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Open Reading Frame (open + reading_frame)
Kinds of Open Reading Frame Terms modified by Open Reading Frame Selected AbstractsSEQUENCE ANALYSIS OF A NOVEL INSECT PHOSPHOGLYCERATE MUTASE GENE FROM THE CHINESE HONEYBEE, APIS CERANA,INSECT SCIENCE, Issue 4 2003Jiang-hong Li Abstract A clone inserted with 1 104 bp fragment containing a 765bp Open Reading Frame(ORF), encoding a putative 2,3-bisphosphoglycerate(2,3BPG) dependent Phosphoglycerate mutase(dPGAM) that catalyzes the transfer of a phosphate group from the C3 carbon atom to the C2 carbon atom of phosphoglycerate, was screened by mass sequencing from the cDNA library of the venom glands of Apis cerana. The deduced amino acid sequence shared high similarities (39% - 88%)with the dPGAM of 7 other organisms, but the similarities with the iPGAM of 4 other organisms were low (10% - 12%). Moreover, the alignment of Ac-PGAM with the dPGAMs from 7 other organisms showed that all the active site amino acid residues were conserved. This result shows that Ac-PGAM is a typical dPGAM. Thus, this is the second PGAM gene reported in Insecta. Furthermore, phylogenetic analysis showed that the evolutionary tree of PGAMs reflects the systematic relationship of species. [source] ABC-type amino acid uptake transporters Bgt and N-II of Anabaena sp. strain PCC 7120 share an ATPase subunit and are expressed in vegetative cells and heterocystsMOLECULAR MICROBIOLOGY, Issue 5 2008Rafael Pernil Summary Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N2 in differentiated cells called heterocysts. Anabaena open reading frames alr4167 and alr3187 encode, respectively, an ATPase subunit, BgtA, and a composite protein bearing periplasmic substrate-binding and transmembrane domains, BgtB, of an ABC-type high-affinity basic amino acid uptake transporter (Bgt). Open reading frame alr4167 is clustered with open reading frames alr4164, alr4165 and alr4166 that encode a periplasmic substrate-binding protein, NatF, and transmembrane proteins NatG and NatH respectively. The NatF, NatG, NatH and BgtA proteins constitute an ABC-type uptake transporter for acidic and neutral polar amino acids (N-II). The Bgt and N-II transport systems thus share the ATPase subunit, BgtA. These transporters together with the previously characterized ABC-type uptake transporter for proline and hydrophobic amino acids (N-I) account for more than 98% of the amino acid transport activity exhibited by Anabaena sp. strain PCC 7120. In contrast to N-I that is expressed only in vegetative cells, the Bgt and N-II systems are present in both vegetative cells and heterocysts. Whereas Bgt is dispensable for diazotrophic growth, N-II appears to contribute together with N-I to the diazotrophic physiology of this cyanobacterium. [source] Cloning and characterization of cDNA for syndecan core protein in sea urchin embryosDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2000Kazuo Tomita The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription,polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae. [source] Purification and cDNA Cloning of Lysozyme II from Cabbage Butterfly, Artogeia rapae LarvaeENTOMOLOGICAL RESEARCH, Issue 4 2005BANG In Seok ABSTRACT Last instar larvae of cabbage butterfly Artogeia rapae respond to injection of bacteria with a set of inducible antibacterial peptides/proteins. The inducible peptides/proteins are related to the known hinnavins (I and II) and lysozymes (I and II). The lysozyme II has been isolated by heat treatment, cation exchange, and reversed-phase chromatography from immunized hemolymph of last instar larvae. The lysozyme II gene of A. rapae was isolated and its nucleotide sequence was determined by the RACE-PCR from immunized fat body with E. coli. It has an open reading frame of 414 bp nucleotide corresponding to 138 amino acids including an 18 amino acid signal sequence. The molecular weight and the isoelectric point of Artogeia lysozyme II without a signal peptide were 13,649.38 Da and 9.11, respectively. It is great similarity with Manduca lysozyme among other lepidopteran. [source] Design and testing of ,genome-proxy' microarrays to profile marine microbial communitiesENVIRONMENTAL MICROBIOLOGY, Issue 2 2008Virginia I. Rich Summary Microarrays are useful tools for detecting and quantifying specific functional and phylogenetic genes in natural microbial communities. In order to track uncultivated microbial genotypes and their close relatives in an environmental context, we designed and implemented a ,genome-proxy' microarray that targets microbial genome fragments recovered directly from the environment. Fragments consisted of sequenced clones from large-insert genomic libraries from microbial communities in Monterey Bay, the Hawaii Ocean Time-series station ALOHA, and Antarctic coastal waters. In a prototype array, we designed probe sets to 13 of the sequenced genome fragments and to genomic regions of the cultivated cyanobacterium Prochlorococcus MED4. Each probe set consisted of multiple 70-mers, each targeting an individual open reading frame, and distributed along each ,40,160 kbp contiguous genomic