Molecular phylogeny of the fungus gnat tribe Exechiini (Mycetophilidae, Diptera)

The phylogenetic relationships within the fungus gnat tribe Exechiini have been left unattended for many years. Recent studies have not shed much light on the intergeneric relationship within the tribe. Here the first attempt to resolve the phylogeny of the tribe Exechiini using molecular markers is presented. The nuclear 18S and the mitochondrial 16S, and cytochrome oxidase subunit I (COI) genes were successfully sequenced for 20 species representing 15 Exechiini genera and five outgroup genera. Bayesian, maximum parsimony and maximum likelihood analyses revealed basically congruent tree topologies and the monophyly of Exechiini, including the genus Cordyla , is confirmed. The molecular data corroborate previous morphological studies in several aspects. Cordyla is found in a basal clade together with Brachypeza , Pseudorymosia and Stigmatomeria . The splitting of the genera Allodiopsis s.l. and Brevicornu s.l. as well as the sistergroup relationship of Exechia and Exechiopsis is also supported. The limited phylogenetic information provided by morphological characters is mirrored in the limited resolution of the molecular markers used in this study. Short internal and long-terminal branches obtained may indicate a rapid radiation of the Exechiini genera during a short evolutionary period.     

Isolation and characterization of acetonitrile utilizing bacteria

Summary Bacteria utilizing high concentrations of acetonitrile as the sole carbon source were isolated and identified asChromobacterium sp. andPseudomonas aeruginosa. Maximum growth was attained after 96 h of incubation andP. aeruginosa grew slightly faster thanChromobacterium sp. The strains were able to grow and oxidize acetonitrile at concentrations as high as 600 mM. However, higher concentrations inhibited growth and oxygen uptake. Degradation studies with (¹⁴C)acetonitrile indicated 57% of acetonitrile was degraded byPseudomonas aeruginosa as compared to 43% byChromobacterium. The isolates utilized different nitrile compounds as carbon substrates.     

An insertion element of the extremely thermophilic archaeon Sulfolobus solfataricus transposes into the endogenous β-galactosidase gene

Three phenotypically stable mutants of the extremely thermophilic archaeon Sulfolobus solfataricus have been isolated by screening for β-galactosidase negative colonies on plates with X-Gal (5-bromo-4-chloro-3-indolyl-(3-d-galactopyranoside). From one of these mutants an insertion element, designated ISC1217, was isolated and characterized. Sequence analysis of ISC1217 and of the regions adjacent to the insertion site in the β-galactosidase gene revealed features typical of a transposable element: ISC1217 contained terminal inverted repeats and was flanked by a direct repeat of 6 bp. The 1147 by sequence contained an open reading frame encoding a putative protein of 354 amino acid residues and, overlapping this, two smaller open reading frames on the opposite strand. There were approximately 8 copies of the insertion element in the S. solfataricus genome. ISC1217 did not cross-hybridize with DNA of other Sulfolobus species. All three independently isolated β-galactosidase mutants of S. solfataricus arose by transposition of ISC1217 or a related element.     

Mechanical Network in Titin Immunoglobulin from Force Distribution Analysis

The role of mechanical force in cellular processes is increasingly revealed by single molecule experiments and simulations of force-induced transitions in proteins. How the applied force propagates within proteins determines their mechanical behavior yet remains largely unknown. We present a new method based on molecular dynamics simulations to disclose the distribution of strain in protein structures, here for the newly determined high-resolution crystal structure of I27, a titin immunoglobulin (IG) domain. We obtain a sparse, spatially connected, and highly anisotropic mechanical network. This allows us to detect load-bearing motifs composed of interstrand hydrogen bonds and hydrophobic core interactions, including parts distal to the site to which force was applied. The role of the force distribution pattern for mechanical stability is tested by in silico unfolding of I27 mutants. We then compare the observed force pattern to the sparse network of coevolved residues found in this family. We find a remarkable overlap, suggesting the force distribution to reflect constraints for the evolutionary design of mechanical resistance in the IG family. The force distribution analysis provides a molecular interpretation of coevolution and opens the road to the study of the mechanism of signal propagation in proteins in general.     

Imaging synaptic inhibition in transgenic mice expressing the chloride indicator, Clomeleon

We describe here a molecular genetic approach for imaging synaptic inhibition. The thy-1 promoter was used to express high levels of Clomeleon, a ratiometric fluorescent indicator for chloride ions, in discrete populations of neurons in the brains of transgenic mice. Clomeleon was functional after chronic expression and provided non-invasive readouts of intracellular chloride concentration ([Cl(-)](i)) in brain slices, allowing us to quantify age-dependent declines in resting [Cl(-)](i) during neuronal development. Activation of hippocampal interneurons caused [Cl(-)](i) to rise transiently in individual postsynaptic pyramidal neurons. [Cl(-)](i) increased in direct proportion to the amount of inhibitory transmission, with peak changes as large as 4 mM. Integrating responses over populations of pyramidal neurons allowed sensitive detection of synaptic inhibition. Thus, Clomeleon imaging permits non-invasive, spatiotemporally resolved recordings of [Cl(-)](i) in a large variety of neurons, opening up new opportunities for imaging synaptic inhibition and other forms of chloride signaling.     

Mutations of the PC2 substrate binding pocket alter enzyme specificity

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.