Mediator Acts Upstream of the Transcriptional Activator Gal4

We show that Mediator, a protein originally isolated on the basis of its ability to respond to transcriptional activators, and thought to be regulated by an activator, can also be the master that controls the activator

Business study mission to Canada chapter on Canada's trade patterns and policy

This report covers the macro-level analysis of the Canadian economy, specifically on the trade issues. The report starts off with a brief history of Canada's trade. The trade policy of Canada is then reviewed with focus on tariffs and non-tariff measures. Tariffs form an important part of the Canadian trade policy. However, with the gradual elimination of tariffs, non-tariff measures are becoming more important in most trade negotiation. Trade agreements and Canada's involvement with some groupings are also discussed.Master of Business Administration (International Business

A galactose-stable Gal80 deletion derivative inhibits galactose induction of the <i>GAL1</i> gene.

<p>(A) <i>BY4742ΔW</i> cells over-expressing the indicated HA-Gal80 deletion derivatives from <i>RS316</i> under the control of the <i>ACT1</i> promoter were 10-fold serially diluted, titrated onto the indicated plates, and incubated for 3 d (Glucose) and 6 d (Galactose+AA = 1 mg/l Antimycin A), respectively. (B) Cells of part A, lines 1, 2 and 6, were grown in glucose liquid media to OD_{600 nm} = 1 and induced with galactose liquid media for the indicated number of hours. Total RNA was isolated and <i>GAL1</i> mRNA was determined relative to <i>ACT1</i> mRNA by quantitative real-time PCR. The value determined for <i>BY4742ΔW</i> cells containing <i>RS316</i> grown with glucose liquid media was set as 1 and the error bars indicate the standard deviations between three replicates. (C) <i>BY4742ΔW</i> cells expressing HA-tagged wild-type Gal80 or Gal80ΔN12 from <i>RS317</i> under the control of the <i>ACT1</i> promoter were grown in glucose liquid media to OD_{600 nm} = 1 and induced with galactose liquid media for 1 h. Cycloheximide was added at time = 0 and the amount of HA-Gal80 and HA-Gal80ΔN12 proteins remaining in the cells after the indicated number of minutes was determined by Western blot with the help of an anti-HA antibody (upper panels). The membranes were stripped and reprobed with an anti-CPY antibody (middle panels), followed by a second stripping and staining with Coomassie Blue as loading controls (lower panels). (D) The ratio of the amount of HA-Gal80 protein to CPY protein (white bars) and of HA-Gal80ΔN12 protein to CPY protein (black bars) in <i>BY4742ΔW</i> cells for each time point in part C was determined with Image J. The ratio of the band intensities before the addition of cycloheximide (time = 0) was set as 1 and the error bars indicate the deviations between duplicates.</p

Galactose induction requires SCF-mediated Gal80 degradation.

<p>(A) <i>JD52</i> cells whose chromosomal <i>SKP1</i> gene had been replaced by <i>HIS3</i> and that expressed Nub-Skp1 (SKP1wt) or Nub-Skp1V90A,E129A (skp1dM) under the control of its own promoter from the single-copy vector <i>PSCNX</i> were transformed with the single-copy vector <i>RS316</i> expressing HA-tagged Gal80 under the control of the <i>ACT1</i> promoter. Cells were grown in glucose liquid media to OD_{600 nm} = 1 and induced with galactose liquid media for 1 h. Cycloheximide was added at time = 0 and the amount of Gal80 protein remaining in the cells after the indicated number of minutes was determined by Western blot with the help of an anti-HA antibody (upper panels). The membranes were stripped and stained with Coomassie Blue as loading controls (lower panels). (B) The ratio of the amount of HA-Gal80 protein to total protein (Coomassie) in <i>SKP1wt</i> cells (white bars) and <i>skp1dM</i> cells (black bars) for each time point in part A was determined with Image J. The ratio of the band intensities before the addition of cycloheximide (time = 0) was set as 1 and the error bars indicate the deviations between duplicates. (C) <i>JD52</i> cells expressing Nub-Skp1wt (line 1) and Nub-Skp1dM (lines 2 to 7) in place of endogenous Skp1 were 10-fold serially diluted, titrated onto the indicated plates, and incubated for 3 d on glucose plates and for 6 d on galactose plates containing 0.1 mg/l of the respiration inhibitor Antimycin A (AA). Cells over-expressed the indicated proteins from the multi-copy vector <i>YEplac112</i> under the control of their own respective promoters. Cells in line 7 expressed Skp1 from the single-copy vector <i>YCplac22</i> under the control of its own promoter. (D) <i>JD52</i> cells of the indicated genotype were grown in glucose liquid media to OD_{600 nm} = 1 and induced with galactose liquid media for 8 h. Cells over-expressed Gal3 from <i>RS314</i> under the control of the <i>ACT1</i> promoter or lacked <i>GAL80</i> as indicated. Total RNA was isolated and <i>GAL1</i> mRNA was determined relative to <i>ACT1</i> mRNA by quantitative real-time PCR. The value determined for <i>SKP1wt</i> cells grown with glucose liquid media was set as 1 and the error bars indicate the standard deviations between three replicates.</p

Gal80 is the main target of Mdm30.

