A Radioimmunoassay for Ependymins β and γ: Two Goldfish Brain Proteins Involved in Behavioral Plasticity

Abstract: A radioimmunoassay (RIA) using ¹²⁵I-labeled antigen was developed for the quantitative determination of two goldfish brain proteins (ependymins β and γ). The proteins were isolated from the cerebrospinal fluid (CSF) and cells of the ependymal zone surrounding goldfish brain ventricles. The turnover rates of β and γ were previously shown to be specifically enhanced after the animals successfully acquired a new pattern of swimming behavior. Femtomole quantities of ependymin β were measurable by the RIA. In applications of the assay, β and γ ependymins were found to have common immunological properties, since ¹²⁵I-β-antigen bound to antibody could be displaced by unlabeled ependymin γ as well as ependymin β but not by a variety of other proteins including several purified glycoproteins isolated from goldfish brain. The ependymins were shown to constitute 14% of the total protein content of the brain extracellular fluid and also to be present as a minor component of the serum proteins (0.3%). Ependymins β and γ have an immunological reactivity in these fractions that can be increased by a factor of 30 on heating. The data suggest that the antigenicity of the molecules is highly masked, and that it may require some unraveling of the quaternary structure of the proteins before maximal interaction with the antisera becomes possible.     

The role of brain extracellular proteins in neuroplasticity and learning

Double labeling studies of the pattern of protein synthesis in goldfish and mouse brain identified a class of glycoproteins (the ependymins) whose turnover rate was enhanced after training. A variety of control experiments indicated that these macromolecules have an important role in the molecular and cell biology of learning. Antisera to the ependymins when injected into the brains of trained goldfish cause amnesia of a newly acquired behavior. Isolation and localization studies by immunocytochemical methods indicate that the ependymins are released into the brain extracellular fluid by a class of neurosecretory cells. In mammalian brain ependymin-containing cells are highly concentrated in the limibic system. The ependymins are constituted from two disulfide-linked acidic polypeptide chains (M.W.37K and 31K). They contain at least 5% covalently bound carbohydrate per chain with mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid as the predominant components. The highly soluble ependymins can rapidly polymerize to form an insoluble fibrous matrix if calcium is removed from solution by the addition of a Ca2+-chelating agent or dialysis. The self-aggregation property of the ependymins can be triggered by the depletion of Ca2+ from the extracellular space. Studies of the kinetics of the aggregation phenomenon by measurements of turbidity changes indicate that the process can be terminated but not reversed by restoring Ca2+ to its normal CSF level. Immunohistochemical studies of the brains of trained goldfish show the presence of punctate statining sites in the perimeter of certain cells located in specific brain regions. This suggests that ependymin aggregation might occur in vivo during learning. A molecular hypothesis relating the aggregation properties of the ependymins to neuroplasticity and learning is proposed.     

Structural and Metabolic Relationships Between Goldfish Brain Glycoproteins Participating in Functional Plasticity of the Central Nervous System

Ependymins beta and gamma (MW 32,000 and 26,000 daltons) are two secreted goldfish brain glycoproteins that exhibit a specifically enhanced turnover rate when the animals successfully acquire a new pattern of swimming behaviour. Both proteins are bound identically to concanavalin A and can be isolated from brain extracellular fluid and from brain cytoplasm by lectin affinity chromatography. Radioimmunoassay data, using purified 125I-labeled ependymins and antisera directed against ependymin beta or ependymin gamma, show complete cross-reactivity between the two proteins. It is demonstrated by Scatchard-plot analysis that the antisera recognize identical immunological determinants in both proteins. The amino acid composition of the ependymins is similar, and several identical polypeptide fragments are obtained after limited proteolysis with Staphylococcus aureus protease. The proteins are capable of forming complexes of the compositions gamma 2, beta gamma, and beta 2. A protease present in the extracellular fluid of goldfish brain promotes proteolysis of ependymin beta to ependymin gamma. The finding that ependymin gamma is physiologically derived from ependymin beta suggests the possibility that ependymin beta might exert its biological function during consolidation of new behavioural patterns via smaller polypeptide fragments.     

