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https://www.readbyqxmd.com/read/28640671/regulation-of-a-muralytic-enzyme-encoding-gene-by-two-non-coding-rnas
#1
Renée J St-Onge, Marie A Elliot
Non-coding regulatory RNAs fine-tune gene expression post-transcriptionally. In the streptomycetes, rpfA - encoding a muralytic enzyme required for establishing and exiting dormancy - is flanked by non-coding regulatory RNA elements both upstream (riboswitch) and downstream [antisense small RNA (sRNA)]. In Streptomyces coelicolor, the upstream riboswitch decreases rpfA transcript abundance in response to the second messenger cyclic di-AMP, itself involved in cell wall metabolism and dormancy. There is, however, no obvious expression platform associated with this riboswitch and consequently, its mechanism of action is entirely unknown...
June 22, 2017: RNA Biology
https://www.readbyqxmd.com/read/28608345/nonradioactive-plant-small-rna-detection-using-biotin-labeled-probes
#2
Jun Hu, Yingguo Zhu
Small noncoding RNAs are essential for gene expression at transcriptional and posttranscriptional levels. Northern blot is the most used method for small RNA detection in tissues. Here we present an improved protocol for the Northern blot-based small RNA detection from plant tissues by using biotin-labeled probes. MicroRNAs and small interfering RNAs derived from Arabidopsis and Oryza sativa, respectively, have been detected with this methodology. Results suggest that this method is sensitive and efficient enough to detect small RNAs from plant tissues by using as low as 5 μg of total RNA...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28608344/nonradioactive-detection-of-small-rnas-using-digoxigenin-labeled-probes
#3
Ariel H Tomassi, Delfina Gagliardi, Damian A Cambiagno, Pablo A Manavella
Small RNAs have been traditionally detected and quantified using small RNA blots, a modified Northern blot technique. The small RNAs are size-fractionated from the rest of the cellular RNA molecules by polyacrylamide gel electrophoresis and transferred by blotting onto a positively charged membrane. A radiolabeled probe was then traditionally used to detect a specific small RNA in the cellular pool. Small RNA blotting is a relatively simple, inexpensive approach to visualize small RNAs without artifacts. However, the radioactive labeling of the probe is sometimes an impediment, especially due to the requirement of specialized facilities...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28586459/fastmir-an-rna-based-sensor-for-in-vitro-quantification-and-live-cell-localization-of-small-rnas
#4
Kun Huang, Francis Doyle, Zachary E Wurz, Scott A Tenenbaum, Reza K Hammond, Jeffrey L Caplan, Blake C Meyers
Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA-mRNA interactions combined with a fluorophore-binding sequence 'Spinach', a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength...
June 6, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28573617/nonradioactive-northern-blot-analysis-to-detect-ebola-virus-minigenomic-mrna
#5
Kristina Brauburger, Tessa Cressey, Elke Mühlberger
In this chapter, we describe the detection of Ebola virus minigenomic mRNA using a nonradioactive Northern hybridization. This protocol comprises all steps beginning with the synthesis of a digoxigenin-labeled riboprobe, harvest of transcribed mRNA from cells transfected with the Ebola virus minigenome system, separation of mRNA species by denaturing RNA gel electrophoresis, transfer of the mRNA to nylon membranes by vacuum blotting, and finally the detection of minigenome-specific mRNA through hybridization with a labeled riboprobe directed against the reporter gene...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28573158/mouse-embryonic-fibroblast-cell-culture-and-stimulation
#6
Lian-Qun Qiu, Wi S Lai, Deborah J Stumpo, Perry J Blackshear
Culture of mouse embryonic fibroblast (MEF) cells represents a powerful system to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments. Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive activities. Because of their ubiquitous distribution as tissue cells, these cells are poised to respond to factors released by newly activated innate immune cells, thus becoming a useful tool to study inflammation and immunity...
