Identical sequences but different expression patterns of Hira gene in gynogenetic and gonochoristic crucian carps

Hir/Hira (histone regulation) genes were first identified in yeast as negative regulators of histone gene expression. It has been confirmed that HIRA is a conserved family of proteins present in various animals and plants. In this paper, the cDNAs of the Hira homolog named CagHira and CaHira were isolated from gynogenetic gibel carp (gyno-carp) and gonochoristic color crucian carp (gono-carp) respectively. The full-length CagHira is 3,860 bp in length with an open reading frame (ORF) of 3,033 bp that encodes 1,011 amino acids, while the full-length CaHira is 3,748 bp in length and also has an ORF of 3,033 bp. The deduced amino acid sequences of both Hira homologs contain seven WD domains and show high identity with other HIRA family members. RT-PCR analyses revealed strong expression of Hira in the ovaries, whereas no expression was detected in the testes of either of the fishes. Hira transcription was not detected in the liver of gyno-carp, but a high level of Hira mRNA was observed in gono-carp. The temporal expression pattern showed that the Hira mRNA is consistently expressed during all embryonic development stages in gyno-carp. However, the abundance of CaHira mRNA significantly decreased (P < 0.05) shortly after fertilization and then increased again and remained stable from gastrula till hatching. The varying spatiotemporal expression patterns of Hira genes in gyno-carp and gono-carp may be associated with the differing reproductive modes used by these two closely related fishes. Our results suggest that Hira may play a role not only in the decondensation of sperm nucleus and the formation of pronucleus during fertilization, but also in gastrulation and the subsequent development of embryos.

Identification of a putative oocyte-specific small nuclear ribonucleoprotein polypeptide C in gibel carp

Previous studies have demonstrated that germinal vesicle of amphibian oocyte contains small nuclear ribonucleoprotein polypeptide C (SNRPC). In this study, a putative member of SNRPC was identified from Carassius auratus gibelio oocyte cDNA library. Its full-length cDNA has an open reading frame of 201 nt for encoding a peptide of 66 an, a short 5'-UTR of 19 nt and a long 3'-UTR of 347 nt including a polyadenylation signal and poly- (A) tail, and the deduced amino acid sequence has 47% identity with the C-terminal of the zebrafish small nuclear ribonucleoprotein polypeptide C. Western blot analysis revealed its oocyte-specific expression. Immunofluorescence localization indicated that its gene product localized to numerous nucleoli within the oocytes and showed dynamic changes with the nucleoli during oocyte maturation. RT-PCR and Western blot analysis further revealed its constant presence in the oocytes and in the embryos until hatching. The data suggested that the newly identified CagOSNRPC might be a nucleolar protein. (c) 2006 Elsevier Inc. All rights reserved.

Molecular cloning and expression pattern of 14 kDa apolipoprotein in orange-spotted grouper, Epinephelus coioides

A novel fish-specific apolipoprotein (apo-14 kDa) has been recently cloned from eel and pufferfish. However, its expression pattern has not been elucidated. in this study, EcApo-14 has been screened from hypothalamic cDNA library of male orange-spotted grouper, which shows 62.9%, 51%, 46.9%, 43.2%, and 31.9% identities to Apo-14 of European flounder, pufferfish, Japanese eel, gibel carp, and grass carp, respectively. RT-PCR analysis reveals that this gene is first transcribed in neurula embryos and maintains a relatively stable expression level during the following embryogenesis. EcApo-14 transcripts are at a very high level during embryonic and early larval development in the yolk syncytial layer (YSL), and decrease in YSL and form intense staining in liver at 3 days after hatching. In adult tissues, EcApo-14 is predominantly expressed in liver and brain. The data suggested that EcApo-14 might play an important role in liver and brain morphogenesis and growth. (c) 2005 Elsevier Inc. All rights reserved.

