Structural and expression analyses of gonadotropin Iβ subunit genes in goldfish (Carassius auratus)

Gene 222 (1998) 257–267

Structural and expression analyses of gonadotropin Ib subunit genes in goldfish (Carassius auratus) Young Chang Sohn *, Hiroaki Suetake, Yasutoshi Yoshiura 1, Makito Kobayashi, Katsumi Aida Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Received 24 July 1998; accepted 7 September 1998; Received by T. Sekiya

Abstract Gonadotropin (GTH ) is a pituitary glycoprotein hormone that regulates gonadal development in vertebrates. In teleosts, it is considered that two types of GTH, GTH I (follicle-stimulating hormone-like GTH ) and GTH II ( luteinizing hormone-like GTH ), are produced in the pituitary, and their molecules are comprised of common a and distinct b subunits. In this study, we describe the complete structure and 5∞-flanking regulatory region of two distinct genes encoding GTH Ib in goldfish, Carassius auratus. The two goldfish GTH Ib genes, gfGTHIb-1 and gfGTHIb-2, span 1719 and 1545 base pairs (bp) nucleotides, respectively, and there is a high sequence identity (92.1%) between the coding regions. Both genes consist of three exons separated by two introns as in mammalian FSH b genes. The locations of the first intron and second intron showed a well-conserved pattern similar to those of mammalian FSH b genes. Inspection of the 5∞-flanking region of the gfGTHIb-1 and gfGTHIb-2 (approximately 1.4 and 1.1 kb, respectively) revealed the presence of several putative cis-acting elements, including the gonadotrope-specific element, gonadotropin-releasing hormone responsive element, and half steroid hormone responsive elements. Interestingly, some of their elements were located contiguously between −187 and −124 bp upstream from a TATAA sequence. Reverse transcription polymerase chain reaction confirmed that these two genes are expressed in the pituitary of individual fish. These results, taken together, demonstrate that there are at least two functional genes encoding GTH Ib, probably due to the tetraploidy of goldfish. The unique locations of the cis-acting elements in the GTH Ib genes suggest they may be involved in the expression of the goldfish GTH Ib gene. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Cyprinids; GTH I; 5∞-flanking region; FSH; Teleost

1. Introduction The vertebrate pituitary produces a family of structurally related glycoproteins including follicle-stimulating hormone ( FSH ), luteinizing hormone (LH ), and thyroid-stimulating hormone ( TSH ) (Pierce and Parsons, 1981). In teleosts, two types of gonadotropins, GTH I * Corresponding author. Tel: +81 3 3812 2111 (Ext. 5289); Fax: +81 3 3812 0529; e-mail: [email protected] 1 Present address: Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan. Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; FSH, follicle-stimulating hormone; gfGTHI, gene (DNA) encoding goldfish GTH I; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/0.015 M Na · citrate pH 7.6; RT-PCR, reverse transcription 3 PCR; UTR, untranslated region(s).

and GTH II, have been purified in several species, and their cDNA structures have also been determined ( Kawauchi et al., 1989; Swanson and Dittman, 1997). The fish GTH I and GTH II are heterodimers composed of a common a and a unique b subunit and are structurally similar to tetrapod FSH and LH, respectively (Que´rat, 1995). The cDNA structures of goldfish GTH Ib and GTH IIb are also similar to tetrapod FSH b and LH b, respectively, as shown in other teleosts ( Yoshiura et al., 1997). In salmonid species, it is suggested that GTH I (FSH-like GTH ) regulates the early stages of gonadal development such as vitellogenesis and spermatogenesis, whereas GTH II (LH-like GTH ) controls ovulation and spermiation at the final stages of gonadal development (Swanson and Dittman, 1997). Growing evidence of structural homology of GTH I and GTH II to mammalian FSH and LH, respectively, in some teleosts has led to the suggestion that the nomenclatures of GTH I and GTH II in fish should be adopted as

0378-1119/98/$ – see front matter © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 50 5 - 8

