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Appl. Environ. Microbiol. 76, 829–42. Lysogeny and sporulation in Bacillus isolates from the Gulf of Mexico. 2010

Mobberley, J., Authement, R.N., Segall, A.M., Edwards, R.A., Slepecky, R.A. and Paul, J.H.

Notes: Certain bacteriophages can promote host cell sporulation under unfavorable conditions to increase survival of the host and prophage. These types of phages, known as spore-converting phages, have been found in terrestrial Bacillus species. In this article the authors examined the effect of prophages on sporulation of 11 Bacillus isolates from the Gulf of Mexico. Potential prophages in the Bacillus isolates were detected by PCR using unique PCR primer sets for each prophage genome and GoTaq® Green Master Mix. One of these isolates, B14905, was examined in more detail; the genome of this isolate was isolated using the Wizard® Genomic DNA Purification Kit, then sequenced. (4091)

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Proc. Natl. Acad. Sci. USA 107, 3293–8. Supramolecular design of self-assembling nanofibers for cartilage regeneration. 2010

Shah, R.N., Shah, N.A., Del Rosario Lim, M.M., Hsieh, C., Nuber, G. and Stupp, S.I.

Notes: The authors developed a novel matrix for use in articular cartilage regeneration and investigated the ability of this matrix to maintain mesenchymal stem cell viability and support chondrogenic differentiation. Expression of the cartilage markers aggrecan and type II collagen were used to assess chondrogenic differentiation. Expression levels were determined using the Plexor® qPCR System and BioRad iQ5™ Real-Time PCR System. (4159)

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J. Am. Soc. Hort. Sci. 135, 291–302. Transcriptional profiling of rapidly growing cucumber fruit by 454-pyrosequencing analysis 2010

Ando, K. and Grumet, R.

Notes: The Wizard® SV Gel and PCR Clean-Up System was used to purify PCR products prior to pyrosequencing. (4546)

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Cereb. Cortex 20, 2333–47. Serotonin 3A receptor subtype as an early and protracted marker of cortical interneuron subpopulations. 2010

Vucurovic, K., Gallopin, T., Ferezou, I., Rancillac, A., Chameau, P., van Hooft, J.A., Geoffroy, H., Monyer, H., Rossier, J. and Vitalis, T.

Notes: The authors characterized mouse neocortical interneurons that express 5-HT3A, a ligand-gated cation channel activated by 5-hydroxytryptamine (serotonin), during embryonic development. Transgenic mice that expressed green fluorescent protein (GFP) under the control of the 5-HT3A promoter were created. Single 5-HT3A-expressing neurons within 300µm brain sections of transgenic mice at various stages of embryonic development were subjected to whole-cell path-clamp recordings to examine their electrophysiological properties. To confirm activation of the 5-HT3A promoter in these cells, GFP expression was visualized by fluorescence microscopy without breaking the patch clamp seal. The contents of these single neurons then were aspirated and expelled into a 10µl reverse transcription reaction. After the reverse transcription, PCR was performed to simultaneously detect mRNAs encoding two isoforms of glutamic acid decarboxylase, three calcium-binding proteins, three neuropeptides, two transcription factors and reelin, a protein thought to be involved in neuronal migration and morphology. Two rounds of PCR using nested primers were required to detect these mRNAs. PCRs were performed using GoTaq® DNA Polymerase. Amplified products were visualized by agarose gel electrophoresis, using the 100bp DNA Ladder as a size standard. (4096)

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Genetics 184, 119–28. Detection, validation, and downstream analysis of allelic variation in gene expression. 2010

Ciobanu, D.C., Lu, L., Mozhui, K., Wang, X., Jagalur, M., Morris, J.A., Taylor, W.L., Dietz, K., Simon, P. and Williams, R.W.

