Journal: bioRxiv

Article Title: Engineering Conjugative Plasmids for Inducible Horizontal DNA Transfer

doi: 10.1101/2025.03.12.642913

Figure Lengend Snippet: Analysis of the inducible cis conjugative system. A) Schematic of plasmids used in cis-conjugation. The conjugative plasmid pSC5GGv1 served as a positive control. Plasmid pSC6 had the trbF gene deleted and relocated to a different position under the pBAD inducible promoter. All plasmids contain a gentamicin selectable marker (GmR; yellow). CAH – elements for replication and maintenance in yeast, including centromere (CEN6), autonomously replication sequence (ARS4), and histidine auxotrophic marker (HIS3). B) Representative final selection plates (Nm/Gm) transconjugant colony frequency. The donor strains had only one plasmid each: pSC5GGv1 or pSC6. Recipient cells have a chromosomally integrated neomycin resistance marker (NmR). All plasmids were tested under three conditions: induced with arabinose, uninduced, and repressed with glucose. Complete figures of all replicates displayed in Supplemental Figure S5. C) Average colony counts for all three conditions across all replicates. Complete colony counts for all replicates displayed in Supplemental Table S4. Error bars represent the standard error of the mean (SEM). Student’s t-test: * p < 0.05; n = 6.

Article Snippet: Saccharomyces cerevisiae VL6−48 (ATCC MYA-3666: MATα, his3-Δ200, trp1-Δ1, ura3-52, lys2, ade2-101, met14, psi + cir 0 ) was grown in 2x yeast extract peptone dextrose media (YPD) supplemented with adenine hemisulfate (200 μg mL -1 ; Sigma-Aldrich, #A2545, St. Louis, MO, USA) (YPAD) and ampicillin (100 μg mL -1 ; BioBasic, Cat #: AB0028, Canada).

Techniques: Conjugation Assay, Plasmid Preparation, Positive Control, Marker, Sequencing, Selection

Journal: bioRxiv

Article Title: Engineering Conjugative Plasmids for Inducible Horizontal DNA Transfer

doi: 10.1101/2025.03.12.642913

Figure Lengend Snippet: Applications of inducible conjugative plasmid. A) Representative transconjugant colonies on final selection plates (Nm/Gm) following cis-conjugation from E. coli to E. coli in liquid media. Recipient cells have a chromosomally integrated neomycin resistance marker (NmR). B) Representative transconjugant colonies on final selection plates (−HIS) following cis-conjugation from E. coli to S. cerevisiae . All plasmids include elements for selection and replication in yeast (HIS3 gene for complementation of yeast auxotrophy). * colony counts were estimated by counting within a 0.5 × 0.5 cm square, and adjusted to plate size assuming equal distribution.

Article Snippet: Saccharomyces cerevisiae VL6−48 (ATCC MYA-3666: MATα, his3-Δ200, trp1-Δ1, ura3-52, lys2, ade2-101, met14, psi + cir 0 ) was grown in 2x yeast extract peptone dextrose media (YPD) supplemented with adenine hemisulfate (200 μg mL -1 ; Sigma-Aldrich, #A2545, St. Louis, MO, USA) (YPAD) and ampicillin (100 μg mL -1 ; BioBasic, Cat #: AB0028, Canada).

Techniques: Plasmid Preparation, Selection, Conjugation Assay, Marker

Journal: Journal of Skin Cancer

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Subversion of the Anti-Inflammatory Response in Human Skin Cells Reveals Correlates of Latency and Disease Pathogenesis

