Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/MoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage. (c) 2005 Wiley-Liss, Inc.

Insulin-like growth factor-binding protein-3 plays an important role in regulating pharyngeal skeleton and inner ear formation and differentiation

Insulin-like growth factor-binding protein (IGFBP)-3 is the major insulin-like growth factor (IGF) carrier protein in the bloodstream. IGFBP-3 prolongs the half-life of circulating IGFs and prevents their potential hypo-glycemic effect. IGFBP-3 is also expressed in many peripheral tissues in fetal and adult stages. In vitro, IGFBP-3 can inhibit or potentiate IGF actions and even possesses IGF-independent activities, suggesting that local IGFBP-3 may also have paracrine/autocrine function(s). The in vivo function of IGFBP-3, however, is unclear. In this study, we elucidate the developmental role of IGFBP-3 using the zebrafish model. IGFBP-3 mRNA expression is first detected in the migrating cranial neural crest cells and subsequently in pharyngeal arches in zebrafish embryos. IGFBP-3 mRNA is also persistently expressed in the developing inner ears. To determine the role of IGFBP-3 in these tissues, we ablated the IGFBP-3 gene product using morpholino-modified antisense oligonucleotides (MOs). The IGFBP-3 knocked down embryos had delayed pharyngeal skeleton morphogenesis and greatly reduced pharyngeal cartilage differentiation. Knockdown of IGFBP-3 also significantly decreased inner ear size and disrupted hair cell differentiation and semicircular canal formation. Furthermore, reintroduction of a MO-resistant form of IGFBP-3 "rescued" the MO-induced defects. These findings suggest that IGFBP-3 plays an important role in regulating pharyngeal cartilage and inner car development and growth in zebrafish.

Characterization of an ETS transcription factor in the sea scallop Chlamys farreri

We have cloned and characterized a cDNA encoding a putative ETS transcription factor, designated Cf-ets. The Cf-ets encodes a 406 amino acid protein containing a conserved ETS domain and a Pointed domain. Phylogenetic analysis revealed that Cf-ets belongs to the ESE group of ETS transcription factor family. Real-time PCR analysis of Cf-ets expression in adult sea scallop tissues revealed that Cf-ets was expressed mainly in gill and hemocytes, in a constitutive manner. Cf-ets mRNA level in hemocytes increased drastically after microbial challenge indicated its indispensable role in the anti-infection process. Simultaneously, the circulating hemocyte number decreased. In mammals, most ETS transcription factors play indispensable roles in blood cell differentiation and linage commitment during hematopoisis. Cf-ets is therefore likely to be a potential biomarker for hematopoiesis studies in scallops. (C) 2009 Elsevier Ltd. All rights reserved.

Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong Scallop Chlamys farreri

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a key signaling adaptor molecule common to the TNFR superfamily and IL-IR/TLR family, is important not only for a diverse array of physiological processes functions of the TNFR superfamily, but also is involved in adaptive immunity and innate immunity. In this report, the first bivalve TRAF6 (named as CfTRAF6) gene is identified and characterized from Zhikong scallop Chlamys farreri. The full-length cDNA of CfTRAF6 is of 2510 bp, consisting of a 5'-terminal untranslated region (UTR) of 337 bp, a 3'-terminal UTR of 208 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame (ORF) encoding a polypeptide of 655 amino acids. The predicted amino acid sequence of CfTRAF6 comprises characteristic motifs of the TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, a coiled-coil region, and a MATH (the meprin and TRAF homology) domain. The overall amino acid sequence identity between CfTRAF6 and other TRAF6s is 28-68%. Phylogenetic analyses of CfTRAF6 sequence with TRAF sequences from other organisms indicate that CfTRAF6 is a true TRAF6 orthologue. The mRNA expression of CfTRAF6 in various tissues is measured by Real-time RT-PCR. The mRNA transcripts are constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill, but the highest expression is observed in the gonad. The temporal expressions of CfTRAF6 mRNA in the mixed primary cultured haemocytes are recorded after treatment with 20 mu g mL(-1) and 0.5 mu g mL(-1) peptido-glycan (PGN). The expression level of CfTRAF mRNA is down-regulated from 1.5 h to 3 h after the treatment with 0.5 mu g mL(-1) PGN, and then recovers to the original level. While the expression of CfTRAF6 is obviously decreased after treatment with 20 mu g mL(-1) PGN, and reach the lowest point (only about 1/9 times to control) at 3 h. The result Suggests that CfTRAF6 can be greatly regulated by PGN and it may be involved in signal transduction and immune response of scallop. (C) 2008 Published by Elsevier Ltd.

