Gene structures and promoter characteristics of interferon regulatory factor 1 (IRF-1), IRF-2 and IRF-7 from snakehead Channa argus

Three interferon regulatory factor (IRF) genes, CaIRF-1, CaIRF-2 and CaIRF-7, and their promoters of snakehead (Channa argus) were cloned and characterized. The CaIRF-1 gene consists of ten exons, spans 4.3 kb and encodes a putative peptide of 299 aa. The CaIRF-2 gene consists of nine exons, spans 8 kb and encodes a putative peptide of 328 aa. The gene organizations of CaIRF-1 and CaIRF-2 are very similar to that of human IRF-1 and IRF-2 except more compact. Comparison of exon-intron organization of the two genes indicated a common evolutionary structure, notably within the exons encoding the DNA binding domain (DBD) of the two factors. The CaIRF-7 gene spans 4.1 kb and encodes a putative peptide of 437 aa. However, the gene organization of CaIRF-7 consisting of ten exons is different to human IRF-7a gene which has an intron in 5' UTR. Three CaIRFs share homology in N-terminal encompassing the DBD that contains a characteristic repeat of tryptophan residues. The promoters of CaIRF-1 and CaIRF-2 genes contain the conserved sites for NF-kappa B and Sp1. The gamma-IFN activation sites (GAS) were found in the promoters of CaIRF-1 and CaIRF-7. The promoter of CaIRF-7 contains conserved interferon stimulating response element (ISRE) which is characteristic of IFN-induced gene promoter, and suggests that there also exist intracellular amplifier circuit in fish IFN signal pathway. Moreover, the element GAAANN oriented in both directions is repeated in CaIRF promoter regions, which confers to further inducibility by IFN. The constitutive expression of CaIRF genes were found to increase obviously in response to induction by the known IFN-inducer poly I:C. (c) 2008 Published by Elsevier Ltd.

Molecular characterization and subcellular localization of Carassius auratus interferon regulatory factor-1

Interferon (IFN)-regulatory transcription factor-1 (IRF-1) has been studied in mammals and fish but little is known about the relationship between its gene structure and nuclear 'ion of IRF-1 protein. In this study, a cDNA encoding Carassius auratus IRF-1 (CaIRF-1) was isolated from an interferon-producing cell line, C. ouratus blastulae embryonic (CAB) cells, exposed to UV-inactivated grass carp hemorrhagic virus (GCHV). The CaIRF-1 genomic locus exhibits exon-intron arrangements similar to those of other vertebrate IRF-1 loci, with nine exons and eight introns, although together with pufferfish IRF-1, CaIRF-1 distinguishes itself from other vertebrate IRF-1 genes by a relatively compact genomic size. Similar to the known IRF-1 genes, CaIRF-1 is ubiquitously expressed, and is upregulated in vitro and in vivo in response to virus, Poty I:C, or CAB INF-containing supernatant (ICS). Subcellular localization analysis confirms the nuclear distribution of CaIRF-1 protein, and reveals two nuclear localization signals (NILS), any one of which is sufficient for nuclear translocation of CaIRF-1. One NLS Locates to amino acids 117-146, and appears to be the structural and functional equivalent of the NLS in mammalian IRF-1. The second NLS (amino acids 73-115) is found within the DNA-binding domain (DBD) of CaIRF-1, and contains two regions rich in basic amino acids (''(KDKSINK101)-K-95" and ''(75)KTWKANFR(82)"). In comparison with mammalian IRF-1, in which the corresponding amino acid stretch does not seem to drive nuclear translocation, five conserved basic amino acids (K-75, K-78, R-82, K-95, and K-101) and one non-conserved basic amino acid (K-97) are present in this NLS from CaIRF-1. This observation suggests that K97 Of CaIRF-1 might be essential for the function of its second NLS, wherein the six basic aminoacids might cooperate to drive CaIRF-1 to the nucleus. Therefore, the current study has revealed a new nuclear localization motif in the DBD of a vertebrate IRF-1. (C) 2007 Elsevier Ltd. All rights reserved.

