p53 33 HON G Yun2Han Manfred SCHARTL 48 (4) : , 2002 DNA ( Kastan et al., ( Martinez et al., 1991) (Lane et al., 1990),

Transcription

1 48 (4) : , 2002 A cta Zoologica S inica 3 p53 33 HON G Yun2Han Manfred SCHARTL (, ) ( Physiological Chemist ry I, U niversity of W uerzburg, W uerzburg, Germany) Long2PCR, 6 p53 DNA 6, 415 kb, 6 PCR p53 p kb, 11 10, p53 p53 p kb, p kb 6 kb ; p53 3 (86 bp) p53 3 (22 bp) ; 4 (170 bp) (280 bp) (260 bp) 4 ; 10 (315 kb) 10 (017 kb 019 kb) SV T K2 neo, SV T K2tk, p53, p53, G418 Ganc, G418 Ganc p53 p53, DNA ( Kastan et al., 1991 ; Lane, 1992), p53 p53, p53, ( Martinez et al., 1991) p53, p53, (Lane et al., 1990), p53 cdna, p53 p53, ( Zakut2Houri et al., 1983 ; Bienz et al., 1984 ; Lamb et al., 1986),,, 3 : (1) ; (2) ( Schartl, 1995),, p53 ; (3),, p53 cdna p53,,, p53 cdna p53 (Caron de Fromentel et al., 1992 ; Kazianis et al., 1998), p53 ( Chen et al., 2001) p , E2mail : ac. cn p53,,,, :

2 (Hong et al., 1996 ; Hong et al., 1998 ; Hong et al., 2000) p53 ( ), 35, min D NA DNA (MES1) pbluescript KS + MES1 Hong (1996) 10 cm, Alfexpress DNA (pharmacia), 3 ml ( 100 mmol/ L Tris2HCl, p H 815 ; 5 mmol/ L ED TA ; 012 % SDS ; 200 mmol/ L NaCl 100 g/ ml K) h DNA ) HSV2tk ( - ) 112 p53 cdna, Ganc) (Chen et al., 2002), 20 PCR, 6, p53, 6 N HSV2tk 40 : N1, 5 2 CA T GGA TCC TGT ACC CGA CCT G23 ; N8, 5 2A TC GAA TTC TGC A GT CGC CGA TTC AAA A TA TT23 ; N14, A TC GAA TTC A GT GTT ACA GTT CCT TA T GA G C23 ; N15, 5 2 A TC GGA TCC CA T TCT CA G GGC CAC TGC GGT CTA23 ; N21, 5 2 A TC GGA TCC TTC TTC TTT TCC CA G A TC AAC CTT23 ; N28, 5 2 A TC GGA TCC GGA AA T TTC A TC A GC TCA GTA AC23 ; C : C1, 5 2A TC TCT A GA CTT TA T TTA ACA A GG AA T TTG GTA C23 ;, 5 2CA G GTC GGG TAC A GG A TC CA T G23 ; C8, 5 2A TC TCT A GA CCA CGT GCT CCG TCT TCT TGT A GA23 ; C9, 5 2A TC TCT A GA GA G CA G GA T GGT GGT CA T CTC A GA23 ; C10, 5 2GGG TCT A GA TTG TTC CA T GCA TGT GTT CTT TAA T23 ; C11, 5 2CTA TCT A GA GCA CA G A TT CTG ACC TCG AA PCR Taq PCR Taq (112 kb), DNA 2 kb, Taqplus Long PCR ( Stratagene) 2 kb PCR : Tris2HCl, 20 mmol/ L, p H 912, 0125 mol/ L, dn TP 012 mmol/ L, DMSO 5 %, KCl 60 mmol/ L, MgCl 2 2 mmol/ L, DNA ng PCR MES min, s, ( ) 30 s, min 114 PCR PCR Q IA (Qiagen), 115, neo ( ( EPC), G418 Gancyclovir (, ( SV T K, 330 bp) B am H S al pmcl neopolya (Stratagene) neo, pbluescript KS + ( Stratagene) pneoa, SV T K pneoa S m a, psv T Kneo ; bp HSV2tk B gl neo psv T Kneo B gl, psv T K2tk SV T K2 neo SV T K2tk, 618 kb p53 DNA p53 DNA 2 kb 418 kb p53 ( p53 1), pbluescript KS +, pm53n32c10 (918 kb), SV T K2 neo pm53 + neo ( 11 kb) ; SV T K2tk (216 kb), pm53 + N2K (1316 kb) 116 (MES1) ( Hong et al., 1996) BioRad Easyject

