The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

PubMed Central

Romero, Lisa C; Nguyen, Thanh V; Deville, Benoit; Ogunjumo, Oluwasanmi; James, Anthony A

2004-01-01

Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development. PMID:15222903

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.

A proposed model for the flowering signaling pathway of sugarcane under photoperiodic control.

PubMed

Coelho, C P; Costa Netto, A P; Colasanti, J; Chalfun-Júnior, A

2013-04-25

Molecular analysis of floral induction in Arabidopsis has identified several flowering time genes related to 4 response networks defined by the autonomous, gibberellin, photoperiod, and vernalization pathways. Although grass flowering processes include ancestral functions shared by both mono- and dicots, they have developed their own mechanisms to transmit floral induction signals. Despite its high production capacity and its important role in biofuel production, almost no information is available about the flowering process in sugarcane. We searched the Sugarcane Expressed Sequence Tags database to look for elements of the flowering signaling pathway under photoperiodic control. Sequences showing significant similarity to flowering time genes of other species were clustered, annotated, and analyzed for conserved domains. Multiple alignments comparing the sequences found in the sugarcane database and those from other species were performed and their phylogenetic relationship assessed using the MEGA 4.0 software. Electronic Northerns were run with Cluster and TreeView programs, allowing us to identify putative members of the photoperiod-controlled flowering pathway of sugarcane.

Putative recombination events and evolutionary history of five economically important viruses of fruit trees based on coat protein-encoding gene sequence analysis.

PubMed

Boulila, Moncef

2010-06-01

To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31beta algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.

AT-rich sequence elements promote nascent transcript cleavage leading to RNA polymerase II termination

PubMed Central

White, Eleanor; Kamieniarz-Gdula, Kinga; Dye, Michael J.; Proudfoot, Nick J.

2013-01-01

RNA Polymerase II (Pol II) termination is dependent on RNA processing signals as well as specific terminator elements located downstream of the poly(A) site. One of the two major terminator classes described so far is the Co-Transcriptional Cleavage (CoTC) element. We show that homopolymer A/T tracts within the human β-globin CoTC-mediated terminator element play a critical role in Pol II termination. These short A/T tracts, dispersed within seemingly random sequences, are strong terminator elements, and bioinformatics analysis confirms the presence of such sequences in 70% of the putative terminator regions (PTRs) genome-wide. PMID:23258704

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.

Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

USDA-ARS?s Scientific Manuscript database

The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

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

A comparative genomics perspective on the genetic content of the alkaliphilic haloarchaeon Natrialba magadii ATCC 43099T

PubMed Central

2012-01-01

Background Natrialba magadii is an aerobic chemoorganotrophic member of the Euryarchaeota and is a dual extremophile requiring alkaline conditions and hypersalinity for optimal growth. The genome sequence of Nab. magadii type strain ATCC 43099 was deciphered to obtain a comprehensive insight into the genetic content of this haloarchaeon and to understand the basis of some of the cellular functions necessary for its survival. Results The genome of Nab. magadii consists of four replicons with a total sequence of 4,443,643 bp and encodes 4,212 putative proteins, some of which contain peptide repeats of various lengths. Comparative genome analyses facilitated the identification of genes encoding putative proteins involved in adaptation to hypersalinity, stress response, glycosylation, and polysaccharide biosynthesis. A proton-driven ATP synthase and a variety of putative cytochromes and other proteins supporting aerobic respiration and electron transfer were encoded by one or more of Nab. magadii replicons. The genome encodes a number of putative proteases/peptidases as well as protein secretion functions. Genes encoding putative transcriptional regulators, basal transcription factors, signal perception/transduction proteins, and chemotaxis/phototaxis proteins were abundant in the genome. Pathways for the biosynthesis of thiamine, riboflavin, heme, cobalamin, coenzyme F420 and other essential co-factors were deduced by in depth sequence analyses. However, approximately 36% of Nab. magadii protein coding genes could not be assigned a function based on Blast analysis and have been annotated as encoding hypothetical or conserved hypothetical proteins. Furthermore, despite extensive comparative genomic analyses, genes necessary for survival in alkaline conditions could not be identified in Nab. magadii. Conclusions Based on genomic analyses, Nab. magadii is predicted to be metabolically versatile and it could use different carbon and energy sources to sustain growth. Nab. magadii has the genetic potential to adapt to its milieu by intracellular accumulation of inorganic cations and/or neutral organic compounds. The identification of Nab. magadii genes involved in coenzyme biosynthesis is a necessary step toward further reconstruction of the metabolic pathways in halophilic archaea and other extremophiles. The knowledge gained from the genome sequence of this haloalkaliphilic archaeon is highly valuable in advancing the applications of extremophiles and their enzymes. PMID:22559199

Cloning and sequence analysis of a cDNA encoding the alpha-subunit of mouse beta-N-acetylhexosaminidase and comparison with the human enzyme.

PubMed Central

Beccari, T; Hoade, J; Orlacchio, A; Stirling, J L

1992-01-01

cDNAs encoding the mouse beta-N-acetylhexosaminidase alpha-subunit were isolated from a mouse testis library. The longest of these (1.7 kb) was sequenced and showed 83% similarity with the human alpha-subunit cDNA sequence. The 5' end of the coding sequence was obtained from a genomic DNA clone. Alignment of the human and mouse sequences showed that all three putative N-glycosylation sites are conserved, but that the mouse alpha-subunit has an additional site towards the C-terminus. All eight cysteines in the human sequence are conserved in the mouse. There are an additional two cysteines in the mouse alpha-subunit signal peptide. All amino acids affected in Tay-Sachs-disease mutations are conserved in the mouse. Images Fig. 1. PMID:1379046

Two different groups of signal sequence in M-superfamily conotoxins.

PubMed

Wang, Qi; Jiang, Hui; Han, Yu-Hong; Yuan, Duo-Duo; Chi, Cheng-Wu

2008-04-01

M-superfamily conotoxins can be divided into four branches (M-1, M-2, M-3 and M-4) according to the number of amino acid residues in the third Cys loop. In general, it is widely accepted that the conotoxin signal peptides of each superfamily are strictly conserved. Recently, we cloned six cDNAs of novel M-superfamily conotoxins from Conus leopardus, Conus marmoreus and Conus quercinus, belonging to either M-1 or M-3 branch. These conotoxins, judging from the putative peptide sequences deducted from cDNAs, are rich in acidic residues and share highly conserved signal and pro-peptide region. However, they are quite different from the reported conotoxins of M-2 and M-4 branches even in their signal peptides, which in general are considered highly conserved for each superfamily of conotoxins. The signal sequences of M-1 and M-3 conotoxins composed of 24 residues start with MLKMGVVL-, while those of M-2 and M-4 conotoxins composed of 25 residues start with MMSKLGVL-. It is another example that different types of signal peptides can exist within a superfamily besides the I-conotoxin superfamily. In addition to the different disulfide connectivity of M-1 conotoxins from that of M-4 or M-2 conotoxins, the sequence alignment, preferential Cys codon usage and phylogenetic tree analysis suggest that M-1 and M-3 conotoxins have much closer relationship, being different from the conotoxins of other two branches (M-4 and M-2) of M-superfamily.

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

Molecular identification of aiiA homologous gene from endophytic Enterobacter species and in silico analysis of putative tertiary structure of AHL-lactonase.

PubMed

Rajesh, P S; Rai, V Ravishankar

2014-01-03

The aiiA homologous gene known to encode AHL- lactonase enzyme which hydrolyze the N-acylhomoserine lactone (AHL) quorum sensing signaling molecules produced by Gram negative bacteria. In this study, the degradation of AHL molecules was determined by cell-free lysate of endophytic Enterobacter species. The percentage of quorum quenching was confirmed and quantified by HPLC method (p<0.0001). Amplification and sequence BLAST analysis showed the presence of aiiA homologous gene in endophytic Enterobacter asburiae VT65, Enterobacter aerogenes VT66 and Enterobacter ludwigii VT70 strains. Sequence alignment analysis revealed the presence of two zinc binding sites, "HXHXDH" motif as well as tyrosine residue at the position 194. Based on known template available at Swiss-Model, putative tertiary structure of AHL-lactonase was constructed. The result showed that novel endophytic strains of Enterobacter genera encode the novel aiiA homologous gene and its structural importance for future study. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular characterization and genomic distribution of Isis: a new retrotransposon of Drosophila buzzatii.

PubMed

García Guerreiro, M P; Fontdevila, A

2007-01-01

A new transposable element, Isis, is identified as a LTR retrotransposon in Drosophila buzzatii. DNA sequence analysis shows that Isis contains three long ORFs similar to gag, pol and env genes of retroviruses. The ORF1 exhibits sequence homology to matrix, capsid and nucleocapsid gag proteins and ORF2 encodes a putative protease (PR), a reverse transcriptase (RT), an Rnase H (RH) and an integrase (IN) region. The analysis of a putative env product, encoded by the env ORF3, shows a degenerated protein containing several stop codons. The molecular study of the putative proteins coded by this new element shows striking similarities to both Ulysses and Osvaldo elements, two LTR retrotransposons, present in D. virilis and D. buzzatii, respectively. Comparisons of the predicted Isis RT to several known retrotransposons show strong phylogenetic relationships to gypsy-like elements, particulary to Ulysses retrotransposon. Studies of Isis chromosomal distribution show a strong hybridization signal in centromeric and pericentromeric regions, and a scattered distribution along all chromosomal arms. The existence of insertional polymorphisms between different strains and high molecular weight bands by Southern blot suggests the existence of full-sized copies that have been active recently. The presence of euchromatic insertion sites coincident between Isis and Osvaldo could indicate preferential insertion sites of Osvaldo element into Isis sequence or vice versa. Moreover, the presence of Isis in different species of the buzzatii complex indicates the ancient origin of this element.