Identification of a novel C1q family member in color crucian carp (Carassius auratus) ovary

Potential roles of Clq/tumor necrosis factor (TNF) superfamily proteins have been observed in vertebrate oogenesis and oocyte maturation, but no ovary-specific member has been identified so far. In this study, we have cloned and identified a novel member of Clq family with a Clq domain in the C-terminal from fully grown oocyte cDNA library of color crucian carp and demonstrated that the gene might be specifically expressed in ovary and therefore designated as Carassius auratus ovary-specific Clq-like factor, CaOClq-like factor. It encodes a 213 amino acid protein with a 17 amino acid signal peptide. There is only one protein band of about 24.5 kDa in the extracts from phase I to phase IV oocytes, but two positive protein bands are detected in the extracts of mature eggs and fertilized eggs. Furthermore, the mobility shift of the smaller target protein band cannot be eliminated by phosphatase treatment, but the larger protein band increases its mobility on the gel after phosphatase treatment, suggesting that the larger protein might be a phosphorylated form. Immunofluorescence localization indicates that the CaOClq-like proteins localize in cytoplasm, cytoplasm membrane and egg envelope of the oocytes at cortical granule stage and vitellogenesis stage, whereas they were compressed to cytoplasm margin in ovulated mature eggs and discharged into perivitelline space between cytoplasm membrane and egg envelope after egg fertilization. Further studies on distribution and translocation mechanism of the CaOClq-like factor will be benefit to elucidate the unique function in oogenesis, oocyte maturation and egg fertilization. (C) 2004 Elsevier Inc. All rights reserved.

cDNA cloning and characterization of a novel SNX gene differentially expressed in previtellogenic oocytes of gibel carp

To understand the molecular events governing fish oogenesis, a multiple technique was used to identify the genes differentially expressed at different phases during fish oogenesis. This technique is a combination of suppression subtractive hybridization, SMART cDNA synthesis and RACE-PCR. Here we report the cDNA cloning and expression characterization of a novel SNX gene based on its differential transcription between previtellogenic and fully mature oocytes in naturally gynogenetic gibel carp. First, a cDNA fragment selectively expressed in previtellogenic oocytes was identified and used to screen a SMART cDNA library prepared from the same mRNA sample by RACE-PCR for cloning fully length cDNA. The full length cDNA was 1392-bp long and coded for a novel SNX protein with 225 amino acids. The 5' UTR had 72 bp and 3' UTR had 642 bp. Unlike most of maternal genes that are transcribed after vitellogenesis and stored in oocytes, this gene is expressed at a higher level in the previtellogenic oocytes and at a much lower level in fully matured oocytes. However, RT-PCR analysis of tissues showed it was ubiquitous transcription. The novel gene is named fish sorting nexin (fSNX), because it contains a conserved PX domain. The fact which major expression of the gene occurs in the previtellogenic oocytes suggests that it might have an important function in the oogenesis. (C) 2003 Elsevier Inc. All rights reserved.

HORMONAL REPLACEMENT THERAPY IN FISH - HUMAN GROWTH-HORMONE GENE-FUNCTION IN HYPOPHYSECTOMIZED CARP

Transgenic common carp, Cyprinus carpio, produced by the microinjection of fertilized eggs with a linearized chimeric plasmid pMThGH, a human growth hormone (hGH) gene with a mouse metallothionein-I (MT) gene promoter in pBR322, were used to produce F1 and F2 transgenics. Following hypophysectomy of the transgenic F2 common carp, non-transgenic common carp and non-transgenic crucian carp, growth was monitored for up to 110 days. In addition, recombinant hGH was injected subcutaenously into a group of the non-transgenic crucian carp. Growth rate analyses indicated that (1) hypophysectomy of non-transgenic common carp and crucian carp results in the cessation of growth, (2) hGH administration can stimulate the growth of hypophysectomized crucian carp and (3) hypophysectomized hGH-transgenic common carp continue to grow in the absence of their own growth hormone, suggesting that the hGH-transgene is being expressed in tissues other than the pituitary.

Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis

Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR Could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A-B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts Occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans. (C) 2009 Published by Elsevier Inc.

Ontogenetic development of digestive enzymes and effect of starvation in miiuy croaker Miichthys miiuy larvae

The ontogenetic development of the digestive enzymes amylase, lipase, trypsin, and alkaline phosphatase and the effect of starvation in miiuy croaker Miichthys miiuy larvae were studied. The activities of these enzymes were detected prior to exogenous feeding, but their developmental patterns differed remarkably. Trypsin activity continuously increased from 2 days after hatching (dah), peaked on 20 dah, and decreased to 25 dah at weaning. Alkaline phosphatase activity oscillated at low levels within a small range after the first feeding on 3 dah. In contrast, amylase and lipase activities followed the general developmental pattern that has been characterized in fish larvae, with a succession of increases or decreases. Amylase, lipase, and trypsin activities generally started to increase or decrease at transitions from endogenous to exogenous feeding or diet changes, suggesting that these enzymatic activities can be modulated by feeding modes. The activities of all the enzymes remained stable from 25 dah onwards, coinciding with the formation of gastric glands and pyloric caecum. These results imply that specific activities of these enzymes underwent changes due to morphological and physiological modifications or diet shift during larval development but that they became stable after the development of the digestive organs and associated glands was fully completed and the organs/glands functioned. Trypsin and alkaline phosphatase were more sensitive to starvation than amylase and lipase because delayed feeding up to 2 days after mouth opening was able to adversely affect their activities. Enzyme activities did not significantly differ among feeding groups during endogenous feeding; however, all activities were remarkably reduced when delayed feeding was within 3 days after mouth opening. Initiation of larvae feeding should occur within 2 days after mouth opening so that good growth and survival can be obtained in the culture.

Cloning, characterization and expression of ferritin subunit from clam Meretrix meretrix in different larval stages

Shell formation is one of the important events during larval development and metamorphosis in bivalves. However, the molecular mechanisms and environmental cues regulating shell initiation and growth are unclear. Here, we report that ferritin, a principal protein for biological iron storage and metabolism, might play a role in larval shell development of the bivalve mollusk Meretrix meretrix. A full-length ferritin subunit cDNA, named as MmeFer, was cloned and characterized. The MmeFer mRNA expression in different developmental stages, from trochophore to post larvae, was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). MmeFer mRNA expression in larvae of later developmental stages increased at least 8-fold following trochophores. Moreover, the temporal and spatial expressions of MmeFer mRNA were examined by whole mount in situ hybridization. In the trochophore stage, MmeFer was detectable where it was supposed to be for shell initiation. In the later developmental stages, MmeFer was found near digestive glands and mantle that secret larval shell. MmeFer expression was also detected in larvae cultured in artificial seawater with different iron concentrations ranging from 0 to 100 mu M. These results suggest that ferritin may play a role in the shell formation of mollusks. (C) 2009 Elsevier Inc. All rights reserved.

Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

The expression of AmphiTCTP, a TCTP orthologous gene in amphioxus related to the development of notochord and somites

The translationally controlled tumor protein (TCTP) is highly conserved and has been widely found in eukaryotic organisms. Here, we report the phylogenetic analysis and developmental expression of AmphiTCTP, a TCTP homologous gene in cephalochordate amphioxus. Phylogenetic analysis indicates that the putative protein of AmphiTCTP is close to its vertebrate orthologs. The mRNA of AmphiTCTP is found in fertilized eggs, early cleavage embryo and most of the early developmental stages by in situ hybridization and RT-PCR, but its expression is not detectable from late cleavage stage to mid-gastrula. The expression of AmphiTCTP in zygotes and early cleavage stages shows that AmphiTCTP may be a maternal gene. From the early neurula stage onward, AmphiTCTP transcript is localized in the presumptive notochord, presomitic mesoderm, and nascent somites. However, its expression is gradually down-regulated after the notochord and somites have been formed. The expression pattern of AmphiTCTP thus coincides with the differentiation of the notochord and somites, this suggests that AmphiTCTP may not be a housekeeping gene and may play an important role in mesoderm development. (c) 2007 Elsevier Inc. All rights reserved.

