- Email: [email protected]
Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos
MARINE ENVIRONMENTAL RESEARCH Marine Environmental Research 58 (2004) 681–685 www.elsevier.com/locate/marenvrev
Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos Yan Li a, Il-Chan Kim a, Young Ja Kim b, Moon Kyoo Kim c, Yong-Dal Yoon c, Yong-Sung Lee a, Jae-Seong Lee b,* a
Department of Biochemistry, College of Medicine, Hanyang University, Seoul 133-791, Republic of Korea b Department of Environmental Science, Graduate School, Hanyang University, Seoul 133-791, Republic of Korea c Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea
Abstract We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus kGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced 7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFjB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 30 UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms’ c-fos genes. We propose its potential use in ecotoxicology. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Rivulus marmoratus; c-fos; Gene; Cloning; Promoter; MRE; XRE
*
Corresponding author. Tel.: +82-2-2290-0769; fax: +82-2-2299-9450. E-mail address: [email protected] (J.-S. Lee).
0141-1136/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2004.03.062
682
Y. Li et al. / Marine Environmental Research 58 (2004) 681–685
c-fos is well-known as an immediate early gene, and its biochemical characteristics and molecular nature have been widely studied (Van der Gucht et al., 2003). However, most research on this gene has been restricted to mammalian species, not aquatic organisms. Its immediate reactivity upon cell damage implies that c-fos gene could be a strong candidate biomarker gene to detect environmental pollutants and to assess their effects at the molecular level. Currently, a few c-fos genes have been reported in fish (e.g., fugu, goldfish, grass carp and carp, rainbow trout) (Matsuoka, Fuyuki, Shoji, & Kurihara, 1998; Trower et al., 1996) but most studies were focused on cDNA sequence information. Exceptionally, in fugu, the extensive sequencing of this gene was carried out to address an evolutionary question by comparative genomics (Trower et al., 1996). Therefore, no fish c-fos genomic clone has been used in an environmentalrelated study. Rivulus marmoratus is a useful species for environmental toxicology studies due to its resistance to extreme environmental conditions and the ease with which it can be maintained in the laboratory (Lee, Chang, Kim, Han, & Lee, 2002, 2000; Rotchell, Lee, Chipman, & Ostrander, 2001). R. marmoratus survives extreme environments (e.g., euryhaline, wide pH range, wide temperature range, chemical exposures). We may therefore be able to study the effects of high doses of toxic compounds. To allow the resistance to extreme environments in this species, we expect that there would be efficient molecular defense mechanisms in R. marmoratus. Therefore, we cloned several defense mechanism-related genes (unpublished data). One of our candidates was the c-fos gene. We have cloned the R. marmoratus c-fos gene and show the genomic structure of this gene. To clone the R. marmoratus c-fos gene, we screened R. marmoratus kGEM-11 genomic DNA library. Briefly, about 3 105 genomic clones were screened by plaque hybriziation using radiolabelled R. marmoratus c-fos probe (PCR product from kR. marmoratus c-fos clone) according to plaque hybridization method. Autoradiography was carried out with Kodak X-ray film and an intensifying screen at )70 °C for 48 h. Positive R. marmoratus c-fos clones were repurified. Nine R. marmoratus cfos clones were isolated from the primary, secondary and tertiary screening of these genomic DNA libraries. Using consensus primers (Fish-fos-F1, 50 -ATG ATG TTT ACG TCT TTT AAC GC-30 /Fish-fos-F2, 50 -CTG CAG TGG ATG GTG CAG CCT TTG G-30 and Fish-fos-R1, 50 -CTG GTG GGC AGC CAG AAT GAA CTC30 /Fish-fos-R2, 50 -CAG CAG GGT CGG GGA GCT GAG GG-30 ) for fish c-fos genes, we amplified PCR products from R. marmoratus c-fos gene and sequenced them. We were then able to amplify long-PCR products of R. marmoratus c-fos kclones using several sets of primers (internal primers obtained from the PCR products and the Sp6/T7 primer from kphage multicloning site) avoiding timeconsuming work in purifying R. marmoratus kc-fos clones. We subcloned them to pCR2.1 vector (Invitrogen) and sequenced with an automated DNA sequencer by continuous primer walking to obtain the full length sequence (about 12 kb). To analyze the gene structure of R. marmoratus c-fos gene, we used the BLAST search program of the NCBI and compared with the existing c-fos sequences of other species for further analysis.
