Notes: These authors used the Maxwell^® 16 System to isolate genomic DNA from whole blood and normal colonic tissue samples. The DNA was used in genotype analysis, testing for wildtype and mutant KRAS genes, and for various EGFR-related polymorphisms. The results were used in a research study testing the relationship between various genotypes and response to different treatment regimens. (3961)

Notes: The human sepiapterin reductase (SPR) gene has been mapped at the PARK3 locus, which is related to the onset of Parkinson disease. The silkworm Bombyx mori body color mutant lemon (lem) has been associated with a lack of SPR activity; lem lethal is a homozygous lethal allele of lem. Genetic linkage analysis was performed with normal silkworm strain p50T, lem strain l70, and lem^l strain a65 to more closely examine the relationship with SPR. DNA from the F1 and F2 crosses were isolated using the Wizard^® SV 96 Genomic DNA Purification System and the genome sequenced. (4017)

Notes: The authors examined how cytotoxicity differed between cancer cell lines treated with drugs that induced either apoptosis or paraptosis, non-apoptotic cell death. HT1080 fibrosarcoma epithelial cells were seeded in 10cm dishes and grown until 40% confluent before transfection with 15µl of FuGENE® 6 Transfection reagent and 4.5µg of wild type or mutant vector + 0.5µg of GFP vector for a 3:1 reagent-to-DNA ratio. After an eight-hour incubation, the cells were trypsinized, seeded into two 24-well plates at 100,000 cells per well), and treated with drugs 24 hours later. Sixteen hours after drug treatment, one set of plates were assessed for viability, the other, cell death. (4381)

Notes: The authors attempted to isolate DNA from samples tested with Hemastix® reagent strips, which are commonly used to test for the presence of blood, using the DNA IQ™ System. Yields from samples that had been tested with Hemastix® strips were dramatically lower than those from untested samples. The experiments suggested that 3,3´,5,5´tetramethylbenzidine (TMB) irreversibly binds to the magnetic DNA IQ™ Resin to prevent DNA recovery. To circumvent this, the authors implemented an effective indirect screening method, where the unknown sample was rubbed with dry filter paper, which was then tested with a prewetted Hemastix® strip. (4035)

Notes: To identify molecules that affect metastasis signaling pathways downstream of HER2-Y1248 phosphorylation, suppression subtractive hybridization assays (SSH) were
performed using MDA-MB-468 cells overexpressing HER2 and control MDA-MB-468 cells expressing HER2 without the Y1248 phosphorylation site. Reactions were cloned
using a T-vector system, transformed and plated. Positive clones from each assay were selected and grown overnight in 2ml deep-well plates. The Wizard^® Magnesil^® Plasmid Purification System was used to isolate plasmids for BigDye™ sequencing. (4134)

Notes: These authors used the Maxwell^® 16 System to isolate DNA from frozen whole blood samples infected with Plasmodium falciparum. The isolated DNA was used in a qPCR-based SNP genotyping assay that sought to uniquely identify the parasites based on their SNP marker profile. (3962)