Notes: Sequence variation within a gene, such as single nucleotide polymorphisms (SNPs), can lead to differences in expressions levels of corresponding mRNAs; genes that are self-regulated by this mechanism (cis modulation) are difficult to identify accurately with existing techniques. The authors used bioinformatic and molecular approaches to estimate error rates when identifying cis-modulated transcripts and developed a simple method to detect these transcripts in C57BL/6J F1 hybrid mice. This method, which they named allelic specific expression, is RT-PCR-based and uses PCR primers that flank an informative SNP to quantify the differential expression levels of transcripts. GoTaq® Flexi DNA Polymerase was used in the PCR. (4051)

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Cancer Res. 70, 9641–9. An illegitimate microRNA target site within the 3' UTR of MDM4 affects ovarian cancer progression and chemosensitivity. 2010

Wynendaele, J., Böhnke, A., Leucci, E., Nielsen, S.J., Lambertz, I., Hammer, S., Sbrzesny, N., Kubitza, D., Wolf, A., Gradhand, E., Balschun, K., Braicu, I., Sehouli, J., Darb-Esfahani, S., Denkert, C., Thomssen, C., Hauptmann, S., Lund, A., Marine, J.C. and Bartel, F.

Notes: The authors identified a single nucleotide polymorphism (SNP) in the 3´ untranslated region (3´ UTR) of MDM4, which promotes tumorigenesis by decreasing p53 tumor suppressor function, in ovarian cancer cells. This A to C transversion creates a putative target site for the hsa-miR-191 microRNA in the MDM4-C allele, but not the wildtype MDM4-A allele. To determine if this SNP affects MDM4 translation efficiency or mRNA stability, the authors cloned a 224-bp fragment of the MDM4 3´UTRs into the psiCHECK™-2 Vector and transfected the ovarian cancer cell line A2780 with the 224A or 224C variants of the MDM4 3´ UTR. The authors used the luciferase-based psiCHECK™-2 Vector and Dual-Glo® Luciferase Assay System to show that the C variant dramatically reduces translation efficiency and/or mRNA stability. They also assessed MDM4 expression levels in A/A, A/C and C/C ovarian cancer cells and tissues using RT-qPCR. qPCR primers for MDM4 were designed using the Plexor® Primer Design Software, and assays were performed using the Plexor® qPCR System. (4157)

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RNA 16, 239–50. Poly(A)-binding protein modulates mRNA susceptibility to cap-dependent miRNA-mediated repression. 2010

Walters, R.W., Bradrick, S.S. and Gromeier, M.

Notes: The authors investigated the mechanism of microRNA (miRNA)-mediated regulation of both endogenous mRNAs and artificial reporter constructs. To determine whether an m7G cap and poly(A) tail are required for repression, the authors created plasmids containing eight synthetic, tandem miR-30 recognition sequences in the 3´ untranslated region (UTR) of a Renilla luciferase gene under the control of a 5´UTR that confers either cap-dependent or cap-independent translation. They used these plasmids as a template for in vitro transcription, then transfected in vitro transcripts with and without an m7G cap and poly(A) tail into 293T cells, along with miR-30 miRNA (and miR-21 as a negative control). Similar experiments were performed by transfecting 293T cell with the Renilla luciferase constructs and miR-30 and miR-21 expression vectors. Finally, the authors exchanged the artificial 3´ UTR of the Renilla luciferase construct with the 3´ UTR of BACH1, a transcription factor that is regulated by miR-155, and cotransfected 293T cells with the BACH1 3´ UTR construct and an miR-115 expression vector to examine regulation via an endogenous 3´ UTR. The Promega Primer Extension System was used to compare miR-21, miR-30 and miR-155 RNA levels in transfected and untransfected 293T cells to determine endogenous levels of these miRNAs. Renilla luciferase activity was determined using the Renilla Luciferase Assay System. The authors used Northern blot analysis and quantitative RT-PCR to determine Renilla luciferase RNA levels (and GAPDH levels for normalization purposes). The Plexor® One-Step qRT-PCR System was used for qRT-PCR. (4048)

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Appl. Environ. Microbiol. 76, 3863–8. Comparison of normalization methods for construction or large multiplex amplicon pools for next-generation sequencing 2010

Harris, J.K., Sahl, J.W., Castoe, T.A., Wagner, B.D., Pollack, D.D. and Spear, J.R.

