FRAGS: estimation of coding sequence substitution rates from fragmentary data

BACKGROUND: Rates of substitution in protein-coding sequences can provide important insights into evolutionary processes that are of biomedical and theoretical interest. Increased availability of coding sequence data has enabled researchers to estimate more accurately the coding sequence divergence of pairs of organisms. However the use of different data sources, alignment protocols and methods to estimate substitution rates leads to widely varying estimates of key parameters that define the coding sequence divergence of orthologous genes. Although complete genome sequence data are not available for all organisms, fragmentary sequence data can provide accurate estimates of substitution rates provided that an appropriate and consistent methodology is used and that differences in the estimates obtainable from different data sources are taken into account. RESULTS: We have developed FRAGS, an application framework that uses existing, freely available software components to construct in-frame alignments and estimate coding substitution rates from fragmentary sequence data. Coding sequence substitution estimates for human and chimpanzee sequences, generated by FRAGS, reveal that methodological differences can give rise to significantly different estimates of important substitution parameters. The estimated substitution rates were also used to infer upper-bounds on the amount of sequencing error in the datasets that we have analysed. CONCLUSION: We have developed a system that performs robust estimation of substitution rates for orthologous sequences from a pair of organisms. Our system can be used when fragmentary genomic or transcript data is available from one of the organisms and the other is a completely sequenced genome within the Ensembl database. As well as estimating substitution statistics our system enables the user to manage and query alignment and substitution data

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic contro

Biochemical, kinetic, and spectroscopic characterization of Ruegeria pomeroyi DddW - A mononuclear iron-dependent DMSP lyase

The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW

Fertility Parameters in Crossbred Sows Treated with Cloprostenol Sodium (Synchromate®) in Zaria, Nigeria

This study was carried out to evaluate fertility parameters in crossbred sows in Zaria, following treatment with Cloprostenol sodium (Synchromate®). Ten (n = 10) apparently healthy crossbred sows were randomly assigned to two equal treatment groups based on number of injections of 500μg Synchromate®. Group 1 received two injections on days 0 and 13, while Group 2 received three injections on days 0, 7 and 13. Oestrus was monitored visually for signs of oestrus twice daily from 0700 – 1000h and 1500 – 1800h. The fertility parameters evaluated were: oestrus response rate (ORR), time to onset of oestrus (TOO), duration of oestrus (DOO), conception rate (CR), pregnancy rate (PR), farrowing rate (FR) and litter size (LS). Data on ORR, CR, PR and FR were expressed in percentages while TOO, DOO and LS were expressed as mean ± SEM. Student t-test and Tukey’s post-hoc test were used to compare the percentages and mean values between the groups. The Graphpad Prism® data package was used for statistical analysis and values of P<0.05 were considered significant. Fertility parameters TOO, DOO, LS, ORR, CR, PR, FR for Group 1 (187.20 ± 8.98h, 87.60 ± 4.49h, 7.25 ± 1.44, 100 %, 60 %, 80 % and 100 % respectively) and Group 2 (176.00 ± 40.00h, 86.60 ± 13.36h, 7.50 ± 1.50, 60 %, 100 %, 80 % and 66.67 % respectively). The differences in fertility parameters between the groups were not statistically significant. It is recommended therefore that double injections of cloprostenol sodium (Synchromate®) should be used for oestrus synchronization as it increases fertility parameters in cross bred sows at 13 days apart.Key words: Fertility parameters, crossbred sows, cloprostenol sodium, Zaria

Chromatin remodeling is required for sRNA-guided DNA elimination in Paramecium.

Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells, given its unique feature of nuclear dimorphism. Here, the formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development-specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development-specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co-immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium

