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PubMed2004

Identification and expression analysis of two IFN-inducible genes in crucian carp (Carassius auratus L.).

Interferon (IFN) exerts its antiviral effects mainly through activation of a subset of IFN-stimulated genes (ISG), but relatively few of fish ISGs have been isolated and characterized so far. Here, we report two fish ISGs, termed CaIFI58 and CaIFI56, cloned from a subtractive cDNA library constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. Database search revealed that both ISGs had a high-level homology with all members of a well conserved gene family with multiple tetratricopeptide repeat (TPR) motifs, including human IFI60, IFI58, IFI56, IFI54 and their homologues in some other mammalian species. The transcripts of CaIFI58 and CaIFI56 were undetectable in CAB cells but could be induced by active GCHV, UV-inactivated GCHV or CAB IFN. Analysis of expression difference between them and IFN signal factors, CaSTAT1 and CaIRF7, indicated that their transcriptions were mediated possibly through JAK-STAT signal pathway, which was further supported by the induction analysis in UV-inactivated GCHV infected, IFN-treated and untreated cells in the presence or absence of cycloheximide (CHX), a potent inhibitor of protein synthesis. In addition, a pufferfish (Fugu rubrides) DNA sequence representing putative FrIFI56 was also revealed when CaIFI58 and CaIFI56 were used to search the pufferfish genome database. Phylogenetic analysis showed that these fish ISGs form a unique clad independent of mammalian homologues, reflecting a distant evolutionary relationship from mammals. These studies identified the first teleost IFI56 and IFI58 orthologues.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Characterisation, expression and promoter analysis of an interleukin 10 homologue in the puffer fish, Fugu rubripes.

Using computer-based tools, an interleukin (IL) 10 homologue has been identified from the puffer fish ( Fugu rubripes) genome database. This is the first report on the existence of an IL-10 homologue in a non-mammalian vertebrate species. The Fugu IL-10 gene is located within a 2790-bp fragment including 549 bp of coding sequence which translates into an 183-amino-acid protein. It is predicted to contain five exons and four introns, sharing the same organization with the mammalian IL-10 genes. The size of the introns in the Fugu IL-10 gene is much smaller compared to mammalian IL-10 genes, whilst the size of the exons is similar. The deduced protein sequence shares 44-50% homology with the mammalian IL-10 sequences, 39-42% with the viral IL-10 sequences and 37-42% with other members of the IL-10 family, IL-20 and IL-22. Southern blot analysis indicates that a single copy of the IL-10 gene is present in the Fugu genome. A very low level of constitutive expression was detected in tissues of healthy fish including liver, kidney, gut and spinal cord, whilst no expression was detectable in spleen, gill, brain, gonad and eye. Analysis of the transcription regulation elements in the promoter region revealed that trans elements are located in the region between 1 bp and 721 bp, cis elements between 934 bp and 1114 bp and a tumour necrosis factor alpha responsive transcription element was located 92 bp upstream of the TATA box.

Amino Acid Sequence↗
PubMed2004

Differential hypoxia response of hsp-16 genes in the nematode.

Small heat shock proteins are induced by various stresses. We here report the differential hypoxia responses of the hsp-16 genes in the nematode. The hsp-16.1 and hsp-16.2 genes in Caenorhabditis elegans responded to hypoxia, while hsp-16.41 and hsp-16.48, which share the promoter regions with hsp-16.1 and hsp-16.2, respectively, did not. For comparative genomic analysis, we identified ten hsp-16 genes in the nematode C.briggsae from the genome database. The comparison of the promoter sequences revealed a new conserved sequence block, CAC(A/T)CT, that was required for the orientation-dependent hypoxia response, but not for other stress responses such as heat or ethanol. We propose a working model for the orientation-dependent promoter usage between two genes sharing the promoter region. We also discuss a possible application of the hypoxia-inducible promoter for conditional gene expression.

Amino Acid Sequence↗
PubMed2005

A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia.

