inducible gal1 promoter  (Addgene inc)

Journal: bioRxiv

Article Title: Disruption of the nascent polypeptide-associated complex leads to reduced polyglutamine aggregation and toxicity

doi: 10.1101/2024.04.19.590245

Figure Lengend Snippet: (A) Yeast 74-D694 WT and NAC deletion strains expressing Gal1-inducible EV, htt25Q, or htt103Q constructs were serially diluted 5-fold and spotted onto ¼ YPD (not shown), SD-ura, and S-ura, 2% galactose (first 3 spots shown) to monitor expanded polyglutamine toxicity and the ability of nac deletion to rescue expanded polyglutamine cytotoxicity (n=3). (B) A yeast 74-D694 WT strain expressing EV, EGD2, or EGD1 constructs in combination with Gal-inducible EV or htt103Q constructs were serially diluted 5-fold on ¼ YPD (not shown), SD-ura-his, and S-ura-his, 2% galactose, 0.1% raffinose (first three spots shown) to evaluate expanded polyglutamine toxicity and the ability of EGD1 and EGD2 overexpression to rescue expanded polyglutamine cytotoxicity (n=3).

Article Snippet: Plasmids expressing alpha-synuclein constructs controlled by the inducible GAL1 promoter (pRS426-Gal1-SNCA WT -GFP, pRS426-Gal1-SNCA A30P -GFP, pRS426-Gal1-SNCA A53T -GFP) were kindly gifted by M. Jackrel. pTH726-CEN-RLuc/minCFLuc and pTH727-CEN-RLuc/staCFLuc were a gift from Tobias von der Haar (Addgene plasmid # 38210 and 38211).

Techniques: Expressing, Construct, Over Expression

Journal: bioRxiv

Article Title: Disruption of the nascent polypeptide-associated complex leads to reduced polyglutamine aggregation and toxicity

doi: 10.1101/2024.04.19.590245

Figure Lengend Snippet: (A) WT and NAC deletion strains expressing Gal1-htt25Q-CFP or Gal1-htt103Q-CFP constructs were grown in selective media in the presence of 2% galactose for 6 and 20 hours. Two-dimensional z-stack images were taken with a Zeiss LSM 880 Airyscan Two-Photon Confocal Microscope with a 63x oil immersion objective. The egd1Δbtt1Δ strain showed a smaller population of cells with aggregates than the WT or egd1Δegd2Δ strains at both time points. (B) Quantification of percent cells expressing Gal1-htt25Q-CFP or Gal1-htt103Q-CFP constructs containing aggregates after 6 hours induction by 2% galactose in selective media. Significance was determined by Fisher’s exact test (****=<0.0001). (C) Quantification of percent cells expressing Gal1-htt25Q-CFP or Gal1-htt103Q-CFP constructs containing aggregates after 20 hours induction by 2% galactose in selective media. Significance was determined by Fisher’s exact test (****=p value<0.0001). (D) Representative images of cells with 0, 1, 2, or 3 aggregates. Microscopy of WT and NAC deletion strains expressing Gal1-htt25Q-CFP or Gal1-htt103Q-CFP constructs for 6 and 20 hours were evaluated based on this scale. (E) WT and NAC deletion strain cells expressing Gal1-htt103Q-CFP constructs for 6 or 20 hours and containing aggregates were evaluated as having 1, 2, or 3 or more aggregates. The resulting population distributions are represented as percentages in pie graphs.

Article Snippet: Plasmids expressing alpha-synuclein constructs controlled by the inducible GAL1 promoter (pRS426-Gal1-SNCA WT -GFP, pRS426-Gal1-SNCA A30P -GFP, pRS426-Gal1-SNCA A53T -GFP) were kindly gifted by M. Jackrel. pTH726-CEN-RLuc/minCFLuc and pTH727-CEN-RLuc/staCFLuc were a gift from Tobias von der Haar (Addgene plasmid # 38210 and 38211).

Techniques: Expressing, Construct, Microscopy

Journal: bioRxiv

Article Title: Disruption of the nascent polypeptide-associated complex leads to reduced polyglutamine aggregation and toxicity

doi: 10.1101/2024.04.19.590245

Figure Lengend Snippet: (A) WT and NAC deletion strains expressing Gal1-FLAG-htt25Q-CFP or Gal1-FLAG-htt103Q-CFP constructs were grown in selective media in the presence of 2% galactose for 6 hours prior to SDS-PAGE and Western blotting for FLAG and PGK1. Western blots are representative images of three independent experiments. (B) WT and NAC deletion strains expressing Gal1-FLAG-htt25Q-CFP (not shown) or Gal1-FLAG-htt103Q-CFP constructs were grown in selective media in the presence of 2% galactose for 6 hours prior to Filter Trap Assay and Western blotting for FLAG. Western blot is a representative image of three independent experiments. (C) Quantification and normalization to WT of 3 independent Filter Trap Assay Western blots represented in 4(B), data are represented as mean ± SEM, significance was determined by paired t-test, *=p value<0.05, and ns=p value>0.05. (D) Western blot image showing the Ubiquitinated proteins in WT and egd1Δbtt1Δ strains co-expressing htt25Q, htt103Q constructs with an Ub-X- LacZ reporter: pGal-Ub-P- LacZ . Pgk1 showing the loading control. Representative image shown here.

Article Snippet: Plasmids expressing alpha-synuclein constructs controlled by the inducible GAL1 promoter (pRS426-Gal1-SNCA WT -GFP, pRS426-Gal1-SNCA A30P -GFP, pRS426-Gal1-SNCA A53T -GFP) were kindly gifted by M. Jackrel. pTH726-CEN-RLuc/minCFLuc and pTH727-CEN-RLuc/staCFLuc were a gift from Tobias von der Haar (Addgene plasmid # 38210 and 38211).