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FSH and LH, respectively (Swanson and Dittman, 1997). However, since full information on the two GTHs including biological functions has not yet been obtained in goldfish (Carassius auratus), we used the nomenclatures of GTH I and GTH II in the present study. Goldfish is often used as a model for investigating the reproductive endocrinology of fish, because of its small body size, and ease of manipulating gonadal development and spawning. Recently, we have found that in female goldfish pituitary, the GTH Ib mRNA level was decreased by sex steroid treatment and increased after ovariectomy, whereas GTH IIb mRNA level showed a slight increase with steroid treatment and almost no changes after ovariectomy (Sohn et al., 1998; Kobayashi et al., unpublished ). With regard to the mechanism of inhibitory effects of the sex steroids on GTH Ib mRNA expression, it is not known whether sex steroids act directly on the pituitary GTH I cells or indirectly via the hypothalamus. In salmonid species, it has been reported that treatment with sex steroids had no effects on GTH Ib mRNA levels, whereas sex steroids increased GTH IIb mRNA levels; the cis-acting DNA sequence in 5∞-flanking region of GTH IIb gene, e.g. estrogen-responsive element ( ERE ), is essential in conferring a cell-type-specific expression of the GTH IIb gene ( Xiong et al., 1994). Since the regulatory region of the GTH Ib gene has not yet been characterized in any teleost, it is of great interest to elucidate the mechanism of the inhibitory effects of sex steroids on GTH Ib gene expression in goldfish. As a first step in exploring the mechanism of the GTH Ib subunit gene expression in teleosts, we have isolated and characterized the goldfish GTH Ib subunit gene, including the 5∞-flanking regions. The present investigation is the first to examine the complete exon–intron structure and the 5∞-flanking region of the GTH Ib or FSH b subunit gene in lower vertebrates. Isolation and characterization of the goldfish GTH Ib gene would not only facilitate the study of the cellular and molecular mechanisms by which the GTH Ib gene expression is regulated in teleosts, but also further define the evolution of glycoprotein hormone genes in vertebrates.

2. Materials and methods

2.1. Construction and screening of a goldfish genomic library Genomic DNA extracted from the hepatopancreas of a female goldfish was partially digested with Sau3A I. Fractions carrying 10- to 20-kb DNA fragments were pooled and ligated into the bacteriophage Lambda EMBL3 vector (Stratagene, La Jolla, CA). After invitro packaging using Gigapak II packaging extract

(Stratagene), the primary library containing 2×106 independent recombinant phages was screened, by plaque hybridization, for clones carrying the GTH Ib gene. After the transfer of the plaques to Hybond N+ nylon membranes (Amersham, South Clearbrook, IL), the membranes were denatured with 1.5 M NaCl/0.5 M NaOH for 1 min, neutralized with 1.5 M NaCl/0.5 M Tris–HCl (pH 8.0) for 3 min, rinsed with 2× SSC for 5 min, air-dried at room temperature, and finally baked at 80°C for 20 min. After prehybridization with a hybridization solution containing 0.5 M Na HPO , 1.0 mM 2 4 EDTA, and 7.0% SDS, at 65°C for 2 h, the membranes were incubated with 50 ml of the hybridization solution containing a previously characterized GTH Ib cDNA ( Yoshiura et al., 1997) as a probe, which was randomly labeled with [a-32P]dCTP (Amersham), at 65°C for 16 h. After hybridization, the membranes were washed twice with low-stringency washing buffer (40 mM Na HPO , 2 4 1.0 mM EDTA, and 5.0% SDS) at room temperature for 5 min and 65°C for 10 min, and twice with highstringency washing buffer (40 mM Na HPO , 1.0 mM 2 4 EDTA, and 1.0% SDS) at 65°C for 30 min. The membranes were air-dried at room temperature and exposed to X-ray film at −80°C for 2 days. The positive clones were picked for second and third rounds of screening. 2.2. Classification of positive clones Several recombinant clones were isolated by repeated plaque hybridization. It is known that the location of the second exon–intron junction is strictly conserved in mammalian FSH b genes (Jameson et al., 1988; Kim et al., 1988; Gharib et al., 1989; Hirai et al., 1990; Guzman et al., 1991; Kumar et al., 1995). To classify the positive clones, the partial nt sequences between exon 2 and exon 3, including the entire intron 2 region, were determined by sequencing (see Section 2.4) using PCR amplification. PCR amplifications were performed with a set of gene-specific oligonucleotide primers (#9 and #12 in Table 1) and a standard PCR mixture (see Section 2.6). The amplification was carried out for 30 cycles of 94°C for 1 min, 55°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. The desired PCR products were then subcloned and sequenced (see Section 2.4). 2.3. Phage recombinant DNA purification and restriction mapping The selected clones (see Section 2.2) were purified using a DNA purification Kit (Qiagen Lambda Midi Kit, Chatsworth, CA). For restriction enzyme mapping, the purified phage DNAs from individual clones were digested with BstXI, and combination with BamHI, EcoRI, HindIII, XbaI, and XhoI, and subjected to electrophoresis in a 0.6% agarose gel (300 ng/lane). The

