Promega Corporation Research Articles

Mitochondrial DNA insertions in the nuclear horse genome

The insertion of mitochondrial DNA in the nuclear genome generates numts, nuclear sequences of mitochondrial origin. In the horse reference genome, we identified 82 numts and showed that the entire horse mitochondrial DNA is represented as numts without gross bias. Numts were inserted in the horse nuclear genome at random sites and were probably generated during the repair of DNA double-strand breaks. We then analysed 12 numt loci in 20 unrelated horses and found that null alleles, lacking the mitochondrial DNA insertion, were present at six of these loci. At some loci, the null allele is prevalent in the sample analysed, suggesting that, in the horse population, the number of numt loci may be higher than 82 present in the reference genome. Contrary to humans, the insertion polymorphism of numts is extremely frequent in the horse population, supporting the hypothesis that the genome of this species is in a rapidly evolving state.

Genetic variability of 15 autosomal STR loci in Russian populations

Allele frequencies for 15 STRs (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, Penta D, Penta E, THO1, TPOX, and vWA) in the PowerPlex 16 System (Promega Corporation) were assessed in 386 individuals from five Russian urban populations. No significant between-population differences in frequencies and molecular variance of 15 microsatellites were revealed. For all 15 loci, the combined matching probability is 3.19 × 10 −18 and the power of exclusion is 99.99989%.

High‐Throughput Assessment of Mammalian Cell Viability by Determination of Adenosine Triphosphate Levels

There are many assays available for high-throughput assessment of mammalian cell viability or cytotoxicity. Approaches include measurement of metabolic capacity, intracellular adenosine triphosphate (ATP) levels, induction of apoptosis, intracellular esterase and protease activity, and cellular membrane integrity. This unit provides an in-depth protocol for measurement of cellular ATP levels as a readout of mammalian cell viability, using the CellTiter-Glo assay from Promega Corporation. A comparison of the key parameters (sensitivity, speed, and cost) for this and other common high-throughput viability assays is also presented. Curr. Protoc. Chem. Biol. 2:153-161 © 2010 by John Wiley & Sons, Inc.

Effect of histone deacetylase inhibitor (HDACI) PCI-24781 on chemotherapy-induced apoptosis in multidrug-resistant sarcoma cell lines.

10089 Background: The antitumor activity of histone deacetylase inhibitors (HDACI) on multidrug-resistant sarcoma cell lines has never been previously described. Methods: We treated chemotherapy-resistant osteosarcoma and chondrosarcoma cell lines with PCI-24781 in combination with chemotherapeutic agents. MTT assay was used to measure relative inhibition of cell growth in each of the conditions. Western blot analysis was used to determine changes in RAD51 and GADD45α levels as a result of treatment with PCI-24781. Caspase activity was measured with the Apo-One Homogeneous Caspase 3/7 assay kit (Promega Corporation). Results: Four multidrug-resistant sarcoma cell lines treated with HDACI PCI-24781 resulted in dose-dependent accumulation of acetylated histones, p21 and PARP cleavage products. Growth of these cell lines was inhibited by single agent PCI-24781 at IC50 of 0.43 to 2.7 μM. When we looked for synergy of PCI-24781 with chemotherapeutic agents, we found that PCI-24781 reverses drug resistance in all four multidrug resistant sarcoma cell lines and synergizes with chemotherapeutic agents to enhance caspase-3/7 activity. Expression of RAD51 (a marker for DNA double-strand break repair) was inhibited and the expression of GADD45α (a marker for growth arrest and DNA-damage) was induced by PCI-24781 in multidrug resistant sarcoma cell lines. Conclusions: HDACI PCI-24781 synergizes with chemotherapeutic drugs to induce apoptosis and reverses drug resistance in multidrug-resistant sarcoma cell lines. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Pharmacyclics

Concordance and population studies along with stutter and peak height ratio analysis for the PowerPlex ® ESX 17 and ESI 17 Systems

The PowerPlex ® ESX 17 and ESI 17 Systems for short tandem repeat (STR) amplification were developed by the Promega Corporation to meet the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling (EDNAP) Group recommendations for increasing the number of STR loci included in the European Standard Set (ESS). The PowerPlex ESX 17 and ESI 17 Systems utilize different PCR primer combinations to co-amplify the following 17 loci: D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, vWA, SE33, and the sex-typing locus amelogenin. A total of 1443 U.S. population samples were evaluated with pre-commercialization versions of both kits. Stutter and heterozygote peak height ratios have been used to characterize kit performance. Typing results have been used to estimate the match probabilities provided by the chosen loci as well as in concordance studies. Full concordance between the typing results for the two kits was observed in 99.994% (49,055 out of 49,062) STR allele calls compared. All genotyping discrepancies were confirmed by DNA sequence analysis. As a result of these comparisons, a second forward primer for the D22S1045 locus has been added to the PowerPlex ESX 17 System to address a primer binding site mutation and the D1S1656 locus reverse primer in the PowerPlex ESI 17 System was modified to eliminate an amplification-efficiency reducing primer dimer.