Y. Li et al. / Marine Environmental Research 58 (2004) 681–685
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The partial sequence information of R. marmoratus c-fos gene was deposited to GenBank under the accession number of AY279212 (R. marmoratus c-fos gene) (Fig. 1). The R. marmoratus c-fos gene contained four exons (excluding a noncoding exon), and they spanned about 2 kb. The accepting and donor sequences of exon/ intron boundary were according to GT/AG rule and were similar to the gene structures of other c-fos genes. The R. marmoratus c-fos gene is moderately conserved in its structure with those of other species. In addition, poly(A) signal sequences of R. marmoratus c-fos gene were located 0.7 and 5.0 kb downstream of termination signal codon for the R. marmoratus c-fos gene. The similarity of amino acid residues of R. marmoratus c-fos gene to those of other species, fugu, goldfish, carp, grass carp, chicken, cat and Xenopus, is shown in Fig. 2. The R. marmoratus c-fos gene showed moderate similarity, but the basic region leucine zipper domain was highly conserved between species, even though 1 kb X
X
M
P M M
M
M M
M P MX X X
N CArG
P1
X
N E2 E2/CArG
X
N
N
Poly(A) signal
1
2
3
4
Poly(A) signal
ERE
Fig. 1. Structure of R. marmoratus c-fos gene. The R. marmoratus c-fos gene contained four exons. The shadow and black boxes indicate a non-coding exon and the coding exons, respectively. X, xenobiotic response element (XRE); M, metal response element (MRE); E2, estradiol binding site; N, NFjB site, P1, pit1; P, prolactin (PRL) motif.
Rivulus Fugu Goldfish Carp Grass carp Chicken Cat Xenopus
MMFTAFNT--ECDS-SSRCSTASPSGDNLGYYPSPAGSYSSIGSPQSQD--FTDLTVSSASFIPTVTAISTSPDLQWMVQP-LISLVAP-SHR-AHPYSS---SPSPAYPRSAMRSAA-MMF TSFNA--ECDS-SSRCS-ASPVGDNL-YYPSPAGSYSSMGSPQSQD--FTDLTASSASFIPTVTAISTSPDLQWMVQP-LISSVAP-SHR-AHPYS-----PSPSYKRTVMRSAA-MMFTSLNA--DCDA-SSRCSTASPSGESVAYYPLN----------QTQE--FTDLSVSSASFVPAVTAISSCPDLQWMVQP-MVSSVAP-SNGAARSYN-----PDP-YPK--MRVTA-MMF TSLNA--DCDA-SSRCSTASAAAESVACYPLN----------QTQK--FTELSVSSASFVPTVTAISSCPDLQWMVQP-MVSSVAP-SNGGARSYN-----PNP-YPK--MRVTG-MMFTSLSA--DCDA-SSRCSTASPSGESVAYYPLN----------QTQE--LTDLSVSSASFVPTVTAISSCPDLQWMVQP-MISSVAP-SNGGARSYN-----PNP-YPK--MRVTG-MMYQGFAG--EYEAPSSRCSSASPAGDSLTYYPSPADSFSSMGSPVNSQDFCTDLAVSSANFVPTVTAISTSPDLQWLVQPTLISSVAPSQNRG-HPYGVPAPAPPAAYSRPAVLKAP-MMF SGFNA--DYEASSSRCSSASPAGDNLSYYHSPADSFSSMGSPVNAQDFCTDLAVSSANFIPTVTAISTSPDLQWLVQPTLVSSVAPSQTRAPHPYGVPAPS-AGAYSRAGVVKTVTA M-YHAFSSNTDYDAASSRCSSASPAGDSLTYYPSSAASFSSMGSPVSPQDFGGD---SSSSFVPTVTAISTSADLQWLVQPTLISSVAPSQSRA-HPYAS-----TPVYSRSGVMKGS-* : .: : :: ***** **. .:.: * . . : **:.*:*:*****:..****:*** ::* *** . :.* * : :
Rivulus Fugu Goldfish Carp Grass carp Chicken Cat Xenopus
