依據本研究室研究證明中樞血清素( 5-hydroxytryptamine, Seroto
nin, 5-HT )系統參與吳郭魚腦部的發育。目前已知在哺乳類動中血清
素接受體( receptor )家族包括有七種家族( 5-HT1-7 )，其中之5-HT1A
接受體能促進神經細胞的增生，在中樞神經發育上扮演重要的角色，
因此本實驗針對5-HT1A接受體基因啟動子( promoter )進行瞭解。為
瞭解5-HT1A接受體的基因調控，我們經由5’ rapid amplification of cD
NA end ( 5’ RACE )與Genome Walker DNA Walking定出轉錄起始點
( transcription start site, TSS )所在位置，並往前定出1633 bp的DNA
序列，於5’端挑選出六個不同長度的片段，分別構築在含有螢火蟲
冷光( firefly luciferase )報導基因的載體，並進行轉染( transfection )至
Human cervical epithelioid carcinoma(HeLa)細胞中表現。藉由冷光(
luciferase )的活性分析選得具啟動基因表現關聯性之片段，經由轉錄
因子結合位之生物資訊分析得知由-52 bp至-46 bp可能有的activator
protein-1 ( AP-1 )結合位置，隨後利用定位點突變( site-direct mutagen
esis )及電泳能力偏移實驗( Electrophoresis mobility shift assay )進行分
析，實驗結果得知在-52 bp至-46 bp確實至少具有兩種蛋白質之鍵結
位，預測可能是AP-1。

The central 5-hydroxytryptamine ( Serotonin, 5-HT ) system play an
important role in the brain sexual differentiation in tilapia,Oreochromis
mossambicus. 　The 5-HT1A receptor is involved in the neural develop-
ment of brain.　The cDNA sequences of 5-HT1A receptor of tilapia, Ore-
ochromis mossambicus have been cloned in our laboratory.　In the
present study, 1633 bp of DNA sequence of 5-HT1A receptor from the
transcription start site ( TSS ) were cloned by 5’ rapid amplification of
cDNA end ( 5’ RACE ) and Genome Walker DNA Walking.　The 1633
bp of DNA sequence divided into six different fragments to detect the
luciferase activity in the HeLa cell.　The bioinformatic analysis was
performed on the fragment which showed the highest luciferase activity
for predicting the transcription factor binding site.　These results showed
that there is a activator protein-1 ( AP-1 ) binding site in the fragment
form -52 bp to -46 bp.　The site-direct mutagenesis and electrophoresis
mobility shift assay was performed on the fragment form -52 bp to -46 bp
.　The present results indicate that there is a transcription factor binding
site in the fragment form -52 bp to -46 bp.　This transcription factor
binding site could be a AP-1 binding site.

致謝---------------------------------------------------------------------------- 1
中文摘要---------------------------------------------------------------------- 2
英文摘要---------------------------------------------------------------------- 4
目錄---------------------------------------------------------------------------- 5
附圖目錄---------------------------------------------------------------------- 6
附表目錄---------------------------------------------------------------------- 7
前言---------------------------------------------------------------------------- 8
實驗材料與方法------------------------------------------------------------- 12
結果---------------------------------------------------------------------------- 44
討論---------------------------------------------------------------------------- 51
參考文獻---------------------------------------------------------------------- 53
附圖---------------------------------------------------------------------------- 61
附表---------------------------------------------------------------------------- 72

Albert, P.R., Zhou, Q.Y., Van Tol, H.H.M(1990). Cloning, functional expression and messenger RNA tissue distribution of the rat 5-hydroxytryptamine1A receptor gene. J. Biol. Chem. 265, 5825–5832.
Albert PR, Storring JM, Charest A, Cheng P.,(1999). TATA-driven transc-
riptional initiation and regulation of the rat serotonin 5-HT1A receptor
gene. J Neurochem. 1999 Jun;72(6):2238-47.
Arnold, A. P. and R. A. Gorski(1984).〝Gonadal steroid induction of
structural sex differences in the central nervous system.〞Ann. Rev.