C-Mannosylation of thrombopoietin receptor (c-Mpl) regulates thrombopoietin-dependent JAK-STAT signaling.

PubMed

Sasazawa, Yukiko; Sato, Natsumi; Suzuki, Takehiro; Dohmae, Naoshi; Simizu, Siro

The thrombopoietin receptor, also known as c-Mpl, is a member of the cytokine superfamily, which regulates the differentiation of megakaryocytes and formation of platelets by binding to its ligand, thrombopoietin (TPO), through Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. The loss-of-function mutations of c-Mpl cause severe thrombocytopenia due to impaired megakaryocytopoiesis, and gain-of-function mutations cause thrombocythemia. c-Mpl contains two Trp-Ser-Xaa-Trp-Ser (Xaa represents any amino acids) sequences, which are characteristic sequences of type I cytokine receptors, corresponding to C-mannosylation consensus sequences: Trp-Xaa-Xaa-Trp/Cys. C-mannosylation is a post-translational modification of tryptophan residue in which one mannose is attached to the first tryptophan residue in the consensus sequence via C-C linkage. Although c-Mpl contains some C-mannosylation sequences, whether c-Mpl is C-mannosylated or not has been uninvestigated. We identified that c-Mpl is C-mannosylated not only at Trp(269) and Trp(474), which are putative C-mannosylation site, but also at Trp(272), Trp(416), and Trp(477). Using C-mannosylation defective mutant of c-Mpl, the C-mannosylated tryptophan residues at four sites (Trp(269), Trp(272), Trp(474), and Trp(477)) are essential for c-Mpl-mediated JAK-STAT signaling. Our findings suggested that C-mannosylation of c-Mpl is a possible therapeutic target for platelet disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae.

PubMed

Albornos, Lucía; Martín, Ignacio; Iglesias, Rebeca; Jiménez, Teresa; Labrador, Emilia; Dopico, Berta

2012-11-07

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

PubMed Central

2012-01-01

Background Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. Results ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. Conclusions We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found. PMID:23134664

Effector profiles distinguish formae speciales of Fusarium oxysporum.

PubMed

van Dam, Peter; Fokkens, Like; Schmidt, Sarah M; Linmans, Jasper H J; Kistler, H Corby; Ma, Li-Jun; Rep, Martijn

2016-11-01

Formae speciales (ff.spp.) of the fungus Fusarium oxysporum are often polyphyletic within the species complex, making it impossible to identify them on the basis of conserved genes. However, sequences that determine host-specific pathogenicity may be expected to be similar between strains within the same forma specialis. Whole genome sequencing was performed on strains from five different ff.spp. (cucumerinum, niveum, melonis, radicis-cucumerinum and lycopersici). In each genome, genes for putative effectors were identified based on small size, secretion signal, and vicinity to a "miniature impala" transposable element. The candidate effector genes of all genomes were collected and the presence/absence patterns in each individual genome were clustered. Members of the same forma specialis turned out to group together, with cucurbit-infecting strains forming a supercluster separate from other ff.spp. Moreover, strains from different clonal lineages within the same forma specialis harbour identical effector gene sequences, supporting horizontal transfer of genetic material. These data offer new insight into the genetic basis of host specificity in the F. oxysporum species complex and show that (putative) effectors can be used to predict host specificity in F. oxysporum. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

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.

Structure and stability of the ankyrin domain of the Drosophila Notch receptor.

PubMed

Zweifel, Mark E; Leahy, Daniel J; Hughson, Frederick M; Barrick, Doug

2003-11-01

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.

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

Comparative analysis of programmed cell death pathways in filamentous fungi.

PubMed

Fedorova, Natalie D; Badger, Jonathan H; Robson, Geoff D; Wortman, Jennifer R; Nierman, William C

2005-12-08

Fungi can undergo autophagic- or apoptotic-type programmed cell death (PCD) on exposure to antifungal agents, developmental signals, and stress factors. Filamentous fungi can also exhibit a form of cell death called heterokaryon incompatibility (HI) triggered by fusion between two genetically incompatible individuals. With the availability of recently sequenced genomes of Aspergillus fumigatus and several related species, we were able to define putative components of fungi-specific death pathways and the ancestral core apoptotic machinery shared by all fungi and metazoa. Phylogenetic profiling of HI-associated proteins from four Aspergilli and seven other fungal species revealed lineage-specific protein families, orphan genes, and core genes conserved across all fungi and metazoa. The Aspergilli-specific domain architectures include NACHT family NTPases, which may function as key integrators of stress and nutrient availability signals. They are often found fused to putative effector domains such as Pfs, SesB/LipA, and a newly identified domain, HET-s/LopB. Many putative HI inducers and mediators are specific to filamentous fungi and not found in unicellular yeasts. In addition to their role in HI, several of them appear to be involved in regulation of cell cycle, development and sexual differentiation. Finally, the Aspergilli possess many putative downstream components of the mammalian apoptotic machinery including several proteins not found in the model yeast, Saccharomyces cerevisiae. Our analysis identified more than 100 putative PCD associated genes in the Aspergilli, which may help expand the range of currently available treatments for aspergillosis and other invasive fungal diseases. The list includes species-specific protein families as well as conserved core components of the ancestral PCD machinery shared by fungi and metazoa.

Pro-apoptotic effect of a Mycoplasma hyopneumoniae putative type I signal peptidase on PK(15) swine cells.

PubMed

Paes, Jéssica A; Virginio, Veridiana G; Cancela, Martín; Leal, Fernanda M A; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Schrank, Irene S; Ferreira, Henrique B

2017-03-01

Mycoplasma hyopneumoniae is an economically significant swine pathogen that causes porcine enzootic pneumonia (PEP). Important processes for swine infection by M. hyopneumoniae depend on cell surface proteins, many of which are secreted by secretion pathways not completely elucidated so far. A putative type I signal peptidase (SPase I), a possible component of a putative Sec-dependent pathway, was annotated as a product of the sipS gene in the pathogenic M. hyopneumoniae 7448 genome. This M. hyopneumoniae putative SPase I (MhSPase I) displays only 14% and 23% of sequence identity/similarity to Escherichia coli bona fide SPase I, and, in complementation assays performed with a conditional E. coli SPase I mutant, only a partial restoration of growth was achieved with the heterologous expression of a recombinant MhSPase I (rMhSPase I). Considering the putative surface location of MhSPase I and its previously demonstrated capacity to induce a strong humoral response, we then assessed its potential to elicit a cellular and possible immunomodulatory response. In assays for immunogenicity assessment, rMhSPase I unexpectedly showed a cytotoxic effect on murine splenocytes. This cytotoxic effect was further confirmed using the swine epithelial PK(15) cell line in MTT and annexin V-flow cytometry assays, which showed that rMhSPase I induces apoptosis in a dose dependent-way. It was also demonstrated that this pro-apoptotic effect of rMhSPase I involves activation of a caspase-3 cascade. The potential relevance of the rMhSPase I pro-apoptotic effect for M. hyopneumoniae-host interactions in the context of PEP is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Ebola virus encodes a miR-155 analog to regulate importin-α5 expression.

PubMed

Liu, Yuanwu; Sun, Jing; Zhang, Hongwen; Wang, Mingming; Gao, George Fu; Li, Xiangdong

2016-10-01

The 2014 outbreak of Ebola virus caused more than 10,000 human deaths. Current knowledge of suitable drugs, clinical diagnostic biomarkers and molecular mechanisms of Ebola virus infection is either absent or insufficient. By screening stem-loop structures from the viral genomes of four virulence species, we identified a novel, putative viral microRNA precursor that is specifically expressed by the Ebola virus. The sequence of the microRNA precursor was further confirmed by mining the existing RNA-Seq database. Two putative mature microRNAs were predicted and subsequently validated in human cell lines. Combined with this prediction of the microRNA target, we identified importin-α5, which is a key regulator of interferon signaling following Ebola virus infection, as one putative target. We speculate that this microRNA could facilitate the evasion of the host immune system by the virus. Moreover, this microRNA might be a potential clinical therapeutic target or a diagnostic biomarker for Ebola virus.

Molecular characterization of infectious pancreatic necrosis virus strains isolated from the three types of salmonids farmed in Chile.