doi: 10.1155/2014/246076

Figure Lengend Snippet: A link between KSHV latency and the MC1-R signaling axis in skin-derived cell lines. (a) Western blot analysis of phosphorylated NF- κ B p65, MC1-R, and TRP-1 in total cell lysates extracted from MeWo (a) or Mel1700 (b) cells either uninfected (control) or acutely infected with KSHV for 0.3 h, 1 h, 3 h, or 6 h. GAPDH was used as loading control. (c) ImageJ quantitation of the p65 band intensities in (a) and (b) relative to GAPDH controls. (d) Mel1700 cells were infected in 6-well plates with increasing volumes (mL/well) of concentrated supernatant containing infectious KSHV, and total RNA from infected cells was subjected to RT-PCR using primer sets for host anti-inflammatory MC1R, POMC, and SLC7A11. (e) Equal aliquots from the same RNA used in (d) were subjected to RT-PCR analysis for select viral latency and cell growth control genes (i.e., GPCR, LANA, and v-FLIP). (f) Western blot analysis of the melanoma cell marker, Melan A, and anti-inflammatory genes MC1-R, TRP1, and SLC7A11 in total cell lysates of uninfected (−) or chronically infected (+) long-term cultures of MeWo-KSHV and Mel1700-KSHV cells. GAPDH was used as an internal control for both the RT-PCR (e) and western blot assays.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): polyclonal anti-GAPDH (L-20), monoclonal anti-TRP-1 (H-90), anti-Melan A (FL-118), anti-MC1R (H-60), and HRP-labeled goat anti-rat secondary antibody.

Techniques: Derivative Assay, Western Blot, Infection, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Journal of Skin Cancer

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Subversion of the Anti-Inflammatory Response in Human Skin Cells Reveals Correlates of Latency and Disease Pathogenesis

doi: 10.1155/2014/246076

Figure Lengend Snippet: KSHV impairment of the melanogenic response is a virologic correlate of latency in skin cells. Uninfected (−) and rKSHV.219-infected (+) MeWo cells (a) or Mel1700-cells (b) were plated on chamber slides without (control) or with α -MSH and allowed to adhere overnight. Cells were then fixed, permeabilized, and stained with anti-TRP-1 antibody and counterstained with DAPI nuclear stain. Shown are representative 63x magnification (oil immersion) images of representative cells from each field of view. White arrows depict ER/Golgi-associated TRP-1 (apparently present in untreated MeWo and in α -MSH-treated MeWo-KSHV cells), whereas green arrows point to outgrowths of promelanogenic dendritic spines induced by α -MSH in MeWos ((a) bottom left) and in KSHV-infected Mel1700, in which they appear to increase even more profoundly upon treatment with α -MSH (b). For wide-view 20x images of the same treatments, please see Figure S11.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): polyclonal anti-GAPDH (L-20), monoclonal anti-TRP-1 (H-90), anti-Melan A (FL-118), anti-MC1R (H-60), and HRP-labeled goat anti-rat secondary antibody.

Techniques: Infection, Staining

Journal: Cell Death & Disease

Article Title: Arginyltransferase1 drives a mitochondria-dependent program to induce cell death

doi: 10.1038/s41419-025-07917-1

Figure Lengend Snippet: A Growth of yeast cells (W303 strain, WT) carrying either the empty expression vectors (pYES2-URA3 and pBF339-TRP1 vectors), or the galactose-inducible mouse BAX (pBM272-pGAL- BAX -URA3) and yeast Ate1 (pYES2-pGAL- ATE1-GFP -Ura3) in the presence of constitutively expressing Bcl-xL (pBF339-ADH- BCLxL -TRP1) or the vector (pBF339-TRP1) was measured by a serial dilution growth assay on either plate containing 2% glucose or 2% galactose, where the expression of Ate1 or BAX is not induced or induced, respectively. Plates were incubated at 30 °C and images were taken after 3 days. B Representative Western blots displaying changes in the distribution of cytochrome c (Cyc) between mitochondria and cytosol in yeast cell where Ate1-6 × His was induced for expression (“+Ate1”) with 2% galactose in Ura-minus liquid media for 6 h at 30 °C. The separated cytosolic fraction and mitochondrial fraction were compensated by buffer to be equal volume before analysis. Alpha-tubulin and Porin (VDAC) were used as markers to display the purity of cytosolic and mitochondrial fractions, respectively. Pgk1 was used as an additional loading control. The quantification of the blot was based on n = 6; error bars represent S.E.M. C Growth of yeast cells (WT or aif1 Δ) carrying either the empty expression pYES2 vector (“Vector”) or pYES-pGAL1: ATE1-GFP (“+Ate1-GFP”) was measured by a serial dilution growth assay on either plate containing 2% glucose or 2% galactose, where the expression of Ate1 is not induced or induced, respectively. Plates were incubated at 30 °C and images were taken after 3–5 days. D Similar to ( C ), except that WT, aif1 Δ, ate1 Δ¸ yca1 Δ, nuc1 Δ, and dnm11 Δ) yeasts were used. E Similar to ( C ), except that WT and cyc3 Δ yeasts were used. See also Suppl Fig. to see the equal growth rate of the involved mutant yeast strains carrying the empty vector on galactose-containing media compared to the WT strain, if they were not already shown in this figure.