Molecular cloning and expression profile of putative antilipopolysaccharide factor in Chinese shrimp (Fenneropenaeus chinensis)

A new antimicrobial protein gene of the anti-lipopolysaccharide factor family (tentatively named as ALFFc) has been cloned from hemocytes of the Chinese shrimp Fenneropenaeus chinensis by rapid amplification of 3' and 5' complementary DNA ends with polymerase chain reaction. The full-length complementary DNA of ALFFc consists of 600 bp with a 369-bp open reading frame, encoding 123 amino acids. The deduced peptide contains a putative signal peptide of 25 amino acids and mature peptide of 98 amino acids. The molecular mass of the deduced mature peptide is 13799.16 Da. It is highly cationic, with a theoretical pI of 10.3. The deduced amino acid sequence of ALFFc showed 56% homology with sequences of Tachypleus tridentatus and L. polyhemus. The tissue expression profile of this gene was studied by Northern blot, and ALFFc transcripts were mainly detected in hemocytes, gill, and intestine. RNA in situ hybridization showed that ALFFc was constitutively expressed in hemocytes. Capillary electrophoresis reverse transcriptase PCR was used to quantify the variation of messenger RNA transcription level during the artificial infection process with Vibrio anguillarum. Significant enhancement of ALFFc transcription appeared during the first 24 hours in response to Vibrio infection. These results provide useful information for understanding the function of ALFFc in shrimp.

Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

Expression of human epidermal growth factor gene in cyanobacteria

The human epidermal growth factor (hEGF) is a small single-chain polypeptide of 53 amino acid residues. It can stimulate the proliferation of many cell types, mainly those of epidermal and epithelial tissues both in vivo and in vitro. A vector pRL-hEGF was constructed using plasmids pRL-489 and pUC-hEGF. The synthetic hEGF gene was recombined into the downstream of strong promoter psbA in plasmids pRL-489. Then, the vector was introduced into Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120 by triparental conjugative transfer. The transformation was confirmed by PCR amplification. The pRL-hEGF is thought to be retained as a plasmid form in the transgenic Anabaena sp. PCC 7120, since it can be recovered. However, it has been integrated into the chromosome of Synechococcus sp. PCC 7002 as there is no duplication origin in the pRL-hEGF in this cyanobacterium. and plasmid cannot be isolated from the Synechococcus sp. PCC 7002 either. The radioimmunoassay (RIA) proved that the hEGF gene has been expressed as the protein existed in these two strains of transgenic cyanobacteria, and the hEGF protein in Anabaena sp. PCC 7002 could be secreted into the medium.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363 bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved.

Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA from Zhikong scallop Chlamys farreri

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter for the TLR/IL-1R family. In this report, the first mollusk Myd88 ortholog (named as CfMyd88) was cloned from Zhikong scallop (Chlamys farreri). The full-length cDNA of CfMyd88 was of 1554 bp, including a 5 '-terminal untranslated region (UTR) of 427 bp, a polyA tail, and an open reading frame (ORF) of 1104 bp encoding a polypeptide of 367 amino acids containing the typical TLR and IL-1R-related (TIR) domain and death domain (DD). Homology analysis revealed that the predicted amino acid sequence of CfMyd88 was homologous to a variety of previously identified Myd88s with more than 30% identity. The temporal expressions of CfMyd88 mRNA in the mixed primary cultured haemocytes stimulated by lipopolysaccharide (LPS) and peptidoglycans (PGN) were measured by real-time RT-PCR system. The mRNA expression of CfMyd88 decreased after stimulation with both LPS and PGN, and the lowest level was about 1/3 times (at 6 h) and 1/10 times (at 9 h) to that in the control group, respectively. The expression then recovered and was upregulated to two-fold at 9 h after LPS stimulation or to the original level at 12 It after PGN stimulation. The results suggest that the MyD88-dependent signaling pathway exists in scallop and was involved in the defense system. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a putative lipopolysaccharide-induced TNF-alpha factor (LITAF) gene homologue from Zhikong scallop Chlamys farreri

