Systematic identification of genes that regulate neuronal wiring in the Drosophila visual system

Forward genetic screens in model organisms are an attractive means to identify those genes involved in any complex biological process, including neural circuit assembly. Although mutagenesis screens are readily performed to saturation, gene identification rarely is, being limited by the considerable effort generally required for positional cloning. Here, we apply a systematic positional cloning strategy to identify many of the genes required for neuronal wiring in the Drosophila visual system. From a large-scale forward genetic screen selecting for visual system wiring defects with a normal retinal pattern, we recovered 122 mutations in 42 genetic loci. For 6 of these loci, the underlying genetic lesions were previously identified using traditional methods. Using SNP-based mapping approaches, we have now identified 30 additional genes. Neuronal phenotypes have not previously been reported for 20 of these genes, and no mutant phenotype has been previously described for 5 genes. The genes encode a variety of proteins implicated in cellular processes such as gene regulation, cytoskeletal dynamics, axonal transport, and cell signalling. We conducted a comprehensive phenotypic analysis of 35 genes, scoring wiring defects according to 33 criteria. This work demonstrates the feasibility of combining large-scale gene identification with large-scale mutagenesis in Drosophila, and provides a comprehensive overview of the molecular mechanisms that regulate visual system wiring.     

Ecological and historical factors affecting distribution pattern and richness of endemic plant species: the case of the Maritime and Ligurian Alps hotspot

The aim of this study was to test a method to locate all the foci, centres, and areas of endemism in a biodiversity hotspot in order to understand the influence of ecological and historical factors on the distribution pattern and to identify priority areas for future conservation projects. The study area was the Maritime and Ligurian Alps hotspot.
Analyses were performed on the presence/absence matrix of 36 vascular plant taxa endemic to the study area. For each operational geographical unit, the number of endemic taxa present was counted. Additionally, the weighted endemism value was calculated. Areas of endemism were distinguished using cluster analysis and parsimony analysis of endemicity. The influence of ecological characteristics and historical factors was evaluated using Multi-Response Permutation Procedure and the Nonparametric Multiplicative Regression. The Indicator Species Analysis (INDVAL) method was used to identify the species characterizing the areas of endemism. Our results show the importance and location of four main areas of endemism within the Maritime and Ligurian Alps and explain the distribution pattern of endemic plants. These areas are easily interpreted by historical and ecological factors, and INDVAL indicates which taxa took part in the history of each endemism area.     

Critical view on the monochlorodimedone assay utilized to detect haloperoxidase activity

The current study aimed to identify the halogenating enzymes involved in the biosynthesis of the ambigols A, B, C and tjipanazole D, isolated from the cyanobacterium Fischerella ambigua. Haloperoxidase (HPO) activity within F. ambigua was therefore assayed spectrophotometrically by using monochlorodimedone (MCD) during protein purification. This strategy revealed the isolation of a protein positive in the MCD-assay, but an involvement in halogenating processes could not be verified. N-terminal sequencing rather demonstrated homology to cytochrome c(6) from other cyanobacteria and green algae. From our findings it thus has to be concluded that the spectrophotometrical MCD-assay routinely used to detect HPO activity may yield false positive results, mainly since the assay focuses on the decline of the educt and not on the formation of the product. Our data indicate that the reaction of MCD with proteins of the cytochrome c- family leads to unspecific products.     

Diversity and Antagonistic Potential of Bacteria Associated with Bryophytes from Nutrient-Poor Habitats of the Baltic Sea Coast

Very little is known about the interaction of bryophytes with bacteria. Therefore, we analyzed bacteria associated with three bryophyte species, Tortula ruralis, Aulacomnium palustre, and Sphagnum rubellum, which represent typical moss species of three nutrient-poor plant communities at the southern Baltic Sea coast in Germany. By use of two cultivation-independent techniques, denaturing gradient gel electrophoresis and single-strand conformation polymorphism analysis of the 16S ribosomal DNA, a high degree of moss specificity was found for associated bacterial communities. This specificity could be further evidenced by a cultivation-dependent approach for the following parameters: (i) plate counts of bacteria on R2A medium, (ii) proportion of antagonistic isolates, (iii) antagonistic activity as well as spectrum against pathogens, and (iv) diversity and richness of antagonistic isolates. The proportion of isolates with antagonistic activity against the pathogenic model fungus Verticillium dahliae was highest for S. rubellum (31%), followed by A. palustre (17%) and T. ruralis (5%). A high percentage (99%) of moss-associated antagonistic bacteria produced antifungal compounds. The high recovery of antagonistic isolates strongly suggests that bryophytes represent an ecological niche which harbors a diverse and hitherto largely uncharacterized microbial population with yet unknown and untapped potential biotechnological applications, e.g., for biological control of plant pathogens.     