region. The targeted organisms or clones, and close relatives, were hybridized to the array both as pure DNA mixtures and as additions of cells to a background of coastal seawater. This prototype array correctly identified the presence or absence of the target organisms and their relatives in laboratory mixes, with negligible cross-hybridization to organisms having , ,75% genomic identity. In addition, the array correctly identified target cells added to a background of environmental DNA, with a limit of detection of ,0.1% of the community, corresponding to ,103 cells ml,1 in these samples. Signal correlated to cell concentration with an R2 of 1.0 across six orders of magnitude. In addition, the array could track a related strain (at 86% genomic identity to that targeted) with a linearity of R2 = 0.9999 and a limit of detection of ,1% of the community. Closely related genotypes were distinguishable by differing hybridization patterns across each probe set. This array's multiple-probe, ,genome-proxy' approach and consequent ability to track both target genotypes and their close relatives is important for the array's environmental application given the recent discoveries of considerable intrapopulation diversity within marine microbial communities. [source] Isolation and characterization of Tn -Dha1, a transposon containing the tetrachloroethene reductive dehalogenase of Desulfitobacterium hafniense strain TCE1ENVIRONMENTAL MICROBIOLOGY, Issue 1 2005Julien Maillard Summary A new 9.9 kb catabolic transposon, Tn -Dha1, containing the gene responsible for tetrachloroethene (PCE) reductive dechlorination activity, was isolated from Desulfitobacterium hafniense strain TCE1. Two fully identical copies of the insertion sequence ISDha1, a new member of the IS256 family, surround the gene cluster pceABCT, a truncated gene for another transposase and a short open reading frame with homology to a member of the twin-arginine transport system (tatA). Evidence was obtained by Southern blot for an alternative form of the transposon element as a circular molecule containing only one copy of ISDha1. This latter structure most probably represents a dead-end product of the transposition of Tn -Dha1. Strong indications for the transposition activity of ISDha1 were given by polymerase chain reaction (PCR) amplification and sequencing of the intervening sequence located between both inverted repeats (IR) of ISDha1 (IR junction). A stable genomic ISDha1 tandem was excluded by quantitative real-time PCR. Promoter mapping of the pceA gene, encoding the reductive dehalogenase, revealed the presence of a strong promoter partially encoded in the right inverted repeat of ISDha1. A sequence comparison with pce gene clusters from Desulfitobacterium sp. strains PCE-S and Y51 and from Dehalobacter restrictus, all of which show 100% identity for the pceAB genes, indicated that both Desulfitobacterium strains seem to possess the same transposon structure, whereas only the pceABCT gene cluster is conserved in D. restrictus. [source] Molecular cloning of CYP1A gene and its expression by benzo(a)pyrene from goldfish (Carassius auratus)ENVIRONMENTAL TOXICOLOGY, Issue 3 2009Seung-Min Oh Abstract We cloned and sequenced the cytochrome P450 1A (CYP1A) gene from goldfish (Carassius auratus). It has a 1581 bp open reading frame that encodes a 526 amino acid protein with a theoretical molecular weight of 59.02 kDa. The CYP1A amino acid sequence clusters in a monophyletic group with other fish CYP1As, and more closely related to zebrafish CYP1A (91% identity) than to other fish CYP1As. Exposure to benzo(a)pyrene (BaP) by intraperitoneal injection increased biliary BaP metabolites and liver CYP1A gene expression. BaP exposure also increased CYP1A gene expression in extrahepatic organs, including intestine, and gill, which are sensitive to aqueous and dietary exposure to Arylhydrocarbon receptor (AhR) agonists. Therefore, goldfish CYP1A identified in this study offers basic information for further research related to biomarker use of CYP1A of goldfish. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009. [source] Molecular cloning of cytochrome P4501A cDNA of medaka (Oryzias latipes) and messenger ribonucleic acid regulation by environmental pollutantsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2004Jisung Ryu Abstract The sequence of cytochrome P4501A (CYP1A) cDNA of medaka (Oryzias latipes) was determined, and its messenger ribonucleic acid (mRNA) regulation by ,-naphthoflavone (,NF) was evaluated. The determined cDNA sequence contained 2,349 base pairs (bp), and the open reading frame contained a total of 1,563 bp encoding 521 predicted amino acids. The induction of CYP1A mRNA in medaka was evaluated using reverse transcription,polymerase chain reaction. The concentration,dependent induction of CYP1A mRNA in the liver was observed after exposure to ,NF at nominal concentrations of 20, 100, and 500 ,g/ L for 2 d. Time-dependent changes of CYP1A mRNA levels were also observed in the liver, gill, gut, and caudal fin tissues of medaka exposed to 100 ,g/L of ,NF for 7 d. Our results showed that the degree of CYP1A mRNA induction in the