Neurosci. 7：413-422.
Azmitia, EC (2001). Modern views on an ancient chemical: serotonin
effects on cell proliferation, maturation, and apoptosis. Brain Res Bull.
2001 Nov 15;56(5):413-24.
Barnes, NM and Sharp, T (1999).A review of central 5-HT receptors and
their function. Neuropharmacology. 1999 Aug;38(8):1083-152.
Blier P, de Montigny C, Chaput Y (1987).Modifications of the serotonin
system by antidepressant treatments; implication for the therapeutic
response in major depression. J Clin Psychopharmacl. 7:24S-35S
Bolliet, V., Perreault, S. and Ali, M.A.(1994).Development of serotonerg-
icneurons in the brain of the mackerel, Scomber scombrus. An immu
ohistochemical study. J. Fish Biol. 44：241-253.Chamey DS, Krystal
JH, Delgado
PL, Heninger CR (1990).Serotonin-specific drugs for anxiety and dresssive
disorders. Annu Rev Med 41:437-446.
Ching-Lin Tasi, Li-Hsueh Wang, Yi-Hua Lin.(2001).Effects of estrogen
and neurotransmitters on the primary culture of tilapia brain from
different age. Developmental Brain Research. 129：111-113.
Curzon G (1992). Serotonin and eating disorders：pharmacological
relationships. In：Serotonin receptor subtypes： pharmacological
sig-nificance and clinical implications & anger SZ, Brunello N, Racagn
i Demchyshyn L, Sunahara RK, Miller K.
Ekstrom, P. and Van Veen, T., (1984).Distribution of 5-hydroxytryptamine
(serotonin) in the brain of teleost, Gasterosteus acukeatus, L.J.Comp.
Neuro., 226：307-320.
Fargin, A., Raymond, J.R., Lohse, M.J., et al.,(1988). The genomic clone
G-21 which resembles a b-adrenergic receptor sequence encodes the
5-HT 1A receptor. Nature. 335, 358–360.
Gould, E.(1999). Serotonin and hippocampal neurogenesis. Neuropsycho
pharmacology. Aug;21(2 Suppl):46S-51S.
Gorski, R.A., Gorden, J.H., Shryne, J.E. and Southam, A.M. (1978). Evid
ence for a morphological sex difference within the medial preoptic area
of the rat brain. Brain Res., 148：333-346.
Guan, X.M., Peroutka, S.J., Kobilka, B.K,(1992). Identification of a
single amino acid residue responsible for the binding of a class of
beta-adrenergic receptor antagonists to 5-hydroxytryptamine1A recepto
rs. Mol. Pharmacol. 41, 695–698.
Haydon PG. McCobb DP, Kater SB. (1984). Serotonin selectively inhitits
growthcone motility and synaptogenesis of specific identified neuron.
Science. 226：561-564.
Hensler JG, Kovachich GB, Frazer A.(1991). A quantitative autoradiogra
phic study of serotonin1A receptor regulation: Effect of 5,7-dihydroxyt
ryptaamine and antidepressant treatments. Neuropsychopharmacology
4,131–44.
Hoyer, D., Clarke, D.E., Fozard, J.R., et al.,(1994). International union
of pharmacological classification of receptors for 5-hydroxytryptamine
(serotonin). Pharmacol. Rev. 46, 157–193.
Hoyer, D., Pazos, A., Probst, A., Palacios, J.M., (1986). Serotonin revep
tors in the human brain. I. Characterisation and autoradiographic locali
sation of 5-HT1A recognition sites, apparent absence of 5-HT1B recog
nition sites. Brain Res. 376, 85–96.
Jarzab B, Kokocinska D, Kaminsiki M, Gubala E, Achtelik W, Wagiel
J,ohler KD.( 1990) Influence of neurotransmitters on sexual diffe
rentiation of the brain：relationship between the volume of the SDN
-POA and functional characteristics. Comp Physiol Biochem,8：41-50.
