Genomic organization, gene duplication, and expression analysis of interleukin-1 beta in channel catfish (Ictalurus punctatus)

Interleukin-1 beta (IL-1 beta) is one of the pivotal early response pro-inflammatory cytokines that enables organisms to respond to infection and induces a cascade of reactions leading to inflammation. In spite of its importance and two decades of studies in the mammalian species, genes encoding IL-1 beta were not identified from non-mammalian species until recently. Recent research, particularly with genomic approaches, has led to sequencing of IL-1 beta from many species. Clinical studies also Suggested IL-1 beta as an immunoreagulatory molecule potentially useful for enhancing vaccination. However, no IL-1 beta genes have been identified from channel catfish, the primary aquaculture species from the United States. In this study, we identified two distinct cDNAs encoding catfish IL-1 beta. Their encoding genes were identified, sequenced, and characterized. The catfish IL-1 beta genes were assigned to bacterial artificial chromosome (BAC) clones. Genomic studies indicated that the IL-1 beta genes were tandemly duplicated on the same chromosome. Phylogenetic analysis of various IL-1 beta genes indicated the possibility of recent species-specific gene duplications in channel catfish, and perhaps also in swine and carp. Expression analysis indicated that both IL-1 beta genes were expressed, but exhibited distinct expression profiles in various catfish tissues, and after bacterial infection with Edwardsiella ictaluri. (c) 2005 Elsevier Ltd. All rights reserved.

NK-lysin of channel catfish: Gene triplication, sequence variation, and expression analysis

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In addition to the previously known four classes of antimicrobial peptides, a fifth class of antimicrobial peptides has been recently identified to include NK-lysins that have a globular three-dimensional structure and are larger with 74-78 amino acid residues. NK-lysin has been shown to harbor antimicrobial activities against a wide spectrum of microorganisms including bacteria, fungi, protozoa, and parasites. To date, NK-lysin genes have been reported from only a limited number of organisms. We previously identified a NK-lysin cDNA in channel catfish. Here we report the identification of two noveltypes of NK-lysin transcripts in channel catfish. Altogether, three distinct NK-lysin transcripts exist in channel catfish. In this work, their encoding genes were identified, sequenced, and characterized. We provide strong evidence that the catfish NK-lysin gene is tripled in the same genomic neighborhood. All three catfish NK-lysin genes are present in the same genomic region and are tightly linked on the same chromosome, as the same BAC clones harbor all three copies of the NK-lysin genes. All three NK-lysin genes are expressed, but exhibit distinct expression profiles in various tissues. In spite of the existence of a single copy of NK-lysin gene in the human genome, and only a single hit from the pufferfish,genome, there are two tripled clusters of NK-lysin genes on chromosome 17 of zebrafish in addition to one more copy on its chromosome 5. The similarity in the genomic arrangement of the tripled NK-lysin genes in channel catfish and zebrafish suggest similar evolution of NK-lysin genes. (c) 2005 Elsevier Ltd. All rights reserved.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrame protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAlL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRFI sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen. (c) 2005 Elsevier B.V. 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.

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.

Expression pattern and developmental behaviour of cellular nucleic acid-binding protein (CNBP) during folliculogenesis and oogenesis in fish

In vertebrates, folliculogeneis establishes an intricate system for somatic cell-oocyte interaction, and ultimately leads to the acquisition of their respective competences. Although the formation process and corresponding interactions are strikingly similar in diverse organisms, knowledge of genes and signaling pathways involved in follicle formation is very incomplete and the underlying molecular mechanisms remain enigmatic. CNBP has been identified for more than ten years, and the highest level of CNBP transcripts has been observed in adult zebrafish ovary, but little is known about its functional significance during folliculogeneis and oogenesis. In this study, we clone CNBP cDNA from gibel carp (Carassius auratus gibelio), and demonstrate its predominant expression in gibel carp ovary and testis not only by RTPCR but also by Western blot. Its full-length cDNA is 1402 bp, and has an ORF of 489 nt for encoding a peptide of 163 aa. And its complete amino acid sequence shared 68.5%-96.8% identity with CNBPs from other vertebrates. Based on the expression characterization, we further analyze its expression pattern and developmental behaviour during folliculogeneis and oogenesis. Following these studies, we reveal an unexpected discovery that the CagCNBP is associated with follicular cells and oocytes, and significant distribution changes have occurred in degenerating and regenerating follicles. More interestingly, the CagCNBP is more highly expressed in some clusters of interconnected cells within ovarian cysts, no matter whether the cell clusters are formed from the original primordial germ cells or from the newly formed cells from follicular cells that invaded into the atretic oocytes. It is the first time to reveal CNBP relevance to folliculogeneis and oogenesis. Moreover, a similar stage-specific and cell-specific expression pattern has also been observed in the gibel carp testis. Therefore, further studies on CNBP expression pattern and developmental behaviour will be of significance for understanding functional roles of CNBP during gametogenests. (c) 2005 Elsevier B.V. All rights reserved.

