Biochimica et Biophysica Acta 949 (1988) 35-42 35 Elsevier

BBA 91769

Molecular cloning and nucleotide sequence of tuna growth hormone cDNA

Nobuyuki Sato b, Kunihiko Watanabe a, Kousaku Murata a, Morihiko Sakaguchi a, Yutaka Kariya b, Shoji Kimura b, Michio Nonaka b

and Akira Kimura a

a Research Institute for Food Science, Kyoto University, Uji, and b Institute for Research and Development, Taiyo Fisheries Co., Ltd., Tokyo (Japan)

(Received 19 May 1987)

Key words: Nudeotide sequence; cDNA library; Growth hormone; (Tuna)

cDNA for mRNA of tuna growth hormone (GH) was cloned by screening a cDNA library constructed from tuna pituitary gland poly(A) + RNA. The nucleotide sequence of cDNA (911 bases) revealed an open reading frame of 615 nucleotides, including a sequence (51 bases) for a possible secretory protein leader peptide. Noncoding regions were found in the nucleotide sequences up- (5'-terminal: 65 bases) and down- (3'-termi- nal: 231 bases) stream of the open reading frame. An amino-acid sequence deduced from the nucleotide sequence of the cDNA was identical with that determined in the purified tuna GH. Tuna GH was composed of 187 amino acids, and had a calculated molecular weight of 21275. Amino-acid sequencing showed that there was one possible N-glycosylation site at Asn (Asn-Cys-Thr). Tuna GH showed amino-acid sequence homologies with chum salmon (67%), yellow tail (90%) and with human (32%) growth hormones.

Introduction

Growth hormones, which are secreted from pituitary glands, are known to function as a stimu- lator of growth, protein synthesis and lipid de- gradation [1,2]. The hormones are also known to modulate a function of insulin through inhibition of glucose utilization [3]. Several kinds of cDNA for growth hormones have been isolated from various sources, and comparative and physiologi- cal studies on the hormones have been done in detail [4-7].

Recently, in a field of fisheries, growth hormones have been receiving increased interest as an efficient feed to bring up fishes. However, the preparation of the hormones from fish pituitary

Correspondence: K. Murata, Research Institute for Food Sci- ence, Kyoto University, Uji, Kyoto 611, Japan.

glands requires tedious and intricate procedures ir~ addition to the problem of low content in the gland. Therefore, an efficient method for mass preparation of the hormones is now being sought. Among the various approaches to this purpose, the recombinant DNA technique is now most easily applied. In fact, by applying this technique, the genes for the growth hormones of chum salmon [8], eel (Saito et al., International Congress on Molecular Biology, Tokyo, 1985) and of yellow tail [9] have been cloned, and the gene from chum salmon has been reported to be highly expressed in microbial cells [8].

Following these studies on the fish growth hormones, we have also cloned a gene for tuna growth hormone. The sequence of the cloned gene has provided some information regarding the potential N-glycosylation site and regions in the primary translation product that may be involved in protein transport and secretion.

0167-4781/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

36

Materials and Methods

Methods Isolation of growth hormone, antibody and

mRNA. Tuna growth hormone was isolated from tuna (Thunnus thynnus) pituitary glands as de- scribed previously [10]. For the isolation of IgG specific to tuna GH, antiserum raised in rabbits was fractionated on a Protein A column [11] and the resulting IgG fraction was passed through a column of Sepharose 4B, attaching tuna GH as a ligand. The IgG bound to tuna GH was eluted with 0.2 M glycine-HC1 (pH 2.5), dialyzed against 20 mM sodium phosphate buffer (pH 7.5), and stored at - 2 0 o C. Total RNA was isolated from 0.7 g of pituitary glands using the guanidium thiocyanate method [12]. Poly(A) + RNA was purified by two chromatographic cycles through oligo(dT)-cellulose [13].

