Identification of Loci Enabling Stable and High-Level Heterologous Gene Expression

International audienceEfficient and reliable genome engineering technologies have yet to be developed for diatoms. The delivery of DNA in diatoms results in the random integration of multiple copies, quite often leading to heterogeneous gene activity, as well as host instability. Transgenic diatoms are generally selected on the basis of transgene expression or high enzyme activity, without consideration of the copy number or the integration locus. Here, we propose an integrated pipeline for the diatom, Phaeodactylum tricornutum , that accurately quantifies transgene activity using a β-glucuronidase assay and the number of transgene copies integrated into the genome through Droplet Digital PCR (ddPCR). An exhaustive and systematic analysis performed on 93 strains indicated that 42% of them exhibited high β-glucuronidase activity. Though most were attributed to high transgene copy numbers, we succeeded in isolating single-copy clones, as well as sequencing the integration loci. In addition to demonstrating the impact of the genomic integration site on gene activity, this study identifies integration sites for stable transgene expression in Phaeodactylum tricornutum

Mitochondrial genomes of the Baltic clam Macoma balthica (Bivalvia: Tellinidae): setting the stage for studying mito-nuclear incompatibilities

International audienceAllopatric divergence across lineages can lead to post-zygotic reproductive isolation upon secondary contact and disrupt coevolution between mitochondrial and nuclear genomes, promoting emergence of genetic incompatibilities. A previous F ST scan on the transcriptome of the Baltic clam Macoma balthica highlighted several genes potentially involved in mito-nuclear incompatibilities (MNIs). As proteins involved in the mitochondrial oxidative phosphorylation (OXPHO) chain are prone to MNIs and can contribute to the maintenance of genetic barriers, the mitochondrial genomes of six Ma. balthica individuals spanning two secondary contact zones were sequenced using the Illumina MiSeq plateform. The mitogenome has an approximate length of 16,806 bp and encodes 13 protein-coding genes, 2 rRNAs and 22 tRNAs, all located on the same strand. atp8, a gene long reported as rare in bivalves, was detected. It encodes 42 amino acids and is putatively expressed and functional. A large unassigned region was identified between rrnS and tRNA Met and could likely correspond to the Control Region. Replacement and synonymous mutations were mapped on the inferred secondary structure of all protein-coding genes of the OXPHO chain. The atp6 and atp8 genes were characterized by background levels of replacement mutations, relative to synonymous mutations. However, most nad genes (notably nad2 and nad5) were characterized by an elevated proportion of replacement mutations.Six nearly complete mitochondrial genomes were successfully assembled and annotated, providing the necessary roadmap to study MNIs at OXPHO loci. Few replacement mutations were mapped on mitochondrial-encoded ATP synthase subunits, which is in contrast with previous data on nuclear-encoded subunits. Conversely, the high population divergence and the prevalence of non-synonymous mutations at nad genes are congruent with previous observations from the nuclear transcriptome. This further suggest that MNIs between subunits of Complex I of the OXPHO chain, coding for NADH deshydrogenase, may play a role in maintaining barriers to gene flow in Ma. balthica

One complete and three draft genome sequences of four Brochothrix thermosphacta strains, CD 337, TAP 175, BSAS1 3 and EBP 3070

Brochothrix thermosphacta is one of the dominant bacterial species associated with spoilage of chilled meat and seafood products through the production of various metabolites responsible for off-odors. However, metabolic pathways leading to meat and seafood spoilage are not all well known. The production of spoiling molecules seems to depend both on strains and on food matrix. Several B. thermosphacta genome sequences have been reported, all issued from meat isolates. Here, we report four genome sequences, one complete and three as drafts. The four B. thermosphacta strains CD 337, TAP 175, BSAS1 3, and EBP 3070 were isolated from different ecological niches (seafood or meat products either spoiled or not and bovine slaughterhouse). These strains known as phenotypically and genetically different were selected to represent intraspecies diversity. CD 337 genome is 2,594,337 bp long, complete and circular, containing 2593 protein coding sequences and 28 RNA genes. TAP 175, BSAS1 3, and EBP 3070 genomes are arranged in 57, 83, and 71 contigs, containing 2515, 2668, and 2611 protein-coding sequences, respectively. These genomes were compared with two other B. thermosphacta complete genome sequences. The main genome content differences between strains are phages, plasmids, restriction/modification systems, and cell surface functions, suggesting a similar metabolic potential but a different niche adaptation capacity

