Agrobacterium

Plant Cell, 1995 Feb, 7(2), 195 - 201
Isolation of the Arabidopsis GA4 locus; Chiang HH et al.; Progeny from a transgenic Arabidopsis plant generated by the Agrobacterium root transformation procedure were found to segregate for a gibberellin (GA)-responsive semidwarf phenotype . Complementation analysis with genetically characterized GA-responsive mutants revealed that the transgenic plant has an insertional mutation (ga4-2) that is an allele of the ga4 locus . The semidwarf phenotype of ga4-2 is inherited as a recessive mutation that cosegregates with both the T-DNA insert and the kanamycin resistance trait . DNA gel blot analysis indicated that the insertion site contains a complex T-DNA unit . A genomic library was constructed with DNA from the tagged ga4 mutant; a DNA clone was isolated from the library that flanks the T-DNA insert . The plant sequence isolated from this clone was used to isolate the corresponding full-length genomic and cDNA clones from wild-type libraries . DNA sequence comparison of the clones to the existing data bases suggests that they encode a hydroxylase . This conclusion is in agreement with a biochemical study that indicated that the ga4 mutant is deficient in 3 beta-hydroxylase in the GA biosynthetic pathway of Arabidopsis . RNA gel blot analysis showed that the message is ubiquitously expressed in different tissues of Arabidopsis but most abundantly in the silique . Unexpectedly, a higher level of transcription was detected in the ethyl methanesulfonate-induced ga4 mutant, and this overexpression was repressed by treatment with exogenous GA.

Plant Mol Biol, 1995 Feb, 27(4), 729 - 41
Promoter analysis of seed storage protein genes from Canavalia gladiata D.C; Yamamoto S et al.; A number of A/T-rich sequences and a CATGCAT/A sequence are contained in the 5'-upstream regions of the genes encoding concanavalin A (Con A) and canavalin, two major seed storage proteins of Canavalia gladiata D.C . To study the role of these sequences in the seed-specific gene expression, we constructed 5'-deletion mutants and examined the transient expression of beta-glucuronidase reporter gene by particle bombardment and the stable expression by Agrobacterium-mediated transformation of tobacco plants . Positive regulatory elements were located in the -894/-602 and -602/-74 regions of the Con A gene, and in the -428/-376, -281/-155 and -155/-50 regions of the canavalin gene . In addition, the results suggested that the A/T-rich sequences in the 5'-upstream region of the Con A gene play a role in transcriptional activation, but that those of the canavalin gene have little effect on the gene expression . The CATGCAT/A sequence was not sufficient by itself for high levels of expression of both the Con A and canavalin genes . The canavalin polypeptide amounted to about 1% of the total extractable protein in the transgenic tobacco seeds, but the Con A polypeptide was not detected in the extractable protein.

Plant Mol Biol, 1995 Feb, 27(4), 651 - 67
Tissue- and cell-specific expression of a cinnamyl alcohol dehydrogenase promoter in transgenic poplar plants; Feuillet C et al.; Cinnamyl alcohol dehydrogenase (CAD) which catalyses the synthesis of the cinnamyl alcohols, the immediate precursors of lignins, from the corresponding cinnamaldehydes is considered to be a highly specific marker for lignification . We have isolated and characterized a CAD genomic clone from eucalyptus, a woody species of economic importance . The full-length promoter (EuCAD, 2.5 kb) and a series of 5' deletions were fused to the beta-glucuronidase (GUS) reporter gene . These constructs were tested in a homologous transient expression system of eucalyptus protoplasts which enabled the identification of several regions involved in transcriptional control . In order to study the spatial and developmental regulation of the CAD gene, the chimeric gene fusion (EuCAD-GUS) was then transferred via Agrobacterium tumefaciens-mediated transformation into poplar, an easily transformable woody angiosperm . Quantitative fluorometric assays conducted on eight independent in vitro transformants showed that GUS activity was highest in roots followed thereafter by stems and leaves . Histochemical staining for GUS activity on both in vitro primary transformants and more mature greenhouse-grown plants indicated a specific expression in the vascular tissues of stems, roots, petioles and leaves . At the onset of xylem differentiation, GUS activity was detected in parenchyma cells differentiating between the xylem-conducting elements . After secondary growth has occurred, GUS activity was localized in xylem ray cells and parenchyma cells surrounding the lignified phloem and sclerenchyma fibers . This first characterization of a woody angiosperm CAD promoter provides functional evidence for the role of CAD in lignification and suggests that parenchyma cells expressing CAD may provide lignin precursors to the adjacent lignified elements (vessels and fibres).

Carbohydr Res, 1995 Feb 1, 267(1), 1 - 15
Sucrose analogues modified at position 3: chemoenzymatic synthesis and inhibition studies of dextransucrases; Simiand C et al.; Conditions for the large-scale (molar) oxidation of sucrose by Agrobacterium tumefaciens were improved, thus leading to homogeneous solutions of 3-ketosucrose in 40% yield . Treatment of this solution with hydroxylamine or methoxylamine afforded the corresponding oximes 3a and 3b (isolated as acetates) in excellent yield . Dissolving-metal reduction of these oximes gave mixtures of amino disaccharides in which the gluco epimer (3-amino-3-deoxysucrose) was predominant . A more efficient approach to this amino sucrose was provided by the highly stereoselective hydrogenation of 3-ketosucrose peracetate (7), which gave exclusively the allo isomer 8 (2,4,6-tri-O-acetyl-alpha-D-allopyranosyl 1,3,4,6-tetra-O-acetyl-beta-D-fructofuranoside) . Upon reaction with lithium azide, the triflate derived from 8, compound 9, afforded 3-azido-3-deoxysucrose peracetate (10) which was converted into 3-amino-3-deoxysucrose (12) . The reaction of triflate 9 with potassium ethylxanthate led to a mixture of products (the expected 3-S-ethoxythiocarbonyl-3-thiosucrose derivative and the peracetates of 3-thiosucrose and of 3-thiosucrose disulfide), which could be all converted into 3-thiosucrose (17) . Sucrose analogues 12 and 17 were not substrates of dextransucrases from various strains of L . mesenteroides, nor did they participate in glycosyl transfer reactions to an acceptor (maltose) . Compounds 3a and 12 were found to be strong competitive inhibitors of the dextran synthesis process (dextransucrase from strain B-1397) . These results indicate that 3a and 12 compete effectively with sucrose for the sucrose binding site but are unable to participate as glycosyl donors in the polymerization or glycosyl-transfer processes.

Appl Environ Microbiol, 1995 Feb, 61(2), 828 - 31
PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains; Sawada H et al.; A universal primer set (VCF/VCR) for PCR analysis based on the sequences of the virC operon located on Ti and Ri plasmids was designed to detect these plasmids from phytopathogenic Agrobacterium strains . With the VCF (sequence, 5'-ATCATTTGTAGCGACT-3') and VCR (sequence, 5'-AGCTCAAACCTGCTTC-3') primer set, DNA fragments of 730 bp in length were amplified from cell lysates of 10 rhizogenic and 65 tumorigenic agrobacteria . DNA sequencing and Southern hybridization analysis confirmed that the amplified fragments corresponded to the target region . The PCR method is considered convenient for routine determination of the potential pathogenicity of Agrobacterium strains.

Appl Environ Microbiol, 1995 Feb, 61(2), 660 - 8
Expression vectors for the use of eukaryotic luciferases as bacterial markers with different colors of luminescence; Cebolla A et al.; An easy way to identify microorganisms is to provide them with gene markers that confer a unique phenotype . Several genetic constructions were developed to use eukaryotic luciferase genes for bacterial tagging . The firefly and click bettle luciferase genes, luc and lucOR, respectively, were cloned under constitutive control and regulated control from different transcriptional units driven by P1, lambda PR, and Ptrc promoters . Comparison of the expression of each gene in Escherichia coli cells from identical promoters showed that bioluminescence produced by luc could be detected luminometrically in a more sensitive manner . In contrast, luminescence from intact lucOR-expressing cells was much more stable and resistant to high temperatures than that from luc-expressing cells . To analyze the behavior of these constructions in other gram-negative bacteria, gene fusions with luc genes were cloned on broad-host-range vectors . Measurements of light emission from Rhizobium meliloti, Agrobacterium tumefaciens, and Pseudomonas putida cells indicated that both luciferases were poorly expressed from P1 in most bacterial hosts . In contrast, the lambda promoter PR yielded constitutively high levels of luciferase expression in all bacterial species tested . PR activity was not regulated by temperature when the thermosensitive repressor cI857 was present in the bacterial species tested, except for E . coli . In contrast, the regulated lacIq-Ptrc::lucOR fusion expression system behaved in a manner similar to that observed in E . coli cells . After IPTG (isopropyl-beta-D-thiogalactopyranoside) induction, this system produced the highest levels of lucOR expression in all bacterial species tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Ecol, 1995 Feb, 4(1), 1 - 10
A phylogenetic analysis of micro-organisms isolated from subsurface environments; Stim KP; Three methods were used to provide information on the identity and phylogenetic relatedness of 19 aerobic, chemoheterotrophic bacteria isolated from topsoil and deep subsurface sediments at a site in South Carolina . These methods were (i) analysis of selected physiological traits, (ii) restriction endonuclease analysis (REA) of genomic DNA, and (iii) analysis of 16S ribosomal RNA sequences . When the 16S rRNA sequences were compared with those for 12 standard strains, two topsoil isolates and six subsurface strains formed a tight group with the high-G+C Gram-positive bacteria and appeared to be most closely related to Arthrobacter globiformis--a coryneform-actinomycete bacterium with unusually effective survival capabilities . The rest of the subsurface isolates were scattered among the standard strains from the Proteobacteria-including the pseudomonads and Agrobacterium tumefaciens--or the low-G+C Gram-positive bacteria.

J Bacteriol, 1995 Feb, 177(3), 750 - 7
The groESL operon of Agrobacterium tumefaciens: evidence for heat shock-dependent mRNA cleavage; Segal G et al.; The heat shock response of the groESL operon of Agrobacterium tumefaciens was studied at the RNA level . The operon was found to be activated under heat shock conditions and transcribed as a polycistronic mRNA that contains the groES and groEL genes . After activation, the polycistronic mRNA appeared to be cleaved between the groES and groEL genes and formed two monocistronic mRNAs . The groES cleavage product appeared to be unstable and subjected to degradation, while the groEL cleavage product appeared to be stable and became the major mRNA representing the groESL operon after long periods of growth at a high temperature . The polycistronic mRNA containing the groES and groEL genes was the major mRNA representing the groESL operon at a low temperature, and it reappeared when the cells were returned to the lower growth temperature after heat shock induction . These findings indicate that the cleavage event is part of the heat shock regulation of the groESL operon in A . tumefaciens.

J Biol Chem, 1995 Jan 20, 270(3), 1269 - 76
Initiation of Agrobacterium tumefaciens T-DNA processing . Purified proteins VirD1 and VirD2 catalyze site- and strand-specific cleavage of superhelical T-border DNA in vitro; Scheiffele P et al.; T-DNA processing during agroinfection of plants is initiated by site- and strand-specific incision at the T-DNA border sequences of the Ti plasmid . Two proteins are required for this reaction: VirD2 (49.6 kDa), catalyzing a site-specific cleaving-joining reaction on single-stranded DNA in vitro (Pansegrau, W., Schoumacher, F., Hohn, B., and Lanka, E . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 11538-11542), and VirD1 (16.1 kDa), an accessory protein required for VirD2-mediated specific cleavage of double-stranded DNA . Following efficient overproduction, VirD1 was isolated in active form from inclusion bodies and purified to near homogeneity . The protein was applied together with purified VirD2 protein for specific cleavage of double-stranded T-DNA border sequences in vitro . The reaction proceeds on negative superhelical DNA and requires Mg2+ ions . Relaxed DNA is not cleaved . The 5' terminus of the broken DNA strand is covalently associated with protein, most probably VirD2, and the cleavage site is located at the same position that is found in vivo, indicating that the in vitro reaction mimics the one that takes place in induced agrobacteria . Relaxation of plasmid DNA occurs only upon addition of protein denaturants, suggesting that the DNA in the VirD1/VirD2 complex is topologically constrained by strong protein-DNA interactions . The characteristics of the VirD1/VirD2-mediated cleavage reaction strongly resemble those observed with relaxosomes of IncP plasmids involved in initiation of transfer DNA replication during bacterial conjugation.

Chin J Biotechnol, 1995, 11(4), 267 - 74
A simple method for the transformation of Agrobacterium tumefaciens by foreign DNA; Cui W et al.; A method for successful introduction of Ti plasmid vector ( > 10 kb) into Agrobacterium tumefaciens is described . Competent A.tumefaciens cells were prepared with 50 mmol/L CaC1(2) at room temperature and introduction was done on ice followed by a 28 degrees C heat pulse . The efficiency of transformation was 10(-4)-10(-5) transformants per total recipient population or 10(6) transformants per microgram DNA . The effects of growth phase of A . tumefaciens cells, concentration of CaC1(2) solution, temperature, liquid N2, heat pulse, recovery time, and storage time of competent cells at 4 degrees C or -20 degrees C (in 15% glycerin) on transformation were studied.

Chin J Biotechnol, 1995, 11(4), 227 - 35
Hairy root culture of Artemisia annua L . by Ri plasmid transformation and biosynthesis of artemisinin; Cai G et al.; The hairy root culture system of the medical plant Artemisia annua L . was established by infection with Agrobacterium rhizogenes R1601 . The transgenic state of transformed roots was confirmed by Southern blot hybridization with TL-DNA of pFw302 . The expression of NPTII gene was confirmed by enzymic assay . The important secondary metabolites-artemisinin was obtained in the hairy root culture . The effects of various physical and chemical factors on the growth of the hairy roots and production of artemisinin were studied . Artemisinin could be detected in hairy roots cultures in the light . The optimum pH value of the medium was 5.4 . Fast growth of the hairy roots and maximal production of artemisinin was observed in the presence of 3% sucrose . Low concentration of naphthylacetic acid (0.025 mg/L) enhanced the growth of the roots but inhibited the production of artemisinin . The growth and artemisinin production in hairy root cultures were greatly promoted by the addition of gibberellin (GA3) to the medium . Its optimum concentration was 4.8 mg/L.

Acta Microbiol Immunol Hung, 1995, 42(4), 373 - 9
Microbial counts and characteristics of Streptoverticillium strains isolated from soils in the Jordan valley; Abussaud MJ; A microbial survey of total bacterial count, agrobacteria, streptomycetes, and fungi was carried out in six locations in the Jordan Valley . The highest microbial counts for the four populations were in spring, the lowest mostly in autumn . No significant differences at p < 0.05 were observed either in the counts between the six locations, or in the seasonal counts of agrobacteria and streptomycetes between the six locations as well as within each location . The total bacterial counts (at p < 0.01) and fungi (at p < 0.05) differed significantly in the six locations . Microbial counts and the studied environmental conditions showed no significant regression . Of 552 streptomycetes strains isolated from soils collected from these locations, 58 belonged to the genus Streptoverticillium . They were distributed in five colour series grey (41%), red (31%), green (17%), blue (7%) and white (3%) . Forty-eight strains had biverticillium spore bearing hyphae while only 10 had monoverticillium . While 28 strains produced distinctive reverse side pigment, only 8 and 6 strains produced soluble pigment and melanin pigment, respectively . Most strains utilized arabinose followed by fructose, mannitol, rhamnose, xylose, sucrose, inositol, and raffinose . Sixty-nine per cent of the strains exhibited activity in vitro test against Pseudomonas aeruginosa, and 45, 41, 28, and 7% against Staphylococcus aureus, Bacillus cereus, Agrobacterium tumefaciens and Escherichia coli, respectively.

Chin J Biotechnol, 1995, 11(2), 137 - 41
Crown gall culture and production of tanshinone in Salvia miltiorrhiza; Zhang Y et al.; Crown galls were induced by direct infection of sterile seedlings with Agrobacterium tumefaciens C58, and the transformation was proved by opine identification . The crown galls grew well in a hormone-free medium . B5 and MS basic media were good for the growth of crown galls (strain Ca), which increased up to 102 and 90 times in a month, respectively . However, 67-V and WP basic media are good for tanshinone production . It has been shown that crown galls can be utilized as a culture system to produce secondary metabolites.

Plant J, 1995 Jan, 7(1), 157 - 64
DNA rearrangement associated with the integration of T-DNA in tobacco: an example for multiple duplications of DNA around the integration target; Ohba T et al.; Transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid is transferred from Agrobacterium tumefaciens to plant cells and is stably integrated into the plant nuclear genome . By the inverse polymerase chain reaction DNA fragments were amplified that contained the T-DNA/plant DNA junctions from the total DNA of a transgenic tobacco plant that had a single copy of the T-DNA in a repetitive region of its genome . A DNA fragment containing the target site was amplified from the total DNA of non-transformed tobacco by the polymerase chain reaction using high-stringency conditions . Comparison of the nucleotide sequence of the target site with those of the T-DNA/plant DNA junctions revealed that various duplications of short stretches of nucleotide sequences around the target and in the incoming T-DNA had accompanied the integration of the T-DNA . A deletion of 16 bp at the target site was also found and the target site was similar, in terms of nucleotide sequence, to regions around the breakpoints of the T-DNA . This finding provides a clear example of the occurrence of complex rearrangements during the integration of T-DNA.

Plant J, 1995 Jan, 7(1), 109 - 19
Targeted recombination in plants using Agrobacterium coincides with additional rearrangements at the target locus; Risseeuw E et al.; The use of Agrobacterium for gene targeting in plants has been investigated . Leaf protoplasts of five transgenic tobacco lines, containing a T-DNA insertion with a defective npt-II gene at different positions in the plant genome, were transformed via Agrobacterium with a T-DNA containing a npt-II repair gene . After selection for kanamycin resistance and PCR analysis six recombinants were derived from four of the target lines . The recombination frequencies were similar for the different target lines with one recombinant from approximately 3 x 10(5) transformants . Apparently gene targeting is more or less independent of the location of the target construct in the plant genome . Molecular analysis revealed that gene targeting had occurred in five of the six recombinant lines . However precise recombination had occurred in only one line, while in the other four lines restoration of the npt-II gene was accompanied by a deletion of part of the target locus . The sixth recombinant line showed restoration of the npt-II gene of the incoming T-DNA construct which was inserted in the plant genome at a position closely linked to the target locus . The different recombination products favour a model in which recombination is via gene conversion followed by reintegration of the synthesized DNA via homologous or illegitimate recombination rather than a reciprocal exchange of DNA between two cross-overs.

Plant Mol Biol, 1995 Jan, 27(2), 225 - 35
Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco; Thomas JC et al.; Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth . Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin . These responses were also associated with environmental stress reactions, such as salt stress, in many plant species . To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv . SR-1 under the control of the light-inducible rbcS-3A promoter from pea . In high light (300 mumol PPFD m-2 s-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mumol PPFD m-2 s-1) . High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants . Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.

Appl Environ Microbiol, 1995 Jan, 61(1), 65 - 73
A new Agrobacterium strain isolated from aerial tumors on Ficus benjamina L; Bouzar H et al.; Crown gall tumors, collected from branches of 1-year-old weeping fig (Ficus benjamina L.) trees, yielded both tumorigenic and nonpathogenic agrobacteria . On the basis of classical diagnostic tests, the nonpathogenic strains were identified as Agrobacterium tumefaciens, whereas the tumorigenic strains could not be assigned to any of the known terrestrial Agrobacterium spp . The tumorigenic strains also differed from other members of the genus by producing more acid from mannitol . According to cluster analysis of carbon substrate oxidation (GN Microplate; Biolog, Inc.) and fatty acid content, the tumorigenic fig strains were distinct from strains of A . tumefaciens, Agrobacterium rhizogenes, Agrobacterium vitis, and Agrobacterium rubi . Furthermore, they had unusual opine metabolism, inducing tumors that synthesized nopaline and three recently discovered opines: chrysopine (d-lactone of N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-L-glutamine, and N-1-deoxy-D-fructosyl-5-oxo-L-proline . The nonpathogenic A . tumefaciens strains present in the same tumors were unable to degrade any of the opines tested . The phylogenetic position of the tumorigenic fig strain AF3.10 was inferred from comparing its rrs (i.e., 16S rRNA gene) sequence with those from the type strains of Agrobacterium and Rhizobium species . The analysis showed that strain AF3.10 clustered with A . tumefaciens and A . rubi but not with A . vitis and was far removed from A . rhizogenes . However, the sequence was significantly different from those of A . tumefaciens and A . rubi to suggest that the tumorigenic fig strain may be a new Agrobacterium species that is as different from A . tumefaciens and A . rubi as these two species are from one another.

Transgenic Res, 1995 Jan, 4(1), 60 - 9
Expression of the Escherichia coli fabA gene encoding beta-hydroxydecanoyl thioester dehydrase and transport to chloroplasts in transgenic tobacco; Saito K et al.; The fabA gene of Escherichia coli encodes beta-hydroxydecanoyl thioester dehydrase (HDDase), a pivotal enzyme in the biosynthesis of the unsaturated fatty acid cis-vaccenic acid, through the anaerobic pathway . This enzyme is specific to bacterial fatty acid biosynthetic pathways, although other enzymes for fatty acid synthesis are very similar in plants and bacteria . We constructed chimaeric plant expression vectors, pfab21 and pfab22, carrying the fabA gene under the transcriptional control of the cauliflower mosaic virus (CaMV) promoter of 35S RNA . In pfab21, fabA was placed directly under the control of the CaMV 35S promoter; whereas in pfab22, the DNA sequence coding for the chloroplast-targeting transit peptide (TP) of the pea ribulose-1,5-bisphosphate carboxylase (RuBisCo) small subunit was fused to the fabA gene in order to allow transport of HDDase to the chloroplast, the organelle responsible for de novo fatty acid biosynthesis in plants . Transgenic plants of Nicotiana tabacum were obtained by Agrobacterium-mediated transformation with pfab21 or pfab22 . Expression of fabA transcripts of sizes expected from the chimaeric constructs was shown by RNA blot hybridization . The HDDase protein derived from pfab22 was correctly processed and transported to chloroplasts in transformed plants . The enzymatic activity of HDDase was also detected in chloroplasts isolated from the transformants derived from pfab22 (but not pfab21) and in total leaf protein of all transformants . However, no significant changes were observed in the fatty acid compositions, including cis-vaccenic acid, of leaf chloroplasts and self-fertilized seeds . These results are discussed in relation with the possible structural organization of plant fatty acid synthase.

Plant Mol Biol, 1995 Jan, 27(1), 41 - 57
Identification of the Agrobacterium tumefaciens C58 T-DNA genes e and f and their impact on crown gall tumour formation; Broer I et al.; DNA sequence analysis of the 4.4 kilobases (kb) Eco RI fragment 14 from T-DNA of Agrobacterium tumefaciens C58 revealed three open reading frames . One of them (945 bp) was supposed to encode the transcript e, the function of which has not been identified to date . Furthermore, a so far undescribed open reading frame (1035 bp) was identified, located in the centre of the Eco RI fragment 14 and termed gene f . The third open reading frame encoded the carboxy-terminal part of the agrocinopine synthase (Acs) . The gene e-encoded protein showed significant homologies to the gene products of the Agrobacterium rhizogenes rolB gene and the Agrobacterium tumefaciens gene 5 . Both gene products are supposed to regulate the plant's reaction on auxin . Depending on the plant species tested, Agrobacterium strains carrying mutations in gene e induced only small or almost no detectable crown gall tumours . According to these mutational studies and the protein homologies observed, the gene e product is suggested to be involved in tumour formation . Infection of several plant species with Agrobacterium carrying a mutated gene f, as well as expression of the gene f in transgenic tobacco plants did not lead to visible morphological changes . Therefore, in contrast to gene e, the gene f seems not to be essential for tumour formation . In order to study whether gene f is an active gene, its expression in agrobacteria and plants was monitored by translational lacZ fusion . In planta, the putative gene f-promoter mediates a tissue-specific expression pattern . Although gene f was expressed in free-living agrobacteria as well as in transgenic plants, the function of the f locus remained unclear . DNA homology studies with the f gene region revealed a mosaic-like DNA structure, indicating that this locus might be the result of genetic exchanges between different Agrobacterium strains during evolution.

Plant Mol Biol, 1995 Jan, 27(1), 1 - 15
Identification of several soybean cytosolic glutamine synthetase transcripts highly or specifically expressed in nodules: expression studies using one of the corresponding genes in transgenic Lotus corniculatus; Marsolier MC et al.; A DNA fragment containing sequences hybridizing to the 5' region of GS15, a gene encoding soybean cytosolic glutamine synthetase, was isolated from a soybean genomic library . Mapping and partial sequence analysis of the genomic clone revealed that it encodes a cytosolic GS gene, GS21, which is different from GS15 . In parallel, a number of cDNA clones encoding cytosolic GS were isolated using the coding region of pGS20 as a probe (pGS20 is a cDNA clone which corresponds to a transcript of the GS15 gene) . Two new full-length cDNAs designated pGS34 and pGS38 were isolated and sequenced . In the 5' non-coding region a strong homology was found between the two clones and the GS21 gene . However, none of these sequences were identical, which suggests that there are at least three members in this group of genes . In order to determine their relative levels of transcription, specific sequences from pGS34, pGS38 and GS21 were used in an RNAse protection assay . This experiment clearly showed that GS21 and the gene encoding pGS38 are specifically expressed in young or mature nodules, whereas the gene encoding pGS34 is highly transcribed in nodules and constitutively expressed at a lower level in other soybean organs . In order to further analyse the molecular mechanisms controlling GS21 transcription, different fragments of the promoter region were fused to the Escherichia coli reporter gene encoding beta-glucuronidase (GUS) and the constructs were introduced into Lotus corniculatus via Agrobacterium rhizogenes-mediated transformation . Analysis of GUS activity showed that the GS21 promoter-GUS constructs were expressed in the vasculature of all vegetative organs . This result is discussed in relation to species-specific metabolic and developmental characteristics of soybean and Lotus.

J Bacteriol, 1995 Jan, 177(2), 449 - 58
A new regulatory element modulates homoserine lactone-mediated autoinduction of Ti plasmid conjugal transfer; Hwang I et al.; Conjugal transfer of the Agrobacterium tumefaciens nopaline-type Ti plasmid pTiC58 is induced by agrocinopines A and B, opines secreted by crown gall tumors induced by the bacterium . This regulation functions through the transcriptional repressor, AccR . However, actual transcription of the tra genes is regulated by autoinduction through the activator TraR and the substituted homoserine lactone second messenger, Agrobacterium autoinducer (AAI) . We have identified a new regulatory element that modulates the response of TraR to AAI . The gene, called traM, suppresses TraR-AAI activation of transcription of tra genes carried on recombinant clones . The suppression could be relieved by increasing the expression of TraR but not by increasing AAI levels . traM is located between traR and traAF on pTiC58 and is transcribed in the clockwise direction . The 306-bp gene encodes an 11.2-kDa protein showing no significant relatedness to other proteins in the databases . Mutations in traM in pTiC58 conferred a transfer-constitutive phenotype, and strains harboring the Ti plasmid produced easily detectable amounts of AAI . These same mutations engineered into the transfer-constitutive Ti plasmid pTiC58 delta accR conferred a hyperconjugal phenotype and very high levels of AAI production . Expression of traM required TraR, indicating that transcription of the gene is regulated by the autoinduction system . TraM had no effect on the expression of traR, demonstrating that the suppressive effect is not due to repression of the gene encoding the activator . These results suggest that TraM is not a direct transcriptional regulator . Since the suppressive effect is demonstrable only when traM is overexpressed with respect to traR, we suggest that TraM functions to sequester TraR from the very small amounts of AAI produced under conditions when the agrocinopines are not present.

J Bacteriol, 1995 Jan, 177(1), 27 - 36
Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence; Stephens KM et al.; virB11, one of the 11 genes of the virB operon, is absolutely required for transport of T-DNA from Agrobacterium tumefaciens into plant cells . Previous studies reported that VirB11 is an ATPase with autophosphorylation activity and localizes to the inner membrane even though the protein does not contain the consensus N-terminal export sequence . In this report, we show that VirB11 localizes to the inner membrane even in the absence of other tumor-inducing (Ti) plasmid-encoded proteins . To facilitate the further characterization of VirB11, we purified this protein from the soluble fraction of an Escherichia coli extract by fusing VirB11 to the maltose-binding protein . The maltose-binding protein-VirB11 fusion was able to complement a virB11 deletion mutant of A . tumefaciens for tumor formation and also localized properly to the inner membrane of A . tumefaciens . The 72-kDa protein, purified from E . coli, exhibited no autophosphorylation, ATPase activity, or ATP-binding activity . To study the importance of the Walker nucleotide-binding site present in VirB11, mutations were generated to replace the conserved lysine residue with either alanine or arginine . Expression of the virB11K175A mutant gene resulted in an avirulent phenotype, and expression of the virB11K175R mutant gene gave rise to an attenuated virulence phenotype . Both mutant proteins were present at levels three to four times higher than that of VirB11 in the wild-type strain . The mutant genes did not exhibit a transdominant phenotype on tumor formation in bacteria that were expressing wild-type virB11 . The mutant proteins also localized properly to the inner membrane of A . tumefaciens, but the VirB11K175R protein appeared to be unstable after lysis of the cells.

Mol Plant Microbe Interact, 1995 Jan-Feb, 8(1), 85 - 91
Broad resistance to tospoviruses in transgenic tobacco plants expressing three tospoviral nucleoprotein gene sequences; Prins M et al.; Transgenic tobacco plants have been obtained expressing nucleoprotein (N) gene sequences of three different tospoviruses known to affect vegetable crops: tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), and groundnut ringspot virus (GRSV) . The chimeric plant transformation vector used comprised the three viral N gene sequences, each with a copy of the CaMV 35S promoter and the nos terminator . Despite the high levels of homology between the different N gene sequences (74-82%) and the presence of repeated promoter and terminator sequences in this construct, unrearranged copies of this triple N gene construct were stably maintained in both Escherichia coli and Agrobacterium tumefaciens plasmids used during the cloning process, as well as in several generations of transgenic tobacco plants . A transgenic tobacco line was obtained that exhibited high levels of resistance to all three tospoviruses, showing the possibility of producing transgenic plants with a broad resistance to tospoviruses by introducing tandemly cloned viral N gene sequences . DNA analysis of this transgenic plant line shows that the multivirus resistance trait is confined to a single genetic locus, which is very convenient for further breeding purposes.

Mol Plant Microbe Interact, 1995 Jan-Feb, 8(1), 58 - 65
Transgenic tobacco plants expressing a truncated form of the PAMV capsid protein (CP) gene show CP-mediated resistance to potato aucuba mosaic virus; Leclerc D et al.; Four constructs of the potato aucuba mosaic virus (PAMV) coat protein (CP) gene were engineered for expression in tobacco plants: The full-length CP sequence (FL CP), two truncated forms--one at the N terminus (46 amino acid residues are deleted) (NT138), one in the conserved core portion (86 amino acids deleted) (CT258) of the gene--and an antisense RNA construct . These constructs were introduced into tobacco plants (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated transformation . The plants transformed with the NT138 and FL CP constructs produced the mRNAs and proteins from the respective transgene . Transformants with the CT258 construct produced the transgenic mRNA, but the modified CP was not detected in the 20 different transformants tested . Transgenic R0 and R1 tobacco plants expressing the full-length, CT258, and the antisense constructs exhibited protection to PAMV infection and a delay in symptom development when inoculated with 0.1 and 0.5 microgram/ml of purified PAMV . Transgenic plants expressing the NT138 construct did not confer any detectable protection to PAMV infection . These results suggest that an engineered coat protein mediated resistance (CPMR) can be obtained from a CP gene truncated in its core region . The role of the N-terminal domain of the CP in the CPMR of PAMV and the implication of either the RNA or the protein in the protection is discussed.

Planta, 1995, 195(3), 369 - 78
Specific reduction of chloroplast glyceraldehyde-3-phosphate dehydrogenase activity by antisense RNA reduces CO2 assimilation via a reduction in ribulose bisphosphate regeneration in transgenic tobacco plants; Price GD et al.; The reduction of 3-phosphoglycerate (PGA) to triose phosphate is a key step in photosynthesis linking the photochemical events of the thylakoid membranes with the carbon metabolism of the photosynthetic carbon-reduction (PCR) cycle in the stroma . Glyceraldehyde-3-phosphate dehydrogenase: NADP oxidoreductase (GAPDH) is one of the two chloroplast enzymes which catalyse this reversible conversion . We report on the engineering of an antisense RNA construct directed against the tobacco (Nicotiana tabacum L.) chloroplast-located GAPDH (A subunit) . The construct was integrated into the tobacco genome by Agrobacterium-mediated transformation of leaf discs . Of the resulting transformants, five plants were recovered with reduced GAPDH activities ranging from 11 to 24% of wild-type (WT) activities . Segregation analysis of the kanamycin-resistance character in self-pollinated T1 seed from each of the five transformants revealed that one plant (GAP-R) had two active DNA inserts and the others had one insert . T1 progeny from GAP-R was used to generate plants with GAPDH activities ranging from WT levels to around 7% of WT levels . These were used to study the effect of variable GAPDH activities on metabolite pools for ribulose-1,5-bisphosphate (RuBP) and PGA, and the accompanying effects on the rate of CO2 assimilation and other gas-exchange parameters . The RuBP pool size was linearly related to GAPDH activity once GAPDH activity dropped below the range for WT plants, but the rate of CO2 assimilation was not affected until RuBP levels dropped to 30-40% of WT levels . That is, the CO2 assimilation rate fell when RuBP per ribulose-1,5-biphosphate carboxylase-oxygenase (Rubisco) site fell below 2 mol.(mol site)-1 while the ratio for WT plants was 4-5 mol.m(mol site)-1 . Leaf conductance was not reduced in leaves with reduced GAPDH activities, resulting in an increase in the ratio of intercellular to ambient CO2 partial pressure . Conductance in plants with reduced GAPDH activities was still sensitive to CO2 and showed a normal decline with increases in CO2 partial pressure . Although PGA levels did not fluctuate greatly, the effect of reduced GAPDH activity on RuBP-pool size and assimilation rate can be interpreted as being due to a blockage in the regeneration of RuBP . Concomitant gas-exchange and chlorophyll alpha fluorescence measurements indicated that photosynthesis changed from being Rubisco-limited to being RuBP-regeneration-limited at a lower CO2 partial pressure in the antisense plants than in WT plants.(ABSTRACT TRUNCATED AT 400 WORDS)

Acta Biochim Pol, 1995, 42(1), 97 - 101
Expression of the plum pox coat protein gene in transgenic Nicotiana tabacum plants; Wypijewski KJ et al.; Plant expression vector pBI 121 containing the gene encoding coat protein of Plum Pox Virus of the Skierniewice isolate (CP PPV-S) was prepared (clone pCM1) . The construct was used for transformation of Nicotiana tabacum plants using an Agrobacterium tumefaciens based system . About 82% of kanamycin resistant plant lines contained a transgene (the sequence of CP PPV-S) but only 81% of them actively expressed the PPV-S coat protein gene as measured by RT-PCR.

Crit Rev Microbiol, 1995, 21(1), 1 - 18
Synthesis of phytohormones by plant-associated bacteria; Costacurta A et al.; The plant hormones, auxins and cytokinins, are involved in several stages of plant growth and development such as cell elongation, cell division, tissue differentiation, and apical dominance . The biosynthesis and the underlying mechanism of auxins and cytokinins action are subjects of intense investigation . Not only plants but also microorganisms can synthesize auxins and cytokinins . The role of phytohormone biosynthesis by microorganisms is not fully elucidated: in several cases of pathogenic fungi and bacteria these compounds are involved in pathogenesis on plants; auxin and cytokinin production may also be involved in root growth stimulation by beneficial bacteria and associative symbiosis . The genetic mechanism of auxin biosynthesis and regulation by Pseudomonas, Agrobacterium, Rhizobium, Bradyrhizobium, and Azospirillum, are well studied; in these bacteria several physiological effects have been correlated to the bacterial phytohormones biosynthesis . The pathogenic bacteria Pseudomonas and Agrobacterium produce indole-3-acetic acid via the indole-3-acetamide pathway, for which the genes are plasmid borne . However, they do possess also the indole-3-pyruvic acid pathway, which is chromosomally encoded . In addition, they have genes that can conjugate free auxins or hydrolyze conjugated forms of auxins and cytokinins . In Agrobacterium there are also several genes, located near the auxin and cytokinin biosynthetic genes, that are involved in the regulation of auxins and cytokinins sensibility of the transformed plant tissue . Symbiotic bacteria Rhizobium and Bradyrhizobium synthesize indole-3-acetic acid via indole-3-pyruvic acid; also the genetic determinants for the indole-3-acetamide pathway have been detected, but their activity has not been demonstrated . In the plant growth-promoting bacterium Azospirillum, as in Agrobacterium and Pseudomonas, both the indole-3-pyruvic acid and the indole-3-acetamide pathways are present, although in Azospirillum the indole-3-pyruvic acid pathway is of major significance . In addition, biochemical evidence for a tryptophan-independent indole-3-acetic acid pathway in Azospirillum has been presented.