FB1, an E2A fusion partner in childhood leukemia, interacts with U19/EAF2 and inhibits its transcriptional activity

Background: U19/EAF2 is a potential tumor suppressor exhibiting frequent down-regulation and allelic loss in advanced human prostate cancer specimens. U 19/EAF2 has also been identified as ELL-associated factor 2 (EAF2) based on its binding to ELL, a fusion partner of MLL in acute myeloid leukemia. U19/EAF2 is a putative transcription factor with a transactivation domain and capability of sequence-specific DNA binding. Methods: Yeast-two-hybrid-screening was used to identify U19/EAF2-binding partners. Co-immunoprecipitation and mammalian 1-hybrid assay were used to characterize a U19/EAF2-binding partner. Results: FB1, an E2A fusion partner in childhood leukemia, was identified as a binding-partner of U19/EAF2. FB1 also binds to EAF1, the only homologue of U19/EAF2. FB1 also interacts and co-localizes with ELL in the nucleus. Interestingly, FB1 inhibited the transcriptional activity of U19/EAF2 but not EAF1. Conclusions: FB1 is an important binding partner and a functional regulator of U19/EAF2, EAF1, and/or ELL. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals. Copyright (C) 2004 John Wiley Sons, Ltd.

alr0117, a two-component histidine kinase gene, is involved in heterocyst development in Anabaena sp PCC 7120

Anabaena sp. PCC; 7120 was mutagenized by transposon Tn5-1087b, generating a mutant whose heterocysts lack the envelope polysaccharide layer. The transposon was located between nucleotides 342 and 343 of alr0117, a 918 bp gene encoding a histidine kinase for a two-component regulatory system. Complementation of the mutant with a DNA fragment containing alr0117 and targeted inactivation of the gene confirmed that alr0117 is involved in heterocyst development. RT-PCR showed that alr0117 was constitutively expressed in the presence or absence of a combined-nitrogen source. hepA and patB, the two genes turned on during wild-type heterocyst development, were no longer activated in an alr0117-null mutant. The two-component signal transduction system involving alr0117 may control the formation of the envelope polysaccharide layer and certain late events essential to the function of heterocysts.

Molecular cloning and characterization of crucian carp (Carassius auratus L.) interferon regulatory factor 7

Interferon (IFN) can induce an antiviral state via interferon-regulatory transcription factors (IRFs), which bind to and control genes directed by the interferon-stimulated response element (ISRE). Here we describe a fish IRF, termed CaIRF7, cloned from a subtractive cDNA library which is constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. CaIRF7 cDNA was found to be 1816 bp in length, with a 42 bp 5' UTR and a 508 bp 3' UTR. The open reading frame translates into 421 amino acids in which a DNA-binding domain (DBD) containing the repeated tryptophan motif and IRFs association domain have been identified. Like chicken GgIRF3, CaIRF7 was most similar to mammalian IRF7 with 27 to 30% identity overall and some 37% identity in their DBDs. A single transcript of 1.9 kb was detected in virally induced CAB cells by virtual Northern blotting. RT-PCR analysis revealed a wide tissue distribution of CaIRF7 constitutive expression, with detectable transcript in non-infected CAB cells and various tissues of healthy crucian carp. In addition, CaIRF7 expression was differentially increased by stimulation of the CAB cells with active GCHV, UV-inactivated GCHV or CAB IFN, indicating that the activation of CaIRF7 was directly regulated by IFN. (C) 2003 Published by Elsevier Ltd.

A conserved family of doublesex-related genes from fishes

The sex-determining gene Mab-3 of C. elegans and the doublesex gene of Drosophila each contain a common DM domain and share a similar role. Human doublesex-related gene DMRT1 also encodes a conserved DM-related DNA-binding domain. We present here the amplification of a broad range of DM domain sequences from three fish species using degenerate PCR. Our results reveal unexpected complexity of the DM domain gene family in vertebrates. (C) 2002 Wiley-Liss, Inc.

Self-assembly of single-stranded RNA on carbon nanotube: Polyadenylic acid to form a duplex structure

All messenger-RNA (mRNA) molecules in eukaryotic cells have a polyadenylic acid [poly (rA)] tail at the 3'-end and human poly (rA) polymerase (PAP) has been considered as a tumor-specific target. A ligand that is capable of recognizing and binding to the poly(M) tail of mRNA might interfere with the full processing of mRNA by PAP and can be a potential therapeutic agent. We report here for the first time that single-walled carbon nanotubes (SWNTs) can cause single-stranded poly (M) to self-structure and form a duplex structure, which is studied by UV melting, atomic force microscopy, circular dichroism spectroscopy, and NMR spectrometry.

Characterization and optimization of AuNPs labeled by Raman reporters on glass based on silver enhancement

In this report, gold nanoparticles (AuNPs) labeled by Raman reporters (AuNPs-R6G) were assembled on glass and used as the seeds to in situ grow silver-coated nanostructures based on silver enhancer solution, forming the nanostructures of AuNPs-R6G@Ag, which were characterized by scanning electron microscopy (SEM) and UV-visible spectroscopy. More importantly, the obtained silver-coated nanostructures can be used as a surface enhancement Raman scattering (SERS) substrate. The different SERS activities can be controlled by the silver deposition time and assembly time of AuNPs-R6G on glass. The results indicate that the maximum SERS activity could be obtained on AuNPs-R6G when these nanostructures were assembled on glass for 2 h with silver deposition for 2 min.