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Y.C. Sohn et al. / Gene 222 (1998) 257–267 Table 1 Oligonucleotides used for nt sequence determination, PCR, and hybridization Primers

Sequences

Directions

Locations

#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13

5∞-GTCCTCATTTGTAAGCTGC-3∞ 5∞-ATATTGGGATTGAGATGC-3∞ 5∞-TTCTGCAGCACAATTCCTGG-3∞ 5∞-GAATATCTGGAATCTGAGGG-3∞ 5∞-GGTCAGTGTTCTGCAGCAC-3∞ 5∞-TTGTCTCAGYGAAACTCC-3∞ 5∞-GCCTGGGCATAAACAAACAGC-3∞ 5∞-CCGATCCTGACATCATTAGC-3∞ 5∞-ATGTGGCAGCTGCATCACAA-3∞ 5∞-CCGGCACAGGCAGTGGTG-3∞ 5∞-CTGTAACTTCAGAGAATGGA-3∞ 5∞-TCTCGTACGTCCATTCTCTG-3∞ 5∞-CCTTGTCTAATGTGCATTGC-3∞

R R R R R F R R F R F R R

gfGTHIb-1, 5∞-flanking gfGTHIb-1, 5∞-flanking gfGTHIb-1, 5∞-flanking gfGTHIb-2, 5∞-flanking gfGTHIb-2, 5∞-flanking gfGTHIb-1 and gfGTHIb-2, gfGTHIb-1, intron 1 gfGTHIb-1 and gfGTHIb-2, gfGTHIb-1 and gfGTHIb-2, gfGTHIb-1 and gfGTHIb-2, gfGTHIb-1 and gfGTHIb-2, gfGTHIb-1 and gfGTHIb-2, gfGTHIb-1, exon 3

exon 1 exon exon exon exon exon

2 2 2 3 3

F, forward direction; R, reverse direction.

digested DNA fragments were transferred to a Hybond N+ nylon membrane (Amersham) and subsequently hybridized with several [c-32P]dATP-labeled oligonucleotide probes for Southern blot analysis. The hybridization and washing procedures are described in Section 2.1. 2.4. DNA sequencing Sequence analysis was carried out with a Model 373A DNA sequencer using dye terminator cycle sequencing kits (Perkin-Elmer Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. Genomic sequencing was performed for either sense or antisense strand of the purified DNA (300–600 ng) with genespecific primers (see Table 1 and Fig. 1). Amplified PCR products were subcloned using pBluescript SK(−) vector system (Stratagene), and both strands of the DNA inserts in the plasmid vector were sequenced in both directions using T and T sequencing primers. 3 7 2.5. RNA preparation for reverse transcription Sexually immature 1-year-old male and female goldfish, body weight 5–8 g, or 2-year-old mature fish, 35–82 g, were anesthetized with 2-phenoxy-ethanol (0.5 ml/l ) prior to decapitation. Mature males were spermiated, and mature females had ovaries with vitellogenic oocytes. The pituitary glands were collected, immediately frozen in liquid nitrogen, and stored at −80°C until the extraction of RNA. Total RNA was extracted from the pituitary glands with a RNA extraction solution, Isogen (Nippon Gene, Toyama, Japan). The concentration of RNA was determined spectrophotometrically by reading absorbance at 260 nm for each sample, and the integrity was verified by ethidium bromide staining of 28S and 18S ribosomal RNA bands on a denaturing agarose gel.

Fig. 1. Restriction maps of the goldfish GTH Ib genes, (A) gfGTHIb1 and (B) gfGTHIb-2, in Lambda EMBL 3. ‘P’ and ‘A’ represent the putative TATAA box and polyadenylation signal, respectively. Open boxes indicate non-coding 5∞- and 3∞-UTR, and solid boxes indicate the coding region. Thin lines between the boxes indicate introns. The arrows and the numbers on the arrows indicate the direction of DNA sequencing and oligonucleotide primers for sequencing (see in Table 1), respectively. The sequenced regions (shown as an asterisk) were used to classify the screened positive clones (see Section 2.2).

