Purification of carp growth hormone and cloning of the complementary DNA

The growth hormone (GH) was isolated and purified from common carp (Cyprinus carpio) pituitary glands by salt precipitation and HPLC on reverse-phase C18 columns. The carp GH cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The positive clones were selected and sequenced. The full-length carp GH cDNA contains 1187 nucleotide basepairs with an open reading frame coding for the precursor form carp GH of 210 amino-acid residues. The partial amino-acid sequence from the protein completely agrees with that derived from the cDNA, with serine as the first residue in mature carp GH preceded by a 22-residue hydrophobic signal peptide. Comparison of the amino-acid sequence of carp GH with those of various species reveals positional identity at 32.4%, 38.8%, 42.0%, 37.2%, 66%, 55% and 49% with GHs of man, rat, duck, bullfrog, salmon, tuna and yellow tail, respectively.     

Cloning and sequencing of bullfrog growth hormone complementary DNA

Total mRNA was isolated from the pituitary glands of bullfrog (Rana catesbeiana), purified by affinity chromatography with oligo(dT)-cellulose columns. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The cDNA library was screened by hybridization with 32P-labeled duck growth hormone (GH) cDNA. A positive clone was selected and sequenced. The full-length bullfrog GH cDNA contains 950 nucleotide pairs with an open reading frame coding for the precursor GH of 215 amino-acid residues. The partial amino-acid sequence from the protein confirms that derived from the cDNA, with Phe as the first residue in the mature bullfrog GH preceded by a 25-residue hydrophobic signal peptide. The bullfrog GH shares sequence homology with those of other vertebrate species in the following order: duck (61% protein sequence homology; 67% cDNA homology), rat (56%; 61%), human (47%; 57%) and salmon (42%; 50%).     

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.     

Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).     

Characterization of a β-D-mannosidase from a marine gastropod, Aplysia kurodai

A β-D-mannosidase (EC 3.2.1.25) with a molecular mass of approximately 100 kDa was purified from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by column chromatographies on TOYOPEARL Butyl-650 M, TOYOPEARL DEAE-650 M, and Superdex 200 10/300 GL. This enzyme, named AkMnsd in the present study, showed optimal activities at pH 4.5 and 40 °C and was stable at the acidic pH range from 2.0 to 6.7 and the temperature below 38 °C. The Km and Vmax values for AkMnsd determined at pH 6.0 and 30 °C with p-nitrophenyl β-d-mannopyranoside were 0.10 mM and 3.75 μmol/min/mg, respectively. AkMnsd degraded various polymer mannans as well as mannooligosaccharides liberating mannose as a major degradation product. Linear mannan from green alga Codium fragile was completely depolymerized by AkMnsd in the presence of AkMan, an endolytic β-mannanase, which we previously isolated from the same animal (Zahura et al., Comp. Biochem. Physiol. B 157, 137-148 (2010)). A cDNA encoding AkMnsd was amplified from the Aplysia hepatopancreas cDNA by the PCR using degenerated primers designed on the basis of N-terminal and internal amino-acid sequences of AkMnsd. The cloned AkMnsd cDNA consisted of 2985 bp and encoded an amino-acid sequence of 931 residues with the calculated molecular mass of 101,970 Da. The deduced sequence of AkMnsd showed 20-43% amino-acid identity to those of glycoside-hydrolase-family 2 (GHF2) β-mannosidases. The catalytically important amino-acid residues determined in GHF2 enzymes were completely conserved in AkMnsd. Thus, AkMnsd is regarded as a new member of GHF2 mannosidase from marine gastropod.     

Cloning and sequence analysis of mink growth hormone cDNA

A cDNA clone for mink growth hormone (GH) was isolated from a mink pituitary cDNA library, employing a part of rat growth hormone cDNA sequence as a probe. According to the nucleotide sequence, mature mink GH consists of 190 amino acids with a calculated molecular weight of 21,720. The amino acid sequence homology between the mature region of mink GH and those of pig GH, rat GH, bovine GH and human GH was 98.4%, 93.7%, 89.0% and 66.7%, respectively.     

