Characterisation of <Emphasis Type="Italic">Solanum lycopersicoides</Emphasis> Dun. root primordia culture with plant recovery

A liquid meristematic root primordia culture (RPC) of Solanum lycopersicoides Dun. based on persistent rhizogenesis in a modified Murashige and Skoog (1962) medium supplemented with NAA (15 mg·l⁻¹) or 2,4-D (1 mg·l⁻¹) was described. The meristematic clumps (2–3 mm in diameter) originating from NAA supplemented medium were capable of regenerating plants through the callus stage (up to 70 %). Efficient direct plant regeneration (up to 21 %) was possible from numerous single globular-shaped root primordia (RP) structures liberated from the parental aggregates in 2,4-D supplemented proliferation medium without NH₄NO₃ and with a 2.5 fold increase in KNO₃. The RP converted into plantlets (artificial seedlings) on solid or liquid media without growth growth regulators through the unipolar followed by the mace-shaped bipolar structure stages. The use of apical shoot bud, root apices or root segments as a primary explants brought about RPC induction and plant regeneration. The plants derived from 2 years old culture were phenotypically identical to their parental S. lycopersicoides plants and possessed the same ploidy.     

Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae

Transformation efficiencies as high as 10⁷ transformants g^–1 DNA have been previously reported for pseudomonads using electroporation protocols established for E. coli with plasmid DNAs prepared from methylation proficient E. coli hosts. We report here a protocol for electroporation of plasmid DNAs into a biocontrol strain of Pseudomonas syringae which could not be electroporated by standard E. coli methods. Transformation efficiencies of 10⁷ or higher were obtained with DNA recovered from initial P. syringae transformation or with DNA prepared from methylation deficient E. coli. Both plasmids used in this study were stably maintained in the absence of selection for at least 50 generations.     

Dynamic NMR structures of [Rp]- and [Sp]-phosphorothioated DNA-RNA hybrids: is flexibility required for RNase H recognition?

Chemically modified DNA oligonucleotides have been crucial to the development of antisense therapeutics. High-resolution structural studies of pharmaceutically relevant derivatives have been limited to only a few molecules. We have used NMR to elucidate the structure in solution of two DNA-RNA hybrids with the sequence d(CCTATAATCC).r(GGAUUAUAGG). The two hybrids contain an unmodified RNA target strand, whereas the DNA strand contains one of two different stereoregular sugar-phosphate backbone linkages at each nucleotide: 1), [Rp]-phosphorothioate or 2), [Sp]-phosphorothioate. Homonuclear two-dimensional spectroscopy afforded nearly complete nonlabile proton assignments. Distance bounds, calculated from the nuclear Overhauser effect (NOE) crosspeak intensities via a complete relaxation matrix approach with the program MARDIGRAS, were used to restrain the structure of the two hybrids during simulations of molecular dynamics. Analysis of restrained molecular dynamics trajectories suggests that both hybrids are flexible, requiring the use of molecular dynamics with time-averaged restraints (MDtar) to generate ensembles of structures capable of satisfying the NMR data. In particular, the deoxyribose sugars of the DNA strand show strong evidence of repuckering. Furthermore, deoxyribose sugar repuckering is accompanied by increased flexibility of overall helical geometry. These observations, together with the analysis of the crystal structure of a hybrid duplex in complex with ribonuclease H (RNase H), suggested that this flexibility may be required for recognition by RNase H.     

Restoration of thrombospondin 1 expression in tumor cells harbouring mutant ras oncogene by treatment with low doses of doxycycline

Oncogenes act as inducers of tumor neovascularization, at least in part through suppression of endogenous angiogenesis inhibitors, e.g., thrombospondin 1 (TSP-1/THSP1). Therefore, restoration of TSP-1 levels can be viewed as a possible means to inhibit tumor angiogenesis. We observed that low concentrations (0.1-10 microg/ml) of doxycycline (but not those of related tetracycline) restore TSP-1 expression in H-ras oncogene-expressing tumor cell lines (528ras1, MT-Ras). Interestingly, this effect was relatively ras-specific, as doxycycline did not alter TSP-1 expression in several cell lines (e.g., 528neu2 fibrosarcoma, B16F1 melanoma, and Lewis lung carcinoma) harbouring other types of transforming alterations. Doxycycline-induced reversal of TSP down-regulation was abrogated under hypoxic conditions. Therefore, we conclude that, in vivo, TSP-1 is likely under dual and/or synergistic control of oncogenes and hypoxia-related pathways. Disruption of both components may be necessary for the 'rescue' of TSP-1 expression in ras-driven cancers.     

pH Dependence of the photocycle kinetics of the E46Q mutant of photoactive yellow protein: protonation equilibrium between I1 and I2 intermediates,chromophore deprotonation by hydroxyl uptake,and protonation relaxation of the dark state

The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH(-)] below about pH 8.3 and constant above about pH 9.5, with a pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'s, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/P(bl) equilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/P(bl) equilibrium is faster than the photocycle time.     