Ependymins are expressed in the meninx of goldfish brain

Summary Ependymins are dimeric glycoproteins found in the extracellular fluid of goldfish brain. They were originally observed because of their enhanced turnover rates after learning. In this paper we present the first investigation concerning the expression of these secretory proteins in goldfish brain via in situ hybridization with synthetic oligonucleotides and cRNA probes. It is shown that ependymin-mRNAs are predominantly expressed in the meninx surrounding the brain and in an invaginated part of the meninx called the cavum cranii. These results have been confirmed by immunhistochemical analysis. This indicates that, in fish, the meninx synthesizes a major protein constituent of the cerebrospinal fluid; furthermore, this suggests that the functional sites of ependymins are removed from the place of their synthesis. Distribution between different compartments may be achieved via the open communication system of the perivascular spaces.     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ependymins from the cerebrospinal fluid of salmonid fish: gene structure and molecular characterization.

So far, ependymins (Epds) have been sequenced only from cypriniform fish, and in the past all attempts have failed to characterize, on a molecular level, homologous Epd proteins in higher vertebrates. Therefore, rainbow trout (Oncorhynchus mykiss) Epds, which represent the predominant proteins of the cerebrospinal fluid, have been N-terminally sequenced and the encoding cDNA subsequently cloned using the polymerase chain reaction. Surprisingly, only 40-42% of the amino acids are identical with the corresponding sequences from goldfish (Carassius auratus), and no convincing immunological cross-reactivity is observed with an antiserum raised against purified Epds from C. auratus. O. mykiss possesses two highly homologous genes encoding Epds (Om-I, Om-II), a feature typical of a quasi-tetraploid species. Western analysis, using two specific antibodies against Epds from O. mykiss, revealed a variety of different glycosylation variants. In contrast to C. auratus, Epds from O. mykiss probably do not form disulfide-linked dimers. The structure of one Epd gene and its flanking regions have been determined for the Atlantic salmon (Salmo salar). Six exons were deduced by comparison with the corresponding cDNA sequence from O. mykiss (almost 98% homology with Om-II).     

Ultrastructural localization of ependymins in the endomeninx of the brain of the rainbow trout: possible association with collagen fibrils of the extracellular matrix

In the rainbow trout, ependymins represent the predominant protein constituents of the cerebrospinal fluid (CSF) and perimeningeal fluid (PMF). Synthesis of these glycoproteins occurs exclusively in the endomeninx. Generally, ependymins share characteristics with proteins mediating cell-contact phenomena. Here, we show that the endomeninx of the rainbow trout is composed of three different layers, viz. an outer layer, an arachnoid-like intermediate barrier layer and an inner layer. This structure is in agreement with a meningeal barrier concept separating the PMF from the CSF. Furthermore, by immuno-electron microscopy, we have localized the majority of intracellular ependymins to the rough endoplasmic reticulum of fibroblast-like cells of the inner layer and to cells to the intermediate barrier layer. This pattern is compatible with the observed distribution of ependymins in both the PMF and CSF. In addition to their intracellular localization, an extracellular association of ependymins with bundles of collagen fibrils is demonstrated; this is particularly pronounced around all blood vessels of the brain.     

Characterization of Basic Proteins from Goldfish Myelin

Abstract: Myelin basic protein (MBP) from common goldfish ( Carassius auratus ) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130–137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with ¹²⁵I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microhet-erogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis. Even from the limited sequence obtained, the sequence ATAST was found in goldfish, which is also present in human, rabbit, and guinea pig MBPs.     

Identification and molecular cloning of germinal vesicle lamin B3 in goldfish (Carassius auratus) oocytes.

A bulk isolation method was developed to collect a large number of germinal vesicles (GV) from postvitellogenic oocytes of goldfish (Carassius auratus). Using this method, we obtained GV lamina which are resistant to high salt and nonionic detergent. 2D PAGE revealed that the goldfish GV lamina contained several spots with similar molecular masses (67 kDa) and slightly different neutral isoelectrofocusing values (pI 5.8-6.2). After trypsin digestion and extraction of a major spot (pI 6.1), the peptide was subjected to RP-HPLC and sequenced. A homology search identified this spot as a nuclear lamin. A cDNA encoding goldfish GV lamin was isolated by RT-PCR using degenerate primers designed from the GV lamin tryptic peptide sequence. The goldfish GV lamin cDNA encodes a predicted molecular mass of 67 455 Da with a pI of 5.84. Phylogenetic analysis indicates that the amino-acid sequence is most similar to Xenopus oocyte-specific GV lamin B3, but differs from somatic lamins (A, B1 or B2). In contrast to somatic lamins, neither goldfish nor Xenopus GV lamin contain conserved phosphorylation sites for nuclear transport, except the nuclear localization sequence. Therefore, we conclude that the goldfish oocyte GV is mainly comprised of GV-type lamin (the homolog of Xenopus lamin B3).     

