Identification of genome organization in the unusual allotetraploid form of Carassius auratus gibelio

The unusual allotetraploid form with unequal contribution of chromosome sets was discovered from the gynogenetic offspring of Carassius auratus gibelio stimulated by red common carp sperm. In this study, genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with 45S rDNA probe are used. The GISH results lead to the identification of species-specific chromosomes, which permits to demonstrate the origin and genome organization in the allotetraploid form. Moreover, chromosome localization of 45S rDNA and co-localizations of 45S rDNA and Cyprinus carpio genomic DNA further confirm that one extra 45S rDNA positive chromosome in the allotetraploid form originates from the paternal haploid genome of C carpio, and other 5 45S rDNA-containing chromosomes are from the maternal genome of Carassius auratus gibelio. And, the correlation between 45 rDNA and the nucleolar organizer regions (NORs) is confirmed by silver nitrate staining. The data provide direct experiment evidence that the allotetraploid actually contains three chromosome sets of Carassius auratus gibelio and one chromosome set of C carpio, and will be a useful genetic material for both basic research and breeding practice. (c) 2006 Elsevier B.V. All rights reserved.

Identification of novel SINEs from Cyprinidae and their evolutionary significance

Short interspersed nuclear elements (SINEs) are widespread among eukaryotic genomes. They are repetitive DNA sequences that have been amplified by retrotransposition. In this study, a class of SINEs were isolated from the Opsariichthys bidens genome, and named Opsar. Sequence analysis confirmed that Opsar is a new class of typical SINEs derived from tRNA molecules. With the tRNA-derived region of Opsar and through BLASTN search, we further identified Zb-SINEs from the zebrafish genome, which includes two groups: Zb-SINE-A and Zb-SINE-B. The Zb-SINE-A group comprises subfamilies of -Al--A5, and the Zb-SINE-B group is a dimer of the tRNA(Ala)-derived region and shares a similar dimeric composition to Alu. Zb-SINEs are composed of three distinct regions: a 5 end tRNA-derived region, a tRNA-unrelated region and a 3 end AT-rich region. The flanking regions are AT rich. The average length of Zb-SINEs elements is about 340 6p. Zb-SINEs account for as much as 0.1% of the whole zebrafish genome. About 70% of the Zb-SINEs are on chromosomes 11, 18, and 19. These Zb-SINEs were characterized by PCR and dot hybridization. The distribution pattern of Zb-SINEs in genome strongly supports the master genes model. The tRNA-derived regions of Opsar and Zb-SINEs were compared with the tRNA(Ala) gene, and they showed 76% similarity, indicating that Opsar and Zb-SINEs originated from an inactive tRNA(Ala) sequence or a tRNA(Ala)-like sequence. In view of the evolutionary status of zebrafish in the Cyprinidae, we deduced that Zb-SINEs were a very old class of interspersed sequences.

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 of growth hormone receptor (GHR) from common carp (Cyprinus carpio L.) and identification of its two forms of mRNA transcripts

The cDNA of growth hormone receptor (GHR) was cloned from the liver of 2-year common carp (Cyprinus carpio L.) by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE). Its open reading frame (ORF) of 1806 nucleotides is translated into a putative peptide of 602 amino acids, including an extracellular ligand-binding domain of 244 amino acids (aa), a single transmembrane domain of 24 aa and an intracellular signal-transduction domain of 334 aa. Sequence analysis indicated that common carp GHR is highly homologous to goldfish (Carassius auratus) GHR at both gene and protein levels. Using a pair of gene-specific primers, a GHR fragment was amplified from the cDNA of 2-year common carp, a 224 bp product was identified in liver and a 321 bp product in other tissues. The sequencing of the products and the partial genomic DNA indicated that the difference in product size was the result of a 97 bp intron that alternatively spliced. In addition, the 321 bp fragment could be amplified from all the tissues of 4-month common carp including liver, demonstrating the occurrence of the alternative splicing of this intron during the development of common carp. Moreover, a semi-quantitative RT-PCR was performed to analyze the expression level of GHR in tissues of 2-year common carp and 4-month common carp. The result revealed that in the tissues of gill, thymus and brain, the expression level of GHR in 2-year common carp was significantly tower than that of 4-month common carp.