In vitro translation and immunoprecipitation. Translation of 0.5-2 ttg mRNA was carried out at 30 °C for 90 min in a 50/tl of rabbit reticulocyte lysate containing 1.5 mCi/ml [35S]methionine. Other in vitro translation conditions were given in the vendor's specification (Amersham Japan). For immunoprecipitation and fluorographic analysis, translated samples were diluted with 4 vol. of 50 mM Tris-HC1 (pH 7.5)/0.2 M NaC1/6.0 mM EDTA/2.5% Triton X-100. The samples were then incubated with affinity-purified tuna GH antibod- ies at 4°C for 60 min. Immunocomplexes were then precipitated by using Protein A Sepharose CL-4B, and washed three times with 20 mM Tris- HC1 (pH 7.0)/1.0 mM EDTA/0.1 M NaC1. The washed pellets were mixed with sample buffer (20% sucrose, 4.0% sodium dodecyl sulfate (SDS)/ 20 mM dithiothreitol/0.1% Bromophenol blue/ 120 mM Tris-HC1 (pH 6.8)), heated at 100 °C for 3 min, cooled and then centrifuged. An aliquot of the supernatants was analyzed by electrophoresis on a 12.5% polyacrylamide gel [14] in the presence of SDS, and labelled translation products were detected by fluorography.

Amino-acid sequence of tuna growth hormone. Amino-acid sequences of NH 2- and COOH-termi- nal regions of the purified tuna growth hormone were determined by Edman degradation using re- versed-phase HPLC, and the sequence analysis was done using an Applied Biosystems 470A [10].

Construction and cloning of cDNA. cDNA library was constructed using the plasmid vectors (pSV7186 primer and pSV1932 linker), which were prepared by Okayama and Berg [15]. Transforma- tion of Escherichia coli strain DH1 was accom- plished by the procedure of Hanahan [16]. Col- onies transformed with cDNAs were grown on LB agar plates (0.1% glucose/0.5 % yeast extract/1.0% Bacto peptone/0.5% NaC1 (pH 7.2)) containing 35 /~g/ml of ampicillin until they were each ap- prox. 0.5-1 mm in diameter and they were then transferred to nylon membranes by placement of a dry, sterile membranes on a top of the agar. The colonies having tuna GH cDNA were screened by colony hybridization [17] using two kinds of oligonucleotide complementary to the NH2-termi- nal (probe 1: 5'-GACTTCTGTAACTCT-3') and COOH-terminal (probe 2: 5'-AAGGACATG- CACAAG-3') regions of the tuna-GH mRNA estimated from amino acid sequence of the puri- fied tuna GH.

Southern blot hybridization, cDNA in positive colonies was extracted, purified and HindlII-AccI fragment of the cDNA was hybridized to probes 1 and 2 after denaturation with 0.2 M NaOH con- taining 0.6 M NaCI. Hybridization was visualized by autoradiography [18].

M13 subcloning and dideoxy sequencing. M13 (mpl8 and mpl9) RF-DNA was digested with the appropriate restriction enzymes, ligated with the digested DNA fragment to be subcloned, and used to transform E. coli strain JM 109. Single- stranded DNA was purified from recombinant M13 phage and sequenced by the chain-termina- tion method of Sanger et al. [19] with the universal M13 sequencing primer.

Materials All the enzymes for DNA manipulation and

M13 sequence kit were purchased from Takara Shuzo Co., Ltd., Kyoto, Japan. [a-3zp]dCTP (3000 Ci/mmol), [y-32p]ATP (3000 Ci/mmol) and L- [35S]methionine (1070 Ci/mmol), rabbit reticulo- cyte lysate and nylon membrane (pore size: 0.45 /~m) were from Amersham Japan. Okayama and Berg plasmid vectors (pSV7186 and pSV1932), Protein A Sepharose CL-4B, CNBr-activated Sepharose 4B and oligo(dT)-cellulose were purchased from Pharmacia Japan. 5'-End-labelled

oligonucleotide probes (6 .106 c p m / m m o l i were synthesized by Appl ied Biosystems Japan. Other chemicals were all analytical grade reagents.

Results

In vitro translation of tuna GH mRNA Total m R N A extracted f rom tuna pitui tary

glands was translated in a rabbit reticulocyte lysate containing [35S]methionine, and labelled transla- t ion products were immunoprecip i ta ted with affin- i ty-purified tuna G H antibody. An examinat ion of the immunoprecipi ta tes on SDS-polyacrylamide gel electrophoresis showed that only one peptide, with molecular mass of 23 kDa, was precipitable with the an t ibody (Fig. 1, lanes 3 and 4), a l though the purified tuna G H peptide showed a molecular mass of 20 kDa on SDS-gel electrophoresis (Fig. 1, lane 6).