-SKAHSSTKRGRMEQLTP-----EEEEKKRIRRERNKQAAAKCRNRRRELTDTLQAETDLLEDEKSSLQNDIANLLKEKERLEFILAAHQPICKIPSELETDFLVSSIS------STVPS -SK AHG--KRSRVEQ----------EEKKRIRRERNKQAAAKCRNRRRELTDTLQAETDQLEDEKSSLQNDIANLLKEKERLEFILAAHQPICKIPSQMDTDFSVVSMSPVHACLSTTVS -TKSPNSNKRLRVEQISPTTPEEEEEEKKRVRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSALQNDIANLLKEKERLEFILAAHKPICKIPG----ELSASSVSP-----IPSDS -TK SPNSNKRARAEQLSP-----EEEEKKRVRRERNKMAAAKCRNRRRELTDTLQAETDELEDEKSALQNDIANLLKEKERLEFILAAHKPICKIP--------SSSVSP-----IPAAS -TKS---NKRSRSEQLSP-----EEEEKKRVRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSALQNDIASLLKEKERLEFILAAHKPICKIPTDMAASFPEPSVSP-----ITSDS GGRGQSIGRRGKVEQLSP-----EEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEEEKSALQAEIANLLKEKEKLEFILAAHRPACKMPEELR-FSEELAAATA---LDLGAP GGR AQSIGRRGKVEQLSP-----EEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPACKIPDDLG-FPEEMSVAS----LDLSGG TGRGQSLGRRGKMEQLSP-----EEEEKKRVRRERNKMAAAKCRNRRRELTDTLQAETDDLEDQKSDLQAEIASLLKEKEKLEFILAAHKPACKIPHDLEGAFQDLTSSFD---LGLISE :. :* : ** ***:*:****** ********************* **::** ** :**.******:********:* **:* : :
215 210 203 194 199 226 227 220
Rivulus Fugu Goldfish Carp Grass carp Chicken Cat Xenopus
SQ-----PVTSAITSSQPTFTSASNSIFSG-------TTVSDSAAVKMADLDASVLEESLDLLAKAEMETARSVPEVDLPS-SIYTSQDWESLHASTSGN---SNDFEPLCTPVVTCTPA TQL QTSIPEATTVTSSHSTFTSTSNSIFSGSSDSLLSTATVSNSVVKMTDLDSSVLEESLDLLAKTEAETARSVPDVNLSN-SLFAAQDWEPLHATIS-----SSDFEPLCTPVVTCTPA VPEIHSITTSVVSTPSTTVMTFSSSSLFSST-----ASIDSFGSTVKISDLEP-TLEESLELLAKAELETARSVPDVDLSS-SLYA-QDWEPLYTAAN------NDLEPLCTPVVTCTPA VPE IHSITTSVVSTANAPVTTSSSSSLFSST-----ASTDSFGSTVEISDLEP-TLEESLELLAKAELETARSVPDVDLSS-SLYA-RDWESLYTPAN------NDLEPLCTPVVTRTPA VPEIHSVTTSVVSTQSAPVTTSSSSSLFSST-----PSTDSFSSTVRISDLEP-SLEESLELLAKAELETARSVPDMDLSS-SLYT-QDWEPLYTPAN------TDLEPLCTPVVTCTPA SPAAA----EEAFALPLMT--EAPPAVPPKEP-------SGSGLELKAEPFDELLFSAGPR-------EASRSVPDMDLPGASSFYASDWEPLGAGSGG------ELEPLCTPVVTCTPC LPE AATPESEEAFTLPLLNDPEPKPSVEPVK--------SISSMELKAEPFDDFLFPASSRPS---GSETARSVPDMDLSG--SFYAADWEPLHGGSLGMGPMATELEPLCTPVVTCTPS TPCSSS--SQEPVAEPLFPFGLSQPSMPDKENT-----QLQVSVELKSEPLDDFLFNSPHAGAG--VSDAARSVPDVDLTS--SLYTSEWEPLYSNLSV------DMEPLCTPVVTCTPT : . :: . :. :: : :::****:::*.. :**.* ::********* **
319 324 309 300 305 320 334 323
Rivulus Fugu Goldfish Carp Grass carp Chicken Cat Xenopus
CTTYTSSFMFTFPEAETFPTCGMAHRRGSNSNDQSSDSLNSPTLLAL CTT LTSSFVFTFPEAETFPTCGVAHRRRSNSNDQSSDSLSSPTLLAL CTTYTSSFMFTYPEND---GCGPVHRRGSSSNDQSSDSLNSPTLLTL CTT YTSSFTFTYPENDVFPSCGPVHRRGSSSNDQSSDSLNSPTLLTL CTTYTSSFMFTYPENDTFPSCGPVHRRGSSSNDQSSDSLNSPTLLTL PSTYTSTFVFTYPEADAFPSCAAAHRKGSSSNEPSSDSLSSPTLLAL CTT YTSSFVFTYPEADSFPSCGAAHRKGSSSNEPSSDSLSSPTLLAL CTTYTTSFVFTYPESDHFPNCGAAHRRGSSSNEQSSDSLNSPTLLAL :* *::* **:** : *. .**: *.**: *****.*****:*
107 103 93 93 93 115 117 108
Basic Region Leucine Zipper
366 371 353 347 352 367 381 370
Fig. 2. Similarity of amino acid residues of the R. mamoratus c-fos protein to others. The GenBank accession numbers of compared sequences are as follows. Fugu P53450; Goldfish AB058417; Carp U81505; Grass carp AF380155; Chicken M18043; Cat AF540373; Xenopus A60089.
