Molecular cloning and characterization of proliferating cell nuclear antigen (PCNA) from Chinese shrimp Fenneropenaeus chinensis

The proliferating cell nuclear antigen gene was cloned from Fenneropenaeus chinensis (FcPCNA). The full-length cDNA sequence of FcPCNA encodes 260 amino acids showing high identity with PCNAs reported in other species. FcPCNA expressed especially high in proliferating tissues of shrimp such as haematopoietic tissue (HPT) and ovary. In order to understand the response of HPT to bacteria and virus challenge, mRNA level of FcPCNA in HPT was analyzed after shrimp were challenged by Vibrio anguillarum and white spot syndrome virus (WSSV). FcPCNA expression in HPT of shrimp was responsive to WSSV and Vibrio challenge, but different expression profiles were obtained after challenge by these two pathogens. The data provide additional information to understand the defense mechanisms of shrimp against virus and bacteria. (c) 2008 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.

cDNA cloning and mRNA expression of a selenium-dependent glutathione peroxidase from Zhikong scallop Chlamys farreri

The glutathione peroxidases are essential enzymes of the cellular antioxidant defence system. In the present study, the full-length cDNA sequence encoding an extracellular glutathione peroxidase (designated CfGPx3) was isolated from Zhikong scallop Chlamys farreri. The complete cDNA was of 1194 bp, containing a 5' untranslated region (UTR) of 50 bp, a 3' UTR of 490 bp and an open reading frame (ORF) of 654 bp encoding a polypeptide of 217 amino acids. CfGPx3 possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase, such as the selenocysteine encoded by stop codon UGA, the GPx signature motif ((96)LGVPCNQFI(103)) and the active site motif ((WNFEKF184)-W-179). The high similarity of CfGPx3 with GPx from other organisms indicated that CfGPx3 should be a new member of the glutathione peroxidase family. By fluorescent quantitative real-time PCR, the CfGPx3 mRNA was universally detected in the tissues of haemocytes, gill, gonad, muscle and hepatopancreas with the highest expression in hepatopancreas. After scallops were challenged by Listonella anguillarum, the expression level of CfGPx3 transcript in haemocytes was significantly up-regulated (P<0.05) at 8 h post challenge. These results suggested that CfGPx3 was potentially involved in the immune response of scallops and perhaps contributed to the protective effects against oxidative stress. (C) 2010 Elsevier Inc. All rights reserved.

Molecular characterization of an ecdysone inducible gene E75 of Chinese shrimp Fenneropenaeus chinensis and elucidation of its role in molting by RNA interference

Ecdysone inducible gene. E75 is a primary target of ecdysone receptor (EcR). and is found to play a critical role in the molting process of arthropods In this study, a cDNA encoding the E75 of Chinese shrimp Fenneropenaeus chinensis (FcE75) was cloned using RT-PCR and RACE techniques FcE75 cDNA was 3611 bp in length with an ORF of 2394 bp. The deduced amino acid sequence of FcE75 had the highest sequence identity to E75 from a land crab Gecarcinus lateral's and E75 of the shrimp Metapenaeus crisis Quantitative real-time PCR revealed a prominently high expression of FcE75 mRNA in the whole body RNA extract of late premolt period (D3) juvenile shrimp. The role of E75 in the process of shrimp molting was investigated using the RNA interference technique Long double-stranded RNA corresponding to the FcE75 (dsE75) efficiently silenced the FcE75 transcript levels in juvenile F. chinensis. Further, injection with dsE75 completely arrested the molting process in experimental shrimp which eventually caused death Setogenic analysis of the uropods from molt-arrested shrimp, showed defective epidermal retraction, poor development of setae and new cuticle. These results indicate that E75 might be related to the molting process and is essential for proper molting and survival of shrimp This is the first report demonstrating the use of double stranded RNA to elucidate the possible role of E75 in the molting of decapod crustaceans (C) 2010 Elsevier Inc All rights reserved

Molecular structure and expression patterns of flounder (Paralichthys olivaceus) Myf-5, a myogenic regulatory factor