Techniques: Expressing, Construct, SDS Page, Western Blot, TRAP Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Evidence for a Conserved Function of Eukaryotic Pantothenate Kinases in the Regulation of Mitochondrial Homeostasis and Oxidative Stress

doi: 10.3390/ijms24010435

Figure Lengend Snippet: Complementation of the thermosensitive phenotype of CAB1 -defective yeast strains by human PanKs. ( A – C ) Equal amounts of serial dilutions of cells (10 4 , 10 3 , 10 2 , 10 1 cells/spot) from exponentially grown cultures of cab1 Δ/ cab1 G351 S ( A ), cab1 Δ/ cab1 N290I ( B ), and cab1 Δ/ cab1 I291T ( C ) mutant strains carrying pFL38/ CAB1 and pESC- URA3 vector containing the different h PANK gene were spotted onto SC medium supplemented with the indicated carbon sources and incubated at 28 °C and 37 °C. Yeast growth was monitored over time and images collected 2 or 3 days post-inoculation. ( D – F ) Growth rates of cab1 Δ/ cab1 G351S ( D ), cab1 Δ/ cab1 N290I ( E ), and cab1 Δ/ cab1 I291T ( F ) strains carrying the empty vector (EV), CAB1 and human PANK s in liquid media on glucose or galactose at 30 °C vs. 37 °C (from top to bottom). Yeast cells were inoculated in minimal medium without uracil and tryptophan but containing either 2% glucose or 2% galactose at a cell density of 10 4 cells/mL. Optical density measurements at 600 nm (OD 600 ) were determined at the indicated time points. Data for liquid assays represent three biological replicates ( n = 3) and the plotted graphs represent the average ± SEM.

Article Snippet: The human PANK genes, PANK1 (Uniprot #Q8TE04), PANK2 (Uniprot # Q9BZ23), and PANK3 (Uniprot # Q9H999) were cloned into the pESC- URA3 vector under the inducible GAL1 promoter (GenScript Inc., Piscataway, NJ, United States), which results in expression of the proteins with an N-terminal Myc tag.

Techniques: Mutagenesis, Plasmid Preparation, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Evidence for a Conserved Function of Eukaryotic Pantothenate Kinases in the Regulation of Mitochondrial Homeostasis and Oxidative Stress

doi: 10.3390/ijms24010435

Figure Lengend Snippet: Analysis of reactive oxygen species (ROS) yeast PanK-deficient mutants expressing human PanK isoforms. ROS measurements were performed for cab1 Δ/ cab1 G351S ( A ), cab1 Δ/ cab1 N290I ( C ), and cab1 Δ/ cab1 I291T ( E ) yeast strains transformed with pFL38/ CAB1 , EV, and pESC- URA3 vector containing different human PanK isoforms. ROS production was determined as the percentage of fluorescent cells using the DHR123 probe. Cytofluorometry analysis of the cab1 Δ/ cab1 G351S ( B ), cab1 Δ/ cab1 N290I ( D ), and cab1 Δ/ cab1 I291T ( F ) yeast strains transformed with either pFL38/ CAB1 , empty vector or pESC- URA3 vector containing different human PanK isoforms following staining with DHR123 probe. Each curve represents the distribution of the measured events (counts) according to their fluorescence intensity expressed in a log unit. The grey spectrum represents background autofluorescence. The strains were grown in YP medium supplemented with 2% galactose at 28 °C. This assay was conducted in triplicate ( n = 3) and the plotted graphs represent the average ± SEM. *: p < 0.05 and **: p < 0.01 relative to mutant strain transformed with CAB1 allele and #: p < 0.05 and ##: p < 0.01 relative to mutant strain transformed with the empty vector.

Article Snippet: The human PANK genes, PANK1 (Uniprot #Q8TE04), PANK2 (Uniprot # Q9BZ23), and PANK3 (Uniprot # Q9H999) were cloned into the pESC- URA3 vector under the inducible GAL1 promoter (GenScript Inc., Piscataway, NJ, United States), which results in expression of the proteins with an N-terminal Myc tag.

Techniques: Expressing, Transformation Assay, Plasmid Preparation, Staining, Fluorescence, Mutagenesis

Journal: Biochemistry

Article Title: Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences.

doi: 10.1021/acs.biochem.2c00250

Figure Lengend Snippet: Figure 4. Leveraging induced dimerization to trigger synthetic condensate formation in living cells. (A) Scheme for encoding and expression of RGG polypeptides in cells, sensitive to small-molecule-induced dimerization and condensation. RGG-GFP-FKBP and RGG-FRB constructs are integrated into the yeast genome controlled by an inducible GAL1 promoter. (B) Representative images for strains described in A, showing addition of 20 μM Rap triggers condensate formation within minutes in live cells. (C) Average number of droplets formed per yeast cell in the n = 105 cells. Shaded area, 95% CI. (D) Optical regulation of condensation in cells following illumination to photouncage dRap. (E) Average number of droplets formed per cell following 10 s of 405 nm laser illumination (n = 66 cells). Shaded area, 95% CI.

Article Snippet: RGG constructs with a coiled coil or FRB and FKBP tags were cloned downstream of a galactose-inducible GAL1 promoter and integrated into the yeast URA3 locus using the Yiplac211 integrating vector or LEU2 locus using the Yiplac128 integrating vector by linearizing plasmid with EcoRV just before transformation.

Techniques: Expressing, Construct