• Title/Summary/Keyword: Reverse transcription

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Pathway Analysis in HEK 293T Cells Overexpressing HIV-1 Tat and Nucleocapsid

  • Lee, Min-Joo;Park, Jong-Hoon
    • Journal of Microbiology and Biotechnology
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    • v.19 no.10
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    • pp.1103-1108
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    • 2009
  • The human immunodeficiency virus (HIV)-l protein Tat acts as a transcription transactivator that stimulates expression of the infected viral genome. It is released from infected cells and can similarly affect neighboring cells. The nucleocapsid is an important protein that has a related significant role in early mRNA expression, and which contributes to the rapid viral replication that occurs during HIV-1 infection. To investigate the interaction between the Tat and nucleocapsid proteins, we utilized cDNA micro arrays using pTat and flag NC cotransfection in HEK 293T cells and reverse transcription-polymerase chain reaction to validate the micro array data. Four upregulated genes and nine downregulated genes were selected as candidate genes. Gene ontology analysis was conducted to define the biological process of the input genes. A proteomic approach using PathwayStudio determined the relationship between Tat and nucleocapsid; two automatically built pathways represented the interactions between the upregulated and downregulated genes. The results indicate that the up- and downregulated genes regulate HIV-1 replication and proliferation, and viral entry.

Effects of 17 β -estradiol, bisphenol A and genistein on the expression of the glutathione peroxidase gene of Philasterides dicentrarchii (Ciliophora: Scuticociliata)

  • Lee, Eun-Hye;Kim, Sung-Mi;Nam, Yoon-Kwon;Kim, Ki-Hong
    • Journal of fish pathology
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    • v.19 no.3
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    • pp.189-195
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    • 2006
  • A subtracted cDNA library of a marine scuticociliate, Philasterides dicentrarchii, in response to 17β-estradiol exposure was constructed using suppression subtractive hybridization (SSH). As a result of SSH, 275 clones were isolated, and among them, only glutathione peroxidase (GPX) gene was isolated as an antioxidative enzyme responding to 17β-estradiol. The semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the transcription of GPX gene of P. dicentrarchii was clearly increased by exposure to 17β-estradiol. The GPX transcription was also clearly increased by exposure to xenoestrogens such as bisphenol A (BPA) and genistein.

Molecular Identification of the Fish 4-Aminobutyrate Aminotransferase from Flounder, Paralichthys olivaceus

  • Sung Bo Kyung;Kim Young Tae
    • Fisheries and Aquatic Sciences
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    • v.4 no.1
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    • pp.25-31
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    • 2001
  • 4-Aminobutyrate aminotransferase plays an essential role in the 4-aminobutyric acid shunt, converting 4-aminobutyrate to succinic semi aldehyde. We isolated and sequenced' a fish cDNA fragment that encodes 4-aminobutyrate aminotransferase. A brain cDNA library from flounder (Paralichthys olivaceus) was constructed using the ZAP- III XR vector and screened for the fish 4-aminobutyrate aminotransferase gene using a probe derived from the conserved sequences of known mammalian 4-aminobutyrate aminotransferases. A partial cDNA for 4-aminobutyrate aminotransferase was cloned and found to be 700 bp in length corresponding to 66 amino acids. Nucleotide sequence of the clone was aligned with NCBI (National Center for Biotechnology Information) DNA sequence data base. The result showed high sequence identity with previously reported mammalian 4-aminobutyrate aminotransferases. The trans­criptional level of flounder 4-aminobutyrate aminotransferase was detected with the presence of mRNA at different flounder tissues by reverse transcription-polymerase chain reaction (RT-PCR). The expression of flounder 4-aminobutyrate aminotransferase was also tested and detected from the flounder tissues of the brain, liver, kidney and pancreas.

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Transcriptional activation of anthocyanin structural genes in torenia cv. Kauai rose by overexpression of anthocyanin regulatory transcription factors

  • Xu, Jun-Ping;Naing, Aung Htay;Kim, Chang-Kil
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2018.10a
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    • pp.33-33
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    • 2018
  • This study was conducted to examine the role of the transcription factors (TFs) (RsMYB1 and mPAP1+B-Peru) in the regulation of anthocyanin biosynthesis in the ornamental torenia cv. Kauai rose. In this study, we could produce several putative transgenic lines overexpressing the TFs via Agrobacterium-mediated transformation, and presence of the TFs in the randomly selected five transgenic lines was confirmed using polymerase chain reaction (PCR). According to results of reverse transcription-PCR analysis (RT-PCR), the expression of the TFs in all transgenic lines and of the anthocyanin structural genes (CHS, F3H, DFR, and ANS) in all transgenic lines and WT plants were distinctly detectable. However, transcript levels of the structural genes expressed in the transgenic lines overexpressing TFs were significantly higher than those expressed in WT plants. Therefore, it is suggested that anthocyanin content in flowers of the transgenic torenia would be significantly higher than that in flowers of WT plants. Moreover, these results indicate that the TFs (RsMYB1 and mPAP1+B-Peru) could be exploited as potential anthocyanin regulatory TFs to enhance anthocyanin content in the other horticultural plants.