<p>(A) HA-tagged Gal80 was expressed in <i>BY4741ΔW</i> cells of the indicated genotype. Cells were grown in glucose liquid media to OD_{600 nm} = 1 and induced with galactose liquid media for 1 h. Cycloheximide was added at time = 0 and the amount of Gal80 protein remaining in the cells after the indicated number of hours was determined by Western blot with the help of an anti-HA antibody (upper panels). The membranes were stripped and stained with Coomassie Blue as loading controls (lower panels). (B) The ratio of the amount of HA-Gal80 protein to total protein (Coomassie) in <i>BY4741ΔW</i> cells (white bars), <i>BY4741ΔWΔMDM30</i> cells (black bars), and <i>BY4741ΔWΔGAL11</i> cells (grey bars) for each time point in part A was determined with Image J. The ratio of the band intensities before the addition of cycloheximide (time = 0) was set as 1 and the error bars indicate the deviations between duplicates. (C) <i>BY4741ΔW</i> cells of the indicated genotype were 10-fold serially diluted, titrated onto the indicated plates, and incubated for 3 d on the glucose plate and for 6 d on the galactose plate. (D) <i>BY4741ΔW</i> cells of the indicated genotype were grown in glucose liquid media to OD_{600 nm} = 1 (Glu) and induced with galactose liquid media for 8 h (Gal). Total RNA was isolated and <i>GAL1</i> mRNA was determined relative to <i>ACT1</i> mRNA by quantitative real-time PCR. The value determined for <i>BY4741ΔW</i> wild-type cells grown with glucose liquid media was set as 1 and the error bars indicate the standard deviations between three replicates. (E) HA3-tagged Mdm30 and GST or GST-Gal80 were co-expressed in <i>BY4741ΔW</i> cells under the control of the <i>ACT1</i> promoter from the multi-copy vectors <i>RS423</i> and <i>RS424</i>, respectively. Cells were grown with glucose liquid media to OD_{600 nm} = 1 (odd lanes) and induced in galactose liquid media for 1 h (even lanes). GST and GST-Gal80 were pulled down from cell extracts with the help of glutathione beads, and Inputs and GST Pulldowns were analyzed by Western blots with the help of an anti-HA antibody (upper panel) and an anti-GST antibody (middle panel). The membrane was stripped and stained with Coomassie in order to compare the amount of protein loaded for Input and GST Pulldown (bottom panel). (F) Histidine-tagged ubiquitinated proteins were precipitated with Ni-beads from extracts of glucose-grown (Glu) and galactose-induced (Gal) <i>SUB288ΔWL</i> (lanes 1, 2, 5, 6, 7) and <i>SUB288ΔWLΔMDM30</i> (lanes 3, 4, 8, 9) cells expressing HA-Gal80 and ubiquitin (lane 5) or histidine-tagged ubiquitin (lanes 1 to 4 and 6 to 9) in place of endogenous ubiquitin. Inputs (lanes 1 to 4) and precipitates (lanes 5 to 9) were analyzed by Western blot with the help of an anti-HA antibody. The ubiquitinated bands appear as doublets, indicating that more than one lysine in Gal80 is subject to ubiquitination.</p

The protein kinase Snf1 is required for galactose-induced protein degradation of Gal80.

