Organization of the histone genes in the rainbow trout (Salmo gairdnerii)

Summary Twelve clones containing histone genes were isolated from a genomic trout library constructed in the vector Charon 4A. Each of the clones was found to contain a conserved 10.2-kb Eco RI fragment that contained one copy of each of the histones in the order H4-H2B-H1-H2A-H3, all of which are transcribed from the same strand. Genomic Southern blots indicate that these clusters are representative of the vast majority of the histone genes in the trout. Tandemly linked clusters were not found. Approximately 145 copies of this cluster are present in a trout sperm cell. Sequence analysis has shown the genes to be without introns and to show strong selection for codons ending in C or G. Consensus signals similar to those found in other histone genes are present in the flanking regions.     

Histone H4 and H2B genes in rainbow trout (Salmo gairdnerii)

Summary The complete nucleotide sequence of the 3.0-kb BamH I-Sst I restriction fragment contained within the rainbow trout genomic clone TH2 has been determined. This region contains the rainbow trout H4 and H2B histone genes and 5 and 3 flanking and spacer sequences, and represents the 5 half of the histone-gene cluster; the remaining half has been characterized previously. The genes are uninterrupted, and are transcribed from the same strand. The protein sequence of H4, as determined from the nucleic acid sequence, is the same as that derived for other vertebrate H4 proteins, although comparison of nucleotide sequences shows a great deal of sequence divergence, especially in the third base position. The amino acid sequence of H2B, though largely homologous to those of other vertebrate H2B proteins, displays some characteristic differences in primary structure. Consensus sequences noted in many other eukaryotic genes, as well as histone-specific consensus sequences, have been identified. An unusual feature of the spacer region between the H4 and H2B genes is the presence of a duplicated sequence 87 bp in length. The 5 and 3 ends of each repeat are complementary, and each repeat contains smaller repeated sequences internally, as well as a possible cruciform structure.     

Main serum protein of rainbow trout (Salmo gairdnerii): its biological properties and significance

• 1.1. Main serum α₁-protein (α₁P) of rainbow trout was purified and its biochemical and physico-pathological properties were studied.
• 2.2. α₁P was suggested to be a primitive protein having both properties of albumin and AFP in serum proteins of mammals according to the following results.
• 3.3. Molecular weight (75,000), two kinds of molecules (pI 4.55 and 5.05) and amino acid composition.
• 4.4. Dye- or ConA binding activity.
• 5.5. Estrogen binding activity and inhibitory effect on lymphoblastoid-forming activity.
• 6.6. Possible osmotic regulator.
• 7.7. Significant elevation of blood α₁P level in the course of hepatoma induction.

     

Identification and expression of natriuretic peptide receptor type-A and -B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.     

Uptake of vitellogenin into oocytes during early vitellogenic development in the rainbow trout, Oncorhynchus mykiss (Walbaum)

Uptake of ³H-vitellogenin (³H-VTG) into oocytes of various sizes was investigated during early vitellogenic development in the rainbow trout, Oncorhynchus mykiss (Walbaum). Females were injected with ³H-VTG and uptake into oocytes of different sizes (<0.4,0.4–0.59, 0.6–0.79, 0.8–0.99 and 1.0 1.2 mm in diameter) measured. Oocytes measuring less than 0.6 mm in diameter appeared unable to sequester VTG and were therefore considered pre-vitellogenic. Oocytes measuring 0.6 mm or more all sequestered VTG. The larger the oocyte, the more ³H-VTG it sequestered, even when uptake was expressed per unit surface area. The latter observation could be due to an increase in the number of VTG receptors per unit surface area, an increase in the rate of turnover of the VTG receptor, greater access of VTG to the receptors as oocytes grow, or a combination of any of these factors. The data suggest that the ability to sequester VTG is developmentally regulated.     

Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression

In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp.     