Characterization of a cDNA for human glutathione-insulin transhydrogenase (protein-disulfide isomerase/oxidoreductase)

A human liver cDNA expression library in lambda-phage gt11 was screened with monoclonal antibodies to rat liver protein-disulfide isomerase/oxidoreductase (EC 5.3.4.1/1.8.4.2), also known as glutathione-insulin transhydrogenase (GIT). The nucleotide sequence of the largest cDNA insert (hgit-1) was determined. It contained approx. 1500 basepairs, representing an estimated 65% of the glutathione-insulin transhydrogenase message. The amino-acid sequence deduced from this cDNA insert contains a 7-amino-acid long polypeptide determined by sequencing the active-site fragment isolated from the rat GIT protein. A comparison of the nucleotide sequence of hgit-1 and a previously reported nucleotide sequence of rat glutathione-insulin transhydrogenase cDNA shows that the human hgit-1 clone corresponds to the middle of the transhydrogenase message at amino-acid residue number 275 of the rat protein, and codes for 206 amino-acid residues, including one of the two active-site regions of glutathione-insulin transhydrogenase, a stop codon (TAA), a long 3'-noncoding region of over 800 bases, a polyadenylation signal (AATAA), and a 29 base poly(A) tail. There exists high homology between the human and rat enzymes (94% in the overall amino-acid sequence, with 100% in the active site region and 81% in the nucleotide sequence within the coding portion of hgit-1). As with the rat enzyme, the human enzyme shows some identity with another dithiol-disulfide-exchange protein, Escherichia coli thioredoxin. Like rat cDNA, the human hgit-1 cDNA hybridized to rat mRNA of 2500 bases on a Northern blot. The relative quantitative abundance of GIT mRNA in nine rat tissues studied using hgit-1 as a hybridization probe was found to be in the same order as previously found with the rat cDNA. Thus, the above studies indicate that glutathione-insulin transhydrogenase is a highly conserved protein and that the human hgit-1 cDNA is suitable for use as a probe for further studies on gene regulation of this enzyme.     

Characterization of a cDNA for human glutathione-insulin transhydrogenase (protein-disulfide isomerase / oxidoreductase)

A human liver cDNA expression library in λ-phage gt11 was screened with monoclonal antibodies to rat liver protein-disulfide isomerase / oxidoreductase (EC 5.3.4.1 / 1.8.4.2), also known as glutathione-insulin transhydrogenase (GIT). The nucleotide sequence of the largest cDNA insert (hgit-1) was determined. It contained approx. 1500 basepairs, representing an estimated 65% of the glutathione-insulin transhydrogenase message. The amino-acid sequence deduced from this cDNA insert contains a 7-amino-acid long polypeptide determined by sequencing the active-site fragment isolated from the rat GIT protein. A comparison of the nucleotide sequence of hgit-1 and a previously reported nucleotide sequence of rat glutathione-insulin transhydrogenase cDNA shows that the human hgit-1 clone corresponds to the middle of the transhydrogenase message at amino-acid residue number 275 of the rat protein, and codes for 206 amino-acid residues, including one of the two active-site regions of glutathione-insulin transhydrogenase, a stop codon (TAA), a long 3′-noncoding region of over 800 bases, a polyadenylation signal (AATAA), and a 29 base poly(A) tail. There exists high homology between the human and rat enzymes (94% in the overall amino-acid sequence, with 100% in the active site region and 81% in the nucleotide sequence within the coding portion of hgit-1). As with the rat enzyme, the human enzyme shows some identity with another dithiol-disulfide-exchange protein, Escherichia coli thioredoxin. Like rat cDNA, the human hgit-1 cDNA hybridized to rat mRNA of 2500 bases on a Northern blot. The relative quantitative abundance of GIT mRNA in nine rat tissues studied using hgit-1 as a hybridization probe was found to be in the same order as previously found with the rat cDNA. Thus, the above studies indicate that glutathione-insulin transhydrogenase is a highly conserved protein and that the human hgit-1 cDNA is suitable for use as a probe for further studies on gene regulation of this enzyme.     

Molecular cloning of bovine beta-lactoglobulin cDNA

A cDNA library from bovine mammary gland mRNA was constructed in pBR322 and screened by hybrid-selected translation and immunoscreening. Several beta-lactoglobulin clones were identified and sequenced. All clones contained cDNA fragments corresponding to the 3' region of beta-lactoglobulin mRNA. The 3' non-translated region of beta-lactoglobulin mRNA consists of 187 nucleotides; the polyadenylation signal AATAAA occurs 17 nucleotides before the poly(A) tail. The amino-acid sequence predicted from the 3' coding region corresponds completely to the previously determined amino-acid sequence of beta-lactoglobulin.     