gill, gut, and caudal fin after exposure to ,NF was relatively higher than that in the liver, possibly because of low basal levels of CYP1A mRNA in the gill, gut, and caudal fin of nonexposed fish. The induction of medaka CYP1A mRNA was also observed after exposure to an environmental sample, landfill leachate. The CYP1A mRNA inductions in the gill, gut, and caudal fin were also higher than that in the liver as shown in the ,NF-treated groups. These results show that CYP1A mRNA determination in the gill, gut, and caudal fin, which are in direct contact with the polluted water, may become a useful method for monitoring CYP1A-inducible chemicals. [source] Characterization of porcine dentin sialoprotein (DSP) and dentin sialophosphoprotein (DSPP) cDNA clonesEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 1 2003Yasuo Yamakoshi Dentin sialophosphoprotein (DSPP) is a chimeric glycoprotein with dentin sialoprotein (DSP) on its N -terminus and dentin phosphoprotein (DPP) on its C -terminus. We have constructed and screened a unidirectional cDNA library derived from the pulp organ of developing pig teeth, and isolated cDNA clones encoding DSP-only, as well as two DSPP clones with alternative sequences in their 3, coding regions. The DSP-only transcript has an open reading frame of 386 codons, and is generated through the use of a polyadenylation signal within intron 4, immediately following the DSP coding region. the use of this polyadenylation signal deletes the DPP coding region and places a TGA translation termination signal as the fourth codon following the exon 4-encoded segment. The DSPP cDNAs contain open reading frames of 593 and 600 codons. Northern blots hybridized to radiolabeled DSP probes showed bands at 1.4, 2.5, 4.4, and 4.8 kb. Cloning and characterization of reverse transcriptase polymerase chain reaction products confirmed the existence of mRNA encoding pDSP386, pDSPP593, and pDSPP600in vivo, but also suggested that DNA sequence redundancies in the DSPP coding region make it prone to cloning artifacts. [source] Novel member of the mouse desmoglein gene family: Dsg1-,EXPERIMENTAL DERMATOLOGY, Issue 1 2003L. Pulkkinen Abstract: Desmosomes are major intercellular adhesion junctions that provide stable cell,cell contacts and mechanical strength to epithelial tissues by anchoring cytokeratin intermediate filaments of adjacent cells. Desmogleins (Dsg) are transmembrane core components of the desmosomes, and belong to the cadherin supergene family of calcium-dependent adhesion molecules. Currently, there are three known isoforms of Dsgs (Dsg1, Dsg2, and Dsg3), encoded by distinct genes that are differentially expressed to determine their tissue specificity and differentiation state of epithelial cells. In this study, we cloned a novel mouse desmoglein gene sharing high homology to both mouse and human Dsg1. We propose to designate the previously published mouse Dsg1 gene as Dsg1- , and the new gene as Dsg1-,. Analysis of intron/exon organization of the Dsg1-, and Dsg1-, genes revealed significant conservation. The full-length mouse Dsg1-, cDNA contains an open reading frame of 3180 bp encoding a precursor protein of 1060 amino acids. Dsg1-, protein shares 94% and 76% identity with mouse Dsg1-, and human DSG1, respectively. RT-PCR using a multitissue cDNA panel demonstrated that while Dsg1-, mRNA was expressed in 15- to 17-day-old embryos and adult spleen and testis, Dsg1-, mRNA was detected in 17-day-old embryos only. To assess subcellular localization, a FLAG-tagged expression construct of Dsg1-, was transiently expressed in epithelial HaCaT cells. Dsg1-,-FLAG was found at the cell,cell border and was recognized by the anti-Dsg1/Dsg2 antibody DG3.10. In summary, we have cloned and characterized a novel member of the mouse desmoglein gene family, Dsg1-,. [source] A simple in vivo assay for measuring the efficiency of gene length-dependent processes in yeast mRNA biogenesisFEBS JOURNAL, Issue 4 2006Macarena Morillo-Huesca We have developed a simple reporter assay useful for detection and analysis of mutations and agents influencing mRNA biogenesis in a gene length-dependent manner. We have shown that two transcription units sharing the same promoter, terminator and open reading frame, but differing in the length of their 3,-untranslated regions, are differentially influenced by mutations affecting factors that play a role in transcription elongation or RNA processing all along the transcription units. In contrast, those mutations impairing the initial steps of transcription, but not affecting later steps of mRNA biogenesis, influence equally the expression of the reporters, independently of the length of their 3,-untranslated regions. The ratio between the product levels of the two transcription units is an optimal parameter with which to estimate the efficiency of gene length-dependent processes in mRNA biogenesis. The presence of a phosphatase-encoding open reading frame in the two transcription units makes it very easy to calculate this ratio in any mutant or physiological condition. Interestingly, using this assay, we have shown that mutations in components of the SAGA complex affect the