The life cycle of Drosophila orphan genes

Funding: Austrian Science Funds (FWF) (P22834).Orphans are genes restricted to a single phylogenetic lineage and emerge at high rates. While this predicts an accumulation of genes, the gene number has remained remarkably constant through evolution. This paradox has not yet been resolved. Because orphan genes have been mainly analyzed over long evolutionary time scales, orphan loss has remained unexplored. Here we study the patterns of orphan turnover among close relatives in the Drosophila obscura group. We show that orphans are not only emerging at a high rate, but that they are also rapidly lost. Interestingly, recently emerged orphans are more likely to be lost than older ones. Furthermore, highly expressed orphans with a strong male-bias are more likely to be retained. Since both lost and retained orphans show similar evolutionary signatures of functional conservation, we propose that orphan loss is not driven by high rates of sequence evolution, but reflects lineage-specific functional requirements.Publisher PDFPeer reviewe

Mapping Accuracy of Short Reads from Massively Parallel Sequencing and the Implications for Quantitative Expression Profiling

BACKGROUND:Massively parallel sequencing offers an enormous potential for expression profiling, in particular for interspecific comparisons. Currently, different platforms for massively parallel sequencing are available, which differ in read length and sequencing costs. The 454-technology offers the highest read length. The other sequencing technologies are more cost effective, on the expense of shorter reads. Reliable expression profiling by massively parallel sequencing depends crucially on the accuracy to which the reads could be mapped to the corresponding genes. METHODOLOGY/PRINCIPAL FINDINGS:We performed an in silico analysis to evaluate whether incorrect mapping of the sequence reads results in a biased expression pattern. A comparison of six available mapping software tools indicated a considerable heterogeneity in mapping speed and accuracy. Independently of the software used to map the reads, we found that for compact genomes both short (35 bp, 50 bp) and long sequence reads (100 bp) result in an almost unbiased expression pattern. In contrast, for species with a larger genome containing more gene families and repetitive DNA, shorter reads (35-50 bp) produced a considerable bias in gene expression. In humans, about 10% of the genes had fewer than 50% of the sequence reads correctly mapped. Sequence polymorphism up to 9% had almost no effect on the mapping accuracy of 100 bp reads. For 35 bp reads up to 3% sequence divergence did not affect the mapping accuracy strongly. The effect of indels on the mapping efficiency strongly depends on the mapping software. CONCLUSIONS/SIGNIFICANCE:In complex genomes, expression profiling by massively parallel sequencing could introduce a considerable bias due to incorrectly mapped sequence reads if the read length is short. Nevertheless, this bias could be accounted for if the genomic sequence is known. Furthermore, sequence polymorphisms and indels also affect the mapping accuracy and may cause a biased gene expression measurement. The choice of the mapping software is highly critical and the reliability depends on the presence/absence of indels and the divergence between reads and the reference genome. Overall, we found SSAHA2 and CLC to produce the most reliable mapping results

Relationships of Cysteine and Lysine residues with the substrate binding site of the mitochondrial ornithine/citrulline carrier: An inhibition kinetic approach combined with the analysis of the homology structural model

AbstractTo gain insights in the relationships of specific amino acid residues with the active site of the mitochondrial ornithine/citrulline carrier, we studied the effect of specific protein modifying reagents on the transport catalysed by the carrier reconstituted into liposomes. It was found that, besides the sulfhydryl reagents NEM, MTSEA, p-hydroxymercuribenzoate, diamide also the lysine reagents PLP, DIDS, SITS, the carboxyl reagents WRK, EDC and the arginine reagent methylglyoxal inhibited the carrier. NEM, MTSEA and PLP inhibited the ornithine/citrulline carrier with a completely competitive type of mechanism. A 1:1 interaction of NEM with the carrier molecule has been demonstrated. The results are in agreement with the localization of one sulfhydryl and at least one amino group in the substrate binding site. On the basis of the interferences between SH reagents and PLP in the transport inhibition, it has been deduced that the distance between the SH and the NH2 residues of the active site should be comparable to the distance between the γ-NH2 and COOH residues of the ornithine molecule. The structural model of the ornithine/citrulline carrier has been obtained by homology modelling using as template the ADP/ATP carrier structure. The combined analysis of the experimental data and the structural model allows to deduce that Cys-132 is located in the substrate binding site, flanked by at least one Lys residue

Identification by Site-directed Mutagenesis and Chemical Modification of Three Vicinal Cysteine Residues in Rat Mitochondrial Carnitine/Acylcarnitine Transporter *

