Structure of a full-length cDNA clone for the pro-alpha1(V/XI) collagen chain of red seabream.

The cDNA of type V/XI collagen alpha1 (rsCOL) chain has been isolated from cells established from eyed-period eggs of red seabream, Pagrus major, and sequenced. The amino acid sequence deduced from red seabream alpha1(V/XI) chain resembles that of type XI collagen alpha1 chain. On the other hand, tissue distribution of rsCOL resembles that of type V collagen based on RT-PCR analysis. This is the first report of the cloning of the full-length cDNA of type V/XI collagen alpha1 chain from fish.

Transgenic medaka overexpressing a melanin-concentrating hormone exhibit lightened body color but no remarkable abnormality.

Melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide that concentrates melanin granules in the melanophores and lightens the body color of a fish. To investigate the utility of MCH as a reporter gene, a transgenic medaka strain overexpressing the MCH gene was established and its phenotypic features were examined. The salmon MCH gene driven by cytomegalovirus promoter was injected into 100 fertilized eggs of the HNI-1 medaka strain, which exhibits black body color. One F(0) female transmitted the transgene and a lightened body color phenotype to the F(1) generation. A homozygous transgenic strain was established by crossing F(2) fish homozygous for the transgene. Expression of the transgene was detected in several organs by Northern blotting. The melanin granules of transgenics were highly shrunk. Bioassay using scales confirmed the secretion of MCH into blood, and the MCH concentration was estimated between 0.5 and 5 microM. Development, growth, feeding behavior, and reproduction of transgenics did not differ significantly among transgenic and nontransgenic siblings. The result whereby enhanced MCH expression induced a change in body color, but no remarkable abnormality, suggests the usefulness of MCH as a novel reporter gene with unique features.

Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cDNA probes.

Up-regulated genes of leucocytes expressing immunoglobulin (Ig+ leucocytes) of hirame rhabdovirus (HRV)-infected Japanese flounder were identified by differential hybridisation, using subtracted and un-subtracted cDNA probes. Ig+ leucocytes were separated from apparently healthy and HRV-infected Japanese flounder by the magnetic beads antibody method using mouse anti-Japanese flounder Ig monoclonal antibody (mab). A cDNA library was constructed from HRV-infected Japanese flounder leucocytes, and was screened with subtracted cDNA probes enriched in genes up-regulated by HRV infection. Fifty cDNAs were isolated for further analysis. These included cDNAs coding for homologues of interferon-inducible 56K protein (IFI56), Stat3, CEF-10, RGS5, inducible poly(A) binding protein, prolylcarboxylpeptidase, basigin III (Ig superfamily), MUC-18 (Ig superfamily), proteasome-nexin 1 (SERPIN), herpes virus entry mediator (TNFR family), collagenase III, gelatinase-b, megakaryocyte stimulating factor, Rab8-interacting protein, IgM, IgD and 20 unknown cDNA clones. The majority of these identified genes are reported for the first time in fish. From leucocytes mRNA for homologues of IFI56, CEF-10, Stat3, SERPIN and inducible poly (A) binding protein expression was shown to increase following HRV infection.

Complete nucleotide sequence of Japanese flounder (Paralichthys olivaceus) mitochondrial genome: structural properties and cue for resolving teleostean relationships.

We cloned and sequenced the complete mitochondrial genome of Japanese flounder (Paralichthys olivaceus). A circular 17,090 bp mitochondrial genome from the flounder contains 37 structural genes as in other vertebrates so far reported. This is the first report of the complete mitochondrial sequence from a higher teleostean fish (Acanthopterygii). The organization including gene order is quite similar to that of other teleostean fishes as well as placental mammals. The putative control region of the Japanese flounder mitochondrial genome contains a length variable region of about a 74 bp tandem repeat cluster. As a preliminary study we adopted the maximum likelihood and neighbor-joining inference methods to examine phylogenetic relationships among teleostean and related fishes. Comparisons of amino acid sequences of protein-coding genes and nucleotide sequences of tRNA genes resolved some middle to deep branches among some teleostean fishes. The flounder mitochondrial genome does not show an indication of evolutionary rate difference among teleosts leading to difficulty in phylogenetic analyses, and our data is useful for future evolutionary studies dealing with higher teleostean fishes.