Distribution of delta-aminolevulinic acid synthetase and delta-aminolevulinic acid dehydratase in liver and kidney of rainbow trout (Salmo gairdnerii)

1. Activities of delta-aminolevulinic acid synthetase (ALA-S) and delta-aminolevulinic acid dehydratase (ALA-D) in trout liver and kidney were compared with those in the mouse. 2. ALA-S activity (per unit tissue fresh weight) exceeded ALA-D activity in trout liver and kidney. 3. In trout kidney, ALA-S activity slightly exceeded, and ALA-D activity far exceeded, their activities in trout liver. 4. In trout, heme synthesis differs from that in mammals in that appreciable synthesis occurs in the kidney, and in that ALA-S activity is not rate limiting.     

Copper exposure and the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss)

Abstract

Sublethal concentrations of copper in water cause the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss). Receptor cell loss has been correlated to the loss of olfaction in fish and may cause difficulties in olfactory mediated behaviors such as migration. This study investigated the effects of three levels of copper (100, 75 and 50 mg L^?1) on the olfactory epithelium of rainbow trout. Twenty fish randomly allocated between three exposure groups and one control were exposed for 24 hours under static renewal conditions. Light and scanning electron microscopic observations of olfactory tissue were taken to determine the extent of degeneration of receptors. In addition, levels of copper and zinc in the brain tissues were analyzed to determine if the olfactory route was a significant route of copper exposure and transfer to fish brain tissue. Results indicate that degeneration of receptors is related to the concentration of copper. Levels of copper in brain were found to be below detection of the instrument. Levels of zinc were extremely variable ranging from 52 to 132 ng zinc g^?1 brain tissue.     

Biochemical polymorphisms in muscle and liver extracts and in the serum of the rainbow trout Salmo gairdneri

The frequency of polymorphic phenotypes determined by starch gel electrophoresis from six enzyme systems was investigated on 664 rainbow trout (stock I) originating equally from six full-sib families. The enzyme systems studied were CA, AKP, G-6-PD, SOD, AEP, and 6-PGD.
On material deriving from six parental matings totalling 212 offspring (stock II) the mode of inheritance of the first four enzymes (AEP and 6-PGD were not polymorphic) and the additional systems IDH, PGM, Alb, and Psta, not analysed in stock I, were investigated.
The G-6-PD system showed no polymorphism in the family material. The CA, PGM, Alb, and Psta systems were easily identifiable. Their mode of inheritance with two alleles each can be considered as proven. For SOD three alleles, in four out of six possible progeny types, were found, for which the postulated mode of inheritance was confirmed.
For IDH the mode of inheritance found by Allendorff & Utter (1973) was confirmed. This pattern shows two disomic gene loci, one of which is monomorphic, while the other carries four different alleles.
The number of alleles and their mode of inheritance for the AEP system, which was not clearly identifiable, could not be elucidated.     

Effects of cyclopenta[c]phenanthrene and its derivatives on zona radiata protein, ERalpha, and CYP1A mRNA expression in liver of rainbow trout (Oncorhynchus mykiss Walbaum)

Despite cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) have been detected in the environment, the ability of CP-PAH to induce cellular and tissue responses remains poorly characterized. In this study, xenoestrogen-associated responses (mRNA levels of estrogen receptor alpha, ERalpha, and zona radiata protein, Zrp) and xenobiotic effects (CYP1A mRNA) have been investigated in liver of juvenile rainbow trout after short-term treatment (8 and 24 h) with following compounds administered singly: cyclopenta[c]phenanthrene (CP[c]Ph); its derivatives, 5A-CP[c]Ph; 5A6M-CP[c]Ph; 5A9M-CP[c]Ph; B[c]Ph, a structurally similar polycyclic aromatic hydrocarbon; B[a]P, a model CYP1A inducer; and zearalenone (ZEA), naturally occurring ligand for ER. The CYP1A mRNA expression after 24 h of exposure with CP[c]Ph or its derivatives, except 5A9M-CP[c]Ph, was 3-9-fold higher compared to controls (P<0.05), but it was less than that caused by B[a]P (65-fold up regulation; P<0.01). Moreover, neither of the CP-PAH compounds modulated liver ERalpha or Zrp mRNA levels as compared to effects associated with ZEA. Interestingly, a treatment with this ER-ligand, caused moderate but significant increase of CYP1A mRNA expression (about 2.5-fold; P<0.05). The finding that ZEA is capable of acting as either estrogenic and xenobiotic compound, should be further explored in a more detailed and differently designed experiment.     

