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