Myf-5, a member of the myogenic regulatory factors (MRF), has been shown to be expressed in muscle precursors in early stage zebrafish embryos. The MRFs, including MyoD, Myf-5, Myogenin and MR-F4, belong to the basic Helix-Loop-Helix transcription factors that contain a conserved basic Helix-Loop-Helix (bHLH) domain. To better understand the role of Myf-5 in the development of fish muscles, we have isolated the Myf-5 genomic sequence and cDNA from Flounder (Paralichthys olivaceus), and analyzed its structures and patterns of expression. Promoter analysis identified several putative transcription factor binding sites such as an E-box, NF-Y sites that might confer muscle-specific expression. Myf-5 transcripts were first detected in the paraxial mesoderm that gives rise to slow muscles. During somitogenesis, Myf-5 expression was found in developing somites. Myf-5 expression decreased gradually in somites in the anterior region, but remained strong in the newly formed somites. In the hatching stage, the expression was also detected in other muscle cells such as head muscle and fin muscle. In the growing fish, RT-PCR results showed that Myf-5 was expressed in the skeletal muscle and intestine. (c) 2006 Elsevier Inc. All rights reserved.

The complete mitochondrial genome of the mantid shrimp Oratosquilla oratoria (Crustacea: Malacostraca: Stomatopoda): Novel non-coding regions features and phylogenetic implications of the Stomatopoda

The complete mitochondrial (mt) genome sequence of Oratosquilla oratoria (Crustacea: Malacostraca: Stomatopoda) was determined; a circular molecule of 15,783 bp in length. The gene content and arrangement are consistent with the pancrustacean ground pattern. The mt control region of O. oratoria is characterized by no GA-block near the 3' end and different position of [TA(A)]n-blocks compared with other reported Stomatopoda species. The sequence of the second hairpin structure is relative conserved which suggests this region may be a synapomorphic character for the Stomatopoda. In addition, a relative large intergenic spacer (101 bp) with higher A + T content than that in control region was identified between the tRNA(Glu) and tRNA(Phe) genes. Phylogenetic analyses based on the current dataset of complete mt genomes strongly support the Stomatopoda is closely related to Euphausiacea. They in turn cluster with Penaeoidea and Caridea clades while other decapods form a separate group, which rejects the monophyly of Decapoda. This challenges the suitability of Stomatopoda as an outgroup of Decapoda in phylogenetic analyses. The basal position of Stomatopoda within Eumalacostraca according to the morphological characters is also questioned. (C) 2010 Elsevier Inc. All rights reserved.

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved

Identification, expression, function and localization of a DUF985 domain-containing hypothetical gene from amphioxus Branchiostoma belcheri

The progress in genome sequencing has led to an increasing submission of uncharacterized hypothetical genes with the domain of unknown function, DUF985, in GenBank, and none of these genes is related to a known protein. We therefore underwent an experimental study to identify the function of a DUF985 domain-containing hypothetical gene BbDUF985 (GenBank Accession No. AY273818) isolated from amphioxus Branchiostoma belcheri (B. belcheri). BbDUF985 was successfully expressed in both prokaryotic and eukaryotic systems, and its recombinant proteins expressed in both systems definitely exhibited an activity of phosphoglucose isomerase (PGI). Both tissue-section in situ hybridization and immunohistochemistry demonstrated that BbDUF985 was expressed in a tissue-specific manner, with most abundant levels in the hepatic caecum and ovary. In CHO cells transfected with the expression plasmid pEGFP-N1/BbDUF985, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that BbDUF985 is a cytosolic protein. In contrast, Western blotting indicated that BbDUF985 was also present in amphioxus humoral fluids, suggesting that it exists as a secreted protein as well. Our study provided a framework for further understanding the biochemical properties and physiological function of DUF985-containing hypothetical proteins in other species. (c) 2008 Elsevier Inc. All rights reserved.

A comparison of future increased CO2 and temperature effects on sympatric Heterosigma akashiwo and Prorocentrum minimum