The intestinal protozoan parasite Giardia lamblia undergoes surface antigenic variation whereby one of a family of structurally related variant-specific surface proteins (VSPs) is replaced in a regulated process by another antigenically distinct VSP. All VSPs are type I membrane proteins that have a conserved hydrophobic sequence terminated by the invariant hydrophilic amino acids, CRGKA. Using transfected Giardia constitutively expressing HA-tagged VSPH7 and incubated with radioactive [3H]palmitate, we demonstrate that the palmitate is attached to the Cys in the conserved CRGKA tail. Surface location of mutant VSPs lacking either the CRGKA tail or its Cys is identical to that of wild-type VSPH7 but non-palmitoylated mutants fail to undergo complement-independent antibody specific cytotoxicity. In addition, membrane localization of non-palmitoylated mutant VSPH7 changes from a pattern similar to rafts to non-rafts. Palmitoyl transferases (PAT), responsible for protein palmitoylation in other organisms, often possess a cysteine-rich domain containing a conserved DHHC motif (DHHC-CRD). An open reading frame corresponding to a putative 50 kDa Giardia PAT (gPAT) containing a DHHC-CRD motif was found in the Giardia genome database. Expression of epitope-tagged gPAT using a tetracycline inducible vector localized gPAT to the plasma membrane, a pattern similar to that of VSPs. Transfection with gPAT antisense producing vectors inhibits gPAT expression and palmitoylation of VSPs in vitro confirming the function of gPAT. These results show that VSPs are palmitoylated at the cysteine within the conserved tail by gPAT and indicate an essential function of palmitoylation in control of VSP-mediated signalling and processing.

Acyltransferases↗
PubMed2006

Nur, a nickel-responsive regulator of the Fur family, regulates superoxide dismutases and nickel transport in Streptomyces coelicolor.

Nickel serves as a cofactor for various microbial enzymes including superoxide dismutase (SOD) found in Streptomyces spp. In Streptomyces coelicolor, nickel represses and induces production of Fe-containing and Ni-containing SODs, respectively, primarily at the transcriptional level. We identified the nickel-responsive regulator (Nur), a Fur (ferric-uptake regulator) homologue, which binds to the promoter region of the sodF gene encoding FeSOD in the presence of nickel. Disruption of the nur gene caused constitutive expression of FeSOD and no induction of NiSOD in the presence of nickel. The intracellular level of nickel was higher in a Deltanur mutant than in the wild type, suggesting that Nur also regulates nickel uptake in S. coelicolor. A putative nickel-transporter gene cluster (nikABCDE) was identified in the genome database. Its transcription was negatively regulated by Nur in the presence of nickel. Purified Nur protein bound to the nikA promoter region in a nickel-dependent way. These results support the action of Nur as a regulator of nickel homeostasis and antioxidative response in S. coelicolor, and add a novel nickel-responsive member to the list of versatile metal-specific regulators of the Fur family.

ATP-Binding Cassette Transporters↗
PubMed2002

Characterization of the repetitive sequences in a 200-kb region around the rice waxy locus: diversity of transposable elements and presence of veiled repetitive sequences.

Repetitive genomic sequences might have various structural features and properties distinct from those of the known transposable elements (TE). Here, the content and properties of the repetitive sequences present in a 200-kb region around the rice waxy locus were analyzed using the available rice genomic database. In our previous Southern blotting analysis, 70% of the segments in this region showed smeared patterns, but according to the present database analysis, the proportion of repetitive sequences in this region was only 15%. The repetitive segments in this 200-kb region comprised 75 repetitive sequences that we classified into 46 subfamilies: 21 subfamilies were known TEs or repetitive sequences and 25 subfamilies consisted of newly identified TEs or novel types of repetitive sequences. The region contains no long terminal repeat (LTR) retrotransposable elements, but miniature inverted repeat transposable elements (MITEs) constituted a major class among the elements identified. These MITEs showed remarkable structural divergence: 12 elements were found to be new members of known MITE superfamilies, while five elements had novel terminal structures, and did not belong to any known TE families. Interestingly, about 10% of the repetitive sequences, including virus-like sequences did not have any of the usual characteristics of TEs, suggesting that a certain proportion of repetitive sequences that might not share the transpositional mechanisms of known elements are dispersed in the compact rice genome.

Base Sequence↗
PubMed2005

Identification of three isoforms for mitochondrial adenine nucleotide translocator in the pufferfish Takifugu rubripes.

Three adenine nucleotide translocator (ANT) genes were identified through in silico data mining of the Fugu genome database along with isolation of their corresponding cDNAs in vivo from the pufferfish (Takifugu rubripes). As a result of phylogenetic analysis, the ANT gene on scaffold_254 corresponded to mammalian ANT1, whereas both of those on scaffold_6 and scaffold_598 to mammalian ANT3. The ANT gene encoded by scaffold_6 was expressed ubiquitously in various tissues, whereas the ANT genes encoded by scaffold_254 and scaffold_598 were predominantly expressed in skeletal muscle and heart, respectively.

