Microarray Analyses of Gene Expression during the Tetrahymena thermophila Life Cycle

Background: The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression. Methodology/Principal Findings: A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation. Conclusions/Significance: Of the,27,000 predicted open reading frames, transcripts homologous to only,5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5x corrected background and 95 genes are expressed at >250x corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.

Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis

P>NF-kappa B is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-kappa B-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor kappa B (I kappa B)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-kappa Bs and phylogenetic analysis suggested that EsRelish was a member of the NF-kappa B family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.

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.

Cloning and characterization of beta-carotene ketolase gene promoter in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates a highly valuable ketocarotenoid, astaxanthin, under various environmental stresses. beta-carotene ketolase (BKT) plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, an approximate 700 bp 5'-flanking region of the bkt gene containing a putative promoter was cloned through walking upstream. The results of the sequence analysis showed that this bkt 5'-flanking region might have cis-acting elements such as sterol regulatory element (SRE-1)-like motifs, the C-repeat/dehydration responsive element (DRE) and al-3 proximal element (APE)-like motifs, except for typical TATA and CCAAT boxes. The results of the P-galactosidase assay and the transient expression of lacZ driven by a series of sequential deletions revealed that a minimal promoter-like region might exist from -630 to -408 bp, and the highest promoter activity was observed to span the positions from -630 to -308 bp. The results of the site-directed mutagenesis of a C-repeat/DRE and two APE-like motifs in a promoter-like region (-630 to -308 bp) suggested that two APE-like motifs might be essential for transcriptional control of the bkt gene.

Effect of lanthanum on expression of recombinant allophycocyanin gene in Pichia pastotis

The enhancing effect of lanthanum on gene expression of recombinant allophycocyanin (rAPC), a potential antitumor medicine, in Pichia pastoris was studied. PCR and sequence analysis were used in order to prove whether the APC gene had integrated into the yeast genome. The expression level of the recombinant allophycocyanin (rAPC) in BMMY medium containing LaCl3 was detected by ELISA method. The recombinant allophycocyanin was determined by Western blot. The results show that the recombinant Pichia pastoris chromosome contained allophycocyanin gene. Expression efficiency of rAPC gene in Pichia pastoris was promoted by proper LaCl3 concentration like 2, 5, 10 mmol (.) L-1, among which 5 mmol (.) L-1 was the most effective. The highest expression yield of rAPC in the BMMY medium containing 5 mmol (.) L-1 LaCl3 was 4.4 mg (.) L-1 at 48 h, that was increased by 110% compared with 2.1 mg (.) L-1 of control, in the meantime, the optimum culture time is shortened from 72 to 48 h. The result of western blot analysis indicates that the rAPC consisted of two kinds of subunits with molecular weight of 19 and 21 kDa respectively.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: Gene cloning, expression and activity analysis

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68 bp, and a long 3'-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis

Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene (Fclectin) was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey (Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have I I amino acids residues, which are relatively invariant in animals' C-type lectin CRDs. Five residues at Ca2+ binding site I are conserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and expression of a Toll receptor gene homologue from Zhikong Scallop, Chlamys farreri

Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors, which show homology with the Drosophila Toll protein and play key roles in detecting various non-self substances and then initiating and activating immune system. In this report, the full length of the first bivalve TLR (named as CfToll-1) is presented. CfToll-1 was originally identified as an EST (expressed sequence tag) fragment from a cDNA library of Zhikong scallop (Chlamys farreri). Its complete sequence was obtained by the construction of Genome Walker library and 5' RACE (rapid amplification of cDNA end) techniques. The full length cDNA of CfToll-1 consisted of 4308 nucleotides with a polyA tail, encoding a putative protein of 1198 amino acids with a 5' UTR (untranslated region) of 211 bp and a 3'UTR of 500 bp. The predicted amino acid sequence comprised an extracellular domain with a potential signal peptide, nineteen leucine-rich repeats (LRR), two LRR-C-terminal (LRRCT) motifs, and a LRR-N-terminal (LRRNT), followed by a transmembrane segment of 20 amino acids, and a cytoplasmic region of 138 amino acids containing the Toll/IL-1R domain (TIR). The deduced amino acid sequence of CfToll-1 was homologous to Drosophila melanogaster Tolls (DmTolls) with 23-35% similarity in the full length amino acids sequence and 30-54% in the TIR domain. Phylogenetic analysis of CfToll-1 with other known TLRs revealed that CfToll-1 was closely related to DmTolls. An analysis of the tissue-specific expression of the CfToll-1 gene by Real-time PCR showed that the transcripts were constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill. The temporal expressions of CfToll-1 in the mixed primary cultured haemocytes were observed after the haemocytes were treated with 1 mu g ml(-1) and 100 ng ml(-1) lipopolysaccharide (LPS), respectively. The expression of CfToll-1 was up-regulated and increased about 2-fold at 6 h with the treatment of 1 mu g ml(-1) LPS. The expression of CfToll-1 was down-regulated with the treatment of 100 ng ml(-1) LPS. The results indicated that the expression of CfToll-1 could be regulated by LPS, and this regulation was dose-dependent. (c) 2006 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.

Identification and characterization of a Cystatin gene from Chinese mitten crab Eriocheir sinensis

Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Enocheir sinensis (designated EsCystain) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5'-terminal untranslated region (UTR) of 92 bp, a 3' UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 548 and the predicted molecular weight of 13 39 kDa. A signal Cystatin-like domain (Gly(25) to Lys(112)) was found in the putative amino acid sequences of EsCystatin Similar to other Cystatins, the conserved central Q(70)VVSG(74) motif was located in the Cystatin-like domain of EsCystatin But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystain were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0 6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01)at 24 h Afterwards. EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2 8-fold of that in blank (P < 0 01)) The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain When the concentration of EsCystatin protein was of 300 mu g mL(-1), almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms. (C) 2010 Elsevier Ltd All rights reserved.

Cloning, characterization, and expression analysis of extracellular copper/zinc superoxide dismutase gene from bay scallop Argopecten irradians

Extracellular superoxide dismutase (ECSOD) is a major extracellular antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned a novel ECSOD from the bay scallop Argopecten irradians (AiECSOD) by 3' and 5' RACE. The full-length cDNA of AiECSOD was 893 bp with a 657 bp open reading frame encoding 218 amino acids. The deduced amino acid sequence contained a putative signal peptide of 20 amino acids, and sequence comparison showed that AiECSOD had low degree of homology to ECSODs of other organisms. The genomic length of the AiECSOD gene was about 5276 bp containing five exons and six introns. The promoter region contained many putative transcription factor binding sites such as c-Myb, Oct-1, Sp1, Kruppel-like, c-ETS, NF kappa B, GATA-1, AP-1, and Ubx binding sites. Furthermore, tissue-specific expressions of AiECSOD and temporal expressions of AiECSOD in haemocytes of bay scallops challenged with bacteria Vibrio anguillarum were quantified using qRT-PCR. High levels of expression were detected in haemocytes, but not in gonad and mantle. The expression of AiECSOD reached the highest level at 12 h post-injection with V. anguillarum and then returned to normal between 24 h and 48 h post-injection. These results indicated that AiECSOD was an inducible protein and that it may play an important role in the immune responses against V anguillarum. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

A role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum

P.M. Hastie and W. Haresign (2006). A role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum. Animal Reproduction Science, 96(1-2), 196-209. Sponsorship: DEFRA RAE2008

The rpf gene of Micrococcus luteus encodes an essential secreted growth factor

Galina V. Mukamolova, Obolbek A. Turapov, Konstantin Kazarian, Miroslav Telkov, Arseny S. Kaprelyants, Douglas B. Kell and Michael Young (2002). The rpf gene of Micrococcus luteus encodes an essential secreted growth factor. Molecular Microbiology, 46 (3), 611-621. Sponsorship: BBSRC / Russian Foundation for Basic Research (grant 00-04-48691)/ WHO Global Programme for Vaccines and Immunization / Wellcome Trust RAE2008

