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Northern blot

Philip P Adams, Carlos Flores Avile, Niko Popitsch, Ivana Bilusic, Renée Schroeder, Meghan Lybecker, Mollie W Jewett
Borrelia burgdorferi, the bacterial pathogen responsible for Lyme disease, modulates its gene expression profile in response to the environments encountered throughout its tick-mammal infectious cycle. To begin to characterize the B. burgdorferi transcriptome during murine infection, we previously employed an in vivo expression technology-based approach (BbIVET). This identified 233 putative promoters, many of which mapped to un-annotated regions of the complex, segmented genome. Herein, we globally identify the 5' end transcriptome of B...
December 1, 2016: Nucleic Acids Research
Dongmei Jiang, Meng Wang, Shifang Li
Cell-to-cell trafficking through different cellular layers is a key process for various RNAs including those of plant viruses and viroids, but the regulatory mechanisms involved are still not fully elucidated and good model systems are important. Here, we analyze the function of a simple RNA motif (termed "loop19") in Potato spindle tuber viroid (PSTVd) which is required for trafficking in Nicotiana benthamiana leaves. Northern blotting, RT-PCR and in situ hybridization analyses demonstrated that unlike wild type PSTVd, which was present in the nuclei in all cell types, the trafficking-defective loop19 mutants were visible only in the nuclei of upper epidermal and palisade mesophyll cells, which shows that PSTVd loop19 plays a role in mediating RNA trafficking from palisade to spongy mesophyll cells in Nicotiana benthamiana leaves...
October 14, 2016: Journal of General Virology
Jacqueline H Finger, Constance M Smith, Terry F Hayamizu, Ingeborg J McCright, Jingxia Xu, Meiyee Law, David R Shaw, Richard M Baldarelli, Jon S Beal, Olin Blodgett, Jeff W Campbell, Lori E Corbani, Jill R Lewis, Kim L Forthofer, Pete J Frost, Sharon C Giannatto, Lucie N Hutchins, Dave B Miers, Howie Motenko, Kevin R Stone, Janan T Eppig, James A Kadin, Joel E Richardson, Martin Ringwald
The Gene Expression Database (GXD; is an extensive and well-curated community resource of mouse developmental expression information. Through curation of the scientific literature and by collaborations with large-scale expression projects, GXD collects and integrates data from RNA in situ hybridization, immunohistochemistry, RT-PCR, northern blot and western blot experiments. Expression data from both wild-type and mutant mice are included. The expression data are combined with genetic and phenotypic data in Mouse Genome Informatics (MGI) and made readily accessible to many types of database searches...
November 28, 2016: Nucleic Acids Research
Hanchao Zhao, Susan Martinis
Aminoacyl-tRNA synthetases (AARS) comprise an enzyme family that generates and maintains pools of aminoacylated tRNAs, which serve as essential substrates for protein synthesis. Many protein synthesis factors, including tRNA and AARS also have non-canonical functions. Particularly in mammalian cells, alternate functions of AARSs have been associated with re-distribution in the cell to sites that are removed from translation. Sub-fractionation methods for E. coli were designed and optimized to carefully investigate re-localization of bacterial AARSs and tRNA that might aid in conferring alternate activities...
November 22, 2016: Methods: a Companion to Methods in Enzymology
Yuli C Chang, Chien-Chih Chiu, Chung-Yee Yuo, Wen-Ling Chan, Ya-Sian Chang, Wen-Hsin Chang, Shou-Mei Wu, Han-Lin Chou, Ta-Chih Liu, Chi-Yu Lu, Wen-Kuang Yang, Jan-Gowth Chang
X-inactive-specific transcript (XIST), a long non-coding RNA, is essential for the initiation of X-chromosome inactivation. However, little is known about other roles of XIST in the physiological process in eukaryotic cells. In this study, the bioinformatics approaches revealed XIST could be processed into a small non-coding RNA XPi2. The XPi2 RNA was confirmed by a northern blot assay; its expression was gender-independent, suggesting the role of XPi2 was beyond X-chromosome inactivation. The pull-down assay combined with LC-MS-MS identified two XPi2-associated proteins, nucleolin and hnRNP A1, connected to the formation of G-quadruplex...
November 17, 2016: Oncotarget
Jin Xu, Deshui Liu, Yongliang Zhang, Ying Wang, Chenggui Han, Dawei Li, Jia-Lin Yu, Xian-Bing Wang
Co-infection of none-coding satellite RNAs (sat-RNAs) usually inhibits replication and attenuates disease symptoms of helper viruses. However, we find that the sat-RNA of Beet black scorch virus (BBSV) and low temperature (18°C) additively enhance the systemic infection of BBSV in Nicotiana benthamiana. Northern blotting hybridization revealed a relatively reduced accumulation of BBSV-derived small interfering RNAs (siRNAs) in presence of sat-RNA as compared to that of BBSV alone. Cloning and sequencing of total small RNAs showed that more than 50% of the total small RNAs sequenced from BBSV-infected plants were BBSV-siRNAs, whereas the abundance of sat-siRNAs were higher than BBSV-siRNAs in the sat-RNA co-infected plants, indicating that the sat-RNA occupies most of the silencing components and possibly relieves the RNA silencing-mediated defense against BBSV...