Porcine growth hormone: molecular cloning of cDNA and expression in bacterial and mammalian cells

Porcine growth hormone (PGH) precursor cDNAs were cloned from a pituitary cDNA library constructed in lambda gt11 by immunoscreening. One of the three clones characterized contained an entire nucleotide sequence for the 216-amino-acid precursor molecule. The deduced amino-acid sequence of PGH confirmed the sequence previously reported for that of the genomic DNA of PGH except for one base difference in the coding sequence. Expression of the full-length PGH cDNA was achieved in bacteria and mammalian cells. The mammalian cell line, COS-1, produced the GH molecule which processed the signal peptide and had the same molecular weight as standard PGH, in contrast to the higher molecular weight of the bacterial product. Radioimmunoassay of the recombinant PGH produced in COS-1 cells also revealed an inhibition curve similar to that of the standard PGH.     

Cloning and sequence analysis of a cDNA from human ovarian granulosa cells encoding the C-terminal part of human elongation factor 2

A cDNA clone, pHGR81, encoding 358 amino-acid residues of the C-terminal region of human elongation factor 2 (EF-2), was isolated from a human ovarian granulosa cell cDNA library. The deduced amino-acid sequence of pHGR81, when compared with the known identical amino-acid sequences of hamster as well as rat EF-2 revealed a substitution of a glutamine by an alanine residue in the partially determined human sequence. The 15 amino-acid-residue sequence comprising the histidine-715, supposed to be of importance for the biological function of EF-2, is preserved in human EF-2. The coding region of the cDNA insert of pHGR81 displays a homology of 87% to hamster and of 88% to rat EF-2 cDNA. In Northern-transfer analysis, pHGR81 specifically hybridizes with an mRNA species of 3.1 kb.     

Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule.

The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.     

Protein structure and cDNA nucleotide sequence of the Japanese quail alpha A globin.

The primary structure of the alpha polypeptide chain (alpha A) of the major component (QII) of Japanese quail hemoglobin was determined by protein and cDNA sequence analysis. The amino-acid sequences of all the soluble tryptic peptides were determined by the conventional protein sequencing technology. The sequence of the remaining portion, which contained an insoluble "core region", was determined through determination of the cDNA nucleotide sequence. The cDNA clones coding for the alpha A globin were isolated from the quail reticulocyte cDNA library, mapped by restriction enzyme digestion, and the nucleotide sequence was determined completely. The primary structure of quail alpha A globin shows a close similarity to that of chicken alpha A globin.     

Cloning and nucleotide sequence of a full-length cDNA for human 14 kDa beta-galactoside-binding lectin

A full-length cDNA for a 14K-type human lung beta-galactoside-binding lectin was cloned. The cDNA includes a 405 bp open reading frame coding 135 amino acids including the initiator methionine, and having a single internal EcoRI site and a polyadenylation signal. The deduced amino-acid sequence agreed completely with the sequence of a human placenta lectin determined by direct amino-acid sequence analysis (Hirabayashi, J. and Kasai, K. (1988) J. Biochem. 104, 1-4). It showed extensive sequence similarity with other vertebrate 14K-type lectins and a 35K-type lectin (carbohydrate-binding protein 35) of mouse 3T3 cell. Search of a Genbank sequence data base revealed significant sequence similarity between the beta-galactoside-binding lectins and the carboxyl-terminal half of an IgE-binding protein, the cDNA of which has been cloned from rat basophilic leukemia cells. Thus, 14K-type lectin, 35K-type lectin and IgE-binding protein appeared to form a superfamily of proteins. Almost all invariant residues are located in the central region of the 14K-type lectins, so this region may constitute an essential part of the lectins, such as the sugar-binding domain.     

Molecular cloning and functional expression of rat liver cytosolic acetyl-CoA hydrolase.

A cytosolic acetyl-CoA hydrolase (CACH) was purified from rat liver to homogeneity by a new method using Triton X-100 as a stabilizer. We digested the purified enzyme with an endopeptidase and determined the N-terminal amino-acid sequences of the two proteolytic fragments. From the sequence data, we designed probes for RT-PCR, and amplified CACH cDNA from rat liver mRNA. The CACH cDNA contains a 1668-bp ORF encoding a protein of 556 amino-acid residues (62 017 Da). Recombinant expression of the cDNA in insect cells resulted in overproduction of functional acetyl-CoA hydrolase with comparable acyl-CoA chain-length specificity and Michaelis constant for acetyl-CoA to those of the native CACH. Database searching shows no homology to other known proteins, but reveals high similarities to two mouse expressed sequence tags (91% and 93% homology) and human mRNA for KIAA0707 hypothetical protein (50% homology) of unknown function.     