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2.6. 5∞-RACE (rapid amplification of cDNA ends) and 3∞-RACE Reverse transcription was carried out in a 25-ml volume for 5∞-RACE. One microgram of the total RNA extracted from the pituitary gland (immature goldfish, see Section 2.5) was used for the first-strand cDNA synthesis with a gene-specific primer (GSP1, #12 in Table 1) for using Superscript II @ reverse transcriptase (GIBCO BRL, Gaithersburg, MD) at 42°C for 60 min. After 3∞-end tailing of the first-strand cDNA for 5∞-RACE with poly(A) using TdT (GIBCO BRL) at 37°C for 10 min, the second strand of cDNA was then synthesized using the 3∞-end-tailed cDNA as a template. Two rounds of PCR amplifications were performed to isolate the 5∞-end of cDNA with dT-adaptor primer [5∞-GACTCGAGTCGACATCGA( T ) -3∞] and GSP1, 17 and adaptor primer (5∞-GACTCGAGTCGACATCGA3∞) and GSP2 (#10 in Table 1), respectively. Each amplification was carried out in a standard PCR mixture of 20 ml: 0.2 mM of dNTP, 15 mM of MgCl , 1× PCR 2 buffer (50 mM of KCl, 10 mM of Tris–HCl, pH 8.3), 0.5 U of Taq DNA polymerase ( Takara, Tokyo, Japan), and 0.5 mM of each primer. The amplification was carried out for 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 min. The desired PCR products were then subcloned and sequenced (see Section 2.4). For 3∞-RACE, 1 mg of the total RNA extracted from the pituitary gland (immature goldfish; see Section 2.5) was reverse-transcribed to cDNA with a dT-adaptor primer [5∞-AACTGGAAGAATTCGCGGCCGCAGGAA( T ) -3∞] using a reverse transcriptase 18 (Ready-To-Go@, Pharmacia Biotech, Piscataway, NJ ) at 37°C for 60 min. Two rounds of PCR amplifications were performed to amplify the 3∞-end of cDNA. The first round of PCR was carried out using the first-strand cDNA as a template with an adaptor primer (RTG, 5∞-AACTGGAAGAATTCGCGGCCG-3∞) and a genespecific primer (#6 in Table 1). The second round of PCR was performed with RTG-nested primer (5∞-TGGAAGAATTCGCGGCCGCAG-3∞) and #6. The PCR reactions were carried out with the same PCR mixture and conditions as in the 5∞-RACE, except for primers. The desired PCR products were then subcloned, and inserts of five clones from both 5∞-RACE and 3∞-RACE reactions were sequenced (see Section 2.4). 2.7. RT-PCR (reverse transcription-polymerase chain reaction) For detection of the expression of GTH Ib mRNA in individual fish, 1 mg of the total RNA extracted from each pituitary gland (immature and mature fish; see Section 2.5) was used for reverse transcription with GSP1 (#12 in Table 1) using Superscript II @ reverse

transcriptase (GIBCO BRL) at 42°C for 60 min. Reverse transcription was carried out in a 25-ml volume. A pair of gene-specific primers (#6 and #10), located in the first exon and second exon of goldfish GTH Ib genes and are common to two genes ( Table 1), were used for subsequent PCR amplification. Two microliters of the synthesized first-strand cDNA solution were used as a template for the following PCR reaction. Each amplification was carried out in the standard PCR mixture as in the 5∞-RACE, except for primers. Twenty-five amplification cycles were set up as follows: 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The PCR products were fractionated on a 2.5% agarose gel.