PubMed

Manríquez, René A; Vera, Tamara; Villalba, Melina V; Mancilla, Alejandra; Vakharia, Vikram N; Yañez, Alejandro J; Cárcamo, Juan G

2017-01-31

The infectious pancreatic necrosis virus (IPNV) causes significant economic losses in Chilean salmon farming. For effective sanitary management, the IPNV strains present in Chile need to be fully studied, characterized, and constantly updated at the molecular level. In this study, 36 Chilean IPNV isolates collected over 6 years (2006-2011) from Salmo salar, Oncorhynchus mykiss, and Oncorhynchus kisutch were genotypically characterized. Salmonid samples were obtained from freshwater, estuary, and seawater sources from central, southern, and the extreme-south of Chile (35° to 53°S). Sequence analysis of the VP2 gene classified 10 IPNV isolates as genogroup 1 and 26 as genogroup 5. Analyses indicated a preferential, but not obligate, relationship between genogroup 5 isolates and S. salar infection. Fifteen genogroup 5 and nine genogroup 1 isolates presented VP2 gene residues associated with high virulence (i.e. Thr, Ala, and Thr at positions 217, 221, and 247, respectively). Four genogroup 5 isolates presented an oddly long VP5 deduced amino acid sequence (29.6 kDa). Analysis of the VP2 amino acid motifs associated with clinical and subclinical infections identified the clinical fingerprint in only genogroup 5 isolates; in contrast, the genogroup 1 isolates presented sequences predominantly associated with the subclinical fingerprint. Predictive analysis of VP5 showed an absence of transmembrane domains and plasma membrane tropism signals. WebLogo analysis of the VP5 BH domains revealed high identities with the marine birnavirus Y-6 and Japanese IPNV strain E1-S. Sequence analysis for putative 25 kDa proteins, coded by the ORF between VP2 and VP4, exhibited three putative nuclear localization sequences and signals of mitochondrial tropism in two isolates. This study provides important advances in updating the characterizations of IPNV strains present in Chile. The results from this study will help in identifying epidemiological links and generating specific biotechnological tools for controlling IPNV outbreaks in Chilean salmon farming.

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal

PubMed Central

Lange, Allison; McLane, Laura M.; Mills, Ryan E.; Devine, Scott E.; Corbett, Anita H.

2010-01-01

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best-characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin-α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9-12 amino acid linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 amino acid linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 amino acid linker. Our studies reveal that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition PMID:20028483

The Arabidopsis SOS5 Locus Encodes a Putative Cell Surface Adhesion Protein and Is Required for Normal Cell Expansion

PubMed Central

Shi, Huazhong; Kim, YongSig; Guo, Yan; Stevenson, Becky; Zhu, Jian-Kang

2003-01-01

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein–like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf. PMID:12509519

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.

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.

Extreme Sensory Complexity Encoded in the 10-Megabase Draft Genome Sequence of the Chromatically Acclimating Cyanobacterium Tolypothrix sp. PCC 7601

PubMed Central

Yerrapragada, Shaila; Shukla, Animesh; Hallsworth-Pepin, Kymberlie; Choi, Kwangmin; Wollam, Aye; Clifton, Sandra; Qin, Xiang; Muzny, Donna; Raghuraman, Sriram; Ashki, Haleh; Uzman, Akif; Highlander, Sarah K.; Fryszczyn, Bartlomiej G.; Fox, George E.; Tirumalai, Madhan R.; Liu, Yamei; Kim, Sun

2015-01-01

Tolypothrix sp. PCC 7601 is a freshwater filamentous cyanobacterium with complex responses to environmental conditions. Here, we present its 9.96-Mbp draft genome sequence, containing 10,065 putative protein-coding sequences, including 305 predicted two-component system proteins and 27 putative phytochrome-class photoreceptors, the most such proteins in any sequenced genome. PMID:25953173

The predicted secretome and transmembranome of the poultry red mite Dermanyssus gallinae.

PubMed

Schicht, Sabine; Qi, Weihong; Poveda, Lucy; Strube, Christina

2013-09-11

The worldwide distributed hematophagous poultry red mite Dermanyssus gallinae (De Geer, 1778) is one of the most important pests of poultry. Even though 35 acaricide compounds are available, control of D. gallinae remains difficult due to acaricide resistances as well as food safety regulations. The current study was carried out to identify putative excretory/secretory (pES) proteins of D. gallinae since these proteins play an important role in the host-parasite interaction and therefore represent potential targets for the development of novel intervention strategies. Additionally, putative transmembrane proteins (pTM) of D. gallinae were analyzed as representatives of this protein group also serve as promising targets for new control strategies. D. gallinae pES and pTM protein prediction was based on putative protein sequences of whole transcriptome data which was parsed to different bioinformatical servers (SignalP, SecretomeP, TMHMM and TargetP). Subsequently, pES and pTM protein sequences were functionally annotated by different computational tools. Computational analysis of the D. gallinae proteins identified 3,091 pES (5.6%) and 7,361 pTM proteins (13.4%). A significant proportion of pES proteins are considered to be involved in blood feeding and digestion such as salivary proteins, proteases, lipases and carbohydrases. The cysteine proteases cathepsin D and L as well as legumain, enzymes that cleave hemoglobin during blood digestion of the near related ticks, represented 6 of the top-30 BLASTP matches of the poultry red mite's secretome. Identified pTM proteins may be involved in many important biological processes including cell signaling, transport of membrane-impermeable molecules and cell recognition. Ninjurin-like proteins, whose functions in mites are still unknown, represent the most frequently occurring pTM. The current study is the first providing a mite's secretome as well as transmembranome and provides valuable insights into D. gallinae pES and pTM proteins operating in different metabolic pathways. Identifying a variety of molecules putatively involved in blood feeding may significantly contribute to the development of new therapeutic targets or vaccines against this poultry pest.

The predicted secretome and transmembranome of the poultry red mite Dermanyssus gallinae

PubMed Central

2013-01-01

Background The worldwide distributed hematophagous poultry red mite Dermanyssus gallinae (De Geer, 1778) is one of the most important pests of poultry. Even though 35 acaricide compounds are available, control of D. gallinae remains difficult due to acaricide resistances as well as food safety regulations. The current study was carried out to identify putative excretory/secretory (pES) proteins of D. gallinae since these proteins play an important role in the host-parasite interaction and therefore represent potential targets for the development of novel intervention strategies. Additionally, putative transmembrane proteins (pTM) of D. gallinae were analyzed as representatives of this protein group also serve as promising targets for new control strategies. Methods D. gallinae pES and pTM protein prediction was based on putative protein sequences of whole transcriptome data which was parsed to different bioinformatical servers (SignalP, SecretomeP, TMHMM and TargetP). Subsequently, pES and pTM protein sequences were functionally annotated by different computational tools. Results Computational analysis of the D. gallinae proteins identified 3,091 pES (5.6%) and 7,361 pTM proteins (13.4%). A significant proportion of pES proteins are considered to be involved in blood feeding and digestion such as salivary proteins, proteases, lipases and carbohydrases. The cysteine proteases cathepsin D and L as well as legumain, enzymes that cleave hemoglobin during blood digestion of the near related ticks, represented 6 of the top-30 BLASTP matches of the poultry red mite’s secretome. Identified pTM proteins may be involved in many important biological processes including cell signaling, transport of membrane-impermeable molecules and cell recognition. Ninjurin-like proteins, whose functions in mites are still unknown, represent the most frequently occurring pTM. Conclusion The current study is the first providing a mite’s secretome as well as transmembranome and provides valuable insights into D. gallinae pES and pTM proteins operating in different metabolic pathways. Identifying a variety of molecules putatively involved in blood feeding may significantly contribute to the development of new therapeutic targets or vaccines against this poultry pest. PMID:24020355

BMP signaling components in embryonic transcriptomes of the hover fly Episyrphus balteatus (Syrphidae)

PubMed Central

2011-01-01

Background In animals, signaling of Bone Morphogenetic Proteins (BMPs) is essential for dorsoventral (DV) patterning of the embryo, but how BMP signaling evolved with changes in embryonic DV differentiation is largely unclear. Based on the extensive knowledge of BMP signaling in Drosophila melanogaster, the morphological diversity of extraembryonic tissues in different fly species provides a comparative system to address this question. The closest relatives of D. melanogaster with clearly distinct DV differentiation are hover flies (Diptera: Syrphidae). The syrphid Episyrphus balteatus is a commercial bio-agent against aphids and has been established as a model organism for developmental studies and chemical ecology. The dorsal blastoderm of E. balteatus gives rise to two extraembryonic tissues (serosa and amnion), whereas in D. melanogaster, the dorsal blastoderm differentiates into a single extraembryonic epithelium (amnioserosa). Recent studies indicate that several BMP signaling components of D. melanogaster, including the BMP ligand Screw (Scw) and other extracellular regulators, evolved in the dipteran lineage through gene duplication and functional divergence. These findings raise the question of whether the complement of BMP signaling components changed with the origin of the amnioserosa. Results To search for BMP signaling components in E. balteatus, we generated and analyzed transcriptomes of freshly laid eggs (0-30 minutes) and late blastoderm to early germband extension stages (3-6 hours) using Roche/454 sequencing. We identified putative E. balteatus orthologues of 43% of all annotated D. melanogaster genes, including the genes of all BMP ligands and other BMP signaling components. Conclusion The diversification of several BMP signaling components in the dipteran linage of D. melanogaster preceded the origin of the amnioserosa. [Transcriptome sequence data from this study have been deposited at the NCBI Sequence Read Archive (SRP005289); individually assembled sequences have been deposited at GenBank (JN006969-JN006986).] PMID:21627820

Modulation of Immune Signaling and Metabolism Highlights Host and Fungal Transcriptional Responses in Mouse Models of Invasive Pulmonary Aspergillosis.

