Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO.

PubMed Central

Jarausch, W; Saillard, C; Dosba, F; Bové, J M

1994-01-01

A 1.8-kb chromosomal DNA fragment of the mycoplasmalike organism (MLO) associated with apple proliferation was sequenced. Three putative open reading frames were observed on this fragment. The protein encoded by open reading frame 2 shows significant homologies with bacterial nitroreductases. From the nucleotide sequence four primer pairs for PCR were chosen to specifically amplify DNA from MLOs associated with European diseases of fruit trees. Primer pairs specific for (i) Malus-affecting MLOs, (ii) Malus- and Prunus-affecting MLOs, and (iii) Malus-, Prunus-, and Pyrus-affecting MLOs were obtained. Restriction enzyme analysis of the amplification products revealed restriction fragment length polymorphisms between Malus-, Prunus, and Pyrus-affecting MLOs as well as between different isolates of the apple proliferation MLO. No amplification with either primer pair could be obtained with DNA from 12 different MLOs experimentally maintained in periwinkle. Images PMID:7916180

DLEU2 encodes an antisense RNA for the putative bicistronic RFP2/LEU5 gene in humans and mouse.

PubMed

Corcoran, Martin M; Hammarsund, Marianne; Zhu, Chaoyong; Lerner, Mikael; Kapanadze, Bagrat; Wilson, Bill; Larsson, Catharina; Forsberg, Lars; Ibbotson, Rachel E; Einhorn, Stefan; Oscier, David G; Grandér, Dan; Sangfelt, Olle

2004-08-01

Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, suggesting that a nonclassical tumor-suppressor mechanism may be involved. Here, we present data showing that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in the mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5. Copyright 2004 Wiley-Liss, Inc.

The organisation and interviral homologies of genes at the 3' end of tobacco rattle virus RNA1

PubMed Central

Boccara, Martine; Hamilton, William D. O.; Baulcombe, David C.

1986-01-01

The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3'-terminal region. The sequence contains three long open reading frames. One of these starts 5' of the cDNA and probably corresponds to the carboxy-terminal sequence of a 170-K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29-K polypeptide, was shown by Northern blot analysis to coincide with a 1.6-kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co-migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29-K polypeptide showed homology with two regions in the 30-K protein of TMV. This homology includes positions in the TMV 30-K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16-K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7-kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor. ImagesFig. 2.Fig. 3. PMID:16453668

The Saccharomyces cerevisiae LOS1 gene involved in pre-tRNA splicing encodes a nuclear protein that behaves as a component of the nuclear matrix.

PubMed

Shen, W C; Selvakumar, D; Stanford, D R; Hopper, A K

1993-09-15

Mutations of the Saccharomyces cerevisiae LOS1 gene cause the accumulation of end matured intron-containing pre-tRNAs at elevated temperatures. In an effort to decipher the role of the LOS1 protein in pre-tRNA splicing, we have analyzed the LOS1 gene and its protein product. The LOS1 gene is located on the left arm of chromosome XI and the order of genes in this area of the chromosome is .... URA1 ... SAC1 TRP3 UBA1 STE6 LOS1 .... FAS1..... The LOS1 open reading frame encodes a putative protein of 1100 amino acids that shows no significant homology to other genes. The LOS1 open reading frame was tagged with the influenza virus hemagglutinin epitope recognized by the 12CA5 antibody. The 12CA5 antibody recognizes an epitope-tagged protein of the size predicted by the LOS1 open reading frame. Using this antibody for indirect immunofluorescence and cell fractionation studies we show that the LOS1 protein is located in nuclei. Los1p cannot be extracted from nuclei by treatment with nucleases, salts, or Triton X-100. This insolubility suggests that Los1p is a component of the nucleoskeleton. We propose that LOS1 mutations may affect pre-tRNA processing via alteration of the nuclear matrix.

Complete sequence of Tvv1, a family of Ty 1 copia-like retrotransposons of Vitis vinifera L., reconstituted by chromosome walking.

PubMed

Pelsy, F.; Merdinoglu, D.

2002-09-01

A chromosome-walking strategy was used to sequence and characterize retrotransposons in the grapevine genome. The reconstitution of a family of retroelements, named Tvv1, was achieved by six successive steps. These elements share a single, highly conserved open reading frame 4,153 nucleotides-long, putatively encoding the gag, pro, int, rt and rh proteins. Comparison of the Tvv1 open reading frame coding potential with those of drosophila copia and tobacco Tnt1, revealed that Tvv1 is closely related to Ty 1 copia-like retrotransposons. A highly variable untranslated leader region, upstream of the open reading frame, allowed us to differentiate Tvv1 variants, which represent a family of at least 28 copies, in varying sizes. This internal region is flanked by two long terminal repeats in direct orientation, sized between 149 and 157 bp. Among elements theoretically sized from 4,970 to 5,550 bp, we describe the full-length sequence of a reference element Tvv1-1, 5,343 nucleotides-long. The full-length sequence of Tvv1-1 compared to pea PDR1 shows a 53.3% identity. In addition, both elements contain long terminal repeats of nearly the same size in which the U5 region could be entirely absent. Therefore, we assume that Tvv1 and PDR1 could constitute a particular class of short LTRs retroelements.

Isolation and molecular identification of Sunshine virus, a novel paramyxovirus found in Australian snakes.

PubMed

Hyndman, Timothy H; Marschang, Rachel E; Wellehan, James F X; Nicholls, Philip K

2012-10-01

This paper describes the isolation and molecular identification of a novel paramyxovirus found during an investigation of an outbreak of neurorespiratory disease in a collection of Australian pythons. Using Illumina® high-throughput sequencing, a 17,187 nucleotide sequence was assembled from RNA extracts from infected viper heart cells (VH2) displaying widespread cytopathic effects in the form of multinucleate giant cells. The sequence appears to contain all the coding regions of the genome, including the following predicted paramyxoviral open reading frames (ORFs): 3'--Nucleocapsid (N)--putative Phosphoprotein (P)--Matrix (M)--Fusion (F)--putative attachment protein--Polymerase (L)--5'. There is also a 540 nucleotide ORF between the N and putative P genes that may be an additional coding region. Phylogenetic analyses of the complete N, M, F and L genes support the clustering of this virus within the family Paramyxoviridae but outside both of the current subfamilies: Paramyxovirinae and Pneumovirinae. We propose to name this new virus, Sunshine virus, after the geographic origin of the first isolate--the Sunshine Coast of Queensland, Australia. Copyright © 2012 Elsevier B.V. All rights reserved.

Functional Analysis of the Gene Cluster Involved in Production of the Bacteriocin Circularin A by Clostridium beijerinckii ATCC 25752

PubMed Central

Kemperman, Robèr; Jonker, Marnix; Nauta, Arjen; Kuipers, Oscar P.; Kok, Jan

2003-01-01

A region of 12 kb flanking the structural gene of the cyclic antibacterial peptide circularin A of Clostridium beijerinckii ATCC 25752 was sequenced, and the putative proteins involved in the production and secretion of circularin A were identified. The genes are tightly organized in overlapping open reading frames. Heterologous expression of circularin A in Enterococcus faecalis was achieved, and five genes were identified as minimally required for bacteriocin production and secretion. Two of the putative proteins, CirB and CirC, are predicted to contain membrane-spanning domains, while CirD contains a highly conserved ATP-binding domain. Together with CirB and CirC, this ATP-binding protein is involved in the production of circularin A. The fifth gene, cirE, confers immunity towards circularin A when expressed in either Lactococcus lactis or E. faecalis and is needed in order to allow the bacteria to produce bacteriocin. Additional resistance against circularin A is conferred by the activity of the putative transporter consisting of CirB and CirD. PMID:14532033

Genome mining of the sordarin biosynthetic gene cluster from Sordaria araneosa Cain ATCC 36386: characterization of cycloaraneosene synthase and GDP-6-deoxyaltrose transferase.

PubMed

Kudo, Fumitaka; Matsuura, Yasunori; Hayashi, Takaaki; Fukushima, Masayuki; Eguchi, Tadashi

2016-07-01

Sordarin is a glycoside antibiotic with a unique tetracyclic diterpene aglycone structure called sordaricin. To understand its intriguing biosynthetic pathway that may include a Diels-Alder-type [4+2]cycloaddition, genome mining of the gene cluster from the draft genome sequence of the producer strain, Sordaria araneosa Cain ATCC 36386, was carried out. A contiguous 67 kb gene cluster consisting of 20 open reading frames encoding a putative diterpene cyclase, a glycosyltransferase, a type I polyketide synthase, and six cytochrome P450 monooxygenases were identified. In vitro enzymatic analysis of the putative diterpene cyclase SdnA showed that it catalyzes the transformation of geranylgeranyl diphosphate to cycloaraneosene, a known biosynthetic intermediate of sordarin. Furthermore, a putative glycosyltransferase SdnJ was found to catalyze the glycosylation of sordaricin in the presence of GDP-6-deoxy-d-altrose to give 4'-O-demethylsordarin. These results suggest that the identified sdn gene cluster is responsible for the biosynthesis of sordarin. Based on the isolated potential biosynthetic intermediates and bioinformatics analysis, a plausible biosynthetic pathway for sordarin is proposed.

The complete nucleotide sequence and genome organization of a novel betaflexivirus infecting Citrullus lanatus.

PubMed

Xin, Min; Zhang, Peipei; Liu, Wenwen; Ren, Yingdang; Cao, Mengji; Wang, Xifeng

2017-10-01

The complete nucleotide sequence of a novel positive single-stranded (+ss) RNA virus, tentatively named watermelon virus A (WVA), was determined using a combination of three methods: RNA sequencing, small RNA sequencing, and Sanger sequencing. The full genome of WVA is comprised of 8,372 nucleotides (nt), excluding the poly (A) tail, and contains four open reading frames (ORFs). The largest ORF, ORF1 encodes a putative replication-associated polyprotein (RP) with three conserved domains. ORF2 and ORF4 encode a movement protein (MP) and coat protein (CP), respectively. The putative product encoded by ORF3, of an estimated molecular mass of 25 kDa, has no significant similarity with other proteins. Identity and phylogenetic analysis indicate that WVA is a new virus, closely related to members of the family Betaflexiviridae. However, the final taxonomic allocation of WVA within the family is yet to be determined.

Identification, cloning, and sequencing of a fragment of Amsacta moorei entomopoxvirus DNA containing the spheroidin gene and three vaccinia virus-related open reading frames.

PubMed Central

Hall, R L; Moyer, R W

1991-01-01

Entomopoxvirus virions are frequently contained within crystalline occlusion bodies, which are composed of primarily a single protein, spheroidin, which is analogous to the polyhedrin protein of baculovirus. The spheroidin gene of Amsacta moorei entomopoxvirus was identified following the microsequencing of polypeptides generated from cyanogen bromide treatment of spheroidin and the subsequent synthesis of oligonucleotide hybridization probes. DNA sequencing of a 6.8-kb region of DNA containing the spheroidin gene showed that the spheroidin protein is derived from a 3.0-kb open reading frame potentially encoding a protein of 115 kDa. Three copies of the heptanucleotide, TTTTTNT, a sequence associated with early gene transcription in the vertebrate poxviruses, and four in-frame translational termination signals were found within 60 bp upstream of the putative spheroidin gene promoter (TAAATG). The spheroidin gene promoter region contains the sequence TAAATG, which is found in many late promoters of the vertebrate poxviruses and which serves as the site of transcriptional initiation, as shown by primer extension. Primer extension experiments also showed that spheroidin gene transcripts contain 5' poly(A) sequences typical of vertebrate poxvirus late transcripts. The 92 bases upstream of the initiating TAAATG are unusually A + T rich and contain only 7 G or C residues. An analysis of open reading frames around the spheroidin gene suggests that the colinear core of "essential genes" typical of the vertebrate poxviruses is absent in A. moorei entomopoxvirus. Images PMID:1942245

Molecular identification and characterization of clustered regularly interspaced short palindromic repeat (CRISPR) gene cluster in Taylorella equigenitalis.

PubMed

Hara, Yasushi; Hayashi, Kyohei; Nakajima, Takuya; Kagawa, Shizuko; Tazumi, Akihiro; Moore, John E; Matsuda, Motoo

2013-09-01

Clustered regularly interspaced short palindromic repeats (CRISPRs), of approximately 10,000 base pairs (bp) in length, were shown to occur in the Japanese Taylorella equigenitalis strain, EQ59. The locus was composed of the putative CRISPRs-associated with 5 (cas5), RAMP csd1, csd2, recB, cas1, a leader region, 13 CRISPR consensus sequence repeats (each 32 bp; 5'-TCAGCCACGTTCGCGTGGCTGTGTGTTTAAAG-3'). These were in turn separated by 12 non repetitive unique spacer regions of similar length. In addition, a leader region, a transposase/IS protein, a leader region, and cas3 were also seen. All seven putative open reading frames carry their ribosome binding sites. Promoter consensus sequences at the -35 and -10 regions and putative intrinsic ρ-independent transcription terminator regions also occurred. A possible long overlap of 170 bp in length occurred between the recB and cas1 loci. Positive reverse transcription PCR signals of cas5, RAMP csd1, csd2-recB/cas1, and cas3 were generated. A putative secondary structure of the CRISPR consensus repeats was constructed. Following this, CRISPR results of the T. equigenitalis EQ59 isolate were subsequently compared with those from the Taylorella asinigenitalis MCE3 isolate.

Tomato golden mosaic virus open reading frame AL4 is genetically distinct from its C4 analogue in monopartite geminiviruses.

PubMed

Pooma, W; Petty, I T

1996-08-01

Tomato golden mosaic virus (TGMV) is a bipartite geminivirus with six well-characterized genes. An additional open reading frame (ORF), AL4, lies within the essential AL1 gene. Recent studies of monopartite, dicot-infecting geminiviruses have revealed that mutations in their analogous C4 ORFs have host-specific effects on infectivity, symptomatology, virus movement and/or viral DNA accumulation. We have investigated whether TGMV has a similar host-specific requirement for AL4. The phenotypes of three TGMV al4 mutants were determined in a range of hosts, which included species that revealed c4 mutant phenotypes for monopartite geminiviruses. Each TGMV al4 mutant was indistinguishable from wild-type TGMV in all hosts tested. Additional analyses of double mutants revealed no evidence for functional redundancy between AL4 and the AL3, or AR1 genes. In contrast to the putative C4 proteins of monpartite geminiviruses, TGMV AL4, if it is expressed, is either non-functional, or functionally redundant with an essential TGMV gene product.

Genomic variation of the fibropapilloma-associated marine turtle herpes virus across seven geographic areas and three host species

USGS Publications Warehouse

Greenblatt, R.J.; Quackenbush, S.L.; Casey, R.N.; Rovnak, J.; Balazs, G.H.; Work, Thierry M.; Casey, J.W.; Sutton, C.A.

2005-01-01

Fibropapillomatosis (FP) of marine turtles is an emerging neoplastic disease associated with infection by a novel turtle herpesvirus, fibropapilloma-associated turtle herpesvirus (FPTHV). This report presents 23 kb of the genome of an FPTHV infecting a Hawaiian green turtle (Chelonia mydas). By sequence homology, the open reading frames in this contig correspond to herpes simplex virus genes UL23 through UL36. The order, orientation, and homology of these putative genes indicate that FPTHV is a member of the Alphaherpesvirinae. The UL27-, UL30-, and UL34-homologous open reading frames from FPTHVs infecting nine FP-affected marine turtles from seven geographic areas and three turtle species (C. mydas, Caretta caretta, and Lepidochelys olivacea) were compared. A high degree of nucleotide sequence conservation was found among these virus variants. However, geographic variations were also found: the FPTHVs examined here form four groups, corresponding to the Atlantic Ocean, West pacific, mid-Pacific, and east Pacific. Our results indicate that FPTHV was established in marine turtle populations prior to the emergence of FP as it is currently known.

Expression of putative pathogenicity-related genes in Xylella fastidiosa grown at low and high cell density conditions in vitro.

PubMed

Scarpari, Leandra M; Lambais, Marcio R; Silva, Denise S; Carraro, Dirce M; Carrer, Helaine

2003-05-16

Xylella fastidiosa is the causal agent of economically important plant diseases, including citrus variegated chlorosis and Pierce's disease. Hitherto, there has been no information on the molecular mechanisms controlling X. fastidiosa-plant interactions. To determine whether predicted open reading frames (ORFs) encoding putative pathogenicity-related factors were expressed by X. fastidiosa 9a5c cells grown at low (LCD) and high cell density (HCD) conditions in liquid modified PW medium, reverse Northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed. Our results indicated that ORFs XF2344, XF2369, XF1851 and XF0125, encoding putative Fur, GumC, a serine-protease and RsmA, respectively, were significantly suppressed at HCD conditions. In contrast, ORF XF1115, encoding putative RpfF, was significantly induced at HCD conditions. Expressions of ORFs XF2367, XF2362 and XF0290, encoding putative GumD, GumJ and RpfA, respectively, were detected only at HCD conditions, whereas expression of ORF XF0287, encoding putative RpfB was detected only at LCD conditions. Bioassays with an Agrobacterium traG::lacZ reporter system indicated that X. fastidiosa does not synthesize N-acyl-homoserine lactones, whereas bioassays with a diffusible signal factor (DSF)-responsive Xanthomonas campestris pv. campestris mutant indicate that X. fastidiosa synthesizes a molecule similar to DSF in modified PW medium. Our data also suggest that the synthesis of the DSF-like molecule and fastidian gum by X. fastidiosa is affected by cell density in vitro.

Transcription of TP0126, Treponema pallidum Putative OmpW Homolog, Is Regulated by the Length of a Homopolymeric Guanosine Repeat

PubMed Central

Brandt, Stephanie L.; Ke, Wujian; Reid, Tara B.; Molini, Barbara J.; Iverson-Cabral, Stefanie; Ciccarese, Giulia; Drago, Francesco; Lukehart, Sheila A.; Centurion-Lara, Arturo

2015-01-01

An effective mechanism for introduction of phenotypic diversity within a bacterial population exploits changes in the length of repetitive DNA elements located within gene promoters. This phenomenon, known as phase variation, causes rapid activation or silencing of gene expression and fosters bacterial adaptation to new or changing environments. Phase variation often occurs in surface-exposed proteins, and in Treponema pallidum subsp. pallidum, the syphilis agent, it was reported to affect transcription of three putative outer membrane protein (OMP)-encoding genes. When the T. pallidum subsp. pallidum Nichols strain genome was initially annotated, the TP0126 open reading frame was predicted to include a poly(G) tract and did not appear to have a predicted signal sequence that might suggest the possibility of its being an OMP. Here we show that the initial annotation was incorrect, that this poly(G) is instead located within the TP0126 promoter, and that it varies in length in vivo during experimental syphilis. Additionally, we show that TP0126 transcription is affected by changes in the poly(G) length consistent with regulation by phase variation. In silico analysis of the TP0126 open reading frame based on the experimentally identified transcriptional start site shortens this hypothetical protein by 69 amino acids, reveals a predicted cleavable signal peptide, and suggests structural homology with the OmpW family of porins. Circular dichroism of recombinant TP0126 supports structural homology to OmpW. Together with the evidence that TP0126 is fully conserved among T. pallidum subspecies and strains, these data suggest an important role for TP0126 in T. pallidum biology and syphilis pathogenesis. PMID:25802057

Discovery of numerous novel small genes in the intergenic regions of the Escherichia coli O157:H7 Sakai genome

PubMed Central

Hücker, Sarah M.; Ardern, Zachary; Goldberg, Tatyana; Schafferhans, Andrea; Bernhofer, Michael; Vestergaard, Gisle; Nelson, Chase W.; Schloter, Michael; Rost, Burkhard; Scherer, Siegfried

2017-01-01

In the past, short protein-coding genes were often disregarded by genome annotation pipelines. Transcriptome sequencing (RNAseq) signals outside of annotated genes have usually been interpreted to indicate either ncRNA or pervasive transcription. Therefore, in addition to the transcriptome, the translatome (RIBOseq) of the enteric pathogen Escherichia coli O157:H7 strain Sakai was determined at two optimal growth conditions and a severe stress condition combining low temperature and high osmotic pressure. All intergenic open reading frames potentially encoding a protein of ≥ 30 amino acids were investigated with regard to coverage by transcription and translation signals and their translatability expressed by the ribosomal coverage value. This led to discovery of 465 unique, putative novel genes not yet annotated in this E. coli strain, which are evenly distributed over both DNA strands of the genome. For 255 of the novel genes, annotated homologs in other bacteria were found, and a machine-learning algorithm, trained on small protein-coding E. coli genes, predicted that 89% of these translated open reading frames represent bona fide genes. The remaining 210 putative novel genes without annotated homologs were compared to the 255 novel genes with homologs and to 250 short annotated genes of this E. coli strain. All three groups turned out to be similar with respect to their translatability distribution, fractions of differentially regulated genes, secondary structure composition, and the distribution of evolutionary constraint, suggesting that both novel groups represent legitimate genes. However, the machine-learning algorithm only recognized a small fraction of the 210 genes without annotated homologs. It is possible that these genes represent a novel group of genes, which have unusual features dissimilar to the genes of the machine-learning algorithm training set. PMID:28902868

Mining of Novel Thermo-Stable Cellulolytic Genes from a Thermophilic Cellulose-Degrading Consortium by Metagenomics

PubMed Central

Xia, Yu; Ju, Feng; Fang, Herbert H. P.; Zhang, Tong

2013-01-01

In this study, metagenomics was applied to characterize the microbial community and to discover carbohydrate-active genes of an enriched thermophilic cellulose-degrading sludge. The 16S analysis showed that the sludge microbiome was dominated by genus of cellulolytic Clostridium and methanogenesis Methanothermobacter. In order to retrieve genes from the metagenome, de novo assembly of the 11,930,760 Illumina 100 bp paired-end reads (totally 1.2 Gb) was carried out. 75% of all reads was utilized in the de novo assembly. 31,499 ORFs (Open Reading Frame) with an average length of 852 bp were predicted from the assembly; and 64% of these ORFs were predicted to present full-length genes. Based on the Hidden Markol Model, 253 of the predicted thermo-stable genes were identified as putatively carbohydrate-active. Among them the relative dominance of GH9 (Glycoside Hydrolase) and corresponding CBM3 (Carbohydrate Binding Module) revealed a cellulosome-based attached metabolism of polysaccharide in the thermophilic sludge. The putative carbohydrate-active genes ranged from 20% to 100% amino acid sequence identity to known proteins in NCBI nr database, with half of them showed less than 50% similarity. In addition, the coverage of the genes (in terms of ORFs) identified in the sludge were developed into three clear trends (112×, 29× and 8×) in which 85% of the high coverage trend (112×) mainly consisted of phylum of Firmicutes while 49.3% of the 29× trend was affiliated to the phylum of Chloroflexi. PMID:23341999

De Novo ORFs in Drosophila Are Important to Organismal Fitness and Evolved Rapidly from Previously Non-coding Sequences

PubMed Central

Reinhardt, Josephine A.; Wanjiru, Betty M.; Brant, Alicia T.; Saelao, Perot; Begun, David J.; Jones, Corbin D.

2013-01-01

How non-coding DNA gives rise to new protein-coding genes (de novo genes) is not well understood. Recent work has revealed the origins and functions of a few de novo genes, but common principles governing the evolution or biological roles of these genes are unknown. To better define these principles, we performed a parallel analysis of the evolution and function of six putatively protein-coding de novo genes described in Drosophila melanogaster. Reconstruction of the transcriptional history of de novo genes shows that two de novo genes emerged from novel long non-coding RNAs that arose at least 5 MY prior to evolution of an open reading frame. In contrast, four other de novo genes evolved a translated open reading frame and transcription within the same evolutionary interval suggesting that nascent open reading frames (proto-ORFs), while not required, can contribute to the emergence of a new de novo gene. However, none of the genes arose from proto-ORFs that existed long before expression evolved. Sequence and structural evolution of de novo genes was rapid compared to nearby genes and the structural complexity of de novo genes steadily increases over evolutionary time. Despite the fact that these genes are transcribed at a higher level in males than females, and are most strongly expressed in testes, RNAi experiments show that most of these genes are essential in both sexes during metamorphosis. This lethality suggests that protein coding de novo genes in Drosophila quickly become functionally important. PMID:24146629

Putative Risk Factors in Developmental Dyslexia: A Case-Control Study of Italian Children

ERIC Educational Resources Information Center

Mascheretti, Sara; Marino, Cecilia; Simone, Daniela; Quadrelli, Ermanno; Riva, Valentina; Cellino, Maria Rosaria; Maziade, Michel; Brombin, Chiara; Battaglia, Marco

2015-01-01

Although dyslexia runs in families, several putative risk factors that cannot be immediately identified as genetic predict reading disability. Published studies analyzed one or a few risk factors at a time, with relatively inconsistent results. To assess the contribution of several putative risk factors to the development of dyslexia, we conducted…

TCOF1 gene encodes a putative nucleolar phosphoprotein that exhibits mutations in Treacher Collins Syndrome throughout its coding region.

PubMed

Wise, C A; Chiang, L C; Paznekas, W A; Sharma, M; Musy, M M; Ashley, J A; Lovett, M; Jabs, E W

1997-04-01

Treacher Collins Syndrome (TCS) is the most common of the human mandibulofacial dysostosis disorders. Recently, a partial TCOF1 cDNA was identified and shown to contain mutations in TCS families. Here we present the entire exon/intron genomic structure and the complete coding sequence of TCOF1. TCOF1 encodes a low complexity protein of 1,411 amino acids, whose predicted protein structure reveals repeated motifs that mirror the organization of its exons. These motifs are shared with nucleolar trafficking proteins in other species and are predicted to be highly phosphorylated by casein kinase. Consistent with this, the full-length TCOF1 protein sequence also contains putative nuclear and nucleolar localization signals. Throughout the open reading frame, we detected an additional eight mutations in TCS families and several polymorphisms. We postulate that TCS results from defects in a nucleolar trafficking protein that is critically required during human craniofacial development.

TCOF1 gene encodes a putative nucleolar phosphoprotein that exhibits mutations in Treacher Collins Syndrome throughout its coding region

PubMed Central

Wise, Carol A.; Chiang, Lydia C.; Paznekas, William A.; Sharma, Mridula; Musy, Maurice M.; Ashley, Jennifer A.; Lovett, Michael; Jabs, Ethylin W.

1997-01-01

Treacher Collins Syndrome (TCS) is the most common of the human mandibulofacial dysostosis disorders. Recently, a partial TCOF1 cDNA was identified and shown to contain mutations in TCS families. Here we present the entire exon/intron genomic structure and the complete coding sequence of TCOF1. TCOF1 encodes a low complexity protein of 1,411 amino acids, whose predicted protein structure reveals repeated motifs that mirror the organization of its exons. These motifs are shared with nucleolar trafficking proteins in other species and are predicted to be highly phosphorylated by casein kinase. Consistent with this, the full-length TCOF1 protein sequence also contains putative nuclear and nucleolar localization signals. Throughout the open reading frame, we detected an additional eight mutations in TCS families and several polymorphisms. We postulate that TCS results from defects in a nucleolar trafficking protein that is critically required during human craniofacial development. PMID:9096354

Sequence and Analysis of the Tomato JOINTLESS Locus1

PubMed Central

Mao, Long; Begum, Dilara; Goff, Stephen A.; Wing, Rod A.

2001-01-01

A 119-kb bacterial artificial chromosome from the JOINTLESS locus on the tomato (Lycopersicon esculentum) chromosome 11 contained 15 putative genes. Repetitive sequences in this region include one copia-like LTR retrotransposon, 13 simple sequence repeats, three copies of a novel type III foldback transposon, and four putative short DNA repeats. Database searches showed that the foldback transposon and the short DNA repeats seemed to be associated preferably with genes. The predicted tomato genes were compared with the complete Arabidopsis genome. Eleven out of 15 tomato open reading frames were found to be colinear with segments on five Arabidopsis bacterial artificial chromosome/P1-derived artificial chromosome clones. The synteny patterns, however, did not reveal duplicated segments in Arabidopsis, where over half of the genome is duplicated. Our analysis indicated that the microsynteny between the tomato and Arabidopsis genomes was still conserved at a very small scale but was complicated by the large number of gene families in the Arabidopsis genome. PMID:11457984

Phage phenomics: Physiological approaches to characterize novel viral proteins

ScienceCinema

Sanchez, Savannah E. [San Diego State Univ., San Diego, CA (United States); Cuevas, Daniel A. [San Diego State Univ., San Diego, CA (United States); Rostron, Jason E. [San Diego State Univ., San Diego, CA (United States); Liang, Tiffany Y. [San Diego State Univ., San Diego, CA (United States); Pivaroff, Cullen G. [San Diego State Univ., San Diego, CA (United States); Haynes, Matthew R. [San Diego State Univ., San Diego, CA (United States); Nulton, Jim [San Diego State Univ., San Diego, CA (United States); Felts, Ben [San Diego State Univ., San Diego, CA (United States); Bailey, Barbara A. [San Diego State Univ., San Diego, CA (United States); Salamon, Peter [San Diego State Univ., San Diego, CA (United States); Edwards, Robert A. [San Diego State Univ., San Diego, CA (United States); Argonne National Lab. (ANL), Argonne, IL (United States); Burgin, Alex B. [Broad Institute, Cambridge, MA (United States); Segall, Anca M. [San Diego State Univ., San Diego, CA (United States); Rohwer, Forest [San Diego State Univ., San Diego, CA (United States)

2018-06-21

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter.

PubMed

Bes, M T; Hernández, J A; Peleato, M L; Fillat, M F

2001-01-15

A gene coding for a Fur (ferric uptake regulation) protein from the cyanobacterium Anabaena PCC 7119 has been cloned and overexpressed in Escherichia coli. DNA sequence analysis confirmed the presence of a 151-amino-acid open reading frame that showed homology with the Fur proteins reported for the unicellular cyanobacteria Synechococcus 7942 and Synechocystis PCC 6803. Two putative Fur-binding sites were detected in the promoter regions of the fur gene from Anabaena. Partially purified recombinant Fur binds to the flavodoxin promoter as well as its own promoter. This suggests that the Fur gene is autoregulated in Anabaena.

Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases.

PubMed

Tanaka, T; Kawata, M

1988-08-01

We have isolated a DNA fragment from Bacillus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities for the exoprotease production in the N-terminal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU.

svviz: a read viewer for validating structural variants.

PubMed

Spies, Noah; Zook, Justin M; Salit, Marc; Sidow, Arend

2015-12-15

Visualizing read alignments is the most effective way to validate candidate structural variants (SVs) with existing data. We present svviz, a sequencing read visualizer for SVs that sorts and displays only reads relevant to a candidate SV. svviz works by searching input bam(s) for potentially relevant reads, realigning them against the inferred sequence of the putative variant allele as well as the reference allele and identifying reads that match one allele better than the other. Separate views of the two alleles are then displayed in a scrollable web browser view, enabling a more intuitive visualization of each allele, compared with the single reference genome-based view common to most current read browsers. The browser view facilitates examining the evidence for or against a putative variant, estimating zygosity, visualizing affected genomic annotations and manual refinement of breakpoints. svviz supports data from most modern sequencing platforms. svviz is implemented in python and freely available from http://svviz.github.io/. Published by Oxford University Press 2015. This work is written by US Government employees and is in the public domain in the US.

An analysis of mobile genetic elements in three Plasmodium species and their potential impact on the nucleotide composition of the P. falciparum genome.

PubMed

Durand, Pierre M; Oelofse, Andries J; Coetzer, Theresa L

2006-11-04

The completed genome sequences of the malaria parasites P. falciparum, P. y. yoelii and P. vivax have revealed some unusual features. P. falciparum contains the most AT rich genome sequenced so far--over 90% in some regions. In comparison, P. y. yoelii is approximately 77% and P. vivax is approximately 55% AT rich. The evolutionary reasons for these findings are unknown. Mobile genetic elements have a considerable impact on genome evolution but a thorough investigation of these elements in Plasmodium has not been undertaken. We therefore performed a comprehensive genome analysis of these elements and their derivatives in the three Plasmodium species. Whole genome analysis was performed using bioinformatic methods. Forty potential protein encoding sequences with features of transposable elements were identified in P. vivax, eight in P. y. yoelii and only six in P. falciparum. Further investigation of the six open reading frames in P. falciparum revealed that only one is potentially an active mobile genetic element. Most of the open reading frames identified in all three species are hypothetical proteins. Some represent annotated host proteins such as the putative telomerase reverse transcriptase genes in P. y. yoelii and P. falciparum. One of the P. vivax open reading frames identified in this study demonstrates similarity to telomerase reverse transcriptase and we conclude it to be the orthologue of this gene. There is a divergence in the frequencies of mobile genetic elements in the three Plasmodium species investigated. Despite the limitations of whole genome analytical methods, it is tempting to speculate that mobile genetic elements might have been a driving force behind the compositional bias of the P. falciparum genome.

Type 2 NADH Dehydrogenases in the Cyanobacterium Synechocystis sp. Strain PCC 6803 Are Involved in Regulation Rather Than Respiration

PubMed Central

Howitt, Crispin A.; Udall, Pacer K.; Vermaas, Wim F. J.

1999-01-01

Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool. PMID:10383967

A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

PubMed

Abraham, S; Solomon, W B

2000-09-19

We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin structure (ARC) are identical to portions of the deduced open reading frame of TIG-1 mRNA.

Individualized Prediction of Reading Comprehension Ability Using Gray Matter Volume.

PubMed

Cui, Zaixu; Su, Mengmeng; Li, Liangjie; Shu, Hua; Gong, Gaolang

2018-05-01

Reading comprehension is a crucial reading skill for learning and putatively contains 2 key components: reading decoding and linguistic comprehension. Current understanding of the neural mechanism underlying these reading comprehension components is lacking, and whether and how neuroanatomical features can be used to predict these 2 skills remain largely unexplored. In the present study, we analyzed a large sample from the Human Connectome Project (HCP) dataset and successfully built multivariate predictive models for these 2 skills using whole-brain gray matter volume features. The results showed that these models effectively captured individual differences in these 2 skills and were able to significantly predict these components of reading comprehension for unseen individuals. The strict cross-validation using the HCP cohort and another independent cohort of children demonstrated the model generalizability. The identified gray matter regions contributing to the skill prediction consisted of a wide range of regions covering the putative reading, cerebellum, and subcortical systems. Interestingly, there were gender differences in the predictive models, with the female-specific model overestimating the males' abilities. Moreover, the identified contributing gray matter regions for the female-specific and male-specific models exhibited considerable differences, supporting a gender-dependent neuroanatomical substrate for reading comprehension.

The genome sequence of Agrotis segetum granulovirus, isolate AgseGV-DA, reveals a new Betabaculovirus species of a slow killing granulovirus.

PubMed

Gueli Alletti, Gianpiero; Eigenbrod, Marina; Carstens, Eric B; Kleespies, Regina G; Jehle, Johannes A

2017-06-01

The European isolate Agrotis segetum granulovirus DA (AgseGV-DA) is a slow killing, type I granulovirus due to low dose-mortality responses within seven days post infection and a tissue tropism of infection restricted solely to the fat body of infected Agrotis segetum host larvae. The genome of AgseGV-DA was completely sequenced and compared to the whole genome sequences of the Chinese isolates AgseGV-XJ and AgseGV-L1. All three isolates share highly conserved genomes. The AgseGV-DA genome is 131,557bp in length and encodes for 149 putative open reading frames, including 37 baculovirus core genes and the per os infectivity factor ac110. Comprehensive investigations of repeat regions identified one putative non-hr like origin of replication in AgseGV-DA. Phylogenetic analysis based on concatenated amino acid alignments of 37 baculovirus core genes as well as pairwise distances based on the nucleotide alignments of partial granulin, lef-8 and lef-9 sequences with deposited betabaculoviruses confirmed AgseGV-DA, AgseGV-XJ and AgseGV-L1 as representative isolates of the same Betabaculovirus species. AgseGV encodes for a distinct putative enhancin, distantly related to enhancins from other granuloviruses. Copyright © 2017. Published by Elsevier Inc.

Accurate indel prediction using paired-end short reads

PubMed Central

2013-01-01

Background One of the major open challenges in next generation sequencing (NGS) is the accurate identification of structural variants such as insertions and deletions (indels). Current methods for indel calling assign scores to different types of evidence or counter-evidence for the presence of an indel, such as the number of split read alignments spanning the boundaries of a deletion candidate or reads that map within a putative deletion. Candidates with a score above a manually defined threshold are then predicted to be true indels. As a consequence, structural variants detected in this manner contain many false positives. Results Here, we present a machine learning based method which is able to discover and distinguish true from false indel candidates in order to reduce the false positive rate. Our method identifies indel candidates using a discriminative classifier based on features of split read alignment profiles and trained on true and false indel candidates that were validated by Sanger sequencing. We demonstrate the usefulness of our method with paired-end Illumina reads from 80 genomes of the first phase of the 1001 Genomes Project ( http://www.1001genomes.org) in Arabidopsis thaliana. Conclusion In this work we show that indel classification is a necessary step to reduce the number of false positive candidates. We demonstrate that missing classification may lead to spurious biological interpretations. The software is available at: http://agkb.is.tuebingen.mpg.de/Forschung/SV-M/. PMID:23442375

Preliteracy signatures of poor-reading abilities in resting-state EEG

PubMed Central

Schiavone, Giuseppina; Linkenkaer-Hansen, Klaus; Maurits, Natasha M.; Plakas, Anna; Maassen, Ben A. M.; Mansvelder, Huibert D.; van der Leij, Aryan; van Zuijen, Titia L.

2014-01-01

The hereditary character of dyslexia suggests the presence of putative underlying neural anomalies already in preliterate age. Here, we investigated whether early neurophysiological correlates of future reading difficulties—a hallmark of dyslexia—could be identified in the resting-state EEG of preliterate children. The children in this study were recruited at birth and classified on the basis of parents' performance on reading tests to be at-risk of becoming poor readers (n = 48) or not (n = 14). Eyes-open rest EEG was measured at the age of 3 years, and the at-risk children were divided into fluent readers (n = 24) and non-fluent readers (n = 24) after reading assessment at their third grade of school. We found that fluent readers and non-fluent readers differed in normalized spectral amplitude. Non-fluent readers were characterized by lower amplitude in the delta-1 frequency band (0.5–2 Hz) and higher amplitude in the alpha-1 band (6–8 Hz) in multiple scalp regions compared to control and at-risk fluent readers. Interestingly, across groups these EEG biomarkers correlated with several behavioral test scores measured in the third grade. Specifically, the performance on reading fluency, phonological and orthographic tasks and rapid automatized naming task correlated positively with delta-1 and negatively with alpha-1. Together, our results suggest that combining family-risk status, neurophysiological testing and behavioral test scores in a longitudinal setting may help uncover physiological mechanisms implicated with neurodevelopmental disorders such as the predisposition to reading disabilities. PMID:25285075

The Apis mellifera Filamentous Virus Genome

PubMed Central

Gauthier, Laurent; Cornman, Scott; Hartmann, Ulrike; Cousserans, François; Evans, Jay D.; de Miranda, Joachim R.; Neumann, Peter

2015-01-01

A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family. PMID:26184284

The Apis mellifera Filamentous Virus Genome.

PubMed

Gauthier, Laurent; Cornman, Scott; Hartmann, Ulrike; Cousserans, François; Evans, Jay D; de Miranda, Joachim R; Neumann, Peter

2015-07-09

A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family.

Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases.

PubMed Central

Tanaka, T; Kawata, M

1988-01-01

We have isolated a DNA fragment from Bacillus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities for the exoprotease production in the N-terminal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU. Images PMID:3136143

Alternate promoter selection within a human cytomegalovirus immediate-early and early transcription unit (UL119-115) defines true late transcripts containing open reading frames for putative viral glycoproteins.

PubMed Central

Leatham, M P; Witte, P R; Stinski, M F

1991-01-01

The human cytomegalovirus open reading frames (ORFs) UL119 through UL115 (UL119-115) are located downstream of the immediate-early 1 and 2 transcription units. The promoter upstream of UL119 is active at all times after infection and drives the synthesis of a spliced 3.1-kb mRNA. The viral mRNA initiates in UL119, contains UL119-117 and UL116, and terminates just downstream of UL115. True late transcripts that are detected only after viral DNA synthesis originate from this transcription unit. True late mRNAs of 2.1 kb, containing ORFs UL116 and UL115, and 1.2 kb, containing ORF UL115 only, are synthesized. The true late viral mRNAs are 3' coterminal with the 3.1-kb mRNA. This transcription unit is an example of late promoters nested within an immediate-early-early transcription unit. The gene products of UL119-117, UL116, and UL115 are predicted to be glycoproteins. Efficient expression of the downstream ORFs at late times after infection may be related to alternate promoter usage and downstream cap site selection. Images PMID:1717716

Two open reading frames (ORF1 and ORF2) within the 2.0-kilobase latency-associated transcript of herpes simplex virus type 1 are not essential for reactivation from latency.

PubMed Central

Fareed, M U; Spivack, J G

1994-01-01

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are dispensable for establishment and maintenance of latent infection. However, the LATs have been implicated in reactivation of the virus from its latent state. Since the reported LAT deletion and/or insertion variants that are reactivation impaired contain deletions in the putative LAT promoter, it is not known which LAT sequences are involved in reactivation. To examine the role of the 2.0-kb LAT in the process of reactivation and the functional importance of the putative open reading frames (ORF1 and ORF2) contained within the 2.0-kb LAT, we have constructed an HSV-1 variant that contains a precise deletion and insertion within the LAT-specific DNA sequences using site-directed mutagenesis. The HSV-1 variant FS1001K contains an 1,186-bp deletion starting precisely from the 5' end of the 2.0-kb LAT and, for identification, a XbaI restriction endonuclease site insertion. The FS1001K genome contains no other deletions and/or insertions as analyzed by a variety of restriction endonucleases. The deletion in FS1001K removes the entire 556-bp intron within the 2.0-kb LAT, the first 229 nucleotides of ORF1, and the first 159 nucleotides of ORF2 without having an affect on the RL2 (ICP0) gene. Explant cocultivation reactivation assays indicated that this deletion had a minimal effect on reactivation of the variant FS1001K compared with the parental wild-type virus using a mouse eye model. As expected, Northern (RNA) blot analyses have shown that the variant virus (FS1001K) does not produce the 2.0-kb LAT or the 1.45- to 1.5-kb LAT either in vitro or in vivo; however, FS1001K produces an intact RL2 transcript in tissue culture. These data suggest that the 2.0-kb LAT putative ORF1 and ORF2 (or the first 1,186 bp of the 2.0-kb LAT) are dispensable for explant reactivation of latent HSV-1. Images PMID:7966597

The putative visual word form area is functionally connected to the dorsal attention network.

PubMed

Vogel, Alecia C; Miezin, Fran M; Petersen, Steven E; Schlaggar, Bradley L

2012-03-01

The putative visual word form area (pVWFA) is the most consistently activated region in single word reading studies (i.e., Vigneau et al. 2006), yet its function remains a matter of debate. The pVWFA may be predominantly used in reading or it could be a more general visual processor used in reading but also in other visual tasks. Here, resting-state functional connectivity magnetic resonance imaging (rs-fcMRI) is used to characterize the functional relationships of the pVWFA to help adjudicate between these possibilities. rs-fcMRI defines relationships based on correlations in slow fluctuations of blood oxygen level-dependent activity occurring at rest. In this study, rs-fcMRI correlations show little relationship between the pVWFA and reading-related regions but a strong relationship between the pVWFA and dorsal attention regions thought to be related to spatial and feature attention. The rs-fcMRI correlations between the pVWFA and regions of the dorsal attention network increase with age and reading skill, while the correlations between the pVWFA and reading-related regions do not. These results argue the pVWFA is not used predominantly in reading but is a more general visual processor used in other visual tasks, as well as reading.

The Putative Visual Word Form Area Is Functionally Connected to the Dorsal Attention Network

PubMed Central

Miezin, Fran M.; Petersen, Steven E.; Schlaggar, Bradley L.

2012-01-01

The putative visual word form area (pVWFA) is the most consistently activated region in single word reading studies (i.e., Vigneau et al. 2006), yet its function remains a matter of debate. The pVWFA may be predominantly used in reading or it could be a more general visual processor used in reading but also in other visual tasks. Here, resting-state functional connectivity magnetic resonance imaging (rs-fcMRI) is used to characterize the functional relationships of the pVWFA to help adjudicate between these possibilities. rs-fcMRI defines relationships based on correlations in slow fluctuations of blood oxygen level–dependent activity occurring at rest. In this study, rs-fcMRI correlations show little relationship between the pVWFA and reading-related regions but a strong relationship between the pVWFA and dorsal attention regions thought to be related to spatial and feature attention. The rs-fcMRI correlations between the pVWFA and regions of the dorsal attention network increase with age and reading skill, while the correlations between the pVWFA and reading-related regions do not. These results argue the pVWFA is not used predominantly in reading but is a more general visual processor used in other visual tasks, as well as reading. PMID:21690259

Phage phenomics: Physiological approaches to characterize novel viral proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

Phylogenetic relationship of Paenibacillus species based on putative replication origin regions and analysis of an yheCD-like sequence found in this region.

PubMed

Iiyama, Kazuhiro; Otao, Masahiro; Mori, Kazuki; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro; Tashiro, Kousuke; Asano, Shin-Ichiro; Yasunaga-Aoki, Chisa

2014-01-01

To determine the phylogenetic relationship among Paenibacillus species, putative replication origin regions were compared. In the rsmG-gyrA region, gene arrangements in Paenibacillus species were identical to those of Bacillus species, with the exception of an open reading frame (orf14) positioned between gyrB and gyrA, which was observed only in Paenibacillus species. The orf14 product was homologous to the endospore-associated proteins YheC and YheD of Bacillus subtilis. Phylogenetic analysis based on the YheCD proteins suggested that Orf14 could be categorized into the YheC group. In the Paenibacillus genome, DnaA box clusters were found in rpmH-dnaA and dnaA-dnaN intergenic regions, known as box regions C and R, respectively; this localization was similar to that observed in B. halodurans. A phylogenetic tree based on the nucleotide sequences of the whole replication origin regions suggested that P. popilliae, P. thiaminolyticus, and P. dendritiformis are closely related species.

Phage phenomics: Physiological approaches to characterize novel viral proteins

DOE PAGES

Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; ...

2015-06-11

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

Transcriptome identification of putative genes involved in protein catabolism and innate immune response in human body louse (Pediculicidae: Pediculus humanus).

PubMed

Pedra, Joao H F; Brandt, Amanda; Li, Hong-Mei; Westerman, Rick; Romero-Severson, Jeanne; Pollack, Richard J; Murdock, Larry L; Pittendrigh, Barry R

2003-11-01

Genomics information relating to human body lice is surprisingly scarce, and this has constrained studies of their physiology, immunology and vector biology. To identify novel body louse genes, we used engorged adult lice to generate a cDNA library. Initially, 1152 clones were screened for inserts, edited for removal of vector sequences and base pairs of poor quality, and viewed for splicing variations, gene families and polymorphism. Computational methods identified 506 inferred open reading frames including the first predicted louse defensin. The inferred defensin aligns well with other insect defensins and has highly conserved cysteine residues, as are known for other defensin sequences. Two cysteine and five serine proteinases were categorized according to their inferred catalytic sites. We also discovered seven putative ubiquitin-pathway genes and four iron metabolizing deduced enzymes. Finally, glutathione-S-transferases and cytochrome P450 genes were among the detoxification enzymes found. Results from this first systematic effort to discover human body louse genes should promote further studies in Phthiraptera and lice.

Molecular cloning of a putative gene encoding isopentenyltransferase from pingyitiancha (Malus hupehensis) and characterization of its response to nitrate.

PubMed

Peng, Jing; Peng, Futian; Zhu, Chunfu; Wei, Shaochong

2008-06-01

A putative isopentenyltransferase (IPT) encoding gene was identified from a pingyitiancha (Malus hupehensis Rehd.) expressed sequence tag database, and the full-length gene was cloned by RACE. Based on expression profile and sequence alignment, the nucleotide sequence of the clone, named MhIPT3, was most similar to AtIPT3, an IPT gene in Arabidopsis. The full-length cDNA contained a 963-bp open reading frame encoding a protein of 321 amino acids with a molecular mass of 37.3 kDa. Sequence analysis of genomic DNA revealed the absence of introns in the frame. Quantitative real-time PCR analysis demonstrated that the gene was expressed in roots, stems and leaves. Application of nitrate to roots of nitrogen-deprived seedlings strongly induced expression of MhIPT3 and was accompanied by the accumulation of cytokinins, whereas MhIPT3 expression was little affected by ammonium application to roots of nitrogen-deprived seedlings. Application of nitrate to leaves also up-regulated the expression of MhIPT3 and corresponded closely with the accumulation of isopentyladenine and isopentyladenosine in leaves.

Genetic and Biochemical Map for the Biosynthesis of Occidiofungin, an Antifungal Produced by Burkholderia contaminans Strain MS14 ▿†

PubMed Central

Gu, Ganyu; Smith, Leif; Liu, Aixin; Lu, Shi-En

2011-01-01

A striking feature of Burkholderia contaminans strain MS14 is the production of a glycolipopeptide named occidiofungin. Occidiofungin has a broad range of antifungal activities against plant and animal pathogens. In this study, a complete covalent structure characterization and identification of the whole genomic DNA region for the occidiofungin gene (ocf) cluster are described. Discovery of the presence of 2,4-diaminobutyric acid and 3-chloro-β-hydroxytyrosine and elucidation of the structure of a novel C18 fatty amino acid residue have been achieved. In addition, seven additional putative open reading frames (the genes from ocfI to ocfN [ocfI-N] and ORF16) were identified. Transcription of all the putative genes ocfI-N identified in the region except ORF16 was regulated by both ambR1 and ambR2. Elucidation of the structure and the ocf gene cluster provides insight into the biosynthesis of occidiofungin and promotes future aims at understanding the biosynthetic machinery. This work provides new avenues for optimizing the production and synthesis of structural analogs of occidiofungin. PMID:21742901

Nucleotide sequences and regulational analysis of genes involved in conversion of aniline to catechol in Pseudomonas putida UCC22(pTDN1).

PubMed Central

Fukumori, F; Saint, C P

1997-01-01

A 9,233-bp HindIII fragment of the aromatic amine catabolic plasmid pTDN1, isolated from a derivative of Pseudomonas putida mt-2 (UCC22), confers the ability to degrade aniline on P. putida KT2442. The fragment encodes six open reading frames which are arranged in the same direction. Their 5' upstream region is part of the direct-repeat sequence of pTDN1. Nucleotide sequence of 1.8 kb of the repeat sequence revealed only a single base pair change compared to the known sequence of IS1071 which is involved in the transposition of the chlorobenzoate genes (C. Nakatsu, J. Ng, R. Singh, N. Straus, and C. Wyndham, Proc. Natl. Acad. Sci. USA 88:8312-8316, 1991). Four open reading frames encode proteins with considerable homology to proteins found in other aromatic-compound degradation pathways. On the basis of sequence similarity, these genes are proposed to encode the large and small subunits of aniline oxygenase (tdnA1 and tdnA2, respectively), a reductase (tdnB), and a LysR-type regulatory gene (tdnR). The putative large subunit has a conserved [2Fe-2S]R Rieske-type ligand center. Two genes, tdnQ and tdnT, which may be involved in amino group transfer, are localized upstream of the putative oxygenase genes. The tdnQ gene product shares about 30% similarity with glutamine synthetases; however, a pUC-based plasmid carrying tdnQ did not support the growth of an Escherichia coli glnA strain in the absence of glutamine. TdnT possesses domains that are conserved among amidotransferases. The tdnQ, tdnA1, tdnA2, tdnB, and tdnR genes are essential for the conversion of aniline to catechol. PMID:8990291

Inactivation of vimF, a Putative Glycosyltransferase Gene Downstream of vimE, Alters Glycosylation and Activation of the Gingipains in Porphyromonas gingivalis W83

PubMed Central

Vanterpool, Elaine; Roy, Francis; Fletcher, Hansel M.

2005-01-01

Regulation/activation of the Porphyromonas gingivalis gingipains is poorly understood. A 1.2-kb open reading frame, a putative glycosyltransferase, downstream of vimE, was cloned, insertionally inactivated using the ermF-ermAM antibiotic resistance cassette, and used to create a defective mutant by allelic exchange. In contrast to the wild-type W83 strain, this mutant, designated P. gingivalis FLL95, was nonpigmented and nonhemolytic when plated on Brucella blood agar. Arginine- and lysine-specific gingipain activities were reduced by approximately 97% and 96%, respectively, relative to that of the parent strain. These activities were unaffected by the growth phase, in contrast to the vimA-defective mutant P. gingivalis FLL92. Expression of the rgpA, rgpB, and kgp gingipain genes was unaffected in P. gingivalis FLL95 in comparison to the wild-type strain. In nonactive gingipain extracellular protein fractions, multiple high-molecular-weight proteins immunoreacted with gingipain-specific antibodies. The specific gingipain-associated sugar moiety recognized by monoclonal antibody 1B5 was absent in FLL95. Taken together, these results suggest that the vimE downstream gene, designated vimF (virulence modulating gene F), which is a putative glycosyltransferase group 1, is involved in the regulation of the major virulence factors of P. gingivalis. PMID:15972484

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in a urease-positive thermophilic Campylobacter sp. (UPTC).

PubMed

Tasaki, E; Hirayama, J; Tazumi, A; Hayashi, K; Hara, Y; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

2012-02-01

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33.

PubMed

Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

2016-08-02

Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F₁-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata.

Nucleotide sequence and transcriptional start site of the Methylobacterium organophilum XX methanol dehydrogenase structural gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Machlin, S.M.; Hanson, R.S.

The nucleotide sequence of a cloned 2.5-kilobase-pair SmaI fragment containing the methanol dehydrogenase (MDH) structural gene from Methylobacterium organophilum XX was determined. A single open reading frame with a coding capacity of 626 amino acids (molecular weight, 66,000) was identified on one stand, and N-terminal sequencing of purified MDH revealed that 27 of these residues constituted a putative signal peptide. Primer extension mapping of in vivo transcripts indicated that the start of mRNA synthesis was 160 to 170 base pairs upstream of the ATG codon. Northern (RNA) blot analysis further demonstrated that the transcript was 2.1 kilobase pairs in lengthmore » and therefore appeared to encode only MDH.« less

Reading Poverty.

ERIC Educational Resources Information Center

Shannon, Patrick

The central purpose of this book is to challenge current social constructions of poverty, reading education, and the putative relationship between the two. It explores how official and popular representations of poverty are bound to specific historical, social, and economic conditions of their own production. The book offers four stances of…

In silico prediction of the G-protein coupled receptors expressed during the metamorphic molt of Sagmariasus verreauxi (Crustacea: Decapoda) by mining transcriptomic data: RNA-seq to repertoire.

PubMed

Buckley, Sean J; Fitzgibbon, Quinn P; Smith, Gregory G; Ventura, Tomer

2016-03-01

Against a backdrop of food insecurity, the farming of decapod crustaceans is a rapidly expanding and globally significant source of food protein. Sagmariasus verreauxi spiny lobster, the subject of this study, are decapods of underdeveloped aquaculture potential. Crustacean neuropeptide G-protein coupled receptors (GPCRs) mediate endocrine pathways that are integral to animal fecundity, growth and survival. The potential use of novel biotechnologies to enhance GPCR-mediated physiology may assist in improving the health and productivity of farmed decapod populations. This study catalogues the GPCRs expressed in the early developmental stages, as well as adult tissues, with a view to illuminating key neuropeptide receptors. De novo assembled contiguous sequences generated from transcriptomic reads of metamorphic and post metamorphic S. verreauxi were filtered for seven transmembrane domains, and used as a reference for iterative re-mapping. Subsequent putative GPCR open reading frames (ORFs) were BLAST annotated, categorised, and compared to published orthologues based on phylogenetic analysis. A total of 85 GPCRs were digitally predicted, that represented each of the four arthropod subfamilies. They generally displayed low-level and non-differential metamorphic expression with few exceptions that we examined using RT-PCR and qPCR. Two putative CHH-like neuropeptide receptors were annotated. Three dimensional structural modelling suggests that these receptors exhibit a conserved extracellular ligand binding pocket, providing support to the notion that these receptors co-evolved with their ligands across Decapoda. This perhaps narrows the search for means to increase productivity of farmed decapod populations. Copyright © 2016 Elsevier Inc. All rights reserved.

Pathogenic and multidrug-resistant Escherichia fergusonii from broiler chicken.

PubMed

Forgetta, V; Rempel, H; Malouin, F; Vaillancourt, R; Topp, E; Dewar, K; Diarra, M S

2012-02-01

An Escherichia spp. isolate, ECD-227, was previously identified from the broiler chicken as a phylogenetically divergent and multidrug-resistant Escherichia coli possessing numerous virulence genes. In this study, whole genome sequencing and comparative genome analysis was used to further characterize this isolate. The presence of known and putative antibiotic resistance and virulence open reading frames were determined by comparison to pathogenic (E. coli O157:H7 TW14359, APEC O1:K1:H7, and UPEC UTI89) and nonpathogenic species (E. coli K-12 MG1655 and Escherichia fergusonii ATCC 35469). The assembled genome size of 4.87 Mb was sequenced to 18-fold depth of coverage and predicted to contain 4,376 open reading frames. Phylogenetic analysis of 537 open reading frames present across 110 enteric bacterial species identifies ECD-227 to be E. fergusonii. The genome of ECD-227 contains 5 plasmids showing similarity to known E. coli and Salmonella enterica plasmids. The presence of virulence and antibiotic resistance genes were identified and localized to the chromosome and plasmids. The mutation in gyrA (S83L) involved in fluoroquinolone resistance was identified. The Salmonella-like plasmids harbor antibiotic resistance genes on a class I integron (aadA, qacEΔ-sul1, aac3-VI, and sulI) as well as numerous virulence genes (iucABCD, sitABCD, cib, traT). In addition to the genome analysis, the virulence of ECD-227 was evaluated in a 1-d-old chick model. In the virulence assay, ECD-227 was found to induce 18 to 30% mortality in 1-d-old chicks after 24 h and 48 h of infection, respectively. This study documents an avian multidrug-resistant and virulent E. fergusonii. The existence of several resistance genes to multiple classes of antibiotics indicates that infection caused by ECD-227 would be difficult to treat using antimicrobials currently available for poultry.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. In thismore » paper, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q 6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Finally, given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.« less

Molecular cloning and expression of rat liver bile acid CoA ligase.

PubMed

Falany, Charles N; Xie, Xiaowei; Wheeler, James B; Wang, Jin; Smith, Michelle; He, Dongning; Barnes, Stephen

2002-12-01

Bile acid CoA ligase (BAL) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5'-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3'-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase. In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates.

Assessment of bacterial and archaeal community structure in Swine wastewater treatment processes.