Characterization of the muscarinic and serotoninergic receptors of the intestine of the rainbow trout (Salmo gairdneri)

The ability of carbachol and 5-hydroxytryptamine (5-HT) to contract isolated segments of rainbow trout intestine in a concentration-dependent manner indicates the presence of muscarinic and serotoninergic receptors in this tissue. The activity of these agonists appears to be directly on the smooth muscle, since ganglionic blockers and inhibitors of neurotransmission did not inhibit contractions. The carbachol-induced contractions were selectively inhibited by atropine and (+-)-3-quinuclidinyl xanthene-9-carboxylate hemioxalate hydrate, an M-2 muscarinic receptor antagonist. However, the inhibition was not competitive. McN-A-343, an M-1 muscarinic agonist had no effect on intrinsic tone. The 5-HT-induced contractions were selectively inhibited by methysergide and the 5-HT2 receptor blockers, ketanserin and 1-(1-naphthyl)piperazine. Again, the inhibition by these agents was not competitive. 5-HT1 and 5-HT3 receptor antagonists did not inhibit contractions. The results thus suggest that the smooth muscle of the rainbow trout intestine contains M-2 muscarinic and 5-HT2 receptors.     

Induction, isolation and a characterization of the lipid content of plasma vitellogenin from two Salmo species: rainbow trout (Salmo gairdneri) and sea trout (Salmo trutta)

Vitellogenin synthesis is induced in juvenile rainbow trout (Salmo gairdneri) and juvenile sea trout (Salmo trutta) by estradiol-17 beta. A purification procedure for vitellogenin from trout plasma by precipitation with MgCl2-EDTA and subsequent anion exchange chromatography on DEAE-Sephacel is described. The total lipid contents of purified rainbow trout and sea trout vitellogenins are 18 and 19%, respectively. Approximately 2/3 of the lipids are phospholipids, while the remainder consists of triglycerides and cholesterol. Phosphorus determinations on delipidated vitellogenin yield a phosphorus content of 0.63% in rainbow trout and 0.58% in sea trout vitellogenin. Native (dimeric) vitellogenins from rainbow trout and sea trout both have an apparent molecular weight of 440,000, when estimated by gel filtration on Sepharose 6B.     

B-naphthoflavone induction and its effect on hepatic phospholipid metabolism in rainbow trout (Salmo gairdneri)

1. B-naphthoflavone (BNF) induction of microsomal cytochrome P-488 and its effect on liver phospholipid metabolism were examined in rainbow trout (Salmo gairdneri) 24 and 96 hr after intraperitoneal injection. 2. Cytochrome P-448 content increased at 96 hr to approximately double the cytochrome content of control fish. 3. Computer-analyzed laser densitometry scans of LDS-polyacrylamide gels of 96 hr BNF-treated liver microsomes showed a 90% increase in cytochrome P-448 levels. 4. A 34% increase in microsomal phospholipid (mumol/mg protein) was observed 24 hr after BNF injection, with a marked increase in choline, ethanolamine and inositol phospholipids. 5. Following 96 hr of exposure to BNF some differences in enzyme activity were noted; choline kinase and cytidylyltransferase activities were reduced, while a marked increase was observed in choline phosphotransferase activity. In light of current information on induction of liver microsomal phospholipid metabolism in mammals, the results of the this study suggest that trout do not respond like mammals to inducers of monooxygenase activity.     