Article Snippet: The inducible BAX-expression vector (pBM272-GAL-BAX-URA), with mouse BAX gene under Gal promoter and a URA3 selection marker, was acquired from Addgene (Plasmid #8770) The constitutively expression vector for Bcl-xL (pBF339-ADH-BCLxL-TRP), with mammalian Bcl-XL gene under ADH1 promoter and a TRP1 selection marker, was acquired from Addgene (Plasmid #8773).

Techniques: Expressing, Plasmid Preparation, Serial Dilution, Growth Assay, Incubation, Western Blot, Control, Mutagenesis

Journal: PLOS ONE

Article Title: A cryptic promoter in the exon of HKR1 drives expression of a truncated form of Hkr1 in Saccharomyces cerevisiae

doi: 10.1371/journal.pone.0314016

Figure Lengend Snippet: (A) Constructs of the plasmids harboring the 2.6-kb fragment of HKR1 ( HKR1 tr ) with its original terminator ( HKR1 ter) introduced into S . cerevisiae A451 cells on the 2-micron multicopy vector, in either the forward or reverse direction relative to the LEU2 marker (pYEHKR1 tr -Fw or pYEHKR1 tr -Rv). The construct of the plasmid pYEHKR1 tr -Rv-ΔMet-1137, harboring HKR1 tr -ΔMet-1137 in the 2-micron multicopy vector, with the ATG codon of Met-1137 replaced with the stop codon TAG. The plasmid vector without the HKR1 tr insert (Vector only) served as a control. The asterisk and the line in red in the pYEHKR1 tr -Rv-ΔMet-1137 construct indicate the position of the ATG codon replaced with the stop codon TAG. (B) Representative photographs of transformants carrying HKR1 tr showing resistance to HM-1 regardless of the direction in which HKR1 tr was inserted into the vector while the transformant carrying the plasmid pYEHKR1 tr -Rv-ΔMet-1137 showed sensitivity to HM-1. H . mrakii cells producing HM-1 were spotted over the lawn of the S . cerevisiae transformants in the center of the plates. The formation of a clear zone around the H . mrakii spot indicates sensitivity to HM-1.

Article Snippet: Throughout this study, Saccharomyces cerevisiae strain A451 (ATCC 200589, MAT α can1 leu2 trp1 ura3 aro7 ) was used for genetic manipulations and as the control strain.

Techniques: Construct, Plasmid Preparation, Marker, Control

Journal: PLOS ONE

Article Title: A cryptic promoter in the exon of HKR1 drives expression of a truncated form of Hkr1 in Saccharomyces cerevisiae