Analysis of the somatic and germline genomes of the ciliate Blepharisma stoltei

Ciliaten sind prototypische, üblicherweise einzellige Eukaryoten mit getrennten Keimbahn- und somatischen Zellkernen. Das somatische Genom entsteht aus dem Keimbahngenom durch einen Prozess der Transposase-vermittelten DNA-Eliminierung und Genom-Neuordnung während der sexuellen Fortpflanzung. Aktuelle Modelle für die Reorganisation des Genoms bei Wimpertierchen gehen davon aus, dass kleine RNAs während der sexuellen Fortpflanzung in den sich entwickelnden somatischen Kern transportiert werden und Transposasen dabei helfen, keimlinienspezifische Sequenzen zu identifizieren und auszuschneiden. Diese Sequenzen, die so genannten intern eliminierten Sequenzen (IES), und ihre Exzisasen werden von einer Maschinerie begleitet, die ihre Entfernung durchführt. Dazu gehören Dicer-ähnliche und Piwi/Argonaute-Proteine, die kleine RNAs erzeugen und transportieren, sowie Proteine, die das Chromatin verändern und die DNA für die Exzision zugänglich machen. Blepharisma gehört zu einer früh divergiereden Klasse von Ciliaten, die als Heterotrichea bekannt sind. Obwohl die Reorganisation des Genoms bei später divergierenden Ciliaten wie den oligohymenophoren Ciliaten Tetrahymena und Paramecium und den spirotrichen Oxytricha untersucht wurde, gibt es deutliche Unterschiede in der Art und Weise, wie sie dies tun. Die Untersuchung dieses Prozesses in einem früh divergiereden Ciliaten wie Blepharisma ist ein wichtiger Beitrag zum Verständnis, wie konserviert die verschiedenen Elemente der Genom- Reorganisationsmaschinerie unter Ciliaten sind. Diese Arbeit bietet den ersten Blick aus genomischer Sicht auf die verschiedenen Teilnehmer und mutmaßlichen Mechanismen der Genomreorganisation in Blepharisma. Mittels Long-Read-Sequenzierung und Annotationsmethoden, die auf die atypischen Genomeigenschaften von Blepharisma zugeschnitten sind, wurden annotierte Referenzgenome für die somatischen und Keimbahnkerne von Blepharisma stoltei (Stamm ATCC 30299) erstellt. Das somatische Genom von B. stoltei ist kompakt (41 Mb), gen-dicht (25710 Gene) und enthält kurze, 15-16 Nukleotide umfassende spliceosomale Introns. Wir haben Schlüsselkomponenten identifiziert, die an der Reorganisation des Genoms im somatischen Genom von Blepharisma beteiligt sind, und sie mit denen der Modell-Ciliaten Paramecium, Tetrahymena und Oxytricha verglichen. Es wurden vier Transposase-Familien gefunden, die in den somatischen und Keimbahn-Genomen kodiert sind, nämlich die PiggyBac-,Tc1/Mariner-, Mutator- und Merlin-Familien. Es ist bekannt, dass PiggyBac-Transposasen die wichtigsten Transposasen sind, die in den Modell-Ciliaten Paramecium und Tetrahymena an der Reorganisation des Genoms beteiligt sind, während sie in Oxytricha, wo vermutlich eine Transposase aus einer anderen Familie verwendet wird, gänzlich fehlen. In Paramecium koordinieren sechs somatisch kodierte PiggyBacs, die nicht zur Katalyse fähig sind, sowie ein katalytisch vollständiges Homolog, namens PiggyMac, die DNA-Exzision. Dies ähnelt der Situation in Blepharisma, wo es dreizehn Homologe der PiggyBac-Transposase gibt, von denen nur eine eine vollständige katalytische Triade besitzt und daher wahrscheinlich die primäre Exzisase ist. Die keimbahnbegrenzten genomischen Regionen von Blepharisma wurden ebenfalls charakterisiert. Die IES von Blepharisma haben zwei wesentliche Merkmale mit den IES von Paramecium gemeinsam, nämlich eine periodische Längenverteilung für kurze IES und überwiegend durch TA-Dinukleotide abgegrenzte IES-Grenzen. Wir haben auch eine Klasse von kleinen RNAs („small RNAs“ ) mit 24 Nukleotiden identifiziert, die mit fortschreitender Entwicklung in Blepharisma zunehmend den IESs zugeordnet werden. Diese Tendenzen ähneln denen, die in Paramecium und Tetrahymena beobachtet wurden, weshalb wir vorschlagen, dass es sich auch hier um so genannte "Scan"-RNAs (scnRNAs) handelt, die die IES-Exzision steuern. Die phylogenetische Analyse der PiggyBac-Homologe von Blepharisma hat gezeigt, dass sie einen gemeinsamen Ursprung mit den PiggyBac-Homologen von Paramecium und Tetrahymena haben, wobei letztere evolutionär stärker divergieren als Blepharisma und auf jüngeren Zweigen des phylogenetischen Stammbaums der Ciliaten zu finden sind. Mehrere Indizien aus diesen Studien deuten daher darauf hin, dass eine PiggyBac-Transposase höchstwahrscheinlich die wichtigste IES-Exzisase in Blepharisma ist und dass der letzte gemeinsame Vorfahre der Ciliaten ebenfalls diesen Transposasetyp besaß.Ciliates are prototypical, conventionally unicellular eukaryotes with separate germline and somatic nuclei. The somatic genome arises from the germline genome through a process of transposase-mediated DNA elimination and genome rearrangement during sexual reproduction. Current models for genome reorganization in ciliates posit that small RNAs are transported to the developing somatic nucleus during sexual reproduction, aiding transposases in identifying and excising germline-specific sequences. Accompanying these sequences, known as Internally Eliminated Sequences (IESs), and their excisases is the machinery to carry out their removal. This includes Dicer-like and Piwi/Argonaute proteins, which generate and transport small RNAs, as well as proteins that alter chromatin, and make DNA accessible for excision. The ciliate Blepharisma belongs to an early diverging class of ciliates known as the Heterotrichea. Though genome reorganization has been studied in later diverging ciliates such the oligohymenophorean ciliates Tetrahymena and Paramecium and the spirotrich Oxytricha there are pronounced differences in how they do so. Studying this process in an early diverging ciliate like Blepharisma is an important contribution to the understanding of how conserved the different elements of the genome reorganization machinery among ciliates are. This thesis provides the first look, from a genomic perspective, at the various participants and putative mechanisms of genome reorganization in Blepharisma. Annotated reference genomes for the somatic and germline nuclei of Blepharisma stoltei (strain ATCC 30299) were generated using long-read sequencing and annotation methods tailored to the atypical genome properties of Blepharisma. The B. stoltei somatic genome is compact (41 Mb), gene-dense (25710 genes) and contains short, 15-16 nucleotide spliceosomal introns. We identified key components involved in genome reorganization in the Blepharisma somatic genome and compared them with those of the model ciliates Paramecium, Tetrahymena and Oxytricha. Four transposase families were found encoded in the somatic and germline genomes, namely the PiggyBac, Tc1/Mariner, Mutator and Merlin families. PiggyBac transposases are known to be the main transposases involved in genome reorganization in the model ciliates Paramecium and Tetrahymena, but are entirely absent in Oxytricha, which is thought to use a transposase from another family. In Paramecium, six somatically encoded PiggyBacs incapable of catalysis, plus one catalytically complete homolog called the PiggyMac, coordinate DNA excision. This resembles the situation in Blepharisma, which has thirteen homologs of the PiggyBac transposase, only one of which has a complete catalytic triad and is hence likely to be the primary excisase. The germline-limited genomic regions of Blepharisma were also characterized. Blepharisma IESs share two key features with the IESs of Paramecium, namely a periodic length distribution for short IESs and predominantly TA-dinucleotide delineated IES boundaries. We also identified a class of 24-nucleotide small RNAs that increasingly map to IESs as development progresses in Blepharisma. These trends are similar to those observed in Paramecium and Tetrahymena, hence we propose that they are also so-called “scan” RNAs (scnRNAs) that guide IES excision. Phylogenetic analysis of the Blepharisma PiggyBac homologs showed that they share common ancestry with the PiggyBac homologs of Paramecium and Tetrahymena, where the latter are evolutionarily more divergent than Blepharisma and are located on more recently diverging branches of the ciliate phylogenetic tree. Several lines of evidence from these studies therefore indicate that a PiggyBac transposase is the most likely the main IES excisase in Blepharisma and that the last ciliate common ancestor also possessed this type of transposase