<p>(A) <i>BY4742ΔW</i> (lanes 1 to 12), <i>BY4742ΔWΔSNF1</i> (lanes 13 to 24), and <i>BY4741ΔWΔSNF4</i> (lanes 25 to 36) cells expressing HA-tagged Gal80 under the control of the <i>ACT1</i> promoter were grown in glucose liquid media to OD_{600 nm} = 1 and induced with galactose liquid media for 1 h. Cycloheximide was added at time = 0 and the amount of Gal80 protein remaining in the cells after the indicated number of minutes was determined by Western blot with the help of an anti-HA antibody (upper panels). The membranes were stripped and reprobed with an anti-CPY antibody (middle panels), followed by a second stripping and staining with Coomassie Blue as loading controls (lower panels). (B) The ratio of the amount of HA-Gal80 protein to CPY protein in <i>BY4742ΔW</i> cells (white bars), <i>BY4742ΔWΔSNF1</i> cells (black bars), and <i>BY4741ΔWΔSNF4</i> cells (grey bars) for each time point in part A was determined with Image J. The ratio of the band intensities before the addition of cycloheximide (time = 0) was set as 1 and the error bars indicate the deviations between duplicates. (C) <i>BY4742ΔW</i> cells (lines 1 to 3) and <i>BY4741ΔW</i> cells (lines 4 to 6) of the indicated genotype were 10-fold serially diluted, dropped onto the depicted plates, and incubated for 6 d at 28°C. The galactose plate contained 1 mg/l Antimycin A. (D) Cells of part C, lines 1 to 3, were grown in glucose liquid media to OD_{600 nm} = 1 and induced with galactose liquid media for the indicated number of hours. Total RNA was isolated and <i>GAL1</i> mRNA was determined relative to <i>ACT1</i> mRNA by quantitative real-time PCR. The value determined for <i>BY4742ΔW</i> cells grown with glucose liquid media was set as 1 and the error bars indicate the standard deviations between three replicates.</p

How Mediator orchestrates its own recruitment to the <i>GAL1</i> promoter.

<p>(A) The protein kinase SNF1 senses the absence of glucose and phosphorylates Mig2. Phosphorylated Mig2 is ubiquitinated by the E3 ubiquitin ligases SCF^Das1/Ufo1<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001290#pbio.1001290-Lim1" target="_blank">[37]</a>. SNF1 also signals Mediator the absence of glucose via the Snf1-Srb11 interaction. Mediator transduces the signal to the E3 ubiquitin ligases SCF^{Mdm30/Ufo1/Das1} via the Srb7-Skp1 interaction. SCF^{Mdm30/Ufo1/Das1} ubiquitinate Gal80 via the interaction of Gal80 with the F-box proteins Mdm30, Ufo1, and Das1. (B) Poly-ubiquitinated Gal80 and Mig2 are degraded by the 26S proteasome. (C) Gal4 recruits chromatin-remodeling and chromatin-modifying complexes as well as the Holoenzyme of Transcription (consisting of RNA Polymerase II, the General Transcription Factors, and Mediator) to the <i>GAL1</i> promoter genes via the Gal4-Gal11 interaction <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001290#pbio.1001290-Lim2" target="_blank">[43]</a>.</p

Gal80 is stable in galactose-induced <i>H₁₀UbD58A</i> cells.

<p>(A) <i>SUB288GAL3ΔWL</i> cells expressing the indicated ubiquitin derivative in place of endogenous ubiquitin were 10-fold serially diluted, titrated onto glucose and onto galactose plates, and incubated at 28°C for 6 d. Cells in lines 3 and 6 over-expressed Gal3 from the <i>ACT1</i> promoter, while cells in lines 4 and 7 lacked <i>GAL80</i>. (B) <i>SUB288GAL3ΔL</i> cells expressing H₁₀Ub and H₁₀UbD58A in place of endogenous ubiquitin as indicated were grown in glucose liquid media to OD_{600 nm} = 1 (Glu) and induced with galactose liquid media for 6 h (Gal). Cells over-expressed Gal3 from the <i>ACT1</i> promoter or lacked <i>GAL80</i> as indicated. Total RNA was isolated and the amount of <i>GAL1</i> mRNA relative to <i>ACT1</i> mRNA was determined with reverse transcription-coupled real-time PCR. The value determined for cells expressing H₁₀Ub grown with glucose was set as 1, and the error bars indicate the standard deviations between three replicates. (C) <i>SUB288GAL3ΔL</i> cells expressing H₁₀Ub (lanes 1 to 8) or H₁₀UbD58A (lanes 9 to 16) in place of endogenous ubiquitin were transformed with the single-copy vector <i>RS316</i> expressing HA-Gal80 under the control of the <i>ACT1</i> promoter. Cells were grown in glucose liquid media to OD_{600 nm} = 1 and induced with galactose liquid media for 1 h. Cycloheximide was added at time = 0 and the amount of Gal80 protein remaining in the cells after the indicated number of hours was determined by Western blot with the help of an anti-hemagglutinin (HA) antibody (upper panels). The membranes were stripped and reprobed with an anti-carboxypeptidase Y (CPY) antibody (middle panels), followed by a second stripping and staining with Coomassie Blue as loading controls (lower panels). (D) The ratio of the amount of HA-Gal80 protein to CPY protein in <i>H₁₀-Ub</i> cells (white bars) and <i>H₁₀-UbD58A</i> cells (black bars) for each time point in part C was determined with Image J. The ratio of the band intensities before the addition of cycloheximide (time = 0) was set as 1, and the error bars indicate the deviations between duplicates.</p