July 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28570637/temperature-mediated-biosynthesis-of-the-phytotoxin-phaseolotoxin-by-pseudomonas-syringae-pv-phaseolicola-depends-on-the-autoregulated-expression-of-the-phtabc-genes
#7
Selene Aguilera, Ariel Alvarez-Morales, Jesús Murillo, José Luis Hernández-Flores, Jaime Bravo, Susana De la Torre-Zavala
Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally synthesized between 18°C and 20°C, while no detectable amounts are present above 28°C. The Pht cluster, involved in the biosynthesis of phaseolotoxin, contains 23 genes that are organized in five transcriptional units. The function of most of the genes from the Pht cluster is still unknown and little information about the regulatory circuitry leading to expression of these genes has been reported...
2017: PloS One
https://www.readbyqxmd.com/read/28552112/endogamous-marriage-and-the-prevalence-of-hemoglobin-e-in-ethnic-groups-of-northern-thailand
#8
Praphat Ruengthanoo, Pathrapol Lithanatudom, Pitsinee Inthi, Janjira Termphiriyakit, Phaivit Laphyai, Daoroong Kangwanpong, Duncan R Smith, Jatupol Kampuansai
OBJECTIVE: To investigate the impact of the endogamous marriage culture on the prevalence of the hemoglobin E (HbE) recessive variant. METHODS: The prevalence of the hemoglobin E (HbE) recessive variant was determined by dot-blot hybridization in 4 endogamous villages (1 Mlabri and 3 Htin ethnic groups) in comparison with 9 other nearby non-endogamous populations. RESULTS: Although the overall HbE prevalence in the population studied (8.44%, 33/391) was not significantly different from that of the general southeast Asian population, a high prevalence and individuals with homozygous HbE were observed in two villages, the Mlabri from Wiang Sa district and the Htin from Thung Chang district of Nan province (26...
April 2017: Asian Pacific Journal of Tropical Medicine
https://www.readbyqxmd.com/read/28548451/molecular-and-functional-characterization-of-the-american-cockroach-periplaneta-americana-rab5-the-first-exopterygotan-low-molecular-weight-ovarian-gtpase-during-oogenesis
#9
Mohamed Elmogy, Amr A Mohamed, Muhammad Tufail, Tomohide Uno, Makio Takeda
The small Rab GTPases are key regulators of membrane vesicle trafficking. Ovaries of Periplaneta americana (Linnaeus) (Blattodea: Blattidae) have small molecular weight GTP/ATP-binding proteins during early and late vitellogenic periods of oogenesis. However, the identification and characterization of the detected proteins have not been yet reported. Herein, we cloned a cDNA encoding Rab5 from the American cockroach, P. americana, ovaries (PamRab5). It comprises 796 bp, encoding a protein of 213 amino acid residues with a predicted molecular weight of 23...
May 26, 2017: Insect Science
https://www.readbyqxmd.com/read/28528880/lyme-borreliae-prevalence-and-genospecies-distribution-in-ticks-removed-from-humans
#10
Patrick Waindok, Sabine Schicht, Volker Fingerle, Christina Strube
Lyme borreliosis (LB) is the most important human tick-borne disease, but Borrelia genospecies cause different clinical manifestations. Ticks of the genus Ixodes removed from humans between 2006 and 2012 were analysed for Borrelia burgdorferi sensu lato (sl) infections. The majority of ticks originated from the Greater Hanover region in Northern Germany. The engorgement status varied over the entire spectrum from unengorged (no evidence of started blood feeding) to fully engorged. In the present study, prevalence data for B...
May 13, 2017: Ticks and Tick-borne Diseases
https://www.readbyqxmd.com/read/28526608/microrna-analysis-of-taenia-crassiceps-cysticerci-under-praziquantel-treatment-and-genome-wide-identification-of-taenia-solium-mirnas
#11
Matías Gastón Pérez, Natalia Macchiaroli, Gabriel Lichtenstein, Gabriela Conti, Sebastián Asurmendi, Diego Humberto Milone, Georgina Stegmayer, Laura Kamenetzky, Marcela Cucher, Mara Cecilia Rosenzvit
MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression and perform critical functions in development and disease. In spite of the increased interest in miRNAs from helminth parasites, no information is available on miRNAs from Taenia solium, the causative agent of cysticercosis, a neglected disease affecting millions of people worldwide. Here we performed a comprehensive analysis of miRNAs from Taenia crassiceps, a laboratory model for T. solium studies, and identified miRNAs in the T...