Notes: GoTaq® Master Mix was used for PCR of bacterial ribosomal RNA genes prior to pyrosequencing. (4529)

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Genome Res. 20, 1590-604.
Next-generation sequencing identifies the natural killer cell microRNA transcriptome
2010

T. A. Fehniger, T. Wylie, E. Germino, et al.

Notes: RNasin was used in the small RNA library preparation step before sequencing on an Illumina platform.


(4532)

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J. Bacteriol. 192, 4720–31. Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function. 2010

Yun, C.S., Suzuki, C., Naito, K., Takeda, T., Takahashi, Y., Sai, F., Terabayashi, T., Miyakoshi, M., Shintani, M., Nishida, H., Yamane, H. and Nojiri, H.

Notes: The authors investigated the expression of genes encoding histone-like (H-NS) proteins from the self-transmissible pCAR1 plasmid and Pseudomonas putida KT2440 genome, as well as the interaction of H-NS family members in vitro. Gene expression was quantified using quantitative RT-PCR and RNA templates that were treated with RQ1 RNase-Free DNase to degrade contaminating DNA. Interactions between Pmr and other H-NS proteins were monitored using pull-down assays. His-tagged Pmr was expressed in E. coli, purified and used as bait for FLAG-tagged H-NS family proteins. Protein purification of His-tagged proteins was performed using the MagneHis™ Protein Purification System. (4119)

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J. Exp. Bot. 61, 191–202. Physiological and molecular changes in Oryza meridionalis Ng., a heat-tolerant species of wild rice. 2010

Scafaro, A.P., Haynes, P.A. and Atwell, B.J.

Notes: The authors compared seedling growth rates, photosynthesis rates and expression levels of heat-responsive genes in the heat-resistant wild rice Oryza meridionalis and the domesticated rice O. sativa when grown at optimal and elevated temperatures. Proteins that were up- or downregulated in response to heat were identified by two-dimensional gel electrophoresis coupled with nano liquid chromatography on line with tandem mass spectrometry (nanoLC-MS/MS). Trypsin was used to cleave proteins prior to nanoLC-MS/MS. Semi-quantitative RT-PCR was performed using the GoTaq® Green Master Mix to determine if the heat-responsive proteins were transcriptionally regulated. (4092)

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J. Biochem. 148, 721–32. A recombinant catalytic domain of matriptase induces detachment and apoptosis of small-intestinal epithelial IEC-6 cells cultured on laminin-coated surface. 2010

Mochida, S., Tsuzuki, S., Inouye, K. and Fushiki, T.

Notes: The authors determined that a recombinant catalytic domain of rat matriptase (His6t-S-CD) caused detachment of small-intestinal epithelial cells (IEC-6 cells) from laminin-coated plates. His6t-S-CD was expressed in the yeast P. pastoris and purified by ammonium sulfate precipitation, gel filtration through a PD-10 column, then Ni2+-chelating chromatography using HisLink™ Resin. The authors also treated IEC-6 cells with purified His6t-S-CD to determine if this domain induced apoptosis by monitoring annexin-V staining, DNA fragmentation and caspase-3 activity. For the DNA fragmentation analysis, IEC-6 cells were treated with His6t-S-CD, then harvested, and genomic DNA was purified using the Wizard® SV Gel and PCR Clean-Up System. DNA fragmentation was assessed by agarose gel electrophoresis and ethidium bromide staining. (4100)

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Appl. Environ. Microbiol. 76, 3590–9. Growth of bacteria on 3-nitropropionic acid as a sole source of carbon, nitrogen, and energy. 2010

Nishino, S.F., Shin, K.A., Payne, R.B. and Spain, J.C.

Notes: The authors identified a bacterial strain that can use the toxin 3-nitropropionic acid (3NPA) as its sole carbon and nitrogen sources and cloned the genes that encode the enzymes responsible for the initial steps in the 3NPA degradation pathway. The Pseudomonas library used to clone these genes was created using genomic DNA isolated using the Wizard® SV Genomic DNA Purification System. Closely related genes were amplified from other bacterial species for phylogenetic analysis. PCRs were performed using the GoTaq® Hot Start Polymerase. (4164)

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Am. J. Bot. 97, 1574–8. Clonal structure of wild populations and origins of horticultural stocks of Illicium parviflorum (Illiciaceae). 2010

Newell, D.L. and Morris, A.B.

