- Email: [email protected]
Sequence of the flounder (Paralichthys olivaceus) growth hormone-encoding gene and its promoter region
Gene, 165 (1995) 321-322 ©1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
321
GENE 09247
Sequence of the flounder (Paralichthys olivaceus) growth hormoneencoding gene and its promoter region * (Gene structure; polymerase chain reaction; cloning)
M i n o r u T a n a k a a, Y a s u a k i T o m a a, T a k e s h i O h k u b o b, S h u S u d o a a n d K u n i o N a k a s h i m a a aDepartment of Biochemistry, Faculty of Medicine, Mie University, Tsu, Mie 514, Japan; and bCenter for Molecular Genetics, Mie University, Tsu, Mie 514, Japan Received by A. Nakazawa: 15 May 1995; Revised/Accepted: 23 June/8 July 1995; Received at publishers: 3 August 1995
SUMMARY
The flounder (Paralichthys olivaceus) growth hormone (GH)-encoding gene (fGH) and its promoter region were cloned and sequenced following amplification of genomic DNA by the polymerase chain reaction. The fGH gene is 2.1-kb long and consists of six exons and five introns. In the Y-flanking region of the determined transcription start point, a potential TATA box is located at -24, and Pit-1/GHF-l-binding site candidates are located in the - 7 0 to - 5 3 and -133 to 141 regions. -
The genomic structures have recently been analyzed for several teleost GHs to reveal that the GH genes of carp species such as common carp (Chiou et al., 1990), grass carp (Ho et al., 1991; Zhu et al., 1992) and silver carp (Hong et al., 1993) consist of five exons and four introns, as is observed with the mammalian and avian GH genes, whereas the GH genes of rainbow trout (Agellon et al., 1988), Atlantic salmon (Johansen et al., 1989; Male et al., 1992), chinoock salmon (Duet al., 1993) and tilapia (Ber and Daniel, 1992) contain the sixth exon. To elucidate the evolutionary trails of teleost GH genes, further investigations in the structures of other teleost GH genes are necessary. We here report the nt sequence of the flounder GH gene (fGH) and its promoter region amplified by PCR. Correspondence to: Dr. K. Nakashima, Department of Biochemistry, Faculty of Medicine, Mie University, Tsu, Mie 514, Japan. Tel. (81-592) 31-5007; Fax (81-592) 32-7944; e-mail: [email protected] * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: aa, amino acid(s); bp, base pair(s); fGH, flounder GH; GH, growth hormone; GH, gene (DNA) encoding GH; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); PCR, polymerase chain reaction; tsp, transcription start point(s). SSDI 0378-1119(95)00532-3
The fGH spans a region of 2.1 kb (Fig. 1), and consists of six exons (exon I, 92 bp: exon II, 134 bp: exon III, 117 bp: exon IV, 144 bp: exon V, 105 bp: exon VI, 243 bp) and five introns (intron I, 874 bp: intron II, 120 bp: intron III, 121 bp: intron IV, 72 bp: intron V, 89 bp), that is structurely similar to that of rainbow trout, Atlantic salmon, chinoock salmon and tilapia. The nt sequence of exons of fGH is completely identical to that of cDNA (Watahiki et al., 1989), except for a silent nt substitution in exon III and a 1-nt insertion in the 3'-noncoding region of exon VI. All the introns are initiated by GT and end with AG, in agreement with the consensus splicesite