although naturally carried by a bacterial plasmid, is programmed as a functional plant gene.

Amino Acid Oxidoreductases↗
PubMed1981

Transcription of T-DNA in octopine and nopaline crown gall tumours is inhibited by low concentrations of alpha-amanitin.

Highly purified and physiologically active nuclei were isolated from four different octopine and nopaline crown gall lines. These nuclei exhibited a high endogenous RNA synthesizing activity involving all three RNA-polymerases I, II and III. Isolated nuclei were shown by Southern blotting to synthesize T-DNA specific RNA. This synthesis was shown to be sensitive to actinomycin D and therefore to be DNA-dependent. The transcription of the T-DNA was also inhibited for more than 90% by low concentrations of alpha-amanitin (0.7 micrograms/ml) indicating that the T-DNA, although from bacterial origin, is transcribed by the host RNA polymerase II.

Amanitins↗
PubMed1981

Nuclear and polysomal transcripts of T-DNA in octopine crown gall suspension and callus cultures.

To establish a detailed map of the transcribed parts of the T-DNA in two octopine crown gall lines grown in suspension culture, T-DNA-derived steady-state nuclear and polysomal RNA as well as RNA synthesized in isolated nuclei purified from the crown gall tissues, was analyzed by southern blot hybridization to specific fragments of the T-region of the octopine plasmid pTi ACH5. In addition total RNA isolated from the same lines grown as callus tissue on solid agar, was analyzed for T-DNA specific transcripts. The results show that all of the T-DNA is transcribed although different segments are transcribed to significantly different extents. Roughly the same hybridization pattern was found for nuclear and polysomal poly-A+ and poly-A- RNA. The transcription pattern was found to be different for cells in the stationary phase of growth compared with actively growing cells.

Cell Line↗
PubMed1981

Mendelian transmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens.

Insertion of the bacterial transposon Tn7 was used to obtain mutants of an octopine Ti plasmid. Crown gall tumours induced on tobacco by an Agrobacterium tumefaciens strain carrying a particular mutant Ti plasmid (pGV2100) were found to give rise to shoots. These shoots were grown in vitro and one of them (rGV-1) was found to contain the T-DNA specific enzyme lysopine dehydrogenase (LpDH) and to form roots. After transfer to soil, rGV-1 developed into a morphologically and functionally normal tobacco plant. All cells of the regenerant and of vegetatively produced offspring were shown, by cloning of leaf protoplasts, to contain T-DNA and LpDH activity, rGV-1 and vegetatively produced offspring flowered normally. Plantlets obtained from haploid anther cultures were tested for LpDH activity. Forty-one percent of these plantlets were LpDH positive. Moreover, both self-pollination of rGV-1 and crosses between rGV-1 and normal tobacco plants showed that the LpDH character was transmitted both through the pollen and through the eggs of rGV-1 as a single dominant factor with Mendelian segregation ratios typical for monohybrid crosses. By repeated selfing, homozygous plants were obtained which bred true with respect to LpDH. The importance of these findings with respect to the use of Agrobacterium tumefaciens and Ti plasmids for genetic engineering in plants is discussed.

Crosses, Genetic↗
PubMed1981

Further insight on the transferred-DNA of octopine crown gall.

Six octopine tumour lines incited by pTiB6S3, pTiAch5 and pTiA6 on tobacco, Arabidopsis and Petunia were studied by the Southern blotting hybridisation technique in order to define accurately the dimensions of the segments of plasmid origin transferred to the tumourous cell and their organisation in the plant genome. Emphasis has been put on the comparison between octopine and nopaline T-DNAs and on the lines presented here compared with those studied previously (Thomashow et al. 1980). The length of the transferred DNA segment does not depend on the plasmids used, nor on the host plants. The octopine T-DNA organisation in the cell nucleus is significantly different from that of nopaline T-DNAs: tandem arrangements of T-DNA segments could not be detected and the T-DNA itself is much shorter. The tumour lines described here can be compared to some extent with those studied by another group (Thomashow et al. 1980) by the same technique. However, some differences were observed. The transferred DNA was seen as a unique stretch of about 11 kb present only once per cell. No amplification of any part was noticed in any of these six lines. Examination of the restriction patterns presented by the boundary fragments of the T-DNA in these lines suggested that some of them were of common origin.

DNA Restriction Enzymes↗
PubMed1981

Site-specific mutagenesis of Agrobacterium Ti plasmids and transfer of genes to plant cells.

A general method is described for the use of tumor-inducing (Ti) plasmids as experimental gene-vectors for plant cells. Intermediate vectors, containing specific Ti plasmid sequences and capable of replication in both E. coli and Agrobacterium strains, were constructed and used for the in vitro introduction of isolated DNA fragments into predetermined sites of the T-region derived fragments. Site-specific inserts and/or deletions-substitutions in Ti plasmids were produced by exchange of the modified T-region sequences for the wild-type sequence by in vivo recombination between intermediate vectors and resident Ti plasmids. This method was applied for the isolation of mutant Ti plasmids affecting either morphogenetic properties or opine synthesis in crown gall tumors. Foreign DNA, inserted at different sites in the T-region of both octopine and nopaline Ti plasmids, was shown to be cotransferred with the T-DNA and to be stably maintained in the transformed plant cells.

Arginine↗
PubMed1980

The interaction of Agrobacterium Ti-plasmid DNA and plant cells.

