Determination of the structure of the DNA binding domain of gamma delta resolvase in solution.

The DNA binding domain (DBD) of gamma delta resolvase (residues 141-183) is responsible for the interaction of this site-specific DNA recombinase with consensus site DNA within the gamma delta transposable element in Escherichia coli. Based on chemical-shift comparisons, the proteolytically isolated DBD displays side-chain interactions within a hydrophobic core that are highly similar to those of this domain when part of the intact enzyme (Liu T, Liu DJ, DeRose EF, Mullen GP, 1993, J Biol Chem 268:16309-16315). The structure of the DBD in solution has been determined using restraints obtained from 2-dimensional proton NMR data and is represented by 17 conformers. Experimental restraints included 458 distances based on analysis of nuclear Overhauser effect connectivities, 17 phi and chi 1 torsion angles based on analysis of couplings, and 17 backbone hydrogen bonds determined from NH exchange data. With respect to the computed average structure, these conformers display an RMS deviation of 0.67 A for the heavy backbone atoms and 1.49 A for all heavy atoms within residues 149-180. The DBD consists of 3 alpha-helices comprising residues D149-Q157, S162-T167, and R172-N183. Helix-2 and helix-3 form a backbone fold, which is similar to the canonical helix-turn-helix motif. The conformation of the NH2-terminal residues, G141-R148, appears flexible in solution. A hydrophobic core is formed by side chains donated by essentially all hydrophobic residues within the helices and turns. Helix-1 and helix-3 cross with a right-handed folding topology. The structure is consistent with a mechanism of DNA binding in which contacts are made by the hydrophilic face of helix-3 in the major groove and the amino-terminal arm in the minor groove. This structure represents an important step toward analysis of the mechanism of DNA interaction by gamma delta resolvase and provides initial structure-function comparisons among the divergent DBDs of related resolvases and invertases.     

Characterization of a rice gene family encoding root-specific proteins

Two cDNA clones (RCc2 and RCc3) corresponding to mRNAs highly expressed only in root tissues of rice (Oryza sativa L.) seedlings were characterized. Respectively, they encode polypeptides of 146 (14.5 kDa) and 133 amino acids (13.4 kDa) that share high (<70%) sequence similarity with a polypeptide encoded by a cDNA (ZRP3) encoding an mRNA preferentially expressed in young maize roots. Genomic DNA blot analysis revealed that they are members of a small gene family and RCg2, the gene corresponding to RCc2, was isolated. A 1656 bp 5-upstream sequence of RCg2 was translationally fused to a -glucuronidase (GUS) reporter gene and stable introduction of the chimeric construct into rice was confirmed by PCR and genomic DNA blot analyses. Histochemical analysis of transgenic rice plants containing the full-length chimeric gene showed high levels of GUS activity in mature cells and the elongation and maturation zones of primary and secondary roots, and in the root caps, but no GUS activity was detected in root meristematic regions. Surprisingly, high GUS activity was also detected in leaves of the same plants. This raises the possibility that the RCg2 5-upstream element may not be sufficient for the proper spatial control of root specificity in transgenic rice.     

Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.

A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin.     

Cyclic AMP and intracellular ionic activities innecturus gallbladder

Summary Open-tip and liquid ion-exchanger microelectrodes were used to study the effects of cAMP (6mm, added to the serosal medium) on apical membrane potential (E _m ) and intracellular sodium, potassium, and chloride activities (a _Na ⁱ ,a _K ⁱ ,a _Cl ⁱ ) inNecturus gallbladder under open-circuit conditions. Transepithelial potential difference (E _Tr ) was also measured. In the presence of cAMP,a _Cl ⁱ fell from about 1.5 times its equilibrium value to a level that corresponded to electrochemical equilibrium across the apical and basolateral cell membranes. Under these conditionsa _Na ⁱ decreased anda _K ⁱ increased,E _m was unchanged andE _Tr increased from virtually zero to a small but significant serosal positive value. The cAMP-induced increase ina _K ⁱ was abolished when Cl^–-free incubation media were used. Addition of the Ca⁺⁺-ionophore A23187 (0.5 g/ml) to the serosal medium had no effect onE _m ,E _Tr , ora _Cl ⁱ . When A23187 was added to the mucosal medium,E _m and the basolateral membrane potential hyperpolarized by about 20 mV and an increase in the outwardly directed electrochemical driving force for Cl^– was observed. These results indicate that cAMP inhibits coupled transapical Na–Cl entry into epithelial cells ofNecturus gallbladder and suggest that this inhibition may not be mediated by an increase in intracellular Ca⁺⁺ concentration.     

A metabolomic,geographic, and seasonal analysis of the contribution of pollen-derived adenosine to allergic sensitization

Background

Studies on ragweed and birch pollen extracts suggested that the adenosine content is an important factor in allergic sensitization. However, exposure levels from other pollens and considerations of geographic and seasonal factors have not been evaluated.

Objective

This study compared the metabolite profile of pollen species important for allergic disease, specifically measured the adenosine content, and evaluated exposure to pollen-derived adenosine.

Methods

An NMR metabolomics approach was used to measure metabolite concentrations in 26 pollen extracts. Pollen count data was analyzed from five cities to model exposure.