sequences. We have reported that flounder GH has the minimal molecular size lacking 14 aa in the C-terminal region (Watahiki et al., 1989). The present genomic analysis indicates that the structural feature of the flounder GH is caused by a deletion in the midst of the fifth exon. The transcription start point (tsp) was determined by the primer extention method. A potential TATA box is located 24 bp upstream from the tsp. Recently, four binding sites for a pituitary-specific transcription factor, Pit-1/GHF-1, have been reported within -238 nt upstream from the tsp of rainbow trout GH gene
322 ccatgttge~tttgttcta[taagttacattttaatttt~attaatttc~atgaaaacct
-177
ggattcatttctgtcctgacgtcacattttaatgttctata~atgttattgtgaaggtaa -117 aacaaacacacacaeacceacacagggttgtttgtccgtttcatgtttctgttgatgaat
41 ttaatgaatttaaacgagttttetgetttaaaaetctgaaetgaaaeeateaeageAeAC
TGAAGAACTGAACCAGTACCTGAACCTGAACCTGAACCAGAATCTGAACCTGAACCAGAA CCTGAACCAGAACCAOCCATGAACAGAOgtaagatcagggttteaattatttttcttgtc M N R
57
4 64 124 3
ccattgaaacagtttgaggttcagtttatatattatgattttatatttgttatgtcaata 184 tataatatatattttctatcatttcttatatateatcaaaatattatatactattatcat 244 a t a c a c c a a t a t t t g a t a t a t c t c t c c c c t t t t t g t c t c c c a c c t g t c t g t c t c t c a c c t 304 g t c t g t c t c t c t t g t c t g t c t c t c t c g c c t g t c t c t c t a c c a c c t g t c c a t c t c t c c t g t 364 c t g t c t c t t a c c t g t c t g t c t g t c t g t c t g t c t a t c t c a c c t g t c t c t c t c c c a c c t g t c 424 c g t c t c t c c t t t c t c t c t c c c a c c t g t c c g t c t c t c c t g t c t g t c t g t c t c t c t t g t c t g 484 t c t c t c t c a c c t g t c t t t c t c c c a c c t g t c c g t c t c t c c t g t c t g t c t c t c a c c t g t c t g 544 tctctcacctgtctgtctgtctctcacctgtccctctcacctgtctctctcccacctgtc 604 c g t c t c t c c t g t c t g t c t c t c a c c t g t c t g t c t c t c a c c t g t c t g t c t g t c t c t c a c c t g 664 t c c c t c t c a c c t g t c t c t c t c c c a c c t g t e c g t c t c t c c t g t c t g t c t g t c t c t c t t g t c 724 t g t c t c t c t c a c c t g t c t t t c t c c c a c c t g t c c g t c t c t c c t g t c t g t c t c t c a c c t g t c 784 tgtctgtctctcacctgtccctctcacctgtctctctcctacctgtccgtctctcctgtc 844 t g t c t c t c a c c t g t c c c t c t c a c c t g t c t c t c t c c t a c c t g t c t g t c t c t c c t g t c c c t c 904 tgacctgtctctctcccacctgtccgtctctcctatctgtctctctcacctgtctgtgtc 964 agTCATCCTCCTGCTGTCAGTCATGTGTGTGGGCGTGTCCTCTCAGCCAATCACAGAGAA 1024 V I L L L S V M C V G V 8 S Q P I T E CCAGCGCCTGTTCTCCATCGCTGTTGGTCGAGTTCAGTATCTTCACCTGGTTGCTAAGAA Q R L F S I A V G R Y Q Y L H L V A K ACTCTTCAGTGACTTTgtaagtgtgttgtgtattattgtgtgtctgttttgtgtaggaga L F S D F
N
23 1084 L 43 1144
48 actctctgcagttggaggattacattgtgtgtgtgtgcgtgtgtgttgtgtattaatgtg 1204 tgtctgtgttgtgta~AGAACTCACTACAGTTGGA~ATCAACGTCTTCTCAACAAAAT1264 E N S L Q L E D Q R L L N K I 63 (G) CGCTTCAAAAGAA~TTGTCATTCAGATAATTTCTTGAGTCCAATCGACAAACACGAGAC A S K E F C H S D N F L S P I D K H E ACAAGGCAGCTCAgtgagttctacacagttctacacaatactaaacattacaatgagttt Q G S s
T
1324 83 1384 87
aattggtaeacacatttgaataagtaacagcegcagttcgattgattgatc~ttattga 1444 ttactctggtcca~TTCAGAAGCTTTTATCGGTCTCTTATCGATTGATTGAGTCCTGGG
V Q K L L S V S Y R L I
E S W