PubMed

Da Silva, Marcio Luis Busi; Cantão, Mauricio Egídio; Mezzari, Melissa Paola; Ma, Jie; Nossa, Carlos Wolfgang

2015-07-01

Microbial communities from two field-scale swine wastewater treatment plants (WWTPs) were assessed by pyrosequencing analyses of bacterial and archaeal 16S ribosomal DNA (rDNA) fragments. Effluent samples from secondary (anaerobic covered lagoons and upflow anaerobic sludge blanket [UASB]) and tertiary treatment systems (open-pond natural attenuation lagoon and air-sparged nitrification-denitrification tank followed by alkaline phosphorus precipitation process) were analyzed. A total of 56,807 and 48,859 high-quality reads were obtained from bacterial and archaeal libraries, respectively. Dominant bacterial communities were associated with the phylum Firmicutes, Bacteroidetes, Proteobacteria, or Actinobacteria. Bacteria and archaea diversity were highest in UASB effluent sample. Escherichia, Lactobacillus, Bacteroides, and/or Prevotella were used as indicators of putative pathogen reduction throughout the WWTPs. Satisfactory pathogen reduction was observed after the open-pond natural attenuation lagoon but not after the air-sparged nitrification/denitrification followed by alkaline phosphorus precipitation treatment processes. Among the archaeal communities, 80% of the reads was related to hydrogeno-trophic methanogens Methanospirillum. Enrichment of hydrogenotrophic methanogens detected in effluent samples from the anaerobic covered lagoons and UASB suggested that CO2 reduction with H2 was the dominant methanogenic pathway in these systems. Overall, the results served to improve our current understanding of major microbial communities' changes downgradient from the pen and throughout swine WWTP as a result of different treatment processes.

Standing Genetic Variation in Contingency Loci Drives the Rapid Adaptation of Campylobacter jejuni to a Novel Host

PubMed Central

Jerome, John P.; Bell, Julia A.; Plovanich-Jones, Anne E.; Barrick, Jeffrey E.; Brown, C. Titus; Mansfield, Linda S.

2011-01-01

The genome of the food-borne pathogen Campylobacter jejuni contains multiple highly mutable sites, or contingency loci. It has been suggested that standing variation at these loci is a mechanism for rapid adaptation to a novel environment, but this phenomenon has not been shown experimentally. In previous work we showed that the virulence of C. jejuni NCTC11168 increased after serial passage through a C57BL/6 IL-10-/- mouse model of campylobacteriosis. Here we sought to determine the genetic basis of this adaptation during passage. Re-sequencing of the 1.64Mb genome to 200-500X coverage allowed us to define variation in 23 contingency loci to an unprecedented depth both before and after in vivo adaptation. Mutations in the mouse-adapted C. jejuni were largely restricted to the homopolymeric tracts of thirteen contingency loci. These changes cause significant alterations in open reading frames of genes in surface structure biosynthesis loci and in genes with only putative functions. Several loci with open reading frame changes also had altered transcript abundance. The increase in specific phases of contingency loci during in vivo passage of C. jejuni, coupled with the observed virulence increase and the lack of other types of genetic changes, is the first experimental evidence that these variable regions play a significant role in C. jejuni adaptation and virulence in a novel host. PMID:21283682

Identification of 28 cytochrome P450 genes from the transcriptome of the marine rotifer Brachionus plicatilis and analysis of their expression.

PubMed

Kim, Hui-Su; Han, Jeonghoon; Kim, Hee-Jin; Hagiwara, Atsushi; Lee, Jae-Seong

2017-09-01

Whole transcriptomes of the rotifer Brachionus plicatilis were analyzed using an Illumina sequencer. De novo assembly was performed with 49,122,780 raw reads using Trinity software. Among the assembled 42,820 contigs, 27,437 putative open reading frame contigs were identified (average length 1235bp; N50=1707bp). Functional gene annotation with Gene Ontology and InterProScan, in addition to Kyoto Encyclopedia of Genes and Genomes pathway analysis, highlighted the metabolism of xenobiotics by cytochrome P450 (CYP). In addition, 28 CYP genes were identified, and their transcriptional responses to benzo[α]pyrene (B[α]P) were investigated. Most of the CYPs were significantly upregulated or downregulated (P<0.05) in response to B[α]P, suggesting that Bp-CYP genes play a crucial role in detoxification mechanisms in response to xenobiotics. This study sheds light on the molecular defense mechanisms of the rotifer B. plicatilis in response to exposure to various chemicals. Copyright © 2017 Elsevier Inc. All rights reserved.

Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence

PubMed Central

2011-01-01

Background Many plants have large and complex genomes with an abundance of repeated sequences. Many plants are also polyploid. Both of these attributes typify the genome architecture in the tribe Triticeae, whose members include economically important wheat, rye and barley. Large genome sizes, an abundance of repeated sequences, and polyploidy present challenges to genome-wide SNP discovery using next-generation sequencing (NGS) of total genomic DNA by making alignment and clustering of short reads generated by the NGS platforms difficult, particularly in the absence of a reference genome sequence. Results An annotation-based, genome-wide SNP discovery pipeline is reported using NGS data for large and complex genomes without a reference genome sequence. Roche 454 shotgun reads with low genome coverage of one genotype are annotated in order to distinguish single-copy sequences and repeat junctions from repetitive sequences and sequences shared by paralogous genes. Multiple genome equivalents of shotgun reads of another genotype generated with SOLiD or Solexa are then mapped to the annotated Roche 454 reads to identify putative SNPs. A pipeline program package, AGSNP, was developed and used for genome-wide SNP discovery in Aegilops tauschii-the diploid source of the wheat D genome, and with a genome size of 4.02 Gb, of which 90% is repetitive sequences. Genomic DNA of Ae. tauschii accession AL8/78 was sequenced with the Roche 454 NGS platform. Genomic DNA and cDNA of Ae. tauschii accession AS75 was sequenced primarily with SOLiD, although some Solexa and Roche 454 genomic sequences were also generated. A total of 195,631 putative SNPs were discovered in gene sequences, 155,580 putative SNPs were discovered in uncharacterized single-copy regions, and another 145,907 putative SNPs were discovered in repeat junctions. These SNPs were dispersed across the entire Ae. tauschii genome. To assess the false positive SNP discovery rate, DNA containing putative SNPs was amplified by PCR from AL8/78 and AS75 and resequenced with the ABI 3730 xl. In a sample of 302 randomly selected putative SNPs, 84.0% in gene regions, 88.0% in repeat junctions, and 81.3% in uncharacterized regions were validated. Conclusion An annotation-based genome-wide SNP discovery pipeline for NGS platforms was developed. The pipeline is suitable for SNP discovery in genomic libraries of complex genomes and does not require a reference genome sequence. The pipeline is applicable to all current NGS platforms, provided that at least one such platform generates relatively long reads. The pipeline package, AGSNP, and the discovered 497,118 Ae. tauschii SNPs can be accessed at (http://avena.pw.usda.gov/wheatD/agsnp.shtml). PMID:21266061

Transcriptome Analysis of Nine Tissues to Discover Genes Involved in the Biosynthesis of Active Ingredients in Sophora flavescens.

PubMed

Han, Rongchun; Takahashi, Hiroki; Nakamura, Michimi; Bunsupa, Somnuk; Yoshimoto, Naoko; Yamamoto, Hirobumi; Suzuki, Hideyuki; Shibata, Daisuke; Yamazaki, Mami; Saito, Kazuki

2015-01-01

Sophora flavescens AITON (kurara) has long been used to treat various diseases. Although several research findings revealed the biosynthetic pathways of its characteristic chemical components as represented by matrine, insufficient analysis of transcriptome data hampered in-depth analysis of the underlying putative genes responsible for the biosynthesis of pharmaceutical chemical components. In this study, more than 200 million fastq format reads were generated by Illumina's next-generation sequencing approach using nine types of tissue from S. flavescens, followed by CLC de novo assembly, ultimately yielding 83,325 contigs in total. By mapping the reads back to the contigs, reads per kilobase of the transcript per million mapped reads values were calculated to demonstrate gene expression levels, and overrepresented gene ontology terms were evaluated using Fisher's exact test. In search of the putative genes relevant to essential metabolic pathways, all 1350 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. By analyzing expression patterns, we proposed some candidate genes involved in the biosynthesis of isoflavonoids and quinolizidine alkaloids. Adopting RNA-Seq analysis, we obtained substantially credible contigs for downstream work. The preferential expression of the gene for putative lysine/ornithine decarboxylase committed in the initial step of matrine biosynthesis in leaves and stems was confirmed in semi-quantitative polymerase chain reaction (PCR) analysis. The findings in this report may serve as a stepping-stone for further research into this promising medicinal plant.

Detailed analysis of metagenome datasets obtained from biogas-producing microbial communities residing in biogas reactors does not indicate the presence of putative pathogenic microorganisms

PubMed Central

2013-01-01

Background In recent years biogas plants in Germany have been supposed to be involved in amplification and dissemination of pathogenic bacteria causing severe infections in humans and animals. In particular, biogas plants are discussed to contribute to the spreading of Escherichia coli infections in humans or chronic botulism in cattle caused by Clostridium botulinum. Metagenome datasets of microbial communities from an agricultural biogas plant as well as from anaerobic lab-scale digesters operating at different temperatures and conditions were analyzed for the presence of putative pathogenic bacteria and virulence determinants by various bioinformatic approaches. Results All datasets featured a low abundance of reads that were taxonomically assigned to the genus Escherichia or further selected genera comprising pathogenic species. Higher numbers of reads were taxonomically assigned to the genus Clostridium. However, only very few sequences were predicted to originate from pathogenic clostridial species. Moreover, mapping of metagenome reads to complete genome sequences of selected pathogenic bacteria revealed that not the pathogenic species itself, but only species that are more or less related to pathogenic ones are present in the fermentation samples analyzed. Likewise, known virulence determinants could hardly be detected. Only a marginal number of reads showed similarity to sequences described in the Microbial Virulence Database MvirDB such as those encoding protein toxins, virulence proteins or antibiotic resistance determinants. Conclusions Findings of this first study of metagenomic sequence reads of biogas producing microbial communities suggest that the risk of dissemination of pathogenic bacteria by application of digestates from biogas fermentations as fertilizers is low, because obtained results do not indicate the presence of putative pathogenic microorganisms in the samples analyzed. PMID:23557021

Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae.

PubMed

Koibuchi, K; Nagasaki, H; Yuasa, A; Kataoka, J; Kitamoto, K

2000-07-01

A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of L-glutamine but also of D-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137-143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5'-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae.

Genetic Diversity of Avian Paramyxovirus Type 6 Isolated from Wild Ducks in the Republic of Korea.

PubMed

Choi, Kang-Seuk; Kim, Ji-Ye; Lee, Hyun-Jeong; Jang, Min-Jun; Kwon, Hyuk-Moo; Sung, Haan-Woo

2018-03-08

Eleven avian paramyxovirus type 6 (APMV-6) isolates from Eurasian Wigeon ( n=5; Anas penelope), Mallards ( n=2; Anas platyrhynchos), and unknown species of wild ducks ( n=4) from Korea were analyzed based on the nucleotide (nt) and deduced amino acid (aa) sequences of the fusion (F) gene. Fecal samples were collected in 2010-2014. Genotypes were assigned based on phylogenetic analyses. Our results revealed that APMV-6 could be classified into at least two distinct genotypes, G1 and G2. The open reading frame (ORF) of the G1 genotype was 1,668 nt in length, and the putative F0 cleavage site sequence was 113 PAPEPRL 119 . The G2 genotype viruses included five isolates from Eurasian wigeons and four isolates from unknown waterfowl species, together with two reference APMV-6 strains from the Red-necked Stint ( Calidris ruficollis) from Japan and an unknown duck from Italy. There was an N-truncated ORF (1,638 nt), due to an N-terminal truncation of 30 nt in the signal peptide region of the F gene, and the putative F0 cleavage site sequence was 103 SIREPRL 109 . The genetic diversity and ecology of APMV-6 are discussed.

Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

PubMed

Ferriol, I; Silva Junior, D M; Nigg, J C; Zamora-Macorra, E J; Falk, B W

2016-11-01

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of the Genome of the Sexually Transmitted Insect Virus Helicoverpa zea Nudivirus 2

PubMed Central

Burand, John P.; Kim, Woojin; Afonso, Claudio L.; Tulman, Edan R.; Kutish, Gerald F.; Lu, Zhiqiang; Rock, Daniel L.

2012-01-01

The sexually transmitted insect virus Helicoverpa zea nudivirus 2 (HzNV-2) was determined to have a circular double-stranded DNA genome of 231,621 bp coding for an estimated 113 open reading frames (ORFs). HzNV-2 is most closely related to the nudiviruses, a sister group of the insect baculoviruses. Several putative ORFs that share homology with the baculovirus core genes were identified in the viral genome. However, HzNV-2 lacks several key genetic features of baculoviruses including the late transcriptional regulation factor, LEF-1 and the palindromic hrs, which serve as origins of replication. The HzNV-2 genome was found to code for three ORFs that had significant sequence homology to cellular genes which are not generally found in viral genomes. These included a presumed juvenile hormone esterase gene, a gene coding for a putative zinc-dependent matrix metalloprotease, and a major facilitator superfamily protein gene; all of which are believed to play a role in the cellular proliferation and the tissue hypertrophy observed in the malformation of reproductive organs observed in HzNV-2 infected corn earworm moths, Helicoverpa zea. PMID:22355451

Genome sequence of an enhancin gene-rich nucleopolyhedrovirus (NPV) from Agrotis segetum: collinearity with Spodoptera exigua multiple NPV.

PubMed

Jakubowska, Agata K; Peters, Sander A; Ziemnicka, Jadwiga; Vlak, Just M; van Oers, Monique M

2006-03-01

The genome sequence of a Polish isolate of Agrotis segetum nucleopolyhedrovirus (AgseNPV-A) was determined and analysed. The circular genome is composed of 147,544 bp and has a G+C content of 45.7 mol%. It contains 153 putative, non-overlapping open reading frames (ORFs) encoding predicted proteins of more than 50 aa, together making up 89.8 % of the genome. The remaining 10.2 % of the DNA constitutes non-coding regions and homologous-repeat regions. One hundred and forty-three AgseNPV-A ORFs are homologues of previously reported baculovirus gene sequences. There are ten unique ORFs and they account for 3 % of the genome in total. All 62 lepidopteran baculovirus genes, including the 29 core baculovirus genes, were found in the AgseNPV-A genome. The gene content and gene order of AgseNPV-A are most similar to those of Spodoptera exigua (Se) multiple NPV and their shared homologous genes are 100 % collinear. Three putative enhancin genes were identified in the AgseNPV-A genome. In phylogenetic analysis, the AgseNPV-A enhancins form a cluster separated from enhancins of the Mamestra species NPVs.

Analysis of expressed sequence tags from Uromyces appendiculatus hyphae and haustoria and their comparison to sequences from other rust fungi.

PubMed

Puthoff, D P; Neelam, A; Ehrenfried, M L; Scheffler, B E; Ballard, L; Song, Q; Campbell, K B; Cooper, B; Tucker, M L

2008-10-01

Hyphae, 2 to 8 days postinoculation (dpi), and haustoria, 5 dpi, were isolated from Uromyces appendiculatus infected bean leaves (Phaseolus vulgaris cv. Pinto 111) and a separate cDNA library prepared for each fungal preparation. Approximately 10,000 hyphae and 2,700 haustoria clones were sequenced from both the 5' and 3' ends. Assembly of all of the fungal sequences yielded 3,359 contigs and 927 singletons. The U. appendiculatus sequences were compared with sequence data for other rust fungi, Phakopsora pachyrhizi, Uromyces fabae, and Puccinia graminis. The U. appendiculatus haustoria library included a large number of genes with unknown cellular function; however, summation of sequences of known cellular function suggested that haustoria at 5 dpi had fewer transcripts linked to protein synthesis in favor of energy metabolism and nutrient uptake. In addition, open reading frames in the U. appendiculatus data set with an N-terminal signal peptide were identified and compared with other proteins putatively secreted from rust fungi. In this regard, a small family of putatively secreted RTP1-like proteins was identified in U. appendiculatus and P. graminis.

Analysis of the genome of the sexually transmitted insect virus Helicoverpa zea nudivirus 2.

PubMed

Burand, John P; Kim, Woojin; Afonso, Claudio L; Tulman, Edan R; Kutish, Gerald F; Lu, Zhiqiang; Rock, Daniel L

2012-01-01

The sexually transmitted insect virus Helicoverpa zea nudivirus 2 (HzNV-2) was determined to have a circular double-stranded DNA genome of 231,621 bp coding for an estimated 113 open reading frames (ORFs). HzNV-2 is most closely related to the nudiviruses, a sister group of the insect baculoviruses. Several putative ORFs that share homology with the baculovirus core genes were identified in the viral genome. However, HzNV-2 lacks several key genetic features of baculoviruses including the late transcriptional regulation factor, LEF-1 and the palindromic hrs, which serve as origins of replication. The HzNV-2 genome was found to code for three ORFs that had significant sequence homology to cellular genes which are not generally found in viral genomes. These included a presumed juvenile hormone esterase gene, a gene coding for a putative zinc-dependent matrix metalloprotease, and a major facilitator superfamily protein gene; all of which are believed to play a role in the cellular proliferation and the tissue hypertrophy observed in the malformation of reproductive organs observed in HzNV-2 infected corn earworm moths, Helicoverpa zea.

Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

PubMed

Adewoye, L O; Worobec, E A

2000-08-08

The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins.

Molecular Characterization of a Novel Species of Capillovirus from Japanese Apricot (Prunus mume)

PubMed Central

Faure, Chantal; Theil, Sébastien; Candresse, Thierry

2018-01-01

With the increased use of high-throughput sequencing methods, new viruses infecting Prunus spp. are being discovered and characterized, especially in the family Betaflexiviridae. Double-stranded RNAs from symptomatic leaves of a Japanese apricot (Prunus mume) tree from Japan were purified and analyzed by Illumina sequencing. Blast comparisons of reconstructed contigs showed that the P. mume sample was infected by a putative novel virus with homologies to Cherry virus A (CVA) and to the newly described Currant virus A (CuVA), both members of genus Capillovirus. Completion of the genome showed the new agent to have a genomic organization typical of capilloviruses, with two overlapping open reading frames encoding a large replication-associated protein fused to the coat protein (CP), and a putative movement protein (MP). This virus shares only, respectively, 63.2% and 62.7% CP amino acid identity with the most closely related viruses, CVA and CuVA. Considering the species demarcation criteria in the family and phylogenetic analyses, this virus should be considered as representing a new viral species in the genus Capillovirus, for which the name of Mume virus A is proposed. PMID:29570605

In silico Prediction, in vitro Antibacterial Spectrum, and Physicochemical Properties of a Putative Bacteriocin Produced by Lactobacillus rhamnosus Strain L156.4

PubMed Central

Oliveira, Letícia de C.; Silveira, Aline M. M.; Monteiro, Andréa de S.; dos Santos, Vera L.; Nicoli, Jacques R.; Azevedo, Vasco A. de C.; Soares, Siomar de C.; Dias-Souza, Marcus V.; Nardi, Regina M. D.

2017-01-01

A bacteriocinogenic Lactobacillus rhamnosus L156.4 strain isolated from the feces of NIH mice was identified by 16S rRNA gene sequencing and MALDI-TOF mass spectrometry. The entire genome was sequenced using Illumina, annotated in the PGAAP, and RAST servers, and deposited. Conserved genes associated with bacteriocin synthesis were predicted using BAGEL3, leading to the identification of an open reading frame (ORF) that shows homology with the L. rhamnosus GG (ATCC 53103) prebacteriocin gene. The encoded protein contains a conserved protein motif associated a structural gene of the Enterocin A superfamily. We found ORFs related to the prebacteriocin, immunity protein, ABC transporter proteins, and regulatory genes with 100% identity to those of L. rhamnosus HN001. In this study, we provide evidence of a putative bacteriocin produced by L. rhamnosus L156.4 that was further confirmed by in vitro assays. The antibacterial activity of the substances produced by this strain was evaluated using the deferred agar-spot and spot-on-the lawn assays, and a wide antimicrobial activity spectrum against human and foodborne pathogens was observed. The physicochemical characterization of the putative bacteriocin indicated that it was sensitive to proteolytic enzymes, heat stable and maintained its antibacterial activity in a pH ranging from 3 to 9. The activity against Lactobacillus fermentum, which was used as an indicator strain, was detected during bacterial logarithmic growth phase, and a positive correlation was confirmed between bacterial growth and production of the putative bacteriocin. After a partial purification from cell-free supernatant by salt precipitation, the putative bacteriocin migrated as a diffuse band of approximately 1.0–3.0 kDa by SDS-PAGE. Additional studies are being conducted to explore its use in the food industry for controlling bacterial growth and for probiotic applications. PMID:28579977

Genomic Organization and Molecular Analysis of Virulent Bacteriophage 2972 Infecting an Exopolysaccharide-Producing Streptococcus thermophilus Strain

PubMed Central

Lévesque, Céline; Duplessis, Martin; Labonté, Jessica; Labrie, Steve; Fremaux, Christophe; Tremblay, Denise; Moineau, Sylvain

2005-01-01

The Streptococcus thermophilus virulent pac-type phage 2972 was isolated from a yogurt made in France in 1999. It is a representative of several phages that have emerged with the industrial use of the exopolysaccharide-producing S. thermophilus strain RD534. The genome of phage 2972 has 34,704 bp with an overall G+C content of 40.15%, making it the shortest S. thermophilus phage genome analyzed so far. Forty-four open reading frames (ORFs) encoding putative proteins of 40 or more amino acids were identified, and bioinformatic analyses led to the assignment of putative functions to 23 ORFs. Comparative genomic analysis of phage 2972 with the six other sequenced S. thermophilus phage genomes confirmed that the replication module is conserved and that cos- and pac-type phages have distinct structural and packaging genes. Two group I introns were identified in the genome of 2972. They interrupted the genes coding for the putative endolysin and the terminase large subunit. Phage mRNA splicing was demonstrated for both introns, and the secondary structures were predicted. Eight structural proteins were also identified by N-terminal sequencing and/or matrix-assisted laser desorption ionization—time-of-flight mass spectrometry. Detailed analysis of the putative minor tail proteins ORF19 and ORF21 as well as the putative receptor-binding protein ORF20 showed the following interesting features: (i) ORF19 is a hybrid protein, because it displays significant identity with both pac- and cos-type phages; (ii) ORF20 is unique; and (iii) a protein similar to ORF21 of 2972 was also found in the structure of the cos-type phage DT1, indicating that this structural protein is present in both S. thermophilus phage groups. The implications of these findings for phage classification are discussed. PMID:16000821

Intrinsic and extrinsic approaches for detecting genes in a bacterial genome.

PubMed Central

Borodovsky, M; Rudd, K E; Koonin, E V

1994-01-01

The unannotated regions of the Escherichia coli genome DNA sequence from the EcoSeq6 database, totaling 1,278 'intergenic' sequences of the combined length of 359,279 basepairs, were analyzed using computer-assisted methods with the aim of identifying putative unknown genes. The proposed strategy for finding new genes includes two key elements: i) prediction of expressed open reading frames (ORFs) using the GeneMark method based on Markov chain models for coding and non-coding regions of Escherichia coli DNA, and ii) search for protein sequence similarities using programs based on the BLAST algorithm and programs for motif identification. A total of 354 putative expressed ORFs were predicted by GeneMark. Using the BLASTX and TBLASTN programs, it was shown that 208 ORFs located in the unannotated regions of the E. coli chromosome are significantly similar to other protein sequences. Identification of 182 ORFs as probable genes was supported by GeneMark and BLAST, comprising 51.4% of the GeneMark 'hits' and 87.5% of the BLAST 'hits'. 73 putative new genes, comprising 20.6% of the GeneMark predictions, belong to ancient conserved protein families that include both eubacterial and eukaryotic members. This value is close to the overall proportion of highly conserved sequences among eubacterial proteins, indicating that the majority of the putative expressed ORFs that are predicted by GeneMark, but have no significant BLAST hits, nevertheless are likely to be real genes. The majority of the putative genes identified by BLAST search have been described since the release of the EcoSeq6 database, but about 70 genes have not been detected so far. Among these new identifications are genes encoding proteins with a variety of predicted functions including dehydrogenases, kinases, several other metabolic enzymes, ATPases, rRNA methyltransferases, membrane proteins, and different types of regulatory proteins. Images PMID:7984428

Transcription of the cottontail rabbit papillomavirus early region and identification of two E6 polypeptides in COS-7 cells.

PubMed Central

Barbosa, M S; Wettstein, F O

1987-01-01

Cottontail rabbit papillomavirus (CRPV) early proteins are present at very low levels in virus-induced tumors and cannot be detected by immunological methods. Furthermore, cells in culture are not readily transformed by the virus. To overcome these difficulties in identifying and characterizing the putative transforming protein(s) coded by the E6 open reading frame, the early cottontail rabbit papillomavirus region was expressed under the control of the late simian virus 40 promoter. Mapping of the transcripts in transiently transfected COS-7 cells indicated that transcription was initiated in the late region of simian virus 40. Two E6-coded polypeptides were identified, representing translation products initiated at the first and second AUG codons. Images PMID:3039182

Expression, Purification And Preliminary X-Ray Analysis of the C-Terminal Domain of An Arginine Repressor Protein From Mycobacterium Tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, G.J.; Garen, C.R.; Cherney, M.M.

2009-06-03

The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 {angstrom}. The crystals belong to space group P1 and the Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of sixmore » molecules per unit cell.« less

Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride

PubMed Central

Matroudi, S.; Zamani, M.R.; Motallebi, M.

2008-01-01

In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli. PMID:24031242

Detection of hepatitis B virus X product using an open reading frame Escherichia coli expression vector.

PubMed Central

Elfassi, E; Haseltine, W A; Dienstag, J L

1986-01-01

The genome of the hepatitis B virus (HBV) contains a sequence, designated X, capable of encoding a protein of 154 amino acids. To determine whether the putative protein synthesized from this region is antigenic, we examined the sera of HBV-infected patients for the ability to react with a hybrid protein that contained 133 amino acids encoded by the X region and portions of the bacterial ompF and beta-galactosidase genes. Some HBV-positive sera tested contained antibodies that specifically recognized the hybrid protein. All sera were from patients diagnosed as suffering from chronic active hepatitis. We conclude that the X region of HBV encodes a protein and that this protein is antigenic in some patients. Images PMID:3515347

Mu-Like Prophage in Serogroup B Neisseria meningitidis Coding for Surface-Exposed Antigens

PubMed Central

Masignani, Vega; Giuliani, Marzia Monica; Tettelin, Hervé; Comanducci, Maurizio; Rappuoli, Rino; Scarlato, Vincenzo

2001-01-01

Sequence analysis of the genome of Neisseria meningititdis serogroup B revealed the presence of an ∼35-kb region inserted within a putative gene coding for an ABC-type transporter. The region contains 46 open reading frames, 29 of which are colinear and homologous to the genes of Escherichia coli Mu phage. Two prophages with similar organizations were also found in serogroup A meningococcus, and one was found in Haemophilus influenzae. Early and late phage functions are well preserved in this family of Mu-like prophages. Several regions of atypical nucleotide content were identified. These likely represent genes acquired by horizontal transfer. Three of the acquired genes are shown to code for surface-associated antigens, and the encoded proteins are able to induce bactericidal antibodies. PMID:11254622

Sequencing and phylogenetic analysis of tobacco virus 2, a polerovirus from Nicotiana tabacum.

PubMed

Zhou, Benguo; Wang, Fang; Zhang, Xuesong; Zhang, Lina; Lin, Huafeng

2017-07-01

The complete genome sequence of a new virus, provisionally named tobacco virus 2 (TV2), was determined and identified from leaves of tobacco (Nicotiana tabacum) exhibiting leaf mosaic, yellowing, and deformity, in Anhui Province, China. The genome sequence of TV2 comprises 5,979 nucleotides, with 87% nucleotide sequence identity to potato leafroll virus (PLRV). Its genome organization is similar to that of PLRV, containing six open reading frames (ORFs) that potentially encode proteins with putative functions in cell-to-cell movement and suppression of RNA silencing. Phylogenetic analysis of the nucleotide sequence placed TV2 alongside members of the genus Polerovirus in the family Luteoviridae. To the best our knowledge, this study is the first report of a complete genome sequence of a new polerovirus identified in tobacco.

Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages.

PubMed Central

Vasala, A; Dupont, L; Baumann, M; Ritzenthaler, P; Alatossava, T

1993-01-01

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module. Images PMID:8497043

Open reading frames in a 4556 nucleotide sequence within MDV-1 BamHI-D DNA fragment: evidence for splicing of mRNA from a new viral glycoprotein gene.

PubMed

Becker, Y; Asher, Y; Tabor, E; Davidson, I; Malkinson, M

1994-01-01

A DNA segment of the MDV-1 BamHI-D fragment was sequenced, and the open reading frames (ORFs) present in the 4556 nucleotide fragment were analyzed by computer programs. Computer analysis identified 19 putative ORFs in the sequence ranging from a coding capacity of 37 amino acids (aa) (ORF-1a) to 684aa (ORF-1). The special properties of four ORFs (1a, 1, 2, and 3) were investigated. Two adjacent ORFs, ORF-1a and ORF-1, were found by computer analysis to have the properties of two introns encoding a glycoprotein: ORF-1a encodes an aa sequence with the properties of a signal peptide, and ORF-1 encodes a polypeptide with a membrane anchor domain and putative N-glycosylation sites in the aa sequence. ORF-1a and ORF-1 were found to be transcribed in MDV-1-infected cells. Two RNA transcripts were detected: a precursor RNA and its spliced form. Both are transcribed from a promoter located 5' to ORF-1a, and splice donor and acceptor sites are used to splice the mRNA after cleavage of a 71-nucleotide sequence. This finding suggest that ORF-1a and ORF-1 are two introns of a new MDV-1 glycoprotein gene. The DNA sequence containing ORF-1 was transiently expressed in COS-1 cells, and the viral protein produced in these cells was found to react with anti-MDV serotype-1 Antigen B-specific monoclonal antibodies. These studies indicate that the protein encoded by ORF-1 has antigenic properties resembling Antigen B of MDV-1. A gene homologous to ORF-1 was detected in the genome of both MDV-2(SB1) and MDV-3(HVT), which serve as commercial vaccine strains. Two additional ORFs were noted in the 4556 nucleotide sequence: ORF-2, which encodes a 333 aa polypeptide initiating in the UL and terminating in the TRL prior to the putative origin of replication, and ORF-3, which encodes a 155 aa polypeptide that is partly homologous to the phosphoprotein pp38 encoded by the BamHI-H sequence. The 65 N-terminal aa of the two gene products are identical, both being derived from the nucleotide sequences in the TRL and IRL, respectively. Additional homologous aa sequences are the hydrophobic aa domain in the middle of both proteins. The functions of ORF-2, ORF-3, and additional ORFs are under study.

A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

PubMed

Romero-Gómez, S; Duarte-Vázquez, M A; García-Almendárez, B E; Mayorga-Martínez, L; Cervantes-Avilés, O; Regalado, C

2008-12-01

A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.

Molecular and functional characterization of novel fructosyltransferases and invertases from Agave tequilana.

PubMed

Cortés-Romero, Celso; Martínez-Hernández, Aída; Mellado-Mojica, Erika; López, Mercedes G; Simpson, June

2012-01-01

Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants.

Molecular and Functional Characterization of Novel Fructosyltransferases and Invertases from Agave tequilana

PubMed Central

Cortés-Romero, Celso; Martínez-Hernández, Aída; Mellado-Mojica, Erika; López, Mercedes G.; Simpson, June

2012-01-01

Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants. PMID:22558253

Putative cross-kingdom horizontal gene transfer in sponge (Porifera) mitochondria.

PubMed

Rot, Chagai; Goldfarb, Itay; Ilan, Micha; Huchon, Dorothée

2006-09-14

The mitochondrial genome of Metazoa is usually a compact molecule without introns. Exceptions to this rule have been reported only in corals and sea anemones (Cnidaria), in which group I introns have been discovered in the cox1 and nad5 genes. Here we show several lines of evidence demonstrating that introns can also be found in the mitochondria of sponges (Porifera). A 2,349 bp fragment of the mitochondrial cox1 gene was sequenced from the sponge Tetilla sp. (Spirophorida). This fragment suggests the presence of a 1143 bp intron. Similar to all the cnidarian mitochondrial introns, the putative intron has group I intron characteristics. The intron is present in the cox1 gene and encodes a putative homing endonuclease. In order to establish the distribution of this intron in sponges, the cox1 gene was sequenced from several representatives of the demosponge diversity. The intron was found only in the sponge order Spirophorida. A phylogenetic analysis of the COI protein sequence and of the intron open reading frame suggests that the intron may have been transmitted horizontally from a fungus donor. Little is known about sponge-associated fungi, although in the last few years the latter have been frequently isolated from sponges. We suggest that the horizontal gene transfer of a mitochondrial intron was facilitated by a symbiotic relationship between fungus and sponge. Ecological relationships are known to have implications at the genomic level. Here, an ecological relationship between sponge and fungus is suggested based on the genomic analysis.

Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33

PubMed Central

Li, You-Hai; Han, Wen-Jin; Gui, Xi-Wu; Wei, Tao; Tang, Shuang-Yan; Jin, Jian-Ming

2016-01-01

Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F1-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata. PMID:27490569

Probability of coding of a DNA sequence: an algorithm to predict translated reading frames from their thermodynamic characteristics.

PubMed Central

Tramontano, A; Macchiato, M F

1986-01-01

An algorithm to determine the probability that a reading frame codifies for a protein is presented. It is based on the results of our previous studies on the thermodynamic characteristics of a translated reading frame. We also develop a prediction procedure to distinguish between coding and non-coding reading frames. The procedure is based on the characteristics of the putative product of the DNA sequence and not on periodicity characteristics of the sequence, so the prediction is not biased by the presence of overlapping translated reading frames or by the presence of translated reading frames on the complementary DNA strand. PMID:3753761

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae*

PubMed Central

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.; Shirasaki, Dyna I.; Wang, Charles; Blaby-Haas, Crysten E.; Merchant, Sabeeha S.; Loo, Joseph A.; Clarke, Catherine F.

2015-01-01

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. Here, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11. PMID:25631044

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae

DOE PAGES

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.; ...

2015-01-28

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. In thismore » paper, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q 6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Finally, given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.« less

Computational Fluids Domain Reduction to a Simplified Fluid Network

DTIC Science & Technology

2012-04-19

readily available read/ write software library. Code components from the open source projects OpenFoam and Paraview were explored for their adaptability...to the project. Both Paraview and OpenFoam read polyhedral mesh. OpenFoam does not read results data. Paraview actually allows for user “filters

Presence of a novel exon 2E encoding a putative transmembrane protein in human IL-33 gene.

PubMed

Tominaga, Shin-ichi; Hayakawa, Morisada; Tsuda, Hidetoshi; Ohta, Satoshi; Yanagisawa, Ken

2013-01-18

Interleukin-33 (IL-33) is a dual-function molecule that regulates gene expression in nuclei and, as a cytokine, conveys proinflammatory signals from outside of cells via its specific receptor ST2L. There are still a lot of questions about localization and processing of IL-33 gene products. In the course of re-evaluating human IL-33 gene, we found distinct promoter usage depending on the cell type, similar to the case in the ST2 gene. Furthermore, we found a novel exon 2E in the conventional intron 2 whose open reading frame corresponded to a transmembrane protein of 131 amino acids. Dependence of exon 2E expression on differentiation of HUVEC cells is of great interest in relation to human IL-33 function. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterisation of a novel enterobacteria phage, CAjan, isolated from rat faeces.

PubMed

Carstens, Alexander B; Kot, Witold; Lametsch, Rene; Neve, Horst; Hansen, Lars H

2016-08-01

In this study, we describe the isolation and characterisation of the novel enterobacteria phage CAjan. This phage belongs to the order Caudovirales and the family Siphoviridae. The phage possesses a linear, double-stranded DNA genome consisting of 59,670 bp with a G+C content of 44.7 % and 91 predicted open reading frames (ORFs). Putative functions were assigned to 39 of the ORFs (37.4 %). The phage structural genes were furthermore functionally characterised by LC MS/MS. CAjan, together with Escherichia phage Seurat and Escherichia phage slur01, represent a novel and genetically distinct clade of Siphoviridae phages that could be considered to constitute a new phage genus. Despite limited sequence similarity, the phages in this group share a number of other common features, including genome structure and the presence of queuosine biosynthesis genes.

Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus.

PubMed

Qiu, T; Lu, R H; Zhang, J; Zhu, Z Y

2001-07-01

The 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 mu1 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 mu1.

Complete genomic sequence of the Lactobacillus temperate phage LF1.

PubMed

Yoon, Bo Hyun; Chang, Hyo Ihl

2011-10-01

Bacteriophage LF1, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild type Lactobacillus fermentum, was found to contain a double-strand DNA of 42,606 base pairs (bp) with a G+C content of 45%. Bioinformatic analysis of the phage genome revealed 57 putative open reading frames (ORFs). The predicted protein products of ORFs were determined and described. According to morphological analysis by transmission electron microscopy (TEM), LF1 has an isometric head and a non-contractile tail, indicating that it belongs to the family Siphoviridae. The temperate phage LF1 has a good genetic mosaic relationship with ΦPYB5 in the packaging module. To our knowledge, this is first report of genomic sequencing and characterization of temperate phage LF1 from wild-type L. fermentum isolated from Kimchi in Korea.

Novel canine circovirus strains from Thailand: Evidence for genetic recombination.

PubMed

Piewbang, Chutchai; Jo, Wendy K; Puff, Christina; van der Vries, Erhard; Kesdangsakonwut, Sawang; Rungsipipat, Anudep; Kruppa, Jochen; Jung, Klaus; Baumgärtner, Wolfgang; Techangamsuwan, Somporn; Ludlow, Martin; Osterhaus, Albert D M E

2018-05-14

Canine circoviruses (CanineCV's), belonging to the genus Circovirus of the Circoviridae family, were detected by next generation sequencing in samples from Thai dogs with respiratory symptoms. Genetic characterization and phylogenetic analysis of nearly complete CanineCV genomes suggested that natural recombination had occurred among different lineages of CanineCV's. Similarity plot and bootscaning analyses indicated that American and Chinese viruses had served as major and minor parental viruses, respectively. Positions of recombination breakpoints were estimated using maximum-likelihood frameworks with statistical significant testing. The putative recombination event was located in the Replicase gene, intersecting with open reading frame-3. Analysis of nucleotide changes confirmed the origin of the recombination event. This is the first description of naturally occurring recombinant CanineCV's that have resulted in the circulation of newly emerging CanineCV lineages.

Identification of the Lomofungin Biosynthesis Gene Cluster and Associated Flavin-Dependent Monooxygenase Gene in Streptomyces lomondensis S015

PubMed Central

Zhang, Chunxiao; Sheng, Chaolan; Wang, Wei; Hu, Hongbo; Peng, Huasong; Zhang, Xuehong

2015-01-01

Streptomyces lomondensis S015 synthesizes the broad-spectrum phenazine antibiotic lomofungin. Whole genome sequencing of this strain revealed a genomic locus consisting of 23 open reading frames that includes the core phenazine biosynthesis gene cluster lphzGFEDCB. lomo10, encoding a putative flavin-dependent monooxygenase, was also identified in this locus. Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin. This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces. PMID:26305803

Detection and assessment of copy number variation using PacBio long-read and Illumina sequencing in New Zealand dairy cattle.

PubMed

Couldrey, C; Keehan, M; Johnson, T; Tiplady, K; Winkelman, A; Littlejohn, M D; Scott, A; Kemper, K E; Hayes, B; Davis, S R; Spelman, R J

2017-07-01

Single nucleotide polymorphisms have been the DNA variant of choice for genomic prediction, largely because of the ease of single nucleotide polymorphism genotype collection. In contrast, structural variants (SV), which include copy number variants (CNV), translocations, insertions, and inversions, have eluded easy detection and characterization, particularly in nonhuman species. However, evidence increasingly shows that SV not only contribute a substantial proportion of genetic variation but also have significant influence on phenotypes. Here we present the discovery of CNV in a prominent New Zealand dairy bull using long-read PacBio (Pacific Biosciences, Menlo Park, CA) sequencing technology and the Sniffles SV discovery tool (version 0.0.1; https://github.com/fritzsedlazeck/Sniffles). The CNV identified from long reads were compared with CNV discovered in the same bull from Illumina sequencing using CNVnator (read depth-based tool; Illumina Inc., San Diego, CA) as a means of validation. Subsequently, further validation was undertaken using whole-genome Illumina sequencing of 556 cattle representing the wider New Zealand dairy cattle population. Very limited overlap was observed in CNV discovered from the 2 sequencing platforms, in part because of the differences in size of CNV detected. Only a few CNV were therefore able to be validated using this approach. However, the ability to use CNVnator to genotype the 557 cattle for copy number across all regions identified as putative CNV allowed a genome-wide assessment of transmission level of copy number based on pedigree. The more highly transmissible a putative CNV region was observed to be, the more likely the distribution of copy number was multimodal across the 557 sequenced animals. Furthermore, visual assessment of highly transmissible CNV regions provided evidence supporting the presence of CNV across the sequenced animals. This transmission-based approach was able to confirm a subset of CNV that segregates in the New Zealand dairy cattle population. Genome-wide identification and validation of CNV is an important step toward their inclusion in genomic selection strategies. The Authors. Published by the Federation of Animal Science Societies and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

PubMed Central

Xuan, J W; Fournier, P; Declerck, N; Chasles, M; Gaillardin, C

1990-01-01

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked. Images PMID:2388625

The crystal structure of Rv1347c, a putative antibiotic resistance protein from Mycobacterium tuberculosis, reveals a GCN5-related fold and suggests an alternative function in siderophore biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Card, G L; Peterson, N A; Smith, C A

2005-02-15

Mycobacterium tuberculosis, the cause of TB, is a devastating human pathogen. The emergence of multi-drug resistance in recent years has prompted a search for new drug targets and for a better understanding of mechanisms of resistance. Here we focus on the gene product of an open reading frame from M. tuberculosis, Rv1347c, which is annotated as a putative aminoglycoside N-acetyltransferase. The Rv1347c protein does not show this activity, however, and we show from its crystal structure, coupled with functional and bioinformatic data, that its most likely role is in the biosynthesis of mycobactin, the M. tuberculosis siderophore. The crystal structuremore » of Rv1347c was determined by MAD phasing from selenomethionine-substituted protein and refined at 2.2 {angstrom} resolution (R = 0.227, R{sub free} = 0.257). The protein is monomeric, with a fold that places it in the GCN5-related N-acetyltransferase (GNAT) family of acyltransferases. Features of the structure are an acylCoA binding site that is shared with other GNAT family members, and an adjacent hydrophobic channel leading to the surface that could accommodate long-chain acyl groups. Modeling the postulated substrate, the N{sup {var_epsilon}}-hydroxylysine side chain of mycobactin, into the acceptor substrate binding groove identifies two residues at the active site, His130 and Asp168, that have putative roles in substrate binding and catalysis.« less

Characterization of the 101-Kilobase-Pair Megaplasmid pKB1, Isolated from the Rubber-Degrading Bacterium Gordonia westfalica Kb1

PubMed Central

Bröker, Daniel; Arenskötter, Matthias; Legatzki, Antje; Nies, Dietrich H.; Steinbüchel, Alexander

2004-01-01

The complete sequence of the circular 101,016-bp megaplasmid pKB1 from the cis-1,4-polyisoprene-degrading bacterium Gordonia westfalica Kb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined. Plasmid pKB1 harbors 105 open reading frames. The predicted products of 46 of these are significantly related to proteins of known function. Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element. Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome. The origin of replication (oriV) of pKB1 was identified and used for construction of Escherichia coli-Gordonia shuttle vectors, which was also suitable for several other Gordonia species and related genera. The metabolic region included the heavy-metal resistance gene cadA, encoding a P-type ATPase. Expression of cadA in E. coli mediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host. When G. westfalica strain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance. Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1. PMID:14679241

Evolutionary Genomics of an Ancient Prophage of the Order Sphingomonadales

PubMed Central

Viswanathan, Vandana; Narjala, Anushree; Ravichandran, Aravind; Jayaprasad, Suvratha

2017-01-01

The order Sphingomonadales, containing the families Erythrobacteraceae and Sphingomonadaceae, is a relatively less well-studied phylogenetic branch within the class Alphaproteobacteria. Prophage elements are present in most bacterial genomes and are important determinants of adaptive evolution. An “intact” prophage was predicted within the genome of Sphingomonas hengshuiensis strain WHSC-8 and was designated Prophage IWHSC-8. Loci homologous to the region containing the first 22 open reading frames (ORFs) of Prophage IWHSC-8 were discovered among the genomes of numerous Sphingomonadales. In 17 genomes, the homologous loci were co-located with an ORF encoding a putative superoxide dismutase. Several other lines of molecular evidence implied that these homologous loci represent an ancient temperate bacteriophage integration, and this horizontal transfer event pre-dated niche-based speciation within the order Sphingomonadales. The “stabilization” of prophages in the genomes of their hosts is an indicator of “fitness” conferred by these elements and natural selection. Among the various ORFs predicted within the conserved prophages, an ORF encoding a putative proline-rich outer membrane protein A was consistently present among the genomes of many Sphingomonadales. Furthermore, the conserved prophages in six Sphingomonas sp. contained an ORF encoding a putative spermidine synthase. It is possible that one or more of these ORFs bestow selective fitness, and thus the prophages continue to be vertically transferred within the host strains. Although conserved prophages have been identified previously among closely related genera and species, this is the first systematic and detailed description of orthologous prophages at the level of an order that contains two diverse families and many pigmented species. PMID:28201618

Are Fireworms Venomous? Evidence for the Convergent Evolution of Toxin Homologs in Three Species of Fireworms (Annelida, Amphinomidae)

PubMed Central

Simpson, Danny

2018-01-01

Abstract Amphinomids, more commonly known as fireworms, are a basal lineage of marine annelids characterized by the presence of defensive dorsal calcareous chaetae, which break off upon contact. It has long been hypothesized that amphinomids are venomous and use the chaetae to inject a toxic substance. However, studies investigating fireworm venom from a morphological or molecular perspective are scarce and no venom gland has been identified to date, nor any toxin characterized at the molecular level. To investigate this question, we analyzed the transcriptomes of three species of fireworms—Eurythoe complanata, Hermodice carunculata, and Paramphinome jeffreysii—following a venomics approach to identify putative venom compounds. Our venomics pipeline involved de novo transcriptome assembly, open reading frame, and signal sequence prediction, followed by three different homology search strategies: BLAST, HMMER sequence, and HMMER domain. Following this pipeline, we identified 34 clusters of orthologous genes, representing 13 known toxin classes that have been repeatedly recruited into animal venoms. Specifically, the three species share a similar toxin profile with C-type lectins, peptidases, metalloproteinases, spider toxins, and CAP proteins found among the most highly expressed toxin homologs. Despite their great diversity, the putative toxins identified are predominantly involved in three major biological processes: hemostasis, inflammatory response, and allergic reactions, all of which are commonly disrupted after fireworm stings. Although the putative fireworm toxins identified here need to be further validated, our results strongly suggest that fireworms are venomous animals that use a complex mixture of toxins for defense against predators. PMID:29293976

Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses.

PubMed

Komatsu, Ken; Hirata, Hisae; Fukagawa, Takako; Yamaji, Yasuyuki; Okano, Yukari; Ishikawa, Kazuya; Adachi, Tatsushi; Maejima, Kensaku; Hashimoto, Masayoshi; Namba, Shigetou

2012-07-01

The first open-reading frame (ORF) of apple stem grooving virus (ASGV), of the genus Capillovirus, encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP). However, our previous study revealed that ASGV mutants with distinct and discontinuous Rep- and CP-coding regions successfully infect plants, indicating that CP expressed via a subgenomic RNA (sgRNA) is sufficient for viability of the virus. Here we identified a transcription start site of the CP sgRNA and revealed that CP translated from the sgRNA is essential for ASGV infection. We mapped the transcription start sites of both the CP and the movement protein (MP) sgRNAs of ASGV and found a hexanucleotide motif, UUAGGU, conserved upstream from both sgRNA transcription start sites. Mutational analysis of the putative CP initiation codon and of the UUAGGU sequence upstream from the transcription start site of CP sgRNA demonstrated their importance for ASGV accumulation. Our results also demonstrated that potato virus T (PVT), an unassigned species closely related to ASGV, produces two sgRNAs putatively deployed for the CP and MP expression and that the same hexanucleotide motif as found in ASGV is located upstream from the transcription start sites of both sgRNAs. This motif, which constituted putative core elements of the sgRNA promoter, is broadly conserved among viruses in the families Alphaflexiviridae and Betaflexiviridae, suggesting that the gene expression strategy of the viruses in both families has been conserved throughout evolution. Copyright © 2012 Elsevier B.V. All rights reserved.

Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes.

PubMed

Azevedo, Analice C; Bento, Cláudia B P; Ruiz, Jeronimo C; Queiroz, Marisa V; Mantovani, Hilário C

2015-10-01

Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on in vitro assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of Streptococcus (66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the Ruminococcus albus strains screened in this study. The in silico analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Putative cross-kingdom horizontal gene transfer in sponge (Porifera) mitochondria

PubMed Central

Rot, Chagai; Goldfarb, Itay; Ilan, Micha; Huchon, Dorothée

2006-01-01

Background The mitochondrial genome of Metazoa is usually a compact molecule without introns. Exceptions to this rule have been reported only in corals and sea anemones (Cnidaria), in which group I introns have been discovered in the cox1 and nad5 genes. Here we show several lines of evidence demonstrating that introns can also be found in the mitochondria of sponges (Porifera). Results A 2,349 bp fragment of the mitochondrial cox1 gene was sequenced from the sponge Tetilla sp. (Spirophorida). This fragment suggests the presence of a 1143 bp intron. Similar to all the cnidarian mitochondrial introns, the putative intron has group I intron characteristics. The intron is present in the cox1 gene and encodes a putative homing endonuclease. In order to establish the distribution of this intron in sponges, the cox1 gene was sequenced from several representatives of the demosponge diversity. The intron was found only in the sponge order Spirophorida. A phylogenetic analysis of the COI protein sequence and of the intron open reading frame suggests that the intron may have been transmitted horizontally from a fungus donor. Conclusion Little is known about sponge-associated fungi, although in the last few years the latter have been frequently isolated from sponges. We suggest that the horizontal gene transfer of a mitochondrial intron was facilitated by a symbiotic relationship between fungus and sponge. Ecological relationships are known to have implications at the genomic level. Here, an ecological relationship between sponge and fungus is suggested based on the genomic analysis. PMID:16972986

High-Molecular-Mass Multi-c-Heme Cytochromes from Methylococcus capsulatus Bath†

PubMed Central

Bergmann, David J.; Zahn, James A.; DiSpirito, Alan A.

1999-01-01

The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6.0. The heme c concentration was estimated to be 8.2 ± 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118,620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94,000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome. PMID:9922265

Plasmid-Encoded MCP Is Involved in Virulence, Motility, and Biofilm Formation of Cronobacter sakazakii ATCC 29544

PubMed Central

Choi, Younho; Kim, Seongok; Hwang, Hyelyeon; Kim, Kwang-Pyo; Kang, Dong-Hyun

2014-01-01

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage. PMID:25332122

Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome, and its gene product

PubMed Central

2010-01-01

In this study, a putative esterase, designated EstMY, was isolated from an activated sludge metagenomic library. The lipolytic gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene estMY contained a 1,083 bp open reading frame (ORF) encoding a polypeptide of 360 amino acids with a molecular mass of 38 kDa. Sequence analysis indicated that it showed 71% and 52% amino acid identity to esterase/lipase from marine metagenome (ACL67845) and Burkholderia ubonensis Bu (ZP_02382719), respectively; and several conserved regions were identified, including the putative active site, GDSAG, a catalytic triad (Ser203, Asp301, and His327) and a HGGG conserved motif (starting from His133). The EstMY was determined to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤C8). This EstMY exhibited the highest activity at 35°C and pH 8.5 respectively, by hydrolysis of p-NP caprylate. It also exhibited the same level of activity over wide temperature and pH spectra and in the presence of metal ions or detergents. The high level of stability of esterase EstMY with unique substrate specificities makes it highly valuable for downstream biotechnological applications. PMID:21054894

Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E, and identification of the active site by site-directed mutagenesis.

PubMed

Mathupala, S P; Lowe, S E; Podkovyrov, S M; Zeikus, J G

1993-08-05

The complete nucleotide sequence of the gene encoding the dual active amylopullulanase of Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum) was determined. The structural gene (apu) contained a single open reading frame 4443 base pairs in length, corresponding to 1481 amino acids, with an estimated molecular weight of 162,780. Analysis of the deduced sequence of apu with sequences of alpha-amylases and alpha-1,6 debranching enzymes enabled the identification of four conserved regions putatively involved in substrate binding and in catalysis. The conserved regions were localized within a 2.9-kilobase pair gene fragment, which encoded a M(r) 100,000 protein that maintained the dual activities and thermostability of the native enzyme. The catalytic residues of amylopullulanase were tentatively identified by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylolytic enzymes. Asp597, Glu626, and Asp703 were individually modified to their respective amide form, or the alternate acid form, and in all cases both alpha-amylase and pullulanase activities were lost, suggesting the possible involvement of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme. These findings substantiate amylopullulanase as a new type of amylosaccharidase.

Leptospiral outer membrane protein LipL41 is not essential for acute leptospirosis but requires a small chaperone protein, lep, for stable expression.

PubMed

King, Amy M; Bartpho, Thanatchaporn; Sermswan, Rasana W; Bulach, Dieter M; Eshghi, Azad; Picardeau, Mathieu; Adler, Ben; Murray, Gerald L

2013-08-01

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp., but knowledge of leptospiral pathogenesis remains limited. However, the development of mutagenesis systems has allowed the investigation of putative virulence factors and their involvement in leptospirosis. LipL41 is the third most abundant lipoprotein found in the outer membranes of pathogenic leptospires and has been considered a putative virulence factor. LipL41 is encoded on the large chromosome 28 bp upstream of a small open reading frame encoding a hypothetical protein of unknown function. This gene was named lep, for LipL41 expression partner. In this study, lipL41 was found to be cotranscribed with lep. Two transposon mutants were characterized: a lipL41 mutant and a lep mutant. In the lep mutant, LipL41 protein levels were reduced by approximately 90%. Lep was shown through cross-linking and coexpression experiments to bind to LipL41. Lep is proposed to be a molecular chaperone essential for the stable expression of LipL41. The roles of LipL41 and Lep in the pathogenesis of Leptospira interrogans were investigated; surprisingly, neither of these two unique proteins was essential for acute leptospirosis.

Isolation and Expression analysis of OsPME1, encoding for a putative Pectin Methyl Esterase from Oryza sativa (subsp. indica).

PubMed

Kanneganti, Vydehi; Gupta, Aditya Kumar

2009-04-01

Pectin Methyl Esterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell walls. Recent studies indicated that PMEs play important role in pollen tube development. In this study, we isolated a 1.3 kb cDNA clone from rice panicle cDNA library. It contained a 1038 bp of open reading frame (ORF) encoding for a putative pectin methyl esterase of 345 aminoacids with a 20 aminoacid signal peptide and was hence designated as OsPME1 (Oryza sativaPectin Methyl Esterase 1). It contained the structural arrangement GXYXE and GXXDFIF, found in the active groups of all PMEs. OsPME1 gene product shared varying identities, ranging from 52 % to 33 % with PMEs from other plant species belonging to Brassicaceae, Fabaceae, Amaranthaceae and Funariaceae. Southern blot analysis indicated that PME1 exists as a single copy in the rice genome. Expression pattern analysis revealed that OsPME1 is expressed only in pollen grains, during the later stages of their development and was also regulated by various abiotic stress treatments and phytohormones. Functional characterization of this pollen specific PME from rice would enable us to understand its role in pollen development.

Plasmid-encoded MCP is involved in virulence, motility, and biofilm formation of Cronobacter sakazakii ATCC 29544.

PubMed

Choi, Younho; Kim, Seongok; Hwang, Hyelyeon; Kim, Kwang-Pyo; Kang, Dong-Hyun; Ryu, Sangryeol

2015-01-01

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Identification, Purification and Characterization of Laterosporulin, a Novel Bacteriocin Produced by Brevibacillus sp. Strain GI-9

PubMed Central

Singh, Pradip Kumar; Chittpurna; Ashish; Sharma, Vikas; Patil, Prabhu B.; Korpole, Suresh

2012-01-01

Background Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology. Methodology/Findings The strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database. Conclusions We have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity. PMID:22403615

Identification, purification and characterization of laterosporulin, a novel bacteriocin produced by Brevibacillus sp. strain GI-9.

PubMed

Singh, Pradip Kumar; Chittpurna; Ashish; Sharma, Vikas; Patil, Prabhu B; Korpole, Suresh

2012-01-01

Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology. The strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database. We have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.

Identification of putative methanol dehydrogenase (moxF) structural genes in methylotrophs and cloning of moxF genes from methylococcus capsulatus bath and Methylomonas albus BG8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephens, R.L.; Haygood, M.G.; Lidstrom, M.E.

An open-reading-frame fragment of a Methylobacterium sp. strain AM1 gene (moxF) encoding a portion of the methanol dehydrogenase structural protein has been used as a hybridization probe to detect similar sequences in a variety of methylotrophic bacteria. This hybridization was used to isolate clones containing putative moxF genes from two obligate methanotrophic bacteria, Methylococcus capsulatus Bath and Methylomonas albus BG8. The identity of these genes was confirmed in two ways. A T7 expression vector was used to produce methanol dehydrogenase protein in Escherichia coli from the cloned genes,a and in each case the protein was identified by immunoblotting with antiserummore » against the Methylomonas albus methanol dehydrogenase. In addition, a moxF mutant of Methylobacterium strain AM1 was complemented to a methanol-positive phenotype that partially restored methanol dehydrogenase activity, using broad-host-range plasmids containing the moxF genes from each methanotroph. The partial complementation of a moxF mutant in a facultative serine pathway methanol utilizer by moxF genes from type I and type X obligate methane utilizers suggests broad functional conservation of the methanol oxidation system among gram-negative methylotrophs.« less

A novel polyomavirus from the nasal cavity of a giant panda (Ailuropoda melanoleuca).

