ABSTRACT: Gene Expression Arrays comparing wild-type Kluyveromyces lactis a, alpha, and a/alpha cells and a comparison between WT a cells and rme1 knock-out a cells
Project description:Gene expression arrays comparing Kluyveromyces lactis wild-type a cells to MATa2 knock-out a cells and wild-type alpha cells to MATalpha2 knock-out alpha cells
Project description:Total RNA versus genomic DNA hybridization on custom arrays designed for all Kluyveromyces lactis genes Total RNA was collected in mid-log phase from Kluyveromyces lactis cells grown in rich medium (abbreviated CM, in house recipe). RNA was then converted to cDNA, Cy3-labeled and hybridized competitively against Cy5 labeled genomic DNA from Kluyveromyces lactis.
Project description:WT and rme1 KO K. lactis cells (a, alpha, and a/alpha) were grown in YEPD, phosphate starvation and phosphate starvation with the addition of alpha pheromone. The goal was to identify cell-type regulated genes and to determine the effects of growth media on the regulation of cell-type regulated genes Expression was measured relative to pooled references, note that only the arrays with matched pooled references can be compared to each other
Project description:WT and rme1 KO K. lactis cells (a, alpha, and a/alpha) were grown in YEPD, phosphate starvation and phosphate starvation with the addition of alpha pheromone. The goal was to identify cell-type regulated genes and to determine the effects of growth media on the regulation of cell-type regulated genes
Project description:ChIP-chip to determine the regulation of the K. lactis hsgs (ChIP of MATa1, MATalpha2, and RME1). The MATa1 and MATalpha2 ChIPs were performed in an a/alpha cell using N-terminally HA-tagged proteins and the RME1 ChIPs were perfomed in an a cell using C-terminally myc-tagged protein. For the RME1 ChIPs, the cells grown with out phosphate. For the MATa1 andMATalpha2 ChIPs the cells were grown in YEPD.
Project description:The transcription factor KlPdr1p, belonging to the Zn2Cys6 family, is a central regulator of efflux pump expression in Kluyveromyces lactis. To better understand how KlPDR1-mediated drug resistance is achieved in K. lactis, we used DNA microarrays to identify genes whose expression was affected by deletion or overexpression of the KlPDR1 gene. All microarray experiments were performed using the 30K Kluyveromyces lactis NRRL Y-1140 microarray (MYcroarray, 5692 Plymouth Road, Ann Arbor, MI 48105, USA). Exponentially growing (1 x 107 cells ml-1) K. lactis PM6-7A cells (wild-type, PM6-7A/pdr1∆ and the wild-type transformed with multicopy plasmid carrying the gain-of-function allele of KlPDR1* gene) (Balazfyova et al. 2013), were collected and total RNA was isolated using RNeasy midi kit (Qiagen GmbH, Germany). 1 mg of total RNA was linearly amplified and labelled using Amino allyl MessageAmpII aRNA Amplification kit (Ambion, USA) with two different fluorescent dyes; AlexaFluor647 and AlexaFluor555 (Life Technologies, Germany). 4 µg of labelled RNA was hybridized (18 h at 45°C) in 6x SSPE with addition of formamide (10%), tween-20 (0,01%) and microarray-specofoc control oligos (1%, MYcroarray, USA). After washing, microarray images and two-color GPR output files were obtained using the microarray scanner InnoScan 900 and Mapix software version 7.3.1 (Inopsys, France). The two-color GPR files were processed using the R version 3.0.2 (R Core Team (2014). R: A language and enviroment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org) and functions available in the limma package (Smyth 2005). Briefly, the two-color GPR files were omported using the read.maimages() function, background-substracted using the “minimum“ method and within-array-normalized using the “loess“ method. The between-array normalisation was achieved using the “Aquantile“ method. For further analysis, only genes with log2FC ˃2 were selected and confirmed using qPCR.
Project description:In the frame of a multispecies project, Kluyveromyces lactis cells were treated with 0.5 mM sodium selenite. Cells were collected 10, 20, 30, 40, 50, 60, 70 and 80 minutes after the treatment and their transcriptomes were compared to those of mock-treated cultures. Four independent biological replicates were performed.
Project description:The goal of the study was to compare the response to Protien Kinase A (PKA) inhibition between Saccharomyces cerevisiae and Kluyveromyces lactis. The ancestor of K. lactis did not undergo the Whole Genome Duplication (or Whole Genome Hybridization) event that S. cerevisiae experienced. We found that many paralog pairs in S. cerevisiae were differentially induced in response to PKA inhibition, and that the shared ortholog for these paralog paris in K. lactis was typically not induced. To inhibit PKA, strains containing point mutations rendering PKA sensitive to inhibition by the ATP analog 1-NM-PP1 were generated. The transcription factors Msn2/4 and Rph1/Gis1 in S. cerevisiae and their shared orthologs in K. lactis were deleted in both species to quantify and compare the effect of those transcription factors on the response to PKA inhibition in each species.
