Multiple quantitative trait loci mapping with cofactors and application of alternative variants of the false discovery rate in an enlarged granddaughter design

The experimental power of a granddaughter design to detect quantitative trait loci (QTL) in dairy cattle is often limited by the availability of progeny-tested sires, by the ignoring of already identified QTL in the statistical analysis, and by the application of stringent experimentwise significance levels. This study describes an experiment that addressed these points. A large granddaughter design was set up that included sires from two countries (Germany and France), resulting in almost 2000 sires. The animals were genotyped for markers on nine different chromosomes. The QTL analysis was done for six traits separately using a multimarker regression that included putative QTL on other chromosomes as cofactors in the model. Different variants of the false discovery rate (FDR) were applied. Two of them accounted for the proportion of truly null hypotheses, which were estimated to be 0.28 and 0.3, respectively, and were therefore tailored to the experiment. A total of 25 QTL could be mapped when cofactors were included in the model-7 more than without cofactors. Controlling the FDR at 0.05 revealed 31 QTL for the two FDR methods that accounted for the proportion of truly null hypotheses. The relatively high power of this study can be attributed to the size of the experiment, to the QTL analysis with cofactors, and to the application of an appropriate FDR.     

Influence of the unusual covalent adduct on the kinetics and formation of radical intermediates in synechocystis catalase peroxidase: a stopped-flow and EPR characterization of the MET275, TYR249, and ARG439 variants

Catalase-peroxidases (KatGs) are heme peroxidases with a catalatic activity comparable to monofunctional catalases. They contain an unusual covalent distal side adduct with the side chains of Trp(122), Tyr(249), and Met(275) (Synechocysis KatG numbering). The known crystal structures suggest that Tyr(249) and Met(275) could be within hydrogen-bonding distance to Arg(439). To investigate the role of this peculiar adduct, the variants Y249F, M275I, R439A, and R439N were investigated by electronic absorption, steady-state and transient-state kinetic techniques and EPR spectroscopy combined with deuterium labeling. Exchange of these conserved residues exhibited dramatic consequences on the bifunctional activity of this peroxidase. The turnover numbers of catalase activity of M275I, Y249F, R439A, and R439N are 0.6, 0.17, 4.9, and 3.14% of wild-type activity, respectively. By contrast, the peroxidase activity was unaffected or even enhanced, in particular for the M275I variant. As shown by mass spectrometry and EPR spectra, the KatG typical adduct is intact in both Arg(439) variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr(249) and Trp(122). In the Y249F variant, the link is absent. EPR studies showed that the radical species formed upon reaction of the Y249F and R439A/N variants with peroxoacetic acid are the oxoferryl-porphyrin radical, the tryptophanyl and the tyrosyl radicals, as in the wild-type enzyme. The dramatic loss in catalase activity of the Y249F variant allowed the comparison of the radical species formed with hydrogen peroxide and peroxoacetic acid. The EPR data strongly suggest that the sequence of intermediates formed in the absence of a one electron donor substrate, is por(.-)(+) --> Trp(.-) (or Trp(.-)(+)) --> Tyr(.-). The M275I variant did not form the Trp(.-) species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp(122)-Tyr(249) adduct. The results are discussed with respect to the bifunctional activity of catalase-peroxidases.     

Comparative genomic hybridization-array analysis enhances the detection of aneuploidies and submicroscopic imbalances in spontaneous miscarriages

Miscarriage is a condition that affects 10%-15% of all clinically recognized pregnancies, most of which occur in the first trimester. Approximately 50% of first-trimester miscarriages result from fetal chromosome abnormalities. Currently, G-banded chromosome analysis is used to determine if large-scale genetic imbalances are the cause of these pregnancy losses. This technique relies on the culture of cells derived from the fetus, a technique that has many limitations, including a high rate of culture failure, maternal overgrowth of fetal cells, and poor chromosome morphology. Comparative genomic hybridization (CGH)-array analysis is a powerful new molecular cytogenetic technique that allows genomewide analysis of DNA copy number. By hybridizing patient DNA and normal reference DNA to arrays of genomic clones, unbalanced gains or losses of genetic material across the genome can be detected. In this study, 41 product-of-conception (POC) samples, which were previously analyzed by G-banding, were tested using CGH arrays to determine not only if the array could identify all reported abnormalities, but also whether any previously undetected genomic imbalances would be discovered. The array methodology detected all abnormalities as reported by G-banding analysis and revealed new abnormalities in 4/41 (9.8%) cases. Of those, one trisomy 21 POC was also mosaic for trisomy 20, one had a duplication of the 10q telomere region, one had an interstitial deletion of chromosome 9p, and the fourth had an interstitial duplication of the Prader-Willi/Angelman syndrome region on chromosome 15q, which, if maternally inherited, has been implicated in autism. This retrospective study demonstrates that the DNA-based CGH-array technology overcomes many of the limitations of routine cytogenetic analysis of POC samples while enhancing the detection of fetal chromosome aberrations.     

T47D breast cancer cell growth is inhibited by expression of VACM-1, a cul-5 gene

Vasopressin-activated calcium-mobilizing (VACM-1), a cul-5 gene, is localized on chromosome 11q22-23 close to the gene for Ataxia Telangiectasia in a region associated with a loss of heterozygosity in breast cancer tumor samples. To examine the biological role of VACM-1, we studied the effect of VACM-1 expression on cellular growth and gene expression in T47D breast cancer cells. Immunocytochemistry studies demonstrated that VACM-1 was expressed in 0.6-6% of the T47D cells and localized to the nucleus of mitotic cells. Overexpressing VACM-1 significantly attenuated cellular proliferation and MAPK phosphorylation when compared to the control cells. In addition, VACM-1 decreased egr-1 and increased Fas-L mRNA levels. Further, egr-1 protein levels were significantly lower in the nuclear fraction from VACM-1 transfected cells when compared to controls. These data indicate that VACM-1 is involved in the regulation of cellular growth.     