Chin J Biotechnol, 1995, 11(1), 1 - 7
Highly insect-resistant transgenic tobacco plants containing both B.t . and CpTI genes; Zhao R et al.; The cowpea trypsin inhibitor (CpTI) gene was synthesized according to its cDNA sequence using DNA synthesizer and confirmed by DNA sequencing . The CpTI gene and modified Bacillus thuringiensis (B.t.) delta-endotoxin gene were cotransformed to tobacco explants mediated by Agrobacterium tumefaciens . The integrations of B.t and CpTI genes were confirmed by PCR and Southern hybridization . Three kinds of transgenic plants were obtained: (1) containing CpTI gene, (2) containing B.t gene, (3) containing both CpTI and B.t genes . Bioassays showed that all these transgenic plants were toxic to the larvae of Heliothis armigera and that the tobacco plants containing both genes had enhanced toxicity to larvae by comparison with plants containing only CpTI or B.t gene.

Science, 1994 Dec 23, 266(5193), 1986 - 8
Splicing of the rolA transcript of Agrobacterium rhizogenes in Arabidopsis; Magrelli A et al.; The rolA gene encoded on the Ri plasmid A4 of Agrobacterium rhizogenes is one of the transferred (TL-DNA) genes involved in the pathogenesis of hairy-root disease in plants . The function of the 100-amino acid protein product of rolA is unknown, although its expression causes physiological and developmental alterations in transgenic plants . The rolA gene of A . rhizogenes contains an intron in its untranslated leader region that has features typical of plant pre-messenger RNA introns . Transcription and splicing of the rolA pre-messenger RNA occur in the plant cell.

Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 12760 - 4
Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation; Nawrath C et al.; In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly{(R)-(-)-3-hydroxybutyrate} (PHB) from acetyl-CoA . In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence . Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses . These plants accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-micron granules within plastids . In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB that accumulated . We conclude that there does not appear to be any biological barrier to high-level production of PHB in higher plants . The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand.

Carbohydr Res, 1994 Dec 16, 265(2), 167 - 79
NMR analysis of succinoglycans from different microbial sources: partial assignment of their 1H and 13C NMR spectra and location of the succinate and the acetate groups; Matulova M et al.; In order to obtain information on the location of succinate and acetate groups, comparative NMR analyses were carried out on succinoglycans from different microbial sources by using conventional and advanced NMR techniques . In particular, one-dimensional, 1H and 13C NMR spectra were recorded for qualitative and quantitative analysis on native high-molecular-weight succinoglycans (both in the Na+ salt and free-acid forms) from Pseudomonas sp . NCIB 11592, Agrobacterium radiobacter A201-25, Rhizobium meliloti YE-2, and Rhizobium sp . isolated from Vicia faba and compared with those of the deacylated and deacylated-depyruvated, partially depolymerised exopolysaccharides from Rhizobium meliloti YE-2 . Moreover, a series of two-dimensional experiments was performed on all the exopolysaccharides aiming at the partial assignment of the NMR spectra . The NMR data showed that succinate is located on O-6 of either one or both of the two side chain 3-linked beta-D-Glc residues, whereas the acetate (when it is present) is located on one of the O-6 of backbone 4-linked beta-D-Glc units, but the specific site could not be determined . In addition, the spectral features of the succinate substituent were found to be sensitive to pH changes.

Mol Gen Genet, 1994 Dec 15, 245(6), 704 - 15
An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA; Fullner KJ et al.; The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells . This study examined one putative component of that complex, VirB4 . A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene . The virB4 gene was found to be necessary for virulence on plants and for the transfer of IncQ plasmids to recipient cells of A . tumefaciens . Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes . Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels . Having shown a role for the VirB4 protein in DNA transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis . virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer . The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown . In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA transfer.

Gene, 1994 Dec 2, 150(1), 117 - 22
A chromosomal cluster of genes encoding ADP-glucose synthetase, glycogen synthase and phosphoglucomutase in Agrobacterium tumefaciens; Uttaro AD et al.; A chromosomal region from Agrobacterium tumefaciens that complements exoC (pgm) mutations was cloned and sequenced . A cluster of three open reading frames (ORF1, ORF2 and ORF3) was identified . These genes are oriented in the same direction and are involved in the synthesis of glycogen and other polysaccharides . ORF1 encodes a 420-amino-acid (aa) protein with 55.9% homology to Escherichia coli GlgC (ADP-glucose synthetase, EC 2.7.7.27) . ORF2 encodes a 480-aa protein with 42.2% homology to E . coli GlgA (glycogen synthase, EC 2.4.1.21) . Based on Tn5 mutagenesis and protein homology, ORF3 was identified as the structural gene encoding phosphoglucomutase (Pgm, EC 2.7.5.1) . ORF3 encodes a 542-aa protein with 52.6% homology to rabbit Pgm . There is no significant homology (less than 20%) to the Xanthomonas campestris XanA protein, which displays phosphomannomutase (Pmm) and Pgm activities {Koplin et al., J . Bacteriol 174 (1992) 191-199} . An A . tumefaciens pgm::Tn5 mutant retains Pmm activity.

Plant Cell, 1994 Dec, 6(12), 1789 - 1803
The elicitor-inducible alfalfa isoflavone reductase promoter confers different patterns of developmental expression in homologous and heterologous transgenic plants; Oommen A et al.; In legumes, the synthesis of infection- and elicitor-inducible antimicrobial phytoalexins occurs via the isoflavonoid branch of the phenylpropanoid pathway . To study transcriptional regulation of isoflavonoid pathway-specific genes, we have isolated the gene encoding isoflavone reductase (IFR), which is the enzyme that catalyzes the penultimate step in the synthesis of the phytoalexin medicarpin in alfalfa . Chimeric gene fusions were constructed between 765- and 436-bp promoter fragments of the IFR gene and the beta-glucuronidase reporter gene and transferred to alfalfa and tobacco by Agrobacterium-mediated transformation . Both promoter fragments conferred elicitor-mediated expression in cell suspension cultures derived from transgenic plants of both species and fungal infection-mediated expression in leaves of transgenic alfalfa . Developmental expression directed by both promoter fragments in transgenic alfalfa was observed only in the root meristem, cortex, and nodules, which is consistent with the accumulation of endogenous IFR transcripts . However, in transgenic tobacco, expression from the 765-bp promoter was observed in vegetative tissues (root meristem and cortex, inner vascular tissue of stems and petioles, leaf tips, and stem peripheries adjacent to petioles) and in reproductive tissues (stigma, placenta, base of the ovary, receptacle, seed, tapetal layer, and pollen grains), whereas the 436-bp promoter was expressed only in fruits, seed, and pollen . These data indicate that infection/elicitor inducibility of the IFR promoter in both species and developmental expression in alfalfa are determined by sequences downstream of position -436, whereas sequences between -436 and -765 confer a complex pattern of strong ectopic developmental expression in the heterologous species that lacks the isoflavonoid pathway.

Plant Mol Biol, 1994 Dec, 26(6), 1995 - 2001
The expression of a chimeric Phaseolus vulgaris nodulin 30-GUS gene is restricted to the rhizobially infected cells in transgenic Lotus corniculatus nodules; Carsolio C et al.; In Phaseolus vulgaris there is a nodulin family, Npv30, of ca . 30 kDa, as detected in an in vitro translation assay {2} . We isolated a gene (npv30-1) for one of the members of this family . The nucleotide sequence of the promoter of npv30-1 contains nodule-specific motifs common to other late nodulin genes . The promoter was fused to the GUS reporter gene; this chimeric fusion was introduced into Lotus corniculatus via Agrobacterium rhizogenes transformation . GUS activity was only detected in the infected cells of the nodules of transgenic plants . By contrast, the expression of a 35S-GUS construct was restricted to the uninfected cells and the vascular tissue.

Plant Mol Biol, 1994 Dec, 26(6), 1759 - 73
Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth; Kuipers AG et al.; Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS) . GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose . The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation . Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones . Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth . This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity . Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter . Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.

Mol Gen Genet, 1994 Nov 15, 245(4), 523 - 7
Interspecies regulation of the recA gene of gram-negative bacteria lacking an E . coli-like SOS operator; Riera J et al.; The recA genes of Agrobacterium tumefaciens, Rhizobium meliloti, Rhizobium phaseoli and Rhodobacter sphaeroides, species belonging to the alpha-group bacteria of the Proteobacteria class, have been fused in vitro to the lacZ gene of Escherichia coli . By using a mini-Tn5 transposon derivative, each of these recA-lacZ fusions was introduced into the chromosome of each of the four species, and into that of E . coli . The recA genes of three of the alpha bacteria are induced by DNA damage when inserted in A . tumefaciens, R . phaseoli or R . meliloti chromosomes . The expression of the recA gene of R . sphaeroides is DNA damage-mediated only when present in its own chromosome; none of the genes is induced in E . coli . Likewise, the recA gene of E . coli is not induced in any of the four alpha species . These data indicate that A . tumefaciens, R . meliloti and R . phaseoli possess a LexA-like repressor, which is able to block the expression of their recA genes, as well as that of R . sphaeroides, but not the recA gene of E . coli . The LexA repressor of R . sphaeroides does not repress the recA gene of A . tumefaciens, R . meliloti, R . phaseoli or E . coli.

Mol Gen Genet, 1994 Nov 15, 245(4), 493 - 505
Agrobacterium vitis nopaline Ti plasmid pTiAB4: relationship to other Ti plasmids and T-DNA structure; Otten L et al.; The Ti plasmid of the Agrobacterium vitis nopaline-type strain AB4 was subcloned and mapped . Several regions of the 157 kb Ti plasmid are similar or identical to parts of the A . vitis octopine/cucumopine (o/c)-type Ti plasmids, and other regions are homologous to the nopaline-type Ti plasmid pTiC58 . The T-DNA of pTiAB4 is a chimaeric structure of recent origin: the left part is 99.2% homologous to the left part of the TA-DNA of the o/c-type Ti plasmids, while the right part is 97.1% homologous to the right part of an unusual nopaline T-DNA recently identified in strain 82.139, a biotype II strain from wild cherry . The 3' noncoding regions of the ipt genes from pTiAB4 and pTi82.139 are different from those of other ipt genes and contain a 62 bp fragment derived from the coding sequence of an ipt gene of unknown origin . A comparison of different ipt gene sequences indicates that the corresponding 62 bp sequence within the coding region of the AB4 ipt gene has been modified during the course of its evolution, apparently by sequence transfer from the 62 bp sequence in the 3' non-coding region . In pTi82.139 the original coding region of the ipt gene has remained largely unmodified . The pTiAB4 6b gene differs from its pTi82.139 counterpart by the lack of a 12 bp repeat in the 3' part of the coding sequence . This leads to the loss of four glutamic acid residues from a series of ten . In spite of these differences, the ipt and 6b genes of pTiAB4 are functional . Our results provide new insight into the evolution of Agrobacterium Ti plasmids and confirm the remarkable plasticity of these genetic elements . Possible implications for the study of bacterial phylogeny are discussed.

Transgenic Res, 1994 Nov, 3(6), 344 - 54
Expression of the pea albumin 1 gene in transgenic white clover and tobacco; Ealing PM et al.; In order to improve the quality of pasture protein for ruminant animal nutrition, we are introducing genes encoding rumen-protected proteins, rich in essential amino acids, into white clover (Trifolium repens L.) . We have introduced a chimaeric gene transcribed from the 35S CaMV promoter, and encoding the pea albumin 1 (PA1) protein, rich in sulphur amino acids, into the white clover genotype WR8 by Agrobacterium-mediated transformation . A transgenic plant with high levels of PA1 mRNA was crossed with a commercial genotype from cv . Regal Ladino and both the parent and progeny plants were analyzed for expression and accumulation of PA1 gene products . Steady-state mRNA levels and transcript sizes in transgenic parent and progeny were comparable . The abundance and stability of the PA1 protein in transgenic white clover plants was examined by immunoselection of in vivo {35S}Na2SO4-labelled plant proteins . Evidence is presented here, that the 11 kDa PA1 proprotein precursor is processed correctly in petiole tissues of newly regenerated white clover plantlets but only the 6 kDa PA1a subunit accumulates in leaflets of tissue-culture-grown and older glasshouse-grown clover plants . Attempts to enhance PA1 abundance by altering its subcellular target in transgenic tobacco plants suggest that the endomembrane system is a relatively stable environment compared with the cytoplasm or chloroplast, for the accumulation of PA1, despite its low abundance there (< 0.001% total cell protein).

Plant J, 1994 Nov, 6(5), 781 - 6
A self-stabilizing Ac derivative and its potential for transposon tagging; Schmitz G et al.; With the goal of developing a self-stabilizing transposon tagging system, a derivative of the maize transposable element Ac (Ds303) harbouring a deletion between bp 246 and 736 has been introduced into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation . The deletion removes the major transcription start site, 84 bp of the putative Ac promoter and part of the untranslated leader . Transpositions from the T-DNA, where Ds303 was inserted between the mannopine synthase 1' promoter and the GUS gene, were observed in four independent transgenic plants analysed . After transposition the transposed Ds303 elements were stably integrated in the genome and could not transactivate a tester Ds element . However, somatic and germinal transpositions of the transposed Ds303 elements occurred in the presence of a transposase source.

Plant Mol Biol, 1994 Nov, 26(3), 923 - 34
Termini and telomeres in T-DNA transformation; Chiurazzi M et al.; A T-DNA vector for plant transformation has been constructed in which the cloning site is located 9 bp from the right-border (RB) end and 27 bp from the left-border (LB) end . In this vector cloned DNA homologous to plant chromosomal sequences is located at the T-DNA termini, and will thus be exposed by even limited exonucleolysis in planta . The arabidopsis ADH (alcohol dehydrogenase) locus was mobilized from Agrobacterium, and integration into the recipient genome was studied . Despite the terminal location of ADH homology in this vector, the T-DNA integrated essentially at random in the Arabidopsis genome rather than at the endogenous ADH locus . T-DNA integration was blocked, however, when Arabidopsis telomeric sequences were added to the construct at each end of the ADH homology . Thus the predominant mode by which incoming T-DNA is integrated into the continuity of chromosomal DNA involves free DNA ends, but, in contrast to modes of recombination such as gap repair, does not involve extensive terminal DNA sequence homology.

Appl Environ Microbiol, 1994 Nov, 60(11), 4192 - 4
Construction of Agrobacterium strains by electroporation of genomic DNA and its utility in analysis of chromosomal virulence mutations; Charles TC et al.; We have extended the technique of electroporation as a genetic tool for manipulating the Agrobacterium tumefaciens chromosome . We used this technique to introduce chromosomal DNA into recipient A . tumefaciens strains by electroporation and constructed isogenic chvE mutants that share the same chromosomal background but differ in their types of pTi (octopine or nopaline) . Both nopaline and octopine pTi-carrying chvE mutants were deficient in vir regulon induction and exhibited similar reductions in host range.

J Bacteriol, 1994 Nov, 176(21), 6418 - 26
Mutational analysis of the transcriptional activator VirG of Agrobacterium tumefaciens; Scheeren-Groot EP et al.; To find VirG proteins with altered properties, the virG gene was mutagenized . Random chemical mutagenesis of single-stranded DNA containing the Agrobacterium tumefaciens virG gene led with high frequency to the inactivation of the gene . Sequence analysis showed that 29% of the mutants contained a virG gene with one single-base-pair substitution somewhere in the open reading frame . Thirty-nine different mutations that rendered the VirG protein inactive were mapped . Besides these inactive mutants, two mutants in which the vir genes were active even in the absence of acetosyringone were found on indicator plates . A VirG protein with an N54D substitution turned out to be able to induce a virB-lacZ reporter gene to a high level even in the absence of the inducer acetosyringone . A VirG protein with an I77V substitution exhibited almost no induction in the absence of acetosyringone but showed a maximum induction level already at low concentrations of acetosyringone.

EMBO J, 1994 Nov 1, 13(21), 5099 - 112
enod40, a gene expressed during nodule organogenesis, codes for a non-translatable RNA involved in plant growth; Crespi MD et al.; Rhizobium meliloti can interact symbiotically with Medicago plants, thereby inducing root nodules . However, certain Medicago plants can form nodules spontaneously, in the absence of rhizobia . A differential screening was performed using spontaneous nodule versus root cDNAs from Medicago sativa ssp . varia . Transcripts of a differentially expressed clone, Msenod40, were detected in all differentiating cells of nodule primordia and spontaneous nodules, but were absent in fully differentiated cells . Msenod40 showed homology to a soybean early nodulin gene, Gmenod40, although no significant open reading frame (ORF) or coding capacity was found in the Medicago sequence . Furthermore, in the sequences of cDNAs and a genomic clone (Mtenod40) isolated from Medicago truncatula, a species containing a unique copy of this gene, no ORFs were found either . In vitro translation of purified Mtenod40 transcripts did not reveal any protein product . Evaluation of the RNA secondary structure indicated that both msenod40 and Gmenod40 transcripts showed a high degree of stability, a property shared with known non-coding RNAs . The Mtenod40 RNA was localized in the cytoplasm of cells in the nodule primordium . Infection with Agrobacterium tumefaciens strains bearing antisense constructs of Mtenod40 arrested callus growth of Medicago explants, while overexpressing Mtenod40 embryos developed into teratomas . These data suggest that the enod40 genes might have a role in plant development, acting as 'riboregulators', a novel class of untranslated RNAs associated with growth control and differentiation.

Mol Microbiol, 1994 Nov, 14(4), 705 - 16
The ccoNOQP gene cluster codes for a cb-type cytochrome oxidase that functions in aerobic respiration of Rhodobacter capsulatus; Thony-Meyer L et al.; The genes for a new type of a haem-copper cytochrome oxidase were cloned from Rhodobacter capsulatus strain 37b4, using the Bradyrhizobium japonicum fixNOQP gene region as a hybridizing probe . Four genes, probably organized in an operon (ccoNOQP), were identified; their products share extensive amino acid sequence similarity with the FixN, O, Q and P proteins that have recently been shown to be the subunits of a cb-type oxidase . CcoN is a b-type cytochrome, CcoO and CcoP are membrane-bound mono- and dihaem c-type cytochromes and CcoQ is a small membrane protein of unknown function . Genes for a similar oxidase are also present in other non-rhizobial bacterial species such as Azotobacter vinelandii, Agrobacterium tumefaciens and Pseudomonas aeruginosa, as revealed by polymerase chain reaction analysis . A ccoN mutant was constructed whose phenotype, in combination with the structural information on the gene products, provides evidence that the CcoNOQP oxidase is a cytochrome c oxidase of the cb type, which supports aerobic respiration in R . capsulatus and which is probably identical to the cbb3-type oxidase that was recently purified from a different strain of the same species . Mutant analysis also showed that this oxidase has no influence on photosynthetic growth and nitrogen-fixation activity.

Mol Microbiol, 1994 Nov, 14(4), 655 - 68
Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens; Pohlman RF et al.; The DNA sequence of a cluster of pKM101 conjugal transfer genes was determined and aligned with the genetic map of the plasmid . Eighteen genes were identified, at least eight and probably 11 of which are required for efficient conjugation . These tra genes are homologous to and colinear with genes found in the virB operon of Agrobacterium tumefaciens Ti plasmids . Seven pKM101 tra genes are also homologous to ptl genes of Bordetella pertussis, which direct the export of pertussis toxin . We used TnphoA to construct translational fusions between pKM101 genes and the Escherichia coli phoA gene, which encodes alkaline phosphatase, and provide evidence that at least 11 of the 18 genes are either fully or partially exported from the cytoplasm.

Mol Plant Microbe Interact, 1994 Nov-Dec, 7(6), 792 - 8
Acquisition and transmission of Agrobacterium by the whitefly Bemisia tabaci; Zeidan M et al.; Whiteflies transmit many different plant pathogens . We show here that the whitefly Bemisia tabaci can also acquire Agrobacterium tumefaciens from liquid cultures and from crown galls, and transmit it to plants . Cloned tomato yellow leaf curl virus (TYLCV) DNA and A . tumefaciens tumor-inducing functions were used as reporter genes . Whiteflies were fed through membranes on cultures of Agrobacterium At::pTY4 containing a dimeric copy of an infectious TYLCV genomic clone between the T-DNA borders of a binary vector . One hour of feeding was sufficient for the insects to be able to transmit TYLCV to tomato test plants . Infectious Agrobacterium was recovered from whiteflies that fed on At::pTY4 cultures, indicating that bacteria was acquired and remained intact in the insects . Whiteflies also acquired the virulent A . tumefaciens strain C58 from crown galls and induced tumors in tomato test plants . PCR and Southern blot analyses indicated that the target plant tissues were transformed . These results show that an insect can transfer foreign genes to plants by acquiring and delivering transforming bacteria.

Mol Plant Microbe Interact, 1994 Nov-Dec, 7(6), 703 - 7
Identification of a novel Rhizobium meliloti nodulation efficiency nfe gene homolog of Agrobacterium ornithine cyclodeaminase; Soto MJ et al.; The nfe genes located on the large plasmid pRmeGR4b are involved in the nodulation efficiency and competitiveness of Rhizobium meliloti GR4 on alfalfa roots . One hundred twenty-eight base-pairs downstream of nfe2 gene we found an open reading frame designated ORFC, 970 bp long and potentially coding for a 320 amino acid long protein . The amino acid sequence of the putatively encoded ORFC product shows similarity with ornithine cyclodeaminase (OCD) of Agrobacterium tumefaciens an unusual enzyme that converts ornithine into proline . The gene product of ORFC was identified as a 37-kDa protein by in vitro-coupled transcription-translation and in vivo by the T7 RNA polymerase/promoter system . DNA hybridization studies showed that strain GR4 carries a single copy of the ocd-like gene . No homologous sequences to GR4 ORFC DNA were found in other R . meliloti strains or Rhizobium spp . assayed . Furthermore, a GR4 derivative mutant obtained by plasmid disruption of ORFC showed an impaired nodulation efficiency as compared to that of the wild-type strain GR4 . Thus, the former locus should be considered a novel nfe gene . We propose to rename the nfe genes, nfe1, 2 and ORFC as nfeA, B, and D, respectively.

J Clin Microbiol, 1994 Nov, 32(11), 2660 - 6
Differentiation of Brucella abortus bv . 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv . 1 by PCR; Bricker BJ et al.; Several PCR assays which identify the genus Brucella but do not discriminate among species have been reported . We describe a PCR assay that comprises five oligonucleotide primers which can identify selected biovars of four species of Brucella . Individual biovars within a species are not differentiated . The assay can identify three biovars (1, 2, and 4) of B . abortus, all three biovars of B . melitensis, biovar 1 of B . suis, and all B . ovis biovars . These biovars include all of the Brucella species typically isolated from cattle in the United States, a goal of the present research . The assay exploits the polymorphism arising from species-specific localization of the genetic element IS711 in the Brucella chromosome . Identity is determined by the size(s) of the product(s) amplified from primers hybridizing at various distances from the element . The performance of the assay with U.S . field isolates was highly effective . When 107 field isolates were screened by the described method, there was 100% agreement with the identifications made by conventional methods . Six closely related bacteria (Agrobacterium radiobacter, Agrobacterium rhizogenes, Ochrobactrum anthropi, Rhizobium leguminosarum, Rhizobium meliloti, and Rhodospirillum rubrum) and two control bacteria (Bordetella bronchiseptica and Escherichia coli) tested negative by the assay.

Plant Physiol, 1994 Nov, 106(3), 887 - 95
Modulation of cysteine biosynthesis in chloroplasts of transgenic tobacco overexpressing cysteine synthase {O-acetylserine(thiol)-lyase}; Saito K et al.; Cysteine synthase {O-acetyl-L-serine(thiol)-lyase, EC 4.2.99.8} (CSase), which is responsible for the terminal step of cysteine biosynthesis, catalyzes the formation of L-cysteine from O-acetyl-L-serine (OAS) and hydrogen sulfide . Three T-DNA vectors carrying a spinach (Spinacia oleracea) cytoplasmic CSase A cDNA (K . Saito, N . Miura, M . Yamazaki, H . Horano, I . Murakoshi {1992} Proc Natl Acad Sci USA 89: 8078-8082) were constructed as follows: pCSK3F, cDNA driven by the cauliflower mosaic virus (CaMV) 35S RNA promoter with a sense orientation; pCSK3R, cDNA driven by the CaMV 355 promoter with an antisense orientation; pCSK4F, cDNA fused with the sequence for chloroplast-targeting transit peptide of pea ribulose-1,5-biphosphate carboxylase small subunit driven by the CaMV 35S promoter with a sense orientation . These chimeric genes were transferred into tobacco (Nicotiana tabacum) with Agrobacterium-mediated transformation, and self-fertilized progeny were obtained . CSase activities in cell-free extracts of pCSK3F and pCSK4F transformants were 2- to 3-fold higher than those of control and pCSK3R plants . CSase activities in chloroplasts of pCSK4F transformants were severalfold higher than those of control and pCSK3F plants, indicating that the foreign CSase protein is transported and accumulated in a functionally active form in chloroplasts of pCSK4F plants . Isolated chloroplasts of a pCSK4F transformant had a more pronounced ability to form cysteine in response to addition of OAS and sulfur compounds than those of a control plant . In particular, feeding of OAS and sulfite resulted in enhanced cysteine formation, which required photoreduction of sulfite in chloroplasts . The enhanced cysteine formation in a pCSK4F plant responding to sulfite was also observed in leaf discs . In addition, these leaf discs were partially resistant to sulfite toxicity, possibly due to metabolic detoxification of sulfite by fixing into cysteine . These results suggested that overaccumulated foreign CSase in chloroplasts could modulate biosynthetic flow of cysteine in response to sulfur stress.

Plant Physiol, 1994 Nov, 106(3), 1195 - 204
Biosynthesis of defense-related proteins in transformed root cultures of Trichosanthes kirilowii Maxim . var japonicum (Kitam.); Savary BJ et al.; We have established transformed ("hairy") root cultures from Trichosanthes kirilowii Maxim . var japonicum Kitam . (Cucurbitaceae) and four related species to study the biosynthesis of the ribosome-inactivating protein trichosanthin (TCN) and other root-specific defense-related plant proteins . Stable, fast-growing root clones were obtained for each species by infecting in vitro grown plantlets with Agrobacterium rhizogenes American Type Culture Collection strain 15834 . Each species accumulated reproducibly a discrete protein pattern in the culture medium . Analysis of the extracellular proteins from T . kirilowii var japonicum root cultures showed differential protein accumulation in the medium during the time course of growth in batch cultures . Maximum protein accumulation, approaching 20 micrograms/mL, was observed at mid-exponential phase, followed by a degradation of a specific protein subset that coincided with the onset of stationary phase . Two major extracellular proteins and one intracellular protein, purified by ion-exchange and reverse-phase high-performance liquid chromatography, were identified as class III chitinases (EC 3.2.1.14) based on N-terminal amino acid sequence and amino acid composition homologies with other class III chitinases . The Trichosanthes chitinases also showed reactivity with a cucumber class III chitinase antiserum and chitinolytic activity in a glycol chitin gel assay . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of intracellular proteins showed that normal and transformed T . kirilowii var japonicum roots accumulated only low levels of TCN (approximately 0.5% total soluble protein) . Storage roots from the plant displayed protein and antigen patterns different from root cultures and produced TCN as the dominant protein . Roots undergoing secondary growth and differentiation exhibited patterns similar to those of storage roots, including increased TCN levels, indicating that high production of TCN is associated with induction of secondary growth in roots.

Mol Gen Genet, 1994 Nov 1, 245(3), 363 - 70
In planta transformation of Arabidopsis thaliana; Katavic V et al.; Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions . After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium . We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure . In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events . Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7-8 weeks . Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A . thaliana ecotypes and mutants recalcitrant to in vitro regeneration . The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector . The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant . Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells . More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes . Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci . The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosci Biotechnol Biochem, 1994 Nov, 58(11), 2024 - 32
Cellular localization and functional analysis of the protein encoded by the chromosomal virulence gene(acvB) of Agrobacterium tumefaciens; Kang HW et al.; A chromosomal virulence gene, acvB, of Agrobacterium tumefaciens {J . Bacteriol., 175, 3208-3212 (1993)} was over-expressed in Escherichia coli . A 47-kDa protein was produced and localized in the periplasmic space of E . coli . Amino acid sequence analysis of its N-terminal demonstrated that a signal peptide of 24 amino acids was cleaved from the pre AcvB protein to produce the mature 47-kDa protein . Western-blot analysis using the antiserum against the AcvB protein detected a 47-kDa protein in the periplasmic space only with strain A208 (acvB+) . The amount of AcvB protein synthesized was not increased in strain A208 by induction with acetosyringone (100 microM) . There was observed no significant difference in induction by acetosyringone of virB::lacZ, virD::lacZ, and virE::lacZ fusion genes regardless of the presence or absence of the acvB gene . The T-strand (lower strand of T-DNA) was detected in strains A208 as well as B119 (acvB-) which were cultured in induction medium containing acetosyringone . AcvB protein bound to single-stranded DNAs with no apparent sequence specificity . The results suggest that AcvB protein binds to the T-strand in periplasm and mediates the transfer of the T-strand from A . tumefaciens to the host plant cell.

Tsitol Genet, 1994 Nov-Dec, 28(6), 27 - 34
{The import of DNA topoisomerase type II from Drosophila melanogaster into the chloroplasts of transgenic Nicotiana tabacum plants}; Storozhenko SV et al.; The construct containing the structural gene of the type II DNA topoisomerase of Drosophila melanogaster fused to the sequence of the chloroplast transit peptide directed by the 35 S promoter was produced on the basis of the binary vector Bin 19 . The construct was introduced into Nicotiana tabacum plants by Agrobacterium-mediated transformation . The integration of the construct into genomic DNA was demonstrated by PCR and Southern blot hybridization . Expression of the chimeric gene at the transcription level was studied by Northern blotting . The protein produce of the expression was identified in chloroplasts by Western blot hybridization with polyclonal anti-type II Drosophila topoisomerase antibodies.

Biol Chem Hoppe Seyler, 1994 Nov, 375(11), 765 - 77
Inhibition of viroid infection by antisense RNA expression in transgenic plants; Matousek J et al.; Complex formation between different antisense RNAs directed against either plus-strand or minus-strand sequences of the potato spindle tuber viroid (PSTVd) was studied using temperature-gradient gel electrophoresis and immunochemical detection with an antibody specific for double-stranded RNA . Short minus-strand sequences were directed against the upper central conserved region (UCCR) of plus-strand viroid replication intermediates, a plus-strand corresponding to the left half of the rod-like secondary structure (VL+) against minus-strand replication intermediates . It was shown that antisense RNA forms complexes with the corresponding target RNA only with low yield during incubation at low (physiological) temperatures but with high yield during in vitro transcription of the target RNA when the antisense RNA is already present in the solution . The antisense RNA sequences were integrated into Solanum tuberosum L . by Agrobacterium tumefaciens transformation . Antisense RNA expression in vivo was analyzed by Northern analysis . Infection tests were performed using the transgenic potato lines in order to evaluate their degree of resistance against PSTVd infection . Although some lines showed a significant inhibition of viroid accumulation, a high variability of viroid infection in different transgenic potato lines was obtained . Since strongly infected plants were observed in all transgenic lines 6 to 8 weeks post inoculation, a threshold concentration of viroid, overcoming the antisense effect has to be assumed . When the rate of viroid accumulation was tested using agroinfection assays on leaf discs, a stronger antisense effect could be achieved.

Appl Environ Microbiol, 1994 Nov, 60(11), 4163 - 6
Expression of tfx and sensitivity to the rhizobial peptide antibiotic trifolitoxin in a taxonomically distinct group of alpha-proteobacteria including the animal pathogen Brucella abortus; Triplett EW et al.; Three phylogenetically distinct groups within the alpha-proteobacteria which differ in trifolitoxin sensitivity are described . Trifolitoxin sensitivity was found in strains of Agrobacterium, Brucella, Mycoplana, Ochrobactrum, Phyllobacterium, Rhodobacter, Rhodopseudomonas, Rhodospirillum, and Rhizobium . Strains of Agrobacterium, Brucella, Phyllobacterium, Rhizobium, and Rhodospirillum were capable of producing trifolitoxin upon conjugal transfer of tfxABCDEFG.

Mol Gen Genet, 1994 Oct 17, 245(1), 1 - 10
NPK15, a tobacco protein-serine/threonine kinase with a single hydrophobic region near the amino-terminus; Ito Y et al.; A cDNA clone (cNPK15) was isolated from tobacco cells in suspension culture, which encodes a predicted protein kinase of 422 amino acids . The predicted NPK15 protein consists of a hydrophobic region near the amino-terminus, a linker domain and the catalytic domain of a protein-serine/threonine kinase in the carboxyl-half . NPK15 was not found to be closely related to any reported protein, but its putative catalytic domain shares some structural similarity with those of receptor-like protein kinases of plants, such as ZmPK1 from Zea mays and TMK1 from Arabidopsis, even though no receptor-like domain is found in NPK15 . Recombinant NPK15 expressed in Escherichia coli as a fusion protein was found capable of autophosphorylation and of phosphorylation of the histone H1 protein on both serine and threonine residues . Upon overexpression of cNPK15 under control of the promoter of cauliflower mosaic virus 35S RNA in tobacco cells, into which it had been introduced by Agrobacterium-mediated transformation, the NPK15 gene acted as a "suicide" gene and blocked proliferation of the host cells . By contrast, such a suicide effect was not observed with the gene for a kinase-negative mutant protein in which the nucleotide sequence for the ATP-binding site had been mutated or with a mutant derivative encoding a protein in which the hydrophobic region had been deleted . Thus, the protein kinase activity of NPK15 and the hydrophobic region of the protein are responsible for the suicide effect . The NPK15 protein kinase seems to be associated with specific cellular functions . Southern blot analysis with cNPK15 as the probe detected several fragments in restriction digests of genomic DNAs from both tobacco and other members of the Solanaceae . This results suggests that NPK15-related genes constitute a small gene family in the genomes of Solanaceae.

Plant Cell, 1994 Oct, 6(10), 1509 - 18
The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis; Sun TP et al.; The first committed step in the gibberellin (GA) biosynthetic pathway is the conversion of geranylgeranyl pyrophosphate (GGPP) through copalyl pyrophosphate (CPP) to ent-kaurene catalyzed by ent-kaurene synthetases A and B . The ga1 mutants of Arabidopsis are gibberellin-responsive male-sterile dwarfs . Biochemical studies indicate that biosynthesis of GAs in the ga1 mutants is blocked prior to the synthesis of ent-kaurene . The GA1 locus was cloned previously using the technique of genomic subtraction . Here, we report the isolation of a nearly full-length GA1 cDNA clone from wild-type Arabidopsis . This cDNA clone encodes an active protein and is able to complement the dwarf phenotype in ga1-3 mutants by Agrobacterium-mediated transformation . In Escherichia coli cells that express both the Arabidopsis GA1 gene and the Erwinia uredovora gene encoding GGPP synthase, CPP was accumulated . This result indicates that the GA1 gene encodes the enzyme ent-kaurene synthetase A, which catalyzes the conversion of GGPP to CPP . Subcellular localization of the GA1 protein was studied using 35S-labeled GA1 protein and isolated pea chloroplasts . The results showed that the GA1 protein is imported into and processed in pea chloroplasts in vitro.

Plant Cell, 1994 Oct, 6(10), 1391 - 400
Overexpression of Arabidopsis COP1 results in partial suppression of light-mediated development: evidence for a light-inactivable repressor of photomorphogenesis; McNellis TW et al.; Arabidopsis seedlings are genetically endowed with the capability to follow two distinct developmental programs: photomorphogenesis in the light and skotomorphogenesis in darkness . The regulatory protein CONSTITUTIVE PHOTO-MORPHOGENIC1 (COP1) has been postulated to act as a repressor of photomorphogenesis in the dark because loss-of-function mutations of COP1 result in dark-grown seedlings phenocopying the light-grown wild-type seedlings . In this study, we tested this working model by overexpressing COP1 in the plant and examining its inhibitory effects on photomorphogenic development . Stable transgenic Arabidopsis lines overexpressing COP1 were generated through Agrobacterium-mediated transformation . Overexpression was achieved using either the strong cauliflower mosaic virus 35S RNA promoter or additional copies of the wild-type gene . Analysis of these transgenic lines demonstrated that higher levels of COP1 can inhibit aspects of photomorphogenic seedling development mediated by either phytochromes or a blue light receptor, and the extent of inhibition correlated quantitatively with the vivo COP1 levels . This result provides direct evidence that COP1 acts as a molecular repressor of photomorphogenic development and that multiple photoreceptors can independently mediate the light inactivation of COP1 . It also suggests that a controlled inactivation of COP1 may provide a basis for the ability of plants to respond quantitatively to changing light signals, such as fluence rate and photoperiod.

Plant Mol Biol, 1994 Oct, 26(1), 465 - 72
Genetic complementation of a floral homeotic mutation, apetala3, with an Arabidopsis thaliana gene homologous to DEFICIENS of Antirrhinum majus; Okamoto H et al.; Among the homeotic mutants with altered floral organs, two mutants of Arabidopsis thaliana, apetala3 and pistillata, and two mutants of Antirrhinum majus, deficiens and globosa, have a homeotic conversion of the floral organs in whorl 2 and 3, namely petals to sepals and stamens to carpels . We have isolated a homologue of the DEFICIENS gene from A . thaliana wild type and shown complete complementation of apetala3 mutation by introducing the isolated gene using Agrobacterium-mediated transformation . These results show that the APETALA3 is a homologue of DEFICIENS structurally and functionally . The 5'-upstream region of APETALA3 contains three SRE-like sequence, where MADS box-containing proteins are assumed to bind and regulate expression in tissue- and stage-specific manner.