Johnsin, M.D. and Crowley, W.R. (1983). Acute effects of estradiol on
circulating luteinizing hormone and prolactin concentrations and on
serotonin turnover in individual brain nuclei. Endocrinology. 113：
1935-1941.
Kah,O and Chambolle, P.,(1983) Serotonin in the brain of goldfish,
Carasi us auratus：An immunohistochemical study. Cell Tissue Res.
234：319-333.
Khan, I.A. and Thomas, P.(1993) Immunocytochemical localization of
serotonin and gonadotropin-releasing hormone in the brain and pituitar
y gland of atlantic coaker. Micropogonias undulatus. Gen.Comp. Endo
crin ol. 91；167-180.
Khawaja, X.(1995) Quantitative autoradiographic characterisation of
the binding of [3H]WAY-100 635, a selective 5-HT1A receptor antag
onist. Brain Res. 673, 217–225.
Kia, H. K.; Brisorgueil, M. J.; Hamon, M.; Calas, A.; Verge, D.(1996).
Ultrastructural localization of 5-hydroxytryptamine1A receptors in the
rat brain. J.Neurosci. Res. Dec 15;46(6):697-708.
Kia HK, Miquel MC, Brisorgueil MJ, Daval G, Riad M, El Mestikawy W
, Hamon M, Verge D.(1996).Immunocyotchemical localization of serot
onin1A receptors in the rat central nervous system. Journal of Compar
ative Neurology.365:289–305.
Kobilka, B.K., Frielle, T., Collins, S., et al.,(1987). An intronless gene
encoding a potential member of the family of receptors coupled to
guanine nucleotide regulatory proteins. Nature. 329, 75–77.
Kung, M.P., Frederick, D., Mu, M., et al.(1995). 4-(2%-Methoxyphenyl)-
1-[2%-(n-2%%-pyridinyl)-p-iodobenzamido]-ethyl-piperazine ( [125I]
p-MPPI ) as a new selective radioligand of serotonin1A sites in rat brai
n: in vitro binding and autoradiographic studies. J.Pharmacol. Exp.
Ther. 272, 429–437.
Lauder JM, Krebs H. (1978). Serotonin as a differentiation signal in early
neurogenesis. Dev Neurosci. 1：15-30.
Lakosky, W. and Gaxiri, L.C.(1970). Brain serotonin and sexuall different
iation of the nervous system. Neuroendocrinology. 3：168-174.
Lauder JM, Wallace JA, Krebs H, Petrusz P, McCarthy K.(1982) In vivo
and in vitro development of serotonergic neurons. Brain Res Bull. Jul-
Dec;9(1-6):605-25.
Li-hsuen Wang and Ching-Lin Tsai .(2000) Temperature affects the deve
lopment of central nrurotransmitter system of tilapia, Oreochromis
mossambicus. Neuroscience Letters. 285：95-98.
M.M. Rapport, A.A. Green, and I.H. Page.(1984). Serum vasoconst
rictor (serotonin). IV. Isolation and characterization J. Biol. Chem. 176:
1243-1251. 1948.
Riad M, Emerit MB, Hamon M.(1994) Neurotrophic effects of ipsapirone
and other 5-HT1A receptor agonists on septal cholinergic neurons in
culture. Brain Res Dev Brain Res. Oct 14;82(1-2):245-58.
Radley JJ and Jacobs BL.(2002) 5-HT1A receptor antagonist administrat-
ion decreases cell proliferation in the dentate gyrus. Brain Res. Nov
15;955(1-2):264-7.
Radja, F., Laporte, A.-M., Daval, G.(1991). Autoradiography of serotonin
receptor subtypes in the central nervous system. Neurochem. Int. 18,
1–15.
Sai, C.-L. ,Wang, L.-H., Chang C.-F. and Kao C.C.(2000). Effects of
Gonadal Steroids on Brain Serotonergic and AromataseActivity During
the Critical Period of Sexual Differentiation in Tilapia, Oreochromis
Mossambicus. Journal of Neuroendocrinology. 49：894-898.