PubMed

Qi, Dunwu; Shan, Tongling; Liu, Zhijian; Deng, Xutao; Zhang, Zhihe; Bi, Wenlei; Owens, Jacob Robert; Feng, Feifei; Zheng, Lisong; Huang, Feng; Delwart, Eric; Hou, Rong; Zhang, Wen

2017-10-27

Polyomaviruses infect a wide variety of mammalian and avian hosts with a broad spectrum of outcomes including asymptomatic infection, acute systemic disease, and tumor induction. Viral metagenomics and general PCR methods were used to detected viral nucleic acid in the samples from a diseased and healthy giant pandas. A novel polyomavirus, the giant panda polyomavirus 1 (GPPyV1) from the nasal cavity of a dead giant panda (Ailuropoda melanoleuca) was characterized. The GPPyV1 genome is 5144 bp in size and reveals five putative open-reading frames coding for the classic small and large T antigens in the early region, and the VP1, VP2 and VP3 capsid proteins in the late region. Phylogenetic analyses of the large T antigen of the GPPyV1 indicated GPPyV1 belonged to a putative new species within genus Deltapolyomavirus, clustering with four human polyomavirus species. The GPPyV1 VP1 and VP2 clustered with genus Alphapolyomavirus. Our epidemiologic study indicated that this novel polyomavirus was also detected in nasal swabs and fecal samples collected from captive healthy giant pandas. A novel polyomavirus was detected in giant pandas and its complete genome was characterized, which may cause latency infection in giant pandas.

The Saccharomyces cerevisiae ETH1 Gene, an Inducible Homolog of Exonuclease III That Provides Resistance to DNA-Damaging Agents and Limits Spontaneous Mutagenesis

PubMed Central

Bennett, Richard A. O.

1999-01-01

The recently sequenced Saccharomyces cerevisiae genome was searched for a gene with homology to the gene encoding the major human AP endonuclease, a component of the highly conserved DNA base excision repair pathway. An open reading frame was found to encode a putative protein (34% identical to the Schizosaccharomyces pombe eth1+ [open reading frame SPBC3D6.10] gene product) with a 347-residue segment homologous to the exonuclease III family of AP endonucleases. Synthesis of mRNA from ETH1 in wild-type cells was induced sixfold relative to that in untreated cells after exposure to the alkylating agent methyl methanesulfonate (MMS). To investigate the function of ETH1, deletions of the open reading frame were made in a wild-type strain and a strain deficient in the known yeast AP endonuclease encoded by APN1. eth1 strains were not more sensitive to killing by MMS, hydrogen peroxide, or phleomycin D1, whereas apn1 strains were ∼3-fold more sensitive to MMS and ∼10-fold more sensitive to hydrogen peroxide than was the wild type. Double-mutant strains (apn1 eth1) were ∼15-fold more sensitive to MMS and ∼2- to 3-fold more sensitive to hydrogen peroxide and phleomycin D1 than were apn1 strains. Elimination of ETH1 in apn1 strains also increased spontaneous mutation rates 9- or 31-fold compared to the wild type as determined by reversion to adenine or lysine prototrophy, respectively. Transformation of apn1 eth1 cells with an expression vector containing ETH1 reversed the hypersensitivity to MMS and limited the rate of spontaneous mutagenesis. Expression of ETH1 in a dut-1 xthA3 Escherichia coli strain demonstrated that the gene product functionally complements the missing AP endonuclease activity. Thus, in apn1 cells where the major AP endonuclease activity is missing, ETH1 offers an alternate capacity for repair of spontaneous or induced damage to DNA that is normally repaired by Apn1 protein. PMID:10022867

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector.

PubMed

Nishida, Takashi; Watanabe, Kenta; Tachibana, Masato; Shimizu, Takashi; Watarai, Masahisa

2017-03-01

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, Km R ), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×10 1 to 1.0×10 5 CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila. Copyright © 2017 Elsevier Inc. All rights reserved.

Bioinformatic analysis suggests that the Orbivirus VP6 cistron encodes an overlapping gene

PubMed Central

Firth, Andrew E

2008-01-01

Background The genus Orbivirus includes several species that infect livestock – including Bluetongue virus (BTV) and African horse sickness virus (AHSV). These viruses have linear dsRNA genomes divided into ten segments, all of which have previously been assumed to be monocistronic. Results Bioinformatic evidence is presented for a short overlapping coding sequence (CDS) in the Orbivirus genome segment 9, overlapping the VP6 cistron in the +1 reading frame. In BTV, a 77–79 codon AUG-initiated open reading frame (hereafter ORFX) is present in all 48 segment 9 sequences analysed. The pattern of base variations across the 48-sequence alignment indicates that ORFX is subject to functional constraints at the amino acid level (even when the constraints due to coding in the overlapping VP6 reading frame are taken into account; MLOGD software). In fact the translated ORFX shows greater amino acid conservation than the overlapping region of VP6. The ORFX AUG codon has a strong Kozak context in all 48 sequences. Each has only one or two upstream AUG codons, always in the VP6 reading frame, and (with a single exception) always with weak or medium Kozak context. Thus, in BTV, ORFX may be translated via leaky scanning. A long (83–169 codon) ORF is present in a corresponding location and reading frame in all other Orbivirus species analysed except Saint Croix River virus (SCRV; the most divergent). Again, the pattern of base variations across sequence alignments indicates multiple coding in the VP6 and ORFX reading frames. Conclusion At ~9.5 kDa, the putative ORFX product in BTV is too small to appear on most published protein gels. Nonetheless, a review of past literature reveals a number of possible detections. We hope that presentation of this bioinformatic analysis will stimulate an attempt to experimentally verify the expression and functional role of ORFX, and hence lead to a greater understanding of the molecular biology of these important pathogens. PMID:18489030

Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria.

PubMed

Cui, Hongli; Wang, Yipeng; Wang, Yinchu; Qin, Song

2012-11-16

Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX) is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS). PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs) encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like) according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC) of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic mechanisms as eukaryotes. All cyanobacterial PRX proteins share highly similar structures, implying that these genes may originate from a common ancestor. In this study, a general framework of the sequence-structure-function connections of the PRXs was revealed, which may facilitate functional investigations of PRXs in various organisms.

Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria

PubMed Central

2012-01-01

Background Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX) is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS). PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Results Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs) encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like) according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC) of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. Conclusions The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic mechanisms as eukaryotes. All cyanobacterial PRX proteins share highly similar structures, implying that these genes may originate from a common ancestor. In this study, a general framework of the sequence-structure-function connections of the PRXs was revealed, which may facilitate functional investigations of PRXs in various organisms. PMID:23157370

Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus cereus group

PubMed Central

2013-01-01

Background Comparatively little information is available on members of the Myoviridae infecting low G+C content, Gram-positive host bacteria of the family Firmicutes. While numerous Bacillus phages have been isolated up till now only very few Bacillus cereus phages have been characterized in detail. Results Here we present data on the large, virulent, broad-host-range B. cereus phage vB_BceM_Bc431v3 (Bc431v3). Bc431v3 features a 158,618 bp dsDNA genome, encompassing 239 putative open reading frames (ORFs) and, 20 tRNA genes encoding 17 different amino acids. Since pulsed-field gel electrophoresis indicated that the genome of this phage has a mass of 155-158 kb Bc431v3 DNA appears not to contain long terminal repeats that are found in the genome of Bacillus phage SPO1. Conclusions Bc431v3 displays significant sequence similarity, at the protein level, to B. cereus phage BCP78, Listeria phage A511 and Enterococcus phage ØEF24C and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Based on these data we suggest that Bc431v3 should be included as a member of the Spounavirinae; however, because of all the diverse taxonomical information has been addressed recently, it is difficult to determine the genus. The Bc431v3 phage contains some highly unusual genes such as gp143 encoding putative tRNAHis guanylyltransferase. In addition, it carries some genes that appear to be related to the host sporulation regulators. These are: gp098, which encodes a putative segregation protein related to FstK/SpoIIIE DNA transporters; gp105, a putative segregation protein; gp108, RNA polymerase sigma factor F/B; and, gp109 encoding RNA polymerase sigma factor G. PMID:23388049

Donkey Orchid Symptomless Virus: A Viral ‘Platypus’ from Australian Terrestrial Orchids

PubMed Central

Wylie, Stephen J.; Li, Hua; Jones, Michael G. K.

2013-01-01

Complete and partial genome sequences of two isolates of an unusual new plant virus, designated Donkey orchid symptomless virus (DOSV) were identified using a high-throughput sequencing approach. The virus was identified from asymptomatic plants of Australian terrestrial orchid Diuris longifolia (Common donkey orchid) growing in a remnant forest patch near Perth, western Australia. DOSV was identified from two D. longifolia plants of 264 tested, and from at least one plant of 129 Caladenia latifolia (pink fairy orchid) plants tested. Phylogenetic analysis of the genome revealed open reading frames (ORF) encoding seven putative proteins of apparently disparate origins. A 69-kDa protein (ORF1) that overlapped the replicase shared low identity with MPs of plant tymoviruses (Tymoviridae). A 157-kDa replicase (ORF2) and 22-kDa coat protein (ORF4) shared 32% and 40% amino acid identity, respectively, with homologous proteins encoded by members of the plant virus family Alphaflexiviridae. A 44-kDa protein (ORF3) shared low identity with myosin and an autophagy protein from Squirrelpox virus. A 27-kDa protein (ORF5) shared no identity with described proteins. A 14-kDa protein (ORF6) shared limited sequence identity (26%) over a limited region of the envelope glycoprotein precursor of mammal-infecting Crimea-Congo hemorrhagic fever virus (Bunyaviridae). The putative 25-kDa movement protein (MP) (ORF7) shared limited (27%) identity with 3A-like MPs of members of the plant-infecting Tombusviridae and Virgaviridae. Transmissibility was shown when DOSV systemically infected Nicotiana benthamiana plants. Structure and organization of the domains within the putative replicase of DOSV suggests a common evolutionary origin with ‘potexvirus-like’ replicases of viruses within the Alphaflexiviridae and Tymoviridae, and the CP appears to be ancestral to CPs of allexiviruses (Alphaflexiviridae). The MP shares an evolutionary history with MPs of dianthoviruses, but the other putative proteins are distant from plant viruses. DOSV is not readily classified in current lower order virus taxa. PMID:24223974

Molecular cloning of Kazal-type proteinase inhibitor of the shrimp Fenneropenaeus chinensis.

PubMed

Kong, Hee Jeong; Cho, Hyun Kook; Park, Eun-Mi; Hong, Gyeong-Eun; Kim, Young-Ok; Nam, Bo-Hye; Kim, Woo-Jin; Lee, Sang-Jun; Han, Hyon Sob; Jang, In-Kwon; Lee, Chang Hoon; Cheong, Jaehun; Choi, Tae-Jin

2009-01-01

Proteinase inhibitors play important roles in host defence systems involving blood coagulation and pathogen digestion. We isolated and characterized a cDNA clone for a Kazal-type proteinase inhibitor (KPI) from a hemocyte cDNA library of the oriental white shrimp Fenneropenaeus chinensis. The KPI gene consists of three exons and two introns. KPI cDNA contains an open reading frame of 396 bp, a polyadenylation signal sequence AATAAA, and a poly (A) tail. KPI cDNA encodes a polypeptide of 131 amino acids with a putative signal peptide of 21 amino acids. The deduced amino acid sequence of KPI contains two homologous Kazal domains, each with six conserved cysteine residues. The mRNA of KPI is expressed in the hemocytes of healthy shrimp, and the higher expression of KPI transcript is observed in shrimp infected with the white spot syndrome virus (WSSV), suggesting a potential role for KPI in host defence mechanisms.

Genetic Characterization and Physiological Role of Endopeptidase O from Lactobacillus helveticus CNRZ32

PubMed Central

Chen, Yo-Shen; Steele, James L.

1998-01-01

A previously identified insert expressing an endopeptidase from a Lactobacillus helveticus CNRZ32 genomic library was characterized. Nucleotide sequence analysis revealed an open reading frame of 1,941 bp encoding a putative protein of 71.2 kDa which contained a zinc-protease motif. Protein homology searches revealed that this enzyme has 40% similarity with endopeptidase O (PepO) from Lactococcus lactis P8-2-47. Northern hybridization revealed that pepO is monocistronic and is expressed throughout the growth phase. CNRZ32 derivatives lacking PepO activity were constructed via gene replacement. Enzyme assays revealed that the PepO mutant had significantly reduced endopeptidase activity when compared to CNRZ32 with two of the three substrates examined. Growth studies indicated that PepO has no detectable effect on growth rate or acid production by Lactobacillus helveticus CNRZ32 in amino acid defined or skim milk medium. PMID:9726890

yadBC of Yersinia pestis, a new virulence determinant for bubonic plague.

PubMed

Forman, Stanislav; Wulff, Christine R; Myers-Morales, Tanya; Cowan, Clarissa; Perry, Robert D; Straley, Susan C

2008-02-01

In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.

Cloning and characterization of a novel zinc finger gene in Xp11.2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Derry, J.M.J.; Jess, U.; Francke, U.

1995-11-20

During a systematic search for open reading frames in chromosome band Xp11.2, a novel gene (ZNF157) that encodes a putative 506-amino-acid protein with the sequence characteristics of a zinc-finger-containing transcription factor was isolated. ZNF157 is encoded by four exons distributed over >20 kb of genomic DNA. The second and third exons contain sequences similar to those of the previously described KRAB-A and KRAB-B domains, motifs that have been shown to mediate transcriptional repression in other members of the protein family. A fourth exon contains 12 zinc finger DNA binding motifs and finger linking regions characteristic of ZNF proteins of themore » Krueppel family. ZNF157 maps to the telomeric end of a cluster of ZNF genes that includes ZNF21, ZNF41, and ZNF81. 19 refs., 2 figs.« less

[Inverse PCR amplification of the complete major capsid protein gene of lymphocystis disease virus isolated from Rachycentron canadum and the phylogenetic analysis of the virus].

PubMed

Fu, Xiao-Zhe; Shi, Cun-Bin; Li, Ning-Qiu; Pan, Hou-Jun; Chang, Ou-Qin; Wu, Shu-Qin

2007-09-01

The major capsid protein of lymphocystis disease virus isolated from Rachycentron canadum (LCDV-rc) was amplified and analysed. The 457bp DNA core fragment was amplified with the degenerate primers designed according to the conserved sequences of MCP gene of iridoviruses, then the flaking sequences adjacent to the core region were amplified by inverse PCR, and the complete sequence was obtained by combining all of them. The open reading frame of the gene is 1380bp in length, encoding a putative protein of 459 aa with molecular weight 51.12 kD and pI 6.87. Constructing the phylogenetic tree for comparing the MCP amino acid of iridoviruses, the results indicated that LCDV-rc is most homologous to the other Lymphocystis viruses and all of them constitute a branch. Accordingly LCDV-rc is identified as Lymphocystivirus.

Identification and Characterization of the Pyridomycin Biosynthetic Gene Cluster of Streptomyces pyridomyceticus NRRL B-2517*

PubMed Central

Huang, Tingting; Wang, Yemin; Yin, Jun; Du, Yanhua; Tao, Meifeng; Xu, Jing; Chen, Wenqing; Lin, Shuangjun; Deng, Zixin

2011-01-01

Pyridomycin is a structurally unique antimycobacterial cyclodepsipeptide containing rare 3-(3-pyridyl)-l-alanine and 2-hydroxy-3-methylpent-2-enoic acid moieties. The biosynthetic gene cluster for pyridomycin has been cloned and identified from Streptomyces pyridomyceticus NRRL B-2517. Sequence analysis of a 42.5-kb DNA region revealed 26 putative open reading frames, including two nonribosomal peptide synthetase (NRPS) genes and a polyketide synthase gene. A special feature is the presence of a polyketide synthase-type ketoreductase domain embedded in an NRPS. Furthermore, we showed that PyrA functioned as an NRPS adenylation domain that activates 3-hydroxypicolinic acid and transfers it to a discrete peptidyl carrier protein, PyrU, which functions as a loading module that initiates pyridomycin biosynthesis in vivo and in vitro. PyrA could also activate other aromatic acids, generating three pyridomycin analogues in vivo. PMID:21454714

Characterization of the Polyoxin Biosynthetic Gene Cluster from Streptomyces cacaoi and Engineered Production of Polyoxin H*S⃞

PubMed Central

Chen, Wenqing; Huang, Tingting; He, Xinyi; Meng, Qingqing; You, Delin; Bai, Linquan; Li, Jialiang; Wu, Mingxuan; Li, Rui; Xie, Zhoujie; Zhou, Huchen; Zhou, Xiufen; Tan, Huarong; Deng, Zixin

2009-01-01

A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics. PMID:19233844

Decoding sORF translation - from small proteins to gene regulation.

PubMed

Cabrera-Quio, Luis Enrique; Herberg, Sarah; Pauli, Andrea

2016-11-01

Translation is best known as the fundamental mechanism by which the ribosome converts a sequence of nucleotides into a string of amino acids. Extensive research over many years has elucidated the key principles of translation, and the majority of translated regions were thought to be known. The recent discovery of wide-spread translation outside of annotated protein-coding open reading frames (ORFs) came therefore as a surprise, raising the intriguing possibility that these newly discovered translated regions might have unrecognized protein-coding or gene-regulatory functions. Here, we highlight recent findings that provide evidence that some of these newly discovered translated short ORFs (sORFs) encode functional, previously missed small proteins, while others have regulatory roles. Based on known examples we will also speculate about putative additional roles and the potentially much wider impact that these translated regions might have on cellular homeostasis and gene regulation.

Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins.

PubMed Central

Aymerich, T; Holo, H; Håvarstein, L S; Hugas, M; Garriga, M; Nes, I F

1996-01-01

A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin. PMID:8633865

Expression, regulation and functional assessment of the 80 amino acid Small Adipocyte Factor 1 (Smaf1) protein in adipocytes.

PubMed

Ren, Gang; Eskandari, Parisa; Wang, Siqian; Smas, Cynthia M

2016-01-15

The gene for Small Adipocyte Factor 1, Smaf1 (also known as adipogenin, ADIG), encodes a ∼600 base transcript that is highly upregulated during 3T3-L1 in vitro adipogenesis and markedly enriched in adipose tissues. Based on the lack of an obvious open reading frame in the Smaf1 transcript, it is not known if the Smaf1 gene is protein coding or non-coding RNA. Using a peptide from a putative open reading frame of Smaf1 as antigen, we generated antibodies for western analysis. Our studies prove that Smaf1 encodes an adipose-enriched protein which in western blot analysis migrates at ∼10 kDa. Rapid induction of Smaf1 protein occurs during in vitro adipogenesis and its expression in 3T3-L1 adipocytes is positively regulated by insulin and glucose. Moreover, siRNA studies reveal that expression of Smaf1 in adipocytes is wholly dependent on PPARγ. On the other hand, use of siRNA for Smaf1 to nearly abolish its protein expression in adipocytes revealed that Smaf1 does not have a major role in adipocyte triglyceride accumulation, lipolysis or insulin-stimulated pAkt induction. However, immunolocalization studies using HA-tagged Smaf1 reveal enrichment at adipocyte lipid droplets. Together our findings show that Smaf1 is a novel small protein endogenous to adipocytes and that Smaf1 expression is closely tied to PPARγ-mediated signals and the adipocyte phenotype. Copyright © 2015 Elsevier Inc. All rights reserved.

Cloning and characterization of an alternatively spliced gene in proximal Xq28 deleted in two patients with intersexual genitalia and myotubular myopathy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laporte, J.; Hu, Ling-Jia; Kretz, C.

1997-05-01

We have identified a novel human gene that is entirely deleted in two boys with abnormal genital development and myotubular myopathy (MTM1). The gene, F18, is located in proximal Xq28, approximately 80 kb centromeric to the recently isolated MTM1 gene. Northern analysis of mRNA showed a ubiquitous pattern and suggested high levels of expression in skeletal muscle, brain, and heart. A transcript of 4.6 kb was detected in a range of tissues, and additional alternate forms of 3.8 and 2.6 kb were present in placenta and pancreas, respectively. The gene extends over 100 kb and is composed of at leastmore » seven exons, of which two are non-coding. Sequence analysis of a 4.6-kb cDNA contig revealed two overlapping open reading frames (ORFs) that encode putative proteins of 701 and 424 amino acids, respectively. Two alternative spliced transcripts affecting the large open reading frame were identified that, together with the Northern blot results, suggest that distinct proteins are derived from the gene. No significant homology to other known proteins was detected, but segments of the first ORF encode polyglutamine tracts and proline-rich domains, which are frequently observed in DNA-binding proteins. The F18 gene is a strong candidate for being implicated in the intersexual genitalia present in the two MTM1-deleted patients. The gene also serves as a candidate for other disorders that map to proximal Xq28. 15 refs., 3 figs., 1 tab.« less

De Novo Assembly of the Japanese Flounder (Paralichthys olivaceus) Spleen Transcriptome to Identify Putative Genes Involved in Immunity

PubMed Central

Huang, Lin; Li, Guiyang; Mo, Zhaolan; Xiao, Peng; Li, Jie; Huang, Jie

2015-01-01

Background Japanese flounder (Paralichthys olivaceus) is an economically important marine fish in Asia and has suffered from disease outbreaks caused by various pathogens, which requires more information for immune relevant genes on genome background. However, genomic and transcriptomic data for Japanese flounder remain scarce, which limits studies on the immune system of this species. In this study, we characterized the Japanese flounder spleen transcriptome using an Illumina paired-end sequencing platform to identify putative genes involved in immunity. Methodology/Principal Findings A cDNA library from the spleen of P. olivaceus was constructed and randomly sequenced using an Illumina technique. The removal of low quality reads generated 12,196,968 trimmed reads, which assembled into 96,627 unigenes. A total of 21,391 unigenes (22.14%) were annotated in the NCBI Nr database, and only 1.1% of the BLASTx top-hits matched P. olivaceus protein sequences. Approximately 12,503 (58.45%) unigenes were categorized into three Gene Ontology groups, 19,547 (91.38%) were classified into 26 Cluster of Orthologous Groups, and 10,649 (49.78%) were assigned to six Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, 40,928 putative simple sequence repeats and 47, 362 putative single nucleotide polymorphisms were identified. Importantly, we identified 1,563 putative immune-associated unigenes that mapped to 15 immune signaling pathways. Conclusions/Significance The P. olivaceus transciptome data provides a rich source to discover and identify new genes, and the immune-relevant sequences identified here will facilitate our understanding of the mechanisms involved in the immune response. Furthermore, the plentiful potential SSRs and SNPs found in this study are important resources with respect to future development of a linkage map or marker assisted breeding programs for the flounder. PMID:25723398

The De Novo Transcriptome and Its Functional Annotation in the Seed Beetle Callosobruchus maculatus.

PubMed

Sayadi, Ahmed; Immonen, Elina; Bayram, Helen; Arnqvist, Göran

2016-01-01

Despite their unparalleled biodiversity, the genomic resources available for beetles (Coleoptera) remain relatively scarce. We present an integrative and high quality annotated transcriptome of the beetle Callosobruchus maculatus, an important and cosmopolitan agricultural pest as well as an emerging model species in ecology and evolutionary biology. Using Illumina sequencing technology, we sequenced 492 million read pairs generated from 51 samples of different developmental stages (larvae, pupae and adults) of C. maculatus. Reads were de novo assembled using the Trinity software, into a single combined assembly as well as into three separate assemblies based on data from the different developmental stages. The combined assembly generated 218,192 transcripts and 145,883 putative genes. Putative genes were annotated with the Blast2GO software and the Trinotate pipeline. In total, 33,216 putative genes were successfully annotated using Blastx against the Nr (non-redundant) database and 13,382 were assigned to 34,100 Gene Ontology (GO) terms. We classified 5,475 putative genes into Clusters of Orthologous Groups (COG) and 116 metabolic pathways maps were predicted based on the annotation. Our analyses suggested that the transcriptional specificity increases with ontogeny. For example, out of 33,216 annotated putative genes, 51 were only expressed in larvae, 63 only in pupae and 171 only in adults. Our study illustrates the importance of including samples from several developmental stages when the aim is to provide an integrative and high quality annotated transcriptome. Our results will represent an invaluable resource for those working with the ecology, evolution and pest control of C. maculatus, as well for comparative studies of the transcriptomics and genomics of beetles more generally.

The De Novo Transcriptome and Its Functional Annotation in the Seed Beetle Callosobruchus maculatus

PubMed Central

Sayadi, Ahmed; Immonen, Elina; Bayram, Helen

2016-01-01

Despite their unparalleled biodiversity, the genomic resources available for beetles (Coleoptera) remain relatively scarce. We present an integrative and high quality annotated transcriptome of the beetle Callosobruchus maculatus, an important and cosmopolitan agricultural pest as well as an emerging model species in ecology and evolutionary biology. Using Illumina sequencing technology, we sequenced 492 million read pairs generated from 51 samples of different developmental stages (larvae, pupae and adults) of C. maculatus. Reads were de novo assembled using the Trinity software, into a single combined assembly as well as into three separate assemblies based on data from the different developmental stages. The combined assembly generated 218,192 transcripts and 145,883 putative genes. Putative genes were annotated with the Blast2GO software and the Trinotate pipeline. In total, 33,216 putative genes were successfully annotated using Blastx against the Nr (non-redundant) database and 13,382 were assigned to 34,100 Gene Ontology (GO) terms. We classified 5,475 putative genes into Clusters of Orthologous Groups (COG) and 116 metabolic pathways maps were predicted based on the annotation. Our analyses suggested that the transcriptional specificity increases with ontogeny. For example, out of 33,216 annotated putative genes, 51 were only expressed in larvae, 63 only in pupae and 171 only in adults. Our study illustrates the importance of including samples from several developmental stages when the aim is to provide an integrative and high quality annotated transcriptome. Our results will represent an invaluable resource for those working with the ecology, evolution and pest control of C. maculatus, as well for comparative studies of the transcriptomics and genomics of beetles more generally. PMID:27442123

ABMapper: a suffix array-based tool for multi-location searching and splice-junction mapping.

PubMed

Lou, Shao-Ke; Ni, Bing; Lo, Leung-Yau; Tsui, Stephen Kwok-Wing; Chan, Ting-Fung; Leung, Kwong-Sak

2011-02-01

Sequencing reads generated by RNA-sequencing (RNA-seq) must first be mapped back to the genome through alignment before they can be further analyzed. Current fast and memory-saving short-read mappers could give us a quick view of the transcriptome. However, they are neither designed for reads that span across splice junctions nor for repetitive reads, which can be mapped to multiple locations in the genome (multi-reads). Here, we describe a new software package: ABMapper, which is specifically designed for exploring all putative locations of reads that are mapped to splice junctions or repetitive in nature. The software is freely available at: http://abmapper.sourceforge.net/. The software is written in C++ and PERL. It runs on all major platforms and operating systems including Windows, Mac OS X and LINUX.

Characterization of the temperate phage vB_RleM_PPF1 and its site-specific integration into the Rhizobium leguminosarum F1 genome.

PubMed

Halmillawewa, Anupama P; Restrepo-Córdoba, Marcela; Perry, Benjamin J; Yost, Christopher K; Hynes, Michael F

2016-02-01

Bacteriophages may play an important role in regulating population size and diversity of the root nodule symbiont Rhizobium leguminosarum, as well as participating in horizontal gene transfer. Although phages that infect this species have been isolated in the past, our knowledge of their molecular biology, and especially of genome composition, is extremely limited, and this lack of information impacts on the ability to assess phage population dynamics and limits potential agricultural applications of rhizobiophages. To help address this deficit in available sequence and biological information, the complete genome sequence of the Myoviridae temperate phage PPF1 that infects R. leguminosarum biovar viciae strain F1 was determined. The genome is 54,506 bp in length with an average G+C content of 61.9 %. The genome contains 94 putative open reading frames (ORFs) and 74.5 % of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5 % (24 ORFs) of the predicted genes. PPF1 was capable of efficiently lysogenizing its rhizobial host R. leguminosarum F1. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence (att core region) it shares with its rhizobial host genome. Phage PPF1 is the first temperate phage infecting members of the genus Rhizobium for which a complete genome sequence, as well as other biological data such as the integration site, is available.

Lexical decision as an endophenotype for reading comprehension: An exploration of an association

PubMed Central

NAPLES, ADAM; KATZ, LEN; GRIGORENKO, ELENA L.

2012-01-01

Based on numerous suggestions in the literature, we evaluated lexical decision (LD) as a putative endophenotype for reading comprehension by investigating heritability estimates and segregation analyses parameter estimates for both of these phenotypes. Specifically, in a segregation analysis of a large sample of families, we established that there is little to no overlap between genes contributing to LD and reading comprehension and that the genetic mechanism behind LD derived from this analysis appears to be more complex than that for reading comprehension. We conclude that in our sample, LD is not a good candidate as an endophenotype for reading comprehension, despite previous suggestions from the literature. Based on this conclusion, we discuss the role and benefit of the endophenotype approach in studies of complex human cognitive functions. PMID:23062302

Identification and characterization of microRNAs related to salt stress in broccoli, using high-throughput sequencing and bioinformatics analysis.

PubMed

Tian, Yunhong; Tian, Yunming; Luo, Xiaojun; Zhou, Tao; Huang, Zuoping; Liu, Ying; Qiu, Yihan; Hou, Bing; Sun, Dan; Deng, Hongyu; Qian, Shen; Yao, Kaitai

2014-09-03

MicroRNAs (miRNAs) are a new class of endogenous regulators of a broad range of physiological processes, which act by regulating gene expression post-transcriptionally. The brassica vegetable, broccoli (Brassica oleracea var. italica), is very popular with a wide range of consumers, but environmental stresses such as salinity are a problem worldwide in restricting its growth and yield. Little is known about the role of miRNAs in the response of broccoli to salt stress. In this study, broccoli subjected to salt stress and broccoli grown under control conditions were analyzed by high-throughput sequencing. Differential miRNA expression was confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). The prediction of miRNA targets was undertaken using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) database and Gene Ontology (GO)-enrichment analyses. Two libraries of small (or short) RNAs (sRNAs) were constructed and sequenced by high-throughput Solexa sequencing. A total of 24,511,963 and 21,034,728 clean reads, representing 9,861,236 (40.23%) and 8,574,665 (40.76%) unique reads, were obtained for control and salt-stressed broccoli, respectively. Furthermore, 42 putative known and 39 putative candidate miRNAs that were differentially expressed between control and salt-stressed broccoli were revealed by their read counts and confirmed by the use of stem-loop real-time RT-PCR. Amongst these, the putative conserved miRNAs, miR393 and miR855, and two putative candidate miRNAs, miR3 and miR34, were the most strongly down-regulated when broccoli was salt-stressed, whereas the putative conserved miRNA, miR396a, and the putative candidate miRNA, miR37, were the most up-regulated. Finally, analysis of the predicted gene targets of miRNAs using the GO and KO databases indicated that a range of metabolic and other cellular functions known to be associated with salt stress were up-regulated in broccoli treated with salt. A comprehensive study of broccoli miRNA in relation to salt stress has been performed. We report significant data on the miRNA profile of broccoli that will underpin further studies on stress responses in broccoli and related species. The differential regulation of miRNAs between control and salt-stressed broccoli indicates that miRNAs play an integral role in the regulation of responses to salt stress.

Fire blight disease reactome: RNA-seq transcriptional profile of apple host plant defense responses to Erwinia amylovora pathogen infection.

PubMed

Kamber, Tim; Buchmann, Jan P; Pothier, Joël F; Smits, Theo H M; Wicker, Thomas; Duffy, Brion

2016-02-17

The molecular basis of resistance and susceptibility of host plants to fire blight, a major disease threat to pome fruit production globally, is largely unknown. RNA-sequencing data from challenged and mock-inoculated flowers were analyzed to assess the susceptible response of apple to the fire blight pathogen Erwinia amylovora. In presence of the pathogen 1,080 transcripts were differentially expressed at 48 h post inoculation. These included putative disease resistance, stress, pathogen related, general metabolic, and phytohormone related genes. Reads, mapped to regions on the apple genome where no genes were assigned, were used to identify potential novel genes and open reading frames. To identify transcripts specifically expressed in response to E. amylovora, RT-PCRs were conducted and compared to the expression patterns of the fire blight biocontrol agent Pantoea vagans strain C9-1, another apple pathogen Pseudomonas syringae pv. papulans, and mock inoculated apple flowers. This led to the identification of a peroxidase superfamily gene that was lower expressed in response to E. amylovora suggesting a potential role in the susceptibility response. Overall, this study provides the first transcriptional profile by RNA-seq of the host plant during fire blight disease and insights into the response of susceptible apple plants to E. amylovora.

Fire blight disease reactome: RNA-seq transcriptional profile of apple host plant defense responses to Erwinia amylovora pathogen infection

PubMed Central

Kamber, Tim; Buchmann, Jan P.; Pothier, Joël F.; Smits, Theo H. M.; Wicker, Thomas; Duffy, Brion

2016-01-01

The molecular basis of resistance and susceptibility of host plants to fire blight, a major disease threat to pome fruit production globally, is largely unknown. RNA-sequencing data from challenged and mock-inoculated flowers were analyzed to assess the susceptible response of apple to the fire blight pathogen Erwinia amylovora. In presence of the pathogen 1,080 transcripts were differentially expressed at 48 h post inoculation. These included putative disease resistance, stress, pathogen related, general metabolic, and phytohormone related genes. Reads, mapped to regions on the apple genome where no genes were assigned, were used to identify potential novel genes and open reading frames. To identify transcripts specifically expressed in response to E. amylovora, RT-PCRs were conducted and compared to the expression patterns of the fire blight biocontrol agent Pantoea vagans strain C9-1, another apple pathogen Pseudomonas syringae pv. papulans, and mock inoculated apple flowers. This led to the identification of a peroxidase superfamily gene that was lower expressed in response to E. amylovora suggesting a potential role in the susceptibility response. Overall, this study provides the first transcriptional profile by RNA-seq of the host plant during fire blight disease and insights into the response of susceptible apple plants to E. amylovora. PMID:26883568

Molecular cloning of a putative divalent-cation transporter gene as a new genetic marker for the identification of Lactobacillus brevis strains capable of growing in beer.

PubMed

Hayashi, N; Ito, M; Horiike, S; Taguchi, H

2001-05-01

Random amplified polymorphic DNA (RAPD) PCR analysis of Lactobacillus brevis isolates from breweries revealed that one of the random primers could distinguish beer-spoilage strains of L. brevis from nonspoilage strains. The 1.1-kb DNA fragment amplified from all beer-spoilers included one open reading frame, termed hitA (hop-inducible cation transporter), which encodes an integral membrane protein with 11 putative trans-membrane domains and a binding protein-dependent transport signature of a non-ATP binding membrane transporter common to several prokaryotic and eukaryotic transporters. The hitA polypeptide is homologous to the natural resistance-associated macrophage protein (Nramp) family characterized as divalent-cation transport proteins in many prokaryotic and eukaryotic organisms. Northern blot analysis indicated that the hitA transcripts are expressed in cells cultivated in MRS broth supplemented with hop bitter compounds, which act as mobile-carrier ionophores, dissipating the trans-membrane pH gradient in bacteria sensitive to the hop bitter compounds by exchanging H+ for cellular divalent cations such as Mn2+. This suggests that the hitA gene products may play an important role in making the bacteria resistant to hop bitter compounds in beer by transporting metal ions such as Mn2+ into cells that no longer maintain the proton gradient.

In Silico Pattern-Based Analysis of the Human Cytomegalovirus Genome

PubMed Central

Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T.; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

2003-01-01

More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/). PMID:12634390

In silico pattern-based analysis of the human cytomegalovirus genome.

PubMed

Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

2003-04-01

More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/).

Gene expression profiles of prohibitin in testes of Octopus tankahkeei (ot-phb) revealing its possible role during spermiogenesis.

PubMed

Mao, Hai-Tao; Wang, Da-Hui; Lan, Zhou; Zhou, Hong; Yang, Wan-Xi

2012-05-01

Prohibitin is essential for intracellular homeostasis and stabilization of mitochondrial respiratory chain complexes. To explore its functions during spermiogenesis of Octopus tankahkeei (O. tankahkeei), we have cloned and sequenced the cDNA of this mammalian PHB homologue (termed ot-PHB) from the testes of O. tankahkeei. The 1165 bp ot-phb cDNA contains a 100 bp 5' UTR, a 882 bp open reading frame and a 183 bp 3' UTR. The putative ot-PHB protein owns a transmembrane domain from 6 to 31 amino acid (aa) and a putative PHB domain from 26 to 178 aa. Protein alignment demonstrated that ot-PHB had 73.3, 73.6, 74.0, 75.1, and 45.4% identity with its homologues in Homo sapiens, Mus muculus, Danio rerio, Xenopus tropicalis and Trypanosoma brucei, respectively. Tissue distribution profile analysis revealed its presence in all the tissues examined. In situ hybridization in spermiogenic cells demonstrated that ot-phb was expressed moderately at the beginning of the spermiogenesis. The abundance of transcripts increased in intermediate spermatids and in drastically remodeling final spermatids. In mature spermatozoa, the residuary transcripts concentrated around the chondriosomal mantle where mitochondria assemble around. In summary, the expression of ot-phb during spermiogenesis implicates a potential function of this protein during mitochondrial ubiquitination. It is the first time to implicate the role of prohibitin in cephalopod spermiogenesis.

Intracellular Localization Map of Human Herpesvirus 8 Proteins▿

PubMed Central

Sander, Gaby; Konrad, Andreas; Thurau, Mathias; Wies, Effi; Leubert, Rene; Kremmer, Elisabeth; Dinkel, Holger; Schulz, Thomas; Neipel, Frank; Stürzl, Michael

2008-01-01

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma. We present a localization map of 85 HHV-8-encoded proteins in mammalian cells. Viral open reading frames were cloned with a Myc tag in expression plasmids, confirmed by full-length sequencing, and expressed in HeLa cells. Protein localizations were analyzed by immunofluorescence microscopy. Fifty-one percent of all proteins were localized in the cytoplasm, 22% were in the nucleus, and 27% were found in both compartments. Surprisingly, we detected viral FLIP (v-FLIP) in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally localized exclusively in the cytoplasm. This suggested that v-FLIP may exert additional or alternative functions compared to cellular FLIPs. In addition, it has been shown recently that the K10 protein can bind to at least 15 different HHV-8 proteins. We noticed that K10 and only five of its 15 putative binding factors were localized in the nucleus when the proteins were expressed in HeLa cells individually. Interestingly, in coexpression experiments K10 colocalized with 87% (13 of 15) of its putative binding partners. Colocalization was induced by translocation of either K10 alone or both proteins. These results indicate active intracellular translocation processes in virus-infected cells. Specifically in this framework, the localization map may provide a useful reference to further elucidate the function of HHV-8-encoded genes in human diseases. PMID:18077714

A novel subviral agent associated with a geminivirus: The first report of a DNA satellite

PubMed Central

Dry, Ian B.; Krake, Leslie R.; Rigden, Justin E.; Rezaian, M. Ali

1997-01-01

Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem–loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication. PMID:9192696

Molecular Cloning, Characterization, and Differential Expression of a Glucoamylase Gene from the Basidiomycetous Fungus Lentinula edodes

PubMed Central

Zhao, J.; Chen, Y. H.; Kwan, H. S.

2000-01-01

The complete nucleotide sequence of putative glucoamylase gene gla1 from the basidiomycetous fungus Lentinula edodes strain L54 is reported. The coding region of the genomic glucoamylase sequence, which is preceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The gla1 gene sequence codes for a putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading frame sequence of the gla1 gene shows strong homology with those of other fungal glucoamylase genes and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity between the Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservation found in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity and levels of gene expression in L. edodes strain L54 grown on different carbon sources (glucose, starch, cellulose, and potato extract) and in various developmental stages (mycelium growth, primordium appearance, and fruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for gla1 gene expression shows that expression of gla1 was induced by starch and increased during the process of fruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesis of the basidiomycetous fungus. PMID:10831434

Open Court Reading©. What Works Clearinghouse Intervention Report. Updated October 2014

ERIC Educational Resources Information Center

What Works Clearinghouse, 2014

2014-01-01

"Open Court Reading©" is a reading program for grades K-6 published by McGraw-Hill Education that is designed to teach decoding, comprehension, inquiry, and writing in a three-part logical progression. Part One of each unit, Preparing to Read, focuses on phonemic awareness, sounds and letters, phonics, fluency, and word knowledge. Part…

Open Court Reading[c]. What Works Clearinghouse Intervention Report

ERIC Educational Resources Information Center

What Works Clearinghouse, 2012

2012-01-01

"Open Court Reading"[c] is a core reading program for grades K-6 developed by SRA/McGraw-Hill that is designed to teach decoding, comprehension, inquiry, and writing in a logical progression. Part 1 of each unit, Preparing to Read, focuses on phonemic awareness, sounds and letters, phonics, fluency, and word knowledge. Part 2, Reading…

Open Court Reading[c]. What Works Clearinghouse Intervention Report

ERIC Educational Resources Information Center

What Works Clearinghouse, 2008

2008-01-01

"Open Court Reading"[c] is an elementary basal reading program for grades K-6 developed by SRA/McGraw-Hill. The program is designed to systematically teach decoding, comprehension, inquiry and investigation, and writing in a logical progression. Part 1 of each unit, Preparing to Read, focuses on phonemic awareness, sounds and letters, phonics,…

Openness to Experience and Night-Sky Watching Interest as Predictors of Reading for Pleasure: Path Analysis of a Mediation Model

ERIC Educational Resources Information Center

Kelly, William E.

2010-01-01

The relation between reading for pleasure, night-sky watching interest, and openness to experience were examined in a sample of 129 college students. Results of a path analysis examining a mediation model indicated that the influence of night-sky interest on reading for pleasure was not mediated by the broad personality domain openness to…

Reading and Reality. Proceedings of the Annual Reading Conference (14th, Terre Haute, Indiana, June 14, 1984).

ERIC Educational Resources Information Center

Gibbs, Vanita M., Comp.; Waterman, David C., Comp.

Intended for reading teachers, this pamphlet contains the presentations of the 14th annual reading conference at Indiana State University, beginning with opening remarks by David C. Waterman and welcoming comments by J. Stephen Hazlett. In the opening address, "What Good is Comprehension without Composition?" by Sharon and David Moore, the role of…

The effect of a specialized dyslexia font, OpenDyslexic, on reading rate and accuracy.

PubMed

Wery, Jessica J; Diliberto, Jennifer A

2017-07-01

A single-subject alternating treatment design was used to investigate the extent to which a specialized dyslexia font, OpenDyslexic, impacted reading rate or accuracy compared to two commonly used fonts when used with elementary students identified as having dyslexia. OpenDyslexic was compared to Arial and Times New Roman in three reading tasks: (a) letter naming, (b) word reading, and (c) nonsense word reading. Data were analyzed through visual analysis and improvement rate difference, a nonparametric measure of nonoverlap for comparing treatments. Results from this alternating treatment experiment show no improvement in reading rate or accuracy for individual students with dyslexia, as well as the group as a whole. While some students commented that the font was "new" or "different", none of the participants reported preferring to read material presented in that font. These results indicate there may be no benefit for translating print materials to this font.

Plastomes of the green algae Hydrodictyon reticulatum and Pediastrum duplex (Sphaeropleales, Chlorophyceae).

PubMed

McManus, Hilary A; Sanchez, Daniel J; Karol, Kenneth G

2017-01-01

Comparative studies of chloroplast genomes (plastomes) across the Chlorophyceae are revealing dynamic patterns of size variation, gene content, and genome rearrangements. Phylogenomic analyses are improving resolution of relationships, and uncovering novel lineages as new plastomes continue to be characterized. To gain further insight into the evolution of the chlorophyte plastome and increase the number of representative plastomes for the Sphaeropleales, this study presents two fully sequenced plastomes from the green algal family Hydrodictyaceae (Sphaeropleales, Chlorophyceae), one from Hydrodictyon reticulatum and the other from Pediastrum duplex . Genomic DNA from Hydrodictyon reticulatum and Pediastrum duplex was subjected to Illumina paired-end sequencing and the complete plastomes were assembled for each. Plastome size and gene content were characterized and compared with other plastomes from the Sphaeropleales. Homology searches using BLASTX were used to characterize introns and open reading frames (orfs) ≥ 300 bp. A phylogenetic analysis of gene order across the Sphaeropleales was performed. The plastome of Hydrodictyon reticulatum is 225,641 bp and Pediastrum duplex is 232,554 bp. The plastome structure and gene order of H. reticulatum and P. duplex are more similar to each other than to other members of the Sphaeropleales. Numerous unique open reading frames are found in both plastomes and the plastome of P. duplex contains putative viral protein genes, not found in other Sphaeropleales plastomes. Gene order analyses support the monophyly of the Hydrodictyaceae and their sister relationship to the Neochloridaceae. The complete plastomes of Hydrodictyon reticulatum and Pediastrum duplex , representing the largest of the Sphaeropleales sequenced thus far, once again highlight the variability in size, architecture, gene order and content across the Chlorophyceae. Novel intron insertion sites and unique orfs indicate recent, independent invasions into each plastome, a hypothesis testable with an expanded plastome investigation within the Hydrodictyaceae.

Bacteriocins (meningocins) in Norwegian isolates of Neisseria meningitidis: possible role in the course of a meningococcal epidemic.

PubMed

Allunans, Juris; Kristiansen, Knut Ivan; Assalkhou, Reza; Bjørås, Magnar

2008-05-01

In the Neisseria meningitidis strain MC58 (serogroup B; ET-5 complex) genome three putative islands of horizontally transferred DNA (IHTs) have been identified. IHT-A2 codes for eight hypothetical proteins and two disrupted open reading frames with similarity to a secretion protein (NMB0097) and an ABC transporter (NMB0098). The strains MC58 and 44/76 (shown here) are meningocin resistant/weakly sensitive. None of these strains are meningocin producers. However, NMB0097 and NMB0098 homologues with open reading frames are found in meningocin producers (N. meningitidis P241 (serogroup A; systemic isolate) and BT878 (serogroup B; carrier isolate), and also in strain FAM18 (serogroup C; ET-37 complex). Knocking out either of the two genes in the strain BT878 yielded mutants that did not secrete meningocin. A similarly disrupted tolC mutant in strain BT878 still released meningocin. Among systemic meningococcal isolates prior to and at the onset (mid-1973 to the end of 1974) of the epidemic peaking in 1975 in North Norway, 12 of 30 (40%) isolates of serogroup A were meningocin producers. However, the rate for serogroup B was 1 of 45 (2.2%). Serogroup B meningocin-resistant/weakly sensitive non-producers dominated in the region from mid-1975 and spread to the rest of the country from then on. No producers were found in selected pharyngeal isolates from healthy carriers collected in Svalbard in the early spring of 1975. Our results suggest that meningocinogeny has played a part in the change from serogroup A to serogroup B among isolates in North Norway during the first half of 1975.

Identification by Subtractive Hybridization of a Novel Insertion Sequence Specific for Virulent Strains of Porphyromonas gingivalis

PubMed Central

Sawada, Koichi; Kokeguchi, Susumu; Hongyo, Hiroshi; Sawada, Satoko; Miyamoto, Manabu; Maeda, Hiroshi; Nishimura, Fusanori; Takashiba, Shogo; Murayama, Yoji

1999-01-01

Subtractive hybridization was employed to isolate specific genes from virulent Porphyromonas gingivalis strains that are possibly related to abscess formation. The genomic DNA from the virulent strain P. gingivalis W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only Bacillus stearothermophilus IS5377, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS1598, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS1598 was detected in P. gingivalis W83, W50, and FDC 381 by Southern hybridization. All three P. gingivalis strains have been shown to possess abscess-forming ability in animal models. However, IS1598 was not detected in avirulent strains of P. gingivalis, including ATCC 33277. The IS1598 may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS1598 isolated in this study is a novel insertion element which might be a specific marker for virulent P. gingivalis strains. PMID:10531208

Patients' Positive and Negative Responses to Reading Mental Health Clinical Notes Online.

PubMed

Denneson, Lauren M; Chen, Jason I; Pisciotta, Maura; Tuepker, Anais; Dobscha, Steven K

2018-05-01

This study describes responses to OpenNotes, clinical notes available online, among patients receiving mental health care and explores whether responses vary by patient demographic or clinical characteristics. Survey data from 178 veterans receiving mental health treatment at a large Veterans Affairs medical center included patient-reported health self-efficacy, health knowledge, alliance with clinicians, and negative emotional responses after reading OpenNotes. Health care data were extracted from the patient care database. Reading OpenNotes helped many participants feel in control of their health care (49%) and have more trust in clinicians (45%), although a few (8%) frequently felt upset after reading their notes. In multivariate models, posttraumatic stress disorder was associated with increased patient-clinician alliance (p=.046) but also with negative emotional responses (p<.01). Patients receiving mental health care frequently reported benefits from reading OpenNotes, yet some experienced negative responses.

Efficient radiologic reading environment by using an open-source macro program as connection software.

PubMed

Lee, Young Han

2012-01-01

The objectives are (1) to introduce an easy open-source macro program as connection software and (2) to illustrate the practical usages in radiologic reading environment by simulating the radiologic reading process. The simulation is a set of radiologic reading process to do a practical task in the radiologic reading room. The principal processes are: (1) to view radiologic images on the Picture Archiving and Communicating System (PACS), (2) to connect the HIS/EMR (Hospital Information System/Electronic Medical Record) system, (3) to make an automatic radiologic reporting system, and (4) to record and recall information of interesting cases. This simulation environment was designed by using open-source macro program as connection software. The simulation performed well on the Window-based PACS workstation. Radiologists practiced the steps of the simulation comfortably by utilizing the macro-powered radiologic environment. This macro program could automate several manual cumbersome steps in the radiologic reading process. This program successfully acts as connection software for the PACS software, EMR/HIS, spreadsheet, and other various input devices in the radiologic reading environment. A user-friendly efficient radiologic reading environment could be established by utilizing open-source macro program as connection software. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

A feasibility study to determine if there is a market for automatic meter-reading devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hilberg, G.R.

1996-08-01

For many utilities the cost of manually reading meters is increasing due to personnel expenses and equipment costs. The current system of manual meters provides little ability for the utility to reduce costs. To reduce meter reading costs the utility must automate the manual system and reduce personnel expenses. A water utility in San Diego county was studied to calculate the cost of reading individual water meters. This would allow for the selective replacement of {open_quotes}high-cost{close_quotes} meters to quickly reduce meter-reading costs while limiting the necessary capital investments. As the {open_quotes}high-cost{close_quotes} meters are selectively replaced, a utility with a significantmore » difference in individual meter reading costs could save three to five dollars per meter per year. This study showed that the {open_quotes}high-cost{close_quotes} meters were six times more expensive to read than the average meter. Additionally, AMR systems increase the information available to consumers and to the utility on usage patterns and problems. The challenge was to cost effectively identify the {open_quotes}high-cost{close_quotes} meters. The costs to collect these data were less than $500.« less

Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content.

PubMed

Xu, Jingyu; Francis, Tammy; Mietkiewska, Elzbieta; Giblin, E Michael; Barton, Dennis L; Zhang, Yan; Zhang, Meng; Taylor, David C

2008-10-01

A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins.

PubMed

Varmanen, P; Rantanen, T; Palva, A

1996-12-01

A proline iminopeptidase gene (pepI) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepI gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepI-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded PepI protein showed 65% identity with the PepI proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepI gene was overexpressed in Escherichia coli. Purified PepI hydrolysed only di and tripeptides with proline in the first position. Optimum PepI activity was observed at pH 7.5 and 40 degrees C. A gel filtration analysis indicated that PepI is a dimer of M(r) 53,000. PepI was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed Km and Vmax values of 0.8 mM and 350 mmol min-1 mg-1, respectively, and a very high turnover number of 135,000 s-1.

Open Reading Frame Phylogenetic Analysis on the Cloud

PubMed Central

2013-01-01

Phylogenetic analysis has become essential in researching the evolutionary relationships between viruses. These relationships are depicted on phylogenetic trees, in which viruses are grouped based on sequence similarity. Viral evolutionary relationships are identified from open reading frames rather than from complete sequences. Recently, cloud computing has become popular for developing internet-based bioinformatics tools. Biocloud is an efficient, scalable, and robust bioinformatics computing service. In this paper, we propose a cloud-based open reading frame phylogenetic analysis service. The proposed service integrates the Hadoop framework, virtualization technology, and phylogenetic analysis methods to provide a high-availability, large-scale bioservice. In a case study, we analyze the phylogenetic relationships among Norovirus. Evolutionary relationships are elucidated by aligning different open reading frame sequences. The proposed platform correctly identifies the evolutionary relationships between members of Norovirus. PMID:23671843

BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA from disk abalone Haliotis discus discus.

PubMed

Kim, Yucheol; De Zoysa, Mahanama; Lee, Youngdeuk; Whang, Ilson; Lee, Jehee

2010-11-01

A BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA was cloned from the disk abalone (Haliotis discus discus) and designated as AbLECT-1. A full-length (705 bp) of AbLECT-1 cDNA was composed of a 576 bp open reading frame that translates into a putative peptide of 192 amino acids. Deduced amino acid sequence of AbLECT-1 had 15.5- and 27.8% identity and similarity to human LECT-1, respectively. Quantitative real-time PCR analysis results showed that the mRNA of AbLECT-1 was constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas in a tissue-specific manner. Moreover, the AbLECT-1 transcription level was induced in hemocytes after challenge with Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes suggesting that it may be involved in immune response reactions in abalone. Copyright 2010 Elsevier Ltd. All rights reserved.

Complete genomic sequence and taxonomic position of eel virus European X (EVEX), a rhabdovirus of European eel.

PubMed

Galinier, Richard; van Beurden, Steven; Amilhat, Elsa; Castric, Jeannette; Schoehn, Guy; Verneau, Olivier; Fazio, Géraldine; Allienne, Jean-François; Engelsma, Marc; Sasal, Pierre; Faliex, Elisabeth

2012-06-01

Eel virus European X (EVEX) was first isolated from diseased European eel Anguilla anguilla in Japan at the end of seventies. The virus was tentatively classified into the Rhabdoviridae family on the basis of morphology and serological cross reactivity. This family of viruses is organized into six genera and currently comprises approximately 200 members, many of which are still unassigned because of the lack of molecular data. This work presents the morphological, biochemical and genetic characterizations of EVEX, and proposes a taxonomic classification for this virus. We provide its complete genome sequence, plus a comprehensive sequence comparison between isolates from different geographical origins. The genome encodes the five classical structural proteins plus an overlapping open reading frame in the phosphoprotein gene, coding for a putative C protein. Phylogenic relationship with other rhabdoviruses indicates that EVEX is most closely related to the Vesiculovirus genus and shares the highest identity with trout rhabdovirus 903/87. Copyright © 2012 Elsevier B.V. All rights reserved.

A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: cloning and expression pattern in pharate first instar larvae in relation to diapause.

PubMed

Yang, Ping; Tanaka, Hiromasa; Kuwano, Eiichi; Suzuki, Koichi

2008-03-01

A new cytochrome P450 gene, CYP4G25, was identified as a differentially expressed gene between the diapausing and post-diapausing pharate first instar larvae of the wild silkmoth Antheraea yamamai, using subtractive cDNA hybridization. The cDNA sequence of CYP4G25 has an open reading frame of 1674 nucleotides encoding 557 amino acid residues. Sequence analysis of the putative CYP4G25 protein disclosed the motif FXXGXRXCXG that is essential for heme binding in P450 cytochromes. Hybridization in situ demonstrated predominant expression of CYP4G25 in the integument of pharate first instar larvae. Northern blotting analysis showed an intensive signal after the initiation of diapause and no or weak expression throughout the periods of pre-diapause and post-diapause, including larval development. These results indicate that CYP4G25 is strongly associated with diapause in pharate first instar larvae.

Cloning and expression analysis of a gene that shows developmental regulation upon tuberization in potato.

PubMed

Jackson, S; Gascón, J; Carrera, E; Monte, E; Prat, S

1997-01-01

Differential screening of a potato leaf cDNA library with cDNA probes made from tuberizing and non-tuberizing Solanum demissum plants led to the identification of a clone that is upregulated in leaves and other tissues upon tuberization. This clone was also shown to have a high level of expression in green tomato fruit, its expression falling off as the fruit turns red. No sucrose or hormonal regulation of the expression of this clone was observed and it did not respond to wounding or heat stress. Clone 32B is 532 bp long and contains an open reading frame encoding a small protein of 98 amino acids. The deduced protein sequence has a putative signal peptide for ER transport and a 10 amino acid domain in the C-terminal region of the protein, both of which are also found in the cotton LEA5, Arabidopsis Di21 and the mungbean Arg2 proteins.

Mobilization of the relaxable Staphylococcus aureus plasmid pC221 by the conjugative plasmid pGO1 involves three pC221 loci.

PubMed Central

Projan, S J; Archer, G L

1989-01-01

The Staphylococcus aureus plasmid pC221, a 4.6-kilobase multicopy chloramphenicol resistance plasmid that forms plasmid-protein relaxation complexes, was mobilized for transfer by the conjugative plasmid pGO1. Two open reading frames on the pC221 genome, now designated mobA and mobB, as well as a cis-acting locus, the putative oriT, were shown to be in involved in pC221 mobilization. The mobA (but not mobB) and oriT loci were required for pC221 relaxation, and relaxation was necessary but not sufficient for pC221 mobilization by pGO1. oriT was cloned onto a pE194 derivative and complemented in trans for both relaxation and mobilization. Mobilization of relaxable plasmids in S. aureus appears to be analogous to mobilization by donation observed in gram-negative bacteria. Images PMID:2703461

Expression, purification and preliminary X-ray analysis of the C-terminal domain of an arginine repressor protein from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, George J.; Garen, Craig R.; Cherney, Maia M.

2007-11-01

The C-terminal portion of the arginine repressor protein from M. tuberculosis H37Rv has been crystallized. The complete transcriptional factor regulates arginine biosynthesis by binding operator DNA when arginine is bound at the C-terminal domain. The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 Å. The crystals belong to space group P1 and themore » Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of six molecules per unit cell.« less

Ngaingan virus, a macropod-associated rhabdovirus, contains a second glycoprotein gene and seven novel open reading frames.

PubMed

Gubala, Aneta; Davis, Steven; Weir, Richard; Melville, Lorna; Cowled, Chris; Walker, Peter; Boyle, David

2010-03-30

Ngaingan virus (NGAV) was isolated from a pool of biting midges that were collected in the tropics of northern Australia. Reported here is the full-length sequence of the NGAV genome, which, at over 15.7 kb, is the largest in any rhabdovirus described to date and contains 13 genes, the highest number of genes observed in any (-) ssRNA virus. Seven of these putative genes show no significant homology to known proteins. Like viruses in the genus Ephemerovirus, NGAV possesses a second glycoprotein gene (G(NS)). Phylogenetic analyses, however, place NGAV within the yet to be classified "Hart Park" group containing Wongabel and Flanders viruses, which do not contain a second glycoprotein gene. Screening of various animal sera from northern Australia has indicated that NGAV is currently circulating in macropods (wallabies, wallaroos and kangaroos), highlighting the need for further studies to determine its potential to cause disease in these species. Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.

An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts.

PubMed

Jaquemar, D; Schenker, T; Trueb, B

1999-03-12

We have identified a novel transformation-sensitive mRNA, which is present in cultured fibroblasts but is lacking in SV40 transformed cells as well as in many mesenchymal tumor cell lines. The corresponding gene is located on human chromosome 8 in band 8q13. The open reading frame of the mRNA encodes a protein of 1119 amino acids forming two distinct domains. The N-terminal domain consists of 18 repeats that are related to the cytoskeletal protein ankyrin. The C-terminal domain contains six putative transmembrane segments that resemble many ion channels. This overall structure is reminiscent of TRP-like proteins that function as store-operated calcium channels. The novel protein with an Mr of 130 kDa is expressed at a very low level in human fibroblasts and at a moderate level in liposarcoma cells. Overexpression in eukaryotic cells appears to interfere with normal growth, suggesting that it might play a direct or indirect role in signal transduction and growth control.