Plant Mol Biol, 1994 Oct, 26(1), 415 - 22
rol genes of Agrobacterium rhizogenes cucumopine strain: sequence, effects and pattern of expression; Serino G et al.; By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA . Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA--which we refer to as rol alpha, beta and gamma--were utilized to infect carrot discs and to obtain transgenic tobacco plants, in order to compare the morphogenetic capabilities to those of the 1855 rol genes . Moreover, a long segment of the 5' non-coding region of rol alpha and rol beta was fused to the GUS reporter gene and the pattern of expression and the responsiveness to auxin of the constructs was analysed in transgenic tobacco . Differences in the auxin requirement for root induction between the 2659 rol genes and their respective 1855 counterparts were pinpointed . These differences are not due to gene regulation and presumably reflect functional differences in the proteins encoded . Differences were also observed in the pattern of expression of rol beta in roots of transgenic plants, as compared to rolB . In addition, the pattern of expression of rol alpha-GUS construct in roots was found to be analogous to that observed for a construct driven by two of the five regulatory domains of the rolB promoter.

Plant Mol Biol, 1994 Oct, 26(1), 189 - 202
Developmental specific expression and organelle targeting of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase in transgenic rape and tobacco seeds; Verwoert II et al.; In both plants and bacteria, de novo fatty acid biosynthesis is catalysed by a type II fatty acid synthetase (FAS) system which consists of a group of eight discrete enzyme components . The introduction of heterologous, i.e . bacterial, FAS genes in plants could provide an alternative way of modifying the plant lipid composition . In this study the Escherichia coli fabD gene, encoding malonyl CoA-ACP transacylase (MCAT), was used as a model gene to investigate the effects of over-producing a bacterial FAS component in the seeds of transgenic plants . Chimeric genes were designed, so as not to interfere with the household activities of fatty acid biosynthesis in the earlier stages of seed development, and introduced into tobacco and rapeseed using the Agrobacterium tumefaciens binary vector system . A napin promoter was used to express the E . coli MCAT in a seed-specific and developmentally specific manner . The rapeseed enoyl-ACP reductase transit peptide was used successfully, as confirmed by immunogold labelling studies, for plastid targeting of the bacterial protein . The activity of the bacterial enzyme reached its maximum (up to 55 times the maximum endogenous MCAT activity) at the end of seed development, and remained stable in mature transgenic seeds . Significant changes in fatty acid profiles of storage lipids and total seed lipid content of the transgenic plants were not found . These results are in support of the notion that MCAT does not catalyse a rate-limiting step in plant fatty acid biosynthesis.

J Biol Chem, 1994 Sep 9, 269(36), 22473 - 6
Expression of active, processed ricin in transgenic tobacco; Sehnke PC et al.; The cDNA encoding the plant toxin precursor preproricin was introduced into tobacco via Agrobacterium tumefaciens-mediated gene transfer . Transgenic plants were assayed for type II ribosome-inactivating protein expression and activity . Western blot analysis of soluble leaf extracts using anti-ricin a-chain (RTA) antibodies identified 34- and 32-kDa proteins, which were electrophoretically indistinguishable from castor seed RTA . Analysis with anti-ricin b-chain (RTB) antibodies identified both a 34-kDa protein major band, which co-migrated with castor seed RTB, and a 30-kDa protein minor band . Enzyme-linked immunoassay of the transgenic leaf extracts with anti-RTA and anti-RTB indicated microgram per gram production on a fresh weight basis of soluble extractable recombinant ricin . Sugar binding enzyme-linked immunoassay employing an immobilized glycoprotein, asialofetuin, and anti-RTB antibodies confirmed the characteristic type II ribosome-inactivating protein galactose binding lectin activity of the recombinant ricin . The enzymatic activity of recombinant ricin was characterized for cell-free translation inhibition, as well as for overall cytotoxicity . A 50% inhibitory dose of 3 x 10(-11) M was observed for the immunoreactive leaf extract material using a rabbit reticulocyte translation inhibition assay, while a 50% lethal dose of 1 x 10(-12) M was calculated with human T-lymphotropic virus-1 infected leukemic T-cells.

J Bacteriol, 1994 Sep, 176(18), 5697 - 703
Inhibition of Agrobacterium tumefaciens oncogenicity by the osa gene of pSa; Chen CY et al.; The IncW plasmid pSa originally derived from Shigella flexneri completely inhibits the tumor-inducing ability of Agrobacterium tumefaciens when it is resident in this organism . Oncogenic inhibition is mediated through the expression of the osa gene on pSa . This gene is part of a 3.1-kb DNA segment of pSa that contains four open reading frames revealed by sequencing . Specific deletions and TnCAT insertions within this segment localized the oncogenic inhibitory activity to the last open reading frame, orf-4, designated osa (for oncogenic suppression activity) . No promoter exists immediately upstream of the coding sequence of osa since TnCAT insertions or deletions into orf-3 caused the loss of oncogenic inhibition . Deletion analysis showed that the promoter of orf-1 is required for osa transcription . The first three orfs have no role in oncogenic inhibition, since osa alone placed under the control of a constitutive Pkm promoter completely inhibited A . tumefaciens oncogenicity . This inhibition of oncogenicity by osa is not limited to a specific host plant but appears to show broad host specificity . Because the osa-encoded product has close homologies to the fiwA-encoded product of the IncP plasmid RP1, osa may be involved in fertility inhibition that would prevent or reduce the formation of stable mating pairs and T-DNA transfer between A . tumefaciens and plants.

J Bacteriol, 1994 Sep, 176(17), 5409 - 13
Rhizobia catabolize nod gene-inducing flavonoids via C-ring fission mechanisms; Rao JR et al.; Gas chromatographic and mass spectrometric analyses of derivatized culture medium extracts were used to identify the products of flavonoid metabolism by rhizobia . A number of Rhizobium species and biovars degraded their nod gene-inducing flavonoids by mechanisms which originated in a cleavage of the C-ring of the molecule and which yielded conserved A- and B-ring products among the metabolites . In contrast, Pseudomonas putida degraded quercetin via an initial fission in its A-ring, and Agrobacterium tumefaciens displayed a nonspecific mode of flavonoid degradation which yielded no conserved A- or B-ring products . When incubated with rhizobia, flavonoids with OH substitutions at the 5 and 7 positions yielded phloroglucinol as the conserved A-ring product, and those with a single OH substitution at the 7 position yielded resorcinol . A wider range of structures was found among the B-ring derivatives, including p-coumaric, p-hydroxybenzoic, protocatechuic, phenylacetic, and caffeic acids . The isoflavonoids genistein and daidzein were also degraded via C-ring fission by Rhizobium fredii and Rhizobium sp . strain NGR234, respectively . Partially characterized aromatic metabolites with potential nod gene-inducing activity were detected among the products of naringenin degradation by Rhizobium leguminosarum bv . viciae . The initial structural modification of nod gene-inducing flavonoids by rhizobia can generate chalcones, whose open C-ring system may have implications for the binding of inducers to the nodD gene product.

J Bacteriol, 1994 Sep, 176(17), 5255 - 61
The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein; Jones AL et al.; The process of T-DNA transfer from Agrobacterium tumefaciens to plant cells is thought to involve passage of a DNA-protein complex through a specialized structure in the bacterial membrane . The virB operon of A . tumefaciens encodes 11 proteins, of which 9 are known to be located in the membranes and 10 have been shown to be essential for virulence . Sequence comparisons between proteins encoded by the virB operon and those encoded by operons from conjugative plasmids indicated that VirB proteins may form a structure similar to a conjugative pilus . Here, we examine the effects of mutations in virB4 on the accumulation and localization of other VirB proteins . VirB4 shares amino acid sequence similarity with the TraC protein of plasmid F, which is essential for pilus formation in Escherichia coli, and with the PtlC protein of Bordetella pertussis, which is required for toxin secretion . Polar and nonpolar virB4 mutants were examined, and all were shown to be unable to accumulate VirB3 protein to wild-type levels . A low level of VirB3 protein which was present in induced NT1RE cells harboring virB4 nonpolar mutant pBM1130 was found to associate with the inner membrane fraction only, whereas in wild-type cells VirB3 associated with both inner and outer membranes . The results indicate that for VirB3 to accumulate in the outer membrane, VirB4 must also be present, and it is possible that one role of VirB4 is in the correct assembly of a VirB protein membrane structure.

Mol Biol (Mosk), 1994 Sep-Oct, 28(5), 1166 - 75
{Expression of a partially modified delta-endotoxin gene from Bacillus thuringiensis var . tenebrionis in transgenic potato plants}; Gulina IV et al.; A modified gene (Bt77) of delta-endotoxin from Bacillus thuringiensis var . tenebrionis was constructed and cloned into pMON505 . This binary transformation vector was introduced into Agrobacterium tumefaciens strains containing different helper disarmed Ti-plasmids, LBA4404, A281, and CBE21 . These Agrobacterium strains were used to transform potato stem segments (S . tuberosum, cv Desiree, Resy, Temp, Granat) . Regenerants were selected on kanamycin-containing media . The presence of the Bt77 sequence in plant genomic DNA was confirmed by PCR analysis . Bt gene expression was studied in regenerated plants . Western blot analysis revealed that transgenic plants produced the Bt protein in the range of 0.005-0.02% of total protein . Total protection against insect damage of leaf tissue from these plants was observed in laboratory bioassays with of Colorado beetle larvae . Transgenic plants showed incomplete protection from CB larvae.

Plant Physiol, 1994 Sep, 106(1), 17 - 23
Herbicide-resistant tobacco plants expressing the fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase; Shiota N et al.; Transgenic tobacco (Nicotiana tabacum cv Xanthi) plants expressing a genetically engineered fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase were produced . The expression plasmid pGFC2 for the fused enzyme was constructed by insertion of the corresponding cDNA into the expression vector pNG01 under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase gene terminator . The fused enzyme cDNA was integrated into tobacco genomes by Agrobacterium infection techniques . In transgenic tobacco plants, the fused enzyme protein was localized primarily in the microsomal fraction . The microsomal monooxygenase activities were approximately 10 times higher toward both 7-ethoxycoumarin and benzo{a}pyrene than in the control plant . The transgenic plants also showed resistance to the herbicide chlortoluron.

Transgenic Res, 1994 Sep, 3(5), 317 - 25
T-DNA-insert-independent mutations induced in transformed plant cells during Agrobacterium co-cultivation; Marton L et al.; Transformation frequencies were determined for 1n, 2n, and 4n Nicotiana plumbaginifolia protoplast cultures in Agrobacterium-mediated gene transfer experiments . An unexpected large drop (50%) in plating efficiencies was observed in the non-selected (control) 1n populations after transformation treatment with virulent strains . This effect was not observed in the 2n or 4n cultures or in the 1n cultures when treated with avirulent bacteria . The mortality was disproportionally high and could not be explained by the low (0.1-0.5%) transformation efficiency in the 1n population, indicating mutagenesis of the cell populations independently from the T-DNA insertions . Mutagenesis was also indicated in gene tagging experiments where nitrate reductase-deficient (NR-) mutants were selected from haploid Nicotiana plumbaginifolia protoplasts, as well as from leaf disc cultures or protoplasts of diploid plants that were heterozygotic for a mutation either in the NR apoenzyme gene (nia/wt) or one of the molybdenum-containing cofactor genes (cnxA/wt), after Agrobacterium co-cultivation . The chlorate-resistant isolates were tested for the T-DNA-specific kanamycin resistance trait only after NR-deficiency had been established . Thirty-nine independent NR-deficient mutants were analysed further by Southern blot hybridization . There was no indication of integrated T-DNA sequences in the mutated NR genes, despite the fact that NR-deficient cells were found more frequently in cell populations which became transformed during the treatment than in the populations which did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant Mol Biol, 1994 Sep, 25(6), 995 - 1009
Production of Agrobacterium-mediated transgenic fertile plants by direct somatic embryogenesis from immature zygotic embryos of Datura innoxia; Ducrocq C et al.; This work describes a new method to obtain transgenic somatic embryos from Agrobacterium-infected immature zygotic embryos of Datura innoxia . It has several advantages over previous transformation methods such as the absence of a callus phase, an average transformation rate of 76% and a high regeneration frequency . Critical steps for optimal transformation were the embryo stage and a short preculture treatment . The marker gene beta-glucuronidase and light microscopy were used to identify the competent embryogenic cells which, after transformation, passed through the classical stages of embryo development . The transgenes were transmitted to the progeny in a Mendelian fashion . The plants regenerated via direct somatic embryogenesis were cytologically and morphologically uniform . We also observed that: (1) wounding or wound-induced divisions were not required for zygotic embryo transformation; (2) epidermal cells were competent for both transformation and regeneration; and (3) competency for Agrobacterium infection was developmental stage-specific . This new method should facilitate the development of new strategies to routinely transform recalcitrant plant species.

Plant Mol Biol, 1994 Sep, 25(6), 989 - 94
The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation; Hajdukiewicz P et al.; The new pPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced . The vectors utilize the pTiT37 T-DNA border regions, the pBR322 bom site for mobilization from Escherichia coli to Agrobacterium, and the ColE1 and pVS1 plasmid origins for replication in E . coli and in Agrobacterium, respectively . Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use in Agrobacterium strains with different drug resistance markers . Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region . A lacZ alpha-peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB) . Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.

Plant Mol Biol, 1994 Sep, 25(6), 977 - 87
Expression of a bacterial gene in transgenic plants confers resistance to the herbicide phenmedipham; Streber WR et al.; Tobacco plants were genetically engineered to express a detoxifying pathway for the herbicide phenmedipham . A gene from Arthrobacter oxidans strain P52 that encodes an enzyme catalysing the hydrolytic cleavage of the carbamate compound phenmedipham has recently been cloned and sequenced . The coding sequence was fused with a cauliflower mosaic virus 35S promoter and introduced into tobacco plants by Agrobacterium-mediated gene transfer . Transgenic plants expressing high levels of phenmedipham hydrolase exhibited resistance when sprayed with the herbicide at up to ten times the usual field application rate.

Plasmid, 1994 Sep, 32(2), 212 - 8
Localization and topology of VirB proteins of Agrobacterium tumefaciens; Beijersbergen A et al.; Agrobacterium tumefaciens causes tumors on plants by transferring part of its Ti (tumor inducing) plasmid, the T-DNA or transferred DNA, to plant cells . The virB operon, the largest operon of the virulence (Vir) region located on the Ti plasmid, is necessary for tumor induction on all plant species . Previously, the complete nucleotide sequences of the virB operons of several Ti plasmids of A . tumefaciens were determined . The 11 predicted proteins mostly have signal sequences and/or hydrophobic domains . Hence, these proteins are thought to be located in or transported over the agrobacterial inner membrane . The VirB proteins are suggested to form a pore structure in the membrane through which the T-DNA-protein complex is transported . To obtain direct evidence for transport of these proteins over the inner membrane, we made fusions between genes of the virB operon and the gene for the enzyme alkaline phosphatase . Here we show the localization of several VirB proteins in the bacterial membrane and predict the topology of the membrane-localized VirB proteins . The finding of a fusion between the VirB7 protein and the enzyme alkaline phosphatase provides the first evidence for the expression of the small virB7 gene . The VirB2 protein shows similarity with the TraA propilin of the Escherichia coli F plasmid . Here we show that the predicted topology of the VirB2 protein in the inner membrane is identical to that of the TraA protein . Therefore, we hypothesize that the VirB2 protein is part of an agrobacterial pilus-like structure.

Mol Microbiol, 1994 Sep, 13(5), 775 - 86
Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coli; Eichler K et al.; The sequence encompassing the cai genes of Escherichia coli, which encode the carnitine pathway, has been determined . Apart from the already identified caiB gene coding for the carnitine dehydratase, five additional open reading frames were identified . They belong to the caiTABCDE operon, which was shown to be located at the first minute on the chromosome and transcribed during anaerobic growth in the presence of carnitine . The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a functional H-NS protein, in relation to the consensus sequences detected in the promoter region of the cai operon . In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicted molecular masses of 56,613 Da (CaiT), 42,564 Da (CaiA), 59,311 Da (CaiC), 32,329 Da (CaiD) and 21,930 Da (CaiE) . Amino acid sequence similarity or enzymatic analysis supported the function assigned to each protein . CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/carnitine CoA ligase . CaiD bears strong homology with enoyl hydratases/isomerases . Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the basal level of carnitine dehydratase activity . It is inferred that CaiE is an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities . Taken together, these data suggest that the carnitine pathway in E . coli resembles that found in a strain situated between Agrobacterium and Rhizobium.

Mol Gen Genet, 1994 Aug 15, 244(4), 367 - 73
The NocR repressor-activator protein regulates expression of the nocB and nocR genes of Agrobacterium tumefaciens; Marincs F et al.; The NocR protein of Agrobacterium tumefaciens was found to regulate expression of the divergently transcribed nocB and nocR genes of the pTiT37 nopaline catabolism (noc) region . Experiments using the firefly luciferase (luc) gene as reporter demonstrated that NocR represses and activates transcription from the nocB promoter in the absence and presence of nopaline, respectively . NocR also negatively autoregulates its own synthesis irrespective of the presence of nopaline . Regulation of expression of both nocB and nocR is mediated by binding of the NocR protein to the nocR promoter . A 12 bp symmetrical sequence, which lies 3 bp downstream of the -10 hexamer of the nocR promoter, was confirmed to be essential for binding of the NocR protein . Functional localization of the nocB and nocR promoters verified that they do not overlap at all, and that the interrupted dyad, at which NocR binds, is 137 bp upstream of the regulated nocB promoter . The in vivo and in vitro results described here and those published previously suggest that a novel type of regulatory mechanism, which may involve changes in DNA topology, controls gene expression in the noc operon of pTiT37.

Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7603 - 7
Constitutive expression of the virulence genes improves the efficiency of plant transformation by Agrobacterium; Hansen G et al.; Inducible virulence (vir) genes of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid are under control of a two-component regulatory system . In response to environmental factors (phenolic compounds, sugars, pH) VirA protein phosphorylates VirG, which in turn interacts with the promoters of other vir genes, causing induction . A mutation of virG, virGN54D (which codes for a Asn-54-->Asp amino acid change in the product), causes constitutive expression of other vir genes independent of virA . We have investigated whether providing Agrobacterium with a plasmid containing virGN54D augments the efficiency of transfer of the T-DNA (transferred DNA) . For both tobacco and cotton, we observed an enhancement of transformation efficiency when the inciting Agrobacterium strain carries the virGN54D mutation . We also tested whether supplying Agrobacterium with a similar plasmid containing wild-type virG affects the efficiency of T-DNA transfer . An intermediate efficiency was observed when this plasmid was employed . Using a beta-glucuronidase (GUS) reporter gene to assess transient expression of T-DNA after transfer to tobacco and maize tissues, we observed a higher frequency of GUS-expressing foci after inoculation with Agrobacterium strains carrying virGN54D than with Agrobacterium carrying the wild-type virG . Gene-transfer efficiency to maize by an octopine strain was greatly improved upon introduction of virGN54D . Multiple copies of wild-type virG were equally effective in promoting transient expression efficiency in tobacco but were virtually ineffective in maize . We propose the use of virGN54D to improve the efficiency of Agrobacterium-mediated transformation, especially for recalcitrant plant species.

Plant Mol Biol, 1994 Aug, 25(5), 899 - 907
Localization of the VirA domain involved in acetosyringone-mediated vir gene induction in Agrobacterium tumefaciens; Turk SC et al.; The VirA protein of Agrobacterium tumefaciens is thought to be a receptor for plant phenolic compounds such as acetosyringone . Although it is not known whether the interaction between VirA and the phenolics is direct or requires other phenolic-binding proteins, it is shown in this study that the first 280 amino acids of the VirA protein are not essential for the acetosyringone mediated vir gene induction response . Considering the fact that the cytoplasmic region between the amino acids 283 and 304 is highly conserved between the different VirA proteins, and that deletion of this region abolishes VirA activity, we suggest that the acetosyringone receptor domain is located in this cytoplasmic domain of the VirA protein.

Plant Mol Biol, 1994 Aug, 25(5), 887 - 97
The tomato gene for the chloroplastic Cu,Zn superoxide dismutase: regulation of expression imposed in transgenic tobacco plants by a short promoter; Kardish N et al.; The chloroplastic Cu,Zn superoxide dismutase (SOD) has an important role in the defense against damage by oxygen radicals in the chloroplasts . Here, for the first time, we report on the isolation of a genomic DNA clone from tomato that contains all the coding sequence for the chloroplastic Cu,Zn SOD as well as a 442 bp DNA fragment upstream of the translational initiation site . The latter upstream sequence has a putative TATA box and a 285 bp promoter region, 5' of the apparent transcriptional initiation and a 157 bp leader region . The coding sequence is composed of 8 exons that are interspaced by 7 introns; we termed this gene SODCp;Le:1 . The 442 bp fragment was cloned into a pBI101 vector, upstream of the uidA (GUS) gene, via transcriptional fusion . Agrobacterium-mediated transformation resulted in transgenic tobacco plants . The progeny (after self-pollination) of 14 transformed plants, which expressed GUS above a threshold of 1 nmol/min per mg protein, were found to fall into two distinct groups . In the seedlings of 10 lines (group A) GUS expression was enhanced by exposure to light . In 4 lines of this group maintenance for 3 days in the dark eliminated GUS activity . The seedlings of group B expressed GUS regardless of the light/dark regime . In plants of group A, GUS expression was also developmentally regulated: high GUS activity in young leaves, low activity in mature leaves and no activity in the roots . The results suggest that this short chloroplastic Cu,Zn SOD promoter contains motifs for developmental (spatial) regulation as well as motifs responsive to light (or to oxygen radicals resulting from light-driven photosynthesis).

J Bacteriol, 1994 Aug, 176(15), 4511 - 7
Octopine and nopaline oxidases from Ti plasmids of Agrobacterium tumefaciens: molecular analysis, relationship, and functional characterization; Zanker H et al.; The occ and noc regions of pTiAch5 (octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of octopine and nopaline in Agrobacterium spp . The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function . The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells . The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors . However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear . Membrane solubilization led to complete loss of enzyme activity . The nopaline oxidase accepted nopaline and octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM) . The octopine oxidase had high activity with octopine (Km = 1 mM) and barely detectable activity with nopaline . The subunits from the occ and the noc regions were exchangeable . The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity . These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity.

J Lipid Res, 1994 Aug, 35(8), 1452 - 61
Occurrence of sulfoquinovosyl diacylglycerol in some members of the family Rhizobiaceae; Cedergren RA et al.; A radiolabeled component of a membrane extract of Rhizobium meliloti 2011 cells grown in the presence of 35S-labeled sulfate was isolated by silica flash chromatography and purified by high performance liquid chromatography (HPLC) . Based on 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric analyses, the structure of the compound was determined to be sulfoquinovosyl diacylglycerol (SQDG) . NMR analyses indicated substantial heterogeneity in the fatty acid composition and that an important group was the cyclopropyl fatty acids . This first report of the occurrence of SQDG outside of the plant kingdom, photosynthetic bacteria or diatoms deserves special attention as, in this case, the bacterium is one that can fix nitrogen in symbiosis with plants . The origins of the bacterium's ability to synthesize this class of membrane lipids is an important question . Membrane extracts of other strains of the family Rhizobiaceae were screened for the presence of SQDG . The occurrence of SQDG in the symbiotic organisms was confirmed, while no SQDG was detected in either the Agrobacterium tumefaciens or the Escherichia coli strains tested . The current function of these lipids in symbiosis and the commonality of the ability of bacteria that function as plant symbionts to synthesize such molecules are all germane to studies of the Rhizobium/legume symbiosis.

Plant Physiol, 1994 Aug, 105(4), 1209 - 15
Alterations of auxin perception in rolB-transformed tobacco protoplasts . Time course of rolB mRNA expression and increase in auxin sensitivity reveal multiple control by auxin; Maurel C et al.; Expression and physiological effects of the root-inducing rolB gene of Agrobacterium rhizogenes T-DNA were studied simultaneously in tobacco (Nicotiana tabacum) mesophyll protoplasts . The kinetic study of the expression of rolB mRNA following exogenous auxin application showed that auxin transiently stimulated rolB expression, with mRNA levels starting to accumulate 6 to 9 h after auxin was supplied and increasing 300-fold after 12 to 18 h . The parallel study of the auxin sensitivity of rolB-transformed protoplasts, as assayed by their electrical response to the hormone, showed that the auxin treatment generated an increase in sensitivity by a factor of up to 100,000, whereas in untransformed protoplasts the same auxin treatment induced an increase in auxin sensitivity that never exceeded 30- to 50-fold . This reflects a strong cooperative effect of auxin and rolB in transformed protoplasts . Surprisingly, the maximal increase in sensitivity was observed several hours before the maximal accumulation of rolB mRNA, suggesting that the dramatic control of auxin sensitivity by auxin in rolB-transformed protoplasts requires only low levels of rolB expression . Antibodies directed against ZmER-abp1, the major auxin-binding protein from maize, differentially altered the auxin sensitivity of the electrical response of rolB-transformed and normal protoplasts . This suggests that alterations of the auxin reception-transduction pathway at the plasma membrane of rolB-transformed protoplasts may account for their increased auxin sensitivity.

Plant J, 1994 Aug, 6(2), 271 - 82
Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA; Hiei Y et al.; A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens . The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium . Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations . Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons . Calli induced from scutella were very good starting materials . A strain of A . tumefaciens that carried a so-called 'super-binary' vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture.

Biosci Biotechnol Biochem, 1994 Aug, 58(8), 1458 - 62
Sensitivity of translation by Brevibacterium lactofermentum ribosomes to type 1 and type 2 ribosome-inactivating proteins; Ferreras JM et al.; An active cell-free translation system was prepared from Brevibacterium lactofermentum, a Gram-positive bacteria used in molecular cloning and protein expression . The system contained high speed postribosomal supernatant (S 370), purified ribosomes and a tRNA mixture from Escherichia coli, and synthesized polyuridylic acid-directed polyphenylalanine once optimized for mono and divalent ions, time, and temperature . The translation system was evaluated for sensitivity to several translational inhibitors including several N-glycosidase ribosome-inactivating proteins (RIPs) isolated from plants . The pattern of inhibition by RIPs resembled that observed recently for Gram-negative bacteria such as Escherichia coli and Agrobacterium tumefaciens {Girbes et al., J . Bacteriol., 175, 6721-6724 (1993)} . A typical inhibitory type 1 RIP such as crotin 2 promoted depurination of the rRNA, which upon treatment with acid aniline released a fragment of approximately 230 nucleotides . On these grounds, we propose that bacterial ribosome sensitivity to plant RIPs depends on the bacterial ribosome-specific presence of protein recognition domains in the RIP present only in some RIP but not in others.

Nucleic Acids Res, 1994 Jul 25, 22(14), 2744 - 51
A plant scaffold attached region detected close to a T-DNA integration site is active in mammalian cells; Dietz A et al.; Integration of foreign genes into plant genomes by the Agrobacterium T-DNA transfer system has been considered to occur at random . It has been speculated that the chromosomal structure of the integration site might affect the expression pattern of the introduced genes . To gain insight into the molecular structure of T-DNA integration sites and its possible impact on gene expression, we have examined plant DNA sequences in the vicinity of T-DNA borders . Analysis of a transgenic petunia plant containing a chloramphenicol acetyltransferase (CAT) gene regulated by the hemoglobin promoter (PAR) from Parasponia andersonii revealed a scaffold attachment region (SAR) close to one T-DNA end . In addition to having strong binding affinities for both animal and plant nuclear scaffolds this petunia SAR element is as active in mammalian cells as the authentic elements from mammalian sources.

Mol Gen Genet, 1994 Jul 8, 244(1), 15 - 22
The rolC promoter of Agrobacterium rhizogenes Ri plasmid is activated by sucrose in transgenic tobacco plants; Yokoyama R et al.; The 5'-upstream region of the rolC gene of the Ri plasmid is expressed specifically in phloem cells of transgenic higher plants . In this study, we demonstrated that the rolC promoter is activated by sucrose in phloem cells of transgenic tobacco seedlings bearing rolC promoter-uidA chimeric fusion gene . Since the rolC promoter is not activated by sorbitol, sucrose metabolism rather than osmotic pressure exerted by the disaccharide may be responsible for induction . Thus, experiments using 5'-upstream deletion mutants, internal deletion mutants, and chimeric constructs with a heterologous promoter (-90 region of the cauliflower mosaic virus 35S promoter) were conducted to define the region of the rolC promoter involved in sucrose activation . The results indicated that a cis-acting sucrose responsive region of the rolC promoter is located between -135 and -94 bp with respect to the transcription initiation site . In phloem cells, high concentrations of sucrose are encountered owing to ongoing translocation of photosynthates from source to sink tissues . Therefore, sucrose as a signal molecule may regulate the phloem-specific expression of the rolC promoter.

Plant Mol Biol, 1994 Jul, 25(4), 721 - 33
Non-recombinant background in gene targeting: illegitimate recombination between a hpt gene and a defective 5' deleted nptII gene can restore a Kmr phenotype in tobacco; de Groot MJ et al.; Previously we have demonstrated gene targeting in plants after Agrobacterium-mediated transformation . In these initial experiments a transgenic tobacco line 104 containing a T-DNA insertion with a defective neomycin phosphotransferase (nptII) gene was transformed with a repair construct containing an otherwise defective nptII gene . Homologous recombination between the chromosomally located target and the incoming complementary defective nptII construct generated an intact nptII gene and led to a kanamycin-resistant (Kmr) phenotype . The gene targeting frequency was 1 x 10(-5) . In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting we transformed the same transgenic tobacco line 104 via electroporation . A total of 1.35 x 10(8) protoplasts were transformed with the repair construct . Out of nearly 221,000 transformed cells 477 Kmr calli were selected . Screening the Kmr calli via PCR for recombination events revealed that in none of these calli gene targeting had occurred . To establish the origin of the high number of Kmr calli in which gene targeting had not occurred we analysed plants regenerated from 24 Kmr calli via PCR and sequence analysis . This revealed that in 21 out of 24 plants analysed the 5'-deleted nptII gene was fused to the hygromycin phosphotransferase (hpt) gene that was also present on the repair construct . Sequence analysis of 7 hpt/nptII gene fusions showed that they all contained a continuous open reading frame . The absence of significant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination . Therefore, illegitimate recombination between an introduced defective gene and another gene present on the repair construct or the chromosome has to be taken into account as a standard byproduct in gene targeting experiments.

Plant Physiol, 1994 Jul, 105(3), 989 - 98
Cell-autonomous cytokinin-independent growth of tobacco cells transformed by Agrobacterium tumefaciens strains lacking the cytokinin biosynthesis gene; Black RC et al.; Mutations at the cytokinin biosynthesis locus (tmr) of Agrobacterium tumefaciens usually result in strains that induce tumors exhibiting the rooty phenotype associated with high auxin-to-cytokinin ratios . However, tobacco (Nicotiana tabacum cv Havana 425) leaf disc explants responded to tmr- mutant strain A356 by producing rapidly growing, unorganized tumors, indicating that these lines can grow in a cytokinin-independent fashion despite the absence of a functional tmr gene . Several methods have been used to characterize the physiological and cellular basis of this phenotype . The results indicate that tmr- tumors have a physiologically distinct mechanism for cytokinin-independent growth in comparison to tumors induced by wild-type bacteria . The cytokinin-independent phenotype of the tmr- transformants appears to be cell autonomous in nature: only the transformed cells and their progeny were capable of cytokinin-independent growth . Specifically, the tmr- tumors did not accumulate cytokinin, and clonal analysis indicated the tmr- transformed cells were not capable of stimulating the growth of neighboring nontransformed cells . Finally, the cytokinin-independent phenotype of the tmr- transformants was shown to be cold sensitive, whereas the wild-type tumors exhibited a cold-resistant cytokinin-independent phenotype . Potential mechanisms for this novel form of cytokinin-independent growth, including the role of the dehydrodiconiferyl alcohol glucosides found in both tumor types, are discussed.

J Biol Chem, 1994 Jul 1, 269(26), 17635 - 41
Novel gene expression system for plant cells based on induction of alpha-amylase promoter by carbohydrate starvation; Chan MT et al.; The 5' regulatory region and putative signal sequence of a rice alpha-amylase gene, alpha Amy8, was fused to a bacterial gene encoding beta-glucuronidase (GUS) and introduced into rice, tobacco, and potato via Agrobacterium-mediated transformation systems . Expression of this chimeric gene in suspension cells of transgenic plants was suppressed by the presence of sucrose in the medium and induced by its absence . Induction or suppression of GUS expression in transgenic rice could be reversed by the deprivation or replenishment, respectively, of sucrose in the medium . The expressed GUS fusion protein was translocated to the endoplasmic reticulum, modified by glycosylation, and secreted into the culture medium of transgenic cells . In the presence of a glycosylation inhibitor, tunicamycin, the enzymatically active form of GUS was assembled in the endoplasmic reticulum . The yield of GUS secreted by transgenic cells was estimated to be as high as 40% of total secreted proteins . The reversible induction of the alpha-amylase promoter in culture cells by sugar level in the medium provides an excellent inducible expression system for basic research in plant science . Combination of the alpha-amylase promoter and signal sequence also offers a novel approach for large scale production of low cost, easily purified, secreted recombinant proteins.

J Bacteriol, 1994 Jul, 176(13), 3966 - 74
Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density; Pierson LS 3rd et al.; We have identified a gene that acts in trans to activate the expression of the phenazine biosynthetic genes in the biological control organism Pseudomonas aureofaciens 30-84 . This gene, phzR (phenazine regulator), is located upstream of and divergently transcribed from the phenazine biosynthetic genes . Thus, the phenazine biosynthetic locus consists of at least two divergently transcribed operons . A functional phzR gene is required for phenazine production . The nucleotide sequence of phzR revealed an open reading frame of 723 nucleotides encoding a protein of ca . 27 kDa . The predicted amino acid sequence of PhzR has homology with other bacterial positive transcriptional activators, including LasR of Pseudomonas aeruginosa, LuxR of Vibrio fischerii, and TraR of Agrobacterium tumefaciens . The addition of cell-free supernatants from late-exponential-phase cultures of strain 30-84 resulted in expression of a genomic phzB:lacZ reporter strain at a lower cell density than normal, indicating the possible presence of an autoinducer . These results indicate that PhzR is a member of a two-component sensor-regulator family with known or predicted carboxy-terminal DNA-binding domains which regulates gene expression in response to environmental and cell density signals.

Plasmid, 1994 Jul, 32(1), 75 - 9
The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids; Mercado-Blanco J et al.; The large plasmid, pRmeGR4a, of Rhizobium meliloti GR4 is a nonsymbiotic, self-transmissible replicon that shows a high degree of stability . Plasmid replication and stabilization functions are located on a 4.8-kb PstI fragment . Analysis of the nucleotide sequence of this DNA region revealed the presence of six open reading frames (ORFs) with coding capabilities; all are transcribed in the same direction . Only one of the ORFs (ORF3) seems to be essential for replication . Its predicted protein sequence shows homology with RepC, a protein that has been suggested to be involved in the replication of Agrobacterium Ri and Ti plasmids.

Mol Biol (Mosk), 1994 Jul-Aug, 28(4), 752 - 60
{The effect of the 5'-leader of potato X-virus on expression of the gene for the potato Y virus membrane protein in transgenic Solanum tuberosum}; Pugin MM et al.; The 5'-leader of potato virus X (PVX) genomic RNA was successfully used to generate potato plants expressing the coat protein of the potato virus YN (Russian isolate) (PVY CP) . Two expression cassettes were constructed that carried the PVY CP coding sequence along with the PVY 3'untranslated region under the control of the CaMV 35S promoter and poly(A) signal . The cassettes contained different sequences between the 35S promoter and the CP artificial AUG initiating codon of the coat protein: either a polylinker or a shortened variant of the PVX leader . Transgenic Belorusskii-3 potato plants generated by agrobacterial transformation were shown to express the PVY CP gene to different extents depending on the cassette integrated into the plant genome . The cassette containing the PVX leader provided detectable amounts of the coat protein in all tested plants, whereas plants carrying the cassette with the polylinker leader produced no coat protein above the Western detection threshold . Northern analysis revealed that the presence of the PVX leader in front of the PVY CP gene initiating codon resulted in a 10-fold increase in the PVY CP mRNA steady-state levels, thus suggesting an influence of the PVX leader on mRNA stability.

Protein Eng, 1994 Jul, 7(7), 905 - 9
The N-terminal domain of VirG of Agrobacterium tumefaciens: modelling and analysis of mutant phenotypes; Rodenburg KW et al.; Fourteen mutants in the N-terminal domain of virulence factor G (VirG) were obtained by random mutagenesis . Two mutants showed an altered phenotype, all others were non-functional . All mutants can still be phosphorylated and bind to DNA . A 3-D model was built based on the coordinates of chemotaxis protein Y (CheY) . Many of the observed phenotypic changes of VirG are explained qualitatively . Combination of model building and biochemical information leads to the conclusion that the active sites of VirG and CheY must partly use different residues to perform the same phosphorylation and dephosphorylation reactions.