3. Results and discussion 3.1. Isolation of two types of goldfish GTH Ib gene Seven recombinant phage clones that hybridized to the goldfish GTH Ib cDNA probe were isolated from a goldfish genomic library. Two distinct types of goldfish GTH Ib subunit gene were characterized from classification of the positive clones by the analysis of partial nt sequencing between exon 2 and exon 3, including the entire intron 2 (Fig. 1). The partial exon sequence in one clone was identical to that of previously characterized goldfish GTH Ib cDNA ( Yoshiura et al., 1997), and the intron 2 spanned 786 bp. We named this type of goldfish GTH Ib-1 gene, gfGTHIb-1. The 8 nt out of 131 nt sequence in the other six clones were different from that of the cDNA, and their full sequences were identical to each other (data not shown). The intron of the new type of goldfish GTH Ib gene, gfGTHIb-2, spanning 700 bp, had a distinct sequence compared to that of gfGTHIb-1 (sequence identity, 53.9%). Restriction patterns were different between gfGTHIb-1 and gfGTHIb-2 ( Fig. 1). The existence of two distinct GTH Ib genes in goldfish concurs well with the results of our previous study in which at least two copies of GTH Ib subunit gene were suggested in goldfish, following genomic Southern blot analysis ( Yoshiura et al., 1997). 3.2. Structure of the two types of goldfish GTH Ib gene The structural organizations of the goldfish GTH Ib genes derived from the sequencing analysis and restriction enzyme mapping are depicted in Fig. 1. The junctions of the exons and introns were defined by comparing the gene sequences with known cDNA sequence ( Yoshiura et al., 1997) and the sequences of RACE products in the present study. The gfGTHIb-1 and gfGTHIb-2 span 1719 and 1545 bp, respectively, and both genes consist of three exons separated by two introns. The sequences of exon–intron boundaries are

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shown in Fig. 2: all the splice junctions conform to the GT–AG rule. Each exon 1 of the two genes encoded 5∞UTR of the mRNAs of GTH Ib-1 and -2, respectively. Introns of the gfGTHIb-1 were longer than those of the gfGTHIb-2: intron 1, 319 vs. 277 bp, and intron 2, 786 vs. 700 bp (Fig. 2). Although there is an overall 67.2% nt sequence identity between the gfGTHIb-1 and gfGTHIb-2 with 56.3% identity between the introns, the coding regions and exons showed 92.1 and 82.6% identity, respectively. In addition, comparison of the deduced aa of the two GTH Ib cDNAs showed 90.0% identity. The high identity of the nt sequences in duplicate cDNAs and genes for GTH Ib in goldfish concurs with the recent tetraploidization in cyprinid fishes (Chang et al., 1992; Kobayashi et al., 1997). A nt sequence of the gfGTHIb-1 differed from our previously reported cDNA sequence ( Yoshiura et al., 1997). At a second nt in the sequence corresponding to exon 2 region, G was substituted by A. Repeated examinations by sequencing the genomic DNA and cDNAs from RACE products confirmed the sequence of A. The different nt may be due to the heterogeneity of goldfish, since fish were obtained from different hatcheries in the previous and present studies. 3.3. Comparison of goldfish GTH Ib and mammalian FSH b genes For comparison, the exon–intron structures of goldfish GTH Ib and mammalian FSH b genes are illustrated in Fig. 3. Both gfGTHIb-1 and gfGTHIb-2 were composed of three exons interrupted by two introns like in mammalian FSH b genes (Jameson et al., 1988; Kim et al., 1988; Gharib et al., 1989; Hirai et al., 1990; Guzman et al., 1991; Kumar et al., 1995). The size of the open reading frame in both gfGTHIb-1 and gfGTHIb-2 was almost the same as those of the mammalian FSH b genes ( Fig. 4). However, the exon 3 of both gfGTHIb-1 and gfGTHIb-2 contained a much shorter 3∞-UTR than those found in the mammalian FSH b genes. In addition, the goldfish GTH Ib genes had shorter introns when compared with the mammalian FSH b genes. It remains to be elucidated whether or not such increases in the size of introns and 3∞-UTR are a general tendency during the evolution of vertebrate FSH b genes. The location of the second exon–intron junction, which is 3 aa downstream from the fifth cysteine residue in the coding region (sixth in goldfish, Yoshiura et al., 1997), is strictly conserved when compared with the pituitary glycoprotein hormone b-subunit genes, reported in mammalian species and in teleosts, except only one case in mouse TSH b gene (Fig. 4). This observation on the second exon–intron junction indicates a strong conservation of gene structure within the glycoprotein family. In contrast to the highly conserved