Identification of a nuclear export signal in MKK6, an activator of the carp p38 mitogen-activated protein kinases.

Carp homologues of p38 mitogen-activated protein kinase (MAPK) and its activator MAPK kinase 6 (MAPKK6, referred to as MKK6) were identified. There exist at least two distinct carp p38s, cp38a and cp38b, both of which consist of 361 amino acids. The transcript of c38a was exclusively expressed in the ovary, whereas that of cp38b was ubiquitously expressed. Western blot analysis with anti-(phosphorylated MAPK) Ig specific to the active p38 or JNK has shown that p38 was activated in response to hypertonic stress (1 M sorbitol) in epithelioma papilosum cyprini carp epithelial cells (EPC) and that the activation of p38 proceeded faster to the maximal level than that of JNK. Carp homologue (cMKK6) of p38 activator MKK6 consists of 404 amino acids. It was expressed ubiquitously but was most abundant in the ovary. An in vitro kinase assay demonstrated that cMKK6 is an upstream activator of cp38 and cp38b in carp because it specifically phosphorylated and activated cp38a and cp38b. Interestingly, we found that cMKK6 has a nuclear export signal (NES) sequence in its N-terminal region although upstream activators of stress-activated MAPKs, p38 and JNK, do not in other animals. The NES sequence facilitated nuclear export of cMKK6 and ovalbumin. Leucine residues in the sequence were crucial for the NES activity, as the activity was lost on replacement of the leucines to alanines. The existence of an NES in cMKK6 implies the requisite of strict regulation of the p38 MAPK pathway in carp. The abundance of these components for the stress-activated pathway in the ovary might be related to ectogenetic early development.

A hyperosmotic stress-induced mRNA of carp cell encodes Na(+)- and Cl(-)-dependent high affinity taurine transporter.

A cDNA clone encoding a Na(+)- and Cl(-)-dependent high affinity taurine transporter was isolated from a common carp cell line, Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78-80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na(+)- and Cl(-)-dependent taurine transport activity with an apparent K(m) of 56 microM. The Na(+)/Cl(-)hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH(2)O. These data suggest the significant role of taurine as an osmolyte in carp cells.

Sequence and expression of a cDNA encoding the red seabream androgen receptor.

The cDNA of the androgen receptor (AR) has been isolated from the ovary of red seabream, Pagrus major, and sequenced. The amino acid sequence of red seabream AR (rsAR) shows about 45% identity with those of Xenopus, rat, mouse, and human ARS. It is shown that rsAR has the ability to trans-activate the responsive gene depending on the presence of androgen.

Novel gelatinolytic activities in rat organs.

Novel gelatinolytic activities in both latent and active forms were detected in the normal organs of rat by gelatin zymography. Multiple active bands were detected in the extracts from the skin, jejunum, muscle, and kidney without any activation. These activities were inhibited by 1,10-phenanthroline or leupeptin, nor by E64, suggesting that these activities were derived from metallo-proteinases or serine-proteinases. Some gelatinolytic active bands were newly induced or enhanced by p-aminophenylmercuric acetate. These results suggest that matrix degrading activities due to metallo- and serine-proteinases were constitutively expressed in various rat normal organs.

Sequence and expression of a cDNA encoding the red sea bream androgen receptor.

The cDNA of the androgen receptor (AR) has been isolated from the ovary of red sea bream, Pagrus major, and sequenced. The amino acid sequence of red sea bream AR (rsAR) shows about 45% identity with that of Xenopus, rat, mouse, and human AR. It is shown that rsAR has the ability to trans-activate the responsive gene depending on the presence of androgen.

Existence of two isoforms of extracellular signal-regulated kinase in fish.