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Y. Li et al. / Marine Environmental Research 58 (2004) 681–685
they are phylogenically very different (Fujiwara et al., 1987; Kindy & Verma, 1990; Mohun, Garrett, & Taylor, 1989; Van der Gucht et al., 2003). In the c-fos promoter, R. marmoratus has seven xenobiotic response elements (XRE) (CACGCW) in 7064 bp, which we expect to mediate the induction of this gene by xenobiotics, including some endocrine-disrupting chemical (EDC) substances. We also found eight metal response elements (MRE) (TGCRCNC) giving rise to the possibility that this gene is also regulated by heavy metals (Fig. 1). The R. marmoratus c-fos gene promoter has more MREs than the Japanese medaka Oryzias latipes metallothionein (MT) gene (unpublished data). Therefore, it may be possible to use R. marmoratus c-fos gene promoter to assess heavy metal contamination in parallel with the MT gene by an in vitro reporter gene assay. In the promoter region, R. marmoratus c-fos gene has additional features such as 2 E2 binding sites (ACCNNNNNNGGT), 4 NFjB (GGGGCTCTC), 2 CarG boxes (CCWWWWWWGG), 2 prolactin (PRL) motifs (CCTGAWWA) and 1 pit1 site (ATGGATAA), while the 30 -UTR of R. marmorauts c-fos gene contains an estrogen response element (ERE) (GGTCAnnnTAACC) as do those of c-fos genes in other species (Fig. 1; see GenBank AY279212). There are also further elements such as an Sp1 binding site and an AP1 site (data not shown). The location of the ERE at 30 UTR is characteristic of various species (Hyder & Stancel, 1994; Hyder, Stancel, Nawaz, McDonnell, & Loose-Mitchell, 1992). The c-fos gene is expressed in muscle tissue with serum response factor via the CarG box in the c-fos promoter (Santoro & Walsh, 1991; Taylor, Treisman, Garrett, & Mohun, 1989). In the R. marmoratus c-fos gene promoter, we found a proximal and a distal CarG box. Therefore, we expect that R. marmoratus c-fos gene would be expressed strongly in muscle tissue. As shown in Fig. 1, the R. marmoratus c-fos gene promoter region includes another estradiol binding site (E2). Thus, the R. marmoratus c-fos gene may respond to estrogen exposure via this element as well as to some EDC substances by XREs. This is the first report to address the gene structure and the usefulness of a fish c-fos gene promoter in an environmental testing species.
Acknowledgements This work was supported by a grant of KRF (2002-005-C00019) to Y.-D.Y. We thank Dr. Tim D. Williams (School of Biosciences, University of Birmingham, UK) for improving the earlier manuscript.
References Fujiwara, K. T., Ashida, K., Nishina, H., Iba, H., Miyajima, N., Nishizawa, M., & Kawai, S. (1987). Journal of Virology, 61, 4012–4018. Hyder, S. M., & Stancel, G. M. (1994). Journal of Steroid Biochemistry and Molecular Biology, 48, 69–79. Hyder, S. M., Stancel, G. M., Nawaz, Z., McDonnell, D. P., & Loose-Mitchell, D. S. (1992). Journal of Biological Chemistry, 267, 18047–18054.