Vliv zkrmování konjugované kyseliny linolové na produkční užitkovost prasniček

The aim of this study was to evaluate whether the supplementation of conjugated linoleic acid (CLA) in feed mixtures influences the parameters of production performance in gilts. The experimental work was realized in operating conditions of a production pig farm. Total number of 40 gilts (20 gilts in control group and 20 gilts in experimental group) of hybrid combination Czech Large White x Czech Landrace (CLW x CL) were included in the observation. During the experiment the gilts kept for rearing were fed with a control compound feed and an experimental compound feed suplemented with 2% of CLA for the time of 65 days. At the beginning and the end of the experiment, performance tests were conducted in accordance with the methodology of ČSN 466164 on Performance and progeny testing in pigs. The gilts in the experimental group reached higher weight 148.05 ± 15.80 kg versus 135.75 ± 17.60 kg (P < 0.05) at the end of the test. The evaluation of mean achieved daily gain for the time from birth to the end of the test confirmed higher values for the gilts in the experimental group 586.05 ± 66.64 g*day-1 against 539.00 ± 62.19 g*day-1 for the gilts in the control group (P < 0.05). Within other parameters of efficiency in gilts, i.e. lean meat content, backfat thickness and depth of MLLT muscle, no significant influence of CLA on their phenotypic manifestation was found. The results of study indicate, that the supplementation of feed mixture for rearing gilts with conjugated linoleic acid can influence the growth ability of gilts.Cílem práce bylo vyhodnotit, zda přídavek konjugované kyseliny linolové (CLA) do krmné směsi ovlivní parametry produkční užitkovosti prasniček. Pokus byl realizován v provozních podmínkách užitkového chovu prasat. Do sledování bylo zařazeno celkem 40 prasniček hybridní kombinace České bílé ušlechtilé x Česká Landrase, přičemž 20 prasniček bylo v kontrolní a 20 prasniček v pokusné skupině. Během sledování byly prasničky ustájeny v odchovně a krmeny kontrolní krmnou směsí a pokusnou směsí s přídavkem 2% CLA, a to po dobu 65 dnů. Na začátku a na konci pokusu byla provedena kontrola užitkovosti prasat dle metodiky České státní normy 466164. Prasničky pokusné skupiny dosáhly na konci testu vyšší hmotnosti 148,05 ± 15,80 kg oproti 135,75 ± 17,60 kg u skupiny kontrolní (P < 0,05). Vyhodnocení průměrného denní přírůstku od narození do konce testu prokázalo vyšší hodnotu u prasniček pokusné skupiny 586,05 ± 66,64 g*den-1 oproti 539,00 ± 6219 g*den-1 u skupiny kontrolní (P < 0,05). V rámci ostatních parametrů produkční užitkovosti prasniček, tj. u podílu libového masa, výšky hřbetního tuku a hloubky MLLT, nebyl zjištěn vliv CLA na jejich fenotypový projev. Získané výsledky ukazují, že přídavek konjugované kyseliny linolové do krmné směsi prasniček během odchovu může ovlivnit jejich růstovou schopnost