Amino Acid Sequence↗
PubMed2003

From immunome to vaccine: epitope mapping and vaccine design tools.

Since the publication of the complete genome of a pathogenic bacterium in 1995, more than 50 bacterial pathogens have been sequenced and at least 120 additional projects are currently underway. Faced with the expanding volume of information now available from genome databases, vaccinologists are turning to epitope mapping tools to screen vaccine candidates. Bioinformatics tools such as EpiMatrix and Conservatrix, which search for unique or multi-HLA-restricted (promiscuous) T cell epitopes and can find epitopes that are conserved across variant strains of the same pathogen, have accelerated the process of epitope mapping. Additional tools for screening epitopes for similarity to 'self' (BlastiMer) and for assembling putative epitopes into strings if they overlap (EpiAssembler) have been developed at EpiVax. Tools that map proteasome cleavage sires are available on the Internet. When used together, these bioinformatics tools offer a significant advantage over traditional methods of vaccine design since high throughput screening and design is performed in silico, followed by confirmatory studies in vitro. These new tools are being used to develop novel vaccines and therapeutics for the prevention and treatment of infectious diseases such as HIV, hepatitis C, tuberculosis, and some cancers. More recent applications of the tools involve deriving novel vaccine candidates directly from whole genomes, an approach that has been named 'genome to vaccine'.

Allergy and Immunology↗
PubMed2002

Structural characterization of GnRH loci in the medaka genome.

To help clarify the origin of a third gonadotropin-releasing hormone (GnRH) paralog found only in the teleost lineage, we have characterized GnRH loci in a teleost species, the medaka Oryzias latipes, and compared corresponding regions of the medaka and human genomes. Three GnRHs for medaka-type GnRH (mdGnRH), chicken-II-type GnRH (cGnRH-II), and salmon-type GnRH (sGnRH) exist as single-copy genes and reside on separate chromosomes in the medaka genome. Both medaka mdGnRH and human mGnRH are closely linked to FLJ20038 encoding a hypothetical protein, and both cGnRH-IIs in the medaka and humans are adjacent to PTP(alpha) for protein tyrosine phosphatase alpha. These conserved syntenies demonstrate that mdGnRH and cGnRH-II in teleosts are orthologous to mGnRH and cGnRH-II in tetrapods, respectively. On the other hand, the third paralogous GnRH in the medaka, sGnRH, is adjacent to PTP(epsilon), a paralog of PTP(alpha). Although humans possess PTP(epsilon) on 10q26, no sGnRH-like sequence was found in the human genome databases. Therefore a gene duplication that gave rise to the third paralogous GnRH likely occurred before the divergence of teleosts and tetrapods, and it has been lost only in the tetrapod lineage. Additionally, together with the prior observations that like GnRH, PTP(alpha)/PTP(epsilon) are strongly expressed in neural and tumor cells and that GnRH can increase PTP activity, the current data suggests that the physically linked cGnRH-II/sGnRH and PTP(alpha)/PTP(epsilon) are also functionally linked.

Amino Acid Sequence↗
PubMed2001

Function-structure analysis of proteins using covarion-based evolutionary approaches: Elongation factors.

The divergent evolution of protein sequences from genomic databases can be analyzed by the use of different mathematical models. The most common treat all sites in a protein sequence as equally variable. More sophisticated models acknowledge the fact that purifying selection generally tolerates variable amounts of amino acid replacement at different positions in a protein sequence. In their "stationary" versions, such models assume that the replacement rate at individual positions remains constant throughout evolutionary history. "Nonstationary" covarion versions, however, allow the replacement rate at a position to vary in different branches of the evolutionary tree. Recently, statistical methods have been developed that highlight this type of variation in replacement rates. Here, we show how positions that have variable rates of divergence in different regions of a tree ("covarion behavior"), coupled with analyses of experimental three-dimensional structures, can provide experimentally testable hypotheses that relate individual amino acid residues to specific functional differences in those branches. We illustrate this in the elongation factor family of proteins as a paradigm for applications of this type of analysis in functional genomics generally.

Amino Acid Sequence↗
PubMed2006

Development of a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (Macaca fascicularis).

To develop a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (Macaca fascicularis), 148 microsatellite markers were selected from the human genome database. The polymorphisms and inheritance of PCR products were determined by screening twenty unrelated monkeys and by analysis of three families, respectively. As a result, 106 primers (72%) gave PCR products of the size expected for humans and rhesus monkeys. Among these products, polymorphism and single-gene inheritance in cynomolgus monkeys was observed for 66 markers (62%). The average number of alleles at the 66 polymorphic loci was 5.86 (range 2-10), and average heterozygosity was 0.63 (range 0.10-0.88). This is the first report of microsatellite markers for cynomolgus monkeys. Chromosomal mapping of these markers is now in progress.