Isolation of cDNA for tuna GH mRNA Starting with 10 #g of poly(A) + R N A extracted

f rom tuna pitui tary gland, a c D N A library of approx. 50 000 recombinants was constructed, and this c D N A l ibrary was first screened by colony hybridizat ion using 32p-labelled oligonucleotide (probe 1: 5 ' - G A C T T C T G T A A C T C T - 3 ' ) (Fig. 2A) complementa ry to the est imated base sequence in

I 2 3 4 5 6 7

Fig. 1. In vitro translation of poly(A) ÷ RNA from tuna pituitary glands. Poly(A) + RNA was translated in a rabbit reticulocyte lysate containing [35S]methionine and labelled translation products were analyzed with SDS-polyacrylamide gel electrophoresis. Lane 1: translation products of total poly(A) + RNA. Lane 2: translation products in the absence of poly(A) ÷ RNA. Lanes 3 and 4: translation products precipi- tated with affinity-purified tuna GH antibody. Lane 6: puri- fied T-GH. Lanes 5 and 7: standard proteins (kDa) (from top): myosin (H-chain), 200; phosphorylase b, 97; bovine serum albumin, 68; ovalbumin, 43; a-chymotrypsinogen, 26; fl-

lactoglobulin, 18 and lysozyme, 14.

37

the NH2-terminal region of tuna GH. By this method, approx. 300 colonies were selected f rom 10 000 recombinants as candidates carrying c D N A for tuna G H m R N A . To obtain a clone having longer insert, the c D N A in these candidates was isolated by an alkaline lysis procedure [20], and sizes of HindlII-AccI digestion products of the c D N A s were compared after agarose gel electro- phoresis. A m o n g 300 clones tested, 40 clones were found to contain about 1.0 kb D N A insert in size, which seemed to be long enough to encode the ho rmone peptide of 23 kDa (Fig. 2B, lane 2). c D N A s in all 40 clones were shown to hybridize

(A)

(B)

2

1 2 3

Fig. 2. Screening of tuna GH cDNA by colony (A) and Southern (B) hybridizations. (A) For the detection of cloned plasmid containing tuna GH cDNA, ampicillin-resistant clones were grown on LB agar plates, transferred to nylon membrane, and then hybridized to 32p-labelled probe 1 complementary to NH2-terminal region of mRNA template. The positive clones were visualized by autoradiography. (B) Plasmid DNA (pTTS339) extracted from a clone hybridized to probe 1 was digested with HindlII-AccI, electrophoresed in 1.2% agarose gel, and visualized by ethidium bromide staining (lane 2). The DNA fragments in lane 2 were transferred to nylon membrane and hybridized to 32 P-labelled probe 2 (lane 3). DNAs in lane

1 are standard markers of Hin dllI-digested A DNA.

38

(A)

H I

P N PPC A Hh Pv

m

O 0.5 I.O (Kb) I , b r , I , , , , l i , , i J

(O)

P H Pv Pv E Pv I I I I i I

o . . . . . . . . i o ( K b !

Fig. 3. Physical maps of cDNA inserts for tuna GH mRNA. (A) Physical map of insert in pTI'S339. The diagram shows (from left to right) G-C tail (thin hne), 5 '-untranslated region (thick line), signal peptide region (hatched box), mature tuna GH peptide region (open box), 3'-untranslated region (thick line) and A.T tail (thin line). The extents and directions of DNA sequencing are indicated by arrows. (B) Physical map of insert in another type of eDNA. Abbreviations used for the restriction sites in physical maps are: H, HindIII; P, PstI; N, NcoI; C, ClaI; A, AccI; Hh, HhaI; Pv, PouII and E, EcoRI.

to 32p-labeUed ol igonucleot ide (probe 2: 5 ' - A A G -

G A C A T G C A C A A G - 3 ' ) complementa ry to the est imated base sequence in COOH- te rmina l re-

gion of tuna G H (Fig. 2B, lane 3). One of the typical clones was selected, designated pTTS339,

a nd was subjected to further analyses. The restric- t ion map of the insert in pTTS339 was con-

structed by restriction endonuclease analysis (Fig. 3A).

