Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA

We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6. Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner. Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and absence of T-DNA sequences. These results demonstrate that DNA transfer events starting at a left border on a native Ti plasmid and moving away from the T-DNA region occur and that they can be detected by designing a suitable selection strategy.     

In vitro and in vivo protein phosphorylation in Avena sativa L. coleoptiles: effects of Ca2+, calmodulin antagonists, and auxin

In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.     

X-ray crystallographic investigation and biological activities of Ru(III) complexes containing Schiff base and triphenyl phosphine/arsine

The crystal structures of the complexes [RuCl(Nap-o-phd)(AsPh₃)] and [RuBr(Nap-o-phd)(PPh₃)] (where H₂-Nap-o-phd = N,N′-bis(2-hydroxy-1-naphthaldehyde) o-phenylenediamine) have been determined by single crystal X-ray diffraction techniques. The antibacterial properties of the complexes have also been examined.     

Structure–activity relationship study of copper(II) complexes with 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (4′-methylbenzoyl) hydrazone: synthesis, structures, DNA and protein interaction studies, antioxidative and cytotoxic activity

Novel 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (4′-methylbenzoyl) hydrazone (H₂L) (1) and its two copper(II) complexes have been synthesized. Single-crystal X-ray diffraction studies revealed that the structure of the new copper(II) chloride complex, [Cu(H₂L)Cl₂]·2H₂O (2), is square pyramidal and that of the copper(II) nitrate complex, [Cu(HL)NO₃]·DMF (3), is square planar. In 2, the copper atom is coordinated by the ligand with ONO donor atoms, one chloride ion in the apical position, and the other chloride in the basal plane. In 3, the ligand coordinates as a uninegative tridentate ONO⁻ species and with one nitrate ion in the basal plane. DNA binding experiments indicated that the ligand and copper(II) complexes can interact with DNA through intercalation. Bovine serum albumin binding studies revealed that the compounds strongly quench the intrinsic fluorescence of bovine serum albumin through a static quenching process. Antioxidative activity tests showed that 1 and its copper(II) complexes have significant radical scavenging activity against free radicals. Cytotoxic activities of the ligand and copper(II) complexes showed that the two copper(II) complexes exhibited more effective cytotoxic activity against HeLa and HEp-2 cells than the corresponding ligand. The entire biological activity results showed that the activity order was 1 < 2 < 3.     

A Cointegrate Ti Plasmid Vector for Agrobacterium tumefaciens - mediated Transformation of indica Rice cv Pusa Basmati 1

An intermediate vector pSSJ1 was constructed by cloning a hph gene and a gus gene with catalase intron in pGV1500. pSSJ1 was cointegrated into a disarmed receptor Ti plasmid pGV2260 harboured in Agrobacterium tumefaciens strain C58C1Rif^R. The resulting A. tumefaciens strain C58C1Rif^R (pGV2260::pSSJ1) stably transformed Oryza sativa L. cv Pusa Basmati 1 scutellum-derived calli at 26% frequency. Introduction of the plasmid pSSJ3 (3′virB, virG and virC of pTiB0542) into A. tumefaciens C58C1Rif^R (pGV2260::pSSJ1) resulted in the elevation of acetosyringone-induced T -strand accumulation. Rice transformation efficiency of the cointegrate plasmid pGV2260::pSSJ1 increased from 26% to 33% in the presence of pSSJ3 and from 26% to 35% in the presence of pToK47 (complete virB, virG and virC). T-DNA integration in To plants was confirmed by Southern hybridization analysis. Inheritance analysis of the T₀ plants with single-copy T-DNA insertions revealed segregation of hygromycin resistance in 3:1 ratio. The feasibility of rice transformation with a cointegrate Ti plasmid vector is clearly established.     

Formation of unusual ruthenium(III) carbonyl complex through ONS tricoordination of salicylaldehyde-N-phenylthiosemicarbazone

An unusual coordination mode of salicylaldehyde-N-phenylthiosemicarbazone (H₂-Sal-Ptsc) ligand was observed in unusual ruthenium(III) carbonyl complex for the first time when it was reacted with [RuHCl(CO)(PPh₃)₃]. The EPR and electrochemical analysis conformed the formation of Ru(III) species.     

Calcium- and calmodulin-regulated phosphorylation of soluble and membrane proteins from corn coleoptiles

In vitro phosphorylation of several membrane polypeptides and soluble polypeptides from corn (Zea mays var. Patriot) coleoptiles was promoted by adding Ca²⁺. Ca²⁺-promoted phosphorylation of the membrane polypeptides was further increased in the presence of calmodulin. Both Ca²⁺-stimulated and Ca²⁺- and calmodulin-stimulated phosphorylations of membrane polypeptides were inhibited by chlorpromazine, a calmodulin antagonist. Ca²⁺-stimulated phosphorylation of soluble polypeptides increased with increasing Ca²⁺ concentration. The calmodulin antagonists chlorpromazine and trifluoperazine inhibited the Ca²⁺-promoted phosphorylation of soluble polypeptides. Added calmodulin promoted the Ca²⁺-dependent phosphorylation of a 98 kilodaltons polypeptide. Both Ca²⁺-dependent and Ca²⁺-independent phosphorylations required Mg²⁺ at an optimal concentration of 5 to 10 millimolar. Cyclic AMP was found to have no stimulatory effect on protein phosphorylation. Sodium molybdate, an inhibitor of protein phosphatase, increased the net phosphorylation of several polypeptides. Rapid loss of radioactivity from the phosphorylated polypeptides following incubation in unlabeled ATP indicated the presence of phosphoprotein phosphatase activity.     

Evaluation of codA,tms2, and ABRIN-A as negative selectable markers in transgenic tobacco and rice

The codA and tms2 genes are used as efficient conditional negative selectable markers (NSMs) in several dicotyledonous plants. We evaluated both genes under control of the CaMV 35S promoter for their effectiveness as conditional NSMs. The ABRIN-A chain gene from Abrus precatorius was evaluated as a nonconditional NSM. The efficacies of codA, tms2, and ABRIN-A as NSMs were compared in transgenic rice and tobacco. Tobacco leaf discs and scutellum-derived callus of rice were transformed with the three genes. Leaf discs of T₀ transgenic tobacco plants and the T₁ seedlings of transgenic rice plants, both transformed with codA, showed a pronounced reduction in growth in the presence of the substrate 5-fluorocytosine. The tms2 gene was inferred to act as a nonconditional NSM in tobacco since all the recovered hygromycin-resistant transgenic tobacco plants harbored only truncated transferred DNAs (T-DNAs) with deletions of the tms2 gene. The T₁ transgenic rice seedlings transformed with tms2 showed a drastic reduction in shoot and root growth in the presence of the substrate naphthaleneacetamide. Both codA and tms2 genes served as good conditional NSMs in rice. The ABRIN-A gene proved to be a good nonconditional NSM in tobacco since all recovered hygromycin-resistant plants harbored only truncated T-DNAs with deletions of the ABRIN-A gene. Twelve transgenic rice plants, which harbored the complete ABRIN-A gene, displayed normal growth suggesting that ABRIN-A is not toxic to rice.