PubMed

Kale, Shiv D; Ayubi, Tariq; Chung, Dawoon; Tubau-Juni, Nuria; Leber, Andrew; Dang, Ha X; Karyala, Saikumar; Hontecillas, Raquel; Lawrence, Christopher B; Cramer, Robert A; Bassaganya-Riera, Josep

2017-12-06

Incidences of invasive pulmonary aspergillosis, an infection caused predominantly by Aspergillus fumigatus, have increased due to the growing number of immunocompromised individuals. While A. fumigatus is reliant upon deficiencies in the host to facilitate invasive disease, the distinct mechanisms that govern the host-pathogen interaction remain enigmatic, particularly in the context of distinct immune modulating therapies. To gain insights into these mechanisms, RNA-Seq technology was utilized to sequence RNA derived from lungs of 2 clinically relevant, but immunologically distinct murine models of IPA on days 2 and 3 post inoculation when infection is established and active disease present. Our findings identify notable differences in host gene expression between the chemotherapeutic and steroid models at the interface of immunity and metabolism. RT-qPCR verified model specific and nonspecific expression of 23 immune-associated genes. Deep sequencing facilitated identification of highly expressed fungal genes. We utilized sequence similarity and gene expression to categorize the A. fumigatus putative in vivo secretome. RT-qPCR suggests model specific gene expression for nine putative fungal secreted proteins. Our analysis identifies contrasting responses by the host and fungus from day 2 to 3 between the two models. These differences may help tailor the identification, development, and deployment of host- and/or fungal-targeted therapeutics.

Maintenance of Paraoxonase 2 Activity as a Strategy to Attenuate P. Aeruginosa Virulence

DTIC Science & Technology

2013-10-01

identify the putative PON2 interacting protein, our approach is to IP the ~300kD BS3 crosslinked complex with a GFP antibody , run the IP on an SDS-PAGE...Dianova) were used at 1:5000. HRP- conjugated secondary antibodies were from Cell Signaling. Stealth-PON2 and control siRNAs (Invitrogen) sequences and...the lactonase paraoxonase 2 (PON2) and induces many immunomodulatory effects in host cells. Because PON2 rapidly inactivates 3OC12, we hypothesized

Bifidobacterium breve C50 secretes lipoprotein with CHAP domain recognized in aggregated form by TLR2.

PubMed

Scuotto, Angelo; Djorie, Serge; Colavizza, Michel; Romond, Pierre-Charles; Romond, Marie-Bénédicte

2014-12-01

Extracellular components secreted by Bifidobacterium breve C50 can induce maturation, high IL-10 production and prolonged survival of dendritic cells via a TLR2 pathway. In this study, the components were isolated from the supernatant by gel filtration chromatography. Antibodies raised against the major compounds with molecular weight above 600 kDa (Bb C50BC) also recognized compounds of lower molecular weight (200–600 kDa). TLR2 and TLR6 bound to the components already recognized by the antibodies. Trypsin digestion of Bb C50BC released three major peptides whose sequences displayed close similarities to a putative secreted protein with a CHAP amidase domain from B. breve. The 1300-bp genomic region corresponding to the hypothetical protein was amplified by PCR. The deduced polypeptide started with an N-terminal signal sequence of 45 amino acids, containing the lipobox motif (LAAC) with the cysteine in position 25, and 2 positively charged residues within the first 14 residues of the signal sequence. Lipid detection in Bb C50BC by GC/MS further supported the implication of a lipoprotein. Sugars were also detected in Bb C50BC. Close similarity with the glucan-binding protein B from Bifidobacterium animalis of two released peptides from Bb C50BC protein suggested that glucose moieties, possibly in glucan form, could be bound to the lipoprotein. Finally, heating at 100 °C for 5 min led to the breakdown of Bb C50BC in compounds of molecular weight below 67 kDa, which suggested that Bb C50BC was an aggregate. One might assume that a basic unit was formed by the lipoprotein bound putatively to glucan. Besides the other sugars and hexosamines recognized by galectin 1 were localized at the surface of the Bb C50BC aggregate. In conclusion, the extracellular components secreted by B. breve C50 were constituted of a lipoprotein putatively associated with glucose moieties and acting in an aggregating form as an agonist of TLR2/TLR6.

Characterization of HIV Transmission in South-East Austria

PubMed Central

Kessler, Harald H.; Haas, Bernhard; Stelzl, Evelyn; Weninger, Karin; Little, Susan J.; Mehta, Sanjay R.

2016-01-01

To gain deeper insight into the epidemiology of HIV-1 transmission in South-East Austria we performed a retrospective analysis of 259 HIV-1 partial pol sequences obtained from unique individuals newly diagnosed with HIV infection in South-East Austria from 2008 through 2014. After quality filtering, putative transmission linkages were inferred when two sequences were ≤1.5% genetically different. Multiple linkages were resolved into putative transmission clusters. Further phylogenetic analyses were performed using BEAST v1.8.1. Finally, we investigated putative links between the 259 sequences from South-East Austria and all publicly available HIV polymerase sequences in the Los Alamos National Laboratory HIV sequence database. We found that 45.6% (118/259) of the sampled sequences were genetically linked with at least one other sequence from South-East Austria forming putative transmission clusters. Clustering individuals were more likely to be men who have sex with men (MSM; p<0.001), infected with subtype B (p<0.001) or subtype F (p = 0.02). Among clustered males who reported only heterosexual (HSX) sex as an HIV risk, 47% clustered closely with MSM (either as pairs or within larger MSM clusters). One hundred and seven of the 259 sequences (41.3%) from South-East Austria had at least one putative inferred linkage with sequences from a total of 69 other countries. In conclusion, analysis of HIV-1 sequences from newly diagnosed individuals residing in South-East Austria revealed a high degree of national and international clustering mainly within MSM. Interestingly, we found that a high number of heterosexual males clustered within MSM networks, suggesting either linkage between risk groups or misrepresentation of sexual risk behaviors by subjects. PMID:26967154

Characterization of HIV Transmission in South-East Austria.

PubMed

Hoenigl, Martin; Chaillon, Antoine; Kessler, Harald H; Haas, Bernhard; Stelzl, Evelyn; Weninger, Karin; Little, Susan J; Mehta, Sanjay R

2016-01-01

To gain deeper insight into the epidemiology of HIV-1 transmission in South-East Austria we performed a retrospective analysis of 259 HIV-1 partial pol sequences obtained from unique individuals newly diagnosed with HIV infection in South-East Austria from 2008 through 2014. After quality filtering, putative transmission linkages were inferred when two sequences were ≤1.5% genetically different. Multiple linkages were resolved into putative transmission clusters. Further phylogenetic analyses were performed using BEAST v1.8.1. Finally, we investigated putative links between the 259 sequences from South-East Austria and all publicly available HIV polymerase sequences in the Los Alamos National Laboratory HIV sequence database. We found that 45.6% (118/259) of the sampled sequences were genetically linked with at least one other sequence from South-East Austria forming putative transmission clusters. Clustering individuals were more likely to be men who have sex with men (MSM; p<0.001), infected with subtype B (p<0.001) or subtype F (p = 0.02). Among clustered males who reported only heterosexual (HSX) sex as an HIV risk, 47% clustered closely with MSM (either as pairs or within larger MSM clusters). One hundred and seven of the 259 sequences (41.3%) from South-East Austria had at least one putative inferred linkage with sequences from a total of 69 other countries. In conclusion, analysis of HIV-1 sequences from newly diagnosed individuals residing in South-East Austria revealed a high degree of national and international clustering mainly within MSM. Interestingly, we found that a high number of heterosexual males clustered within MSM networks, suggesting either linkage between risk groups or misrepresentation of sexual risk behaviors by subjects.

Analysis of Putative Apoplastic Effectors from the Nematode, Globodera rostochiensis, and Identification of an Expansin-Like Protein That Can Induce and Suppress Host Defenses

PubMed Central

Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Côté, Olivier; Stare, Barbara Gerič; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

2015-01-01

The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses. PMID:25606855

Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

PubMed

Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Côté, Olivier; Stare, Barbara Gerič; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

2015-01-01

The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.

Comparison of different signal peptides for secretion of heterologous proteins in fission yeast

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

Kjaerulff, Soren; Jensen, Martin Roland

2005-10-28

In the fission yeast Schizosaccharomyces pombe, there are relatively few signal peptides available and most reports of their activity have not been comparative. Using sequence information from the S. pombe genome database we have identified three putative signal peptides, designated Cpy, Amy and Dpp, and compared their ability to support secretion of green fluorescent protein (GFP). In the comparison we also included the two well-described secretion signals derived from the precursors of, respectively, the Saccharomyces cerevisiae {alpha}-factor and the S. pombe P-factor. The capability of the tested signal peptides to direct secretion of GFP varied greatly. The {alpha}-factor signal didmore » not confer secretion to GFP and all the produced GFP was trapped intracellular. In contrast, the Cpy signal peptide supported efficient secretion of GFP with yields approximating 10 mg/L. We also found that the use of an attenuated version of the S. cerevisiae URA3 marker substantially increases vector copy number and expression yield in fission yeast.« less

Sequence analyses and evolutionary relationships among the energy-coupling proteins Enzyme I and HPr of the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

PubMed Central

Reizer, J.; Hoischen, C.; Reizer, A.; Pham, T. N.; Saier, M. H.