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Kindy, M. S., & Verma, I. M. (1990). Cell, Growth and Differentiation, 1, 27–37. Lee, J.-S., Chang, S. Y., Kim, I.-C., Han, M.-S., & Lee, Y.-S. (2002). Teratogenesis, Carcinogenesis, and Mutagenesis, 22, 363–367. Lee, J.-S., Park, E.-H., Choe, J., & Chipman, J. K. (2000). Teratogenesis, Carcinogenesis, and Mutagenesis, 20, 1–9. Matsuoka, I., Fuyuki, K., Shoji, T., & Kurihara, K. (1998). Biochimica et Biophysica Acta, 1395, 220–227. Mohun, T. J., Garrett, N., & Taylor, M. V. (1989). Development, 107, 835–846. Rotchell, J. M., Lee, J.-S., Chipman, J. K., & Ostrander, G. K. (2001). Aquatic Toxicology, 55, 1–21. Santoro, I. M., & Walsh, K. (1991). Molecular and Cellular Biology, 11, 6296–6305. Taylor, M., Treisman, R., Garrett, N., & Mohun, T. (1989). Development, 106, 67–78. Trower, M. K., Orton, S. M., Purvis, I. J., Sanseau, P., Riley, J., Christodoulou, C., Burt, D., See, C. G., Elgar, G., Sherrington, R., Rogaev, E. I., St. George-Hyslop, P., Brenner, S., & Dykes, C. W. (1996). In Proceeding of the national academy of sciences of United States of America (Vol. 93, pp. 1366–1369). Van der Gucht, E., Massie, A., De Klerck, B., Peeters, K., Winters, K., Gerets, H. H. J., Clerens, S., Vandesande, F., & Arckens, L. (2003). Molecular Brain Research, 111, 198–210.
Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos Yan Li a, Il-Chan Kim a, Young Ja Kim b, Moon Kyoo Kim c, Yong-Dal Yoon c, Yong-Sung Lee a, Jae-Seong Lee b,* a
Department of Biochemistry, College of Medicine, Hanyang University, Seoul 133-791, Republic of Korea b Department of Environmental Science, Graduate School, Hanyang University, Seoul 133-791, Republic of Korea c Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea
Abstract We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus kGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced 7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFjB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 30 UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms’ c-fos genes. We propose its potential use in ecotoxicology. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Rivulus marmoratus; c-fos; Gene; Cloning; Promoter; MRE; XRE
*
Corresponding author. Tel.: +82-2-2290-0769; fax: +82-2-2299-9450. E-mail address: [email protected] (J.-S. Lee).
0141-1136/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2004.03.062
682
Y. Li et al. / Marine Environmental Research 58 (2004) 681–685
c-fos is well-known as an immediate early gene, and its biochemical characteristics and molecular nature have been widely studied (Van der Gucht et al., 2003). However, most research on this gene has been restricted to mammalian species, not aquatic organisms. Its immediate reactivity upon cell damage implies that c-fos gene could be a strong candidate biomarker gene to detect environmental pollutants and to assess their effects at the molecular level. Currently, a few c-fos genes have been reported in fish (e.g., fugu, goldfish, grass carp and carp, rainbow trout) (Matsuoka, Fuyuki, Shoji, & Kurihara, 1998; Trower et al., 1996) but most studies were focused on cDNA sequence information. Exceptionally, in fugu, the extensive sequencing of this gene was carried out to address an evolutionary question by comparative genomics (Trower et al., 1996). Therefore, no fish c-fos genomic clone has been used in an environmentalrelated study. Rivulus marmoratus is a useful species for environmental toxicology studies due to its resistance to extreme environmental conditions and the ease with which it can be maintained in the laboratory (Lee, Chang, Kim, Han, & Lee, 2002, 2000; Rotchell, Lee, Chipman, & Ostrander, 2001). R. marmoratus survives extreme environments (e.g., euryhaline, wide pH range, wide temperature range, chemical exposures). We may therefore be able to study the effects of high doses of toxic compounds. To allow the resistance to extreme environments in this species, we expect that there would be efficient molecular defense mechanisms in R. marmoratus. Therefore, we cloned several defense mechanism-related genes (unpublished data). One of our candidates was the c-fos gene. We have cloned the R. marmoratus c-fos gene and show the genomic structure of this gene. To clone the R. marmoratus c-fos gene, we screened R. marmoratus kGEM-11 genomic DNA library. Briefly, about 3 105 genomic clones were screened by plaque hybriziation using radiolabelled R. marmoratus c-fos probe (PCR product from kR. marmoratus c-fos clone) according to plaque hybridization method. Autoradiography was carried out with Kodak X-ray film and an intensifying screen at )70 °C for 48 h. Positive R. marmoratus c-fos clones were repurified. Nine R. marmoratus cfos clones were isolated from the primary, secondary and tertiary screening of these genomic DNA libraries. Using consensus primers (Fish-fos-F1, 50 -ATG ATG TTT ACG TCT TTT AAC GC-30 /Fish-fos-F2, 50 -CTG CAG TGG ATG GTG CAG CCT TTG G-30 and Fish-fos-R1, 50 -CTG GTG GGC AGC CAG AAT GAA CTC30 /Fish-fos-R2, 50 -CAG CAG GGT CGG GGA GCT GAG GG-30 ) for fish c-fos genes, we amplified PCR products from R. marmoratus c-fos gene and sequenced them. We were then able to amplify long-PCR products of R. marmoratus c-fos kclones using several sets of primers (internal primers obtained from the PCR products and the Sp6/T7 primer from kphage multicloning site) avoiding timeconsuming work in purifying R. marmoratus kc-fos clones. We subcloned them to pCR2.1 vector (Invitrogen) and sequenced with an automated DNA sequencer by continuous primer walking to obtain the full length sequence (about 12 kb). To analyze the gene structure of R. marmoratus c-fos gene, we used the BLAST search program of the NCBI and compared with the existing c-fos sequences of other species for further analysis.