The enhanced immune protection of Zhikong scallop Chlamys farreri on the secondary encounter with Listonella anguillarum

It is well known that invertebrates are devoid of adaptive immune components and rely primarily on innate immunity to defend against pathogens, but recent studies have demonstrated the existence of enhanced secondary immune protection in some invertebrates. in the present study, the cumulative mortality of scallops received two successive Listonella anguillarum stimulations was recorded, and variations of immune parameters including phagocytosis (phagocytic rate and phagocytic index), phenoloxidase-like enzyme, acid phosphatase and superoxide dismutase activities were also examined. The scallops received a previous short-term L anguillarum stimulation were protected against a long-term stimulation of L. anguillarum. Significantly higher level of phagocytic activities and acid phosphatase activity were observed in the scallops received twice stimulations compared with those only received the secondary stimulation. These results indicated that a short-term immersion with L. anguillarum modulated the scallops' immune system and endowed the scallops with enhanced resistance to the secondary bacterial stimulation: phagocytosis and acid phosphatase were suspected to be involved in the protection. (C) 2008 Elsevier Inc. All rights reserved.

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32%similar to 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, A)Dnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana. (C) 2007 Published by Elsevier Inc.

cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. The full-length cDNA of Zhikong scallop Chlamysfarreri HSP90 (designated CfHSP90) was cloned by EST and rapid RACE techniques. It was of 2710 bp, including an open reading frame (ORF) of 2181 bp encoding a polypeptide of 726 amino acids with all the five HSP90 family signatures. BLAST analysis revealed that the CfHSP90 gene shared high similarity with other known HSP90 genes. Fluorescent real-time quantitative RT-PCR was used to examine the expression pattern of CfHSP90 mRNA in haemocytes of scallops exposed to Cd2+, Pb2+ and Cu2+ for 10 and 20 days, respectively. All the three heavy metals could induce CfHSP90 expression. There was a clear dose-dependent expression pattern of CfHSP90 after heavy metals exposure for 10 days or 20 days. Different concentrations of the same metal resulted in different effects on CfHSP90 expression. The results indicated that CfHSP90 responded to various heavy metal stresses with a dose-dependent expression pattern as well as exposure time effect, and could be used as a molecular biomarker in a heavy metal polluted environment. (c) 2007 Elsevier Inc. All rights reserved.

Cloning and expression analysis of myogenin from flounder (Paralichthys olivaceus) and promoter analysis of muscle-specific expression

Myogenin is a bHLH transcription factor of the MyoD family. It plays a crucial role in myoblast differentiation and maturation. We report here the isolation of flounder myogenin gene and the characterization of its expression patterns. Sequence analysis indicated that flounder myogenin shared a similar structure and the conserved bHLH domain with other vertebrate myogenin genes. Flounder myogenin gene contains 3 exons and 2 introns. Sequence alignment and phylogenetic showed that flounder myogenin was more homologous with halibut (Hippoglossus hippoglossus) myogenin and striped bass (Morone saxatilis) myogenin. Whole-mount embryo in situ hybridization revealed that flounder myogenin was first detected in the medial region of somites that give rise to slow muscles, and expanded later to the lateral region of the somite that become fast muscles. The levels of myogenin transcripts dropped significantly in matured somites at the trunk region. Its expression could only be detected in the caudal somites, which was consistent with the timing of somite maturation. Transient expression analysis showed that the 546 bp flounder myogenin promoter was sufficient to direct muscle-specific GFP expression in zebrafish embryos. (c) 2007 Elsevier Inc. All rights reserved.