Functional significance of nerve growth factor and its receptor (TrkA) in inflammatory arthritis

We read the article by Barthel and colleagues [1] in this is sue of Arthritis Research & Th erapy with great interest. Th ey reported that neurotrophin receptors/ligands and specifi cally nerve growth factor (NGF) and NGF receptor (NGF-R) (TrkA and p75) are expressed in synovial fl uid (SF) cells and synovial tissue (ST) of patients with rheuma toid arthritis (RA) or spondyloarthritis (SpA). Th e authors also looked for the cellular source of NGF and demonstrated that the T cells and monocytes/macrophages derived from SF of patients with RA/SpA are enriched with NGF. Furthermore, they reported that STderived fi broblast-like synoviocytes (FLSs) did not produce NGF in vitro. In a recent publication, we observed similarly that NGF levels in SF were signifi cantly higher in patients with psoriatic arthritis (PsA) (365.5 ± 85.2 pg/ mL) or RA (120 ± 35 pg/mL) than in patients with osteoarthritis (OA) (30 ± 6 pg/mL) [2]. However, in regard to the source of NGF, we observed that FLSs produced a signifi cant amount of NGF. Here, we would like to share our observations about the NGF/TrkA system in human FLSs and its function. Synovial biopsies from patients with meniscal injury without any other joint diseases or PsA, OA, or RA were collected. FLSs were isolated and examined for NGF/ NGF-R expression in accordance with our standardized protocols [2]. Using a highly sensitive enzyme-linked immuno sorbent assay (ELISA) for human NGF (NGFEmax assay; Promega Corporation, Madison, WI, USA) and Hi-D fl uorescence-activated cell sorting analyses, we observed that under basal conditions FLSs from healthy individuals express low levels of NGF/TrkA. However, there was a marked upregulation of NGF and TrkA in these FLSs following stimulation with tumor necrosis factor-alpha and interleukin-1-beta (Table 1). A critical observation was that FLSs from patients with PsA or RA produced spontaneously higher levels of NGF and had increased expression of TrkA compared with FLSs of patients with OA (Table 1). Furthermore, we observed that NGF signifi cantly stimulated the proliferation of FLSs derived from PsA synovial tissue (Figure 1). Barthel and colleagues [1] cultured FLSs from only one patient with RA and one patient with SpA and noticed that FLSs did not produce NGF, but the authors do mention that they can’t rule out the production of NGF by FLS. We cultured FLSs from 15 subjects (Table 1), and it is also possible that the NGF ELISA kit that we used is more sensitive. A fully formed pannus is characterized by proliferation of FLSs, infl ammatory infi ltrates, and a marked angiogenesis. NGF and its receptor system are known to

The presence of tri-allelic TPOX genotypes in Dominican Population

The presence of the tri-allelic TPOX genotypes represents a new finding about the Dominican Population origins. The STRs in our population have been characterized in our previous investigation, now we are beginning the process to confirm the true components of our ethnical structure. About 90% of the contemporary Dominican Population has African ancestry or has African roots, for this reason we strongly consider the nature of the tri-allelic TPOX genotypes as African origins. In this work, we report tri-allelic TPOX genotypes by routine relationship testing using PowerPlex 16 (Promega Corporation) kits.

Assembly of a large Y-STR haplotype database for the Czech population and investigation of its substructure

Twelve Y-chromosomal short tandem repeats (Y-STR) (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a, DYS385b, DYS437, DYS438, and DYS439) included in the PowerPlex Y™ Kit (Promega Corporation, Madison, USA) were studied for 1750 unrelated males living in 14 regions of the Czech Republic. A total of 1148 different haplotypes were found. The overall haplotype diversity (HD) was determined as 0.998. Analysis of Molecular Variance (AMOVA) reveals non-significant distances between regions concerning their haplotype distribution, thus allowing to use the whole sample as a representative reference database of the Czech Republic. Median network analysis shows a remarkable bipartite composition of the Czech haplotypes, falling in distinct clusters with Eastern and Western European roots.

