Splicing factor SRp30c interaction with Y-box protein-1 confers nuclear YB-1 shuttling and alternative splice site selection

The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5' splicing site selection and formation of the 12 S isoform.     

Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gram-negative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 microm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low microg/ml range, whereas eukaryotic cells were not affected up to 60 microg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.     

CD and NMR studies of prion protein (PrP) helix 1. Novel implications for its role in the PrPC-->PrPSc conversion process

The conversion of prion helix 1 from an alpha-helical into an extended conformation is generally assumed to be an essential step in the conversion of the cellular isoform PrPC of the prion protein to the pathogenic isoform PrPSc. Peptides encompassing helix 1 and flanking sequences were analyzed by nuclear magnetic resonance and circular dichroism. Our results indicate a remarkably high instrinsic helix propensity of the helix 1 region. In particular, these peptides retain significant helicity under a wide range of conditions, such as high salt, pH variation, and presence of organic co-solvents. As evidenced by a data base search, the pattern of charged residues present in helix 1 generally favors helical structures over alternative conformations. Because of its high stability against environmental changes, helix 1 is unlikely to be involved in the initial steps of the pathogenic conformational change. Our results implicate that interconversion of helix 1 is rather representing a barrier than a nucleus for the PrPC-->PrPSc conversion.     

Patterns and dynamics of sex-biased dispersal in a nocturnal primate, the grey mouse lemur, Microcebus murinus

We examined predictions on the proportion of dispersing natal males and females, dispersal distances, the age at dispersal and the potential for inbreeding over a 6-year period in a free-living population of grey mouse lemurs. We used monthly mark-recapture procedures to determine individual locations and interindividual distances. The analysis of seven polymorphic microsatellite markers for 213 (130 males, 83 females) individuals allowed us to estimate relatedness coefficients and kinship relationships. Closely related males ranged further from each other than closely related females and natal males were found further from their potential mothers than were females. Natal males were more likely to disperse from their birth sites than females, although male dispersal was not universal. Male breeding dispersal was detected in half of the long-term observations. Males therefore seem to be the predominant vectors for gene flow between populations and social units. Females usually stayed within one to two home range diameters of their potential mother, facilitating the evolution of cooperative behaviour by kin selection among females. Most dispersal took place before the mating season, indicating an age of less than 7 months for natal dispersal. The analysis of spatiotemporal coexistence revealed the potential for inbreeding in only 3.8% of the potential mother-son dyads, but in 21.9% of the potential father-daughter dyads and in 41.7% of other closely related male-female dyads. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour      

Choline head groups stabilize the matrix loop regions of the ATP/ADP carrier ScAAC2

ATP/ADP carriers (AACs) are essential to the cell as they exchange ATP produced in mitochondria for cytosolic ADP. Monoclonal antibodies against the isoform 2 of Saccharomyces cerevisiae AAC (ScAAC2) were used to probe the accessibility of the matrix loops 1 and 3 depending on the environment of the carrier. In mitochondrial membranes ScAAC2 was not recognized, whereas in dodecylmaltoside the antibodies bound to the carrier, suggesting that the epitopes are hidden in the native environment. Exposure of the epitopes by detergents was reversed by reconstitution of the carrier in phospholipids or by exchanging with detergents having a choline or a trimethylammonium head group. Circular dichroism spectroscopy on peptides representing the C-terminal regions of all three matrix loops showed that only phosphocholine detergents induced a structural reorganization. Since in addition phosphatidylcholine was found to be tightly associated with the purified carrier, the matrix loop regions are likely to be associated to the membrane by phosphatidylcholine.     

Genomic structure of the gene for mouse germ-cell nuclear factor (GCNF). II. Comparison with the genomic structure of the human GCNF gene

Background  

Germ-cell nuclear factor (GCNF, NR6AI) is an orphan nuclear receptor. Its expression pattern suggests it functions during embryogenesis, in the placenta and in germ-cell development. Mouse GCNF cDNA codes for a protein of 495 amino acids, whereas the four reported human cDNA variants code for proteins of 454 to 480 amino acids. Apart from this size difference, there is sequence conservation of up to 98.7%. To elucidate the genomic structure that gives rise to the different human GCNF mRNAs, the sequence information of the human GCNF locus is compared to the previously reported structure of the mouse locus.     

