J Bacteriol, 1991 May, 173(9), 3021 - 4 Biosynthesis and excretion of cyclic glucans by Rhizobium meliloti 1021; Geiger O et al.; Cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species play an important role in the interaction of these bacteria with plant hosts . In this study, we show that (i) the neutral cyclic glucans are the biosynthetic precursors of anionic cyclic glucans; (ii) the conversion of neutral to anionic glucans is much more rapid and more extensive in exponentially growing cultures than in cultures in the stationary phase, although the latter synthesize large amounts of glucan; and (iii) the excretion of glucan, as well as the total amount synthesized, is strongly influenced by the medium. J Bacteriol, 1991 May, 173(9), 2833 - 41 Mannopine and mannopinic acid as substrates for Arthrobacter sp . strain MBA209 and Pseudomonas putida NA513; Nautiyal CS et al.; The characteristics of mannopine and mannopinic acid utilization by Agrobacterium tumefaciens B6S3, Arthrobacter sp . strain MBA209, and Pseudomonas putida NA513 were studied . Strain B6S3 utilized the four mannityl opines, mannopine, mannopinic acid, agropine, and agropinic acid . It also utilized several mannityl opine analogs, which were modified in either the sugar or the amino acid moiety . It utilized mannopine more rapidly after preincubation on mannopine, mannopinic acid, or glutamine than after pregrowth on glucose, mannose, or mannitol . Strains MBA209 and NA513 utilized mannopine and mannopinic acid, but not the other two mannityl opines . They utilized few mannityl opine analogs, sometimes because of failure to utilize the products of initial cleavage of the analog . Utilization of mannopine and mannopinic acid by strain NA513 was strictly dependent on prior growth on these substrates . A spontaneous regulatory variant of strain NA513 remained unable to utilize most of the mannityl opine analogs . Glutamine, mannose, and several analogs had no inhibitory effect on {14C}mannopine utilization by strain NA513. Eur J Clin Microbiol Infect Dis, 1991 May, 10(5), 450 - 2 Isolation of Agrobacterium radiobacter from a central venous catheter; Hammerberg O et al.; A case of septicemia caused by Agrobacterium radiobacter is reported in a patient undergoing chemotherapy treatment who had recently been neutropenic . Agrobacterium radiobacter was isolated from the Hickman line blood culture . The patient responded favorably to removal of the Hickman catheter and treatment with amikacin and piperacillin . The molecular and biochemical characteristics of the isolate are presented. Plant Mol Biol, 1991 May, 16(5), 853 - 64 35S-beta-glucuronidase gene blocks biological effects of cotransferred iaa genes; Tinland B et al.; The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease . The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM) . IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene . In functional studies on the activity of the iaa genes of the TB region of the A . tumefaciens biotype III strain Tm4, the frequently used 35S-beta-glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes . To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene . The 35S promoter alone is sufficient to cause the inhibitory effect. Plant Cell, 1991 May, 3(5), 461 - 71 Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2; Wilkinson JQ et al.; Chlorate, the chlorine analog of nitrate, is a herbicide that has been used to select mutants impaired in the process of nitrate assimilation . In Arabidopsis thaliana, mutations at any one of eight distinct loci confer resistance to chlorate . The molecular identities of the genes at these loci are not known; however, one of these loci--chl3--maps very near the nitrate reductase structural gene NIA2 . Through the isolation, characterization, and genetic analysis of new chlorate-resistant mutants generated by gamma irradiation, we have been able to demonstrate that the CHL3 gene and the NIA2 gene are identical . Three new chlorate-resistant mutants were identified that had deletions of the entire NIA2 gene . These nia2 null mutants were viable and still retained 10% of wild-type nitrate reductase activity in the leaves of the plants . All three deletion mutations were found to be new alleles of chl3 . Introduction of the NIA2 gene back into these chl3 mutants by Agrobacterium-mediated transformation partially complemented their mutant phenotype . From these data, we conclude that Arabidopsis has at least two functional nitrate reductase genes and that the NIA2 gene product accounts for the majority of the leaf nitrate reductase activity and chlorate sensitivity of Arabidopsis plants. Plant Mol Biol, 1991 May, 16(5), 807 - 20 Transgenic rice cell lines and plants: expression of transferred chimeric genes; Meijer EG et al.; Cell suspension-derived rice (Oryza sativa L.) protoplasts were transformed by direct gene uptake . PEG-mediated transformation was more efficient than electroporation . Plasmid DNA containing a hygromycin phosphotransferase (HPT) gene (which confers hygromycin resistance) driven by the CaMV 35S promoter and a beta-D-glucuronidase (GUS) gene under control of the 1', 2' double promoter of the mannopine synthase (mas) locus of Agrobacterium tumefaciens was introduced into rice protoplasts . Southern analysis of DNA from transformed cell lines showed that the HPT and GUS genes were present intact . Both genes were expressed in transgenic cell suspensions . GUS activity was detected by histochemical staining of the cells and by enzyme assays . During a 12-day culture period the proportion of stained cells rose to a maximum and then decreased again . Considerably higher numbers of blue-stained cells were obtained when the transgenic cell lines were grown in the presence of 5-azacytidine . Transcripts of the GUS gene could not be detected, in contrast with the HPT gene . Plantlets were regenerated from one transgenic cell line . GUS activity was found in both leaf and root tissues of these plants, particularly, but not exclusively, in vascular bundles . A mouse dihydrofolate reductase coding sequence (DHFR), conferring methotrexate resistance, fused to the CaMV 35S promotor and the wild-type nopaline synthase (NOS) gene of A . tumefaciens were also introduced into rice protoplasts . Stable integration of both genes was confirmed by Southern analysis . Expression of the DHFR gene was demonstrated by high levels of resistance to methotrexate of the transgenic cell suspensions and by the presence of DHFR transcripts . Expression of the NOS gene at enzyme or RNA level was not detected . Southern analysis suggests that this gene was probably either methylated or scrambled in these lines. Mol Gen Genet, 1991 Apr, 226(1-2), 250 - 6 Cloning and nucleotide sequence of the hemA gene of Agrobacterium radiobacter; Drolet M et al.; The hemA gene of Agrobacterium radiobacter ATCC4718 was identified by hybridization with a hemA probe from Rhizobium meliloti and cloned by complementation of a hemA mutant of Escherichia coli K12 . E . coli hemA transformants carrying the hemA gene of Agrobacterium showed delta-aminolevulinic acid synthetase (delta-ALAS) activity in vitro . The hemA gene was carried on a 4.4 kb EcoRI fragment which could be reduced to a 2.6 kb EcoRI-SstI fragment without affecting its complementing or delta-ALAS activity . The sequence of the hemA gene showed an open reading frame of 1215 nucleotides, which could code for a protein of 44,361 Da . This is very close to the molecular weight of the HemA protein obtained using an in vitro coupled transcription-translation system (45,000 Da) . Comparison of amino acid sequences of the delta-ALAS of A . radiobacter and Bradyrhizobium japonicum showed strong homology between the two enzymes; less, but still significant, homology was observed when A . radiobacter and human delta-ALAS were compared . Primer extension experiments enabled us to identify two promoters for the hemA gene of A . radiobacter . One of these promoters shows some similarity to the first promoter of the hemA gene of R . meliloti. J Bacteriol, 1991 Apr, 173(8), 2608 - 16 The virC and virD operons of the Agrobacterium Ti plasmid are regulated by the ros chromosomal gene: analysis of the cloned ros gene; Cooley MB et al.; The ros chromosomal gene is present in octopine and nopaline strains of Agrobacterium tumefaciens as well as in Rhizobium meliloti . This gene encodes a 15.5-kDa protein that specifically represses the virC and virD operons in the virulence region of the Ti plasmid . The ros gene was cloned from a genomic bank by electroporation and complementation in Agrobacterium cells . Reporter fusion to the ros gene indicates that the level of transcription is controlled in part by autoregulation . A consensus inverted repeat sequence present in the ros promoter and in the virC and virD promoters of pTiC58, pTiA6, and pRiA4b suggests that a specific Ros binding site exists in these promoters . In the virC and virD promoter region, this binding site is within a cluster of vir box consensus sequences in which the VirG protein binds . This suggests possible binding competition between Ros and VirG at the virC and virD promoters . That the Ros protein binds DNA is suggested by the presence of a 'zinc finger' consensus sequence in the protein. J Bacteriol, 1991 Apr, 173(7), 2155 - 9 Occurrence of lipid A variants with 27-hydroxyoctacosanoic acid in lipopolysaccharides from members of the family Rhizobiaceae; Bhat UR et al.; Lipopolysaccharides (LPSs) isolated from several strains of Rhizobium, Bradyrhizobium, Agrobacterium, and Azorhizobium were screened for the presence of 27-hydroxyoctacosanoic acid . The LPSs from all strains, with the exception of Azorhizobium caulinodans, contained various amounts of this long-chain hydroxy fatty acid in the lipid A fractions . Analysis of the lipid A sugars revealed three types of backbones: those containing glucosamine (as found in Rhizobium meliloti and Rhizobium fredii), those containing glucosamine and galacturonic acid (as found in Rhizobium leguminosarum bv . phaseoli, trifolii, and viciae), and those containing 2,3-diamino-2,3-dideoxyglucose either alone or in combination with glucosamine (as found in Bradyrhizobium japonicum and Bradyrhizobium sp . {Lupinus} strain DSM 30140) . The distribution of 27-hydroxyoctacosanoic acid as well as analysis of lipid A backbone sugars revealed the taxonomic relatedness of various strains of the Rhizobiaceae. Plant Mol Biol, 1991 Apr, 16(4), 601 - 14 Sequence of the iaa and ipt region of different Agrobacterium tumefaciens biotype III octopine strains: reconstruction of octopine Ti plasmid evolution; Paulus F et al.; The TA regions of biotype III octopine/cucumopine (OC) Ti plasmids are closely related to the TL region of the biotype I octopine Ti plasmids pTiAch5 and pTi15955 . Sequence analysis shows that the limited and wide host range biotype III OC TA regions are derived from a common ancestor structure which lacked the 6a gene found in the biotype I octopine TL region . The TA region of the wide host range OC Ti plasmids has conserved most of the original TL-like structure . In most wide host range OC isolates the TA-iaaH gene is inactivated by the insertion of an IS866 element . However, the TA region of the wide host range isolate Hm1 carries an intact TA-iaaH gene . This gene encodes a biologically active product, as shown by root induction tests and indole-3-acetic acid measurements . The limited host range OC Ti plasmids pTiAB3 and pTiAg57 have shorter TA regions which are derived from a wide host range TA region . The AB3 type arose by an IS868-mediated, internal TA region deletion which removed the iaa genes and part of the ipt gene and left a copy of IS868 at the position of the deleted fragment . The pTiAB3 iaa/ipt deletion was followed by insertion of a second IS element, IS869, immediately 3' of the ipt gene . pTiAg57 underwent the same iaa-ipt deletion as pTiAB3, but lacks the IS868 and IS869 elements . Analysis of the various TA region structures provides a detailed insight into the evolution of the biotype III OC strains. Mol Gen Genet, 1991 Apr, 226(1-2), 337 - 40 Homology of the ligand-binding regions of Rhizobium symbiotic regulatory protein NodD and vertebrate nuclear receptors; Gyorgypal Z et al.; The signal specificity and structure of sensor-activator proteins from different species (NodD of Rhizobium bacteria and vertebrate nuclear receptors) were compared . Several compounds (including flavonoids, coumestrol and estradiol) that bind to mammalian receptors also interact with NodD proteins . NodD-dependent synergism of the signal compounds luteolin and catechin was observed suggesting that these compounds bind directly to NodD . Two regions comprising 63 and 37 amino acids in NodD showed 45% and 36% homology, respectively, with the estrogen receptor . These regions, designated as modules M1 and M2, coincide with conserved parts of the ligand-binding domains of the nuclear receptors . A part of NodD overlapping with the M1 module was predicted to be membrane associated and was 46% homologous to a membrane-spanning sensory segment of the Agrobacterium VirA protein . We suggest that the homologous