Cellular Source and Mechanisms of High Transcriptome Complexity in the Mammalian Testis

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Reconfiguration of a Multi-oscillator Network by Light in the Drosophila Circadian Clock

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A Bacillus megaterium Plasmid System for the Production, Export, and One-Step Purification of Affinity-Tagged Heterologous Levansucrase from Growth Medium

A multiple vector system for the production and export of recombinant affinity-tagged proteins in Bacillus megaterium was developed. Up to 1 mg/liter of a His₆-tagged or Strep-tagged Lactobacillus reuteri levansucrase was directed into the growth medium, using the B. megaterium esterase LipA signal peptide, and recovered by one-step affinity chromatography.     

Activity and Diversity of Sulfate-Reducing Bacteria in a Petroleum Hydrocarbon-Contaminated Aquifer

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO₄²⁻ reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day⁻¹ for sulfate reduction and from 0.13 to 0.60 day⁻¹ for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.     

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.     

Characterization of a mutation that results in independence of oxidosqualene cyclase (Erg7) activity from the downstream 3-ketoreductase (Erg27) in the yeast ergosterol biosynthetic pathway

In yeast, deletion of ERG27, which encodes the sterol biosynthetic enzyme, 3-keto-reductase, results in a concomitant loss of the upstream enzyme, Erg7p, an oxidosqualene cyclase (OSC). However, this phenomenon occurs only in fungi, as mammalian Erg27p orthologues are unable to rescue yeast Erg7p activity. In this study, an erg27 mutant containing the mouse ERG27 orthologue was isolated that was capable of growing without sterol supplementation (FGerg27). GC/MS analysis of this strain showed an accumulation of squalene epoxides, 3-ketosterones, and ergosterol. This strain which was crossed to a wildtype and daughter segregants showed an accumulation of squalene epoxides as well as ergosterol indicating that the mutation entailed a leaky block at ERG7. Upon sequencing the yeast ERG7 gene an A598S alteration was found in a conserved alpha helical region. We theorize that this mutation stabilizes Erg7p in a conformation that mimics Erg27p binding. This mutation, while decreasing OSC activity still retains sufficient residual OSC activity such that the strain in the presence of the mammalian 3-keto reductase enzyme functions and no longer requires the yeast Erg27p. Because sterol biosynthesis occurs in the ER, a fusion protein was synthesized combining Erg7p and Erg28p, a resident ER protein and scaffold of the C-4 demethyation complex. Both FGerg27 and erg27 strains containing this fusion plasmid and the mouse ERG27 orthologue showed restoration of ergosterol biosynthesis with minimal accumulation of squalene epoxides. These results indicate retention of Erg7p in the ER increases its activity and suggest a novel method of regulation of ergosterol biosynthesis.     

A novel 145 kd brain cytosolic protein reconstitutes Ca(2+)-regulated secretion in permeable neuroendocrine cells.

The regulated secretory pathway is activated by elevated cytoplasmic Ca2+; however, the components mediating Ca2+ regulation have not been identified. In semi-intact neuroendocrine cells, Ca(2+)-activated secretion is ATP- and cytosol protein-dependent. We have identified a novel brain protein, p145, as a cytosolic factor that reconstitutes Ca(2+)-activated secretion in two neuroendocrine cell types. The protein is a dimer of 145 kd subunits, exhibits Ca(2+)-dependent interaction with a hydrophobic matrix, and binds phospholipid vesicles, suggesting a membrane-associated function. A p145-specific antibody inhibits the reconstitution of Ca(2+)-activated secretion by cytosol, indicating an essential role for p145. The restricted expression of p145 in tissues exhibiting a regulated secretory pathway suggests a key role for this protein in the transduction of Ca2+ signals into vectorial membrane fusion events.     

Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: Redirecting carbon flow

Abstract Threonine and lysine are two of the economically most important essential amino acids. They are produced industrially by species of the genera Corynebacterium and Brevibacterium . The branched biosynthetic pathway of these amino acids in corynebacteria is unusual in gene organization and in the control of key enzymatic steps with respect to other microorganisms. This article reviews the molecular control mechanisms of the biosynthetic pathways leading to threonine and lysine in corynebacteria, and their implications in the production of these amino acids. Carbon flux can be redirected at branch points by gene disruption of the competing pathways for lysine or threonine. Removal of bottlenecks has been achieved by amplification of genes which encode feedback resistant aspartokinase and homoserine dehydrogenase (obtained by in vitro directed mutagenesis).     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

Energy minimization in low-frequency normal modes to efficiently allow for global flexibility during systematic protein-protein docking

Protein-protein association can frequently involve significant backbone conformational changes of the protein partners. A computationally rapid method has been developed that allows to approximately account for global conformational changes during systematic protein-protein docking starting from many thousands of start configurations. The approach employs precalculated collective degrees of freedom as additional variables during protein-protein docking minimization. The global collective degrees of freedom are obtained from normal mode analysis using a Gaussian network model for the protein. Systematic docking searches were performed on 10 test systems that differed in the degree of conformational change associated with complex formation and in the degree of overlap between observed conformational changes and precalculated flexible degrees of freedom. The results indicate that in case of docking searches that minimize the influence of local side chain conformational changes inclusion of global flexibility can significantly improve the agreement of the near-native docking solutions with the corresponding experimental structures. For docking of unbound protein partners in several cases an improved ranking of near native docking solutions was observed. This was achieved at a very modest ( approximately 2-fold) increase of computational demands compared to rigid docking. For several test cases the number of docking solutions close to experiment was also significantly enhanced upon inclusion of soft collective degrees of freedom. This result indicates that inclusion of global flexibility can facilitate in silico protein-protein association such that a greater number of different start configurations results in favorable complex formation.