title: Expression vectors for C-terminal fusions with fluorescent proteins and epitope tags in Candida glabrata.
abstract: Candida glabrata is a haploid yeast considered the second most common of the Candida species found in nosocomial infections, accounting for approximately 18% of candidemias worldwide. Even though molecular biology methods are easily adapted to study this organism, there are not enough vectors that will allow probing the transcriptional and translational activity of any gene of interest in C. glabrata. In this work we have generated a set of expression vectors to systematically tag any gene of interest at the carboxy-terminus with three different fluorophores (CFP, YFP and mCherry) or three epitopes (HA, FLAG or cMyc) independently. This system offers the possibility to generate translational fusions in three versions: under the gene's own promoter integrated in its native locus in genome, on a replicative plasmid under its own promoter, or on a replicative plasmid under a strong promoter to overexpress the fusions. The expression of these translational fusions will allow determining the transcriptional and translational activity of the gene of interest as well as the intracellular localization of the protein. We have tested these expression vectors with two biosynthetic genes, HIS3 and TRP1. We detected fluorescence under the microscope and we were able to immunodetect the fusions using the three different versions of the system. These vectors permit coexpression of several different fusions simultaneously in the same cell, which will allow determining protein-protein and protein-DNA interactions. This set of vectors adds a new toolbox to study expression and protein interactions in the fungal pathogen C. glabrata.
authors.affiliation: IPICYT, Instituto Potosino de Investigación Científica y Tecnológica, División de Biología Molecular, Camino a la Presa San José # 2055, Lomas 4a sección, San Luis Potosí, San Luis Potosí 78216, Mexico.
authors.forename: Patricia
authors.initials: P
authors.lastname: Yáñez-Carrillo
authors.affiliation: IPICYT, Instituto Potosino de Investigación Científica y Tecnológica, División de Biología Molecular, Camino a la Presa San José # 2055, Lomas 4a sección, San Luis Potosí, San Luis Potosí 78216, Mexico.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Orta-Zavalza
authors.affiliation: IPICYT, Instituto Potosino de Investigación Científica y Tecnológica, División de Biología Molecular, Camino a la Presa San José # 2055, Lomas 4a sección, San Luis Potosí, San Luis Potosí 78216, Mexico.
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Gutiérrez-Escobedo
authors.affiliation: IPICYT, Instituto Potosino de Investigación Científica y Tecnológica, División de Biología Molecular, Camino a la Presa San José # 2055, Lomas 4a sección, San Luis Potosí, San Luis Potosí 78216, Mexico.
authors.forename: Araceli
authors.initials: A
authors.lastname: Patrón-Soberano
authors.affiliation: IPICYT, Instituto Potosino de Investigación Científica y Tecnológica, División de Biología Molecular, Camino a la Presa San José # 2055, Lomas 4a sección, San Luis Potosí, San Luis Potosí 78216, Mexico.
authors.forename: Alejandro
authors.initials: A
authors.lastname: De Las Peñas
authors.affiliation: IPICYT, Instituto Potosino de Investigación Científica y Tecnológica, División de Biología Molecular, Camino a la Presa San José # 2055, Lomas 4a sección, San Luis Potosí, San Luis Potosí 78216, Mexico. Electronic address: icastano@ipicyt.edu.mx.
authors.forename: Irene
authors.initials: I
authors.lastname: Castaño
mesh_terms: D020413:3' Untranslated Regions; D041221:Candida glabrata / Q000235:genetics* / Q000378:metabolism; D000939:Epitopes / Q000235:genetics*; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; D000939:Epitopes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins
keywords: Candida glabrata; Epitope tag; Fluorescent tag; Integrative vector; Overexpression vector; Translational fusion