Biochemical research on oogenesis. Transfer RNA is fully charged in the 42-S storage particles of Xenopus laevis oocytes.

1. Transfer RNA makes up 30-40% of total RNA in previtellogenic oocytes of Xenopus laevis. The bulk of tRNA is associated with 5-S RNA and two proteins in a high-molecular-weight complex sedimenting at 42S. 2. We show here that all kinds of tRNA are present in the 42-S particles and all of them sediment coincidently. Particle tRNA is fully charged in vivo. During purification of the 42-S particles tRNA becomes partially uncharged. When purified particles are incubated in vitro with amino acids and ATP a charging reaction occurs without disruption of the nucleoprotein complex. Many aminoacyl-tRNA synthetases can be shown to co-sediment with the 42-S particles. We conclude that complete aminoacylation of tRNA within the storage particles results from the activity of particle-bound aminoacyl-tRNA synthetases.     

Cloning, physical mapping and genome organization of mitochondrial DNA from Cyprinus carpio oocytes

Summary The mitochondrial genome from Cyprinus carpio oocytes is a 10.5 megadalton, circular DNA molecule. The carp mitochondrial DNA was cloned in pBR325. Three recombinant plasmids accounted for the entire genome. Mapping of this DNA using 11 different restriction endonucleases is reported here. Both the large and small rRNA genes were then localized using Southern blot analysis. The subunit I of the cytochrome oxidase, the cytochrome b, the tRNA^Glu and the URF 4 genes were localized by nucleotide sequence analysis and homology studies with human mtDNA.Our results suggest that a similar gene order has been maintained in the mitochondrial genomes of Chordata and support the hypothesis of a common ancestor for all vertebrate organelle genomes.This study constitutes the first report on the genome organization of a fish mtDNA and provides information for further investigation in connection with sequence determination, replication, and gene expression in carp mitochondria.This work was supported by proyect RS-82-21 from the Universidad Austral de Chile and Grant N^o 1116 from Fondo Nacional de Desarrollo Cientifico y Tecnologico     

Characterization of 20-S and 40-S non-polysomal cytoplasmic messenger ribonucleoprotein particles from rat liver

Two populations of free messenger ribonucleoprotein (mRNP) particles, sedimenting at 20 S and 40 S respectively, were isolated from a rat liver postpolysomal supernatant. After treatment with 0.5 M KCl and recentrifugation through a sucrose layer, the mRNP particles were characterized with respect to their low-molecular-weight RNA and protein components. 40-S and 20-S particles show very different RNA patterns. Four distinct low-molecular-weight RNA species of approximately 105, 139, 187 and 256 nucleotides were found as components of the 40-S mRNPs. The 20-S mRNP particles contain one major low-Mr RNA species of approximately 243 nucleotides and a characteristic pattern of low-Mr RNAs similar to the one found in nuclear ribonucleoprotein particles. In contrast to the low-Mr RNAs found in nuclear RNP particles most of the low-Mr RNA species present in 20-S and 40-S mRNP particles are rapidly labeled after [3H]orotate administration. Whereas the low-Mr RNA composition of 20-S and 40-S mRNP particles is very different, the protein patterns of both mRNP complexes are very similar. Six major polypeptides with the following molecular weights of 117000, 79800, 76700, 53800, 43900, 36300 and several minor ones were found in both 20-S and 40-S mRNPs. In a cell-free system from wheat germs neither 20-S nor 40-S mRNP particles stimulated the incorporation of [3H]leucine into proteins. However, phenol-extracted RNA from 20-S and 40-S mRNPs stimulated total protein synthesis 16-fold and 3-fold, respectively. Furthermore, the RNA from both mRNP pools directed the synthesis of albumin in vitro.     

Purification and Characterization of Serum Serpin from Carp (Cyprinus carpio)

A serine proteinase inhibitor, termed serpin62, was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 6.2-fold and a 3% recovery rate after separation from α₁-antitrypsin. Specific inhibitory activity of serpin62 against bovine pancreatic trypsin was less than half of the specific antitryptic activity of α₁-antitrypsin. Under both reducing and nonreducing conditions, serpin62 was estimated to have a molecular weight (62,000) apparently larger than that of α₁-antitrypsin (55,000). They both consist of single polypeptide chains, but serpin62 differs from serine proteinase inhibitors from muscles of carp and white croaker in molecular weight and structure. Antibody raised against serpin62 immunologically crossreacted with serpin62 and had no crossreactivity with fish serum α₁-antitrypsin and muscular analogues. The antibody was susceptible to both serpin62 and its derivatives, which were widely distributed in carp tissues. Serpin62 is most likely distinct from other fish serine proteinase inhibitors expressing antitryptic activity physicochemically and immunologically. Received June 4, 1998; accepted September 10, 1998.     