Besides these 40 clones (pTTS339), another type

(4 clones) of e D N A could be detected in the 300

clones men t ioned above. These four clones were found to conta in about 1.5 kb D N A insert in size and were different from pTTS339 in the restric-

t ion pa t te rn by HindIII-AccI endonuclease, al- though they hybridized with bo th probes 1 and 2

(data not shown). The restriction sites of these clones were two for PouII, one for HindIII and

EcoRI. No sites were found for ClaI, AccI and HhaI (Fig. 3B).

Nucleotide sequence of tuna GH cDNA The strategy used for the sequence analysis of

the insert in pTTS339 is shown in Fig. 3A. The nucleot ide sequence of the insert, along with the sequence of the 204 amino acids composing the p r imary structure of the tuna G H polypept ide coded by the in terna l open reading frame is shown

in Fig. 4. The amino-acid coding sequence begins at nucleot ide 66 and extends to nucleotide 680,

TABLE I

CODON USAGE IN TUNA GH eDNA

The numbers indicate the frequency with which the codons are used in the coding region of T-GH eDNA. OC, OP and AM designate the terminators ochre, opal and amber, respectively.

TIT Phe 2 TCT Ser 11 TAT Tyr 4 TGT Cys 1 TTC Phe 7 TCC Ser 4 TAC Tyr 3 TGC Cys 3 TrA Leu 0 TCA Ser 3 TAA OC 0 TGA OP 0 TTG Leu 2 TCG Ser 2 TAG AM 1 TGG Trp 1

CTT Leu 1 CCT Pro 0 CAT His 1 CGT Arg 3 CTC Leu 8 CCC Pro 1 CAC His 4 CGC Arg 1 CTA Leu 0 CCA Pro 4 CAA Gin 4 CGA Arg 2 CTG Leu 20 CCG Pro 2 CAG Gin 9 CGG Arg 1

ATT lie 0 ACT Thr 0 AAT Asn 1 AGT Ser 1 ATC lie 10 ACC Thr 2 AAC Asn 5 AGC Ser 8 ATA Ile 0 ACA Thr 4 AAA Lys 3 AGA Arg 4 ATG Met 3 ACG Thr 1 AAG Lys 6 AGG Arg 2

GTT Val 1 GCT Ala 7 GAT Asp 3 GGT Gly 2 GTC Val 4 GCC Ala 4 CAG Asp 7 GGC Gly 0 GTA Val 0 GCA Ala 1 GAA Glu 3 GGA Gly 5 GTG Val 4 GCG Ala 0 GAG glu 9 GGG Gly 0

39

i 50 5' ...... CTGCAGGGGGGGGGGAGATCGCACTGAAGAACTGAGCTCAGATCAGATTAACCAGAACCAGAACTGAACCCAGACCAGCC

i00 ATG GAC AGA GTC TTT CTC TTG CTG TCA GTC CTG TCT CTG GGT GTC TCC TCT CAG CCA ATC ACA GAC AGC CAG CGT Met Asp Arg Val Phe Leu Leu Leu Ser Val Leu Ser Leu Gly Val Ser Ser Gln Pro Ile Thr Asp Ser Gln Arg

-i0 -I 1 150 200

CTG TTC TCC ATC GCT GTC AGC AGA GTG CAA CAC CTC CAC CTG CTC GCC CAG AGA CTC TTC TCT GAC TTT GAG AGC Leu Phe Ser Ile Ala Val Ser Arg Val Gln His Leu His Leu Leu Ala Gln Arg Leu Phe Set Asp Phe GIu Ser

I0 20 30 250 *** *** *** *** ***

TCT CTG CAG ACA GAG GAG CAG CGT CAG CTC AAC AAA ATC TTC CTG CAG GAT TTC TGC AAC TCT GAT TAC ATC ATC Ser Leu Gln Thr Glu Glu Gln Arg Gln Leu Asn Lys Ile Phe Leu Gln Asp Phe Cys Asn Ser Asp Tyr Ile Ile