Notes: In this paper, researchers were looking for efficient chromophores for singlet oxygen generation used for chromophore-assisted light inactivation (CALI) of proteins. The HaloTag^® protein and firefly luciferase were used to test how well the chromophores performed in crude extracts and living cells. The expression vector for an epitope-tagged Luciferase-HTP protein, 3X Flag-Luc-HTP-Myc, was constructed using firefly luciferase amplified from the pGL3-Basic Vector and HaloTag^® (HTP) amplified from the pHT2 Vector. The fusion protein was tested for labeling with a HaloTag^® biotin ligand by transfecting HeLa cells with 8μg of 3X Flag-Luc-HTP-Myc plasmid and 80ng of pRL-SV40 Vector. After transfection, cells were lysed with Passive Lysis Buffer and 2μl of HeLa cell lysate was diluted in 48μl of PBS + BSA and incubated for 30 minutes at room temperature with increasing concentrations of a biotin-HT ligand. The samples then were incubated with streptavidin-agarose for 30 minutes at room temperature, centrifuged and the luciferase activity of 20μl of supernatant was measured using the Dual-Luciferase^® Reporter Assay System. The fusion protein was also tested using two chromophore ligands, ruthenium(II)tris-bipyridyl (Ru-HaloTag^®[HT]) and fluoroscein-HT at a concentration of 100nM, and both were successful as measured by the Dual-Luciferase^® Reporter Assay System. An in vivo CALI was performed by transfecting HeLa cells with 100ng of 3X Flag-HTP-Luc-Myc plasmid and 1ng of pRL-SV40 Vector for 15 hours, and treating the cells with Ru-HT or F-HT for 3 hours. The cells were then irradiated for 30 minutes, placed in the dark for 30 minutes, then the cells were lysed and analyzed with the DLR Assay. (3954)

Notes: The authors of this study investigated the mechanism of action of syringolin A (SylA), which is secreted by virulent strains of the plant pathogen Pseudomonas syringae. They show that SylA inhibits all three activities of the proteasome in vitro. They also used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to show that SylA inhibits the chymotrypsin-like activity of the proteasome in SK-N-HS neuroblastoma cells. (3846)

Notes: To examine the role of the membrane form of macrophage colony–stimulating factor(mM-CSF) in the hematopoietic system, RT-PCR was used amplify the cDNA of human mM-CSF from J6-1 cells, a human leukemia cell line. The PCR product was digested and cloned into the pTargeT™ Mammalian Expression Vector. After sequencing to verify the sequence, the construct and empty pTargeT™ Mammalian Expression Vector were purified and used to transfect Namalwa and Ramos cells, human Burkitt’s lymphoma cell lines, in 24-well plates. The transfected cells were then selected for stable expression of the transfected vector using 1.4 mg/ml G418. Expression of mM-CSF and neomycin (in the empty vector) was confirmed using RT-PCR. These cells were injected into mice and the oncogenicity of the cells determined using antibody staining of tissues. (3989)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to explore the dimerization of epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases without ligand. The intracellular domains of EGFR, ErbB2, ErbB3 and ErbB4 were amplified and cloned into both the pACT and pBIND Vectors. Transfection into NIH3T3 cells in 12-well plates occurred with 0.3 μg of pG5luc Vector (the reporter vector), 0.2 μg of pACT Vector or an equimolar amount of pACT construct, and 0.1 μg of pBIND Vector or an equimolar amount of pBIND construct. After 24 hours, the cells were lysed and luciferase activity assessed using the Dual-Luciferase® Reporter Assay System. (3993)

Notes: The authors examined protein interactions between splice variants of the SH3A domain of intersection 1 (ITSN1) and the main ITSN1 protein partners using protein pull-down assays. In one set of pull-down assays, SH3A splice variants were expressed as polyhistidine-tagged proteins, and the proline-rich domain of dynamin 1, a known ITSN1 protein partner, was expressed as a glutathione-S-transferase (GST) fusion protein. In a second set of experiments, the SH3A domain variants were expressed as GST-fusion proteins, immobilized, then used to capture endogenous dynamin 1, SOS1, c-Cbl and Cbl-b from cell lysates. Recombinant GST-fusion proteins were purified using glutathione-Sepharose^® 4B or the HisLink™ Protein Purification Resin. Based on the data, the authors concluded that alternative splicing of ITSN1 can change the binding properties and its protein partners. (3950)

Notes: These authors amplified and characterized a putative epoxide hydrolase gene from the jewel wasp Nasonia vitripennis. PCR fragments were amplified from genomic DNA, purified from gels using the Wizard^® SV Gel and PCR Clean Up System and then subcloned into the pGEM^®-T Easy Vector. The plasmid DNA was purified using the PureYield™ Midiprep System. Linearized plasmids were used for in vitro transcription of RNA for use in RNA interference experiments. (3903)