May 17, 2017: International Journal for Parasitology
https://www.readbyqxmd.com/read/28522867/movement-protein-of-apple-chlorotic-leaf-spot-virus-is-genetically-unstable-and-negatively-regulated-by-ribonuclease-e-in-e-coli
#12
Rahul Mohan Singh, Dharam Singh, Vipin Hallan
Movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) belongs to "30 K" superfamily of proteins and members of this family are known to show a wide array of functions. In the present study this gene was found to be genetically unstable in E. coli when transformed DH5α cells were grown at 28 °C and 37 °C. However, genetic instability was not encountered at 20 °C. Heterologous over expression failed despite the use of different transcriptional promoters and translational fusion constructs...
May 18, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28521851/regulation-of-interleukin-11-expression-in-ovulatory-follicles-of-the-rat-ovary
#13
You-Jee Jang, You-Jee Jang, Jae-Il Park, Jae-Il Park, Seong-Eun Jeong, Seong-Eun Jeong, You-Mi Seo, You-Mi Seo, Phuong T M Dam, Phuong T M Dam, Young-Woo Seo, Young-Woo Seo, Bum-Chae Choi, Bum-Chae Choi, Sang-Jin Song, Sang-Jin Song, Sang-Young Chun, Sang-Young Chun, Moon-Kyoung Cho, Moon-Kyoung Cho
The aim of the present study was to examine the regulation of interleukin (IL)-11 expression, as well as the role of IL-11, during ovulation in gonadotropin-primed immature rats. Injection of equine chorionic gonadotropin (eCG), followed by human CG (hCG) to induce superovulation stimulated expression of the Il11 gene in theca cells within 6h, as revealed by northern blot and in situ hybridisation analyses. Real-time reverse transcription-polymerase chain reaction analysis showed that the IL-11 receptor, α subunit gene was expressed in granulosa and theca cells and that injection of hCG had no effect on its expression...
May 19, 2017: Reproduction, Fertility, and Development
https://www.readbyqxmd.com/read/28516114/measurement-of-mrna-decay-in-mouse-embryonic-fibroblasts
#14
Lian-Qun Qiu, Wi S Lai, Deborah J Stumpo, Perry J Blackshear
mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative transcript abundance using semi-quantitative real time RT-PCR...
July 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28507067/the-global-transcriptional-response-to-organic-hydroperoxide-and-the-role-of-ohrr-in-the-control-of-virulence-traits-in-chromobacterium-violaceum
#15
Maristela Previato Mello, Diogo de Abreu Meireles, Luis Eduardo Soares Netto, José Freire da Silva Neto
A major pathway for detoxification of organic hydroperoxides, such as cumene hydroperoxide (CHP), involves the MarR family transcriptional regulator OhrR and the peroxidase OhrA. However, the effect of these peroxides on the global transcriptome and the contribution of the OhrA/OhrR system on bacterial virulence remain poorly explored. Here, we analyzed the transcriptome profiles of Chromobacterium violaceum exposed to CHP and after deletion of ohrR, and we showed that OhrR controls virulence of this human opportunistic pathogen...
May 15, 2017: Infection and Immunity
https://www.readbyqxmd.com/read/28494343/gene-end-like-sequences-within-the-3-non-coding-region-of-the-nipah-virus-genome-attenuate-viral-gene-transcription
#16
Akihiro Sugai, Hiroki Sato, Misako Yoneda, Chieko Kai
The regulation of transcription during Nipah virus (NiV) replication is poorly understood. Using a bicistronic minigenome system, we investigated the involvement of non-coding regions (NCRs) in the transcriptional re-initiation efficiency of NiV RNA polymerase. Reporter assays revealed that attenuation of NiV gene expression was not constant at each gene junction, and that the attenuating property was controlled by the 3' NCR. However, this regulation was independent of the gene-end, gene-start and intergenic regions...