Mechanism of Cph1 phytochrome assembly from stopped-flow kinetics and circular dichroism

The kinetics and mechanism of the autocatalytic assembly of holo-Cph1 phytochrome (from Synechocystis) from the apoprotein and the bilin chromophores phycocyanobilin (PCB) and phycoerythrobilin (PEB) were investigated by stopped flow and circular dichroism. At 1:1 stoichiometry, pH 7.9, and 10 degrees C, SVD analysis of the kinetic data for PCB revealed three spectral components involving three transitions with time constants tau(1) approximately 150 ms, tau(2) approximately 2.5 s, and tau(3) approximately 50 s. Tau(1) was associated with a major red shift and transfer of oscillator strength from the Soret region to the 680 nm region. When the sulfhydryl group of cysteine 259 was blocked with iodoacetamide, preventing the formation of a covalent adduct, a noncovalent red-shifted complex (680 nm) was formed with a time constant of 200 ms. Tau(1) could thus be assigned to the formation of a noncovalent complex. The absorption changes during tau(1) are due to the formation of the extended conformation of the linear tetrapyrrole and to its protonation in the binding pocket. From the concentration and pH dependence of the kinetics we obtained a value of 1.5 microM for the K(D) of this noncovalent complex and a value of 8.4 for the pK(a) of the proton donor. The tau(2) component was associated with a blue shift of about 25 nm and was attributed to the formation of the covalent bond (P(r)), accompanied with the loss of the 3-3' double bond to ring A. Tau(3) was due to photoconversion to P(fr). For PEB, which is not photochromic, the formation of the noncovalent complex is faster (tau(1) = 70 ms), but the covalent bond formation is about 80 times slower (tau(2) = 200 s) than with the natural chromophore PCB. The CD spectra of the PCB adduct in the 250-800 nm range show that the chromophore geometries in P(r) and P(fr) are similar to those in plant phytochrome. The opposite rotational strengths of P(r) and P(fr) in the longest wavelength band suggest that the photoisomerization induces a reversal of the chirality. The Cph1 complex with noncovalently bound PCB was still photochromic when cysteine 259 was blocked with IAA or with the bulkier IAF. The covalent linkage to cysteine 259 is thus not required for photoconversion. The CD spectra of the noncovalently bound PCB in P(r)- and P(fr)-like states are qualitatively similar to those of the covalent adducts, suggesting analogous structures in the binding pocket. The noncovalent interactions with the binding pocket are apparently sufficient to hold the chromophore in the appropriate geometry for photoisomerization.     

An isocratic separation of underivatized gentamicin components, 1H NMR assignment and protonation pattern

A simple method for the separation of the major components of commercial gentamicin sulfate (C-1, C-1a, C-2, C-2a) by high-performance liquid chromatography (HPLC) on an analytical and a semipreparative scale was developed. The method utilized ion-pair reversed-phase chromatography, isocratic elution with an aqueous solution containing 9% trifluoroacetic acid and 2.5% acetonitrile as the mobile phase at a flow rate of 2 and 9 mL/min for analytical and semipreparative columns, respectively. Detection was carried out at 213 nm without derivatization. The protonation pattern of the separated gentamicins was determined by potentiometry and 15N and 1H NMR. The full proton NMR assignment for gentamicin C-1 was obtained through the use of 1H 1D and 2D 1H-1H COSY measurements.     

Ca2+ differently affects hydrophobic properties of guanylyl cyclase-activating proteins (GCAPs) and recoverin

Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca(2+)-binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca(2+). Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca(2+)-dependent binding of recoverin to Phenyl-Sepharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca(2+)](free) (10 microM) than at low [Ca(2+)](free) (10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca(2+)](free) in recoverin and at low [Ca(2+)](free) in GCAP2. Such different changes of hydrophobicity evoked by Ca(2+) appear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.     

Comparison of rice and Arabidopsis annotation

Several versions of the rice genome were published in 2002, providing a first overview of the genome content of this model monocot. At the same time, the genome of the model dicot, Arabidopsis thaliana, reached a new level of annotation as thousands of full-length cDNA sequences were integrated with the genome sequence.