Identification of a WSSV neutralizing scFv antibody by phage display technology and in vitro screening

White spot syndrome virus (WSSV) is one of the most significant viral pathogens causing high mortality and economic damage in shrimp aquaculture. Although intensive efforts were undertaken to detect and characterize WSSV infection in shrimp during the last decade, we still lack methods either to prevent or cure white spot disease. Most of the studies on neutralizing antibodies from sera have been performed using in vivo assays. For the first time, we report use of an in vitro screening method to obtain a neutralizing scFv antibody against WSSV from a previously constructed anti-WSSV single chain fragment variable region (scFv) antibody phage display library. From clones that were positive for WSSV by ELISA, 1 neutralizing scFv antibody was identified using an in vitro screening method based on shrimp primary lymphoid cell cultures. The availability of a neutralizing antibody against the virus should accelerate identification of infection-related genes and the host cell receptor, and may also enable new approaches to the prevention and cure of white spot disease.

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. (c) 2006 Published by Elsevier B.V.

Isolation and identification of a novel WSSV nucleocapsid protein by cDNA phage display using an scFv antibody

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A I with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A I scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the prefer-red selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv. (c) 2006 Elsevier B.V. All rights reserved.

EST-based identification of genes expressed in the hypothalamus of male orange-spotted grouper (Epinephelus coioides)

The orange-spotted grouper, Epinephelus coioides, is an important marine aquaculture fish, but its large-scale aquaculture has been hindered by the rarity of natural males because it is a protogynous hermaphroditic fish. Hypothalamus-pituitary-gonad is an important endocrine axis in regulating reproduction and sex differentiation. To reveal the molecular mechanism of hypothalamic physiological functions, we performed he studies on identification of genes expressed in the hypothalamus of male orange-spotted grouper using EST and RT-PCR strategy. A total of 1006 ESTs were sequenced, and 402 (39.96%) clones were identified as known genes and 604 (60.04%) as unknown genes. The 402 clones of known gene products represent transcripts of 18 1 genes. Moreover, the expression patterns of 26 unknown genes were analyzed in various tissues, such as liver, kidney, spleen, fat, heart, muscle, pituitary, hypothalamus, telencephalon, cerebellum, midbrain, medulla oblongata, ovary and testes. Five different categories of expression patterns were observed from them. Several unknown ESTs, such as DN551996, DN551998, DN552082, and DN552070, were detected to be hypothalamus-specific, brains-specific, or hypothalamus and gonad-specific genes. Interestingly, DN551996, not only exhibiting expression differences between ovary and testis, but also showing sex-dependent differences in hypothalamus of grouper, might play significant role in grouper reproduction or sex inversion. Further functional studies on these genes will provide more information on molecule regulation mechanism of sex inversion in groupers. (c) 2006 Elsevier B.V All fights reserved.

Identification of differentially expressed genes in Tetrahmena thermophila in response to dichlorodiphenyltrichloroethane (DDT) by suppression subtractive hybridization

The insecticide dichlorodiphenyltrichloroethane (DDT) is persistent in the environment, and continues to cause health problems. Tetrahymena has potential as a model organism for assaying low levels of DDT and for analysing the mechanisms of its toxicity. We constructed the suppression subtractive hybridization library of T thermophila exposed to DDT, and screened out 90 Expressed Sequence Tags whose expressions were significantly up- or downregulated with DDT treatment. From this, a series of important genes related to the DDT metabolism and detoxification were discovered, such as P450 gene, glutathione S-transferase gene and sterol carrier protein 2 gene. Furthermore, their expressions under different concentrations of DDT treatment were detected by real-time fluorescent quantitative PCR. The results show that Tetrahymena is a relevant and useful model organism for detecting DDT in the environment and for discovering biomarkers that can be used to develop specific bio-reporters at the molecular and genomic levels.