Chicken growth hormone: cDNA-synthesis and base sequence

1. Growth hormone (GH)-cDNA was synthesized from poly A(+)-mRNA extracts of chicken pituitary glands. 2. Chicken-cDNA library was cloned into E. coli. 3. Base sequence analysis of chicken GH-cDNA revealed only 70% similarity compared with duck GH-cDNA, and 97% similarity with a previously published chicken GH-cDNA sequence. 4. Dissimilarities in base sequences are primarily observed in the 3'-non-coding region of GH-cDNAs (chicken and duck). 5. Comparisons of amino acid sequences of chicken and duck GH exhibit only three substitutions, while the amino acid sequences of GHs of chicken are identical.     

Mouse and human CD14 (myeloid cell-specific leucine-rich glycoprotein) primary structure deduced from cDNA clones

cDNA clones complementary to MS7-4 (Setoguchi et al. (1988) Somat. Cell Mol. Genet. 14, 427-438) from a mouse macrophage cDNA library were separated. Sequence analysis of these clones demonstrated that the longest cDNA clone, MS7X, had a 1366 bp insert and high homology with that of the human CD14 gene (Ferrero and Goyert (1988) Nucleic Acids Res. 16, 4173). Using the MS7X cDNA probe, cDNA clones were separated from cDNA libraries constructed from a human macrophage cell line and macrophages. The total cDNA sequence was 1364 bp in length, with an open reading frame of 1125 nucleotides matching that of the human CD14 gene except for one nucleotide difference. The amino-acid sequence (mouse CD14), deduced from the nucleotide sequence of the MS7X insert consisted of 351 amino-acid residues with a high leucine content (17.66%) and five putative N-glycosylation sites, and in vitro translation predicted a protein of molecular mass of 37.5 kDa. Human CD14 had 356 amino-acid residues, with high leucine content (15.5%), and contained four putative N-glycosylation sites. Mouse CD14 showed 13 building blocks, of which internal nine blocks have a conserved leucine motif and significant homology with human leucine-rich alpha 2-glycoprotein.     

Cloning of a calcitonin gene-related peptide receptor and a novel calcitonin receptor-like receptor from the gill of flounder, Paralichthys olivaceus

For the first time in non-mammalian vertebrates, cDNA encoding CGRPR was isolated from the gill cDNA library of flounder. The nucleotide sequence consists of a 237bp 5'-UTR, a 1398bp coding sequence for a 465-amino-acid protein, and a 981bp 3'-UTR. The predicted amino-acid sequence has a high degree of identity to hCGRPR (72.3%) and rCGRPR (71.6%) and, to a lesser degree, to hCTR (55.6%) and rCTR (59.3%). In addition, a different type of receptor cDNA was also obtained from the gill cDNA. The nucleotide sequence contains an open-reading frame of 1380bp to produce a 459-amino-acid protein. The open-reading frame of this receptor shows the same degree of identity to mammalian CTR (60.2% to hCTR; 62.3% to rCTR) and CGRPR (64.6% to hCGRPR; 64.4% to rCGRPR). However, the predicted amino-acid sequence was more homologous to hCGRPR (60.2%) and rCGRPR (61.3%) than to hCTR (48.8%) and rCTR (49.5%). The identity of this receptor to fCGRPR is 66.6% at the nucleotide level and 64.2% at the amino-acid level, indicating that the receptor is not likely to be an isoform of CGRPR. The receptor, but not fCGRPR, is expressed in bones, suggesting the possibility that this receptor corresponds to the flounder CTR.     

鲤鱼(Cyprinus carpio)生长激素基因克隆及原核表达

采用逆转录—聚合酶链式反应（ＲＴ－ＰＣＲ）方法,从鲤鱼脑垂体总ＲＮＡ中扩增出编码鲤鱼生长激素（ＧＨ）成熟肽基因序列．定向克隆至质粒ｐＵＣ１８,克隆的鲤鱼ＧＨｃＤＮＡ不含信号肽序列并以新的起始密码子ＡＴＧ取代鲤鱼ＧＨｃＤＮＡ第１个密码子ＴＣＡ．序列分析表明,与Ｋｏｒｅｎ报道的鲤鱼ＧＨｃＤＮＡ相比有两个碱基差异,但推断的氨基酸序列完全一致．将鲤鱼ＧＨｃＤＮＡ定向克隆至原核表达载体ｐＢＶ２２０,构建成重组鲤鱼ＧＨ基因表达载体ｐＢＶｃＧＨ８．ＳＤＳ－ＰＡＧＥ和薄层扫描分析表明：经４２℃诱导,ｐＢＶｃＧＨ８在大肠杆菌中可表达一分子量约２２０００的特异蛋白,表达量占细胞总蛋白的２９．２％．该基因重组的鲤鱼ＧＨ添加到饲料中投喂罗非鱼,证实有明显的促进生长作用