Shenk, T. and Parks C.L., (1996). The serotonin 1a receptor gene contains
a TATA-less promoter that responds to MAZ and Sp1. J Biol Chem.
Feb 23;271(8):4417-30.
Simerly, R.B, Swanson, L.M. and Gorski, R.A.(1985). Reversal of the
sexually dimorphic distrution of serotonin-immunoreactivity fibers in
the medical preoptic nucleus by treatment with perinatal androgen.
Brain Res. 340：139-143
Parent, A., Dube, L., Braford, M.R. and Northcutt, R.G. (1978). The
organiz ation of monoamine-containing neurons in the brain of the
sunfish(Lepo mis gibbosus) as revealed by fluorescence micrscopy. J.
Comp. Neurol. 182：495-516.
Pazos, A., Cortes, R., Palacios, J.M.(1985). Quantitative autoradiogra
phic mapping of serotonin receptors in the rat brain. II. Serotonin-2
receptors. Brain Res. 346, 231–249.
Somoza, G.M., Yu, K.L. and Peter, R.E.(1988). Serotonin stimulates
gonadotropin release in female and male goldfish, Carassius auratus.
L. Gen. Copm. Endocrin, 72：374-382.
Somoza, G.M. and Peter, R.E.(1991). Effects of serotonin on gonadotr-
opin and growth hormone release form perifused goldfish pituitary
fragment. Gen. Copm. Endocrin. 82：103-110.
Tsai CL, Jang TH, Wang LH.(1995) Effects of mercury on serotonin
concentration in the brain of tilapia, Oreochromis mossambicus. Neur
osci Lett. Jan 30;184(3):208-11.
Tsai CL, Wang LH.(1997). Effects of androgens replacement on the
serotonin and norepinepherine content in the brain of male tilapia,
Oreochromis mossambicus. Biogenic Amines. Mar 14；13(2)：109-122.
Tsai CL, Wang LH. (1997). Effects of Estradiol on the Serotonin and
Turnover in the Hypothalamus of Male Tilapia, Oreochromis mossam
bicus, in Vitro. General and Comparative Endocrimology. 106：175-180.
Tsai CL, Wang LH.(1999) Effects of gonadal steroids on the serotonin
synthesisand metabolism in the early developing tilapia brain. Neuros
cience Letters. 264：45-48.
Tsai CL, Wang LH.(1997). Effects of thermal acclimation on the neurotr
ansmitters, serotonin and norepinephrine in the discrete brain of male
and female tilapia, Oreochromis mossambicus. Neurosci Lett. Sep
19;233(2-3):77-80.
Tsai, C.L. ,Wang, L.H. and WEN Z.H.(2000). Alteration of norepineph-
riine and serotonin content in the discrete brain of male and female
tilapia, Oreochromis mossambicus, during the low temperature acclima
tion. Biogenic Amines. Vol 15, No. 6, pp. 655-667.
Verge, D., Daval, G., Marcinkiewicz, M., et al.(1986).Quantitative autora
diography of multiple 5-HT1 receptor subtypes in the brain of control
or 5,7-dihydroxytryptamine-treated rats. J. Neurosci. 6, 3473–3482.
Verge´ D, Daval G, Marcinkiewicz M, Patey A, El Mestikawy S, Gozlan H, Hamon M.(1986). Quantitative autoradiography of multiple 5-HT
1 receptor subtypes in the brain of control or 5,7-dihydroxy tryptamine
-treated rats. Journal of Neuroscience. 6:3474–82.
Weissmann-Nanopoulus, D., Mach, E., Margre, J., et al.(1985).Evidence
for the localisation of 5-HT1A binding sites on serotonin containing
neurones in the raphe dorsalis and raphe centralis nuclei of the rat brain.
Neurochem. Int. 7, 1061–1072.
Yu, K.L., Rosenblum, P.M. and Peter, R.E., 1991. In vitro release of gonad
otropin-releasing hormone and pituitary of female goldfish, Carassius
auratus. L. Gen. Copm. Endocrin. 81：256-267.