Notes: In this paper, the researchers describe and demonstrate a new method for creating precise genome modifications in Saccharomyces cerevisiae. The mutagenic inverted repeat assisted genome engineering (MIRAGE) was tested in S. cerevisiae W303a by deleting gal7 as well as point and frameshift mutations. Genomic DNA was isolated using the Wizard^® Genomic DNA Purification Kit, amplified and modifications verified by gel analysis or DNA sequencing. (4014)

Notes: The authors of this study used the HRE-CRE-luciferase reporter cell line (Glo-Response™ Cells) to test HSV constructs for activity. Cells were infected with HSV-CREB, HSV-mCREB (dominant negative) or HSV-LacZ control vector. Comparisons indicated that cells transfected with HSV-CREB showed increase in CRE-mediated activity, while those transfected with HSV-mCREB showed attenuation of CRE-mediated cellular activity. (3956)

Notes: Human boule (BOLL) gene was subcloned into the HaloTag^® pHT2 Vector and transformed into JM109 and HeLa cells. (4060)

Notes: To examine the prevalence of Campylobacter species in asymptomatic carriers that can pass the bacteria onto humans, rectal swabs were collected from 147 dogs and 35 cats in Ireland and cultured on various diagnostic plates. The Wizard^® Genomic DNA Purification System was used to isolate DNA from any suspect Campylobacter cultures. The purified DNA was used in multiplex PCR and RFLP to determine which species of Campylobacter was present. (4016)

Notes: In this paper, the role of prostaglandin E2 (PGE2) stimulation of integrin-linked kinase (ILK) in human lung carcinoma was explored. Mutations of Sp1 and NF-κB cis-acting elements in an ILK promoter-pGL3-Basic Vector construct were created using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The mutations were confirmed via sequencing. Human non–small cell lung carcinoma (NSCLC) cells were plated at a density of 5 × 10⁵ cells per well in six-well plates and transfected with 2µg of ILK promoter reporter vectors with or without 0.2µg of the phRL-TK Renilla Luciferase Reporter Vector. After 24 hours, the transfected cells were exposed to PGE2 and the cells lysed for assessment using the Dual-Luciferase^® Reporter Assay System. NSCLC cells were transfected with inactive (ILK-S343A) and superactive ILK (ILK-S343D) cDNA, incubated for 24 hours, treated with or without exogenous PGE2 or with an Sp1 inhibitor for 2 hours. The numbers of viable cells were measured using the CellTiter-Glo^® Luminescent Cell Viability Assay. (4026)

Notes: These authors compared 6 different detection strategies for protein-protein interactions on protein arrays. They expressed HaloTag^® labeled bait proteins in a cell-free expression system, and captured these bait proteins onto coated glass slides using the HaloLink™ Array System. They then compared detection strategies using prey proteins labeled as follows: 1)³⁵S methionine, 2) fluorescence (BODIPY-FL) and 3) biotin labeling of lysine residues using modified Lys tRNA, 4) chemical labeling after expression, 5) HaloTag^® fusion, and 6) N-terminal FLAG tag. The authors evaluated signal:background ratios, adaptability to high-throughput screening, and ease of use. (3999)

Notes: The authors used the Halotag^® Labeling Technology in pulse-chase experiments. They pulse labeled proteins in cultured mammalian cells. Using the HaloTag^® Technology, they were able to monitor the degradation of the labeled protein, Smad1, that was induced by coexpression of Smurf1. They conclude that the HaloTag^® Technology could be used to monitor the regulation of SMAD1 degradation. (4055)

Notes: In this paper, the role of the KAE1/osgep/ygjD gene family, a universally conserved gene set without an assigned function, was investigated in yeast and Caenorhabditis elegans. Genomic DNA was isolated from Saccharomyces cerevisiae using the Wizard^® SV Genomic DNA Purification System. This purified DNA was then used in PCR. (4065)

Notes: This study examined the expression levels of each UGT isoform in human liver and evaluated the variability between individuals. Total RNA from appropriate human tissues or various cell lines was used for RT-PCR of various human UDP-glucuronosyltransferases (UGT) cDNAs. The amplimers were cloned into the pTARGET™ Mammalian Expression Vector and verified by sequencing. The UGT vectors were linearized by restriction enzyme digestion and used for standards in real-time RT-PCR analysis. (4034)

Notes: The association of thiopurines (anticancer drugs) with acute skin sensitivity to ultraviolet A (UVA) radiation and a high risk of skin cancer was tested using six human fibroblast and lymphoid cell lines. The thiopurine 6-thioguanine (6-TG) was added at 0.8 or 0.6mM to each of six cell lines and incubated for 48 hours to ensure incorporation. DNA and RNA were extracted and 40µg of nucleic acid were digested to nucleosides, separated by HPLC, and the 6-TG 20-deoxy and ribonucleosides quantified by absorbance at 342 nm. The same DNA isolation and digestion method was used when the cells were treated with 1µM of 6-TG and then irradiated with 5 kJ/m² UVA after 48 hours. The Wizard^® Genomic DNA Purification Kit was used for DNA extraction. (4013)