AGTTTTTCAGTCGCTTCCTGGTCGCAAGTTTTGCTGTGAGGACCCAGGTTACATCCAAAC E F F S R F L V A S F A V R T Q V T S K TGTCAGAACTGAAGATGGGTCTCCTGAAGCTGATAGAGgtgagaacacatcgacactctc L 8 E L K M 6 L L K L I E ttgatctgacgaatcagaacagctgtctgactgaccaactcacccttaagGCCAATCAGG A N Q ATGGAGCAGGTGGATTCTCTGAGAGTTCGGTGCTCCAGCTCACGCCGTATGGAAACTCTG D G A G G F S E S S V L Q L T P Y G N S AACTGTTCGCCTGCTTTAAGAAGGATATGCACAAGgtgggggttaatgaaaatgtaatga E L F A C F K K D M H K tgatgtaatgatgatgtagttatgtagtaatcatatgtaataatgatgacatctgtgttt gcagGTGGAGACGTACCTGACCGTGGCCAAATGCCGACTCTTTCCAGAAGCTAACTGCAC Y E T Y L T V h K C R L F P E h N C T
1504
102 1564 122 1624 135 1684 138
1744 158 1804. 170 1864
1924 189
CCTGTAGCCCCGCCTCTCCGCCAAGAAGTACCTCCCCGCAGATGACATCATATGCATTCT 1984
L * (-) GTAGCCCGACCCCTGTGGTTGCCGAGTCTGCTGACTAGCA~AATGTTAGCATCTGTTGG
190 2044
TTCTGCATTCCACCTGATGATGTCATTGTGATGTCATACTGTCAGCAAATAAAGATTTCA 2104 TTCAGTC
2111
Fig. I. The nt sequence of fGH. The fGH was amplified by 30 cycles of PCR using primer 1 (5'-ACACTGAAGAACTGAACCAGT)derived from the 5' region of the flounder GH cDNA (Watahiki et al., 1989) and primer 2, 5'-GACTGAATGAAATCTTTATTT, complementary to the 3' region of the cDNA. Each PCR cycle consisted of 1 min of denaturation at 95°C, 2 min of annealing at 58°C, and 3 min of extension at 72°C. The antisense strand of 5'-flanking region of fGH was synthesized by the Tth DNA polymerase using primer 3, 5'-ATTGAAACCCTGATCTTACC, and the testis genomic DNA, and a poly(A) tail was added to the 3' end with terminal deoxynucleotidyl transferase. The poly(A)-tailed DNA was amplified by 30 cycles of PCR using primer 4, 5'-TACCTCTGTTCATGGCTGGT and primer 5, 5'-GACTCGAGTCGACATCG(dT)lv, containing the sites for XhoI, SalI and ClaI. The PCR-amplified DNA was digested with restriction enzymes, cloned into p U C l l 8 plasmid vector and sequenced. The nt in exons are shown in capitals, and introns and the 5'-flanking region are shown in lower-case letters. The deduced aa residues are shown in the one-letter code under the nt codons. The asterisk indicates the stop codon. The numbering is given on the right margin and begins at the tsp (+ 1} determined by primer extention analysis of pituitary poly(A)+RNA. Potential TATA box is overlined. The candidate sequences for the Pit-1/GHF-1-binding sites are shown by dotted overlines. The polyadenylation signal is underlined. G and hyphen in parentheses above the genomic sequence indicate nt substitution and deletion in the cDNA sequence, respectively. This nt sequence will appear in the EMBL/Genbank/DDBJ databases under accession No. D29737.
(Yamada et al., 1993). Although, the 5'-flanking region up to - 2 3 6 of fGH has no overall sequence homology with the promoter regions suggested for the rainbow trout GH gene, the sequence from - 7 0 to - 5 3 nt of fGH shows high homology (78%) with the sequence from - 6 3 to - 4 6 nt in the rainbow trout GH gene containing one of the Pit-1/GHF-l-binding sites. Another sequence, TCTATACAT, from - 133 to - 144 nt, is also similar to the rainbow trout Pit-1/GHF-l-binding sites. However, the significance of these sequences in the regulation of fGH expression is yet to be elucidated.
ACKNOWLEDGEMENT
This study was partly supported by a Grant-in-Aid from the Fisheries Agency.