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location of the second intron, the location of the first intron is more variable in different glycoprotein hormone subunit genes, occurring either in the signal sequences (mammalian LHb; carp GTH IIb; goldfish GTH IIb, our unpublished data) or upstream from the translational start point (goldfish GTH Ib in the present study; mammalian FSHb; salmon GTH IIb; mammalian TSH b; goldfish TSH b, our unpublished data). The location of the first introns of gfGTHIb-1 and gfGTHIb-2 belongs to the latter category, including all the FSH b genes in mammalian species. From the structural homology and physiological functions, it was suggested that teleost GTH I and GTH II should be adopted to FSH and LH, respectively (Que´rat, 1995; Swanson and Dittman, 1997). The structure of the goldfish GTH Ib genes presented here supports their suggestion. 3.4. Expression of gfGTHIb-1 and gfGTHIb-2 genes To determine whether both goldfish GTH Ib-1 and GTH Ib-2 genes are expressed, the amplification of the 5∞-proximal region by RT-PCR analysis was conducted using the total RNA extracted from the pituitary of individual fish. From the nt sequences of the cDNAs and genes of GTH Ib-1 and GTH Ib-2, we noticed that 43 nt of exon 2 in gfGTHIb-1 encode 5∞-UTR of the GTH Ib-1 mRNA, whereas the 43 nt correspond to the distal nt of intron 1 in gfGTHIb-2 (double-underlined in Fig. 2). The differences of 43 bases in length between goldfish GTH Ib-1 and GTH Ib-2 mRNAs were used in the experiment to determine the differential expression of the two genes. As shown in Fig. 5, RT-PCR generated two cDNA fragments with a difference of 43 bp in length (224 bp for GTH Ib-1 and 181 bp for GTHIb-2). In addition, two amplified fragments were confirmed by PCR amplification using genomic DNA extracted from the hepatopancreas (almost 540 bp for GTH Ib-1 and 460 bp for GTHIb-2) (data not shown). The relative amounts of GTHIb-2 products seem to be larger than those of GTHIb-1 products in sexually immature goldfish ( Fig. 5, lanes 2, 3, 4, and 5), whereas those of two products were almost the same in mature fish (Fig. 5, lanes 6, 7, 8, and 9). The efficiency of the amplification is considered to be equal for GTHIb-1 and GTHIb-2, since the identical primers were used. Therefore, it is suggested that the transcript levels of the two GTH Ib genes are differentially regulated by sexual maturity. However, the details of the expression of these genes during the reproductive cycle remain to be studied. 3.5. Analysis of the 5∞-flanking regions of gfGTHIb-1 and gfGTHIb-2 genes In both gfGTHIb-1 and gfGTHIb-2, a TATAA sequence is located 30 bp upstream from the first nt of the sequences of 5∞-RACE products (Fig. 6). Further

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Fig. 2. Nt and deduced aa sequences of goldfish GTH Ib genes, (A) gfGTHIb-1 and (B) gfGTHIb-2, contained in the transcribed portion. The numbers on the right margin refer to the last nt on the corresponding line and to the distance from the first nt of 5∞-UTR, revealed by 5∞-RACE products. The exons are shown in upper-case letters, and introns and flanking regions are shown in lower-case letters. A 5∞-UTR of gfGTHIb-1, revealed as an intron in gfGTHIb-2, is double-underlined. Identical nt between gfGTHIb-1 and gfGTHIb-2 are represented by dots, and dashes indicate a gap that was inserted to maximize identity. Encoded aa residues, represented by one-letter symbols, are placed above the second nt of each codon and numbered with the first aa (glycine, G) of the putative mature GTH Ib subunit as 1; only the aa residues of gfGTHIb-2 that differ

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Fig. 3. Comparison of goldfish GTH Ib and mammalian FSH b genes. The open reading frame and the UTR of exons are represented by the solid and open boxes, respectively. Thin lines between the boxes indicate introns. Numbers below the introns show the approximate length in kb. E, exon; In, intron. References: mouse, [ Kumar et al. (1995)]; rat, [Gharib et al. (1989)]; ovine, [Guzman et al. (1991)]; bovine, [ Kim et al. (1988)]; porcine, [ Hirai et al. (1990)]; human, [Jameson et al. (1988)].

TATAA sequences were also found in the 5∞-flanking regions, located at −1253, −693, −684, and −480 bp in the gfGTHIb-1, and at −551 and −456 bp in the gfGTHIb-2 (underlined in Fig. 6). However, it is most likely that the putative TATAA boxes are those located at −30 bp, and the transcription start points lay just upstream of the exon 1 in the two genes presented here, since the length of the two GTH Ib cDNAs obtained by RACE reactions (614 bp for GTH Ib-1 and 571 bp for GTH Ib-2) is similar to that of the GTH Ib mRNA determined by Northern blot analysis ( Yoshiura et al., 1997). Although sequencing of other 5∞-RACE products was performed, we could not obtain a longer sequence than those of the GTH Ib cDNAs presented here. In mammalian FSH b genes, it is known that the TATAA box is located to be 31 or 32 bp upstream from the transcription start point (Jameson et al., 1988; Kim et al., 1988; Gharib et al., 1989; Hirai et al., 1990; Guzman et al., 1991; Kumar et al., 1995). Recently, we have found an inhibitory effect of sex steroids on goldfish GTH Ib gene expression in vivo. Pituitary GTH Ib mRNA levels were decreased by sex steroid treatment and increased after ovariectomy, whereas GTH IIb mRNA levels showed a slight increase with steroid treatment and almost no changes after ovariectomy (Sohn et al., 1998; Kobayashi et al., unpublished ). In salmonid species, it was suggested that the

