Molecular cloning and expression of salmon prolactin cDNA

Prolactin was purified from chum salmon pituitaries. It was resolved into two variants by reverse-phase high-performance liquid chromatography. A cDNA library was prepared from Pacific chinook salmon pituitaries. Salmon prolactin gene was screened using a synthetic oligonucleotide based on partial protein sequence. A positive clone (PRL-10) was identified and sequenced. It is a full-size clone containing 1.1 kb and coding for a preprolactin of 211 amino acids. A modified prolactin plasmid (PRL-10A), in which the 5' untranslated sequence and the nucleotide sequence coding for the signal peptide of prolactin were deleted, was reconstructed into an expression vector using the heat-inducible lambda pL promotor. Mature prolactin, a single polypeptide of 22 kDa, was efficiently expressed in the bacteria at an elevated temperature.     

Isolation and characterization of chum salmon growth hormone

Two molecular forms of salmon growth hormone (sGH), sGH I and II, have been isolated from the pituitary glands of the chum salmon (Oncorhynchus keta); a two-step extraction procedure, under alkaline (pH 10) conditions, subsequent to acid-acetone extraction was employed for extraction of the sGHs. They were then purified by iso-electric precipitation at pH 5.6, gel filtration on Sephadex G-100, and high-performance liquid chromatography on ODS. Intraperitoneal injection of sGH I and a combination of sGH I and II at doses of 0.01 microgram/g body wt at different intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The GH producing cells in the pituitary of mature chum salmon were identified in the proximal pars distalis immunocytochemically with a specific antiserum; no cross-reactivity was seen in the prolactin cells in the rostral pars distalis. A molecular weight of 22,000 was estimated for both sGHs by gel electrophoresis in sodium dodecyl sulfate. Isoelectric points, by gel electrofocusing, of 5.6 and 6.0 were estimated for sGH I and II, respectively, with differences present in the amino acid composition and the N-terminal residue, suggesting that they may be genetic variants coded on two separate genes. The partial amino acid sequences of sGH I at both terminal regions have been determined.     

Structures of two genes coding for melanin-concentrating hormone of chum salmon

Two MCH genes coding for melanin-concentrating hormone (MCH) were isolated from a chum salmon liver DNA library and characterized. They were shown to be intronless genes with 0.63-kb exons, each of which commonly consisted of an about 80-bp 5'-untranslated region, a region coding for 132 amino acids (aa) MCH precursor protein and an approx. 160-bp 3'-untranslated region. About 20 bp upstream from the putative cap site, sequences were found corresponding to the TATA box. The two genes were 86% identical at the nucleotide sequence level. Sequences homologous to the chum salmon MCH genes were present in the genomes of other fish such as catfish, carp and Chinese grass carp, whereas no highly homologous sequence could be detected in other vertebrate genomes.     

Molecular cloning and sequencing of coho salmon growth hormone cDNA

A cDNA library was constructed using mRNA isolated from coho salmon pituitaries. By employing rainbow trout growth hormone cDNA as a probe, the coho salmon cDNA was isolated and the complete nucleotide (nt) sequence determined. The coding region contains 630 nt while the 5'- and 3'-untranslated regions are 64 and 489 nt in length, respectively. Comparison of the noncoding regions of coho and chum salmon cDNAs reveal identity at the 5' end but significant variation in the 3' end. Chum salmon and rainbow trout have identical amino acid (aa) sequences, but coho salmon growth hormone has a sequence that differs by 6 of the 188 predicted aa. Since salmonids are tetraploid, this difference may be the result of either divergence of the same growth hormone locus or of variation between different loci. Comparisons of the cDNA restriction maps of these three fish species suggest the former possibility.     

Molecular cloning of eel growth hormone cDNA and its expression in Escherichia coli

cDNA clones coding for growth hormone (eGH) of Japanese eel (Anguilla japonica) have been isolated from a cDNA library prepared from pituitary gland poly(A)+ RNA. The nucleotide sequence of the eGH cDNA was determined. It codes for the prehormone of 209 amino acids (aa) including a putative signal peptide of 19 aa. The deduced amino acid sequence was identical with that determined for eGH protein. The primary structure of eGH was compared with those of other species growth hormones (chum salmon, chicken, rat, and human). Mature eGH was expressed in Escherichia coli harboring a plasmid in which the eGH cDNA was under control of the phage lambda pL promoter. Recombinant eGH polypeptide was immunoreactive to rabbit antiserum against natural eGH. Furthermore, eGH derivative with amino-terminal deletion (delta 1-3 eGH) was produced in E. coli reaching up to 5% of total cellular proteins.     

鲈鱼生长激素的分离及其生物活性鉴定

运用葡聚糖凝胶Ｇ－１００过滤和反相高儿液相色谱纯化两步法,首镒从鲈鱼脑垂体中分离出鲈鱼生长激素,通过ＳＤＳ－聚丙烯凝胶电泳测得鲈鱼非还原性的和还原性的生长激素分子量分别为１９．２和２０．７ｋＤ;等电聚焦证实鲈鱼生长激素等电点为７．１５。Ｗｅｓｔｅｒｎ免疫印迹反应证实,鲈鱼生长激素具有与大麻哈鱼生长激素抗体发生特异性免疫交叉反应的特性,而与大麻哈鱼催乳素和生长催乳素抗体无免疫交叉反应。     

Isolation and characterization of a novel growth hormone cDNA from chum salmon (Oncorhynchus keta)

A cDNA clone which codes for a novel growth hormone has been isolated from the library of chum salmon pituitaries. The clone encodes a polypeptide of 210 amino-acid residues including 22 amino-acid residues of signal peptide, which is identical in length with known chum salmon growth hormone. In the coding region, there are 30 base substitutions, some of which result in 12 amino-acid substitutions. There are 8 base changes in the 5' untranslated region, and large insertions/deletions are in the 3' non-coding region. These results clearly indicate that there are at least two species of mRNAs for growth hormone in chum salmon pituitary.     