polypeptide modules detected in NodD and the nuclear receptors originate from a common ancestor protein and may be directly involved in ligand binding. Biotechnology (N Y), 1991 Apr, 9(4), 373 - 7 The development of virus-resistant alfalfa, Medicago sativa L; Hill KK et al.; We have generated more than 100 transgenic alfalfa plants, via Agrobacterium-mediated gene transfer, from genotypes selected from five alfalfa cultivars . These plants express the genes for kanamycin resistance and for the coat protein of alfalfa mosaic virus (AMV) . The strongest expressers accumulated nearly 500 ng coat protein per mg soluble leaf protein . AMV inoculation of protoplasts from these strong expressers indicated that they were resistant to infection by AMV, while protoplasts from plants containing about a hundred-fold less coat protein and from control untransformed plants were not . Transgenic alfalfa plants containing large amounts of coat protein were, likewise, resistant to AMV . These plants did not develop systemic infections following inoculation with up to 50 micrograms/ml AMV, while inoculated control plants developed systemic infections following inoculation with as little as 10 micrograms/ml AMV . These results demonstrate that expression of the AMV coat protein gene confers resistance to AMV infection in transgenic alfalfa plants. Microbiol Rev, 1991 Mar, 55(1), 35 - 58 Cellulose biosynthesis and function in bacteria; Ross P et al.; The current model of cellulose biogenesis in plants, as well as bacteria, holds that the membranous cellulose synthase complex polymerizes glucose moieties from UDP-Glc into beta-1,4-glucan chains which give rise to rigid crystalline fibrils upon extrusion at the outer surface of the cell . The distinct arrangement and degree of association of the polymerizing enzyme units presumably govern extracellular chain assembly in addition to the pattern and width of cellulose fibril deposition . Most evident for Acetobacter xylinum, polymerization and assembly appear to be tightly coupled . To date, only bacteria have been effectively studied at the biochemical and genetic levels . In A . xylinum, the cellulose synthase, composed of at least two structurally similar but functionally distinct subunits, is subject to a multicomponent regulatory system . Regulation is based on the novel nucleotide cyclic diguanylic acid, a positive allosteric effector, and the regulatory enzymes maintaining its intracellular turnover: diguanylate cyclase and Ca2(+)-sensitive bis-(3',5')-cyclic diguanylic acid (c-di-GMP) phosphodiesterase . Four genes have been isolated from A . xylinum which constitute the operon for cellulose synthesis . The second gene encodes the catalytic subunit of cellulose synthase; the functions of the other three gene products are still unknown . Exclusively an extracellular product, bacterial cellulose appears to fulfill diverse biological roles within the natural habitat, conferring mechanical, chemical, and physiological protection in A . xylinum and Sarcina ventriculi or facilitating cell adhesion during symbiotic or infectious interactions in Rhizobium and Agrobacterium species . A . xylinum is proving to be most amenable for industrial purposes, allowing the unique features of bacterial cellulose to be exploited for novel product applications. Mol Gen Genet, 1991 Mar, 225(3), 369 - 78 High-level expression of a sweet potato sporamin gene promoter: beta-glucuronidase (GUS) fusion gene in the stems of transgenic tobacco plants is conferred by multiple cell type-specific regulatory elements; Ohta S et al.; Genes coding for sporamin, the most abundant protein of the tuberous root of the sweet potato, are expressed at a high levels in the stems of plantlets cultured axenically on sucrose-containing medium . Their expression is also induced in leaf-petiole explants by high concentrations of sucrose . A fusion gene comprising of the 1 kb 5' upstream region of the gSPO-A1 gene coding for the A-type sporamin and the coding sequence of bacterial beta-glucuronidase (GUS) was introduced into the tobacco genome by Agrobacterium-mediated transformation . Transgenic tobacco plants cultured axenically on sucrose-containing medium expressed GUS activity predominantly in their stems . Histochemical examination of GUS activity using a chromogenic substrate showed a distinct spatial pattern of GUS staining in the stem . Strong GUS activity was detected in the internal phloem of the vascular system and at the node, especially at the base of the axillary bud . Relatively weaker GUS activity was also detected in pith parenchyma . A 5' deletion of the promoter to nucleotide -305, relative to the transcription start site, did not alter significantly the level of GUS activity or the spatial pattern of GUS staining in the stem . However, further deletions to -237 and -192 resulted in a decrease in the level of GUS activity in the stem that occurred simultaneously with the loss of GUS staining in both the internal phloem and at the base of the axillary bud . However, plants with these deletion constructs still exhibited the predominant expression pattern of GUS activity in the stem and GUS staining in the pith parenchyma cells . Deletion to -94 completely abolished the expression of GUS activity . These results indicate that a sequence between -305 and -237 contains a cis-regulatory element(s) that is required for expression of the GUS reporter gene in both the internal phloem and at the base of the axillary bud, while a sequence between -192 and -94 contains a cis-acting element(s) that is required for expression in pith parenchyma cells. J Bacteriol, 1991 Mar, 173(6), 1867 - 72 A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens; Zhang LH et al.; Several octopine strains of Agrobacterium tumefaciens were tested for Ti plasmid (pTi) transfer after induction by 400 micrograms of octopine per ml for 24 h . The strains could be divided into two groups, transfer efficient (Trae) and transfer inefficient (Traie); the respective rates of transfer were 0.77 x 10(-2) to 1.14 x 10(-2) and 0.33 x 10(-6) to 9.8 x 10(-6) plasmid transconjugant per donor cell . Transfer efficiencies of Traie strains were greatly increased when the time of induction was 72 h . A diffusible conjugation factor (CF) that can enhance conjugal transfer of pTi in A . tumefaciens was discovered when both Trae and Traie donor strains were induced in the same plate . The evidence indicates that CF is a key factor affecting transfer efficiency of pTi but is not sufficient by itself to induce transfer . Trac mutants can produce CF constitutively, and Trae strains can produce it after induction by low octopine concentrations . The transfer efficiency of Traie strains was greatly increased by adding CF to the induction medium . The thermosensitive strain B6S, which normally cannot conjugate at temperatures above 30 degrees C, could transfer pTi efficiently at 32 and 34 degrees C in the presence of CF . Production of CF is dependent on the presence of pTi but appears to be common for different opine strains; it was first detected in octopine strains, but nopaline strains also produced the same or a similar compound . CF is very biologically active, affecting donor but not recipient bacterial cells, but CF does not promote aggregation . Data suggest that CF might be an activator or derepressor in the conjugation system of A . tumefaciens . CF is a dialyzable small molecule and is resistant to DNase, RNase, protease, and heating to 100 degrees C for 10 min, but autoclaving (121 degrees C for 15 min) and alkaline treatment removed all activity. EMBO J, 1991 Mar, 10(3), 697 - 704 T-DNA integration: a mode of illegitimate recombination in plants; Mayerhofer R et al.; Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants . Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp . In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides . Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences . Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions . Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination . The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration. Mol Plant Microbe Interact, 1991 Mar-Apr, 4(2), 163 - 72 Role of T-region borders in Agrobacterium host range; Paulus F et al.; The limited host range AB3 strain of Agrobacterium tumefaciens induces tumors by transferring two T-regions, TA and TB . TA is a deleted version of the well-known biotype I octopine TL-region that lacks the iaa and ipt genes, but carries an intact oncogene, gene 6b, and typical left and right border sequences . TB carries two iaa genes that together code for the synthesis of indoleacetic acid . Gene 6b and the iaa gene act synergistically when transferred in a coinoculation experiment . The TA-region of the limited host range isolate Ag57 is related to the TA-region of AB3, but differs from it at several positions . The most significant difference is the absence of the right border region . In spite of this, Ag57 and the exconjugant strain C58C9(pTiAg57) induce normal tumors on Nicotiana rustica and Vitis vinifera . Various experiments indicate that gene 6b of the Ag57 TA-region is active and transferred in spite of the absence of the right border . On N . tabacum, C58C9(pTiAg57) is nononcogenic but becomes oncogenic when the pTiAg57 TA-region is restored by the right TA border sequence of pTiAB3 . Thus, the right TA border sequence of the biotype III limited host range strains is required for tumor induction on some hosts, but not on others. Mol Plant Microbe Interact, 1991 Mar-Apr, 4(2), 155 - 62 The TR-DNA region carrying the auxin synthesis genes of the Agrobacterium rhizogenes agropine-type plasmid pRiA4: nucleotide sequence analysis and introduction into tobacco plants; Camilleri C et al.; We have determined the nucleotide sequence of a 6-kilobase fragment of the Agrobacterium rhizogenes plasmid pRiA4 TR-region that carries genes (aux1 and aux2) responsible for auxin biosynthesis in transformed plant cells . Sequence analysis revealed two open reading frames corresponding to proteins of 749 amino acids for the aux1 gene and 466 amino acids for the aux2 gene . We observed significant similarity between the amino acid sequences deduced from the pRiA4 aux genes and those of the auxin biosynthesis genes of A . tumefaciens octopine-type Ti plasmids, the iaaM and iaaH genes of Pseudomonas savastanoi, and different genes of the pRiA4 TL-region; however, the 5'-flanking regions of the pRi and pTi auxin biosynthesis genes were found to be completely different . Transgenic tobacco plants containing this entire 6-kilobase fragment of the pRiA4 TR-region have been obtained . Regenerated plants are phenotypically normal . The aux1 gene is not or is very weakly expressed in these plants, but expression of the aux2 gene leads to a modified root phenotype when plants are grown on medium containing an auxin precursor (naphthalene acetamide). Plant Mol Biol, 1991 Mar, 16(3), 427 - 36 Different promoter regions control level and tissue specificity of expression of Agrobacterium rhizogenes rolB gene in plants; Capone I et al.; Expression of the rolB gene of A . rhizogenes T-DNA triggers root differentiation in transformed plant cells . In order to study the regulation of this morphogenetic gene, the GUS reporter gene was placed under the control of several deleted fragments of the rolB 5' non-coding region: carrot disc transformations and the analysis of transgenic tobacco plants containing these constructions identified the presence of distinct regulatory domains in the rolB promoter . Two regions (located from positions -623 to -471 and from -471 to -341, from the translation start codon) control the level but not the tissue specificity of rolB expression: progressive deletions of the rolB promoter starting from position -1185 to -341, although at different levels, maintained the same pattern of GUS expression-maximal in root meristems and less pronounced in the vascular tissue of aerial organs . Further deletion of 35 bp, from -341 to -306, drastically affected tissue specificity: GUS activity was still clearly detectable in the vascular tissue of the aerial organs while expression in the root meristem was totally suppressed . Analysis of transgenic embryos and seedlings confirmed that distinct promoter domains are responsible for meristematic (root) and differentiated (vascular) expression of rolB . Finally, we present data concerning the effects of plant hormones on the expression of rolB-GUS constructions. Mol Plant Microbe Interact, 1991 Mar-Apr, 4(2), 190 - 7 Limited host range Ti plasmids: recent origin from wide host range Ti plasmids and involvement of a novel IS element, IS868; Paulus F et al.; Agrobacterium tumefaciens biotype III octopine strains have been isolated from grapevine tumors worldwide . They comprise limited and wide host range (LHR and WHR) strains that carry related tumor-inducing (Ti) plasmids with two T-regions, TA and TB . The WHR TA-region resembles the biotype I octopine region, whereas the LHR TA-region is a recent deletion derivative of the WHR TA-region, which lacks the iaa genes and part of the ipt gene . Sequencing of the TA-region of the ubiquitous LHR strain AB3 showed that the deleted region is replaced by an insertion sequence (IS) element, IS868, which resembles the IS51 element of Pseudomonas syringae subsp . savastanoi . The Ti plasmid of LHR strain Ag57 carries essentially the same iaa gene deletion as pTiAB3, but lacks IS868 . We propose that the LHR Ti plasmids arose by the recent insertion of an IS868 element into the TA-region of a WHR-type Ti plasmid, followed by transposition to a nearby site . The deletion was caused during the second transposition or by later recombination between the two IS868 copies . Biotype III octopine strains also carry an IS51-like sequence close to the TB iaa genes . Our results confirm and extend earlier observations indicating that IS51-like elements in Pseudomonas and Agrobacterium are associated with iaa genes and played a major role in Ti plasmid evolution. Mol Microbiol, 1991 Mar, 5(3), 737 - 45 nolC, a Rhizobium fredii gene involved in cultivar-specific nodulation of soybean, shares homology with a heat-shock gene; Krishnan HB et al.; Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall . Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene-reduction activity is absent . We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176bp open reading frame that we designate nolC . nolC encodes a protein of deduced molecular weight 43564 . Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea . nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat-shock protein . Both R . fredii USDA257 and mutant 257DH5 produce heat-shock proteins of 78, 70, 22, and 16kDa . A 4.3kb EcoRI-HindIII subclone containing nolC expresses a single 43kDa polypeptide in mini-cells . A longer, 9.4kb EcoRI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat-shock protein . Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat-shock gene, it does not directly function in the heat-shock response. Biochim Biophys Acta, 1991 Feb 16, 1088(2), 225 - 33 Cloning and characterization of an amidase gene from Rhodococcus species N-774 and its expression in Escherichia coli; Hashimoto Y et al.; For investigation of an unknown open reading frame which is present upstream of the nitrile hydratase (NHase) gene from Rhodococcus sp . N-774, a longer DNA fragment covering the entire gene was cloned in Escherichia coli . Nucleotide sequencing and detailed subcloning experiments predicted a single open reading frame consisting of 521 amino acid residues of Mr 54,671 . The amino acid sequence, especially its NH2-terminal portion, showed significant homology with those of indoleacetamide hydrolases from Pseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase from Aspergillus nidulans . The 521-amino acid coding region was therefore expressed by use of the E . coli lac promoter in E . coli, and was found to direct a considerable amidase activity . This amidase hydrolyzed propionamide efficiently, and also hydrolyzed, at a lower efficiency, acetamide, acrylamide and indoleacetamide . These data clearly show that the unknown open reading frame present upstream of the NHase coding region encodes an amidase . Because the TAG translational stop codon of the amidase is located only 75 base pairs apart from the ATG start codon of the alpha-subunit of NHase, these genes are probably translated in a polycistronic manner. Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1456 - 60 Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids; Waters VL et al.; The IncP antibiotic-resistance plasmids transfer to a broad range of bacterial species . The RK2 origin of DNA transfer (oriT) consists of a 250-base-pair segment including the single-stranded cleavage site (nic) needed to generate the DNA strand believed to be transferred . Deletion derivatives and a bank of hydroxylamine-generated oriT mutants were screened for loss of transferability . DNA regions flanking both sides of nic are required for optimal transfer of the oriT clone . Of the chemically induced mutants, critical base-pair changes that dramatically reduced transfer frequency were found in a 10-base-pair region adjacent to nic . Relaxation (nicking) assays performed with these point mutants using protein-DNA complexes reconstituted in vitro revealed a correlation between DNA nicking and transfer frequency . Base-pair changes within the proximal arm of an inverted repeat upstream from the nick site resulted in reduced binding of the essential transfer protein TraJ and correspondingly reduced transfer frequencies . The results support a model of relaxosome formation involving at least two essential proteins: TraI and TraJ . The nick region defined by the point mutants was located in a segment known to be nearly identical in the related plasmid R751 . This sequence was also found to be highly conserved in both border junctions of the transfer DNA (T-DNA) of plant tumor-inducing plasmids of Agrobacterium tumefaciens, indicating a relationship between IncP-mediated broad-host-range bacterial conjugation and T-DNA transfer to plants. Gene, 1991 Feb 1, 98(1), 113 - 6 Nucleotide sequence analysis of a chloramphenicol-resistance determinant from Agrobacterium tumefaciens and identification of its gene product; Tennigkeit J et al.; The nucleotide sequence of a chloramphenicol-resistance (CmR) determinant from the Gram- soil bacterium Agrobacterium tumefaciens was determined, and its gene product was identified as Cm acetyltransferase (CAT) . Comparison of the amino acid sequences of the A . tumefaciens CAT and various CAT proteins of Gram+ and Gram- origin shows no homology between this and the other enzymes. Mol Gen Genet, 1991 Feb, 225(2), 289 - 96 Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs; Visser RG et al.; Granule-bound starch synthase {GBSS; EC 24.1.21} determines the presence of amylose in reserve starches . Potato plants were transformed to produce antisense RNA from a gene construct containing a full-length granule-bound starch synthase cDNA in reverse orientation, fused between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator . The construct was integrated into the potato genome by Agrobacterium rhizogenes-mediated transformation . Inhibition of GBSS activity in potato tuber starch was found to vary from 70% to 100% . In those cases where total suppression of GBSS activity was found both GBSS protein and amylose were absent, giving rise to tubers containing amylose-free starch . The variable response of the transformed plants indicates that position effects on the integrated sequences might be important . The results clearly demonstrate that in tubers of potato plants which constitutively synthesize antisense RNA the starch composition is altered. Genes Dev, 1991 Feb, 5(2), 287 - 97 Illegitimate recombination in plants: a model for T-DNA integration; Gheysen G et al.; Agrobacterium tumefaciens is a soil bacterium capable of transferring DNA (the T-DNA) to the genome of higher plants, where it is then stably integrated . Six T-DNA inserts and their corresponding preinsertion sites were cloned from Arabidopsis thaliana and analyzed . Two T-DNA integration events from Nicotiana tabacum were included in the analysis . Nucleotide sequence comparison of plant target sites before and after T-DNA integration showed that the T-DNA usually causes only a small (13-28 bp) deletion in the plant DNA, but larger target rearrangements can occur . Short homologies between the T-DNA ends and the target sites, as well as the presence of filler sequences at the junctions, indicate that T-DNA integration is mediated by illegitimate recombination and that these processes in plants are very analogous to events in mammalian cells . We propose a model for T-DNA integration on the basis of limited base-pairing for initial synapsis, followed by DNA repair at the junctions . Variations of the model can explain the formation of filler DNA at the junctions by polymerase slipping and template switching during DNA repair synthesis and the presence of larger plant target DNA rearrangements. Proc Natl Acad Sci U S A, 1991 Feb 1, 88(3), 834 - 8 Propeptide of a precursor to a plant vacuolar protein required for vacuolar targeting; Matsuoka K et al.; Sporamin is a protein without glycans that accumulates in large quantities in the vacuoles of the tuberous root of the sweet potato . It is synthesized as a prepro precursor with an N-terminal extension composed of a 21-amino-acid signal peptide and a 16-amino-acid propeptide . A total of 48 base pairs, corresponding to the nucleotide sequence that encodes the propeptide, was deleted from a cDNA clone for sporamin . This delta pro mutant sequence, as well as the sequence of the wild-type sporamin cDNA, was placed downstream from the promoter of the 35S transcript from cauliflower mosaic virus and introduced into the genome of suspension-cultured tobacco cells by Agrobacterium-mediated transformation . In contrast to the vacuolar localization of sporamin in cells that expressed the wild-type precursor, sporamin was secreted into the culture medium from cells in which the delta pro precursor was expressed . The secreted form of sporamin was shorter by two amino acids at its N terminus than authentic sporamin; it migrated anomalously during electrophoresis on SDS/polyacrylamide gel as a result of the presence of intramolecular disulfide bridges, as does authentic sporamin . The kinetics of secretion of sporamin from the cell were similar to those of proteins normally secreted by the host tobacco cells . These results indicate that the propeptide of the precursor to sporamin is required for correct targeting of sporamin to the vacuole and that proteins can be secreted from plant cells by a bulk-flow default pathway in the absence of a functional sorting signal. J Bacteriol, 1991 Feb, 173(3), 1139 - 44 Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter; Chen CY et al.; The Agrobacterium VirG protein is normally expressed from two promoters in response to multiple stimuli, including plant-released phenolics (at promoter P1) and acidic growth media (at promoter P2) . To simplify the analysis of vir gene induction, we sought to create Agrobacterium strains in which virG could be expressed in a controllable fashion . To study the possibility of using the lac promoter and repressor, we constructed a plasmid containing the lac promoter fused to the lacZ structural gene . A derivative of this plasmid containing the lacIq gene was also constructed . The plasmid not containing lacIq expressed high levels of beta-galactosidase . The plasmid containing lacIq expressed beta-galactosidase at very low levels in the absence of o-nitrophenyl-beta-D-galactoside (IPTG) and at moderate levels in the presence of IPTG . We also fused the lac promoter to a virG::lacZ translational fusion and found that IPTG elevated expression of this translational fusion to moderate levels, though not to levels as high as from the stronger of the two native virG promoters . Finally, the lac promoter was used to express the native virG gene in strains containing a virB::lacZ translational fusion . virB expression in this strain depended on addition of IPTG as well as the vir gene inducer acetosyringone . In a similar strain lacking lacIq, virB expression was greater than in a strain in which virG was expressed from its native promoters . Expression of virG from the lac promoter did not alter the acidic pH optimum for vir gene induction, indicating that the previously observed requirement for acidic media was not due solely to the need to induce P2. Plant Mol Biol, 1991 Feb, 16(2), 263 - 9 New patterns of gene activity in plants detected using an Agrobacterium vector; Goldsbrough A et al.; A vector has been designed that contains a truncated CaMV (cauliflower mosaic virus) 35S promoter fused to a receptor gene encoding beta-glucuronidase (GUS), placed adjacent to the left border sequence of an Agrobacterium vector . In potato plants transformed with this vector, different patterns of transcription were detected at high frequency using in situ assays for GUS activity . Previous studies in Drosophila using analogous vectors have shown that the new patterns of transcription in many cases reflect the patterns of expression of genes adjacent to the site of vector insertion . If this is also the case in plants, the vector described here will be useful in identifying the activity of genes in different cell types and will assist in determining their function. J Bacteriol, 1991 Feb, 173(3), 1080 - 7 Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes; Metts J et al.; Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis . Two of the mutants were avirulent on all hosts tested . The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N . debneyi, N . glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato) . That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange . The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome . The effects of the mutations on various steps in tumor formation were examined . All three mutants showed no alteration in binding to carrot cells . However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction . When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS) . LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain . Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins . The third mutant, Ivr-225, was missing a 79-kDa surface peptide . The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown. Enzyme Microb Technol, 1991 Jan, 13(1), 59 - 65 Enzyme immobilization using a cellulose-binding domain: properties of a beta-glucosidase fusion protein; Ong E et al.; Using molecular genetic techniques, a fusion protein has been produced which contains the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi fused to a beta-glucosidase (Abg) from Agrobacterium sp . The CBD functions as an affinity tag for the simultaneous purification and immobilization of the enzyme on cellulose . Binding to cellulose was stable for prolonged periods at temperatures from 4 degrees C to at least 50 degrees C, at ionic strengths from 10 mM to greater than 1 M, and at pH values below 8 . The fusion protein can be desorbed from cellulose with distilled water or at pH greater than 8 . Immobilized enzyme columns of the fusion protein bound to cotton fibers exhibited stable beta-glucosidase activity for at least 10 days of continuous operation at temperatures up to 37 degrees C . At higher temperatures, the bound enzyme lost activity . The thermal stability of the fusion protein was greatly improved by immobilization . Immobilization did not alter the pH stability . Except for its ability to bind to cellulose, the properties of the fusion protein were virtually the same as those of the native enzyme. Sci China B, 1991 Jan, 34(1), 71 - 7 Studies on heterologous expression of Rhizobium meliloti nifA gene and oxygen sensitivity of its product; Wang SP et al.; When the R . meliloti (Rm) nifA'-lacZ fusion-carrying plasmids were introduced into the strains of Agrobacterium tumefaciens, R . trifolii and R . astragalus, beta-galactosidase activity was demonstrated . However, activity was not induced by microaerobiosis . Furthermore, R . meliloti nifA'-lacZ fusion was also not expressed in the nodule bacteroids of R . trifolii and R . astragalus . We speculate that some factor(s) important for the induction of Rm nifA presumed to be the fixLJ regulatory system would not be operative in these bacteria . Experiments using R . meliloti nifH'-lacZ/K . Pneumoniae nifH'-lacZ fusion and the constitutive Rm nifA system to test the nifA-dependent expression of nifH'-lacZ under aerobic and microaerobic conditions in E . coli were performed . The inhibition of the Rm nifA activation of nifH'-lacZ expression in the bacteria grown in the aerobic condition was shown . Assays on the Rm nifA-m RNA produced by the constitutive Rm nifA in E . coli under aerobic and microaerobic conditions with the cloned nifA as a probe for dot blot hybridization showed a marked decrease of Rm nifA mRNA when the bacteria were grown under aeration. Mol Gen Genet, 1991 Jan, 225(1), 148 - 57 Developmental and environmental regulation of pea legumin genes in transgenic tobacco; Rerie WG et al.; Two distinct legumin genes (LegA1 and LegA2) which encode a major class of seed storage protein in pea were isolated from a genomic library . The cloned fragments were introduced into tobacco via Agrobacterium-mediated transformation and the regenerated plants were used to study the expression characteristics of the genes in a heterologous host . It was found that both LegA1 and LegA2 were functional members of the pea legumin gene family and that their expression was similar in both pea and transgenic tobacco . Legumin was detected only in the seed of tobacco where the primary translation products were processed in a manner analogous to that which occurs in pea . Legumin gene expression was also shown to be temporally regulated during seed development . Legumin polypeptides and mRNA began to accumulate 16 days after flowering (DAF), in contrast to the endogenous tobacco storage proteins which were apparent at 13 DAF . It was also demonstrated that the legumin genes in tobacco were environmentally regulated to the nutritional status of the plant . As has been previously shown in pea, legumin accumulation in transgenic tobacco seed was progressively reduced when the plants were grown under conditions of increasing severity of sulphur-nutrient stress . The reduced accumulation of protein was correlated with lower levels of legumin mRNA in the developing seed . Despite encoding nearly identical subunits, nucleotide sequence data for LegA1 and LegA2 showed that the similarity of their respective 5'-flanking regions was restricted to several short elements mostly within 240 bp from the start of transcription . However, a deletion series using the LegA1 gene demonstrated that 237 bp of 5'-flanking sequence was insufficient to permit the expression of the legumin gene in tobacco . The data indicated that an as yet unidentified sequence element(s) located between positions -668 and -237 was essential in re-establishing the high level of regulated gene expression observed with the full-length LegA1 gene. J Bacteriol, 1991 Jan, 173(2), 903 - 5 Transport of nonmetabolizable opines by Agrobacterium tumefaciens; Krishnan M et al.; We have examined the uptake of {14C}octopine and {14C}nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes . All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium . The transport of these opines required active cellular metabolism . Nonradioactive octopine, nopaline, and arginine competed effectively with {14C}octopine and {14C}nopaline for transport into A . tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid. Virology, 1991 Jan, 180(1), 70 - 80 Molecular characterization of two bipartite geminiviruses causing squash leaf curl disease: role of viral replication and movement functions in determining host range; Lazarowitz SG; The genomes of two distinct, but highly homologous, bipartite geminiviruses have been identified in and cloned from extracts of squash leaf curl diseased field squash . These two squash leaf curl viruses (SqLCVs) have covalently closed, circular single-stranded DNA genomes with the same bipartite component organization characteristic of other whitefly-transmitted geminiviruses . Infectivity studies using virus preparations or cloned viral genomic components on different potential host plants demonstrated that these two SqLCVs have different host range phenotypes which can be explained by specific interactions among the different viral genomic components that act to influence viral replication and systemic movement in the plant . Analysis of Agrobacterium-inoculated leaf discs demonstrated that replication of the restricted virus was rescued in trans by the nonrestricted virus, providing an explanation for the mixtures of viral DNA components often found in particular hosts in the field . Sequence analysis of the common regions of these two SqLCVs identified a 13-base deletion in the restricted virus as compared to the nonrestricted virus, suggesting a potential sequence alteration likely to be involved in their host range phenotypic differences and strengthening the conclusion based on hybridization studies of their close evolutionary relationship . Also identified in the original field squash was a defective viral component which appeared to interfere with movement of the restricted SqLCV in its normally permissive hosts and accounted for another aspect of host range variation observed for this virus. Virology, 1991 Jan, 180(1), 58 - 69 Infectivity and complete nucleotide sequence of the cloned genomic components of a bipartite squash leaf curl geminivirus with a broad host range phenotype; Lazarowitz SG et al.; Through cloning and molecular analysis we have identified two highly homologous bipartite geminiviruses as causing squash leaf curl disease . Mechanical and Agrobacterium-mediated inoculation of plants with cloned viral DNA components identified the two genomic components of SqLCV-E, a squash leaf curl virus with an unexpectedly broad host range for a whitefly-transmitted geminivirus . Nucleotide sequence analysis of the genome of this virus showed it to have the same bipartite component organization characteristic of other whitefly-transmitted geminiviruses . Sequence comparison with the genomic components of tomato golden mosaic virus and bean golden mosaic virus revealed a close evolutionary relationship with these two bipartite geminiviruses, with which SqLCV-E shares common hosts . These studies provide clear molecular evidence for the assignment of SqLCV to the subfamily of bipartite geminiviruses. Plant Mol Biol, 1991 Jan, 16(1), 117 - 28 Normal and lysine-containing zeins are unstable in transgenic tobacco seeds; Ohtani T et al.; Chimeric genes composed of the beta-phaseolin promoter, an alpha-zein coding sequence and its modified versions containing lysine codons, and a beta-zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation . alpha-Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population . At 25 days after pollination the 19 kDa alpha-zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants . The transgenic plant with the highest level of zein gene expression had an alpha-zein content that was approximately 0.003% of the total seed protein . The amount of alpha-zein in other transgenic plants varied between 1 x 10(-4)% and 1 x 10(-5)% of the total seed protein . The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the alpha-zein protein . Polysomes translating alpha-zein mRNA isolated from tobacco seeds contained fever ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis . In vivo labelling and immunoprecipitation indicated that newly synthesized alpha-zein was degraded in tobacco seeds with a half-life of less than 1 hour. Plant Mol Biol, 1991 Jan, 16(1), 49 - 58 Analysis of the 5' flanking region responsible for the endosperm-specific expression of a rice glutelin chimeric gene in transgenic tobacco; Takaiwa F et al.; The 5' upstream region of the rice storage protein type II glutelin gene was examined for its regulatory function in transgenic tobacco . Chimeric genes containing 5' flanking regions of the glutelin gene transcriptionally fused to the beta-glucuronidase (GUS) reporter gene were introduced into the tobacco genome by Agrobacterium tumefaciens-mediated gene transfer . The chimeric genes were expressed specifically in developing seeds, as opposed to leaves and stems, of the transgenic tobacco . Histochemical analysis revealed that the GUS activity was restricted to the endosperm tissue . A deletion series of the 5' flanking region was created from position -1329 to -74 relative to the transcriptional initiation site and similarly examined in transgenic tobacco . Measurement of GUS activity of the seeds from the transgenic plants bearing the chimeric genes indicated that the region between positions -441 and -237 was required for the temporal and endosperm-specific expression of the GUS activity in tobacco . RNA analysis by northern blotting confirmed the importance of the -441 to -237 region . Addition of up to 888 bp to the -441 deletion resulted in little increase in GUS activity, although all constructs expressing the GUS gene showed a similar tissue and temporal regulation pattern. Plant Mol Biol, 1991 Jan, 16(1), 105 - 15 Cytokinin content and tissue distribution in plants transformed by a reconstructed isopentenyl transferase gene; Smigocki AC; The cytokinin gene, isopentenyl transferase (ipt), was placed under the control of a heat-inducible promoter from the Drosophila melanogaster hsp70 gene and introduced into Nicotiana plumbaginifolia by cocultivation with Agrobacterium tumefaciens . Transformants were analyzed for organ-specific expression, cytokinin levels and effects on plant development before and after the heat induction . The ipt gene transcripts were detected in leaves and stems but not roots of transgenic plants following a 2 hour, 45 degrees C treatment . Maximum mRNA levels observed occurred 2 hours after heat treatment and 46 hours later were detected only in leaves . Zeatin and zeatinriboside concentrations 2 hours after heat shock ranged from over 900 to 2000 pmol/g, representing a greater than 140- to 200-fold increase over uninduced levels . After 46 hours, approximately 50% of the cytokinins are still present in the leaves as opposed to much reduced levels in the stems . Transgenic plants were greener, shorter, had an underdeveloped root system, reduced leaf width, and increased growth of axillary buds . After a single heat treatment, plants exhibited a darker green pigment and continued growth of lateral buds . Transient accumulations of endogenous cytokinins following thermal induction did not appear to alter the plant's preprogrammed pattern of differentiation. Plasmid, 1991 Jan, 25(1), 27 - 39 Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes; Bouchez D et al.; The agropine/mannopine synthesis region of the TR region of the Ri plasmid of Agrobacterium rhizogenes strain A4 was localized on the basis of sequence similarity with probes from Ti plasmids of Agrobacterium tumefaciens and analysis of transposon insertions . The nucleotide sequence of the right part of the TR-DNA of pRiA4, encompassing the three genes involved in mannityl-opine synthesis, was determined and compared