Genomic Run On (GRO): determination of the nascent transcriptional rates and mRNA levels in several yeast mutants (SAC3, SUS1, SRC1)
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ABSTRACT: We analyzed the effect of SAC3, SUS1 and SRC1 on the transcription rates, mRNA stabilities and mRNA levels by doing GRO experiments in a deletion mutants and the partial C-truncated version of Sac3 comparing with a wild type. Some data for mRNA amounts (sus1 ans src1 mutants) are not included because were already in GEO database: GSE920 and GSE6370 accession numbers. This study focus on the transcriptional activity of RNA pol II in yeast genes. S. cerevisiae cells grown in YPD to exponential phase were subjected to Genomic Run On. Data were normalized by the ArrayStat software. Home-made macroarrays containing entire ORFs were used.
Project description:In order to maintain the appropriate level of mRNA it is necessary coordinate simultaneously all the steps along the mRNA life cycle. It has been shown that several factors act in the regulation of gene expression as global coordinators. Thus, some kind of information is transferred from the nucleus to the cytoplasm, imprinted in the mRNA. In this way, it is conceivable the existence of mechanisms that ensure the balance between mRNA synthesis and degradation through the information flow from the cytoplasm to the nucleus and vice versa, as a crosstalk among both process to ensure the proper mRNA homeostasis in the cell. In this study, we have used null mutants related with transcription and mRNA degradation and we find that the lack of those factors impacts both over transcription and RNA stability at genome wide levels to compensate the impaired effect produce by the loss of each studied factor. This study focus on the transcriptional activity of RNA pol II in yeast genes. S. cerevisiae cells grown in YPD or YPGal to exponential phase were subjected to Genomic Run On. Data were normalized by the ArrayStat software. Home-made macroarrays containing 6035 ORF were used.
Project description:We analyzed the effect of SPT16 on the transcription rates, mRNA stabilities and mRNA levels by doing GRO experiments in a thermosensitive spt16 point mutant (G132D) mutant at 35 ºC comparing with permisive temperature (30 ºC) and a wt treated in the same way. Cells (wt or spt16 mutant) were grown in SC-ura at 30 ºC to 0.25 OD600. At that time α factor was added and let to stand for 160 min in the same conditions. Then each sample (wt or mutant) was separated in two aliquots. One of them remain at 30ºC and the other was incubated at 35ºC. Both aliquots in the presence of α factor for 1h more. Finally, samples were processed for GRO experiment as described in Garcia-Marinez et al (2004). Three replicates of the whole experiment were done.
Project description:The goal of the project was to check the effect on transcription rate of a sudden depletion of Mex67 protein by using a thermosensitive mutant: mex67-5 Cells were grown in YPD at 26ºC to 0.5 OD600. At that time cells were shifted to 37ºC to induce Mex67 protein inactivation and recovered after, 0, 5, 20, 40, 60 and 150 min.
Project description:We analyzed the effect of SAC3, SUS1 and SRC1 on the transcription rates, mRNA stabilities and mRNA levels by doing GRO experiments in a deletion mutants and the partial C-truncated version of Sac3 comparing with a wild type. Some data for mRNA amounts (sus1 ans src1 mutants) are not included because were already in GEO database: GSE920 and GSE6370 accession numbers.
Project description:The goal of the project was to check the differential effect on transcription rate of a sudden degron-induced depletion of Hpr1 protein and its comparison towards the effect of a constitutive absence of Hpr1 protein in a deletion strain. Cells were grown in YP-Raffinose + 0.1 mM CuSO4 at 26ºC to 0.5 OD600. At that cells were spun-down and changed to YP-Galactose without CuSO4 for 30 min at 26ºC. Then cells were shifted to 37ºC to induce degron-mediated proteolysis and recovered after 30 min. Samples include wt, hpr1delta and hpr1-degron after 0 or 30 min growth at 30ºC.
Project description:We analyzed the effect of PUB1 and NGR1 on the transcription rates, mRNA stabilities and mRNA levels by doing GRO experiments in a deletion mutants comparing with a wild type in both glucose (YPD) and galactose (YPGal) media. Some data for the wt in YPGal are not included because were already in GEO database: GSE1002 accession number. This study focus on the transcriptional activity of RNA pol II in yeast genes. S. cerevisiae cells grown in YPD or YPGal to exponential phase were subjected to Genomic Run On. Data were normalized by the ArrayStat software. Home-made macroarrays containing 6035 ORF were used.
Project description:The goal of the project was to study the effects on transctiptome of some Rpb1 foot mutations that block Rpb4/7 assembly Cells were grown in YPD at 30ºC to 0.5 OD600. At that cells were spun-down or changed to 37ºC by adding hot YPD. These aliquots were cultured for 30 min and collected. Thus, wt and mutants have two samples: 30 and 37ºC. Each sample was done in triplicate.
Project description:GRO (Genomic run-on) experiments with different mutants that affect to the accumulation of non active RNA pol II along the yeast genome. Keywords: Genomic run-on GRO There are 4 different strains: rap1-sil (without the silencing domain), RAP1(both from Graham I.R. et al 1999) and tpk1 & tpk2 mutants (Euroscarf). For each experiment there are GRO data (Transcription Rate) and gDNA data used for normalizing the GRO signals. The last number of the GRO filters correspond to the number of gDNA filters.There are 3 independent biological replicates of Rap1 and rap1-sil experiments and 2 for the tpk1 & tpk2 experiments.
Project description:Genomic Run-on analysis of topoisomerase mutants (top1-delta/top2ts or top2ts) has been conducted at reference temperature for wt strain and at non-permissive temperature for wt and mutant strains. Keywords: Genomic Run On The analysis includes 3 repeats of the wild type strain (JCW25) at 30ºC and 3 more at 37ºC, 3 repeats of the top2ts mutant (JCW26) at 37ºC and 3 repeats of the double top1 (delta) top2ts mutant at 37ºC. A single genomic DNA hybridization on one of the filters can be used for normalization between gene probes if needed.
Project description:Transcription rate (TR) analysis of W303 hog1 mutant yeast strain growing in exponential phase in YPD subjected to osmotic stress Keywords: Time course Transcription rate analysis by means of GRO of three independent replicates the yeast strain growing in exponential phase. Each time point replicate has been hybridized on a different macroarray (F18-F24).