Aptamer-guided gene targeting in yeast and human cells

Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting.     

Rap1 activation is required for Fc gamma receptor-dependent phagocytosis

Phagocytosis of IgG-opsonized microbes via the Fc gamma receptor (Fc gammaR) requires the precise coordination of a number of signaling molecules, including the low-molecular mass GTPases. Little is known about the Ras-family GTPase Rap1 in this process. We therefore investigated its importance in mediating Fc gammaR-dependent phagocytosis in NR8383 rat alveolar macrophages. Pulldown of active Rap1 and fluorescence microscopic analysis of GFP-RalGDS (Ral guanine dissociation stimulator)-transfected macrophages revealed that Rap1 is indeed activated by Fc gammaR crosslinking. Inhibition of Rap1 activity, both by Rap1GAP (GTPase-activating protein) expression and liposome-delivered blocking Ab, severely impaired the ability of cells to ingest IgG-opsonized targets. Fc gammaR-induced Rap1 activation was found to be independent of both cAMP and Ca(2+), suggesting a role for the second messenger-independent guanosine exchange factor, C3G. This was supported by the facts that 1) liposome-delivered blocking Ab against C3G inhibited both Fc gammaR-dependent phagocytosis and Rap1 activation, and 2) both active Rap1GTP and C3G were found to translocate to the phagosome. Taken together, our data demonstrate a novel role for Rap1 and its exchange factor C3G in mediating Fc gammaR-dependent phagocytosis.     

Protein-DNA interactions: structural, thermodynamic and clustering patterns of conserved residues in DNA-binding proteins

Amino acid residues, which play important roles in protein function, are often conserved. Here, we analyze thermodynamic and structural data of protein-DNA interactions to explore a relationship between free energy, sequence conservation and structural cooperativity. We observe that the most stabilizing residues or putative hotspots are those which occur as clusters of conserved residues. The higher packing density of the clusters and available experimental thermodynamic data of mutations suggest cooperativity between conserved residues in the clusters. Conserved singlets contribute to the stability of protein-DNA complexes to a lesser extent. We also analyze structural features of conserved residues and their clusters and examine their role in identifying DNA-binding sites. We show that about half of the observed conserved residue clusters are in the interface with the DNA, which could be identified from their amino acid composition; whereas the remaining clusters are at the protein-protein or protein-ligand interface, or embedded in the structural scaffolds. In protein-protein interfaces, conserved residues are highly correlated with experimental residue hotspots, contributing dominantly and often cooperatively to the stability of protein-protein complexes. Overall, the conservation patterns of the stabilizing residues in DNA-binding proteins also highlight the significance of clustering as compared to single residue conservation.     

Life forms and strategies of lichen-feeding darkling beetles (Coleoptera,Tenebrionidae: Helopini)

Trophic relations of lichen-feeding darkling beetles of the tribe Helopini in Turkey, the Caucasus, and Anti-Atlas are considered. This group is the most diverse in relict Anatolian forests of Cedrus libani, Abies cilicica, Pinus nigra, Juniperus spp., and Quercus cerris, where multitaxon lichenophagous assemblages exist. Three life strategies for attenuation of interspecific competition are distinguished: (1) differentiation of the consumed lichens, (2) separation of the life forms based on the types of the lichens consumed, and (3) segregation of circadian activity in adults. We assume that adaptation of the Helopini larvae to the soil life promoted their wide distribution in different landscapes by elimination of the limiting factor for completing their life cycles, namely the need for ligneous substrate of old rotting trees. The possibility of using lichen-feeding beetles of the tribe Helopini in ecosystem and lichen indication is considered.     

Herbivory in global climate change research: direct effects of rising temperature on insect herbivores

This review examines the direct effects of climate change on insect herbivores. Temperature is identified as the dominant abiotic factor directly affecting herbivorous insects. There is little evidence of any direct effects of CO₂ or UVB. Direct impacts of precipitation have been largely neglected in current research on climate change. Temperature directly affects development, survival, range and abundance. Species with a large geographical range will tend to be less affected. The main effect of temperature in temperate regions is to influence winter survival; at more northerly latitudes, higher temperatures extend the summer season, increasing the available thermal budget for growth and reproduction. Photoperiod is the dominant cue for the seasonal synchrony of temperate insects, but their thermal requirements may differ at different times of year. Interactions between photoperiod and temperature determine phenology; the two factors do not necessarily operate in tandem. Insect herbivores show a number of distinct life‐history strategies to exploit plants with different growth forms and strategies, which will be differentially affected by climate warming. There are still many challenges facing biologists in predicting and monitoring the impacts of climate change. Future research needs to consider insect herbivore phenotypic and genotypic flexibility, their responses to global change parameters operating in concert, and awareness that some patterns may only become apparent in the longer term.     

Diversity and distribution of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in Turkey

The diversity and distribution of entomopathogenic nematodes in thefamilies Steinernematidae and Heterorhabditidae were assessed throughout anextensive soil survey in Turkey during 1999 and 2000. Entomopathogenic nematodeswere recovered from six out of seven regions sampled, with 22 positive sites(2%) out of 1080 sites sampled. A single nematode isolate was recovered at eachof the positive sites, of which 15 were steinernematid isolates and seven wereheterorhabditid isolates representing a total of four species. Based onmorphometric and molecular data, the nematode species were identified asHeterorhabditis bacteriophora, Steinernemafeltiae, S. affine, andSteinernema n. sp. The most common species was S.feltiae, which was isolated from 10 sites in six regions, followed byH. bacteriophora from seven sites in five regions,S. affine from four sites in two regions, andSteinernema n. sp. from one site. Heterorhabditisbacteriophora and S. feltiae have been found inmany parts of the world, whereas S. affine, so far, hasonly been recovered in Europe until our survey. Steinernemaaffine was isolated from the European (Marmara) as well as theAsiatic region (Middle Anatolia) of Turkey. A new undescribedSteinernema sp. was isolated from the most eastern region(East Anatolia) of Turkey. Soils of the positive sites were classified as sandy,sandy loam, or loam (68.2%) and sandy–clay–loam or clay loam (31.8%) and the pHranged from 5.6 to 7.9. The habitats from which the entomopathogenic nematodeswere isolated were broadly classified as disturbed (59.1%), which includedagricultural fields and poplar planted for lumber and wind breaks, andundisturbed (40.9%), which included pine forest, grassland, marsh and reed sites.Steinernema feltiae, S. affine, andH. bacteriophora were recovered from both disturbed andundisturbed habitats. The new Steinernema sp. was recoveredfrom grassland. Our survey showed that these nematodes occur widely throughoutTurkey, but at a frequency below that reported for other parts of the world.