Very little is known about how global anthropogenic changes will affect major harmful algal bloom groups. Shifts in the growth and physiology of HAB species like the raphidophyte Heterosigma akashiwo and the dinoflagellate Prorocentrum minimum due to rising CO2 and temperature could alter their relative abundance and environmental impacts in estuaries where both form blooms, such as the Delaware Inland Bays (DIB). We grew semi-continuous cultures of sympatric DIB isolates of these two species under four conditions: (1) 20 degrees C and 375 ppm CO2 (ambient control), (2)20 degrees C and 750 ppm CO2 (high CO2),(3) 24 degrees C and 375 ppm CO2 (high temperature), and (4) 24 degrees C and 750 ppm CO2 (combined). Elevated CO2 alone or in concert with temperature stimulated Heterosigma growth, but had no significant effect on Prorocentrum growth. P-Bmax (the maximum biomass-normalized light-saturated carbon fixation rate) in Heterosigma was increased only by simultaneous CO2 and temperature increases, whereas P-Bmax in Prorocentrum responded significantly to CO2 enrichment, with or without increased temperature. CO2 and temperature affected photosynthetic parameters alpha, Phi(max), E-k, and Delta F/F'(m) in both species. Increased temperature decreased and increased the Chl a content of Heterosigma and M Prorocentrum, respectively. CO2 availability and temperature had pronounced effects on cellular quotas of C and N in Heterosigma, but not in Prorocentrum. Ratios of C:P and N:P increased with elevated carbon dioxide in Heterosigma but not in Prorocentrum. These changes in cellular nutrient quotas and ratios imply that Heterosigma could be more vulnerable to N limitation but less vulnerable to P-limitation than Prorocentrum under future environmental conditions. In general, Heterosigma growth and physiology showed a much greater positive response to elevated CO2 and temperature compared to Prorocentrum, consistent with what is known about their respective carbon acquisition mechanisms. Hence, rising temperature and CO2 either alone or in combination with other limiting factors could significantly alter the relative dominance of these two co-existing HAB species over the next century. (c) 2007 Elsevier B.V. All rights reserved.

Phenotypic plasticity of gut structure and function during periods of inactivity in Apostichopus japonicus

Apostichopus japonicus is a common sea cucumber that undergoes seasonal inactivity phases and ceases feeding during the summer months. We used this sea cucumber species as a model in which to examine phenotypic plasticity of the digestive tract in response to food deprivation. We measured the body mass, gross gut morphology and digestive enzyme activities of A. japonicus before, during, and after the period of inactivity to examine the effects of food deprivation on the gut structure and function of this animal. Individuals were sampled semi-monthly from June to November (10 sampling intervals over 178 days) across temperature changes of more than 18 degrees C. On 5 September, which represented the peak of inactivity and lack of feeding, A. japonicus decreased its body mass, gut mass and gut length by 50%, 85%, and 70%, respectively, in comparison to values for these parameters preceding the inactive period. The activities of amylase, cellulase and lipase decreased by 77%, 98%, and 35% respectively, in comparison to mean values for these enzymes in June, whereas pepsin activity increased two-fold (luring the inactive phase. Alginase and trypsin activities were variable and did not change significantly across the 178-day experiment. With the exception of amylase and cellulase, all body size indices and digestive enzyme activities recovered and even surpassed the mean values preceding the inactive phase during the latter part of the experiment (October-November). Principal Component Analysis (PCA) utilizing the digestive enzyme activity and body size index data divided the physiological state of this cucumber into four phases: an active stage, prophase of inactivity peak inactivity, and a reversion phase. These phases are all consistent with previously suggested life stages for this species, but our data provide more defined characteristics of each phase. A. japonicus clearly exhibits phenotypic plasticity (or life-cycle staging) of the digestive tract during its annual inactive period. (C) 2008 Elsevier Inc. All rights reserved.

Photoperiodic regulation in energy intake, thermogenesis and body mass in root voles (Microtus oeconomus)

The present study was designed to examine whether photoperiod alone was effective to induce seasonal regulations in physiology in root voles (Microtus oeconomus) from the Qinghai-Tibetan plateau noted for its extreme cold environment. Root voles were randomly assigned into either long photoperiod (LD; 16L: 8D) or short photoperiod (SD; 8L: 16D) for 4 weeks at constant temperature (20 degrees C). At the end of acclimation, SD voles showed lower body mass and body fat coupled with higher energy intake than LD voles. SD greatly enhanced thermogenic capacities in root voles, as indicated by elevated basal metabolic rate (BMR), nonshivering thermogenesis (NST), mitochondrial protein content and uncoupling protein-1 (UCP1) content in brown adipose tissue (BAT). Although no variations in serum leptin levels were found between SD and LD voles, serum leptin levels were positively correlated with body mass and body fat mass, and negatively correlated with energy intake and UCP1 content in BAT, respectively. To summarize, SD alone is effective in inducing higher thermogenic capacities and energy intake coupled with lower body mass and body fat mass in root voles. Leptin is potentially involved in the photoperiod induced body mass regulation and thermogenesis in root voles. (c) 2006 Elsevier Inc. All rights reserved.

