Yeast drug resistance gene mutants in glucose or ammonium limited chemostats
ABSTRACT: Homozygous deletion mutants of drug resistance genes QDR3 and PDR3 and the homozygous deletion of HO (control) were grown in ammonium limited or glucose limited synthetic defined medium in aerated, pH controlled chemostats at a dilution rate of 0.1hr-1. Samples were collected at steady state.
Project description:Wild type S. cerevisiae cells were grown under glucose or ammonium limitation in fully controlled aerobic chemostat cultures. At steady state, the limited nutrient was introduced into the growth medium in an impulse like manner to recover the medium to its non-limiting conditions. Samples were collected at steady state and following the impulse (20s, 40s, 60s, 8 min, 16 min, 24 min, 32 min, 1 hr, 2hr, 3hr, 4hr, 5hr, 7 hr) until the effect of the perturbation ceased to exist. The last sample was collected the second time the chemostat reached steady state.
Project description:Deletion mutants of S. cerevisiae 4743 were grown in glucose-limited continuous chemostats (D = 0.2h-1). The deletion mutants used were: Homozygous deletion mutants of HAP4, OXA1, BCS1, RIP1, MBA1 and MIG1. Heterozygous deletion mutants of HAP4, RIP1 and MIG1.
Project description:The fungal pathogen Candida albicans produces dark-pigmented melanin when grown in a basal medium containing 1 mM l-DOPA as melanin substrate. In the widely used C. albicans strain SC5314, melanin appeared after 3-4 days of incubation in l-DOPA medium. The experiment was designed to reveal cadidate genes associated with melanin biosynthesis by expression profiling at different times of growth with and without L-DOPA added to the medium. Expression profiling of C. albicans revealed very few genes significantly up- or down-regulated by growth in l-DOPA. Sixteen paired samples were submitted to dual channel analysis. Four biological replicate samples of RNA were prepared from cells of C. albicans strain SC5314 at four time points, from cultures grown with and without addition of 1 mM L-DOPA, thus generating an initial total of 32 samples for analysis: four experimental and four control samples made after 6 h, 24 h, 48 h and 72 h of growth. For each time point the control (no L-DOPA) samples were pooled and labelled with Cy3-dUTP by reverse transcription. The four individual biological replicates for each time point were separately labelled with Cy5-dUTP by reverse transcription and independently hybridized against the pooled controls on the microarray chips. Results were read with a ScanArray 4000 Microarray Analysis System.
Project description:Gata6 ChIP-seq on mouse posterior branchial arches connected to outflow tract of the heart (PBA/OFT) at embryonic day (E) 11.5. H3K27Ac ChIP-seq on mouse second branchial arch (BA2) and posterior branchial arches connected to outflow tract of the heart (PBA/OFT) at embryonic day (E) 11.5.
Project description:In this study we have tried to utilize the unique aspects of the T. ruralis response to desiccation and rehydration to design a strategy to identify rehydrins that are of low abundance and perhaps completely novel to the desiccated or rehydration transcriptomes. We have constructed two Subtractive Suppression Hybridization (SSH) libraries (Diatchenko et al., 1996) that are designed to enrich for differentially expressed low-abundance transcripts contained within gametophytic cells either in the slow-dried state (mRNP sequestrated rehydrin transcripts) or cells that have been rapidly dried, rehydrated and sampled at 2h of hydration (rehydrin and recovery transcripts) when the translational change in gene expression is at its peak (Oliver 1991). To achieve this aim we constructed SSH libraries using PolyA RNA isolated from the polysomal (mRNP) fractions from the slow-dried and 2h rehydrated rapid dried gametophytes selected against PolyA RNA from hydrated control gametophytes as the source for driver cDNA. Collections of cDNA clones from each library were sequenced and used to generate a small T. ruralis SSH cDNA microarray for expression profiling of both total RNA extracts for transcript accumulation assessments and polysomal RNA extracts for transcript sequestration and recruitment assessments. To assess the expression characteristics of the transcripts represented by the SSH contigs we established a cDNA microarray containing the inserts (PCR derived fragment) from each of the 768 individual SSH ESTs, with the exception of thirteen that failed to generate a PCR fragment. Twelve of the missing thirteen SSH EST cDNAs were replaced with PCR fragments from previously isolated T. ruralis cDNAs four of which, representing the ribosomal proteins S14, S16, L23 and L15 (Wood et al., 2000, Zeng and Wood 2000), were previously reported to be constitutively expressed and were added to serve as normalization genes. The remaining eight clones, Tr155, Tr217, Tr403, Tr416, Tr421 (described by Scott and Oliver, 1994), and TrCDPK (U82087) were added as either positive “up-regulated” (Tr155, Tr403, Tr421), negative “down-regulated (Tr217, Tr416), or neutral (TrCDPK) controls based on previous northern analyses. The cDNAs were printed from two 384 well plates in 12 blocks (two columns of 6) of 24 x 8 spots such that each SSH EST and controls were represented in triplicate. Each of the triplicate cDNAs was separated within the blocks to eliminate possible spatial hybridization bias. All hybridizations were duplicated as dye swaps with two separate RNA preparations, from large populations of individual gametophytes (isolated from a minimum of three separate clumps), serving as the source for the sscDNA Cy3 and Cy5 labeled probes. The RNA preparations for the Total polyA RNA were by necessity separate samples from those used to isolate Polysomal poly A RNA.
Project description:We demonstrated that RORa-deficient staggerer mice (RORasg/sg) fed with a high fat diet (HFD) showed reduced adiposity and hepatic triglyceride levels compared to wild type (WT) littermates and were resistant to the development of hepatic steatosis, adipose-associated inflammation, and insulin resistance. Gene expression profiling showed that many genes involved in triglyceride synthesis and storage, including Cidec, Cidea, and Mogat1, were expressed at much lower levels in liver of RORasg/sg mice. In addition to reduced lipid accumulation, inflammation was greatly diminished in white adipose tissue (WAT) of RORasg/sg mice fed with a HFD. The infiltration of macrophages and the expression of many immune-response and pro-inflammatory genes, including those encoding various chemo/cytokines, toll-like receptors, and TNF signaling proteins, were significantly reduced in RORasg/sg WAT. Moreover, RORasg/sg mice fed with a HFD were protected from the development of insulin resistance. Together, these results indicate that RORa plays a critical role in the regulation of several aspects of metabolic syndrome. Therefore, RORa may provide a novel therapeutic target in the management of obesity and associated metabolic diseases. Liver and white adipose tissue (WAT) total RNAs were purified from 5 WT and 5 RORasg/sg (natural deletion of RORa gene in mice) mice fed with a high fat diet for 6 weeks. Then samples were applied on Agilent mouse genome chip.
Project description:Bisulfite padlock probe technique was used to examine DNA modifications at the lactase gene region for human and mouse tissues DNA modifications were investigated in human sperm, blood, jejunal enterocytes and jejunum lacking enterocytes, as well as mouse jejunal enterocytes and jejunum lacking enterocytes. For WGA samples, a genome devoid of DNA modifications was used to verify the efficiency of the bisulfite conversion reactions (the tissue sources were human or mouse intestine).