Int J Syst Bacteriol, 1994 Jul, 44(3), 511 - 22
Rhizobium ciceri sp . nov., consisting of strains that nodulate chickpeas (Cicer arietinum L.); Nour SM et al.; The taxonomic status of 16 collection strains of chickpea (Cicer arietinum L.) rhizobia which were previously determined to belong to two groups (groups A and B) were compared with reference strains belonging to different genera and species of the family Rhizobiaceae . We used the following taxonomic, phylogenetic, and phenotypic characteristics and approaches to study these organisms: DNA homology, guanine-plus-cytosine content, restriction fragment length polymorphism of the amplified 16S-intergenic spacer rRNA gene, partial 16S rRNA sequencing, and auxanographic tests performed with 147 carbon sources . Similar groups of chickpea strains were identified by the different approaches . The chickpea strains were found to belong to the genus Rhizobium regardless of the phylogenetic group to which they belonged (group A or B) . All strains fell into a tight cluster which included Rhizobium loti and Rhizobium galegae, and the group B strains were closely related to R . loti . An analysis of partial 16S ribosomal DNA sequences revealed identical nucleotide sequences for the slowly growing strains and fast-growing strains that were used as representatives of groups A and B, respectively, and these organisms fell into the Rhizobium-Agrobacterium lineage . When the sequences of these organisms were compared with the partial sequences of Rhizobium huakuii and R . loti, one- and two-nucleotide mismatches were observed, respectively, indicating that the chickpea rhizobia are closely related to these two species . The DNA-DNA hybridization data revealed that the chickpea rhizobia exhibited low levels of homology (less than 17%) to previously described Rhizobium and Bradyrhizobium species . Moreover, when we compared chickpea strains to R . loti and R . huakuii, the most closely related species as determined by the partial 16S rRNA sequence analysis, the homology values ranged from 21 to 52% and the delta Tm values were greater than 5 degrees C (delta Tm is the difference between the denaturation temperatures of the heterologous and homologous duplexes) . These results confirmed that the rhizobia that nodulate chickpeas cannot be assigned to a previously described species . Within the chickpea rhizobia, the DNA homology values obtained when members of groups A and B were compared were less than 38%, indicating that the group A and group B organisms belong to different species . Furthermore, these organisms can be distinguished from each other by the results of phenotypic tests . We propose that the group B chickpea rhizobia should be assigned to a new species, Rhizobium ciceri; UPM-Ca7 is the type strain of R . ciceri.

Mol Gen Genet, 1994 Jun 15, 243(6), 706 - 10
Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumours of N . glauca x N . langsdorffii; Aoki S et al.; A region homologous to the TL-DNA of Agrobacterium rhizogenes was previously detected in the genome of untransformed Nicotiana glauca and designated cellular T-DNA (cT-DNA) . Subsequently, part of this region was sequenced and two genes, which corresponded to rolB and rolC and were named NgrolB and NgrolC, were found . We have now sequenced a region of the cT-DNA other than the region that includes NgrolB and C and we have found two other open reading frames (ORFs), NgORF13 and NgORF14 . These ORFs correspond to ORFs 13 and 14 of the TL-DNA of A . rhizogenes and exhibit a high degree of homology to these ORFs, without having a nonsense codon . We have not found any sequence homologous to rolD (ORF15) . The two genes, NgORF13 and 14, as well as the NgrolB and C genes, are expressed in genetic tumors of hybrids between N . glauca and N . langsdorffii but not in leaf tissues of the hybrid.

Mol Gen Genet, 1994 Jun 15, 243(6), 666 - 73
Transgenic tomato lines containing Ds elements at defined genomic positions as tools for targeted transposon tagging; Knapp S et al.; We have introduced a genetically marked Dissociation transposable element (DsHPT) into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation . Probes for the flanking regions of the T-DNA and transposed DsHPT elements were obtained with the inverse polymerase chain reaction (IPCR) technique and used in RFLP linkage analyses . The RFLP map location of 11 T-DNAs carrying DsHPT was determined . The T-DNAs are distributed on 7 of the 12 tomato chromosomes . To explore the feasibility of gene tagging strategies in tomato using DsHPT, we examined the genomic distribution of DsHPT receptor sites relative to the location of two different, but very closely linked, T-DNA insertion sites . After crosses with plants expressing Ac transposase, the hygromycin phosphotransferase (HPT) marker on the Ds element and the excision markers beta-glucuronidase (GUS) and Basta resistance (BAR) facilitated the identification of plants bearing germinally transposed DsHPT elements . RFLP mapping of 21 transposed DsHPT elements originating from the two different T-DNA insertions revealed distinct patterns of reintegration sites.

Proc Natl Acad Sci U S A, 1994 Jun 7, 91(12), 5538 - 42
Identification of plant genetic loci involved in a posttranscriptional mechanism for meiotically reversible transgene silencing; Dehio C et al.; Numerous reports describe phenomena of transgene silencing in plants, yet the underlying genetic and molecular mechanisms are poorly understood . We observed that regeneration of Arabidopsis thaliana plants transgenic for the rolB gene of Agrobacterium rhizogenes results in a selection for transgene silencing . Transgene silencing could be monitored in this system by reversion of the visible RolB phenotype . We report a phenotypic, molecular, and genetic characterization of a meiotically reversible transgene silencing phenomenon observed in a rolB transgenic line . In this line, the rolB gene is expressed strongly and uniformly in seedlings, but in the course of further development, the rolB gene is silenced erratically at a frequency that depends on the dosage of rolB . The silenced state is mitotically stable, while complete resetting of rolB gene expression occurs in seedlings of the following generation . The silencing of rolB correlates with a dramatic reduction of steady-state rolB transcripts, while rolB nuclear run-off transcripts are only moderately reduced . Therefore, rolB gene silencing seems to act predominantly at the posttranscriptional level . The process of rolB gene silencing was found to be affected by two extragenic modifier loci that influence both the frequency and the timing of rolB gene silencing during plant development . These genetic data demonstrate a direct involvement of defined plant genes in this form of gene silencing.

J Bacteriol, 1994 Jun, 176(12), 3816 - 9
Rhicadhesin-mediated attachment and virulence of an Agrobacterium tumefaciens chvB mutant can be restored by growth in a highly osmotic medium; Swart S et al.; Cyclic beta-1,2-glucan is considered to play a role in osmoadaptation of members of the family Rhizobiaceae in hypotonic media . Agrobacterium tumefaciens chvB mutants, lacking beta-1,2-glucan, exhibit a pleiotropic phenotype, including nonmotility, attachment deficiency, and avirulence . Here we report that by growth of chvB mutant cells in tryptone-yeast extract medium supplemented with 7 mM CaCl2 and 100 mM NaCl, the mutant cells become motile, attach to pea root hair tips, and are virulent on Kalanchoe leaves . Moreover, whereas chvB mutants grown in tryptone-yeast extract medium containing 7 mM CaCl2 do not produce active rhicadhesin, addition of 100 mM NaCl to this medium resulted in restoration of rhicadhesin activity . The presence of CaCl2 appeared to be required for attachment, virulence, and activity of rhicadhesin . The results support a role for cyclic beta-1,2-glucan in osmoadaptation and strengthen the notion that rhicadhesin is required for attachment and virulence of A . tumefaciens.

J Bacteriol, 1994 Jun, 176(12), 3646 - 60
Genetic complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essential virulence genes; Berger BR et al.; The Agrobacterium tumefaciens virB gene products are proposed to assemble into a transport system capable of exporting complexes of DNA and protein across the bacterial envelope en route to plant cells . Nonpolar null mutations were constructed in each of the 11 virB genes of the A . tumefaciens pTiA6NC plasmid . In tumorigenicity assays, delta virB1 mutants exhibited severely attenuated virulence and delta virB2 through delta virB11 mutants exhibited avirulence . NdeI restriction sites introduced at the predicted translational start sites of the virB genes were used to subclone each of the virB genes downstream of the lacZ or virB promoter on broad-host-range plasmids . virB gene expression plasmids were used to define promoter and general sequence requirements for genetic complementation of the deletion mutations . Whereas virB1 and virB2 complemented delta virB1 and delta virB2, respectively, only when expressed in trans from the virB promoter, virB3 through virB11 complemented the corresponding deletion mutations when expressed in trans from either the lacZ or virB promoter . Several virB genes required additional upstream or downstream sequences for complementation: (i) virB2 complemented the delta virB2 mutation only when the complementing plasmid coexpressed virB1 and virB2, (ii) virB6 and virB9 complemented the delta virB6 and delta virB9 mutations only when the complementing plasmids carried at most 55 and 230 bp of sequences residing 5' of these genes, respectively, and (iii) virB7 and virB8 complemented the delta virB7 and delta virB8 mutations only when the complementing plasmid coexpressed virB7 and virB8 . These studies established that virB1 is an accessory virulence determinant and virB2 through virB11 are absolutely essential for the A . tumefaciens infection process.

J Bacteriol, 1994 Jun, 176(12), 3576 - 83
Functional role of the Ti plasmid-encoded catabolic mannopine cyclase in mannityl opine catabolism by Agrobacterium spp; Hong SB et al.; Catabolic mannopine (MOP) cyclase encoded by Ti or Ri plasmids lactonizes MOP to agropine (AGR) . The gene of the octopine-type Ti plasmid pTi15955 encoding the catabolic MOP cyclase enzyme previously was localized to a 1.6-kb segment within a cosmid clone, pYDH208 . A subclone containing only this region complemented the AGR catabolism-negative phenotype conferred by a derivative of the octopine-type plasmid pTiB6S3 containing a Tn7 insertion in the region encoding the MOP cyclase enzyme . Uptake assays of strains harboring pRiA4 or pArA4a, along with complementation analyses, indicate that MOP cyclase is not sufficient for catabolism of AGR but that the strains must also express an AGR transport system . To determine the requirement for MOP cyclase in opine catabolism unequivocally, a site-specific, nonpolar deletion mutation abolishing only MOP cyclase activity was introduced into pYDH208, a cosmid clone that confers utilization of MOP, AGR, and mannopinic acid (MOA) . Strains harboring this MOP cyclase-negative mutant clone, pYDPH208, did not utilize AGR but continued to utilize MOP . Growth on AGR was restored in this strain upon introduction of clones encoding the pTi15955-derived catabolic or anabolic MOP cyclase genes . The induction pattern of MOA catabolism shown by strain NT1 harboring the MOP cyclase-deficient pYDPH208 suggests that AGR is converted into MOP by MOP cyclase and that MOP, but not AGR, induces catabolism of MOA . Genetic and biochemical analyses of MOP and AGR metabolism suggest that only the conversion of AGR to MOP is directly involved in catabolism of AGR, even though the reaction catalyzed by MOP cyclase predominantly lies in the lactonization of MOP to AGR.

J Bacteriol, 1994 Jun, 176(11), 3242 - 9
Glu-255 outside the predicted ChvE binding site in VirA is crucial for sugar enhancement of acetosyringone perception by Agrobacterium tumefaciens; Banta LM et al.; Transcriptional activation of the Agrobacterium tumefaciens vir regulon is regulated by phenolics such as acetosyringone (AS), certain monosaccharides, and acidic conditions produced by wounded plant cells . The transmembrane protein VirA acts as an environmental sensor, mediating signal transduction upon perception of these stimuli . Although the periplasmic domain of VirA is not absolutely required for AS-dependent vir gene induction, it is needed for interactions with the periplasmic sugar-binding protein ChvE that result in sugar-induced enhancement of phenolic sensitivity . In this report, we demonstrate that mutations within the periplasmic domain but outside the predicted ChvE binding region can drastically alter the sensitivity of VirA to As . Using site-directed mutagenesis, we have characterized the roles of three individual amino acids in sugar-dependent AS sensitivity and have correlated the induction phenotype with the tumorigenic capacity of strains expressing mutant versions of VirA . Substitution of leucine for Glu-255 abolishes sugar enhancement while replacement with aspartic acid results in a wild-type phenotype . This residue lies outside the predicted ChvE binding site and thus identifies a new region of the VirA periplasmic domain crucial for the enhancement of vir gene induction by carbohydrates . In the absence of inducing sugar, wild-type VirA protein appears to be subject to some form of inhibition that suppresses the maximal level of transcriptional activation; deletions within the periplasmic region relieve this suppression.

Clin Infect Dis, 1994 Jun, 18(6), 914 - 20
Infections with the unusual human pathogens Agrobacterium species and Ochrobactrum anthropi; Alnor D et al.; Agrobacterium species and Ochrobactrum anthropi are generally considered innocuous in clinical settings, yet during the last decade a number of sporadic cases of human infection due to these organisms have been reported . We studied nine cases of infection (septicemia and peritonitis) caused by Agrobacterium-like microorganisms in eight patients . All patients were immunocompromised and had permanent central venous or peritoneal dialysis catheters in place . Seven patients were women, and eight infections were community acquired . Six isolates were identified as Agrobacterium species and three as O . anthropi . These two groups of strains differed in the production of beta-galactosidase and of acid from lactose, erythritol, salicin, and cellobiose . All strains were strictly aerobic, peritrichous, gram-negative bacilli that produced oxidase, urease, and acid from glucose, fructose, arabinose, xylose, mannitol, inositol, and ethanol . The in vitro adherence of isotope-labeled bacteria to silicone tubes was similar to that of staphylococci . We conclude that Agrobacterium species and O . anthropi can be pathogenic in immunocompromised patients with permanent catheters.

Microbiol Rev, 1994 Jun, 58(2), 145 - 61
Cyclic beta-glucans of members of the family Rhizobiaceae; Breedveld MW et al.; Cyclic beta-glucans are low-molecular-weight cell surface carbohydrates that are found almost exclusively in bacteria of the Rhizobiaceae family . These glucans are major cellular constituents, and under certain culture conditions their levels may reach up to 20% of the total cellular dry weight . In Agrobacterium and Rhizobium species, these molecules contain between 17 and 40 glucose residues linked solely by beta-(1,2) glycosidic bonds . In Bradyrhizobium species, the cyclic beta-glucans are smaller (10 to 13 glucose residues) and contain glucose linked by both beta-(1,6) and beta-(1,3) glycosidic bonds . In some rhizobial strains, the cyclic beta-glucans are unsubstituted, whereas in other rhizobia these molecules may become highly substituted with moieties such as sn-1-phosphoglycerol . To date, two genetic loci specifically associated with cyclic beta-glucan biosynthesis have been identified in Rhizobium (ndvA and ndvB) and Agrobacterium (chvA and chvB) species . Mutants with mutations at these loci have been shown to be impaired in their ability to grow in hypoosmotic media, have numerous alterations in their cell surface properties, and are also impaired in their ability to infect plants . The present review will examine the structure and occurrence of the cyclic beta-glucans in a variety of species of the Rhizobiaceae . The possible functions of these unique molecules in the free-living bacteria as well as during plant infection will be discussed.

Plant Physiol, 1994 Jun, 105(2), 483 - 90
Species-dependent expression of the hyoscyamine 6 beta-hydroxylase gene in the pericycle; Kanegae T et al.; The tropane alkaloid scopolamine is synthesized in the pericycle of branch roots in certain species of the Solanaceae . The enzyme responsible for the synthesis of scopolamine from hyoscyamine is hyoscyamine 6 beta-hydroxylase (H6H) . The gene for H6H was isolated from Hyoscyamus niger . It has an exon/intron organization very similar to those for ethylene-forming enzymes, suggesting a common evolutionary origin . The 827-bp 5' flanking region of the H6H gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to three solanaceous species by Agrobacterium-mediated transformation systems: H . niger and belladonna (Atropa belladonna), which have high and low levels, respectively, of H6H mRNA in the root, and tobacco (Nicotiana tabacum), which has no endogenous H6H gene . Histochemical analysis showed that GUS expression occurred in the pericycle and at the root meristem of transgenic H . niger hairy roots, but only at the root meristem of transgenic H . niger hairy roots, but only at the root meristem of hairy roots and plants of transgenic tobacco . In transgenic hairy roots and regenerated plants of belladonna, the root meristem was stained with GUS activity, except for a few transformants in which the vascular cylinder was also stained . These studies indicate that the cell-specific expression of the H6H gene is controlled by some genetic regulation specific to scopolamine-producing plants.

Plant J, 1994 Jun, 5(6), 895 - 903
Identification of molecular markers of embryogenesis in Arabidopsis thaliana by promoter trapping; Topping JF et al.; The technique of promoter trapping has been exploited to identify markers of embryogenesis in Arabidopsis thaliana . A population of transgenic A . thaliana was generated containing the promoter trap vector pdeltagusBin19, following Agrobacterium tumefaciens-mediated transformation . This vector contains at the T-DNA left border, a promoterless gusA gene, which is activated following integration downstream of a native gene promoter that directs transgene transcription . Screening of a population of 430 independent transgenic lines revealed that 74 lines (17.2%) exhibited GUS activity in siliques, as determined by fluorimetric assay . Histochemical GUS analysis was used to identify lines that expressed GUS in embryos, and three lines that were demonstrated to contain single T-DNA inserts were analysed in detail . Each line showed a distinct pattern of GUS fusion activity . Fusion transcripts were identified, demonstrating that transcription was initiated in the genomic DNA flanking the T-DNA left border . Inverse PCR was used to clone the T-DNA flanking sequences, and for one line a corresponding cDNA was identified, demonstrating that the tagged sequences are transcribed . The markers represent the earliest embryonic genes known to be expressed in plants.

Mol Microbiol, 1994 Jun, 12(5), 811 - 7
Mutational analysis of Agrobacterium tumefaciens pTiA6 virD1: identification of functionally important residues; Vogel AM et al.; Mutagenesis experiments were used to identify functionally important regions of Agrobacterium tumefaciens pTiA6 VirD1 . Random mutations were introduced by using Taq polymerase in a mutagenic reaction buffer containing manganese and altered nucleotide ratios to increase errors during the polymerase chain reaction (PCR) . The mutants were assayed for VirD1-, VirD2-dependent border-nicking activity in Escherichia coli harbouring a border-containing substrate plasmid . Analysis of the mutants led to the identification of a region from amino acids 45-60 that is important for VirD1 activity . This region corresponds to a previously postulated potential DNA-binding domain . Deletion mutagenesis indicated that amino acids 2-16 could be deleted without affecting VirD1 function, whereas a larger deletion, amino acids 5-27, completely inactivated VirD1.

Appl Environ Microbiol, 1994 Jun, 60(6), 2137 - 46
In vivo nuclear magnetic resonance study of the osmoregulation of phosphocholine-substituted beta-1,3;1,6 cyclic glucan and its associated carbon metabolism in Bradyrhizobium japonicum USDA 110; Pfeffer PE et al.; A phosphocholine-substituted beta-1,3;1,6 cyclic glucan (PCCG), an unusual cyclic oligosaccharide, has been isolated from Bradyrhizobium japonicum USDA 110 (D . B . Rolin, P . E . Pfeffer, S . F . Osman, B . S . Swergold, F . Kappler, and A . J . Benesi, Biochim . Biophys . Acta 1116:215-225, 1992) . Data presented here suggest that PCCG synthesis is dependent on the carbon metabolism and that osmotic regulation of its biosynthesis parallels regulation of membrane-derived oligosaccharide biosynthesis observed in Escherichia coli (E . P . Kennedy, M . K . Rumley, H . Schulman, and L . M . G . van Golde, J . Biol . Chem . 251:4208-4213, 1976) and Agrobacterium tumefaciens (G . A . Cangelosi, G . Martinetti, and E . W . Nester, J . Bacteriol . 172:2172-2174, 1990) . Growth of B . japonicum USDA 110 cells in the reference medium at relatively low osmotic pressures (LO) (65 mosmol/kg of H2O) caused a large accumulation of PCCG and unsubstituted beta-1,3;1,6 cyclic glucans (CG) . Sucrose and polyethylene glycol, nonionic osmotica, reduce all growth rates and inhibit almost completely the production of PCCG at high osmotic pressures (HO) above 650 and 400 mosmol/kg of H2O), respectively . We used in vivo 13C nuclear magnetic resonance spectroscopy to identify the active osmolytes implicated in the osmoregulation process . The level of alpha,alpha-trehalose in B . japonicum cells grown in autoclaved or filter-sterilized solutions remained constant in HO (0.3 M sucrose or 250 g of polyethylene glycol 6000 per liter) medium . Significant amounts of glycogen and extracellular polysaccharides were produced only when glucose was present in the autoclaved HO 0.3 M sucrose media . The results of hypo- and hyperosmotic shocking of B . japonicum USDA 110 cells were monitored by using in vivo 31P and 13C nuclear magnetic resonance spectroscopy . The first observed osmoregulatory response of glycogen-containing cells undergoing hypoosmotic shock was release of P(i) into the medium . Within 7 h, reabsorption of P(i) was complete and production of PCCG was initiated . After 12 h, the PCCG content had increased by a factor of 7 . Following the same treatment, cells containing little or no glycogen released trehalose and failed to produce PCCG . Thus the production of PCCG/CG in response to hypoosmotic shocking of stationary-phase cells was found to be directly linked to the interconversion of stored glycogen . Hyperosmotic shocking of LO-grown stationary-phase cells with sucrose had no effect on the content of previously synthesized CG/PCCG . The PCCG/CG content and its osmotically induced biosynthesis are discussed in terms of carbon metabolism and a possible role in hypoosmotic adaptation in B . japonicum USDA 110.

Res Microbiol, 1994 Jun-Aug, 145(5-6), 461 - 73
Host recognition by the VirA, VirG two-component regulatory proteins of agrobacterium tumefaciens; Winans SC et al.; Agrobacterium tumefaciens contains about 25 vir genes localized on a 200-kb tumour-inducing (Ti) plasmid that direct a conjugation-like transfer of tumorigenic DNA from the bacterium to the nuclei of infected plant cells . These genes are strongly and coordinately induced during infection in response to three different classes of stimuli which are thought to be key chemical features of a typical wound site . These stimuli are (i) guaiacol and syringol derivatives such as acetosyringone, (ii) sugars such as glucose and glucuronic acid, and (iii) acidic pH . The sensing of these compounds is carried out by the VirA, VirG and ChvE proteins . VirA is a four-domain histidine protein kinase, while VirG is a transcriptional activator which is activated by VirA-mediated phosphorylation . ChvE is a chromosomally encoded periplasmic sugar binding protein which is required for sensing sugars but dispensable for sensing the other two stimuli . Here we will review the nature of these chemical stimuli, the structure and function of the three regulatory proteins, their similarity to sensors found in human and animal pathogens, the factors influencing their pool size, and their role in the host range of different strains of A . tumefaciens.

Biosci Biotechnol Biochem, 1994 Jun, 58(6), 1045 - 9
Purification and characterization of arginine amidinohydrolase from Bacillus brevis TT02-8; Shimotohno KW et al.; A bacterial arginase was purified to homogeneity from a strain of Bacillus brevis . The native enzyme, with an estimated MW of 143,000, migrated on SDS-PAGE as a single polypeptide of estimated MW of 33,000 . The enzyme, highly specific to L-arginine, showed the maximum activity at pH 11.0 in the presence of Mn2+ ions and the pI was 4.8 by isoelectric focusing . The enzyme activity was increased significantly by the addition of Mn2+, Ni2+, or Co2+ ions, and inhibited potently by chemicals such as HgCl2, N-bromosuccinimide, or glutathione . The Kms for L-arginine and L-canavanine were 0.69 and 22.2 mM, respectively . The enzyme was inhibited competitively by gamma-guanidinobutyric acid, and non-competitively by L-lysine, L-ornithine, creatine, blasticidin S, and edeine B1 . Analysis of the N-terminal amino acid sequence of the purified bacterial enzyme found 33-36% homologies with the Agrobacterium, yeast, rat, and human enzymes.

Proc Natl Acad Sci U S A, 1994 May 24, 91(11), 4639 - 43
TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N-acylhomoserine lactone autoinducer; Hwang I et al.; Conjugal transfer of the nopaline-type Agrobacterium Ti plasmid pTiC58 is regulated by a transcriptional activator, TraR, and a diffusible signal molecule, conjugation factor (CF) . CF is a member of a family of substituted homoserine lactones (HSLs) that act as coinducers for regulating gene expression in diverse Gram-negative bacteria by a mechanism called autoinduction . In Vibrio fischeri HSL production is conferred by the luxI gene . Homologues of this gene are responsible for HSL production by other Gram-negative bacteria . A gene that we call traI, conferring production of material with CF activity, was localized to a 1-kb region at the upstream end of tra3 of pTiC58 . Spectroscopy showed that the activity was authentic CF . Sequence analysis showed that traI could encode a protein of 211 amino acids, TraI, that is related to the proteins responsible for HSL production by other bacteria . A second, partial open reading frame immediately downstream of traI could encode a protein related to TrbB of plasmid RP4, which is required for conjugal transfer . Transcription of traI and of the downstream tra3 genes requires TraR and CF and initiates from the traI promoter . The results show that traI is responsible for CF production, that it is the first gene of the tra3 operon, and that expression of this operon is regulated by autoinduction.

Virology, 1994 May 15, 201(1), 36 - 45
Agrobacterium-mediated inoculation of PSTVd cDNAs onto tomato reveals the biological effect of apparently lethal mutations; Hammond RW; Potato spindle tuber viroid (PSTVd) mutants which contain alterations in the terminal loops of the rod-like native structure have previously been reported from our laboratory . PSTVd-P contains mutations at positions 2, 4, and 6 in the left terminal loop; PSTVd-R+, a sequence permutation of PSTVd-R, contains the same mutations at positions 177 and 178 in the right terminal loop as PSTVd-R and contains in addition a 1-nucleotide G insertion at position 176 . PSTVd-P, PSTVd-R, and PSTV-R+ were noninfectious when either cDNA or SP6-generated RNA transcripts were used as inoculum onto tomato cotyledons . In the current study, mutant and wild-type PSTVd constructs were mobilized into Agrobacterium tumefaciens and used for stem inoculation of tomato plants . Agrobacterium-mediated inoculation of the mutant and wild-type constructs has confirmed the inability of the PSTVd-P mutant to establish an infection . The PSTV-R+ mutant and/or sequence variants derived in vivo can establish an infection, although PSTVd-R+ progeny and replicative intermediates appear to be primarily restricted to the gall and root tissues of the plant and only occasionally are progeny detectable in the newly developing leaves . The reduced level of viroid accumulation from the PSTVd-R+ mutant appears to be consistent with the mutant viroid replicating/accumulating only in a limited number of cells or cell types . The mutations in the right terminal loop may alter interactions with specific host components and thereby disrupt the normal pattern of intercellular transport of the viroid or limit its replication to a cell type but not abolish replication per se.

J Bacteriol, 1994 May, 176(10), 2796 - 806
A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite; Fuqua WC et al.; Conjugal transfer of Agrobacterium octopine-type Ti plasmids is activated by octopine, a metabolite released from plant tumors . Octopine causes conjugal donors to secrete a pheromone, Agrobacterium autoinducer (AAI), and exogenous AAI further stimulates conjugation . The putative AAI synthase and an AAI-responsive transcriptional regulator were found to be encoded by the Ti plasmid traI and traR genes, respectively, and the expression of traR was induced by octopine . The octopine-type traR gene product is highly homologous to the TraR protein recently characterized from a nopaline-type Ti plasmid . TraR and TraI are homologous to the LuxR and LuxI regulatory proteins of Vibrio fischeri, and AAI is similar in structure to the diffusable V . fischeri autoinducer, the inducing ligand of LuxR . TraR activated target genes in the presence of AAI and also activated traR and traI themselves, creating two positive-feedback loops . TraR-AAI-mediated activation in wild-type Agrobacterium strains was dramatically enhanced by culturing on solid media, suggesting a possible role in cell density sensing.

Plant Physiol, 1994 May, 105(1), 259 - 68
Analysis of the involvement of ocs-like bZip-binding elements in the differential strength of the bidirectional mas1'2' promoter; Feltkamp D et al.; The ocs-like elements of the bidirectional mas1'2' promoter of Agrobacterium tumefaciens, mas1' and mas2', were analyzed to elucidate their role in the expression conferred by this promoter . Tetramers of the elements were cloned upstream of the beta-glucuronidase-coding region linked to the 35S promoter deleted at -54 . Transient expression assays with tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) protoplasts showed that tetramers of the mas1' element had 3- to 8-fold enhancing activity, respectively . Enhancement obtained by tetramers of the mas2' element was higher, suggesting that this element plays a role in the stronger promoter activity from the 2' side . Three cDNA clones with higher homology to the tobacco transcription factor TGA1a were isolated from a potato root expression library . Overexpression of the proteins encoded by these cDNA clones in Escherichia coli and analysis of DNA-binding activity in bacterial extracts showed that all three factors could bind strongly to the mas1' ocs-like element . In contrast, only two of the mas-binding factors exhibited significant binding to the mas2' element . Southern analysis revealed the presence of a small, multigene family encoding the mas-binding factors in potato.

Plant Mol Biol, 1994 May, 25(2), 193 - 205
Regulation of an Arabidopsis oleosin gene promoter in transgenic Brassica napus; Plant AL et al.; Progressive deletions of the 5'-flanking sequences of an Arabidopsis oleosin gene were fused to beta-glucuronidase (GUS) and introduced into Brassica napus plants using Agrobacterium-mediated transformation . The effect of these deletions on the quantitative level of gene expression, organ specificity and developmental regulation was assessed . In addition, the influence of abscisic acid (ABA), jasmonic acid (JA), sorbitol and a combined ABA/sorbitol treatment on gene expression was investigated . Sequences that positively regulate quantitative levels of gene expression are present between -1100 to -600 and -400 to -200 of the promoter . In addition, sequences present between -600 and -400 down-regulate quantitative levels of expression . In transgenic B . napus plants, the oleosin promoter directs seed-specific expression of GUS which is present at early stages of seed development and increases throughout seed maturation . Sequences present between -2500 and -1100 of the promoter are involved in modulating the levels of expression at early stages of embryo development . Histochemical staining of embryos demonstrated that expression is uniform throughout the tissues of the embryo . Sequences involved in the response to ABA and sorbitol are present between -400 and -200 . The induction of GUS activity by a combined ABA/sorbitol treatment is additive suggesting that ABA is not the sole mediator of osmotically induced oleosin gene expression . A response to JA was only observed when the oleosin promoter was truncated to -600 suggesting that the reported effect of JA on oleosin gene expression may be at a post-transcriptional level.

Biotechnology (N Y), 1994 May, 12(5), 500 - 4
Transgenic tomato plants expressing the tomato yellow leaf curl virus capsid protein are resistant to the virus; Kunik T et al.; The tomato yellow leaf curl virus (TYLCV) gene that encodes the capsid protein (V1) was placed under transcriptional control of the cauliflower mosaic virus 35S promoter and cloned into an Agrobacterium Ti-derived plasmid and used to transform plants from an interspecific tomato hybrid, Lycopersicon esculentum X L . pennellii (F1), sensitive to the TYLCV disease . When transgenic F1 plants, expressing the V1 gene, were inoculated with TYLCV using whiteflies fed on TYLCV-infected plants, they responded either as untransformed tomato or showed expression of delayed disease symptoms and recovery from the disease with increasingly more resistance upon repeated inoculation . Transformed plants that were as sensitive to inoculation as untransformed controls expressed the V1 gene at the RNA level only . All the transformed plants that recovered from disease expressed the TYLCV capsid protein.

Gene, 1994 Apr 20, 141(2), 201 - 5
A 43-kDa nuclear tobacco protein interacts with a specific single-stranded DNA sequence from the 5'-upstream region of the Agrobacterium rhizogenes rolC gene; Matsuki R et al.; We identified in the nuclear extract of tobacco seedlings a 43-kDa DNA-binding protein RCS2 that interacted with the 5'-upstream region of the rolC gene of the Agrobacterium rhizogenes Ri plasmid . DNA-protein gel shift and competition assays demonstrated that RCS2 bound to single-stranded DNA in a sequence-specific manner . A five-base direct repeat is important for the DNA binding of RCS2 . South-Western blot analysis was employed to determine the size of RCS2 which appears to be approx . 43 kDa.

Proc Natl Acad Sci U S A, 1994 Apr 12, 91(8), 3210 - 4
Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco; Citovsky V et al.; Previously, we have shown that Agrobacterium-plant cell transferred DNA (T-DNA) transport into the host cell nucleus is likely mediated by two specific bacterial proteins, VirD2 and VirE2 . Here, we used these proteins to study molecular pathways of nuclear import . First, the role of VirE2 nuclear localization signals (NLSs) in the T-DNA transport pathway was examined by using tobacco plants transgenic for deletion mutants of VirE2 . In these plants, the virulence of wild-type Agrobacterium was reduced possibly by competition for the cellular nuclear import machinery . Second, we analyzed the nuclear localization of VirE2 and VirD2 in the nonhost monocot maize . Part of the known recalcitrance of monocots to transformation by Agrobacterium could be due to a potential selectivity in nuclear import pathways in monocotyledonous and dicotyledonous plants . Nuclear transport of VirD2 and VirE2 in maize leaves and roots was compared to that in tobacco protoplasts and roots . Both proteins accumulated in maize leaf and tobacco protoplast nuclei as well as in nuclei of immature root cells . In contrast, VirD2 and VirE2 expressed in mature roots of maize and tobacco remained cytoplasmic . Point mutations of VirE2 nuclear localization signals, NSE 1 and NSE 2, also revealed that, in maize, the NSE 1 signal was mainly responsible for nuclear import; in contrast, both signals functioned independently in tobacco protoplasts.

Proc Natl Acad Sci U S A, 1994 Apr 12, 91(8), 2994 - 8
Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells; Yusibov VM et al.; During the inception of crown gall tumorigenesis, the transferred DNA (T-DNA) is processed from the Ti (tumor inducing) plasmid of Agrobacterium tumefaciens and is transferred to plant cells . T-DNA processing and transfer require the induction of vir (virulence) genes by phenolic compounds secreted by wounded plant cells . After vir gene induction, both single-stranded (T-strands) and double-stranded forms of processed T-DNA accumulate in the bacteria . Although current models favor the transfer of T-strands to plants, there has yet been no experimental evidence to show this . In this paper, we show that T-strands disappear from acetosyringone-induced A . tumefaciens within 30 min of bacterial cocultivation with tobacco protoplasts . PCR analysis of T-DNA associated with protoplasts indicates that single-stranded, but not double-stranded, T-DNA can be detected in the plant cells within 30 min of bacterial cocultivation . Control experiments show that this T-DNA does not originate from lysed contaminating bacterial cells . T-DNA transfer depends on a functional bacterial virB operon . Protoplast infections using an A . tumefaciens virE mutant result in a low level of accumulation of T-strands in the plant cells.

Nucleic Acids Res, 1994 Apr 11, 22(7), 1167 - 71
Evidence for a group II intron in Escherichia coli inserted into a highly conserved reading frame associated with mobile DNA sequences; Knoop V et al.; The distribution of group II introns in the living world is an important aspect of the hypothesis which postulates their evolutionary relation to the nuclear spliceosome . As an alternative to the restricted experimental approaches towards their identification we devised a strategy to recognize group II introns in sequence data . By this approach we identified a locus on a plasmid in the bacterium Escherichia coli . Modelling of the derived RNA secondary structure reveals the presence of perfectly conserved domains V and VI as typical features of group II introns . An intron internal reading frame upstream of domain V is homologous to group II intron encoded maturases . A reading frame downstream of the predicted 3'-splice site is highly similar to a small polypeptide encoded in the central part of the Agrobacterium tumefaciens T-DNA . With the TBLASTN algorithm a set of plasmid-borne insertion sequences in Agrobacteria and Rhizobia and surprisingly also in a Yersinia pseudotuberculosis strain was identified which contain this highly conserved reading frame.

Gene, 1994 Apr 8, 141(1), 91 - 6
Nucleotide and deduced amino acid sequences of Rhizobium meliloti 102F34 lacZ gene: comparison with prokaryotic beta-galactosidases and human beta-glucuronidase; Fanning S et al.; The nucleotide (nt) sequence of a 2.57-kb Sau3A fragment carrying the Rhizobium meliloti beta-galactosidase (beta Gal)-encoding gene (RmlacZ) was determined . An open reading frame (ORF) of 2.26 kb was identified which encoded a 755-amino-acid (aa) polypeptide with a calculated molecular mass of 84,141 Da, in fair agreement with the value of 88 kDa determined by SDS-PAGE . The deduced N-terminal aa sequence was confirmed by direct sequencing of electrophoretically purified R . meliloti beta Gal . The size of the native R . meliloti beta Gal was approx . 174 kDa . Similarities were found between the aa sequence of the R . meliloti beta Gal and those from Clostridium thermosulfurogenes EM1 and Agrobacterium radiobacter, as well as human beta-glucuronidase (beta Glu) . Comparisons with beta Gal from Escherichia coli, Klebsiella pneumoniae, Lactobacillus bulgaricus and Kluyveromyces lactis found only weak similarities; however, the putative active site residues appear to be conserved . The RmlacZ sequence is flanked by two partially sequenced ORFs, which show aa sequence and organisational similarities to the previously reported lac operon in A . radiobacter.

FEBS Lett, 1994 Apr 4, 342(2), 145 - 8
Changing root system architecture through inhibition of putrescine and feruloyl putrescine accumulation; Ben-Hayyim G et al.; Plant roots provide anchorage and absorb the water and minerals necessary for photosynthesis in the aerial parts of the plant . Since plants are sessile organisms, their root systems must forage for resources in heterogeneous soils through differential branching and elongation {(1988) Funct . Ecol . 2, 345-351; (1991) Plant Roots: The Hidden Half, pp . 3-25, Marcel Dekker, NY} . Adaptation to drought, for instance, can be facilitated by increased root growth and penetration . Root systems thus develop as a function of environmental variables and the needs of the plant {(1988) Funct . Ecol . 2, 345-351; (1986) Bot . Gaz . 147, 137-147; (1991) Plant Roots: The Hidden Half, pp . 309-330, Marcel Dekker, NY} . We show, in a model system consisting of excised tobacco roots, that both alpha-DL-difluoromethylornithine (an inhibitor of putrescine biosynthesis) and the rolA gene (from the root-inducing transferred DNA of Agrobacterium rhizogenes) stimulate overall root growth and cause a conversion in the pattern of root system formation, producing a dominant or 'tap' root . These morphological changes are correlated with a depression in the accumulation of polyamines and their conjugates.