Identification and characterization of a novel family of mammalian ependymin-related proteins (MERPs) in hematopoietic, nonhematopoietic, and malignant tissues.

Evidence is presented for a family of mammalian homologs of ependymin, which we have termed the mammalian ependymin-related proteins (MERPs). Ependymins are secreted glycoproteins that form the major component of the cerebrospinal fluid in many teleost fish. We have cloned the entire coding region of human MERP-1 and mapped the gene to chromosome 7p14.1 by fluorescence in situ hybridization. In addition, three human MERP pseudogenes were identified on chromosomes 8, 16, and X. We have also cloned the mouse MERP-1 homolog and an additional family member, mouse MERP-2. Then, using bioinformatics, the mouse MERP-2 gene was localized to chromosome 13, and we identified the monkey MERP-1 homolog and frog ependymin-related protein (ERP). Despite relatively low amino acid sequence conservation between piscine ependymins, toad ERP, and MERPs, several amino acids (including four key cysteine residues) are strictly conserved, and the hydropathy profiles are remarkably alike, suggesting the possibilities of similar protein conformation and function. As with fish ependymins, frog ERP and MERPs contain a signal peptide typical of secreted proteins. The MERPs were found to be expressed at high levels in several hematopoietic cell lines and in nonhematopoietic tissues such as brain, heart, and skeletal muscle, as well as several malignant tissues and malignant cell lines. These findings suggest that MERPs have several potential roles in a range of cells and tissues.     

The carp–goldfish nucleocytoplasmic hybrid has mitochondria from the carp as the nuclear donor species

It is widely accepted that mitochondria and its DNA (mtDNA) exhibit strict maternal inheritance, with sperm contributing no or non-detectable mitochondria to the next generation. In fish, nuclear transfer (NT) through the combination of a donor nucleus and an enucleated oocyte can produce fertile nucleocytoplasmic hybrids (NCHs) even between different genera and subfamilies. One of the best studied fish NCHs is CyCa produced by transplanting the nuclei plus cytoplasm from the common carp (Cyprinus carpio var. wuyuanensis) into the oocytes of the wild goldfish (Carassius auratus), which has been propagated by self-mating for three generations. These NCH fish thus provide a unique model to study the origin of mitochondria. Here we report the complete mtDNA sequence of the CyCa hybrid and its parental species carp and goldfish as nuclear donor and cytoplasm host, respectively. Interestingly, the mtDNA of NCH fish CyCa is 99.69% identical to the nuclear donor species carp, and 89.25% identical to the oocyte host species goldfish. Furthermore, an amino acid sequence comparison of 13 mitochondrial proteins reveals that CyCa is 99.68% identical to the carp and 87.68% identical to the goldfish. On an mtDNA-based phylogenetic tree, CyCa is clustered with the carp but separated from the goldfish. A real-time PCR analysis revealed the presence of carp mtDNA but the absence of goldfish mtDNA. These results demonstrate – for the first time to our knowledge – that the mtDNA of a NCH such as CyCa fish may originate from its nuclear donor rather than its oocyte host.     

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.     

Extracellular Fluid Proteins of Goldfish Brain: Evidence for the Presence of Proteases and Esterases

Preparations of enriched fractions of extracellular fluid (ECF) proteins from goldfish brain were found to contain protease(s) and esterase(s). The N-substituted furanacryloyl (FA) peptides FA-Phe-Gly-Gly and FA-Phe-OMe were used as model substrates for determining protease and esterase activity, respectively, in a spectrophotometric assay. Studies of the profile of substrate specificity and identification of the types of compounds that were effective as inhibitors showed that these ECF enzymes have some distinctive properties. GSH, but not GSSG, and EDTA inhibited the protease(s) without influencing the esterase(s), whereas L-1-tosylamide-2-phenylethylchloromethyl ketone blocked both protease and esterase activities of ECF. Most of the protease and esterase properties of ECF could be bound to concanavalin A-Sepharose affinity chromatographic columns in association with ependymin--a brain extracellular protein. These observations indicate that ECF may contain a metalloprotease(s) and raise the possibility that the ependymins might be a substrate for these ECF enzymes.     