Augmenting the Traditional Bacterial Transformation Lab

[ILLUSTRATION OMITTED] Using plasmid DNA to transform competent E. coli is a standard laboratory in many biology curriculum, including the AP Curriculum (Laboratory 6, Molecular Biology) and many general biology laboratory and genetics lab curricula (such as Mertens & Hammersmith, 2007; Vodopich & Moore, 2008). The standard transformation protocol of adding a plasmid coding for antibiotic resistance to competent cells is straightforward and elegantly demonstrates the concept of bacterial genetic exchange as well as illustrates the potential for transfer of antibiotic resistance to bacteria in the environment. Although most students take for granted that nucleic acid is the genetic material, in the early part of the 20th century, there was an intense debate raging at that time about what constituted the genetic material. Many scientists believed that DNA was too simple to be the genetic material and instead proposed the protein was the genetic material. In 1944, researchers, including Oswald Avery, Colin MacLeod, and Maclyn McCarty, were interested in determining which biological molecule is the genetic material. The genetic material was also called the "transforming factor" because in previous experiments performed by Franklin Griffith it could transform bacteria of one genotype to another. In their experiment, Avery, MacLeod, and McCarty used enzymes to degrade one component of a bacterial extraction and then added the remainder of the biological material to bacteria to determine which component would change or transform the genotype of the starting bacteria (Avery et al., 1944; McCarty, 1985). Here I describe an extension of the general transformation lab in which the students mimic the hypothesis driven experiment of Avery, MacLeod, and McCarty to determine the "transforming factor." * Materials Teachers need any type of transformation kit, such as: * Bio-Rad's pGLO kit * Carolina Biological Supply's pBLU Colony Transformation Kit * those from Modern Biology (Producing a Strain of E. coli that Glows in the Dark; or E. coli Transformation Kit) * Ward's Natural Science (Transformation of E.coli with Green and Blue Fluorescent Protein Lab Activity) * their own homemade version (for examples see BIOTECH Project, The University of Arizona, 2002; Slish, 1999). Additional reagents needed are amylase, DNase, RNase, protease, and Luria Bertani plates with the appropriate antibiotic. 1. Amylase is available for free from human saliva (Jones & Cooper, 2005; Marini, 2005); appropriate cautions should be employed with human saliva. Bacterial amylase can be purchased (Carolina 202360) and has little to no DNAse activity. 2. DNase is available from many sources. The least expensive I have found is from Promega (1000 units for $43, M6101). 3. RNase A 5mg/ml (500 [micro]l) is available from Carolina (211745). 4. Trypsin protease is available from Carolina (25 g, FA-89-7000) or Wards (5 g, 947 V 2400; or 10 g, 947 V 2401). Dissolve trypsin at a 1mg/ml concentration in double distilled water and freeze small aliquots for use. 5. Pre-poured Luria Bertani plates with ampicillin can be purchased from Ward's (88 V 0904) or Carolina (216601). Alternatively you can make your own (10 g tryptone, 5 g yeast extract, 10 g NaCl, in a total of 1 L of water. Autoclave to sterilize; cool to around 50[degrees]C; add appropriate antibiotics and other supplements, such as X-Gal and IPTG, if needed; and then pour). * Reagent Companies ** Bio-Rad www.bio-rad.com 800-424-6723 ** Carolina Biological Supply www.carolina.com 800-334-5551 ** Modern Biology, Inc. www.modernbio.com 800-733-6544 ** Promega Corporation www. …

UV Radiation Regulates Mi-2 through Protein Translation and Stability

Dermatomyositis (DM) is an autoimmune disease, which is often accompanied by the development of disease-specific autoantibodies directed against the SNF2-superfamily helicase, Mi-2. Recent evidence suggests that ultraviolet radiation exposure may be an important risk factor for the development of not only the disease but also specific autoimmunity against Mi-2. Consequently, we investigated the effects of ultraviolet radiation on Mi-2 protein expression. We observed an increase in protein levels upon ultraviolet radiation exposure in cell culture systems. These changes in expression occur quite rapidly, are maximized just 1 h following exposure, and are unique to Mi-2 when compared with other members of the NuRD complex. Changes in protein levels are not mediated through transcriptional mechanisms. Treatment results in a more efficiently translated message through regulatory elements in the 5'-UTR region of the transcript. Investigation into protein half-life further demonstrated increased stability of Mi-2 following UV exposure. Taken together, we describe a system by which Mi-2 protein expression can be quickly increased following UV exposure and then maintained up to 16 h later. These data provide a novel regulation of an important transcriptional regulator and provide insight into the possible mechanisms of the development of DM and associated autoantibodies.