Microscale multiple biomolecules printing in one step using a PDMS macrostamp

International audienceWe have developed a novel method for multiplexing the Micro-Contact Printing (μCP) technique which targets the deposition of 100–1000 different molecules in one step. For this purpose we have fabricated so-called macrostamps containing millimetric pads arranged in a periodic array compatible with titration plates. Through a very simple procedure of molding, each of this pad includes topographical micro and nanoscale features suitable for μCP on its surface. Then by inking the macrostamp in a single step through a titration plate, it becomes possible to print different inks in one step. The validations of this process are presented with the latest results showing that without any trace of contamination up to 800 different biological inks can be printed in one step. This method could be efficiently exploited for generating at low cost new slides for DNA Micro-arrays

Exposure to high static or pulsed magnetic fields does not affect cellular processes in the yeast Saccharomyces cerevisiae

International audienc

An overview of gene expression dynamics during early ovarian folliculogenesis: specificity of follicular compartments and bi-directional dialog

Chantier qualité GABackground: Successful early folliculogenesis is crucial for female reproductive function. It requires appropriate gene specific expression of the different types of ovarian cells at different developmental stages. To date, most gene expression studies on the ovary were conducted in rodents and did not distinguish the type of cell. In mono-ovulating species, few studies have addressed gene expression profiles and mainly concerned human oocytes. Results: We used a laser capture microdissection method combined with RNA-seq technology to explore the transcriptome in oocytes and granulosa cells (GCs) during development of the sheep ovarian follicle. We first documented the expression profile of 15 349 genes, then focused on the 5 129 genes showing differential expression between oocytes and GCs. Enriched functional categories such as oocyte meiotic arrest and GC steroid synthesis reflect two distinct cell fates. We identified the implication of GC signal transduction pathways such as SHH, WNT and RHO GTPase. In addition, signaling pathways (VEGF, NOTCH, IGF1, etc.) and GC transzonal projections suggest the existence of complex cell-cell interactions. Finally, we highlighted several transcription regulators and specifically expressed genes that likely play an important role in early folliculogenesis. Conclusions: To our knowledge, this is the first comprehensive exploration of transcriptomes derived from in vivo oocytes and GCs at key stages in early follicular development in sheep. Collectively, our data advance our understanding of early folliculogenesis in mono-ovulating species and will be a valuable resource for unraveling human ovarian dysfunction such as premature ovarian failure (POF)

Oocytomics: combining transcriptomics and proteomics to understand post-transcriptional regulation in bovine oocytes

National audienc

How to Design a good RNA-Seq experiment in an interdisciplinary context?

How to Design a good RNA-Seq experiment in an interdisciplinary context?. European conference on Computational Biolog

Transcriptome-wide investigation of genomic imprinting in chicken

Session : Genetic diversity and polymorphismsThe question of evolution of imprinting in vertebrates and its existence in birds is evoked in the literature, but not yet definitely answered. Genomic imprinting is an epigenetic modification leading to parent-oforigin- specific expression of certain genes. It has been observed in eutherian mammals and marsupials, but not in birds. So far, the allelic expression of imprinted gene orthologs has been analyzed in the chicken, without any reliable evidence of imprinting. Several imprinted QTL have been found in poultry; as some of them may finally be considered as not relevant to genomic imprinting, others appeared to be consistent, when using appropriate animal design and methodology. Our main objectives are to detect genes for which variation in expression is observed according to the allele, either because of an allele-specific expression or a parent-of-origin dependent expression. We screened the entire genome for allele-specific differential expression on whole embryonic transcriptomes by using high-throughput sequencing. Two chicken lines were used, as inbred and as genetically distant as possible, to unquestionably identify the parental origin of each observed haplotype. Two families from 2 reciprocal crosses were produced and transcripts from 20 embryos (4.5 d) have been tagged and sequenced through 6 HiSeq2000 lanes. About 200 Gb have been generated and are under analysis

Y a-t-il de l'empreinte chez la poule ?

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