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MOLECULAR CLONING OF CHICKEN INTERFERON-GAMMA (닭 인터페론 유전자의 클로닝에 관한 연구)

  • ;Hyun Lillehoj
    • Proceedings of the Korea Society of Poultry Science Conference
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    • 1999.11a
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    • pp.34-50
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    • 1999
  • A cDNA encoding chicken interferon-gamma (chIFN-${\gamma}$) was amplified from P34, a CD4$^{+}$ T-cell hybridoma by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pUC18. THe sequences of cloned PCR products were determined to confirm the correct cloning. Using this cDNA as probe, chicken genomic library from White Leghorn spleen was screened. Phage clones harboring chicken interferon-gamma (chIFN-${\gamma}$) were isolated and their genomic structure elucidated. The chIFN-${\gamma}$ contains 4 exons and 3 introns spanning over 14 kb, and follows the GT/AG rule for correct splicing at the exon/intron boundaries. The four exons encode 41, 26, 57 and 40 amino acids, respectively, suggesting that the overall structure of IFN-${\gamma}$ is evolutionairly conserved in mammalian and avian species. The 5’-untranslated region and signal sequences are located in exon 1. Several AT-rich sequences located in the fourth exon may indicate a role in mRNA turnover. The 5’-flanking region contains sequences homologous to the potential binding sites for the mammalian transcription factors, activator protein-1(AP-1) activator protein-2(AP-2) cAMP-response element binding protein(CREB), activating transcription factor(ATF), GATA-binding fator(GATA), upstream stimulating factor(USF), This suggests that the mechanisms underlying transcriptional regulation of chicken and mammalian IFN-${\gamma}$ genes may be similar.r.

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G-Networks Based Two Layer Stochastic Modeling of Gene Regulatory Networks with Post-Translational Processes

  • Kim, Ha-Seong;Gelenbe, Erol
    • Interdisciplinary Bio Central
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    • v.3 no.2
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    • pp.8.1-8.6
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    • 2011
  • Background: Thanks to the development of the mathematical/statistical reverse engineering and the high-throughput measuring biotechnology, lots of biologically meaningful genegene interaction networks have been revealed. Steady-state analysis of these systems provides an important clue to understand and to predict the systematic behaviours of the biological system. However, modeling such a complex and large-scale system is one of the challenging difficulties in systems biology. Results: We introduce a new stochastic modeling approach that can describe gene regulatory mechanisms by dividing two (DNA and protein) layers. Simple queuing system is employed to explain the DNA layer and the protein layer is modeled using G-networks which enable us to account for the post-translational protein interactions. Our method is applied to a transcription repression system and an active protein degradation system. The steady-state results suggest that the active protein degradation system is more sensitive but the transcription repression system might be more reliable than the transcription repression system. Conclusions: Our two layer stochastic model successfully describes the long-run behaviour of gene regulatory networks which consist of various mRNA/protein processes. The analytic solution of the G-networks enables us to extend our model to a large-scale system. A more reliable modeling approach could be achieved by cooperating with a real experimental study in synthetic biology.

Sinensetin Inhibits Interleukin-6 in Human Mast Cell - 1 Via Signal Transducers and Activators of the Transcription 3 (STAT3) and Nuclear Factor Kappa B (NF-κB) Pathways

  • Chae, Hee-Sung;Kim, Young-Mi;Chin, Young-Won
    • Natural Product Sciences
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    • v.23 no.1
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    • pp.1-4
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    • 2017
  • Sinensetin, a pentamethoxyflavone, is known to exert various pharmacological activities including anti-angiogenesis, anti-diabetic and anti-inflammatory activities. However, its effects on the human mast cell - 1 (HMC-1) mediated inflammatory mechanism remain unknown. To explore the mediator and cellular inflammatory response of sinensetin, we examined its influence on phorbol 12-myristate 13-acetate (PMA) plus A23187 induced inflammatory mediator production in a human mast cell line. In this study, interleukin (IL)-6 production was measured using the enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. Sinensetin inhibited PMA plus A23187 induced IL-6 production in a dose-dependent manner as well as IL-4, IL-5 and IL-8 mRNA expression. Furthermore, sinensetin inhibited signal transducer and activator of transcription 3 (STAT3) phosphorylation, suggesting that sinensetin inhibits the production of inflammatory mediators by blocking STAT3 phosphorylation. Moreover, sinensetin was found to inhibit nuclear factor kappa B activation. These findings suggest that sinensetin may be involved in the regulation of mast cell-mediated inflammatory responses.