Results

A principal component analysis of the various metabolites identified by NMR showed that pollen extracts could be differentiated primarily by sugar content: glucose, fructose, sucrose, and myo-inositol. In extracts of 10 mg of pollen/ml, the adenosine was highest for grasses (45 µM) followed by trees (23 µM) and weeds (19 µM). Pollen count data showed that tree pollen was typically 5–10 times the amount of other pollens. At the daily peaks of tree, grass, and weed season the pollen-derived adenosine exposure per day is likely to be only 1.1, 0.11, and 0.12 µg, respectively. Seasonal models of pollen exposure and respiration suggest that it would be a rare event limited to tree pollen season for concentrations of pollen-derived adenosine to approach physiological levels.

Conclusion

Sugar content and other metabolites may be useful in classifying pollens. Unless other factors create localized exposures that are very different from these models, pollen-derived adenosine is unlikely to be a major factor in allergic sensitization.

     

NMR assignment of polymerase β labeled with 2H, 13C, and 15N in complex with substrate DNA

DNA Polymerase β is a multifunctional enzyme involved in base excision repair of nuclear DNA in vertebrate cells. We present here the first assignments of the full-length protein (335 residues, 39 kDa) in the presence of a double gap—double hairpin DNA (22 nucleotides, 7 kDa).     

Meta-analysis Reveals Different Competition Effects on Tree Growth Resistance and Resilience to Drought

Ecosystems - Drought will increasingly threaten forest ecosystems worldwide. Understanding how competition influences tree growth response to drought is essential for forest management aiming at...     

The evolution of trade-offs: effects of inbreeding on fecundity relationships in the cricket Gryllus firmus

The evolution of traits is modulated by their interrelationships with each other, particularly when those relationships result in a fitness trade-off. In this paper we explore the consequences of genetic architecture on functional relationships between traits. Specifically, we address the consequences of inbreeding on these relationships. We show that the linear regression between two traits will not be affected if there is no dominance genetic variance in either trait, whereas the intercept but not the slope of the regression will change if there is dominance genetic variance in one trait only. We test the latter hypothesis using fecundity relationships in the cricket Gryllus firmus. Data from pedigree analysis and an inbreeding experiment show that there is significant dominance genetic variance in fecundity, but not head width (an index of body size) or dorsal longitudinal muscle (DLM) mass. Fecundity increases with head width, but decreases with DLM mass. As predicted, the intercepts of the regressions of fecundity on these two morphological traits decrease with inbreeding, but there is little or no change in slope. Gryllus firmus is wing dimorphic, with the macropterous (LW) morph having a lower fecundity than the micropterous (SW) morph. We hypothesize that the difference in fecundity arises primarily because of a competition for resources in the LW females between DLM maintenance (i.e., mass) and egg production. As a consequence, we predict that the fecundity within each morph should decline linearly with the inbreeding coefficient at the same rate in both morphs. The result of this will be a change in the relative fitness of the two morphs, that of the SW morph increasing with inbreeding. This prediction is supported. These results indicate that trade-offs will evolve and such changes will affect evolutionary trajectories by altering the pattern of relationships among fitness components.     

The inter-ligand Overhauser effect: A powerful new NMR approach for mapping structural relationships of macromolecular ligands

NMR experiments that transfer conformational information from the bound to the uncomplexed state via exchange have been utilized for many years. It is demonstrated here that inter-ligand NOEs (ILOEs), which exist in ternary complexes with enzymes or other macromolecular receptors, can be transferred via exchange to pairs of uncomplexed ligands. This approach is illustrated by studies of glycolate + NAD⁺ in the presence of porcine heart lactate dehydrogenase, and by glucose-6-phosphate + NADPH in the presence of L. mesenteroides glucose-6-phosphate dehydrogenase. This strategy opens up a general methodology for exploring the active sites of enzymes and for the development of artificial ligands which can function as inhibitors, or more generally as modifiers of protein function.     

Structural consequences of metallothionein dimerization: solution structure of the isolated Cd4-alpha-domain and comparison with the holoprotein dimer

The NMR determination of the structure of Cd(7)-metallothionein was done previously using a relatively large protein concentration that favors dimer formation. The reactivity of the protein is also affected under this condition. To examine the influence of protein concentration on metallothionein conformation, the isolated Cd(4)-alpha-domain was prepared from rabbit metallothionein-2 (MT 2), and its three-dimensional structure was determined by heteronuclear, (1)H-(111)Cd, and homonuclear, (1)H-(1)H NMR, correlation experiments. The three-dimensional structure was refined using distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. The backbone superposition of the alpha-domain from rabbit holoprotein Cd(7)-MT 2 and the isolated rabbit Cd(4)-alpha was measured at a RMSD of 2.0 A. Nevertheless, the conformations of the two Cd-thiolate clusters were distinctly different at two of the cadmium centers. In addition, solvent access to the sulfhydryl ligands of the isolated Cd(4)-alpha cluster was 130% larger due to this small change in cluster geometry. To probe whether these differences were an artifact of the structure calculation, the Cd(4)-alpha-domain structure in rabbit Cd(7)-MT 2 was redetermined, using the previously defined set of NOEs and the present calculation protocol. All calculations employed the same ionic radius for Cd(2+) and same cadmium-thiolate bond distance. The newly calculated structure matched the original with an RMSD of 1.24 A. It is hypothesized that differences in the two alpha-domain structures result from a perturbation of the holoprotein structure because of head-to-tail dimerization under the conditions of the NMR experiments.