Y. Li et al. / Marine Environmental Research 58 (2004) 681–685
683
The partial sequence information of R. marmoratus c-fos gene was deposited to GenBank under the accession number of AY279212 (R. marmoratus c-fos gene) (Fig. 1). The R. marmoratus c-fos gene contained four exons (excluding a noncoding exon), and they spanned about 2 kb. The accepting and donor sequences of exon/ intron boundary were according to GT/AG rule and were similar to the gene structures of other c-fos genes. The R. marmoratus c-fos gene is moderately conserved in its structure with those of other species. In addition, poly(A) signal sequences of R. marmoratus c-fos gene were located 0.7 and 5.0 kb downstream of termination signal codon for the R. marmoratus c-fos gene. The similarity of amino acid residues of R. marmoratus c-fos gene to those of other species, fugu, goldfish, carp, grass carp, chicken, cat and Xenopus, is shown in Fig. 2. The R. marmoratus c-fos gene showed moderate similarity, but the basic region leucine zipper domain was highly conserved between species, even though 1 kb X
X
M
P M M
M
M M
M P MX X X
N CArG
P1
X
N E2 E2/CArG
X
N
N
Poly(A) signal
1
2
3
4
Poly(A) signal
ERE
Fig. 1. Structure of R. marmoratus c-fos gene. The R. marmoratus c-fos gene contained four exons. The shadow and black boxes indicate a non-coding exon and the coding exons, respectively. X, xenobiotic response element (XRE); M, metal response element (MRE); E2, estradiol binding site; N, NFjB site, P1, pit1; P, prolactin (PRL) motif.
Rivulus Fugu Goldfish Carp Grass carp Chicken Cat Xenopus
MMFTAFNT--ECDS-SSRCSTASPSGDNLGYYPSPAGSYSSIGSPQSQD--FTDLTVSSASFIPTVTAISTSPDLQWMVQP-LISLVAP-SHR-AHPYSS---SPSPAYPRSAMRSAA-MMF TSFNA--ECDS-SSRCS-ASPVGDNL-YYPSPAGSYSSMGSPQSQD--FTDLTASSASFIPTVTAISTSPDLQWMVQP-LISSVAP-SHR-AHPYS-----PSPSYKRTVMRSAA-MMFTSLNA--DCDA-SSRCSTASPSGESVAYYPLN----------QTQE--FTDLSVSSASFVPAVTAISSCPDLQWMVQP-MVSSVAP-SNGAARSYN-----PDP-YPK--MRVTA-MMF TSLNA--DCDA-SSRCSTASAAAESVACYPLN----------QTQK--FTELSVSSASFVPTVTAISSCPDLQWMVQP-MVSSVAP-SNGGARSYN-----PNP-YPK--MRVTG-MMFTSLSA--DCDA-SSRCSTASPSGESVAYYPLN----------QTQE--LTDLSVSSASFVPTVTAISSCPDLQWMVQP-MISSVAP-SNGGARSYN-----PNP-YPK--MRVTG-MMYQGFAG--EYEAPSSRCSSASPAGDSLTYYPSPADSFSSMGSPVNSQDFCTDLAVSSANFVPTVTAISTSPDLQWLVQPTLISSVAPSQNRG-HPYGVPAPAPPAAYSRPAVLKAP-MMF SGFNA--DYEASSSRCSSASPAGDNLSYYHSPADSFSSMGSPVNAQDFCTDLAVSSANFIPTVTAISTSPDLQWLVQPTLVSSVAPSQTRAPHPYGVPAPS-AGAYSRAGVVKTVTA M-YHAFSSNTDYDAASSRCSSASPAGDSLTYYPSSAASFSSMGSPVSPQDFGGD---SSSSFVPTVTAISTSADLQWLVQPTLISSVAPSQSRA-HPYAS-----TPVYSRSGVMKGS-* : .: : :: ***** **. .:.: * . . : **:.*:*:*****:..****:*** ::* *** . :.* * : :
Rivulus Fugu Goldfish Carp Grass carp Chicken Cat Xenopus