Full-length cDNAs for extracellular signal-regulated kinases (ERK1 and ERK2) were isolated from a carp ovary cDNA library. The deduced amino acid sequences of carp ERK1 (cERK1) and ERK2 (cERK2) exhibited high degrees of homology to the known sequences of the ERK group. Northern blot analysis showed that cERK1 mRNA was not expressed in a tissue-specific manner, though the level of expression of cERK2 mRNA varied among tissues. Western blot analysis of the brain, kidney, and ovary confirmed the expression of cERK1 and cERK2 in carp. Our findings indicate that two isoforms of ERK, ERK1 and ERK2, exist in fish.

Structure and expression of carp mitogen-activated protein kinases homologous to mammalian JNK/SAPK.

Two distinct stress-activated protein kinase (JNKa and b) cDNAs were isolated from a carp ovary cDNA library. These cDNAs contained a full-length open reading frame encoding 427 amino acid residues with a predicted mass of 48.6 kDa. The deduced amino acid sequences of JNKa and b were 95.8% identical, with 18 residues replaced, and showed a high degree of sequence similarity to mammalian JNK/SAPK subgroup including the common dual phosphorylation motif of TPY. By Northern blot analysis, the carp JNKs were found to be abundant in the brain and ovary. Detailed study by RT-PCR assay revealed ubiquitous expression of JNKb, although expression of JNKa was specific to the brain and ovary. The high level expression of both JNKa and b in the ovary implies that JNKs play an important role in egg maturation or ectogenetic early development.

Molecular cloning and growth hormone-regulated gene expression of carp insulin-like growth factor-I.

Two distinct insulin-like growth factor-I (IGF-I) cDNAs were isolated from a juvenile carp liver cDNA library. Both of the cDNAs encoded a full length prepro IGF-I with 161 amino acids: a mature peptide (70 residues), its signal peptide (44 residues), and a carboxy-terminal E domain (47 residues). The similarity of the two cDNA in the open reading frame was 95.1%. The amino acid sequences of E domain predicted by the two cDNAs were different while those of the signal peptide and the mature peptide were the same. By Northern blot analysis, four different sizes (1, 1.5, 2, and 4.5 kb) of IGF-I mRNA were recognized in the liver of juvenile carp. The two smaller species (1 kb and 1.5 kb) were not detected in adult carp. The expression of these two species was preferably induced by exogenous growth hormone administration in the liver of juvenile carp.

Fish glucocorticoid receptor with splicing variants in the DNA binding domain.

Here we describe the isolation of a rainbow trout cDNA containing an entire GR coding region. Although the encoded protein is highly homologous to other GRs, especially in its DNA binding domain, it contains a nine amino acid insertion between the two zinc fingers. This novel form is found in all rainbow trout tissues examined; however, the testis also contains a splice variant lacking this insert, making it completely continuous to other GRs. In transient transfection assays of cultured cells, the two rainbow trout GR variants activated transcription from the glucocorticoid-responsive mouse mammary tumor virus promoter to comparable levels.

Transgenic expression of L-gulono-gamma-lactone oxidase in medaka (Oryzias latipes), a teleost fish that lacks this enzyme necessary for L-ascorbic acid biosynthesis.

Transfer of the gene for L-gulono-gamma-lactone oxidase, the missing enzyme in L-ascorbic acid biosynthesis in scurvy-prone animals, into medaka (Oryzias latipes) was successfully done. The expression plasmid pSVL-GLO, carrying rat liver L-gulono-gamma-lactone oxidase cDNA, was microinjected into the cytoplasm of fertilized eggs during the one-cell stage. Four male F0 fish having the transgene in their germ cells came to maturity, and F1 progeny derived from one of the F0 fish possessed L-gulono-gamma-lactone oxidase activity, indicating that the transgene was functionally expressed in the fish. Genomic Southern blot analysis demonstrated that the transgene existed in both chromosome-integrated and extrachromosomal forms.

Effect of maturation on activities of various proteases and protease inhibitors in the muscle of Ayu (Plecoglossus altivelis).