Studies on the Interaction between Single-strand Oligonucleotide and Ginsenosides by Electrospray Ionization Mass Spectrometry

Oligonucleotide from SARS virus was selected as a target molecule in the paper. The noncovalent complexes of ginsenosides with the target molecule were investigated by electrospray ionization mass spectrometry. The effects of experimental conditions were examined firstly on the formation of noncovalent complexes. Based on the optimized experimental conditions, the interaction of different ginsenosides with the target molecule was researched, finding that the interaction orders are relative with the structure of aglycons, the length and terminal sugar types of saccharide chains in the ginsenosides. There are certain rules for the interaction between the ginsenosides and DNA target molecule. For different type ginsenosides, the interaction intensity takes the orders 20-S-protopanaxatriol > 20-S-protopanaxadiol, and panaxatriol ginsenosides > panaxadiol ginsenosides. For the ginsenosides with the same type aglycone, tri-saccharide chain > di-saccharide chain > tetra-saccharide chain and single-saccharide chain > panaxatriol. For the ginsenosides with the same tetra-saccharide chain, the ginsenosides with smaller molecule masses > the ginsenosides with larger molecule masses.

Multi-armed poly(L-glutamic acid)-graft-oligoethylenimine copolymers as efficient nonviral gene delivery vectors

Background The application of polyethylenimine (PEI) in gene delivery has been severely limited by significant cytotoxicity that results from a nondegradable methylene backbone and high cationic charge density. It is therefore necessary to develop novel biodegradable PEI derivates for low-toxic, highly efficient gene delivery.Methods A series of novel cationic copolymers with various charge density were designed and synthesized by grafting different kinds of oligoethylenimine (OEI) onto a determinate multi-armed poly(L-glutamic acid) backbone. The molecular structures of multi-armed poly(L-glutamic acid)-graft-OEI (MP-g-OEI) copolymers were characterized using nuclear magnetic resonance, viscosimetry and gel permeation chromatography. Moreover, the MP-g-OEI/DNA complexes were measured by a gel retardation assay, dynamic light scattering and atomic force microscopy to determine DNA binding ability, particle size, zeta potential, complex formation and shape, respectively. MP-g-OEI copolymers were also evaluated in Chinese hamster ovary and human embryonic kidney-293 cells for their cytotoxicity and transfection efficiency.

Investigations on the adsorption behavior of Neutral Red on mercaptoethane sulfonate protected gold nanoparticles

A detailed investigation on the adsorption behavior of Neutral Red (NR) molecules on mercaptoethane sulfonate-monolayer protected gold clusters (MES-MPCs) has been conducted by the spectroscopic method. It is found that cationic NR molecules are adsorbed on the negatively charged MPCs surfaces via electrostatic attractive forces. The absorption study shows that the optical properties of NR molecules are significantly influenced upon the adsorption. Based on the electrostatic adsorption nature and the excellent stability of MES-MPCs against the electrolytes, this association can be released by the addition of electrolyte salts, which can be monitored by both absorption and fluorescence spectroscopy. In addition, dication Ca2+ is found to be more effective in the release of NR than univalent Na+. Moreover, the MES-MPCs exert energy transfer quenching of NR fluorescence by both static and dynamic quenching. However, static quenching seems to be the dominating quenching mechanism. Furthermore, this energy transfer quenching exhibits strong dependence of Au core size, and 5.0 nm MPCs show stronger ability in quenching the NR fluorescence than that of 2.7 nm MPCs.

Poly(L-lysine)-graft-chitosan copolymers: Synthesis, characterization, and gene transfection effect

Polypeptide/polysaccharide graft copolymers poly(L-lysine)-graft-chitosan (PLL-g-Chi) were prepared by ring-opening polymerization (ROP) of epsilon-benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of 6-O-triphenylmethyl chitosan. The PLL-g-Chi copolymers were thoroughly characterized by H-1 NMR, C-13 NMR, Fourier transform infrared (FT-IR), and gel permeation chromatography (GPC). The number-average degree of polymerization of PLL grafted onto the chitosan backbone could be adjusted by controlling the feed ratio of NCA to 6-O-triphenylmethyl chitosan. The particle size of the complexes formed from the copolymer and calf thymus DNA was measured by dynamic light scattering (DLS). It was found in the range of 120 similar to 340 nm. The gel retardation electrophoresis showed that the PLL-g-Chi copolymers possessed better plasmid DNA-binding ability than chitosan. The gene transfection effect in HEK 293T cells of the copolymers was evaluated, and the results showed that the gene transfection ability of the copolymer was better than that of chitosan and was dependent on the PLL grafting ratio. The PLL-g-Chi copolymers could be used as effective gene delivery vectors.

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.