胸腺素原(Prothymosin alpha, Ptma) 是一種約110 個胺基酸，12.5 kDa，等電點3.5的高度酸性小型核蛋白。在細胞培養的實驗中，胸腺素原被認為與細胞增生、抗細胞凋亡，以及細胞附著相關，而在許多癌症的診斷上也能作為一個有價值的標記。但既使如此，胸腺素原的確切功能仍然不甚清楚。本實驗以斑馬魚為模式，探討胸腺素原在胚胎發育時期的表現與生物體內的功能。在比較了數種已知的胸腺素原胺基酸序列後，發現它在各物種間的相似度相當高，斑馬魚的胸腺素原只有105個胺基酸，比起其他物種要少，但與牛、豬等哺乳類有五成以上的相似度，和人類是六成，而與兩棲類的相似度更高達七成。而在反轉錄聚合酶連鎖反應與全胚胎原位雜交實驗中，發現胸腺素原為母系遺傳基因，在發育最初的單一細胞期就有表現，並且持續到發育後期；表現的位置隨著發育階段的改變，從最初未分化的囊胚細胞團，到發育初期神經管、腦部、晶體、三叉神經前驅、原腎管、皮膚、胸鰭芽胞、鰓弧、後部側線神經前驅細胞、血管，到發育後期的心臟、胸腺、消化道、泳鰾，除了在骨骼、肌肉和體節上沒有發現，基本上其表現與器官組織發育的進程大致相同，顯示胸腺素原在細胞增生與器官與組織的發育上扮演重要角色。將胸腺素原與紅螢光蛋白RFP結合，並以keratin 18 驅動過度表現於斑馬魚的表皮，建立轉殖品系Tg(k18:ptma:rfp)，簡稱KPR，發現螢光表現明顯集中於細胞核。BrdU的標記與紅螢光的位置重疊顯示出過度表現胸腺素原的細胞正在增生中，而轉殖品系KPR表現出較多的細胞增生反應，並產生較厚的皮膚。而在紫外線UV-B照射的實驗中，發現過量表現胸腺素原的轉殖品系在較低劑量的紫外線照射下，與野生種相比會產生略微嚴重的細胞凋亡現象，但是在較高劑量的照射後轉殖品系KPR產生的凋亡現象反而比野生種輕微許多，表示胸腺素原在細胞凋亡的途徑中同時具有促進與抑制的作用。由以上結果，我認為胸腺素原可能促進細胞增生，並且控制細胞凋亡。

Prothymosin alpha (ptma) is a small (110 a.a., 12.5 kDa) and highly acidic (pI 3.5) nuclear protein. In cell culture experiments, ptma shows to be involved with proliferation, anti-apoptosis, and cell attachment. It also could be a useful marker in cancer diagnosis. However, its biological functions in vivo are still unclear. Here, I used zebrafish as a model to investigate the developmental expression and biological function of Ptma. Zebrafish ptma cDNA was encoded a 105-amino-acid-polypeptide. This sequence identified more than 50% with reported Ptma of mammalians, 60% with human, and up to 70% with amphibian. In RT-PCR and whole-mount in situ hybridization showed that ptma was a maternally inherited gene expressed at 1-cell stage, and continued to 7 dpf. At early blustrula stage, it expressed in all blastomeres. At segmentation stages, the ptma transcripts were restricted in the future neural tube, retina, otic placode, trigeminal nerve soma, pronephric ducts and brains, but were not found in somites, notochord or muscles. During pharyngeal period, ptma signals were observed in pectoral fin buds, skin, dorsal aorta and primordial of the posterior lateral lines. At early larval period (3-7 dpf), the expression was observed in aortic arches, heart, liver, thymus, swim bladder and guts. Ptma expression was basically conformed to the process of tissues and organs development except skeletons and muscles. These data suggested that Ptma played a role in cell proliferation that might be important for organogenesis. The germ line Tg(k18:ptma:rfp) (KPR) was established to over-expressed Ptma by promoter keratin 18 restricted in zebrafish epidermis labeled with red fluorescence. This transgenic line did not have exterior defects. The RFP signal was limited in nucleus and co-localized with BrdU signal. It suggested that Ptma over-expressed-cell was proliferating. KPR had more BrdU signals in the epidermis and thicker skin than WT did (KPR 0.0179 mm; WT 0.0125 mm), that meant over-expressed Ptma would activate cell proliferation. After low-doseage of UV-B irradiation, KPR showed a little more apoptotic signals compared to WT embryos. However, WT had much more serious damages than KPR after high-doesage UB-B exposure. On the basis of these observations, I propose that Ptma might promote proliferation and regulate apoptosis.