The genome of the Lactobacillus sanfranciscensis temperate phage EV3

PubMed Central

2013-01-01

Background Bacteriophages infection modulates microbial consortia and transduction is one of the most important mechanism involved in the bacterial evolution. However, phage contamination brings food fermentations to a halt causing economic setbacks. The number of phage genome sequences of lactic acid bacteria especially of lactobacilli is still limited. We analysed the genome of a temperate phage active on Lactobacillus sanfranciscensis, the predominant strain in type I sourdough fermentations. Results Sequencing of the DNA of EV3 phage revealed a genome of 34,834 bp and a G + C content of 36.45%. Of the 43 open reading frames (ORFs) identified, all but eight shared homology with other phages of lactobacilli. A similar genomic organization and mosaic pattern of identities align EV3 with the closely related Lactobacillus vaginalis ATCC 49540 prophage. Four unknown ORFs that had no homologies in the databases or predicted functions were identified. Notably, EV3 encodes a putative dextranase. Conclusions EV3 is the first L. sanfranciscensis phage that has been completely sequenced so far. PMID:24308641

Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins

PubMed Central

Delcourt, Vivian; Lucier, Jean-François; Gagnon, Jules; Beaudoin, Maxime C; Vanderperre, Benoît; Breton, Marc-André; Motard, Julie; Jacques, Jean-François; Brunelle, Mylène; Gagnon-Arsenault, Isabelle; Fournier, Isabelle; Ouangraoua, Aida; Hunting, Darel J; Cohen, Alan A; Landry, Christian R; Scott, Michelle S

2017-01-01

Recent functional, proteomic and ribosome profiling studies in eukaryotes have concurrently demonstrated the translation of alternative open-reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by these altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and contain functional domains. Evolutionary analyses indicate that altORFs often show more extreme conservation patterns than their CDSs. Thousands of alternative proteins are detected in proteomic datasets by reanalysis using a database containing predicted alternative proteins. This is illustrated with specific examples, including altMiD51, a 70 amino acid mitochondrial fission-promoting protein encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting mitochondrial fission. Our results suggest that many genes are multicoding genes and code for a large protein and one or several small proteins. PMID:29083303

Molecular variability analysis of five new complete cacao swollen shoot virus genomic sequences.

PubMed

Muller, E; Sackey, S

2005-01-01

Cacao swollen shoot virus (CSSV), a member of the family Caulimovi-ridae, genus Badnavirus occurs in all the main cacao-growing areas of West Africa. We amplified, cloned and sequenced complete genomes of five new isolates, two originating from Togo and three originating from Ghana. The genome of these five newly sequenced isolates all contain the five putative open reading frames I, II, III, X and Y described for the first sequenced CSSV isolate, Agou1 originating from Togo. Their genomes have been aligned with the genome of Agou1. The nucleotide and amino acid sequence identities between isolates have been calculated and a phylogenetic analysis has been made including other pararetroviruses. Maximum nucleotide sequence variability between complete genomes of CSSV isolates was 29.4%. Geographical differentiation between isolates appears more important than differentiation between mild and severe isolates. ORF X differs greatly in size and sequence between the Togolese isolates Nyongbo2 and Agou1, and the four other isolates, its functional role is therefore clearly questionable.

Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans.

PubMed Central

Chernin, L S; De la Fuente, L; Sobolev, V; Haran, S; Vorgias, C E; Oppenheim, A B; Chet, I

1997-01-01

The gene chiA, which codes for endochitinase, was cloned from a soilborne Enterobacter agglomerans. Its complete sequence was determined, and the deduced amino acid sequence of the enzyme designated Chia_Entag yielded an open reading frame coding for 562 amino acids of a 61-kDa precursor protein with a putative leader peptide at its N terminus. The nucleotide and polypeptide sequences of Chia_Entag showed 86.8 and 87.7% identity with the corresponding gene and enzyme, Chia_Serma, of Serratia marcescens, respectively. Homology modeling of Chia_Entag's three-dimensional structure demonstrated that most amino acid substitutions are at solvent-accessible sites. Escherichia coli JM109 carrying the E. agglomerans chiA gene produced and secreted Chia_Entag. The antifungal activity of the secreted endochitinase was demonstrated in vitro by inhibition of Fusarium oxysporum spore germination. The transformed strain inhibited Rhizoctonia solani growth on plates and the root rot disease caused by this fungus in cotton seedlings under greenhouse conditions. PMID:9055404

Characterization of a putative periplasmic transport system for octopine accumulation encoded by Agrobacterium tumefaciens Ti plasmid pTiA6.

PubMed Central

Valdivia, R H; Wang, L; Winans, S C

1991-01-01

Neoplastic crown gall tumors incited by Agrobacterium tumefaciens release novel amino acid or sugar derivatives known as opines, whose synthesis is directed by genes transferred to plant cells. Agrobacterium cells can transport and catabolize these compounds as sources of carbon and nitrogen. This article describes a region of the pTiA6 plasmid which is required for catabolism of the opine octopine and whose transcription is induced by octopine. This region of the plasmid contains four open reading frames, occQ, occM, occP, and occJ, which show homology to the family of so-called shock-sensitive permeases. TnphoA mutagenesis demonstrated that the OccJ and OccM proteins lie fully or partly in the periplasmic space. The OccJ protein was identified by electrophoresis and found to be fully localized in the periplasmic space. When these proteins were expressed in Escherichia coli, radiolabeled octopine became cell-associated. Images FIG. 6 PMID:1655707

Genetic variability of the equine casein genes.

PubMed

Brinkmann, J; Jagannathan, V; Drögemüller, C; Rieder, S; Leeb, T; Thaller, G; Tetens, J

2016-07-01

The casein genes are known to be highly variable in typical dairy species, such as cattle and goat, but the knowledge about equine casein genes is limited. Nevertheless, mare milk production and consumption is gaining importance because of its high nutritive value, use in naturopathy, and hypoallergenic properties with respect to cow milk protein allergies. In the current study, the open reading frames of the 4 casein genes CSN1S1 (αS1-casein), CSN2 (β-casein), CSN1S2 (αS2-casein), and CSN3 (κ-casein) were resequenced in 253 horses of 14 breeds. The analysis revealed 21 nonsynonymous nucleotide exchanges, as well as 11 synonymous nucleotide exchanges, leading to a total of 31 putative protein isoforms predicted at the DNA level, 26 of which considered novel. Although the majority of the alleles need to be confirmed at the transcript and protein level, a preliminary nomenclature was established for the equine casein alleles. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

TTSV1, a new virus-like particle isolated from the hyperthermophilic crenarchaeote Thermoproteus tenax.

PubMed

Ahn, Dae-Gyun; Kim, Se-Il; Rhee, Jin-Kyu; Kim, Kwang Pyo; Pan, Jae-Gu; Oh, Jong-Won

2006-08-01

A new virus-like particle TTSV1 was isolated from the hyperthermophilic crenarchaeote Thermoproteus tenax sampled at a hot spring region in Indonesia. TTSV1 had a spherical shape with a diameter of approximately 70 nm and was morphologically similar to the PSV isolated from a strain of Pyrobaculum. The 21.6 kb linear double-stranded DNA genome of TTSV1 had 38 open reading frames (ORFs), of which 15 ORFs were most similar to those of PSV. The remaining 23 ORFs showed little similarity to proteins in the public databases. Southern blot analysis demonstrated that the viral genome is not integrated into the host chromosome. TTSV1 consisted of three putative structural proteins of 10, 20, and 35 kDa in size, and the 10-kDa major protein was identified by mass spectrometry as a TTSV1 gene product. TTSV1 could be assigned as a new member of the newly emerged Globuloviridae family that includes so far only one recently characterized virus PSV.

Identification of the WBSCR9 gene, encoding a novel transcriptional regulator, in the Williams-Beuren syndrome deletion at 7q11.23.

PubMed

Peoples, R J; Cisco, M J; Kaplan, P; Francke, U

1998-01-01

We have identified a novel gene (WBSCR9) within the common Williams-Beuren syndrome (WBS) deletion by interspecies sequence conservation. The WBSCR9 gene encodes a roughly 7-kb transcript with an open reading frame of 1483 amino acids and a predicted protein product size of 170.8 kDa. WBSCR9 is comprised of at least 20 exons extending over 60 kb. The transcript is expressed ubiquitously throughout development and is subject to alternative splicing. Functional motifs identified by sequence homology searches include a bromodomain; a PHD, or C4HC3, finger; several putative nuclear localization signals; four nuclear receptor binding motifs; a polyglutamate stretch and two PEST sequences. Bromodomains, PHD motifs and nuclear receptor binding motifs are cardinal features of proteins that are involved in chromatin remodeling and modulation of transcription. Haploinsufficiency for WBSCR9 gene products may contribute to the complex phenotype of WBS by interacting with tissue-specific regulatory factors during development.

A third genotype of the human parvovirus PARV4 in sub-Saharan Africa.

PubMed

Simmonds, Peter; Douglas, Jill; Bestetti, Giovanna; Longhi, Erika; Antinori, Spinello; Parravicini, Carlo; Corbellino, Mario

2008-09-01

PARV4 is a recently discovered human parvovirus widely distributed in injecting drug users in the USA and Europe, particularly in those co-infected with human immunodeficiency virus (HIV). Like parvovirus B19, PARV4 persists in previously exposed individuals. In bone marrow and lymphoid tissue, PARV4 sequences were detected in two sub-Saharan African study subjects with AIDS but without a reported history of parenteral exposure and who were uninfected with hepatitis C virus. PARV4 variants infecting these subjects were phylogenetically distinct from genotypes 1 and 2 (formerly PARV5) that were reported previously. Analysis of near-complete genome sequences demonstrated that they should be classified as a third (equidistant) PARV4 genotype. The availability of a further near-complete genome sequence of this novel genotype facilitated identification of conserved novel open reading frames embedded in the ORF2 coding sequence; one encoded a putative protein with identifiable homology to SAT proteins of members of the genus Parvovirus.

Differentiation of closely related but biologically distinct cherry isolates of Prunus necrotic ringspot virus by polymerase chain reaction.

PubMed

Hammond, R W; Crosslin, J M; Pasini, R; Howell, W E; Mink, G I

1999-07-01

Prunus necrotic ringspot ilarvirus (PNRSV) exists as a number of biologically distinct variants which differ in host specificity, serology, and pathology. Previous nucleotide sequence alignment and phylogenetic analysis of cloned reverse transcription-polymerase chain reaction (RT-PCR) products of several biologically distinct sweet cherry isolates revealed correlations between symptom type and the nucleotide and amino acid sequences of the 3a (putative movement protein) and 3b (coat protein) open reading frames. Based upon this analysis, RT-PCR assays have been developed that can identify isolates displaying different symptoms and serotypes. The incorporation of primers in a multiplex PCR protocol permits rapid detection and discrimination among the strains. The results of PCR amplification using type-specific primers that amplify a portion of the coat protein gene demonstrate that the primer-selection procedure developed for PNRSV constitutes a reliable method of viral strain discrimination in cherry for disease control and will also be useful for examining biological diversity within the PNRSV virus group.

The chloroplast tRNALys(UUU) gene from mustard (Sinapis alba) contains a class II intron potentially coding for a maturase-related polypeptide.

PubMed

Neuhaus, H; Link, G

1987-01-01

The trnK gene endocing the tRNALys(UUU) has been located on mustard (Sinapis alba) chloroplast DNA, 263 bp upstream of the psbA gene on the same strand. The nucleotide sequence of the trnK gene and its flanking regions as well as the putative transcription start and termination sites are shown. The 5' end of the transcript lies 121 bp upstream of the 5' tRNA coding region and is preceded by procaryotic-type "-10" and "-35" sequence elements, while the 3' end maps 2.77 kb downstream to a DNA region with possible stemloop secondary structure. The anticodon loop of the tRNALys is interrupted by a 2,574 bp intron containing a long open reading frame, which codes for 524 amino acids. Based on conserved stem and loop structures, this intron has characteristic features of a class II intron. A region near the carboxyl terminus of the derived polypeptide appears structurally related to maturases.

Antisense and sense poly(A)-RNAs from the Xenopus laevis pyruvate dehydrogenase gene loci are regulated with message production during embryogenesis.

PubMed

Islam, N; Poitras, L; Gagnon, F; Moss, T

1996-10-17

The structure and temporal expression of two Xenopus cDNAs encoding the beta subunit of pyruvate dehydrogenase (XPdhE1 beta) have been determined. XPdhE1 beta was 88% homologous to mature human PdhE1 beta, but the putative N-terminal mitochondrial signal peptide was poorly conserved. Zygotic expression of XPdhE1 beta mRNA was detected at neural tube closure and increased until stage 40. RT-PCR cloning identified a short homology to a protein kinase open reading frame within the 3' non-coding sequence of the XPdhE1 beta cDNAs. This homology, which occurred on the antisense cDNA strand, was shown by strand specific RT-PCR to be transcribed in vivo as part of an antisense RNA. Northern analysis showed that this RNA formed part of an abundant and heterogeneous population of antisense and sense poly(A)-RNAs transcribed from the XPdhE1 beta loci and coordinately regulated with message production.

Types of Aphasia

MedlinePlus

... Auditory Overload Aphasia vs Apraxia Reading, Writing and Math Reading Rehab (PDF opens in new window) Putting ... on Paper (PDF opens in new window) Acalculia - Math Challenges After Stroke Maximizing Communication Recovery & Independence Talking ...

Oral Language and Reading; Proceedings of the Annual Reading Conference of the Department of Elementary Education at Indiana State University (3rd, Terre Haute, June 14-15, 1973).

ERIC Educational Resources Information Center

Waterman, David C., Ed.; Gibbs, Vanita M., Ed.

This pamphlet is a collection of the speeches given at the Third Annual Reading Conference at Indiana State University, Terre Haute. The theme of the conference was "Oral Language and Reading." The contents include: "Official Program"; opening remarks, "They Led and Followed," by William G. McCarthy; opening address, "Strategies for Reading…

Teaching in an Open Classroom: Informal Checks, Diagnoses, and Learning Strategies for Beginning Reading and Math.

ERIC Educational Resources Information Center

Langstaff, Nancy

This book, intended for use by inservice teachers, preservice teachers, and parents interested in open classrooms, contains three chapters. "Beginning Reading in an Open Classroom" discusses language development, sight vocabulary, visual discrimination, auditory discrimination, directional concepts, small muscle control, and measurement of…

Origins of Media Exposure: Linking Personality Traits to TV, Radio, Print, and Film Use.

ERIC Educational Resources Information Center

Finn, Seth

1997-01-01

Investigates the five-factor model of personality (neuroticism, extroversion, openness, agreeableness, and conscientiousness) as a correlate of mass media use. Shows strongest relationships for mass media use between openness and pleasure reading, extroversion and negative pleasure reading, and openness and negative TV viewing. Finds that…

Clustering single cells: a review of approaches on high-and low-depth single-cell RNA-seq data.

PubMed

Menon, Vilas

2017-12-11

Advances in single-cell RNA-sequencing technology have resulted in a wealth of studies aiming to identify transcriptomic cell types in various biological systems. There are multiple experimental approaches to isolate and profile single cells, which provide different levels of cellular and tissue coverage. In addition, multiple computational strategies have been proposed to identify putative cell types from single-cell data. From a data generation perspective, recent single-cell studies can be classified into two groups: those that distribute reads shallowly over large numbers of cells and those that distribute reads more deeply over a smaller cell population. Although there are advantages to both approaches in terms of cellular and tissue coverage, it is unclear whether different computational cell type identification methods are better suited to one or the other experimental paradigm. This study reviews three cell type clustering algorithms, each representing one of three broad approaches, and finds that PCA-based algorithms appear most suited to low read depth data sets, whereas gene clustering-based and biclustering algorithms perform better on high read depth data sets. In addition, highly related cell classes are better distinguished by higher-depth data, given the same total number of reads; however, simultaneous discovery of distinct and similar types is better served by lower-depth, higher cell number data. Overall, this study suggests that the depth of profiling should be determined by initial assumptions about the diversity of cells in the population, and that the selection of clustering algorithm(s) is subsequently based on the depth of profiling will allow for better identification of putative transcriptomic cell types. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Association of coral algal symbionts with a diverse viral community responsive to heat shock.

PubMed

Brüwer, Jan D; Agrawal, Shobhit; Liew, Yi Jin; Aranda, Manuel; Voolstra, Christian R

2017-08-17

Stony corals provide the structural foundation of coral reef ecosystems and are termed holobionts given they engage in symbioses, in particular with photosynthetic dinoflagellates of the genus Symbiodinium. Besides Symbiodinium, corals also engage with bacteria affecting metabolism, immunity, and resilience of the coral holobiont, but the role of associated viruses is largely unknown. In this regard, the increase of studies using RNA sequencing (RNA-Seq) to assess gene expression provides an opportunity to elucidate viral signatures encompassed within the data via careful delineation of sequence reads and their source of origin. Here, we re-analyzed an RNA-Seq dataset from a cultured coral symbiont (Symbiodinium microadriaticum, Clade A1) across four experimental treatments (control, cold shock, heat shock, dark shock) to characterize associated viral diversity, abundance, and gene expression. Our approach comprised the filtering and removal of host sequence reads, subsequent phylogenetic assignment of sequence reads of putative viral origin, and the assembly and analysis of differentially expressed viral genes. About 15.46% (123 million) of all sequence reads were non-host-related, of which <1% could be classified as archaea, bacteria, or virus. Of these, 18.78% were annotated as virus and comprised a diverse community consistent across experimental treatments. Further, non-host related sequence reads assembled into 56,064 contigs, including 4856 contigs of putative viral origin that featured 43 differentially expressed genes during heat shock. The differentially expressed genes included viral kinases, ubiquitin, and ankyrin repeat proteins (amongst others), which are suggested to help the virus proliferate and inhibit the algal host's antiviral response. Our results suggest that a diverse viral community is associated with coral algal endosymbionts of the genus Symbiodinium, which prompts further research on their ecological role in coral health and resilience.

Post-genome research on the biosynthesis of ergot alkaloids.

PubMed

Li, Shu-Ming; Unsöld, Inge A

2006-10-01

Genome sequencing provides new opportunities and challenges for identifying genes for the biosynthesis of secondary metabolites. A putative biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine type, was identified in the genome sequence of ASPERGILLUS FUMIGATUS by a bioinformatic approach. This cluster spans 22 kb of genomic DNA and comprises at least 11 open reading frames (ORFs). Seven of them are orthologous to genes from the biosynthetic gene cluster of ergot alkaloids in CLAVICEPS PURPUREA. Experimental evidence of the identified cluster was provided by heterologous expression and biochemical characterization of two ORFs, FgaPT1 and FgaPT2, in the cluster of A. FUMIGATUS, which show remarkable similarities to dimethylallyltryptophan synthase from C. PURPUREA and function as prenyltransferases. FgaPT2 converts L-tryptophan to dimethylallyltryptophan and thereby catalyzes the first step of ergot alkaloid biosynthesis, whilst FgaPT1 catalyzes the last step of the fumigaclavine C biosynthesis, i. e., the prenylation of fumigaclavine A at C-2 position of the indole nucleus. In addition to information obtained from the gene cluster of ergot alkaloids from C. PURPUREA, the identification of the biosynthetic gene cluster of fumigaclavine C in A. FUMIGATUS opens an alternative way to study the biosynthesis of ergot alkaloids in fungi.

An evaluation of emergency medicine investigators' views on open access to medical literature.

PubMed

Rodriguez, R M; Wong, J; Hardy, J; Frankel, E

2006-12-01

Scientists and governmental agencies have called for free universal access to research publications via the internet--open access. To examine the current medical literature reading practices of emergency medicine investigators (EMIs) and their views towards open access. Surveys were mailed to the 212 corresponding authors of all original research articles published in years 2002 and 2003 in the Annals of Emergency Medicine, Academic Emergency Medicine and The Journal of Emergency Medicine. The most commonly read forms of medical literature reported by the 129 (61%) EMI respondents were hard-copy medical journals and online literature review services. 59% of EMIs were in favour of open access; 58% stated they would read a wider variety of medical literature; 21% believed open access would improve the quality of publications and 39% thought it would decrease the quality. When asked how a US 1500 dollars fee for open access would affect their ability to publish research, 69% said it would greatly impede and 19% said it would slightly impede their research. Despite concerns that open access may impede their ability to publish research and decrease the quality of publications, most EMIs surveyed favoured open access. They believed open access would increase and broaden their medical literature reading.

Cis-regulatory element based targeted gene finding: genome-wide identification of abscisic acid- and abiotic stress-responsive genes in Arabidopsis thaliana.

PubMed

Zhang, Weixiong; Ruan, Jianhua; Ho, Tuan-Hua David; You, Youngsook; Yu, Taotao; Quatrano, Ralph S

2005-07-15

A fundamental problem of computational genomics is identifying the genes that respond to certain endogenous cues and environmental stimuli. This problem can be referred to as targeted gene finding. Since gene regulation is mainly determined by the binding of transcription factors and cis-regulatory DNA sequences, most existing gene annotation methods, which exploit the conservation of open reading frames, are not effective in finding target genes. A viable approach to targeted gene finding is to exploit the cis-regulatory elements that are known to be responsible for the transcription of target genes. Given such cis-elements, putative target genes whose promoters contain the elements can be identified. As a case study, we apply the above approach to predict the genes in model plant Arabidopsis thaliana which are inducible by a phytohormone, abscisic acid (ABA), and abiotic stress, such as drought, cold and salinity. We first construct and analyze two ABA specific cis-elements, ABA-responsive element (ABRE) and its coupling element (CE), in A.thaliana, based on their conservation in rice and other cereal plants. We then use the ABRE-CE module to identify putative ABA-responsive genes in A.thaliana. Based on RT-PCR verification and the results from literature, this method has an accuracy rate of 67.5% for the top 40 predictions. The cis-element based targeted gene finding approach is expected to be widely applicable since a large number of cis-elements in many species are available.

The Bordetella bhu Locus Is Required for Heme Iron Utilization

PubMed Central

Vanderpool, Carin K.; Armstrong, Sandra K.

2001-01-01

Bordetella pertussis and Bordetella bronchiseptica are capable of obtaining iron from hemin and hemoglobin. Genes encoding a putative bacterial heme iron acquisition system (bhu, for Bordetella heme utilization) were identified in a B. pertussis genomic sequence database, and the corresponding DNA was isolated from a virulent strain of B. pertussis. A B. pertussis bhuR mutant, predicted to lack the heme outer membrane receptor, was generated by allelic exchange. In contrast to the wild-type strain, bhuR mutant PM5 was incapable of acquiring iron from hemin and hemoglobin; genetic complementation of PM5 with the cloned bhuRSTUV genes restored heme utilization to wild-type levels. In parallel studies, B. bronchiseptica bhu sequences were also identified and a B. bronchiseptica bhuR mutant was constructed and confirmed to be defective in heme iron acquisition. The wild-type B. bronchiseptica parent strain grown under low-iron conditions produced the presumptive BhuR protein, which was absent in the bhuR mutant. Furthermore, production of BhuR by iron-starved B. bronchiseptica was markedly enhanced by culture in hemin-supplemented medium, suggesting that these organisms sense and respond to heme in the environment. Analysis of the genetic region upstream of the bhu cluster identified open reading frames predicted to encode homologs of the Escherichia coli ferric citrate uptake regulators FecI and FecR. These putative Bordetella regulators may mediate heme-responsive positive transcriptional control of the bhu genes. PMID:11418569

Molecular cloning, sequence characterization and expression analysis of a CD63 homologue from the coleopteran beetle, Tenebrio molitor.

PubMed

Patnaik, Bharat Bhusan; Kang, Seong Min; Seo, Gi Won; Lee, Hyo Jeong; Patnaik, Hongray Howrelia; Jo, Yong Hun; Tindwa, Hamisi; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Bang, In Seok; Han, Yeon Soo

2013-10-15

CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic "Cys-Cys-Gly" motif and "Cys188" residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Molecular Cloning, Sequence Characterization and Expression Analysis of a CD63 Homologue from the Coleopteran Beetle, Tenebrio molitor

PubMed Central

Patnaik, Bharat Bhusan; Kang, Seong Min; Seo, Gi Won; Lee, Hyo Jeong; Patnaik, Hongray Howrelia; Jo, Yong Hun; Tindwa, Hamisi; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Bang, In Seok; Han, Yeon Soo

2013-01-01

CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens. PMID:24132157

Deep sequencing and in silico analysis of small RNA library reveals novel miRNA from leaf Persicaria minor transcriptome.

PubMed

Samad, Abdul Fatah A; Nazaruddin, Nazaruddin; Murad, Abdul Munir Abdul; Jani, Jaeyres; Zainal, Zamri; Ismail, Ismanizan

2018-03-01

In current era, majority of microRNA (miRNA) are being discovered through computational approaches which are more confined towards model plants. Here, for the first time, we have described the identification and characterization of novel miRNA in a non-model plant, Persicaria minor ( P . minor ) using computational approach. Unannotated sequences from deep sequencing were analyzed based on previous well-established parameters. Around 24 putative novel miRNAs were identified from 6,417,780 reads of the unannotated sequence which represented 11 unique putative miRNA sequences. PsRobot target prediction tool was deployed to identify the target transcripts of putative novel miRNAs. Most of the predicted target transcripts (mRNAs) were known to be involved in plant development and stress responses. Gene ontology showed that majority of the putative novel miRNA targets involved in cellular component (69.07%), followed by molecular function (30.08%) and biological process (0.85%). Out of 11 unique putative miRNAs, 7 miRNAs were validated through semi-quantitative PCR. These novel miRNAs discoveries in P . minor may develop and update the current public miRNA database.

Draft Genome Sequencing and Comparative Analysis of Aspergillus sojae NBRC4239

PubMed Central

Sato, Atsushi; Oshima, Kenshiro; Noguchi, Hideki; Ogawa, Masahiro; Takahashi, Tadashi; Oguma, Tetsuya; Koyama, Yasuji; Itoh, Takehiko; Hattori, Masahira; Hanya, Yoshiki

2011-01-01

We conducted genome sequencing of the filamentous fungus Aspergillus sojae NBRC4239 isolated from the koji used to prepare Japanese soy sauce. We used the 454 pyrosequencing technology and investigated the genome with respect to enzymes and secondary metabolites in comparison with other Aspergilli sequenced. Assembly of 454 reads generated a non-redundant sequence of 39.5-Mb possessing 13 033 putative genes and 65 scaffolds composed of 557 contigs. Of the 2847 open reading frames with Pfam domain scores of >150 found in A. sojae NBRC4239, 81.7% had a high degree of similarity with the genes of A. oryzae. Comparative analysis identified serine carboxypeptidase and aspartic protease genes unique to A. sojae NBRC4239. While A. oryzae possessed three copies of α-amyalse gene, A. sojae NBRC4239 possessed only a single copy. Comparison of 56 gene clusters for secondary metabolites between A. sojae NBRC4239 and A. oryzae revealed that 24 clusters were conserved, whereas 32 clusters differed between them that included a deletion of 18 508 bp containing mfs1, mao1, dmaT, and pks-nrps for the cyclopiazonic acid (CPA) biosynthesis, explaining the no productivity of CPA in A. sojae. The A. sojae NBRC4239 genome data will be useful to characterize functional features of the koji moulds used in Japanese industries. PMID:21659486

Sequencing, Analysis, and Annotation of Expressed Sequence Tags for Camelus dromedarius

PubMed Central

Al-Swailem, Abdulaziz M.; Shehata, Maher M.; Abu-Duhier, Faisel M.; Al-Yamani, Essam J.; Al-Busadah, Khalid A.; Al-Arawi, Mohammed S.; Al-Khider, Ali Y.; Al-Muhaimeed, Abdullah N.; Al-Qahtani, Fahad H.; Manee, Manee M.; Al-Shomrani, Badr M.; Al-Qhtani, Saad M.; Al-Harthi, Amer S.; Akdemir, Kadir C.; Otu, Hasan H.

2010-01-01

Despite its economical, cultural, and biological importance, there has not been a large scale sequencing project to date for Camelus dromedarius. With the goal of sequencing complete DNA of the organism, we first established and sequenced camel EST libraries, generating 70,272 reads. Following trimming, chimera check, repeat masking, cluster and assembly, we obtained 23,602 putative gene sequences, out of which over 4,500 potentially novel or fast evolving gene sequences do not carry any homology to other available genomes. Functional annotation of sequences with similarities in nucleotide and protein databases has been obtained using Gene Ontology classification. Comparison to available full length cDNA sequences and Open Reading Frame (ORF) analysis of camel sequences that exhibit homology to known genes show more than 80% of the contigs with an ORF>300 bp and ∼40% hits extending to the start codons of full length cDNAs suggesting successful characterization of camel genes. Similarity analyses are done separately for different organisms including human, mouse, bovine, and rat. Accompanying web portal, CAGBASE (http://camel.kacst.edu.sa/), hosts a relational database containing annotated EST sequences and analysis tools with possibility to add sequences from public domain. We anticipate our results to provide a home base for genomic studies of camel and other comparative studies enabling a starting point for whole genome sequencing of the organism. PMID:20502665

From reads to regions: a Bioconductor workflow to detect differential binding in ChIP-seq data

PubMed Central

Lun, Aaron T. L.; Smyth, Gordon K.

2016-01-01

Chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) is widely used to identify the genomic binding sites for protein of interest. Most conventional approaches to ChIP-seq data analysis involve the detection of the absolute presence (or absence) of a binding site. However, an alternative strategy is to identify changes in the binding intensity between two biological conditions, i.e., differential binding (DB). This may yield more relevant results than conventional analyses, as changes in binding can be associated with the biological difference being investigated. The aim of this article is to facilitate the implementation of DB analyses, by comprehensively describing a computational workflow for the detection of DB regions from ChIP-seq data. The workflow is based primarily on R software packages from the open-source Bioconductor project and covers all steps of the analysis pipeline, from alignment of read sequences to interpretation and visualization of putative DB regions. In particular, detection of DB regions will be conducted using the counts for sliding windows from the csaw package, with statistical modelling performed using methods in the edgeR package. Analyses will be demonstrated on real histone mark and transcription factor data sets. This will provide readers with practical usage examples that can be applied in their own studies. PMID:26834993

STORMSeq: an open-source, user-friendly pipeline for processing personal genomics data in the cloud.

PubMed

Karczewski, Konrad J; Fernald, Guy Haskin; Martin, Alicia R; Snyder, Michael; Tatonetti, Nicholas P; Dudley, Joel T

2014-01-01

The increasing public availability of personal complete genome sequencing data has ushered in an era of democratized genomics. However, read mapping and variant calling software is constantly improving and individuals with personal genomic data may prefer to customize and update their variant calls. Here, we describe STORMSeq (Scalable Tools for Open-Source Read Mapping), a graphical interface cloud computing solution that does not require a parallel computing environment or extensive technical experience. This customizable and modular system performs read mapping, read cleaning, and variant calling and annotation. At present, STORMSeq costs approximately $2 and 5-10 hours to process a full exome sequence and $30 and 3-8 days to process a whole genome sequence. We provide this open-access and open-source resource as a user-friendly interface in Amazon EC2.

Developmental rearrangement of cyanobacterial nif genes: nucleotide sequence, open reading frames, and cytochrome P-450 homology of the Anabaena sp. strain PCC 7120 nifD element.

PubMed Central

Lammers, P J; McLaughlin, S; Papin, S; Trujillo-Provencio, C; Ryncarz, A J

1990-01-01

An 11-kbp DNA element of unknown function interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. In developing heterocysts the nifD element excises from the chromosome via site-specific recombination between short repeat sequences that flank the element. The nucleotide sequence of the nifH-proximal half of the element was determined to elucidate the genetic potential of the element. Four open reading frames with the same relative orientation as the nifD element-encoded xisA gene were identified in the sequenced region. Each of the open reading frames was preceded by a reasonable ribosome-binding site and had biased codon utilization preferences consistent with low levels of expression. Open reading frame 3 was highly homologous with three cytochrome P-450 omega-hydroxylase proteins and showed regional homology to functionally significant domains common to the cytochrome P-450 superfamily. The sequence encoding open reading frame 2 was the most highly conserved portion of the sequenced region based on heterologous hybridization experiments with three genera of heterocystous cyanobacteria. Images PMID:2123860

The Effect of a Specialized Dyslexia Font, Opendyslexic, on Reading Rate and Accuracy

ERIC Educational Resources Information Center

Wery, Jessica J.; Diliberto, Jennifer A.

2017-01-01

A single-subject alternating treatment design was used to investigate the extent to which a specialized dyslexia font, OpenDyslexic, impacted reading rate or accuracy compared to two commonly used fonts when used with elementary students identified as having dyslexia. OpenDyslexic was compared to Arial and Times New Roman in three reading tasks:…

Cultural Awareness and Reading. Proceedings of the Annual Reading Conference (12th, Terre Haute, Indiana, June 17-18, 1982).

ERIC Educational Resources Information Center

Gibbs, Vanita M., Comp.; Pabst, Robert L., Comp.

Reflecting the expertise of the speakers and providing a rich resource of information within the conference theme, the articles in these proceedings explore the relationship between cultural awareness and reading. The proceedings begin with a copy of the conference program and opening remarks by the conference cochair. Following an opening address…

PRGdb: a bioinformatics platform for plant resistance gene analysis

PubMed Central

Sanseverino, Walter; Roma, Guglielmo; De Simone, Marco; Faino, Luigi; Melito, Sara; Stupka, Elia; Frusciante, Luigi; Ercolano, Maria Raffaella

2010-01-01

PRGdb is a web accessible open-source (http://www.prgdb.org) database that represents the first bioinformatic resource providing a comprehensive overview of resistance genes (R-genes) in plants. PRGdb holds more than 16 000 known and putative R-genes belonging to 192 plant species challenged by 115 different pathogens and linked with useful biological information. The complete database includes a set of 73 manually curated reference R-genes, 6308 putative R-genes collected from NCBI and 10463 computationally predicted putative R-genes. Thanks to a user-friendly interface, data can be examined using different query tools. A home-made prediction pipeline called Disease Resistance Analysis and Gene Orthology (DRAGO), based on reference R-gene sequence data, was developed to search for plant resistance genes in public datasets such as Unigene and Genbank. New putative R-gene classes containing unknown domain combinations were discovered and characterized. The development of the PRG platform represents an important starting point to conduct various experimental tasks. The inferred cross-link between genomic and phenotypic information allows access to a large body of information to find answers to several biological questions. The database structure also permits easy integration with other data types and opens up prospects for future implementations. PMID:19906694

STORMSeq: An Open-Source, User-Friendly Pipeline for Processing Personal Genomics Data in the Cloud

PubMed Central

Karczewski, Konrad J.; Fernald, Guy Haskin; Martin, Alicia R.; Snyder, Michael; Tatonetti, Nicholas P.; Dudley, Joel T.

2014-01-01

The increasing public availability of personal complete genome sequencing data has ushered in an era of democratized genomics. However, read mapping and variant calling software is constantly improving and individuals with personal genomic data may prefer to customize and update their variant calls. Here, we describe STORMSeq (Scalable Tools for Open-Source Read Mapping), a graphical interface cloud computing solution that does not require a parallel computing environment or extensive technical experience. This customizable and modular system performs read mapping, read cleaning, and variant calling and annotation. At present, STORMSeq costs approximately $2 and 5–10 hours to process a full exome sequence and $30 and 3–8 days to process a whole genome sequence. We provide this open-access and open-source resource as a user-friendly interface in Amazon EC2. PMID:24454756

76 FR 4659 - Science Advisory Board Staff Office; Notification of a Public Teleconference of the Science...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-26

...://yosemite.epa.gov/sab/ sabproduct.nsf/fedrgstr--activities/ FL%20Estuaries%20TSD?OpenDocument'' should read ``http:// yosemite.epa.gov/sab/sabproduct.nsf/fedrgstr--activites/ FL%20Estuaries%20TSD?OpenDocument.'' 2.../sabproduct.nsf/fedrgstr--activities/ FL%20Estuaries%20TSD?OpenDocument'' should read ``http:// yosemite.epa...

Findings from a Multiyear Scale-Up Effectiveness Trial of Open Court Reading

ERIC Educational Resources Information Center

Vaden-Kiernan, Michael; Borman, Geoffrey; Caverly, Sarah; Bell, Nance; Sullivan, Kate; Ruiz de Castilla, Veronica; Fleming, Grace; Rodriguez, Debra; Henry, Chad; Long, Tracy; Hughes Jones, Debra

2018-01-01

This multiyear scale-up effectiveness study of Open Court Reading (OCR) involved approximately 4,500 students and more than 1,000 teachers per year in Grades K-5 from 49 elementary schools in seven districts across the country. Using a school-level cluster randomized trial design, we assessed the implementation and effectiveness of Open Court…

Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

PubMed Central

Brown, D P; Idler, K B; Katz, L

1990-01-01

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks. Images FIG. 2 FIG. 3 PMID:2180909

The Varicella-zoster virus DNA encapsidation genes: Identification and characterization of the putative terminase subunits

PubMed Central

Visalli, Robert J.; Nicolosi, Denise M.; Irven, Karen L.; Goshorn, Bradley; Khan, Tamseel; Visalli, Melissa A.

2007-01-01

The putative DNA encapsidation genes encoded by open reading frames (ORFs) 25, 26, 30, 34, 43, 45/42 and 54 were cloned from Varicella-zoster virus (VZV) strain Ellen. Sequencing revealed that the Ellen ORFs were highly conserved at the amino acid level when compared to those of nineteen previously published VZV isolates. Additionally, RT-PCR provided the first evidence that ORF45/42 was expressed as a spliced transcript in VZV-infected cells. All seven ORFs were expressed in vitro and full length products were identified using a C-terminal V5 epitope tag. The in vitro products of the putative VZV terminase subunits encoded by ORFs 30 and 45/42 proved useful in protein-protein interaction assays. Previous studies have reported the formation of a heterodimeric terminase complex involved in DNA encapsidation for both herpes simplex virus-type 1 (HSV-1) and human cytomegalovirus (HCMV). Here we report that the C-terminal portion of exon II of ORF45/42 (ORF42-C269) interacted in GST-pull down experiments with in vitro synthesized ORF30 and ORF45/42. The interactions were maintained in the presence of anionic detergents and in buffers of increasing ionic strength. Cells transiently transfected with epitope tagged ORF45/42 or ORF30 showed primarily cytoplasmic staining. In contrast, an antiserum directed to the N-terminal portion of ORF45 showed nearly exclusive nuclear localization of the ORF45/42 gene product in infected cells. An ORF30 specific antiserum detected an 87 kDa protein in both the cytoplasmic and nuclear fractions of VZV infected cells. The results were consistent with the localization and function of herpesviral terminase subunits. This is the first study aimed at the identification and characterization of the VZV DNA encapsidation gene products. PMID:17868947

Identification of a CYP3A form (CYP3A126) in fathead minnow (Pimephales promelas) and characterisation of putative CYP3A enzyme activity.

PubMed

Christen, Verena; Caminada, Daniel; Arand, Michael; Fent, Karl

2010-01-01

Cytochrome P450-dependent monooxygenases (CYPs) are involved in the metabolic defence against xenobiotics. Human CYP3A enzymes metabolise about 50% of all pharmaceuticals in use today. Induction of CYPs and associated xenobiotic metabolism occurs also in fish and may serve as a useful tool for biomonitoring of environmental contamination. In this study we report on the cloning of a CYP3A family gene from fathead minnows (Pimephales promelas), which has been designated as CYP3A126 by the P450 nomenclature committee (GenBank no. EU332792). The cDNA was isolated, identified and characterised by extended inverse polymerase chain reaction (PCR), an alternative to the commonly used method of rapid amplification of cDNA ends. In a fathead minnow cell line we identified a full-length cDNA sequence (1,863 base pairs (bp)) consisting of a 1,536 bp open reading frame encoding a 512 amino acid protein. Genomic analysis of the identified CYP3A isoenzyme revealed a DNA sequence consisting of 13 exons and 12 introns. CYP3A126 is also expressed in fathead minnow liver as demonstrated by reverse transcription PCR. Exposure of fathead minnow (FHM) cells with the CYP3A inducer rifampicin leads to dose-dependent increase in putative CYP3A enzyme activity. In contrast, inhibitory effects of diazepam treatment were observed on putative CYP3A enzyme activity and additionally on CYP3A126 mRNA expression. This indicates that CYP3A is active in FHM cells and that CYP3A126 is at least in part responsible for this CYP3A activity. Further investigations will show whether CYP3A126 is involved in the metabolism of environmental chemicals.

A simplified financial model for automatic meter reading

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, S.M.

1994-01-15

The financial model proposed here (which can be easily adapted for electric, gas, or water) combines aspects of [open quotes]life cycle,[close quotes] [open quotes]consumer value[close quotes] and [open quotes]revenue based[close quotes] approaches and addresses intangible benefits. A simple value tree of one-word descriptions clarifies the relationship between level of investment and level of value, visually relating increased value to increased cost. The model computes the numerical present values of capital costs, recurring costs, and revenue benefits over a 15-year period for the seven configurations: manual reading of existing or replacement standard meters (MMR), manual reading using electronic, hand-held retrievers (EMR),more » remote reading of inaccessible meters via hard-wired receptacles (RMR), remote reading of meters adapted with pulse generators (RMR-P), remote reading of meters adapted with absolute dial encoders (RMR-E), offsite reading over a few hundred feet with mobile radio (OMR), and fully automatic reading using telephone or an equivalent network (AMR). In the model, of course, the costs of installing the configurations are clearly listed under each column. The model requires only four annualized inputs and seven fixed-cost inputs that are rather easy to obtain.« less

39 CFR 3004.12 - Reading room.

Code of Federal Regulations, 2010 CFR

2010-07-01

... reading room at its offices is open during business hours. (b) The records available for public inspection... Reading room. (a) The Commission maintains a public reading room at its offices (901 New York Avenue, NW...

Skill dependent audiovisual integration in the fusiform induces repetition suppression.

PubMed

McNorgan, Chris; Booth, James R

2015-02-01

Learning to read entails mapping existing phonological representations to novel orthographic representations and is thus an ideal context for investigating experience driven audiovisual integration. Because two dominant brain-based theories of reading development hinge on the sensitivity of the visual-object processing stream to phonological information, we were interested in how reading skill relates to audiovisual integration in this area. Thirty-two children between 8 and 13 years of age spanning a range of reading skill participated in a functional magnetic resonance imaging experiment. Participants completed a rhyme judgment task to word pairs presented unimodally (auditory- or visual-only) and cross-modally (auditory followed by visual). Skill-dependent sub-additive audiovisual modulation was found in left fusiform gyrus, extending into the putative visual word form area, and was correlated with behavioral orthographic priming. These results suggest learning to read promotes facilitatory audiovisual integration in the ventral visual-object processing stream and may optimize this region for orthographic processing. Copyright © 2014 Elsevier Inc. All rights reserved.

Skill Dependent Audiovisual Integration in the Fusiform Induces Repetition Suppression

PubMed Central

McNorgan, Chris; Booth, James R.

2015-01-01

Learning to read entails mapping existing phonological representations to novel orthographic representations and is thus an ideal context for investigating experience driven audiovisual integration. Because two dominant brain-based theories of reading development hinge on the sensitivity of the visual-object processing stream to phonological information, we were interested in how reading skill relates to audiovisual integration in this area. Thirty-two children between 8 and 13 years of age spanning a range of reading skill participated in a functional magnetic resonance imaging experiment. Participants completed a rhyme judgment task to word pairs presented unimodally (auditory- or visual-only) and cross-modally (auditory followed by visual). Skill-dependent sub-additive audiovisual modulation was found in left fusiform gyrus, extending into the putative visual word form area, and was correlated with behavioral orthographic priming. These results suggest learning to read promotes facilitatory audiovisual integration in the ventral visual-object processing stream and may optimize this region for orthographic processing. PMID:25585276

On the Importance of Anchoring and the Consequences of Its Impairment in Dyslexia

ERIC Educational Resources Information Center

Banai, Karen; Ahissar, Merav

2010-01-01

One of the main impediments of individuals with reading difficulties and individuals with language difficulties is poor working memory. Typically measured using verbal stimuli, working memory deficits have often been considered as one aspect of the phonological difficulty putatively underlying dyslexia. Over the years it has been shown that a…

Triterpenoid Saponin Biosynthetic Pathway Profiling and Candidate Gene Mining of the Ilex asprella Root Using RNA-Seq

PubMed Central

Zheng, Xiasheng; Xu, Hui; Ma, Xinye; Zhan, Ruoting; Chen, Weiwen

2014-01-01

Ilex asprella, which contains abundant α-amyrin type triterpenoid saponins, is an anti-influenza herbal drug widely used in south China. In this work, we first analysed the transcriptome of the I. asprella root using RNA-Seq, which provided a dataset for functional gene mining. mRNA was isolated from the total RNA of the I. asprella root and reverse-transcribed into cDNA. Then, the cDNA library was sequenced using an Illumina HiSeq™ 2000, which generated 55,028,452 clean reads. De novo assembly of these reads generated 51,865 unigenes, in which 39,269 unigenes were annotated (75.71% yield). According to the structures of the triterpenoid saponins of I. asprella, a putative biosynthetic pathway downstream of 2,3-oxidosqualene was proposed and candidate unigenes in the transcriptome data that were potentially involved in the pathway were screened using homology-based BLAST and phylogenetic analysis. Further amplification and functional analysis of these putative unigenes will provide insight into the biosynthesis of Ilex triterpenoid saponins. PMID:24722569

Sustaining Composition: Studying Content-Based, Ecological, and Economical Sustainability of Open-Source Textbooks through "Writing Spaces: Readings on Writing"

ERIC Educational Resources Information Center

Munson, Margaret

2013-01-01

Writing programs in institutions of higher education work to prepare students for real-world writing within any field of study. The composition of "Writing Spaces: Readings on Writing" offers an open-source text for students, teachers, and policy-makers at all levels. Exposure to an open space for learning encourages access to information,…

Open chromatin encoded in DNA sequence is the signature of ‘master’ replication origins in human cells

PubMed Central

Audit, Benjamin; Zaghloul, Lamia; Vaillant, Cédric; Chevereau, Guillaume; d'Aubenton-Carafa, Yves; Thermes, Claude; Arneodo, Alain

2009-01-01

For years, progress in elucidating the mechanisms underlying replication initiation and its coupling to transcriptional activities and to local chromatin structure has been hampered by the small number (approximately 30) of well-established origins in the human genome and more generally in mammalian genomes. Recent in silico studies of compositional strand asymmetries revealed a high level of organization of human genes around 1000 putative replication origins. Here, by comparing with recently experimentally identified replication origins, we provide further support that these putative origins are active in vivo. We show that regions ∼300-kb wide surrounding most of these putative replication origins that replicate early in the S phase are hypersensitive to DNase I cleavage, hypomethylated and present a significant enrichment in genomic energy barriers that impair nucleosome formation (nucleosome-free regions). This suggests that these putative replication origins are specified by an open chromatin structure favored by the DNA sequence. We discuss how this distinctive attribute makes these origins, further qualified as ‘master’ replication origins, priviledged loci for future research to decipher the human spatio-temporal replication program. Finally, we argue that these ‘master’ origins are likely to play a key role in genome dynamics during evolution and in pathological situations. PMID:19671527

Cloning and characterization of an inulinase gene from the marine yeast Candida membranifaciens subsp. flavinogenie W14-3 and its expression in Saccharomyces sp. W0 for ethanol production.

PubMed

Zhang, Lin-Lin; Tan, Mei-Juan; Liu, Guang-Lei; Chi, Zhe; Wang, Guang-Yuan; Chi, Zhen-Ming

2015-04-01

The INU1 gene encoding an exo-inulinase from the marine-derived yeast Candida membranifaciens subsp. flavinogenie W14-3 was cloned and characterized. It had an open reading frame of 1,536 bp long encoding an inulinase. The coding region of it was not interrupted by any intron. The cloned gene encoded 512 amino acid residues of a protein with a putative signal peptide of 23 amino acids and a calculated molecular mass of 57.8 kDa. The protein sequence deduced from the inulinase gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP FS and Q. The protein also had six conserved putative N-glycosylation sites. The deduced inulinase from the yeast strain W14-3 was found to be closely related to that from Candida kutaonensis sp. nov. KRF1, Kluyveromyces marxianus, and Cryptococcus aureus G7a. The inulinase gene with its signal peptide encoding sequence was subcloned into the pMIRSC11 expression vector and expressed in Saccharomyces sp. W0. The recombinant yeast strain W14-3-INU-112 obtained could produce 16.8 U/ml of inulinase activity and 12.5 % (v/v) ethanol from 250 g/l of inulin within 168 h. The monosaccharides were detected after the hydrolysis of inulin with the crude inulinase (the yeast culture). All the results indicated that the cloned gene and the recombinant yeast strain W14-3-INU-112 had potential applications in biotechnology.

Heterologous expression of a Tpo1 homolog from Arabidopsis thaliana confers resistance to the herbicide 2,4-D and other chemical stresses in yeast.

PubMed

Cabrito, Tânia R; Teixeira, Miguel C; Duarte, Alexandra A; Duque, Paula; Sá-Correia, Isabel

2009-10-01

The understanding of the molecular mechanisms underlying acquired herbicide resistance is crucial in dealing with the emergence of resistant weeds. Saccharomyces cerevisiae has been used as a model system to gain insights into the mechanisms underlying resistance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The TPO1 gene, encoding a multidrug resistance (MDR) plasma membrane transporter of the major facilitator superfamily (MFS), was previously found to confer resistance to 2,4-D in yeast and to be transcriptionally activated in response to the herbicide. In this work, we demonstrate that Tpo1p is required to reduce the intracellular concentration of 2,4-D. ScTpo1p homologs encoding putative plasma membrane MFS transporters from the plant model Arabidopsis thaliana were analyzed for a possible role in 2,4-D resistance. At5g13750 was chosen for further analysis, as its transcript levels were found to increase in 2,4-D stressed plants. The functional heterologous expression of this plant open reading frame in yeast was found to confer increased resistance to the herbicide in Deltatpo1 and wild-type cells, through the reduction of the intracellular concentration of 2,4-D. Heterologous expression of At5g13750 in yeast also leads to increased resistance to indole-3-acetic acid (IAA), Al(3+) and Tl(3+). At5g13750 is the first plant putative MFS transporter to be suggested as possibly involved in MDR.

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114.

PubMed

Adams, C; Dowling, D N; O'Sullivan, D J; O'Gara, F

1994-06-03

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 bp was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined -16 to -25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.

Gene silencing reveals multiple functions of Na+/K+-ATPase in the salmon louse (Lepeophtheirus salmonis).

PubMed

Komisarczuk, Anna Z; Kongshaug, Heidi; Nilsen, Frank

2018-02-01

Na + /K + -ATPase has a key function in a variety of physiological processes including membrane excitability, osmoregulation, regulation of cell volume, and transport of nutrients. While knowledge about Na + /K + -ATPase function in osmoregulation in crustaceans is extensive, the role of this enzyme in other physiological and developmental processes is scarce. Here, we report characterization, transcriptional distribution and likely functions of the newly identified L. salmonis Na + /K + -ATPase (LsalNa + /K + -ATPase) α subunit in various developmental stages. The complete mRNA sequence was identified, with 3003 bp open reading frame encoding a putative protein of 1001 amino acids. Putative protein sequence of LsalNa + /K + -ATPase revealed all typical features of Na + /K + -ATPase and demonstrated high sequence identity to other invertebrate and vertebrate species. Quantitative RT-PCR analysis revealed higher LsalNa + /K + -ATPase transcript level in free-living stages in comparison to parasitic stages. In situ hybridization analysis of copepodids and adult lice revealed LsalNa + /K + -ATPase transcript localization in a wide variety of tissues such as nervous system, intestine, reproductive system, and subcuticular and glandular tissue. RNAi mediated knock-down of LsalNa + /K + -ATPase caused locomotion impairment, and affected reproduction and feeding. Morphological analysis of dsRNA treated animals revealed muscle degeneration in larval stages, severe changes in the oocyte formation and maturation in females and abnormalities in tegmental glands. Thus, the study represents an important foundation for further functional investigation and identification of physiological pathways in which Na + /K + -ATPase is directly or indirectly involved. Copyright © 2018 Elsevier Inc. All rights reserved.

Transcriptome analysis of the couch potato (CPO) protein reveals an expression pattern associated with early development in the salmon louse Caligus rogercresseyi.

PubMed

Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Nuñez-Acuña, Gustavo; Chávez-Mardones, Jacqueline; Maldonado-Aguayo, Waleska

2014-02-15

The couch potato (CPO) protein is a key biomolecule involved in regulating diapause through the RNA-binding process of the peripheral and central nervous systems in insects and also recently discovered in a few crustacean species. As such, ectoparasitic copepods are interesting model species that have no evidence of developmental arrest. The present study is the first to report on the cloning of a putative CPO gene from the salmon louse Caligus rogercresseyi (CrCPO), as identified by high-throughput transcriptome sequencing. In addition, the transcription expression in larvae and adults was evaluated using quantitative real-time PCR. The CrCPO cDNA sequence showed 3261 base pairs (bp), consisting of 713bp of 5' UTR, 1741bp of 3' UTR, and an open reading frame of 807bp encoding for 268 amino acids. The highly conserved RNA binding regions RNP2 (LFVSGL) and RNP1 (SPVGFVTF), as well the dimerization site (LEF), were also found. Furthermore, eight single nucleotide polymorphisms located in the untranslated regions and one located in the coding region were detected. Gene transcription analysis revealed that CrCPO has ubiquitous expression across larval stages and in adult individuals, with the highest expression from nauplius to copepodid stages. The present study suggests a putative biological function of CrCPO associated with the development of the nervous system in salmon lice and contributes molecular evidence for candidate genes related to host-parasite interactions. Copyright © 2013 Elsevier B.V. All rights reserved.

Complete nucleotide sequences and genome characterization of a novel double-stranded RNA virus infecting Rosa multiflora.

PubMed

Salem, Nidá M; Golino, Deborah A; Falk, Bryce W; Rowhani, Adib

2008-01-01

The three double-stranded (ds) RNAs were detected in Rosa multiflora plants showing rose spring dwarf (RSD) symptoms. Northern blot analysis revealed three dsRNAs in preparations of both dsRNA and total RNA from R. multiflora plants. The complete sequences of the dsRNAs (referred to as dsRNA 1, dsRNA 2 and dsRNA 3) were determined based on a combination of shotgun cloning of dsRNA cDNAs and reverse transcription-polymerase chain reaction (RT-PCR). The largest dsRNA (dsRNA 1) was 1,762 bp long with a single open reading frame (ORF) that encoded a putative polypeptide containing 479 amino acid residues with a molecular mass of 55.9 kDa. This polypeptide contains amino acid sequence motifs conserved in the RNA-dependent RNA polymerases (RdRp) of members of the family Partitiviridae. Both dsRNA 2 (1,475 bp) and dsRNA 3 (1,384 bp) contained single ORFs, encoding putative proteins of unknown function. The 5' untranslated regions (UTR) of all three segments shared regions of high sequence homology. Phylogenetic analysis using the RdRp sequences of the various partitiviruses revealed that the new sequences would constitute the genome of a virus in family Partitiviridae. This virus would cluster with Fragaria chiloensis cryptic virus and Raphanus sativus cryptic virus 2. We suggest that the three dsRNA segments constitute the genome of a novel cryptic virus infecting roses; we propose the name Rosa multiflora cryptic virus (RMCV). Detection primers were developed and used for RT-PCR detection of RMCV in rose plants.

Characterization of the response to zinc deficiency in the cyanobacterium Anabaena sp. strain PCC 7120.

PubMed

Napolitano, Mauro; Rubio, Miguel Ángel; Santamaría-Gómez, Javier; Olmedo-Verd, Elvira; Robinson, Nigel J; Luque, Ignacio

2012-05-01

Zur regulators control zinc homeostasis by repressing target genes under zinc-sufficient conditions in a wide variety of bacteria. This paper describes how part of a survey of duplicated genes led to the identification of the open reading frame all2473 as the gene encoding the Zur regulator of the cyanobacterium Anabaena sp. strain PCC 7120. All2473 binds to DNA in a zinc-dependent manner, and its DNA-binding sequence was characterized, which allowed us to determine the relative contribution of particular nucleotides to Zur binding. A zur mutant was found to be impaired in the regulation of zinc homeostasis, showing sensitivity to elevated concentrations of zinc but not other metals. In an effort to characterize the Zur regulon in Anabaena, 23 genes containing upstream putative Zur-binding sequences were identified and found to be regulated by Zur. These genes are organized in six single transcriptional units and six operons, some of them containing multiple Zur-regulated promoters. The identities of genes of the Zur regulon indicate that Anabaena adapts to conditions of zinc deficiency by replacing zinc metalloproteins with paralogues that fulfill the same function but presumably with a lower zinc demand, and with inducing putative metallochaperones and membrane transport systems likely being involved in the scavenging of extracellular zinc, including plasma membrane ABC transport systems and outer membrane TonB-dependent receptors. Among the Zur-regulated genes, the ones showing the highest induction level encode proteins of the outer membrane, suggesting a primary role for components of this cell compartment in the capture of zinc cations from the extracellular medium.

Chaperone-Usher Pili Loci of Colonization Factor-Negative Human Enterotoxigenic Escherichia coli

PubMed Central

Del Canto, Felipe; O'Ryan, Miguel; Pardo, Mirka; Torres, Alexia; Gutiérrez, Daniela; Cádiz, Leandro; Valdés, Raul; Mansilla, Aquiles; Martínez, Rodrigo; Hernández, Daniela; Caro, Benjamin; Levine, Myron M.; Rasko, David A.; Hill, Christopher M.; Pop, Mihai; Stine, O. Colin; Vidal, Roberto

2017-01-01

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea worldwide. Among the 25 different ETEC adhesins, 22 are known as “colonization factors” (CFs), of which 17 are assembled by the chaperone-usher (CU) mechanism. Currently, there is no preventive therapy against ETEC, and CFs have been proposed as components for vaccine development. However, studies of diarrhea-causing ETEC strains worldwide indicate that between 15 and 50% of these are negative for known CFs, hindering the selection of the most widespread structures and suggesting that unknown adhesins remain to be identified. Here, we report the result of a comprehensive analysis of 35 draft genomes of ETEC strains which do not carry known adhesin genes; our goal was to find new CU pili loci. The phylogenetic profiles and serogroups of these strains were highly diverse, a majority of which produced only the heat-labile toxin. We identified 10 pili loci belonging to CU families β (1 locus), γ2 (7 loci), κ (1 locus), and π (1 locus), all of which contained the required number of open reading frames (ORFs) to encode functional structures. Three loci were variants of previously-known clusters, three had been only-partially described, and four are novel loci. Intra-loci genetic variability identified would allow the synthesis of up to 14 different structures. Clusters of putative γ2-CU pili were most common (23 strains), followed by putative β-CU pili (12 strains), which have not yet been fully characterized. Overall, our findings significantly increase the number of ETEC adhesion genes associated with human infections. PMID:28111618

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

NASA Technical Reports Server (NTRS)

Cho, G.; Keefe, A. D.; Liu, R.; Wilson, D. S.; Szostak, J. W.