Characterization of a novel cell line from the caudal fin of koi carp Cyprinus carpio

A continuous cell line (KF‐101) derived from the caudal fin of the koi carp Cyprinus carpio was established and characterized. The KF‐101 cell line multiplied abundantly in Leibovitz's L‐15 medium containing 10% foetal bovine serum at 25° C, and was subcultured for >90 passages over a period of 3 years. Immunocytochemistry revealed that the KF‐101 cells contain keratin, junction proteins connexin‐43 and occludin, and ectodermal stem‐cell marker Pax‐6, but not vimentin. Furthermore, the KF‐101 cells reacted with anti‐human DARPP‐32 and anti‐human GATA‐4 antibodies, and the labelling was regulated according to the cell cycle. The labels of the DARPP‐32 and GATA‐4 antibodies in the KF‐101 cells were the suggested phosphatase‐1 inhibitor‐1 and GATA‐3, respectively. In addition, the KF‐101 cells were susceptible to koi herpesvirus but were resistant to eel herpesvirus, iridovirus, grouper nodavirus and chum salmon (Oncorhynchus keta) virus. The results indicate that the KF‐101 cells are suitable materials for investigating biological and virological development.     

Biochemical research on oogenesis. Binding of tRNA to the nucleoprotein particles of Xenopus laevis previtellogenic oocytes

In previtellogenic oocytes of Xenopus laevis, nearly all tRNA is included in nucleoprotein particles (thesaurisomes) sedimenting at 42 S. We evaluate the possibility of a tRNA exchange between the particles and the ribosomes during protein synthesis. We find that the particles take up tRNA after a very short incubation in vitro. In the absence of ATP, the particles preferentially bind charged tRNA. In the presence of ATP, more tRNA binds to the particles, and the sedimentation coefficient of the integrated tRNA is displaced to 45 S. When added to nonfractionated homogenates of oocytes together with ATP, poly(U) strongly stimulates the incorporation of radioactive phenylalanine into tRNA and protein. The labeled protein (polyphenylalanine) cosediments with the ribosomes, whereas most of phenylalanyl tRNA cosediments with the thesaurisomes. These data suggest that the thesaurisomes participate to some extent in protein synthesis. They release charged tRNA, thereby supplying the ribosomes with activated amino acids. Discharged tRNA is then taken up, reacylated, and stored in the particles until the next round of peptide bond formation. The aminoacylation and storage functions are probably carried out by two very unequal populations of particles. The main subclass of particles (42 S) binds and stores tRNA in an ATP-independent manner. A much smaller subclass of particles (45 S) is responsible for reacylation of discharged tRNA.     

Biochemical research on oogenesis. Aminoacyl tRNA turns over in the 42-S particles of Xenopus laevis oocytes, but its ester bond is protected against hydrolysis

The ester bond aminoacyl tRNA is protected against hydrolysis in the 42-S particles (thesaurisomes) present in Xenopus laevis previtellogenic oocytes. Deacylation of tRNA is very slow in vitro, unless ATP is present. ATP causes a partial turnover of aminoacyl tRNA in vitro, with no detectable decrease in the overall aminoacylation level of tRNA, which remains close to 100%. tRNA in the particles turns over rapidly in vivo. Since the ester bond of aminoacyl tRNA is stabilized inside the 42-S particles, this turnover cannot be a consequence of spontaneous deacylation of tRNA, followed by reacylation by the aminoacyl-tRNA synthetases associated with the particles. We rather consider this turnover as reflecting a true metabolic activity of the particles, and a direct or indirect involvement of these particles in the oocyte's protein-synthesizing system.     

鲤鱼cDNA-AFLP技术反应体系的建立

将cDNA扩增片段长度多态性(cDNA-amplified fragment length polymorphism)技术应用在鲤鱼基因的转录表达研究中,本文通过对cDNA-AFLP主要步骤的优化和改进,建立起鲤鱼cDNA-AFLP反应体系.结果表明,采用MseⅠ和 BstYⅠ酶切鲤鱼cDNA 100 ng,分别处理2 h和3 h后,以接头浓度分别为2 pmol/μL和0.2 pmol/μL、T4连接酶浓度为0.12 U/μL的反应体系进行连接.以稀释50倍的预扩增产物为模板、BstYⅠ和MseⅠ比例为1:4的引物配比进行选择扩增可得到稳定的扩增结果.本研究建立的cDNA-AFLP技术将为鲤鱼转录组研究建立基础和技术支持.     