Notes: The authors developed a new human prostate cancer cell line, CWR22Pc, that is both androgen-dependent and able to produce tumors in dihydrotestosterone-supplemented nude mice. To confirm that CWR22Pc cells are derived from primary CWR22 human prostate xenograft tumors, the authors performed genotyping at 8 STR loci and amelogenin using the PowerPlex^® 1.2 System. DNA purification from the cell line and original tumor samples was performed using the Wizard^® Genomic DNA Purification Kit. (4041)

Notes: The authors wanted to examine the role of plasma membrane protein Na+/H+ exchanger isoform 1 (NHE1) in apoptosis. API cells, a NHE1-deficient Chinese hamster ovary cell line, was cotransfected with wild-type NHE1 or mutant NHE1 constructs and destabilized yellow fluorescent protein (YFP). Cells were plated at a density of 1 × 10⁴ cells/well in a 96-well plate with or without FBS. To induce apoptosis in the cells, serum was withdrawn for 24 hours. The ratio of dead-to-live cells was measured using the MultiTox-Fluor Multiplex Cytotoxicity Assay. Cell death was also determined by examining the loss of YFP fluorescence under a microscope. (3937)

Notes: The authors of this study investigated the role of the genes CLV1 and CLV3 stem cell renewal and differentiation in self-renewing shoot apical meristem (SAM) in Arabidopsis. CLV1 encodes a receptor kinase that is a negative regulator of cell growth, and therefore cannot be overexpressed in plant cells. CLV3, which encodes a protein that is processed to a 12-amino acid peptide, is believed to be a ligand for CLV1. In this study, the authors made a construct in which the CLV1 kinase domain was replaced with the HaloTag™ protein and expressed the fusion protein in tobacco BY-2 cells. The fusion protein was detected using the HaloTag™ TMR ligand. They next incubated membrane extracts from wildtype and transgenic BY-2 cells (expressing the HaloTag™-CLV1 fusion) with tritium-labeled CLV3. The transgenic cells showed significantly higher CLV3 binding than wildtype, and the authors show that CLV3 specifically labels a 130-kd band that corresponds to the HaloTag™-CLV1 fusion protein, indicating that CLV3 binds the ectodomain of CLV1. (3763)

Notes: These authors showed that the stomatal initiating factor SPEECHLESSS (SPCH) is a substrate of the kinases MPK3 and MPK6 in vitro, that specific phosphorylation sites on SPCH regulate its activity in vivo, and that components of the stomatal development signaling network modulate SPCH. As part of the study, coomassie-stained gel bands containing the SPCH protein were excised and digested using trypsin and ProteaseMax Surfactant. Mass spectrometry was used to assess phosphorylation at several functionally critical sites. (4087)

Notes: The authors wanted to understand the role of ASAP3, an Arf GTPase-activating protein in cancer cells. FLAG- or hemagglutinin-tagged ASAP3 and GAP-deficient mutants of ASAP3 were cloned in the pCI Mammalian Expression Vector. The constructs were transfected into cells and used in immunofluorescence and Western blotting experiments. (3987)

Notes: The authors examined the different ATP sensitivities of inositol (1,4,5)-triphosphate receptor (InsP₃R) isoforms InsP₃R1, InsP₃R2 and InsP₃R3. To compare the ATP-binding properties of InsP₃R2 and InsP₃R3, nucleotide sequences encompassing the ATP-binding domains were amplified by PCR and cloned into the pFN2A (GST) Flexi^® Vector. The ATP-binding sites were expressed as glutathione-S-transferase (GST) fusion proteins in BL21(DE3)pLysS cells. Fusion proteins were purified, and the GST tag removed by cleavage with tobacco etch virus (TEV) protease. Purified proteins were then used in a fluorescent ATP-binding assay. (3901)