The tumour-inducing plasmids of Agrobacterium tumefaciens (Ti-plasmids) reveal several interesting properties. They are catabolic plasmids, which, instead of rendering Agrobacterium strains capable of catabolizing compounds found in Nature, force a plant to synthesize these catabolites (denoted 'opines'). This situation is obtained by insertion of a segment of the Ti-plasmid (the T-DNA) into the plant nucleus, where T-DNA genes become expressed and intervene in the biosynthesis of these opines. Cells containing the T-DNA behave as neoplasms (crown gall cells). Southern blotting shows that the insertion process responsible for T-DNA transfer probably recognizes special sequences on the T-DNA since the length of the T-DNA segment observed in different, independently isolated tumour lines was found to be similar. For the nopaline Ti-plasmids both left-hand and right-hand borders were found to be constant. For the octopine plasmid the left border was constant and at least two classes of right-hand borders were found. Upon redifferentiation of the transformed plant cells, the T-DNA was found to be conserved in all somatic cells examined. However, small deletions at the border fragments of the T-DNA have been observed. The exact arrangement and copy number of the T-DNA in a nucleus is still under study, but genomic cloning has already revealed that an interspersed tandem arrangement is dominant in nopaline tumours. Clones containing both the right border of one T-DNA and the left border of the neighbouring tandem T-DNA were isolated. In order to identify the different T-plasmid encoded functions an extensive use was made of transposon insertion mutagenesis. When an antibiotic resistance transposon was inserted into the non-essential regions of the T-DNA, a linked transfer to the plant DNA of the transposon together with the T-DNA was observed. This indicates that Ti-plasmids are possible vectors for genetic engineering in plants. A strategy is described for insertion of any cloned DNA segment into the T-DNA.

Amino Acids↗
PubMed1980

Tumor DNA structure in plant cells transformed by A. tumefaciens.

Crown gall tumors are induced in plants by infection with the soil bacterium Agrobacterium tumefaciens. Because the tumor induction involves transfer of a portion of the tumor-inducing (Ti) plasmid DNA from the bacterium to the plant cells, this system is of interest for the study of genetic exchange as well as tumor induction. The boundaries of the transferred DNA (T-DNA) have been cloned from transformed plant cells of tobacco. Detailed mapping with restriction enzymes and nucleotide sequence analysis of two independent clones were used to study the molecular structure of the ends of the T-DNA. One clone contains the two ends of the T-DNA joined together; the other contains one end of the T-DNA joined to repetitive plant DNA sequences. These studies provide direct evidence that the T-DNA can be integrated into the plant genome. In addition, the data suggest that in the plant, T-DNA can be tandemly repeated. Sequence analysis of the junction of crown gall clone 1 reveals several direct repeats as well as an inverted repeat; these structures may be involved in the transfer of the DNA from Agrobacterium to plant cells.

Base Sequence↗
PubMed1980

The ral gene of phage lambda. I. Identification of a non-essential gene that modulates restriction and modification in E. coli.

Host controlled restriction in Escherichia coli can be relieved by pre-infecting restricting cells with modified lambda helper phages. This process, in which intact unmodified phage genomes are allowed to escape restriction attack, is mediated by a newly identified lambda function called ral. The ral gene has been located by deletion mapping between cIII and N. Efficient expression of the ral gene requires the product of the regulator gene N. Polyacrylamide gel analysis of the lambda proteins specified by the cIII-N region failed to reveal the product of the ral gene, but demonstrated that protein Ea10 is encoded by a gene located immediately to the left of ral. From these results the map order cIII-Ea10-ral-TL1-N was deduced. Ral specifically alleviates restriction in E. coli K and E. coli B, but does not affect restriction systems EcoRI, EcoRII and EcoP1. In addition, ral enhances the modification activity of the EcoK and EcoB restriction enzymes: we observed that efficient modification of progeny phages obtained by propagating unmodified lambda phages in r-m+ hosts, is dependent upon the presence of ral. We thus conclude that the ral gene product acts by modulating the restriction and modification activities of the type I restriction systems in E. coli, and the possible mechanisms will be discussed.

Bacteriophage lambda↗
PubMed1980

The ral gene of phage lambda. II. Isolation and characterization of ral deficient mutants.

The lambda ral function modulates the restriction and modification activities of the Escherichia coli K12 and B restriction enzymes (Zabeau et al., 1980). In order to further analyse this function, ral deficient mutants have been isolated, using a method which exploits the property of the strong mutagen N-methyl-N'-nitro-N-nitrosoguanidine (N.G.) to induce multiple closely linked mutations. Hence, mutagenized phages carrying mutations in one locus were frequently found to contain additional mutations in adjacent loci. This very efficient mutagenesis procedure enabled us to isolate 27 independent Ral deficient mutants. Seven mutants were found to affect the ral gene directly and were located between the genes N anc cIII. Detailed mapping of two of these mutants showed that the lambda ral gene is located at position 70.6-70.9% on the physical map. The isolation and characterization of these mutants further supports the conclusion that ral is a gene different from the N gene, and demonstrates that the ral gene product is responsible for both counteracting restriction and enhancing modification.

Bacteriophage lambda↗
PubMed1980

The ral gene of phage lambda. III. Interference with E. coli ATP dependent functions.

The ral gene of phage lambda has previously been shown to counteract host controlled restriction and to enhance DNA modification in Escherichia coli (Zabeau et al., 1980). The studies presented in this paper demonstrate that although ral plays only a minor role in the lytic development of phage lambda, it counteracts different E. coli functions, which, like the E. coli restriction system, are ATP dependent. First, ral was found to specifically decrease the efficiency of recombination mediated by the RecBC pathway. Secondly, we observed that E. coli strains in which ral is constitutively expressed, exhibit phenotypes analogous to those of strains carrying mutations in the transcription termination factor rho. In addition, in rho deficient strains general recombination and host controlled restriction are both reduced. These findings strongly suggest that ral might be a second anti-termination function, which in contrast to the lambda N gene product directly antagonizes rho.

Adenosine Triphosphate↗