Notes: The authors investigated genetic diversity in a Florida population of Illicium parviflorum, an endangered evergreen shrub, by amplifying intersimple sequence repeats (ISSRs). Amplifications were performed using GoTaq® Hot Start Polymerase. (4161)

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Nucl. Acids Res. 38, 6985-96. Targeted next-generation sequencing of DNA regions proximal to a conserved CXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer 2010

Chmielecki,J., Peifer, M., Socci, N.D., Hutchinson, K., Viale, A., Zhao, Z., Thomas, R.K. and Pao, W.

Notes: Human Genomic DNA:Male was used as a negative control in standard PCR and Sanger Sequencing to confirm fusion genomic breakpoints identified by NGS experiments. (4534)

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Am. J. Pathol. 177, 2347–56. Development of sporadic microsatellite instability in colorectal tumors involves hypermethylation at methylated-in-tumor loci in adenoma. 2010

de Maat, M.F., Narita, N., Benard, A., Yoshimura, T., Kuo, C., Tollenaar, R.A., de Miranda, N.F., Turner, R.R., van de Velde, C.J., Morreau, H. and Hoon, D.S.

Notes: The authors examined the methylation status of methylated-in-tumor (MINT) loci and the degree of microsatellite instability (MSI) in colorectal cancer to determine if methylation of MINT loci during the progression of adenoma to cancer was linked to MSI. They used on-slide sodium bisulfite modification and methylation-specific PCR to examine the methylation index in paraffin-embedded tissue blocks containing normal, adenoma and cancer tissues. MSI status was determined using the MSI Analysis System. Patients with instability at more than 4 markers were classified as MSI-high, and patients with instability at more than 1 marker were considered microsatellite-stable. (4106)

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Development 137, 901–11. SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. 2010

Hattori, T., Müller, C., Gebhard, S., Bauer, E., Pausch, F., Schlund, B., Bösl, M.R., Hess, A., Surmann-Schmitt, C., von der Mark, H., de Crombrugghe, B. and von der Mark, K.

Notes: To study the role of the transcription factor Sox9 in the transition from cartilage to bone in newborn mice, the authors performed chromatin immunoprecipitation using the HaloCHIP™ System. Primary rib chondrocytes were transfected with a vector expressing full-length Sox9 with a HaloTag® protein tag, then proteins and DNA were cross-linked. DNA was isolated and sonicated, and the Sox9:DNA complexes were precipitated using the HaloLink™ Resin. The precipitated DNA then was amplified by PCR to determine that SOX9 binds to the SRY sites in the Vegfa gene. (4054)

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Appl. Environ. Microbiol. 76, 2783–90. Revelation by single-nucleotide polymorphism genotyping that mutations leading to a premature stop codon in inlA are common among Listeria monocytogenes isolates from ready-to-eat foods but not human listeriosis cases. 2010

Van Stelten, A., Simpson, J.M., Ward, T.J. and Nightingale, K.K.

Notes: Listeria monocytogenes uses the internalin A protein (InlA) to cross the intestinal barrier and cause foodborne illness. Mutations in inlA can introduce a premature stop codon, producing a truncated InlA protein that is secreted rather than associated with the bacterial cell wall. Strains with these inlA mutations have reduced virulence. The authors describe an inlA single nucleotide polymorphism (SNP)-based genotyping assay to distinguish isolates with the inlA mutations and use this assay to screen >1,000 L. monocytogenes isolates from ready-to-eat foods and human listeriosis cases. The assay involves amplification of the full-length inlA gene using GoTaq® Colorless Master Mix, purification of amplified products, then single-base-pair extension reactions. (4099)

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Clin. Can. Res. 16, 1391–401. High-resolution array comparative genomic hybridization in sporadic and celiac disease-related small bowel adenocarcinomas. 2010

Diosdado, B., Buffart, T.E., Watkins, R., Carvalho, B., Ylstra, B., Tijssen, M., Bolijn, A.S., Lewis, F., Maude, K., Verbeke, C., Nagtegaal, I.D., Grabsch, H., Mulder, C.J., Quirke, P., Howdle, P. and Meijer, G.A.