Mol Gen Genet, 1994 Apr, 243(1), 32 - 8
Efficiency of the tetracycline-dependent gene expression system: complete suppression and efficient induction of the rolB phenotype in transgenic plants; Roder FT et al.; We have investigated the use of the tetracycline-dependent gene expression system to regenerate and propagate tobacco plants transformed with a gene whose product--when highly expressed--interferes with regeneration and/or further reproduction . Plants transformed with the Agrobacterium rhizogenes rolB gene under the control of the tetracycline-dependent expression system were phenotypically indistinguishable from wild type owing to efficient repression of the promoter . Induction of the rolB gene with tetracycline led to high-level expression of the rolB mRNA, which resulted in extremely stunted plants with necrotic and wrinkled leaves that did not develop a floral meristem . Upon cessation of tetracycline treatment healthy shoots developed even from severely affected meristems . Data on the dose response of the rolB phenotype as a function of tetracycline concentration demonstrate that the tetracycline-dependent gene expression system can be used to modulate the manifestation of a particular phenotype.

Mol Microbiol, 1994 Apr, 12(1), 23 - 30
A mutation in the receiver domain of the Agrobacterium tumefaciens transcriptional regulator VirG increases its affinity for operator DNA; Han DC et al.; We fused the wild-type Agrobacterium tumefaciens virG gene and the constitutive virGN54D allele to the malE gene of Escherichia coli, and studied the binding of MBP-VirG fusions to the autoregulated virG promoter . MBP-VirGN54D protein bound this promoter with 10-fold higher affinity than MBP-VirG, and bound to vir box I with eightfold higher affinity than to vir box III . Disruption of vir box III did not alter the affinity for vir box I, suggesting a lack of cooperativity between these sites . We provide evidence that protein bound at a single vir box may have a higher oligomeric state than non-bound protein, and that a DNA distortion adjacent to vir box I may occur during activation.

Biotechniques, 1994 Apr, 16(4), 664 - 8, 670
Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection; Chen H et al.; Freeze-thaw transformation provides a simple and rapid method to transform Agrobacterium tumefaciens directly with plasmid DNA . Competent A . tumefaciens cells of strains LBA4404, GV3850 and EHA101 were transformed with four to nine plasmids differing in size, size of insert and in some cases sensitivity to antibiotics . A threefold to fourfold increase in transformed colonies per microgram of DNA was obtained by freezing cells with liquid nitrogen vs . dry ice/ethanol . Freezing cells in liquid nitrogen followed by incubation of transformed cells in a low concentration of appropriate antibiotics prior to plating resulted in a ninefold increase in colonies obtained compared with the procedure of freezing cells in dry ice/ethanol without the incubation period in the low concentration of antibiotics prior to plating . Restriction fragments of the expected sizes from the plasmids indicated that the procedural modifications did not cause apparent recombinations in the region of the inserts.

Appl Environ Microbiol, 1994 Apr, 60(4), 1068 - 77
Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv . vesicatoria; Leite RP Jr et al.; Three pairs of oligonucleotide primers specific for different regions of the hrp gene (hypersensitive reaction and pathogenicity) cluster of Xanthomonas campestris pv . vesicatoria were designed and tested for amplification of DNA isolated from a large number of different bacteria . DNA sequences related to the hrp genes were successfully amplified from X . fragariae and from 28 pathovars of X . campestris . No DNA amplification occurred with genomic DNA from phytopathogenic strains of X . campestris pv . secalis, X . campestris pv . translucens, and X . albilineans or from nonpathogenic opportunistic xanthomonads and phytopathogenic strains of the genera Acidovorax, Agrobacterium, Clavibacter, Erwinia, Pseudomonas, and Xylella . The DNA from those bacteria also failed to hybridize to hrp-specific fragments in Southern blot analysis . DNA fragments amplified with a particular primer pair were of identical size from each of the different phytopathogenic xanthomonads . However, restriction analysis of these fragments by using frequently cutting endonucleases revealed variation in the pattern for these hrp-related fragments amplified from the different Xanthomonas strains . The restriction patterns generated for the different fragments allowed distinction of the strains representing a pathovar or species of phytopathogenic xanthomonads . We believe that DNA amplification with hrp-specific oligonucleotide primers is a highly sensitive and specific method that can be applied for detection and identification of phytopathogenic xanthomonads.

Plant Mol Biol, 1994 Apr, 25(1), 83 - 90
Oncogene arrangement in a shooty strain of Agrobacterium tumefaciens; Drevet C et al.; The Agrobacterium tumefaciens nopaline strain 82.139 induces non-teratogenic shooty tumours on several plant species . We have determined the position of the T-region oncogenes in a 11.4 kb Xba I fragment which shows a general organization similar to its pTiC58 counterpart . Sequence analysis of the 4.7 kb right part of this fragment allowed us to identify the pTi82.139 ipt, 6b and nos coding sequences . pTi82.139 lacks the 6a gene, which lies between the ipt and 6b genes in pTiC58 . The intervening region between the 6b and the nos genes contains an additional ORF with homology to ORF 21 (transcript 3') from the TR-DNA of octopine strain pTi15955.

Mol Microbiol, 1994 Apr, 12(1), 17 - 22
Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis; Kado CI; Conjugative transfer of DNA that occurs between bacteria also operates between bacteria and higher organisms . The transfer of DNA between Gram-negative bacteria requires initial contact by a sex pilus followed by DNA traversing four membranes (donor plus recipient) using a transmembrane pore . Accumulating evidence suggests that transfer of the T-DNA from Agrobacterium tumefaciens to plants may also occur via a conjugative mechanism . The virB operon of the Ti plasmid exhibits close homologies to genes that are known to encode the pilin subunits and pilin assembly proteins . The proteins encoded by the PilW operon of IncW plasmid R388 share strong similarities (average similarity = 50.8%) with VirB proteins . Similarly, the TraA, TraL and TraC proteins of IncF plasmid F have similarities to VirB2, VirB3 and VirB4 respectively (average similarity = 45.3%) . VirB2 protein (12.3 kDa) contains a signal peptidase-I cleavage sequence that generates a polypeptide of 7.2 kDa . Likewise, the 12.8 kDa propilin protein TraA of plasmid F also possesses a peptidase-I cleavage site that generates the 7.2 kDa pilin structural protein . Similar amino acid sequences of the conjugative transfer genes of F, R388 as well as plasmid RP4 and the genes of the ptI operon of Bortedella pertussis suggest the existence of a superfamily of transmembrane proteins adapted to the promiscuous transfer of DNA-protein complexes.

Proc Natl Acad Sci U S A, 1994 Mar 29, 91(7), 2507 - 11
ocs element promoter sequences are activated by auxin and salicylic acid in Arabidopsis; Zhang B et al.; ocs elements are a group of promoter elements that have been exploited by two distinct groups of plant pathogens, Agrobacterium and certain viruses, to express genes in plants . We examined the activity of single and multiple ocs elements linked to a minimal plant promoter and the uidA reporter gene in transgenic Arabidopsis . beta-Glucuronidase activity was detected only in root tips and in callus tissue after auxin treatment . A more sensitive assay revealed that auxin treatment also increased ocs element activity in aerial parts of the plant, although the absolute levels of ocs element activity were greater in roots . The response of ocs elements to exogenous auxin began within 1 h . Salicylic acid, a disease-resistance signal in plants, also increased ocs element activity in both roots and aerial parts of the plant . The question of whether the induction in ocs element activity is mediated through auxin and/or salicylic acid signal transduction pathways or is part of a more general stress response is discussed.

Transgenic Res, 1994 Mar, 3(2), 138 - 40
A plant transformation vector with a minimal T-DNA; During K; Plant transformation, via Agrobacterium tumefaciens, is usually performed with binary vectors . Most of the available binary vectors contain within the T-DNA (which is transferred to the plant genome) components not required for the intended modification . These additional sequences may cause potential risks during field testing of the transgenic plants or even more in the case of commercialization . The aim of this study was to produce a plant transformation vector which only contains a selectable and screenable marker gene and a multiple cloning site for insertion of promoter::foreign gene::terminator cassettes from other plasmids.

Mol Biol (Mosk), 1994 Mar-Apr, 28(2), 437 - 43
{Creation of transgenic plants Nicotiana tabacum and Solanum tuberosum, resistant to the herbicide phosphinothricin}; Padegimas L et al.; The expression cassette for phosphinothricin acetyltransferase gene (PAT or bar) from S . hygroscopicus has been constructed on the basis of the pBI121 plasmid . Leaf disks of N . tabacum cv . SR1 and steam segments of S . tuberosum cv . Prigozhii-2 have been transformed using Agrobacterium . Selection of the transformed plants carried out by PCR analysis revealed 12 transgenic tobacco plants and 3 transgenic potato plants . bar gene expression and plant resistance to phosphinothricin treatment have been studied in regenerated plants . All the transgenic plants were found to be resistant to the herbicide . Mendelian inheritance of the inserted gene has been proved by analysis of the F1 progeny of transformed tobacco plants.

Plant J, 1994 Mar, 5(3), 343 - 51
Hypocotyl expression and light downregulation of the soybean tubulin gene, tubB1; Tonoike H et al.; The tubB1 beta-tubulin gene of Glycine max (previously named s beta 1) is highly expressed only in rapidly elongating regions of etiolated seedling hypocotyls and this expression is strongly downregulated when the seedlings are exposed to light . Primer extension demonstrated that the gene was transcribed in these tissues and contained two sites of transcriptional initiation . To determine the mechanism regulating tubB1 expression, a chimeric reporter gene was constructed by fusing 5' upstream regions of tubB1 to a promoterless beta-glucuronidase (GUS) gene and these constructs were introduced into protoplasts by electroporation . Strong transient expression of the reporter gene was obtained after electroporation of chimeric constructs containing 1 kb of tubB1 5' upstream sequence into tobacco protoplasts . Deletion of the distal most 300 bp from the 5' sequence of tubB1 enhanced expression, suggesting the possibility of a negative transcriptional regulator in this region . Additional deletions of the 5' sequence reduced expression substantially . Constructs containing a tubB1 3' terminus were expressed at much lower levels than those containing a nopaline synthase (NOS) 3' terminus . The tubB1-GUS chimeric gene also was introduced into tobacco by Agrobacterium-mediated Ti plasmid transformation and the organ-specific expression pattern of the chimeric gene was determined in seedlings of the transgenic plants . Hypocotyls exhibited strong GUS activity when the seedlings were germinated in darkness, but lacked the GUS enzyme when the seedlings were germinated in the light.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant Physiol, 1994 Mar, 104(3), 1067 - 71
The herpes simplex virus thymidine kinase gene as a conditional negative-selection marker gene in Arabidopsis thaliana; Czako M et al.; The human herpes simplex virus thymidine kinase type 1 gene (HSVtk) acts as a conditional lethal marker in mammalian cells . The HSVtk-encoded enzyme is able to phosphorylate certain nucleoside analogs (e.g . ganciclovir, an antiherpetic drug), thus converting them to toxic DNA replication inhibitors . The utility of HSVtk as a conditional negative-selection marker was explored in Arabidopsis thaliana (L.) Heynh . HSVtk was introduced into Arabidopsis by Agrobacterium-mediated transformation . Transgenic plants were morphologically indistinguishable from wild type and exhibited normal fertility . Ganciclovir at 10(-5) to 10(-4) M drastically reduced shoot regeneration on transgenic, HSVtk+ root explants or callus formation on HSVtk+ leaf explants but did not affect the wild-type cultures . There was a 35-fold reduction in shoot regeneration 8 d after transfer to shoot-induction medium . Negative selection against HSVtk activity along with kanamycin selection was also efficient in Agrobacterium-mediated gene transfer experiments . Shoot regeneration was 25 times lower on double-selective (ganciclovir plus kanamycin) plates than in the kanamycin control . This regeneration rate in double-selective plates is in the range of the frequency of shoots normally escaping kanamycin selection in Arabidopsis cultures.

Appl Environ Microbiol, 1994 Mar, 60(3), 913 - 20
A novel gene tag for identifying microorganisms released into the environment; Hwang I et al.; A novel method using a moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955 was developed as a tag to identify genetically modified microorganisms released into the environment . Pseudomonas fluorescens 1855.344, a plant-growth-promoting rhizosphere bacterium, was chosen as the organism in which to develop and test the system . moc genes carried by pYDH208, a cosmid clone containing a 20-kb segment of the octopine-mannityl opine-type Ti plasmid, conferred on P . fluorescens strains the capacity to utilize mannopine and agropine (AGR) as a sole source of carbon and energy . Modified P . fluorescens strains containing moc or moc::nptII inserted into a chromosomal site were constructed by marker exchange . One such modified strain, PF5MT12, utilized AGR as a sole carbon source and contained detectable levels of mannopine cyclase, an easily assayable enzyme encoded by the moc region . Catabolism of AGR could be used to recover selectively the marked strain from mixed populations containing a large excess of closely related bacteria . Nucleic acid-based detection strategies were developed on the basis of the unique fusion region between Agrobacterium DNA and Pseudomonas DNA in strain PF5MT12 . The specificity and sensitivity of detection of PF5MT12 were enhanced by amplifying the fused DNA region by using PCR . The target fragment could be detected at levels of sensitivity comparable to those of other described PCR-based gene tags, even in the presence of high levels of Agrobacterium, Pseudomonas, or Escherichia coli DNA . This gene tag strategy gives a method for direct selection and enumeration of the marked strain from mixtures containing a large excess of closely related bacteria and a sensitive and highly specific system for detection by PCR amplification of the target fragment even in the presence of large amounts of DNA from related or unrelated organisms.

Mol Gen Genet, 1994 Mar, 242(6), 666 - 74
Alpha-domain of human metallothionein IA can bind to metals in transgenic tobacco plants; Pan A et al.; With a view to exploring its use as a metal-binding factor in transgenic plants we prepared the alpha-domain of metallothionein by reconstitution of rabbit apometallothionein and proteolysis of MT-1 and MT-2 with subtilisin . The isolated alpha-domains were characterised by UV and CD spectroscopy Double-Stranded . DNA encoding the alpha-domain (106 bp) of the human MT-IA was constructed from chemically synthesized oligomers by repair synthesis and enzymatic ligation, cloned into pUC19 and sequenced . A expression construct containing the cloned alpha-domain was introduced into tobacco cells on a disarmed Agrobacterium tumefaciens Ti-plasmid . Transformed tobacco cells were selected and regenerated on medium containing cadmium and kanamycin . The growth of roots and shoots of transformants was unaffected by up to 100 microM cadmium, whereas control plants showed severe inhibition of root and shoot growth, and chlorosis of leaves on medium containing only 10 microM cadmium . Southern hybridization confirmed the presence of the transgene in the transformed plant tissues . The concentration of human alpha-domain peptides in transgenic tobacco leaves was determined by the Cd/hemoglobin saturation assay and polarography using the rabbit alpha-domain as standard . The results indicate that the alpha-domain, one of two domains in MT molecules, is not only stable in vitro, but is also expressed efficiently and functions independently in transgenic plant cells.

J Bacteriol, 1994 Mar, 176(6), 1711 - 7
The essential virulence protein VirB8 localizes to the inner membrane of Agrobacterium tumefaciens; Thorstenson YR et al.; Agrobacterium tumefaciens genetically transforms plant cells by transferring a specific DNA fragment from the bacterium through several biological membranes to the plant nucleus where the DNA is integrated . This complex DNA transport process likely involves membrane-localized proteins in both the plant and the bacterium . The 11 hydrophobic or membrane-localized proteins of the virB operon are excellent candidates to have a role in DNA export from agrobacteria . Here, we show by TnphoA mutagenesis and immunogold electron microscopy that one of the VirB proteins, VirB8, is located at the inner membrane . The observation that a virB8::TnphoA fusion restores export of alkaline phosphatase to the periplasm suggests that VirB8 spans the inner membrane . Immunogold labeling of VirB8 was detected on the inner membrane of vir-induced A . tumefaciens by transmission electron microscopy . Compared with that of the controls, VirB8 labeling was significantly greater on the inner membrane than on the other cell compartments . These results confirm the inner membrane localization of VirB8 and strengthen the hypothesis that VirB proteins help form a transfer DNA export channel or gate.

Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1969 - 73
Relocation of the plastid rbcL gene to the nucleus yields functional ribulose-1,5-bisphosphate carboxylase in tobacco chloroplasts; Kanevski I et al.; The conserved plastid localization of rbcL suggests that biosynthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase {Rubisco; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39} in chloroplasts is required to obtain functional enzyme . To examine the validity of this hypothesis, we relocated the plastid rbcL gene to the nucleus . First, we deleted the rbcL gene from the tobacco plastid genome by targeted insertion of a selectable aadA gene encoding spectinomycin resistance . The rbcL coding region was then inserted into an expression cassette and introduced into the nuclear genome of these plants by Agrobacterium-mediated transformation . We report that the nuclear rbcL functionally complements the defective plastids when the Rubisco large subunit is targeted to chloroplasts by a transit peptide . Therefore, the evolutionary process that relocates functional plastid genes to the nucleus has not yet occurred in the case of the rbcL gene . Targeted deletion of plastid genes, combined with their allotopic expression, will provide opportunities for studying the function of plastid enzyme complexes.

Proc Natl Acad Sci U S A, 1994 Mar 1, 91(5), 1726 - 30
A visible marker for antisense mRNA expression in plants: inhibition of chlorophyll synthesis with a glutamate-1-semialdehyde aminotransferase antisense gene; Hofgen R et al.; Glutamate 1-semialdehyde aminotransferase {(S)-4-amino-5-oxopentanoate 4,5-aminomutase, EC 5.4.3.8} catalyzes the last step in the conversion of glutamate to delta-aminolevulinate of which eight molecules are needed to synthesize a chlorophyll molecule . Two full-length cDNA clones that probably represent the homeologous Gsa genes of the two tobacco (Nicotiana tabacum) genomes have been isolated . The deduced amino acid sequences of the 468-residue-long precursor polypeptides differ by 10 amino acids . The cDNA sequence of isoenzyme 2 was inserted in reverse orientation under the control of a cauliflower mosaic virus 35S promoter derivative in an expression vector and was introduced by Agrobacterium-mediated transformation into tobacco plants . Antisense gene expression decreased the steady-state mRNA level of glutamate 1-semialdehyde aminotransferase, the translation of the enzyme, and chlorophyll synthesis . Remarkably, partial or complete suppression of the aminotransferase mimics in tobacco a wide variety of chlorophyll variegation patterns caused by nuclear or organelle gene mutations in different higher plants . The antisense gene is inherited as a dominant marker.

Sci China B, 1994 Mar, 37(3), 286 - 92
Genetic transformation of Lycium barbarum L . via A . tumefaciens; Du LQ et al.; A system for transformation and regeneration of Lycium barbarum L., an important Chinese medical plant, has been established . Young stem segments from Lycium barbarum L . were infected with Agrobacterium tumefaciens C58cl(pGV3850::neo1103), and the transformed calli selected from the callus induction medium containing 50 micrograms/ml kanamycin could regenerate buds on differentiation medium containing 25 micrograms/ml kanamycin . 30% of the regenerated buds were normal in morphology . The normal buds could develop into whole plantlets after they were transferred to the rooting medium to induce roots . Nopaline detection, NPT-II enzyme activity assay and Southern blotting hybridization indicated that the foreign genes had been integrated into the genome of Lycium barbarum L . and expressed in the plant . In the processes of experiments, it was found that (i) after the pre-processes, the explants which formed callus quickly were easy to transform; (ii) the rate of normal regenerated plants from transgenic calli was higher than that from the untransgenic ones.

Sci China B, 1994 Mar, 37(3), 280 - 5
Integration and expression of human growth hormone gene in Caladium bicolor; Li BJ et al.; Human growth hormone (hGH) gene has been inserted into the plasmid pLGV1103 to give the recombinant plasmid pLB-9 . It has been introduced into the agrobacterium containing plasmid pGV3850 . The recombinant Ti plasmid pGL198(hGH) has been obtained by homologous recombination . The monocotyledon Caladium bicolor has been transferred with pGL198 (hGH) with the leaf-disk co-cultivation method, and transgenic plants have been regenerated . The results of nopaline analysis, NPT II detection Southern blot and Western blot show that the hGH gene was integrated into the genome of Caladium bicolor, and a 22-kD protein was synthesized in the transgenic plants.

Mol Plant Microbe Interact, 1994 Mar-Apr, 7(2), 164 - 72
Natural instability of Agrobacterium vitis Ti plasmid due to unusual duplication of a 2.3-kb DNA fragment; Fournier P et al.; The octopine/cucumopine (o/c) Ti plasmids of Agrobacterium vitis carry two T regions, TA and TB . The TA region resembles the octopine TL region . The TB region contains the auxin synthesis genes TB-iaaM and TB-iaaH and the cucumopine synthesis gene cus . Within the group of o/c isolates, strains 2608 and 2641 are closely related . However, 2641 lacks the TB region . The restriction maps of pTi2608 and pTi2641 were established and showed that the TB deletion resulted from intramolecular recombination between two directly repeated sequences separated by 66 kb in pTi2608 . The 2,294-bp repeated sequence lacks inverted repeats and does not duplicate its target site, indicating that it is not a classical bacterial insertion sequence (IS element) . It was therefore called an RSAv element (repeated sequence of A . vitis) . The RSAv element carries two open reading frames: ORF234 is homologous to the traR gene of the A . tumefaciens nopaline Ti plasmid pTiC58; ORF488 is homologous to the sucrose phosphorylase gene of Leuconostoc mesenteroides and the glucosyl transferase A gene of Streptococcus mutans . The RSAv repeat starts precisely at the start codon of ORF488 and ends two base pairs 3' of the stop codon of ORF234 . The structural organization of the RSAv element suggests that the amplification event did not result from a random amplification process . A study of the distribution of the two RSAv copies (RSAv-1 and RSAv-2) in pTi2608 and other o/c isolates indicates that the ancestor o/c Ti plasmid contained only RSAv-1 and that this sequence was duplicated at one point during the divergent evolution of the o/c Ti plasmids.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Pharm Bull (Tokyo), 1994 Mar, 42(3), 730 - 2
Bryonolic acid production in hairy roots of Trichosanthes kirilowii Max . var Japonica Kitam . Transformed with Agrobacterium rhizogenes and its cytotoxic activity; Takeda T et al.; The hairy roots of Trichosanthes kirilowii Max, var . japonica Kitam . were induced by Agrobacterium rhizogenes (TCC 15834) on sterile shoots . The axenic culture of hairy roots proliferated 30 to 60-fold based on the initial fresh weight after three weeks of culture in Murashige & Skoog liquid media . Bryonolic acid as the main triterpenoid was isolated in a high yield, together with chondrillasterol from the hairy roots of this plant . Bryonolic acid showed strong inhibition of the growth of B-16 melanoma cells.

Carbohydr Res, 1994 Feb 17, 254, 183 - 94
Regioselective synthesis of new sucrose derivatives via 3-ketosucrose; Pietsch M et al.; 3-Ketosucrose (alpha-D-ribo-hexopyranosyl-3-ulose-beta-D-fructofuranoside), obtained from sucrose via microbial oxidation with Agrobacterium tumefaciens, was shown to be an appropriate and versatile synthon for regioselective syntheses . Condensation with hydroxylamine and its derivatives with allyl and benzyl groups leads to the oxime and the corresponding substituted products . By reductive amination 3-amino-3-deoxy-alpha-D-allopyranosyl-beta-D-fructofuranoside is obtained which can readily be submitted to further functionalization to methacryloyl and fatty acid derivatives . After silylation of 3-ketosucrose the 3-allyl and butylene-substituted as well as decyl- and dodecyl-substituted sucrose can be obtained via Grignard reaction, the side chains being C-C linked to the saccharide.

Cell, 1994 Feb 11, 76(3), 567 - 76
RNA-directed de novo methylation of genomic sequences in plants; Wassenegger M et al.; One monomeric and three oligomeric potato spindle tuber viroid (PSTVd) cDNA units were introduced into the tobacco genome via the Agrobacterium-mediated leaf-disc transformation . Southern analysis of the integrates revealed that only their PSTVd-specific sequences become fully methylated, whereas the flanking T-DNA and the genomic plant DNA remain unaltered . Viroid cDNA methylation could only be observed after autonomous viroid RNA-RNA replication had taken place in these plants . These findings demonstrate that a mechanism of de novo methylation of genes might exist that can be induced and targeted in a sequence-specific manner by their own mRNA.

Mol Microbiol, 1994 Feb, 11(3), 581 - 8
An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes; Shirasu K et al.; The 9.5 kb virB operon is the largest of the six major operons in the Ti plasmid vir region . This operon contains eleven genes, the largest of which is virB4 . This gene encodes an 84 kDa protein whose function has not been identified . Its roles in conferring virulence on Agrobacterium tumefaciens and in the T-DNA transfer process were determined by generating non-polar mutants by using the Tn5pvirB transposon in which the virB promoter is transcribed downstream of its position of insertion . Several independent mutants were isolated and each insertion site in virB4 was confirmed by nucleotide sequence analysis . These mutants were tested for T-DNA transfer ability by agroinfection and for tumorigenicity by inoculation in Brassica and Datura . All mutants were agroinfection- and tumorigenicity-negative . These data strongly suggest that virB4 is essential for both the interkingdom transfer of the T-DNA and virulence . Furthermore, by using anti-VirB4 serum, the protein product of virB4 was localized to the inner-membrane fraction of A . tumefaciens . Purified VirB4 protein hydrolyses ATP and this activity was quenched by the anti-VirB4 serum . The energy generated by VirB4 ATPase therefore may be used to transfer T-DNA or to assemble the T-DNA transfer apparatus on the bacterial membrane . Protein sequence analyses revealed striking similarities between VirB4 protein and the proteins required for conjugative transfer, which include TraC, TrwK, and TrbE of plasmids F, R388, and RP4, respectively . These findings suggest that VirB proteins play a direct role in the assembly of a conjugative transfer apparatus required for the transfer of the T-DNA from A . tumefaciens to plant cells.

Mol Gen Genet, 1994 Feb, 242(3), 327 - 36
Mapping and genetic organization of pTiChry5, a novel Ti plasmid from a highly virulent Agrobacterium tumefaciens strain; Kovacs LG et al.; Agrobacterium tumefaciens Chry5, a wild-type strain originally isolated from chrysanthemum, is unusually tumorigenic, particularly on soybean . We have mapped the Chry5 Ti plasmid by genomic walking and restriction endonuclease analysis, and have located its virulence, T-DNA, plasmid incompatibility, and L,L-succinamopine utilization loci . Southern analysis has revealed that about 85% of the Chry5 Ti plasmid is highly homologous to another Ti plasmid, pTiBo542 . Although all the functions that we have located on pTiChry5 are encoded by pTiBo542-homologous regions, the two Ti plasmids differ in their genetic organization . The overall patterns of restriction sites in the plasmids also differ, with the exception of an approximately 12 kb segment of the virulence region, where the BamHI sites appear to be conserved . Complementation analysis has shown that deletion of a DNA segment which flanks the oncogenic T-DNA results in severe attenuation of virulence . This region also contains a sequence that is repeated in the Chry5 genome outside the Ti plasmid, and that is widely distributed in the Rhizobiaceae.

C R Acad Sci III, 1994 Feb, 317(2), 141 - 8
Multiple group II self-splicing introns in mobile DNA from Escherichia coli; Ferat JL et al.; By PCR (polymerase chain reaction) amplification and cloning, we have identified four group II self-splicing introns encoding proteins related to reverse transcriptases in natural Escherichia coli isolates belonging to the ECOR collection . One intron, IntD, interrupts a DNA sequence virtually identical to that of the previously described IS3411 Insertion Sequence . A second intron, IntC, is located within an open reading frame that is closely related to a reading frame in the T-DNA of Agrobacterium tumefaciens . Finally, introns IntA and IntB are inserted at two distinct sites in one of the Rhs elements of E . coli . A comparison of their open reading frames shows that the two Rhs introns are more closely related to each other than to any other known group II intron: this suggests that transposition of group II introns may occur preferentially in cis, along the same piece of DNA.

FEBS Lett, 1994 Jan 31, 338(2), 127 - 32
The binding site of the transcriptional activator VirG from Agrobacterium comprises both conserved and specific nonconserved sequences; Roitsch T et al.; Virulence genes of Agrobacterium tumefaciens are transcriptionally activated in response to phenolic compounds and certain sugars . The transcription activator VirG specifically binds to fragments containing the conserved vir box sequence present in the promoter region of all vir genes . This study shows that both the vir box as well as specific nonconserved sequences downstream of the vir box are required for VirG binding and transcriptional activation . Insertion of the identified VirG binding site into the lac promoter resulted in transcriptional activation of this heterologous promoter in response to the plant phenolic signal molecule acetosyringone.

J Biol Chem, 1994 Jan 28, 269(4), 2645 - 51
Mutants of Agrobacterium VirA that activate vir gene expression in the absence of the inducer acetosyringone; McLean BG et al.; In the presence of inducer molecules produced by wounded plants, the VirA/VirG two-component positive regulatory system of Agrobacterium tumefaciens initiates transcription of virulence genes required for crown gall tumor formation . Exactly how this system enables the bacterium to respond to an environmental signal is not known, but phosphorylation of VirA and VirG plays a role . To analyze further the function of VirA, we chemically mutagenized the virA gene . Two mutants that activate vir transcription without the plant inducer acetosyringone were found; these mutants alter VirA function by distinct mechanisms . One mutant functions entirely independently of acetosyringone, whereas the activity of the second mutant is enhanced by acetosyringone . Both mutants function best at acid pH, but respond differently to specific monosaccharides that stimulate induction by wild-type VirA . Both mutant phenotypes are dominant over wild-type VirA, and both need the conserved histidine at the autophosphorylation site for strong inducer-independent vir transcription.

Proc Natl Acad Sci U S A, 1994 Jan 18, 91(2), 694 - 8
Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein; Jasper F et al.; Virulence proteins VirD1 and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants . The VirD1-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conjugative DNA strand (the T-strand) at the third base of these repeats . Nuclear localization signals present in VirD2 target the T-strand to plant cell nuclei . In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination . Whereas Agrobacterium transformation of dicots is very efficient, T-DNA integration in most monocots can barely be detected . To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirD1 and VirD2 proteins purified from overexpressing Escherichia coli strains . The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirD1 protein . During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs . In contrast, VirD2, alone or in complex with VirD1, fails to nick linear double-stranded DNA templates in vitro.

J Mol Biol, 1994 Jan 14, 235(2), 448 - 64
Genetic organization of the conjugal DNA processing region of the IncW plasmid R388; Llosa M et al.; The region of the IncW plasmid R388 involved in conjugal DNA metabolism and mobilization (MOBw) has been analyzed by Tn5tac1 insertion mutagenesis, genetic complementation and DNA sequencing . Three genes (trwA, trwB and trwC) were mapped within MOBw . They are transcribed from the same strand and away from oriT . The predicted products of trwA, trwB and trwC are proteins of 121, 507 and 966 amino acids, respectively . The three proteins were visualized in a minicell expression system, showing apparent molecular masses of 13.5, 55 and 105 kDa, respectively . The deduced amino acid sequence of TrwA shows significant similarity to TraJ of the IncP plasmids RP4 and R751, to NikA of the IncI plasmid R64 and to MobB of plasmid pTF-FC2 . The amino acid sequence of TrwB predicts an integral membrane protein which contains an NTP-binding motif . It shows 28% to 29% identity with TraD of plasmids F and R100, 23% identity with TraG of plasmids RP4 and R751 and 20% identity with VirD4 of the Ti plasmids of Agrobacterium tumefaciens . The amino acid sequence of TrwC shows the characteristic motifs of the Rep family of DNA helicases . It shows 33% identity with the sequence of helicase I (TraI) of plasmid F . The similarity is highest in the N-terminal segments of the proteins, which show conservation of characteristic amino acid motifs of a family of DNA-relaxases, including VirD2 of the Ti plasmid . The conserved features of these three proteins among the different transfer systems suggest that a very widespread conjugal DNA mobilization mechanism is shared by the transfer apparatuses of IncF, IncI, IncP, IncW and Ti plasmids.

J Biol Chem, 1994 Jan 7, 269(1), 668 - 75
Two binding sites for the plant transcription factor ASF-1 can respond to auxin treatments in transgenic tobacco; Liu X et al.; The hormone, auxin, plays an important role in the differentiation and growth of plant cells . A number of auxin-responsive genes have been characterized but until now minimal auxin-responsive cis-elements within these promoter regions have not been identified . Here we show that two related DNA sequences of 21 base pairs can respond to auxin treatment in transgenic tobacco . In contrast, treatments with cytokinin or abscisic acid do not cause any apparent increase in promoter activity of these cis-acting elements . These sequences are present in the promoter regions of the nopaline synthase gene from the T-DNA of Agrobacterium tumefaciens and the 35 S promoter from cauliflower mosaic virus . Both sequences have been shown to be binding sites for the tobacco transcription factor ASF-1 . Pretreatment of leaves with cycloheximide does not inhibit the response to auxin treatment, suggesting that hormone sensitivity of these promoter elements does not involve de novo synthesis of ASF-1 . In addition, promoter elements from some auxin-responsive plant genes can bind ASF-1 in vitro . Based on these results, we propose that transcriptional activation by ASF-1 may be modulated by auxin through modification of pre-existing factors . Our results also suggest a role for ASF-1 in mediating some of the effects of auxin in vivo.

J Bacteriol, 1994 Jan, 176(2), 495 - 503
Opine-regulated promoters and LysR-type regulators in the nopaline (noc) and octopine (occ) catabolic regions of Ti plasmids of Agrobacterium tumefaciens; von Lintig J et al.; Essential steps in the uptake and catabolism of the plant tumor metabolites nopaline and octopine in Agrobacterium spp . are performed by proteins encoded in the nopaline catabolic (noc) and octopine catabolic (occ) regions of Ti plasmids . We investigated the opine activation of the genes by using (i) promoter studies of Agrobacterium spp . and (ii) analysis of the promoter interaction with the regulatory proteins NocR (noc) and OccR (occ) . The noc region contained two nopaline-induced promoters (Pi1{noc} and Pi2{noc}) and one autogenously regulated promoter (Pr {control of NocR expression}) . Pi2 and Pr overlapped and were divergently oriented (Pi2 {noc}) . DNA binding studies and DNase I footprints indicated that NocR bound specifically to single binding sites in Pi1{noc} and Pi2/Pr{noc} and that Pi2 and Pr were regulated from the same binding site . The binding was independent of the inducer nopaline, and nopaline caused small changes in the footprint . The promoters in the noc and occ regions shared sequence motif and contained the sequence T-N11-A, which is characteristic for LysR-type-regulated promoters . The occ region contained one octopine-induced and one autogenously regulated promoter (Pi/Pr{occ}) in the same arrangement as Pi2/Pr{noc} in the noc region . Promoter deletions indicated that sequences flanking the OccR binding site determined the extent of induction, although they did not bind OccR . The promoter bound OccR in the absence and presence of octopine . The opine caused a change in the mobility of the DNA-protein complex with the complete promoter . The resected fragments did not reveal this opine-induced shift, and it was also not detectable with the DNA-NocR complexes with the two promoters of the noc region.

Mol Gen Genet, 1994 Jan, 242(1), 116 - 20
Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides; Calero S et al.; The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA- mutant of Pseudomonas aeruginosa . Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids . The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium meliloti, Rhizobium phaseoli, and Agrobacterium tumefaciens . An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 bp upstream region of the R . sphaeroides recA gene . Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R . sphaeroides is inducible by DNA damage . A recA-defective strain of R . sphaeroides was obtained by replacement of the active recA gene by a gene copy inactivated in vitro . The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression . This is the first recA gene from a Gram-negative bacterium that lacks an E . coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.

Mol Plant Microbe Interact, 1994 Jan-Feb, 7(1), 151 - 3
An open reading frame downstream of Rhizobium meliloti nodQ1 shows nucleotide sequence similarity to an Agrobacterium tumefaciens insertion sequence; Schwedock J et al.; We sequenced a small uncharacterized region in the Rhizobium meliloti nod gene cluster downstream of nodQ1 . We found the beginning of a large open reading frame (260 amino acids) in this fragment . The sequence reported here has striking similarity to IS66 (Y . Machida, M . Sakurai, S . Kiyokawa, A . Ubasawa, and S . Yasushiro, 1984, Proc . Natl . Acad . Sci . USA 81:7495-7499), an insertion element found in an Agrobacterium tumefaciens mutant.

Mol Gen Genet, 1994 Jan, 242(2), 226 - 36
A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertholletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes; Saalbach I et al.; The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S . This was used for transformation of tobacco (Nicotiana tabacum L . cv . Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101 . Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and beta-glucuronidase (GUS) activities . Transgenic plants were grown until flowering and fruiting occurred . The presence of the foreign gene was confirmed by Southern analysis . GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds . In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots . 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco . The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting . Analysis of seeds from the R2 progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene . Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUC1-2S was also used to transform Pisum sativum L . and Vicia faba L . Hairy roots expressed the 2S albumin-specific gene . Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.