40 50 300 350

AGC CCG ATC GAC AAG CAT GAG ACA CAA CGC AGC TCT GTT CTG AAG CTG CTG TCG ATC TCC TAT CGA TTG GTG GAA Ser Pro Ile Asp Lys His Glu Thr Gln Arg Ser Ser Val Leu Lys Leu Leu Ser Ile Ser Tyr Arg Leu Val Glu

60 70 80 400

TCG TGG GAG TTC CCC AGC CGT TCT CTG TCC GGA GGT TCT GCT CCA AGG AAC CAA ATC TCA CCA AAA CTG TCT GAA Ser Trp Glu Phe Pro Ser Arg Ser Leu Ser Gly Gly Ser Ala Pro Arg Asn Gln Ile Ser Pro Lys Leu Ser Glu

90 I00 450 500

CTG AAG ACA GGA ATC CAC CTG CTG ATC AGG GCC AAT CAG GAT GGA GAC GAG ATG TTC GCT GAC AGC TCT GCC CTC Leu Lys Thr Gly Ile His Leu Leu Ile Arg Ala Asn Gin Asp Gly Ala Glu Met Phe Ala Asp Ser Ser Ala Leu

110 120 130 55O

CAG CTC GCT CCG TAT GGA AAC TAT TAT CAA AGT CTG GGA GCT GAC GAG TCA CTG AGA CGG AGC TAC GAG CTG CTT Gin Leu Ala Pro Tyr Gly ASh Tyr Tyr Gln Ser Leu Gly Ala Asp Glu Ser Leu Arg Arg Ser Tyr Glu Leu Leu

140 150 600 *** *** *** *** *** 650

GCC TGC TTC AAG AAG GAC ATG CAC AAG GTG GAG ACC TAC CTG ACG GTG GCT AAA TGT CGA CTC TCT CCA GAA GCT Ala Cys Phe Lys Lys Asp Met His Lys Val Glu Thr Tyr Leu Thr Val Ala Lys Cys Arg Leu Ser Pro Glu Ala

160 170 180 700 750

AAC TGC ACC CTG TAG CCCCGCCTCTCTGATGACGTCATCCTGTGTGTTCTGGAGCCCCGCCTCTCTGATGACGTAATCATGTGTGTTCTGTAGC _A_sn.Cys_ _rh_E Leu

8.00 850 CC CGCC TCCATGTTC TCTTTGCTGGTTAGCATTAGCCTGTGATGGTTTTCTGATGTCAT~ATCAGATAAATAGTACAAGCTATGAACAGGAAGTGATGT

900 CAGACTGTC GGCC TTTTCTCAGCATGTG~ TGCGCTGAGTTGCATTCAAAAAAAAAAAAAAAAAA .......... 3 '

Fig. 4. Nucleotide sequence of tuna GH cDNA and amino-acid sequence of precursor tuna GH. The base sequence was determined

as described in Materials and Methods. Precursor tuna GH codon sequence including leader peptide (amino acid: -17-, -I) and

mature tuna GH peptide (amino acid: 1-187) starts at nucleotide position 66 and ends at nucleotide position 677. The sequences corresponding to those of probes 1 and 2 are indicated by asterisks (nucleotide position 264-278 for probe 1 and nucleotide position 60_3-617 for probe 2). Polyadenylation signal (nucleotide position 888-893) is boxed. Potential N-glycosylation site is marked by

dashed line and three direct repeats are by underlines.

4 I , . z / i ' 1

o ~,-I " I"

0 50 I00 150 200 Residue No.

Fig. 5. Hydropathy of tuna GH peptide. Hydrophobicity val- ues obtained by the method of Kyte and Doolitfle [23] were plotted with respect to the positions of amino-acid sequence. The values for 14 sequential amino acids were averaged and assigned to the middle residue of the span. The arrow indicates

the N-terminus of the mature tuna GH peptide.

leaving 65 basepair (bp) and 231 bp untranslated sequences at the 5' and 3' ends of the cDNA, respectively.