GTH IIb gene expression is stimulated by a complex interplay between the cis-acting regulatory elements in the 5∞-flanking region and the tissue-specific factors, e.g. estrogen receptor, steroidogenic factor-1 (SF-1), gonadotropin-releasing hormone (GnRH ), sex steroids, and others ( Xiong et al., 1994; Dre´an et al., 1996). It is tempting, therefore, to search for the potential regulatory DNA sequences that may be located at the 5∞-flanking region of GTH Ib gene in goldfish, and to speculate on a different fashion of gene expression compared to GTH IIb. Inspection of the 5∞-flanking region of goldfish GTH Ib genes yielded several putative cis-acting elements, including an estrogen-responsive element ( ERE; Kato et al., 1992), androgen-responsive element (ARE; Faisst and Meyer, 1992), GnRH-responsive element (GnRH-RE; Schoderbek et al., 1993), and gonadotrope-specific element (GSE or SF-1 binding element; Dre´an et al., 1996) with sequence similarity. The position of these elements in the 5∞-flanking regions of gfGTHIb-1 and gfGTHIb-2 is shown in Fig. 6. Although consensus sequences for ARE and ERE were not found in the 5∞-flanking region, several half-palindromic motifs, 5∞-TGTYCT-3∞ for ARE and 5∞-TGACC-3∞ for ERE, were contained in the regions ( Fig. 6). Interestingly, some of the putative regulatory sequences were located contiguously and overlapped between −218 and −162 bp in gfGTHIb-1, and −210

from the gfGTHIb-1 are shown in parentheses. The stop codon and putative polyadenylation signals are indicated by an asterisk and underline, respectively. The nt sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nt sequence databases with the Accession Nos AB015482 and AB015483 for gfGTHIb-1 and gfGTHIb-2, respectively.

Fig. 4. Alignment of the aa sequences of the pituitary glycoprotein b subunits including putative signal sequences. The signal sequences are shown in lower-case letters. Gaps (shown by a dash) were inserted to maximize alignment of the cysteine (C ) residues among the b subunits. The C residues are numbered accordingly, and A1 indicates an additional C residue in goldfish. The putative N-linked glycosylation site is marked by an asterisk. The location of intron is indicated by a triangle. References are as follows. GTH Ib and FSH b genes: goldfish, the present study and [ Yoshiura et al. (1997)]; mouse, [ Kumar et al. (1995)]; rat, [Gharib et al. (1989)]; ovine, [Guzman et al. (1991)]; bovine, [ Kim et al. (1988)];porcine, [ Hirai et al. (1990)]; human, [Jameson et al. (1988)]. GTH IIb and LH b genes: rat, [Jameson et al. (1984)]; bovine, [ Virgin et al. (1985)]; porcine, [Ezashi et al. (1990)]; human, [ Talmadge et al. (1984)]; carp, [Chang et al. (1992)]; salmon, [ Xiong and Hew (1991)]. TSH b genes: rat, [Carr et al. (1987)]; mouse, [Gordon et al. (1988)]; human, [ Wondisford et al. (1988)].

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Fig. 5. Electrophoresis of RT-PCR products of gfGTHIb-1 and gfGTHIb-2. One microgram of total RNA from a single pituitary gland of immature or mature fish was used for the first-strand cDNA synthesis. After reverse transcription, 2 ml out of 25 ml of the first-strand cDNA solution was used as the template for PCR amplification. A pair of common primers (#6 and #10 in Table 1) for both GTH Ib cDNAs, which match the 5∞-proximal regions, were used in the amplification. The PCR products were fractionated on a 2.5% agarose gel and stained with ethidium bromide. Lane 1, DNA mass standards; lane 2 and 3, immature males; lane 4 and 5, immature females; lane 6 and 7, mature males; lane 8 and 9, mature females; lanes 10 and 11, positive controls with the cDNAs encoding GTH Ib-1 and GTH Ib-2, respectively; lane 12, negative control with no template.