Altered turnover of allelic variants of hypoxanthine phosphoribosyltransferase is associated with N-terminal amino acid sequence variation

The results of our previous studies suggested that differences in the primary structures of the hypoxanthine phosphoribosyltransferase (HPRT) A and B proteins (EC 2.4.2.8) of mice are associated with altered turnover of these proteins in reticulocytes. On the basis of nucleotide sequence comparisons of their corresponding cDNAs, we show here that the HPRT A and B proteins differ at two positions; there is an alanine/proline substitution at amino acid position 2 and a valine/alanine substitution at amino acid position 29 (HPRT A/B proteins, respectively; total protein length, 218 amino acids). On the basis of results obtained from sequencing of the N termini of the purified HPRT A and B proteins, we also show that these amino acid substitutions are associated with differences in processing of the proteins; HPRT B, which is encoded as N-terminal Met-Pro, has a free N-terminal proline residue; HPRT A, which is encoded as N-terminal Met-Ala, lacks a free N-terminal alpha-amino group and is presumed to be acetylated following removal of the N-terminal methionine (i.e. AcO-Ala). These observations are discussed in reference to the idea that the N terminus of a protein plays a role in determining the rate at which the protein is degraded in erythroid cells.     

鲫Kaiso基因cDNA的克隆和表达分析

在爪蟾和斑马鱼中, Kaiso是一种在整个基因组范围内与甲基化CpG序列特异性结合的转录抑制因子, 在调控被甲基化基因表达的时间模式中起重要的作用。为深入研究DNA甲基化对我国重要养殖鱼类生殖和发育的影响, 我们克隆了鲫Kaiso基因的cDNA序列, 并对其时空表达模式进行了分析。该cDNA全长3145 bp, 5′-非翻译区132 bp, 3′-非翻译区1117 bp, 开放阅读框1896 bp, 编码631个氨基酸。鲫Kaiso蛋白与其他物种Kaiso蛋白的同源性分析表明, 与其他物种一样, 其 N端和C端分别有高保守性的BTB/POZ结构域和锌指结构域。整胚原位杂交结果显示, Kaiso mRNA在早期胚胎发育的各个时期均广泛表达, 信号均一, 但从尾芽期开始出现组织特异性表达差异。对不同发育阶段胚胎的实时定量PCR检测结果表明: 卵子中有高丰度的母源Kaiso mRNA存在; 在卵裂期至囊胚中期胚胎中Kaiso mRNA的丰度逐渐降低; 从囊胚中期至原肠早期都维持在最低水平状态; 原肠后期其表达水平又逐渐升高, 至尾芽期达到与未受精卵中相当的高水平后在器官发生期的整体水平又稍有下降。Kaiso mRNA丰度在胚胎发育早期的这种变化过程提示在卵裂期检测到的mRNA可能都是母源mRNA, 合子核Kaiso基因可能是在囊胚晚期后才开始转录。对成体不同组织的实时定量PCR检测结果表明Kaiso的表达存在明显的组织特异性差异, 在鲫肌肉、视网膜、心脏和脑中表达水平较高, 而在肾、胰、肝等器官中表达水平很低。Kaiso表达的时间和组织特异性提示其作为甲基化基因的转录抑制因子参与了胚胎和成体基因表达时空模式的调控。这些结果为进一步研究Kaiso和DNA甲基化修饰在鲫发育调控和遗传育种中的作用提供了基础资料。     

In vivo import experiments in protoplasts reveal the importance of the overall context but not specific amino acid residues of the transit peptide during import into chloroplasts

The N-terminal transit peptide of chloroplast proteins is necessary and sufficient to direct proteins to the chloroplasts. However, the requirement of the transit peptide of chloroplast proteins is not fully understood. In this study we investigated the requirement of a transit peptide at the level of amino acid sequence using an in vivo targeting approach. Targeting experiments with green fluorescent protein (GFP) fusion proteins containing varying lengths of the N-terminal region of the small subunit of rubisco complex (RbcS) revealed that at least 73 amino acid residues of the N-terminal region is required to direct GFP to the chloroplasts without affecting the efficiency. Even a small deletion from the C- or N-termini of the minimal length of the transit peptide results in strong inhibition of targeting. Also, a small internal deletion within the minimal transit peptide strongly affected targeting of GFP fusion proteins. However, when we replaced one or two amino acid residues of the transit peptide with corresponding numbers of alanine residues sequentially, all the mutants were imported into chloroplasts with 80 to 100% efficiency. Together these results suggest that the overall context of amino acid sequence, but not any specific amino acid residue, of the transit peptide is critical for targeting to the chloroplasts.     

