Plasma levels of gonadotropin and 17α,20β-dihydroxy-4-pregnen-3-one in relation to spawning behaviour of rainbow trout, Oncorhynchus mykiss(Walbaum)

Manipulation of the opportunity to spawn was used to investigate the relationship between endocrine events, egg viability and spawning behaviour in female rainbow trout. Females were prevented from spawning by isolating them from males and gravel for up to 21 days after ovula- tion. Blood samples were taken before pairing with a male, at the onset of nesting activity, and at the completion of spawning. Plasma hormone levels of gonadotropin (GtH) and 17α,20β-dihydroxy-4-pregnen-3-one (17,2OP) were measured by specific radioimmunoassays. There were no qualitative or quantitative differences in the spawning behaviour of females paired on the day of ovulation or 7. 14, or 21 days after ovulation. There was a general decrease in the viability of eggs with increasing retention times. In females paired on the day of ovulation, or after 7 or 14 days, GtH levels increased with the onset of nesting behaviour and declined as fish reached the post-spawning condition. By day 21, GtH levels before pairing were significantly higher than prepairing levels in the other three treatment groups, and did not increase at the onset of nesting, or decrease in post-spawning fish. Plasma 17,20P remained high in prepairing and nesting samples of all four groups and declined to low levels in fish in post-spawning condition. In females paired on the day of ovulation there was a significant increase in 17,20P from the prepairing to the nesting stage. These results suggest that 17,20P plays a key role in the synchronization of behavioural and maturational events at the time of spawning.     

Single base mismatches in DNA. Long- and short-range structure probed by analysis of axis trajectory and local chemical reactivity

We have devised a procedure to generate any single base mismatch in a constant sequence context, and have studied these from two points of view. (1) We have examined electrophoretic mobility of 458 base-pair fragments containing approximately centrally located single mismatches, in polyacrylamide gels, compared to fully matched DNA fragments. We found that no single mismatch caused a significant perturbation of gel mobility, and we conclude that all the mismatches may be accommodated within a helical geometry such that there is no alteration of the path of the helix axis in a straight DNA molecule. (2) We have studied all the single mismatches with respect to reactivity to a number of chemical probes. We found that: (a) No mispaired adenine bases are reactive to diethyl pyrocarbonate and are therefore not simply unpaired such that N-7 is exposed. (b) A number of mispaired thymine bases are reactive to osmium tetroxide, and cytosine bases to hydroxylamine. (c) Where crystal or nuclear magnetic resonance structures are available, the reactivity correlates with exposure of the pyrimidine 5,6 double bonds to attack in the major groove as a result of wobble base-pair formation. This is particularly clear for G.T and I.T base-pairs. (d) Reactivity of bases in mismatched pairs can be dependent on sequence context. (e) Reactivity of the C.C mismatch to hydroxylamine is suppressed at low pH, suggesting that a rearrangement of base-pairing occurs on protonation. The results overall are consistent with the formation of stacked intrahelical base-pairs wherever possible, resulting in no global distortion of the DNA structure, but specific enhancement of chemical reactivity in some cases.     

The role of metal ions in the conformation of the four-way DNA junction.

Metal ions fold DNA junctions into a compact conformation that confers protection of all thymine bases to modification by osmium tetroxide. In the absence of the cation the arms of the junction are fully extended in an approximately square-planar configuration. Group IIa cations are effective in achieving a folded conformation of the junction at 80-100 microM, and there is an excellent agreement between the ionic concentrations that fold the junctions as deduced from gel electrophoretic experiments, and those that prevent osmium tetroxide reaction at the junction. Hexamminecobalt(III) achieves full folding at 2 microM, while spermine and spermidine are effective at 25 microM. Some transition metal ions such as Ni(II) may replace the group IIA cations. Monovalent ions of group IA are only partially effective in folding the junctions. Very much higher concentrations are necessary, gel electrophoretic mobilities suggest that a less symmetrical conformation is adopted and thymine bases at the junction remain reactive to osmium tetroxide. Charge-charge interactions at the centre of the junction are structurally extremely important. Substitution of junction phosphate groups by uncharged methyl phosphonates severely perturbs the structure of the junction. If just two phosphates are substituted, diametrically facing across the junction, the structure always folds in order to place the electrically neutral phosphate on the exchanging strands. We suggest that folding of the junction into the stacked X-structure generates electronegative clefts that can selectively bind metal ions, depending on the chemistry, size and charge of the ion. Moreover, occupation of these cavities is essential for junction folding, in order to reduce electrostatic repulsion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Large-scale opening of A + T rich regions within supercoiled DNA molecules is suppressed by salt.