Characterization of transcription factor genes related to cold tolerance in Brassica napus

  • Sharma, Mayur Mukut Murlidhar;Ramekar, Rahul Vasudeo;Park, Nam-Il;Choi, Ik-Young;Choi, Seon-Kang;Park, Kyong-Cheul
    • Genomics & Informatics
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    • v.19 no.4
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    • pp.45.1-45.8
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    • 2021
  • Brassica napus is the third most important oilseed crop in the world; however, in Korea, it is greatly affected by cold stress, limiting seed growth and production. Plants have developed specific stress responses that are generally divided into three categories: cold-stress signaling, transcriptional/post-transcriptional regulation, and stress-response mechanisms. Large numbers of functional and regulatory proteins are involved in these processes when triggered by cold stress. Here, our objective was to investigate the different genetic factors involved in the cold-stress responses of B. napus. Consequently, we treated the Korean B. napus cultivar Naehan at the 4-week stage in cold chambers under different conditions, and RNA and cDNA were obtained. An in silico analysis included 80 cold-responsive genes downloaded from the National Center for Biotechnology Information (NCBI) database. Expression levels were assessed by reverse transcription polymerase chain reaction, and 14 cold-triggered genes were identified under cold-stress conditions. The most significant genes encoded zinc-finger proteins (33.7%), followed by MYB transcription factors (7.5%). In the future, we will select genes appropriate for improving the cold tolerance of B. napus.

Dimethyloxaloylglycine promotes spermatogenesis activity of spermatogonial stem cells in Bama minipigs

  • Cao, Yaqi;Dai, ZiFu;Lao, Huizhen;Zhao, Huimin
    • Journal of Veterinary Science
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    • v.23 no.2
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    • pp.35.1-35.13
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    • 2022
  • Background: The testis has been reported to be a naturally O2-deprived organ, dimethyloxaloylglycine (DMOG) can inhibit hypoxia inducible factor-1alpha (HIF-1α) subject to degradation under normal oxygen condition in cells. Objectives: The objective of this study is to detect the effects of DMOG on the proliferation and differentiation of spermatogonial stem cells (SSCs) in Bama minipigs. Methods: Gradient concentrations of DMOG were added into the culture medium, HIF-1α protein in SSCs was detected by western blot analysis, the relative transcription levels of the SSC-specific genes were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Six days post-induction, the genes related to spermatogenesis were detected by qRT-PCR, and the DNA content was determined by flow cytometry. Results: Results revealed that the levels of HIF-1α protein increased in SSCs with the DMOG treatment in a dose-dependent manner. The relative transcription levels of SSC-specific genes were significantly upregulated (p < 0.05) by activating HIF-1α expression. The induction results showed that DMOG significantly increased (p < 0.05) the spermatogenesis capability of SSCs, and the populations of haploid cells significantly increased (p < 0.05) in DMOG-treated SSCs when compared to those in DMOG-untreated SSCs. Conclusion: We demonstrate that DMOG can promote the spermatogenesis activity of SSCs.

A genetic approach to comprehend the complex and dynamic event of floral development: a review

  • Jatindra Nath Mohanty;Swayamprabha Sahoo;Puspanjali Mishra
    • Genomics & Informatics
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    • v.20 no.4
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    • pp.40.1-40.8
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    • 2022
  • The concepts of phylogeny and floral genetics play a crucial role in understanding the origin and diversification of flowers in angiosperms. Angiosperms evolved a great diversity of ways to display their flowers for reproductive success with variations in floral color, size, shape, scent, arrangements, and flowering time. The various innovations in floral forms and the aggregation of flowers into different kinds of inflorescences have driven new ecological adaptations, speciation, and angiosperm diversification. Evolutionary developmental biology seeks to uncover the developmental and genetic basis underlying morphological diversification. Advances in the developmental genetics of floral display have provided a foundation for insights into the genetic basis of floral and inflorescence evolution. A number of regulatory genes controlling floral and inflorescence development have been identified in model plants such as Arabidopsis thaliana and Antirrhinum majus using forward genetics, and conserved functions of many of these genes across diverse non-model species have been revealed by reverse genetics. Transcription factors are vital elements in systems that play crucial roles in linked gene expression in the evolution and development of flowers. Therefore, we review the sex-linked genes, mostly transcription factors, associated with the complex and dynamic event of floral development and briefly discuss the sex-linked genes that have been characterized through next-generation sequencing.