An Updated Validation of Promega’s PowerPlex® 16 System: High Throughput Databasing Under Reduced PCR Volume Conditions on Applied Biosystem’s 96 Capillary 3730xl DNA Analyzer*

The PowerPlex 16 System from Promega Corporation allows single tube multiplex amplification of sixteen short tandem repeat (STR) loci including all 13 core combined DNA index system STRs. This report presents an updated validation of the PowerPlex 16 System on Applied Biosystem's 96 capillary 3730xl DNA Analyzer. The validation protocol developed in our laboratory allows for the analysis of 1536 loci (96 x 16) in c. 50 min. We have further optimized the assay by decreasing the reaction volume to one-quarter that recommended by the manufacturer thereby substantially reducing the total cost per sample without compromising reproducibility or specificity. This reduction in reaction volume has the ancillary benefit of dramatically increasing the sensitivity of the assay allowing for accurate analysis of lower quantities of DNA. Due to its substantially increased throughput capability, this extended validation of the PowerPlex 16 System should be useful in reducing the backlog of unanalyzed DNA samples currently facing public DNA forensic laboratories.

Asp330 and Tyr331 in the C-terminal Cysteine-rich Region of the Luteinizing Hormone Receptor Are Key Residues in Hormone-induced Receptor Activation

The luteinizing hormone (LH) receptor plays an essential role in male and female gonadal function. Together with the follicle-stimulating hormone (FSH) and thyroid stimulating hormone (TSH) receptors, the LH receptor forms the family of glycoprotein hormone receptors. All glycoprotein hormone receptors share a common modular topography, with an N-terminal extracellular ligand binding domain and a C-terminal seven-transmembrane transduction domain. The ligand binding domain consists of 9 leucine-rich repeats, flanked by N- and C-terminal cysteine-rich regions. Recently, crystal structures have been published of the extracellular domains of the FSH and TSH receptors. However, the C-terminal cysteine-rich region (CCR), also referred to as the "hinge region," was not included in these structures. Both structure and function of the CCR therefore remain unknown. In this study we set out to characterize important domains within the CCR of the LH receptor. First, we mutated all cysteines and combinations of cysteines in the CCR to identify the most probable disulfide bridges. Second, we exchanged large parts of the LH receptor CCR by its FSH receptor counterparts, and characterized the mutant receptors in transiently transfected HEK 293 cells. We zoomed in on important regions by focused exchange and deletion mutagenesis followed by alanine scanning. Mutations in the CCR specifically decreased the potencies of LH and hCG, because the potency of the low molecular weight agonist Org 41841 was unaffected. Using this unbiased approach, we identified Asp(330) and Tyr(331) as key amino acids in LH/hCG mediated signaling.

Biochemical and structural characterisation of membrane-containing icosahedral dsDNA bacteriophages infecting thermophilic Thermus thermophilus

Icosahedral dsDNA viruses isolated from hot springs and proposed to belong to the Tectiviridae family infect the Gram-negative thermophilic Thermus thermophilus bacterium. Seven such viruses were obtained from the Promega Corporation collection. The structural protein patterns of three of these viruses, growing to a high titer, appeared very similar but not identical. The most stable virus, P23-77, was chosen for more detailed studies. Analysis of highly purified P23-77 by thin layer chromatography for neutral lipids showed lipid association with the virion. Cryo-EM based three-dimensional image reconstruction of P23-77 to 1.4 nm resolution revealed an icosahedrally-ordered protein coat, with spikes on the vertices, and an internal membrane. The capsid architecture of P23-77 is most similar to that of the archaeal virus SH1. These findings further complicate the grouping of icosahedrally-symmetric viruses containing an inner membrane. We propose a single superfamily or order with members in several viral families.

Enzymological Analysis of Mutant Protein Kinase Cγ Causing Spinocerebellar Ataxia Type 14 and Dysfunction in Ca2+ Homeostasis

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cgamma (PKCgamma). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCgamma kinase activity. To determine the effect of these mutations on PKCgamma function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCgammas. We found that wild-type PKCgamma, but not C1 domain mutants, inhibits Ca2+ influx in response to muscarinic receptor stimulation. The sustained Ca2+ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKCgamma. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKCgamma. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKCgammas was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca2+ entry. Such an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.

