Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tryptophan hydroxylase (TPH) is the rate limiting enzyme in serotonin biosynthesis [D.G. Grahame-Smith, Tryptophan hydroxylation in brain, Biochem. Biophys. Res. Commun. 16 (1964) 586-592 [19]]. As such, the TPH gene is a likely target for modulation of serotonergic function, which has been associated with several psychiatric disorders [E.C. Azmitia, P.M. Whitaker-Azmitia, Awakening the sleeping giant: anatomy and plasticity of the brain serotonergic system, J. Clin. Psychiatry 52 (12, Suppl.) (1991) 4-16 [1]; R.P. Hart, R. Yang, L.A. Riley., T.L. Green, Post-transcriptional control of tryptophan hydroxylase gene expression in rat brain stem and pineal gland, Mol. Cell. Neurosci. 2 (1991) 71-77 [20]; M.J. Owens, C.B. Numeroff, Role of serotonin in the pathophysiology of depression: focus on the serotonin transporter, Clin. Chem. 40 (1994) 288-295 [24]]. Unfortunately, it has been technically difficult to measure TPH mRNA levels in central serotonergic neurons due to its low levels. For example, detection with ribonuclease protection assays requires pooling of 5-10 dissected brainstems [M.C. Darmon, B. Guibert, V. Leviel, M. Ehret, M. Maitre, J. Mallet, Sequence of two mRNAs encoding active rat tryptophan hydroxylase, J. Neurochem. 51 (1988) 312-316 [15]; B.L. Jacobs, E.C. Azmitia, Structure and function of the brain serotonin system, Physiol. Rev. 72 (1992) 165-229 [21]]. This protocol describes the use of competitive RT-PCR to measure TPH mRNA levels from rat brain. First described in 1988, competitive RT-PCR has become an accepted method of measuring RNA abundance [M. Clementi, S. Menzo, P. Bagnarelli, A. Manzin, A. Valenza, P.E. Varaldo, Quantitative PCR and RT-PCR in virology, PCR Methods Appl. 2 (1994) 191-196 [12]; N.C.P. Cross, Quantitative PCR techniques and applications, Br. J. Haematol. 89 (1995) 693-697 [14]; K.P. Foley, M.W. Leonard, J.D. Engel, Quantitation of RNA using the polymerase chain reaction, Trends Genet. 9 (1993) 380-385 [17]; P.D. Siebert, J.W. Larrick, Competitive PCR, Nature 359 (1992) 558 [27]]. Competitive RT-PCR uses co-amplification with a known quantity of an in vitro transcribed RNA which amplifies using the same primers and thus competes for reactants with the product of interest. As the two products amplify with the same efficiency, the relative abundance of the two amplification products remains constant, and thus can be used to determine initial tissue TPH mRNA levels [G. Gilliland, S. Perrin, K. Blanchard, H.F. Bunn, Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 2725-2729 [18]; A.M. Wang, M. V. Doyle, D.F. Mark, Quantitation of mRNA by the polymerase chain reaction, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 9717-9721 [31]]. We first demonstrate equivalent results between RNA slot blots and competitive RT-PCR using the CA77 thyroid C cell line [M.S. Clark, A. F. Russo, Tissue-specific glucocorticoid regulation of tryptophan hydroxylase mRNA levels, Mol. Brain Res. 48 (1997) 346-354 [9]]. We then describe the use of competitive RT-PCR to measure TPH mRNA levels in RNA isolated from rat brain poly-A+ RNA.
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PMID:Measurement of tryptophan hydroxylase mRNA levels by competitive RT-PCR. 963 Jun 72

Tryptophan hydroxylase-2 (TPH2) catalyzes the synthesis of neuronal serotonin, a major neurotransmitter involved in many brain functions and psychiatric disorders. We have previously revealed a critical role of the human TPH2 (hTPH2) 5'-UTR in gene expression regulation. This study aimed to further characterize mechanism(s) by which the hTPH2 5'-UTR regulates gene expression. An internal ribosome entry site (IRES) activity in hTPH2 5'-UTR was suggested by the conventional bicistronic reporter assay; however, further stringent experiments, including in vitro translation, quantitative real-time PCR, Northern blot, ribonuclease protection assay, and monocistronic reporter assay, demonstrated that the hTPH2 5'-UTR harbors a bidirectional promoter, but not IRES, within its downstream segment (61-141). The antisense promoter is much stronger than the sense promoter, but the strength of both promoters are cell-line dependent, with the highest and lowest activities being observed in HEK-293T and SK-N-MC cells, respectively. In accordance with our previous findings, the upstream segment (1-60) of hTPH2 5'-UTR suppresses the neighboring promoter of both direction, independent of the cell line and its location in the 5'- or 3'-flanking regions of the gene. In summary, this study demonstrates that no IRES but an asymmetric bidirectional promoter is present in the downstream segment of hTPH2 5'-UTR, and this promoter is susceptible to a gene silencing effect caused by the upstream segment (1-60) of hTPH2 5'-UTR. Our findings point to the potential involvement of antisense transcription and non-coding RNA in the regulation of TPH2 gene expression.
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PMID:5'-Untranslated region of the tryptophan hydroxylase-2 gene harbors an asymmetric bidirectional promoter but not internal ribosome entry site in vitro. 1934 41