level of mRNA in a transcript length-dependent fashion, suggesting a role for SAGA in transcription elongation. The use of this assay allows the identification and/or characterization of new mutants and drugs affecting transcription elongation and other related processes. [source] Cloning and expression of murine enzymes involved in the salvage pathway of GDP- l -fucoseFEBS JOURNAL, Issue 1 2004GDP- l -fucose pyrophosphorylase, l -fucokinase In the salvage pathway of GDP- l -fucose, free cytosolic fucose is phosphorylated by l -fucokinase to form l -fucose-1-phosphate, which is then further converted to GDP- l -fucose in the reaction catalyzed by GDP- l -fucose pyrophosphorylase. We report here the cloning and expression of murine l -fucokinase and GDP- l -fucose pyrophosphorylase. Murine l -fucokinase is expressed as two transcripts of 3057 and 3270 base pairs, encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant of l -fucokinase was enzymatically active when expressed in COS-7 cells. Murine GDP- l -fucose pyrophosphorylase has an open reading frame of 1773 base pairs encoding a protein of 591 amino acids with a predicted molecular mass of 65.5 kDa. GDP- l -fucose, the reaction product of GDP- l -pyrophosphorylase, was identified by HPLC and MALDI-TOF MS analysis. The tissue distribution of murine l -fucokinase and GDP- l -fucose pyrophosphorylase was investigated by quantitative real time PCR, which revealed high expression of l -fucokinase and GDP- l -fucose pyrophosphorylase in various tissues. The wide expression of both enzymes can also be observed from the large amount of data collected from a number of expressed sequence tag libraries, which indicate that not only the de novo pathway alone, but also the salvage pathway, could have a significant role in the synthesis of GDP- l -fucose in the cytosol. [source] The interferon alpha induced protein ISG12 is localized to the nuclear membraneFEBS JOURNAL, Issue 22 2001Pia M. Martensen Interferons exert their biological function mainly through the activation of interferon-stimulated genes (ISGs). ISG12 (originally designated p27) belongs to a family of small, interferon , inducible genes of unknown function. We have determined the 5, end sequence of ISG12 cDNA from the human cell lines HeLa and AMA by RACE. Comparing this sequence to ISG12 sequences in the expressed sequence tag (EST) database revealed the presence of two alternative splice variants of ISG12 in human cells exhibiting the same open reading frame. We have sequenced the promoter region of the ISG12 gene and found ISRE, IRF1/IRF2, and STAT elements correlating to the interferon , inducibility of the gene. Subsequently, we have expressed human ISG12, a 12-kDa hydrophobic protein in the baculovirus expression system and with a C-terminal FLAG-tag in the human cell line 293. Recombinant ISG12 sediments in the nuclear envelope in both cell types. Finally, we have been able to demonstrate the prevalence of the ISG12 gene product in the nuclear envelope of HeLa cells treated with interferon , by immunocytochemical analyses. ISG12 is the first interferon induced protein found localizing to the nuclear envelope. [source] Expression of the gene and processed pseudogenes encoding the human and rabbit translationally controlled tumour protein (TCTP)FEBS JOURNAL, Issue 17 2000Holger Thiele In humans and rabbits, the TPT1 gene encoding the translationally controlled tumour protein TCTP generates two mRNAs (TCTP mRNA1 and TCTP mRNA2) which differ in the length of their 3, untranslated regions. The distribution of these mRNAs was investigated in 10 rabbit and 50 human tissues. They were transcribed in all tissues investigated, but differed considerably in their quantity and ratio of expression. This indicates an extensive transcriptional control and involvement of tissue-specific factors. In the rabbit genome numerous processed, intronless pseudogenes were detected. Four, corresponding to both types of mRNAs, were sequenced and analysed in detail; all displayed only few mutations and were either preserved completely in the original amino acid sequence of the intron containing gene, or contained only minor mutations in the coding region which did not interrupt the open reading frame. In the mRNA population of rabbit reticulocytes two additional TCTP RNAs of the TCTP mRNA2 type were detected, which have the characteristics of pseudogene transcripts. Pseudogene transcription was supported further by CAT reporter gene assays showing substantial promoter activity of 5,-flanking regions of two TPT1 pseudogenes. [source] Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomalleiFEMS MICROBIOLOGY LETTERS, Issue 1 2000May-Ann Lee Abstract Burkholderia pseudomallei, a Gram-negative bacterium is found in the soil and water, mainly in Southeast Asia and Northern Australia. It is responsible for melioidosis in human and animals. The bacteria produce several potential virulent factors such as extracellular protease, hemolysin, lipase and lecithinase. The isolation of virulence genes and the study of their functions will contribute to our understanding of bacterial pathogenesis. Previous studies have