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      Molecular cloning and expression analysis of the STAT1 gene from olive flounder, Paralichthys olivaceus

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          Abstract

          Background

          Signal transducer and activator of transcription 1 (STAT1) is a critical component of interferon (IFN)-alpha/beta and IFN-gamma signaling. Although seven isoforms of STAT proteins have been reported from mammals, limited information is available for the STAT genes in fish. We isolated complementary DNA with high similarity to mammalian STAT1 from the olive flounder, Paralichthys olivaceus.

          Results

          A DNA fragment containing the conserved SH2 domain was amplified by RT-PCR using degenerate primers designed based on the highly conserved sequences in the SH2 domains of the zebrafish and mammalian STAT1. The complete cDNA sequence was obtained by 5' and 3' RACE. The flounder STAT1 transcript consisted of 2,909 bp that encoded a polypeptide of 749 amino acids. The overall similarity between flounder STAT1 and other STATs was very high, with the highest amino acid sequence identity to snakehead (89%). Phylogenetic analyses reveal that flounder STAT1 is in the same monophyletic group with snakehead STAT1. Quantitative real time RT-PCR and in situ hybridization revealed that STAT1 was expressed in almost all examined organs and tissues, with high expression in gill, spleen, kidney, and heart. The accumulation of STAT1 mRNA in different developmental stages, as determined by real time RT-PCR, increased with development.

          Conclusion

          Recent cloning of various cytokine genes and the STAT1 gene of olive flounder here suggest that fish also use the highly specialized JAK-STAT pathway for cytokine signaling. Identification of other STAT genes will elucidate in detail the signal transduction system in this fish.

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          Most cited references27

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          Antiviral actions of interferons.

          C Samuel (2001)
          Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2',5'-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2'-5'-oligoadenylate-dependent RNase L, respectively, and also in RNA editing by the IFN-inducible RNA-specific adenosine deaminase (ADAR1). IFN also induces a form of inducible nitric oxide synthase (iNOS2) and the major histocompatibility complex class I and II proteins, all of which play important roles in immune response to infections. Several additional genes whose expression profiles are altered in response to IFN treatment and virus infection have been identified by microarray analyses. The availability of cDNA and genomic clones for many of the components of the IFN system, including IFN-alpha, IFN-beta, and IFN-gamma, their receptors, Jak and Stat and IRF signal transduction components, and proteins such as PKR, 2',5'-OAS, Mx, and ADAR, whose expression is regulated by IFNs, has permitted the generation of mutant proteins, cells that overexpress different forms of the proteins, and animals in which their expression has been disrupted by targeted gene disruption. The use of these IFN system reagents, both in cell culture and in whole animals, continues to provide important contributions to our understanding of the virus-host interaction and cellular antiviral response.
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            The JAK-STAT signaling pathway: input and output integration.

            Universal and essential to cytokine receptor signaling, the JAK-STAT pathway is one of the best understood signal transduction cascades. Almost 40 cytokine receptors signal through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Despite intense study, there remain substantial gaps in understanding how the cascades are activated and regulated. Using the examples of the IL-6 and IL-10 receptors, I will discuss how diverse outcomes in gene expression result from regulatory events that effect the JAK1-STAT3 pathway, common to both receptors. I also consider receptor preferences by different STATs and interpretive problems in the use of STAT-deficient cells and mice. Finally, I consider how the suppressor of cytokine signaling (SOCS) proteins regulate the quality and quantity of STAT signals from cytokine receptors. New data suggests that SOCS proteins introduce additional diversity into the JAK-STAT pathway by adjusting the output of activated STATs that alters downstream gene activation.
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              An overview of real-time quantitative PCR: applications to quantify cytokine gene expression.

              The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues. Copyright 2001 Elsevier Science (USA).
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                Author and article information

                Journal
                BMC Immunol
                BMC Immunology
                BioMed Central
                1471-2172
                2008
                26 June 2008
                : 9
                : 31
                Affiliations
                [1 ]Biotechnology Research Institute, National Fisheries Research and Development Institute, 408-1, Sirang-Ri, Gijang-Eup, Gijang-Gun, Busan, 619-902, Republic of Korea
                [2 ]Pathology Division, National Fisheries Research and Development Institute, 408-1, Sirang-Ri, Gijang-Eup, Gijang-Gun, Busan, 619-902, Republic of Korea
                [3 ]Department of Microbiology, Pukyong National University, 599-1 Daeyeon-Dong, Nam-Gu, Busan, 608-737, Republic of Korea
                [4 ]Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, 305-701, Republic of Korea
                Article
                1471-2172-9-31
                10.1186/1471-2172-9-31
                2443792
                18578892
                8ea41c0b-439e-4c37-90c4-4d19c0f8577a
                Copyright © 2008 Park et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 20 February 2008
                : 26 June 2008
                Categories
                Research Article

                Immunology
                Immunology

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