Cloning and sequence analyses of vasotocin and isotocin precursor cDNAs in the masu salmon, Oncorhynchus masou: evolution of neurohypophysial hormone precursors.

We have cloned and determined the nucleotide sequences of cDNAs encoding precursors of neurohypophysial hormones, vasotocin (VT) and isotocin (IT), from the hypothalamus of masu salmon, Oncorhynchus masou. The deduced amino acid sequences of masu salmon VT and IT precursors (proVT-I and proIT-I) are highly homologous to those of chum salmon proVT-I and proIT-I, respectively. The VT and IT precursors are composed of a signal peptide, hormone and neurophysin (NP), the middle portion of which is highly conserved among vertebrates. Both the NPs extend about 30 amino acids at the C-terminal. The extended C-terminals have a leucin-rich segment in the carboxyl-terminal, as copeptin of vasopressin precursor. Southern bot analysis showed the presence of two types of proVT genes (proVT-I and proVT-II) and proIT genes (proIT-I and proIT-II) in an individual masu salmon, as in a chum salmon. Southern blot analysis with proVT probes further suggested that at least two different types of proVT-I genes exist in a single masu salmon. Northern blot analysis indicated that proVT-I and proIT-I genes are expressed in the hypothalamus, whereas proVT-II and proIT-II genes are not expressed. Evolutionary distance between proVT-I and proIT-I genes was statistically estimated based on synonymous nucleotide substitution in the coding region of the cDNAs. The magnitude of distance between masu salmon proVT-I and proIT-I genes suggested that the highly conserved central portion of NPs resulted from a gene conversion event. Between masu salmon and chum salmon, evolutionary distance for proVT-I genes is about 6-fold larger than that for proIT-I genes.     

Chemical identification of catfish growth hormone and prolactin.

Isolation and primary structure of growth hormone (GH) and prolactin (PRL) from the pituitary gland of catfish (Ictalurus punctatus) are described. Alkaline extract of the pituitary glands was fractionated by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE-cellulose, and reversed-phase high-performance liquid chromatography on Octadecyl silica ODS. Catfish GH and PRL were identified by Western blotting with antisera against chum salmon GH and PRL. The catfish GH consists of 178 residues and is the most similar to carp GH, with sequence identity of 77%, although there is an uninterrupted deletion of 10 amino acid residues that corresponds to carp GH (90-99). The PRL is composed of 187 residues, which also exhibits the highest identity (79%) with carp PRL. Sequence identity between catfish GH and PRL is only 27%.     

Structure of the rat prolactin gene

The organization and sequence of the rat preprolactin gene has been investigated. Analysis of two different plasmids containing pituitary cDNA inserts has provided the complete 681-nucleotide coding sequence of preprolactin as well as 17 nucleotides preceding the initiation codon and 90 nucleotides following the termination codon. Digestion of rat chromosomal DNA with the restriction endonuclease Eco RI followed by size fractionation and hybridization to a labeled prolactin cDNA probe has demonstrated that prolactin genomic sequences are located on 6.0-, 3.9-, and 2.9-kilobase fragments. The 6.0- and 3.9-kilobase fragments were isolated from a library of cloned rat DNA fragments. The sequence of more than 1800 nucleotides of the cloned DNA has been determined. The sequenced region contains coding regions of 180 and 189 nucleotides which specify the COOH-terminal 123 amino acids of the 227-amino-acid sequence of rat preprolactin. These coding regions are separated by an intervening sequence of 597 nucleotides. At least one other large intervening sequence separates this region from the region coding for the NH2-terminal portion of preprolactin. Hybridization experiments suggested that the intervening sequences of the rat prolactin gene contain DNA sequences which are repeated elsewhere in the rat genome.     

Molecular cloning and nucleotide sequence of tuna growth hormone cDNA

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′-terminal: 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.     

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.     

Properties of lactate dehydrogenase from the isopod, Saduria entomon

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.     

Molecular cloning and characterization of complementary DNA encoding for the gonadotropin α-subunit of yellowfin porgy (Acanthopagrus latus)

Recombinant phage clones containing the complementary DNA (cDNA) coding for the a-subunit of gonadotropin (GTH) were isolated from a pituitary gland cDNA library of yellowfin porgy ( Acanthopagrus latus ) by using a degenerate oligonucleotide based on the conserved amino acids sequence of GTH from other species as a probe. This section of 735 base pairs (bp) cDNA contained a 351 bp open reading frame with 20 bp and 345 bp flanking regions at the 5'- and 3'-ends, respectively. The deduced amino acid sequence revealed a 23 amino acid signal peptide and a 94 amino acid mature α-subunit of GTH polypeptide. The latter polypeptide showed 66, 62 and 61% amino acid identity with chum salmon, pike eel and carp GTH peptide hormone α-subunit, respectively.