A comparative study of the hepatotoxicity of monochlorobenzene in the rainbow trout (Salmo gairdnerii) and the Sprague-Dawley rat

Chlorobenzene (CB) was less hepatotoxic to trout than to rats. This difference could not be totally accounted for by reduced absorption in the trout and CB was metabolized in the trout. Glutathione concentrations in trout livers were 1/3 of those of the rat and prior depletion of the tripeptide led to irreversible binding of CB to trout liver protein equivalent to that of rats suffering extensive necrosis. No consistent correlation between glutathione content or protein binding and liver damage was seen in either species.     

Uptake of the fish pathogen, Aeromonas salmonicida, by rainbow trout (Salmo gairdneri L.)

Aeromonas salmonicida was detected in the blood, kidney and spleen of rainbow trout within 2 min of immersion in a suspension containing 10⁴ cells/ml. Uptake into fish was enhanced by culturing the pathogen in low levels of nutrient, i.e., 0.1% (w/v) brain heart infusion (BHI) broth and by the addition of latex particles to the bacterial suspensions. However, there was no apparent difference in the uptake of pathogenic or non-pathogenic isolates. Moreover, the fish did not succumb to clinical signs of disease.     

Characterization of transferrin‐linked microsatellites in brown trout (Salmo trutta) and Atlantic salmon (Salmo salar)

The transferrin (TF) gene has recently received increased interest in fish given its fitness relevance as a resistance gene against pathogenic bacteria. In this study, we characterized five TF‐linked microsatellites in brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). Interestingly, each marker amplified duplicated loci and linkage analysis revealed that the TF gene has most likely experienced a tandem duplication during salmonid evolution. In addition, the amplification of all five markers across a wide range of salmonid species suggests that they may be of general interest for the genetic analysis of the TF gene(s) in this teleost family.     

Characterization of tetranucleotide microsatellites for Rio Grande cutthroat trout and rainbow trout,and their cross‐amplification in other cutthroat trout subspecies

We describe the isolation and characterization of 12 tetranucleotide microsatellites for Rio Grande cutthroat trout (Oncorhynchus clarkii virginalis) and rainbow trout (Oncorhynchus mykiss), and subsequently investigate their performance in Colorado River cutthroat trout (Oncorhynchus clarkii pleuriticus), greenback cutthroat trout (Oncorhynchus clarkii stomias) and Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri). All 12 loci are polymorphic in all subspecies of O. clarkii examined.     

Evaluation of glutamic acid and glycine as sources of nonessential amino acids for lake trout (Salvelinus namaycush) and rainbow trout (Salmo gairdnerii)

A semi-purified test diet which contained either glutamic acid or glycine as the major source of nonessential amino acids (NEAA) was fed to lake and rainbow trout. Trout fed the diet containing glutamic acid consistently showed better growth and feed conversion efficiencies than those fed the diets containing glycine. The data indicate that these trout utilize glutamic acid more efficiently than glycine when no other major sources of NEAA are present.     

Biosynthesis of triacylglycerols in the intestines of rainbow trout (Salmo gairdnerii) fed marine zooplankton rich in wax esters

Intestinal preparations from rainbow trout fed a diet rich in wax esters incorporated [1(-14)C]hexadecanoic acid and [1(-14)C]hexadecanol into triacylglycerols at the same rate. The ratio of the number of H atoms from C1 of hexadecanol to the number of molecules of hexadecanol incorporated into triacylglycerols was 1.6 : 3.0. [U-14C]Glucose was incorporated much faster into the glycerol moiety of triacylglycerols than was [U-14C]aspartic acid. We conclude that the oxidation of absorbed fatty alcohol to fatty acid and its subsequent incorporation into triacylglycerols is closely linked with the reductive formation of triacylglycerol-glycerol from glucose. The ability of trout intestines to metabolise fatty alcohol to triacylglycerols was the same in fish fed wax esters as in those fed triacylglycerols.