2016: Frontiers in Microbiology
Yaqiong Li, Zhijie Liu, Junlong Liu, Jifei Yang, Qian Li, Pengfei Guo, Guiquan Guan, Guangyuan Liu, Jianxun Luo, Hong Yin, Youquan Li
BACKGROUND: Bovine theileriosis is a common disease transmitted by ticks, and can cause loss of beef and dairy cattle worldwide. Here, an indirect enzyme-linked immunosorbent assay (iELISA) based on Theileria luwenshuni surface protein (TlSP) was developed and used to carry out a seroepidemiological survey of bovine theileriosis in northern China. METHODS: We used the BugBuster Ni-NTA His•Bind Purification Kit to purify recombinant TlSP (rTlSP), which was subsequently analyzed by Western Blotting to evaluate cross-reactivity with other pathogen-positive sera...
November 18, 2016: Parasites & Vectors
Marcus J O Johansson
Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control mechanism which triggers decay of mRNAs harboring premature translation termination codons. In this chapter, I describe methods for monitoring the influence of NMD on mRNA abundance and decay rates in Saccharomyces cerevisiae. The descriptions include detailed methods for growing yeast cells, total RNA isolation, and Northern blotting. Although the chapter focuses on NMD, the methods can be easily adapted to assess the effect of other mRNA decay pathways...
2017: Methods in Molecular Biology
Xiyang Zhao, Tangchun Zheng, Longting Shao, Zhenhai Xiao, Fuwei Wang, Shuchun Li, Lina Zang, Mi Zheng, Ying Li, Guan-Zheng Qu
The aim of this study was to determine whether transgenic birch (Betula platyphylla) ectopic overexpressing a late embryogenesis abundant (LEA) gene and a basic leucine zipper (bZIP) gene from the salt-tolerant genus Tamarix (salt cedar) show increased tolerance to salt (NaCl) stress. Co-transfer of TaLEA and ThbZIP in birch under the control of two independent CaMV 35S promoters significantly enhanced salt stress. PCR and northern blot analyses indicated that the two genes were ectopically overexpressed in several dual-gene transgenic birch lines...
2016: PloS One
Ben Fan, Yu-Long Li, Aruljothi Mariappan, Anke Becker, Xiao-Qin Wu, Rainer Borriss
The alternative sigma factor D is known to be involved in at least three biological processes in Bacilli: flagellin synthesis, methyl-accepting chemotaxis, and autolysin synthesis. Although many Bacillus genes have been identified as SigD regulon, the list may be not completed. With a microarray-based systemic screening, we found a set of genes down-regulated in the sigD knock-out mutant of the plant growth promoting rhizobacterium B. amyloliquefaciens subsp. plantarum FZB42. Eight genes (appA, blsA, dhaS, spoVG, yqgA, RBAM_004640, RBAM_018080, and ytk) were further confirmed by quantitative PCR and/or Northern blot to be controlled by SigD at the transcriptional level...
October 26, 2016: Biology Open
Ji-Woong Choi, Tae-Yub Kwon, Su-Hyung Hong, Heon-Jin Lee
MicroRNAs in eukaryotic cells are thought to control highly complex signal transduction and other biological processes by regulating coding transcripts, accounting for their important role in cellular events in eukaryotes. Recently, a novel class of bacterial RNAs similar in size [18-22 nucleotides (nt)] to microRNAs has been reported. Herein, we describe microRNAs, small RNAs from the oral pathogen Streptococcus sanguinis. The bacteria are normally present in the oral cavities and cause endocarditis by contaminating bloodstreams...
October 31, 2016: Cell Biochemistry and Biophysics
Barend L Penzhorn, Ilse Vorster, Gernot Redecker, Marinda C Oosthuizen
Although there is evidence of high seroprevalence of antibodies to Babesia spp. in dogs in central Namibia, clinical babesiosis is rarely diagnosed. Rhipicephalus sanguineus sensu lato, the vector of Babesia vogeli, is common in Namibia while Haemaphysalis elliptica, the vector of the highly virulent but morphologically indistinguishable Babesia rossi, has rarely been recorded, mainly in northern Namibia. On the basis of vector occurrence, clinical cases of canine babesiosis in Windhoek, central Namibia, have been ascribed to B...
October 26, 2016: Journal of the South African Veterinary Association
Si Liu, Jiatao Xie, Jiasen Cheng, Bo Li, Tao Chen, Yanping Fu, Guoqing Li, Manqun Wang, Huanan Jin, Hu Wan, Daohong Jiang
Mycoviruses are usually transmitted horizontally via hyphal anastomosis and vertically via sexual/asexual spores. Previously, we reported that a gemycircularvirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), could infect its fungal host extracellularly. Here, we discovered that SsHADV-1 could infect a mycophagous insect, Lycoriella ingenua, and use it as a transmission vector. Virus acquired by larvae feeding on colonies of a virus-infected strain of S. sclerotiorum was replicated and retained in larvae, pupae, adults, and eggs...