3 4 : p g Not pm53 + N2K DNA 10 7, SV T K2 neo, 250 V,600 F 10 cm SV T K2tk, -, h,, pn K pn K G418 (500 g/ ml) G418 (500 g/ ml) Gancyclovir (015 g/ ml), EPC, G ,, EPC, - ( ), Giemsa, neo tk, p53 PCR p53, p53 ( 5) 4, N82C kb, N12C8 2 kb, N152C9 014 kb N142C11 1 kb, G418 Ganc ( 1), 7,, 7 11, N21, N21 C kb, 8 11 ; 10, N28, N28 C1 015 kb 3 ( 1) 6 p p53, p , bp ( 2) p53 p53, p53 4 p53 : (1) 1 (846 bp) (611 kb) (10 kb) 1 ; (2) 3 (86 bp) 3 (22 bp) ; (3) 4 (171 bp) 4 (260 bp 280 bp) ; (4) 10 (315 kb) (700 bp) (900 bp) 10 p53 p p53 4, GT 1 p53 PCR ( A) ( B), A G, Fig. 1 PCR fragments ( A) and structure of the medaka p53 gene and locations of PCR primers ( B) (Mount, 1982), p53, G418 Ganc 214 p53 G418 Ganc, 618 kb p53, SV T K2 neo, SV T K2tk,, G418 Gancyclovir 1, G418 M : DNA 1 : Primer N8 C2 2 : Primer N1 C8 3 : Primer N15 C9 4 : Primer N14 C11 5 : Primer N21 C10 6 : Primer N28 C1 ( Intron) ( Translated exon) (Untranslated exon)

4 p53 Fig. 2 Sequence of medaka p53 gene (Capital letters mean exons) (Lower case letters mean introns)

5 4 : p p53 Fig. 3 Comparison of the structure of exons and introns of p53 gene between medaka, mouse and human 3 Bienz (1984) Lamb (1986) (Data of mouse and human were from Bienz et al., 1984 and Lamb et al., 1986) 4 p53 - Fig. 4 Nucleotide sequence around the exon2intron junctions and sizes ( bp) of intron sequences within the medaka p53 gene (Weinberg, 1991) p53, cdna, (, Greenblatt et al., 1994), [Splice junctions between exon and intron sequences are indicated with colons. The cdna location of these sites are shown by the number above the corresponding letters. Nucleotides deviating from the consensus sequence ( shown at the bottom of the figure) are underlined. ] 5 p53 Fig. 5 Medaka p53 gene targeting vector 3 p53,, p53,, p53 cdna ( Kazianis et al1, 1998), p53 ( Chen et al., 2001), p53, p53 p53, p53 p53 G418 Gancyclovir, 11 10