Identification of a novel C2 domain factor in ovaries of orange-spotted grouper (Epinephelus coioides)

Follicle consists of an oocyte and a lot of surrounding follicular cells, and significant interactions exist between the oocyte and the somatic cells. In this study, a novel cDNA has been screened from a subtractive cDNA library between tail bud embryos and blastula embryos in the protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Its full-length cDNA is 821 bp, and has an ORF of 414 by for encoding a peptide of 137 aa, which shows 38%, 37%, 33%, and 33% homology with 4 putative proteins screened from zebrafish (Danio rerio). Conserved domain search in NCBI reveals a single C2 domain existing in the C2 domain superfamily proteins, and has only 7 beta strands in comparison with 8 beta strands of C2 domains in other C2 domain superfamily proteins. Artificial sex reversal, RT-PCR analysis and Western blot detection demonstrated ovary-specific expression of the C2 domain factor, and therefore the novel gene was designated as E. coioides ovary-specific C2 domain factor, EcOC2 factor. Moreover, predominant expression of EcOC2 factor was further revealed in grouper mature ovary, and its strong immunofluorescence signals were located between granulosa cells and oocyte zona radiata in grouper mature follicles. The data indicate that the novel EcOC2 factor might be a main component that associates between granulosa cells and the oocyte during oocyte maturation, and might play significant roles in regulating oocyte maturation and ovulation. Further studies on its developmental behaviour and physiological functions will elucidate the interactions between oocyte and the surrounding somatic cells and the underlying molecular mechanisms. (C) 2005 Elsevier Inc. All rights reserved.

Molecular identification and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from Japanese flounder (Paralichthys olivaceus)

Natural resistance associated macrophage protein (Nramp) controls partially innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from Japanese flounder (Paratichthys olivaceus). The full-length cDNA of the Nramp is 3066 bp in length, including 224 bp 5' terminal UTR, 1662 bp encoding region and 1180 bp 3' terminal UTR. The 1662-nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that Japanese flounder Nramp consists of 12 transmembrane (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of Japanese flounder had 77.30%, 82.71%, 82.67%, 79.64%, 80.72%, 90.97%, 91.16%, 60.14%, 71.48%, 61.69%, 72.37% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, striped sea bass Nramp, red sea bream Nramp, mouse Nramp 1 and 2, human Nramp 1 and 2, respectively. RT-PCR indicated that Nramp transcripts were highly abundant in spleen, head kidney, abundant in intestine, liver and gill, and less abundant in heart. The level of Nramp mRNA in embryos gradually increases during embryogenesis from 4 h (8 cell stage) to 80 h (hatched stage) after fertilization. (c) 2005 Elsevier Ltd. All rights reserved.

Identification of one intron loss and phylogenetic evolution of Dfak gene in the Drosophila melanogaster species group

Intron loss and its evolutionary significance have been noted in Drosophila. The current study provides another example of intron loss within a single-copy Dfak gene in Drosophila. By using polymerase chain reaction (PCR), we amplified about 1.3 kb fragment spanning intron 5-10, located in the position of Tyr kinase (TyK) domain of Dfak gene from Drosophila melanogaster species group, and observed size difference among the amplified DNA fragments from different species. Further sequencing analysis revealed that D. melanogaster and D. simulans deleted an about 60 bp of DNA fragment relative to other 7 Drosophila species, such as D. elegans, D. ficusphila, D. biarmipes, D. takahashii, D. jambulina, D. prostipennis and D. pseudoobscura, and the deleted fragment located precisely in the position of one intron. The data suggested that intron loss might have occurred in the Dfak gene evolutionary process of D. melanogaster and D. simulans of Drosophila melanogaster species group. In addition, the constructed phylogenetic tree based on the Dfak TyK domains clearly revealed the evolutionary relationships between subgroups of Drosophila melanogaster species group, and the intron loss identified from D. melanogaster and D. simulans provides a unique diagnostic tool for taxonomic classification of the melanogaster subgroup from other group of genus Drosophila.

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides(CT)(10) and (GT)(10) served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.

Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss

We report the cloning of a novel antimicrobial peptide gene, termed rtCATH_1, found in the rainbow trout, Oncorhynchus mykiss. The predicted 216-residue rtCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. The rtCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5' flanking region. By Northern blot analysis, the expression of rtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I (.) C) also expressed riCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus, rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.

Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.