Jumping spiders attend to context during learned avoidance of aposematic prey

A large number of studies on both animals and humans have demonstratedthat learning is influenced by context or secondary cues thatare present when an association is formed. Few studies, however,have examined the functional value of attending to context.We first demonstrated that jumping spiders, Phidippus princeps,could be trained to avoid aposematic, distasteful milkweed bugs,Oncopeltus fasciatus. Spiders readily attacked bugs on firstexposure but were significantly less likely to do so after eighttrials, although they subsequently attacked and ate crickets.Spiders exposed to nontoxic milkweed bugs reared on sunflowerseeds did not show the same decline in attack rate. We nextexamined the effects of secondary contextual cues on spiderlearning by training spiders to avoid milkweed bugs in one oftwo environments. When spiders were tested in an environmentdifferent from the one in which they were trained, attack ratesincreased, and spiders no longer demonstrated retention of theassociation. Spiders tested in the same environment in whichthey were trained continued to avoid attacking the bugs. Theseresults have potential consequences for the evolution of bothpredator and prey and point to the importance of studying context-dependentlearning.     

Effects of Karenia brevis diet on RNA:DNA ratios and egg production of Acartia tonsa

Karenia brevis is a harmful alga associated with deleterious effects on zooplankton, but the exact cause (e.g. toxin, nutritional inadequacy or starvation) of these adverse effects is not clear. RNA:DNA ratios, fecundity and fecal pellet production of Acartia tonsa were measured on mono-algal and mixed-algal culture diets of K. brevis and Peridinium foliaceum to examine the usefulness of RNA:DNA ratios as an indicator of nutrition and to determine if adverse effects of K. brevis are due to the presence of toxins, poor nutritional quality or starvation. RNA:DNA ratios and egg production values were significantly higher for 100% P. foliaceum diet compared to 100% K. brevis diet. Significant differences in egg production, but not RNA:DNA ratios, were found between the various mixed diets, suggesting egg production is a more sensitive indicator of nutritional quality than RNA:DNA ratios. Changes in RNA:DNA ratios, fecundity and fecal pellet production of copepods fed two different toxic K. brevis strains were nearly identical, indicating that the presence of brevetoxins has little affect on A. tonsa. The similarity in RNA:DNA ratios, egg production, percent hatching and fecal production between the 100% K. brevis diet and starved copepods suggests that A. tonsa does not consume K. brevis when offered as its sole food source.     

Synthesis,binding and bioactivity of γ-methylene γ-lactam ecdysone receptor ligands: Advantages of QSAR models for flexible receptors

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or ‘attractors’. We describe the synthesis, in vitro binding and selected in vivo toxicity data for γ-methylene γ-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.     

Small within-day increases in temperature affects boldness and alters personality in coral reef fish

Consistent individual differences in behaviour, termed personality, are common in animal populations and can constrain their responses to ecological and environmental variation, such as temperature. Here, we show for the first time that normal within-daytime fluctuations in temperature of less than 3°C have large effects on personality for two species of juvenile coral reef fish in both observational and manipulative experiments. On average, individual scores on three personality traits (PTs), activity, boldness and aggressiveness, increased from 2.5- to sixfold as a function of temperature. However, whereas most individuals became more active, aggressive and bold across temperature contexts (were plastic), others did not; this changed the individual rank order across temperatures and thus altered personality. In addition, correlations between PTs were consistent across temperature contexts, e.g. fish that were active at a given temperature also tended to be both bold and aggressive. These results (i) highlight the importance of very carefully controlling for temperature when studying behavioural variation among and within individuals and (ii) suggest that individual differences in energy metabolism may contribute to animal personality, given that temperature has large direct effects on metabolic rates in ectotherms.     

Improved automated monitoring and new analysis algorithm for circadian phototaxis rhythms in Chlamydomonas

Automated monitoring of circadian rhythms is an efficient way of gaining insight into oscillation parameters like period and phase for the underlying pacemaker of the circadian clock. Measurement of the circadian rhythm of phototaxis (swimming towards light) exhibited by the green alga Chlamydomonas reinhardtii has been automated by directing a narrow and dim light beam through a culture at regular intervals and determining the decrease in light transmittance due to the accumulation of cells in the beam. In this study, the monitoring process was optimized by constructing a new computer-controlled measuring machine that limits the test beam to wavelengths reported to be specific for phototaxis and by choosing an algal strain, which does not need background illumination between test light cycles for proper expression of the rhythm. As a result, period and phase of the rhythm are now unaffected by the time a culture is placed into the machine. Analysis of the rhythm data was also optimized through a new algorithm, whose robustness was demonstrated using virtual rhythms with various noises. The algorithm differs in particular from other reported algorithms by maximizing the fit of the data to a sinusoidal curve that dampens exponentially. The algorithm was also used to confirm the reproducibility of rhythm monitoring by the machine. Machine and algorithm can now be used for a multitude of circadian clock studies that require unambiguous period and phase determinations such as light pulse experiments to identify the photoreceptor(s) that reset the circadian clock in C. reinhardtii.