Investigation of toll-like receptor tolerance in the regulation of inflammation

Inflammation is a complex and highly organised immune response to microbes and tissue injury. Recognition of noxious stimuli by pathogen recognition receptor families including Toll-like receptors results in the expression of hundreds of genes that encode cytokines, chemokines, antimicrobials and regulators of inflammation. Regulation of TLR activation responses is controlled by TLR tolerance which induces a global change in the cellular transcriptional expression profile resulting in gene specific suppression and induction of transcription. In this thesis the plasticity of TLR receptor tolerance is investigated using an in vivo, transcriptomics and functional approach to determine the plasticity of TLR tolerance in the regulation of inflammation. Firstly, using mice deficient in the negative regulator of TLR gene transcription, Bcl-3 (Bcl-3-/-) in a model of intestinal inflammation, we investigated the role of Bcl-3 in the regulation of intestinal inflammatory responses. Our data revealed a novel role for Bcl-3 in the regulation of epithelial cell proliferation and regeneration during intestinal inflammation. Furthermore this data revealed that increased Bcl-3 expression contributes to the development of inflammatory bowel disease (IBD). Secondly, we demonstrate that lipopolysaccharide tolerance is transient and recovery from LPS tolerance results in polarisation of macrophages to a previously un-described hybrid state (RM). In addition, we identified that RM cells have a unique transcriptional profile with suppression and induction of genes specific to this polarisation state. Furthermore, using a functional approach to characterise the outcomes of TLR tolerance plasticity, we demonstrate that cytokine transcription is uncoupled from cytokine secretion in macrophages following recovery from LPS tolerance. Here we demonstrate a novel mechanism of regulation of TLR tolerance through suppression of cytokine secretion in macrophages. We show that TNF-α is alternatively trafficked towards a degradative intracellular compartment. These studies demonstrate that TLR tolerance is a complex immunological response with the plasticity of this state playing an important role in the regulation of inflammation.

An investigation into the role of Bcl-3 in toll-like receptor signalling

Through the recognition of potentially harmful stimuli, Toll-like receptors (TLRs) initiate the innate immune response and induce the expression of hundreds of immune and pro-inflammatory genes. TLRs are critical in mounting a defence against invading pathogens however, strict control of TLR signalling is vital to prevent host damage from excessive or prolonged immune activation. In this thesis the role of the IκB protein Bcl (B-cell lymphoma)-3 in the regulation of TLR signalling is investigated. Bcl3-/- mice and cells are hyper responsive to TLR stimulation and are defective in LPS tolerance. Bcl-3 interacts with and blocks the ubiquitination of homodimers of the NF-κB subunit, p50. Through stabilisation of inhibitory p50 homodimers, Bcl-3 negatively regulates NF-κB dependent inflammatory gene transcription following TLR activation. Firstly, we investigated the nature of the interaction between Bcl-3 and p50 and using peptide array technology. Key amino acids required for the formation of the p50:Bcl-3 immunosuppressor complex were identified. Furthermore, we demonstrate for the first time that interaction between Bcl-3 and p50 is necessary and sufficient for the anti-inflammatory properties of Bcl-3. Using the data generated from peptide array analysis we then generated cell permeable peptides designed to mimic Bcl-3 function and stabilise p50 homodimers. These Bcl-3 derived peptides are potent inhibitors of NF-κB dependent transcription activity in vitro and provide a solid basis for the development of novel gene-specific approaches in the treatment of inflammatory diseases. Secondly, we demonstrate that Bcl-3 mediated regulation of TLR signalling is not limited to NF-κB and identify the MAK3K Tumour Progression Locus (Tpl)-2 as a new binding partner of Bcl-3. Our data establishes role for Bcl-3 as a negative regulator of the MAPK-ERK pathway.