2000-01-01

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. Copyright 2000 Academic Press.

Characterization of the Tupaia rhabdovirus genome reveals a long open reading frame overlapping with P and a novel gene encoding a small hydrophobic protein.

PubMed

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-06-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.

Characterization of the Tupaia Rhabdovirus Genome Reveals a Long Open Reading Frame Overlapping with P and a Novel Gene Encoding a Small Hydrophobic Protein

PubMed Central

Springfeld, Christoph; Darai, Gholamreza; Cattaneo, Roberto

2005-01-01

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses. PMID:15890917

VP3 is crucial for the stability of Nora virus virions.

PubMed

Sadanandan, Sajna Anand; Ekström, Jens-Ola; Jonna, Venkateswara Rao; Hofer, Anders; Hultmark, Dan

2016-09-02

Nora virus is an enteric virus that causes persistent, non-pathological infection in Drosophila melanogaster. It replicates in the fly gut and is transmitted via the fecal-oral route. Nora virus has a single-stranded positive-sense RNA genome, which is translated in four open reading frames. Reading frame three encodes the VP3 protein, the structure and function of which we have investigated in this work. We have shown that VP3 is a trimer that has an α-helical secondary structure, with a functionally important coiled-coil domain. In order to identify the role of VP3 in the Nora virus life cycle, we constructed VP3-mutants using the cDNA clone of the virus. Our results show that VP3 does not have a role in the actual assembly of the virus particles, but virions that lack VP3 or harbor VP3 with a disrupted coiled coil domain are incapable of transmission via the fecal-oral route. Removing the region downstream of the putative coiled coil appears to have an effect on the fitness of the virus but does not hamper its replication or transmission. We also found that the VP3 protein and particularly the coiled coil domain are crucial for the stability of Nora virus virions when exposed to heat or proteases. Hence, we propose that VP3 is imperative to Nora virus virions as it confers stability to the viral capsid. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, P.J.; Walthers, E.A.; Richmond, K.L.

1997-04-01

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five Polymorphisms differed by the presence Of two to six copies of the 12-bp tandem repeat 5{prime}-CAATATCAACAA-3{prime}. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats aremore » generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations. 22 refs., 4 figs., 3 tabs.« less

iPS models of Parkin and PINK1.

PubMed

Rakovic, Aleksandar; Seibler, Philip; Klein, Christine

2015-04-01

Parkinson disease (PD) is a degenerative disorder of the central nervous system resulting from depletion of dopaminergic neurons and currently remains incurable despite enormous international research efforts. The development of induced pluripotent stem cell (iPSC) technology opened up the unique possibility of studying disease mechanisms in human tissue that was otherwise not accessible, such as the brain. Of particular interest are the monogenetic forms of PD as they closely resemble the more common 'idiopathic' PD and, through the mutated protein, provide a clear research target in iPSC-derived neurons. Recessively inherited Parkin and PTEN-induced putative kinase 1 (PINK1) mutations have been investigated in this context and the present review describes the first insights gained from studies in iPSC-derived dopaminergic neurons, which comprise abnormalities in mitochondrial and dopamine homoeostasis, microtubular stability and axonal outgrowth. These new models of PD have a high translational potential that includes the identification of druggable targets, testing of known and novel therapeutic agents in the disease-relevant tissue using well-defined read-outs and potential regenerative approaches.

Identification and Characterization of a Novel Alpaca Respiratory Coronavirus Most Closely Related to the Human Coronavirus 229E

PubMed Central

Crossley, Beate M.; Mock, Richard E.; Callison, Scott A.; Hietala, Sharon K.

2012-01-01

In 2007, a novel coronavirus associated with an acute respiratory disease in alpacas (Alpaca Coronavirus, ACoV) was isolated. Full-length genomic sequencing of the ACoV demonstrated the genome to be consistent with other Alphacoronaviruses. A putative additional open-reading frame was identified between the nucleocapsid gene and 3'UTR. The ACoV was genetically most similar to the common human coronavirus (HCoV) 229E with 92.2% nucleotide identity over the entire genome. A comparison of spike gene sequences from ACoV and from HCoV-229E isolates recovered over a span of five decades showed the ACoV to be most similar to viruses isolated in the 1960’s to early 1980’s. The true origin of the ACoV is unknown, however a common ancestor between the ACoV and HCoV-229E appears to have existed prior to the 1960’s, suggesting virus transmission, either as a zoonosis or anthroponosis, has occurred between alpacas and humans. PMID:23235471

Fe(II)-dependent, uridine-5'-monophosphate α-ketoglutarate dioxygenases in the synthesis of 5'-modified nucleosides.

PubMed

Yang, Zhaoyong; Unrine, Jason; Nonaka, Koichi; Van Lanen, Steven G

2012-01-01

Several nucleoside antibiotics from various actinomycetes contain a high-carbon sugar nucleoside that is putatively derived via C-5'-modification of the canonical nucleoside. Two prominent examples are the 5'-C-carbamoyluridine- and 5'-C-glycyluridine-containing nucleosides, both families of which were discovered using screens aimed at finding inhibitors of bacterial translocase I involved in the assembly of the bacterial peptidoglycan cell wall. A shared open reading frame was identified whose gene product is similar to enzymes of the nonheme, Fe(II)-, and α-ketoglutarate-dependent dioxygenases. The enzyme LipL from the biosynthetic pathway for A-90289, a 5'-C-glycyluridine-containing nucleoside, was functionally characterized as an UMP:α-ketoglutarate dioxygenase, providing the enzymatic imperative for the generation of a nucleoside-5'-aldehdye that serves as a downstream substrate for an aldol or aldol-type reaction leading to the high-carbon sugar scaffold. The functional assignment of LipL and the homologous enzymes-including bioinformatic analysis, iron detection and quantification, and assay development for biochemical characterization-is presented herein. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2.

PubMed

Rodriguez, P L; Leube, M P; Grill, E

1998-11-01

We report the cloning of both the cDNA and the corresponding genomic sequence of a new PP2C from Arabidopsis thaliana, named AtP2C-HA (for homology to ABI1/ABI2). The AtP2C-HA cDNA contains an open reading frame of 1536 bp and encodes a putative protein of 511 amino acids with a predicted molecular mass of 55.7 kDa. The AtP2C-HA protein is composed of two domains, a C-terminal PP2C catalytic domain and a N-terminal extension of ca. 180 amino acid residues. The deduced amino acid sequence is 55% and 54% identical to ABI1 and ABI2, respectively. Comparison of the genomic structure of the ABI1, ABI2 and AtP2C-HA genes suggests that they belong to a multigene family. The expression of the AtP2C-HA gene is up-regulated by abscisic acid (ABA) treatment.

Identification of a differentially-expressed gene in fatty liver of overfeeding geese.

PubMed

Zhao, Ayong; Tang, Huachun; Lu, Sufang; He, Ruiguo

2007-09-01

In response to overfeeding, geese develop fatty liver. To understand the fattening mechanism, mRNA differential display reverse transcription PCR was used to study the gene expression differences between French Landes grey geese and Xupu white geese in conditions of overfeeding and normal feeding. One gene was found to be up-regulated in the fatty liver in both breeds, and it has a 1797 bp cDNA with 83% identity to chicken SELENBP1. The sequence analysis revealed that its open reading frame of 1413 bp encodes a protein of 471 amino acids, which contains a putative conserved domain of 56 kDa selenium binding protein with high homology to its homologues of chicken (95%), rat (86%), mouse (84%), human (86%), monkey (86%), dog (86%), and cattle (86%). The function of this protein has been briefly reviewed based on published information. In tissue expression analysis, the expression of geese SELENBP1 mRNA was found to be higher in liver or kidney than in other tested tissues. The results showed that overfeeding could increase the mRNA expression level of geese SELENBP1.

The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

PubMed

Sirena, Dominique; Ruzsics, Zsolt; Schaffner, Walter; Greber, Urs F; Hemmi, Silvio

2005-12-20

Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.

Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR.

PubMed

Chen, Jin-Zhong; Wang, Shu; Tang, Rong; Yang, Quan-Sheng; Zhao, Enpeng; Chao, Yaoqiong; Ying, Kang; Xie, Yi; Mao, Yu-Min

2002-09-01

A cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The 2390 bp cDNA with an open reading fragment (ORF) of 816 bp encodes a 272 amino acids putative protein with a thrombospondin type I repeat (TSR) domain and a cysteine-rich region at the N-terminus, so it is named hPWTSR. We used Northern blot detected two bands with length of about 3 kb and 4 kb respectively, which expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were expressed ubiquitously in fetal tissues and human tumor tissues too. However, the transcript was detected neither in ovarian carcinoma GI-102 nor in lung carcinoma LX-1. Blast analysis against NCBI database revealed that the new gene contained at least 5 exons and located in human chromosome 6q22.33. Our results demonstrate that the gene is a novel member of TSR supergene family.

Complete genome sequence of maize yellow striate virus, a new cytorhabdovirus infecting maize and wheat crops in Argentina.

PubMed

Maurino, Fernanda; Dumón, Analía D; Llauger, Gabriela; Alemandri, Vanina; de Haro, Luis A; Mattio, M Fernanda; Del Vas, Mariana; Laguna, Irma Graciela; Giménez Pecci, María de la Paz

2018-01-01

A rhabdovirus infecting maize and wheat crops in Argentina was molecularly characterized. Through next-generation sequencing (NGS) of symptomatic leaf samples, the complete genome was obtained of two isolates of maize yellow striate virus (MYSV), a putative new rhabdovirus, differing by only 0.4% at the nucleotide level. The MYSV genome consists of 12,654 nucleotides for maize and wheat virus isolates, and shares 71% nucleotide sequence identity with the complete genome of barley yellow striate mosaic virus (BYSMV, NC028244). Ten open reading frames (ORFs) were predicted in the MYSV genome from the antigenomic strand and were compared with their BYSMV counterparts. The highest amino acid sequence identity of the MYSV and BYSMV proteins was 80% between the L proteins, and the lowest was 37% between the proteins 4. Phylogenetic analysis suggested that the MYSV isolates are new members of the genus Cytorhabdovirus, family Rhabdoviridae. Yellow striate, affecting maize and wheat crops in Argentina, is an emergent disease that presents a potential economic risk for these widely distributed crops.

In silico cloning, expression of Rieske-like apoprotein gene and protein subcellular localization in the Pacific oyster, Crassostrea gigas.

PubMed

He, Xiaocui; Zhang, Yang; Yu, Ziniu

2010-10-01

Rieske protein gene in the Pacific oyster Crassostrea gigas was obtained by in silico cloning for the first time, and its expression profiles and subcellular localization were determined, respectively. The full-length cDNA of Cgisp is 985 bp in length and contains a 5'- and 3'-untranslated regions of 35 and 161 bp, respectively, with an open reading frame of 786 bp encoding a protein of 262 amino acids. The predicted molecular weight of 30 kDa of Cgisp protein was verified by prokaryotic expression. Conserved Rieske [2Fe-2S] cluster binding sites and highly matched-pair tertiary structure with 3CWB_E (Gallus gallus) were revealed by homologous analysis and molecular modeling. Eleven putative SNP sites and two conserved hexapeptide sequences, box I (THLGC) and II (PCHGS), were detected by multiple alignments. Real-time PCR analysis showed that Cgisp is expressed in a wide range of tissues, with adductor muscle exhibiting the top expression level, suggesting its biological function of energy transduction. The GFP tagging Cgisp indicated a mitochondrial localization, further confirming its physiological function.

Porcine parvovirus: DNA sequence and genome organization.

PubMed

Ranz, A I; Manclús, J J; Díaz-Aroca, E; Casal, J I

1989-10-01

We have determined the nucleotide sequence of an almost full-length clone of porcine parvovirus (PPV). The sequence is 4973 nucleotides (nt) long. The 3' end of virion DNA shows a Y-shaped configuration homologous to rodent parvoviruses. The 5' end of virion DNA shows a repetition of 127 nt at the carboxy terminus of the capsid proteins. The overall organization of the PPV genome is similar to those of other autonomous parvoviruses. There are two large open reading frames (ORFs) that almost entirely cover the genome, both located in the same frame of the complementary strand. The left ORF encodes the non-structural protein NS1 and the right ORF encodes the capsid proteins (VP1, VP2 and VP3). Promoter analysis, location of splicing sites and putative amino acid sequences for the viral proteins show a high homology of PPV with feline panleukopenia virus and canine parvoviruses (FPV and CPV) and rodent parvovirus. Therefore we conclude that PPV is related to the Kilham rat virus (KRV) group of autonomous parvoviruses formed by KRV, minute virus of mice, Lu III, H-1, FPV and CPV.

The complete genome sequence of freesia mosaic virus and its relationship to other potyviruses.

PubMed

Choi, H I; Lim, H R; Song, Y S; Kim, M J; Choi, S H; Song, Y S; Bae, S C; Ryu, K H

2010-07-01

We have completed the genomic sequence of a potyvirus, freesia mosaic virus (FreMV), and compared it to those of other known potyviruses. The full-length genome sequence of FreMV consists of 9,489 nucleotides. The large protein contains 3,077 amino acids, with an AUG start codon and UAA stop codon, containing one open reading frame typical of a potyvirus polyprotein. The polyprotein of FreMV-Kr gives rise to eleven proteins (P1, HC-pro, P3, PIPO, 6K1, CI, 6K2, VPg, NIa, NIb and CP), and putative cleavage sites of each protein were identified by sequence comparison to those of other known potyviruses. Phylogenetic analysis of the polyprotein revealed that FreMV-Kr was most closely related to PeMoV and was related to BtMV, BaRMV and PeLMV, which belong to the BCMV subgroup. This is the first information on the complete genome structure of FreMV, and the sequence information clearly supports the status of FreMV as a member of a distinct species in the genus Potyvirus.

Human retina-specific amine oxidase (RAO): cDNA cloning, tissue expression, and chromosomal mapping

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imamura, Yutaka; Kubota, Ryo; Wang, Yimin

In search of candidate genes for hereditary retinal disease, we have employed a subtractive and differential cDNA cloning strategy and isolated a novel retina-specific cDNA. Nucleotide sequence analysis revealed an open reading frame of 2187 bp, which encodes a 729-amino-acid protein with a calculated molecular mass of 80,644 Da. The putative protein contained a conserved domain of copper amine oxidase, which is found in various species from bacteria to mammals. It showed the highest homology to bovine serum amine oxidase, which is believed to control the level of serum biogenic amines. Northern blot analysis of human adult and fetal tissuesmore » revealed that the protein is expressed abundantly and specifically in retina as a 2.7-kb transcript. Thus, we considered this protein a human retina-specific amine oxidase (RAO). The RAO gene (AOC2) was mapped by fluorescence in situ hybridization to human chromosome 17q21. We propose that AOC2 may be a candidate gene for hereditary ocular diseases. 38 refs., 4 figs.« less

Pseudo-polyprotein translated from the full-length ORF1 of capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication.

PubMed

Hirata, Hisae; Yamaji, Yasuyuki; Komatsu, Ken; Kagiwada, Satoshi; Oshima, Kenro; Okano, Yukari; Takahashi, Shuichiro; Ugaki, Masashi; Namba, Shigetou

2010-09-01

The first open-reading frame (ORF) of the genus Capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP), while other viruses in the family Flexiviridae have separate ORFs encoding these proteins. To investigate the role of the full-length ORF1 polyprotein of capillovirus, we generated truncation mutants of ORF1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative Rep- and CP-coding regions. These mutants were capable of systemic infection, although their pathogenicity was attenuated. In vitro translation of ORF1 produced both the full-length polyprotein and the smaller Rep protein. The results of in vivo reporter assays suggested that the mechanism of this early termination is a ribosomal -1 frame-shift occurring downstream from the conserved Rep domains. The mechanism of capillovirus gene expression and the very close evolutionary relationship between the genera Capillovirus and Trichovirus are discussed. Copyright (c) 2010. Published by Elsevier B.V.

First venom gland transcriptomic analysis of Iranian yellow scorpion "Odonthubuthus doriae" with some new findings.

PubMed

NaderiSoorki, Maryam; Galehdari, Hamid; Baradaran, Masomeh; Jalali, Amir

2016-09-15

Scorpion venom contains mixture of biologic molecules including selective toxins with medical capability. Odonthubuthus doriae (O. doriae) belonged to Buthidae family of scorpions and gained more interest among Iranian dangerous scorpion since 2005. We constructed the first cDNA library to explore the transcriptomic composition of this Iranian scorpiontelson. Then by used of bioinformatic software each expression sequence taq (EST) from the library analyzed and its quiddity was clear. Analysis showed that toxins (42%) had more venom transcript than other component such as antimicrobial peptides, venom peptides and cell proteins. Over 16% of transcripts didn't have any open reading frames (ORF), however their sequences showed similarity by other scorpion sequences. One EST didn't have any similarity by known scorpion peptides. For the first time; we report a comprehensive study of an Iranian scorpion with interesting and novel findings. We characterized a new putative sodium channel modifier in scorpions by some bioinformatics software, and then predicted its structure and function. Copyright © 2016. Published by Elsevier Ltd.

Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1.

PubMed

Atago, Yuki; Shimodaira, Jun; Araki, Naoto; Bin Othman, Nor'azizi; Zakaria, Zuriati; Fukuda, Masao; Futami, Junichiro; Hara, Hirofumi

2016-05-01

Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

Isolation and characterization of a novel mycovirus from Penicillium digitatum.

PubMed

Niu, Yuhui; Zhang, Tingfu; Zhu, Ying; Yuan, Yongze; Wang, Shengqiang; Liu, Jing; Liu, Deli

2016-07-01

A novel double-stranded RNA virus designated Penicillium digitatum virus 1 (PdV1) was isolated from the citrus fruit rot pathogen P. digitatum (HS-RH1). The full-length cDNA sequence of the dsRNA/PdV1 (5211bp) possesses two partially overlapping open reading frames, which encode a coat protein (CP) and a putative RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis based on multiple alignments of the amino acid sequences of the RdRp and CP indicated that PdV1 tentatively belongs to the genus Victorivirus in the Totiviridae family. Electron micrographs of negatively stained viral particles purified from the peak fraction of sucrose density gradient centrifugation showed spherical particles ~35nm in diameter. Transfection experiments with purified virions indicated that PdV1 could reduce the vegetative growth and virulence of P. digitatum strain HS-F6. In summary, we report the first isolation and characterization of a mycovirus from P. digitatum that contributes to the hypovirulence phenotypes of the host strain. Copyright © 2016 Elsevier Inc. All rights reserved.

Comprehensive analysis of single molecule sequencing-derived complete genome and whole transcriptome of Hyposidra talaca nuclear polyhedrosis virus.

PubMed

Nguyen, Thong T; Suryamohan, Kushal; Kuriakose, Boney; Janakiraman, Vasantharajan; Reichelt, Mike; Chaudhuri, Subhra; Guillory, Joseph; Divakaran, Neethu; Rabins, P E; Goel, Ridhi; Deka, Bhabesh; Sarkar, Suman; Ekka, Preety; Tsai, Yu-Chih; Vargas, Derek; Santhosh, Sam; Mohan, Sangeetha; Chin, Chen-Shan; Korlach, Jonas; Thomas, George; Babu, Azariah; Seshagiri, Somasekar

2018-06-12

We sequenced the Hyposidra talaca NPV (HytaNPV) double stranded circular DNA genome using PacBio single molecule sequencing technology. We found that the HytaNPV genome is 139,089 bp long with a GC content of 39.6%. It encodes 141 open reading frames (ORFs) including the 37 baculovirus core genes, 25 genes conserved among lepidopteran baculoviruses, 72 genes known in baculovirus, and 7 genes unique to the HytaNPV genome. It is a group II alphabaculovirus that codes for the F protein and lacks the gp64 gene found in group I alphabaculovirus viruses. Using RNA-seq, we confirmed the expression of the ORFs identified in the HytaNPV genome. Phylogenetic analysis showed HytaNPV to be closest to BusuNPV, SujuNPV and EcobNPV that infect other tea pests, Buzura suppressaria, Sucra jujuba, and Ectropis oblique, respectively. We identified repeat elements and a conserved non-coding baculovirus element in the genome. Analysis of the putative promoter sequences identified motif consistent with the temporal expression of the genes observed in the RNA-seq data.

Molecular and characterization of NnPPO cDNA from lotus (Nelumbo nucifera) in rhizome browning.

PubMed

Dong, C; Yu, A Q; Yang, M G; Zhou, M Q; Hu, Z L

2016-04-30

The complete cDNA (NnPPO) of polyphenol oxidase in Nelumbo nucifera was successfully isolated, using Rapid amplification cDNA end (RACE) assays. The full-length cDNA of NnPPO was 2069 bp in size, containing a 1791 bp open reading frame coding 597 amino acids. The putative NnPPO possessed the conserved active sites and domains for PPO function. Phylogenetic analysis revealed that NnPPO shared high homology with PPO of high plants, and the homology modeling proved that NnPPO had the typical structure of PPO family. In order to characterize the role of NnPPO, Real-time PCR assay demonstrated that NnPPO mRNA was expressed in different tissues of N. nucifera including young leave, rhizome, flower, root and leafstalk, with the highest expression in rhizome. Patterns of NnPPO expression in rhizome illustrated its mRNA level was significantly elevated, which was consistent with the change of NnPPO activity during rhizome browning. Therefore, transcriptional activation of NnPPO was probably the main reason causing rhizome browning.

Mouse Mesenchyme forkhead 2 (Mf2): expression, DNA binding and induction by sonic hedgehog during somitogenesis.

PubMed

Wu, S C; Grindley, J; Winnier, G E; Hargett, L; Hogan, B L

1998-01-01

Cloning and sequencing of mouse Mf2 (mesoderm/mesenchyme forkhead 2) cDNAs revealed an open reading frame encoding a putative protein of 492 amino acids which, after in vitro translation, binds to a DNA consensus sequence. Mf2 is expressed at high levels in the ventral region of newly formed somites, in sclerotomal derivatives, in lateral plate and cephalic mesoderm and in the first and second branchial arches. Other regions of mesodermal expression include the developing tongue, meninges, nose, whiskers, kidney, genital tubercule and limb joints. In the nervous system Mf2 is transcribed in restricted regions of the mid- and forebrain. In several tissues, including the early somite, Mf2 is expressed in cell populations adjacent to regions expressing sonic hedgehog (Shh) and in explant cultures of presomitic mesoderm Mf2 is induced by Shh secreted by COS cells. These results suggest that Mf2, like other murine forkhead genes, has multiple roles in embryogenesis, possibly mediating the response of cells to signaling molecules such as SHH.

Rat Humanin is encoded and translated in mitochondria and is localized to the mitochondrial compartment where it regulates ROS production.

PubMed

Paharkova, Vladislava; Alvarez, Griselda; Nakamura, Hiromi; Cohen, Pinchas; Lee, Kuk-Wha

2015-09-15

Evidence for the putative mitochondrial origin of the Humanin (HN) peptide has been lacking, although its cytoprotective activity has been demonstrated in a variety of organismal and cellular systems. We sought to establish proof-of-principle for a mitochondria-derived peptide (MDP) in a rat-derived cellular system as the rat HN sequence is predicted to lack nuclear insertions of mitochondrial origin (NUMT). We found that the rat HN (Rattin; rHN) homologue is derived from the mitochondrial genome as evidenced by decreased production in Rho-0 cells, and that peptide translation occurs in the mitochondria as it is unaffected by cycloheximide. Rat HN localizes to the mitochondria in cellular subfractionation and immunohistochemical studies. Addition of a HN analogue to isolated mitochondria from rat INS-1 beta cells reduced hydrogen peroxide production by 55%. In summary, a locally bioactive peptide is derived and translated from an open reading frame (ORF) within rat mitochondrial DNA encoding 16S rRNA. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization.

PubMed

Wang, Hualei; Li, Guinan; Li, Mingyang; Wei, Dongzhi; Wang, Xuedong

2014-01-01

In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V max and K m toward 3-cyanopyridine were 77.5 μmol min(-1) mg(-1) and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.

The complete DNA sequence of lymphocystis disease virus.

PubMed

Tidona, C A; Darai, G

1997-04-14

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease, which has been reported to occur in over 100 different fish species worldwide. LCDV is a member of the family Iridoviridae and the type species of the genus Lymphocystivirus. The virions contain a single linear double-stranded DNA molecule, which is circularly permuted, terminally redundant, and heavily methylated at cytosines in CpG sequences. The complete nucleotide sequence of LCDV-1 (flounder isolate) was determined by automated cycle sequencing and primer walking. The genome of LCDV-1 is 102.653 bp in length and contains 195 open reading frames with coding capacities ranging from 40 to 1199 amino acids. Computer-assisted analyses of the deduced amino acid sequences led to the identification of several putative gene products with significant homologies to entries in protein data banks, such as the two major subunits of the viral DNA-dependent RNA polymerase, DNA polymerase, several protein kinases, two subunits of the ribonucleoside diphosphate reductase, DNA methyltransferase, the viral major capsid protein, insulin-like growth factor, and tumor necrosis factor receptor homolog.

Identification of sodium chloride-regulated genes in Burkholderia cenocepacia.

PubMed

Bhatt, Shantanu; Weingart, Christine L

2008-05-01

Previous studies have suggested that the airways of cystic fibrosis (CF) patients have elevated sodium chloride (NaCl) levels due to the malfunctioning of the CF transmembrane conductance regulator protein. For bacteria to survive in this high-salt environment, they must adjust by altering the regulation of gene expression. Among the different bacteria inhabiting the airways of CF patients is the opportunistic pathogen Burkholderia cenocepacia. Previous studies have indicated that B. cenocepacia produces a toxin and cable pili under high osmolar conditions. We used transposon mutagenesis to identify NaCl-regulated genes in the clinical strain B. cenocepacia K56-2. Six transconjugants were induced with increasing NaCl concentration. The DNA flanking the transposon was sequenced and five distinct open reading frames were identified encoding the following putative proteins: an integrase, an NAD-dependent deacetylase, TolB, an oxidoreductase, and a novel hypothetical protein. The collective results of this study provide important information about the physiology of B. cenocepacia when faced with osmotic stress and suggest the identity of significant virulence mechanisms in this opportunistic pathogen.

Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme.

PubMed Central

Fu, C; Javedan, S; Moshiri, F; Maier, R J

1994-01-01

Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product. PMID:8197192

Nucleotide sequence and genetic organization of barley stripe mosaic virus RNA gamma.

PubMed

Gustafson, G; Hunter, B; Hanau, R; Armour, S L; Jackson, A O

1987-06-01

The complete nucleotide sequences of RNA gamma from the Type and ND18 strains of barley stripe mosaic virus (BSMV) have been determined. The sequences are 3164 (Type) and 2791 (ND18) nucleotides in length. Both sequences contain a 5'-noncoding region (87 or 88 nucleotides) which is followed by a long open reading frame (ORF1). A 42-nucleotide intercistronic region separates ORF1 from a second, shorter open reading frame (ORF2) located near the 3'-end of the RNA. There is a high degree of homology between the Type and ND18 strains in the nucleotide sequence of ORF1. However, the Type strain contains a 366 nucleotide direct tandem repeat within ORF1 which is absent in the ND18 strain. Consequently, the predicted translation product of Type RNA gamma ORF1 (mol wt 87,312) is significantly larger than that of ND18 RNA gamma ORF1 (mol wt 74,011). The amino acid sequence of the ORF1 polypeptide contains homologies with putative RNA polymerases from other RNA viruses, suggesting that this protein may function in replication of the BSMV genome. The nucleotide sequence of RNA gamma ORF2 is nearly identical in the Type and ND18 strains. ORF2 codes for a polypeptide with a predicted molecular weight of 17,209 (Type) or 17,074 (ND18) which is known to be translated from a subgenomic (sg) RNA. The initiation point of this sgRNA has been mapped to a location 27 nucleotides upstream of the ORF2 initiation codon in the intercistronic region between ORF1 and ORF2. The sgRNA is not coterminal with the 3'-end of the genomic RNA, but instead contains heterogeneous poly(A) termini up to 150 nucleotides long (J. Stanley, R. Hanau, and A. O. Jackson, 1984, Virology 139, 375-383). In the genomic RNA gamma, ORF2 is followed by a short poly(A) tract and a 238-nucleotide tRNA-like structure.

The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora.

PubMed

Nowrousian, Minou; Cebula, Patricia

2005-11-03

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Deltatap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.

The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora

PubMed Central

Nowrousian, Minou; Cebula, Patricia

2005-01-01

Background The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. Results Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Δtap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. Conclusion The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora. PMID:16266439

Page turning system

NASA Technical Reports Server (NTRS)

Kerley, James J. (Inventor); Eklund, Wayne D. (Inventor)

1992-01-01

A device for holding reading materials for use by readers without arm mobility is presented. The device is adapted to hold the reading materials in position for reading with the pages displayed to enable turning by use of a rubber tipped stick that is held in the mouth and has a pair of rectangular frames. The frames are for holding and positioning the reading materials opened in reading posture with the pages displayed at a substantially unobstructed sighting position for reading. The pair of rectangular frames are connected to one another by a hinge so the angle between the frames may be varied thereby varying the inclination of the reading material. A pair of bent spring mounted wires for holding opposing pages of the reading material open for reading without substantial visual interference of the pages is mounted to the base. The wires are also adjustable to the thickness of the reading material and have a variable friction adjustment. This enables the force of the wires against the pages to be varied and permits the reader to manipulate the pages with the stick.

Vampiremania Sparks Developmental Readers (Open to Suggestion).

ERIC Educational Resources Information Center

Kaiden, Ellen Beth

1994-01-01

Describes how using Anne Rice's vampire novel "The Queen of the Damned" as supplementary reading in a developmental college reading course motivated students to enjoy reading and to improve their skills. (SR)

4 CFR 81.8 - Public reading facility.

Code of Federal Regulations, 2011 CFR

2011-01-01

....8 Public reading facility. GAO maintains a public reading facility in the Law Library at the Government Accountability Office Building, 441 G Street, NW., Washington, DC. The facility shall be open to...

4 CFR 81.8 - Public reading facility.

Code of Federal Regulations, 2010 CFR

2010-01-01

....8 Public reading facility. GAO maintains a public reading facility in the Law Library at the Government Accountability Office Building, 441 G Street, NW., Washington, DC. The facility shall be open to...

Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals.

PubMed

Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2004-05-01

The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.

Moving from Recitation to Open-Format Literature Discussion in Chinese Classrooms

ERIC Educational Resources Information Center

Cheng, Yahua; Zhang, Jie; Li, Hong; Anderson, Richard; Ding, Fengjiao; Nguyen-Jahiel, Kim; Shu, Hua; Wu, Xinchun

2015-01-01

A study involving 106 fourth graders and two teachers from a school in Beijing investigated the impact of a peer-led, open-format discussion approach, called collaborative reasoning (CR), on students' reading comprehension and teacher's professional learning. Mixed results of effects of CR on children's reading comprehension were found. After…

Results with Open Court Reading.

ERIC Educational Resources Information Center

McGraw-Hill Companies, New York, NY. Educational and Professional Publishing Group.

This publication tells the stories of eight schools from around the nation that have used the Open Court Reading program, describing the history of the schools, the challenges they faced, and their attempts to meet those challenges. The schools are located in California, Florida, Texas, and New York. Each of the school stories includes a focus on…

Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA.

PubMed Central

Mariottini, P; Chomyn, A; Riley, M; Cottrell, B; Doolittle, R F; Attardi, G

1986-01-01

In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH2-terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH2-terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells. Images PMID:3456601

Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

PubMed Central

Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

2012-01-01

Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

ORFer--retrieval of protein sequences and open reading frames from GenBank and storage into relational databases or text files.

PubMed

Büssow, Konrad; Hoffmann, Steve; Sievert, Volker

2002-12-19

Functional genomics involves the parallel experimentation with large sets of proteins. This requires management of large sets of open reading frames as a prerequisite of the cloning and recombinant expression of these proteins. A Java program was developed for retrieval of protein and nucleic acid sequences and annotations from NCBI GenBank, using the XML sequence format. Annotations retrieved by ORFer include sequence name, organism and also the completeness of the sequence. The program has a graphical user interface, although it can be used in a non-interactive mode. For protein sequences, the program also extracts the open reading frame sequence, if available, and checks its correct translation. ORFer accepts user input in the form of single or lists of GenBank GI identifiers or accession numbers. It can be used to extract complete sets of open reading frames and protein sequences from any kind of GenBank sequence entry, including complete genomes or chromosomes. Sequences are either stored with their features in a relational database or can be exported as text files in Fasta or tabulator delimited format. The ORFer program is freely available at http://www.proteinstrukturfabrik.de/orfer. The ORFer program allows for fast retrieval of DNA sequences, protein sequences and their open reading frames and sequence annotations from GenBank. Furthermore, storage of sequences and features in a relational database is supported. Such a database can supplement a laboratory information system (LIMS) with appropriate sequence information.

Development of an Action Plan for the Creation of a Capacity Building Cell within the National Institute of Open Schooling, India. Contract No. C10A-159

ERIC Educational Resources Information Center

Mishra, Sanjaya

2010-01-01

In order to understand the Open Schooling system in India, the National Institute of Open Schooling (NIOS), and its operations, the author scanned and read a large number of documents in a short span of time. These readings formed the basis of his discussions with a range of stakeholders with whom he conducted interviews/interaction to gather…

Open the Door for Reading (Motivational Activities).

ERIC Educational Resources Information Center

Voorhees, Roxy

Designed to help elementary teachers motivate students to read, this illustrated booklet presents a store of classroom ideas that promote and enrich reading. Materials presented include (1) instructions for making a "bookworm" bookmark for each student; (2) various animated bulletin board games intended to accompany the reading process and to help…

Open to Suggestion.

ERIC Educational Resources Information Center

Journal of Reading, 1987

1987-01-01

Recounts the use of: (1) a game of reading trivia to review a unit in reading, (2) a reading-related art activity that emphasized the importance of following directions, and (3) the assignment of a research paper in a remedial curriculum. (NKA)

Single nucleotide polymorphism discovery in bovine liver using RNA-seq technology.

PubMed

Pareek, Chandra Shekhar; Błaszczyk, Paweł; Dziuba, Piotr; Czarnik, Urszula; Fraser, Leyland; Sobiech, Przemysław; Pierzchała, Mariusz; Feng, Yaping; Kadarmideen, Haja N; Kumar, Dibyendu

2017-01-01

RNA-seq is a useful next-generation sequencing (NGS) technology that has been widely used to understand mammalian transcriptome architecture and function. In this study, a breed-specific RNA-seq experiment was utilized to detect putative single nucleotide polymorphisms (SNPs) in liver tissue of young bulls of the Polish Red, Polish Holstein-Friesian (HF) and Hereford breeds, and to understand the genomic variation in the three cattle breeds that may reflect differences in production traits. The RNA-seq experiment on bovine liver produced 107,114,4072 raw paired-end reads, with an average of approximately 60 million paired-end reads per library. Breed-wise, a total of 345.06, 290.04 and 436.03 million paired-end reads were obtained from the Polish Red, Polish HF, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed that 81.35%, 82.81% and 84.21% of the mapped sequencing reads were properly paired to the Polish Red, Polish HF, and Hereford breeds, respectively. This study identified 5,641,401 SNPs and insertion and deletion (indel) positions expressed in the bovine liver with an average of 313,411 SNPs and indel per young bull. Following the removal of the indel mutations, a total of 195,3804, 152,7120 and 205,3184 raw SNPs expressed in bovine liver were identified for the Polish Red, Polish HF, and Hereford breeds, respectively. Breed-wise, three highly reliable breed-specific SNP-databases (SNP-dbs) with 31,562, 24,945 and 28,194 SNP records were constructed for the Polish Red, Polish HF, and Hereford breeds, respectively. Using a combination of stringent parameters of a minimum depth of ≥10 mapping reads that support the polymorphic nucleotide base and 100% SNP ratio, 4,368, 3,780 and 3,800 SNP records were detected in the Polish Red, Polish HF, and Hereford breeds, respectively. The SNP detections using RNA-seq data were successfully validated by kompetitive allele-specific PCR (KASPTM) SNP genotyping assay. The comprehensive QTL/CG analysis of 110 QTL/CG with RNA-seq data identified 20 monomorphic SNP hit loci (CARTPT, GAD1, GDF5, GHRH, GHRL, GRB10, IGFBPL1, IGFL1, LEP, LHX4, MC4R, MSTN, NKAIN1, PLAG1, POU1F1, SDR16C5, SH2B2, TOX, UCP3 and WNT10B) in all three cattle breeds. However, six SNP loci (CCSER1, GHR, KCNIP4, MTSS1, EGFR and NSMCE2) were identified as highly polymorphic among the cattle breeds. This study identified breed-specific SNPs with greater SNP ratio and excellent mapping coverage, as well as monomorphic and highly polymorphic putative SNP loci within QTL/CGs of bovine liver tissue. A breed-specific SNP-db constructed for bovine liver yielded nearly six million SNPs. In addition, a KASPTM SNP genotyping assay, as a reliable cost-effective method, successfully validated the breed-specific putative SNPs originating from the RNA-seq experiments.

Findings from a Multi-Year Scale-Up Effectiveness Trial of Open-Court Reading (Imagine It!)

ERIC Educational Resources Information Center

Vaden-Kiernan, Michael; Borman, Geoffrey; Caverly, Sarah; Bell, Nance; de Castilla, Veronica Ruiz; Sullivan, Kate; Fleming, Grace

2016-01-01

This study addresses the effectiveness of a widely used core reading program that reflects the research-based practices recommended by the National Reading Panel. This and other similar programs are increasingly used to prevent reading difficulties and ensure that all children are reading at or above grade level by the end of third grade.…

Beach Books: 2014-2016. What Do Colleges and Universities Want Students to Read outside Class?

ERIC Educational Resources Information Center

Randall, David

2016-01-01

Hundreds of American colleges and universities continue to assign a summer reading to entering freshmen--typically one book, which the students are asked to read outside their courses. Many institutions embed the common reading in a larger program of campus activities: typically, they invite the common reading author to help open the academic year…

Emerging Concerns in Reading; Highlights of the 21st Annual Reading Conference of Lehigh University.

ERIC Educational Resources Information Center

Kender, Joseph P., Ed.

Addressing the question, "How can we teach all educable children to read effectively?" the papers in the first part of this volume look at several topical concerns in reading: "On Relocating the Reading Program: Finding a Better Barn for Our Sacred Cow,""The Transitional Open Classroom,""A Computer Program for Initial Screening of Problem…

Preliminary Findings from a Multi-Year Scale-Up Effectiveness Trial of Open-Court Reading (Imagine It!)

ERIC Educational Resources Information Center

Borman, Geoffrey; Vaden-Kiernan, Michael; Caverly, Sarah; Bell, Nance; de Castilla, Veronica Ruiz; Sullivan, Kate

2015-01-01

This study addresses the effectiveness of a nationally used core reading program that reflects the research-based practices recommended by the National Reading Panel. This and other similar programs are increasingly used to prevent reading difficulties and ensure that all children are reading at or above grade level by the end of third grade. The…

The Reading Environment; Proceedings of the Annual Reading Conference (8th, Terre Haute, Indiana, June 15-16, 1978).

ERIC Educational Resources Information Center

Waterman, David C., Comp.; Gibbs, Vanita M., Comp.

The papers presented in this annual reading conference report focus on various aspects of the topic, "The Reading Environment." The opening address advocates establishing reading programs that correspond to the developmental stages of individual children--programs that consider the needs of the whole child. The other papers delivered at the…

19 CFR 145.3 - Opening of letter class mail; reading of correspondence prohibited.

Code of Federal Regulations, 2011 CFR

2011-04-01

... letter class mail subject to Customs examination which appears to contain matter in addition to, or other... 19 Customs Duties 2 2011-04-01 2011-04-01 false Opening of letter class mail; reading of correspondence prohibited. 145.3 Section 145.3 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF...

19 CFR 145.3 - Opening of letter class mail; reading of correspondence prohibited.

Code of Federal Regulations, 2014 CFR

2014-04-01

... letter class mail subject to Customs examination which appears to contain matter in addition to, or other... 19 Customs Duties 2 2014-04-01 2014-04-01 false Opening of letter class mail; reading of correspondence prohibited. 145.3 Section 145.3 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF...

19 CFR 145.3 - Opening of letter class mail; reading of correspondence prohibited.

Code of Federal Regulations, 2013 CFR

2013-04-01

... letter class mail subject to Customs examination which appears to contain matter in addition to, or other... 19 Customs Duties 2 2013-04-01 2013-04-01 false Opening of letter class mail; reading of correspondence prohibited. 145.3 Section 145.3 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF...

19 CFR 145.3 - Opening of letter class mail; reading of correspondence prohibited.

Code of Federal Regulations, 2012 CFR

2012-04-01

... letter class mail subject to Customs examination which appears to contain matter in addition to, or other... 19 Customs Duties 2 2012-04-01 2012-04-01 false Opening of letter class mail; reading of correspondence prohibited. 145.3 Section 145.3 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF...

Identification of a non-LTR retrotransposon from the gypsy moth

Treesearch

K.J. Garner; J.M. Slavicek

1999-01-01

A family of highly repetitive elements, named LDT1, has been identified in the gypsy moth, Lymantria dispar. The complete element is 5.4 kb in length and lacks long-terminal repeats, The element contains two open reading frames with a significant amino acid sequence similarity to several non-LTR retrotransposons. The first open reading frame contains...

Expression and strain variation of the novel “Small Open Reading Frame” 3 (smorf) multigene family in Babesia bovis

USDA-ARS?s Scientific Manuscript database

Small open reading frame (smorf) genes comprise the second largest Babesia bovis multigene family. All known 44 variant smorf genes are located in close chromosomal proximity to ves1 genes, which encode proteins that mediate cytoadhesion and contribute to immune evasion. In this study, we characte...

A Multisite Cluster Randomized Field Trial of Open Court Reading

ERIC Educational Resources Information Center

Borman, Geoffrey D.; Dowling, N. Maritza; Schneck, Carrie

2008-01-01

In this article, the authors report achievement outcomes of a multisite cluster randomized field trial of Open Court Reading 2005 (OCR), a K-6 literacy curriculum published by SRA/McGraw-Hill. The participants are 49 first-grade through fifth-grade classrooms from predominantly minority and poor contexts across the nation. Blocking by grade level…

U.S. Global Change Research Program

MedlinePlus

... Announcing... Read more The Deepening Story of How Climate Change Threatens Human Health Read more Celebrating the 25th Anniversary of the U.S. Global Change Research... Read more Nomination Period Open for ... more Connecting America’s Communities with Actionable Climate ...

MetAMOS: a modular and open source metagenomic assembly and analysis pipeline

PubMed Central

2013-01-01

We describe MetAMOS, an open source and modular metagenomic assembly and analysis pipeline. MetAMOS represents an important step towards fully automated metagenomic analysis, starting with next-generation sequencing reads and producing genomic scaffolds, open-reading frames and taxonomic or functional annotations. MetAMOS can aid in reducing assembly errors, commonly encountered when assembling metagenomic samples, and improves taxonomic assignment accuracy while also reducing computational cost. MetAMOS can be downloaded from: https://github.com/treangen/MetAMOS. PMID:23320958

Readings and Experiences of Multimodality

ERIC Educational Resources Information Center

Leander, Kevin M.; Aziz, Seemi; Botzakis, Stergios; Ehret, Christian; Landry, David; Rowsell, Jennifer

2017-01-01

Our understanding of reading--including reading multimodal texts--is always constrained or opened up by what we consider to be a text, what aspects of a reader's embodied activity we focus on, and how we draw a boundary around a reading event. This article brings together five literacy researchers who respond to a human-scale graphic novel,…

Defense Threat Reduction Agency > Research > DTRIAC > FAQ Sheet

Science.gov Websites

FOIA Electronic Reading Room Privacy Impact Assessment DTRA No Fear Act Reporting Nuclear Test . Q: Does DTRIAC have a public reading room? A: No, DTRIAC does not have a reading room open to the

Sacred Cows That Should Be Put to Pasture.

ERIC Educational Resources Information Center

Artley, A. Sterl

This paper examines some of the problems associated with unquestioned teaching practices and theories ("sacred cows") in the field of reading. Topics discussed include phonics, pronunciation, oral reading, teacher accountability and behavioral objectives, individualized reading, and the open classroom. (KS)

Overexpression of a novel thermostable and chloride-tolerant laccase from Thermus thermophilus SG0.5JP17-16 in Pichia pastoris and its application in synthetic dye decolorization.

PubMed

Liu, Huiping; Cheng, Yu; Du, Bing; Tong, Chaofan; Liang, Shuli; Han, Shuangyan; Zheng, Suiping; Lin, Ying

2015-01-01

Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (LacTT) from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), guaiacol, and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0-11.0 and thermostable at 40°C-90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters.

Overexpression of a Novel Thermostable and Chloride-Tolerant Laccase from Thermus thermophilus SG0.5JP17-16 in Pichia pastoris and Its Application in Synthetic Dye Decolorization

PubMed Central

Liu, Huiping; Cheng, Yu; Du, Bing; Tong, Chaofan; Liang, Shuli; Han, Shuangyan; Zheng, Suiping; Lin, Ying

2015-01-01

Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (LacTT) from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), guaiacol, and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0–11.0 and thermostable at 40°C–90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters. PMID:25790466

Comparative Genetic Analyses of Human Rhinovirus C (HRV-C) Complete Genome from Malaysia.

PubMed

Khaw, Yam Sim; Chan, Yoke Fun; Jafar, Faizatul Lela; Othman, Norlijah; Chee, Hui Yee

2016-01-01

Human rhinovirus-C (HRV-C) has been implicated in more severe illnesses than HRV-A and HRV-B, however, the limited number of HRV-C complete genomes (complete 5' and 3' non-coding region and open reading frame sequences) has hindered the in-depth genetic study of this virus. This study aimed to sequence seven complete HRV-C genomes from Malaysia and compare their genetic characteristics with the 18 published HRV-Cs. Seven Malaysian HRV-C complete genomes were obtained with newly redesigned primers. The seven genomes were classified as HRV-C6, C12, C22, C23, C26, C42, and pat16 based on the VP4/VP2 and VP1 pairwise distance threshold classification. Five of the seven Malaysian isolates, namely, 3430-MY-10/C22, 8713-MY-10/C23, 8097-MY-11/C26, 1570-MY-10/C42, and 7383-MY-10/pat16 are the first newly sequenced complete HRV-C genomes. All seven Malaysian isolates genomes displayed nucleotide similarity of 63-81% among themselves and 63-96% with other HRV-Cs. Malaysian HRV-Cs had similar putative immunogenic sites, putative receptor utilization and potential antiviral sites as other HRV-Cs. The genomic features of Malaysian isolates were similar to those of other HRV-Cs. Negative selections were frequently detected in HRV-Cs complete coding sequences indicating that these sequences were under functional constraint. The present study showed that HRV-Cs from Malaysia have diverse genetic sequences but share conserved genomic features with other HRV-Cs. This genetic information could provide further aid in the understanding of HRV-C infection.

Genetic heterogeneity of the dnaK gene locus including transcription terminator region (TTR) in Campylobacter lari.

PubMed

Shitara, M; Tsuboi, Y; Sekizuka, T; Tazumi, A; Moorei, J E; Millar, B C; Taneike, I; Matsuda, M

2008-01-01

Nucleotide sequences of approximately 3.1 kbp consisting of the full-length open reading frame (ORF) for grpE, a non-coding (NC) region and a putative ORF for the full-length dnaK gene (1860 bp) were identified from a urease-positive thermophilic Campylobacter (UPTC) CF89-12 isolate. Then, following the construction of a new degenerate polymerase chain reaction (PCR) primer pair for amplification of the dnaK structural gene, including the transcription terminator region of C. lari isolates, the dnaK region was amplified successfully, TA-cloned and sequenced in nine C. lari isolates. The dnaK gene sequences commenced with an ATG and terminated with a TAA in all 10 isolates, including CF89-12. In addition, the putative ORFs for the dnaK gene locus from seven UPTC isolates consisted of 1860 bases, and the four urease-negative (UN) C. lari isolates included C. lari RM2100 reference strain 1866. Interestingly, different probable ribosome binding sites and hypothetically intrinsic p-independent terminator structures were identified between the seven UPTC and four UN C. lari isolates, respectively. Moreover, it is interesting to note that 20 out of a total of 28 polymorphic sites occurred among amino acid sequences of the dnaK ORF from 11 C. lari isolates, identified to be alternatively UPTC-specific or UN C. lari-specific. In the neighbour-joining tree based on the nucleotide sequence information of the dnaK gene, C. lari forms two major distinct clusters consisting of UPTC and UN C. lari isolates, respectively, with UN C. lari being more closely related to other thermophilic campylobacters than to UPTC.

First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia.

PubMed

Reis, Marta I R; do Vale, Ana; Pinto, Cristina; Nascimento, Diana S; Costa-Ramos, Carolina; Silva, Daniela S P; Silva, Manuel T; Dos Santos, Nuno M S

2007-03-01

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924bp cDNA of sea bass caspase-9 was obtained, consisting of 1308bp open reading frame coding for 435 amino acids, 199bp of the 5'-UTR and 417bp of the 3'-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp(121), Asp(325) and Asp(343). The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida (Phdp) strain PP3, showed increased expression from 0 to 12h returning to control levels at 24h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48h post-infection, when the fish were with advanced septicaemia.

OI-57, a Genomic Island of Escherichia coli O157, Is Present in Other Seropathotypes of Shiga Toxin-Producing E. coli Associated with Severe Human Disease▿

PubMed Central

Imamovic, Lejla; Tozzoli, Rosangela; Michelacci, Valeria; Minelli, Fabio; Marziano, Maria Luisa; Caprioli, Alfredo; Morabito, Stefano

2010-01-01

Strains of Shiga toxin-producing Escherichia coli (STEC) are a heterogeneous E. coli group that may cause severe disease in humans. STEC have been categorized into seropathotypes (SPTs) based on their phenotypic and molecular characteristics and the clinical features of the associated diseases. SPTs range from A to E, according to a decreasing rank of pathogenicity. To define the virulence gene asset (“virulome”) characterizing the highly pathogenic SPTs, we used microarray hybridization to compare the whole genomes of STEC belonging to SPTs B, C, and D with that of STEC O157 (SPT A). The presence of the open reading frames (ORFs) associated with SPTs A and B was subsequently investigated by PCR in a larger panel of STEC and in other E. coli strains. A genomic island termed OI-57 was present in SPTs A and B but not in the other SPTs. OI-57 harbors the putative virulence gene adfO, encoding a factor enhancing the adhesivity of STEC O157, and ckf, encoding a putative killing factor for the bacterial cell. PCR analyses showed that OI-57 was present in its entirety in the majority of the STEC genomes examined, indicating that it represents a stable acquisition of the positive clonal lineages. OI-57 was also present in a high proportion of the human enteropathogenic E. coli genomes assayed, suggesting that it could be involved in the attaching-and-effacing colonization of the intestinal mucosa. In conclusion, OI-57 appears to be part of the virulome of pathogenic STEC and further studies are needed to elucidate its role in the pathogenesis of STEC infections. PMID:20823207

Molecular characterization of a KIF3B-like kinesin gene in the testis of Octopus tankahkeei (Cephalopoda, Octopus).

PubMed

Dang, Ran; Zhu, Jun-Quan; Tan, Fu-Qing; Wang, Wei; Zhou, Hong; Yang, Wan-Xi

2012-05-01

KIF3B is known for maintaining and assembling cilia and flagellum. To date, the function of KIF3B and its relationship with KIF3A during spermiogenesis in the cephalopod Octopus tankahkeei remains unknown. In the present study, we characterized a gene encoding a homologue of rat KIF3B in the O. tankahkeei testis and examined its temporal and spatial expression pattern during spermiogenesis. The cDNA of KIF3B was obtained with degenerate and RACE PCR and the distribution pattern of ot-kif3b were observed with RT-PCR. The morphological development during spermiogenesis was illustrated by histological and transmission electron microscopy and mRNA expression of ot-kif3b was observed by in situ hybridization. The 2,365 nucleotides cDNA consisted of a 102 bp 5' untranslated region (UTR), a 2,208 bp open reading frame (ORF) encoding a protein of 736 amino acids, and a 55 bp 3' UTR. Multiple alignments revealed that the putative Ot-KIF3B shared 68, 68, 69, 68, and 67% identity with that of Homo sapiens, Mus musculus, Gallus gallus, Danio rerio, and Xenopus laevis, respectively, along with high identities with Ot-KIF3A in fundamental structures. Ot-kif3b transcripts appeared gradually in early spermatids, increased in intermediate spermatids and maximized in drastically remodeled and final spermatids. The kif3b gene is identified and its expression pattern is demonstrated for the first time in O. tankahkeei. Compared to ot-kif3a reported by our laboratory before, our data suggested that the putative heterodimeric motor proteins Ot-KIF3A/B may be involved in intraspermatic transport and might contribute to structural changes during spermiogenesis.

Molecular cloning and heterologous expression of a biosynthetic gene cluster for the antitubercular agent D-cycloserine produced by Streptomyces lavendulae.

PubMed

Kumagai, Takanori; Koyama, Yusuke; Oda, Kosuke; Noda, Masafumi; Matoba, Yasuyuki; Sugiyama, Masanori

2010-03-01

In the present study, we successfully cloned a 21-kb DNA fragment containing a d-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyces lavendulae strain, ATCC 11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated dcsA to dcsJ. This cluster includes two ORFs encoding D-alanyl-D-alanine ligase (dcsI) and a putative membrane protein (dcsJ) as the self-resistance determinants of the producer organism, indicated by our previous work. When the 10 ORFs were introduced into DCS-nonproducing Streptomyces lividans 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of dcsG, seen in the DCS biosynthetic gene cluster, made it possible for the strain ATCC 11924 to lose its DCS production. We here propose the DCS biosynthetic pathway. First, L-serine is O acetylated by a dcsE-encoded enzyme homologous to homoserine O-acetyltransferase. Second, O-acetyl-L-serine accepts hydroxyurea via an O-acetylserine sulfhydrylase homolog (dcsD product) and forms O-ureido-L-serine. The hydroxyurea must be supplied by the catalysis of a dcsB-encoded arginase homolog using the L-arginine derivative, N(G)-hydroxy-L-arginine. The resulting O-ureido-L-serine is then racemized to O-ureido-D-serine by a homolog of diaminopimelate epimerase. Finally, O-ureido-D-serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the dcsG or dcsH product, which belongs to the ATP-grasp fold family of protein.

The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism.

PubMed

Auernik, Kathryne S; Maezato, Yukari; Blum, Paul H; Kelly, Robert M

2008-02-01

Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, approximately 2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized.

Genomic characterisation of Wongabel virus reveals novel genes within the Rhabdoviridae.

PubMed

Gubala, Aneta J; Proll, David F; Barnard, Ross T; Cowled, Chris J; Crameri, Sandra G; Hyatt, Alex D; Boyle, David B

2008-06-20

Viruses belonging to the family Rhabdoviridae infect a variety of different hosts, including insects, vertebrates and plants. Currently, there are approximately 200 ICTV-recognised rhabdoviruses isolated around the world. However, the majority remain poorly characterised and only a fraction have been definitively assigned to genera. The genomic and transcriptional complexity displayed by several of the characterised rhabdoviruses indicates large diversity and complexity within this family. To enable an improved taxonomic understanding of this family, it is necessary to gain further information about the poorly characterised members of this family. Here we present the complete genome sequence and predicted transcription strategy of Wongabel virus (WONV), a previously uncharacterised rhabdovirus isolated from biting midges (Culicoides austropalpalis) collected in northern Queensland, Australia. The 13,196 nucleotide genome of WONV encodes five typical rhabdovirus genes N, P, M, G and L. In addition, the WONV genome contains three genes located between the P and M genes (U1, U2, U3) and two open reading frames overlapping with the N and G genes (U4, U5). These five additional genes and their putative protein products appear to be novel, and their functions are unknown. Predictive analysis of the U5 gene product revealed characteristics typical of viroporins, and indicated structural similarities with the alpha-1 protein (putative viroporin) of viruses in the genus Ephemerovirus. Phylogenetic analyses of the N and G proteins of WONV indicated closest similarity with the avian-associated Flanders virus; however, the genomes of these two viruses are significantly diverged. WONV displays a novel and unique genome structure that has not previously been described for any animal rhabdovirus.

Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H2-Oxidizing Bacteria ▿†

PubMed Central

Constant, Philippe; Chowdhury, Soumitra Paul; Hesse, Laura; Pratscher, Jennifer; Conrad, Ralf

2011-01-01

Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H2 possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H2-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H2 oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 106 to 108 hhyL gene copies g (dry weight)−1. Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H2-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H2 oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H2 by soil, because high-affinity H2 oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur. PMID:21742924

Molecular Cloning and Characterization of cDNA Encoding a Putative Stress-Induced Heat-Shock Protein from Camelus dromedarius

PubMed Central

Elrobh, Mohamed S.; Alanazi, Mohammad S.; Khan, Wajahatullah; Abduljaleel, Zainularifeen; Al-Amri, Abdullah; Bazzi, Mohammad D.

2011-01-01

Heat shock proteins are ubiquitous, induced under a number of environmental and metabolic stresses, with highly conserved DNA sequences among mammalian species. Camelus dromedaries (the Arabian camel) domesticated under semi-desert environments, is well adapted to tolerate and survive against severe drought and high temperatures for extended periods. This is the first report of molecular cloning and characterization of full length cDNA of encoding a putative stress-induced heat shock HSPA6 protein (also called HSP70B′) from Arabian camel. A full-length cDNA (2417 bp) was obtained by rapid amplification of cDNA ends (RACE) and cloned in pET-b expression vector. The sequence analysis of HSPA6 gene showed 1932 bp-long open reading frame encoding 643 amino acids. The complete cDNA sequence of the Arabian camel HSPA6 gene was submitted to NCBI GeneBank (accession number HQ214118.1). The BLAST analysis indicated that C. dromedaries HSPA6 gene nucleotides shared high similarity (77–91%) with heat shock gene nucleotide of other mammals. The deduced 643 amino acid sequences (accession number ADO12067.1) showed that the predicted protein has an estimated molecular weight of 70.5 kDa with a predicted isoelectric point (pI) of 6.0. The comparative analyses of camel HSPA6 protein sequences with other mammalian heat shock proteins (HSPs) showed high identity (80–94%). Predicted camel HSPA6 protein structure using Protein 3D structural analysis high similarities with human and mouse HSPs. Taken together, this study indicates that the cDNA sequences of HSPA6 gene and its amino acid and protein structure from the Arabian camel are highly conserved and have similarities with other mammalian species. PMID:21845074

Sequence analysis and expression of the M1 and M2 matrix protein genes of hirame rhabdovirus (HIRRV)

USGS Publications Warehouse

Nishizawa, T.; Kurath, G.; Winton, J.R.