implicated protease as a contributing virulence factor in the pathogenesis of some bacteria. Three out of 5000 clones screened from a genomic DNA library of B. pseudomallei were found to express protease activity. The clones were found to have the same sequence. The nucleotide sequence revealed an open reading frame (designated as metalloprotease A, mprA) encoding a 500-amino acid protein, MprA, with an estimated molecular mass of 50,241 Da. The predicted amino acid sequence shares homology with the subtilisin family of serine proteases. [source] The iron dependent regulatory protein IdeR (DtxR) of Rhodococcus equiFEMS MICROBIOLOGY LETTERS, Issue 1 2000Clara A. Boland Abstract This paper reports the presence of an ideR gene, which encodes an iron-dependent regulatory protein, in Rhodococcus erythropolis and in the intracellular pathogen Rhodococcus equi. The ideR gene of the latter encoded a protein of 230 amino acids with a molecular mass of 25,619. The ,-helices forming the helix-turn-helix motif of the R. equi protein were identical to those of the DtxR protein of Corynebacterium diphtheriae, which is an IdeR homologue. This indicates that the two proteins bind to the same DNA binding site. This was confirmed following expression of IdeR in Escherichia coli, which showed that the IdeR protein could repress transcription of the tox promoter of C. diphtheriae in an iron dependent manner. An open reading frame specifying a 283-amino acid polypeptide similar to galE encoding UDP-galactose 4-epimerase was present downstream of the ideR gene. [source] Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxiiFEMS YEAST RESEARCH, Issue 4 2003Hawk-Bin Kwon Abstract In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively. [source] A single nucleotide polymorphism at the splice donor site of the human MYH base excision repair gene results in reduced translation efficiency of its transcriptsGENES TO CELLS, Issue 5 2002Satoru Yamaguchi Background: Adenine paired with 8-hydroxyguanine, a major oxidatively damaged DNA lesion, is excised by mutY homologue (MYH) base excision repair protein in human cells. Since genetic polymorphisms of DNA repair genes associated with the activities and the expression levels of their products may modulate cancer susceptibility of individuals, we investigated the effect of a single nucleotide polymorphism (SNP) in the MYH gene on the difference in the expression levels of its products. Results: An aberrant size of the , type nuclear form transcript was detected in a lung cancer cell line, VMRC-LCD, by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The transcript contained the intron 1 sequence, and it was due to alternative splicing resulting from IVS1+5G/C SNP. The presence of the upstream open reading frame (ORF) on the 5,-side of the native ORF in the , type transcript from the IVS1+5C allele could reduce the translation efficiency of the transcript into the nuclear form protein. Thus, expression vectors bearing the 5,-untranslated region sequence of either the IVS1+5G or 5C allele were constructed. In vitro translation analysis, as well as Western blot and quantitative RT-PCR analyses of the H1299 lung cancer cell line transfected with these vectors, revealed that the translation efficiency of the IVS1+5C transcript into MYH protein was much lower (, 30%) than that of the IVS1+5G transcript. Conclusions: The SNP at the splice donor site of the MYH gene resulted in reduced translation efficiency of its transcripts. This is the fourth case of single nucleotide variations that cause alterations in translation initiation sites and translation efficiencies in human cells. [source] Relevance of translocation type in myxoid liposarcoma and identification of a novel EWSR1-DDIT3 fusionGENES, CHROMOSOMES AND CANCER, Issue 11 2007B. Bode-Lesniewska The clinical course of myxoid/round cell liposarcoma (MRCL) is characterized by frequent local recurrences and metastases at unusual sites. MRCLs carry specific translocations, t(12;16) or rarely t(12;22), linking the FUS or the EWSR1 gene with the DDIT3 gene, respectively. Nine FUS/DDIT3 and three EWSR1/DDIT3 variants of fusion transcripts have been described thus far. In search of prognostic markers for MRCL, we analyzed the translocation types of 31 patients and related them to the event free and overall survival. Using break-apart FISH and RT-PCR combined with DNA sequencing, we detected FUS/DDIT3 fusions in 30 sarcomas, while an EWSR1/DDIT3 translocation was identified in one tumor. FUS/DDIT3 type II (exons 5-2) was most commonly detected (20 cases), followed by type I (7-2) (7 cases) and type III (8-2) (3 cases). A single tumor carrying a t(12;22) translocation expressed a hitherto unknown EWSR1-DDIT3 fusion transcript (13-3) linking the complete RNA-binding domain of EWSR1 with a short piece of the 5,-UTR and the entire open reading frame of the DDIT3 gene. Interestingly, five of six patients with type I (7-2) FUS/DDIT3 fusions displayed local recurrences and/or metastatic spread within the first 3 years, generally requiring chemotherapeutical treatment (median disease-free survival 17 months). In contrast, 9 of 13 patients