Animals↗
PubMed2004

Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization.

BACKGROUND: Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH) was used to identify specific chromosomal sequences unique to B. anthracis. RESULTS: Two SSH libraries were generated. Genomic DNA from plasmid-cured B. anthracis was used as the tester DNA in both libraries, while genomic DNA from either B. cereus or B. thuringiensis served as the driver DNA. Progressive screening of the libraries by colony filter and Southern blot analyses identified 29 different clones that were specific for the B. anthracis chromosome relative not only to the respective driver DNAs, but also to seven other different strains of B. cereus and B. thuringiensis included in the process. The nucleotide sequences of the clones were compared with those found in genomic databases, revealing that over half of the clones were located into 2 regions on the B. anthracis chromosome. CONCLUSIONS: Genes encoding potential cell wall synthesis proteins dominated one region, while bacteriophage-related sequences dominated the other region. The latter supports the hypothesis that acquisition of these bacteriophage sequences occurred during or after speciation of B. anthracis relative to B. cereus and B. thuringiensis. This study provides insight into the chromosomal differences between B. anthracis and its closest phylogenetic relatives.

Bacillus anthracis↗
PubMed1999

Identification of three additional femAB-like open reading frames in Staphylococcus aureus.

Three new proteins, FmhA, FmhB and FmhC, with significant identities to FemA and FemB were identified in the Staphylococcus aureus (ATCC 55748) genome database. They were mapped to the SmaI-C, SmaI-H and SmaI-A fragments of the S. aureus 8325 chromosome, respectively. Whereas insertional inactivation of fmhA and fmhC had no effects on growth, antibiotic susceptibility, lysostaphin resistance, or peptidoglycan composition of the strains, fmhB could not be inactivated, strongly suggesting that fmhB may be an essential gene. As deduced from the functions of FemA and FemB which are involved in the synthesis of the peptidoglycan pentaglycine interpeptide, FmhB may be a candidate for the postulated FemX thought to add the first glycine to the nascent interpeptide.

Bacterial Proteins↗
PubMed2000

Hunting for disease genes in multi-functional diseases.

Disease genes may be identified through functional, positional, and candidate gene approaches. Although extensive and often labor-intensive studies such as family linkage analysis, functional investigation of gene products and genome database searches are usually involved, thousands of human disease genes, especially for monogenic diseases with Mendelian transmission, have been identified. However, in diseases caused by more than one gene, or by a combination of genetic and environmental factors, identification of the genes is even more difficult. Common examples include atherosclerosis, cancer, Alzheimer's disease, asthma, diabetes, glaucoma, and age-related macular degeneration. There have been conflicting reports on the roles of associated genes. Even with population-based case-control studies and new statistical methods such as the sib-ship disequilibrium test and the discordant alleles test, there is no agreement on whether alpha2-macroglobulin (A2M) is a gene for Alzheimer's disease. Another example is the inconsistent association between age-related macular degeneration and ATP-binding cassette transporter (ABCR). Ethnic variation causes further complications. In our investigation of LDL-receptor variants in familial hypercholesterolemia, and the trabecular meshwork inducible glucocorticoid response protein, or myocillin (TIGR-MYOC) mutation pattern in primary open angle glaucoma, we did find dissimilar results in Chinese compared to Caucasians. New information from the Human Genome Project and advancements in technologies will aid the search for and confirm identification of disease genes despite such challenges.

ATP-Binding Cassette Transporters↗
PubMed2000

Genomic organization of alpha1 and beta1 subunits of the mammalian soluble guanylyl cyclase genes.

The structures of the genes encoding the alpha(1) and beta(1) subunits of murine soluble guanylyl cyclase (sGC) were determined. Full-length cDNAs isolated from mouse lungs encoding the alpha(1) (2.5 kb) and beta(1) (3.3 kb) subunits are presented in this report. The alpha(1) sGC gene is approximately 26.4 kb and contains nine exons, whereas the beta(1) sGC gene spans 22 kb and consists of 14 exons. The positions of exon/intron boundaries and the sizes of introns for both genes are described. Comparison of mouse genomic organization with the Human Genome Database predicted the exon/intron boundaries of the human genes and revealed that human and mouse alpha1 and beta1 sGC genes have similar structures. Both mouse genes are localized on the third chromosome, band 3E3-F1, and are separated by a fragment that is 2% of the chromosomal length. The 5' untranscribed regions of alpha(1) and beta(1) subunit genes were subcloned into luciferase reporter constructs, and the functional analysis of promoter activity was performed in murine neuroblastoma N1E-115 cells. Our results indicate that the 5' untranscribed regions for both genes possess independent promoter activities and, together with the data on chromosomal localization, suggest independent regulation of both genes.