The proximity of the Cys residues present in the mitochondrial rat carnitine/acylcarnitine carrier (CAC) primary structure was studied by using site-directed mutagenesis in combination with chemical modification. CAC mutants, in which one or more Cys residues had been replaced with Ser, were overexpressed in Escherichia coli and reconstituted into liposomes. The effect of SH oxidizing, cross-linking, and coordinating reagents was evaluated on the carnitine/carnitine exchange catalyzed by the recombinant reconstituted CAC proteins. All the tested reagents efficiently inhibited the wild-type CAC. The inhibitory effect of diamide, Cu(2+)-phenanthroline, or phenylarsine oxide was largely reduced or abolished by the double substitutions C136S/C155S, C58S/C136S, and C58S/C155S. The decrease in sensitivity to these reagents was much lower in double mutants in which Cys(23) was substituted with Cys(136) or Cys(155). No decrease in inhibition was found when Cys(89) and/or Cys(283) were replaced with Ser. Sb(3+), which coordinates three cysteines, inhibited only the Cys replacement mutants containing cysteines 58, 136, and 155 of the six native cysteines. In addition, the mutant C23S/C89S/C155S/C283S, in which double tandem fXa recognition sites were inserted in positions 65-72, i.e. between Cys(58) and Cys(136), was not cleaved into two fragments by fXa protease after treatment with diamide. These results are interpreted in light of the homology model of CAC based on the available x-ray structure of the ADP/ATP carrier. They indicate that Cys(58), Cys(136), and Cys(155) become close in the tertiary structure of the CAC during its catalytic cycle

Volumetric expression of palynological spectra for nutritional studies

When pollen is studied from nutritional point of view, it would be desirable to correct its raw palynological spectrum in order to express the mass contribution of each pollen type. Different approaches applied for this correction through volumetric coefficients are discussed, and a simple and reliable procedure is proposed

Microsatellite Analysis of Geographically Close Isolates of Cystoisospora suis

Microsatellites are short repetitive DNA sequences of 2–6 repeats interspersed in the genome that display a rapid mutation rate and consequently show high variation between individuals or populations. They have been used to characterize population diversity and structure and the level of variation between different isolates of a number of different organisms, including apicomplexan protozoa. Currently nothing is known about the genetic variability and population structure of Cystoisospora suis (Apicomplexa: Coccidia), the causative agent of piglet coccidiosis, and we made use of the recently available genome of C. suis (strain Wien-I) to amplify microsatellite regions (ca. 300–550 bp) and evaluate the applicability of fluorescence-labeled primers to investigate amplicon length variation at high resolution using capillary electrophoresis (CE). Two phenotypically characterized isolates (Wien-I, toltrazuril susceptible; Holl 1 toltrazuril resistant) and six field isolates from Europe were compared by conventional PCR followed by agar-gel electrophoresis, Sanger sequencing, and CE (fluorescence labeling and fragment length analysis) to evaluate the applicability of the method. Four primer pairs could be identified that amplified bands of the expected size and were labeled for CE analysis. High resolution CE for size determination of PCR amplicons proved to be a reliable and simple method. It revealed high diversity of the analyzed strains, with marked differences even between two strains from neighboring swine farms. In follow-up studies, adaptation of the PCR assay to multiplexing and amplification of small DNA quantities will provide a cost-effective tool to analyse field strains to reveal geographic diversity that could be mapped to phenotypic traits

Do BRAF inhibitors select for populations with different disease progression kinetics?

Ipilimumab, an anti-CTLA-4 monoclonal antibody, has been shown to improve overall survival in patients with metastatic melanoma. Preliminary data suggest that patients who fail BRAF inhibitor treatment experience a very rapid progression of disease. Such selectivity for more rapid disease progression may mean these patients do not receive the same benefit from subsequent treatment with ipilimumab as patients without prior BRAF inhibitor treatment. The current challenge is focused on how to identify and approach the two populations of fast and slow progressors and recent hypothesis suggest that treatment choice could be guided by baseline risk factors. However, no data have yet defined which the best sequence is and more research is needed to identify predictors of response in patients with metastatic melanoma to help guide whether a BRAF inhibitor or ipilimumab should be used first in sequential therapy

Poly-L-lactic acid beta-tricalcium phosphate screws: a preliminary in vivo biocompatibility study.

The aim of this study is to assess the biocompatibility of two types of Poly-L-lactic acid (PLLA) screws (with either hydroxiapatite (HA) or β-tricalcium phosphate (β-TCP)) implanted in the left femur of four sheep euthanized at 42, 50, 57 and 84 days after surgery. Titanium screws were also implanted for comparison purposes. No signs of inflammation were seen in the 240 specimens. A rating of "+/-"for macrophages and "-"for neutrophils was assigned to all specimens. All specimens were assigned a rating which ranged from "+/-" to "+++" for fibroblasts and osteoblasts. The presence of macrophages, neutrophils and fibroblasts/osteoblasts was not statistically different for the four implantation periods. PLLA implants with β-TCP have a biocompatibility comparable to PLLA implants with HA