to the sequence of the corresponding region of the octopine-type Ti plasmid pTi15955 . The organization of this region is strongly conserved between Ri and Ti plasmids, but the similarity is restricted to the coding sequences: no homology was detected in the 5' and 3' flanking sequences . The mas1' and ags proteins are the most conserved, showing more than 68% amino acid conservation, whereas the mas2' proteins are only 59% identical . Significant G/C content and codon usage differences are observed between pTi15955 and pRiA4 . An open reading frame strongly similar to that of bacterial repressors is situated immediately to the right of the TR region. J Biochem (Tokyo), 1991 Jan, 109(1), 66 - 9 Direct evidence of the Entner-Doudoroff pathway operating in the metabolism of D-glucosamine in bacteria; Iwamoto R et al.; Pseudomonas fluorescens (Migula) (IFO 14808) has both a membrane-bound PQQ-dependent D-glucose (D-Glc) dehydrogenase {EC 1.1.99.17} {which also acts on D-glucosamine (D-GlcN)} and a PLP-dependent D-glucosaminate (D-GlcNA) dehydratase {EC 4.2.1.26} . Further, these two enzymes were induced when D-GlcN was added to the culture medium . However, D-glucosamine-6-phosphate (D-GlcN-6-P) isomerase {EC 5.3.1.10}, another enzyme involved in the metabolism of D-GlcN, was only present at a low level in this bacterium . The bacterium was able to grow in a minimal medium containing D-GlcN or D-GlcNA as the sole source of carbon and nitrogen . Intact cells of P . fluorescens (Migula) converted D-GlcN to D-GlcNA and then to 2-keto-3-deoxy-D-gluconate (KDGA) . These results demonstrate that D-GlcN is metabolized via D-GlcNA to KDGA in P . fluorescens (Migula) (Entner-Doudoroff pathway) . In contrast, Enterobacter cloacae(IFO 13535) and Agrobacterium radiobacter (IAM 1526) have significant amounts of D-GlcN-6-P isomerase with low levels of the D-Glc dehydrogenase and D-GlcNA dehydratase . Further, only the isomerase activity was induced on the addition of D-GlcN to the culture medium . These results demonstrate that there is a new route (Entner-Doudoroff pathway), i.e., in addition to the known one (Embden-Meyerhof pathway), for the metabolism of D-GlcN in bacteria and one of the two routes is predominant in the each of bacteria examined. Arch Microbiol, 1991, 156(4), 270 - 6 Codon usage and G + C content in Bradyrhizobium japonicum genes are not uniform; Ramseier TM et al.; To date, the sequences of 45 Bradyrhizobium japonicum genes are known . This provides sufficient information to determine their codon usage and G + C content . Surprisingly, B . japonicum nodulation and NifA-regulated genes were found to have a less biased codon usage and a lower G + C content than genes not belonging to these two groups . Thus, the coding regions of nodulation genes and NifA-regulated genes could hardly be identified in codon preference plots whereas this was not difficult with other genes . The codon frequency table of the highly biased genes was used in a codon preference plot to analyze the RSRj alpha 9 sequence which is an insertion sequence (IS)-like element . The plot helped identify a new open reading frame (ORF355) that escaped previous detection because of two sequencing errors . These were now corrected . The deduced gene product of ORF355 in RSRj alpha 9 showed extensive similarity to a putative protein encoded by an ORF in the T-DNA of Agrobacterium rhizogenes . The DNA sequences bordering both ORFs showed inverted repeats and potential target site duplications which supported the assumption that they were IS-like elements. Bioessays, 1991 Jan, 13(1), 25 - 9 Swimming against the tide: chemotaxis in Agrobacterium; Shaw CH; Chemotaxis in bacteria is an excellent model for signal transduction processes . In Agrobacterium tumefaciens, the causative agent of crown gall tumour on wounded plants, it is a vital part of the organism's biology . A chromosomally-determined chemotaxis system causes the bacterium to be attracted into the rhizosphere by chemoattractants in plant exudates . By interfacing with this system, the multifunctional products of two Ti-plasmid encoded genes, virA and virG, allow the sensing of specific wound phenolics such as acetosyringone . This attracts Ti-plasmid harbouring A . tumefaciens to wound sites, where the higher acetosyringone concentrations lead to virA and virG-mediated induction of the vir-genes . The products of the induced genes, act in concert to effect transfer of the T-DNA to the plant cell. Plant Cell, 1991 Jan, 3(1), 11 - 22 Ammonia-regulated expression of a soybean gene encoding cytosolic glutamine synthetase in transgenic Lotus corniculatus; Miao GH et al.; A full-length cDNA clone encoding cytosolic glutamine synthetase (GS), expressed in roots and root nodules of soybean, was isolated by direct complementation of an Escherichia coli gln A- mutant . This sequence is induced in roots by the availability of ammonia . A 3.5-kilobase promoter fragment of a genomic clone (lambda GS15) corresponding to this cDNA was isolated and fused with a reporter {beta-glucuronidase (GUS)} gene . The GS-GUS fusion was introduced into a legume (Lotus corniculatus) and a nonlegume (tobacco) plant by way of Agrobacterium-mediated transformations . This chimeric gene was found to be expressed in a root-specific manner in both tobacco and L . corniculatus, the expression being restricted to the growing root apices and the vascular bundles of the mature root . Treatment with ammonia increased the expression of this chimeric gene in the legume background (i.e., L . corniculatus); however, no induction was observed in tobacco roots . Histochemical localization of GUS activity in ammonia-treated transgenic L . corniculatus roots showed a uniform distribution across all cell types . These data suggest that the tissue specificity of the soybean cytosolic GS gene is conserved in both tobacco and L . corniculatus; however, in the latter case, this gene is ammonia inducible . Furthermore, the ammonia-enhanced GS gene expression in L . corniculatus is due to an increase in transcription . That this gene is directly regulated by externally supplied or symbiotically fixed nitrogen is also evident from the expression of GS-GUS in the infection zone, including the uninfected cells, and the inner cortex of transgenic L . corniculatus nodules, where a flux of ammonia is encountered by this tissue . The lack of expression of GS-GUS in the outer cortex of the nodules suggests that ammonia may not be able to diffuse outside the endodermis. Plasmid, 1991 Jan, 25(1), 3 - 15 Polymorphism of Nopaline-type T-DNAs from Agrobacterium tumefaciens; Wabiko H et al.; The structure of several T-DNAs of Agrobacterium tumefaciens was determined by molecular cloning and Southern hybridization . The T-DNAs cloned in Escherichia coli vectors from four different nopaline type strains (PyTE1, PO31, PO22, and AKE10) showed various sizes of restriction enzyme fragments . Comparative analysis of the restriction maps revealed that the T-DNAs were composed of three distinct structural domains: (1) the region proximal to the right border (Domain I) containing the portion essential for tumorigenicity, (2) the proximity to the left border (Domain II), and (3) the region between the two domains (Domain III) to both of which no functional assignments have yet been made . The restriction map indicated that the Domains I and II were conserved in the most clones, including the well-characterized T37 T-DNA . The only exception was AKK1 (obtained from AKE10) which differed in Domain I . In the Domain III, insertions of 1.5- or 1.6-kb DNA were found in four clones, whereas an additional 2.5-kb insertion was found in one clone (PO22P1) . The individual T-DNAs including Domain III with insertions was demonstrated in petunia and poplar tumors induced by the referred A . tumefaciens strains . However, resulting tumors differed in morphology and growth . These results suggest that the length polymorphism of the nopaline type T-DNA can be accounted by DNA insertions, and that diverse T-DNAs reflect their different roles in tumorigenicity. Symp Soc Exp Biol, 1991, 45, 63 - 75 Development of an efficient transposon tagging system in Arabidopsis thaliana; Dean C et al.; We are currently developing a transposon tagging system in Arabidopsis thaliana using the maize transposable elements Ac and Ds . In order to make the system as efficient as possible, four different antibiotic resistance markers have been tested for their usefulness in monitoring excision and reinsertion of transposons in the Arabidopsis genome . Owing to the low transposition frequency of wild-type Ac in Arabidopsis we have also tested a number of modifications to the Ac element . Deletion of the CpG-rich region in the transposase 5' untranslated leader was found to significantly increase the activity of Ac in Arabidopsis . In our first non-targeted tagging experiment, 200 individuals with an inherited transposed Ac element have been collected and their progeny are being screened in families of twelve for segregation of mutant phenotypes . A two-element system, using Ac and Ds, is also being developed and a way of stabilising the Ds-induced mutations by counter selecting plants carrying the transposase source is being investigated . The Agrobacterium T-DNA insertions carrying the different transposons are currently being mapped onto the Arabidopsis RFLP map . Inverse polymerase chain reaction (IPCR) is being used to generate flanking DNA probes, that will be used on the RFLP blots to map the T-DNA relative to the other known markers . Once the first few have been mapped the first targeted tagging experiment will be initiated. Chin J Biotechnol, 1991, 7(3), 185 - 9 Regeneration of transgenic Lycium barbarum L; Wang H et al.; A simple and effective system for the transformation and regeneration of Lycium barbarum L . has been developed . Young stem segments from Lycium barbarum L . were infected by Agrobacterium tumefaciens harboring a vector containing neomycin phosphotransferase II (npt-II) gene derived from non-oncogenic Ti plasmid . Calli originating from young stem segments on selective induction medium could differentiate into buds on selective differentiation medium rapidly and finally developed into whole plants . NPT-II enzyme activity assay and DNA hybridization indicated that the foreign gene had been integrated into the genome of Lycium barbarum L . and could be expressed in plants. DNA Seq, 1991, 2(3), 163 - 72 Nucleotide sequence of Rhizobium meliloti insertion sequence ISRm1: homology to IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens; Watson RJ et al.; Nucleotide sequencing of Rhizobium meliloti insertion sequence ISRm1 showed that it is 1319 nucleotides long and includes 32/31 nucleotide terminal inverted repeats . Analysis of five different insertion sites using sequencing primers complementary to sequences within the left and right ends demonstrated that ISRm1 generates five bp direct repeats at the sites of insertion . Although ISRm1 has shown a target preference for certain short regions (hot spots), there was no apparent similarity in the DNA sequences near the insertion sites . On one strand ISRm1 contains two contiguous open reading frames (ORFs) spanning most of its length . ISRm1 was found to have over 50% sequence homology to insertion sequences IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens . Their sizes, the sequences of their inverted repeats, and the characteristics of their insertion sites are also comparable, indicating that ISRm1, IS2 and IS426 belong to a class of related insertion sequences . Comparison of the proteins potentially encoded by these insertion sequences showed that the two ORFs found in ISRm1 are also present in IS2 and IS426, suggesting that they may be functional genes. DNA Seq, 1991, 1(5), 303 - 27 Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4; Ziegelin G et al.; The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined . These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes . A seventh gene completely overlaps another one in a different reading frame . Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred . The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon . Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor . The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids . The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons . This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences . Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation . Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship. Nucleic Acids Res, 1990 Dec 11, 18(23), 6953 - 8 VirD2 gene product from the nopaline plasmid pTiC58 has at least two activities required for virulence; Steck TR et al.; Virulence genes virD1 and virD2 are required for T-DNA processing in Agrobacterium tumefaciens . The regions within virD2 contributing to T-DNA processing and virulence were investigated . Some insertional mutations in virD2 prevented T-DNA border endonucleolytic cleavage and produced an avirulent phenotype . However, a non-polar insertion immediately after bp 684 of the 1344 bp open reading frame of virD2 did not inhibit endonucleolytic cleavage but still caused a loss of virulence . This suggested that in addition to T-DNA border cleaving activity, the VirD2 protein has another virulence function which resides in the C-terminal half of the protein . Comparative nucleotide sequence analyses of virD2 showed that the first 684 bp were 81% homologous to virD2 of an octopine Ti plasmid whereas the remaining 660 bp were only 44% homologous . A plasmid containing the virD region from octopine Ti plasmid could restore both virulence and processing to a nopaline virD2 mutant . No complementation resulted when a nopaline virD2 clone containing a region similar to eukaryotic nuclear envelope transport sequences was deleted from the 3' end . These results suggest that virD1 and only the first half of virD2 are required to encode for the T-DNA processing endonuclease, and that the 3'-half of virD2 encodes a function separate from endonuclease activity that is required for virulence. Nucleic Acids Res, 1990 Dec 11, 18(23), 6909 - 13 Characterization of the VirG binding site of Agrobacterium tumefaciens; Pazour GJ et al.; Expression of Agrobacterium tumefaciens virulence (vir) genes is dependent on the presence of a conserved 'vir box' sequence in their 5' nontranscribed regions . The location and number of these sequences vary considerably in different vir genes . Site-directed mutagenesis was used to identify the functional vir box(es) of virB, virC and virD . For virB expression both vir box B1 and B2 are required but only the vir box B1 is absolutely essential . Of the five vir boxes of virC and virD two are required for virC expression while only one vir box is required for virD expression . To investigate the minimum sequences necessary for vir gene induction a deletion derivative of virE that lacks the vir box region was used . This mutant is not induced by acetosyringone . The inducibility of this promoter was restored when a synthetic deoxyoligonucleotide dGTTTCAATTGAAAC was introduced at a location analogous to that of the wild type vir box sequence . Mutational analysis indicate that the functional vir box sequence is 14 residues in length, contains a dyad symmetry and has the consensus sequence d ryTncAaTTGnAaY {corrected} (r = purine, y = pyrimidine). Mol Gen Genet, 1990 Dec, 224(3), 309 - 16 Integration of Agrobacterium T-DNA into a tobacco chromosome: possible involvement of DNA homology between T-DNA and plant DNA; Matsumoto S et al.; We established tobacco tumour cell lines from crown galls induced by Agrobacterium . Restriction fragments containing T-DNA/plant DNA junctions were cloned from one of the cell lines, which has a single copy of the T-DNA in a unique region of its genome . We also isolated a DNA fragment that contained the integration target site from nontransformed tobacco cells . Nucleotide sequence analyses showed that the right and left breakpoints of the T-DNA mapped ca . 7.3 kb internal to the right 25 bp border and ca . 350 bp internal to the left border respectively . When the nucleotide sequences around these breakpoints were compared with the sequence of the target, significant homology was seen between the region adjacent to the integration target site and both external regions of the T-DNA breakpoints . In addition, a short stretch of plant DNA in the vicinity of the integration site was deleted . This deletion seems to have been promoted by homologous recombination between short repeated sequences that were present on both sides of the deleted stretch . Minor rearrangements, which included base substitutions, insertions and deletions, also took place around the integration site in the plant DNA . These results, together with previously reported results showing that in some cases sequences homologous to those in T-DNA are present in plant DNA regions adjacent to left recombinational junctions, indicate that sequence homology between the incoming T-DNA and the plant chromosomal DNA has an important function in T-DNA integration.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1990 Dec, 172(12), 6849 - 55 Osmoregulation in Agrobacterium tumefaciens: accumulation of a novel disaccharide is controlled by osmotic strength and glycine betaine; Smith LT et al.; We have investigated the mechanism of osmotic stress adaptation (osmoregulation) in Agrobacterium tumefaciens biotype I (salt-tolerant) and biotype II (salt-sensitive) strains . Using natural-abundance 13C nuclear magnetic resonance spectroscopy, we identified all organic solutes that accumulated to significant levels in osmotically stressed cultures . When stressed, biotype I strains (C58, NT1, and A348) accumulated glutamate and a novel disaccharide, beta-fructofuranosyl-alpha-mannopyranoside, commonly known as mannosucrose . In the salt-sensitive biotype II strain K84, glutamate was observed but mannosucrose was not . We speculate that mannosucrose confers the extra osmotic tolerance observed in the biotype I strains . In addition to identifying the osmoregulated solutes that this species synthesizes, we investigated the ability of A . tumefaciens to utilize the powerful osmotic stress protectant glycine betaine when it is supplied in the medium . Results from growth experiments, nuclear magnetic resonance spectroscopy, and a 14C labeling experiment demonstrated that in the absence of osmotic stress, glycine betaine was metabolized, while in stressed cultures, glycine betaine accumulated intracellularly and conferred enhanced osmotic stress tolerance . Furthermore, when glycine betaine was taken up in stressed cells, its accumulation caused the intracellular concentration of mannosucrose to drop significantly . The possible role of osmoregulation of A . tumefaciens in the transformation of plants is discussed. J Bacteriol, 1990 Dec, 172(12), 6764 - 73 Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp . strain R312: structural evidence for genetic coupling with nitrile hydratase; Mayaux JF et al.; An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp . strain R312 . Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA . Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans . Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon . Our results also showed that Rhodococcus sp . strain N-774 and Brevibacterium sp . strain R312 are probably identical, or at least very similar, microorganisms . The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 mumol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide . Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30 degrees C under the control of an E . coli promoter and ribosome-binding site. Plant Cell, 1990 Dec, 2(12), 1239 - 48 Analysis of tomato polygalacturonase expression in transgenic tobacco; Osteryoung KW et al.; Tomato polygalacturonase is a cell wall enzyme secreted in large amounts during tomato fruit ripening . Polygalacturonase is synthesized as a glycoprotein precursor that undergoes numerous cotranslational and post-translational processing steps during its maturation, yielding three isozymes in tomato fruit, PG1, PG2A, and PG2B . To investigate the physiological roles of the three isozymes and the functional significance of the polygalacturonase processing domains in its intracellular transport and activity, we have examined polygalacturonase expression in transgenic tobacco plants . A full-length polygalacturonase cDNA was placed under control of the cauliflower mosaic virus 35S promoter and introduced into tobacco by way of Agrobacterium-mediated transformation . Analysis of transgenic tobacco plants indicated that (1) immunologically detectable polygalacturonase can be extracted from leaves, roots, and stems of transgenic tobacco plants; (2) only PG2A and PG2B were detectable in transgenic tobacco; (3) the polygalacturonase isozymes present in transgenic tobacco were electrophoretically indistinguishable from the tomato isozymes; (4) the N-terminal sequence, degree of N-linked glycosylation, and extent of oligosaccharide processing were similar in polygalacturonase from transgenic tobacco and tomato; (5) polygalacturonase was properly localized in cell walls of transgenic tissue; (6) the protein was enzymatically active in vitro; however, (7) accumulation of PG2A and PG2B in cell walls of transgenic tobacco did not result in pectin degradation in vivo . These results indicated that tomato polygalacturonase was properly processed and transported to the cell wall of tobacco . However, accumulation of the two polygalacturonase isozymes expressed in this heterologous host was insufficient to promote polyuronide degradation in tobacco leaf tissue. Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9534 - 7 A bacterial peptide acting as a plant nuclear targeting signal: the amino-terminal portion of Agrobacterium VirD2 protein directs a beta-galactosidase fusion protein into tobacco nuclei; Herrera-Estrella A et al.; Agrobacterium tumefaciens is a soil bacterium capable of transferring DNA to the genome of higher plants . Of the virulence region-encoded proteins of the tumor-inducing (Ti) plasmid of A . tumefaciens, the VirD1 and VirD2 proteins are essential for T-DNA transfer to plant cells . These two proteins have been shown to be directly responsible for the formation of T-strands . VirD2 was also shown to be firmly attached to the 5' termini of T-strands; these facts have led to its postulation as a pilot protein in the T-DNA transfer process and as a nucleus-targeting signal in plants . We have constructed a chimeric gene by fusing the virD2 gene and the Escherichia coli lacZ gene . Cell fractionation and electron microscopy studies with transgenic tobacco plants containing the VirD2-LacZ fusion protein indicate that the first 292 amino acids of VirD2 are able to direct the cytoplasmic protein beta-galactosidase to the plant nucleus . This provides an example of cross-kingdom nuclear localization between two free-living organisms: a bacterial peptide is capable of acting as a eukaryotic (plant) nuclear targeting signal. Plant Mol Biol, 1990 Dec, 15(6), 851 - 64 The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants; Hobbs SL et al.; Inter-transformant variability in the expression of introduced genes was studied in the R1 and R2 generations of 10 tobacco transformants, produced by Agrobacterium-mediated transformation . In replicated and physiologically equivalent material, tranformants showed considerable variability in the expression of the reporter gene uidA as shown by transcript levels and beta-glucuronidase (GUS) activity . However, homozygous R2 material could be investigated for seven of the transformants and among these, and in one line in which two inserts could segregate independently, this inter-transformant variability was reduced to simple bimodal expression . The two levels of expression for GUS activity in leaves were high or low (approximately 2.5 or 0.3 nmol cm-2 min-1 respectively), with no continuous variation . Transformants in the high group had single T-DNA insertions, while those in the low group had multiple T-DNA insertions, at the same or different loci . Within each group, although T-DNA was apparently integrated at different sites in the plant genome, there was no evidence of position effects . GUS activity levels of the transformants were very similar in the field and in environmentally controlled conditions under high or low light . Plants with multiple insertions and low expression also tended to have increased methylation of the integrated T-DNA. Mol Microbiol, 1990 Dec, 4(12), 2159 - 66 Specific binding of VirG to the vir box requires a C-terminal domain and exhibits a minimum concentration threshold; Powell BS et al.; The positive regulatory protein VirG from the virulence region of the Ti plasmid of Agrobacterium tumefaciens was first demonstrated to possess DNA-binding capabilities using chromatographically purified protein and in vitro assays (Powell et al., 1989) . This paper is an extension of that research and presents evidence on the in vivo DNA-binding properties of VirG using a transcription interference assay . VirG protein bound specifically to a 'vir box' response element and repressed transcription of a lacZ reporter gene, but increased transcription in the absence of a vir box . A biphasic response in specific DNA-binding was observed upon increasing virG expression, suggesting that specific binding was co-operatively affected by protein concentration . Certain TrpE'-'VirG hybrid proteins also bound the vir box, but required sequences distal to amino acid Arg-118 of the VirG polypeptide . These data further localize a DNA-binding domain within VirG, and support a modified model for the regulation of virulence genes in which transphosphorylation by the coregulator VirA functions to stabilize specific DNA-binding by low concentrations of VirG, resulting in gene activation . Otherwise, at high concentrations, VirG promotes expression of the virulence regulon without assistance from VirA as was shown previously (Rogowsky et al., 1987). Plant Mol Biol, 1990 Dec, 15(6), 827 - 33 Heat-inducible hygromycin resistance in transgenic tobacco; Severin K et al.; We have constructed a chimaeric gene consisting of the promoter of the soybean heat shock (hs) gene Gmhsp17, 6-L, the coding