Abstract (Chinese)......................................I
Abstract (English)	.....................................II
Content...............................................III
Chart list..............................................V

1. Introduction.........................................1
1.1 Discovery of Prothymosin alpha......................1
1.2 Structure of Prothymosin alpha......................1
1.3 Subcellular localization of prothymosin alpha.......2
1.4 Prothymosin alpha is regulated by several genes.....3
1.5 Prothymosin alpha is involved in cell 
    proliferation.......................................3
1.6 Prothymosin alpha plays different roles in 
    apoptosis...........................................4
1.7 Prothymosin alpha in tumor is used as a marker......5
1.8 Specific aims of this thesis........................6
2. Materials and Methods................................8
2.1 Materials...........................................8
2.2 Methods.............................................9
3. Results.............................................17
3.1 Isolation and nucleotide sequence analysis of 
   zebrafish ptma......................................17
3.2 Amino acid sequence analysis of zebrafish Ptma.....17
3.3 Spatiotemporal expression pattern of ptma during
   zebrafish development...............................18
3.4 Germ line Tg(k18:ptma:rfp) establishment...........20
3.5 Overexpression of ptma in skin does not cause 
   obvious morphological changes.......................21
3.6 Ptma expressed in the proliferating cells..........22
3.7 Overexpression of Ptma in skin led to a thicker
   epidermis...........................................22
3.8 Over-expressed Ptma in skin could anti-apoptosis
   via UV irradiation..................................23
3.9 Injection of Ptma mRNA did not cause abnormal 
   phenotype...........................................23
4. Discussion..........................................25
4.1 Ptma expressed at precursor cells ready for 
   organogenesis.......................................25
4.2 This is the first report about Ptma expression 
   at later developmental stages in zebrafish..........25
4.3 Ptma over-expression might promote
   proliferation.......................................26
4.4 Ptma over-expression could requlate apoptosis......28
5. Reference...........................................30



Fig. 1a Alignment of Ptma amino acid sequences of 
      9 organisms......................................37
Fig. 1b Phylogenetic tree of evolutionary relationship based on alignments of Ptma............................38
Fig. 2 Expression pattern of ptma in zebrafish embryos.39
Fig. 3 Tg(k18:ptma:rfp) (KPR) establishment............41
Fig. 4 Ptma was located in nucleus.....................42
Fig. 5 Cell-cell adhesion in KPR was normal as in WT...43
Fig. 6 BrdU treatment at 3 dpf.........................44
Fig. 7 H&E stain.......................................45
Fig. 8 UV irradiated 4 dpf KPR had broken nuclei.......46
Fig. 9a AO treatment labeled UV-induced apoptosis at 
      4 dpf............................................47
Fig. 9b UV irradiation with TUNEL treatment............47
Fig. 10 ptma mRNA injection............................48


Table 1 Primers........................................49
Table 2 Plasmids.......................................49
Table 3 Chemical reagents preparation..................50
Table 4 Amino acid sequences similarity (%)............51
Table 5 Germ line transmission.........................52



Appendix I Putative mimic domains of Ptma..............53 
Appendix II Selective model for Ptma target genes  
        implicated in cell death and survival pathways.54
Appendix III Camera lucida sketches of the embryo at 
        selected stages................................55

Aniello F, Branno M, De Rienzo G, Ferrara D, Palmiero C, Minucci S. First evidence of prothymosin alpha in a non-mammalian vertebrate and its involvement in the spermatogenesis of the frog Rana esculenta. Mech Dev. 2002. 110(1-2):213-7.

Barbini L, Gonzalez R, Dominguez F, Vega F. Apoptotic and proliferating hepatocytes differ in prothymosin alpha expression and cell localization. Mol Cell Biochem. 2006. 291(1-2):83-91. 

Bianco NR, Montano MM. Regulation of prothymosin alpha by estrogen receptor alpha: molecular mechanisms and relevance in estrogen-mediated breast cell growth.
Oncogene. 2002. 21(34):5233-44. 

Chen YH, Wang YH, Chang MY, Lin CY, Weng CW, Westerfield M, Tsai HJ. Multiple upstream modules regulate zebrafish myf5 expression. BMC Dev Biol. 2007. 7:1.

Clinton M, Graeve L, el-Dorry H, Rodriguez-Boulan E, Horecker BL. Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ. Proc Natl Acad Sci U S A. 1991. 88(15):6608-12.