1993-01-01

We have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) (Reizer, J., et al., 1992, J. Biol. Chem. 267, 9158-9169). We now report the sequencing of the ptsI gene of B. subtilis encoding Enzyme I (570 amino acids and 63,076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject to carbon source-dependent regulation. Multiple alignments of the B. subtilis Enzyme I with (1) six other sequenced Enzymes I of the PTS from various bacterial species, (2) phosphoenolpyruvate synthase of Escherichia coli, and (3) bacterial and plant pyruvate: phosphate dikinases (PPDKs) revealed regions of sequence similarity as well as divergence. Statistical analyses revealed that these three types of proteins comprise a homologous family, and the phylogenetic tree of the 11 sequenced protein members of this family was constructed. This tree was compared with that of the 12 sequence HPr proteins or protein domains. Antibodies raised against the B. subtilis and E. coli Enzymes I exhibited immunological cross-reactivity with each other as well as with PPDK of Bacteroides symbiosus, providing support for the evolutionary relationships of these proteins suggested from the sequence comparisons. Putative flexible linkers tethering the N-terminal and the C-terminal domains of protein members of the Enzyme I family were identified, and their potential significance with regard to Enzyme I function is discussed. The codon choice pattern of the B. subtilis and E. coli ptsI and ptsH genes was found to exhibit a bias toward optimal codons in these organisms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7686067

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

Extreme Sensory Complexity Encoded in the 10-Megabase Draft Genome Sequence of the Chromatically Acclimating Cyanobacterium Tolypothrix sp. PCC 7601.

PubMed

Yerrapragada, Shaila; Shukla, Animesh; Hallsworth-Pepin, Kymberlie; Choi, Kwangmin; Wollam, Aye; Clifton, Sandra; Qin, Xiang; Muzny, Donna; Raghuraman, Sriram; Ashki, Haleh; Uzman, Akif; Highlander, Sarah K; Fryszczyn, Bartlomiej G; Fox, George E; Tirumalai, Madhan R; Liu, Yamei; Kim, Sun; Kehoe, David M; Weinstock, George M

2015-05-07

Tolypothrix sp. PCC 7601 is a freshwater filamentous cyanobacterium with complex responses to environmental conditions. Here, we present its 9.96-Mbp draft genome sequence, containing 10,065 putative protein-coding sequences, including 305 predicted two-component system proteins and 27 putative phytochrome-class photoreceptors, the most such proteins in any sequenced genome. Copyright © 2015 Yerrapragada et al.

Two host cytoplasmic effectors are required for pathogenesis of Phytophthora sojae by suppression of host defenses.

PubMed

Liu, Tingli; Ye, Wenwu; Ru, Yanyan; Yang, Xinyu; Gu, Biao; Tao, Kai; Lu, Shan; Dong, Suomeng; Zheng, Xiaobo; Shan, Weixing; Wang, Yuanchao; Dou, Daolong

2011-01-01

Phytophthora sojae encodes hundreds of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling- and necrosis-inducing proteins (CRN) or Crinkler. Their functions and mechanisms in pathogenesis are mostly unknown. Here, we identify a group of five P. sojae-specific CRN-like genes with high levels of sequence similarity, of which three are putative pseudogenes. Functional analysis shows that the two functional genes encode proteins with predicted nuclear localization signals that induce contrasting responses when expressed in Nicotiana benthamiana and soybean (Glycine max). PsCRN63 induces cell death, while PsCRN115 suppresses cell death elicited by the P. sojae necrosis-inducing protein (PsojNIP) or PsCRN63. Expression of CRN fragments with deleted signal peptides and FLAK motifs demonstrates that the carboxyl-terminal portions of PsCRN63 or PsCRN115 are sufficient for their activities. However, the predicted nuclear localization signal is required for PsCRN63 to induce cell death but not for PsCRN115 to suppress cell death. Furthermore, silencing of the PsCRN63 and PsCRN115 genes in P. sojae stable transformants leads to a reduction of virulence on soybean. Intriguingly, the silenced transformants lose the ability to suppress host cell death and callose deposition on inoculated plants. These results suggest a role for CRN effectors in the suppression of host defense responses.

Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes.

PubMed

Noh, Ju Young; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Seo, Gi Won; Kim, Dong Hyun; Patnaik, Hongray Howrelia; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Han, Yeon Soo

2014-01-25

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5'-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic alpha helices, including a short helix 3'. The 'helix-short helix-helix' motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly upregulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor. Copyright © 2013 Elsevier B.V. All rights reserved.

Are algal genes in nonphotosynthetic protists evidence of historical plastid endosymbioses?

PubMed

Stiller, John W; Huang, Jinling; Ding, Qin; Tian, Jing; Goodwillie, Carol

2009-10-20

How photosynthetic organelles, or plastids, were acquired by diverse eukaryotes is among the most hotly debated topics in broad scale eukaryotic evolution. The history of plastid endosymbioses commonly is interpreted under the "chromalveolate" hypothesis, which requires numerous plastid losses from certain heterotrophic groups that now are entirely aplastidic. In this context, discoveries of putatively algal genes in plastid-lacking protists have been cited as evidence of gene transfer from a photosynthetic endosymbiont that subsequently was lost completely. Here we examine this evidence, as it pertains to the chromalveolate hypothesis, through genome-level statistical analyses of similarity scores from queries with two diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, and two aplastidic sister taxa, Phytophthora ramorum and P. sojae. Contingency tests of specific predictions of the chromalveolate model find no evidence for an unusual red algal contribution to Phytophthora genomes, nor that putative cyanobacterial sequences that are present entered these genomes through a red algal endosymbiosis. Examination of genes unrelated to plastid function provide extraordinarily significant support for both of these predictions in diatoms, the control group where a red endosymbiosis is known to have occurred, but none of that support is present in genes specifically conserved between diatoms and oomycetes. In addition, we uncovered a strong association between overall sequence similarities among taxa and relative sizes of genomic data sets in numbers of genes. Signal from "algal" genes in oomycete genomes is inconsistent with the chromalveolate hypothesis, and better explained by alternative models of sequence and genome evolution. Combined with the numerous sources of intragenomic phylogenetic conflict characterized previously, our results underscore the potential to be mislead by a posteriori interpretations of variable phylogenetic signals contained in complex genome-level data. They argue strongly for explicit testing of the different a priori assumptions inherent in competing evolutionary hypotheses.

Systematic analysis and evolution of 5S ribosomal DNA in metazoans.

PubMed

Vierna, J; Wehner, S; Höner zu Siederdissen, C; Martínez-Lage, A; Marz, M

2013-11-01

Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12,766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades.

Systematic analysis and evolution of 5S ribosomal DNA in metazoans

PubMed Central

Vierna, J; Wehner, S; Höner zu Siederdissen, C; Martínez-Lage, A; Marz, M

2013-01-01

Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12 766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades. PMID:23838690

PUTATIVE GENE PROMOTER SEQUENCES IN THE CHLORELLA VIRUSES

PubMed Central

Fitzgerald, Lisa A.; Boucher, Philip T.; Yanai-Balser, Giane; Suhre, Karsten; Graves, Michael V.; Van Etten, James L.

2008-01-01

Three short (7 to 9 nucleotides) highly conserved nucleotide sequences were identified in the putative promoter regions (150 bp upstream and 50 bp downstream of the ATG translation start site) of three members of the genus Chlorovirus, family Phycodnaviridae. Most of these sequences occurred in similar locations within the defined promoter regions. The sequence and location of the motifs were often conserved among homologous ORFs within the Chlorovirus family. One of these conserved sequences (AATGACA) is predominately associated with genes expressed early in virus replication. PMID:18768195

Functional characterization of putative cilia genes by high-content analysis

PubMed Central

Lai, Cary K.; Gupta, Nidhi; Wen, Xiaohui; Rangell, Linda; Chih, Ben; Peterson, Andrew S.; Bazan, J. Fernando; Li, Li; Scales, Suzie J.

2011-01-01

Cilia are microtubule-based protrusions from the cell surface that are involved in a number of essential signaling pathways, yet little is known about many of the proteins that regulate their structure and function. A number of putative cilia genes have been identified by proteomics and comparative sequence analyses, but functional data are lacking for the vast majority. We therefore monitored the effects in three cell lines of small interfering RNA (siRNA) knockdown of 40 of these genes by high-content analysis. We assayed cilia number, length, and transport of two different cargoes (membranous serotonin receptor 6-green fluorescent protein [HTR6-GFP] and the endogenous Hedgehog [Hh] pathway transcription factor Gli3) by immunofluorescence microscopy; and cilia function using a Gli-luciferase Hh signaling assay. Hh signaling was most sensitive to perturbations, with or without visible structural cilia defects. Validated hits include Ssa2 and mC21orf2 with ciliation defects; Ift46 with short cilia; Ptpdc1 and Iqub with elongated cilia; and Arl3, Nme7, and Ssna1 with distinct ciliary transport but not length defects. Our data confirm various ciliary roles for several ciliome proteins and show it is possible to uncouple ciliary cargo transport from cilia formation in vertebrates. PMID:21289087

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.

Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus.

PubMed

Camicia, Federico; Paredes, Rodolfo; Chalar, Cora; Galanti, Norbel; Kamenetzky, Laura; Gutierrez, Ariana; Rosenzvit, Mara C

2008-03-31

We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of approximately 60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.