region of a hygromycin phosphotransferase (hpt) gene, and the termination sequence of the nopaline synthase (nos) gene . This gene fusion was introduced into tobacco by Agrobacterium-mediated gene transfer . Heat-inducible synthesis of mRNA was shown by northern hybridization, and translation of this RNA into a functional protein was indicated by plant growth on hygromycin-containing media in a temperature-dependent fashion . One hour incubation at 40 degrees C per day, applied for several weeks, was sufficient to express the resistant phenotype in transgenic plants containing the chimaeric hs-hpt gene . These data suggest that the hygromycin resistance gene is functional and faithfully controlled by the soybean hs promoter . The suitability of these transgenic plants for selection of mutations that alter the hs response is discussed. Genetika, 1990 Dec, 26(12), 2111 - 21 {Design of an expression integrative vector and its application for introducing the human recombinant alfa interferon gene into plants}; Smirnov SP et al.; Integrative expression vector pST6 was developed for dicote plant transformation . The vector contains neomycin phosphotransferase gene under control of PNOS promoter which confers kanamycin resistance to transformed plant cells . The vector also includes expression cassette with strong constitutive 35S CaMV promoter for cloning of foreign genes . The human recombinant alfa interferon gene has been inserted into the expression cassette of pST6 . Recombinant plasmid obtained was transferred into Agrobacterium tumefaciens cells, where the plasmid integrated into disarmed Ti plasmid pVG2260 . Transgenic tobacco plants were obtained via transformation of leaf discs . It was determined that the interferon gene was inserted into plant genome . Transcription and translation of human interferon was observed in the plants transformed. Gene, 1990 Nov 30, 96(1), 43 - 9 The virD genes from the vir region of the Ti plasmid: T-region border dependent processing steps in different rec mutants of Escherichia coli; Alt-Morbe J et al.; We evaluated the substrate requirements for virD-mediated T-circle formation in an in vivo binary test system in Escherichia coli . Two copies of the 25-bp sequence which defines the right border of the T-DNA (transferred DNA) are sufficient, and the right and the left copy of the border are equivalent in function in this system . Experiments with different rec mutants show that the occurrence and frequency of circular double-stranded and single-stranded T-DNA equivalents strongly depend on rec functions of the host . These results are discussed in the context of processing of the tumor-inducing Ti plasmid preceding the T-DNA transfer from agrobacteria to plants. J Bacteriol, 1990 Nov, 172(11), 6182 - 8 Agrobacterium rhizogenes mutants that fail to bind to plant cells; Crews JL et al.; Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells . A light microscope binding assay was used . The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks . The mutants did not appear to be altered in cellulose production . The composition of the medium affected the ability of the parent and mutant bacteria to bind to carrot cells . The parent strain bound to carrot cells in greatest numbers in low-ionic-strength media such as 4% sucrose but still showed significant binding in Murashige-Skoog tissue culture medium . All of the mutants showed reduced binding in 4% sucrose after 2 h of incubation with carrot cells . One mutant was delayed in binding in 4% sucrose . This mutant and one other mutant also showed reduced binding to carrot cells in Murashige-Skoog medium . To determine whether the Tn5 insertion was responsible for the mutant phenotype, DNA containing the Tn5 insertion was cloned from the mutant bacteria and used to introduce Tn5 into the parent strain in the same location as in the original mutant by marker exchange . The resulting transconjugants had the same avirulent, nonattaching phenotype as the original mutants, suggesting that the mutant phenotype was due to the Tn5 insertion . The cloned DNA containing the Tn5 insertion was also tested for homology to DNA of known genes that affect attachment of Agrobacterium tumefaciens to plant cells by DNA hybridization . No homology to chv, att, or pscA clones was observed . In addition, cloned chv, att, and pscA genes from A . tumefaciens were unable to complement the attachment-minus A . rhizogenes mutants . Thus, the A . rhizogenes nonattaching mutants appear to be different from the previously described A . tumefaciens mutants. Mol Gen Genet, 1990 Nov, 224(2), 248 - 56 Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events; Herman L et al.; During Agrobacterium tumefaciens infection, the T-DNA flanked by 24 bp imperfect direct repeats is transferred and stably integrated into the plant chromosome at random positions . Here we measured the frequency with which a promoterless reporter gene is activated after insertion into the Nicotiana tabacum SR1 genome . When adjacent to the right or left T-DNA border sequences, at least 35% of the transformants express the marker gene, suggesting preferential T-DNA insertion (greater than 70%) in transcriptionally active regions of the plant genome . When the promoterless neomycin phosphotransferase II (nptII) gene is located internally in the T-DNA, the activation frequency drops to 1% since gene activation requires T-DNA truncation . These truncation events in the nptII upstream region occur independently of the nature of the upstream sequence and of the T-DNA length . Deletion of the right border region prevents the detection of activated marker genes . Therefore, T-DNA truncation probably occurs after synthesis of a normal T-DNA intermediate during the transfer and/or integration process . In the absence of border regions, expression of the nptII selectable marker directed by the nopaline synthase promoter was detected in 1 out of 10(5) regenerated calli, suggesting the possibility that any DNA sequence from the Ti plasmid can be transformed into the plant genome, albeit at a low frequency. J Bacteriol, 1990 Nov, 172(11), 6442 - 6 Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides; Ankenbauer RG et al.; The virulence genes of Agrobacterium tumefaciens are induced by specific plant phenolic metabolites and sugars (G . A . Cangelosi, R . G . Ankenbauer, and E . W . Nester, Proc . Natl . Acad . Sci . USA, in press) . In this report, monosaccharides, derivatives, and analogs which induce the vir regulon have been identified and the structural requirements for monosaccharide-mediated induction have been determined . Pyranose sugars with equatorial hydroxyls at C-1, C-2, and C-3 displayed strong vir gene-inducing activity; the C-4 hydroxyl could be epimeric and a wide variety of substitutions at C-5 were permissible . The acidic monosaccharide derivatives D-galacturonic acid and D-glucuronic acid were the strongest inducers among the monosaccharides tested . Eight of the 11 inducing compounds are known plant metabolites, and 7 are monomers of major plant cell wall polysaccharides . A role for monosaccharides and plant phenolic compounds as wound-specific plant metabolites which signal the ChvE/VirA/VirG regulatory system is proposed. Plant Mol Biol, 1990 Nov, 15(5), 747 - 53 The non-conserved region of cucumopine-type Agrobacterium rhizogenes T-DNA is responsible for hairy root induction; Failla MC et al.; The T-DNA regions of three strains of Ri plasmids 1855, 8196, 2659 (agropine, mannopine and cucumopine type respectively) share two highly conserved regions flanking a non-homologous central part . We have cloned segments of the cucumopine Ri plasmid 2659 T-DNA in the binary vector system Bin 19 and infected carrot discs with recombinant Agrobacterium strains . We show here that the central non-conserved region is crucial in hairy root induction as it is sufficient to induce rooting on the apical (auxin-rich) surface of carrot discs; in order to observe rooting on the basal (auxin-depleted) side of the discs, a longer T-DNA fragment, also encompassing part of the right conserved region, had to be utilized in conjunction with a Agrobacterium strain carrying aux genes . Differences of growth properties in culture are exhibited by roots transformed with different fragments of pRi 2659 T-DNA, although all transformed roots show the plagiotropic behaviour typical of hairy roots. Plasmid, 1990 Nov, 24(3), 227 - 34 Nucleotide sequence analysis of IS427 and its target sites in Agrobacterium tumefaciens T37; De Meirsman C et al.; We have determined the nucleotide sequence of IS427, an insertion sequence from Agrobacterium tumefaciens T37, IS427 is 1271 bp long, contains 16-bp imperfect terminal inverted repeats, and generates a 2-bp target sequence duplication . It is present at three sites in the pTiT37 plasmid and is absent from the chromosome of A . tumefaciens T37 . Each of the IS427 elements sequenced was near a site with sequence homology to integration host factor (IHF)-binding sites which suggested that IHF may be involved in IS427 transposition. J Bacteriol, 1990 Nov, 172(11), 6316 - 22 Complementation of an RNase P RNA (rnpB) gene deletion in Escherichia coli by homologous genes from distantly related eubacteria; Waugh DS et al.; We report the construction of a strain of Escherichia coli in which the only functional gene for the RNA moiety of RNase P (rnpB) resides on a plasmid that is temperature sensitive for replication . The chromosomal RNase P RNA gene was replaced with a chloramphenicol acetyltransferase gene . The conditionally lethal phenotype of this strain was suppressed by plasmids that carry RNase P RNA genes from some distantly related eubacteria, including Alcaligenes eutrophus, Bacillus subtilis, and Chromatium vinosum . Thus, the rnpB genes from these organisms are capable of functioning as the sole source of RNase P RNA in E . coli . The rnpB genes of some other organisms (Agrobacterium tumefaciens, Pseudomonas fluorescens, Bacillus brevis, Bacillus megaterium, and Bacillus stearothermophilus) could not replace the E . coli gene . The significance of these findings as they relate to RNase P RNA structure and function and the utility of the described strain for genetic studies are discussed. J Mol Biol, 1990 Oct 20, 215(4), 537 - 47 Binding of the regulatory protein VirG to the phased signal sequences upstream from virulence genes on the hairy-root-inducing plasmid; Tamamoto S et al.; The VirG protein is a positive regulator for the virulence genes of which expression is induced by a plant factor, and is essential for Agrobacterium pathogenicity on dicotyledonous plants . The VirG protein of the hairy-root-inducing plasmid A4 was overproduced in Escherichia coli cells, and purified to homogeneity . DNase I footprinting experiments revealed that the purified VirG protein was bound to the upstream region of virulence genes including the phased vir box sequences, which had been presumed to be the VirG recognition signal from the sequence analysis . In dimethyl sulfate footprinting, the VirG protein specifically protected the guanine residues within every vir box sequence . It was concluded that the VirG protein was bound to the phased vir box sequences from the major groove along one side of double-helical DNA. Gene, 1990 Oct 15, 94(2), 155 - 63 Cloning and sequence analysis of truncated T-DNA inserts from Nicotiana tabacum; Gheysen G et al.; Transgenic plants produced by Agrobacterium-mediated transformation usually have one or a few stable and intact T-DNA insertions . However, in a significant number of the transformants Southern blot analysis has revealed the occurrence of aberrant T-DNA insertions missing one or both ends . During the study of this phenomenon, we obtained KmR Nicotiana tabacum clones after cocultivation with an Agrobacterium strain containing a promoterless nptII gene located internally in the T-DNA . Expression of this nptII gene requires a break in the T-DNA region upstream from the nptII-coding sequence and insertion of the truncated T-DNA in a transcriptionally active plant DNA region . The most conspicuous result from Southern analyses on four such KmR plant clones is that they contain several T-DNAs truncated at other positions besides the upstream region of the nptII sequence . Four truncated T-DNA insertions have been cloned . Two insertions contain the nptII gene fused to plant expression signals and are missing the right part of the T-DNA . Another is missing the left T-DNA part and the last T-DNA is lacking both ends . Sequence analysis of the T-DNA::plant junctions has shown that the T-DNA breakpoints are randomly distributed and do not show obvious homologies to one another or to the border consensus sequence . S1-type mapping of the most strongly expressed plant genome::nptII fusion revealed a specific transcription start point and putative TATA and CAAT boxes in the upstream plant DNA region; the steady-state nptII mRNA in these plants is about 20 times more abundant than in transgenic Pnos-nptII plants. J Biol Chem, 1990 Oct 5, 265(28), 16772 - 7 Structure, genetic mapping, and expression of the gene for pyruvate, orthophosphate dikinase from maize; Matsuoka