-SKAHSSTKRGRMEQLTP-----EEEEKKRIRRERNKQAAAKCRNRRRELTDTLQAETDLLEDEKSSLQNDIANLLKEKERLEFILAAHQPICKIPSELETDFLVSSIS------STVPS -SK AHG--KRSRVEQ----------EEKKRIRRERNKQAAAKCRNRRRELTDTLQAETDQLEDEKSSLQNDIANLLKEKERLEFILAAHQPICKIPSQMDTDFSVVSMSPVHACLSTTVS -TKSPNSNKRLRVEQISPTTPEEEEEEKKRVRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSALQNDIANLLKEKERLEFILAAHKPICKIPG----ELSASSVSP-----IPSDS -TK SPNSNKRARAEQLSP-----EEEEKKRVRRERNKMAAAKCRNRRRELTDTLQAETDELEDEKSALQNDIANLLKEKERLEFILAAHKPICKIP--------SSSVSP-----IPAAS -TKS---NKRSRSEQLSP-----EEEEKKRVRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSALQNDIASLLKEKERLEFILAAHKPICKIPTDMAASFPEPSVSP-----ITSDS GGRGQSIGRRGKVEQLSP-----EEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEEEKSALQAEIANLLKEKEKLEFILAAHRPACKMPEELR-FSEELAAATA---LDLGAP GGR AQSIGRRGKVEQLSP-----EEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPACKIPDDLG-FPEEMSVAS----LDLSGG TGRGQSLGRRGKMEQLSP-----EEEEKKRVRRERNKMAAAKCRNRRRELTDTLQAETDDLEDQKSDLQAEIASLLKEKEKLEFILAAHKPACKIPHDLEGAFQDLTSSFD---LGLISE :. :* : ** ***:*:****** ********************* **::** ** :**.******:********:* **:* : :
215 210 203 194 199 226 227 220
Rivulus Fugu Goldfish Carp Grass carp Chicken Cat Xenopus
SQ-----PVTSAITSSQPTFTSASNSIFSG-------TTVSDSAAVKMADLDASVLEESLDLLAKAEMETARSVPEVDLPS-SIYTSQDWESLHASTSGN---SNDFEPLCTPVVTCTPA TQL QTSIPEATTVTSSHSTFTSTSNSIFSGSSDSLLSTATVSNSVVKMTDLDSSVLEESLDLLAKTEAETARSVPDVNLSN-SLFAAQDWEPLHATIS-----SSDFEPLCTPVVTCTPA VPEIHSITTSVVSTPSTTVMTFSSSSLFSST-----ASIDSFGSTVKISDLEP-TLEESLELLAKAELETARSVPDVDLSS-SLYA-QDWEPLYTAAN------NDLEPLCTPVVTCTPA VPE IHSITTSVVSTANAPVTTSSSSSLFSST-----ASTDSFGSTVEISDLEP-TLEESLELLAKAELETARSVPDVDLSS-SLYA-RDWESLYTPAN------NDLEPLCTPVVTRTPA VPEIHSVTTSVVSTQSAPVTTSSSSSLFSST-----PSTDSFSSTVRISDLEP-SLEESLELLAKAELETARSVPDMDLSS-SLYT-QDWEPLYTPAN------TDLEPLCTPVVTCTPA SPAAA----EEAFALPLMT--EAPPAVPPKEP-------SGSGLELKAEPFDELLFSAGPR-------EASRSVPDMDLPGASSFYASDWEPLGAGSGG------ELEPLCTPVVTCTPC LPE AATPESEEAFTLPLLNDPEPKPSVEPVK--------SISSMELKAEPFDDFLFPASSRPS---GSETARSVPDMDLSG--SFYAADWEPLHGGSLGMGPMATELEPLCTPVVTCTPS TPCSSS--SQEPVAEPLFPFGLSQPSMPDKENT-----QLQVSVELKSEPLDDFLFNSPHAGAG--VSDAARSVPDVDLTS--SLYTSEWEPLYSNLSV------DMEPLCTPVVTCTPT : . :: . :. :: : :::****:::*.. :**.* ::********* **
319 324 309 300 305 320 334 323
Rivulus Fugu Goldfish Carp Grass carp Chicken Cat Xenopus
CTTYTSSFMFTFPEAETFPTCGMAHRRGSNSNDQSSDSLNSPTLLAL CTT LTSSFVFTFPEAETFPTCGVAHRRRSNSNDQSSDSLSSPTLLAL CTTYTSSFMFTYPEND---GCGPVHRRGSSSNDQSSDSLNSPTLLTL CTT YTSSFTFTYPENDVFPSCGPVHRRGSSSNDQSSDSLNSPTLLTL CTTYTSSFMFTYPENDTFPSCGPVHRRGSSSNDQSSDSLNSPTLLTL PSTYTSTFVFTYPEADAFPSCAAAHRKGSSSNEPSSDSLSSPTLLAL CTT YTSSFVFTYPEADSFPSCGAAHRKGSSSNEPSSDSLSSPTLLAL CTTYTTSFVFTYPESDHFPNCGAAHRRGSSSNEQSSDSLNSPTLLAL :* *::* **:** : *. .**: *.**: *****.*****:*
107 103 93 93 93 115 117 108
Basic Region Leucine Zipper
366 371 353 347 352 367 381 370