Identification of a novel prophage-like gene cluster actively expressed in both virulent and avirulent strains of Leptospira interrogans serovar Lai.

PubMed

Qin, Jin-Hong; Zhang, Qing; Zhang, Zhi-Ming; Zhong, Yi; Yang, Yang; Hu, Bao-Yu; Zhao, Guo-Ping; Guo, Xiao-Kui

2008-06-01

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.

The mitochondrial genome of Pocillopora (Cnidaria: Scleractinia) contains two variable regions: the putative D-loop and a novel ORF of unknown function.

PubMed

Flot, Jean-François; Tillier, Simon

2007-10-15

The complete mitochondrial genomes of two individuals attributed to different morphospecies of the scleractinian coral genus Pocillopora have been sequenced. Both genomes, respectively 17,415 and 17,422 nt long, share the presence of a previously undescribed ORF encoding a putative protein made up of 302 amino acids and of unknown function. Surprisingly, this ORF turns out to be the second most variable region of the mitochondrial genome (1% nucleotide sequence difference between the two individuals) after the putative control region (1.5% sequence difference). Except for the presence of this ORF and for the location of the putative control region, the mitochondrial genome of Pocillopora is organized in a fashion similar to the other scleractinian coral genomes published to date. For the first time in a cnidarian, a putative second origin of replication is described based on its secondary structure similar to the stem-loop structure of O(L), the origin of L-strand replication in vertebrates.

The current status and portability of our sequence handling software.

PubMed Central

Staden, R

1986-01-01

I describe the current status of our sequence analysis software. The package contains a comprehensive suite of programs for managing large shotgun sequencing projects, a program containing 61 functions for analysing single sequences and a program for comparing pairs of sequences for similarity. The programs that have been described before have been improved by the addition of new functions and by being made very much easier to use. The major interactive programs have 125 pages of online help available from within them. Several new programs are described including screen editing of aligned gel readings for shotgun sequencing projects; a method to highlight errors in aligned gel readings, new methods for searching for putative signals in sequences. We use the programs on a VAX computer but the whole package has been rewritten to make it easy to transport it to other machines. I believe the programs will now run on any machine with a FORTRAN77 compiler and sufficient memory. We are currently putting the programs onto an IBM PC XT/AT and another micro running under UNIX. PMID:3511446

Complete Genome Sequence of a Putative Densovirus of the Asian Citrus Psyllid, Diaphorina citri.

PubMed

Nigg, Jared C; Nouri, Shahideh; Falk, Bryce W

2016-07-28

Here, we report the complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri Diaphorina citri densovirus (DcDNV) was originally identified through metagenomics, and here, we obtained the complete nucleotide sequence using PCR-based approaches. Phylogenetic analysis places DcDNV between viruses of the Ambidensovirus and Iteradensovirus genera. Copyright © 2016 Nigg et al.

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins.

PubMed

Wolffe, E J; Gause, W C; Pelfrey, C M; Holland, S M; Steinberg, A D; August, J T

1990-01-05

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.

Identification and molecular characterization of five putative toxins from the venom gland of the snake Philodryas chamissonis (Serpentes: Dipsadidae).

PubMed

Urra, Félix A; Pulgar, Rodrigo; Gutiérrez, Ricardo; Hodar, Christian; Cambiazo, Verónica; Labra, Antonieta

2015-12-15

Philodryas chamissonis is a rear-fanged snake endemic to Chile. Its bite produces mild to moderate symptoms with proteolytic and anti-coagulant effects. Presently, the composition of the venom, as well as, the biochemical and structural characteristics of its toxins, remains unknown. In this study, we cloned and reported the first full-length sequences of five toxin-encoding genes from the venom gland of this species: Type III snake venom metalloprotease (SVMP), snake venom serine protease (SVSP), Cysteine-rich secretory protein (CRISP), α and β subunits of C-type lectin-like protein (CLP) and C-type natriuretic peptide (NP). These genes are highly expressed in the venom gland and their sequences exhibited a putative signal peptide, suggesting that these are components of the venom. These putative toxins had different evolutionary relationships with those reported for some front-fanged snakes, being SVMP, SVSP and CRISP of P. chamissonis closely related to the toxins present in Elapidae species, while NP was more related to those of Viperidae species. In addition, analyses suggest that the α and β subunits of CLP of P. chamissonis might have a α-subunit scaffold in common with Viperidae species, whose highly variable C-terminal region might have allowed the diversification in α and β subunits. Our results provide the first molecular description of the toxins possibly implicated in the envenomation of prey and humans by the bite of P. chamissonis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Association of candidate genes with drought tolerance traits in diverse perennial ryegrass accessions

PubMed Central

Jiang, Yiwei

2013-01-01

Drought is a major environmental stress limiting growth of perennial grasses in temperate regions. Plant drought tolerance is a complex trait that is controlled by multiple genes. Candidate gene association mapping provides a powerful tool for dissection of complex traits. Candidate gene association mapping of drought tolerance traits was conducted in 192 diverse perennial ryegrass (Lolium perenne L.) accessions from 43 countries. The panel showed significant variations in leaf wilting, leaf water content, canopy and air temperature difference, and chlorophyll fluorescence under well-watered and drought conditions across six environments. Analysis of 109 simple sequence repeat markers revealed five population structures in the mapping panel. A total of 2520 expression-based sequence readings were obtained for a set of candidate genes involved in antioxidant metabolism, dehydration, water movement across membranes, and signal transduction, from which 346 single nucleotide polymorphisms were identified. Significant associations were identified between a putative LpLEA3 encoding late embryogenesis abundant group 3 protein and a putative LpFeSOD encoding iron superoxide dismutase and leaf water content, as well as between a putative LpCyt Cu-ZnSOD encoding cytosolic copper-zinc superoxide dismutase and chlorophyll fluorescence under drought conditions. Four of these identified significantly associated single nucleotide polymorphisms from these three genes were also translated to amino acid substitutions in different genotypes. These results indicate that allelic variation in these genes may affect whole-plant response to drought stress in perennial ryegrass. PMID:23386684

Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

PubMed

Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

1993-06-25

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

Discriminative prediction of mammalian enhancers from DNA sequence

PubMed Central

Lee, Dongwon; Karchin, Rachel; Beer, Michael A.

2011-01-01

Accurately predicting regulatory sequences and enhancers in entire genomes is an important but difficult problem, especially in large vertebrate genomes. With the advent of ChIP-seq technology, experimental detection of genome-wide EP300/CREBBP bound regions provides a powerful platform to develop predictive tools for regulatory sequences and to study their sequence properties. Here, we develop a support vector machine (SVM) framework which can accurately identify EP300-bound enhancers using only genomic sequence and an unbiased set of general sequence features. Moreover, we find that the predictive sequence features identified by the SVM classifier reveal biologically relevant sequence elements enriched in the enhancers, but we also identify other features that are significantly depleted in enhancers. The predictive sequence features are evolutionarily conserved and spatially clustered, providing further support of their functional significance. Although our SVM is trained on experimental data, we also predict novel enhancers and show that these putative enhancers are significantly enriched in both ChIP-seq signal and DNase I hypersensitivity signal in the mouse brain and are located near relevant genes. Finally, we present results of comparisons between other EP300/CREBBP data sets using our SVM and uncover sequence elements enriched and/or depleted in the different classes of enhancers. Many of these sequence features play a role in specifying tissue-specific or developmental-stage-specific enhancer activity, but our results indicate that some features operate in a general or tissue-independent manner. In addition to providing a high confidence list of enhancer targets for subsequent experimental investigation, these results contribute to our understanding of the general sequence structure of vertebrate enhancers. PMID:21875935

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis.

PubMed

Qin, L; Overmars, H; Helder, J; Popeijus, H; van der Voort, J R; Groenink, W; van Koert, P; Schots, A; Bakker, J; Smant, G

2000-08-01

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

The nagA gene of Penicillium chrysogenum encoding beta-N-acetylglucosaminidase.

PubMed

Díez, Bruno; Rodríguez-Sáiz, Marta; de la Fuente, Juan Luis; Moreno, Miguel Angel; Barredo, José Luis

2005-01-15

We purified the beta-N-acetylglucosaminidase from the filamentous fungus Penicillium chrysogenum and its N-terminal sequence was determined, showing the presence of a mixture of two proteins (P1 and P2). A genomic DNA fragment was cloned by using degenerated oligonucleotides from the Nt sequences. The nucleotide sequence showed the presence of an ORF (nagA gene) lacking introns, with a length of 1791 bp, and coding for a protein of 66.5 kDa showing similarity to acetylglucosaminidases. The NagA deduced protein includes P1 and P2 as incomplete forms of the mature protein, and contains putative features for protein maturation: an 18-amino acid signal peptide, a KEX2 processing site, and four glycosylation motifs. The sequence just after the signal peptide corresponds to P2 and that after the KEX2 site to P1. The nagA transcript has a size of about 2.1 kb and is present until the end of the fermentation process for penicillin production. NagA is one of the most largely represented proteins in P. chrysogenum, increasing along the fermentation process. The suitability of the nagA promoter (PnagA) for gene expression in fungi was demonstrated by expressing the bleomycin resistance gene (ble(R)) from Streptoalloteichus hindustanus in P. chrysogenum.