Association of Mobiluncus curtisii With Recurrence of Bacterial Vaginosis

Association of Mobiluncus curtisii With Recurrence of Bacterial Vaginosis

Automated DNA extraction from large volumes

Automation of DNA extraction with DNA-IQ™ (Promega Corporation), requires special adaptations when trace amounts of DNA must be recovered from substrates requiring large volumes of extraction buffer (e.g. clothing). We have developed a method consisting of in situ cell lysis with DNA extraction buffer (containing SDS or Sarkosyl, Proteinase K, DNA IQ lysis buffer) followed by batch DNA recovery through incubation with DNA-binding magnetic beads. Tests were performed with blood, semen, saliva and manipulated or worn objects on cotton, polyester and nylon substrates. The process was optimized for incubation time, temperature, detergent and Proteinase K concentrations. Non-probative forensic samples were tested and the procedure integrated into the robotic scripts.

The distribution of allele frequencies of 15 STRs in Dominican population

The most effective tools for individual and population genetic characterization that we will use today are STRs. This work presents the results of a population study of 15 autosomal STRs loci included in PowerPlex 16 (Promega Corporation) that has the aim of creating a local database. Buccal swabs and blood samples were obtained from more than two thousands unrelated individuals were born in Dominican Republic and lived there for at least two generations.

The distribution of Y-chromosome STRs in Dominican population

We have studied the distribution of Y-chromosome STRs and established a database within population samples from Dominican Republic by analyzing male-specific markers. Laboratorio Clinico Lic Patria Rivas collected in the population samples and this population is truly representative of the entire country. We analyzed 12 Y-STRs loci included in PowerPlex Y (Promega Corporation).

Gastrointestinal Disease in Simian Immunodeficiency Virus-Infected Rhesus Macaques Is Characterized by Proinflammatory Dysregulation of the Interleukin-6-Janus Kinase/Signal Transducer and Activator of Transcription3 Pathway

Gastrointestinal disease and inflammation are common sequelae of human and simian immunodeficiency virus (SIV) infection. Nevertheless, the molecular mechanisms that lead to gastrointestinal dysfunction remain unclear. We investigated regulation of the interleukin (IL)-6-JAK-STAT3 pathway in jejunum and colon, collected at necropsy, from 10 SIV-infected macaques with diarrhea (group 1), 10 non-SIV-infected macaques with diarrhea (group 2), and 7 control uninfected macaques (group 3). All group 1 and 2 macaques had chronic diarrhea, wasting, and colitis, but group 1 animals had more frequent and severe lesions in the jejunum. A significant increase in IL-6 and SOCS-3 gene expression along with constitutive STAT3 activation was observed in the colon of all group 1 and 2 macaques and in the jejunum of only group 1 macaques compared to controls. Further, in colon, histopathology severity scores correlated significantly with IL-6 (groups 1 and 2) and SOCS-3 (group 2) gene expression. In jejunum, a similar correlation was observed only in group 1 animals. Phosphorylated STAT3 (p-STAT3) was localized to lymphocytes (CD3+) and macrophages (CD68+), with fewer CD3+ lymphocytes expressing p-STAT3 in group 1 macaques. Despite high SOCS-3 expression, STAT3 remained constitutively active, providing a possible explanation for persistent intestinal inflammation and immune activation that may favor viral replication and disease pro-gression.

Allosteric Transinhibition by Specific Antagonists in CCR2/CXCR4 Heterodimers

Chemokine receptors are presently used as targets for candidate drugs in the frame of inflammatory diseases and human immunodeficiency virus infection. They were shown to dimerize, but the functional relevance of dimerization in terms of drug action remains poorly understood. We reported previously the existence of negative binding cooperativity between the subunits of CCR2/CCR5 heterodimers. In the present study, we extend these observations to heterodimers formed by CCR2 and CXCR4, which are more distantly related. We also show that specific antagonists of one receptor inhibit the binding of chemokines to the other receptor as a consequence of their heterodimerization, both in recombinant cell lines and primary leukocytes. This resulted in a significant functional cross-inhibition in terms of calcium mobilization and chemotaxis. These data demonstrate that chemokine receptor antagonists regulate allosterically the functional properties of receptors on which they do not bind directly, with important implications on the effects of these potential therapeutic agents.