Covelo G, Sarandeses CS, Diaz-Jullien C, Freire M. Prothymosin alpha interacts with free core histones in the nucleus of dividing cells. J Biochem. 2006. 140(5):627-37.

Desbarats L, Gaubatz S, Eilers M. Discrimination between different E-box-binding proteins at an endogenous target gene of c-myc. Genes Dev. 1996. 10(4):447-60.

Donizetti A, Liccardo D, Esposito D, Del Gaudio R, Locascio A, Ferrara D, Minucci S, Aniello F. Differential expression of duplicated genes for prothymosin alpha during zebrafish development. Dev Dyn. 2008. 237(4):1112-8.

Economou M, Seferiadis K, Frangou-Lazaridis M, Horecker BL, Tsolas O.  Isolation and partial characterization of prothymosin alpha from porcine tissues. FEBS Lett. 1988. 233(2):342-6.

Eilers M, Schirm S, Bishop JM. The MYC protein activates transcription of the alpha-prothymosin gene. EMBO J. 1991. 10(1):133-41.

Eschenfeldt WH, Berger SL. The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA. Proc Natl Acad Sci U S A. 1986. 83(24):9403-7.

Evstafieva AG, Belov GA, Kalkum M, Chichkova NV, Bogdanov AA, Agol VI, Vartapetian AB. Prothymosin alpha fragmentation in apoptosis. FEBS Lett. 2000. 467(2-3):150-4.

Evstafieva AG, Belov GA, Rubtsov YP, Kalkum M, Joseph B, Chichkova NV, Sukhacheva EA, Bogdanov AA, Pettersson RF, Agol VI, Vartapetian AB. Apoptosis-related fragmentation, translocation, and properties of human prothymosin alpha. Exp Cell Res. 2003. 284(2):211-23.

Franco del Amo F, Freire M. The prothymosin alpha gene is specifically expressed in ectodermal and mesodermal regions during early postimplantation mouse embryogenesis. FEBS Lett. 1995. 359(1):15-9.

Frillingos S, Frangou-Lazaridis M, Seferiadis K, Hulmes JD, Pan YC, Tsolas O. Isolation and partial sequence of goat spleen prothymosin alpha. Mol Cell Biochem. 1991. 108(1):85-94.

Gast K, Damaschun H, Eckert K, Schulze-Forster K, Maurer HR, Müller-Frohne M, Zirwer D, Czarnecki J, Damaschun G. Prothymosin alpha: a biologically active protein with random coil conformation. Biochemistry. 1995. 34(40):13211-8.

Haritos AA, Goodall GJ, Horecker BL. Prothymosin alpha: isolation and properties of the major immunoreactive form of thymosin alpha 1 in rat thymus. Proc Natl Acad Sci U S A. 1984. 81(4):1008-11.

Haritos AA, Blacher R, Stein S, Caldarella J, Horecker BL. Primary structure of rat thymus prothymosin alpha. Proc Natl Acad Sci U S A. 1985. 82(2):343-6.


Karetsou Z, Kretsovali A, Murphy C, Tsolas O, Papamarcaki T. Prothymosin alpha interacts with the CREB-binding protein and potentiates transcription. EMBO Rep. 2002. 3(4):361-6. 

Karetsou Z, Sandaltzopoulos R, Frangou-Lazaridis M, Lai CY, Tsolas O, Becker PB, Papamarcaki T. Prothymosin alpha modulates the interaction of histone H1 with chromatin. Nucleic Acids Res. 1998. 26(13):3111-8.

Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Dev Dyn. 1995. 203(3):253-310.

Kobayashi T, Wang T, Maezawa M, Kobayashi M, Ohnishi S, Hatanaka K, Hige S, Shimizu Y, Kato M, Asaka M, Tanaka J, Imamura M, Hasegawa K, Tanaka Y, Brachmann RK. Overexpression of the oncoprotein prothymosin alpha triggers a p53 response that involves p53 acetylation. Cancer Res. 2006. 66(6):3137-44.

Komiyama T, Pan LX, Haritos AA, Wideman JW, Pan YC, Chang M, Rogers I, Horecker BL. The primary structure of rat parathymosin. Proc Natl Acad Sci U S A. 1986. 83(5):1242-5.

Lee HC, Huang HY, Lin CY, Chen YH, Tsai HJ. Foxd3 mediates zebrafish myf5 expression during early somitogenesis. Dev Biol. 2006. 290(2):359-72.

Lele Z, Krone PH. The zebrafish as a model system in developmental, toxicological and transgenic research. Biotechnol Adv. 1996;14(1):57-72.