Inductive expression and characterization analysis of Paralichthys olivaceus pigment epithelium-derived factor in a virally infected cell line

Pigment epithelium-derived factor (PEDF) is acknowledged to be a non-inhibitory member of the serine protease inhibitor (serpin) superfamily, with antiangiogenesis, and neuroprotective and immumoregulatory function, mainly in the tissues of nervous system. Here, A PEDF gene homolog, Paralichthys olivaceus PEDF (PoPEDF), was isolated from flounder embryonic cells (FEC) treated with UV-inactivated Grass carp hemorrhage virus (GCHV) and subsequently identified as a differentially expressed gene. The full length of PoPEDF cDNA is 1803 bp with an open reading frame of 1212 bp encoding a 403-amino-acid protein. This deduced protein contains an N-terminal signal peptide, a glycosylation site, a consensus serpin motif, and a 34-mer and a 44-mer fragment, all of which are very conserved in the PEDF family. PoPEDF gene exhibits a conserved exon-intron arrangement with 8 exons and 7 introns. This conserved evolutionary relationship was further confirmed by a phylogenetic analysis, where fish PEDFs and mammalian members formed a well-supported clade. Constitutive expression of PoPEDF was widely detected in many tissues. In response to UV-inactivated GCHV or poly(I:C), PEDF mRNA was upregulated in FEC cells with time. This is the first report on the transcriptional induction of PEDF in virally infected cells. (C) 2005 Elsevier Inc. All rights reserved.

Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate

The RNA helicase Vasa is a germ cell marker in animals, and its homolog in vertebrates to date has been limited to bisexual reproduction. We cloned and characterized CagVasa, a Vasa homolog from the gibel carp, a fish that reproduces bisexually or gynogenetically. CagVasa possesses 14 RGG repeats and eight conserved motifs of Vasa proteins. In bisexually reproducing gibel carp, vasa is maternally supplied and its zygotic expression is restricted to gonads. By in situ hybridization on testicular sections, vasa is low in spermatogonia, high in primary spermatocytes, reduced in secondary spermatocytes, but disappears in spermatids and sperm. In contrast, vasa persists throughout oogenesis, displaying low-high-low levels from oogonia over vitellogenic oocytes to maturing oocytes. A rabbit anti-Vasa antibody (alpha Vasa) was raised against the N-terminal CagVasa for fluorescent immunohistochemistry. On testicular sections, Vasa is the highest in spermatogonia, reduced in spermatocytes, low in spermatids, and absent in sperm. In the ovary, Vasa is the highest in oogonia but persists throughout oogenesis. Subcellular localization of vasa and its protein changes dynamically during oogenesis. The aVasa stains putative primordial germ cells in gibel carp fry. It detects gonadal germ cells also in several other teleosts. Therefore, Cagvasa encodes a Vasa ortholog that is differentially expressed in the testis and ovary. Interestingly, the alpha Vasa in combination with a nuclear dye can differentiate critical stages of spermatogenesis and oogenesis in fish. The cross-reactivity and the ability to stain stage-specific germ cells make this antibody a useful tool to identify fish germ cell development and differentiation. (c) 2005 Wiley-Liss, Inc.

Effects of 2,3,7,8-TCDD and benzo[a]pyrene on modulating vitellogenin expression in primary culture of crucian carp (Carassius auratus) hepatocytes

Vitellogenin (Vtg) is the precursor of yolk protein. Its expression and secretion are estrogen-regulated and are crucial for oocyte maturation. An in vitro xenoestrogen screening model was established by measuring Vtg induction in cultured primary hepatocytes from crucian carp. Vtg production was detected by biotin-avidin sandwich ELISA method while Vtg and cytochrome P4501A1 (CYP1A1) mRNA induction were measured by semi- quantitative PCR-primer dropping technique. Vtg and Vtg mRNA were dose-dependently induced by diethylstilbestrol (DES, 0.2-200 ng/mL) in hepatocytes of crucian carp. Co-treatment of the DES-induced hepatocytes with either 2,3,7,8-TCDD (TCDD, 0.1-4 pg/mL) or benzo[a]pyrene (B[a]P, 5-1000 ng/mL) resulted in a reduction of Vtg production and an increment of CYP1A1 mRNA expression both in a dose dependent manner, indicating the anti-estrogenic effects of the compounds. However, at lower tested concentrations, TCDD (0.1, 0.2 pg/mL), B[a]P (5 ng/mL) seemed to have a potentiating effect on Vtg expression and secretion, although by their own these compounds had no observable estrogenic effect on Vtg induction. Tamoxifen (a selective estrogen receptor modulators, 1 nmol/L-1 mumol/L), and P-naphtho-flavone (beta-NF, an aryl hydrocarbon receptor inducing compounds, 2.5-1000 ng/mL) also were employed to study the possible interactions in DES-induced Vtg expression. In co-treatment of the DES-induced hepatocytes with beta-NF or tamoxifen, the decrease in Vtg production did parallel induction of CYP1A1 for beta-NF, but tamoxifen inhibited Vtg induction did not parallel induced CYP1A1 expression in all test concentrations. On the contrary, it was found that in co-treatment of the TCDD-induced hepatocytes with DES, TCDD induced CYP1A1 mRNA production was inhibited by DES also. These results implicated a possible cross talk between estrogen receptor- and aryl hydrocarbon receptor-mediated pathways in the hepatocytes.

Expression and molecular analysis of phbB gene in Chlamydomonas reinhardtii

The expression vector containing phbB and ble genes was constructed and transformed into cell-wall-deficient strain Chlamydomonas reinhardtii CC-849 by the glass-head method. The transgenic alga was selected and maintained in the TAP agar plates containing 10 mug/mL Zeomycin. Transgenic alga, which could express phbB at the transcriptional level, was obtained and further confirmed with PCR, Southern blot and RT-PCR-DNA hybridization analysis.