A stretch of more than 30 adenosine residues is found at the Y-end of the insert. An eukaryotic polyadenylation signal, AATAAA, was present 19 bases upstream from the begining of poly(A) tail. The open reading frame coded a polypeptide with a molecular weight of 23 093, which represents a precursor polypeptide containing a leader or sig- nal peptide sequence that is subsequently cleaved off during processing a n d / o r secretion of the ma- ture tuna G H protein with a molecular weight of 20 kDa (Fig. 1, lane 6). Slightly higher hydropathy

40

H-GH 1 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu Cys Leu Pro Trp Leu Gln Glu Gly Ser 25

T-GH 1 Met Asp Arg Val Phe -- Leu Leu Leu Set Val Leu Set Leu Gly Val Ser 16

S-GH 1 Met Gly Gln Val Phe -- Leu Leu Met Pro Val Leu Leu Val Ser Cys Phe 16

Y-GH 1 Met Asp Arg Val Val -- Leu Leu Leu Ser Val Leu Ser Leu Gly Val Ser 16

H-GH 26 Ala Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp ASh Ala Met Leu Arg Ala His Arg 45

T-GH 17 Set Gln Pro Ile Thr Asp Ser Gln -- --Arg Leu Phe Ser Ile Ala Val Ser Arg Val Gln His 36

S-GH 17 Leu Set Gln Gly Ala Ala Ile Glu Asn -- Gln -- --Arg Leu Phe Asn Ile Ala Val Set Arg Val Gln His 38

Y-GH 17 Set Gln Pro Ile Thr Asp Set Gln -- -- His Leu Phe Ser Ile Ala Val Set Arg Ile Gln Asn 36

H-GH 46

T-GH 37

S-Gh 39

Y-GH 37

H-GH 71

T-GH 61

S-GH 63

Y-GH 61

H-GH 96

T-GH 86

S-GH 88

Y-GH 86

iLeu His Gln Leu Ala L Gln Asp Thr Tyr Glu Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Set Phe 70

Leu His Leu Leu Ala L Arg Leu Phe Ser Asp Phe Glu Ser Ser Leu Gln Thr Glu Glu Gln Arg Gln Leu -- 60

Phe

Leu His Leu Leu AlaJGln Lys Met Phe Asn Asp Phe Asp Gly Thr Leu Leu Pro Asp Glu Arg Arg Gln Leu -- 62

I Leu His Leu Leu Ala~ Gln Arg Leu Phe Ser Asn Phe Glu Ser Thr Leu Gln Thr Glu Asp Gln Arg Gln Leu -- 60

Leu Gln Asn Pro Gln Thr Set Leu Cys Phe Set Glu Set Ile Pro Thr Pro Set Asn Arg GIu GIu Thr Gln Gln 95

Asn Lys Ile Phe Leu Gln Asp Phe Cys Asn Set Asp Tyr Ile Ile Ser Pro Ile Asp Lys HislGlu Thr Gln[Arg 85

Ash Lys Ile Phe Leu Leu Asp Phe Cys Asn Ser Asp Ser Ile Val Ser Pro Val Asp Lys His Glu Thr Gln Lys 87

Asn Lys Ile Phe Leu Gln Asp Phe Cys Asn Set Asp Tyr ,11e Ile Set Pro Ile Asp Lys His GIu Thr Gln Arg 85

Trp"

Lys Ser ASh Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg 120

Set Set Val Leu Lys Leu Leu Set Ile Set Tyr Arg Leu Val GIu Ser Glu Phe Pro Set Arg Ser Leu -- 108

Set Set Val Leu Lys Leu Leu His Ile Ser Phe ArglLeu Ile GIu Ser TrpIGlu Tyr Pro Set Gln Thr Leu Ile 112 ! I

Ser Ser Val Leu Lys Leu Leu Ser Ile Ser Tyr ArgILeu Val Glu Ser TrPIGlu Phe Ser Set Arg Phe Leu -- 108

H-GH 121

T-GH 109

S-GH 113

Y-GH 109

Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Set Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu 145

Ser Gly Gly Ser -- Ala Pro Arg Asn Gln Ile Ser Pro Lys Leu Set GIu Leu Lys Thr 128

Ile Ser Asn -- -- Ser Leu Met Val -- Arg Asn Ala Asn Gln Ile Ser Glu Lys Leu Ser Asp Leu Lys Val 134

Ser Gly Gly Ser -- Ala Leu Arg ASh Gln Ile Ser Pro Arg Leu Ser Glu Leu Lys Thr 128