Large-scale cooperative helix opening has been previously observed in A + T rich sequences contained in supercoiled DNA molecules at elevated temperatures. Since it is well known that helix melting of linear DNA is suppressed by addition of salt, we have investigated the effects of added salts on opening transitions in negatively supercoiled DNA circles. We have found that localised large-scale stable melting in supercoiled DNA is strongly suppressed by modest elevation of salt concentration, in the range 10 to 30 mM sodium. This has been shown in a number of independent ways: 1. The temperature required to promote cruciform extrusion by the pathway that proceeds via the coordinate large-scale opening of an A + T rich region surrounding the inverted repeat (the C-type pathway, first observed in the extrusion of the ColE1 inverted repeat) is elevated by addition of salt. The temperature required for extrusion was increased by about 4 deg for an addition of 10 mM NaCl. 2. A + T rich regions in supercoiled DNA exhibit hyperreactivity towards osmium tetroxide as the temperature is raised; this reactivity is strongly suppressed by the addition of salt. At low salt concentrations of added NaCl (10 mM) we observe that there is an approximate equivalence between reducing the salt concentration, and the elevation of temperature. Above 30 mM NaCl the reactivity of the ColE1 sequences is completely supressed at normal temperatures. 3. Stable helix opening transitions in A + T rich sequences may be observed with elevated temperature, using two-dimensional gel electrophoresis; these transitions become progressively harder to demonstrate with the addition of salt. With the addition of low concentrations of salt, the onset of opening transitions shifts to higher superhelix density, and by 30 mM NaCl or more, no transitions are visible up to a temperature of 50 degrees C. Statistical mechanical simulation of the data indicate that the cooperativity free energy for the transition is unaltered by addition of salt, but that the free energy cost for opening each basepair is increased. These results demonstrate that addition of even relatively low concentrations of salt strongly suppress the large-scale helix opening of A + T rich regions, even at high levels of negative supercoiling. While the opening at low salt concentrations may reveal a propensity for such transitions, spontaneous opening is very unlikely under physiological conditions of salt, temperature and superhelicity, and we conclude that proteins will therefore be required to facilitate opening transitions in cellular DNA.     

Structures of bulged three-way DNA junctions.

We have studied a series of three-way DNA junctions containing unpaired bases on one strand at the branch-point of the junctions. The global conformation of the arms of the junctions has been analysed by means of polyacrylamide gel electrophoresis, as a function of conditions. We find that in the absence of added metal ions, all the results for all the junctions can be accounted for by extended structures, with the largest angle being that between the arms defined by the strand containing the extra bases. Upon addition of magnesium (II) or hexamine cobalt (III) ions, the electrophoretic patterns change markedly, indicative of ion-dependent folding transitions for some of the junctions. For the junction lacking the unpaired bases, the three inter-arm angles appear to be quite similar, suggesting an extended structure. However, the addition of unpaired bases permits the three-way junction to adopt a significantly different structure, in which one angle becomes smaller than the other two. These species also exhibit marked protection against osmium addition to thymine bases at the point of strand exchange. These results are consistent with a model in which two of the helical arms undergo coaxial stacking in the presence of magnesium ions, with the third arm defining an angle that depends upon the number of unpaired bases.     

The essential active-site lysines of clostridial glutamate dehydrogenase. A study with pyridoxal-5'-phosphate.