Transgenic Res, 1994 Jan, 3(1), 26 - 35
Expression of a human S-adenosylmethionine decarboxylase cDNA in transgenic tobacco and its effects on polyamine biosynthesis; Noh EW et al.; S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key regulatory enzyme in the polyamine biosynthetic pathway . Numerous studies have shown that the enzyme activity and polyamine levels are generally correlated with cellular growth in plants, animals and bacteria . In order to gain more insight into the role of polyamines in plants, human SAMDC cDNA under control of 35S promoter of cauliflower mosaic virus, along with a neomycin phosphotransferase gene, was transferred to tobacco (Nicotiana tabacum cv.Xanthi) via Agrobacterium tumefaciens . Transgenic plants showed the presence of SAMDC mRNA and a 2-4-fold increase in SAMDC activity . In the transformed tissues, putrescine levels were significantly reduced, while spermidine content was 2-3 times higher than the control tissues . Cellular spermine content was either increased or remained unchanged . Excised leaf segments from transformed plants frequently produced shoots even on callus inducing medium.

Yakugaku Zasshi, 1994 Jan, 114(1), 1 - 20
{Molecular genetics and biotechnology in medicinal plants: studies by transgenic plants}; Saito K; The advances in molecular genetics and biotechnology in the field of medicinal plant research are discussed with focusing on the works using transgenic plants . Differentiated organ cultures and transgenic teratomas, incited by the infection with mutants of Agrobacterium Ti and Ri plasmids, were established in quinolizidine-alkaloid producing plants and Solanaceae plants . These cultured cells were used for the production and bioconversion of specific alkaloids produced in these plants . The methods of integration of foreign genes into medicinal plants were developed using an Ri binary vector . The mode of gene expression driven by TR1'-2' promoters was elucidated in transgenic medicinal plants, e.g., Nicotiana tabacum, Glycyrrhiza uralensis, Digitalis purpurea and Atropa belladonna . The genes for herbicide resistance, mammalian cytochrome P450 and bacterial beta-hydroxydecanoylthioester dehydrase were transferred and expressed in plants either to confer herbicide-resistant trait or to change the pattern of metabolites . The cDNA clones encoding cysteine synthase responsible for sulfur assimilation and biosynthesis of non-protein amino acids were isolated and characterized from Spinacea oleracea and Citrullus vulgaris . The functional lysine residue was identified by site-directed mutagenesis experiments . An over-expression system in Escherichia coli was constructed for the bacterial production of the plant specific non-protein amino acids . We made transgenic N . tabacum integrated with sense- and antisense-constructs of cysteine synthase cDNA driven by cauliflower mosaic virus 35S promoter for the purpose of genetic manipulation of biosynthetic flow of cysteine in plants . The future prospects of medicinal plant research are also discussed in the context of modern plant molecular biology.

Plant Cell, 1994 Jan, 6(1), 25 - 41
A FUSCA gene of Arabidopsis encodes a novel protein essential for plant development; Castle LA et al.; Arabidopsis fusca mutants display striking purple coloration due to anthocyanin accumulation in their cotyledons . We describe six recessive fusca mutants isolated from Agrobacterium-transformed Arabidopsis families . These mutants first become defective during embryogenesis and exhibit limited seedling development . Double mutant constructs revealed that developmental defects were not simply a consequence of anthocyanin accumulation . fusca seedlings showed altered responses to several environmental and endogenous factors . Allelism tests established that three fusca loci are represented by mutants previously described as defective in light-regulated responses . To study the molecular basis of the fusca phenotype, we cloned the FUS6 gene . FUS6 encodes a novel protein that is hydrophilic, alpha-helical, and contains potential protein kinase C phosphorylation sites . The FUSCA proteins appear to act in a network of signal transduction pathways critical for plant development.

Plant Mol Biol, 1994 Jan, 24(2), 341 - 51
Expression of mouse metallothionein-I gene confers cadmium resistance in transgenic tobacco plants; Pan A et al.; Transgenic tobacco plants containing a mouse metallothionein-I (MT-I) gene fused to the cauliflower mosaic virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming tobacco leaf discs with an Agrobacterium tumefaciens strain carrying the chimaeric gene . Transformants were directly selected and rooted on medium containing cadmium and kanamycin . A total of 49 individual transgenic tobacco plants were regenerated . Among them 20% showed a very high expression level and their growth was unaffected by up to 200 microM cadmium, whereas the growth of control plants was severely affected leaf chlorosis occurred on medium containing only 10 microM cadmium . The concentration of microM cadmium . The concentration of MT-I in leaves of control and transgenic tobacco was determined with Cd/haemoglobin saturation assay, a polarographic method and western blotting . In addition, seeds from self-fertilized transgenic plants were germinated on medium containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal . The ratio of tolerant to susceptible plants was 3:1 indicating that the metallothionein gene is inherited as a single locus.

Plant Mol Biol, 1994 Jan, 24(1), 171 - 83
Purification and partial characterization of a glycoprotein from pea (Pisum sativum) with receptor activity for rhicadhesin, an attachment protein of Rhizobiaceae; Swart S et al.; Attachment of Rhizobium and Agrobacterium bacteria to cells of their host plants is a two-step process . The first step, direct attachment of bacteria to the plant cell wall, is mediated by the bacterial protein rhicadhesin . A putative plant receptor molecule for rhicadhesin was purified from cell walls of pea roots using a bioassay based on suppression of rhicadhesin activity . This molecule appeared to be sensitive to treatments with pronase or glycosidase . Its isoelectric point is 6.4, and its apparent molecular mass was estimated to be 32 kDa before and 29 kDa after glycosidase treatment, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ultrafiltration . The sequence of the first 29 N-terminal amino acids was determined: A-D-A-D-A-L-Q-D-L-C(?)-V-A-D-Y-A-S-V-I-L- V-N-G-F-A-S-K(Q)-(P/Q)-(L)-(I) . No homology with known proteins was found . In the course of this research project the extracellular matrix protein vitronectin was reported to inhibit attachment of A . tumefaciens to carrot cells {29} . A variety of adhesive proteins, including vitronectin, contain a common cell attachment determinant with the sequence R-G-D . Since we could not detect other cell wall components able to suppress rhicadhesin activity, and since an R-G-D containing hexapeptide was also active as a receptor, we speculate that the plant receptor for rhicadhesin is a glycoprotein containing an R-G-D attachment site.

Plant Mol Biol, 1994 Jan, 24(1), 119 - 27
Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants; Garbarino JE et al.; A genomic clone encoding the potato homolog of the yeast ubiquitin-ribosomal protein fusion gene ubi3 was isolated and characterized . Chimeric genes containing the ubi3 promoter (920 bp of 5' to the ubiquitin start codon) were constructed in which the reporter gene beta-glucuronidase (GUS) was either fused directly to the promoter, or introduced as a translational fusion to the ubiquitin-coding region . After introduction into the potato by Agrobacterium-mediated transformation, GUS activities were measured in leaves and in tubers of transgenic clones . GUS activity was 5- to 10-fold higher in clones expressing the ubiquitin-GUS translational fusion than in clones containing GUS fused directly to the ubi3 promoter . For both types of constructs, GUS activity was highest in meristematic leaves and declined during leaf expansion, then rose again to near the meristematic levels during senescence . GUS activity in tubers was similar to that in young leaves . In contrast to the native ubi3 genes, the chimeric ubi3-GUS transgenes were not activated in the tuber by wounding.

Sci China B, 1994 Jan, 37(1), 37 - 41
Expression of human hepatitis B virus surface antigen gene in transgenic tobacco; Liu YL et al.; Expression of Human hepatitis B virus surface antigen (HBsAg) gene in plant was reported for the first time . The recombinant plasmid pRoKII-HBsAg was constructed by inserting HBsAg gene into the downstream of CaMV 35S promoter of binary vector pRoKII and then introduced into Agrobacterium tumefaciens LBA4404 . The kanamycin-resistant plants were obtained by Agrobacterium-mediated transformation system . It was shown that HBsAg gene was expressed in transgenic tobacco plants and their progenies by ELISA . The spherical particles of psi 22 nm is the leaf extract of transgenic tobacco were observed by immunosorbent electron microscopy.

Arch Virol Suppl, 1994, 9, 41 - 50
Principles and background for the construction of transgenic plants displaying multiple virus resistance; Truve E et al.; We investigated the possibility of reconstructing the 2'-5' oligoadenylate (2-5A) pathway into the plant kingdom to achieve multiple virus resistance . Differently phosphorylated 2-5A trimers and tetramers inhibited TMV RNA translation in cell-free systems . In wheat germ extracts the most potent inhibitors were nonphosphorylated forms of 2-5A . Triphosphorylated forms of 2-5A were deposphorylated and hydrolysed in plant extracts . Since we could not detect homologous DNA to mammalian 2-5A synthetase cDNA in tobacco or potato, we cloned rat 2-5A synthetase cDNA and transformed it by the Agrobacterium-mediated mechanism into tobacco and potato . Transformed tobacco plants were resistant to PVS infection and propagation of PVX was reduced . In transgenic potatoes tolerance to PVX and, in one transgenic clone, also to PVY was observed.

Appl Biochem Biotechnol, 1994 Spring, 45-46, 811 - 22
The effect of media composition on EDTA degradation by Agrobacterium sp; Palumbo AV et al.; EDTA degradation by an Agrobacterium sp . has been examined by quantifying 14C-labeled CO2 produced from iron-{2-14C} EDTA and by measured loss of nonlabeled EDTA by HPLC . Fe-EDTA degradation resulted in a rise in pH, nitrate concentration, and ammonia concentration . Addition of glycerol resulted in suppression of Fe-EDTA degradation and in a decrease in pH and NH4+ concentration in the media . Addition of peptone or yeast extract did not affect degradation . Some of the components (e.g., biotin) of the media are not necessary for growth and biodegradation . Although cobalt-EDTA cannot be degraded, ferrous iron can be added to displace the cobalt.

C R Acad Sci III, 1994 Jan, 317(1), 49 - 53
A new open reading frame, encoding a putative regulatory protein, in Agrobacterium rhizogenes T-DNA; Hansen G et al.; We report the presence of an open reading frame, named ORF13a, encoding a putative regulatory protein on the T-DNA of Agrobacterium rhizogenes 8196 Ri plasmid . Homologous ORFs are present at the same location in two other types of Ri plasmids . We present evidence that ORF13a is transcriptionally active . Expression of ORF13a was investigated by analysis of glucuronidase (GUS) activity in transgenic tobacco containing an ORF13aGUS fusion . The gene fusion was expressed at higher level in roots than in leaves . The putative protein encoded by ORF13a has an isoelectric point of 11.55 and carries SPXX repeated motifs suggesting a possible regulatory function for this gene.

Rev Environ Contam Toxicol, 1994, 138, 73 - 145
Glufosinate (phosphinothricin), a natural amino acid with unexpected herbicidal properties; Hoerlein G; Glufosinate ammonium (phosphinothricin ammonium) (GLA) is the active ingredient of Basta and several other herbicides used worldwide . It is produced as part of the tripeptide L-phosphinothricyl-L-alanyl-L-alanin, which was first isolated from Streptomyces viridichromogenes or Streptomyces hygroscopicus . Its structure is confirmed by degradation and synthesis . Several processes for the preparation of D,L- and L-phosphinothricin are described . Glufosinate is a structural analog of glutamate and inhibits the glutamine synthetase . The result is a rapid build-up of a high ammonia level and a concomitant depletion of glutamine and several other amino acids in the plant . These effects are accompanied by a rapid decline of photosynthetic CO2-fixation and are followed by chlorosis and desiccation . The results of numerous toxicological studies show that glufosinate ammonium and its commercial formulations are safe for users and consumers under the conditions of recommended use . The fast and complete degradation in soil and surface water prevents movement of residues into groundwater . The toxicological threshold levels for all the nontarget organisms tested are well above the potential exposure levels and therefore do not reflect any hazard for nontarget organisms in the ecosystem . Basta is a nonselective foliar applied herbicide for the control of undesirable mono- and dicotyledonous plants in orchards, vineyards, and plantations for minimum tillage, and as a harvest aid . A synthetic phosphinothricin acetyltransferase (PAT) gene has been introduced via Agrobacterium tumefaciens into dicot crops, such as like tobacco, tomato, spring and winter rapeseed, alfalfa, and several horticultural crops . The PAT gene was also successfully introduced into maize protoplasts that could be regenerated into fertile plants . All transgenic crop plants tolerated a two- to threefold field dosage of Basta.

Arch Microbiol, 1994, 161(4), 300 - 9
Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions; Ponsonnet C et al.; Chromosomes and Ti plasmids of 41 Agrobacterium strains, belonging to biovars 1, 2, 3, and Agrobacterium rubi species were characterized by the restriction fragment length polymorphism of PCR-amplified DNAs . Profiles that were obtained by the analysis of the amplified 16S rDNA confirmed the grouping of the strains according to their species . Higher polymorphism was detected in the intergenic spacer between the 16S rDNA and 23S rDNA genes, allowing efficient discrimination of strains . Identification of most strains was possible, and the genetic relatednesses of Agrobacterium strains could be estimated . The analysis of the plasmid Ti encoded regions between the tmr and nos genes, and the virA and virB2 genes, allowed fingerprinting of Ti plasmids . Genomic typing by the rapid PCR-RFLP method is thus shown to be useful for an independent identification of strains and of the conjugative Ti plasmids.

Genetica, 1994, 94(2-3), 203 - 11
Bacterial plant oncogenes: the rol genes' saga; Costantino P et al.; The rol genes are part of the T-DNA which is transferred by Agrobacterium rhizogenes in plant cells, causing neoplastic growth and differentiation . Each of these bacterial oncogenes deeply influences plant development and is finely regulated once transferred into the plant host . Both from the study of the effects and biochemical function of the rol genes and from the analysis of their regulation, important insight in plant development can be derived . Some of the most intriguing aspects of past, current and future research on this gene system are highlighted and discussed.

Chin J Biotechnol, 1994, 10(3), 203 - 9
Suspension culture of Catharanthus roseus crown gall cell induced by Agrobacterium C58; Wang N et al.; In comparison with calli from leaf or stem, Catharanthus roseus crown gall cell cultured on MS basic medium was superior in growth, total indole alkaloids and ajmalicine contents . The effects of illumination, cultural temperature, sucrose level of the medium and exogenous L-Trp on the growth, total indole alkaloids and ajmalicine contents of C . roseus crown gall cell cultures were studied . The results will provide a theoretical basis for the attempt of using suspension cultures of C . roseus crown gall cells to produce indole alkaloids.

Chin J Biotechnol, 1994, 10(2), 129 - 34
Studies on the extracellular polysaccharide from Agrobacterium radiobacter biovar I S-1231; Yu N et al.; A strain S-1231 isolated from specimen of soil around Beijing area is gram-negative, non-sporing, motile by peritrichous flagella . It produces exopolysaccharide succinoglycan from carbohydrates as its carbon source but not starch and cellulose . Acid is produced during fermentation of glucose . Growing for 12-24 hr, the cells are rods 0.7-0.8 x 1.3-1.5 microns, round ended, single or in pairs . Colonies on nutrient agar plate are unpigmented, circular, raised, smooth and moist-glistening, edge entire . The organism produces 3-ketolactose and is unable to invade sunflower tissue . The G+C content of DNA is 62.8-63.4 mol% . The organism is referred to as Agrobacterium radiobacter . Moreover, the strain is oxidase-positive, catalase-positive, H2S-produce and can grow at 35 degrees C and 2% NaCl also . Litmus milk is alkalified . Thus, the organism was renamed Agrobacterium radiobacter biovar I . Component analyses showed that the exopolysaccharide (Agran-S) from A . radiobacter biovar I S-1231 consisted of D-glucose (69.1%), D-galactose (8.6%), pyruvic acid (9.5%) and succinic acid (10.5%) . Methylation analyses revealed that the polysaccharide Agran-S contained following main structural units: (1-->3)-linked D-glucose (21.2%), (1-->3)-linked D-galactose (11.4%), (1-->6)-linked D-glucose (10.5%), (1-->4)-linked D-glucose (30.4%), (1-->4, 1-->6)-linked D-glucose (22.2%) and terminal D-glucose (4.3%) . The -1H-NMR spectrum of the polysaccharide indicated that the linkages in the polymer are all beta-glycosidic . The IR spectra of the polysaccharide revealed the presence of ester linkage in polysaccharide Agran-S.

Planta, 1994, 193(3), 438 - 45
A 17-kDa Nicotiana tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform of acid invertase; Weil M et al.; When cell-wall invertase (CWI) from Nicotiana tabacum L . cell-suspension cultures, either non-transformed or transformed with Agrobacterium tumefaciens, was salt-eluted from intact cells and purified on Sulfopropyl-Sephadex (SPS) by pH-gradient elution, the enzyme lost about 50% of its activity during a 1-h incubation at pH 4.8 . However, Western-blot analysis indicated no appreciable enzyme degradation . Re-chromatography of CWI peak fractions on SPS using NaCl-gradient elution showed the presence of a 17-kDa peptide (p17) in fractions with low CWI activity but strong CWI immunosignal (Weil and Rausch 1994, Planta 193, 430-437) . When separating CWI from p17 by Concanavalin A (Con A)-Sepharose chromatography, inhibition could be restored by incubating the inhibitor-containing fraction with inhibitor-free CWI . More than 90% of CWI could be inhibited, suggesting that all CWI was susceptible to p17 binding . The presence of divalent metal ions (Ca2+, Mg2+, Zn2+) during pre-incubation of CWI with p17 reduced CWI inhibition substantially . Also, sucrose protected CWI against inhibition by p17 (half-maximum protection at 1.3 mM) . Binding of p17 to CWI during a 1-h pre-incubation was pH-dependent, pH 4.5 causing maximum inhibition, whereas at pH 6.5 no inhibition was observed . Gel-permeation chromatography revealed that the native inhibitor acts as a monomer . Immunoprecipitation of CWI co-precipitated p17, confirming direct binding of p17 to CWI . When fractions containing CWI and p17 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting a diffuse immunosignal of 86-90 kDa was observed (in addition to the prominent CWI signal at 69 kDa).(ABSTRACT TRUNCATED AT 250 WORDS)

Curr Microbiol, 1994 Jan, 28(1), 41 - 7
Novel narrow-host-range vectors for direct cloning of foreign DNA in Pseudomonas; Boivin R et al.; Narrow-host-range vectors, based on an indigenous replicon and containing a multiple cloning site, have been constructed in a Pseudomonas host capable of growth on unusual substrates . The new cloning vectors yield sufficient amounts of DNA for preparative purposes and belong to an incompatibility group different from that of the incP and incQ broad-host-range vectors . One of these vectors, named pDB47F, was used to clone, directly in Pseudomonas, DNA fragments from Agrobacterium, Pseudomonas, and Rhizobium . A clone containing Agrobacterium and KmR gene sequences was transformed with a higher efficiency than an RSF1010-derived vector (by as much as 1250-fold) in four out of five Pseudomonas strains tested . The considerable efficiency obtained with this system makes possible the direct cloning and phenotypic selection of foreign DNA in Pseudomonas.

Gene, 1993 Dec 22, 136(1-2), 87 - 94
Transgenic plants that express genes including the 3' untranslated region of the turnip yellow mosaic virus (TYMV) genome are partially protected against TYMV infection; Zaccomer B et al.; In order to evaluate new possibilities for protecting plants against virus infection by interference with viral replication, two chimeric genes were constructed in which the (+) strand 3'-terminal 100 nucleotides (nt) of the noncoding region of the turnip yellow mosaic virus (TYMV) genome were placed downstream from the sense or antisense cat coding region . The two chimeric genes were then introduced into the genome of rapeseed (Brassica napus) using an Agrobacterium rhizogenes vector system . Plants expressing high levels of either chimeric gene showed partial protection against infection by TYMV RNA or virions . One interesting feature of the protection is that a proportion of the inoculated transgenic plants does not become infected . Protection was overcome when the inoculum concentration was increased . RNA complementary to the initial transcript was detected after infection.

Gene, 1993 Dec 22, 136(1-2), 13 - 25
Analysis of a transfer region from the staphylococcal conjugative plasmid pSK41; Firth N et al.; The nucleotide sequence of a 14.4-kb region (tra) associated with DNA transfer of the staphylococcal conjugative plasmid, pSK41, has been determined . Analysis of the sequence revealed the presence of 15 genes potentially involved in the conjugative process . Polypeptide products likely to correspond to ten of these genes have been identified, of which one was found to be a lipoprotein . Comparison of the deduced tra products to the protein databases revealed several interesting similarities, one of which suggests an evolutionary link between this Gram+ bacterial conjugation system and DNA transfer systems of Gram- bacteria, such as Escherichia coli and Agrobacterium tumefaciens . The nt sequence also provided an insight into the transcriptional organisation and regulation of the region.

Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11538 - 42
Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation; Pansegrau W et al.; As an early stage of plant transformation by Agrobacterium tumefaciens, the Ti plasmid is nicked at the border sequences that delimit the T-DNA . Cleavage results in covalent attachment of VirD2 to the 5' terminal of the nicked strand by a process resembling initiation of DNA transfer that occurs in the donor cell during bacterial conjugation . We demonstrate that this cleavage can be reproduced in vitro: VirD2 protein, the border-cleaving enzyme, was overproduced and purified . Cleavage assays were performed with single-stranded oligodeoxyribonucleotides encompassing the Ti plasmid border region or the transfer origin's nick region of the conjugative plasmid RP4 . VirD2 of pTiC58 cleaves both border- and nick region-containing oligonucleotides . However, the relaxase TraI of RP4 can cut only the cognate nick regions . The respective proteins remain covalently bound to the 5' end of the cleavage sites, leaving the 3' termini unmodified . VirD2-mediated oligonucleotide cleavage was demonstrated to be an equilibrium reaction that allows specific joining of cleavage products restoring border and nick regions, respectively . The possible role of VirD2 in T-DNA integration into the plant cell's genome is discussed in terms of less stringent target-sequence requirements.

J Biol Chem, 1993 Dec 15, 268(35), 26552 - 8
Genetic evidence for an interaction between the VirA sensor protein and the ChvE sugar-binding protein of Agrobacterium; Shimoda N et al.; Most vir genes of Agrobacterium, which are required for tumorigenicity of the bacterium, are expressed in response to plant phenolics . The induction of vir is markedly enhanced by specific monosaccharides . Signals generated by both types of compound are transduced into Agrobacterium cells via the functions of the VirA membrane-bound sensor protein . A putative sugar-binding protein, known as ChvE, also functions at a step of the enhancement of vir induction by monosaccharides . To investigate the signal pathway of the enhancement by the sugars, we first isolated a virA mutant of Agrobacterium with a base substitution mutation that caused a single amino acid change in the periplasmic domain . The mutant exhibited no enhancement of vir induction by sugar and had severely attenuated tumorigenicity on Kalanchoe leaves . We then isolated two chvE mutants that restored sugar enhancement on the background of this virA mutation . One chvE mutant, which exhibited a higher level of sugar enhancement, restored the tumorigenicity of the virA mutant . Wild-type and suppressor ChvE proteins were localized in the periplasmic space . These results provide genetic evidence for the physical interaction between VirA and ChvE proteins in the periplasmic space of Agrobacterium, which enhances the cytoplasmic signal generated by phenolics . We also discuss the molecular architecture of the operon to which the chvE gene belongs.

Microbiol Rev, 1993 Dec, 57(4), 995 - 1017
ABC transporters: bacterial exporters; Fath MJ et al.; The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain . ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters . We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems . The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates . We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates . Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family . The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti . Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches . One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides . Differences in substrate specificity do not correlate with evolutionary relatedness . A complete survey of the known and putative bacterial ABC exporters is included at the end of the review.

Plant Mol Biol, 1993 Dec, 23(6), 1199 - 210
Phenotype and hormonal status of transgenic tobacco plants overexpressing the rolA gene of Agrobacterium rhizogenes T-DNA; Dehio C et al.; The rolA gene of the TL-DNA of Agrobacterium rhizogenes Ri-plasmid plays a major role in establishing the hairy root syndrome in transgenic plants . Transgenic tobacco plants (Nicotiana tabacum L.) expressing constitutively the rolA gene under the transcriptional control of the 35S RNA promoter show pronounced phenotypical alterations . P35S-rolA transgenic tobacco plants are characterized by stunted growth, dark green wrinkled leaves with an altered length-to-width ratio, condensed influorescences, retarded onset of flowering, a reduced number of flowers and shortened styles . To investigate whether the pleiotropic alterations of growth and development are linked to an altered hormonal status we have compared the immunoreactive content of indole-3-acetic acid, cytokinins, abscisic acid, gibberellin and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) of seedlings and different tissues of P35S-rolA transgenic plants, transgenic plants expressing the rolA gene under control of its own phloem-specific promoter and wild-type plants . Multiple tissue-specific alterations of phytohormone concentrations are the consequence of rolA gene activity . Changes of phytohormonal content can explain part of the rolA-induced phenotypic alterations . Most strikingly, in young and fully developed leaves of rolA and P35S-rolA transgenic clones a 40-60% reduction of immunoreactive gibberellin A1 was found, as compared to wild-type leaves . Treatment of wild-type tobacco plants with inhibitors of gibberellin biosynthesis phenotypic alterations similar to those of rolA transgenic plants . This suggests that the reduction of gibberellic acid content is indirectly but causally involved in rolA-induced alterations of stem elongation and planar leaf blade growth.

Plant Physiol, 1993 Dec, 103(4), 1155 - 63
Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.); McKersie BD et al.; Activated oxygen or oxygen free radicals have been implicated in a number of physiological disorders in plants including freezing injury . Superoxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H2O2 and thereby reduces the titer of activated oxygen molecules in the cell . To further examine the relationship between oxidative and freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago sativa L.) . The Mn-SOD cDNA from Nicotiana plumbaginifolia under the control of the cauliflower mosaic virus 35S promoter was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation . Two plasmid vectors, pMitSOD and pChlSOD, contained a chimeric Mn-SOD construct with a transit peptide for targeting to the mitochondria or one for targeting to the chloroplast, respectively . The putatively transgenic plants were selected for resistance to kanamycin and screened for neomycin phosphotransferase activity and the presence of an additional Mn-SOD isozyme . Detailed analysis of a set of four selected transformants indicated that some had enhanced SOD activity, increased tolerance to the diphenyl ether herbicide, acifluorfen, and increased regrowth after freezing stress . The F1 progeny of one line, RA3-ChlSOD-30, were analyzed by SOD isozyme activity, by polymerase chain reaction for the Mn-SOD gene, and by polymerase chain reaction for the neo gene . RA3-ChlSOD-30 had three sites of insertion of pChlSOD, but only one gave a functional Mn-SOD isozyme; the other two were apparently partial insertions . The progeny with a functional Mn-SOD transgene had more rapid regrowth following freezing stress than those progeny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD serves a protective role by minimizing oxygen free radical production after freezing stress.

Mol Gen Genet, 1993 Dec, 241(5-6), 504 - 14
Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis; Castle LA et al.; Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants . One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified . Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail) . Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome . Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes . Chromosomal rearrangements may therefore be widespread following seed transformation . DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns . Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation . The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants . Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.

J Bacteriol, 1993 Dec, 175(24), 7880 - 6
The chromosomal virulence gene, chvE, of Agrobacterium tumefaciens is regulated by a LysR family member; Doty SL et al.; Certain plant phenolic compounds and monosaccharides induce the transcription of virulence (vir) genes of Agrobacterium tumefaciens through the VirA-VirG two-component regulatory system . The product of the chromosomal virulence gene chvE is homologous to galactose-binding protein of Escherichia coli and is required for vir gene induction by sugars . Adjacent to, but divergent in transcription from, chvE is an open reading frame, now termed gbpR (galactose-binding protein regulator), that is homologous to the LysR family of transcriptional regulators . chvE::lacZ expression was induced by L-arabinose, D-galactose, and D-fucose when gbpR was present . In the absence of inducer, GbpR repressed chvE::lacZ expression . In addition, GbpR negatively regulated its own expression.

J Bacteriol, 1993 Dec, 175(24), 7869 - 74
Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome; Allardet-Servent A et al.; Analysis of the entire Agrobacterium tumefaciens C58 genome by pulsed-field gel electrophoresis (PFGE) reveals four replicons: two large molecules of 3,000 and 2,100 kb, the 450-kb cryptic plasmid, and the 200-kb Ti plasmid . Digestion by PacI or SwaI generated 12 or 14 fragments, respectively . The two megabase-sized replicons, used as probes, hybridize with different restriction fragments, showing that these replicons are two independent genetic entities . A 16S rRNA probe and genes encoding functions essential to the metabolism of the organism were found to hybridize with both replicons, suggesting their chromosomal nature . In PFGE, megabase-sized circular DNA does not enter the gel . The 2.1-Mb chromosome always generated an intense band, while the 3-Mb band was barely visible . After linearization of the DNA by X-irradiation, the intensity of the 3-Mb band increased while that of the 2.1-Mb remained constant . This suggests that the 3-Mb chromosome is circular and that the 2.1-Mb chromosome is linear . To confirm this hypothesis, genomic DNA, trapped in an agarose plug, was first submitted to PFGE to remove any linear DNA present . The plug was then recovered, and the remaining DNA was digested with either PacI or SwaI and then separated by PFGE . The fragments corresponding to the small chromosome were found to be absent, while those corresponding to the circular replicon remained, further proof of the linear nature of the 2.1-Mb chromosome.

J Bacteriol, 1993 Dec, 175(23), 7715 - 9
Altered-function mutations in the Agrobacterium tumefaciens OccR protein and in an OccR-regulated promoter; Cho K et al.; OccR is a LysR-type transcriptional activator that controls the occQ and traR promoters of octopine-type Ti plasmids . The opine octopine converts OccR from a repressor to an activator of occQ, shortens the protein's DNase I footprint, and decreases the angle of an OccR-caused DNA bend at the occQ promoter . In this study we first localized the cis-acting DNA sequences required for regulated expression of occQ . To understand better the mechanism of activation of OccR, we isolated mutations both in the occQ promoter and in the occR gene which function differently from the wild type . An occQ promoter mutation that changes the putative -35 region of occQ from TTGACC to TTGACA increases the basal expression of occQ about 15-fold . Three mutations in occR were also identified, one of which activates occQ at fully constitutive levels in both the absence and presence of octopine . This mutation (E23G) is located in the first helix of a putative helix-turn-helix DNA-binding motif . The other two occR mutations cause the protein to detect much lower concentrations of octopine than wild-type OccR protein does . These mutations (F113L and G148D) are located in a region of the protein that is predicted to contain the ligand-binding site.

J Gen Microbiol, 1993 Dec, 139 ( Pt 12), 3265 - 73
Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies; Ouahrani S et al.; An insertion sequence (IS) element of Brucella ovis, named IS6501, was isolated and its complete nucleotide sequence determined . IS6501 is 836 bp in length and occurs 20-35 times in the B . ovis genome and 5-15 times in other Brucella species . Analysis of the junctions at the sites of insertion revealed a small target site duplication of four bases and inverted repeats of 17 bp with one mismatch . IS6501 presents significant similarity (53.4%) with IS427 identified in Agrobacterium tumefaciens, suggesting a common ancestral sequence . A long ORF of 708 bp was identified encoding a protein with a predicted molecular mass of 26 kDa and sharing sequence identity with the hypothetical protein 1 of A . tumefaciens and with the transposase of Mycobacterium tuberculosis . IS6501 is present in all Brucella strains we have tested . Restriction fragment length polymorphism of reference and field strains of two species (B . melitensis and B . ovis) was studied using either pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA or hybridization of EcoRI-digested DNA using IS6501 as a probe . The genome of B . melitensis biovar 3 contains about 10 IS copies per genome and field strains of the same species could not be distinguished either by IS hybridization or by XbaI (PFGE) restriction patterns . In contrast, the number of IS copies in the B . ovis genome is around 30 and the different field strains can be differentiated by both methods.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Microbiol, 1993 Dec, 139 ( Pt 12), 2915 - 20
Comparison of pathways for biodegradation of monomethyl sulphate in Agrobacterium and Hyphomicrobium species; Higgins TP et al.; Different mechanisms have been proposed previously for the biodegradation of monomethyl sulphate (MMS) in Agrobacterium sp . and Hyphomicrobium sp . Sulphate liberation from MMS in Agrobacterium sp . M3C was previously shown to be O2-dependent, whereas in several Hyphomicrobium spp . the initiating step has been considered hitherto to be hydrolytic and catalysed by methyl sulphatase . In the present study, Agrobacterium and Hyphomicrobium strains were compared for their ability to oxidize MMS and its potential metabolites in the oxygen electrode . MMS-grown Agrobacterium sp . M3C and Hyphomicrobium sp . MS223 oxidized MMS with consumption of 0.5 mol O2 per mol of substrate, but they were unable to oxidize methanol . By repeatedly challenging MMS-grown Hypomicrobium with MMS in the electrode chamber, all the O2 in the electrode became exhausted, at which point SO4(2-) liberation stopped although excess MMS was available . SO4(2-) release resumed immediately when O2 was re-admitted to the electrode chamber . Thus liberation of SO4(2-) from MMS in the oxygen electrode was dependent on the continuing availability of O2 . Hyphomicrobium sp . MS223 therefore closely resembled Agrobacterium sp . M3C in its obligatory requirement for O2 in MMS degradation . Unlike Agrobacterium sp . M3C, Hyphomicrobium sp . MS223 was able to grow on methanol and methanol-grown cells oxidized methanol (0.5 mol O2 per mol of substrate) but not MMS . Cyclopropanol, an inhibitor of methanol dehydrogenase, abolished oxidation of methanol by methanol-grown Hyphomicrobium sp . MS223 but did not affect oxidation of MMS by MMS-grown cells . Thus Hyphomicrobium sp . MS223 expresses enzymes for oxidation of methanol when needed for growth on this compound, but not when grown on MMS.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1993 Nov 25, 268(33), 24926 - 32
Essentiality of the three carboxyl-terminal amino acids of the plasmid RK2 replication initiation protein TrfA for DNA binding and replication activity in gram-negative bacteria; Cereghino JL et al.; In a previous study of mutations in trfA, the gene encoding the replication initiation protein of the broad host-range plasmid RK2, a carboxyl-terminal deletion of 3 amino acids of the TrfA protein was found to be completely nonfunctional for RK2 replication in Escherichia coli and other Gram-negative bacteria . In this work site-directed mutagenesis of the trfA gene was carried out to construct TrfA proteins altered in the three carboxyl-terminal positions . Specifically, TrfA proteins with deletions or substitutions of the terminal cysteine, lysine, and arginine (codons 380-382, respectively) were constructed and characterized for their ability to initiate replication from an RK2 origin in vivo in E . coli, Azotobacter vinelandii, Pseudomonas putida, and Agrobacterium tumefaciens and for binding activity to the iterons at the replication origin . Substitutions of the cysteine at position 380 with a glycine or an arginine resulted in a TrfA protein defective in binding to the RK2 origin and, therefore, defective in replication initiation activity in all four Gram-negative bacteria . Substitution of a serine at that position preserved limited function in replication and DNA binding . The lysine at position 381 could be changed to a glutamine without any obvious change in TrfA function . Deletion of the terminal arginine at position 382 did not affect the ability of TrfA to bind to origin iterons but caused a complete loss of replication activity in all four bacteria . Substitution of this terminal arginine with alanine, serine, or glutamic acid also produced replication-defective TrfA protein in all four bacterial hosts while not affecting iteron binding activity . However, substitution of this arginine with a lysine resulted in a loss of replication activity in E . coli and A . vinelandii but had no effect in P . putida and A . tumefaciens . These observations suggest that the terminal arginine plays an essential role in the activity of the TrfA protein, possibly interaction with host proteins, which can be separated from its iteron binding activity.

FEBS Lett, 1993 Nov 22, 334(3), 277 - 80
Transcription in vitro promoted by the Agrobacterium VirG protein; Endoh H et al.; Expression of the virulence genes (vir) on the hairy-root-inducing plasmid pRiA4 is induced by plant signals in Agrobacterium cells through a two-component regulatory system, the VirA-VirG system . We constructed an in vitro transcription system that consisted of the purified VirG protein and the Agrobacterium RNA polymerase holoenzyme . Both versions of VirG, the non-phosphorylated form and the VirA-phosphorylated form, were active but showed different patterns of the pH-dependency for transcriptional activation.

Plant Mol Biol, 1993 Nov, 23(4), 847 - 60
Single-copy T-DNA insertions in Arabidopsis are the predominant form of integration in root-derived transgenics, whereas multiple insertions are found in leaf discs; Grevelding C et al.; Different patterns of T-DNA integration in Arabidopsis were obtained that depended on whether a root or a leaf-disc transformation method was used . An examination of 82 individual transgenic Arabidopsis plants, derived from 15 independent Agrobacterium-mediated transformations in which different cointegrate and binary constructs were used, indicated that the transformation method had a significant influence on the type and copy number of T-DNA integration events . Southern hybridizations showed that most of the transgenic plants produced by a leaf-disc method contained multiple T-DNA insertions (89%), the majority of which were organized as right-border inverted repeat structures (58%) . In contrast, a root transformation method mostly resulted in single T-DNA insertions (64%), with fewer right-border inverted repeats (38%) . The transformation vectors, including cointegrate and binary types, and the plant selectable markers, hygromycin phosphotransferase and dihydrofolate reductase, did not appear to influence the T-DNA integration patterns.