Animals↗
PubMed2005

New types of mouse centromeric satellite DNAs.

Genomic databases do not contain complete sequences of the centromeric regions. We created a pUC19-based library of DNA fragments from isolated chromocentres of interphase nuclei. In this library we have found major satellite (MaSat) and two new satellite sequences - MS3 and MS4. The computer analysis of MS3 and MS4 sequences by alignment, fragment curved state and search for MAR motifs in comparison with the mouse major and minor satellite (MiSat) DNA has shown them to be new satellite fragments. Southern blot of MS3 and MS4 with total DNA digested by restriction enzymes shows the ladder characteristic of satellite DNA. 2.2% of the total DNA consists of MS3, the monomer of which is 150 bp long. The MS4 monomer is 300 bp long and accounts for 1.6% of the total DNA. On metaphase chromosomes MS3 and MS4 are located at the centromeric region. FISH analysis of L929 nuclei during the cell cycle showed relative positions of MaSat, MiSat, MS3, and MS4. All mapped satDNA fragments except MaSat belong to the outer layer of the chromocentres in the G0/G1 phase. MS3 is likely to be involved in the centromere formation. The mouse genome contains at least four satDNA types: AT-rich (MaSat and MiSat), and CG-rich (MS3 and MS4).

Animals↗
PubMed2002

Identification and characterization of a novel human DNA glycosylase for repair of cytosine-derived lesions.

Two candidate human orthologs of Escherichia coli MutM/Nei were recently identified in the human genome database, and one of these, NEH1, was characterized earlier (Hazra, T. K., Izumi, T., Boldogh, I., Imhoff, B., Kow, Y. W., Jaruga, P., and Dizdaroglu, M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3523-3528). Here we report characterization of the second protein, originally named NEH2 and now renamed NEIL2 (Nei-like). The 37-kDa wild-type NEIL2 expressed in and purified from E. coli has DNA glycosylase/AP lyase activity, primarily for excising oxidative products of cytosine, with highest activity for 5-hydroxyuracil, one of the most abundant and mutagenic lesions induced by reactive oxygen species, and with lower activity for 5,6-dihydrouracil and 5-hydroxycytosine. It has negligible or undetectable activity with 8-oxoguanine, thymine glycol, 2-hydroxyadenine, hypoxanthine, and xanthine. NEIL2 is similar to NEIL1 in having N-terminal Pro as the active site. However, unlike NEIL1, its expression was independent of the cell cycle stage in fibroblasts, and its highest expression was observed in the testes and skeletal muscle. Despite the absence of a putative nuclear localization signal, NEIL2 was predominantly localized in the nucleus. These results suggest that NEIL2 is involved in global genome repair mainly for removing oxidative products of cytosine.

Cell Cycle↗
PubMed2000

Listeriolysin O as a reporter to identify constitutive and in vivo-inducible promoters in the pathogen Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. Bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin O (LLO) encoded by hly. LLO-negative mutants of L. monocytogenes are avirulent in the mouse model. We have developed a genetic system with hly as a reporter gene allowing the identification of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting L. monocytogenes chromosomal fragments upstream of the promoterless hly gene cloned into gram-positive and gram-negative shuttle vectors and expressed in an LLO-negative mutant strain. With this hly-based promoter trap system, combined with access to the L. monocytogenes genome database, we identified 20 in vitro-transcribed genes, including genes encoding (i) p60, a previously known virulence gene, (ii) a putative new hemolysin, and (iii) two proteins of the general protein secretion pathway. By using the hly-based system as an in vivo expression technology tool, nine in vivo-induced loci of L. monocytogenes were identified, including genes encoding (i) the previously known in vivo-inducible phosphatidylinositol phospholipase C and (ii) a putative N-acetylglucosamine epimerase, possibly involved in teichoic acid biosynthesis. The use of hly as a reporter is a simple and powerful alternative to classical methods for transcriptional analysis to monitor promoter activity in L. monocytogenes.

Amino Acid Sequence↗