Ledent V. Postembryonic development of the posterior lateral line in zebrafish. Development. 2002. 129(3):597-604.

Letsas KP, Frangou-Lazaridis M. Surfing on prothymosin alpha proliferation and anti-apoptotic properties. Neoplasma. 2006;53(2):92-6.

Leys CM, Nomura S, LaFleur BJ, Ferrone S, Kaminishi M, Montgomery E, Goldenring JR. Expression and prognostic significance of prothymosin-alpha and ERp57 in human gastric cancer. Surgery. 2007. 141(1):41-50. 

Magdalena C, Dominguez F, Loidi L, Puente JL. Tumour prothymosin alpha content, a potential prognostic marker for primary breast cancer. Br J Cancer. 2000, 82(3):584-90.

Manrow RE, Sburlati AR, Hanover JA, Berger SL. Nuclear targeting of prothymosin alpha. J Biol Chem. 1991. 266(6):3916-24.

Markova OV, Evstafieva AG, Mansurova SE, Moussine SS, Palamarchuk LA, Pereverzev MO, Vartapetian AB, Skulachev VP. Cytochrome c is transformed from anti- to pro-oxidant when interacting with truncated oncoprotein prothymosin alpha.
Biochim Biophys Acta. 2003. 1557(1-3):109-17.

Martini PG, Delage-Mourroux R, Kraichely DM, Katzenellenbogen BS.  Prothymosin alpha selectively enhances estrogen receptor transcriptional activity by interacting with a repressor of estrogen receptor activity. Mol Cell Biol. 2000. 20(17):6224-32.

Nowak M, Ko:ster C, Hammerschmidt M. Perp is required for tissue-specific cell survival during zebrafish development. Cell Death Differ. 2005. 12(1):52-64.

Ojima E, Inoue Y, Miki C, Mori M, Kusunoki M. Effectiveness of gene expression profiling for response prediction of rectal cancer to preoperative radiotherapy. J Gastroenterol. 2007. 2(9):730-6.

Orre RS, Cotter MA 2nd, Subramanian C, Robertson ES. Prothymosin alpha functions as a cellular oncoprotein by inducing transformation of rodent fibroblasts in vitro. J Biol Chem. 2001. 276(3):1794-9. 

Piacentini M, Evangelisti C, Mastroberardino PG, Nardacci R, Kroemer G. Does prothymosin-alpha act as molecular switch between apoptosis and autophagy?
Cell Death Differ. 2003. 10(9):937-9. 

Pineiro A, Cordero OJ, Nogueira M. Fifteen years of prothymosin alpha: contradictory past and new horizons. Peptides. 2000. 21(9):1433-46. Review.

Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, Farber SA, Ekker SC. p53 activation by knockdown technologies. PLoS Genet. 2007. 3(5):e78.

Rodriguez P, Viñuela JE, Alvarez-Ferna'ndez L, Buceta M, Vidal A, Domínguez F, Gómez-Márquez J. Overexpression of prothymosin alpha accelerates proliferation and retards differentiation in HL-60 cells. Biochem J. 1998. 331 (3):753-61.


Rubtsov YP, Zolotukhin AS, Vorobjev IA, Chichkova NV, Pavlov NA, Karger EM, Evstafieva AG, Felber BK, Vartapetian AB. Mutational analysis of human prothymosin alpha reveals a bipartite nuclear localization signal. FEBS Lett. 1997. 413(1):135-41.

Sarandeses CS, Covelo G, Díaz-Jullien C, Freire M. Prothymosin alpha is processed to thymosin alpha 1 and thymosin alpha 11 by a lysosomal asparaginyl endopeptidase. J Biol Chem. 2003. 278(15):13286-93.

Sasaki H, Nonaka M, Fujii Y, Yamakawa Y, Fukai I, Kiriyama M, Sasaki M. Expression of the prothymosin-a gene as a prognostic factor in lung cancer. Surg Today. 2001. 31(10):936-8.

Sburlati AR, De La Rosa A, Batey DW, Kurys GL, Manrow RE, Pannell LK, Martin BM, Sheeley DM, Berger SL. Phosphorylation of human and bovine prothymosin alpha in vivo. Biochemistry. 1993. 32(17):4587-96.


Sburlati AR, Manrow RE, Berger SL. Prothymosin alpha antisense oligomers inhibit myeloma cell division. Proc Natl Acad Sci U S A. 1991. 88(1):253-7.

