Amplification biases: possible differences among deviating gene expressions.

International audienceBACKGROUND: Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT) and polymerase chain reaction (PCR), the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. RESULTS: Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3) and somatic tissues (n = 2), we proceeded to moderate amplifications starting from 1 mug of total RNA (global PCR or IVT one round). Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number) but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70%) and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID). However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions) and relevant (biologically validated). In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples. CONCLUSION: Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i) the sample used: brain, ovary or embryos, (ii) the enzymatic properties initially inferred (exponential or linear) and (iii) the preliminary optimization of the protocols. Moreover the use of an in-house developed array, small-sized but well suited to the tissues we worked with, was of real interest for the search of differential expressions

Comparative cytogenetic mapping reveals chromosome rearrangements between the X chromosomes of two closely related mammalian species (cattle and goats)

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Characterization of 2 novel H2O2 producing glyoxal oxidases from Pycnoporus cinnabarinus implicated in the breakdown of lignocelluloses

Lignin is one of the most abundant and recalcitrant natural polymers. Consequently, lignin degradation is important for completing the carbon cycle in forest ecosystems and a central challenge towards more environmentally-friendly and economically performing second-generation lignocellulosic-based biorefineries. The monokaryotic cell-line of the white-rot fungus Pycnoporus cinnabarinus BRFM 137 was shown to be an outstanding model to study the enzymatic machinery involved in the degradation and transformation of lignocellulosic materials [1]. Seven AA5_1 Carbohydrate-Active enzymes (Cazymes) have been identified including three glyoxal oxidases (GLOX) [2]. These GLOX are naturally secreted into the extracellular medium around the growing hyphal tip during fungal growth on lignin-rich substrates and acts as one of the sources of extracellular hydrogen peroxide that is mandatory for the oxidation reactions catalyzed by lignolytic peroxidases involved in lignin degradation [3]. In this work, two novel hydrogen peroxide-producing glyoxal oxidases from Pycnoporus cinnabarinus (PciGLOX1 and PciGLOX2) were for the first time cloned and successfully heterologously expressed in Aspergillus niger in Erlenmeyer flasks and in a 10 L bioreactor, and biochemically characterized. The wide variety of aldehydes that were oxidized makes these GLOX a promising tool not only for the deconstruction of plant biomass and the valorization of industrial lignin, but also for the production of valuable molecules for Green Chemistry applications. These enzymes and other oxidative enzymes produced in the framework of the EU-FP7 INDOX project will now be used simultaneously in order to study their synergistic interactions on lignocellulosic materials with the aim to get insights into the lignin-degrading strategies employed by Pycnoporus cinnabarinus

Expression profiles and localization of genes involved in ovarian differentiation in sheep

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Molecular characterization of genomic activities at the onset of zygotic transcription in mammals

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Effect of time interval from insemination to first cleavage on the developmental characteristics, sex ratio and pregnancy rate after transfer of bovine embryos

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Expression profiles and chromosomal localization of genes controlling meiosis and follicular development in the sheep ovary

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Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus: Glyoxal Oxidases from Pycnoporus cinnabarinus

International audienceThe genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2 and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced in Aspergillus niger strain D15#26 (pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for PciGLOX1 and PciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but PciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyd

Utilisation de la génomique pour l'étude de la phase d'élongation de l'embryon bovin

Chez les bovins, plus de 30 % des pertes embryonnaires observées après insémination artificielle, semblent avoir une origine précoce, liée à la croissance exponentielle du trophoblaste. Durant cette phase d’élongation qui se produit entre le 13ème et le 19ème jour de gestation, se mettent en place entre l’embryon et l’utérus, des interactions physiologiques indispensables pour le maintien de la gestation. Nos travaux en cours portent sur l’identification de transcrits impliqués dans la croissance de l’embryon avant l’implantation. Dans le cadre de cette étude, nous avons utilisé un réseau sur filtre de nylon, comprenant 1920 inserts ordonnés provenant d’une banque d’ADNc établie à partir d’embryons bovins en début d’élongation (14ème jour), pour comparer les profils d’expression de trophoblastes provenant d’embryons de taille croissante : ovoïde (10-18mm), tubulaire (50-60mm) et filamenteux (140-160mm). Une première analyse statistique fait apparaître des gènes jusqu’alors inconnus dont l’expression est différentielle entre ces stades

Gene expression profiling on sheep brain reveals differential transcripts in scrapie-affected/not-affected animals.

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