1. Increases in activities of muscle muticatalytic proteinase, modori-inducing proteinase (latent trypsin-like proteinase), cathepsin B and L-like proteases and cathepsin D were observed more markedly for male fish than female fish, in the spawning stage. 2. Decreases in inhibitory activities of muscle serine and cysteine protease inhibitors were observed more markedly for male fish than female fish in the spawning stage.

A group of novel latent serine proteinases degrading myosin heavy chain in fish muscle.

Through a study on thermal degradation of fish jelly products, the existence of a group of latent trypsin-like serine proteinases was demonstrated in fish muscle. These proteinases share common properties in existing as latent forms (being activated by heating around neutral pH in the presence of NaCl), having trypsin-like serine proteinase properties and showing strong myosin heavy chain degrading activity. This group of proteinases could be classified into four subtypes according to the intracellular localization (sarcoplasmic and myofibril-associated types) and the optimum temperature range (50 and 60 degrees C types).

Purification and properties of a novel latent proteinase showing myosin heavy chain-degrading activity from threadfin-bream muscle.

A novel latent proteinase of which activity was induced by heating in the presence of NaCl was purified to homogeneity from threadfin-bream muscle by a combination of DEAE-cellulose, Con A-Sepharose, Arg-Sepharose, and Shim-pack HAC chromatographies. This proteinase was a glycoprotein having a monomeric subunit structure; Mr was estimated to be 77,000 on SDS-PAGE analysis. The proteinase hydrolyzed Boc-Leu-Thr-Arg-MCA as well as myosin heavy chain in the presence of 2-4% NaCl at pH 7.0 and at 60 degrees C, optimally. The proteinase was classified as serine proteinase based on the effects of soybean trypsin inhibitor, leupeptin, and antipain.

Significant amount of multicatalytic proteinase identified on membrane from human erythrocyte.

Multicatalytic proteinase (MCP) was solubilized from human erythrocyte membrane with 0.1% Triton X-100 and purified to homogeneity using a combination of DEAE-cellulose, hydroxylapatite, and Ultrogel AcA34 chromatographies. This membranous MCP had similar properties to MCP purified in parallel from the cytosol. Both MCPs had a molecular mass of 570 kDa, were composed of apparently nine subunits of 22-36 kDa and had trypsin- and chymotrypsin-like activities. These activities were latent and required heating for the induction. However, slight differences were observed in the effects of reagents (DFP, monoiodoacetic acid, Mg2+, and Ca2+) between membranous and cytosolic MCP. The amount of MCP identified on membranes was estimated to be three-quarters or one-half of that found in the cytosol based on its trypsin- or chymotrypsin-like activity, respectively.

Effect of NaCl on the heating activation of the heat-stable alkaline proteinases from various animal muscles.

1. Heat-stable alkaline proteinase (HAP) showed a wide distribution in fish, avian and mammalian muscles, while the total activity varied among animal species. 2. Total activity of HAP was changeable according to the degree of maturation in the case of chum salmon. 3. Effect of NaCl on HAPs varied among animal species. 4. It seems likely that the different sensitivity of HAPs to NaCl reflects the difference in the living circumstances of each animal species. 5. It is also postulated that the different sensitivity of HAPs to NaCl reflects the conformational diversity of the regulatory structure of HAP among animal species.

Comparison of calpain I and calpain II from carp muscle.

1. The content of calpain II is 3.4 times more than that of calpain I when estimated by the elution profiles from a column of DEAE-cellulose. 2. Calpain I required 1 mM Ca2+ and calpain II required 5 mM Ca2+ to show the full activities. These data demonstrated that Ca2+-sensitivities of both calpains were lower than those of mammalian calpains, respectively. 3. The optimum caseinolytic activity was pH 7.2 for calpain I and pH 7.5 for calpain II. 4. The molecular weight of calpain I was estimated to be 110 k and that of calpain II to be 120 k by gel filtration. 5. Calpain I was much more heat-stable than calpain II around 50-60 degrees C. 6. Both calpains were sensitive to calpastatin, an endogenous inhibitor for calpain.