Notes: To better examine the molecular mechanisms of small bowel adenocarcinomas, DNA was extracted from paraffin-embedded tissue and tested for microsatellite instability (MSI) using the MSI Analysis System, Version 1.2. The PCR products were run on the Applied Biosystems 3130 Genetic Analyzer and analyzed using GeneScan® software. If two or more of the the BAT-25, BAT-26, NR-21, NR-24 or MONO-27 monomorphic markers had altered lengths, the tumors were designated as MSI unstable. (4112)

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Clin. Chem. 55, 748–56. Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR. 2009

Li, J., Wang, L., Jänne, P.A. and Makrigiorgos, GM.

Notes: The authors describe a new form of PCR, co-amplification at lower denaturation temperature PCR (COLD-PCR), to detect low-level somatic mutations. This technique is based on the facts that a) each DNA sequence has a critical denaturation temperature (Tc), which is lower than the melting temperature (Tm) and below which PCR efficiency decreases dramatically and b) Tc depends on DNA sequence. The authors used GoTaq® Flexi DNA Polymerase and mutation-specific TaqMan® probes for tumor protein 53 (TP53) and epidermal growth factor receptor (EGFR) to detect low-level somatic mutations in a mixture of wildtype and mutant DNAs. Conventional TaqMan® technology can detect mutant alleles at an abundance of 10–20% of that of the wildtype allele; using COLD-PCR the authors were able to increase selectivity 15- to 30-fold, detecting as little as 0.8% mutuant alleles. (4038)

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Appl. Environ. Microbiol. 75, 2275–83. Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by gacA. 2009

Shi, X.Y., Dumenyo, C.K., Hernandez-Martinez, R., Azad, H. and Cooksey, D.A.

Notes: To gain a better understanding of how Xylella fastidiosa causes diseases in grapes, the authors mutated conserved regulatory genes, including gacA, that affect expression of virulence-related factors in other species. The relative expression levels of gacA in wildtype and mutated strains were examined using RT-PCR. The authors also identified and quantified a number of genes that were regulated by GacA by microarray analysis. Microarray results were confirmed using RT-PCR. RT-PCR was performed using the AccessQuick™ RT-PCR System. (4052)

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Neuropsychopharmacology Feb. 11, (epub ahead of print). Nucleus accumbens CREB activity is necessary for nicotine conditioned place preference. 2009

Brunzell, D.H., Mineur, Y.S, Neve, R.L. and Picciotto, M.R.

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)

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Clin. Can. Res. 15, 7562–70. Smoking-related gene expression in laser capture-microdissected human lung. 2009

Tan, X.L., Wang, T., Xiong, S., Kumar, S.V., Han, W. and Spivack, S.D.

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)

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Otolaryngol. Head Neck Surg. 140, 55–60. Microsatellite instability analysis of sinonasal carcinomas. 2009

Martínez, J.G., Pérez-Escuredo, J., López, F., Suárez, C., Alvarez-Marcos, C., Llorente, J.L. and Hermsen, M.A.

Notes: Because intestinal-type sinonasal adenocarcinoma (ITAC) and squamous cell carcinoma of the nasal cavity (SCCNC) are histopathologically similar to microsatellite-unstable colorectal adenocarcinoma or laryngeal squamous cell carcinoma, respectively, the microsatellite instability (MSI) state of the nasal tumors were of interest to researchers. Two nanograms of purified DNA from 41 ITACs and 24 SCCNCs were amplified for shifts in five mononucleotide microsatellite loci using the MSI Analysis System, Version 1.2. The multiplex PCR products were analyzed by capillary electrophoresis and noted as MSI positive if there was a size shift of at least one marker. (4117)

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J. Biol. Chem. 284, 19402–11. Structural Determinants of G-protein α Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2) 2009

Kimple, A.J., Soundararajan, M., Hutsell, S.Q., Roos, A.K., Urban, D.J., Setola, V., Temple, B.R.S., Roth, B.L., Knapp, S.K., Willard, F.S. and Siderovsk, D.P.

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 Gαq or Gαi/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 Gi-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 Gαi subunit binding responsible for enhanced GTPase activity.
(4148)

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