Plant Mol Biol, 1993 Nov, 23(4), 749 - 57
Constitutive or light-regulated expression of the rolC gene in transgenic potato plants has different effects on yield attributes and tuber carbohydrate composition; Fladung M et al.; Tetraploid potato clones, transgenic for the rolC gene of Agrobacterium rhizogenes under control of the light-inducible ribulose bisphosphate carboxylase small subunit promoter (rbcS-rolC), were compared, with respect to yield attributes and tuber carbohydrates, with transformed and untransformed controls and with 35S-rolC transgenic potato plants . In rbcS-rolC plants, the expression of the rolC gene was located mainly in leaves, while in 35S-rolC plant transcripts were detected as well in shoots and roots . Phenotypically, rbcS-rolC transgenic plants were found to be slightly reduced in plant size with a few more tillers than control plants . Photosynthetic rate and chlorophyll content were significantly lower in all rolC transgenic plants irrespective of the type of construct used . Tuber yield was not significantly different between controls and rbcS-rolC transgenic plants, but was reduced in the 35S-rolC transformants . Sucrose level was unchanged in all rolC clones investigated, whereas fructose content was significantly enhanced in 35S-rolC transformants, but not in the plants expressing the rolC gene in aerial plant parts only . In both types of rolC transgenic plants, glucose content was lower than in controls, resulting in a significant reduction of reducing sugar in tubers . The results suggest a hormonal influence on the carbohydrate composition of potato tubers.

Mol Gen Genet, 1993 Nov, 241(3-4), 359 - 66
Stable expression of a single-copy rolA gene in transgenic Arabidopsis thaliana plants allows an exhaustive mutagenic analysis of the transgene-associated phenotype; Dehio C et al.; Several publications have documented the instability of transgene expression in plants . Previous genetic approaches to the study of transgene-associated phenotypes in plants were limited by this phenomenon . Here we show that a transgene can be expressed in plants with sufficient stability to allow an exhaustive mutagenic analysis of the resulting phenotype . We have expressed the morphogenic rolA gene from the TL-DNA of Agrobacterium rhizogenes Ri plasmid in transgenic Arabidopsis thaliana plants . The resulting pleiotropic RolA phenotype allows a visual screen for reversion to detect germinal as well as somatic instability of transgene expression . However no spontaneous reversions of the Ro-1A phenotype were observed in 65,000 progeny of two independent transgenic A . thaliana lines, each carrying a single homozygous rolA locus . In contrast, 12 revertants of the RolA phenotype were isolated from 360,000 ethyl methane sulphonate (EMS)-mutagenized M2 progeny . All revertants were shown genetically to carry stable recessive mutations in the rolA locus, thus establishing a series of loss-of-function alleles . Molecular characterization revealed that the loss-of-function alleles were structurally intact and expressed in all rolA mutants . A wildtype rolA locus and two loss-of-function alleles were reisolated and sequenced; base pair substitutions were found in each loss-of-function allele leading to single amino acid substitutions in the rolA open reading frame . Therefore no instability of expression of the rolA locus was detected in any of the 425,000 individuals studied in this analysis . Furthermore even under conditions of saturation mutagenesis, no extragenic suppressor locus was detected.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9939 - 43
Preformed dimeric state of the sensor protein VirA is involved in plant--Agrobacterium signal transduction; Pan SQ et al.; Plant signal molecules such as acetosyringone and certain monosaccharides induce the expression of Agrobacterium tumefaciens virulence (vir) genes, which are required for the processing, transfer, and possibly integration of a piece of the bacterial plasmid DNA (T-DNA) into the plant genome . Two fo the vir genes, virA and virG, belonging to the bacterial two-component regulatory system family, control the induction of vir genes by plant signals . virA encodes a membrane-bound sensor kinase protein and virG encodes a cytoplasmic regulator protein . Although it is well established from in vitro studies that the signal transduction process involves VirA autophosphorylation and subsequent phosphate transfer to VirG, the structural state of the VirA protein involved in signal transduction is not understood . In this communication, we describe an in vivo crosslinking approach which provides physical evidence that VirA exists as a homodimer in its native configuration . The dimerization of VirA neither requires nor is stimulated by the plant signal molecule acetosyringone . We also present genetic data which support the hypothesis that VirA exists as a homodimer which is the functional state transducing the plant signal in an intersubunit mechanism . To our knowledge, this report provides the first evidence that a bacterial membrane-bound sensor kinase exists and functions as a homodimer in vivo.

J Bacteriol, 1993 Nov, 175(21), 6830 - 5
The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures; Jin S et al.; Previous studies have shown that Agrobacterium tumefaciens causes tumors on plants only at temperatures below 32 degrees C, and virulence gene expression is specifically inhibited at temperatures above 32 degrees C . We show here that this effect persists even when the virA and virG loci are expressed under the control of a lac promoter whose activity is temperature independent . This finding suggests that one or more steps in the signal transduction process mediated by the VirA and VirG proteins are temperature sensitive . Both the autophosphorylation of VirA and the subsequent transfer of phosphate to VirG are shown to be sensitive to high temperatures (> 32 degrees C), and this correlates with the reduced vir gene expression observed at these temperatures . At temperatures of 32 degrees C and higher, the VirA molecule undergoes a reversible inactivation while the VirG molecule is not affected . vir gene induction is temperature sensitive in an acetosyringone-independent virA mutant background but not in a virG constitutive mutant which is virA and acetosyringone independent . These observations all support the notion that the VirA protein is responsible for the thermosensitivity of vir gene expression . However, an Agrobacterium strain containing a constitutive virG locus still cannot cause tumors on Kalanchoe plants at 32 degrees C . This strain induces normal-size tumors at temperatures up to 30 degrees C, whereas the wild-type Agrobacterium strain produces almost no tumors at 30 degrees C . These results suggest that at temperatures above 32 degrees C, the plant becomes more resistant to infection by A . tumefaciens and/or functions of some other vir gene products are lost in spite of their normal levels of expression.

EMBO J, 1993 Nov, 12(11), 4125 - 35
Roles of plant homologs of Rab1p and Rab7p in the biogenesis of the peribacteroid membrane, a subcellular compartment formed de novo during root nodule symbiosis; Cheon CI et al.; The peribacteroid membrane (PBM) in legume root nodules is derived from plasma membrane following endocytosis of Rhizobium by fusion of newly synthesized vesicles . We studied the roles of plant Rab1p and Rab7p homologs, the small GTP-binding proteins involved in vesicular transport, in the biogenesis of the PBM . Three cDNAs encoding legume homologs of mammalian Rab1p and Rab7p were isolated from soybean (sRab1p, sRab7p) and Vigna aconitifolia (vRab7p) . sRab1p was confirmed to be a functional counterpart of yeast Ypt1p (Rab1p) by complementation of a yeast ypt1-1 mutant . Both srab1 and vrab7 genes are induced during nodulation with the level of vrab7 mRNA being 12 times higher than that in root meristem and leaves . This induction directly correlates with membrane proliferation in nodules . Antisense constructs of srab1 and vrab7, under a nodule-specific promoter (leghemoglobin, Lbc3), were made in a binary vector and transgenic nodules were developed on soybean hairy roots obtained through Agrobacterium rhizogenes-mediated transformation . Both antisense srab1 and vrab7 nodules were smaller in size and showed lower nitrogenase activity than controls . The antisense srab1 nodules showed lack of expansion of infected cells, fewer bacteroids per cell and their frequent release into vacuoles . In contrast, antisense vrab7 expressing nodules showed accumulation of late endosomal structure and multivesicular bodies in the perinuclear region . These data suggest that both Rab1p and Rab7p are essential for the development of the PBM compartment in effective symbiosis.

Sci China B, 1993 Nov, 36(11), 1307 - 14
5'-flanking region responsible for endosperm-specific expression of rice prolamine chimeric gene in transgenic tobacco; Zhou XJ et al.; The 5' upstream region (-680-(+)40) containing the complete signal peptide coding sequence of the rice seed storage prolamine gene was amplified in vitro with polymerase chain reaction from the genome of Chinese super rice cultivar Zhonghua 8 . Physical map and DNA sequence analysis showed strong homology with the 5'-flanking region of rice prolamine gene reported by Kim in 1988 . No changes in the signal peptide coding sequence and a long leader sequence with several small ORFs were found . Chimeric gene containing 5'-flanking region of the prolamine gene has been transcriptionally fused with the beta-glucuronidase reporter gene . The fusion junction was confirmed by both physical map and DNA sequence analysis . The resultant chimeric gene was used to transform the tobacco explants by Ti binary system of Agrobacterium tumefaciens LBA4404 . With dot and Southern blotting hybridization, three transgenic tobacco plants with the copies of chimeric GUS genes as many as 20 were obtained . Histochemical analysis revealed that the GUS activity in the endosperm tissues of tobacco seeds at the developmental stage was about 20 DAP . No GUS activity was found in leaves, stems, roots and flowers of the transgenic tobacco plants . Therefore, we concluded that the 5'-upstream cis-elements from -680 to -18 were enough to confer the endosperm-specific and temporal expression of rice prolamine gene.

Mol Microbiol, 1993 Nov, 10(3), 597 - 605
Restoration of attachment, virulence and nodulation of Agrobacterium tumefaciens chvB mutants by rhicadhesin; Swart S et al.; In contrast to wild-type Agrobacterium tumefaciens strains, beta-1,2-glucan-deficient chvB mutants were found to be unable to attach to pea root hair tips . The mutants appeared to produce rhicadhesin, the protein that mediates the first step in attachment of Rhizobiaceae cells to plant root hairs, but the protein was inactive . Both attachment to root hairs and virulence of the chvB mutants could be restored by treatment of the plants with active rhicadhesin, whereas treatment of plants with beta-1,2-glucan had no effect on attachment or virulence . Moreover, nodulation ability of a chvB mutant carrying a Sym plasmid could be restored by pretreatment of the host plant with rhicadhesin . Apparently the attachment-minus and avirulence phenotype of chvB mutants is caused by lack of active rhicadhesin, rather than directly being caused by a deficiency in beta-1,2-glucan synthesis . The results strongly suggest that rhicadhesin is essential for attachment and virulence of A . tumefaciens cells . They also indicate that the mechanisms of binding of Agrobacterium and Rhizobium bacteria to plant target cells are similar, despite differences between these target cells.

Gene, 1993 Oct 29, 133(1), 1 - 8
Mutations in the gene encoding the replication-initiation protein of plasmid RK2 produce elevated copy numbers of RK2 derivatives in Escherichia coli and distantly related bacteria; Fang FC et al.; Mini-replicons of the broad-host-range plasmid RK2 with increased copy number (cn) due to mutations in the gene encoding the essential replication initiation protein TrfA are described . The cn of these derivatives have been determined in Escherichia coli, Pseudomonas aeruginosa and Agrobacterium tumefaciens and were found to be elevated in all three bacterial hosts . One of the cn mutations was introduced into the intact 60-kb RK2 plasmid by homologous recombination in vivo, resulting in an approximately twofold cn increase . The expression of trfA from this mutant RK2 plasmid did not respond to the cn change as predicted by a simple transcription rate-limitation, replication control model . Implications for the model of RK2 replication control and the potential use of mutant RK2 mini-replicons as high-copy broad-host-range gene cloning vectors are discussed.

Nucleic Acids Res, 1993 Oct 25, 21(21), 4867 - 72
Genetic and biochemical analysis of an endonuclease encoded by the IncN plasmid pKM101; Pohlman RF et al.; The IncN plasmid pKM101 nuc gene encodes a periplasmically localized endonuclease . DNA sequence analysis indicates that this gene encodes a hydrophilic protein of about 19.5 kDa containing a hydrophobic signal sequence . nuc is homologous to a partially sequenced open reading frame adjacent to the sog gene of the plasmid CollB-P9, a plasmid known to encode an endonuclease similar to that of pKM101 . A partially sequenced tra gene directly upstream of nuc is homologous to the virB11 gene of Agrobacterium tumefaciens . We have partially purified the pKM101 nuclease by osmotic shock and cation exchange chromatography, and used this enzyme preparation to sequence the protein's amino terminus . The first 13 amino acids of the mature protein match amino acids 23 to 35 of the predicted sequence, indicating that the protein is proteolytically processed to a molecular mass of approximately 17 kDa, probably during export to the periplasmic space . The enzyme was able to attack many sites along an end labelled duplex DNA substrate, but showed clearly preferred cleavage sites, and may cleave preferentially at purine-rich regions.

J Bacteriol, 1993 Oct, 175(20), 6721 - 4
Effects of ribosome-inactivating proteins on Escherichia coli and Agrobacterium tumefaciens translation systems; Girbes T et al.; The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system . The depurinating activity of RIPs on E . coli ribosomes was also evaluated . Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E . coli lysates, with submicromolar 50% inhibitory concentrations . Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E . coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A . tumefaciens ribosomes, with submicromolar 50% inhibitory concentrations.

J Bacteriol, 1993 Oct, 175(20), 6626 - 36
The chromosomal response regulatory gene chvI of Agrobacterium tumefaciens complements an Escherichia coli phoB mutation and is required for virulence; Mantis NJ et al.; In an effort to identify the Agrobacterium tumefaciens phosphate regulatory gene(s), we isolated a clone from an A . tumefaciens cosmid library that restored regulated alkaline phosphatase activity to an Escherichia coli phoB mutant . The gene that complemented phoB was localized by subcloning and deletion analysis, and the DNA sequence was determined . An open reading frame, denoted chvI, was identified that encoded a predicted protein with amino acid similarity to the family of bacterial response regulators and 35% identify to PhoB . Surprisingly, an A . tumefaciens chvI mutant showed normal induction of phosphatase activity and normal virG expression when grown in phosphate-limiting media . However, this mutant was unable to grow in media containing tryptone, peptone, or Casamino Acids and was also more sensitive than the wild type to acidic extracellular pH . This mutant was avirulent on Kalanchoee diagremontiana and was severely attenuated in vir gene expression . The pH-inducible expression of virG was also abolished . Growth of the chvI mutant was inhibited by K . diagremontiana wound sap, suggesting that avirulence may be due, in part, to the inability of this mutant to survive the plant wound environment.

J Bacteriol, 1993 Oct, 175(20), 6614 - 25
A chromosomally encoded two-component sensory transduction system is required for virulence of Agrobacterium tumefaciens; Charles TC et al.; TnphoA mutagenesis of Agrobacterium tumefaciens identified new extracytoplasmic protein-encoding virulence loci . Mutations in these loci conferred increased sensitivity to detergents and several antibiotics . Clones carrying these loci were isolated from an A . tumefaciens cosmid library by complementation of the detergent sensitivities of the mutants . The locus on one complementing clone was delineated by Tn5 and TnphoA mutagenesis . DNA sequence analysis of the delineated region revealed that this locus is made up of two transcriptional units, chvG and chvI, which were predicted, on the basis of amino acid sequence homology, to encode the members of a two-component sensory transduction system . The membrane-spanning sensor, a histidine protein kinase, was designated ChvG, and the response regulator, presumably a transcriptional activator, was designated ChvI . Surprisingly, ChvG was also predicted to contain a Walker type A consensus nucleotide binding site, which is unusual for sensor histidine protein kinases . Site-specific insertion mutations in either chvG or chvI abolished tumor formation ability, as well as the ability to grow on complex media . Neither the genes which are regulated nor the inducing signal is known yet for this system.

J Bacteriol, 1993 Oct, 175(20), 6553 - 61
Isolation and characterization of a DNA replication origin from the 1,700-kilobase-pair symbiotic megaplasmid pSym-b of Rhizobium meliloti; Margolin W et al.; A 4-kb fragment active as an autonomously replicating sequence (ARS) from the Rhizobium meliloti symbiotic megaplasmid pSym-b was isolated by selecting for sequences that allowed a normally nonreplicative pBR322 derivative to replicate in R . meliloti . The resulting Escherichia coli-R . meliloti shuttle plasmid (mini-pSym-b) containing the ARS also replicated in the closely related Agrobacterium tumefaciens, but only in strains carrying pSym-b, suggesting that a megaplasmid-encoded trans-acting factor is required . The copy number of mini-pSym-b was approximately the same as that of the resident megaplasmid, and mini-pSym-b was unstable in the absence of antibiotic selection . An 0.8-kb DNA subfragment was sufficient for replication in both R . meliloti and A . tumefaciens . The minimal ARS exhibited several sequence motifs common to other replication origins, such as an AT-rich region, three potential DnA binding sites, a potential 13-mer sequence, and several groups of short direct repeats . Hybridization experiments indicated that there may be a related ARS on the other megaplasmid, pSym-a . The pSym-b ARS was mapped near exoA, within a region nonessential for pSym-b replication . These results suggest that the R . meliloti megaplasmids share conserved replication origins and that pSym-b contains multiple replication origins . Since the mini-pSym-b shuttle vector can coexist with IncP-1 broad-host-range plasmids, it is also now possible to use two compatible plasmids for cloning and genetic manipulation in R . meliloti.

J Bacteriol, 1993 Oct, 175(20), 6415 - 25
The mating pair formation system of plasmid RP4 defined by RSF1010 mobilization and donor-specific phage propagation; Lessl M et al.; Transfer functions of the conjugative plasmid RP4 (IncP alpha) are distributed among distinct regions of the genome, designated Tra1 and Tra2 . By deletion analyses, we determined the limits of the Tra1 region, essential for intraspecific Escherichia coli matings . The Tra1 core region encompasses approximately 5.8 kb, including the genes traF, -G, -H, -I, -J, and -K as well as the origin of transfer . The traM gene product, however, is not absolutely required for conjugation but significantly increases transfer efficiency . To determine the transfer phenotype of genes encoded by the Tra2 core region, we generated a series of defined Tra2 mutants . This revealed that at least trbB, -C, -E, -G, and -L are essential for RP4 conjugation . To classify these transfer functions as components of the DNA transfer and replication (Dtr) or of the mating pair formation (Mpf) system, we analyzed the corresponding derivatives with respect to mobilization of IncQ plasmids and donor-specific phage propagation . We found that all of the Tra2 genes listed above and the traG and traF genes of Tra1 are required for RSF1010 mobilization . Expression of traF from Tra1 in conjunction with the Tra2 core was sufficient for phage propagation . This implies that the TraG protein is not directly involved in pilus formation and potentially connects the relaxosome with proteins enabling the membrane passage of the DNA . The proposed roles of the RP4 transfer gene products are discussed in the context of virulence functions encoded by the evolutionarily related Ti T-DNA transfer system of agrobacteria.

Antimicrob Agents Chemother, 1993 Oct, 37(10), 2119 - 25
Identification of the satA gene encoding a streptogramin A acetyltransferase in Enterococcus faecium BM4145; Rende-Fournier R et al.; Enterococcus faecium BM4145, a clinical isolate from urine, was resistant to streptogramin group A antibiotics by inactivation . The strain harbored a plasmid containing a gene, satA, responsible for this resistance; this gene was cloned and sequenced . It encoded SatA, a protein deduced to be 23,634 Da in mass and homologous with a new family of chloramphenicol acetyltransferases described in Agrobacterium tumefaciens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus . The similarity of SatA to other acetyltransferases, LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) from E . coli, and to two putative acetyltransferases, NodL from Rhizobium leguminosarum and Urf1 from E . coli, was also observed in a region considered to be the enzyme's active site . Acetylation experiments indicated that acetyl coenzyme A was necessary for SatA activity and that a single acetylated derivative of pristinamycin IIA was produced . Other members of the streptogramin A group such as virginiamycin M and RP54476 were also substrates for the enzyme . We conclude that resistance to the streptogramin A group of antibiotics in E . faecium BM4145 is due to acetylation by an enzyme related to the novel chloramphenicol acetyltransferase family.

Plant J, 1993 Oct, 4(4), 711 - 6
Isolation and characterization of two related Arabidopsis ocs-element bZIP binding proteins; Zhang B et al.; Ocs-elements are a group of related, bipartite promoter elements which have been exploited by two distinct groups of plant pathogens, Agrobacterium and certain viruses to express genes in plants . The genes for two Arabidopsis bZIP (basic region-leucine zipper) proteins that bind to ocs-elements have been isolated and characterized . The genes, called OBF4 and OBF5, were isolated by screening an Arabidopsis genomic library with degenerate oligonucleotides complementary to the DNA-binding domains of other plant ocs-element-binding proteins . The OBF4 and OBF5 proteins show 53% amino acid identity but low DNA homology . Southern blot analysis demonstrated that each of the OBF genes is a member of a small family . OBF4 is more similar to the tobacco TGA1a and Arabidopsis TGA1 proteins, while OBF5 is more similar to the maize OBF3.1, wheat HBP1b and Arabidopsis aHBP1b proteins . The DNA-binding properties of OBF4 and OBF5 were similar although OBF5 was able to bind simultaneously to both halves of the ocs-element more efficiently than OBF4 . This difference in binding to the ocs-element between two closely related proteins from the same species is potentially significant since binding to both halves of the ocs-element is a pre-requisite for in vivo transcriptional activity.

Int J Syst Bacteriol, 1993 Oct, 43(4), 826 - 31
Molecular systematics of the genus Zoogloea and emendation of the genus; Shin YK et al.; Phylogenetic relationships among strains of Zoogloea and related taxa were determined by 16S rDNA sequencing and genomic DNA hybridization techniques . The 16S rRNA gene was amplified by the polymerase chain reaction with a pair of eubacterial consensus primers and sequenced directly by using an automated fluorescent DNA sequencer . Sequence comparisons and distance matrix tree analysis revealed that Zoogloea ramigera IAM 12136 (= N . C . Dondero 106, type strain) and Zoogloea sp . ATCC 19324 formed a lineage with Rhodocyclus purpureus in the beta subclass of Proteobacteria . Z . ramigera IAM 12670 (= P . R . Dugan 115) was shown to belong to another cluster with Alcaligenes eutrophus and Pseudomonas cepacia in the beta subclass . In contrast, Z . ramigera IAM 12669 (= K . Crabtree I-16-M) proved to be a member of the alpha subclass of the Proteobacteria, closely related to Agrobacterium tumefaciens . Genomic DNA hybridization studies also showed that there is genetic diversity among the strains currently designated Z . ramigera, but typical Zoogloea strains, characterized by their production of rhodoquinones, are highly related to each other and can be regarded as a single species . On the basis of the molecular data, together with the early phenotypic and chemotaxonomic information, we have emended the generic description of Zoogloea.

Int J Syst Bacteriol, 1993 Oct, 43(4), 694 - 702
Proposal for rejection of Agrobacterium tumefaciens and revised descriptions for the genus Agrobacterium and for Agrobacterium radiobacter and Agrobacterium rhizogenes; Sawada H et al.; The 16S rRNA sequences of seven representative Agrobacterium strains, eight representative Rhizobium strains, and the type strains of Azorhizobium caulinodans and Bradyrhizobium japonicum were determined . These strains included the type strains of Agrobacterium tumefaciens, Agrobacterium rhizogenes, Agrobacterium radiobacter, Agrobacterium vitis, Agrobacterium rubi, Rhizobium fredii, Rhizobium galegae, Rhizobium huakuii, Rhizobium leguminosarum, Rhizobium loti, Rhizobium meliloti, and Rhizobium tropici . A phylogenetic analysis showed that the 15 strains of Agrobacterium and Rhizobium species formed a compact phylogenetic cluster clearly separated from the other members of the alpha subclass of the Proteobacteria . However, Agrobacterium species and Rhizobium species are phylogenetically entwined with one another, and the two genera cannot be separated . In the Agrobacterium species, the strains of biovar 1, biovar 2, Agrobacterium rubi, and Agrobacterium vitis were clearly separated . The two biovars exhibited homogeneity in their phenotypic, chemotaxonomic, and phylogenetic characteristics, and two species should be established for the two biovars . We considered the nomenclature of the two biovars, and revised descriptions of Agrobacterium radiobacter (for the biovar 1 strains) and Agrobacterium rhizogenes (for the biovar 2 strains) are proposed . The name Agrobacterium tumefaciens is rejected because the type strain of this species was assigned to Agrobacterium radiobacter, and consequently the description of the genus Agrobacterium is revised.

Mol Gen Genet, 1993 Oct, 241(1-2), 65 - 72
Nopaline causes a conformational change in the NocR regulatory protein-nocR promoter complex of Agrobacterium tumefaciens Ti plasmid pTiT37; Marincs F et al.; The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37 . We have cloned and sequenced nocR, which encodes a DNA-binding protein . The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins . Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline . The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex . Sequence analysis of the NocR binding site indicated the presence immediately downstream of the -10 sequence of the nocR promoter of a 12 bp putative operator overlapping a consensus gyrase recognition sequence and an 18 bp long alternating purine-pyrimidine sequence . These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.

Plant Mol Biol, 1993 Sep, 22(6), 995 - 1005
Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes; Czosnek H et al.; The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes . This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance . Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid of Agrobacterium tumefaciens . In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2-5 days after inoculation . Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs . This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects . Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant . Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation, L . chilense LA1969 and L . hirsutum LA1777, did not support TYLCV DNA replication . Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.

FEMS Microbiol Rev, 1993 Sep, 12(1-3), 149 - 63
Structure and function of proteins of the phosphotransferase system and of 6-phospho-beta-glycosidases in gram-positive bacteria; Hengstenberg W et al.; New information about the proteins of the phosphotransferase system (PTS) and of phosphoglycosidases of homofermentative lactic acid bacteria and related species is presented . Tertiary structures were elucidated from soluble PTS components . They help to understand regulatory processes and PTS function in lactic acid bacteria . A tertiary structure of a membrane-bound enzyme II is still not available, but expression of Gram-positive genes encoding enzymes II can be achieved in Escherichia coli and enables the development of effective isolation procedures which are necessary for crystallization experiments . Considerable progress was made in analysing the functions of structural genes which are in close vicinity of the genes encoding the sugar-specific PTS components, such as the genes encoding the tagatose-6-P pathway and the 6-phospho-beta-glycosidases . These phosphoglycosidases belong to a subfamily of the beta-glycosidase family I among about 300 different glycosidases . The active site nucleophile was recently identified to be Glu 358 in Agrobacterium beta-glucosidase . This corresponds to Glu 375 in staphylococcal and lactococcal 6-phospho-beta-galactosidase . This enzyme is inactivated by mutating Glu 375 to Gln . Diffracting crystals of the lactococcal 6-P-beta-galactosidase allow the elucidation of its tertiary structure which helps to derive the structures for the entire glycosidase family 1 . In addition, a fusion protein with 6-phospho-beta-galactosidase and staphylococcal protein A was constructed.

J Bacteriol, 1993 Sep, 175(17), 5575 - 84
Genetic analysis of the agrocinopine catabolic region of Agrobacterium tumefaciens Ti plasmid pTiC58, which encodes genes required for opine and agrocin 84 transport; Hayman GT et al.; The acc region, subcloned from pTiC58 of classical nopaline and agrocinopine A and B Agrobacterium tumefaciens C58, allowed agrobacteria to grow using agrocinopine B as the sole source of carbon and energy . acc is approximately 6 kb in size . It consists of at least five genes, accA through accE, as defined by complementation analysis using subcloned fragments and transposon insertion mutations of acc carried on different plasmids within the same cell . All five regions are required for agrocin 84 sensitivity, and at least four are required for agrocinopine and agrocin 84 uptake . The complementation results are consistent with the hypothesis that each of the five regions is separately transcribed . Maxicell experiments showed that the first of these genes, accA, encodes a 60-kDa protein . Analysis of osmotic shock fractions showed this protein to be located in the periplasm . The DNA sequence of the accA region revealed an open reading frame encoding a predicted polypeptide of 59,147 Da . The amino acid sequence encoded by this open reading frame is similar to the periplasmic binding proteins OppA and DppA of Escherichia coli and Salmonella typhimurium and OppA of Bacillus subtilis.

J Bacteriol, 1993 Sep, 175(17), 5403 - 10
Melanin production by Rhizobium meliloti GR4 is linked to nonsymbiotic plasmid pRmeGR4b: cloning, sequencing, and expression of the tyrosinase gene mepA; Mercado-Blanco J et al.; Melanin production by Rhizobium meliloti GR4 is linked to nonsymbiotic plasmid pRmeGR4b (140 MDa) . Transfer of this plasmid to GR4-cured derivatives or to Agrobacterium tumefaciens enables these bacteria to produce melanin . Sequence analysis of a 3.5-kb PstI fragment of plasmid pRmeGR4b has revealed the presence of a open reading frame 1,481-bp that codes for a protein whose sequence shows strong homology to two conserved regions involved in copper binding in tyrosinases and hemocyanins . In vitro-coupled transcription-translation experiments showed that this open reading frame codes for a 55-kDa polypeptide . Melanin production in GR4 is not under the control of the RpoN-NifA regulatory system, unlike that in R . leguminosarum bv . phaseoli 8002 . The GR4 tyrosinase gene could be expressed in Escherichia coli under the control of the lacZ promoter . For avoiding confusion with mel genes (for melibiose), a change of the name of the previously reported mel genes of R . leguminosarum bv . phaseoli and other organisms to mep genes (for melanin production) is proposed.

Mol Plant Microbe Interact, 1993 Sep-Oct, 6(5), 673 - 5
The persistence of engineered Agrobacterium tumefaciens in agroinfected plants; Mogilner N et al.; Several plant species, including tomato (Lycopersicon esculentum), Gynura aurantiaca, avocado (Persea americana), and grapefruit (Citrus paradisi) grafted on Troyer citrange (Poncirus trifoliata x C . sinensis) were "agro-infected" with Agrobacterium tumefaciens strain LBA-4404, carrying a mini-Ti plasmid with a dimeric cDNA of citrus exocortis viroid (CEVd) . Extracts prepared from tissues of the agroinfected plants 38-90 days after inoculation were plated on selective media and found to contain large amounts of the engineered bacteria . These observations suggest the need for more stringent quarantine measures when handling A . tumefaciens cells harboring constructs for "agroinoculation" with plant viruses or viroids.

Protein Eng, 1993 Sep, 6(7), 755 - 62
Construction of multiple copy of alpha-domain gene fragment of human liver metallothionein IA in tandem arrays and its expression in transgenic tobacco plants; Pan A et al.; Metallothioneins (MT) are low molecular weight, cysteine-rich, metal-binding proteins . An MT molecule contains two domains which appear to act independently--an alpha-domain, which is characterized by cadmium-binding, and a beta-domain, which binds preferentially to copper . Based on this conception, DNA duplex encoding the alpha-domain (106 bp) of human MT-IA was constructed from a chemically-synthesized oligomer by repair synthesis and enzymatic ligation and cloned into pUC19 . The genes cloned were sequenced and found to be in the correct order as designed . Synthetic directional adapters were attached to the terminals of the alpha-domain gene fragment of human MT-IA to establish complete control over fragment orientation during ligation . The use of these directional adapters thereby ensured the production of multiple copies of the alpha-domain in tandem arrays . The successive alpha-domains were linked by a peptide linker consisting of 10 residues . A chimeric gene containing 12 cloned tandemly repeated copies of the 106 bp alpha-domain DNA was introduced into tobacco cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens . A total of 10 different transgenic tobacco plants were generated, of which two showed root and shoot growth unaffected by up to 200 mg/l kanamycin and 100 microM cadmium, whereas root growth of control plants was severely inhibited and leaf chlorosis developed on media containing only 10 microM cadmium.

Plant J, 1993 Sep, 4(3), 525 - 34
Interaction between the tobacco DNA-binding activity CBF and the cyt-1 promoter element of the Agrobacterium tumefaciens T-DNA gene T-CYT correlates with cyt-1 directed gene expression in multiple tobacco tissue types; Neuteboom ST et al.; A novel DNA-binding activity, designated CBF, has been identified in nuclear extracts from tobacco leaf, stem and root tissue . CBF interacts specifically with a 30 bp promoter fragment, referred to as cyt-1, of the Agrobacterium tumefaciens T-DNA cytokinin (T-cyt) gene . The T-cyt promoter, although of bacterial origin is active in planta and the 30 bp cyt-1 element is located within a region that is essential for T-cyt promotor activity in leaf, stem and root cells of tobacco plants . Gel retardation assays using different synthetic oligonucleotides and methylation interference experiments pinpointed the binding site of CBF to a GC-rich sequence ATGCCCCACA within the cyt-1 element . Site-directed mutagenesis of the CBF binding site within the T-cyt promoter by using PCR resulted in an almost complete loss of T-cyt promoter activity in transgenic tobacco plants . In a gain-of-function experiment a hexamer of cyt-1 was shown to be able to confer leaf, stem and root expression when fused upstream of a TATA box containing -55 derivative of the T-cyt promoter . A mutant cyt-1 hexamer, defective in CBF binding, did not show activity above background levels . These results indicate that binding of CBF to the cyt-1 element is required for cyt-1 directed gene expression, suggesting that CBF might act as a transcriptional activator . Apart from the ASF-1 binding site of the CaMV 35S promoter, which is also present in the T-DNA nopaline and octopine synthase genes, the cyt-1 element is the only other identified element reported until now that in combination with a TATA box is sufficient to drive gene expression in multiple tobacco tissue types.

Plant J, 1993 Sep, 4(3), 495 - 505
Tissue-specific and wound-inducible pattern of expression of the mannopine synthase promoter is determined by the interaction between positive and negative cis-regulatory elements; Guevara-Garcia A et al.; In transgenic tobacco plants the regulatory region of the Agrobacterium genes involved in mannopine synthesis (pmas) directs expression in vascular tissues and is induced upon wounding . To identify cis-acting sequences required for the cell-type-specific and wound-inducible expression of this regulatory region, the expression pattern of chimeric genes in which 5' upstream deletions of the pmas 1' promoter direct the expression of the beta-glucuronidase coding sequence, was analyzed in transgenic tobacco plants . It was found that the pmas1' promoter is specifically expressed in phloem cells and that this tissue-specific pattern of expression is the result of the interaction of at least two negative regulatory elements with a positive element that directs expression in many cell types . It was also found that wound induction is mediated by two different mechanisms, one of them involving a dramatic change in tissue-specific expression.

Plant J, 1993 Sep, 4(3), 433 - 43
Does the ocs-element occur as a functional component of the promoters of plant genes?
Ellis JG, Tokuhisa JG, Llewellyn DJ, Bouchez D, Singh K, Dennis ES, Peacock WJ.
The structural requirements of the ocs-element, a promoter element in several genes transferred to the host plant nucleus by Agrobacterium tumefaciens and certain DNA viruses, have been further characterized both in vitro and in vivo . Two adjacent and functionally identical protein-binding sites separated by an exact number of nucleotides are required for in vivo activity of the ocs-element . Plant pathogens have presumably recruited cellular transcription factors that interact with these binding sites to drive the high-level expression of their essential genes . Our functional analyses of the ocs-elements from two pathogen promoters define the structure of a sequence motif that might also be expected to occur in plant nuclear genes, and a search of the plant gene database has identified a number of plant gene promoters that contain sequences that resemble the ocs-element . These sequences were analysed for their ability both to bind the maize nuclear protein OCSTF and to activate transcription of an inactive promoter . A functional ocs-element was identified in only one of the plant genes, the soybean heat-shock gene, Gmhsp26-A . The apparent rarity of the ocs-element in plant genes contrasts with its frequent use by pathogens that transform the plant nucleus . Sequences resembling half of an ocs-element, on the other hand, are common in plant promoters and may form part of multi-element control motifs with a variety of regulatory functions . Plant pathogens may, therefore, have evolved to circumvent tight regulatory control of their promoters by the host by duplicating the half ocs-element promoter motifs to take advantage of the ubiquitous ocs-element-binding transcription factors in plants.

Mikrobiol Z, 1993 Sep-Oct, 55(5), 59 - 68
{The antimicrobial spectrum of the action of bacteriocins and bacteriophages from Klebsiella strains}; Sharga BM et al.; The percentage of bacteriocin-producing and phage-producing Klebsiella strains was as follows: K . pneumoniae-10%, K . ozaenae-7%, K . rhinoscleromatis-9% . The antimicrobial spectrum of the studied inducible particler was broad and was not limited by the frames of the genus and family . Bacteriocins and bacteriophages from Klebsiella were active to Klebsiella, Enterobacter, Escherichia, Shigella and Proteus representatives significant in medicine . Klebocins and Klebsiella phages exhibited antagonistic effects to phytopathogenic bacteria . Some strains of Erwinia and Pseudomonas were sensitive to phages or bacteriocins from Klebsiella . Bacteriocins protected corn and tomato seeds from contamination by erwinioses agents . All cultures of Agrobacterium, Corynebacterium, Micrococcus, Staphylococcus, Streptococcus were resistant to action of phages and klebocins . Bacteriocins from Klebsiella were assayed for their sensitivity to trypsin, chymotrypsin, lysozyme, ribonuclease, deoxyribonuclease . Action of klebocins was associated with a protein component . Proceeding from data of diffusion through the disc ultrafiltration membranes molecular weight of klebocins was in the range of 30,000 and 50,000 Da.

Mol Microbiol, 1993 Sep, 9(6), 1123 - 30
Processes at the nick region link conjugation, T-DNA transfer and rolling circle replication; Waters VL et al.; Data from prokaryotic replicative and conjugative systems, which interrelate DNA processing events initiated by a site-specific nick, are reviewed . While the replicative systems have been established in accordance with the rolling circle replication model, the mechanism of conjugative replication has not been elucidated experimentally . We summarize data involving random point mutagenesis of the RK2 transfer origin (oriT), which yielded relaxation-deficient and transfer-deficient derivatives having mutations exclusively in a 10bp region defined as the nick region . Features of the RK2 (IncP) nick region, including the DNA sequence, nick site position, and 5' covalent attachment of the nicking protein, have striking parallels in other systems involving nicking and mobilization of single-stranded DNA from a supercoiled substrate . These other systems include T-DNA transfer occurring in Agrobacterium tumefaciens Ti plasmid-mediated tumorigenesis in plants, and the rolling circle replication of plasmids of Gram-positive bacteria and of phi X174-like bacteriophage . The structural and functional similarities suggest that IncP conjugative replication, originating at the oriT, and T-DNA transfer replication, originating at the T-DNA border, produce continuous strands via a rolling circle-type replication.