REFERENCES Agellon, L.B., Davies, S.L., Chen, T.T. and Powers, D.A.: Structure of a fish (rainbow trout) growth hormone gene and its evolutionary implications. Proc. Natl. Acad. Sci. USA 85 (1988) 5136-5140. Ber, R. and Daniel, V.: Structure and sequence of the growth hormoneencoding gene from Tilapia nilotica. Gene 113 (1992) 245 250. Chiou, C.S., Chen, H.-T. and Chang, W.-C.: The complete nucleotide sequence of the growth-hormone gene from the common carp (Cyprinus carpio}. Biochim. Biophys. Acta 1087 (1990) 91 94. Du, S.J., Devlin, R.H. and Hew, C.L.: Genomic structure of growth hormone genes in chinook salmon (Oncorhynchus tshawytscha): presence of two functional genes, GH-I and GH-II, and a male specific pseudogene, GH-q J. DNA Cell Biol. 12 (1993) 739-751. Ho, W.K.K., Wong, M.W. and Chan, A.P.Y.: Cloning and sequencing of the grass carp (Ctenopharyngodon idellus) growth hormone gene. Biochim. Biophys. Acta 1090 (1991) 245 248. Hong, Y. and Schartl, M.: Sequence of the growth hormone (GH) gene from the silver carp (Hypophthalmichthys molitrix) and evolution of GH genes in vertebrates. Biochim. Biophys. Acta 1174 (1993) 285-288. Johansen, B., Johnsen, O.C. and Valla, S.: The complete nucleotide sequence of the growth-hormone gene from Atlantic salmon (Salmo salar). Gene 77 (1989) 317 324. Male, R., Nerland, A.H., Lorens, J.B., Telle, W., Lossius, I. and Totland, G.K.: The complete nucleotide sequence of the Atlantic salmon growth hormone I gene. Biochim. Biophys. Acta 1130 (1992) 345 348. Watahiki, M., Yamamoto, M., Yamakawa, M., Tanaka, M. and Nakashima, K.: Conserved and unique amino acid residues in the domains of the growth hormones: flounder growth hormone deduced from the cDNA sequence has the minimal size in the growth hormone prolactin gene family. J. Biol. Chem. 264 (1989) 312-316. Yamada, S., Hata, J. and Yamashita, S.: Molecular cloning of fish Pit-1 cDNA and its functional binding to promoter of gene expressed in pituitary. J. Biol. Chem. 268 (1993) 24361 24366. Zhu, Z., He, L. and Chen, T.T.: Primary-structural and evolutionary analyses of the growth-hormone gene from grass carp (Ctenopharyngodon idellus). Eur. J. Biochem. 207 (1992) 643 648.
321
GENE 09247
Sequence of the flounder (Paralichthys olivaceus) growth hormoneencoding gene and its promoter region * (Gene structure; polymerase chain reaction; cloning)
M i n o r u T a n a k a a, Y a s u a k i T o m a a, T a k e s h i O h k u b o b, S h u S u d o a a n d K u n i o N a k a s h i m a a aDepartment of Biochemistry, Faculty of Medicine, Mie University, Tsu, Mie 514, Japan; and bCenter for Molecular Genetics, Mie University, Tsu, Mie 514, Japan Received by A. Nakazawa: 15 May 1995; Revised/Accepted: 23 June/8 July 1995; Received at publishers: 3 August 1995
SUMMARY
The flounder (Paralichthys olivaceus) growth hormone (GH)-encoding gene (fGH) and its promoter region were cloned and sequenced following amplification of genomic DNA by the polymerase chain reaction. The fGH gene is 2.1-kb long and consists of six exons and five introns. In the Y-flanking region of the determined transcription start point, a potential TATA box is located at -24, and Pit-1/GHF-l-binding site candidates are located in the - 7 0 to - 5 3 and -133 to 141 regions. -