Notes: The authors examined the role of p53 in modulating dUTPase promoter activity. Base substitution mutations of Sp1- and E2F-binding sites in the dUTPase promoter were performed using the GeneEditor™ in vitro Site-Directed Mutagenesis System. Each mutant was confirmed by DNA sequencing. To determine growth inhibition, HCT116 human colon cancer cells were seeded in 96-well plates at 3 × 10³ cells/well and treated with 5-fluorouracil (5-FU), fluorodeoxyuridine (FUdR), oxaliplatin or in combination. After 72 hours, the CellTiter^® 96 AQ_ueous One Solution was dispensed into each well and absorbance measured. RNA was isolated from HCT116 p53+/+ and HCT116 p53-/- cells. cDNA was reverse transcribed from 200ng total RNA followed by multiplex qPCR using the Plexor™ qPCR System to amplify dUTPase, thymidylate synthase and GAPDH, a housekeeping gene. The 1.2 kb region of the dUTPase promoter upstream of the transcriptional start site was amplified by PCR and the fragment cloned into the pGL3-Basic Vector. Truncated promoters were also generated by PCR and cloned into the same vector. Drosophila SL-2 cells and HCT116 cell lines were seeded in a 24-well plate and transfected with dUTPase pGL3 promoter constructs or with pCI-Neo:p53WT, pCI-Neo:p53MUT and the empty pCI-neo Mammalian Expression Vector; all transfections included the pRL-TK Vector at a ratio of 1:10. After six hours, the cells were incubated in either fresh medium or medium containing a cytotoxic agent at the appropriate concentration. Thirty hours later, the cells were lysed, quantitated by Western blotting and 20µl of lysate analyzed with the Dual-Luciferase^® Reporter Assay System. Electrophoretic mobility shift analyses (EMSA) were performed using –64 to –91 of the dUTPase-nuclear isoform transcriptional start site in the Gel Shift Assay System. (4031)

Notes: These authors expressed recombinant dynactin and dynein in Saccharomyces cerevisiae and investigated their interactions in motility assays. They created a c-terminal Halotag-Dynactin fusion, and were able to site-specifically label the fusion protein with the fluorescent dye tetramethylrhodamine (TMR). They studied the effect of the purified dynactin fusion protein on the motility of dynein complexes using total internal reflection fluorescence microscopy. Dynactin alone did not interact with microtubules. However, when coincubated with recombinant dynein, the TMR-labeled dynactin moved processively along microtubules. The authors then used truncation mutants of dynactin to identify the region of the dynactin molecule required for localization and enhanced processivity of dynein. (3960)

Notes: Mutations in the extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) cause skeletal dysplasia. This study evaluated the efficacy of a ribozyme (a short catalytic RNA oligonucleotide) to knock down COMP expression. As part of the study, COS7 cells were transfected with plasmids that expressed mutant or wild-type COMP, as well as with a ribozyme targeting COMP. Transfection conditions were as follows: Cells were grown to 80%–90% confluency in 6-well plates, then transfected with plasmid DNA using a 3:2 ratio of FuGENE 6 reagent:DNA. Ribozyme treatment reduced expression of COMP mRNA in the transfected cells. (4357)

Notes: To try to understand the mechanism by which certain strains of Shiga-toxigenic E.coli adhere to host intestinal epithelium, the authors characterized the novel autotransporter protein Sab. Expression levels and protein localization were examined by Western blot analysis and enzyme-linked immunosorbent assay. The mouse anti-Sab antibody used in these studies was raised against an N-terminal His₆-Sab fusion protein purified using the HisLink™ Resin. (4102)

Notes: In this study, FuGENE® 6 reagent was used to transiently transfect Jurkat T lymphocyte cells and HEK 293 adenovirus-transformed  HEK 293 cells with 0.25µg DNA at a 6:1 ratio of FuGENE:DNA. The Jurkat cells were plated at 1 × 10e6 cells/plate and the HEK cells were plated at 5 × 10e5 cells/plate. (4375)

Notes: The authors developed a single nucleotide polymorphism (SNP)-based assay to distinguish four Sclerotinia species. The assay consisted of amplification of a 300bp intergenic spacer and portions of the calmodulin and ras genes, followed by Southern blot using species-specific, radiolabeled probes. Amplifications were performed using the GoTaq® Colorless Master Mix, 0.2µM of each primer and 10–20ng of template DNA. (4098)

Notes: The authors characterized differential expression of several carcinogen metabolism genes in human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissues in smokers, former smokers and people who have never smoked. They combined laser capture microdissection (LCM) and quantitative RT-PCR. RNA was isolated from paired microdissected malignant and nonmalignant lung tissue, 100ng of total RNA was reverse transcribed in a 20µl reaction, then 1µl of cDNA was amplified by real-time PCR using an ABI PRISM® 7500HT sequence detection system, GoTaq® Flexi DNA Polymerase and gene-specific primers. The expression level for each gene was normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results showed that expression of cytochrome P450 1B1 and glutathione-S-transferase P1 in AC, but not BEC, tissue was strongly associated with exposure to tobacco. (4095)