with type II FUS/DDIT3 translocations remained at 3 years disease-free (median disease-free survival 75 months). Since the total number of patients is still limited, further studies are required to verify a putative association of type I FUS/DDIT3 -fusion transcripts with a prognosis of MRCL. © 2007 Wiley-Liss, Inc. [source] Identification of novel alternatively spliced BRCA1-associated RING domain (BARD1) messenger RNAs in human peripheral blood lymphocytes and in sporadic breast cancer tissuesGENES, CHROMOSOMES AND CANCER, Issue 9 2007Grazia Lombardi BARD1 (BRCA1-associated RING domain) is the dominant binding partner of BRCA1 in vivo. The BARD1 gene has been reported to be mutated in a subset of breast and ovarian cancer patients and BARD1 germ-line mutations have been identified in breast cancer patients negative for BRCA1 or BRCA2 gene alterations. In the present study, we show by RT-PCR and direct sequencing analysis the occurrence of seven novel and one previously identified BARD1 splicing variants in human lymphocytes and breast cancers. Two of the eight variants (BARD1, and BARD1 ,RIN) preserve a correct open reading frame and could encode BARD1 internally deleted proteins, while the remaining six variants display premature stop codons. Characterization of the relative expression of BARD1 FL, BARD1,, and BARD1 ,RIN using quantitative PCR analysis indicated that the mean expression levels of BARD1 FL, BARD1,, and BARD1 ,RIN were significantly higher in tumors than in morphologically normal tissues and lymphocytes. However, we were unable to identify either qualitatively or quantitatively tumor-specific expression patterns of the identified BARD1 splicing variants. © 2007 Wiley-Liss, Inc. [source] Cloning and sequence analysis of a zebrafish cDNA encoding DNA (cytosine-5)-methyltransferase-1GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 4 2001Aizeddin A. Mhanni Abstract Summary: The zebrafish has become a well-established animal model for the analysis of development and of several disease phenotypes. Several of the favorable traits that make it a popular model organism would also be beneficial for the study of normal and abnormal vertebrate development in which DNA methylation may play a role. We report the determination of the full-length cDNA sequence corresponding to the zebrafish DNA (cytosine-5-) methyltransferase gene, Dnmt1. It is 4,907 bases long and has an open reading frame predicted to encode a 1,499 amino acid protein that is similar in size and sequence to a number of other methyltransferases identified in other organisms. genesis 30:213,219, 2001. © 2001 Wiley-Liss, Inc. [source] Stage-specific gene expression in early differentiating oligodendrocytesGLIA, Issue 2 2002Francesca Blasi Abstract The screening of a differential library from precursor and differentiated oligodendrocytes, obtained through the representational difference analysis (RDA) technique, has generated a number of cDNA recombinants corresponding to mRNA coding for known and unknown proteins: (1) mRNA coding for proteins involved in protein synthesis, (2) mRNA coding for proteins involved in the organization of the cytoskeleton, and (3) mRNA coding for proteins of unknown function. The expression profile of the mRNA was studied by Northern blot hybridization to the poly-A+ mRNA from primary rat progenitor and differentiated oligodendrocytes. In most cases, hybridization to the precursor was higher than hybridization to the differentiated mRNA, supporting the validity of the differential screening. Hybridization of the cDNA to rat cerebral hemisphere and brain stem poly-A+ mRNA, isolated from 1- to 90-day-old rats, confirms the results obtained with the mRNA from differentiating oligodendrocytes. The intensity of the hybridization bands decreases as differentiation proceeds. The pattern of expression observed in oligodendrocytes is different from that found in the brain only in the case of the nexin-1 mRNA, the level of which remains essentially constant throughout differentiation both in the brain stem and in the cerebral hemispheres, in agreement with the published data. In contrast, the intensity of hybridization to the oligodendrocyte mRNA is dramatically lower in the differentiated cells compared with the progenitor oligodendrocyte cells. Some of the recombinant cDNA represent mRNA sequences present at high frequency distribution in the cells, while others belong to the rare sequences group. Six recombinants code for proteins of the ribosomal family, suggesting that of approximately 70 known ribosomal proteins, only a few are upregulated during oligodendrocyte differentiation. The third category of open reading frame (ORF) is represented by rare messengers coding for proteins of unknown functions and includes six clones: RDA 279, 11, 95, 96, 254, and 288. GLIA 39:114,123, 2002. © 2002 Wiley-Liss, Inc. [source] Zoonotic risk of hepatitis E virus (HEV): A study of HEV infection in animals and humans in suburbs of BeijingHEPATOLOGY RESEARCH, Issue 12 2009Yibin Chang Aim:, To investigate hepatitis E virus (HEV) infection among different animals and workers in pig farms and slaughterhouses, and analyze