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

Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

PubMed Central

Takeshita, S; Kikuno, R; Tezuka, K; Amann, E

1993-01-01

A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8363580

Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies

PubMed Central

Santiago-Rodriguez, Tasha M.; Luciani, Stefania; Toranzos, Gary A.; Marota, Isolina; Giuffra, Valentina; Cano, Raul J.

2017-01-01

Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era. PMID:29112136

Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies.

PubMed

Santiago-Rodriguez, Tasha M; Fornaciari, Gino; Luciani, Stefania; Toranzos, Gary A; Marota, Isolina; Giuffra, Valentina; Cano, Raul J

2017-11-07

Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase.

PubMed Central

Charbonneau, H; Tonks, N K; Walsh, K A; Fischer, E H

1988-01-01

A major protein tyrosine phosphatase (PTPase 1B) has been isolated in essentially homogeneous form from the soluble and particulate fractions of human placenta. Unexpectedly, partial amino acid sequences displayed no homology with the primary structures of the protein Ser/Thr phosphatases deduced from cDNA clones. However, the sequence is strikingly similar to the tandem C-terminal homologous domains of the leukocyte common antigen (CD45). A 157-residue segment of PTPase 1B displayed 40% and 33% sequence identity with corresponding regions from cytoplasmic domains I and II of human CD45. Similar degrees of identity have been observed among the catalytic domains of families of regulatory proteins such as protein kinases and cyclic nucleotide phosphodiesterases. On this basis, it is proposed that the CD45 family has protein tyrosine phosphatase activity and may represent a set of cell-surface receptors involved in signal transduction. This suggests that the repertoire of signal transduction mechanisms may include the direct control of an intracellular protein tyrosine phosphatase, offering the possibility of a regulatory balance with those protein tyrosine kinases that act at the internal surface of the membrane. Images PMID:2845400

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

PubMed Central

Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

2013-01-01

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

Origin, evolution, and divergence of plant class C GH9 endoglucanases.

PubMed

Kundu, Siddhartha; Sharma, Rita

2018-05-30

Glycoside hydrolases of the GH9 family encode cellulases that predominantly function as endoglucanases and have wide applications in the food, paper, pharmaceutical, and biofuel industries. The partitioning of plant GH9 endoglucanases, into classes A, B, and C, is based on the differential presence of transmembrane, signal peptide, and the carbohydrate binding module (CBM49). There is considerable debate on the distribution and the functions of these enzymes which may vary in different organisms. In light of these findings we examined the origin, emergence, and subsequent divergence of plant GH9 endoglucanases, with an emphasis on elucidating the role of CBM49 in the digestion of crystalline cellulose by class C members. Since, the digestion of crystalline cellulose mandates the presence of a well-defined set of aromatic and polar amino acids and/or an attributable domain that can mediate this conversion, we hypothesize a vertical mode of transfer of genes that could favour the emergence of class C like GH9 endoglucanase activity in land plants from potentially ancestral non plant taxa. We demonstrated the concomitant occurrence of a GH9 domain with CBM49 and other homologous carbohydrate binding modules, in putative endoglucanase sequences from several non-plant taxa. In the absence of comparable full length CBMs, we have characterized several low strength patterns that could approximate the CBM49, thereby, extending support for digestion of crystalline cellulose to other segments of the protein. We also provide data suggestive of the ancestral role of putative class C GH9 endoglucanases in land plants, which includes detailed phylogenetics and the presence and subsequent loss of CBM49, transmembrane, and signal peptide regions in certain populations of early land plants. These findings suggest that classes A and B of modern vascular land plants may have emerged by diverging directly from CBM49 encompassing putative class C enzymes. Our detailed phylogenetic and bioinformatics analysis of putative GH9 endoglucanase sequences across major taxa suggests that plant class C enzymes, despite their recent discovery, could function as the last common ancestor of classes A and B. Additionally, research into their ability to digest or inter-convert crystalline and amorphous forms of cellulose could make them lucrative candidates for engineering biofuel feedstock.

The Red Imported Fire Ant (Solenopsis invicta Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor

PubMed Central

Bajracharya, Prati; Lu, Hsiao-Ling; Pietrantonio, Patricia V.

2014-01-01

Neuropeptides and their receptors play vital roles in controlling the physiology and behavior of animals. Short neuropeptide F (sNPF) signaling regulates several physiological processes in insects such as feeding, locomotion, circadian rhythm and reproduction, among others. Previously, the red imported fire ant (Solenopsis invicta) sNPF receptor (S. invicta sNPFR), a G protein-coupled receptor, was immunolocalized in queen and worker brain and queen ovaries. Differential distribution patterns of S. invicta sNPFR protein in fire ant worker brain were associated both with worker subcastes and with presence or absence of brood in the colony. However, the cognate ligand for this sNPFR has not been characterized and attempts to deorphanize the receptor with sNPF peptides from other insect species which ended in the canonical sequence LRLRFamide, failed. Receptor deorphanization is an important step to understand the neuropeptide receptor downstream signaling cascade. We cloned the full length cDNA of the putative S. invicta sNPF prepropeptide and identified the putative “sNPF” ligand within its sequence. The peptide ends with an amidated Tyr residue whereas in other insect species sNPFs have an amidated Phe or Trp residue at the C-terminus. We stably expressed the HA-tagged S. invicta sNPFR in CHO-K1 cells. Two S. invicta sNPFs differing at their N-terminus were synthesized that equally activated the sNPFR, SLRSALAAGHLRYa (EC50 = 3.2 nM) and SALAAGHLRYa (EC50 = 8.6 nM). Both peptides decreased the intracellular cAMP concentration, indicating signaling through the Gαi-subunit. The receptor was not activated by sNPF peptides from other insect species, honey bee long NPF (NPY) or mammalian PYY. Further, a synthesized peptide otherwise identical to the fire ant sequence but in which the C-terminal amidated amino acid residue ‘Y’ was switched to ‘F’, failed to activate the sNPFR. This discovery will now allow us to investigate the function of sNPY and its cognate receptor in fire ant biology. PMID:25310341

Analysis of a library of macaque nuclear mitochondrial sequences confirms macaque origin of divergent sequences from old oral polio vaccine samples.

PubMed

Vartanian, Jean-Pierre; Wain-Hobson, Simon

2002-05-28

Nuclear mtDNA sequences (numts) are a widespread family of paralogs evolving as pseudogenes in chromosomal DNA [Zhang, D. E. & Hewitt, G. M. (1996) TREE 11, 247-251 and Bensasson, D., Zhang, D., Hartl, D. L. & Hewitt, G. M. (2001) TREE 16, 314-321]. When trying to identify the species origin of an unknown DNA sample by way of an mtDNA locus, PCR may amplify both mtDNA and numts. Indeed, occasionally numts dominate confounding attempts at species identification [Bensasson, D., Zhang, D. X. & Hewitt, G. M. (2000) Mol. Biol. Evol. 17, 406-415; Wallace, D. C., et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14900-14905]. Rhesus and cynomolgus macaque mtDNA haplotypes were identified in a study of oral polio vaccine samples dating from the late 1950s [Blancou, P., et al. (2001) Nature (London) 410, 1045-1046]. They were accompanied by a number of putative numts. To confirm that these putative numts were of macaque origin, a library of numts corresponding to a small segment of 12S rDNA locus has been made by using DNA from a Chinese rhesus macaque. A broad distribution was found with up to 30% sequence variation. Phylogenetic analysis showed that the evolutionary trajectories of numts and bona fide mtDNA haplotypes do not overlap with the signal exception of the host species; mtDNA fragments are continually crossing over into the germ line. In the case of divergent mtDNA sequences from old oral polio vaccine samples [Blancou, P., et al. (2001) Nature (London) 410, 1045-1046], all were closely related to numts in the Chinese macaque library.

Comparative analysis of Leishmania exoproteomes: implication for host-pathogen interactions.

PubMed

Peysselon, Franck; Launay, Guillaume; Lisacek, Frédérique; Duclos, Bertrand; Ricard-Blum, Sylvie

2013-12-01

Leishmaniasis is a vector-borne disease caused by the protozoa Leishmania. We have analyzed and compared the sequences of three experimental exoproteomes of Leishmania promastigotes from different species to determine their specific features and to identify new candidate proteins involved in interactions of Leishmania with the host. The exoproteomes differ from the proteomes by a decrease in the average molecular weight per protein, in disordered amino acid residues and in basic proteins. The exoproteome of the visceral species is significantly enriched in sites predicted to be phosphorylated as well as in features frequently associated with molecular interactions (intrinsic disorder, number of disordered binding regions per protein, interaction and/or trafficking motifs) compared to the other species. The visceral species might thus have a larger interaction repertoire with the host than the other species. Less than 10% of the exoproteomes contain heparin-binding and RGD sequences, and ~30% the host targeting signal RXLXE/D/Q. These latter proteins might thus be exported inside the host cell during the intracellular stage of the infection. Furthermore we have identified nine protein families conserved in the three exoproteomes with specific combinations of Pfam domains and selected eleven proteins containing at least three interaction and/or trafficking motifs including two splicing factors, phosphomannomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, the paraflagellar rod protein-1D and a putative helicase. Their role in host-Leishmania interactions warrants further investigation but the putative ATP-dependent DEAD/H RNA helicase, which contains numerous interaction motifs, a host targeting signal and two disordered regions, is a very promising candidate. © 2013.