The genomic structures have recently been analyzed for several teleost GHs to reveal that the GH genes of carp species such as common carp (Chiou et al., 1990), grass carp (Ho et al., 1991; Zhu et al., 1992) and silver carp (Hong et al., 1993) consist of five exons and four introns, as is observed with the mammalian and avian GH genes, whereas the GH genes of rainbow trout (Agellon et al., 1988), Atlantic salmon (Johansen et al., 1989; Male et al., 1992), chinoock salmon (Duet al., 1993) and tilapia (Ber and Daniel, 1992) contain the sixth exon. To elucidate the evolutionary trails of teleost GH genes, further investigations in the structures of other teleost GH genes are necessary. We here report the nt sequence of the flounder GH gene (fGH) and its promoter region amplified by PCR. Correspondence to: Dr. K. Nakashima, Department of Biochemistry, Faculty of Medicine, Mie University, Tsu, Mie 514, Japan. Tel. (81-592) 31-5007; Fax (81-592) 32-7944; e-mail: [email protected] * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: aa, amino acid(s); bp, base pair(s); fGH, flounder GH; GH, growth hormone; GH, gene (DNA) encoding GH; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); PCR, polymerase chain reaction; tsp, transcription start point(s). SSDI 0378-1119(95)00532-3
The fGH spans a region of 2.1 kb (Fig. 1), and consists of six exons (exon I, 92 bp: exon II, 134 bp: exon III, 117 bp: exon IV, 144 bp: exon V, 105 bp: exon VI, 243 bp) and five introns (intron I, 874 bp: intron II, 120 bp: intron III, 121 bp: intron IV, 72 bp: intron V, 89 bp), that is structurely similar to that of rainbow trout, Atlantic salmon, chinoock salmon and tilapia. The nt sequence of exons of fGH is completely identical to that of cDNA (Watahiki et al., 1989), except for a silent nt substitution in exon III and a 1-nt insertion in the 3'-noncoding region of exon VI. All the introns are initiated by GT and end with AG, in agreement with the consensus splicesite sequences. We have reported that flounder GH has the minimal molecular size lacking 14 aa in the C-terminal region (Watahiki et al., 1989). The present genomic analysis indicates that the structural feature of the flounder GH is caused by a deletion in the midst of the fifth exon. The transcription start point (tsp) was determined by the primer extention method. A potential TATA box is located 24 bp upstream from the tsp. Recently, four binding sites for a pituitary-specific transcription factor, Pit-1/GHF-1, have been reported within -238 nt upstream from the tsp of rainbow trout GH gene
322 ccatgttge~tttgttcta[taagttacattttaatttt~attaatttc~atgaaaacct
-177
ggattcatttctgtcctgacgtcacattttaatgttctata~atgttattgtgaaggtaa -117 aacaaacacacacaeacceacacagggttgtttgtccgtttcatgtttctgttgatgaat
41 ttaatgaatttaaacgagttttetgetttaaaaetctgaaetgaaaeeateaeageAeAC
TGAAGAACTGAACCAGTACCTGAACCTGAACCTGAACCAGAATCTGAACCTGAACCAGAA CCTGAACCAGAACCAOCCATGAACAGAOgtaagatcagggttteaattatttttcttgtc M N R
57
4 64 124 3
ccattgaaacagtttgaggttcagtttatatattatgattttatatttgttatgtcaata 184 tataatatatattttctatcatttcttatatateatcaaaatattatatactattatcat 244 a t a c a c c a a t a t t t g a t a t a t c t c t c c c c t t t t t g t c t c c c a c c t g t c t g t c t c t c a c c t 304 g t c t g t c t c t c t t g t c t g t c t c t c t c g c c t g t c t c t c t a c c a c c t g t c c a t c t c t c c t g t 364 c t g t c t c t t a c c t g t c t g t c t g t c t g t c t g t c t a t c t c a c c t g t c t c t c t c c c a c c t g t c 424 c g t c t c t c c t t t c t c t c t c c c a c c t g t c c g t c t c t c c t g t c t g t c t g t c t c t c t t g t c t g 484 t c t c t c t c a c c t g t c t t t c t c c c a c c t g t c c g t c t c t c c t g t c t g t c t c t c a c c t g t c t g 544 tctctcacctgtctgtctgtctctcacctgtccctctcacctgtctctctcccacctgtc 604 c g t c t c t c c t g t c t g t c t c t c a c c t g t c t g t c t c t c a c c t g t c t g t c t g t c t c t c a c c t g 664 t c c c t c t c a c c t g t c t c t c t c c c a c c t g t e c g t c t c t c c t g t c t g t c t g t c t c t c t t g t c 724 t g t c t c t c t c a c c t g t c t t t c t c c c a c c t g t c c g t c