LPS-induced TNF-alpha factor (LITAF) is a novel transcriptional factor that was first discovered in LPS-stimulated human macrophage cell line THP-1. LITAF can bind to TNF-a promoter to regulate its expression. The first scallop LITAF (named as CfLITAF) was cloned from Zhikong scallop Chlamys farreri by Expressed Sequence Tag (EST) and Polymerase Chain Reaction (PCR) techniques. The cDNA of CfLITAF was of 1240 bp and consisted of a 5' untranslated region (UTR) of 112 bp, a 3' UTR of 678 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 16.08 kDa and theoretical isoelectric point of 6.77. A typical conserved LITAF-domain was identified in CfLITAF by SMART analysis. Homology analysis of the deduced amino acid sequence of CfLITAF with other known sequences by using the BLAST program revealed that CfLITAF was homologous to the LITAF from human and rat (Identity = 46%), cattle, horse, mouse and chicken (Identity = 48%), western clawed frog (Identity = 42%), and zebrafish (Identity = 50%). The mRNA expression of CfLITAF in different tissues including haemocytes, muscle, mantle, heart, gill and gonad, and the temporal expression in haemocytes challenged by LPS or peptidoglycan (PGN) were measured by Real-time RT-PCR. CfLITAF mRNA transcripts could be detected in all tissues examined and be up-regulated in haemocytes after LPS challenge. No significant changes were observed after PGN stimulation. All these data indicated the existence of LITAF in scallop and also provided clue on the presence of TNF-alpha-like molecules in invertebrates. (C) 2007 Elsevier Ltd. All rights reserved.

A novel tumor necrosis factor ligand superfamily member (CsTL) from Ciona savignyi: Molecular identification and expression analysis

A novel invertebrate TNF ligand was identified and characterized in Ciona savignyi. The CsTL cDNA consisted of 995 nucleotides and encoded 281 amino acids. A conserved TNF family signature and several motifs of TNF ligand superfamily were identified in deduced amino acid sequence of CsTL. Phylogenetic analysis grouped CsTL, CiTNF (predicted TNF ligand superfamily homolog in Ciona intestinalis) and urchin TL1A with their own cluster apart from mammalian TNF alpha, LTA, TNFSF15 and fish TNFa proteins. Expression studies demonstrated that CsTL mRNA is present in all tested tissues from unchallenged ascidians and its expression was significantly upregulated in hemocytes following LIPS injection. The recombinant CsTL protein expressed using a baculovirus expression system showed potential cytotoxic activity in L929 cells. Present results indicated that TNF ligand superfamity molecules are present in marine invertebrates. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus

Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab. (C) 2010 Elsevier Ltd. 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 second anti-lipopolysaccharide factor (EsALF-2) with antimicrobial activity from Eriocheir sinensis

The anti-lipopolysaccharide factor CALF) is a small basic protein that can bind and neutralize lipopolysaccharide (LPS), mediating degranulation and activation of an intracellular coagulation cascade. In the present study, cDNA of the second Eriocheir sinensis ALF (designated as EsALF-2) was cloned and the full-length cDNA of EsALF-2 was of 724 bp, consisting of an open reading frame (ORF) of 363 bp encoding a polypeptide of 120 amino acids. The deduced amino acid of EsALF-2 shared 82% similarity with EsALF-1 from E. sinensis and about 53-65% similarity with ALFs from other crustaceans. The potential tertiary structures of EsALF-1 and EsALF-2 contained two highly conserved-cysteine residues to define the LPS binding site, but the N-terminal of EsALF-1 formed a single additional alpha-helix compared to EsALF-2, implying that EsALF-1 and EsALF-2 might represent different biological functions in E. sinensis. The mRNA transcript of EsALF-2 was detected in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad, which suggested that EsALF-2 could be a multifunctional molecule for the host immune defense responses and thereby provided systemic protection against pathogens. The mRNA expression of EsALF-2 was up-regulated after Listonelln anguillarum and Pichia pastoris challenge and the recombinant protein of EsALF-2 showed antimicrobial activity against L. anguillarum and P. pastoris. indicating that EsALF-2 was involved in the immune defense responses in Chinese mitten crab against L. anguillarum and P. pastoris. These results together indicated that there were abundant and diverse ALFs in E. sinensis with various biological functions and these ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control of crab aquaculture. (C) 2010 Elsevier Ltd. All rights reserved.