1997-01-01

We have cloned and sequenced a 2318 nucleotide region of the genomic RNA of hirame rhabdovirus (HIRRV), an important viral pathogen of Japanese flounder Paralichthys olivaceus. This region comprises approximately two-thirds of the 3' end of the nucleocapsid protein (N) gene and the complete matrix protein (M1 and M2) genes with the associated intergenic regions. The partial N gene sequence was 812 nucleotides in length with an open reading frame (ORF) that encoded the carboxyl-terminal 250 amino acids of the N protein. The M1 and M2 genes were 771 and 700 nucleotides in length, respectively, with ORFs encoding proteins of 227 and 193 amino acids. The M1 gene sequence contained an additional small ORF that could encode a highly basic, arginine-rich protein of 25 amino acids. Comparisons of the N, M1, and M2 gene sequences of HIRRV with the corresponding sequences of the fish rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) or viral hemorrhagic septicemia virus (VHSV) indicated that HIRRV was more closely related to IHNV than to VHSV, but was clearly distinct from either. The putative consensus gene termination sequence for IHNV and VHSV, AGAYAG(A)(7), was present in the N-M1, M1-M2, and M2-G intergenic regions of HIRRV as were the putative transcription initiation sequences YGGCAC and AACA. An Escherichia coli expression system was used to produce recombinant proteins from the M1 and M2 genes of HIRRV. These were the same size as the authentic M1 and M2 proteins and reacted with anti-HIRRV rabbit serum in western blots. These reagents can be used for further study of the fish immune response and to test novel control methods.

A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

PubMed Central

Ohkawa, T; Majima, K; Maeda, S

1994-01-01

Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

Cloning and Expression of a Phloretin Hydrolase Gene from Eubacterium ramulus and Characterization of the Recombinant Enzyme

PubMed Central

Schoefer, Lilian; Braune, Annett; Blaut, Michael

2004-01-01

Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37°C and 7.0, respectively. The Km for phloretin was 13 ± 3 μM and the kcat was 10 ± 2 s−1. The enzyme did not transform phloretin-2′-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl2 to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively. PMID:15466559

Transcriptional and Functional Studies of Acidithiobacillus ferrooxidans Genes Related to Survival in the Presence of Copper▿

PubMed Central

Navarro, Claudio A.; Orellana, Luis H.; Mauriaca, Cecilia; Jerez, Carlos A.

2009-01-01

The acidophilic Acidithiobacillus ferrooxidans can resist exceptionally high copper (Cu) concentrations. This property is important for its use in biomining processes, where Cu and other metal levels range usually between 15 and 100 mM. To learn about the mechanisms that allow A. ferrooxidans cells to survive in this environment, a bioinformatic search of its genome showed the presence of at least 10 genes that are possibly related to Cu homeostasis. Among them are three genes coding for putative ATPases related to the transport of Cu (A. ferrooxidans copA1 [copA1Af], copA2Af, and copBAf), three genes related to a system of the resistance nodulation cell division family involved in the extraction of Cu from the cell (cusAAf, cusBAf, and cusCAf), and two genes coding for periplasmic chaperones for this metal (cusFAf and copCAf). The expression of most of these open reading frames was studied by real-time reverse transcriptase PCR using A. ferrooxidans cells adapted for growth in the presence of high concentrations of Cu. The putative A. ferrooxidans Cu resistance determinants were found to be upregulated when this bacterium was exposed to Cu in the range of 5 to 25 mM. These A. ferrooxidans genes conferred to Escherichia coli a greater Cu resistance than wild-type cells, supporting their functionality. The results reported here and previously published data strongly suggest that the high resistance of the extremophilic A. ferrooxidans to Cu may be due to part or all of the following key elements: (i) a wide repertoire of Cu resistance determinants, (ii) the duplication of some of these Cu resistance determinants, (iii) the existence of novel Cu chaperones, and (iv) a polyP-based Cu resistance system. PMID:19666734

The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon Metallosphaera sedula Provides Insights into Bioleaching-Associated Metabolism▿ †

PubMed Central

Auernik, Kathryne S.; Maezato, Yukari; Blum, Paul H.; Kelly, Robert M.

2008-01-01

Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ∼2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized. PMID:18083856

Comparative genomic sequence analysis of novel Helicoverpa armigera nucleopolyhedrovirus (NPV) isolated from Kenya and three other previously sequenced Helicoverpa spp. NPVs.

PubMed

Ogembo, Javier Gordon; Caoili, Barbara L; Shikata, Masamitsu; Chaeychomsri, Sudawan; Kobayashi, Michihiro; Ikeda, Motoko

2009-10-01

A newly cloned Helicoverpa armigera nucleopolyhedrovirus (HearNPV) from Kenya, HearNPV-NNg1, has a higher insecticidal activity than HearNPV-G4, which also exhibits lower insecticidal activity than HearNPV-C1. In the search for genes and/or nucleotide sequences that might be involved in the observed virulence differences among Helicoverpa spp. NPVs, the entire genome of NNg1 was sequenced and compared with previously sequenced genomes of G4, C1 and Helicoverpa zea single-nucleocapsid NPV (Hz). The NNg1 genome was 132,425 bp in length, with a total of 143 putative open reading frames (ORFs), and shared high levels of overall amino acid and nucleotide sequence identities with G4, C1 and Hz. Three NNg1 ORFs, ORF5, ORF100 and ORF124, which were shared with C1, were absent in G4 and Hz, while NNg1 and C1 were missing a homologue of G4/Hz ORF5. Another three ORFs, ORF60 (bro-b), ORF119 and ORF120, and one direct repeat sequence (dr) were unique to NNg1. Relative to the overall nucleotide sequence identity, lower sequence identities were observed between NNg1 hrs and the homologous hrs in the other three Helicoverpa spp. NPVs, despite containing the same number of hrs located at essentially the same positions on the genomes. Differences were also observed between NNg1 and each of the other three Helicoverpa spp. NPVs in the diversity of bro genes encoded on the genomes. These results indicate several putative genes and nucleotide sequences that may be responsible for the virulence differences observed among Helicoverpa spp., yet the specific genes and/or nucleotide sequences responsible have not been identified.

Identification of a Novel Dioxygenase Involved in Metabolism of o-Xylene, Toluene, and Ethylbenzene by Rhodococcus sp. Strain DK17

PubMed Central

Kim, Dockyu; Chae, Jong-Chan; Zylstra, Gerben J.; Kim, Young-Soo; Kim, Seong-Ki; Nam, Myung Hee; Kim, Young Min; Kim, Eungbin

2004-01-01

Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the ORFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediately downstream of one copy of the dioxygenase genes (akbA1a and akbA2a) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1b and akbA2b) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1a-AkbA2a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1a-AkbA2a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively. PMID:15574904

One precursor, three apolipoproteins: the relationship between two crustacean lipoproteins, the large discoidal lipoprotein and the high density lipoprotein/β-glucan binding protein.

PubMed

Stieb, Stefanie; Roth, Ziv; Dal Magro, Christina; Fischer, Sabine; Butz, Eric; Sagi, Amir; Khalaila, Isam; Lieb, Bernhard; Schenk, Sven; Hoeger, Ulrich

2014-12-01

The novel discoidal lipoprotein (dLp) recently detected in the crayfish, differs from other crustacean lipoproteins in its large size, apoprotein composition and high lipid binding capacity, We identified the dLp sequence by transcriptome analyses of the hepatopancreas and mass spectrometry. Further de novo assembly of the NGS data followed by BLAST searches using the sequence of the high density lipoprotein/1-glucan binding protein (HDL-BGBP) of Astacus leptodactylus as query revealed a putative precursor molecule with an open reading frame of 14.7 kb and a deduced primary structure of 4889 amino acids. The presence of an N-terminal lipid bind- ing domain and a DUF 1943 domain suggests the relationship with the large lipid transfer proteins. Two-putative dibasic furin cleavage sites were identified bordering the sequence of the HDL-BGBP. When subjected to mass spectroscopic analyses, tryptic peptides of the large apoprotein of dLp matched the N-terminal part of the precursor, while the peptides obtained for its small apoprotein matched the C-terminal part. Repeating the analysis in the prawn Macrobrachium rosenbergii revealed a similar protein with identical domain architecture suggesting that our findings do not represent an isolated instance. Our results indicate that the above three apolipoproteins (i.e HDL-BGBP and both the large and the small subunit of dLp) are translated as a large precursor. Cleavage at the furin type sites releases two subunits forming a heterodimeric dLP particle, while the remaining part forms an HDL-BGBP whose relationship with other lipoproteins as well as specific functions are yet to be elucidated.

Transcription activation mediated by a cyclic AMP receptor protein from Thermus thermophilus HB8.

PubMed

Shinkai, Akeo; Kira, Satoshi; Nakagawa, Noriko; Kashihara, Aiko; Kuramitsu, Seiki; Yokoyama, Shigeyuki

2007-05-01

The extremely thermophilic bacterium Thermus thermophilus HB8, which belongs to the phylum Deinococcus-Thermus, has an open reading frame encoding a protein belonging to the cyclic AMP (cAMP) receptor protein (CRP) family present in many bacteria. The protein named T. thermophilus CRP is highly homologous to the CRP family proteins from the phyla Firmicutes, Actinobacteria, and Cyanobacteria, and it forms a homodimer and interacts with cAMP. CRP mRNA and intracellular cAMP were detected in this strain, which did not drastically fluctuate during cultivation in a rich medium. The expression of several genes was altered upon disruption of the T. thermophilus CRP gene. We found six CRP-cAMP-dependent promoters in in vitro transcription assays involving DNA fragments containing the upstream regions of the genes exhibiting decreased expression in the CRP disruptant, indicating that the CRP is a transcriptional activator. The consensus T. thermophilus CRP-binding site predicted upon nucleotide sequence alignment is 5'-(C/T)NNG(G/T)(G/T)C(A/C)N(A/T)NNTCACAN(G/C)(G/C)-3'. This sequence is unique compared with the known consensus binding sequences of CRP family proteins. A putative -10 hexamer sequence resides at 18 to 19 bp downstream of the predicted T. thermophilus CRP-binding site. The CRP-regulated genes found in this study comprise clustered regularly interspaced short palindromic repeat (CRISPR)-associated (cas) ones, and the genes of a putative transcriptional regulator, a protein containing the exonuclease III-like domain of DNA polymerase, a GCN5-related acetyltransferase homolog, and T. thermophilus-specific proteins of unknown function. These results suggest a role for cAMP signal transduction in T. thermophilus and imply the T. thermophilus CRP is a cAMP-responsive regulator.

RegA, an AraC-like protein, is a global transcriptional regulator that controls virulence gene expression in Citrobacter rodentium.

PubMed

Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J; Frankel, Gad; Hartland, Elizabeth L; Robins-Browne, Roy M

2008-11-01

Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5alpha, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization.

RegA, an AraC-Like Protein, Is a Global Transcriptional Regulator That Controls Virulence Gene Expression in Citrobacter rodentium▿

PubMed Central

Hart, Emily; Yang, Ji; Tauschek, Marija; Kelly, Michelle; Wakefield, Matthew J.; Frankel, Gad; Hartland, Elizabeth L.; Robins-Browne, Roy M.

2008-01-01

Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5α, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization. PMID:18765720

Molecular identification and characterisation of catalase and catalase-like protein genes in urease-positive thermophilic Campylobacter (UPTC).

PubMed

Nakajima, T; Kuribayashi, T; Moore, J E; Millar, B C; Yamamoto, S; Matsuda, Motoo

2016-01-01

Thermophilic Campylobacter are important bacterial pathogens of foodborne diseases worldwide. These organisms' physiology requires a microaerophilic atmosphere. To date, little is known about the protective catalase mechanism in urease-positive thermophilic campylobacters (UPTC); hence, it was the aim of this study to identify and characterise catalase and catalase-like protein genes in these organisms. Catalase (katA) and catalase (Kat)-like protein genes from the Japanese UPTC CF89-12 strain were molecularly analysed and compared with C. lari RM2100 and other C. lari and thermophilic Campylobacter reference isolates. A possible open reading frame of 1,422 base pairs, predicted to encode a peptide of 474 amino acid residues, with calculated molecular weight of 52.7 kilo Daltons for katA, was identified within UPTC CF89-12. A probable ribosome binding site, two putative promoters and a putative ρ-independent transcription terminator were also identified within katA. A similar katA cluster also existed in the C. lari RM2100 strain, except that this strain carries no DcuB genes. However, the Kat-like protein gene or any other homologue(s) were never identified in the C. lari RM2100 strain, or in C. jejuni and C. upsaliensis. This study demonstrates the presence of catalase/catalase-like protein genes in UPTC organisms. These findings are significant in that they suggest that UPTC organisms have the protective genetic capability of helping protect the organisms from toxic oxygen stress, which may help them to survive in physiologically harsh environments, both within human and animal hosts, as well as in the natural environment.

Comparative Genetic Analyses of Human Rhinovirus C (HRV-C) Complete Genome from Malaysia

PubMed Central

Khaw, Yam Sim; Chan, Yoke Fun; Jafar, Faizatul Lela; Othman, Norlijah; Chee, Hui Yee

2016-01-01

Human rhinovirus-C (HRV-C) has been implicated in more severe illnesses than HRV-A and HRV-B, however, the limited number of HRV-C complete genomes (complete 5′ and 3′ non-coding region and open reading frame sequences) has hindered the in-depth genetic study of this virus. This study aimed to sequence seven complete HRV-C genomes from Malaysia and compare their genetic characteristics with the 18 published HRV-Cs. Seven Malaysian HRV-C complete genomes were obtained with newly redesigned primers. The seven genomes were classified as HRV-C6, C12, C22, C23, C26, C42, and pat16 based on the VP4/VP2 and VP1 pairwise distance threshold classification. Five of the seven Malaysian isolates, namely, 3430-MY-10/C22, 8713-MY-10/C23, 8097-MY-11/C26, 1570-MY-10/C42, and 7383-MY-10/pat16 are the first newly sequenced complete HRV-C genomes. All seven Malaysian isolates genomes displayed nucleotide similarity of 63–81% among themselves and 63–96% with other HRV-Cs. Malaysian HRV-Cs had similar putative immunogenic sites, putative receptor utilization and potential antiviral sites as other HRV-Cs. The genomic features of Malaysian isolates were similar to those of other HRV-Cs. Negative selections were frequently detected in HRV-Cs complete coding sequences indicating that these sequences were under functional constraint. The present study showed that HRV-Cs from Malaysia have diverse genetic sequences but share conserved genomic features with other HRV-Cs. This genetic information could provide further aid in the understanding of HRV-C infection. PMID:27199901

Cloning and expression of a CYP720B orthologue involved in the biosynthesis of diterpene resin acids in Pinus brutia.

PubMed

Semiz, Asli; Sen, Alaattin

2015-03-01

Cytochrome P450 monooxygenases mediate a broad range of oxidative reactions involved in the biosynthesis of both primary and secondary metabolites in plants. Until now, only two P450 genes, CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, have been functionally characterised and described in the literature. The purpose of this study was to describe the cloning and expression of CYP720B from Pinus brutia due to its suggested role in the synthesis of bioactive compounds used for chemical defence against insects. A PCR product of the P. brutia CYP720B gene was cloned into the pCR8/GW/TOPO cloning vector. After optimising the sequence for codon usage in yeast, it was transferred into the inducible expression vector pYES-DEST52 and transfected into the S. cerevisiae INVSc1 strain. Sequence analysis showed that the P. brutia CYP720B gene contains an open reading frame of 1,464 nucleotides, which encodes a 53,570 Da putative protein of 487 amino acid residues. The putative protein contains the classic heme-binding sequence motif that is conserved in all P450 enzymes. It shares 99 and 61% identity with the deduced amino acid sequences of CYP720B1 from Pinus taeda and CYP720B4 from Picea sitchensis, respectively. Recombinant CYP720B protein expression was confirmed using western blot analysis. Furthermore, recombinant CYP720B was functionally active, showing a Soret peak at approximately 448 nm in the reduced CO difference spectra. These data suggest that the cloned gene is an orthologue of CYP720B in P. brutia and might be involved in DRA biosynthesis.

Horizontal transfer of archaeal genes into the deinococcaceae: detection by molecular and computer-based approaches

NASA Technical Reports Server (NTRS)

Olendzenski, L.; Liu, L.; Zhaxybayeva, O.; Murphey, R.; Shin, D. G.; Gogarten, J. P.

2000-01-01

Members of the Deinococcaceae (e.g., Thermus, Meiothermus, Deinococcus) contain A/V-ATPases typically found in Archaea or Eukaryotes which were probably acquired by horizontal gene transfer. Two methods were used to quantify the extent to which archaeal or eukaryotic genes have been acquired by this lineage. Screening of a Meiothermus ruber library with probes made against Thermoplasma acidophilum DNA yielded a number of clones which hybridized more strongly than background. One of these contained the prolyl tRNA synthetase (RS) gene. Phylogenetic analysis shows the M. ruber and D. radiodurans prolyl RS to be more closely related to archaeal and eukaryal forms of this gene than to the typical bacterial type. Using a bioinformatics approach, putative open reading frames (ORFs) from the prerelease version of the D. radiodurans genome were screened for genes more closely related to archaeal or eukaryotic genes. Putative ORFs were searched against representative genomes from each of the three domains using automated BLAST. ORFs showing the highest matches against archaeal and eukaryotic genes were collected and ranked. Among the top-ranked hits were the A/V-ATPase catalytic and noncatalytic subunits and the prolyl RS genes. Using phylogenetic methods, ORFs were analyzed and trees assessed for evidence of horizontal gene transfer. Of the 45 genes examined, 20 showed topologies in which D. radiodurans homologues clearly group with eukaryotic or archaeal homologues, and 17 additional trees were found to show probable evidence of horizontal gene transfer. Compared to the total number of ORFs in the genome, those that can be identified as having been acquired from Archaea or Eukaryotes are relatively few (approximately 1%), suggesting that interdomain transfer is rare.

Molecular cloning and characterization of an acetylcholinesterase cDNA in the brown planthopper, Nilaparvata lugens.

PubMed

Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

2010-01-01

A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain.

A survey of endogenous retrovirus (ERV) sequences in the vicinity of multiple sclerosis (MS)-associated single nucleotide polymorphisms (SNPs).

PubMed

Brütting, Christine; Emmer, Alexander; Kornhuber, Malte; Staege, Martin S

2016-08-01

Although multiple sclerosis (MS) is one of the most common central nervous system diseases in young adults, little is known about its etiology. Several human endogenous retroviruses (ERVs) are considered to play a role in MS. We are interested in which ERVs can be identified in the vicinity of MS associated genetic marker to find potential initiators of MS. We analysed the chromosomal regions surrounding 58 single nucleotide polymorphisms (SNPs) that are associated with MS identified in one of the last major genome wide association studies. We scanned these regions for putative endogenous retrovirus sequences with large open reading frames (ORFs). We observed that more retrovirus-related putative ORFs exist in the relatively close vicinity of SNP marker indices in multiple sclerosis compared to control SNPs. We found very high homologies to HERV-K, HCML-ARV, XMRV, Galidia ERV, HERV-H/env62 and XMRV-like mouse endogenous retrovirus mERV-XL. The associated genes (CYP27B1, CD6, CD58, MPV17L2, IL12RB1, CXCR5, PTGER4, TAGAP, TYK2, ICAM3, CD86, GALC, GPR65 as well as the HLA DRB1*1501) are mainly involved in the immune system, but also in vitamin D regulation. The most frequently detected ERV sequences are related to the multiple sclerosis-associated retrovirus, the human immunodeficiency virus 1, HERV-K, and the Simian foamy virus. Our data shows that there is a relation between MS associated SNPs and the number of retroviral elements compared to control. Our data identifies new ERV sequences that have not been associated with MS, so far.

The temperate marine phage PhiHAP-1 of Halomonas aquamarina possesses a linear plasmid-like prophage genome.

PubMed

Mobberley, Jennifer M; Authement, R Nathan; Segall, Anca M; Paul, John H

2008-07-01

A myovirus-like temperate phage, PhiHAP-1, was induced with mitomycin C from a Halomonas aquamarina strain isolated from surface waters in the Gulf of Mexico. The induced cultures produced significantly more virus-like particles (VLPs) (3.73 x 10(10) VLP ml(-1)) than control cultures (3.83 x 10(7) VLP ml(-1)) when observed with epifluorescence microscopy. The induced phage was sequenced by using linker-amplified shotgun libraries and contained a genome 39,245 nucleotides in length with a G+C content of 59%. The PhiHAP-1 genome contained 46 putative open reading frames (ORFs), with 76% sharing significant similarity (E value of <10(-3)) at the protein level with other sequences in GenBank. Putative functional gene assignments included small and large terminase subunits, capsid and tail genes, an N6-DNA adenine methyltransferase, and lysogeny-related genes. Although no integrase was found, the PhiHAP-1 genome contained ORFs similar to protelomerase and parA genes found in linear plasmid-like phages with telomeric ends. Southern probing and PCR analysis of host genomic, plasmid, and PhiHAP-1 DNA indicated a lack of integration of the prophage with the host chromosome and a difference in genome arrangement between the prophage and virion forms. The linear plasmid prophage form of PhiHAP-1 begins with the protelomerase gene, presumably due to the activity of the protelomerase, while the induced phage particle has a circularly permuted genome that begins with the terminase genes. The PhiHAP-1 genome shares synteny and gene similarity with coliphage N15 and vibriophages VP882 and VHML, suggesting an evolutionary heritage from an N15-like linear plasmid prophage ancestor.

Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila.

PubMed

Lüneberg, E; Mayer, B; Daryab, N; Kooistra, O; Zähringer, U; Rohde, M; Swanson, J; Frosch, M

2001-03-01

We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.

Structural and functional studies of a family of Dictyostelium discoideum developmentally regulated, prestalk genes coding for small proteins.

PubMed

Vicente, Juan J; Galardi-Castilla, María; Escalante, Ricardo; Sastre, Leandro

2008-01-03

The social amoeba Dictyostelium discoideum executes a multicellular development program upon starvation. This morphogenetic process requires the differential regulation of a large number of genes and is coordinated by extracellular signals. The MADS-box transcription factor SrfA is required for several stages of development, including slug migration and spore terminal differentiation. Subtractive hybridization allowed the isolation of a gene, sigN (SrfA-induced gene N), that was dependent on the transcription factor SrfA for expression at the slug stage of development. Homology searches detected the existence of a large family of sigN-related genes in the Dictyostelium discoideum genome. The 13 most similar genes are grouped in two regions of chromosome 2 and have been named Group1 and Group2 sigN genes. The putative encoded proteins are 87-89 amino acids long. All these genes have a similar structure, composed of a first exon containing a 13 nucleotides long open reading frame and a second exon comprising the remaining of the putative coding region. The expression of these genes is induced at10 hours of development. Analyses of their promoter regions indicate that these genes are expressed in the prestalk region of developing structures. The addition of antibodies raised against SigN Group 2 proteins induced disintegration of multi-cellular structures at the mound stage of development. A large family of genes coding for small proteins has been identified in D. discoideum. Two groups of very similar genes from this family have been shown to be specifically expressed in prestalk cells during development. Functional studies using antibodies raised against Group 2 SigN proteins indicate that these genes could play a role during multicellular development.

Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

PubMed Central

Campos, Cristine G.; Borst, Luke

2013-01-01

Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3′ to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen. PMID:23340315

Characterization of BcaA, a putative classical autotransporter protein in Burkholderia pseudomallei.

PubMed

Campos, Cristine G; Borst, Luke; Cotter, Peggy A

2013-04-01

Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3' to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

The Oenococcus oeni clpX Homologue Is a Heat Shock Gene Preferentially Expressed in Exponential Growth Phase

PubMed Central

Jobin, Michel-Philippe; Garmyn, Dominique; Diviès, Charles; Guzzo, Jean

1999-01-01

Using degenerated primers from conserved regions of previously studied clpX gene products, we cloned the clpX gene of the malolactic bacterium Oenococcus oeni. The clpX gene was sequenced, and the deduced protein of 413 amino acids (predicted molecular mass of 45,650 Da) was highly similar to previously analyzed clpX gene products from other organisms. An open reading frame located upstream of the clpX gene was identified as the tig gene by similarity of its predicted product to other bacterial trigger factors. ClpX was purified by using a maltose binding protein fusion system and was shown to possess an ATPase activity. Northern analyses indicated the presence of two independent 1.6-kb monocistronic clpX and tig mRNAs and also showed an increase in clpX mRNA amount after a temperature shift from 30 to 42°C. The clpX transcript is abundant in the early exponential growth phase and progressively declines to undetectable levels in the stationary phase. Thus, unlike hsp18, the gene encoding one of the major small heat shock proteins of Oenococcus oeni, clpX expression is related to the exponential growth phase and requires de novo protein synthesis. Primer extension analysis identified the 5′ end of clpX mRNA which is located 408 nucleotides upstream of a putative AUA start codon. The putative transcription start site allowed identification of a predicted promoter sequence with a high similarity to the consensus sequence found in the housekeeping gene promoter of gram-positive bacteria as well as Escherichia coli. PMID:10542163

FmLC5, a putative galactose-binding C-type lectin with two QPD motifs from the hemocytes of Fenneropenaeus merguiensis participates in shrimp immune defense.

PubMed

Senghoi, Wilaiwan; Runsaeng, Phanthipha; Utarabhand, Prapaporn

2017-11-01

Crustaceans are deficient in adaptive immune system. They depend completely on an innate immunity to protect themselves from invading microorganisms. One kind of pattern recognition receptors that contribute roles in the innate immunity is lectin. A new C-type lectin gene designated as FmLC5 was isolated from Fenneropenaeus merguiensis. Its full-length cDNA is composed of 1526bp and one open reading frame of 852bp encoding a peptide of 284 amino acids. The deduced amino acid sequence of FmLC5 comprises a signal peptide of 20 contiguous amino acids with a molecular mass of 31.47kDa and an isoelectric point of 4.35. The primary structure of FmLC5 consists of two similar carbohydrate recognition domains (CRDs), each CRD contains a Ca 2+ binding site-2 and a QPD motif specific for galactose-binding. The FmLC5 transcripts were detected only in the hemocytes analyzed by RT-PCR and in situ hybridization. The FmLC5 expression was significantly up-regulated after challenge with Vibrio harveyi, white spot syndrome virus (WSSV) or lipopolysaccharide. RNAi-based silencing with co-injection by V. harveyi or WSSV resulted in critical suppression of the FmLC5 expression, increasing in mortality and reduction of the median lethal time. These results conclude that FmLC5 is unique putative galactose-binding C-type lectin in F. merguiensis that may contribute as receptor molecule in the immune response to defend the shrimp from pathogenic bacteria and viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in Enterococcus faecium DPC1146

PubMed Central

O’Keeffe, Triona; Hill, Colin; Ross, R. Paul

1999-01-01

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10,879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA, entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. Immediately downstream of the entT and entD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene of Bacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the control of a constitutive promoter resulted in heterologous enterocin A production in both E. faecalis and Lactococcus lactis. PMID:10103244

Complete genome sequence of lymphocystis disease virus isolated from China.

PubMed

Zhang, Qi-Ya; Xiao, Feng; Xie, Jian; Li, Zheng-Qiu; Gui, Jian-Fang

2004-07-01

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.

Complete Genome Sequence of Lymphocystis Disease Virus Isolated from China

PubMed Central

Zhang, Qi-Ya; Xiao, Feng; Xie, Jian; Li, Zheng-Qiu; Gui, Jian-Fang

2004-01-01

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1. PMID:15194775

78 FR 21120 - Registration Review; Pesticide Dockets Opened for Review and Comment and Other Docket Acts...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-09

... Constitution Ave. NW., Washington, DC 20460-0001. The Public Reading Room is open from 8:30 a.m. to 4:30 p.m., Monday through Friday, excluding legal holidays. The telephone number for the Public Reading Room is (202... the visitor instructions and additional information about the docket available at http://www.epa.gov...

Regulation of hTERT Expression and Function in Newly Immortalized p53(+) Human Mammary Epithelial Cell Lines

DTIC Science & Technology

2008-06-01

proteins during embryogene- sis, neurodevelopment and cancer. Part of their function is through the repression of CKIs, including p16. Some functions...protein 2 Other AUTS2 autism susceptibility candidate 2 C1orf24 chromosome 1 open reading frame 24 C20orf97 chromosome 20 open reading frame 97

Regulation of hTERT Expression and Function in Newly Immortalized p53(+) Human Mammary Epithelial Cell Lines

DTIC Science & Technology

2007-06-01

embryogene- sis, neurodevelopment and cancer. Part of their function is through the repression of CKIs, including p16. Some functions have been attributed to...AUTS2 autism susceptibility candidate 2 C1orf24 chromosome 1 open reading frame 24 C20orf97 chromosome 20 open reading frame 97 DKFZP566B183

Children's Literature in the Basals.

ERIC Educational Resources Information Center

O'Brien, Maureen A.

Three basal reading series, levels kindergarten through grade three, were studied to categorize the types of literature each contained. The following series were analyzed: "The Headway Program" (Open Court Publishing Company), "Series r Macmillan Reading," and "Basics in Reading" (Scott, Foresman and Company). It was…

Applying Research in Reading Instruction for Adults: First Steps for Teachers

ERIC Educational Resources Information Center

McShane, Susan

2005-01-01

Reading is the most basic of skills. Reading provides access to other skills and knowledge, facilitates life-long learning, and opens doors to opportunity. The National Institute for Literacy is authorized by the U.S. Congress to collect and disseminate information on the components of reading and the findings from scientific research. The…

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

PubMed

Ustav, M; Stenlund, A

1991-02-01

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.

Search strings for the study of putative occupational determinants of disease

PubMed Central

Mattioli, Stefano; Zanardi, Francesca; Baldasseroni, Alberto; Schaafsma, Frederieke; Cooke, Robin MT; Mancini, Gianpiero; Fierro, Mauro; Santangelo, Chiara; Farioli, Andrea; Fucksia, Serenella; Curti, Stefania; Verbeek, Jos

2010-01-01

Objective To identify efficient PubMed search strategies to retrieve articles regarding putative occupational determinants of conditions not generally considered to be work related. Methods Based on MeSH definitions and expert knowledge, we selected as candidate search terms the four MeSH terms describing ‘occupational disease’, ‘occupational exposure’, ‘occupational health’ and ‘occupational medicine’ (DEHM) alongside 22 other promising terms. We first explored overlaps between the candidate terms in PubMed. Using random samples of abstracts retrieved by each term, we estimated the proportions of articles containing potentially pertinent information regarding occupational aetiology in order to formulate two search strategies (one more ‘specific’, one more ‘sensitive’). We applied these strategies to retrieve information on the possible occupational aetiology of meningioma, pancreatitis and atrial fibrillation. Results Only 20.3% of abstracts were retrieved by more than one DEHM term. The more ‘specific’ search string was based on the combination of terms that yielded the highest proportion (40%) of potentially pertinent abstracts. The more ‘sensitive’ string was based on the use of broader search fields and additional coverage provided by other search terms under study. Using the specific string, the numbers of abstracts needed to read to find one potentially pertinent article were 1.2 for meningioma, 1.9 for pancreatitis and 1.8 for atrial fibrillation. Using the sensitive strategy, the numbers needed to read were 4.4 for meningioma, 8.9 for pancreatitis and 10.5 for atrial fibrillation. Conclusions The proposed strings could help health care professionals explore putative occupational aetiology for diseases that are not generally thought to be work related. PMID:19819858

Motivating Remedial Readers (Open to Suggestion).

ERIC Educational Resources Information Center

Glowacki, Joan

1990-01-01

Describes the effect on remedial reading students of engaging in reading activities with trainable mentally handicapped children. Reports that the students requested additional opportunities to share reading activities with the mentally handicapped students. Notes that students developed a Big Book that described their experiences during the…

Comprehensive Analysis of the Triterpenoid Saponins Biosynthetic Pathway in Anemone flaccida by Transcriptome and Proteome Profiling

PubMed Central

Zhan, Chuansong; Li, Xiaohua; Zhao, Zeying; Yang, Tewu; Wang, Xuekui; Luo, Biaobiao; Zhang, Qiyun; Hu, Yanru; Hu, Xuebo

2016-01-01

Background: Anemone flaccida Fr. Shmidt (Ranunculaceae), commonly known as ‘Di Wu’ in China, is a perennial herb with limited distribution. The rhizome of A. flaccida has long been used to treat arthritis as a tradition in China. Studies disclosed that the plant contains a rich source of triterpenoid saponins. However, little is known about triterpenoid saponins biosynthesis in A. flaccida. Results: In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from A. flaccida. Among the A. flaccida transcripts, 36,617 (77.97%) showed significant similarity (E-value < 1e-5) to the known proteins in the public database. Of the total 46,962 unigenes, 36,617 open reading frame (ORFs) were predicted. By the fragments per kilobases per million reads (FPKM) statistics, 14,004 isoforms/unigenes were found to be upregulated, and 14,090 isoforms/unigenes were down-regulated in the rhizomes as compared to those in the leaves. Based on the bioinformatics analysis, all possible enzymes involved in the triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA) and the plastidial methylerythritol pathway (MEP). Additionally, a total of 126 putative cytochrome P450 (CYP450) and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, four of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome analysis, as well as quantitative RT-PCR verification, the expression level of gene and protein committed to triterpenoids biosynthesis in the leaf versus the rhizome was compared. Conclusion: A combination of the de novo transcriptome and proteome profiling based on the Illumina HiSeq 2000 sequencing platform and iTRAQ technique was shown to be a powerful method for the discovery of candidate genes, which encoded enzymes that were responsible for the biosynthesis of novel secondary metabolites in a non-model plant. The transcriptome data of our study provides a very important resource for the understanding of the triterpenoid saponins biosynthesis of A. flaccida. PMID:27504115

Improving Reading Comprehension in Reading and Listening Settings: The Effect of Two Training Programmes Focusing on Metacognition and Working Memory

ERIC Educational Resources Information Center

Carretti, Barbara; Caldarola, Nadia; Tencati, Chiara; Cornoldi, Cesare

2014-01-01

Background: Metacognition and working memory (WM) have been found associated with success in reading comprehension, but no studies have examined their combined effect on the training of reading comprehension. Another open question concerns the role of listening comprehension: In particular, it is not clear whether training to improve reading…

Factors That Facilitate or Hinder the Use of Computer-Assisted Reading in the L2 Reading Classroom

ERIC Educational Resources Information Center

Al-Seghayer, Khalid

2016-01-01

This study examined factors associated with whether instructors of English as a second or foreign language (ESL/EFL) incorporate computer-assisted reading (CAR) into their second-language (L2) reading classrooms. To achieve this goal, 70 ESL/EFL instructors completed a survey containing 37 items and 1 open-ended question concerning their…

Cloning of oligopeptide transport carrier PepT1 and comparative analysis of PepT1 messenger ribonucleic acid expression in response to dietary nitrogen levels in yak () and indigenous cattle () on the Qinghai-Tibetan plateau.

PubMed

Wang, H C; Shi, F Y; Hou, M J; Fu, X Y; Long, R J

2016-08-01

The gastrointestinal lumen can directly absorb all di- and tripeptide protein degradation products, and oligopeptide absorption depends on the specific peptide transport carriers, which are located in gastrointestinal epithelial cells on the brush border membrane. Yak () use N more efficiently than cattle do, which implies that yak have a specific mechanism of nonprotein utilization including a peptide absorption mechanism. However, this mechanism has not been clarified. Our objective was to explore whether yak possess any adaptive mechanisms of peptide absorption to survive in the harsh foraging environment of the Qinghai-Tibetan plateau. Twelve castrated males of each of 2 genotypes, yak () and indigenous cattle (), were fed diets of various N levels. The yak PepT1 (yPepT1) cDNA was cloned in omasum epithelial tissue. Our results showed that the full-length yPepT1 cDNA contains 2,805 bp, and a 2,121-bp open reading frame encodes a putative protein of 707 AA residues. The yPepT1 AA sequence identified 5 putative extracellular N-glycosylation sites (Asn, Asn, Asn, Asn, and Asn), 2 putative intracellular protein kinase A sites (Ser and Thr), and 3 intracellular putative protein kinase C sites (Ser, Ser, and Ser). The yPepT1 AA sequence was 99, 95, 86, and 83% identical to PepT1 from cattle (), sheep (), pigs (), and humans (), respectively. The relative PepT1 mRNA expression for indigenous cattle was greater than yak in the rumen, omasum, duodenum, ileum, and liver ( < 0.001); however, it was lower in jejunum tissue ( < 0.01). The relative PepT1 mRNA expression in response to increasing dietary N for both genotypes were linear in the rumen and jejunum ( < 0.10); quadratic or cubic in the reticulum ( < 0.01); linear or quadratic in the duodenum, ileum, and liver ( ≤ 0.01); and linear, quadratic, or cubic in the omasum ( < 0.001). Moreover, there were significant interactions between genotype and dietary N in rumen, reticulum, omasum, duodenum, jejunum, ileum, and liver tissues. In conclusion, the PepT1 profile and expression in gastrointestinal epithelial cells of yak varied from those of cattle, implying that yak have evolved a peptide transport mechanism to adapt the environment of the Qinghai-Tibetan plateau.

Characterization of the Basic Replicon of Rhodococcus Plasmid pSOX and Development of a Rhodococcus-Escherichia coli Shuttle Vector†

PubMed Central

Denis-Larose, Claude; Bergeron, Hélène; Labbé, Diane; Greer, Charles W.; Hawari, Jalal; Grossman, Matthew J.; Sankey, Bruce M.; Lau, Peter C. K.

1998-01-01

The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS−, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose. PMID:9797291

A naturally occurring cowpox virus with an ectromelia virus A-type inclusion protein gene displays atypical A-type inclusions.

PubMed

Okeke, Malachy Ifeanyi; Hansen, Hilde; Traavik, Terje

2012-01-01

Human orthopoxvirus (OPV) infections in Europe are usually caused by cowpox virus (CPXV). The genetic heterogeneity of CPXVs may in part be due to recombination with other OPV species. We describe the characterization of an atypical CPXV (CPXV-No-H2) isolated from a human patient in Norway. CPXV-No-H2 was characterized on the basis of A-type inclusion (ATI) phenotype as well as the DNA region containing the p4c and atip open reading frames. CPXV-No-H2 produced atypical V(+/) ATI, in which virions are on the surface of ATI but not within the ATI matrix. Phylogenetic analysis showed that the atip gene of CPXV-No-H2 clustered closely with that of ectromelia virus (ECTV) with a bootstrap support of 100% whereas its p4c gene is diverged compared to homologues in other OPV species. By recombination analysis we identified a putative crossover event at nucleotide 147, downstream the start of the atip gene. Our results suggest that CPXV-No-H2 originated from a recombination between CPXV and ECTV. Our findings are relevant to the evolution of OPVs. Copyright © 2011 Elsevier B.V. All rights reserved.

Comparative analysis of prophages in Streptococcus mutans genomes

PubMed Central

Fu, Tiwei; Fan, Xiangyu; Long, Quanxin; Deng, Wanyan; Song, Jinlin

2017-01-01

Prophages have been considered genetic units that have an intimate association with novel phenotypic properties of bacterial hosts, such as pathogenicity and genomic variation. Little is known about the genetic information of prophages in the genome of Streptococcus mutans, a major pathogen of human dental caries. In this study, we identified 35 prophage-like elements in S. mutans genomes and performed a comparative genomic analysis. Comparative genomic and phylogenetic analyses of prophage sequences revealed that the prophages could be classified into three main large clusters: Cluster A, Cluster B, and Cluster C. The S. mutans prophages in each cluster were compared. The genomic sequences of phismuN66-1, phismuNLML9-1, and phismu24-1 all shared similarities with the previously reported S. mutans phages M102, M102AD, and ϕAPCM01. The genomes were organized into seven major gene clusters according to the putative functions of the predicted open reading frames: packaging and structural modules, integrase, host lysis modules, DNA replication/recombination modules, transcriptional regulatory modules, other protein modules, and hypothetical protein modules. Moreover, an integrase gene was only identified in phismuNLML9-1 prophages. PMID:29158986

Molecular cloning of pepsinogens A and C from adult newt (Cynops pyrrhogaster) stomach.

PubMed

Inokuchi, Tomofumi; Ikuzawa, Masayuki; Yamazaki, Shin; Watanabe, Yukari; Shiota, Koushiro; Katoh, Takuma; Kobayashi, Ken-Ichiro

2013-08-01

The full-length cDNAs of three pepsinogens (Pgs) were cloned from the stomach of newt, Cynops pyrrhogaster, and nucleotide sequences of the full-length cDNAs were determined. Molecular phylogenetic analysis showed that two Pgs, named PgC1 and PgC2, belong to the pepsinogen C group, and one Pg, named PgA, belongs to the pepsinogen A group. The sequences contain an open reading frame (ORF) encoding 385 amino acid residues for PgC1, 383 amino acid residues for PgC2 and 377 amino acid residues for PgA. In addition, all of the three amino acid sequences conserve some unique characteristics such as six cysteine residues and putative active site two aspartic acid residues. All of the pepsinogen mRNAs were detected in the stomach by RT-PCR but not in other organs. Although a slight difference at the time of the start of expression was seen among the three pepsinogen genes, all of them were expressed in the larval stage after hatching. This is the first report on cloning of pepsinogens from urodele stomach. Copyright © 2013 Elsevier Inc. All rights reserved.

A Bacteriophage-Related Chimeric Marine Virus Infecting Abalone

PubMed Central

Zhuang, Jun; Cai, Guiqin; Lin, Qiying; Wu, Zujian; Xie, Lianhui

2010-01-01

Marine viruses shape microbial communities with the most genetic diversity in the sea by multiple genetic exchanges and infect multiple marine organisms. Here we provide proof from experimental infection that abalone shriveling syndrome-associated virus (AbSV) can cause abalone shriveling syndrome. This malady produces histological necrosis and abnormally modified macromolecules (hemocyanin and ferritin). The AbSV genome is a 34.952-kilobase circular double-stranded DNA, containing putative genes with similarity to bacteriophages, eukaryotic viruses, bacteria and endosymbionts. Of the 28 predicted open reading frames (ORFs), eight ORF-encoded proteins have identifiable functional homologues. The 4 ORF products correspond to a predicted terminase large subunit and an endonuclease in bacteriophage, and both an integrase and an exonuclease from bacteria. The other four proteins are homologous to an endosymbiont-derived helicase, primase, single-stranded binding (SSB) protein, and thymidylate kinase, individually. Additionally, AbSV exhibits a common gene arrangement similar to the majority of bacteriophages. Unique to AbSV, the viral genome also contains genes associated with bacterial outer membrane proteins and may lack the structural protein-encoding ORFs. Genomic characterization of AbSV indicates that it may represent a transitional form of microbial evolution from viruses to bacteria. PMID:21079776

Purification, characterization, and cDNA cloning of a novel acidic endoglycoceramidase from the jellyfish, Cyanea nozakii.

PubMed

Horibata, Y; Okino, N; Ichinose, S; Omori, A; Ito, M

2000-10-06

Endoglycoceramidase (EC ) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here the purification, characterization, and cDNA cloning of a novel endoglycoceramidase from the jellyfish, Cyanea nozakii. The purified enzyme showed a single protein band estimated to be 51 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed a pH optimum of 3.0 and was activated by Triton X-100 and Lubrol PX but not by sodium taurodeoxycholate. This enzyme preferentially hydrolyzed gangliosides, especially GT1b and GQ1b, whereas neutral glycosphingolipids were somewhat resistant to hydrolysis by the enzyme. A full-length cDNA encoding the enzyme was cloned by 5'- and 3'-rapid amplification of cDNA ends using a partial amino acid sequence of the purified enzyme. The open reading frame of 1509 nucleotides encoded a polypeptide of 503 amino acids including a signal sequence of 25 residues and six potential N-glycosylation sites. Interestingly, the Asn-Glu-Pro sequence, which is the putative active site of Rhodococcus endoglycoceramidase, was conserved in the deduced amino acid sequences. This is the first report of the cloning of an endoglycoceramidase from a eukaryote.

Identification and Analysis of the Biosynthetic Gene Cluster Encoding the Thiopeptide Antibiotic Cyclothiazomycin in Streptomyces hygroscopicus 10-22▿ †

PubMed Central

Wang, Jiang; Yu, Yi; Tang, Kexuan; Liu, Wen; He, Xinyi; Huang, Xi; Deng, Zixin

2010-01-01

Thiopeptide antibiotics are an important class of natural products resulting from posttranslational modifications of ribosomally synthesized peptides. Cyclothiazomycin is a typical thiopeptide antibiotic that has a unique bridged macrocyclic structure derived from an 18-amino-acid structural peptide. Here we reported cloning, sequencing, and heterologous expression of the cyclothiazomycin biosynthetic gene cluster from Streptomyces hygroscopicus 10-22. Remarkably, successful heterologous expression of a 22.7-kb gene cluster in Streptomyces lividans 1326 suggested that there is a minimum set of 15 open reading frames that includes all of the functional genes required for cyclothiazomycin production. Six genes of these genes, cltBCDEFG flanking the structural gene cltA, were predicted to encode the enzymes required for the main framework of cyclothiazomycin, and two enzymes encoded by a putative operon, cltMN, were hypothesized to participate in the tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure. This rigorous bioinformatics analysis based on heterologous expression of cyclothiazomycin resulted in an ideal biosynthetic model for us to understand the biosynthesis of thiopeptides. PMID:20154110

cDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

1999-05-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

cDNA encoding a polypeptide including a hev ein sequence

DOEpatents

Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

2000-07-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

cDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

1999-05-04

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 12 figs.

CDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

1995-03-21

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

Molecular Characterization of a Catalase from Hydra vulgaris

PubMed Central

Dash, Bhagirathi; Phillips, Timothy D.

2012-01-01

Catalase, an antioxidant and hydroperoxidase enzyme protects the cellular environment from harmful effects of hydrogen peroxide by facilitating its degradation to oxygen and water. Molecular information on a cnidarian catalase and/or peroxidase is, however, limited. In this work an apparent full length cDNA sequence coding for a catalase (HvCatalase) was isolated from Hydra vulgaris using 3’- and 5’- (RLM) RACE approaches. The 1859 bp HvCatalase cDNA included an open reading frame of 1518 bp encoding a putative protein of 505 amino acids with a predicted molecular mass of 57.44 kDa. The deduced amino acid sequence of HvCatalase contained several highly conserved motifs including the heme-ligand signature sequence RLFSYGDTH and the active site signature FXRERIPERVVHAKGXGA. A comparative analysis showed the presence of conserved catalytic amino acids [His(71), Asn(145), and Tyr(354)] in HvCatalase as well. Homology modeling indicated the presence of the conserved features of mammalian catalase fold. Hydrae exposed to thermal, starvation, metal and oxidative stress responded by regulating its catalase mRNA transcription. These results indicated that the HvCatalase gene is involved in the cellular stress response and (anti)oxidative processes triggered by stressor and contaminant exposure. PMID:22521743

Polypeptide p41 of a Norwalk-Like Virus Is a Nucleic Acid-Independent Nucleoside Triphosphatase

PubMed Central

Pfister, Thomas; Wimmer, Eckard

2001-01-01

Southampton virus (SHV) is a member of the Norwalk-like viruses (NLVs), one of four genera of the family Caliciviridae. The genome of SHV contains three open reading frames (ORFs). ORF 1 encodes a polyprotein that is autocatalytically processed into six proteins, one of which is p41. p41 shares sequence motifs with protein 2C of picornaviruses and superfamily 3 helicases. We have expressed p41 of SHV in bacteria. Purified p41 exhibited nucleoside triphosphate (NTP)-binding and NTP hydrolysis activities. The NTPase activity was not stimulated by single-stranded nucleic acids. SHV p41 had no detectable helicase activity. Protein sequence comparison between the consensus sequences of NLV p41 and enterovirus protein 2C revealed regions of high similarity. According to secondary structure prediction, the conserved regions were located within a putative central domain of alpha helices and beta strands. This study reveals for the first time an NTPase activity associated with a calicivirus-encoded protein. Based on enzymatic properties and sequence information, a functional relationship between NLV p41 and enterovirus 2C is discussed in regard to the role of 2C-like proteins in virus replication. PMID:11160659

cDNA encoding a polypeptide including a hevein sequence

DOEpatents

Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

1995-03-21

A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 11 figures.

The Bradyrhizobium japonicum nolA gene and its involvement in the genotype-specific nodulation of soybeans.

PubMed Central

Sadowsky, M J; Cregan, P B; Gottfert, M; Sharma, A; Gerhold, D; Rodriguez-Quinones, F; Keyser, H H; Hennecke, H; Stacey, G

1991-01-01

Several soybean genotypes have been identified which specifically exclude nodulation by members of Bradyrhizobium japonicum serocluster 123. We have identified and sequenced a DNA region from B. japonicum strain USDA 110 which is involved in genotype-specific nodulation of soybeans. This 2.3-kilobase region, cloned in pMJS12, allows B. japonicum serocluster 123 isolates to form nodules on plants of serogroup 123-restricting genotypes. The nodules, however, were ineffective for symbiotic nitrogen fixation. The nodulation-complementing region is located approximately 590 base pairs transcriptionally downstream from nodD2. The 5' end of pMJS12 contains a putative open reading frame (ORF) of 710 base pairs, termed nolA. Transposon Tn3-HoHo mutations only within the ORF abolished nodulation complementation. The N terminus of the predicted nolA gene product has strong similarity with the N terminus of MerR, the regulator of mercury resistance genes. Translational lacZ fusion experiments indicated that nolA was moderately induced by soybean seed extract and the isoflavone genistein. Restriction fragments that hybridize to pMJS12 were detected in genomic DNAs from both nodulation-restricted and -unrestricted strains. PMID:1988958

Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T).

PubMed

Iwamoto, Kazuaki; Tsuruta, Hiroki; Nishitaini, Yosuke; Osawa, Ro

2008-09-01

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.

Discovery of sex-related genes through high-throughput transcriptome sequencing from the salmon louse Caligus rogercresseyi.

PubMed

Farlora, Rodolfo; Araya-Garay, José; Gallardo-Escárate, Cristian

2014-06-01

Understanding the molecular underpinnings involved in the reproduction of the salmon louse is critical for designing novel strategies of pest management for this ectoparasite. However, genomic information on sex-related genes is still limited. In the present work, sex-specific gene transcription was revealed in the salmon louse Caligus rogercresseyi using high-throughput Illumina sequencing. A total of 30,191,914 and 32,292,250 high quality reads were generated for females and males, and these were de novo assembled into 32,173 and 38,177 contigs, respectively. Gene ontology analysis showed a pattern of higher expression in the female as compared to the male transcriptome. Based on our sequence analysis and known sex-related proteins, several genes putatively involved in sex differentiation, including Dmrt3, FOXL2, VASA, and FEM1, and other potentially significant candidate genes in C. rogercresseyi, were identified for the first time. In addition, the occurrence of SNPs in several differentially expressed contigs annotating for sex-related genes was found. This transcriptome dataset provides a useful resource for future functional analyses, opening new opportunities for sea lice pest control. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of the Operon for the Sorbitol (Glucitol) Phosphoenolpyruvate:Sugar Phosphotransferase System in Streptococcus mutans

PubMed Central

Boyd, David A.; Thevenot, Tracy; Gumbmann, Markus; Honeyman, Allen L.; Hamilton, Ian R.

2000-01-01

Transposon mutagenesis and marker rescue were used to isolate and identify an 8.5-kb contiguous region containing six open reading frames constituting the operon for the sorbitol P-enolpyruvate phosphotransferase transport system (PTS) of Streptococcus mutans LT11. The first gene, srlD, codes for sorbitol-6-phosphate dehydrogenase, followed downstream by srlR, coding for a transcriptional regulator; srlM, coding for a putative activator; and the srlA, srlE, and srlB genes, coding for the EIIC, EIIBC, and EIIA components of the sorbitol PTS, respectively. Among all sorbitol PTS operons characterized to date, the srlD gene is found after the genes coding for the EII components; thus, the location of the gene in S. mutans is unique. The SrlR protein is similar to several transcriptional regulators found in Bacillus spp. that contain PTS regulator domains (J. Stülke, M. Arnaud, G. Rapoport, and I. Martin-Verstraete, Mol. Microbiol. 28:865–874, 1998), and its gene overlaps the srlM gene by 1 bp. The arrangement of these two regulatory genes is unique, having not been reported for other bacteria. PMID:10639465

Molecular characterization of a novel luteovirus infecting apple by next-generation sequencing.

PubMed

Shen, Pan; Tian, Xin; Zhang, Song; Ren, Fang; Li, Ping; Yu, Yun-Qi; Li, Ruhui; Zhou, Changyong; Cao, Mengji

2018-03-01

A new single-stranded positive-sense RNA virus, which shares the highest nucleotide (nt) sequence identity of 53.4% with the genome sequence of cherry-associated luteovirus South Korean isolate (ChALV-SK, genus Luteovirus), was discovered in this work. It is provisionally named apple-associated luteovirus (AaLV). The complete genome sequence of AaLV comprises 5,890 nt and contains eight open reading frames (ORFs), in a very similar arrangement that is typical of members of the genus Luteovirus. When compared with other members of the family Luteoviridae, ORF1 of AaLV was found to encompass another ORF, ORF1a, which encodes a putative 32.9-kDa protein. The ORF1-ORF2 region (RNA-dependent RNA polymerase, RdRP) showed the greatest amino acid (aa) sequence identity (59.7%) to that of cherry-associated luteovirus Czech Republic isolate (ChALV-CZ, genus Luteovirus). The results of genome sequence comparisons and phylogenetic analysis, suggest that AaLV should be a member of a novel species in the genus Luteovirus. To our knowledge, it is the sixth member of the genus Luteovirus reported to naturally infect rosaceous plants.

Genomic comparison of Escherichia coli K1 strains isolated from the cerebrospinal fluid of patients with meningitis.

PubMed

Yao, Yufeng; Xie, Yi; Kim, Kwang Sik

2006-04-01

Escherichia coli is a major cause of enteric/diarrheal diseases, urinary tract infections, and sepsis. E. coli K1 is the leading gram-negative organism causing neonatal meningitis, but the microbial basis of E. coli K1 meningitis is incompletely understood. Here we employed comparative genomic hybridization to investigate 11 strains of E. coli K1 isolated from the cerebrospinal fluid (CSF) of patients with meningitis. These 11 strains cover the majority of common O serotypes in E. coli K1 isolates from CSF. Our data demonstrated that these 11 strains of E. coli K1 can be categorized into two groups based on their profile for putative virulence factors, lipoproteins, proteases, and outer membrane proteins. Of interest, we showed that some open reading frames (ORFs) encoding the type III secretion system apparatus were found in group 2 strains but not in group 1 strains, while ORFs encoding the general secretory pathway are predominant in group 1 strains. These findings suggest that E. coli K1 strains isolated from CSF can be divided into two groups and these two groups of E. coli K1 may utilize different mechanisms to induce meningitis.

Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase.

PubMed

Naum-Onganía, Gabriela; Gago-Zachert, Selma; Peña, Eduardo; Grau, Oscar; Garcia, Maria Laura

2003-10-01

Citrus psorosis virus (CPsV), the type member of genus Ophiovirus, has three genomic RNAs. Complete sequencing of CPsV RNA 1 revealed a size of 8184 nucleotides and Northern blot hybridization with chain specific probes showed that its non-coding strand is preferentially encapsidated. The complementary strand of RNA 1 contains two open reading frames (ORFs) separated by a 109-nt intergenic region, one located near the 5'-end potentially encoding a 24K protein of unknown function, and another of 280K containing the core polymerase motifs characteristic of viral RNA-dependent RNA polymerases (RdRp). Comparison of the core RdRp motifs of negative-stranded RNA viruses, supports grouping CPsV, Ranunculus white mottle virus (RWMV) and Mirafiori lettuce virus (MiLV) within the same genus (Ophiovirus), constituting a monophyletic group separated from all other negative-stranded RNA viruses. Furthermore, RNAs 1 of MiLV, CPsV and RWMV are similar in size and those of MiLV and CPsV also in genomic organization and sequence.

Molecular anatomy of lymphocystis disease virus.

PubMed

Tidona, C A; Darai, G

1997-01-01

Lymphocystis disease (LD) has been reported to occur in over one hundred different species of fish worldwide. The disease is caused by lymphocystis disease virus (LCDV), a member of the iridovirus family. Numerous fish species that play an important role in fishery and fish farming are highly susceptible to LCDV infection. The infected animals develop disseminated clusters of aberrant hypertrophied cells within their connective tissue, the so-called lymphocystis cells. In the cytoplasm of these cells a massive accumulation of virions can be observed. As a first step towards understanding the mechanisms of viral infection and pathogenesis the complete genomic nucleotide sequence of lymphocystis disease virus type 1 (LCDV-1; flounder isolate) was determined. LCDV-1 is the type species of the genus Lymphocystivirus within the family Iridoviridae. The virions contain a single linear double-stranded DNA molecule that is circularly permuted, terminally redundant and heavily methylated. Since there is no convenient cell system for virus replication we determined the complete nucleotide sequence of the viral genome (102,653 base pairs). Computer assisted analyses of 195 potential open reading frames resulted in the identification of a number of putative gene products with significant homology to functionally characterized proteins of other species.

A novel monopartite dsRNA virus isolated from the phytopathogenic fungus Ustilaginoidea virens and ancestrally related to a mitochondria-associated dsRNA in the green alga Bryopsis.

