41 resultados para Single-gene


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Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.

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The population genetic structure of fish parasitic nematode, Camallanus cotti, collected from the Yangtze River, Pearl River and Minjiang River in China was investigated. From these parasites, the similar to 730 bp of the first internal transcribed spacer of ribosomal DNA (ITS1 rDNA) and the 428 bp of mitochondrial cytochrome c oxidase subunit I (COI) gene were sequenced. For the ITS1 rDNA data set, highly significant Fst values and low rates of migration were detected between the Pearl River group and both the Yangtze River (Fst = 0.70, P < 0.00001; Nm = 0.21) and Minjiang River (Fst = 0.73, P < 0.00001; Nm = 0.18) groups, while low Fst value (Fst = 0.018, P > 0.05) and high rate of migration (Nm = 28.42) were found between the Minjiang and the Yangtze rivers. When different host/locality populations (subpopulations) within each river were considered, subpopulations between the Yangtze River and Minjiang River had low Fst values (<= 0.12) and high Nm values (>3.72), while Pearl River subpopulations were significantly different from the Yangtze River and Minjiang River subpopulations (Fst >= 0.59; Nm < 1). The COI gene data set revealed a similar genetic structure. Both phylogenetic analyses and a statistical parsimony network grouped the Pearl River haplotypes into one phylogroup, while the Yangtze River and Minjiang River haplotypes formed a second group. These results suggested that the Yangtze River and Minjiang River subpopulations constituted a single reproductive pool that was distinct from the Pearl River subpopulations. In addition, the present study did not find host-related genetic differentiation occurring in the same drainage. (C) 2009 Published by Elsevier B.V.

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Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

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Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the TNF superfamily members, participating in many biological processes including cell proliferation and apoptotic death. In this study, a TRAIL gene was cloned from a perciform fish, the mandarin fish Siniperca chuatsi, a major cultured fish in China's aquaculture, and is named as SCTRAIL for S. chuatsi TRAIL. The full-length cDNA of SCTRAIL is 1359 bp, encoding a 283-amino-acid protein. This deduced protein contains the CYS231, a 23-mer fragment of transmembrane region, a glycosylation site and a TNF family signature, all of which are conserved among TRAIL members. SCTRAIL gene consists of six exons, with five intervening introns, spaced over approximately 9 kb of genomic sequence. Southern blotting demonstrated that the SCTRAIL gene is present as a single copy in mandarin fish genome. A 620 bp promoter region obtained by genome walking contains a number of putative transcription factor binding sites, such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPaLp, NF-kappa B and AP-1. The SCTRAIL is constitutively expressed in all the analyzed tissues, as revealed by RT-PCR, which is confirmed by Western blotting analysis using polyclonal antibody against bacteria-derived recombinant SCTRAIL protein. As an apoptosis-inducing ligand, the overexpression of SCTRAIL but not the mutant SCTRAIL-C203S in HeLa cells induced changes characteristic of apoptosis, including chromatin condensation, nucleus fragmentation, DNA ladder, and increase of sub-G0/G1 cells in FACS analysis. (c) 2007 Elsevier Ltd. All rights reserved.

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Wild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5' and 3' portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5' end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, P-hetC-gfp was very weakly expressed, and in hetZ::Tn5-1087b, P-hetR-gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.

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With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

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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.

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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.

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The fp25k gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) was studied. HearNPV fp25k gene transcription was found starting from about 18 h post-infection, and protein could be detected from the same time with antiserum against FP25K. To study the function of HearNPV fp25k, a recombinant HearNPV (HaBacWD11) with an enhanced green fluorescent protein (GFP) gene replacing the fp25k was constructed using HaBacHZ8, a bacmid of HearNPV that lacks the polyhedrin gene. Growth curve analysis showed that HaBacWD11 produced higher titres of budded viruses (BVs) than its wild-type counterpart HaBacHZ8-GFP. Electron microscopic analysis indicated that at the late stage of infection, the number of intranuclear enveloped nucleocapsids in HaBacWD11-infected cells was much less than that of HaBacHZ8-GFP. A rescue recombinant virus HaBacWD14 was constructed by reintroducing fp25k gene into HaBacWD11. The growth curve and electron microscopic analysis of the rescued recombinant confirmed that the increase of BV yield and the decrease of the virion production in infected cells were the result of fp25k deletion. The expression of membrane fusion protein (Ha133) and ODV-E66 were studied using the FP25K mutants HaBacWD11 and HaBacHZ8-GFP. Unlike FP25K mutants in Autographa californica multicapsid NPV (AcMNPV), which caused an increase in the expression of membrane fusion protein GP64 and a decrease of ODV-E66, no obvious changes at the expression level of Ha133 and ODV-E66 were observed in HearNPV FP25K mutant.