High-resolution mapping of the human 4q21 and the mouse 5E3 SCYB chemokine cluster by fiber-fluorescence in situ hybridization

The CXC chemokine or small inducible cytokine B (SCYB) subfamily includes the T-cell chemoattractants MIG (CXCL9, SCYB9), IP-10 (CXCL10, SCYB10), and I-TAC (CXCL11, SCYB11). These three highly homologous chemokines lack the glutamic acid-leucine-arginine (ELR) motif and signal via the CXCR3 receptor. Previous work showed that the genes encoding these chemokines are localized in an individual mini-cluster on human Chromosome (Chr) 4 at position 4q21.2. Recently, we identified mouse Scyb11 and mapped this gene by fluorescence in situ hybridization (FISH) to mouse Chr 5E3, the orthologous locus to human 4q21 where the other two homologous mouse genes, Scyb9 and Scyb10, have also been localized. Since SCYB10 and SCYB11 are not represented in the recently published draft sequence of the human genome, we wanted to clarify exactly the order and distances of the three chemokine genes using two-color FISH on stretched DNA fiber preparations. Here, we report the simultaneous localization of all three genes and provide high-resolution visual maps of this chemokine cluster from both mouse and human. The three chemokine genes were found within a range of 32 kb on mouse and 29 kb on human DNA fiber targets. The precise physical distances were defined, and an almost identical arrangement of the human and mouse homologues was identified, indicating that this CXC chemokine mini-cluster has been completely conserved evolutionarily since the divergence of mouse and human. Our results refine previous maps of the three genes, support the hypothesis that they resulted from gene duplication that took place in a common ancestor of mouse and human, and provide complementary information on a region of the draft sequence of human Chr 4 that is not yet covered.     

Widespread occurrence of glyceryl ether monooxygenase activity in rat tissues detected by a novel assay

An assay was set up for glyceryl ether monooxygenase activity in tissue samples using the novel substrate 1-O-pyrenedecyl-sn-glycerol and high-performance liquid chromatographic analysis of reaction mixtures with fluorescence detection, allowing robust detection of enzymatic activity in microgram amounts of tissue homogenates. The activity partially purified from rat liver strictly depended on the presence of a tetrahydropteridine. Tetrahydrobiopterin-dependent glyceryl ether monooxygenase activity was observed in all rat tissues tested except female heart, with highest activities in liver, intestine, and cerebellum. Activity was not uniformly distributed in brain: it was higher in cerebellum than in striatum or cortex. These data demonstrate that tetrahydrobiopterin-dependent glyceryl ether monooxygenase is found not only in liver and the gastrointestinal tract but also in brain and other organs of the rat and provide an additional goal for tetrahydrobiopterin biosynthesis in these organs.     

Mhc polymorphisms fail to explain the heritability of phytohaemagglutinin-induced skin swelling in a wild passerine

Genetic estimates of the variability of immune responses are rarely examined in natural populations because of confounding environmental effects. As a result, and because of the difficulty of pinpointing the genetic determinants of immunity, no study has to our knowledge examined the contribution of specific genes to the heritability of an immune response in wild populations. We cross-fostered nestling house sparrows to disrupt the association between genetic and environmental effects and determine the heritability of the response to a classic immunological test, the phytohaemagglutinin (PHA)-induced skin swelling. We detected significant heritability estimates of the response to PHA, of body mass and tarsus length when nestlings were 5 and 10 days old. Variation at Mhc genes, however, did not explain a significant portion of the genetic variation of nestling swelling to PHA. Our results suggest that while PHA-induced swelling is influenced by the nest of origin, the importance of additive genetic variation relative to non-additive genetic variation and the genetic factors that influence the former in wild populations still need to be identified for this trait.     

Docking studies on PARP-1 inhibitors: insights into the role of a binding pocket water molecule

The binding mode of a series of competitive PARP-1 inhibitors was investigated employing a molecular docking approach by using Autodock 3.0. A particular attention was given to the role played by a water molecule present in some but not all the so far available crystal structures of the catalytic domain of PARP-1. Good correlation between calculated binding energies and experimental inhibitory activities was obtained either by including (r2=0.87) or not (r2=0.84) the structural water molecule. Closer inspection of our results suggested that this water molecule should be considered part of the hydration shell of polar inhibitors and not as a structural water.     

Separation and quantitation of metallothionein isoforms from liver of untreated rats by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry

A sensitive method for determination of metallothionein (MT) isoform levels in rat liver by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry was developed. Critical steps in sample preparation, like MT extraction, MT saturation with Cd and protein separation, were optimized. This method is capable of measuring levels of 2.0 μg/g liver for metallothionein-1 (MT-1) and 1.3 μg/g liver for metallothionein-2 (MT-2), respectively, with a high recovery of 103% on average. The method described, thus, proved suitable for analyzing metallothionein isoform concentrations even in untreated animals. The ratio of MT-1 to MT-2 was found to be 1:1 on average. MT decomposition during storage was very high in whole livers, but could be reduced by about 80% when extracted liver samples were used.