the genotype of HEV isolated in this study. Methods:, Serum samples were collected from adult swine, cows, sheep, younger swine (< 3 months), and workers in pig farms and slaughterhouses (professional group). Fecal samples were collected from younger swine in the south suburbs of Beijing. Anti-HEV antibody was evaluated by direct sandwich enzyme immunoassay. HEV RNA was extracted from fecal samples and amplified by nested reverse transcription polymerase chain reaction (RT-nPCR). The PCR products were sequenced, and the sequence homology and phylogenetics of the HEV strains isolated from swine were analyzed. Results:, The anti-HEV positivity rates in adult swine, cows, sheep, younger swine, professional group and general population were 98.23% (222/226), 29.35% (54/184), 9.80% (20/207), 60.73% (99/164), 42.51% (105/247) and 20.29% (522/2572), respectively. The HEV RNA positivity rate of fecal samples was 22.89% (19/83) and 16/19 samples were positive for HEV RNA amplified with both primers, HEV open reading frame (ORF)1 and HEV ORF2. Sequence analysis of these 16 samples showed that there were two groups, designated BJ-1 and BJ-2. The nucleotide homology of BJ-1 and BJ-2 was 99%. Phylogenetic analysis indicated that both of these groups belonged to genotype 4d. Conclusion:, Workers in pig farms and slaughterhouses were more likely to contract HEV infection than the general population because of close contact with swine with a high prevalence of anti-HEV. [source] Differential functional effects of novel mutations of the transcription factor FOXL2 in BPES patients,HUMAN MUTATION, Issue 8 2008Jeyabalan Nallathambi Abstract Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development lead to the Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome (BPES) in human. Here, we describe nine mutations in the open reading frame of FOXL2. Six of them are novel: c.292T>A (p.Trp98Arg), c.323T>C (p.Leu108Pro), c.650C>G (p.Ser217Cys) and three frameshifts. We have performed localization and functional studies for three of them. We have observed a strong cytoplasmic mislocalization induced by the missense mutation p.Leu108Pro located in the forkhead (FKH) domain of FOXL2. In line with this, transcriptional activity assays confirmed the loss-of-function induced by this variant. Interestingly, the novel mutation p.Ser217Cys, mapping between the FKH and the polyalanine domain of FOXL2 and producing a mild eyelid phenotype, led to normal localization and transactivation. We have also modeled the structure of the FKH domain to explore the potential structural impact of the mutations reported here and other previously reported ones. This analysis shows that mutants can be sorted into two classes: those that potentially alter protein-protein interactions and those that might disrupt the interactions with DNA. Our findings reveal new insights into the molecular effects of FOXL2 mutations, especially those affecting the FKH binding domain. © 2008 Wiley-Liss, Inc. [source] Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy,HUMAN MUTATION, Issue 2 2007Christophe Béroud Abstract Approximately two-thirds of Duchenne muscular dystrophy (DMD) patients show intragenic deletions ranging from one to several exons of the DMD gene and leading to a premature stop codon. Other deletions that maintain the translational reading frame of the gene result in the milder Becker muscular dystrophy (BMD) form of the disease. Thus the opportunity to transform a DMD phenotype into a BMD phenotype appeared as a new treatment strategy with the development of antisense oligonucleotides technology, which is able to induce an exon skipping at the pre-mRNA level in order to restore an open reading frame. Because the DMD gene contains 79 exons, thousands of potential transcripts could be produced by exon skipping and should be investigated. The conventional approach considers skipping of a single exon. Here we report the comparison of single- and multiple-exon skipping strategies based on bioinformatic analysis. By using the Universal Mutation Database (UMD)-DMD, we predict that an optimal multiexon skipping leading to the del45-55 artificial dystrophin (c.6439_8217del) could transform the DMD phenotype into the asymptomatic or mild BMD phenotype. This multiple-exon skipping could theoretically rescue up to 63% of DMD patients with a deletion, while the optimal monoskipping of exon 51 would rescue only 16% of patients. Hum Mutat 28(2), 196,202, 2007. © 2006 Wiley-Liss, Inc. [source] piggyBac- like elements in cotton bollworm, Helicoverpa armigera (Hübner)INSECT MOLECULAR BIOLOGY, Issue 1 2008Z. C. Sun Abstract Two piggyBac -like elements (PLEs) were identified in the cotton bollworm, Helicoverpa armigera, and were designated as HaPLE1 and HaPLE2. HaPLE1 is flanked by 16 bp inverted terminal repeats (ITRs) and the duplicated TTAA tetranucleotide, and contains an open reading frame (ORF) of 1794 bp with the presumed DDD domain, indicating that this element may be an active autonomously mobile element. HaPLE2 was found with the same ITRs, but lacks the majority of an ORF-encoding transposase. Thus, this element was thought to be a non-autonomous