Notes: The authors identified the N-terminal Src homology 3 (SH3) domain-binding motif of Crk and CrkL as the preferred binding partner of nonstructural protein 1 (NS1), an important virulence factor of the influenza A virus. Interaction of NS1 with Crk, CrkL and other SH3 domain-containing proteins p85, p85β and Eps8L1 was investigated by protein pull-down assays. Expression constructs for biotinylated Crk, CrkL, p85, p85β and Eps8L1 were created by amplifying the 123 amino acid biotin acceptor domain from Propionibacterium shermanii from the PinPoint Xa-T Vector and inserting it upstream of the protein-coding sequences in a pGEX vector derivative. These constructs and a construct encoding Myc-tagged NS1 were transfecting into 293FT cells, and the biotinylated proteins were immobilized from the cell lysate using TetraLink™ Tetrameric Avidin Resin. Any Myc-NS1 that bound to the immobilized protein was detected using Western Blot analysis and an anti-Myc antibody. The authors also investigated the ability of wildtype NS1 or NS1 mutants to inhibit interferon-induced gene expression. A reporter plasmid was created by cloning an interferon-stimulated response element upstream of a minimal thymidine kinase promoter driving firefly luciferase expression. A vector containing Renilla luciferase was used as a control to normalize transfection efficiency. Huh-7 cells were cotransfected with the firefly and Renilla luciferase reporter constructs (0.2µg and 5ng, respectively), treated with interferon-β and lysed using the Passive Lysis Buffer. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System. (3803)

Notes: The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase. (3926)

Notes: The authors investigated gene transcription within periplasmic flagella of Borrelia burgdorferi, which are composed of a basal body, hook and filament, to determine if hook formation influences flagellin gene expression. They used insertion mutagenesis to construct strains with mutated versions of the hook structural gene flgE that were disrupted by a kanamycin-resistance cassette. The flgE gene and antibiotic-resistance cassette were amplified by PCR and cloned into the pGEM^®-T Vector. To assess the effect of flgE disruption on the transcription of filament proteins FlaA and FlaB, quantitative RT-PCR was performed; enolase was used as an internal control. Negative controls without the reverse transcriptase were included for each sample. (3885)

Notes: To determine whether oocytes are able to select X-bearing or Y-bearing spermatozoa, the authors performed in vitro fertilization of bovine oocytes with X-sorted semen, Y-sorted semen, a mixture of X- and Y-sorted semen, and unsorted semen. The gender of the resulting embryos was determined by amplifying two DNA targets: a Y chromosome-specific target for gender assignment and a bovine-specific satellite sequence as a control. PCRs were performed using GoTaq^® Flexi DNA Polymerase (1 unit per 25µl reaction), and amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. (3881)

Notes: The Plautia stali virus contains two open reading frames and includes a 5´ internal ribosome entry site (IRES) and an intergenic IRES region. These authors showed that the 5´ IRES was functional and initiated translation in insect cell lysate, but not in rabbit reticulocyte lysate or wheat germ extract. The efficiency of translation mediated by the 5´ IRES region was tested with and without cap analogue using various firefly and Renilla luciferase reporter constructs. They also used deletion mutants to identify the specific regions required for translation initiation. (3942)

Notes: These authors investigated the amo operon of the marine ammonia oxidizer Nitrosococcus oceani. The bacteria were grown at 30°C for 3 weeks in 200-400ml batch cultures in artificial seawater in the dark without shaking. Genomic DNA was isolated from cells in stationary phase using the Wizard^® Genomic DNA Purification Kit. The isolated DNA was then used for PCR analysis. (3740)

Notes: The authors of this study investigated the role of Lyn, a Src-family tyrosine kinase, in regulating the formation of the phagosome in alveolar macrophages in response to Psuedomonas aeruginosa (PA) infection. The Kinase-Glo^® Assay was used to assess Lyn activity, using acid-denatured enolase as the substrate. The authors found that Lyn kinase activity was increased following infection with PA. (3929)