H-GH 146

T-GH 129

S-GH 135

Y-GH 129

H-GH 168

T-GH 154

S-GH 160

Y-GH 154

H-GH 193

T-GH 179

S-GH 185

Y-GH 179

H-GH 217

T-GH 204

S-GH 210

Y-GH 204

Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly -- -- Ser Pro Arg Thr Gly Gln lle Phe Lys Gln Thr 167

Gly Ile His Leu Leu Ile Arg Ala Asn Gln Asp Gly Ala Glu Met Phe Ala Asp Set Ser Ala Leu Gln Leu Ala 153

Gly Ile Asn Leu Leu Ile Thr Gly Ser Gln Asp Gly Val Leu Ser Leu Asp Asp Asn Asp Set Gln Gln Leu Pro 159

Gly Ile Asn Leu Leu Ile Thr Gly Ser Gln Asp Gly Ala Glu Met Phe Ser Asp Val Set Ala Leu Gln Leu Ala 153

-- Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys ASh Tyr Gly Leu Leu Tyr Cys Phe 192

Pro Tyr Gly Asn Tyr Tyr Gln Set Leu Gly Ala Asp Glu Ser Leu Arg Arg SerlTyr GIu Leu Leu Ala Cys Phe 178

I Pro Tyr Gly Asn Tyr Tyr Gln Asn Leu Gly Gly Asp Gly ASh Val Arg Arg Asn~Tyr Glu Leu Leu Ala Cys Phe 184

r Pro Tyr Gly Asn Phe Tyr Gln Ser Leu Gly Gly Glu Glu Leu Leu Arg Arg AsnITyr Glu Leu Leu Ala Cys Phe 178

Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg -- Set Val Glu Gly Ser Cys Gly 216

Lys Lys Asp Met His Lys Val Glu Thr Tyr Leu Thr Val Ala Lys Cys Arg Leu Ser Pro Glu Ala Asn Cys Thr 203

Lys Lys Asp Met His Lys Val Glu Thr Tyr Leu Thr Val Ala Lys Cys Arg Lys Ser Leu Glu Ala Asn Cys Thr 209

Lys Lys Asp Met His Lys Val Glu Thr Tyr Leu Thr Val Ala Lys Cys Arg Leu Ser Pro Glu Ala Asn Cys Thr 203

Phe

Leu

Leu

Leu

Fig. 6. Comparison of amino acid sequence between tuna (T-GH), human (H-GH), salmon (S-GH) and yellow tail (Y-GH) growth hormones. Gaps were introduced in the sequences to maximize the potential homologies. The amino-acid residues largely conserved in four species are boxed in with solid lines. The NH2-terminal amino acid (methionine) of precursor hormones are taken at

position 1.

was found in a leader or signal peptide sequence region (Fig. 5). The analysis of the amino-acid composition of tuna GH polypeptide indicated that the mature protein of tuna GH starts from glutamine residue at position 18 (Gin-18). One possible site for N-glycosylation (Asn-Xaa-Thr or Ser) [20] was located in Asn-201 (Asn-201-Cys- 202-Thr-203) (Fig. 4). The direct-repeat sequences can be located at nucleotide positions 675, 718 and 753, all of which were included in the 3'- untranslated terminal region of tuna GH cDNA. The amino-acid composition deduced from base sequence was as follows: Ala, 12; Arg, 13; Asn, 6; Asp, 10; Cys, 4; Gly, 7; His, 5; Ile, 10; Leu, 31; Lys, 9; Met, 3; Phe, 9; Pro, 7; Ser, 29; Thr, 7; Trp, 1; Tyr, 7; and Val, 9. Approx. 30% of the total amino acid residues in tuna GH was occupied by leucine and serine. In analogy with other growth hormones hitherto purified, tuna GH contained 4 cysteine (Cys-52, Cys-160, Cys-177, Cys-185) and 1 tryptophan (Trp-85) residues in its molecule. Codon usage of tuna GH is shown in Table I. It was rather nonrandom.