Notes: In this study, the effect of solar disinfection on Shigella flexneri and Salmonella typhimurium in drinking water samples was evaluated. A variety of viability indicators were used to investigate the effectiveness of the disinfection method, including measurement of cellular ATP levels. The BacTiter-Glo Assay was used for ATP detection. (4104)

Notes: This study examined the effect of lactic acid bacteria on gene expression of Staphylococcus aureus in mixed cultures. Total RNA was isolated from a mixed culture of S. aureus and Lactococcus lactis, labeled with 5µg of RNA with Cy^®3- or Cy^®5-dCTP using the ChipShot™ Labeling and Cleanup System, the cDNA was dried and stored at –20°C. Genomic DNA was isolated from S. aureus and L. lactis cultures, digested with HinPII, purified, and 400ng of the digested gDNA labeled with Cy^®3- or Cy^®5-dCTP and the Prime-a-Gene^® Labeling System. The level of gene expression was assessed using a S. aureus microarray. (4068)

Notes: The authors created a triple mutant of Regulator of G-Protein Signaling Protein 2 (RGS2) to characterize the structural features responsible for its selectivity in binding to the Gα_q or Gα_i/o subunits of GTPase-accelerating protein (GAP). The RGS2 enhances the termination of G-protein coupled signaling by enhancing GAP. RGS proteins are considered key modulators of G Protein-Coupled Receptor (GPCR) signaling based on their ability to accelerate GTP hydrolysis. The GloSensor™ cAMP assay was used to assess the level of GPCR activity and indicate which structural determinants of RGS2 affect binding to Gα subunits of GAP.

HEK293T cells were transiently co-transfected with expression vectors for the GloSensor™ cAMP biosensor and the G_i-coupled dopamine D2-receptor with empty vector, wild type RGS2, or the RGS2(triple) mutant. Treatment of transfected cells with forskolin produced an increase in luminescence from the cAMP sensor, reflecting direct activation of adenylyl cyclase by forskolin. Quinpirole, a dopamine D2 receptor agonist, produced a dose-dependent inhibition of cAMP production. Inhibition of forskolin-stimulated cAMP production was assessed after activation of the D2 receptor with various concentrations of quinpirole to compare IC₅₀ values for the empty vector, wild type RGS2 and triple mutant RGS2. Cellular expression of the triple mutant resulted in a significantly higher IC₅₀ for quinpirole (762nM versus 18 nM for empty vector), indicating that the three point mutations weaken Gα_i subunit binding responsible for enhanced GTPase activity.
(4148)

Notes: To better understand which lysine residues in monocarboxylate transporter 1 (MCT1) were involved with binding di-isothiocyanostilbene disulfonate (DIDS), a transport inhibitor, and crosslinking to embigin, rat MCT1 and embigin were subcloned into the pCI-neo Mammalian Expression Vector via the EcoRI site. The vector was then subjected to site-directed mutatgenesis of each of six lysines either singly or in combination, and the effect on the binding activity or inhibition was tested. (4069)

Notes: The authors were wanted to study the upstream open reading frame 2 (uORF2) of the 5’ leader of bZIP11 mRNA, which has a role in sucrose regulation. The whole 5’ leader fragment of bZIP11 was subcloned into the pALTER^® Vector and amino acid substitutions were introduced using the Altered Sites^® II in vitro Mutagenesis System. The pGEM^®-T Easy Vector was used to clone two PCR fragments that were then subcloned using restriction enzymes to create a fusion of uORF2 to a different 5’ leader. Arabidopsis seedlings were transformed via particle bombardment. 20mg of plant tissue was ground in Passive Lysis Buffer, centrifuged, and 20µl of the supernatant was assessed for reporter gene expression using the Dual-Luciferase^® Reporter Assay System. (4023)

Notes: The authors used a systems biology approach to examine the transcriptional regulation of water channel aquaporin-2 (AQP2). A 1,511bp fragment from the 5´-flanking region of the mouse AQP2 gene was amplified from mouse tail DNA and cloned into the pGEM^®-T Vector. This construct was then digested with two restriction enzymes and cloned into a double-digested pGL3-Basic Vector. Full length Elf3, Elf5 and Ehf cDNA, members of the ETS family of transcriptional regulators, were amplified, sequenced and ligated into the pTARGET™ Mammalian Expression Vector. LLCPK1 cells were cotransfected with AQP2-pGL3 reporter and one of the pTARGET™ constructs. Reporter activity was measured using 20µl of cell lysate in a luciferase assay. (4033)

Notes: MCF7 cells were grown in a 35mm dish to 80% confluence. Four micrograms pCMV6-XL5/RNF146 or pCMV6-XL5 (Origen Technologies Inc.) was mixed with 8 μL FuGene HD transfection reagent and added to the cultured cells. After 48 or 72 h transfection, the cells were washed with 1X PBS and lysed. The lysates were centrifuged at 13,200 rpm and the supernatants used in Western blot analysis. (4424)

Notes: These authors examined conformation of DNA bound to the DNA-gate of Bacillus subtilis gyrase as well as the conformation of the DNA-gate itself. Negatively supercoiled pUC18 plasmid was purified using the PureYield™ Plasmid Midiprep System and used in single-molecule FRET experiments. (4061)