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A toolbox for epitope-tagging and genome-wide location analysis in Candida albicans

ABSTRACT: Background : Candida albicans is a diploid pathogenic fungus not yet amenable to routine genetic investigations. Understanding aspects of the regulation of its biological functions and the assembly of its protein complexes would lead to further insight into the biology of this common disease-causing microbial agent. Results: We have developed a toolbox allowing in vivo protein tagging by PCR-mediated homologous recombination with TAP, HA and MYC tags. The transformation cassettes were designed to accommodate a common set of integration primers. The tagged proteins can be used to perform tandem affinity purification (TAP) or chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP). Tandem affinity purification of C. albicans Nop1 revealed the high conservation of the small processome composition in yeasts. Data obtained with in vivo TAP-tagged Tbf1, Cbf1 and Mcm1 recapitulates previously published genome-wide location profiling by ChIP-CHIP. We also designed a new reporter system for in vivo analysis of transcriptional activity of gene loci in C. albicans. Conclusion: This toolbox provides a basic setup to perform purification of protein complexes and increase the number of annotated transcriptional regulators and genetic circuits in C. albicans.

ORGANISM(S): Candida albicans

PROVIDER: GSE13427 | GEO | 2008/11/15

SECONDARY ACCESSION(S): PRJNA110007

REPOSITORIES: GEO

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A toolbox for epitope-tagging and genome-wide location analysis in Candida albicans

Project description:Background : Candida albicans is a diploid pathogenic fungus not yet amenable to routine genetic investigations. Understanding aspects of the regulation of its biological functions and the assembly of its protein complexes would lead to further insight into the biology of this common disease-causing microbial agent. Results: We have developed a toolbox allowing in vivo protein tagging by PCR-mediated homologous recombination with TAP, HA and MYC tags. The transformation cassettes were designed to accommodate a common set of integration primers. The tagged proteins can be used to perform tandem affinity purification (TAP) or chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP). Tandem affinity purification of C. albicans Nop1 revealed the high conservation of the small processome composition in yeasts. Data obtained with in vivo TAP-tagged Tbf1, Cbf1 and Mcm1 recapitulates previously published genome-wide location profiling by ChIP-CHIP. We also designed a new reporter system for in vivo analysis of transcriptional activity of gene loci in C. albicans. Conclusion: This toolbox provides a basic setup to perform purification of protein complexes and increase the number of annotated transcriptional regulators and genetic circuits in C. albicans. Two independent biological replicates of ChIP-CHIP of Mcm1-TAP in yeast and hyphal states. ChIP-CHIP of Cbf1-TAP and Tbf1-TAP.

2010-05-16 | E-GEOD-13427 | biostudies-arrayexpress

Using ChIP-chip to map genome-wide binding of GalR

Project description:We used ChIP-chip to map the binding of C-terminally TAP (tandem affinity purification)-tagged GalR (tagged at its native locus in an unmarked strain) across the E. coli genome.

2016-07-01 | E-MTAB-4903 | biostudies-arrayexpress

Purification of TAP::ALG-1 complexes and associated RNA from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2) animals

Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein We purified TAP::ALG-1 complexes from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2, serving as a mock control) animals and hybridized the associated mRNAs to two-color microarrays

2012-03-01 | GSE32941 | GEO

Trf4p in vivo crosslinking and ribonucleoprotein-immunopurification-chip analysis (X-RIP-Chip)

Project description:In vivo cross-linking and ribonucleoprotein-immunopurification experiments followed by microarray analysis of bound RNAs (X-RIP-chip). Cells expressing recombinant tandem-affinity purification (TAP)-tagged Trf4 protein were cross-linked with formaldehyde, and Trf4-containing ribonucleoprotein complexes were recovered by affinity selection on IgG-coupled beads (see linked protocol). As a control for non-specifically enriched RNAs, the same experiment was done with untagged WT cells and with cells expressing Fpr1-TAP, a peptidyl-prolyl-cis-trans-isomerase not expected to bind RNA. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Antigenic peptide used in IP: Protein A derivative

2009-07-15 | GSE16105 | GEO

Trf4p in vivo crosslinking and ribonucleoprotein-immunopurification-chip analysis (X-RIP-Chip)

2010-06-24 | E-GEOD-16105 | biostudies-arrayexpress

Expression analysis of WS4303 (wild type) vs WS5041 (mir-58 mutants) total mRNA

Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as “wild type” and the transgenic WS5041 animals as “mir-58”. In addition to immunopurifying the TAP::ALG-1 and associated RNAs from these strains, we also compared total mRNA levels between these two strains.

2012-03-01 | GSE32943 | GEO

RIP-Seq analysis of Trypanosoma brucei ELAV-like protein Tb927.8.6650

Project description:A tagged ectopic version of the ELAV-like protein Tb927.8.6650 of T. brucei was expressed in stable cell lines and pulled down. Co-purifying transcripts were analyzed by sequencing to identify RNAs associated with Tb927.8.6650. Stable procyclic form cell lines expressing tetracycline-inducible TAP-tagged Tb927.8.6650 were created. Cells were harvested 48h after tet-induction, followed by tandem affinity purification of Tb927.8.6650, extraction of co-purified RNA, and sequencing.

2013-04-18 | E-GEOD-46160 | biostudies-arrayexpress

Genome-wide identification of PPD2 target genes

Project description:To identify the direct target genes of PPD2, tandem chromatin affinity purification (TChAP, Verkest et al. (2014)), a variant of chromatin immunoprecipitation (ChIP) in which tandem affinity tags are used instead of epitope tags, followed by sequencing (TChAP-Seq) was performed. An Arabidopsis cell suspension culture overexpressing an HBH-tagged PPD2 was used for the purification of the chromatin bound by PPD2.

2015-07-07 | E-MTAB-3046 | biostudies-arrayexpress

Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants

Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as wild typeand the transgenic WS5041 animals as mir-58. We compared the mRNA population that coimmunopurified with TAP::ALG-1 from synchronized L4 stage wild-type animals with that from synchronized L4 stage mir-58 mutant animals by one-color Affymetrix gene arrays. miR-58 target mRNAs should be specifically underrepresented in the latter samples.

2012-03-01 | GSE32942 | GEO

H3K36me3 Reader NPAC/GLYR1 Recruits H3K4 Demethylase LSD2/KDM1B to Modulate Active Gene Transcription

Project description:NPAC ChIP were performed by anti-Flag and anti-HA tandem affinity purification from HeLa stably expressing Flag-HA tagged NPAC from pOZ-N vector, and enrichement on chromosome 3, 21, and 22 were determined by chip microarray analysis using Affymatrix HumanTiling 2.0 arrays

2021-10-31 | GSE160129 | GEO

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