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A tumor necrosis factor receptor-associated factor 2 binding protein (T2BP) gene was isolated from the grass carp (Ctenopharyngodon idellus) by utilizing suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The grass carp T2BP (GT2BP) gene contains an open reading frame of 579 nucleotide(s) (nt), encoding 193 amino acids, with 23 nt 5'-untranslated region and a long 3'-untranslated region of 434 nt including poly (A), 1 AUUUA motif and 4 AUUUUA motifs. No signal peptide has been detected in the predicted GT2BP, but a characteristic forkhead associated domain is present. The GT2BP mRNA shares 83% identity with the zebrafish DNA sequence, and they both have no introns in the genomic DNA. The putative transcription factor binding sites of GT2BP include two C/EBP alpha binding sites, and one c-Jun binding, one AP-1 binding, and one nuclear factor kappa B (NF kappa B) binding sites. Southern blot analysis revealed that the GT2BP was a single-copy gene. Individual difference was observed in GT2BP expression in examined organs of healthy grass carp. However, the expression of GT2BP in all examined organs in a fish with the highest copepod infection level and the significantly higher expression level in spleen and liver in infected fish may indicate its up-regulation with the parasite infection. (c) 2005 Elsevier B.V. All rights reserved.

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Using degenerate primers based on conserved regions of the UDP-glucose dehydrogenase (UDPGDH) gene, an initial 476-bp DNA fragment was amplified from the water-bloom forming cyanobacterium, Microcystis aeruginosa FACHB 905. TAIL-PCR and ligation-mediated PCR were used to amplify the flanking regions to isolate an about 2.5-kb genomic DNA fragment. Sequence analysis revealed an ORF encoding a putative 462 amino acid protein, designated Mud for Microcystis UDPGDH. The Mud amino acid sequence is closely related to UDPGDH sequences from cyanobacterium Synechocystis PCC6803 (73% identity, 81% similarity), and bacterium Bacillus subtilis (51% identity and 67% similarity). The cloned mud gene was expressed in Escherichia coli using the pGEX-4T-1 fusion expression vector system to generate a GST-Mud fusion protein that exhibited UDPGDH activity. The cytosolic fraction of M aeruginosa FACHB 905 was subjected to Western analysis with an anti-Mud antibody, which revealed a single band of approximately 49 kD, consistent with the deduced molecular mass of the enzyme. The Mud protein could thus be characterized as a UDP-glucose dehydrogenase, which was a key enzyme for polysaccharide synthesis and has, for the first time, been studied in algae.

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An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates. (C) 2003 Elsevier B.V. All rights reserved.

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Gloeobacter violaceus, a cyanobacterium lack of thylakoids, is refractory to genetic manipulations because its cells are enveloped by a thick gelatinous sheath and in colonial form. In this study, a large number of single cells were obtained by repeated pumping with a syringe with the gelatinous sheath removed. And an exogenous broad host range plasmid pKT210 was conjugatively transferred into G. violaceus. Analyses with dot-blot hybridization and restriction mapping showed that the exogenous plasmid pKT210 had been introduced into G. violaceus and stably maintained with no alteration in its structure. pKT210 extracted from G. violaceus exconjugants could be transformed into the mcr - mrr - E. coli strain DH10B but not the mcr(+) mrr(+) strain DH5alpha, which suggests that a methylase system may be present in G. violaceus.

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Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.

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7The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined. It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa. Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mul of mammalian reovirus. The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY). The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mul.