Multitrophic interactions of the silverleaf whitefly,host plants,competing herbivores,and phytopathogens

Our laboratory found that silverleaf whitefly (SLW; Bemisia argentifolii Bellows & Perring) feeding alters host plant physiology and chemistry. The SLW induces a number of host plant defenses, including pathogenesis-related (PR) protein accumulation (e.g., chitinases, beta-1,3-glucanases, peroxidases, chitosanases, etc.). Induction of the PR proteins by SLW feeding occurs in various plant species and varieties. The extent and type of induction is dependent on a number of factors that include host plant growing conditions, the length of time the host plant is exposed to SLW feeding, the plant variety, and SLW population densities. The appearance of PR proteins correlates well with reduced infestations of conspecific insect herbivore competitors. Greenhouse and field experiments in which herbivore competitors (cabbage looper, Trichoplusia ni; leaf miner, Liromyza trifolii) were placed on plants previously exposed to SLW feeding demonstrated behavioral differences (oviposition, feeding preferences) and reduced survival rates and development times of these insects. The interaction was asymmetrical, i.e., SLW infestations of plants previously exposed to leaf miners had little or no effect on SLW behavior (oviposition). Induction of plant-defensive proteins by SLW feeding was both local (at the feeding site) and systemic (uninfested leaves distant to the feeding site). There are interactions between diseases such as tomato mottle virus (ToMoV; a geminivirus) and the host plant and SLW. PR proteins were induced in tomato plants infected with ToMoV much as they were via non-viruliferous SLW feeding. The presence of ToMoV in tomato plants significantly increased the number of eggs produced by SLW females. Experiments using tomato plants, powdery mildew (PM), and tobacco mosaic virus (TMV) show that whitefly infestations can affect plant pathogen relationships but the effects vary among pathogen types. Enzyme analyses prior to pathogen inoculation showed that whitefly treatment significantly increased the activities of foliar chitinase and peroxidase. Evaluation of pathogen growth 3 weeks after inoculation showed that whitefly feeding significantly reduced the incidence of PM. However, TMV levels evaluated by ELISA were not significantly affected by whitefly feeding. Six weeks after inoculation with pathogens, the chitinase and peroxidase activities were still elevated in plants initially fed on by whiteflies but continuing pathogen infection had no effect on these enzymes. The possibility that geminivirus infection and/or SLW infestations isolate the host plant for the selected reproduction of the virus and the insect is discussed. Multitrophic cascade effects may contribute to the successful eruptive appearance of SLW on various crops, ranking them as a major pest. They may explain the general observation that when SLW infest a host plant there are few if any competing insect herbivores and pathogens found in the host. However, the results indicate that certain SLW-virus relationships could be mutualistic.     

Evolution of Duplicated <Emphasis Type="BoldItalic">reggie</Emphasis> Genes in Zebrafish and Goldfish

Invertebrates, tetrapod vertebrates, and fish might be expected to differ in their number of gene copies, possibly due the occurrence of genome duplication events during animal evolution. Reggie (flotillin) genes code for membrane-associated proteins involved in growth signaling in developing and regenerating axons. Until now, there appeared to be only two reggie genes in fruitflies, mammals, and fish. The aim of this research was to search for additional copies of reggie genes in fishes, since a genome duplication might have increased the gene copy number in this group. We report the presence of up to four distinct reggie genes (two reggie-1 and two reggie-2 genes) in the genomes of zebrafish and goldfish. Phylogenetic analyses show that the zebrafish and goldfish sequence pairs are orthologous, and that the additional copies could have arisen through a genome duplication in a common ancestor of bony fish. The presence of novel reggie mRNAs in fish embryos indicates that the newly discovered gene copies are transcribed and possibly expressed in the developing and regenerating nervous system. The intron/exon boundaries of the new fish genes characterized here correspond with those of human genes, both in location and phase. An evolutionary scenario for the evolution of reggie intron-exon structure, where loss of introns appears to be a distinctive trait in invertebrate reggie genes, is presented. Received: 24 January 2001 / Accepted: 27 July 2001