element. Transposable element displays and distribution of the two PLEs in individuals from three different H. armigera populations suggest that transmobilization of HaPLE2 by the transposase of HaPLE1 may be likely, and mobilization of HaPLE1 might occur not only within the same individual, but also among different individuals. In addition, horizontal transfer was probably involved in the evolution of PLEs between H. armigera and Trichoplusia ni. [source] A p38 MAP kinase regulates the expression of the Aedes aegypti defensin gene in mosquito cellsINSECT MOLECULAR BIOLOGY, Issue 4 2007R. Chen-Chih Wu Abstract An Aedes aegypti p38 (Aap38) mitogen-activated protein kinase was isolated and characterized in this study. The 1761 bp long full-length Aap38 cDNA encodes an open reading frame of 358 amino acids, exhibiting characteristics of Thr/Tyr dual kinase specificities. We showed that bacteria activate both the kinase activity of Aap38 and the expression of the Aedes aegypti defensin A (AaDefA) gene, which is inhibited by a p38 kinase inhibitor SB203580 and dsRNA interference of Aap38. A similar result was obtained by a reporter construct containing the AaDefA regulatory region linked to Ds-Red. The lipopolysaccharide-activated reporter gene was inhibited by SB203580. In addition, Aap38 translocated to the nucleus after lipopolysaccharide induction. Our findings suggest that the p38 protein kinase pathway is involved in the antibacterial peptide synthesis in mosquitoes. [source] A tapeworm molecule manipulates vitellogenin expression in the beetle Tenebrio molitorINSECT MOLECULAR BIOLOGY, Issue 4 2006E. Warr Abstract Metacestodes of Hymenolepis diminuta secrete a molecule that decreases vitellogenin (Vg) synthesis in the beetle host, Tenebrio molitor. The 5608 bp T. molitor Vg cDNA represents a single-copy gene encoding a single open reading frame of 1821 amino acids with a predicted molecular mass of 206 kDa. Northern blot analysis revealed detectable levels of transcripts only in adult females. In vivo, Vg mRNA abundance was significantly higher in fat bodies from infected females compared with control females at all but the earliest time point. In vitro, Vg mRNA abundance was significantly increased in fat bodies incubated with live stage I,II parasites. The apparent conflict between increased Vg mRNA abundance and decreased Vg protein in fat bodies from infected females is discussed. [source] Cloning and characterization of a trypsin-encoding cDNA of the human body louse Pediculus humanusINSECT MOLECULAR BIOLOGY, Issue 1 2004A. H. Kollien Abstract From a cDNA library of the whole insect, a trypsin gene of Pediculus humanus has been cloned and sequenced. The 908 bp clone has an open reading frame of 759 bp, which encodes a pre-proenzyme with 253 amino acid residues. A sixteen-residue N-terminal signal peptide is followed by a twelve-residue activation peptide with putative cleavage sites at Gly16 and Tyr28. The deduced amino acid sequence has several features typical of trypsin proteases and an overall identity of 35,43% with the trypsins of several haematophagous Diptera. The 1.0 kb genomic trypsin gene contains three introns of 102, 79 and 80 nucleotides following the codons for Gly16, Gln74 and Ala155, respectively. Only a single gene seems to be present. In Northern blot analysis, unfed first instar larvae have an identical or slightly lower level of trypsin mRNA than fed adult lice, and in adults 2,24 h after the bloodmeal this gene shows a constitutive expression. After in vitro transcription and translation, the activation peptide is cleaved by chymotrypsin, a so far unreported phenomenon in trypsin activation. [source] Isolation and molecular characterization of Musca domestica delta-9 desaturase sequencesINSECT MOLECULAR BIOLOGY, Issue 6 2002A. L. Eigenheer Abstract We have isolated fatty acyl-CoA desaturase cDNA (Mdomd9) and genomic sequences from the housefly, Musca domestica. Two ,1.66 kb cDNAs were recovered. They had identical coding regions and 3, untranslated regions (UTRs), but differed in their 5, UTRs. The open reading frame encodes a 380 amino acid (aa) protein with 82% identity to Drosophila melanogaster desat1, and significant (> 50%) identity with other insect delta-9 desaturases. Functional analyses in a yeast expression system confirmed the cDNA encodes a ,9 desaturase. Northern analysis indicated two transcripts of 1.7 and 2.9 kb that hybridized specifically to the open reading frame. PCR amplification of genomic templates revealed three intron sites that are conserved among other insect species. Southern analysis of genomic DNA indicated at least two desaturase gene copies per haploid genome. There is a high degree of polymorphism, most of which appears to be due to variable intron sequences; curiously, individual flies had varying morphs of intron II and intron III. Together, the data suggest that there are more ,9 desaturase alleles within the population studied than there are loci within the genome, and support other studies suggesting that insect fatty acyl-CoA desaturases are a dynamically evolving gene family. [source] | ||||||||||||||||||||||||||||||||||||||||