Fig. 2. Similarity of amino acid residues of the R. mamoratus c-fos protein to others. The GenBank accession numbers of compared sequences are as follows. Fugu P53450; Goldfish AB058417; Carp U81505; Grass carp AF380155; Chicken M18043; Cat AF540373; Xenopus A60089.
684
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they are phylogenically very different (Fujiwara et al., 1987; Kindy & Verma, 1990; Mohun, Garrett, & Taylor, 1989; Van der Gucht et al., 2003). In the c-fos promoter, R. marmoratus has seven xenobiotic response elements (XRE) (CACGCW) in 7064 bp, which we expect to mediate the induction of this gene by xenobiotics, including some endocrine-disrupting chemical (EDC) substances. We also found eight metal response elements (MRE) (TGCRCNC) giving rise to the possibility that this gene is also regulated by heavy metals (Fig. 1). The R. marmoratus c-fos gene promoter has more MREs than the Japanese medaka Oryzias latipes metallothionein (MT) gene (unpublished data). Therefore, it may be possible to use R. marmoratus c-fos gene promoter to assess heavy metal contamination in parallel with the MT gene by an in vitro reporter gene assay. In the promoter region, R. marmoratus c-fos gene has additional features such as 2 E2 binding sites (ACCNNNNNNGGT), 4 NFjB (GGGGCTCTC), 2 CarG boxes (CCWWWWWWGG), 2 prolactin (PRL) motifs (CCTGAWWA) and 1 pit1 site (ATGGATAA), while the 30 -UTR of R. marmorauts c-fos gene contains an estrogen response element (ERE) (GGTCAnnnTAACC) as do those of c-fos genes in other species (Fig. 1; see GenBank AY279212). There are also further elements such as an Sp1 binding site and an AP1 site (data not shown). The location of the ERE at 30 UTR is characteristic of various species (Hyder & Stancel, 1994; Hyder, Stancel, Nawaz, McDonnell, & Loose-Mitchell, 1992). The c-fos gene is expressed in muscle tissue with serum response factor via the CarG box in the c-fos promoter (Santoro & Walsh, 1991; Taylor, Treisman, Garrett, & Mohun, 1989). In the R. marmoratus c-fos gene promoter, we found a proximal and a distal CarG box. Therefore, we expect that R. marmoratus c-fos gene would be expressed strongly in muscle tissue. As shown in Fig. 1, the R. marmoratus c-fos gene promoter region includes another estradiol binding site (E2). Thus, the R. marmoratus c-fos gene may respond to estrogen exposure via this element as well as to some EDC substances by XREs. This is the first report to address the gene structure and the usefulness of a fish c-fos gene promoter in an environmental testing species.
Acknowledgements This work was supported by a grant of KRF (2002-005-C00019) to Y.-D.Y. We thank Dr. Tim D. Williams (School of Biosciences, University of Birmingham, UK) for improving the earlier manuscript.
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