6 , p53 Table 1 Transfection and positive2negative, selection of medaka ES cells G418 r G418 r 2 G418 r Treat2 Gc r G418 r 2Gc r ment Numbers of Numbers of G418 electropo2 surviving r G418 r 2 Ratio of colonies rated electropor2 Gc r G418 r to cells ation colonies G418 r 2Gc r ( ) Mansour (1988) -, Chen (2002) -, SV T K2 neo SV T K2tk ( EPC), -,, p53 1, SV T K2 neo (0185 kb) (10 kb) (6 kb) SV T K2tk 1, p53, G4182Gancyclovir (Lozano et al., 1991), (7 10), p53 ( ) p53 ( Horie et al., 1994 ; Johnson et al., 1989), p53 Bienz, B., R. Zakut2Houri, D ( References) Givol and M. Oren 1984 Analysis of the gene encoding the murine cellular tumour antigen p53. EMBO J. 3 : Caron de Fromentel, C., F. Pakdel, A. Chapes, C. Baney, P. May and T. Soussi 1992 Rainbow trout p53 : cdna cloning and biochemical characterization. Gene 112 : Chen, S. L., Y. Hong, S. J. Scherer and M. Schartl 2001 Lack of ultraviolet2light inducibility of the medakafish ( Oryzias latipes) tumor suppressor gene p53. Gene 264 (2) : Chen, S. L., Y. Hong and M. Schartl 2002 Development of positive2negative selection procedure for gene targeting in fish. press). A quacult ure (in Greenblatt, M. S., W. P. Bennett and M. Hollstein 1994 Mutations in the p53 tumour suppression gene : clues to cancer etiology and molecular pathogenesis. Cancer Res. 54 : Hong, Y., C. Winkler and M. Schartl 1996 Pluripotency and differentiation of embryonic stem cell lines from the medakafish ( Oryzias latipes). Mech. Dev. 60 : Hong, Y., C. Winkler and M. Schartl 1998 Production of medakafish chimeras from a stable embryonic stem cell line. Proc. N atl. Acad. Sci. USA 95 : Hong, Y., S. L. Chen and M. Schartl 2000 Embryonic stem cells in fish : current status and perspectives. Fish Physiol. Biochem. 22 : Horie, K, S., S. Nishiguchi, S. Maeda and K. Shimada 1994 Structure of replacement vectors for efficient gene targeting. J. Biochem. 115 : Johnson, R. S., M. Sheng, M. E. Greenberg, R. E. Kolodner, V. E. Papaioannou and B. M. Spiegelman 1989 Targeting of nonexpressed genes in embryonic stem cells via homologous recombination. Science 245 : Kastan, M. B., O. Onyekwere, D. Sidransky, B. Vogelstein and R. W. Craig 1991 Participation of p53 in the cellular response to DNA damage. Cancer Res. 51 : Kazianis, S., L. Gan, L. D. Coletta, B. Santi, C. Morizot and R. S. Nairn 1998 Cloning and comparative sequence analysis of TP53 in Xiphophorus fish hybrid melanoma models. gene 212 : Lamb, P. and L. Crawford 1986 Characterization of the human p53 gene. Mol. Cell. Biol. 6 : Lane, D. P p53, guardian of the genome. N at ure 358 :

7 4 : p Lane, D. P. and S. Benchimol 1990 p53 : oncogene or antioncogene. Genes Dev. 4 : 1 8. Lozano, G. and A. J. Levine 1991 Tissue2specific expression of p53 in transgenic mice is regulated by intron sequences. Mol. Carci nogenesis 4 : 3 9. Mansour, S. L., K. R. Thomas and M. R. Capecchi 1988 Disruption of the proto2oncogene int22 in mouse embryo2derived stem cells : a general strategy for targeting mutations to non2selectable gene. N at ure 336 : Martinez, J., I. Georgoff, J. Martinez and A. J. Levine 1991 Cellular localization and cell cycle regulation by a temperature2sensitive mutant of p53. Genes Dev. 5 : Mount, S A catalogue of splice junction sequences. N ucleic Aci ds Res. 10 : Schartl, M Platyfish and swordtails : a genetic system for the analysis of molecular mechanisms in tumor formation. Trends Genet. 11 : Weinberg, R. A Tumour suppression genes. Science 254 : Zakut2Houri, R., M. Oren, B. Bienz, V. Lavie, S. Hazum and D. Givol 1983 A single gene and a pseudogene for the cellular tumour antigen p53. N at ure 306 : ( Abstract) CLONING, STRUCTURAL ANALYSIS AND CONSTRUCTION OF HOMOLOGOUS RECOMBINATION VECTOR OF P53 GENE IN MEDAKA FISH ( O R YZIAS L A TI P ES) 3 CHEN Song2Lin 3 3 HON G Yun2Han and Manfred SCHARTL ( Yellow Sea Fisheries Research Instit ute, Chi nese Academy of Fishery Sciences, Qi ngdao , S handong, China) ( Physiological Chemist ry I, Biocenter of the U niversity of W uerzburg, A m Hubland, W uerzburg, Germany) The tumor suppressor p53 is critical for guarding the genome from the incorporation of damaged DNA. In mammals p53 functions in the regulation of the cell cycle, cell death and differentiation. Mutation of the p53 gene can lead to cell t ransformation and neoplasia. Similarly, cancer is also a f requent disease in fish. In analogy to the mammalian situation fish p53 was expected to play a similar crucial role in tumorigenesis. However, by inspecting p53 cdna sequences f rom t umors in different fish species, surprisingly not a single mutation was found. We have t herefore considered t he possibility t hat p53, unlike many ot her molecules which appear to play the same role in tumor development in higher and lower vertebrates, might have a different function in fish. The p53 gene was cloned from the medaka fish ( O ryzias lati pes ) using the long PCR technique. Genomic DNA was isolated from the medaka embryonic stem cell2like cell line MES1 according to the standard procedure. Six pairs of overlapping primers (N82C2, N12C8, N152C9, N142C11, N212C10 and N282C1) were used to amplify the medaka p53 gene were as follows : N1, 5 2 CA T GGA TCC TGT ACC CGA CCT G23 ; N8, 5 2A TC GAA TTC TGC A GT CGC CGA TTC AAA A TA TT23 ; N14, A TC GAA TTC A GT GTT ACA GTT CCT TA T GA G C23 ; N15, 5 2 A TC GGA TCC CA T TCT CA G GGC CAC TGC GGT CTA23 ; N21, 5 2 A TC GGA TCC TTC TTC TTT TCC CA G A TC AAC CTT23 ; N28, 5 2A TC GGA TCC GGA AA T TTC A TC A GC TCA GTA AC23 ; C1, 5 2 A TC TCT A GA CTT TA T TTA ACA A GG AA T TTG GTA C23 ;, 5 2CA G GTC GGG TAC A GG A TC CA T G23 ; C8, 5 2A TC TCT A GA CCA CGT GCT CCG TCT TCT TGT A GA23 ; C9, 5 2A TC TCT A GA GA G CA G GA T GGT GGT CA T CTC A GA23 ; C10, 5 2GGG TCT A GA TTG TTC CA T GCA TGT GTT CTT TAA T23 ; C11, 5 2CTA TCT A GA GCA CA G A TT CTG ACC TCG AA23. PCR products were cloned into the pbluescript KS + vector and sequenced with the automated Alfexpress DNA analysis system. Sequence analysis revealed that the medaka p53 gene is bp in length and consists of 11 3 This work was partially supported by a grant for returned scientists founded by State Ministry of Personnel 33 Corresponding author E2mail : ac. cn