PubMed

Zhang, Tingting; Jiang, Yinhui; Dong, Wubei

2014-08-01

In this study, we describe a novel mycovirus isolated from Ustilaginoidea virens, which was designated Ustilaginoidea virens nonsegmented virus 1 (UvNV-1). The sequence analysis revealed that UvNV-1 has two open reading frames (ORFs). ORF1 encodes an unknown protein, which is similar to the hypothetical protein BN7_5177 of Wickerhamomyces ciferrii. ORF2 encodes a putative RNA-dependent RNA polymerase (RdRp), which is most closely related to Bryopsis mitochondria-associated dsRNA (BDRM) and is likely expressed by a +1 ribosomal frameshift within the sequence CCC_UUU_CGA. The phylogenetic analysis of the RdRp of UvNV-1 showed that UvNV-1 represents a new virus taxon of mycoviruses with a partitivirus-like lineage that is classified into the family of picorna-like viruses. Based on northern hybridization, UvNV-1 was found to be common to U. virens from different geographic locations in China. The biological comparison of virus-free and infected fungal strains revealed that UvNV-1 is likely to be cryptic to its host. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

PubMed Central

Ustav, M; Stenlund, A

1991-01-01

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells. Images PMID:1846806

Single nucleotide polymorphism discovery in bovine liver using RNA-seq technology

PubMed Central

Pareek, Chandra Shekhar; Błaszczyk, Paweł; Dziuba, Piotr; Czarnik, Urszula; Fraser, Leyland; Sobiech, Przemysław; Pierzchała, Mariusz; Feng, Yaping; Kadarmideen, Haja N.; Kumar, Dibyendu

2017-01-01

Background RNA-seq is a useful next-generation sequencing (NGS) technology that has been widely used to understand mammalian transcriptome architecture and function. In this study, a breed-specific RNA-seq experiment was utilized to detect putative single nucleotide polymorphisms (SNPs) in liver tissue of young bulls of the Polish Red, Polish Holstein-Friesian (HF) and Hereford breeds, and to understand the genomic variation in the three cattle breeds that may reflect differences in production traits. Results The RNA-seq experiment on bovine liver produced 107,114,4072 raw paired-end reads, with an average of approximately 60 million paired-end reads per library. Breed-wise, a total of 345.06, 290.04 and 436.03 million paired-end reads were obtained from the Polish Red, Polish HF, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed that 81.35%, 82.81% and 84.21% of the mapped sequencing reads were properly paired to the Polish Red, Polish HF, and Hereford breeds, respectively. This study identified 5,641,401 SNPs and insertion and deletion (indel) positions expressed in the bovine liver with an average of 313,411 SNPs and indel per young bull. Following the removal of the indel mutations, a total of 195,3804, 152,7120 and 205,3184 raw SNPs expressed in bovine liver were identified for the Polish Red, Polish HF, and Hereford breeds, respectively. Breed-wise, three highly reliable breed-specific SNP-databases (SNP-dbs) with 31,562, 24,945 and 28,194 SNP records were constructed for the Polish Red, Polish HF, and Hereford breeds, respectively. Using a combination of stringent parameters of a minimum depth of ≥10 mapping reads that support the polymorphic nucleotide base and 100% SNP ratio, 4,368, 3,780 and 3,800 SNP records were detected in the Polish Red, Polish HF, and Hereford breeds, respectively. The SNP detections using RNA-seq data were successfully validated by kompetitive allele-specific PCR (KASPTM) SNP genotyping assay. The comprehensive QTL/CG analysis of 110 QTL/CG with RNA-seq data identified 20 monomorphic SNP hit loci (CARTPT, GAD1, GDF5, GHRH, GHRL, GRB10, IGFBPL1, IGFL1, LEP, LHX4, MC4R, MSTN, NKAIN1, PLAG1, POU1F1, SDR16C5, SH2B2, TOX, UCP3 and WNT10B) in all three cattle breeds. However, six SNP loci (CCSER1, GHR, KCNIP4, MTSS1, EGFR and NSMCE2) were identified as highly polymorphic among the cattle breeds. Conclusions This study identified breed-specific SNPs with greater SNP ratio and excellent mapping coverage, as well as monomorphic and highly polymorphic putative SNP loci within QTL/CGs of bovine liver tissue. A breed-specific SNP-db constructed for bovine liver yielded nearly six million SNPs. In addition, a KASPTM SNP genotyping assay, as a reliable cost-effective method, successfully validated the breed-specific putative SNPs originating from the RNA-seq experiments. PMID:28234981

SNP discovery by high-throughput sequencing in soybean

PubMed Central

2010-01-01

Background With the advance of new massively parallel genotyping technologies, quantitative trait loci (QTL) fine mapping and map-based cloning become more achievable in identifying genes for important and complex traits. Development of high-density genetic markers in the QTL regions of specific mapping populations is essential for fine-mapping and map-based cloning of economically important genes. Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation existing between any diverse genotypes that are usually used for QTL mapping studies. The massively parallel sequencing technologies (Roche GS/454, Illumina GA/Solexa, and ABI/SOLiD), have been widely applied to identify genome-wide sequence variations. However, it is still remains unclear whether sequence data at a low sequencing depth are enough to detect the variations existing in any QTL regions of interest in a crop genome, and how to prepare sequencing samples for a complex genome such as soybean. Therefore, with the aims of identifying SNP markers in a cost effective way for fine-mapping several QTL regions, and testing the validation rate of the putative SNPs predicted with Solexa short sequence reads at a low sequencing depth, we evaluated a pooled DNA fragment reduced representation library and SNP detection methods applied to short read sequences generated by Solexa high-throughput sequencing technology. Results A total of 39,022 putative SNPs were identified by the Illumina/Solexa sequencing system using a reduced representation DNA library of two parental lines of a mapping population. The validation rates of these putative SNPs predicted with low and high stringency were 72% and 85%, respectively. One hundred sixty four SNP markers resulted from the validation of putative SNPs and have been selectively chosen to target a known QTL, thereby increasing the marker density of the targeted region to one marker per 42 K bp. Conclusions We have demonstrated how to quickly identify large numbers of SNPs for fine mapping of QTL regions by applying massively parallel sequencing combined with genome complexity reduction techniques. This SNP discovery approach is more efficient for targeting multiple QTL regions in a same genetic population, which can be applied to other crops. PMID:20701770

Keeping It Simple: The Case for E-Mail.

ERIC Educational Resources Information Center

Haimovic, Gila

The Open University of Israel (OUI) is a distance education institution that offers over 250 computer-mediated courses through the Internet. All OUI students must pass an English reading comprehension exemption exam or take the University's English reading comprehension courses. Because reading instruction differs from content instruction,…

Viewing Reading Recovery as a Restructuring Phenomenon

ERIC Educational Resources Information Center

Rinehart, James S.; Short, Paula Myrick

2010-01-01

This study investigated components of Reading Recovery that relate to a restructuring paradigm. Specifically, Reading Recovery was analyzed as a way to redesign teachers' work, empower teachers, and affect the core technology of teaching. Data were collected by a survey that consisted of open-ended questions and of categorical response items.…

Reading with Love: Reading of Life Narrative of a Mother of a Child with Cerebral Palsy

ERIC Educational Resources Information Center

Mercieca, Daniela; Mercieca, Duncan P.

2014-01-01

This paper draws upon Deleuze and Guattari's ideas to suggest a different kind of reading of a narrative of a mother of a child with severe disability, and thus a different kind of ethical response to them. This reading gives readers the possibility of opening up experiences of parents and children with disability, rather than…

Comparing comprehension measured by multiple-choice and open-ended questions.

PubMed

Ozuru, Yasuhiro; Briner, Stephen; Kurby, Christopher A; McNamara, Danielle S

2013-09-01

This study compared the nature of text comprehension as measured by multiple-choice format and open-ended format questions. Participants read a short text while explaining preselected sentences. After reading the text, participants answered open-ended and multiple-choice versions of the same questions based on their memory of the text content. The results indicated that performance on open-ended questions was correlated with the quality of self-explanations, but performance on multiple-choice questions was correlated with the level of prior knowledge related to the text. These results suggest that open-ended and multiple-choice format questions measure different aspects of comprehension processes. The results are discussed in terms of dual process theories of text comprehension. PsycINFO Database Record (c) 2013 APA, all rights reserved

Open to Suggestion.

ERIC Educational Resources Information Center

Journal of Reading, 1981

1981-01-01

Contributors offer teaching ideas, including: using Sherlock Holmes mysteries to teach character and plot development, using materials supplied by the athletic coaches in the reading laboratory, and using individualized reading in the content areas. (AEA)

Frozen shoulder

MedlinePlus

... Changes in your hormones, such as during menopause Shoulder injury Shoulder surgery Open heart surgery Cervical disk disease ... and the A.D.A.M. Editorial team. Shoulder Injuries and Disorders Read more NIH MedlinePlus Magazine Read ...

The Right to Read: An Open Letter to the Citizens of Our Country from the National Council of Teachers of English.

ERIC Educational Resources Information Center

National Council of Teachers of English, Champaign, IL.

Democracy can exist only in a climate in which teachers are free to teach and students are free to learn, a climate conducive to open inquiry and responsible discussion of any and all questions related to the ethical and cultural welfare of mankind. The right of an individual to read rests on the fundamental democratic assumption that an educated…

Reading Quizzes Improve Exam Scores for Community College Students

ERIC Educational Resources Information Center

Pape-Lindstrom, Pamela; Eddy, Sarah; Freeman, Scott

2018-01-01

To test the hypothesis that adding course structure may encourage self-regulated learning skills resulting in an increase in student exam performance in the community college setting, we added daily preclass online, open-book reading quizzes to an introductory biology course. We compared three control terms without reading quizzes and three…

Tobacco chloroplast tRNALys(UUU) gene contains a 2.5-kilobase-pair intron: An open reading frame and a conserved boundary sequence in the intron

PubMed Central

Sugita, Mamoru; Shinozaki, Kazuo; Sugiura, Masahiro

1985-01-01

The nucleotide sequence of a tRNALys(UUU) gene on tobacco (Nicotiana tabacum) chloroplast DNA has been determined. This gene is located 215 base pairs upstream from the gene for the 32,000-dalton thylakoid membrane protein on the same DNA strand and has a 2526-base-pair intron in the anticodon loop. The intron boundary sequence does not follow the G-U/A-G rule but is similar to those of tobacco chloroplast split genes for tRNAGly(UCC) and ribosomal proteins L2 and S12. The intron contains one major open reading frame of 509 codons. The codon usage in the open reading frame resembles those observed in the genes for tobacco chloroplast proteins so far analyzed. The primary transcript of this tRNA gene is 2.7 kilobases long. Images PMID:16593561

Tobacco chloroplast tRNA(UUU) gene contains a 2.5-kilobase-pair intron: An open reading frame and a conserved boundary sequence in the intron.

PubMed

Sugita, M; Shinozaki, K; Sugiura, M

1985-06-01

The nucleotide sequence of a tRNA(Lys)(UUU) gene on tobacco (Nicotiana tabacum) chloroplast DNA has been determined. This gene is located 215 base pairs upstream from the gene for the 32,000-dalton thylakoid membrane protein on the same DNA strand and has a 2526-base-pair intron in the anticodon loop. The intron boundary sequence does not follow the G-U/A-G rule but is similar to those of tobacco chloroplast split genes for tRNA(Gly)(UCC) and ribosomal proteins L2 and S12. The intron contains one major open reading frame of 509 codons. The codon usage in the open reading frame resembles those observed in the genes for tobacco chloroplast proteins so far analyzed. The primary transcript of this tRNA gene is 2.7 kilobases long.

Production of Candida antaractica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell & Expression in Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production

USDA-ARS?s Scientific Manuscript database

A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was produced using an automated PCR assembly and DNA purification protocol on an integrated robotic workcell. The lycotoxin-1 (Lyt-1) C3 variant gene ORF was added in-frame with the CALB ORF to pote...

Measuring College Students' Reading Comprehension Ability Using Cloze Tests

ERIC Educational Resources Information Center

Williams, Rihana Shiri; Ari, Omer; Santamaria, Carmen Nicole

2011-01-01

Recent investigations challenge the construct validity of sustained silent reading tests. Performance of two groups of post-secondary students (e.g. struggling and non-struggling) on a sustained silent reading test and two types of cloze test (i.e. maze and open-ended) was compared in order to identify the test format that contributes greater…

Rose City Reading: Towards an Open Educational Resource with a Place-Based Curriculum

ERIC Educational Resources Information Center

Twenge-Jinings, Fidelia; Sullivan, Joanna

2016-01-01

Realizing that ESOL students often do not integrate naturally into their new communities, we developed a reading class that focuses only on topics such as history, culture, arts, nature and entertainment that pertain to Portland or Oregon. In addition to readings, we asked that students go out and "live" what they learned through a…

An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani

PubMed Central

Das, Aditi; Mandal, Chhabinath; Dasgupta, Arindam; Sengupta, Tanushri; Majumder, Hemanta K.

2002-01-01

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes. PMID:11809893

Oximetry in children recovering from deep hypothermia for cardiac surgery.

PubMed

Macnab, A J; Baker-Brown, G; Anderson, E E

1990-10-01

Although pulse oximetry is a potentially useful diagnostic tool in the treatment of children after major open heart surgery, there are concerns regarding its reliability for measuring oxygen saturation (SaO2) in hypothermic or low perfusion states. To test pulse oximeter reliability in children under these conditions, our study compared 187 SaO2 pulse oximeter readings (Biox 3700) with simultaneous hemoximeter (OSM2, Radiometer) readings from 56 children rewarming after open heart surgery. Ages ranged from 4 months to 18 yr; temperatures ranged from 23.5 degrees to 38 degrees C (toe) and 31.3 degrees to 40.8 degrees C (core). The mean pulse oximeter SaO2 reading was 94.90% (SD 7.18, range 54% to 100%), mean hemoximeter reading was 96.07% (SD 7.06; minimum 54%; maximum 100%). The correlation between the readings was high (r = .88, p less than .005), and was not affected by low core temperature. When oximeter and cardiac monitor pulse rates coincided, the oximeter SaO2 value was within +/- 5% (p less than .05). We conclude that the Biox 3700 oximeter is reliable for noninvasive SaO2 monitoring in mild to moderately hypothermic children after open heart surgery, particularly when oximeter and cardiac heart rates coincide. Further studies are needed to confirm our findings in children with core temperatures less than 31.3 degrees C, and when other oximeters are used.

Federal Reading Rooms for Risk Management Plans (RMP)

EPA Pesticide Factsheets

Reading Rooms, listed here by state, are open to the public by either appointment or walk-in. You may access Off-Site Consequence Analysis (scenarios) portions of RMPs, and take notes but not remove or reproduce materials.

The activated sludge ecosystem contains a core community of abundant organisms

PubMed Central

Saunders, Aaron M; Albertsen, Mads; Vollertsen, Jes; Nielsen, Per H

2016-01-01

Understanding the microbial ecology of a system requires that the observed population dynamics can be linked to their metabolic functions. However, functional characterization is laborious and the choice of organisms should be prioritized to those that are frequently abundant (core) or transiently abundant, which are therefore putatively make the greatest contribution to carbon turnover in the system. We analyzed the microbial communities in 13 Danish wastewater treatment plants with nutrient removal in consecutive years and a single plant periodically over 6 years, using Illumina sequencing of 16S ribosomal RNA amplicons of the V4 region. The plants contained a core community of 63 abundant genus-level operational taxonomic units (OTUs) that made up 68% of the total reads. A core community consisting of abundant OTUs was also observed within the incoming wastewater to three plants. The net growth rate for individual OTUs was quantified using mass balance, and it was found that 10% of the total reads in the activated sludge were from slow or non-growing OTUs, and that their measured abundance was primarily because of immigration with the wastewater. Transiently abundant organisms were also identified. Among them the genus Nitrotoga (class Betaproteobacteria) was the most abundant putative nitrite oxidizer in a number of activated sludge plants, which challenges previous assumptions that Nitrospira (phylum Nitrospirae) are the primary nitrite-oxidizers in activated sludge systems with nutrient removal. PMID:26262816

Identification of Putative Chemosensory Receptor Genes from the Athetis dissimilis Antennal Transcriptome

PubMed Central

Dong, Junfeng; Song, Yueqin; Li, Wenliang; Shi, Jie; Wang, Zhenying

2016-01-01

Olfaction plays a crucial role in insect population survival and reproduction. Identification of the genes associated with the olfactory system, without the doubt will promote studying the insect chemical communication system. In this study, RNA-seq technology was used to sequence the antennae transcriptome of Athetis dissimilis, an emerging crop pest in China with limited genomic information, with the purpose of identifying the gene set involved in olfactory recognition. Analysis of the transcriptome of female and male antennae generated 13.74 Gb clean reads in total from which 98,001 unigenes were assembled, and 25,930 unigenes were annotated. Total of 60 olfactory receptors (ORs), 18 gustatory receptors (GRs), and 12 ionotropic receptors (IRs) were identified by Blast and sequence similarity analyzes. One obligated olfactory receptor co-receptor (Orco) and four conserved sex pheromone receptors (PRs) were annotated in 60 ORs. Among the putative GRs, five genes (AdisGR1, 6, 7, 8 and 94) clustered in the sugar receptor family, and two genes (AdisGR3 and 93) involved in CO2 detection were identified. Finally, AdisIR8a.1 and AdisIR8a.2 co-receptors were identified in the group of candidate IRs. Furthermore, expression levels of these chemosensory receptor genes in female and male antennae were analyzed by mapping the Illumina reads. PMID:26812239

Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures[OPEN

PubMed Central

Zhu, Bo; Zhang, Wenli; Jiang, Jiming

2015-01-01

Enhancers are important regulators of gene expression in eukaryotes. Enhancers function independently of their distance and orientation to the promoters of target genes. Thus, enhancers have been difficult to identify. Only a few enhancers, especially distant intergenic enhancers, have been identified in plants. We developed an enhancer prediction system based exclusively on the DNase I hypersensitive sites (DHSs) in the Arabidopsis thaliana genome. A set of 10,044 DHSs located in intergenic regions, which are away from any gene promoters, were predicted to be putative enhancers. We examined the functions of 14 predicted enhancers using the β-glucuronidase gene reporter. Ten of the 14 (71%) candidates were validated by the reporter assay. We also designed 10 constructs using intergenic sequences that are not associated with DHSs, and none of these constructs showed enhancer activities in reporter assays. In addition, the tissue specificity of the putative enhancers can be precisely predicted based on DNase I hypersensitivity data sets developed from different plant tissues. These results suggest that the open chromatin signature-based enhancer prediction system developed in Arabidopsis may serve as a universal system for enhancer identification in plants. PMID:26373455

Test Performance Vis a Vis Test-Taking Strategies in Reading Comprehension of English as a Second Language.

ERIC Educational Resources Information Center

Roizen, Merle A.

A study of students' self-expressed test-taking strategies for reading comprehension tests in English as a second language (ESL) had as subjects 86 Hebrew-speaking university students in Israel. The students were given a reading comprehension test, half with open-ended questions and half with multiple choice. Half the responses were anonymous and…

The Stickybear Reading Comprehension Series: Science. School Version with Lesson Plans for Ages 7-10. Volume 1. Stickybear Software.

ERIC Educational Resources Information Center

1996

This software product presents multi-level stories to capture the interest of children in grades two through five, while teaching them crucial reading comprehension skills. With stories touching on everything from the invention of velcro to the journey of food through the digestive system, the open-ended reading comprehension program is versatile…

Processing and Memory of Central versus Peripheral Information as a Function of Reading Goals: Evidence from Eye-Movements

ERIC Educational Resources Information Center

Yeari, Menahem; van den Broek, Paul; Oudega, Marja

2015-01-01

The present study examined the effect of reading goals on the processing and memory of central and peripheral textual information. Using eye-tracking methodology, we compared the effect of four common reading goals--entertainment, presentation, studying for a close-ended (multiple-choice) questions test, and studying for an open-ended questions…

Rhythm, Rhyme, and Reason; Proceedings of the Annual Reading Conference (9th, Terre Haute, Indiana, June 14, 1979).

ERIC Educational Resources Information Center

Waterman, David C., Comp.; Gibbs, Vanita M., Comp.

Part of a series on selected aspects of curriculum development, this monograph contains reading conference proceedings that include an opening address by author Marguerite Henry in which she shares personal experiences in her evolution as a writer and six papers on remedial reading and language arts. The first paper describes the neurological…

Multidimensional Aspects of Literacy Research, Theory, and Practice. Forty-Third Yearbook of the National Reading Conference.

ERIC Educational Resources Information Center

Kinzer, Charles K., Ed.; Leu, Donald J., Ed.

The 55 conference papers, the annual review of research, and the student-award-winning paper in this yearbook cover such diverse topics in the field of reading as assessment, content-area reading, early literacy, teacher education, teacher behavior, and construction of meaning through writing. The yearbook opens with the presidential address to…

Single Nucleotide Polymorphism Discovery in Bovine Pituitary Gland Using RNA-Seq Technology

PubMed Central

Pareek, Chandra Shekhar; Smoczyński, Rafał; Kadarmideen, Haja N.; Dziuba, Piotr; Błaszczyk, Paweł; Sikora, Marcin; Walendzik, Paulina; Grzybowski, Tomasz; Pierzchała, Mariusz; Horbańczuk, Jarosław; Szostak, Agnieszka; Ogluszka, Magdalena; Zwierzchowski, Lech; Czarnik, Urszula; Fraser, Leyland; Sobiech, Przemysław; Wąsowicz, Krzysztof; Gelfand, Brian; Feng, Yaping; Kumar, Dibyendu

2016-01-01

Examination of bovine pituitary gland transcriptome by strand-specific RNA-seq allows detection of putative single nucleotide polymorphisms (SNPs) within potential candidate genes (CGs) or QTLs regions as well as to understand the genomics variations that contribute to economic trait. Here we report a breed-specific model to successfully perform the detection of SNPs in the pituitary gland of young growing bulls representing Polish Holstein-Friesian (HF), Polish Red, and Hereford breeds at three developmental ages viz., six months, nine months, and twelve months. A total of 18 bovine pituitary gland polyA transcriptome libraries were prepared and sequenced using the Illumina NextSeq 500 platform. Sequenced FastQ databases of all 18 young bulls were submitted to NCBI-SRA database with NCBI-SRA accession numbers SRS1296732. For the investigated young bulls, a total of 113,882,3098 raw paired-end reads with a length of 156 bases were obtained, resulting in an approximately 63 million paired-end reads per library. Breed-wise, a total of 515.38, 215.39, and 408.04 million paired-end reads were obtained for Polish HF, Polish Red, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed 93.04%, 94.39%, and 83.46% of the mapped sequencing reads were properly paired to the Polish HF, Polish Red, and Hereford breeds, respectively. Constructed breed-specific SNP-db of three cattle breeds yielded at 13,775,885 SNPs. On an average 765,326 breed-specific SNPs per young bull were identified. Using two stringent filtering parameters, i.e., a minimum 10 SNP reads per base with an accuracy ≥ 90% and a minimum 10 SNP reads per base with an accuracy = 100%, SNP-db records were trimmed to construct a highly reliable SNP-db. This resulted in a reduction of 95,7% and 96,4% cut-off mark of constructed raw SNP-db. Finally, SNP discoveries using RNA-Seq data were validated by KASP™ SNP genotyping assay. The comprehensive QTLs/CGs analysis of 76 QTLs/CGs with RNA-seq data identified KCNIP4, CCSER1, DPP6, MAP3K5 and GHR CGs with highest SNPs hit loci in all three breeds and developmental ages. However, CAST CG with more than 100 SNPs hits were observed only in Polish HF and Hereford breeds.These findings are important for identification and construction of novel tissue specific SNP-db and breed specific SNP-db dataset by screening of putative SNPs according to QTL db and candidate genes for bovine growth and reproduction traits, one can develop genomic selection strategies for growth and reproductive traits. PMID:27606429

Single Nucleotide Polymorphism Discovery in Bovine Pituitary Gland Using RNA-Seq Technology.

PubMed

Pareek, Chandra Shekhar; Smoczyński, Rafał; Kadarmideen, Haja N; Dziuba, Piotr; Błaszczyk, Paweł; Sikora, Marcin; Walendzik, Paulina; Grzybowski, Tomasz; Pierzchała, Mariusz; Horbańczuk, Jarosław; Szostak, Agnieszka; Ogluszka, Magdalena; Zwierzchowski, Lech; Czarnik, Urszula; Fraser, Leyland; Sobiech, Przemysław; Wąsowicz, Krzysztof; Gelfand, Brian; Feng, Yaping; Kumar, Dibyendu

2016-01-01

Examination of bovine pituitary gland transcriptome by strand-specific RNA-seq allows detection of putative single nucleotide polymorphisms (SNPs) within potential candidate genes (CGs) or QTLs regions as well as to understand the genomics variations that contribute to economic trait. Here we report a breed-specific model to successfully perform the detection of SNPs in the pituitary gland of young growing bulls representing Polish Holstein-Friesian (HF), Polish Red, and Hereford breeds at three developmental ages viz., six months, nine months, and twelve months. A total of 18 bovine pituitary gland polyA transcriptome libraries were prepared and sequenced using the Illumina NextSeq 500 platform. Sequenced FastQ databases of all 18 young bulls were submitted to NCBI-SRA database with NCBI-SRA accession numbers SRS1296732. For the investigated young bulls, a total of 113,882,3098 raw paired-end reads with a length of 156 bases were obtained, resulting in an approximately 63 million paired-end reads per library. Breed-wise, a total of 515.38, 215.39, and 408.04 million paired-end reads were obtained for Polish HF, Polish Red, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed 93.04%, 94.39%, and 83.46% of the mapped sequencing reads were properly paired to the Polish HF, Polish Red, and Hereford breeds, respectively. Constructed breed-specific SNP-db of three cattle breeds yielded at 13,775,885 SNPs. On an average 765,326 breed-specific SNPs per young bull were identified. Using two stringent filtering parameters, i.e., a minimum 10 SNP reads per base with an accuracy ≥ 90% and a minimum 10 SNP reads per base with an accuracy = 100%, SNP-db records were trimmed to construct a highly reliable SNP-db. This resulted in a reduction of 95,7% and 96,4% cut-off mark of constructed raw SNP-db. Finally, SNP discoveries using RNA-Seq data were validated by KASP™ SNP genotyping assay. The comprehensive QTLs/CGs analysis of 76 QTLs/CGs with RNA-seq data identified KCNIP4, CCSER1, DPP6, MAP3K5 and GHR CGs with highest SNPs hit loci in all three breeds and developmental ages. However, CAST CG with more than 100 SNPs hits were observed only in Polish HF and Hereford breeds.These findings are important for identification and construction of novel tissue specific SNP-db and breed specific SNP-db dataset by screening of putative SNPs according to QTL db and candidate genes for bovine growth and reproduction traits, one can develop genomic selection strategies for growth and reproductive traits.

Open Admissions: A Bibliography for Research and Application.

ERIC Educational Resources Information Center

Shrier, Irene; Lavin, David E.

This bibliography presents materials for research and application of open admissions policies in higher education. Sections cover: open admissions; factors influencing high school graduates to attend college; disadvantaged and minority students; precollege and special programs; English and reading skills; general compensatory programs; dropouts;…

A Pilot Comparison of 18F-fluorocholine PET/CT, Ultrasonography and 123I/99mTc-sestaMIBI Dual-Phase Dual-Isotope Scintigraphy in the Preoperative Localization of Hyperfunctioning Parathyroid Glands in Primary or Secondary Hyperparathyroidism

PubMed Central

Michaud, Laure; Balogova, Sona; Burgess, Alice; Ohnona, Jessica; Huchet, Virginie; Kerrou, Khaldoun; Lefèvre, Marine; Tassart, Marc; Montravers, Françoise; Périé, Sophie; Talbot, Jean-Noël

2015-01-01

Abstract We compared 18F-fluorocholine hybrid positron emission tomography/X-ray computed tomography (FCH-PET/CT) with ultrasonography (US) and scintigraphy in patients with hyperparathyroidism and discordant, or equivocal results of US and 123I/99mTc-sesta-methoxyisobutylisonitrile (sestaMIBI) dual-phase parathyroid scintigraphy. FCH-PET/CT was performed in 17 patients with primary (n = 11) lithium induced (n = 1) or secondary hyperparathyroidism (1 dialyzed, 4 renal-transplanted). The reference standard was based on results of surgical exploration and histopathological examination. The results of imaging modalities were evaluated, on site and by masked reading, on per-patient and per-lesion bases. In a first approach, equivocal images/foci were considered as negative. On a per-patient level, the sensitivity was for US 38%, for scintigraphy 69% by open and 94% by masked reading, and for FCH-PET/CT 88% by open and 94% by masked reading. On a per-lesion level, sensitivity was for US 42%, for scintigraphy 58% by open and 83% by masked reading, and for FCH-PET/CT 88% by open and 96% by masked reading. One ectopic adenoma was missed by the 3 imaging modalities. Considering equivocal images/foci as positive increased the accuracy of the open reading of scintigraphy or of FCH-PET/CT, but not of US. FCH-PET/CT was significantly superior to US in all approaches, whereas it was more sensitive than scintigraphy only for open reading considering equivocal images/foci as negative (P = 0.04). FCH uptake was more intense in adenomas than in hyperplastic parathyroid glands. Thyroid lesions were suspected in 9 patients. They may induce false-positive results as in one case of oncocytic thyroid adenoma, or false-negative results as in one case of intrathyroidal parathyroid adenoma. Thyroid cancer (4 cases) can be visualized with FCH as with 99mTc-sestaMIBI, but the intensity of uptake was moderate, similar to that of parathyroid hyperplasia. This pilot study confirmed that FCH-PET/CT is an adequate imaging tool in patients with primary or secondary hyperparathyroidism, since both adenomas and hyperplastic parathyroid glands can be detected. The sensitivity of FCH-PET/CT was better than that of US and was not inferior to that of dual-phase dual-isotope 123I/99mTc-scintigraphy. Further studies should evaluate whether FCH could replace 99mTc-sestaMIBI as the functional agent for parathyroid imaging, but US would still be useful to identify thyroid lesions. PMID:26469908

Transposition of an intron in yeast mitochondria requires a protein encoded by that intron.

PubMed

Macreadie, I G; Scott, R M; Zinn, A R; Butow, R A

1985-06-01

The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.

Protein expression of preferred human codon-optimized Gaussia luciferase genes with an artificial open-reading frame in mammalian and bacterial cells.

PubMed

Inouye, Satoshi; Suzuki, Takahiro

2016-12-01

The protein expressions of three preferred human codon-optimized Gaussia luciferase genes (pGLuc, EpGLuc, and KpGLuc) were characterized in mammalian and bacterial cells by comparing them with those of wild-type Gaussia luciferase gene (wGLuc) and human codon-optimized Gaussia luciferase gene (hGLuc). Two synthetic genes of EpGLuc and KpGLuc containing the complete preferred human codons have an artificial open-reading frame; however, they had the similar protein expression levels to those of pGLuc and hGLuc in mammalian cells. In bacterial cells, the protein expressions of pGLuc, EpGLuc, and KpGLuc with approximately 65% GC content were the same and showed approximately 60% activities of wGLuc and hGLuc. The artificial open-reading frame in EpGLuc and KpGLuc did not affect the protein expression in mammalian and bacterial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Transcriptomic analysis of the interaction between Helianthus annuus and its obligate parasite Plasmopara halstedii shows single nucleotide polymorphisms in CRN sequences.

PubMed

As-sadi, Falah; Carrere, Sébastien; Gascuel, Quentin; Hourlier, Thibaut; Rengel, David; Le Paslier, Marie-Christine; Bordat, Amandine; Boniface, Marie-Claude; Brunel, Dominique; Gouzy, Jérôme; Godiard, Laurence; Vincourt, Patrick

2011-10-11

Downy mildew in sunflowers (Helianthus annuus L.) is caused by the oomycete Plasmopara halstedii (Farl.) Berlese et de Toni. Despite efforts by the international community to breed mildew-resistant varieties, downy mildew remains a major threat to the sunflower crop. Very few genomic, genetic and molecular resources are currently available to study this pathogen. Using a 454 sequencing method, expressed sequence tags (EST) during the interaction between H. annuus and P. halstedii have been generated and a search was performed for sites in putative effectors to show polymorphisms between the different races of P. halstedii. A 454 pyrosequencing run of two infected sunflower samples (inbred lines XRQ and PSC8 infected with race 710 of P. halstedii, which exhibit incompatible and compatible interactions, respectively) generated 113,720 and 172,107 useable reads. From these reads, 44,948 contigs and singletons have been produced. A bioinformatic portal, HP, was specifically created for in-depth analysis of these clusters. Using in silico filtering, 405 clusters were defined as being specific to oomycetes, and 172 were defined as non-specific oomycete clusters. A subset of these two categories was checked using PCR amplification, and 86% of the tested clusters were validated. Twenty putative RXLR and CRN effectors were detected using PSI-BLAST. Using corresponding sequences from four races (100, 304, 703 and 710), 22 SNPs were detected, providing new information on pathogen polymorphisms. This study identified a large number of genes that are expressed during H. annuus/P. halstedii compatible or incompatible interactions. It also reveals, for the first time, that an infection mechanism exists in P. halstedii similar to that in other oomycetes associated with the presence of putative RXLR and CRN effectors. SNPs discovered in CRN effector sequences were used to determine the genetic distances between the four races of P. halstedii. This work therefore provides valuable tools for further discoveries regarding the H. annuus/P. halstedii pathosystem.

Transcriptomic analysis of the interaction between Helianthus annuus and its obligate parasite Plasmopara halstedii shows single nucleotide polymorphisms in CRN sequences

PubMed Central

2011-01-01

Background Downy mildew in sunflowers (Helianthus annuus L.) is caused by the oomycete Plasmopara halstedii (Farl.) Berlese et de Toni. Despite efforts by the international community to breed mildew-resistant varieties, downy mildew remains a major threat to the sunflower crop. Very few genomic, genetic and molecular resources are currently available to study this pathogen. Using a 454 sequencing method, expressed sequence tags (EST) during the interaction between H. annuus and P. halstedii have been generated and a search was performed for sites in putative effectors to show polymorphisms between the different races of P. halstedii. Results A 454 pyrosequencing run of two infected sunflower samples (inbred lines XRQ and PSC8 infected with race 710 of P. halstedii, which exhibit incompatible and compatible interactions, respectively) generated 113,720 and 172,107 useable reads. From these reads, 44,948 contigs and singletons have been produced. A bioinformatic portal, HP, was specifically created for in-depth analysis of these clusters. Using in silico filtering, 405 clusters were defined as being specific to oomycetes, and 172 were defined as non-specific oomycete clusters. A subset of these two categories was checked using PCR amplification, and 86% of the tested clusters were validated. Twenty putative RXLR and CRN effectors were detected using PSI-BLAST. Using corresponding sequences from four races (100, 304, 703 and 710), 22 SNPs were detected, providing new information on pathogen polymorphisms. Conclusions This study identified a large number of genes that are expressed during H. annuus/P. halstedii compatible or incompatible interactions. It also reveals, for the first time, that an infection mechanism exists in P. halstedii similar to that in other oomycetes associated with the presence of putative RXLR and CRN effectors. SNPs discovered in CRN effector sequences were used to determine the genetic distances between the four races of P. halstedii. This work therefore provides valuable tools for further discoveries regarding the H. annuus/P. halstedii pathosystem. PMID:21988821

Deep Sequencing-Based Analysis of the Cymbidium ensifolium Floral Transcriptome

PubMed Central

Li, Xiaobai; Luo, Jie; Yan, Tianlian; Xiang, Lin; Jin, Feng; Qin, Dehui; Sun, Chongbo; Xie, Ming

2013-01-01

Cymbidium ensifolium is a Chinese Cymbidium with an elegant shape, beautiful appearance, and a fragrant aroma. C. ensifolium has a long history of cultivation in China and it has excellent commercial value as a potted plant and cut flower. The development of C. ensifolium genomic resources has been delayed because of its large genome size. Taking advantage of technical and cost improvement of RNA-Seq, we extracted total mRNA from flower buds and mature flowers and obtained a total of 9.52 Gb of filtered nucleotides comprising 98,819,349 filtered reads. The filtered reads were assembled into 101,423 isotigs, representing 51,696 genes. Of the 101,423 isotigs, 41,873 were putative homologs of annotated sequences in the public databases, of which 158 were associated with floral development and 119 were associated with flowering. The isotigs were categorized according to their putative functions. In total, 10,212 of the isotigs were assigned into 25 eukaryotic orthologous groups (KOGs), 41,690 into 58 gene ontology (GO) terms, and 9,830 into 126 Arabidopsis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 9,539 isotigs into 123 rice pathways. Comparison of the isotigs with those of the two related orchid species P. equestris and C. sinense showed that 17,906 isotigs are unique to C. ensifolium. In addition, a total of 7,936 SSRs and 16,676 putative SNPs were identified. To our knowledge, this transcriptome database is the first major genomic resource for C. ensifolium and the most comprehensive transcriptomic resource for genus Cymbidium. These sequences provide valuable information for understanding the molecular mechanisms of floral development and flowering. Sequences predicted to be unique to C. ensifolium would provide more insights into C. ensifolium gene diversity. The numerous SNPs and SSRs identified in the present study will contribute to marker development for C. ensifolium. PMID:24392013

Directional RNA-seq reveals highly complex condition-dependent transcriptomes in E. coli K12 through accurate full-length transcripts assembling.

PubMed

Li, Shan; Dong, Xia; Su, Zhengchang

2013-07-30

Although prokaryotic gene transcription has been studied over decades, many aspects of the process remain poorly understood. Particularly, recent studies have revealed that transcriptomes in many prokaryotes are far more complex than previously thought. Genes in an operon are often alternatively and dynamically transcribed under different conditions, and a large portion of genes and intergenic regions have antisense RNA (asRNA) and non-coding RNA (ncRNA) transcripts, respectively. Ironically, similar studies have not been conducted in the model bacterium E coli K12, thus it is unknown whether or not the bacterium possesses similar complex transcriptomes. Furthermore, although RNA-seq becomes the major method for analyzing the complexity of prokaryotic transcriptome, it is still a challenging task to accurately assemble full length transcripts using short RNA-seq reads. To fill these gaps, we have profiled the transcriptomes of E. coli K12 under different culture conditions and growth phases using a highly specific directional RNA-seq technique that can capture various types of transcripts in the bacterial cells, combined with a highly accurate and robust algorithm and tool TruHMM (http://bioinfolab.uncc.edu/TruHmm_package/) for assembling full length transcripts. We found that 46.9 ~ 63.4% of expressed operons were utilized in their putative alternative forms, 72.23 ~ 89.54% genes had putative asRNA transcripts and 51.37 ~ 72.74% intergenic regions had putative ncRNA transcripts under different culture conditions and growth phases. As has been demonstrated in many other prokaryotes, E. coli K12 also has a highly complex and dynamic transcriptomes under different culture conditions and growth phases. Such complex and dynamic transcriptomes might play important roles in the physiology of the bacterium. TruHMM is a highly accurate and robust algorithm for assembling full-length transcripts in prokaryotes using directional RNA-seq short reads.

Directional RNA-seq reveals highly complex condition-dependent transcriptomes in E. coli K12 through accurate full-length transcripts assembling

PubMed Central

2013-01-01

Background Although prokaryotic gene transcription has been studied over decades, many aspects of the process remain poorly understood. Particularly, recent studies have revealed that transcriptomes in many prokaryotes are far more complex than previously thought. Genes in an operon are often alternatively and dynamically transcribed under different conditions, and a large portion of genes and intergenic regions have antisense RNA (asRNA) and non-coding RNA (ncRNA) transcripts, respectively. Ironically, similar studies have not been conducted in the model bacterium E coli K12, thus it is unknown whether or not the bacterium possesses similar complex transcriptomes. Furthermore, although RNA-seq becomes the major method for analyzing the complexity of prokaryotic transcriptome, it is still a challenging task to accurately assemble full length transcripts using short RNA-seq reads. Results To fill these gaps, we have profiled the transcriptomes of E. coli K12 under different culture conditions and growth phases using a highly specific directional RNA-seq technique that can capture various types of transcripts in the bacterial cells, combined with a highly accurate and robust algorithm and tool TruHMM (http://bioinfolab.uncc.edu/TruHmm_package/) for assembling full length transcripts. We found that 46.9 ~ 63.4% of expressed operons were utilized in their putative alternative forms, 72.23 ~ 89.54% genes had putative asRNA transcripts and 51.37 ~ 72.74% intergenic regions had putative ncRNA transcripts under different culture conditions and growth phases. Conclusions As has been demonstrated in many other prokaryotes, E. coli K12 also has a highly complex and dynamic transcriptomes under different culture conditions and growth phases. Such complex and dynamic transcriptomes might play important roles in the physiology of the bacterium. TruHMM is a highly accurate and robust algorithm for assembling full-length transcripts in prokaryotes using directional RNA-seq short reads. PMID:23899370

Pteros: fast and easy to use open-source C++ library for molecular analysis.

PubMed

Yesylevskyy, Semen O

2012-07-15

An open-source Pteros library for molecular modeling and analysis of molecular dynamics trajectories for C++ programming language is introduced. Pteros provides a number of routine analysis operations ranging from reading and writing trajectory files and geometry transformations to structural alignment and computation of nonbonded interaction energies. The library features asynchronous trajectory reading and parallel execution of several analysis routines, which greatly simplifies development of computationally intensive trajectory analysis algorithms. Pteros programming interface is very simple and intuitive while the source code is well documented and easily extendible. Pteros is available for free under open-source Artistic License from http://sourceforge.net/projects/pteros/. Copyright © 2012 Wiley Periodicals, Inc.

75 FR 39528 - Farm Credit Administration Board; Amendment to Sunshine Act Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-09

... item to the open session to read as follows: Open Session B. New Business Proposed Bookletter--Farm... amend the agenda by adding an item to the open session of that meeting. FOR FURTHER INFORMATION CONTACT... INFORMATION: Parts of this meeting of the Board will be open to the public (limited space available), and...

10 CFR 1703.103 - Requests for board records available through the public reading room.

Code of Federal Regulations, 2011 CFR

2011-01-01

... Public Reading Room will be maintained at the Board's headquarters and will be open between 8:30 a.m. and... interested person's comments, data, views, or arguments to the Board concerning its recommendations and the...

Analysis of the Macaca mulatta transcriptome and the sequence divergence between Macaca and human.

PubMed

Magness, Charles L; Fellin, P Campion; Thomas, Matthew J; Korth, Marcus J; Agy, Michael B; Proll, Sean C; Fitzgibbon, Matthew; Scherer, Christina A; Miner, Douglas G; Katze, Michael G; Iadonato, Shawn P

2005-01-01

We report the initial sequencing and comparative analysis of the Macaca mulatta transcriptome. Cloned sequences from 11 tissues, nine animals, and three species (M. mulatta, M. fascicularis, and M. nemestrina) were sampled, resulting in the generation of 48,642 sequence reads. These data represent an initial sampling of the putative rhesus orthologs for 6,216 human genes. Mean nucleotide diversity within M. mulatta and sequence divergence among M. fascicularis, M. nemestrina, and M. mulatta are also reported.

The Stickybear Reading Comprehension Series: Sports. School Version with Lesson Plans. For Ages 7-10. Volume 2. Stickybear Software.

ERIC Educational Resources Information Center

1996

This software product presents multi-level stories to capture the interest of children in grades two through five, while teaching them crucial reading comprehension skills. With stories touching on everything from superstars to sports facts, the open-ended reading comprehension program is versatile and easy to use for educators and children alike.…

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

PubMed

Mattow, J; Jungblut, P R; Schaible, U E; Mollenkopf, H J; Lamer, S; Zimny-Arndt, U; Hagens, K; Müller, E C; Kaufmann, S H

2001-08-01

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Non-contiguous genome sequence of Mycobacterium simiae strain DSM 44165(T.).

PubMed

Sassi, Mohamed; Robert, Catherine; Raoult, Didier; Drancourt, Michel

2013-01-01

Mycobacterium simiae is a non-tuberculosis mycobacterium causing pulmonary infections in both immunocompetent and imunocompromized patients. We announce the draft genome sequence of M. simiae DSM 44165(T). The 5,782,968-bp long genome with 65.15% GC content (one chromosome, no plasmid) contains 5,727 open reading frames (33% with unknown function and 11 ORFs sizing more than 5000 -bp), three rRNA operons, 52 tRNA, one 66-bp tmRNA matching with tmRNA tags from Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium marinum, and Mycobacterium africanum and 389 DNA repetitive sequences. Comparing ORFs and size distribution between M. simiae and five other Mycobacterium species M. simiae clustered with M. abscessus and M. smegmatis. A 40-kb prophage was predicted in addition to two prophage-like elements, 7-kb and 18-kb in size, but no mycobacteriophage was seen after the observation of 10(6) M. simiae cells. Fifteen putative CRISPRs were found. Three genes were predicted to encode resistance to aminoglycosides, betalactams and macrolide-lincosamide-streptogramin B. A total of 163 CAZYmes were annotated. M. simiae contains ESX-1 to ESX-5 genes encoding for a type-VII secretion system. Availability of the genome sequence may help depict the unique properties of this environmental, opportunistic pathogen.

Isolation and Abiotic Stress Resistance Analyses of a Catalase Gene from Ipomoea batatas (L.) Lam.

PubMed

Yong, Bin; Wang, Xiaoyan; Xu, Pan; Zheng, Haiyan; Fei, Xueting; Hong, Zixi; Ma, Qinqin; Miao, Yuzhi; Yuan, Xianghua; Jiang, Yusong; Shao, Huanhuan

2017-01-01

As an indicator of the antioxidant capability of plants, catalase can detoxify reactive oxygen species (ROS) generated by environmental stresses. Sweet potato is one of the top six most important crops in the world. However, its catalases remain largely unknown. In this study, a catalase encoding gene, IbCAT2 (accession number: KY615708), was identified and cloned from sweet potato cv. Xushu 18. It contained a 1479 nucleotides' open reading frame (ORF). S-R-L, Q-K-L, and a putative calmodulin binding domain were located at the C-terminus of IbCAT2, which suggests that IbCAT2 could be a peroxisomal catalase. Next-generation sequencing (NGS) based quantitative analyses showed that IbCAT2 was mainly expressed in young leaves and expanding tuberous roots under normal conditions. When exposed to 10% PEG6000 or 200 mmol/L NaCl solutions, IbCAT2 was upregulated rapidly in the first 11 days and then downregulated, although different tissues showed different degree of change. Overexpression of IbCAT2 conferred salt and drought tolerance in Escherichia coli and Saccharomyces cerevisiae . The positive response of IbCAT2 to abiotic stresses suggested that IbCAT2 might play an important role in stress responses.

Tula and Puumala hantavirus NSs ORFs are functional and the products inhibit activation of the interferon-beta promoter.

PubMed

Jääskeläinen, Kirsi M; Kaukinen, Pasi; Minskaya, Ekaterina S; Plyusnina, Angelina; Vapalahti, Olli; Elliott, Richard M; Weber, Friedemann; Vaheri, Antti; Plyusnin, Alexander

2007-10-01

The S RNA genome segment of hantaviruses carried by Arvicolinae and Sigmodontinae rodents encodes the nucleocapsid (N) protein and has an overlapping (+1) open reading frame (ORF) for a putative nonstructural protein (NSs). The aim of this study was to determine whether the ORF is functional. A protein corresponding to the predicted size of Tula virus (TULV) NSs was detected using coupled in vitro transcription and translation from a cloned S segment cDNA, and a protein corresponding to the predicted size of Puumala virus (PUUV) NSs was detected in infected cells by Western blotting with an anti-peptide serum. The activities of the interferon beta (IFN-beta) promoter, and nuclear factor kappa B (NF-kappaB)- and interferon regulatory factor-3 (IRF-3) responsive promoters, were inhibited in COS-7 cells transiently expressing TULV or PUUV NSs. Also IFN-beta mRNA levels in IFN-competent MRC5 cells either infected with TULV or transiently expressing NSs were decreased. These data demonstrate that Tula and Puumala hantaviruses have a functional NSs ORF. The findings may explain why the NSs ORF has been preserved in the genome of most hantaviruses during their long evolution and why hantavirus-infected cells secrete relatively low levels of IFNs. (c) 2007 Wiley-Liss, Inc.

The Nucleotide Sequence and Spliced pol mRNA Levels of the Nonprimate Spumavirus Bovine Foamy Virus

PubMed Central

Holzschu, Donald L.; Delaney, Mari A.; Renshaw, Randall W.; Casey, James W.

1998-01-01

We have determined the complete nucleotide sequence of a replication-competent clone of bovine foamy virus (BFV) and have quantitated the amount of splice pol mRNA processed early in infection. The 544-amino-acid Gag protein precursor has little sequence similarity with its primate foamy virus homologs, but the putative nucleocapsid (NC) protein, like the primate NCs, contains the three glycine-arginine-rich regions that are postulated to bind genomic RNA during virion assembly. The BFV gag and pol open reading frames overlap, with pro and pol in the same translational frame. As with the human foamy virus (HFV) and feline foamy virus, we have detected a spliced pol mRNA by PCR. Quantitatively, this mRNA approximates the level of full-length genomic RNA early in infection. The integrase (IN) domain of reverse transcriptase does not contain the canonical HH-CC zinc finger motif present in all characterized retroviral INs, but it does contain a nearby histidine residue that could conceivably participate as a member of the zinc finger. The env gene encodes a protein that is over 40% identical in sequence to the HFV Env. By comparison, the Gag precursor of BFV is predicted to be only 28% identical to the HFV protein. PMID:9499074

Rearrangement of a large novel Pseudomonas aeruginosa gene island in strains isolated from a patient developing ventilator-associated pneumonia.

PubMed

Singh, G; Srinivasan, R; Cheng, J; Peng, Z; Fujimura, K; Baek, M S; Panzer, A R; Tringe, S G; Chen, F; Sorek, R; Weng, L; Bristow, J; Wiener-Kronish, J P; Lynch, S V

2014-07-01

Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative β-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNA(lys) recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Rearrangement of a Large Novel Pseudomonas aeruginosa Gene Island in Strains Isolated from a Patient Developing Ventilator-Associated Pneumonia

PubMed Central

Singh, G.; Srinivasan, R.; Cheng, J.; Peng, Z.; Fujimura, K.; Baek, M. S.; Panzer, A. R.; Tringe, S. G.; Chen, F.; Sorek, R.; Weng, L.; Bristow, J.; Wiener-Kronish, J. P.

2014-01-01

Bacterial gene islands add to the genetic repertoire of opportunistic pathogens. Here, we perform comparative analyses of three Pseudomonas aeruginosa strains isolated sequentially over a 3-week period from a patient with ventilator-associated pneumonia (VAP) who received clindamycin and piperacillin-tazobactam as part of their treatment regime. While all three strains appeared to be clonal by standard pulsed-field gel electrophoresis, whole-genome sequencing revealed subtle alterations in the chromosomal organization of the last two strains; specifically, an inversion event within a novel 124-kb gene island (PAGI 12) composed of 137 open reading frames [ORFs]. Predicted ORFs in the island included metabolism and virulence genes. Overexpression of a gene island-borne putative β-lactamase gene was observed following piperacillin-tazobactam exposure and only in those strains that had undergone the inversion event, indicating altered gene regulation following genomic remodeling. Examination of a separate cohort of 76 patients with VAP for integration at this tRNAlys recombination site demonstrated that patients exhibiting evidence of integration at this site had significantly higher 28-day mortality. These findings provide evidence that P. aeruginosa can integrate, rapidly remodel, and express exogenous genes, which likely contributes to its fitness in a clinical setting. PMID:24789195

Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.

PubMed Central

Hong, S W; Jon, J H; Kwak, J M; Nam, H G

1997-01-01

A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1-1, abi2-1, and abi3-1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed. PMID:9112773

Isolation and Abiotic Stress Resistance Analyses of a Catalase Gene from Ipomoea batatas (L.) Lam

PubMed Central

Yong, Bin; Wang, Xiaoyan; Xu, Pan; Zheng, Haiyan; Fei, Xueting; Hong, Zixi; Ma, Qinqin; Miao, Yuzhi; Yuan, Xianghua; Jiang, Yusong

2017-01-01

As an indicator of the antioxidant capability of plants, catalase can detoxify reactive oxygen species (ROS) generated by environmental stresses. Sweet potato is one of the top six most important crops in the world. However, its catalases remain largely unknown. In this study, a catalase encoding gene, IbCAT2 (accession number: KY615708), was identified and cloned from sweet potato cv. Xushu 18. It contained a 1479 nucleotides' open reading frame (ORF). S-R-L, Q-K-L, and a putative calmodulin binding domain were located at the C-terminus of IbCAT2, which suggests that IbCAT2 could be a peroxisomal catalase. Next-generation sequencing (NGS) based quantitative analyses showed that IbCAT2 was mainly expressed in young leaves and expanding tuberous roots under normal conditions. When exposed to 10% PEG6000 or 200 mmol/L NaCl solutions, IbCAT2 was upregulated rapidly in the first 11 days and then downregulated, although different tissues showed different degree of change. Overexpression of IbCAT2 conferred salt and drought tolerance in Escherichia coli and Saccharomyces cerevisiae. The positive response of IbCAT2 to abiotic stresses suggested that IbCAT2 might play an important role in stress responses. PMID:28638833

Secrets of soil survival revealed by the genome sequence of Arthrobacter aurescens TC1.

PubMed

Mongodin, Emmanuel F; Shapir, Nir; Daugherty, Sean C; DeBoy, Robert T; Emerson, Joanne B; Shvartzbeyn, Alla; Radune, Diana; Vamathevan, Jessica; Riggs, Florenta; Grinberg, Viktoria; Khouri, Hoda; Wackett, Lawrence P; Nelson, Karen E; Sadowsky, Michael J

2006-12-01

Arthrobacter sp. strains are among the most frequently isolated, indigenous, aerobic bacterial genera found in soils. Member of the genus are metabolically and ecologically diverse and have the ability to survive in environmentally harsh conditions for extended periods of time. The genome of Arthrobacter aurescens strain TC1, which was originally isolated from soil at an atrazine spill site, is composed of a single 4,597,686 basepair (bp) circular chromosome and two circular plasmids, pTC1 and pTC2, which are 408,237 bp and 300,725 bp, respectively. Over 66% of the 4,702 open reading frames (ORFs) present in the TC1 genome could be assigned a putative function, and 13.2% (623 genes) appear to be unique to this bacterium, suggesting niche specialization. The genome of TC1 is most similar to that of Tropheryma, Leifsonia, Streptomyces, and Corynebacterium glutamicum, and analyses suggest that A. aurescens TC1 has expanded its metabolic abilities by relying on the duplication of catabolic genes and by funneling metabolic intermediates generated by plasmid-borne genes to chromosomally encoded pathways. The data presented here suggest that Arthrobacter's environmental prevalence may be due to its ability to survive under stressful conditions induced by starvation, ionizing radiation, oxygen radicals, and toxic chemicals.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

NASA Astrophysics Data System (ADS)

Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

2015-09-01

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

Construction and characterization of a recombinant invertebrate iridovirus.

PubMed

Ozgen, Arzu; Muratoglu, Hacer; Demirbag, Zihni; Vlak, Just M; van Oers, Monique M; Nalcacioglu, Remziye

2014-08-30

Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral replication dynamics. We showed that homologous recombination is a valid method to make CIV gene knockouts and to insert foreign genes. The CIV 157L gene, putatively encoding a non-functional inhibitor of apoptosis (IAP), was chosen as target for foreign gene insertion. The gfp open reading frame preceded by the viral mcp promoter was inserted into the 157L locus by homologous recombination in Anthonomus grandis BRL-AG-3A cells. Recombinant virus (rCIV-Δ157L-gfp) was purified by successive rounds of plaque purification. All plaques produced by the purified recombinant virus emitted green fluorescence due to the presence of GFP. One-step growth curves for recombinant and wild-type CIV were similar and the recombinant was fully infectious in vivo. Hence, CIV157L can be inactivated without altering the replication kinetics of the virus. Consequently, the CIV 157L locus can be used as a site for insertion of foreign DNA, e.g. to modify viral properties for insect biocontrol. Copyright © 2014 Elsevier B.V. All rights reserved.

A 38-Kilobase Pathogenicity Island Specific for Mycobacterium avium subsp. paratuberculosis Encodes Cell Surface Proteins Expressed in the Host

PubMed Central

Stratmann, Janin; Strommenger, Birgit; Goethe, Ralph; Dohmann, Karen; Gerlach, Gerald-F.; Stevenson, Karen; Li, Ling-ling; Zhang, Qing; Kapur, Vivek; Bull, Tim J.

2004-01-01

We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe3+ and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island. PMID:14977927

Complement component 3: characterization and association with mastitis resistance in Egyptian water buffalo and cattle.

PubMed

El-Halawany, Nermin; Abd-El-Monsif, Shawky A; Al-Tohamy Ahmed, F M; Hegazy, Lamees; Abdel-Shafy, Hamdy; Abdel-Latif, Magdy A; Ghazi, Yasser A; Neuhoff, Christiane; Salilew-Wondim, Dessie; Schellander, Karl

2017-03-01

Mastitis is an infectious disease of the mammary gland that leads to reduced milk production and change in milk composition. Complement component C3 plays a major role as a central molecule of the complement cascade involving in killing of microorganisms, either directly or in cooperation with phagocytic cells. C3 cDNA were isolated, from Egyptian buffalo and cattle, sequenced and characterized. The C3 cDNA sequences of buffalo and cattle consist of 5025 and 5019 bp, respectively. Buffalo and cattle C3 cDNAs share 99% of sequence identity with each other. The 4986 bp open reading frame in buffalo encodes a putative protein of 1661 amino acids-as in cattle-and includes all the functional domains. Further, analysis of the C3 cDNA sequences detected six novel single-nucleotide polymorphisms (SNPs) in buffalo and three novel SNPs in cattle. The association analysis of the detected SNPs with milk somatic cell score as an indicator of mastitis revealed that the most significant association in buffalo was found in the C>A substitution (ss: 1752816097) in exon 27, whereas in cattle it was in the C>T substitution (ss: 1752816085) in exon 12. Our findings provide preliminary information about the contribution of C3 polymorphisms to mastitis resistance in buffalo and cattle.

Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

PubMed Central

Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

1995-01-01

Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

Characterization of three terpenoid glycosyltransferase genes in 'Valencia' sweet orange (Citrus sinensis L. Osbeck).

PubMed

Fan, Jing; Chen, Chunxian; Yu, Qibin; Li, Zheng-Guo; Gmitter, Frederick G

2010-10-01

Three putative terpenoid UDP-glycosyltransferase (UGT) genes, designated CsUGT1, CsUGT2, and CsUGT3, were isolated and characterized in 'Valencia' sweet orange (Citrus sinensis L. Osbeck). CsUGT1 consisted of 1493 nucleotides with an open reading frame encoding 492 amino acids, CsUGT2 consisted of 1727 nucleotides encoding 504 amino acids, and CsUGT3 consisted of 1705 nucleotides encoding 468 amino acids. CsUGT3 had a 145 bp intron at 730-874, whereas CsUGT1 and CsUGT2 had none. The three deduced glycosyltransferase proteins had a highly conserved plant secondary product glycosyltransferase motif in the C terminus. Phylogenetic analysis showed that CsUGT1 and CsUGT3 were classified into group L of glycosyltransferase family 1, and CsUGT2 was classified into group D. Through Southern blotting analysis, CsUGT1 was found to have two copies in the sweet orange genome, whereas CsUGT2 and CsUGT3 had at least seven and nine copies, respectively. CsUGT1, CsUGT2, and CsUGT3 were constitutively expressed in leaf, flower, and fruit tissues. The results facilitate further investigation of the function of terpenoid glycosyltransferases in citrus and the biosynthesis of terpenoid glycosides in vitro.

Identification and characterization of a phospholipid scramblase encoded by planarian Dugesia japonica.