Karyotypes of Cyprinus carpio and Leuciscus cephalus

Karyological investigations on the common carp (Cyprinus carpio) and chub (Leuciscus cephalus) are described. The diploid chromosome complement in the carp was found to be 98, with 50 meta- to submetacentric chromosomes, 48 subtelo- to telocentric chromosomes, and an arm number of 148. Chromosomal studies on the chub revealed the diploid chromosome complement to be 50, with 34 meta- to submetacentric chromosomes, 16 subtelo- to telocentric chromosomes, and an arm number of 84.     

Preliminary characterization of chelation-sensitive nucleoprotein particles

Nucleoprotein particles (B2), isolated following digestion of calf thymus chromatin with micrococcal nuclease, are resolved on a non-chelating Bio-Gel A-5m column. B2 protein electrophoresis showed the presence of several H1 species and several nonhistone proteins but was depleted in core histones. DNA electrophoresis demonstrated that native B2 DNA has a length of about 46 base pairs. On DNA sequencing gels, the length distribution of denatured B2 DNA ranged from 12 to 35 bases with a weighted average chain length of about 26 bases. Depletion of a 20 base band in B2 DNA suggested specific protection of internucleosomal DNA sites during the nuclease digestion.     

NADPH-Diaphorase in the Olfactory System of the Carp Cyprinus carpio

Ultrastructural distribution of NADPH-diaphorase (NADPH-d) in olfactory epithelium and bulb of the carp Cyprinus carpio L. was studied using light and electron microscopy. The diaphorase staining was revealed in the supranuclear area of the sensory and indifferent epithelium, in the olfactory nerve, as well as in the outer layers of the olfactory bulb—in fibers and glomeruli. NADPH-d-positive neurons were found in the interglomerular neuropil. Electron microscopy showed that NADPH-d in the olfactory lining epithelium was related only to receptor cells and ciliary supporting cells and was present in submembranous structures. Besides, in both parts of the olfactory system the main, cytosolic part of the enzyme is bound to cytoskeleton and is also present in membranes of endoplasmic reticulum and in mitochondria. In general, the NADPH-d of the carp olfactory system is characterized by predominantly intracellular localization and widespread contacts of the enzyme with cytosol.     

鲤鱼mtDNA酶切图谱的构建

采用密度梯度离心法及ＲＮａｓｅ消化法制备并纯化了鲤（ＧｙｐｒｉｎｕｓｃａｒｐｉｏＬｉｎｎａｅｕｓ）肝脏线粒体ＤＮＡ（ｍｔＤＮＡ）,用１０种限制性内切酶对ｍｔＤＮＡ进行了分析,鲤鱼ｍｔＤＮＡ分子量约１０．１２×１０￣６,约１６．４９ｋｂ．ＳａｌⅠ、ＰｓｔⅠ、ＢａｍＨⅠ、ＸｂａⅠ、ＢｇｌⅠ、ＰｖｕⅡ、ＸｈｏⅠ、ＥｃｏＲⅠ、ＤｒａⅠ和ＨｉｎｄⅢ分别为１、１、３、３、３、４、１、４、４、和６个切点。根据单酶解及双酶解结果,构建了鲤ｍｔＤＮＡ１０种具酶３０个切点的限制性酶切图谱。     

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis,with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5–ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5–ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.     

Characterization of gonadotropin-releasing hormone (GnRH) receptors in the ovary of common carp (Cyprinus carpio).

Gonadotropin-releasing hormone (GnRH) binding sites have been characterized in the fully mature common carp ovary, using an analog of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]-GnRH; sGnRH-A) as a labeled ligand. Binding of sGnRH-A to carp follicular membrane preparation was found to be time-, temperature-, and pH-dependent. Optimal binding was achieved after 40 min of incubation at 4 degrees C at pH 7.6; binding was found to be unstable at room temperature. Binding of radioligand was a function of tissue concentration, with a linear correlation over the range of 8.0-40.0 micrograms membrane protein per tube. Incubation of membrane preparations with increasing levels of [125I]sGnRH-A revealed saturable binding at radioligand concentrations greater than 400 nM. The binding of [125I]sGnRH-A to the carp ovary was also found to be reversible; addition of unlabeled sGnRH-A (10(-6) M) after reaching equilibrium resulted in complete dissociation of [125I]sGnRH-A within 30 min, and the log dissociation plot indicated the existence of a single class of binding sites. Addition of unlabeled sGnRH-A displaced the bound [125I]sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis suggested the presence of one class of high affinity GnRH binding sites. Bound [125I]sGnRH-A was also found to be displaceable by other GnRH peptides, including sGnRH ([Trp7,Leu8]-GnRH), cGnRH-II ([His5,Trp7,Tyr8]-GnRH) and a GnRH antagonist ([D-pGlu1,D-Phe2,D-PTrp3,6]-GnRH; GnRH-ANT) in a parallel fashion, indicating that these peptides bind to the same class of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)