Glutamate dehydrogenase (GDH) of Clostridium symbiosum, like GDH from other species, is inactivated by pyridoxal 5'-phosphate (pyridoxal-P). This inactivation follows a similar pattern to that for beef liver GDH, in which a non-covalent GDH-pyridoxal-P complex reacts slowly to form a covalent complex in which pyridoxal-P is in a Schiff's-base linkage to lysine residues. [formula: see text] The equilibrium constant of this first-order reaction on the enzyme surface determines the final extent of inactivation observed [S. S. Chen and P. C. Engel (1975) Biochem. J. 147, 351-358]. For clostridial GDH, the maximal inactivation obtained was about 70%, reached after 10 min with 7 mM pyridoxal-P at pH 7. In keeping with the model, (a) inactivation became irreversible after reduction with NaBH4. (b) The NaBH4-reduced enzyme showed a new absorption peak at 325 nm. (c) Km values for NAD+ and glutamate were unaltered, although Vmax values were decreased by 70%. Kinetic analysis of the inactivation gave values of 0.81 +/- 0.34 min-1 for k3 and 3.61 +/- 0.95 mM for k2/k1. The linear plot of 1/(1-R) against 1/[pyridoxal-P], where R is the limiting residual activity reached in an inactivation reaction, gave a slightly higher value for k2/k1 of 4.8 +/- 0.47 mM and k4 of 0.16 +/- 0.01 min-1. NADH, NAD+, 2-oxoglutarate, glutarate and succinate separately gave partial protection against inactivation, the biggest effect being that of 40 mM succinate (68% activity compared with 33% in the control). Paired combinations of glutarate or 2-oxoglutarate and NAD+ gave slightly better protection than the separate components, but the most effective combination was 40 mM 2-oxoglutarate with 1 mM NADH (85% activity at equilibrium). 70% inactivated enzyme showed an incorporation of 0.7 mM pyridoxal-P/mol subunit, estimated spectrophotometrically after NaBH4 reduction, in keeping with the 1:1 stoichiometry for the inactivation. In a sample protected with 2-oxoglutarate and NADH, however, incorporation was 0.45 mol/mol, as against 0.15 mol/mol expected (85% active). Tryptic peptides of the enzyme, modified with and without protection, were purified by HPLC. Two major peaks containing phosphopyridoxyllysine were unique to the unprotected enzyme. These peaks yielded three peptide sequences clearly homologous to sequences of other GDH species. In each case, a gap at which no obvious phenylthiohydantoin-amino-acid was detected, matched a conserved lysine position. The gap was taken to indicate phosphopyridoxyllysine which had prevented tryptic cleavage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proteomics in Drosophila melanogaster.

To be able to understand cellular mechanisms, we require fully integrated data sets combining information about gene expression, protein expression, post-translational modification states, sub-cellular location and complex formation. Proteomics is a very powerful technique that can be applied to interrogate changes at the protein level. Studying this effectively requires specialised facilities within research institutes. Here, we describe the setting up and operation of such a facility, providing a resource for the Arabidopsis and Drosophila research communities.     

Subsaturating Ribulose-1,5-Bisphosphate Concentration Promotes Inactivation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) (Studies Using Continuous Substrate Addition in the Presence and Absence of Rubisco Activase)

We developed a continuous-addition method for maintaining subsaturating concentrations of ribulose-1,5-bisphosphate (RuBP) for several minutes, while simultaneously monitoring its consumption by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This method enabled us to observe the effects of subsaturating RuBP and CO2 concentrations on the activity of Rubisco during much longer periods than previously studied. At saturating CO2, the activity of the enzyme declined faster when RuBP was maintained at concentrations near its Km value than when RuBP was saturating. At saturating RuBP, activity declined faster at limiting than at saturating CO2, in accordance with previous observations. The most rapid decline in activity occurred when both CO2 and RuBP concentrations were subsaturating. The activity loss was accompanied by decarbamylation of the enzyme, even though the enzyme was maintained at the same CO2 concentration before and after exposure to RuBP. Rubisco activase ameliorated the decline in activity at subsaturating CO2 and RuBP concentrations. The results are consistent with a proposed mechanism for regulating the carbamylation of Rubisco, which postulates that Rubisco activase counteracts Rubisco's unfavorable carbamylation equilibrium in the presence of RuBP by accelerating, in an ATP-dependent manner, the release of RuBP from its complex with uncarbamylated sites.