doi: 10.1371/journal.pone.0314016

Figure Lengend Snippet: (A) Diagrams of the reporter construct harboring the 160-bp fragment from the HKR1 exon (from the nucleotide position #3249 to #3408, HKR1 exon) cloned upstream of the reporter gene mUkG1 encoding the fluorescent protein mUkG1 in the multicopy plasmid vector, resulting in pYE-exon160UkG1. The plasmid was then introduced into S . cerevisiae A451 cells. The S . cerevisiae ADH1 promoter (pro ADH1 ) was used as a positive control (pYE-ADHUkG1). Fluorescence of exponentially growing transformants carrying pYE-exon160UkG1 ( HKR1 exon), a promoterless mUkG1 construct (No promoter), or pYE-ADHUkG1 (pro ADH1 ) was observed and photographed under a fluorescence microscope. The scale bar indicates a length of 20 μm. (B) Construct harboring 160-bp HKR1 exon or its 5′ deletion series variants ligated upstream of the reporter gene lacZ . A resulting cassette was inserted into the YCp-based centromeric plasmid vector. β-Galactosidase activity was measured with o -nitrophenyl β-D-galactopyranoside (ONPG) as a substrate and is shown as Miller units . Region 1 is a sequence important for sufficient transcription, and Region 2 is necessary for basal transcription. Eight independent primary transformants were tested for each construct. Mean values are given with standard deviation.

Article Snippet: Throughout this study, Saccharomyces cerevisiae strain A451 (ATCC 200589, MAT α can1 leu2 trp1 ura3 aro7 ) was used for genetic manipulations and as the control strain.

Techniques: Construct, Clone Assay, Plasmid Preparation, Positive Control, Fluorescence, Microscopy, Activity Assay, Sequencing, Standard Deviation

Journal: PLOS ONE

Article Title: A cryptic promoter in the exon of HKR1 drives expression of a truncated form of Hkr1 in Saccharomyces cerevisiae

doi: 10.1371/journal.pone.0314016

Figure Lengend Snippet: (A) Fold-changes in the number of fluorescent-positive cells harboring the constructs illustrated in cultured with NaCl, sorbitol, or HM-1, as determined by flow cytometry. Each construct was introduced into S . cerevisiae A451 cells, and the transformants were grown under control growth conditions or cultured with 250 mM NaCl for 18 h. The number of fluorescent cells upon NaCl treatment among all transformants was normalized to that under control conditions. In addition, the same experiment was carried out with 1 M sorbitol or the 50% lethal dose of HM-1 treatment. The mUkG1 reporter gene driven by the ADH1 promoter (pro ADH1 ) was used as a control. Six independent primary transformants were tested for each construct. Values are means with standard deviation. * p < 0.01 based on the paired samples t-test. (B) Representative fluorescence microscope photographs of cells carrying the mUkG1 gene driven by the exonic HKR1 promoter sequence 4, 5, or 6 treated with 250 mM NaCl (+NaCl), 1 M sorbitol (+Sorbitol), or maintained under control conditions (No treatment). The ADH1 promoter (pro ADH1 ) was used as a control. The scale bar indicates a length of 10 μm.

Article Snippet: Throughout this study, Saccharomyces cerevisiae strain A451 (ATCC 200589, MAT α can1 leu2 trp1 ura3 aro7 ) was used for genetic manipulations and as the control strain.

Techniques: Construct, Cell Culture, Flow Cytometry, Control, Standard Deviation, Fluorescence, Microscopy, Sequencing

Journal: PLOS ONE

Article Title: A cryptic promoter in the exon of HKR1 drives expression of a truncated form of Hkr1 in Saccharomyces cerevisiae

doi: 10.1371/journal.pone.0314016

Figure Lengend Snippet: Diagrams of S . cerevisiae Hkr1, human MUC1, and C . albicans Msb2. Met-1137 of Hkr1 may serve as an internal translation initiation site following transcription from the exonic HKR1 promoter ( HKR1 exon) to produce the C-terminal portion of the protein, Hkr1 tr , which is assumed to participate as a signal transduction messenger. Proteolytic cleavage of MUC1 and Msb2 produces short C-terminal fragments. For the domain names, please see .

Article Snippet: Throughout this study, Saccharomyces cerevisiae strain A451 (ATCC 200589, MAT α can1 leu2 trp1 ura3 aro7 ) was used for genetic manipulations and as the control strain.

Techniques: Transduction