Browsing by Author "Gutiérrez, Rodrigo A."

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    Genome-wide analysis in response to nitrogen and carbon identifies regulators for root AtNRT2 transporters
    (2021) Ruffel, Sandrine; Chaput, Valentín; Przybyla Toscano, Jonathan; Fayos, Ian; Ibarra, Catalina; Moyano, Tomás; Fizames, Cécile; Tillard, Pascal; O’Brien, José Antonio; Gutiérrez, Rodrigo A.; Gojon, Alain; Lejay, Laurence
    In Arabidopsis (Arabidopsis thaliana), the High-Affinity Transport System (HATS) for root nitrate (⁠NO3−⁠) uptake depends mainly on four NRT2 NO3− transporters, namely NRT2.1, NRT2.2, NRT2.4, and NRT2.5. The HATS is the target of many regulations to coordinate nitrogen (N) acquisition with the N status of the plant and with carbon (C) assimilation through photosynthesis. At the molecular level, C and N signaling pathways control gene expression of the NRT2 transporters. Although several regulators of these transporters have been identified in response to either N or C signals, the response of NRT2 gene expression to the interaction of these signals has never been specifically investigated, and the underlying molecular mechanisms remain largely unknown. To address this question we used an original systems biology approach to model a regulatory gene network targeting NRT2.1, NRT2.2, NRT2.4, and NRT2.5 in response to N/C signals. Our systems analysis of the data identified three transcription factors, TGA3, MYC1, and bHLH093. Functional analysis of mutants combined with yeast one-hybrid experiments confirmed that all three transcription factors are regulators of NRT2.4 or NRT2.5 in response to N or C signals. These results reveal a role for TGA3, MYC1, and bHLH093 in controlling the expression of root NRT2 transporter genes.
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    Loop assembly: a simple and open system for recursive fabrication of DNA circuit
    (2019) Pollak, Bernardo; Cerda, Ariel; Delmans, Mihails; Álamos, Simón; Moyano, Tomás; West, Anthony; Gutiérrez, Rodrigo A.; Patron, Nicola J.; Federici, Fernán; Haseloff, Jim
    High-efficiency methods for DNA assembly have enabled the routine assembly of syntheticDNAs of increased size and complexity. However, these techniques require customization,elaborate vector sets or serial manipulations for the different stages of assembly. We have developed Loop assembly based on a recursive approach to DNA fabrication. Thesystem makes use of two Type IIS restriction endonucleases and corresponding vector sets forefficient and parallel assembly of large DNA circuits. Standardized level 0 parts can be assem-bled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets.The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly enables efficient and versatile DNA fabrication for plant transformation. Weshow the construction of plasmids up to 16 genes and 38 kb with high efficiency (> 80%).We have characterized Loop assembly on over 200 different DNA constructs and validatedthe fidelity of the method by high-throughput Illumina plasmid sequencing. Our method provides a simple generalized solution for DNA construction with standardizedparts. The cloning system is provided under an OpenMTA license for unrestricted sharing andopen access.
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    Step-by-Step Construction of Gene Co-expression Networks from High-Throughput Arabidopsis RNA Sequencing Data
    (Humana Press, 2018) Contreras López, Orlando; Moyano, Tomás C.; Soto, Daniela C.; Gutiérrez, Rodrigo A.
    The rapid increase in the availability of transcriptomics data generated by RNA sequencing represents both a challenge and an opportunity for biologists without bioinformatics training. The challenge is handling, integrating, and interpreting these data sets. The opportunity is to use this information to generate testable hypothesis to understand molecular mechanisms controlling gene expression and biological processes (Fig. 1). A successful strategy to generate tractable hypotheses from transcriptomics data has been to build undirected network graphs based on patterns of gene co-expression. Many examples of new hypothesis derived from network analyses can be found in the literature, spanning different organisms including plants and specific fields such as root developmental biology. In order to make the process of constructing a gene co-expression network more accessible to biologists, here we provide step-by-step instructions using published RNA-seq experimental data obtained from a public database. Similar strategies have been used in previous studies to advance root developmental biology. This guide includes basic instructions for the operation of widely used open source platforms such as Bio-Linux, R, and Cytoscape. Even though the data we used in this example was obtained from Arabidopsis thaliana, the workflow developed in this guide can be easily adapted to work with RNA-seq data from any organism.
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    Transcriptomic profiles reveal differences in zinc metabolism, inflammation, and tight junction proteins in duodenum from cholesterol gallstone subjects
    (2020) Riveras Hernández, Eleodoro Javier; Azócar, Lorena; Moyano, Tomás C.; Ocares, Marcia; Molina, Héctor; Romero, Diego; Roa Strauch, Juan Carlos Enrique; Valbuena Mora, José Rafael; Gutiérrez, Rodrigo A.; Miquel P., Juan Francisco
    Cholesterol Gallstone Disease (GSD) is a common multifactorial disorder characterized by crystallization and aggregation of biliary cholesterol in the gallbladder. The global prevalence of GSD is similar to 10-20% in the adult population but rises to 28% in Chile (17% among men and 30% among women). The small intestine may play a role in GSD pathogenesis, but the molecular mechanisms have not been clarified. Our aim was to identify the role of the small intestine in GSD pathogenesis. Duodenal biopsy samples were obtained from patients with GSD and healthy volunteers. GSD status was defined by abdominal ultrasonography. We performed a transcriptome study in a discovery cohort using Illumina HiSeq. 2500, and qPCR, immunohistochemistry and immunofluorescence were used to validate differentially expressed genes among additional case-control cohorts. 548 differentially expressed genes between GSD and control subjects were identified. Enriched biological processes related to cellular response to zinc, and immune and antimicrobial responses were observed in GSD patients. We validated lower transcript levels of metallothionein, NPC1L1 and tight junction genes and higher transcript levels of genes involved in immune and antimicrobial pathways in GSD patients. Interestingly, serum zinc and phytosterol to cholesterol precursor ratios were lower in GSD patients. A significant association was observed between serum zinc and phytosterol levels. Our results support a model where proximal small intestine plays a key role in GSD pathogenesis. Zinc supplementation, modulation of proximal microbiota and/or intestinal barrier may be novel targets for strategies to prevent GSD.