PubMed

Han, Yu; Li, Ao; Gao, Lili; Wu, Weiwei; Deng, Hongkuan; Hu, Wenjing; Li, Na; Sun, Shimin; Zhang, Xiufang; Zhao, Bosheng; Liu, Baohua; Pang, Qiuxiang

2017-02-20

Phospholipid scramblases (PLSCRs) are the conserved calcium-binding, type II transmembrane proteins synthesized in all eukaryotic organisms. In mammals, these proteins play essential roles in various physiological processes, especially in the immune responses. However, the existence of PLSCRs and their biological functions in planarian are still unknown at present. In this study, a new member of PLSCRs was identified in planarian Dugesia japonica (D. japonica), named DjPLSCR. The sequence analysis revealed that it contains an opening reading frame consisting of 726bp encoding a putative protein of 241 amino acids with a predicted molecular mass of ~28.7kDa and an isoelectric point of 6.21. Whole-mount in situ hybridization showed that mRNAs of DjPLSCR are predominantly expressed in adult and regenerative pharynx which is an important organ of immune system in planarians. Importantly, we found that the transcription level of DjPLSCR was significantly upregulated when planarians were stimulated with the pathogen-associated molecular patterns [polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and β-glucan], suggesting that DjPLSCR is involved in the immune response upon pathogen invasion. Our findings provide the first experimental insights into the characteristics and potential functions of PLSCR in planarians. Copyright © 2016 Elsevier B.V. All rights reserved.

GDR (Genome Database for Rosaceae): integrated web-database for Rosaceae genomics and genetics data

PubMed Central

Jung, Sook; Staton, Margaret; Lee, Taein; Blenda, Anna; Svancara, Randall; Abbott, Albert; Main, Dorrie

2008-01-01

The Genome Database for Rosaceae (GDR) is a central repository of curated and integrated genetics and genomics data of Rosaceae, an economically important family which includes apple, cherry, peach, pear, raspberry, rose and strawberry. GDR contains annotated databases of all publicly available Rosaceae ESTs, the genetically anchored peach physical map, Rosaceae genetic maps and comprehensively annotated markers and traits. The ESTs are assembled to produce unigene sets of each genus and the entire Rosaceae. Other annotations include putative function, microsatellites, open reading frames, single nucleotide polymorphisms, gene ontology terms and anchored map position where applicable. Most of the published Rosaceae genetic maps can be viewed and compared through CMap, the comparative map viewer. The peach physical map can be viewed using WebFPC/WebChrom, and also through our integrated GDR map viewer, which serves as a portal to the combined genetic, transcriptome and physical mapping information. ESTs, BACs, markers and traits can be queried by various categories and the search result sites are linked to the mapping visualization tools. GDR also provides online analysis tools such as a batch BLAST/FASTA server for the GDR datasets, a sequence assembly server and microsatellite and primer detection tools. GDR is available at http://www.rosaceae.org. PMID:17932055

Molecular Cloning and Characterization of a P-Glycoprotein from the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae)

PubMed Central

Tian, Lixia; Yang, Jiaqiang; Hou, Wenjie; Xu, Baoyun; Xie, Wen; Wang, Shaoli; Zhang, Youjun; Zhou, Xuguo; Wu, Qingjun

2013-01-01

Macrocyclic lactones such as abamectin and ivermectin constitute an important class of broad-spectrum insecticides. Widespread resistance to synthetic insecticides, including abamectin and ivermectin, poses a serious threat to the management of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), a major pest of cruciferous plants worldwide. P-glycoprotein (Pgp), a member of the ABC transporter superfamily, plays a crucial role in the removal of amphiphilic xenobiotics, suggesting a mechanism for drug resistance in target organisms. In this study, PxPgp1, a putative Pgp gene from P. xylostella, was cloned and characterized. The open reading frame (ORF) of PxPgp1 consists of 3774 nucleotides, which encodes a 1257-amino acid peptide. The deduced PxPgp1 protein possesses structural characteristics of a typical Pgp, and clusters within the insect ABCB1. PxPgp1 was expressed throughout all developmental stages, and showed the highest expression level in adult males. PxPgp1 was highly expressed in midgut, malpighian tubules and testes. Elevated expression of PxPgp1 was observed in P. xylostella strains after they were exposed to the abamectin treatment. In addition, the constitutive expressions of PxPgp1 were significantly higher in laboratory-selected and field-collected resistant strains in comparison to their susceptible counterpart. PMID:24264038

Molecular cloning and structural characterization of Ecdysis Triggering Hormone from Choristoneura fumiferana.

PubMed

P, Bhagath Kumar; K, Kasi Viswanath; S, Tuleshwori Devi; R, Sampath Kumar; Doucet, Daniel; Retnakaran, Arthur; Krell, Peter J; Feng, Qili; Ampasala, Dinakara Rao

2016-07-01

At the end of each stadium, insects undergo a precisely orchestrated process known as ecdysis which results in the replacement of the old cuticle with a new one. This physiological event is necessary to accommodate growth in arthropods since they have a rigid chitinous exoskeleton. Ecdysis is initiated by the direct action of Ecdysis Triggering Hormones on the central nervous system. Choristoneura fumiferana is a major defoliator of coniferous forests in Eastern North America. It is assumed that, studies on the ecdysis behavior of this pest might lead to the development of novel pest management strategies. Hence in this study, the cDNA of CfETH was cloned. The open reading frame of the cDNA sequence was found to encode three putative peptides viz., Pre-Ecdysis Triggering Hormone (PETH), Ecdysis Triggering Hormone (ETH), and Ecdysis Triggering Hormone Associated Peptide (ETH-AP). The CfETH transcript was detected in the epidermal tissue of larval and pupal stages, but not in eggs and adults. In order to explore the structural conformation of ETH, ab initio modelling and Molecular Dynamics (MD) Simulations were performed. Further, a library of insecticides was generated and virtual screening was performed to identify the compounds displaying high binding capacity to ETH. Copyright © 2016 Elsevier B.V. All rights reserved.

Genomic and proteomic characterization of a thermophilic Geobacillus bacteriophage GBSV1.

PubMed

Liu, Bin; Zhou, Fengfeng; Wu, Suijie; Xu, Ying; Zhang, Xiaobo

2009-03-01

Phages are present wherever life is found, and play roles in many biogeochemical and ecological processes. The thermophilic bacteriophages, however, have not been well studied. In this study, phage GBSV1 was obtained from a thermophilic bacterium Geobacillus sp. 6k51 isolated from a hot spring. GBSV1 contains a double-stranded linear DNA of 34,683bp, which encodes 54 putative open reading frames (ORFs). Thirty three of these 54 ORFs exhibit sequence similarities to genes from 7 species of Geobacillus or Bacillus bacteria, as well as of bacteriophages infecting these bacteria. Twenty-two ORFs have been functionally annotated based on both their sequence similarities to known genes and predicted Pfam protein domains. Five structural proteins of the purified GBSV1 virion have been identified by proteomic analyses. Surprisingly, 7 of the GBSV1 ORFs share sequence similarities with genes from bacteria relevant to human diseases. This is the first report that genes of human disease-inducing bacteria are found in a thermophilic phage. It is suggested that thermophilic phages may be the potential evolutionary link between thermophiles and human pathogens. The characterization of GBSV1 may possibly lead to new insights into virus-host interactions and to a better understanding of gene transfers and evolution of life on earth in general.

Molecular cloning of an inducible serine esterase gene from human cytotoxic lymphocytes.

PubMed Central

Trapani, J A; Klein, J L; White, P C; Dupont, B

1988-01-01

A cDNA clone encoding a human serine esterase gene was isolated from a library constructed from poly(A)+ RNA of allogeneically stimulated, interleukin 2-expanded peripheral blood mononuclear cells. The clone, designated HSE26.1, represents a full-length copy of a 0.9-kilobase mRNA present in human cytotoxic cells but absent from a wide variety of noncytotoxic cell lines. Clone HSE26.1 contains an 892-base-pair sequence, including a single 741-base-pair open reading frame encoding a putative 247-residue polypeptide. The first 20 amino acids of the polypeptide form a leader sequence. The mature protein is predicted to have an unglycosylated Mr of approximately equal to 26,000 and contains a single potential site for N-linked glycosylation. The nucleotide and predicted amino acid sequences of clone HSE26.1 are homologous with all murine and human serine esterases cloned thus far but are most similar to mouse granzyme B (70% nucleotide and 68% amino acid identity). HSE26.1 protein is expressed weakly in unstimulated peripheral blood mononuclear cells but is strongly induced within 6-hr incubation in medium containing phytohemagglutinin. The data suggest that the protein encoded by HSE26.1 plays a role in cell-mediated cytotoxicity. Images PMID:3261871

Massive gene acquisitions in Mycobacterium indicus pranii provide a perspective on mycobacterial evolution

PubMed Central

Saini, Vikram; Raghuvanshi, Saurabh; Khurana, Jitendra P.; Ahmed, Niyaz; Hasnain, Seyed E.; Tyagi, Akhilesh K.; Tyagi, Anil K.

2012-01-01

Understanding the evolutionary and genomic mechanisms responsible for turning the soil-derived saprophytic mycobacteria into lethal intracellular pathogens is a critical step towards the development of strategies for the control of mycobacterial diseases. In this context, Mycobacterium indicus pranii (MIP) is of specific interest because of its unique immunological and evolutionary significance. Evolutionarily, it is the progenitor of opportunistic pathogens belonging to M. avium complex and is endowed with features that place it between saprophytic and pathogenic species. Herein, we have sequenced the complete MIP genome to understand its unique life style, basis of immunomodulation and habitat diversification in mycobacteria. As a case of massive gene acquisitions, 50.5% of MIP open reading frames (ORFs) are laterally acquired. We show, for the first time for Mycobacterium, that MIP genome has mosaic architecture. These gene acquisitions have led to the enrichment of selected gene families critical to MIP physiology. Comparative genomic analysis indicates a higher antigenic potential of MIP imparting it a unique ability for immunomodulation. Besides, it also suggests an important role of genomic fluidity in habitat diversification within mycobacteria and provides a unique view of evolutionary divergence and putative bottlenecks that might have eventually led to intracellular survival and pathogenic attributes in mycobacteria. PMID:22965120

Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase.

PubMed

Shao, Zhiyong; Graf, Shannon; Chaga, Oleg Y; Lavrov, Dennis V

2006-10-15

The 16,937-nuceotide sequence of the linear mitochondrial DNA (mt-DNA) molecule of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa) - the first mtDNA sequence from the class Scypozoa and the first sequence of a linear mtDNA from Metazoa - has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs. In addition, two open reading frames of 324 and 969 base pairs in length have been found. The deduced amino-acid sequence of one of them, ORF969, displays extensive sequence similarity with the polymerase [but not the exonuclease] domain of family B DNA polymerases, and this ORF has been tentatively identified as dnab. This is the first report of dnab in animal mtDNA. The genes in A. aurita mtDNA are arranged in two clusters with opposite transcriptional polarities; transcription proceeding toward the ends of the molecule. The determined sequences at the ends of the molecule are nearly identical but inverted and lack any obvious potential secondary structures or telomere-like repeat elements. The acquisition of mitochondrial genomic data for the second class of Cnidaria allows us to reconstruct characteristic features of mitochondrial evolution in this animal phylum.

Isolation, characterization, and complementation of a motility mutant of Spiroplasma citri.

PubMed Central

Jacob, C; Nouzières, F; Duret, S; Bové, J M; Renaudin, J

1997-01-01

The helical mollicute Spiroplasma citri, when growing on low-agar medium, forms fuzzy colonies with occasional surrounding satellite colonies due to the ability of the spiroplasmal cells to move through the agar matrix. In liquid medium, these helical organisms flex, twist, and rotate rapidly. By using Tn4001 insertion mutagenesis, a motility mutant was isolated on the basis of its nondiffuse, sharp-edged colonies. Dark-field microscopy observations revealed that the organism flexed at a low frequency and had lost the ability to rotate about the helix axis. In this mutant, the transposon was shown to be inserted into an open reading frame encoding a putative polypeptide of 409 amino acids for which no significant homology with known proteins was found. The corresponding gene, named scm1, was recovered from the wild-type strain and introduced into the motility mutant by using the S. citri oriC plasmid pBOT1 as the vector. The appearance of fuzzy colonies and the observation that spiroplasma cells displayed rotatory and flexional movements showed the motile phenotype to be restored in the spiroplasmal transformants. The functional complementation of the motility mutant proves the scm1 gene product to be involved in the motility mechanism of S. citri. PMID:9244268

Lipoxygenase in Caragana jubata responds to low temperature, abscisic acid, methyl jasmonate and salicylic acid.

PubMed

Bhardwaj, Pardeep Kumar; Kaur, Jagdeep; Sobti, Ranbir Chander; Ahuja, Paramvir Singh; Kumar, Sanjay

2011-09-01

Lipoxygenase (LOX) catalyses oxygenation of free polyunsaturated fatty acids into oxylipins, and is a critical enzyme of the jasmonate signaling pathway. LOX has been shown to be associated with biotic and abiotic stress responses in diverse plant species, though limited data is available with respect to low temperature and the associated cues. Using rapid amplification of cDNA ends, a full-length cDNA (CjLOX) encoding lipoxygenase was cloned from apical buds of Caragana jubata, a temperate plant species that grows under extreme cold. The cDNA obtained was 2952bp long consisting of an open reading frame of 2610bp encoding 869 amino acids protein. Multiple alignment of the deduced amino acid sequence with those of other plants demonstrated putative LH2/ PLAT domain, lipoxygenase iron binding catalytic domain and lipoxygenase_2 signature sequences. CjLOX exhibited up- and down-regulation of gene expression pattern in response to low temperature (LT), abscisic acid (ABA), methyl jasmonate (MJ) and salicylic acid (SA). Among all the treatments, a strong up-regulation was observed in response to MJ. Data suggests an important role of jasmonate signaling pathway in response to LT in C. jubata. Copyright © 2011 Elsevier B.V. All rights reserved.

Pyrosequencing the Bemisia tabaci Transcriptome Reveals a Highly Diverse Bacterial Community and a Robust System for Insecticide Resistance

PubMed Central

Wu, Qing-jun; Wang, Shao-li; Yang, Xin; Yang, Ni-na; Li, Ru-mei; Jiao, Xiao-guo; Pan, Hui-peng; Liu, Bai-ming; Su, Qi; Xu, Bao-yun; Hu, Song-nian; Zhou, Xu-guo; Zhang, You-jun

2012-01-01

Background Bemisia tabaci (Gennadius) is a phloem-feeding insect poised to become one of the major insect pests in open field and greenhouse production systems throughout the world. The high level of resistance to insecticides is a main factor that hinders continued use of insecticides for suppression of B. tabaci. Despite its prevalence, little is known about B. tabaci at the genome level. To fill this gap, an invasive B. tabaci B biotype was subjected to pyrosequencing-based transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes. Methodology and Principal Findings Using Roche 454 pyrosequencing, 857,205 reads containing approximately 340 megabases were obtained from the B. tabaci transcriptome. De novo assembly generated 178,669 unigenes including 30,980 from insects, 17,881 from bacteria, and 129,808 from the nohit. A total of 50,835 (28.45%) unigenes showed similarity to the non-redundant database in GenBank with a cut-off E-value of 10–5. Among them, 40,611 unigenes were assigned to one or more GO terms and 6,917 unigenes were assigned to 288 known pathways. De novo metatranscriptome analysis revealed highly diverse bacterial symbionts in B. tabaci, and demonstrated the host-symbiont cooperation in amino acid production. In-depth transcriptome analysis indentified putative molecular markers, and genes potentially involved in insecticide resistance and nutrient digestion. The utility of this transcriptome was validated by a thiamethoxam resistance study, in which annotated cytochrome P450 genes were significantly overexpressed in the resistant B. tabaci in comparison to its susceptible counterparts. Conclusions This transcriptome/metatranscriptome analysis sheds light on the molecular understanding of symbiosis and insecticide resistance in an agriculturally important phloem-feeding insect pest, and lays the foundation for future functional genomics research of the B. tabaci complex. Moreover, current pyrosequencing effort greatly enriched the existing whitefly EST database, and makes RNAseq a viable option for future genomic analysis. PMID:22558125

A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination.

PubMed

Mu, Changkao; Song, Xiaoyan; Zhao, Jianmin; Wang, Lingling; Qiu, Limei; Zhang, Huan; Zhou, Zhi; Wang, Mengqiang; Song, Linsheng; Wang, Chunlin

2012-05-01

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae.

PubMed

Stevenson, Andrew; Macdonald, Julie; Roberts, Mark

2003-03-20

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumoniae. Little is known about the mechanisms by which A. pleuropneumoniae colonises the respiratory tract. Fimbriae are common mediators of bacterial adherence to mucosal epithelia and have been observed on the surface of A. pleuropneumoniae cells. Here we report the identification and characterisation of the type 4 fimbrial structural gene (apfA) from A. pleuropneumoniae. In addition a number of open reading frames were identified in A. pleuropneumoniae that have significant homology to type 4 fimbrial biogenesis genes from other species, including a putative leader specific peptidase (apfD). A. pleuropneumoniae apfA codes for a predicted polypeptide of approximately 16kDa, removal of the leader sequence at the predicted cleavage site would yield a 14.5kDa polypeptide. The first 30 residues of the mature polypeptide are well conserved with other members of the group A type 4 fimbriae family. The signal sequence of ApfA is 13 amino acids in length and, unusually, the residue that precedes the cleavage site is alanine rather than glycine which is found in most other type 4 fimbriae. The C-terminus of ApfA possesses cysteine residues that are conserved in type 4 fimbriae of many species. In other type 4 fimbriae the distal C-terminal cysteines form a disulphide bond that produces a loop, which is important for the function of fimbriae and also comprises a major antigenic determinant. A motif within the predicted loop in ApfA was found to be highly conserved in type 4 fimbriae of other HAP organisms (Haemophilus, Actinobacillus, Pasteurella). The A. pleuropneumoniae type 4 fimbrial biogenesis genes showed the strongest homology to putative type 4 fimbrial genes of Haemophilus ducreyi. A. pleuropneumoniae apfA gene was shown to be present and highly conserved in different serotypes of A. pleuropneumoniae. Recombinant ApfA was produced and used to raise anti-ApfA antisera.

CaAP2 transcription factor is a candidate gene for a flowering repressor and a candidate for controlling natural variation of flowering time in Capsicum annuum.

PubMed

Borovsky, Yelena; Sharma, Vinod K; Verbakel, Henk; Paran, Ilan

2015-06-01

The APETALA2 transcription factor homolog CaAP2 is a candidate gene for a flowering repressor in pepper, as revealed by induced-mutation phenotype, and a candidate underlying a major QTL controlling natural variation in flowering time. To decipher the genetic control of transition to flowering in pepper (Capsicum spp.) and determine the extent of gene function conservation compared to model species, we isolated and characterized several ethyl methanesulfonate (EMS)-induced mutants that vary in their flowering time compared to the wild type. In the present study, we report on the isolation of an early-flowering mutant that flowers after four leaves on the primary stem compared to nine leaves in the wild-type 'Maor'. By genetic mapping and sequencing of putative candidate genes linked to the mutant phenotype, we identified a member of the APETALA2 (AP2) transcription factor family, CaAP2, which was disrupted in the early-flowering mutant. CaAP2 is a likely ortholog of AP2 that functions as a repressor of flowering in Arabidopsis. To test whether CaAP2 has an effect on controlling natural variation in the transition to flowering in pepper, we performed QTL mapping for flowering time in a cross between early and late-flowering C. annuum accessions. We identified a major QTL in a region of chromosome 2 in which CaAP2 was the most significant marker, explaining 52 % of the phenotypic variation of the trait. Sequence comparison of the CaAP2 open reading frames in the two parents used for QTL mapping did not reveal significant variation. In contrast, significant differences in expression level of CaAP2 were detected between near-isogenic lines that differ for the flowering time QTL, supporting the putative function of CaAP2 as a major repressor of flowering in pepper.

Transcript profiles of mitochondrial and cytoplasmic manganese superoxide dismutases in Exopalaemon carinicauda under ammonia stress

NASA Astrophysics Data System (ADS)

Ren, Hai; Li, Jian; Li, Jitao; Liu, Ping; Liang, Zhongxiu; Wu, Jianhua

2015-05-01

Superoxide dismutase (SOD) is one of the most important antioxidant defense enzymes, and is considered as the first line against oxidative stress. In this study, we cloned a mitochondrial manganese (Mn) SOD ( mMnSOD) cDNA from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) methods. The full-length cDNA for mMnSOD was 1 014-bp long, containing a 5'-untranslated region (UTR) of 37-bp, a 3'-UTR of 321-bp with a poly (A) tail, and included a 657-bp open reading frame encoding a protein of 218 amino acids with a 16-amino-acid signal peptide. The protein had a calculated molecular weight of 23.87 kDa and a theoretical isoelectric point of 6.75. The mMnSOD sequence included two putative N-glycosylation sites (NHT and NLS), the MnSOD signature sequence 180DVWEHAYY187, and four putative Mn binding sites (H48, H96, D180, and H184). Sequence comparison showed that the mMnSOD deduced amino acid sequence of E. carinicauda shared 97%, 95%, 89%, 84%, 82%, 72%, and 69% identity with that of Macrobrachium rosenbergii, Macrobrachium nipponense, Fenneropeneaus chinensis, Callinectes sapidus, Perisesarma bidens, Danio rerio, and Homo sapiens, resectively. Quantitative real-time RT-PCR analysis showed that mMnSOD transcripts were present in all E. carinicauda tissues examined, with the highest levels in the hepatopancreas. During an ammonia stress treatment, the transcript levels of mMnSOD and cMnSOD were up-regulated at 12 h in hemocytes and at 24 h in the hepatopancreas. As the duration of the ammonia stress treatment extended to 72 h, the transcript levels of mMnSOD and cMnSOD significantly decreased both in hemocytes and hepatopancreas. These findings indicate that the SOD system is induced to respond to acute ammonia stress, and may be involved in environmental stress responses in E. carinicauda.

Genomic analysis and temperature-dependent transcriptome profiles of the rhizosphere originating strain Pseudomonas aeruginosa M18

PubMed Central

2011-01-01

Background Our previously published reports have described an effective biocontrol agent named Pseudomonas sp. M18 as its 16S rDNA sequence and several regulator genes share homologous sequences with those of P. aeruginosa, but there are several unusual phenotypic features. This study aims to explore its strain specific genomic features and gene expression patterns at different temperatures. Results The complete M18 genome is composed of a single chromosome of 6,327,754 base pairs containing 5684 open reading frames. Seven genomic islands, including two novel prophages and five specific non-phage islands were identified besides the conserved P. aeruginosa core genome. Each prophage contains a putative chitinase coding gene, and the prophage II contains a capB gene encoding a putative cold stress protein. The non-phage genomic islands contain genes responsible for pyoluteorin biosynthesis, environmental substance degradation and type I and III restriction-modification systems. Compared with other P. aeruginosa strains, the fewest number (3) of insertion sequences and the most number (3) of clustered regularly interspaced short palindromic repeats in M18 genome may contribute to the relative genome stability. Although the M18 genome is most closely related to that of P. aeruginosa strain LESB58, the strain M18 is more susceptible to several antimicrobial agents and easier to be erased in a mouse acute lung infection model than the strain LESB58. The whole M18 transcriptomic analysis indicated that 10.6% of the expressed genes are temperature-dependent, with 22 genes up-regulated at 28°C in three non-phage genomic islands and one prophage but none at 37°C. Conclusions The P. aeruginosa strain M18 has evolved its specific genomic structures and temperature dependent expression patterns to meet the requirement of its fitness and competitiveness under selective pressures imposed on the strain in rhizosphere niche. PMID:21884571

Transcriptional and functional studies of Acidithiobacillus ferrooxidans genes related to survival in the presence of copper.

PubMed

Navarro, Claudio A; Orellana, Luis H; Mauriaca, Cecilia; Jerez, Carlos A

2009-10-01

The acidophilic Acidithiobacillus ferrooxidans can resist exceptionally high copper (Cu) concentrations. This property is important for its use in biomining processes, where Cu and other metal levels range usually between 15 and 100 mM. To learn about the mechanisms that allow A. ferrooxidans cells to survive in this environment, a bioinformatic search of its genome showed the presence of at least 10 genes that are possibly related to Cu homeostasis. Among them are three genes coding for putative ATPases related to the transport of Cu (A. ferrooxidans copA1 [copA1(Af)], copA2(Af), and copB(Af)), three genes related to a system of the resistance nodulation cell division family involved in the extraction of Cu from the cell (cusA(Af), cusB(Af), and cusC(Af)), and two genes coding for periplasmic chaperones for this metal (cusF(Af) and copC(Af)). The expression of most of these open reading frames was studied by real-time reverse transcriptase PCR using A. ferrooxidans cells adapted for growth in the presence of high concentrations of Cu. The putative A. ferrooxidans Cu resistance determinants were found to be upregulated when this bacterium was exposed to Cu in the range of 5 to 25 mM. These A. ferrooxidans genes conferred to Escherichia coli a greater Cu resistance than wild-type cells, supporting their functionality. The results reported here and previously published data strongly suggest that the high resistance of the extremophilic A. ferrooxidans to Cu may be due to part or all of the following key elements: (i) a wide repertoire of Cu resistance determinants, (ii) the duplication of some of these Cu resistance determinants, (iii) the existence of novel Cu chaperones, and (iv) a polyP-based Cu resistance system.

Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

PubMed Central

Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

2015-01-01

Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

The Complete Genomic Sequence of Pepper Yellow Leaf Curl Virus (PYLCV) and Its Implications for Our Understanding of Evolution Dynamics in the Genus Polerovirus

PubMed Central

Dombrovsky, Aviv; Glanz, Eyal; Lachman, Oded; Sela, Noa; Doron-Faigenboim, Adi; Antignus, Yehezkel

2013-01-01

We determined the complete sequence and organization of the genome of a putative member of the genus Polerovirus tentatively named Pepper yellow leaf curl virus (PYLCV). PYLCV has a wider host range than Tobacco vein-distorting virus (TVDV) and has a close serological relationship with Cucurbit aphid-borne yellows virus (CABYV) (both poleroviruses). The extracted viral RNA was subjected to SOLiD next-generation sequence analysis and used as a template for reverse transcription synthesis, which was followed by PCR amplification. The ssRNA genome of PYLCV includes 6,028 nucleotides encoding six open reading frames (ORFs), which is typical of the genus Polerovirus. Comparisons of the deduced amino acid sequences of the PYLCV ORFs 2-4 and ORF5, indicate that there are high levels of similarity between these sequences to ORFs 2-4 of TVDV (84-93%) and to ORF5 of CABYV (87%). Both PYLCV and Pepper vein yellowing virus (PeVYV) contain sequences that point to a common ancestral polerovirus. The recombination breakpoint which is located at CABYV ORF3, which encodes the viral coat protein (CP), may explain the CABYV-like sequences found in the genomes of the pepper infecting viruses PYLCV and PeVYV. Two additional regions unique to PYLCV (PY1 and PY2) were identified between nucleotides 4,962 and 5,061 (ORF 5) and between positions 5,866 and 6,028 in the 3' NCR. Sequence analysis of the pepper-infecting PeVYV revealed three unique regions (Pe1-Pe3) with no similarity to other members of the genus Polerovirus. Genomic analyses of PYLCV and PeVYV suggest that the speciation of these viruses occurred through putative recombination event(s) between poleroviruses co-infecting a common host(s), resulting in the emergence of PYLCV, a novel pathogen with a wider host range. PMID:23936244

The complete genomic sequence of pepper yellow leaf curl virus (PYLCV) and its implications for our understanding of evolution dynamics in the genus polerovirus.

PubMed

Dombrovsky, Aviv; Glanz, Eyal; Lachman, Oded; Sela, Noa; Doron-Faigenboim, Adi; Antignus, Yehezkel

2013-01-01

We determined the complete sequence and organization of the genome of a putative member of the genus Polerovirus tentatively named Pepper yellow leaf curl virus (PYLCV). PYLCV has a wider host range than Tobacco vein-distorting virus (TVDV) and has a close serological relationship with Cucurbit aphid-borne yellows virus (CABYV) (both poleroviruses). The extracted viral RNA was subjected to SOLiD next-generation sequence analysis and used as a template for reverse transcription synthesis, which was followed by PCR amplification. The ssRNA genome of PYLCV includes 6,028 nucleotides encoding six open reading frames (ORFs), which is typical of the genus Polerovirus. Comparisons of the deduced amino acid sequences of the PYLCV ORFs 2-4 and ORF5, indicate that there are high levels of similarity between these sequences to ORFs 2-4 of TVDV (84-93%) and to ORF5 of CABYV (87%). Both PYLCV and Pepper vein yellowing virus (PeVYV) contain sequences that point to a common ancestral polerovirus. The recombination breakpoint which is located at CABYV ORF3, which encodes the viral coat protein (CP), may explain the CABYV-like sequences found in the genomes of the pepper infecting viruses PYLCV and PeVYV. Two additional regions unique to PYLCV (PY1 and PY2) were identified between nucleotides 4,962 and 5,061 (ORF 5) and between positions 5,866 and 6,028 in the 3' NCR. Sequence analysis of the pepper-infecting PeVYV revealed three unique regions (Pe1-Pe3) with no similarity to other members of the genus Polerovirus. Genomic analyses of PYLCV and PeVYV suggest that the speciation of these viruses occurred through putative recombination event(s) between poleroviruses co-infecting a common host(s), resulting in the emergence of PYLCV, a novel pathogen with a wider host range.

Delineation of the Caffeine C-8 Oxidation Pathway in Pseudomonas sp. Strain CBB1 via Characterization of a New Trimethyluric Acid Monooxygenase and Genes Involved in Trimethyluric Acid Metabolism

PubMed Central

Mohanty, Sujit Kumar; Yu, Chi-Li; Das, Shuvendu; Louie, Tai Man; Gakhar, Lokesh

2012-01-01

The molecular basis of the ability of bacteria to live on caffeine via the C-8 oxidation pathway is unknown. The first step of this pathway, caffeine to trimethyluric acid (TMU), has been attributed to poorly characterized caffeine oxidases and a novel quinone-dependent caffeine dehydrogenase. Here, we report the detailed characterization of the second enzyme, a novel NADH-dependent trimethyluric acid monooxygenase (TmuM), a flavoprotein that catalyzes the conversion of TMU to 1,3,7-trimethyl-5-hydroxyisourate (TM-HIU). This product spontaneously decomposes to racemic 3,6,8-trimethylallantoin (TMA). TmuM prefers trimethyluric acids and, to a lesser extent, dimethyluric acids as substrates, but it exhibits no activity on uric acid. Homology models of TmuM against uric acid oxidase HpxO (which catalyzes uric acid to 5-hydroxyisourate) reveal a much bigger and hydrophobic cavity to accommodate the larger substrates. Genes involved in the caffeine C-8 oxidation pathway are located in a 25.2-kb genomic DNA fragment of CBB1, including cdhABC (coding for caffeine dehydrogenase) and tmuM (coding for TmuM). Comparison of this gene cluster to the uric acid-metabolizing gene cluster and pathway of Klebsiella pneumoniae revealed two major open reading frames coding for the conversion of TM-HIU to S-(+)-trimethylallantoin [S-(+)-TMA]. The first one, designated tmuH, codes for a putative TM-HIU hydrolase, which catalyzes the conversion of TM-HIU to 3,6,8-trimethyl-2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (TM-OHCU). The second one, designated tmuD, codes for a putative TM-OHCU decarboxylase which catalyzes the conversion of TM-OHCU to S-(+)-TMA. Based on a combination of enzymology and gene-analysis, a new degradative pathway for caffeine has been proposed via TMU, TM-HIU, TM-OHCU to S-(+)-TMA. PMID:22609920

Molecular Cloning and Characterization of an Acetylcholinesterase cDNA in the Brown Planthopper, Nilaparvata lugens

PubMed Central

Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

2010-01-01

A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain. PMID:20874389

Identification of a Linked Set of Genes in Serpulina hyodysenteriae (B204) Predicted To Encode Closely Related 39-Kilodalton Extracytoplasmic Proteins

PubMed Central

Gabe, Jeffrey D.; Dragon, Elizabeth; Chang, Ray-Jen; McCaman, Michael T.

1998-01-01

A tandem pair of nearly identical genes from Serpulina hyodysenteriae (B204) were cloned and sequenced. The full open reading frame of one gene and the partial open reading frame of the neighboring gene appear to encode secreted proteins which are homologous to, yet distinct from, the 39-kDa extracytoplasmic protein purified from the membrane fraction of S. hyodysenteriae. We have designated these newly identified genes vspA and vspB (for variable surface protein). PMID:9440540

An Expert System Interfaced with a Database System to Perform Troubleshooting of Aircraft Carrier Piping Systems

DTIC Science & Technology

1988-12-01

interval of four feet, and are numbered sequentially bow to stem. * "wing tank" is a tank or void, outboard of the holding bulkhead, away from the center...system and DBMS simultaneously with a multi-processor, allowing queries to the DBMS without terminating the expert system. This method was judged...RECIRC). eductor -strip("Y"):- ask _ques _read_ans(OVBD,"ovbd dis open"),ovbd dis-open(OVBD). eductor-strip("N"):- ask_ques read_ans( LINEUP , "strip lineup

Marco Said I Look like Charcoal: A Puerto Rican's Exploration of Her Ethnic Identity

ERIC Educational Resources Information Center

Vargas, Yarma Velazquez

2008-01-01

When race is so self-evident that it becomes "unspoken," it becomes a text that can be arbitrarily read or not read at all. When race is thought of as shades or textures of color that paint the already loaded canvas of our ethnicities, it is the reader who has the power of interpretation, opening the possibility for multiple readings that will…

Terrain - Umbra Package v. 1.0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oppel, Fred; Hart, Brian; Rigdon, James Brian

This library contains modules that read terrain files (e.g., OpenFlight, Open Scene Graph IVE, GeoTIFF Image) and to read and manage ESRI terrain datasets. All data is stored and managed in Open Scene Graph (OSG). Terrain system accesses OSG and provides elevation data, access to meta-data such as soil types and enables linears, areals and buildings to be placed in a terrain, These geometry objects include boxes, point, path, and polygon (region), and sector modules. Utilities have been made available for clamping objects to the terrain and accessing LOS information. This assertion includes a managed C++ wrapper code (TerrainWrapper) tomore » enable C# applications, such as OpShed and UTU, to incorporate this library.« less

Genome‐wide gene expression dynamics of the fungal pathogen Dothistroma septosporum throughout its infection cycle of the gymnosperm host Pinus radiata

PubMed Central

Guo, Yanan; Sim, Andre D.; Kabir, M. Shahjahan; Chettri, Pranav; Ozturk, Ibrahim K.; Hunziker, Lukas; Ganley, Rebecca J.; Cox, Murray P.

2015-01-01

Summary We present genome‐wide gene expression patterns as a time series through the infection cycle of the fungal pine needle blight pathogen, Dothistroma septosporum, as it invades its gymnosperm host, Pinus radiata. We determined the molecular changes at three stages of the disease cycle: epiphytic/biotrophic (early), initial necrosis (mid) and mature sporulating lesion (late). Over 1.7 billion combined plant and fungal reads were sequenced to obtain 3.2 million fungal‐specific reads, which comprised as little as 0.1% of the sample reads early in infection. This enriched dataset shows that the initial biotrophic stage is characterized by the up‐regulation of genes encoding fungal cell wall‐modifying enzymes and signalling proteins. Later necrotrophic stages show the up‐regulation of genes for secondary metabolism, putative effectors, oxidoreductases, transporters and starch degradation. This in‐depth through‐time transcriptomic study provides our first snapshot of the gene expression dynamics that characterize infection by this fungal pathogen in its gymnosperm host. PMID:25919703

De Novo Assembly and Characterization of the Transcriptome of the Chinese Medicinal Herb, Gentiana rigescens

PubMed Central

Zhang, Xiaodong; Allan, Andrew C.; Li, Caixia; Wang, Yuanzhong; Yao, Qiuyang

2015-01-01

Gentiana rigescens is an important medicinal herb in China. The main validated medicinal component gentiopicroside is synthesized in shoots, but is mainly found in the plant’s roots. The gentiopicroside biosynthetic pathway and its regulatory control remain to be elucidated. Genome resources of gentian are limited. Next-generation sequencing (NGS) technologies can aid in supplying global gene expression profiles. In this study we present sequence and transcript abundance data for the root and leaf transcriptome of G. rigescens, obtained using the Illumina Hiseq2000. Over fifty million clean reads were obtained from leaf and root libraries. This yields 76,717 unigenes with an average length of 753 bp. Among these, 33,855 unigenes were identified as putative homologs of annotated sequences in public protein and nucleotide databases. Digital abundance analysis identified 3306 unigenes differentially enriched between leaf and root. Unigenes found in both tissues were categorized according to their putative functional categories. Of the differentially expressed genes, over 130 were annotated as related to terpenoid biosynthesis. This work is the first study of global transcriptome analyses in gentian. These sequences and putative functional data comprise a resource for future investigation of terpenoid biosynthesis in Gentianaceae species and annotation of the gentiopicroside biosynthetic pathway and its regulatory mechanisms. PMID:26006235

Literature Especially with Remedial Readers (Open to Suggestion).

ERIC Educational Resources Information Center

Glowacki, Joan

1990-01-01

Argues that incorporating literature into daily reading instruction opens up avenues that junior high remedial students may never think exist for them. Presents eight techniques to help teachers get started using literature in the remedial classroom. (RS)

Examinations and Grading: The System of Open-Book Examination is Excellent

ERIC Educational Resources Information Center

Ching-yen, Chang

1973-01-01

Positive support for the system of open-book examination is presented. A case study of an open-book examination in organic chemistry is cited as a basis for theories that testing method improves capacity for analyzing and solving problems, deepens knowledge, and helps to overcome shortcomings of casual readings. (SM)

The Impact of Open Licensing on the Early Reader Ecosystem

ERIC Educational Resources Information Center

Butcher, Neil; Hoosen, Sarah; Levey, Lisbeth; Moore, Derek

2016-01-01

The Impact of Open Licensing on the Early Reader Ecosystem examines how to use open licensing to promote quality learning resources for young children that are relevant and interesting. Research in early reading tends to focus on traditional publishing value and supply chains, without taking much consideration of new approaches and solutions…

Balancing Open Access with Academic Standards: Implications for Community College Faculty

ERIC Educational Resources Information Center

Gabbard, Anita; Mupinga, Davison M.

2013-01-01

Community colleges act as the gateway for students to higher education. Many of these colleges realize this mission through open-door policies where students lacking in basic reading, writing, and mathematics skills can enroll. But, this open-access policy often creates challenges when meeting academic standards. Based on data collected from…

Liberty Centennial Student Campaign Manual.

ERIC Educational Resources Information Center

Statue of Liberty-Ellis Island Foundation, New York, NY.

Background readings and project ideas to involve elementary and secondary school students in fundraising for the restoration of the Statue of Liberty are provided. Materials can be divided into two sections. The first section, background readings, contains President Ronald Reagan's opening remarks at the ceremony introducing the Statue of…

Continuing Literacy.

ERIC Educational Resources Information Center

Clarke, Ronald F., Ed.

The aim of this conference was to stimulate joint action among organizations involved with adult education in east and central Africa, as well as publishers, journalists, librarians and book distributors; it focused on the problems of keeping people reading and improving their ability to read in any language. Opening addresses covered reading…

Identification and Characterization of the Anti-Methicillin-Resistant Staphylococcus aureus WAP-8294A2 Biosynthetic Gene Cluster from Lysobacter enzymogenes OH11 ▿ †

PubMed Central

Zhang, Wei; Li, Yaoyao; Qian, Guoliang; Wang, Yan; Chen, Haotong; Li, Yue-Zhong; Liu, Fengquan; Shen, Yuemao; Du, Liangcheng

2011-01-01

Lysobactor enzymogenes strain OH11 is an emerging biological control agent of fungal and bacterial diseases. We recently completed its genome sequence and found it contains a large number of gene clusters putatively responsible for the biosynthesis of nonribosomal peptides and polyketides, including the previously identified antifungal dihydromaltophilin (HSAF). One of the gene clusters contains two huge open reading frames, together encoding 12 modules of nonribosomal peptide synthetases (NRPS). Gene disruption of one of the NRPS led to the disappearance of a metabolite produced in the wild type and the elimination of its antibacterial activity. The metabolite and antibacterial activity were also affected by the disruption of some of the flanking genes. We subsequently isolated this metabolite and subjected it to spectroscopic analysis. The mass spectrometry and nuclear magnetic resonance data showed that its chemical structure is identical to WAP-8294A2, a cyclic lipodepsipeptide with potent anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and currently in phase I/II clinical trials. The WAP-8294A2 biosynthetic genes had not been described previously. So far, the Gram-positive Streptomyces have been the primary source of anti-infectives. Lysobacter are Gram-negative soil/water bacteria that are genetically amendable and have not been well exploited. The WAP-8294A2 synthetase represents one of the largest NRPS complexes, consisting of 45 functional domains. The identification of these genes sets the foundation for the study of the WAP-8294A2 biosynthetic mechanism and opens the door for producing new anti-MRSA antibiotics through biosynthetic engineering in this new source of Lysobacter. PMID:21930890

Genome Sequence, Structural Proteins, and Capsid Organization of the Cyanophage Syn5: A “Horned” Bacteriophage of Marine Synechococcus

PubMed Central

Pope, Welkin H.; Weigele, Peter R.; Chang, Juan; Pedulla, Marisa L.; Ford, Michael E.; Houtz, Jennifer M.; Jiang, Wen; Chiu, Wah; Hatfull, Graham F.; Hendrix, Roger W.; King, Jonathan

2010-01-01

Marine Synechococcus spp and marine Prochlorococcus spp are numerically dominant photoautotrophs in the open oceans and contributors to the global carbon cycle. Syn5 is a short-tailed cyanophage isolated from the Sargasso Sea on Synechococcus strain WH8109. Syn5 has been grown in WH8109 to high titer in the laboratory and purified and concentrated retaining infectivity. Genome sequencing and annotation of Syn5 revealed that the linear genome is 46,214bp with a 237bp terminal direct repeat. Sixty-one open reading frames (ORFs) were identified. Based on genomic organization and sequence similarity to known protein sequences within GenBank, Syn5 shares features with T7-like phages. The presence of a putative integrase suggests access to a temperate life-cycle. Assignment of eleven ORFs to structural proteins found within the phage virion was confirmed by mass-spectrometry and N-terminal sequencing. Eight of these identified structural proteins exhibited amino acid sequence similarity to enteric phage proteins. The remaining three virion proteins did not resemble any known phage sequences in GenBank as of August 2006. Cryoelectron micrographs of purified Syn5 virions revealed that the capsid has a single “horn”, a novel fibrous structure protruding from the opposing end of the capsid from the tail of the virion. The tail appendage displayed an apparent three-fold rather than six-fold symmetry. An 18Å-resolution icosahedral reconstruction of the capsid revealed a T=7 lattice, but with an unusual pattern of surface knobs. This phage/host system should allow detailed investigation of the physiology and biochemistry of phage propagation in marine photosynthetic bacteria. PMID:17383677

Teachers' Perceptions of the Efficacy of the Open Court Program for English Proficient and English Language Learners

ERIC Educational Resources Information Center

Lee, Steven K.; Ajayi, Lasisi; Richards, Rachel

2007-01-01

The purpose of the study is to examine teachers' perceptions of the Open Court language program. Open Court is published by McGraw Hill and has been approved by the "No Child Left Behind Act" as an appropriate research-based reading program. The Open Court program was adopted as part of the efforts to provide all elementary school…

Words in Context: The Effects of Length, Frequency, and Predictability on Brain Responses During Natural Reading

PubMed Central

Schuster, Sarah; Hawelka, Stefan; Hutzler, Florian; Kronbichler, Martin; Richlan, Fabio

2016-01-01

Word length, frequency, and predictability count among the most influential variables during reading. Their effects are well-documented in eye movement studies, but pertinent evidence from neuroimaging primarily stem from single-word presentations. We investigated the effects of these variables during reading of whole sentences with simultaneous eye-tracking and functional magnetic resonance imaging (fixation-related fMRI). Increasing word length was associated with increasing activation in occipital areas linked to visual analysis. Additionally, length elicited a U-shaped modulation (i.e., least activation for medium-length words) within a brain stem region presumably linked to eye movement control. These effects, however, were diminished when accounting for multiple fixation cases. Increasing frequency was associated with decreasing activation within left inferior frontal, superior parietal, and occipito-temporal regions. The function of the latter region—hosting the putative visual word form area—was originally considered as limited to sublexical processing. An exploratory analysis revealed that increasing predictability was associated with decreasing activation within middle temporal and inferior frontal regions previously implicated in memory access and unification. The findings are discussed with regard to their correspondence with findings from single-word presentations and with regard to neurocognitive models of visual word recognition, semantic processing, and eye movement control during reading. PMID:27365297

Reading Quizzes Improve Exam Scores for Community College Students.

PubMed

Pape-Lindstrom, Pamela; Eddy, Sarah; Freeman, Scott

2018-06-01

To test the hypothesis that adding course structure may encourage self-regulated learning skills resulting in an increase in student exam performance in the community college setting, we added daily preclass online, open-book reading quizzes to an introductory biology course. We compared three control terms without reading quizzes and three experimental terms with online, open-book reading quizzes; the instructor of record, class size, and instructional time did not vary. Analyzing the Bloom's taxonomy level of a random sample of exam questions indicated a similar cognitive level of high-stakes assessments across all six terms in the study. To control for possible changes in student preparation or ability over time, we calculated each student's grade point average in courses other than biology during the term under study and included it as a predictor variable in our regression models. Our final model showed that students in the experimental terms had significantly higher exam scores than students in the control terms. This result shows that online reading quizzes can boost achievement in community college students. We also comment on the importance of discipline-based education research in community college settings and the structure of our community college/4-year institution collaboration.

Upstream open reading frames regulate the expression of the nuclear Wnt13 isoforms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang Tao; Rector, Kyle; Barnett, Corey D.

2008-02-22

Wnt proteins control cell survival and cell fate during development. Although Wnt expression is tightly regulated in a spatio-temporal manner, the mechanisms involved both at the transcriptional and translational levels are poorly defined. We have identified a downstream translation initiation codon, AUG(+74), in Wnt13B and Wnt13C mRNAs responsible for the expression of Wnt13 nuclear forms. In this report, we demonstrate that the expression of the nuclear Wnt13C form is translationally regulated in response to stress and apoptosis. Though the 5'-leaders of both Wnt13C and Wnt13B mRNAs have an inhibitory effect on translation, they did not display an internal ribosome entrymore » site activity as demonstrated by dicistronic reporter assays. However, mutations or deletions of the upstream AUG(-99) and AUG(+1) initiation codons abrogate these translation inhibitory effects, demonstrating that Wnt13C expression is controlled by upstream open reading frames. Since long 5'-untranslated region with short upstream open reading frames characterize other Wnt transcripts, our present data on the translational control of Wnt13 expression open the way to further studies on the translation control of Wnt expression as a modulator of their subcellular localization and activity.« less

A comparative analysis of minimally invasive and open spine surgery patient education resources.

PubMed

Agarwal, Nitin; Feghhi, Daniel P; Gupta, Raghav; Hansberry, David R; Quinn, John C; Heary, Robert F; Goldstein, Ira M

2014-09-01

The Internet has become a widespread source for disseminating health information to large numbers of people. Such is the case for spine surgery as well. Given the complexity of spinal surgeries, an important point to consider is whether these resources are easily read and understood by most Americans. The average national reading grade level has been estimated to be at about the 7th grade. In the present study the authors strove to assess the readability of open spine surgery resources and minimally invasive spine surgery resources to offer suggestions to help improve the readability of patient resources. Online patient education resources were downloaded in 2013 from 50 resources representing either traditional open back surgery or minimally invasive spine surgery. Each resource was assessed using 10 scales from Readability Studio Professional Edition version 2012.1. Patient education resources representing traditional open back surgery or minimally invasive spine surgery were all found to be written at a level well above the recommended 6th grade level. In general, minimally invasive spine surgery materials were written at a higher grade level. The readability of patient education resources from spine surgery websites exceeds the average reading ability of an American adult. Revisions may be warranted to increase quality and patient comprehension of these resources to effectively reach a greater patient population.

A retrotransposable element from the mosquito Anopheles gambiae .

PubMed Central

Besansky, N J

1990-01-01

A family of middle repetitive elements from the African malaria vector Anopheles gambiae is described. Approximately 100 copies of the element, designated T1Ag, are dispersed in the genome. Full-length elements are 4.6 kilobase pairs in length, but truncation of the 5' end is common. Nucleotide sequences of one full-length, two 5'-truncated, and two 5' ends of T1Ag elements were determined and aligned to define a consensus sequence. Sequence analysis revealed two long, overlapping open reading frames followed by a polyadenylation signal, AATAAA, and a tail consisting of tandem repetitions of the motif TGAAA. No direct or inverted long terminal repeats (LTRs) were detected. The first open reading frame, 442 amino acids in length, includes a domain resembling that of nucleic acid-binding proteins. The second open reading frame, 975 amino acids long, resembles the reverse transcriptases of a category of retrotransposable elements without LTRs, variously termed class II retrotransposons, class III elements or non-LTR retrotransposons. Similarity at the sequence and structural levels places T1Ag in this category. Images PMID:1689457

A versatile and efficient high-throughput cloning tool for structural biology.

PubMed

Geertsma, Eric R; Dutzler, Raimund

2011-04-19

Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.

An All-School Library Challenge

ERIC Educational Resources Information Center

Quirk, Connie

2005-01-01

The library media center is hosting an all-school team challenge, designed to celebrate reading and library skills. Students could choose from the contest categories like "Lord of the Rings", "Harry Potter", Author Facts Challenge and Opening Lines Challenge for the competition and those students who read more challenging books show their…

Connecting Libraries and Schools with CLASP.

ERIC Educational Resources Information Center

Del Vecchio, Stephen

1993-01-01

Describes the Dewitt Wallace-Reader's Digest Connecting Libraries and Schools Project (CLASP) of the New York Public Library, a cooperative pilot project to encourage reading among children and youth. Sample projects described include summer reading lists, open school night outreach, and outreach to parents. The importance of materials support is…

75 FR 77697 - Action To Ensure Authority To Issue Permits Under the Prevention of Significant Deterioration...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-13

... Reading Room is open from 8:30 a.m. to 4:30 p.m., Monday through Friday, excluding legal holidays. The telephone number for the Public Reading Room is (202) 566-1744, and the telephone number for the Air Docket...

Developing Literacy for the Vocational-Technical Trades.

ERIC Educational Resources Information Center

Penisten, John

The University of Hawaii at Hilo offers a vocational-technical occupational reading curriculum that encourages literacy for technology. The program is geared to meet the special needs of enrollees in the university's trade and industry division, most of whom are postsecondary open-admission students. Most have poor reading skills, averaging a…

Opening a Window into Reading Development: Eye Movements' Role within a Broader Literacy Research Framework

ERIC Educational Resources Information Center

Miller, Brett; O'Donnell, Carol

2013-01-01

insight into how readers process text. The heart of this work spans roughly 40 years reflecting the maturity of both the topics under study and experimental approaches used to investigate reading. Recent technological advancements offer increased flexibility to the field, providing…

INSaFLU: an automated open web-based bioinformatics suite "from-reads" for influenza whole-genome-sequencing-based surveillance.

PubMed

Borges, Vítor; Pinheiro, Miguel; Pechirra, Pedro; Guiomar, Raquel; Gomes, João Paulo

2018-06-29

A new era of flu surveillance has already started based on the genetic characterization and exploration of influenza virus evolution at whole-genome scale. Although this has been prioritized by national and international health authorities, the demanded technological transition to whole-genome sequencing (WGS)-based flu surveillance has been particularly delayed by the lack of bioinformatics infrastructures and/or expertise to deal with primary next-generation sequencing (NGS) data. We developed and implemented INSaFLU ("INSide the FLU"), which is the first influenza-oriented bioinformatics free web-based suite that deals with primary NGS data (reads) towards the automatic generation of the output data that are actually the core first-line "genetic requests" for effective and timely influenza laboratory surveillance (e.g., type and sub-type, gene and whole-genome consensus sequences, variants' annotation, alignments and phylogenetic trees). By handling NGS data collected from any amplicon-based schema, the implemented pipeline enables any laboratory to perform multi-step software intensive analyses in a user-friendly manner without previous advanced training in bioinformatics. INSaFLU gives access to user-restricted sample databases and projects management, being a transparent and flexible tool specifically designed to automatically update project outputs as more samples are uploaded. Data integration is thus cumulative and scalable, fitting the need for a continuous epidemiological surveillance during the flu epidemics. Multiple outputs are provided in nomenclature-stable and standardized formats that can be explored in situ or through multiple compatible downstream applications for fine-tuned data analysis. This platform additionally flags samples as "putative mixed infections" if the population admixture enrolls influenza viruses with clearly distinct genetic backgrounds, and enriches the traditional "consensus-based" influenza genetic characterization with relevant data on influenza sub-population diversification through a depth analysis of intra-patient minor variants. This dual approach is expected to strengthen our ability not only to detect the emergence of antigenic and drug resistance variants but also to decode alternative pathways of influenza evolution and to unveil intricate routes of transmission. In summary, INSaFLU supplies public health laboratories and influenza researchers with an open "one size fits all" framework, potentiating the operationalization of a harmonized multi-country WGS-based surveillance for influenza virus. INSaFLU can be accessed through https://insaflu.insa.pt .

Transcription and Regulation of the Bidirectional Hydrogenase in the Cyanobacterium Nostoc sp. Strain PCC 7120▿

PubMed Central

Sjöholm, Johannes; Oliveira, Paulo; Lindblad, Peter

2007-01-01

The filamentous, heterocystous cyanobacterium Nostoc sp. strain PCC 7120 (Anabaena sp. strain PCC 7120) possesses an uptake hydrogenase and a bidirectional enzyme, the latter being capable of catalyzing both H2 production and evolution. The completely sequenced genome of Nostoc sp. strain PCC 7120 reveals that the five structural genes encoding the bidirectional hydrogenase (hoxEFUYH) are separated in two clusters at a distance of approximately 8.8 kb. The transcription of the hox genes was examined under nitrogen-fixing conditions, and the results demonstrate that the cluster containing hoxE and hoxF can be transcribed as one polycistronic unit together with the open reading frame alr0750. The second cluster, containing hoxU, hoxY, and hoxH, is transcribed together with alr0763 and alr0765, located between the hox genes. Moreover, alr0760 and alr0761 form an additional larger operon. Nevertheless, Northern blot hybridizations revealed a rather complex transcription pattern in which the different hox genes are expressed differently. Transcriptional start points (TSPs) were identified 66 and 57 bp upstream from the start codon of alr0750 and hoxU, respectively. The transcriptions of the two clusters containing the hox genes are both induced under anaerobic conditions concomitantly with the induction of a higher level of hydrogenase activity. An additional TSP, within the annotated alr0760, 244 bp downstream from the suggested translation start codon, was identified. Electrophoretic mobility shift assays with purified LexA from Nostoc sp. strain PCC 7120 demonstrated specific interactions between the transcriptional regulator and both hox promoter regions. However, when LexA from Synechocystis sp. strain PCC 6803 was used, the purified protein interacted only with the promoter region of the alr0750-hoxE-hoxF operon. A search of the whole Nostoc sp. strain PCC 7120 genome demonstrated the presence of 216 putative LexA binding sites in total, including recA and recF. This indicates that, in addition to the bidirectional hydrogenase gene, a number of other genes, including open reading frames connected to DNA replication, recombination, and repair, may be part of the LexA regulatory network in Nostoc sp. strain PCC 7120. PMID:17630298

Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida.

PubMed Central

Irie, S; Doi, S; Yorifuji, T; Takagi, M; Yano, K

1987-01-01

The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined. Five open reading frames were found in the sequence. Four corresponding protein molecules were detected by a DNA-directed in vitro translation system. Escherichia coli cells containing the fragment with the four open reading frames transformed benzene to cis-benzene glycol, which is an intermediate of the oxidation of benzene to catechol. The relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed. Images PMID:3667527

An in silico pipeline to filter the Toxoplasma gondii proteome for proteins that could traffic to the host cell nucleus and influence host cell epigenetic regulation.

PubMed

Syn, Genevieve; Blackwell, Jenefer M; Jamieson, Sarra E; Francis, Richard W

2018-01-01

Toxoplasma gondii uses epigenetic mechanisms to regulate both endogenous and host cell gene expression. To identify genes with putative epigenetic functions, we developed an in silico pipeline to interrogate the T. gondii proteome of 8313 proteins. Step 1 employs PredictNLS and NucPred to identify genes predicted to target eukaryotic nuclei. Step 2 uses GOLink to identify proteins of epigenetic function based on Gene Ontology terms. This resulted in 611 putative nuclear localised proteins with predicted epigenetic functions. Step 3 filtered for secretory proteins using SignalP, SecretomeP, and experimental data. This identified 57 of the 611 putative epigenetic proteins as likely to be secreted. The pipeline is freely available online, uses open access tools and software with user-friendly Perl scripts to automate and manage the results, and is readily adaptable to undertake any such in silico search for genes contributing to particular functions.

Photometry Using Kepler "Superstamps" of Open Clusters NGC 6791 & NGC 6819

NASA Astrophysics Data System (ADS)

Kuehn, Charles A.; Drury, Jason A.; Bellamy, Beau R.; Stello, Dennis; Bedding, Timothy R.; Reed, Mike; Quick, Breanna

2015-09-01

The Kepler space telescope has proven to be a gold mine for the study of variable stars. Usually, Kepler only reads out a handful of pixels around each pre-selected target star, omitting a large number of stars in the Kepler field. Fortunately, for the open clusters NGC 6791 and NGC 6819, Kepler also read out larger "superstamps" which contained complete images of the central region of each cluster. These cluster images can be used to study additional stars in the open clusters that were not originally on Kepler's target list. We discuss our work on using two photometric techniques to analyze these superstamps and present sample results from this project to demonstrate the value of this technique for a wide variety of variable stars.

De novo Genome Assembly of the Fungal Plant Pathogen Pyrenophora semeniperda

PubMed Central

Soliai, Marcus M.; Meyer, Susan E.; Udall, Joshua A.; Elzinga, David E.; Hermansen, Russell A.; Bodily, Paul M.; Hart, Aaron A.; Coleman, Craig E.

2014-01-01

Pyrenophora semeniperda (anamorph Drechslera campulata) is a necrotrophic fungal seed pathogen that has a wide host range within the Poaceae. One of its hosts is cheatgrass (Bromus tectorum), a species exotic to the United States that has invaded natural ecosystems of the Intermountain West. As a natural pathogen of cheatgrass, P. semeniperda has potential as a biocontrol agent due to its effectiveness at killing seeds within the seed bank; however, few genetic resources exist for the fungus. Here, the genome of P. semeniperda isolate assembled from sequence reads of 454 pyrosequencing is presented. The total assembly is 32.5 Mb and includes 11,453 gene models encoding putative proteins larger than 24 amino acids. The models represent a variety of putative genes that are involved in pathogenic pathways typically found in necrotrophic fungi. In addition, extensive rearrangements, including inter- and intrachromosomal rearrangements, were found when the P. semeniperda genome was compared to P. tritici-repentis, a related fungal species. PMID:24475219