Homology of tuna GH with other growth hormones Comparison of amino-acid sequence of tuna

GH with other growth hormones is shown in Fig. 6. The tuna GH showed 67% amino-acid and 66% nucleotide sequence homologies with salmon growth hormone, 90% amino-acid and 88% nucleotide sequence homologies with yellow tail growth hormone. The homologies of tuna GH with human growth hormone were rather low and were 32% and 46% for amino-acid and nucleotide sequences, respectively.

Discussion

To clone a gene for tuna growth hormone, we have constructed a cDNA library according to the procedure given by Okayama and Berg [15]. By colony and Southern hybridizations [17,18], two types of positive clone were identified from 10000 recombinants in the cDNA library. These clones hybridized to both oligonucleotide probes that were synthesized based on the amino-acid se- quence of NH 2- and COOH-terminal regions of the purified tuna GH. This result suggested that tuna pituitary gland contains two kinds of tuna

41

GH. The occurrence of two types of growth hormone has also been reported in salmon [8,22] and eel [23] pituitary glands. The abundant tuna GH cDNA clone in the cDNA library was desig- nated pTTS339 and was used for the sequence analyses.

The amino-acid sequence deduced from the cDNA sequence for the tuna GH clone is con- sistent with that determined in the purified tuna GH (Fig. 4). The ATG at nucleotide sequence position 66 is the start codon and the peptide region having the least number of charged amino acid is located at amino-acid positions 1-17. The secretory protein leader peptide may be in this region. A characteristic base arrangement of the tuna GH cDNA was found in direct repeats in 3'-untranslated region. The tuna GH had three direct repeats with a fundamental sequence of CTGTAGCCCCGCCTCTCTGATGACGT, al- though the function of these repeats is not yet clear. Such direct repeats have not been observed in cDNA base sequences for salmon, yellow tail or human growth hormones [8,9,5]. As has been indi- cated for the eukaryotic mRNAs by Martial et al. [5], codon usage in tuna GH was nonrandom and there was a tendency to use guanine and cytosine bases preferentially in the third position of the codons in tuna GH cDNA (Table I). A similar tendency has also been observed in many codons for eukaryotic mRNAs [4,22]. The amino-acid se- quence of mature tuna GH showed 90%, 67% and 32% homology with that of yellow tail, salmon and human GH, respectively, indicating that the tuna GH is rather closer to yellow tail GH than to salmon GH (Fig. 6). Our results on the analyses of tuna GH cDNA add some evidence as to the unique structural features among four growth hormones (tuna, yellow tail, salmon and human). Firstly, highly conserved sequences exist in four regions. They are Leu-37-Ala-41, Glu-82-Gln-84, Leu-98-Trp-102 and Tyr-172-Leu-189 (Fig. 6). Secondly, these four growth hormones contain four cysteine residues and one tryptophan residues at nearly identical positions in their molecules. The four cysteine residues may be essential for the stabilization of peptide in a biological active form through the disulfide linkage. A consensus amino-acid sequence for the N-glycosylation site (Asn-Xaa-Thr or Ser) was found in the COOH-

42

terminal region of tuna GH (Asn-201-Cys- 202-Thr-203) and the site was located at exactly the same position with respect to the COOH- terminal (4th amino acid from COOH-terminal) (Fig. 6) as that found in other fish growth hormones. The result agreed well with the recent finding of Wagner et al. [25] that a sugar chain is linked with the COOH-terminal region of salmon GH. The number of N-glycosylation sites varies depending on the sources of the growth hormones. Salmon GH has been reported to have two N-gly- cosylation sites, at the positions Asn-153-Asp- 154-Ser-155 and Asn-207-Cys-208-Thr-209. On the other hand, no N-glycosylation sites have been found in human GH [5]. Growth hormone, pro- lactin and somatomammotropin are closely re- lated hormonal peptides [4,22,26-29] and recently such peptides have been purified from fishes [22,23,30-33]. The analyses of these peptides would be useful for a better understanding of the interrelationship between the structure, function and evolution of fish hormones.

The cDNA prepared from tuna pituitary gland poly(A) ÷ RNA has already been cloned onto a expression vector for E. coli cells and study of the microbial production of tuna GH in large quantity is in progress.

Acknowledgement

We thank Dr. H. Kawauchi, Professor at Kitasato University, for his kind help in de- termination of the amino-acid sequence of tuna growth hormone.

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