t c t c c t g t c t g t c t c t c a c c t g t c 784 tgtctgtctctcacctgtccctctcacctgtctctctcctacctgtccgtctctcctgtc 844 t g t c t c t c a c c t g t c c c t c t c a c c t g t c t c t c t c c t a c c t g t c t g t c t c t c c t g t c c c t c 904 tgacctgtctctctcccacctgtccgtctctcctatctgtctctctcacctgtctgtgtc 964 agTCATCCTCCTGCTGTCAGTCATGTGTGTGGGCGTGTCCTCTCAGCCAATCACAGAGAA 1024 V I L L L S V M C V G V 8 S Q P I T E CCAGCGCCTGTTCTCCATCGCTGTTGGTCGAGTTCAGTATCTTCACCTGGTTGCTAAGAA Q R L F S I A V G R Y Q Y L H L V A K ACTCTTCAGTGACTTTgtaagtgtgttgtgtattattgtgtgtctgttttgtgtaggaga L F S D F
N
23 1084 L 43 1144
48 actctctgcagttggaggattacattgtgtgtgtgtgcgtgtgtgttgtgtattaatgtg 1204 tgtctgtgttgtgta~AGAACTCACTACAGTTGGA~ATCAACGTCTTCTCAACAAAAT1264 E N S L Q L E D Q R L L N K I 63 (G) CGCTTCAAAAGAA~TTGTCATTCAGATAATTTCTTGAGTCCAATCGACAAACACGAGAC A S K E F C H S D N F L S P I D K H E ACAAGGCAGCTCAgtgagttctacacagttctacacaatactaaacattacaatgagttt Q G S s
T
1324 83 1384 87
aattggtaeacacatttgaataagtaacagcegcagttcgattgattgatc~ttattga 1444 ttactctggtcca~TTCAGAAGCTTTTATCGGTCTCTTATCGATTGATTGAGTCCTGGG
V Q K L L S V S Y R L I
E S W
AGTTTTTCAGTCGCTTCCTGGTCGCAAGTTTTGCTGTGAGGACCCAGGTTACATCCAAAC E F F S R F L V A S F A V R T Q V T S K TGTCAGAACTGAAGATGGGTCTCCTGAAGCTGATAGAGgtgagaacacatcgacactctc L 8 E L K M 6 L L K L I E ttgatctgacgaatcagaacagctgtctgactgaccaactcacccttaagGCCAATCAGG A N Q ATGGAGCAGGTGGATTCTCTGAGAGTTCGGTGCTCCAGCTCACGCCGTATGGAAACTCTG D G A G G F S E S S V L Q L T P Y G N S AACTGTTCGCCTGCTTTAAGAAGGATATGCACAAGgtgggggttaatgaaaatgtaatga E L F A C F K K D M H K tgatgtaatgatgatgtagttatgtagtaatcatatgtaataatgatgacatctgtgttt gcagGTGGAGACGTACCTGACCGTGGCCAAATGCCGACTCTTTCCAGAAGCTAACTGCAC Y E T Y L T V h K C R L F P E h N C T
1504
102 1564 122 1624 135 1684 138
1744 158 1804. 170 1864
1924 189
CCTGTAGCCCCGCCTCTCCGCCAAGAAGTACCTCCCCGCAGATGACATCATATGCATTCT 1984
L * (-) GTAGCCCGACCCCTGTGGTTGCCGAGTCTGCTGACTAGCA~AATGTTAGCATCTGTTGG
190 2044
TTCTGCATTCCACCTGATGATGTCATTGTGATGTCATACTGTCAGCAAATAAAGATTTCA 2104 TTCAGTC
2111
Fig. I. The nt sequence of fGH. The fGH was amplified by 30 cycles of PCR using primer 1 (5'-ACACTGAAGAACTGAACCAGT)derived from the 5' region of the flounder GH cDNA (Watahiki et al., 1989) and primer 2, 5'-GACTGAATGAAATCTTTATTT, complementary to the 3' region of the cDNA. Each PCR cycle consisted of 1 min of denaturation at 95°C, 2 min of annealing at 58°C, and 3 min of extension at 72°C. The antisense strand of 5'-flanking region of fGH was synthesized by the Tth DNA polymerase using primer 3, 5'-ATTGAAACCCTGATCTTACC, and the testis genomic DNA, and a poly(A) tail was added to the 3' end with terminal deoxynucleotidyl transferase. The poly(A)-tailed DNA was amplified by 30 cycles of PCR using primer 4, 5'-TACCTCTGTTCATGGCTGGT and primer 5, 5'-GACTCGAGTCGACATCG(dT)lv, containing the sites for XhoI, SalI and ClaI. The PCR-amplified DNA was digested with restriction enzymes, cloned into p U C l l 8 plasmid vector and sequenced. The nt in exons are shown in capitals, and introns and the 5'-flanking region are shown in lower-case letters. The deduced aa residues are shown in the one-letter code under the nt codons. The asterisk indicates the stop codon. The numbering is given on the right margin and begins at the tsp (+ 1} determined by primer extention analysis of pituitary poly(A)+RNA. Potential TATA box is overlined. The candidate sequences for the Pit-1/GHF-1-binding sites are shown by dotted overlines. The polyadenylation signal is underlined. G and hyphen in parentheses above the genomic sequence indicate nt substitution and deletion in the cDNA sequence, respectively. This nt sequence will appear in the EMBL/Genbank/DDBJ databases under accession No. D29737.