M; Pyruvate, orthophosphate dikinase is a key enzyme in photosynthesis in some plants that exploit the C4 photosynthetic pathway for the fixation of CO2 . The gene for this enzyme has been cloned and its primary structure has been analyzed . The sequence of the cloned genes spans about 12 kilobase pairs and consists of 19 exons . The site of initiation of transcription is located 211 nucleotides upstream from the first nucleotide of the initiation codon (position -211) . Typical TATA and inverted CCAAT elements are present in the anticipated regions, as well as a sequence that is homologous to the binding site of Sp-1 protein (-51 to -42) . Three long, direct-repeated sequences are present contiguously in the 5'-flanking region (-286 to -172), and two of the repeated sequences include sequences homologous to the core sequence of SV40 enhancer in the 3'-end portions . Southern blot analysis, coupled with mapping by analysis of restriction fragment length polymorphism, indicates that the gene for the enzyme involved in C4 photosynthesis is encoded by a single-copy gene and is located on chromosome 6 . To test the promoter activity of the isolated gene, a chimeric gene composed of the 5'-flanking region of the gene and a structural gene for bacterial beta-glucuronidase was constructed and introduced into tobacco protoplasts by electroporation or used to transform tobacco plants by Agrobacterium-mediated transfer of the gene . In both cases, expression of the beta-glucuronidase gene was observed, indicating that the 5'-flanking region of the gene can act as a promoter in tobacco cells. FEMS Microbiol Lett, 1990 Oct, 60(1-2), 1 - 4 Involvement of Azospirillum brasilense plasmid DNA in the production of indole acetic acid; Katzy EI et al.; Indole acetic acid (IAA) production in Azospirillum brasilense strain Sp245 is controlled by a 85 MDa plasmid naturally present in this bacterium . In the presence of L-tryptophan, anthranilic acid production and almost no IAA production occurs in a derivative strain harbouring a Tn5-Mob insertion in the 85 MDa plasmid . Agrobacterium tumefaciens strain GM19023, upon transfer of Tn5-Mob labelled 85 MDa plasmid of A . brasilense Sp245, gains the ability to produce anthranilic acid. FEBS Lett, 1990 Oct 1, 271(1-2), 28 - 32 Characterization of the virA gene of the agropine-type plasmid pRiA4 of Agrobacterium rhizogenes; Endoh H et al.; We sequenced a 4.2-kb DNA region encompassing the vir A locus of the hairy-root-inducing plasmid pRiA4, and compared its sequence with the published vir A region sequences of four tumor-inducing plasmids . An open reading frame capable of coding for 829 amino acids was identified for vir A . Deletion mutants of vir A constructed by fusing to lacZ, but not the wild-type game itself, were efficiently expressed in Escherichia coli when they were put downstream front the lac promoter . These fused gene products became soluble or insoluble depending on the length of their lacZ moieties. J Bacteriol, 1990 Oct, 172(10), 6054 - 60 Mutational analysis of the VirG protein, a transcriptional activator of Agrobacterium tumefaciens virulence genes; Roitsch T et al.; The VirG protein of Agrobacterium tumefaciens is required in conjunction with the VirA protein for transcriptional activation of the virulence (vir) genes in response to plant phenolic compounds . These proteins are members of a family of two component regulatory systems . vir genes are activated via a cascade of phosphorylation reactions involving a specific aspartic acid residue of the VirG protein . We have conducted a mutational analysis of the VirG protein . By mutating conserved and nonconserved aspartic acid residues in the N-terminal domain, we demonstrated that two of three conserved aspartic acid residues located in two different regions are important for the phosphorylation of VirG by VirA phosphate . A third conserved N-terminal region was also shown to be critical for the biological function of VirG as a transcriptional activator . The identification of phosphorylatable but biologically inactive mutated VirG proteins suggests that not only phosphorylation but also a conformational change is necessary for its activity . We further demonstrated that phosphorylation is not required for sequence-specific binding to a vir gene regulatory sequence (vir box) and that the C-terminal domain is sufficient for DNA binding . The data support the model of a two-domain structure for the VirG protein and demonstrate that the sequence homologies to other two-component regulatory systems reflect both functional and structural homologies. J Bacteriol, 1990 Oct, 172(10), 5742 - 9 Inhibition by Agrobacterium tumefaciens and Pseudomonas savastanoi of development of the hypersensitive response elicited by Pseudomonas syringae pv . phaseolicola; Robinette D et al.; Injection into tobacco leaves of biotype 1 Agrobacterium tumefaciens or of Pseudomonas savastanoi inhibited the development of a visible hypersensitive response to the subsequent injection at the same site of Pseudomonas syringae pv . phaseolicola . This interference with the hypersensitive response was not seen with injection of bacterial growth medium or Escherichia coli cells . Live A . tumefaciens cells were required for the inhibitory effect . Various mutants and strains of A . tumefaciens were examined to determine the genes involved . Known chromosomal mutations generally had no effect on the ability of A . tumefaciens to inhibit the hypersensitive response, except for chvB mutants which showed a reduced (but still significant) inhibition of the hypersensitive response . Ti plasmid genes appeared to be required for the inhibition of the hypersensitive response . The bacteria did not need to be virulent in order to inhibit the hypersensitive response . Deletion of the vir region from pTi had no effect on the inhibition . However, the T region of the Ti plasmid was required for inhibition . Studies of transposon mutants suggested that the tms but not tmr or ocs genes were required . These genes were not acting after transfer to plant cells since they were effective in strains lacking vir genes and thus unable to transfer DNA to plant cells . The results suggest that the expression of the tms genes in the bacteria may inhibit the development of the hypersensitive response by the plant . An examination of the genes required in P . savastanoi for the inhibition of the hypersensitive response suggested that bacterial production of auxin was also required for the inhibition of the hypersensitive response by these bacteria. EMBO J, 1990 Oct, 9(10), 3077 - 84 Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation; Offringa R et al.; We determined whether T-DNA molecules introduced into plant cells using Agrobacterium are suitable substrates for homologous recombination . For the detection of such recombination events different mutant versions of a NPTII construct were used . In a first set of experiments protoplasts of Nicotiana tabacum SR1 were cocultivated with two Agrobacterium tumefaciens strains . Each strain contained a different T-DNA, one carrying a 5' deleted NPTII gene and the other a NPTII gene with a 3' deletion . A restored NPTII gene was found in 1-4% of the protoplasts that had been cotransformed with both T-DNAs . Restoration of the NPTII gene could only be the consequence of homologous recombination between the two different T-DNAs in the plant cell, since the possibility of recombination in Agrobacterium was excluded in control experiments . In subsequent experiments was investigated the potential use of Agrobacterium for gene targeting in plants . A transgenic tobacco line with a T-DNA insertion carrying a defective NPTII gene with a 3' deletion was transformed via Agrobacterium with a T-DNA containing a defective NPTII repair gene . Several kanamycin resistant plant lines were obtained with an intact NPTII gene integrated in their genome . In one of these lines the defective NPTII gene at the target locus had been properly restored . Our results show that in plants recombination can occur between a chromosomal locus and a homologous T-DNA introduced via A . tumefaciens . This opens the possibility of using the Agrobacterium transformation system for site directed mutagenesis of the plant genome. EMBO J, 1990 Oct, 9(10), 3033 - 44 Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants; von Schaewen A et al.; Chimeric genes consisting of the coding sequence of the yeast invertase gene suc 2 and different N-terminal portions of the potato-derived vacuolar protein proteinase inhibitor II fused to the 35S CaMV promoter and the poly-A site of the octopine synthase gene were transferred into tobacco and Arabidopsis thaliana plants using Agrobacterium based systems . Regenerated transgenic plants display a 50- to 500-fold higher invertase activity compared to non-transformed control plants . This invertase is N-glycosylated and efficiently secreted from the plant cell leading to its apoplastic location . Whereas expression of the invertase does not lead to drastic changes in transgenic Arabidopsis thaliana plants, transgenic tobacco plants show dramatic changes with respect to development and phenotype . Expression of the invertase leads to stunted growth due to reduction of internodal distances, to development of bleached and/or necrotic regions in older leaves and to suppressed root formation . In mature leaves, high levels of soluble sugars and starch accumulate . These carbohydrates do not show a diurnal turnover . The accumulation of carbohydrate is accompanied by an inhibition of photosynthesis, and in tobacco, by an increase in the rate of respiration . Measurements in bleached versus green areas of the same leaf show that the bleached section contains high levels of carbohydrates and has lower photosynthesis and higher respiration than green sections . It is concluded that expression of invertase in the cell wall interrupts export and leads to an accumulation of carbohydrates and inhibition of photosynthesis. Mol Gen Genet, 1990 Oct, 224(1), 17 - 23 Excision of a Ds-like maize transposable element (Ac delta) in a transient assay in Petunia is enhanced by a truncated coding region of the transposable element Ac; Houba-Herin N et al.; The excision of a Ds-like transposable element (Ac delta) is mediated in trans by the transposable element Ac or its derivatives in Petunia protoplasts cotransfected with two plasmid DNAs . Excision restores the activity of the beta-glucuronidase (GUS) gene that is otherwise shut off by the presence of Ac delta in its leader sequence . A transient expression assay (histochemical test) is used to detect the beta-glucuronidase activity at the protoplast to detect the beta-glucuronidase activity at the protoplast level . The number of blue-stained protoplasts is a measure of the excision frequency . With Ac delta alone a near-zero background of GUS activity is detected, which is weakly enhanced by the presence, in trans, of either the wild-type Ac or the coding region (ORFa) transcribed from the 2' promoter of Agrobacterium tumefaciens TR-DNA . A strong enhancement is observed when a truncated Ac coding region, also under the control of the 2' promoter, is supplied in trans . The truncated version has ATG10 at codon 103 in frame with ORFa and is preceded by 7 out-of-frame ATGs . The assay is quick and well suited for detection of excision frequencies above the value obtained with the wild-type Ac . The presence of empty donor sites following excision can be demonstrated by PCR amplification and direct sequencing of the appropriate DNA fragment. J Bacteriol, 1990 Oct, 172(10), 5828 - 36 picA, a novel plant-inducible locus on the Agrobacterium tumefaciens chromosome; Rong L et al.; We used the transposon Mu dI1681 to identify genes on the Agrobacterium tumefaciens chromosome that are inducible by extracts from carrot roots . One such locus (picA, for plant inducible chromosomal), harbored by A . tumefaciens At156, was inducible 10- to 50-fold by these extracts . Mutation of picA had no detectable effect upon bacterial growth or virulence under laboratory assay conditions . However, A . tumefaciens cells harboring a mutated picA locus aggregated into long "ropes" when incubated with pea root tip cells . Such aggregation was not displayed by the parental strain A . tumefaciens A136 . A preliminary characterization of the inducing compound in the carrot root extract suggests that the active substance is an acidic polysaccharide that is most likely derived from the pectic portion of the plant cell wall. Plant Mol Biol, 1990 Oct, 15(4), 605 - 22 Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore development; Albani D et al.; In this paper we describe the isolation and characterization of a genomic clone (Bp4) from Brassica napus which contains three members of a pollen-specific multigene family . This family is composed of 10 to 15 closely related genes which are expressed in early stages of microspore development . The complete nucleotide sequence of the clone Bp4 and of three homologous cDNA clones is reported . One of the genes (Bp4B) contained in the genomic clone is believed to be non-functional because of sequence rearrangemen