Fig. 6. Sequence features of the 5∞-flanking region of goldfish GTH Ib genes, (A) gfGTHIb-1 and (B) gfGTHIb-2. The numbers on the right margin refer to the last nt with respect to the distance from the first nt (+1) of 5∞-UTR, revealed by 5∞-RACE products. The putative TATAA sequences are underlined. The putative cis-acting regulatory sequences are double-underlined, and the sequences in reverse orientation are indicated by asterisks. The following abbreviations are used for consensus sequences: 1/2ARE, half androgen responsive element (5∞-TGTYCT-3∞; [Faisst and Meyer, 1992]); 1/2ERE, half estrogen responsive element (5∞-TGACC-3∞; [ Kato et al., 1992]); GSE, gonadotrope-specific element (5∞-YMRAGGYCR-3∞; [Dre´an et al., 1996 ]); GnRH-RE, gonadotropin-releasing hormone responsive element (5∞-TTCCTGTT-3∞; [Schoderbek et al., 1993]). The sequence, 5∞-TGABTCA-3∞ ([Strahl et al., 1997]), which is known as activating protein-1 (AP-1) binding elements, was not observed in the 5∞-flanking regions of goldfish GTH Ib genes.

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and −154 bp in gfGTHIb-2. Taken together, the results obtained from an analysis of the 5∞-flanking region suggest that in goldfish, contiguously overlapped regulatory elements, containing half ARE/ERE and other elements, may be partly involved in the negative effects of the sex steroids on GTH Ib gene expression, by interfering with the action of other stimulating regulatory factors, e.g. GnRH and SF-1. In fact, ARE overlaps another binding site required for basal transcriptional activity in human glycoprotein hormone a-subunit gene, leading to an attenuation in transcription (Clay et al., 1993). Further studies on this 5∞-flanking region in goldfish are needed to elucidate the molecular basis of the sex steroids, SF-1, and GnRH actions on the GTH Ib gene expression. Fig. 7 shows the organization of putative cis-acting elements in the 5∞-flanking regions (−400 bp) of goldfish GTH Ib and mammalian FSH b subunit genes. In goldfish, the location of these elements between −218 and −154 bp is unique when compared with those of the mammalian FSH b genes. The contiguously overlapped regions with half ARE/ERE, GnRH-RE, and GSE in goldfish were not observed in the other compared mammalian species. However, the putative activating

protein-1 (AP-1) binding element was absent in the 5∞-flanking regions (about 1.4 and 1.1 kb) in goldfish GTH Ib genes ( Figs. 6 and 7). The AP-1 element shows a sequence conservation among mammalian FSH b genes (Strahl et al., 1997). This observation suggests that the 5∞-flanking region of goldfish GTH Ib genes is partly involved in unique gene expression, in some manner different from those of the mammalian FSH b genes. 3.6. Conclusions We have determined the complete exon–intron structures of goldfish GTH Ib subunit genes, indicating that fish GTH I is similar to FSH. In addition, it is clearly demonstrated that two distinct genes encoding GTH Ib are present and expressed in the goldfish pituitary, probably due to tetraploidy in this species. The nt sequences of 5∞-flanking regions in the goldfish GTH Ib genes were determined and compared with the corresponding regions of the mammalian FSH b genes. The comparison has revealed a unique region consisting of overlapped and continued cis-acting elements such as half ARE/ERE, GnRH-RE, and GSE in the goldfish.

Fig. 7. Comparison of the organization of the cis-acting regulatory elements in the 5∞-flanking region of goldfish GTH Ib and mammalian FSH b genes. The solid boxes and open boxes represent the potential cis-acting elements and TATAA boxes, respectively. The numbers in parentheses and the asterisks on the elements indicate the location and the sequences in reverse orientation, respectively. The abbreviations of the elements and the references are described in Figs. 3 and 6. PRE (progesterone responsive element), showing a binding ability to progesterone receptor in rat ([O’Conner et al., 1997]), is known to be identical with ARE and GRE (glucocorticoid responsive element) ([Faisst and Meyer, 1992]).

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This structure may imply the diversity of FSH b (including GTH Ib) gene expression in vertebrates.

Acknowledgement This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.

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