Molecular cloning of gefiltin (ON1): serial expression of two new neurofilament mRNAs during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins of this pathway do not match the intermediate filament protein composition of adult higher vertebrate neurons, which lack the capacity for growth and development. Using a goldfish retina lambda gt10 library we isolated cDNA clones representing the predominant goldfish optic nerve neurofilament protein, ON1. The mRNA for this protein is abundant in retinal ganglion cells, and its level increases slowly during optic nerve regeneration. The rate of ON1 mRNA accumulation after optic nerve crush was compared with that of plasticin, a previously described novel type III neurofilament from goldfish retinal ganglion cells. Plasticin mRNA is normally expressed at low steady state levels, but accumulates dramatically and rapidly, preceding gefiltin mRNA, in response to optic nerve crush. The predicted amino acid sequence for ON1 indicates that it is a novel intermediate filament protein. We have named it gefiltin, for goldfish eye intermediate filament protein. The serial expression of plasticin and gefiltin is discussed with respect to the diversity of neurofilament proteins during neurogenesis.     

Identification of a novel heterodimeric outer membrane protein of Porphyromonas gingivalis by two-dimensional gel electrophoresis and peptide mass fingerprinting.

Porphyromonas gingivalis is a Gram-negative, anaerobic bacterium associated with chronic periodontitis. A 2D electrophoretic analysis of the outer membrane of P. gingivalis W50 revealed a dominant train of spots at 40-41 kDa. The proteins in the train of spots were digested in-gel with trypsin and identified by MS. The train of spots represented two proteins, designated Omp40 and Omp41 that share 47% sequence identity. Preparation of outer membranes in the absence of protease inhibitors resulted in partial cleavage of Omp40 and Omp41 to produce an N-terminal and C-terminal fragment of both proteins. The N-terminal fragments displayed the same isoelectric heterogeneity as the intact proteins. Almost 100% of the amino-acid sequence of these N-terminal fragments in each 2D gel spot was verified suggesting lack of post-translational modification. Re-subjecting a single N-terminal domain spot to 2D electrophoresis resulted in the complete series of spots being reproduced, suggesting that the heterogeneity was related to conformational equilibria. Under reduced conditions and without heating, Omp40 and Omp41 migrated as 34- to 35-kDa proteins in SDS/PAGE whereas under nonreduced conditions the proteins migrated as 70-kDa proteins, suggesting the formation of dimers through intersubunit disulfide bonds. The proteins each contain two cysteine residues in the conserved sequence RPVSCPECPE. Tryptic peptides generated from the nonreduced forms of the proteins confirmed the presence of heterodimers stabilized through intersubunit disulfide bond formation. With the exception of heterodimer formation, the two proteins share several similarities with OmpA-like porins of other Gram-negative bacteria including consensus sequence, abundance, modification by heat, overall length and positioning of domains.     

Molecular cloning of cDNA encoding APC11, a catalytic component of anaphase-promoting-complex (APC/C), from goldfish (Carassius auratus), and establishment of in vitro ubiquitinating system

Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin-degrading system, including anaphase-promoting-complex/cyclosome (APC/C), has been shown to be responsible for cyclin B destruction. Here we present the cloning, sequencing, and expression analysis of goldfish (Carassius auratus) APC11, which encodes the catalytic component of APC/C from goldfish ovary. The cloned cDNA is 348 bp long and encodes 88 amino acids. The deduced amino acid sequence is highly homologous to APC11 from other species. The expression of mRNA for APC11 was ubiquitous among tissues, as opposed to that of mRNA for E2-C, which occurred at a very high level in the ovary. Recombinant goldfish APC11 possesses ubiquitinating activity against cyclin B. We established an in vitro ubiquitinating system of proteins composed of purified recombinant E1, E2-C, and APC11 from goldfish. The reconstructed system for these ubiquitinating enzymes makes it feasible to elucidate the molecular mechanism of cyclin B degradation.