(Yamada et al., 1993). Although, the 5'-flanking region up to - 2 3 6 of fGH has no overall sequence homology with the promoter regions suggested for the rainbow trout GH gene, the sequence from - 7 0 to - 5 3 nt of fGH shows high homology (78%) with the sequence from - 6 3 to - 4 6 nt in the rainbow trout GH gene containing one of the Pit-1/GHF-l-binding sites. Another sequence, TCTATACAT, from - 133 to - 144 nt, is also similar to the rainbow trout Pit-1/GHF-l-binding sites. However, the significance of these sequences in the regulation of fGH expression is yet to be elucidated.
ACKNOWLEDGEMENT
This study was partly supported by a Grant-in-Aid from the Fisheries Agency.
REFERENCES Agellon, L.B., Davies, S.L., Chen, T.T. and Powers, D.A.: Structure of a fish (rainbow trout) growth hormone gene and its evolutionary implications. Proc. Natl. Acad. Sci. USA 85 (1988) 5136-5140. Ber, R. and Daniel, V.: Structure and sequence of the growth hormoneencoding gene from Tilapia nilotica. Gene 113 (1992) 245 250. Chiou, C.S., Chen, H.-T. and Chang, W.-C.: The complete nucleotide sequence of the growth-hormone gene from the common carp (Cyprinus carpio}. Biochim. Biophys. Acta 1087 (1990) 91 94. Du, S.J., Devlin, R.H. and Hew, C.L.: Genomic structure of growth hormone genes in chinook salmon (Oncorhynchus tshawytscha): presence of two functional genes, GH-I and GH-II, and a male specific pseudogene, GH-q J. DNA Cell Biol. 12 (1993) 739-751. Ho, W.K.K., Wong, M.W. and Chan, A.P.Y.: Cloning and sequencing of the grass carp (Ctenopharyngodon idellus) growth hormone gene. Biochim. Biophys. Acta 1090 (1991) 245 248. Hong, Y. and Schartl, M.: Sequence of the growth hormone (GH) gene from the silver carp (Hypophthalmichthys molitrix) and evolution of GH genes in vertebrates. Biochim. Biophys. Acta 1174 (1993) 285-288. Johansen, B., Johnsen, O.C. and Valla, S.: The complete nucleotide sequence of the growth-hormone gene from Atlantic salmon (Salmo salar). Gene 77 (1989) 317 324. Male, R., Nerland, A.H., Lorens, J.B., Telle, W., Lossius, I. and Totland, G.K.: The complete nucleotide sequence of the Atlantic salmon growth hormone I gene. Biochim. Biophys. Acta 1130 (1992) 345 348. Watahiki, M., Yamamoto, M., Yamakawa, M., Tanaka, M. and Nakashima, K.: Conserved and unique amino acid residues in the domains of the growth hormones: flounder growth hormone deduced from the cDNA sequence has the minimal size in the growth hormone prolactin gene family. J. Biol. Chem. 264 (1989) 312-316. Yamada, S., Hata, J. and Yamashita, S.: Molecular cloning of fish Pit-1 cDNA and its functional binding to promoter of gene expressed in pituitary. J. Biol. Chem. 268 (1993) 24361 24366. Zhu, Z., He, L. and Chen, T.T.: Primary-structural and evolutionary analyses of the growth-hormone gene from grass carp (Ctenopharyngodon idellus). Eur. J. Biochem. 207 (1992) 643 648.