Segade F, Gómez-Márquez J. Prothymosin alpha. Int J Biochem Cell Biol. 1999. 31(11):1243-8. Review.

Skopeliti M, Voutsas IF, Klimentzou P, Tsiatas ML, Beck A, Bamias A, Moraki M, Livaniou E, Neagu M, Voelter W, Tsitsilonis OE. The immunologically active site of prothymosin alpha is located at the carboxy-terminus of the polypeptide. Evaluation of its in vitro effects in cancer patients. Cancer Immunol Immunother. 2006. 55(10):1247-57.

Suzuki S, Takahashi S, Takahashi S, Takeshita K, Hikosaka A, Wakita T, Nishiyama N, Fujita T, Okamura T, Shirai T. Expression of prothymosin alpha is correlated with development and progression in human prostate cancers. Prostate. 2006. 66(5):463-9.

Tsay HJ, Wang YH, Chen WL, Huang MY, Chen YH. Treatment with sodium benzoate leads to malformation of zebrafish larvae. Neurotoxicol Teratol. 2007. 29(5):562-9.


Tzai TS, Tsai YS, Shiau AL, Wu CL, Shieh GS, Tsai HT. Urine prothymosin-alpha as novel tumor marker for detection and follow-up of bladder cancer.
Urology. 2006. 67(2):294-9.

Vareli K, Frangou-Lazaridis M. Prothymosin alpha is localized in mitotic spindle during mitosis. Biol Cell. 2004. 96(6):421-8.

Vareli K, Frangou-Lazaridis M, van der Kraan I, Tsolas O, van Driel R. Nuclear distribution of prothymosin alpha and parathymosin: evidence that prothymosin alpha is associated with RNA synthesis processing and parathymosin with early DNA replication. Exp Cell Res. 2000. 257(1):152-61.

Vareli K, Tsolas O, Frangou-Lazaridis M. Regulation of prothymosin alpha during the cell cycle. Eur J Biochem. 1996. 238(3):799-806.

Wang M, Pan JY, Song GR, Chen HB, An LJ, Qu SX. Altered expression of estrogen receptor alpha and beta in advanced gastric adenocarcinoma: correlation with prothymosin alpha and clinicopathological parameters. Eur J Surg Oncol. 2007. 33(2):195-201.

Wang YH, Chen YH, Lin YJ, Tsai HJ. Spatiotemporal expression of zebrafish keratin 18 during early embryogenesis and the establishment of a keratin 18:RFP transgenic line. Gene Expr Patterns. 2006a. 6(4):335-9. 

Wang YH, Chen YH, Wu TN, Lin YJ, Tsai HJ. A keratin 18 transgenic zebrafish Tg(k18(2.9):RFP) treated with inorganic arsenite reveals visible overproliferation of epithelial cells. Toxicol Lett. 2006b. 163(3):191-7.

Wang YH, Li CK, Lee GH, Tsay HJ, Tsai HJ, Chen YH. Inactivation of zebrafish mrf4 leads to myofibril misalignment and motor axon growth disorganization. Dev Dyn. 2008. 237(4):1043-50.

Westerfield M. The zebrafish book. Third edition. University of Oregon Press; 1995.

Wilson CL, Monteith WB, Danell AS, Burns CS. Purification and characterization of the central segment of prothymosin-alpha: methodology for handling highly acidic peptides. J Pept Sci. 2006. 12(11):721-5.

Wu CG, Boers W, Reitsma PR, van Deventer SJ, Chamuleau RA. Overexpression of prothymosin alpha, concomitant with c-myc, during rat hepatic carcinogenesis. Biochem Biophys Res Commun. 1997. 232(3):817-21.

Wu CL, Shiau AL, Lin CS. Prothymosin alpha promotes cell proliferation in NIH3T3 cells. Life Sci. 1997. 61: 2091–2101 

Yang CH, Murti A, Baker SJ, Frangou-Lazaridis M, Vartapetian AB, Murti KG, Pfeffer LM. Interferon induces the interaction of prothymosin-alpha with STAT3 and results in the nuclear translocation of the complex. Exp Cell Res. 2004. 298(1):197-206.

Yu TH, Chen YH. Sonic hedgehog signaling promotes cell cycle progression and consequently causes epidermis dysplasia of the zebrafish embryos. Tamkang University. 2007.

Zhao R, Gish K, Murphy M, Yin Y, Notterman D, Hoffman WH, Tom E, Mack DH, Levine AJ. Analysis of p53-regulated gene expression patterns using oligonucleotide arrays. Genes Dev. 2000. 14(8):981-93.