Biotechnology (N Y), 1993 Sep, 11(9), 1048 - 52
Transgenic potato plants expressing mammalian 2'-5' oligoadenylate synthetase are protected from potato virus X infection under field conditions; Truve E et al.; We cloned and sequenced a rat cDNA encoding the 2'-5' oligoadenylate synthetase, a component of the mammalian interferon-induced antiviral response, and used Agrobacterium-mediated transformation to generate transgenic potato clones expressing this mammalian enzyme . In transgenic plants infected with potato virus X and followed under field conditions, virus concentrations in leaves and in tubers were significantly lower than in nontransgenic controls . Additionally, virus concentration in the leaves of five transgenic clones and in tubers of one clone was also lower than in transgenic potatoes expressing potato virus X coat protein.

Cell Mol Biol (Noisy-le-grand), 1993 Sep, 39(6), 575 - 81
Preparation, optimization and characterization of a polyphenylalanine synthetizing system from Agrobacterium tumefaciens; Alegre C et al.; A very active cell-free translation system was prepared from Agrobacterium tumefaciens, a bacterium broadly used to transfect plant cells to introduce foreign genes and one that produces tumours in plants . Once optimized for Mg2+, NH4+, high speed supernatant S 370, purified ribosomes and time, the system translates polyuridylic acid very efficiently . A . tumefaciens purified ribosomes were inhibited in vitro by several well-known translational inhibitors including some ribosome-inactivating proteins . Treatment of A . tumefaciens purified ribosomes with type 1-RIP crotin 2 lead to the depurination of the 23S rRNA which, upon treatment with acid aniline, released a diagnostic RNA fragment of about 235 nucleotides.

Gene, 1993 Aug 16, 130(1), 91 - 8
Sequence of a staphylococcal gene, vat, encoding an acetyltransferase inactivating the A-type compounds of virginiamycin-like antibiotics; Allignet J et al.; The Staphylococcus aureus plasmids, pIP680 and pIP1156, which confer resistance to A-type compounds of virginiamycin-like antibiotics (Vml: streptogramin A, pristinamycin IIA, virginiamycin M) and to synergistic mixtures of the A and B compounds of Vml antibiotics, were shown to direct the modification of A-type compounds by acetylation . The vat gene, encoding the acetyltransferase modifying A-type compounds, was isolated from plasmid pIP680 and sequenced . This gene potentially encodes a 219-amino-acid (aa) protein, VAT, of 24 330 Da showing at least 38% aa identity with two chloramphenicol acetyltransferases encoded by cat genes isolated from Escherichia coli and Agrobacterium tumefaciens . Resistance to A-type compounds of Vml antibiotics conferred to S . aureus by vat was not expressed in E . coli, although a protein having a M(r) similar to that encoded by this gene was detected in E . coli minicells . The vat gene was detected by the polymerase chain reaction in two chromosomally located staphylococcal conjugative elements and in the conjugative plasmid, pIP1156, conferring resistance to A-type compounds.

J Gen Microbiol, 1993 Aug, 139 ( Pt 8), 1767 - 72
Characterization of an IS-like element from Mycobacterium tuberculosis; Mariani F et al.; A DNA sequence, present in members of the Mycobacterium tuberculosis complex, has been identified and characterized . The distribution of this DNA sequence among mycobacterial species was analysed by DNA hybridization and PCR experiments . As the sequence was detected only in bacteria belonging to the M . tuberculosis complex, it may be useful for the rapid discrimination of mycobacteria . Interestingly, the sequence has some characteristics of an insertion element (IS) and codes for a hypothetical protein with significant homologies to proteins encoded by several IS elements of other organisms, namely IS427 and IS869 from Agrobacterium tumefaciens, IS402 from Pseudomonas cepacia, Tn4811 from Streptomyces lividans and ISRm4 from Rhizobium meliloti . Together, these elements form a previously unrecognized family of transposable elements . This finding suggests the possibility of horizontal gene transfer between pathogenic mycobacteria and other organisms including Gram-negative plant-pathogenic bacteria.

FEMS Microbiol Lett, 1993 Aug 1, 111(2-3), 287 - 94
Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens; Shirasu K et al.; The virB operon of the Agrobacterium tumefaciens Ti plasmid encodes 11 proteins . Specific antisera to VirB2, VirB3 and VirB9 were used to locate these virulence proteins in the A . tumefaciens cell . Immunoblot analysis located VirB2 protein to the inner and outer membranes; VirB3 and VirB9 were likewise associated with both membranes, but mainly in the outer membrane . VirB2 is processed from a 12.3-kDa protein into a 7.2-kDa polypeptide . Such sized protein results from cleavage at residue Ala47, upstream of which two additional alanine residues Ala45-Ala46 are contained and bearing resemblance to a signal peptide peptidase-I cleavage sequence . VirB2 and VirB3 sequences are strikingly similar to the pilin biosynthetic proteins TraA and TraL encoded by the tra operon of F and R1-19 plasmids . Since traA encodes a propilin that is cleaved into a 7.2-kDa conjugative pilin product and since this cleavage site is present in both TraA and VirB2, we propose that virB2 encodes a pilin-like protein which together with VirB3 and VirB9 as well as other VirB proteins may be used for interkingdom T-DNA transfer between bacteria and plants.

Plant Mol Biol, 1993 Aug, 22(5), 923 - 9
In planta analysis of the Agrobacterium tumefaciens T-cyt gene promoter: identification of an upstream region essential for promoter activity in leaf, stem and root cells of transgenic tobacco; Neuteboom ST et al.; The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a beta-glucuronidase (gusA) reporter gene and introduced into tobacco plants . Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants . Developmental stage-dependent promoter activity was observed in seedlings . Analysis of 5'-deleted promoter fragments showed that sequences located between positions -185 and -139 with respect to the T-cyt translational start codon are essential for T-cyt promoter activity in transfected tobacco protoplasts as well as in transformed tobacco plants.

Plant Mol Biol, 1993 Aug, 22(5), 907 - 12
A chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant cells; Goddijn OJ et al.; A novel selection system for plant genetic transformation was developed based on the enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus . This enzyme converts the toxic tryptophan analogue 4-methyl tryptophan (4-mT) into the non-toxic compound 4-methyl tryptamine . Expression of tdc in transgenic plants that have no endogenous TDC-activity allows selection on 4-mT . A vector was constructed containing a tdc cDNA clone under control of the constitutively expressed cauliflower mosaic virus 35S promoter . This vector was used in Agrobacterium-mediated tobacco leaf disc transformation experiments . The optimal concentration for selection with 4-mT was found to be 0.1 mM . The transformed nature of shoots obtained after tdc gene transfer and subsequent selection on 0.1 mM 4-mT was confirmed by northern blot analysis.

Plant Mol Biol, 1993 Aug, 22(5), 751 - 65
Investigation of the mechanism underlying the inhibitory effect of heterologous ras genes in plant cells; Liu ZR et al.; The ras genes from yeast and mammalian cells were fused to plant expression promoters, and introduced into plant cells via Agrobacterium, to study their effect on cell growth and development . All introduced ras genes had a strong inhibitory effect on callus and shoot regeneration from plant tissues . This is consistent with earlier findings that heterologous ras genes were highly lethal to protoplasts following direct DNA uptake . These effects could not be reversed by increasing exogenous or endogenous cytokinin levels . These effects were also independent of the v-Ha-ras mutations in functionally important regions of Ras proteins such as effector-binding and membrane-binding sites . Similarly, co-transformation with the genes encoding the Ras-negative regulators, GTPase-activating protein and neurofibromin did not affect the ras inhibitory effect, indicating that the mechanism of ras inhibition of plant cells is not related to normal ras cellular functions . This conclusion was supported by further studies in which ras gene expression was modified using various promoters and antisense constructs . The introduced ras sequences remained fully inhibitory regardless of which promoters (inducible or tissue-specific) or which orientations (sense or antisense) were tested . This strongly suggests that the ras DNA sequence itself, rather than the Ras protein or ras mRNA, is directly involved in the inhibitory effect . The mechanism underlying this novel phenomenon remains unknown . Introduced ras genes may inhibit plant cell growth by inducing co-suppression of unknown endogenous ras or ras-related genes, thereby leading to the arrest of cell growth.

Plant Mol Biol, 1993 Aug, 22(5), 731 - 49
Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants; Wyatt RE et al.; Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes beta-galactosidase, and a polyadenylation 3'-untranslated nopaline synthase sequence from Agrobacterium . These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses . A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5' promoter tested, a 3.0 kb fragment . Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin . LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments . Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament . The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature . Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypocotyls . The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation . Staining was evident in cell types that develop lignified cell walls, e.g . trichomes, anther endothecial cells, and especially developing xylem . The chimeric gene was not expressed in primary meristems . While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged . Transgenic plants with a 600 bp promoter construct (-0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct . Transgenic plants with a -500 bp promoter had levels of expression similar to the -3.0 kb construct . The -0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 x 10(-6) to 5 x 10(-5) M 2,4-D and was responsive to as little as 5 x 10(-8) M . The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.

J Bacteriol, 1993 Aug, 175(16), 5233 - 41
Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure; Thorstenson YR et al.; Plant cell transformation by Agrobacterium tumefaciens involves the transfer of a single-stranded DNA-protein complex (T-complex) from the bacterium to the plant cell . One of the least understood and important aspects of this process is how the T-complex exits the bacterium . The eleven virB gene products have been proposed to specify the DNA export channel on the basis of their predicted hydrophobicity . To determine the cellular localization of the VirB proteins, two different cell fractionation methods were employed to separate inner and outer membranes . Seven VirB-specific antibodies were used on Western blots (immunoblots) to detect the proteins in the inner and outer membranes and soluble (containing cytoplasm and periplasm) fractions . VirB5 was in both the inner membrane and cytoplasm . Six of the VirB proteins were detected in the membrane fractions only . Three of these, VirB8, VirB9, and VirB10, were present in both inner and outer membrane fractions regardless of the fractionation method used . Three additional VirB proteins, VirB1, VirB4, and VirB11, were found mainly in the inner membrane fraction by one method and were found in both inner and outer membrane fractions by a second method . These results confirm the membrane localization of seven VirB proteins and strengthen the hypothesis that VirB proteins are involved in the formation of a T-DNA export channel or gate . That most of the VirB proteins analyzed are found in both inner and outer membrane fractions suggest that they form a complex pore structure that spans both membranes, and their relative amounts in the two membrane fractions reflect their differential sensitivity to the experimental conditions.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7346 - 50
Nonreciprocal homologous recombination between Agrobacterium transferred DNA and a plant chromosomal locus; Offringa R et al.; Previously, we demonstrated the occurrence of gene targeting in tobacco cells after Agrobacterium-mediated transformation . In these experiments a defective kanamycin resistance (Kmr) gene residing at a chromosomal location was restored via homologous recombination with an incoming transferred DNA (T-DNA) repair construct (pSDM101) containing a different defective Kmr gene . In this article we describe gene targeting experiments with the same target line, but using an improved repair construct, pSDM321 . In one of the Kmr calli obtained after transformation with pSDM321 (line A) the product of homologous recombination was detected using PCR . Further molecular analysis revealed that the defective Kmr gene present on the incoming T-DNA had been restored via homologous recombination with the target locus . The target locus was left unchanged and the corrected T-DNA was found to be inserted on the same chromosome but not close to the target locus . This paper presents molecular evidence in plants for the conversion of an introduced DNA molecule (in this case, T-DNA) by a homologous chromosomal locus.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 7089 - 93
Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein; Lodge JK et al.; Exogenous application of pokeweed antiviral protein (PAP), a ribosome-inhibiting protein found in the cell walls of Phytolacca americana (pokeweed), protects heterologous plants from viral infection . A cDNA clone for PAP was isolated and introduced into tobacco and potato plants by transformation with Agrobacterium tumefaciens . Transgenic plants that expressed either PAP or a double mutant derivative of PAP showed resistance to infection by different viruses . Resistance was effective against both mechanical and aphid transmission . Analysis of the vacuum infiltrate of leaves expressing PAP showed that it is enriched in the intercellular fluid . Analysis of resistance in transgenic plants suggests that PAP confers viral resistance by inhibiting an early event in infection . Previous methods for creating virus-resistant plants have been specific for a particular virus or closely related viruses . To protect plants against more than one virus, multiple genes must be introduced and expressed in a single transgenic line . Expression of PAP in transgenic plants offers the possibility of developing resistance to a broad spectrum of plant viruses by expression of a single gene.

J Bacteriol, 1993 Aug, 175(15), 4790 - 9
Dynamic structure of Agrobacterium tumefaciens Ti plasmids; Fortin C et al.; Agrobacterium tumefaciens C58F is a variant of strain C58 which generates a high proportion of avirulent mutants in the presence of the virulence (vir) gene inducer acetosyringone . These mutants are altered in the Ti plasmid and do not respond to the acetosyringone signal (C . Fortin, E . W . Nester, and P . Dion, J . Bacteriol . 174:5676-5685, 1992) . The physical organization of the Ti plasmid was compared in strain C58 and its variant . One feature distinguishing pTiC58F from its parent plasmid was the presence of the insertion element IS426 . Three copies of this element were detected in the strain C58 chromosome, whereas two additional copies were found in strain C58F, including one copy in the Ti plasmid . This particular copy of IS426 was associated with the region of arginine and nopaline catabolism of pTiC58F . Most of the avirulent mutants recovered following growth of strain C58F in the presence of acetosyringone were complemented by clones carrying either virA or virG . Element IS426 was no longer found in the arginine and nopaline catabolism region of the Ti plasmids from the virA and virG mutants, but it resided in the particular KpnI fragment containing the modified vir locus . Behavior of a strain C58F derivative, which was inactivated in a chromosomal component required for the response to acetosyringone, was consistent with the possibility that vir gene induction is essential to the massive production of avirulent mutants.

Mol Microbiol, 1993 Aug, 9(4), 803 - 12
The virD4 gene is required for virulence while virD3 and orf5 are not required for virulence of Agrobacterium tumefaciens; Lin TS et al.; The virD operon of the resident Ti plasmid of Agrobacterium tumefaciens contains loci involved in T-DNA processing and undefined virulence functions . Nucleotide sequence of the entire virD operon of pTiC58 revealed similarities to the virD operon of the root-inducing plasmid pRiA4b and to that of the octopine-type plasmid pTiA6NC . However, comparative sequence data show that virD of pTiC58 is more akin to that of the pRiA4b than to that of the pTiA6NC . T7f10::virD gene fusions were used to generate polypeptides that confirm the presence of four open reading frames virD1, virD2, virD3, and virD4 within virD which have a coding capacity for proteins of 16.1, 49.5, 72.6, and 73.5 kDa, respectively . virD3 therefore encodes a polypeptide 3.4 times larger (72.6 versus 21.3 kDa) than that encoded by virD3 of octopine Ti plasmids . Non-polar virD4 mutants could not be complemented by a distant homologue, TraG protein of plasmid RP4 . An independently regulated fifth ORF (orf5) is located immediately downstream of 3' end of virD4 and encodes a polypeptide of 97.4 kDa . The expression of orf5 is dependent on its own promoter and is independent of acetosyringone induction in A . tumefaciens . Recently, it has been shown that virD3 of octopine Ri or Ti plasmids is not required for virulence . In this report, we confirm and extend these findings on a nopaline Ti plasmid by using several virD non-polar mutants that were tested for virulence . virD3 and orf5 non-polar mutants showed no effect on tumorigenicity on 14 different plant species, while virD4 mutants lost their tumorigenicity completely on all these test plants . These data suggest that virD3 and orf5 are not essential for virulence whereas virD4 is absolutely required on a wide range of host plants.

Mol Biol (Mosk), 1993 Jul-Aug, 27(4), 947 - 51
{Testing transgenic plants using the polymerase chain reaction}; Padegimas L et al.; A test system for selecting transgenic plants based on polymerase chain reaction (PCR) has been proposed . It is applicable to primary screening of transgenic plants obtained by cocultivation with Agrobacterium which contains any vector carrying neomycin phosphotransferase genes from transposon Tn5 and Streptococcus (for example pBIN19) . These genes confer kanamycin resistance in plants and bacteria respectively . The absence of strong homology between these two genes allows one to perform two PCRs in the same reaction mixture . Thus simultaneous selection of transgenic plants and test for contamination with Agrobacterium are possible . We have also proposed a simple procedure for preparing small samples of plant DNA suitable for PCR detection.

Appl Environ Microbiol, 1993 Jul, 59(7), 2112 - 20
Fate of Agrobacterium radiobacter K84 in the environment; Stockwell VO et al.; Agrobacterium radiobacter K84 is an effective, commercially applied, biological control agent for the plant disease crown gall, yet little is known about the survival and dissemination of K84 . To trace K84 in the environment, spontaneous antibiotic-resistant mutants were used . Growth rates and phenotypes of streptomycin- or rifampin-resistant K84 were similar to those of the parental K84, except the rifampin-resistant mutant produced less agrocin 84 as determined by bioassay . K84 and a strain of Agrobacterium tumefaciens established populations averaging 10(5) CFU/g in the rhizosphere of cherry and persisted on roots for 2 years . K84 established rhizosphere populations between 10(4) and 10(6) CFU/g on cherry, ryegrass, and 11 other herbaceous plants . Populations of K84 declined substantially in fallow soil or water over a 16-week period . K84 was detected in the rhizosphere of ryegrass located up to 40 cm from an inoculum source, indicating lateral dissemination of K84 in soil . In gall tissue on cherry, K84 established populations of 10(5) CFU/g, about 10- to 100-fold less than that of the pathogen . These data demonstrate that K84 persists for up to 2 years in a field environment as a rhizosphere inhabitant or in association with crown gall tissue.

Plant J, 1993 Jul, 4(1), 29 - 40
Analysis of a desiccation and ABA-responsive promoter isolated from the resurrection plant Craterostigma plantagineum; Michel D et al.; The resurrection plant Craterostigma plantagineum can recover from severe desiccation within 24 h of contact with water, and it is used as a model system to analyse desiccation tolerance in higher plants . During drying or ABA treatment a specific set of transcripts accumulates rapidly in leaves and other tissues . In order to study transcriptional mechanisms of stress-induced gene expression one gene (CDeT27-45) was selected for promoter analysis . Chimeric gene fusions were constructed of the CDeT27-45 promoter and beta-glucuronidase or luciferase . These constructs were tested in a homologous transient expression system which allowed the identification of promoter elements conferring ABA inducibility . By introducing the chimeric gene fusions into tobacco via Agrobacterium-mediated transformation we found that the promoter activity is under strict tissue-specific and developmental control . In tobacco the promoter was only active in developing embryos and in mature pollen grains-two tissues which are naturally desiccation tolerant in tobacco . The specific temporal expression pattern was attributed to particular 5' upstream sequences . The promoter analysis presented here should allow the separation of important regulatory components as a first step in dissecting events in the signal transduction chain.

Mol Gen Genet, 1993 Jul, 240(1), 49 - 57
Rapid divergence of Agrobacterium vitis octopine-cucumopine Ti plasmids from a recent common ancestor; van Nuenen M et al.; The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules . Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHm1 . Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures . One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9-2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type . The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes . These results have important implications for studies on bacterial evolution.

J Bacteriol, 1993 Jun, 175(11), 3486 - 90
Analysis of the Ros repressor of Agrobacterium virC and virD operons: molecular intercommunication between plasmid and chromosomal genes; D'Souza-Ault MR et al.; The virulence genes of the Agrobacterium tumefaciens Ti plasmid are regulated both positively and negatively . The products of the genes of the virC and virD operons play an important role in host specificity and T-DNA processing . These operons are transcribed in opposite directions and therefore bear diametrically oriented promoters . These promoters are positively regulated by the VirG protein, which is believed to be activated through phosphorylation by a histidine kinase encoded by the virA gene . The virC and virD operons are also regulated by a 15.5-kDa repressor protein encoded by the ros chromosomal gene . A mutation in ros causes the constitutive expression of virC and virD in the complete absence of the VirG protein . It appears, therefore, that the Ros repressor interacts with the regulatory region of these operons . The Ros repressor is shown here to bind to an upstream sequence (Ros box) comprising 40 bp bearing a 9-bp inverted repeat, TATATTTCA/TGTAATATA, in the promoter region of these operons . The affinity for this sequence is specific and tenacious, since the addition of at least a 20,000-fold excess of competitor DNA failed to remove the Ros protein coding sequence from the Ros box . DNase I footprint analysis showed that the Ros box overlaps the binding site of VirG (Vir box) . This result suggests that virC and virD transcription is modulated by Ros and VirG proteins.

Plant Mol Biol, 1993 Jun, 22(3), 491 - 506
Agrobacterium-mediated production of transgenic rice plants expressing a chimeric alpha-amylase promoter/beta-glucuronidase gene; Chan MT et al.; We have successfully transferred and expressed a reporter gene driven by an alpha-amylase promoter in a japonica type of rice (Oryza sativa L . cv . Tainung 62) using the Agrobacterium-mediated gene transfer system . Immature rice embryos (10-12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of beta-glucuronidase (uidA) and neomycin phosphotransferase (nptII) . Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice . The uidA and nptII genes, which are under the control of promoters of a rice alpha-amylase gene (alpha Amy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants . Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis . Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice alpha-amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves . This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant . Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis . These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.

Mol Gen Genet, 1993 Jun, 239(3), 345 - 53
The VirD2 protein of Agrobacterium tumefaciens carries nuclear localization signals important for transfer of T-DNA to plant; Rossi L et al.; Agrobacterium tumefaciens is able to transfer a piece of DNA, the T-DNA, to the nucleus of the plant cell . The VirD2 protein is required for the production of the T-DNA, it is tightly linked to the T-DNA and it is thought to direct it to the plant genome . Two nuclear localization signals (NLS), one in the N-terminal part and one in the C-terminal part of the VirD2 protein, have been shown to be able to target marker proteins to the plant nucleus . Here we analyze nuclear entry of the T-DNA complex using a new and very sensitive assay for T-DNA transfer . We show that optimal T-DNA transfer requires the VirD2 NLS located in the C-terminal part of the protein, whereas mutations in the N-terminal NLS coding sequence seem to have no effect on T-DNA transfer.

Nature, 1993 May 6, 363(6424), 69 - 71
Transgenic N . glauca plants expressing bacterial virulence gene virF are converted into hosts for nopaline strains of A . tumefaciens; Regensburg-Tuink AJ et al.; Tumours are induced by Agrobacterium tumefaciens on a variety of plants . The virulence determinants of A . tumefaciens reside on a large tumour-inducing (Ti) plasmid . This plasmid carries two regions essential for tumour induction, namely the T region and the Vir region . During infection the T region is transferred to the plant cell, where it becomes stably integrated in one of the host chromosomes as T-DNA . Expression of T-DNA leads to the production of the plant hormones auxin and cytokinin, as well as to the synthesis of specific amino-acid derivatives termed opines . Agrobacterium strains are classified according to the types of opines produced by the tumours they induce . The Vir region contains genes that are expressed in the bacterium and are required for T-DNA transfer to plant cells, and several other genes that affect the efficiency of transfer and the host range . Vir regions from different Ti plasmids may vary slightly in the genes they contain: for instance, the virF gene, which is present in the Vir-region of octopine Ti plasmids, is absent from nopaline Ti plasmids . Mutation of the virF gene leads to a weakened virulence of octopine strains on tomato and Nicotiana glauca (shrub tobacco) . Nopaline strains are strongly attenuated in N . glauca compared with octopine strains because of the absence of the virF virulence gene from the Ti plasmid in nopaline strains . The virF gene product may be transferred to and be active in plant cells . Here we isolate transgenic N . glauca plants in which the virF coding sequence is expressed using the cauliflower mosaic virus 35S promoter . The presence of the VirF protein converts the non-host N . glauca into a host for tumour formation by A . tumefaciens nopaline strains and octopine virF mutants . Our results indicate that certain virulence gene products such as the VirF protein may be transferred to plant cells during tumour induction, where they function as mediators of T-DNA transfer.

J Virol Methods, 1993 May, 42(2-3), 227 - 39
Transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA of tobacco mosaic virus; Turpen TH et al.; We engineered cDNA of tobacco mosaic tobamovirus (TMV) into Agrobacterium tumefaciens for inoculation of plant cells . The resulting bacterial strains were used to transfect tobacco (Nicotiana tabacum cv . Xanthi and Xanthi/nc) with wild type and a defective virus . Lesion formation on Xanthi/nc tobacco was used to measure the timing and efficiency of transfection . Infections mediated by Agrobacterium produced lesions an average of two days later than infections produced by inoculation with virions . The addition of approximately 80 bp of non-viral sequences to the 5'-end of TMV transcripts abolished transfection . Transcripts with non-viral sequences at the 3'-end initiated infections, while precise transcript termination with a synthetic ribozyme sequence increased transfection frequencies two-fold . Culture conditions reported to induce genes of the vir region of the Agrobacterium Ti plasmid also increased the transfection frequency approximately two-fold . Therefore, in addition to the pararetroviruses and geminiviruses previously described, 'agroinoculation' may be used to infect plants with plus-sense RNA viruses.

Mol Gen Genet, 1993 May, 239(1-2), 225 - 34
Multiple regions of a divergent promoter control the expression of the Agrobacterium rhizogenes aux1 and aux2 plant oncogenes; Gaudin V et al.; The two auxin biosynthesis genes, aux1 and aux2 of Agrobacterium rhizogenes strain A4, are located on opposite DNA strands with a short integenic region (394 bp) between their coding sequences . A functional analysis of this divergent promoter is presented . The transcription initiation sites of the two aux genes were determined and regions important for promoter activity were identified by deletion and transient expression analyses in tobacco protoplasts . The promoter activity of the aux intergenic region was demonstrated . A strong enhancer element contained within an 84 bp promoter fragment was identified . Far upstream regions were shown to have negative effects on the promoter activity of the short intergenic region . Interactions between positive elements in the intergenic region and negative effects of the upstream sequences may be the basis of strict control of the auxin biosynthesis necessary for the induction and maintenance of hairy root growth.

Eur J Biochem, 1993 May 1, 213(3), 1315 - 24
Purification and biochemical characterization of the hydantoin hydrolyzing enzyme from Agrobacterium species . A hydantoinase with no 5,6-dihydropyrimidine amidohydrolase activity; Runser SM et al.; A soluble hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from a newly isolated Agrobacterium species . This hydrolase consists of about 578 aminoacyl residues and is a slightly acidic protein with an isoelectric point of 6.5 . The first 22 N-terminal amino acid residues were determined by Edman degradation . Determination of the relative molecular mass of the protein by gel-filtration chromatography gave an apparent value of 250,000 . The subunit M(r) was 62,000, as estimated by analytical SDS/PAGE and 66,500, as estimated by denaturing gel-filtration chromatography . The pure hydantoinase exhibits the following hydrodynamic properties: a sedimentation coefficient of 8.8 S as determined by sedimentation velocity experiments; a Stokes radius of 6.8 nm; a diffusion coefficient of 31.5 microns2.s-1 as determined by analytical gel-filtration chromatography . From these experimental data, the following physical constants could be calculated: a theoretical M(r) of 265,000, a frictional ratio, f/fo, of 1.59, a maximal axial ratio, a/b, of 3.1; a Perrin shape factor, F, of 1.37 . As shown by different Km values, the preferred substrates of this hydrolase were 5-monosubstituted hydantoins bearing aromatic substituents . 5,5-Dimethylhydantoin and different thio analogs of the 5-p-hydroxyphenylhydantoin molecule are competitive inhibitors of this hydrolase . The classification of this microbial hydantoinase, which exhibits no hydrolytic activity with all the dihydropyrimidines tested, under the systematic name of 5,6-dihydropyrimidine amidohydrolase, and its putative metabolic role are further discussed.

J Bacteriol, 1993 May, 175(10), 3208 - 12
Isolation and characterization of a new chromosomal virulence gene of Agrobacterium tumefaciens; Wirawan IG et al.; A mutant (strain B119) of Agrobacterium tumefaciens with a chromosomal mutation was isolated by transposon (Tn5) mutagenesis . The mutant exhibited growth rates on L agar and minimal medium (AB) plates similar to those of the parent strain (strain A208 harboring a nopaline-type Ti plasmid) . The mutant was avirulent on all host plants tested: Daucus carota, Cucumis sativus, and Kalanchoe diagremontiana . The mutant was not impaired in attachment ability to carrot cells . The mutant had one insertion of Tn5 in its chromosome . The avirulent phenotype of B119 was shown to be due to the Tn5 insertion in the chromosome by the marker exchange technique . A wild-type target chromosomal segment (3.0 kb) which included the site of mutation was cloned and sequenced . Two open reading frames, ORF-1 (468 bp) and ORF-2 (995 bp), were identified in the 3.0-kb DNA segment . Tn5 was inserted in the middle of ORF-2 (acvB gene) . Introduction of the acvB gene into the mutant B119 strain complemented the avirulent phenotype of the strain . Homology search found no genes homologous to acvB, although it had some similarity to the open reading frame downstream of the virA gene on the Ti plasmid . Thus, the acvB gene identified in this study seems to be a new chromosomal virulence gene of A . tumefaciens.

J Bacteriol, 1993 May, 175(10), 3026 - 30
Cloning, sequencing, and transcriptional analysis of the gene coding for the vegetative sigma factor of Agrobacterium tumefaciens; Segal G et al.; The sigA gene of Agrobacterium tumefaciens was cloned and sequenced . Comparison with previously analyzed sigA genes revealed a high degree of similarity in nucleotide and amino acid sequences of regions two, three, and four of vegetative sigma factors . However, the upstream regulatory region shows no sequence homology with the Escherichia coli heat shock (sigma 32) promoters . It also does not contain the hairpin-loop structure (inverted repeat sequence) that was found in the upstream region of the groE operon in A . tumefaciens . The transcription initiation site of the gene was determined and found to be at the same position during normal growth and under heat shock conditions . Furthermore, no heat shock activation was observed at the transcriptional level.

J Bacteriol, 1993 May, 175(10), 3151 - 60
IS870 requires a 5'-CTAG-3' target sequence to generate the stop codon for its large ORF1; Fournier P et al.; The TB regions of the Agrobacterium vitis octopine/cucumopine Ti plasmids constitute a family of related structures . All contain a bacterial insertion element downstream of the TB-iaaM gene, IS870.1 . Whereas 43 isolates with octopine/cucumopine Ti plasmids carry only one IS870 copy, strain Ag57 carries a second copy (IS870.2) 3.9 kb to the right of IS870.1 and part of the same TB region . Two other octopine/cucumopine strains carry an IS870 copy on their chromosome (IS870.3) . A study of the unmodified insertion sites of IS870.2 and IS870.3, cloned from closely related strains, enabled us to delimit the IS870 elements . IS870 has a size of 1,152 bp and is terminated by inverted repeats . It contains a large open reading frame without a stop codon . However, a stop codon is generated by insertion into the target sequence 5'-CTAG-3' . IS870 is related to five other insertion sequence elements . For two of these, the stop codon of the largest open reading frame is also created by insertion into a CTAG target site.

Mol Microbiol, 1993 May, 8(5), 915 - 26
Formation of a putative relaxation intermediate during T-DNA processing directed by the Agrobacterium tumefaciens VirD1,D2 endonuclease; Filichkin SA et al.; During the initial stages of crown gall tumorigenesis, the T-DNA region of the Agrobacterium tumefaciens Ti-plasmid is processed, resulting in the production of T-DNA molecules that are subsequently transferred to the plant cell . Processing of the T-DNA in the bacterium involves the nicking of T-DNA border sequences by an endonuclease encoded by the virD locus, and the subsequent tight (possibly covalent) association of the VirD2 protein with the 5' end of the processed single-stranded or double-stranded T-DNA molecule . To investigate the interaction of the VirD1,D2 endonuclease with a right T-DNA border, a set of plasmids containing both the border and virD sequences on the same high-copy-number replicon has been constructed and introduced into Escherichia coli . In this model system a tight nucleoprotein complex is formed between the relaxed double-stranded substrate plasmid and the VirD2 protein . This putative T-DNA processing complex may be analogous to the covalent relaxation complex formed between the pilot protein and plasmid DNA during bacterial conjugation . VirD2 attachment to the relaxed substrate plasmid was resistant to denaturing agents but sensitive to S1 nuclease digestion, indicating a single-stranded region near the site of protein attachment . We speculate that this structure may be an intermediate formed prior to T-strand unwinding from the substrate plasmid in a host bacterium.

Transgenic Res, 1993 May, 2(3), 170 - 80
Insect-resistant chrysanthemum calluses by introduction of a Bacillus thuringiensis crystal protein gene; van Wordragen MF et al.; A 3'-end truncated crystal protein gene, derived from Bacillus thuringiensis (Bt) subsp . aizawai 7.21, encoding the toxic fragment of the insecticidal protein cryIA(b), was constructed . The gene was inserted into a transformation vector, also carrying the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (gus) gene, and introduced in the oncogenic Agrobacterium tumefaciens strain A281, harbouring the Ti-plasmid pTiBO542 . The recombinant Agrobacterium strain was used to transform leaf explants of chrysanthemum (Dendranthema grandiflora) cultivar Parliament . The resulting tumours were kanamycin-resistant, exhibited beta-glucuronidase activity and produced agropine and mannopine . In most tumours, all simultaneously transferred genes were expressed, owing to selection for the presence of both T-DNAs, but no correlation was found between the level of expression of the various genes . A bioassay was developed, in which larvae were fed with tumorous chrysanthemum tissue, in order to detect the effect of the transferred toxin gene on larval development . Using this bioassay with second instar larvae of Heliothis virescens (tobacco budworm), 17 tumour lines were tested . Several of these lines proved to be strongly inhibitory to larval growth . These results indicate that Bt-based insect resistance might be used as a tool in reducing the amount of pesticides used in chrysanthemum culture.

Transgenic Res, 1993 May, 2(3), 141 - 6
The endosperm-specific expression of a rice prolamin chimaeric gene in transgenic tobacco plants; Zhou X et al.; The 5' upstream region (-680 to +40), containing the potential promoter and complete signal peptide coding sequence of the rice seed storage prolamin gene was amplified in vitro using the polymerase chain reaction from the genome of Chinese rice cultivar Zhonghua 8 . The physical map and DNA sequence analysis show strong homology with the 5' flanking region of the rice prolamin gene published by Kim and Okita (1988a) . No change in the signal peptide coding sequence and a long leader sequence with several small open reading frames were found . The chimaeric gene containing the 5' flanking region of the prolamin gene (-680 to -18) was transcriptionally fused with the beta-glucuronidase (GUS) reporter gene and the fusion junction was confirmed by both physical mapping and DNA sequence analysis . The resultant chimaeric gene was used to transform tobacco explants, using the Ti binary system of Agrobacterium tumefaciens LBA4404 . Three transgenic tobacco plants with as many as 20 copies of the chimaeric GUS gene (confirmed by dot and Southern hybridization) were analysed further . Histochemical analysis revealed GUS activity in the endosperm tissue of tobacco seed at the developmental stage about 20 days after flowering (DAF) . No GUS activity was found in leaves, stems, roots and flowers of the transgenic tobacco plants . Therefore, we conclude that the 5' upstream region from -680 to -18 was sufficient to confer the endosperm-specific expression of the rice prolamin gene.

Mol Microbiol, 1993 May, 8(3), 443 - 56
Molecular cloning and characterization of 13 out genes from Erwinia carotovora subspecies carotovora: genes encoding members of a general secretion pathway (GSP) widespread in gram-negative bacteria; Reeves PJ et al.; The chemical mutagen ethylmethanesulphonate (EMS) has been used to generate mutants of Erwinia carotovora subspecies carotovora which are defective in the secretion of pectinases (Pel) and cellulases (Cel) but unaltered for protease (Prt) secretion . Such mutants, called Out-, still synthesize Pel and Cel but these enzymes accumulate within the periplasm . Cosmid clones carrying wild-type E . carotovora ssp . carotovora DNA, identified by their ability to restore the Out+ phenotype when transferred to some Out- mutants, were classified into six complementation groups using cosmids and cosmid derivatives . Analysis of the nucleotide sequence of a 12.7 kb DNA fragment, encompassing complementing cosmid inserts, revealed a coding capacity for 13 potential open reading frames (ORFs), and these were designated outC-outO . Some of the out gene products were visualized using a T7 gene 10 expression system . The predicted Out proteins are highly similar to components of extracellular enzyme secretion systems from a diverse range of eubacteria including Erwinia chrysanthemi, Klebsiella oxytoca, Aeromonas hydrophila, Pseudomonas aeruginosa and Xanthomonas campestris . Lower levels of similarity exist between Ecc Out proteins and components of macromolecular trafficking systems from Bacillus subtilis, Haemophilus influenzae, Agrobacterium tumefaciens, Yersinia pestis and a protein involved in the morphogenesis of filamentous bacteriophages such as M13.

Mol Microbiol, 1993 May, 8(3), 429 - 34
Pertussis toxin export requires accessory genes located downstream from the pertussis toxin operon; Covacci A et al.; Pertussis toxin, a major virulence factor of Bordetella pertussis, is an oligomeric protein composed of five different subunits that are exported individually to the periplasmic space by the signal peptide-dependent pathway . After assembly, the protein is exported from the periplasm to the extracellular compartment . We show that pertussis toxin secretion across the outer membrane requires the gene product of at least one gene (ptlC) that is located downstream from the pertussis toxin operon . The amino acid sequence of PtlC shows a high degree of homology to VirB4, a protein encoded by the virB operon, which contains 11 open reading frames that are involved in the transfer of T-DNA from Agrobacterium tumefaciens to the plant cells . This is a novel mechanism of protein export in Gram-negative bacteria.

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