8 exons. Comparison of t he genomic organization of t he p53 gene uncovered several differences between medaka and mammalian p53 genes : (1) Intron 1 (0185 kb) of the medaka p53 gene is much smaller than that from the mouse (6 kb) and human (10 kb) p53 gene ; (2) exon 3 (86 bp) is significantly larger than that of mammals (22 bp) ; (3) intron 10 (315 kb) in medaka p53 is 4 to 5 times larger than that in mice and humans. These data suggest that the medaka p53 gene differs from that in mice and humans in gene structure, especially in intron size. As the introns of the p53 gene in mammals contain important regulation elements and define some species specificity, t hese st ruct ural differences in t he sizes of int rons might point to a f unctional difference in t ranscriptional cont rol. For studying the function of p53 in fish, homologous recombination vectors were constructed on the basis of the neo and tk cassettes and a genomic fragment of p53 in multiple steps. First, two fragments (2 kb and 418 kb) of medaka p53 gene were amplified by Long2PCR, and ligated into basic plasmid pbluescript KS +, resulting in plasmid pm53n32c10 (918 kb). Secondly, t he 1122kb neo cassette gene was inserted at t he unique B am H site between the 2 kb and the 418 kb fragments in pm53n32c10 as positive selection marker. insertion disrupts the open reading frame of the p53 gene. The plasmid in which the orientation of neo was the same as t hat of t he p53 gene was designated as pm53 + neo ( 11 kb). The Finally, t he 2162kb tk cassette was inserted at the Xba site in pm53 + neo as a negative selection marker, generating homologous recombination vector pm53 + N2K (1316 kb). For transfection experiments, the plasmids pm53 + N2K were linearized with Not and transfected by electroporation. MES1 cells were plated in 10 cm2dishes at a density of 80 % confluence the day before electroporation. Cells of 350 l at cells/ ml were electroporated in the presence of 48 of plasmid DNA using a single pulse delivered from an Easyject electroporator set at 250 V, 600 F. Immediately after electroporation, the cells were diluted in 15 ml growth medium and plated in three 102cm dishes. For drug selection experiments, 48 h after electrotroporation, the cells were subjected to drug selection by G418 or G418 plus Ganc. Positive selection of pm53 + N2K t ransfected M ES1 cells resulted in G418 r clones, while dual selection with G418 plus Ganc resulted in G418 r 2Gc r clones. A ratio of between G418 r and G418 r 2Gc r colonies was obtained, demonstrating the feasibility of the p53 gene homologous recombination vector in M ES1 cells. Key words Medaka ( O ryzias lati pes), p53 gene, Structure, Homologous recombination vector, ES cell