May 8, 2017: Virology
https://www.readbyqxmd.com/read/28483524/%C3%AE-12-fatty-acid-desaturase-is-involved-in-growth-at-low-temperature-in-yeast-yarrowia-lipolytica
#17
Satoshi Tezaki, Ryo Iwama, Satoshi Kobayashi, Yuh Shiwa, Hirofumi Yoshikawa, Akinori Ohta, Hiroyuki Horiuchi, Ryouichi Fukuda
We investigated the role of FAD2, which was predicted to encode a fatty acid desaturase of the n-alkane-assimilating yeast Yarrowia lipolytica. Northern blot analysis suggested that FAD2 transcription was upregulated at low temperature or in the presence of n-alkanes or oleic acid. The FAD2 deletion mutant grew as well as the wild-type strain on glucose, n-alkanes, or oleic acid at 30 °C, but grew at a slower rate at 12 °C, when compared to the wild-type strain. The growth of the FAD2 deletion mutant at 12 °C was restored by the addition of 18:2, but not 18:1, fatty acids...
June 17, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28479482/embryonic-expression-of-festina-lente-fel-a-novel-maternal-gene-in-the-oligochaete-annelid-tubifex-tubifex
#18
Takuma Nakamura, Inori Shiomi, Takashi Shimizu
We have cloned and characterized the expression of a novel maternal gene festina lente (designated Ttu-fel) from the clitellate annelid Tubifex tubifex. Northern blot analyses have shown that Ttu-fel mRNA is approximately 8 kbp in length and that its expression is restricted to oocytes undergoing maturation division and early embryos up to 22-cell stage. Maternal transcripts of Ttu-fel are first detected in oocytes in the ovary of young adults (ca. 40 days after hatching); its expression continues in growing oocytes in the ovisac...
May 4, 2017: Gene Expression Patterns: GEP
https://www.readbyqxmd.com/read/28442397/molecular-cloning-characterization-and-expression-analysis-of-heat-shock-protein-90-in-albino-northern-snakehead-channa-argus
#19
Aiguo Zhou, Shaolin Xie, Zhenlu Wang, Muhammad Junaid, Lanfen Fan, Chao Wang, Qiao Ye, Yanfeng Chen, De-Sheng Pei, Jixing Zou
The great albino northern snakehead Channa argus is habitual to only the Sichuan Jialing Rivers System in China, making its introduction difficult to other riverine systems. Here, we characterized heat shock protein 90 (AcaHSP90) and probed its molecular responses toward the environmental stressors that C. argus can face during its introduction and breeding in the other southern latitudes of China. To serve the purpose, cDNA encoding of AcaHSP90 were cloned and characterized in albino C. argus. The cDNA was 2752bps that contained an open reading frame (ORF), encoding a 726-amino-acid polypeptide of 83...
April 22, 2017: Gene
https://www.readbyqxmd.com/read/28439822/improved-denaturation-of-small-rna-duplexes-and-its-application-for-northern-blotting
#20
C Jake Harris, David C Baulcombe, Attila Molnar
Small RNAs (sRNAs) are short (18-30 nucleotide) noncoding RNA molecules, which control gene expression and pathogen response in eukaryotes. They are associated with and guide nucleases to target nucleic acids by nucleotide base pairing. We found that current techniques for small RNA detection are adversely affected by the presence of complementary RNA. Thus we established FDF-PAGE (fully denaturing formaldehyde polyacrylamide gel electrophoresis), which dramatically improves denaturation efficiency and subsequently the detection of sequestered sRNAs...
2017: Methods in Molecular Biology
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