八角科植物系统学研究 — 比较木材解剖学及花被片表皮比较形态学

本文回顾了八角科（Illiciaceae）的分类历史，对其现状进行了介绍，并提出了存在的一些问题，针对这些问题，我们对八角科及其近缘类群 [五味子科（Schisandraceae）和木兰科（Magnoliaceae）] 的木材解剖特征，以及对组成八角目(Illiciales)的八角科和五味子科的花被片表皮形态进行了研究，进一步确定了八角科在离生心皮类中的原始地位，且为其属的次级划分提供了更多的性状支持，更详尽地阐述了它与近缘类群间的系统学关系，确定了八角目的独立地位。 １　比较木材解剖学 在光学显微镜和扫描电子显微镜下，对八角科八角属（Illicium）2组19种1亚种43个材料、五味子科2属4种7个材料和木兰科1属3种4个材料的木材解剖结构进行了比较研究。 因八角科导管单独、多角形、密集、细长且具螺纹加厚，穿孔板梯状且细密，射线异形等特征，本文支持建立单属的八角科。在八角属内，由于八角组（Sect. Illicium）的导管分子密度较高和直径较窄，易损度为0.090，射线较高，而八角茴香组（Sect. Cymbostemon）的导管分子密度较低和直径较宽，易损度为0.194，射线较矮，因而本文支持将八角属分为八角组和八角茴香组，且八角组比八角茴香组原始。根据欧式距离，八角科与五味子科之间的相似性（d=4.690）比八角科与木兰科（d=4.899）之间的相似性大，本文支持建立八角目（Illiciales）。 2　花被片表皮比较形态学 在光学显微镜和扫描电子显微镜下，观察了八角科八角属2组11种20个样品、五味子科南五味子属（Kadsura）2亚属2组8种15个样品和五味子属（Schisandra）2亚属4组6种17个样品的花被片表皮形态特征。 首次报道了八角目2个科（八角科和五味子科）3个属（八角属、南五味子属和五味子属）植物花被片表皮细胞的形状、分泌细胞的形状及分布、气孔器的形状及分布、花被片表面的纹饰，在五味子科中发现2个新的性状（气孔对和环列型气孔）。 通过与八角目2个科3个属植物的叶表皮形态比较，发现花被片表皮气孔器外拱盖均为单层，与叶表皮气孔器外拱盖层数（常绿种类为双层和落叶种类为单层）之间没有相关性。通过对两性花、雌花和雄花花被片表皮观察，发现花被片表皮形态与花的性别之间没有相关性。通过对八角属、南五味子属和五味子属花被片表皮比较，发现五味子属花被片表皮形态比南五味子属花被片表皮形态表现出更多的衍生性状，南五味子属花被片表皮形态比八角属花被片表皮形态表现出更多的衍生性状;南五味子属花被片表皮形态与五味子属花被片表皮形态之间的相似性程度较大，与八角属花被片表皮形态之间的相似性程度较小，支持建立含南五味子属和五味子属的五味子科，支持建立只含八角属的八角科。

Ectopic Six3 expression in the dragon eye goldfish

For goldfish (Carassius auratus), there are many varieties with different eye phenotypes due to artificial selection and adaptive evolution. Dragon eye is a variant eye characterized by a large-size eyeball protruding out of the socket similar to the eye of dragon in Chinese legends. In this study, anatomical structure of the goldfish dragon eye was compared with that of the common eye, and a stretching of the retina was observed in the enlarged dragon eye. Moreover, the homeobox-containing transcription factor Six3 cDNAs were cloned from the two types of goldfish, and the expression patterns were analyzed in both normal eye and dragon eye goldfish. No amino acid sequence differences were observed between the two deduced peptides, and the expression pattern of Six3 protein in dragon eye is quite similar to common eye during embryogenesis, but from 2 days after hatching, ectopic Six3 expression began to occur in the dragon eye, especially in the outer nuclear layer cells. With eye development, more predominant Six3 distribution was detected in the outer nuclear layer cells of dragon eye than that of normal eye, and fewer cell-layers in outer nuclear layer were observed in dragon eye retina than in normal eye retina. The highlight of this study is that higher Six3 expression occurs in dragon eye goldfish than in normal eye goldfish during retinal development of larvae. (C) 2007 Elsevier Inc. All rights reserved.

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.