Hybrids and fruit set in a mixed flowering-time population of Gymnadenia conopsea (Orchidaceae)

We have recently found that the morphologically determined subspecies Gymnadenia conopsea ssp conopsea in Sweden includes early and late flowering individuals. We were interested in the interactions between the flowering time groups; if there were gene flow between them and if so this was detrimental or advantageous. A spatially mixed population of early and late flowering individuals was studied using three microsatellite loci. We measured patterns in genetic differentiation and inferred occurrence of hybridisation and introgression. Variation in flowering time, fertility and relative and absolute fruit set was measured. The pattern of introgression between flowering-time groups differed between loci. In two of the three investigated loci, allele separation was distinct between early and late flowering plants and one genetically obvious hybrid was infertile. In the third locus, several alleles were shared between the two flowering time variants. The degree of introgression was associated to fruit set failure, which was higher in the late flowering plants and lower in early flowering plants. A small group of early flowering individuals with somewhat delayed flowering compared to the main group was genetically distinct and had lower relative and absolute fruit set. This group was not genetically intermediate, but rather constituting an independent group, with lower fruit set possibly caused by absence of pollinators. There seem to be a strong barrier against introgression into the late flowering group which is kept genetically distinct and less diverse. The early flowering group is diverse, includes two subgroups and seems to benefit from gene flow.     

Tetranuclear homoleptic copper and silver aryls: 2,4,6-Triethylphenylcopper and 2,4,6-triethylphenylsilver

The ethyl analogues of mesitylcopper and mesitylsilver, 2,4,6-triethylphenylcopper and 2,4,6-triethylphenylsilver, have been prepared and characterised by means of crystal structure determination. Three different compounds, [Cu₄(Et₃Ph)₄] · 2C₆H₅CH₃ (1), and , have thus been obtained by crystallisation from toluene or diethylether/tetrahydrofuran. All three show the square-planar metal core, most characteristic of homoleptic copper and silver aryls. The ordered (1) or disordered (2 and 3) solvent molecules are situated in the channels formed between the organometallic complexes, there being no strong directional interactions between the solvent and 2,4,6-triethylphenylcopper or 2,4,6-triethylphenylsilver.     

Salting the charged surface: pH and salt dependence of protein G B1 stability

This study shows significant effects of protein surface charges on stability and these effects are not eliminated by salt screening. The stability for a variant of protein G B1 domain was studied in the pH-range of 1.5-11 at low, 0.15 M, and 2 M salt. The variant has three mutations, T2Q, N8D, and N37D, to guarantee an intact covalent chain at all pH values. The stability of the protein shows distinct pH dependence with the highest stability close to the isoelectric point. The stability is pH-dependent at all three NaCl concentrations, indicating that interactions involving charged residues are important at all three conditions. We find that 2 M salt stabilizes the protein at low pH (protein net charge is +6 and total number of charges is 6) but not at high pH (net charge is or=18). Furthermore, 0.15 M salt slightly decreases the stability of the protein over the pH range. The results show that a net charge of the protein is destabilizing and indicate that proteins contain charges for reasons other than improved stability. Salt seems to reduce the electrostatic contributions to stability under conditions with few total charges, but cannot eliminate electrostatic effects in highly charged systems.     

Predicting the solvent accessibility of transmembrane residues from protein sequence

In this study, we propose a novel method to predict the solvent accessible surface areas of transmembrane residues. For both transmembrane alpha-helix and beta-barrel residues, the correlation coefficients between the predicted and observed accessible surface areas are around 0.65. On the basis of predicted accessible surface areas, residues exposed to the lipid environment or buried inside a protein can be identified by using certain cutoff thresholds. We have extensively examined our approach based on different definitions of accessible surface areas and a variety of sets of control parameters. Given that experimentally determining the structures of membrane proteins is very difficult and membrane proteins are actually abundant in nature, our approach is useful for theoretically modeling membrane protein tertiary structures, particularly for modeling the assembly of transmembrane domains. This approach can be used to annotate the membrane proteins in proteomes to provide extra structural and functional information.     

Experimental investigation of an optimization approach to microwave tomography

Microwave imaging is an interesting and growing research field with a number of medical applications. This paper is based on the first series of experimental results using an iterative gradient algorithm based on the finite difference time domain (FDTD) method and synthetic pulses. Using our method, the permittivity and conductivity of an object are reconstructed layer by layer by minimizing a functional consisting of the difference between the measured and calculated electric field surrounding the object. This is done by surrounding the object with a number of antennas which are all in turn transmitting and receiving. The dielectric profiles used in the calculations are then iteratively updated until the functional is minimized. Results are presented demonstrating the ability to detect metallic and dielectric material in air and water.     

How a lateralized brain supports symmetrical bimanual tasks

A large repertoire of natural object manipulation tasks require precisely coupled symmetrical opposing forces by both hands on a single object. We asked how the lateralized brain handles this basic problem of spatial and temporal coordination. We show that the brain consistently appoints one of the hands as prime actor while the other assists, but the choice of acting hand is flexible. When study participants control a cursor by manipulating a tool held freely between the hands, the left hand becomes prime actor if the cursor moves directionally with the left-hand forces, whereas the right hand primarily acts if it moves with the opposing right-hand forces. In neurophysiological (electromyography, transcranial magnetic brain stimulation) and functional magnetic resonance brain imaging experiments we demonstrate that changes in hand assignment parallels a midline shift of lateralized activity in distal hand muscles, corticospinal pathways, and primary sensorimotor and cerebellar cortical areas. We conclude that the two hands can readily exchange roles as dominant actor in bimanual tasks. Spatial relationships between hand forces and goal motions determine hand assignments rather than habitual handedness. Finally, flexible role assignment of the hands is manifest at multiple levels of the motor system, from cortical regions all the way down to particular muscles.     

Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis

Currently, in real-time PCR, one often has to choose between using a sequence-specific probe and a nonspecific double-stranded DNA (dsDNA) binding dye for the detection of amplified DNA products. The sequence-specific probe has the advantage that it only detects the targeted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplification, which reveals what kind of products have been formed. Here we present a new strategy based on combining a sequence-specific probe and a nonspecific dye, BOXTO, in the same reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan probes and locked nucleic acid (LNA) probes without interfering with the PCR. The probe signal reflect formation of target product, while melting curve analysis of the BOXTO signal reveals primer-dimer formation and the presence of any other anomalous products.     

Novel conserved hydrolase domain in the CLCA family of alleged calcium-activated chloride channels

Advanced protein structure prediction methods combined with structure modeling show that the mammalian proteins, described until now as calcium-activated chloride channels (CLCAs), appear in fact to be membrane anchored metal-dependent hydrolases, possibly proteases. A metallohydrolase structural domain was predicted, unexpectedly, in the CLCA sequences. The well-conserved active site in the modeled structure of this hydrolase domain allows the prediction of catalytic action similar to that of metalloproteases. A number of protein structure prediction methods suggest the overall fold of the N-terminal hydrolase domain to be most similar to that of zinc metalloproteases (zincins), notably matrixins. This is confirmed by analysis of the three-dimensional structure model of the predicted CLCA1 hydrolase domain built using the known structure of the MMP-11 catalytic domain. Fragments of CLCA1 corresponding to the modeled hydrolase domain were expressed in Escherichia coli, and the resulting proteins were readily refolded into monomeric soluble protein, indicating formation of stable independent domains. The homology model was used to predict putative substrate sequences. Homologs of mammalian CLCA genes were detected in the genomes of a vast array of multicellular animals: lower vertebrates, tunicates, insects, crustaceans, echinoderms, and flatworms. The hydrolase prediction is discussed in the context of published experimentally determined effects of CLCA proteins on chloride conductance. Altered proteolytic processing of full-length CLCA1 containing a mutation abolishing the predicted hydrolase activity is shown as initial experimental evidence for a role of the hydrolase domain in processing of mature full-length CLCA1. The hydrolase prediction together with the presented experimental data add to doubts about the function of CLCAs as chloride channels and strengthen the hypothesis of channel-activating and/or channel-accessory roles.     

Substitution Rate Heterogeneity and the Male Mutation Bias

Germline mutation rates have been found to be higher in males than in females in many organisms, a likely consequence of cell division being more frequent in spermatogenesis than in oogenesis. If the majority of mutations are due to DNA replication error, the male-to-female mutation rate ratio (α_m) is expected to be similar to the ratio of the number of germ line cell divisions in males and females (c), an assumption that can be tested with proper estimates of α_m and c. α_m is usually estimated by comparing substitution rates in putatively neutral sequences on the sex chromosomes. However, substantial regional variation in substitution rates across chromosomes may bias estimates of α_m based on the substitution rates of short sequences. To investigate regional substitution rate variation, we estimated sequence divergence in 16 gametologous introns located on the Z and W chromosomes of five bird species of the order Galliformes. Intron ends and potentially conserved blocks were excluded to reduce the effect of using sequences subject to negative selection. We found significant substitution rate variation within Z chromosome (G₁₅ = 37.6, p = 0.0010) as well as within W chromosome introns (G₁₅ = 44.0, p = 0.0001). This heterogeneity also affected the estimates of α_m, which varied significantly, from 1.53 to 3.51, among the introns (ANOVA: F_13,14 =2.68, p = 0.04). Our results suggest the importance of using extensive data sets from several genomic regions to avoid the effects of regional mutation rate variation and to ensure accurate estimates of α_m. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Mr. Martin Kreitman] Nick G.C. Smith Deceased     

Essential role of the B23/NPM core domain in regulating ARF binding and B23 stability

How cells coordinate inhibition of growth and division during genotoxic events is fundamental to our understanding of the origin of cancer. Despite increasing interest and extensive study, the mechanisms that link regulation of DNA synthesis and ribosomal biogenesis remain elusive. Recently, the tumor suppressor p14(ARF) (ARF) has been shown to interact functionally with the nucleolar protein B23/NPM (B23) and inhibit rRNA biogenesis. However, the molecular basis of the ARF-B23 interaction is hitherto unclear. Here we show that a highly conserved motif in the B23 oligomerization domain is essential for mediating ARF binding in vivo. Mutagenesis of conserved B23 core residues (L102A, G105A, G107A) prevented B23 from interacting with ARF. Modeling of the B23 core indicated that substitutions in the GSGP loop motif could trigger conformational changes in B23 thereby obstructing ARF binding. Interestingly, the GSGP loop mutants were unstable, defective for oligomerization, and delocalized from the nucleolus to the nucleoplasm. B23 core mutants displayed increased ubiquitination and proteasomal degradation. We conclude that the functional integrity of the B23 core motif is required for stability, efficient nucleolar localization as well as ARF binding.