October 24, 2016: Proceedings of the National Academy of Sciences of the United States of America
Shujing Li, Ke Shui, Ying Zhang, Yongqiang Lv, Wankun Deng, Shahid Ullah, Luoying Zhang, Yu Xue
We report a database of circadian genes in eukaryotes (CGDB,, containing ∼73 000 circadian-related genes in 68 animals, 39 plants and 41 fungi. Circadian rhythm is ∼24 h rhythm in behavioral and physiological processes that exists in almost all organisms on the earth. Defects in the circadian system are highly associated with a number of diseases such as cancers. Although several databases have been established for rhythmically expressed genes, a comprehensive database of cycling genes across phyla is still lacking...
October 26, 2016: Nucleic Acids Research
Julia Hahn, Olga V Tsoy, Sebastian Thalmann, Jelena Čuklina, Mikhail S Gelfand, Elena Evguenieva-Hackenberg
Small open reading frames (sORFs) and genes for non-coding RNAs are poorly investigated components of most genomes. Our analysis of 1391 ORFs recently annotated in the soybean symbiont Bradyrhizobium japonicum USDA 110 revealed that 78% of them contain less than 80 codons. Twenty-one of these sORFs are conserved in or outside Alphaproteobacteria and most of them are similar to genes found in transposable elements, in line with their broad distribution. Stabilizing selection was demonstrated for sORFs with proteomic evidence and bll1319_ISGA which is conserved at the nucleotide level in 16 alphaproteobacterial species, 79 species from other taxa and 49 other Proteobacteria...
2016: PloS One
Silvia Monticelli, Tarmo Äijö, Sara Trifari
MicroRNAs (miRNAs) are crucial components of the molecular networks regulating differentiation and responses of T lymphocytes in health and disease. It is therefore essential to rely on robust methods of qualitative and quantitative investigation of miRNA expression in T cell subsets, and during T cell activation and differentiation. Here, we focus on different methods for miRNA analysis, including Northern blots, quantitative RT-PCR, and next-generation sequencing, and we discuss advantages and disadvantages of each method...
2017: Methods in Molecular Biology
Carla Motta, Gabriella Lupo, Dario Rusciano, Melania Olivieri, Liliana Lista, Mario De Rosa, Vincenzo Pavone, Carmelina Daniela Anfuso
Purpose: To investigate the molecular mechanisms of the antiangiogenic activity of UPARANT, an antagonist of the urokinase-type plasminogen activator receptor (uPAR), on primary human retinal endothelial cells (HREC) as a model of in vitro angiogenesis. Methods: The antiangiogenic activity of UPARANT was evaluated on endothelial cell migration, invasion, and tube formation. Human REC were further analyzed for viability, transendothelial electrical resistance (TEER), and tight junction (TJ) expression at the protein and mRNA levels...
October 1, 2016: Investigative Ophthalmology & Visual Science
Todd Blevins
Cells have evolved intricate RNA-directed mechanisms that destroy viruses, silence transposons, and regulate gene expression. These nucleic acid surveillance and gene silencing mechanisms rely upon the selective base-pairing of ~19-25 nt small RNAs to complementary RNA targets. This chapter describes northern blot hybridization techniques for the detection of such small RNAs. Blots spiked with synthetic standards are used to illustrate the detection specificity and sensitivity of DNA oligonucleotide probes...
2017: Methods in Molecular Biology
Subir K Nagdas, Virginia P Winfrey, Gary E Olson
Nevertheless, a nonviable sperm population is present in the cauda epididymidis of many species. Degenerating spermatozoa release enzymes that could have detrimental effects on the viability of neighboring cells, and they are source of autoantigens that induce an autoimmune response if they escape the blood-epididymis barrier. Does the epididymis have specialized protective mechanism(s) to segregate the viable sperm population from defective spermatozoa? Previously, we identified a fibrinogen-like protein-2 (fgl2) that specifically binds to and polymerizes into a cocoon-like complex coating defective spermatozoa and sperm fragments...
October 9, 2016: International Journal of Biochemistry & Cell Biology
Ying Xiong, Te Liu, Suwei Wang, Huiying Chi, Chuan Chen, Jin Zheng
The dysfunction of ovarian granulosa cells (OGCs) directly affects the premature ovarian failure (POF). In vivo experiments showed that cyclophosphamide significantly induced mouse ovarian atrophy and proliferation inhibition of OGCs. The expressions of p53, p66Shc and p16 were significantly higher in OGCs of the cyclophosphamide treatment group. MTT assay showed that cyclophosphamide effectively inhibited the proliferation of OGCs in vitro. SA-β-Gal staining showed that the OGCs in the cyclophosphamide treatment group had many senescent cells...
October 8, 2016: Gene
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