In-silico and in-vivo analyses of EST databases unveil conserved miRNAs from Carthamus tinctorius and Cynara cardunculus

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are small RNAs (21-24 bp) providing an RNA-based system of gene regulation highly conserved in plants and animals. In plants, miRNAs control mRNA degradation or restrain translation, affecting development and responses to stresses. Plant miRNAs show imperfect but extensive complementarity to mRNA targets, making their computational prediction possible, useful when data mining is applied on different species. In this study we used a comparative approach to identify both miRNAs and their targets, in artichoke and safflower. Results Two complete expressed sequence tags (ESTs) datasets from artichoke (3.6·104 entries) and safflower (4.2·104), were analysed with a bioinformatic pipeline and in vitro experiments, identifying 17 potential miRNAs. For each EST, using RNAhybrid program and 953 non redundant miRNA mature sequences, available in mirBase as reference, we searched matching putative targets. 8730 out of 42011 ESTs from safflower and 7145 of 36323 ESTs from artichoke showed at least one predicted miRNA target. BLAST analysis showed that 75% of all ESTs shared at least a common homologous region (E-value < 10-4) and about 50% of these displayed 400 bp or longer aligned sequences as conserved homologous/orthologous (COS) regions. 960 and 890 ESTs of safflower and artichoke organized in COS shared 79 different miRNA targets, considered functionally conserved, and statistically significant when compared with random sequences (signal to noise ratio > 2 and specificity ≥ 0.85). Four highly significant miRNAs selected from in silico data were experimentally validated in globe artichoke leaves. Conclusions Mature miRNAs and targets were predicted within EST sequences of safflower and artichoke. Most of the miRNA targets appeared highly/moderately conserved, highlighting an important and conserved function. In this study we introduce a stringent parameter for the comparative sequence analysis, represented by the identification of the same target in the COS region. After statistical analysis 79 targets, found on the COS regions and belonging to 60 miRNA families, have a signal to noise ratio > 2, with ≥ 0.85 specificity. The putative miRNAs identified belong to 55 dicotyledon plants and to 24 families only in monocotyledon. PMID:22536958

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis

PubMed Central

2011-01-01

Background Sessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M. galloprovincialis, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes. Results We have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in M. galloprovincialis. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with Vibrio splendidus at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the Vibrio-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways. Conclusions The Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on Vibrio-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the Mytilus species to an evolving microbial world. PMID:21269501

Influence of Molecular Resolution on Sequence-Based Discovery of Ecological Diversity among Synechococcus Populations in an Alkaline Siliceous Hot Spring Microbial Mat ▿ †

PubMed Central

Melendrez, Melanie C.; Lange, Rachel K.; Cohan, Frederick M.; Ward, David M.

2011-01-01

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel. PMID:21169433

Complete Genome Sequence of Diaphorina citri-associated C virus, a Novel Putative RNA Virus of the Asian Citrus Psyllid, Diaphorina citri.

PubMed

Nouri, Shahideh; Salem, Nidà; Falk, Bryce W

2016-07-21

We present here the complete nucleotide sequence and genome organization of a novel putative RNA virus identified in field populations of the Asian citrus psyllid, Diaphorina citri, through sequencing of the transcriptome followed by reverse transcription-PCR (RT-PCR). We tentatively named this virus Diaphorina citri-associated C virus (DcACV). DcACV is an unclassified positive-sense RNA virus. Copyright © 2016 Nouri et al.

Complete genome sequence of an avian paramyxovirus representative of putative new serotype 13

USDA-ARS?s Scientific Manuscript database

Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15- 02/2011. The genomic characterization of the isolate s...

Biochemical and Genetic Evidence that Enterococcus faecium L50 Produces Enterocins L50A and L50B, the sec-Dependent Enterocin P, and a Novel Bacteriocin Secreted without an N-Terminal Extension Termed Enterocin Q

PubMed Central

Cintas, Luis M.; Casaus, Pilar; Herranz, Carmen; Håvarstein, Leiv Sigve; Holo, Helge; Hernández, Pablo E.; Nes, Ingolf F.

2000-01-01

Enterococcus faecium L50 grown at 16 to 32°C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47°C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Håvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321–4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47°C and maximally detected at 47 and 37 to 47°C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25°C and are not detected at 37°C or above. PMID:11073927

Transcriptome analysis of Spodoptera frugiperda Sf9 cells reveals putative apoptosis-related genes and a preliminary apoptosis mechanism induced by azadirachtin.

PubMed

Shu, Benshui; Zhang, Jingjing; Sethuraman, Veeran; Cui, Gaofeng; Yi, Xin; Zhong, Guohua

2017-10-16

As an important botanical pesticide, azadirachtin demonstrates broad insecticidal activity against many agricultural pests. The results of a previous study indicated the toxicity and apoptosis induction of azadirachtin in Spodoptera frugiperda Sf9 cells. However, the lack of genomic data has hindered a deeper investigation of apoptosis in Sf9 cells at a molecular level. In the present study, the complete transcriptome data for Sf9 cell line was accomplished using Illumina sequencing technology, and 97 putative apoptosis-related genes were identified through BLAST and KEGG orthologue annotations. Fragments of potential candidate apoptosis-related genes were cloned, and the mRNA expression patterns of ten identified genes regulated by azadirachtin were examined using qRT-PCR. Furthermore, Western blot analysis showed that six putative apoptosis-related proteins were upregulated after being treated with azadirachtin while the protein Bcl-2 were downregulated. These data suggested that both intrinsic and extrinsic apoptotic signal pathways comprising the identified potential apoptosis-related genes were potentially active in S. frugiperda. In addition, the preliminary results revealed that caspase-dependent or caspase-independent apoptotic pathways could function in azadirachtin-induced apoptosis in Sf9 cells.

Sequence of a cDNA encoding pancreatic preprosomatostatin-22.

PubMed Central

Magazin, M; Minth, C D; Funckes, C L; Deschenes, R; Tavianini, M A; Dixon, J E

1982-01-01

We report the nucleotide sequence of a precursor to somatostatin that upon proteolytic processing may give rise to a hormone of 22 amino acids. The nucleotide sequence of a cDNA from the channel catfish (Ictalurus punctatus) encodes a precursor to somatostatin that is 105 amino acids (Mr, 11,500). The cDNA coding for somatostatin-22 consists of 36 nucleotides in the 5' untranslated region, 315 nucleotides that code for the precursor to somatostatin-22, 269 nucleotides at the 3' untranslated region, and a variable length of poly(A). The putative preprohormone contains a sequence of hydrophobic amino acids at the amino terminus that has the properties of a "signal" peptide. A connecting sequence of approximately 57 amino acids is followed by a single Arg-Arg sequence, which immediately precedes the hormone. Somatostatin-22 is homologous to somatostatin-14 in 7 of the 14 amino acids, including the Phe-Trp-Lys sequence. Hybridization selection of mRNA, followed by its translation in a wheat germ cell-free system, resulted in the synthesis of a single polypeptide having a molecular weight of approximately 10,000 as estimated on Na-DodSO4/polyacrylamide gels. Images PMID:6127673

Structure of the horseradish peroxidase isozyme C genes.

PubMed

Fujiyama, K; Takemura, H; Shibayama, S; Kobayashi, K; Choi, J K; Shinmyo, A; Takano, M; Yamada, Y; Okada, H

1988-05-02

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.

The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains

PubMed Central

1996-01-01

Mutations in the Caenorhabditis elegans gene unc-89 result in nematodes having disorganized muscle structure in which thick filaments are not organized into A-bands, and there are no M-lines. Beginning with a partial cDNA from the C. elegans sequencing project, we have cloned and sequenced the unc-89 gene. An unc-89 allele, st515, was found to contain an 84-bp deletion and a 10-bp duplication, resulting in an in- frame stop codon within predicted unc-89 coding sequence. Analysis of the complete coding sequence for unc-89 predicts a novel 6,632 amino acid polypeptide consisting of sequence motifs which have been implicated in protein-protein interactions. UNC-89 begins with 67 residues of unique sequences, SH3, dbl/CDC24, and PH domains, 7 immunoglobulins (Ig) domains, a putative KSP-containing multiphosphorylation domain, and ends with 46 Ig domains. A polyclonal antiserum raised to a portion of unc-89 encoded sequence reacts to a twitchin-sized polypeptide from wild type, but truncated polypeptides from st515 and from the amber allele e2338. By immunofluorescent microscopy, this antiserum localizes to the middle of A-bands, consistent with UNC-89 being a structural component of the M-line. Previous studies indicate that myofilament lattice assembly begins with positional cues laid down in the basement membrane and muscle cell membrane. We propose that the intracellular protein UNC-89 responds to these signals, localizes, and then participates in assembling an M-line. PMID:8603916

Multiple abiotic stress tolerance of the transformants yeast cells and the transgenic Arabidopsis plants expressing a novel durum wheat catalase.

PubMed

Feki, Kaouthar; Kamoun, Yosra; Ben Mahmoud, Rihem; Farhat-Khemakhem, Ameny; Gargouri, Ali; Brini, Faiçal

2015-12-01

Catalases are reactive oxygen species scavenging enzymes involved in response to abiotic and biotic stresses. In this study, we described the isolation and functional characterization of a novel catalase from durum wheat, designed TdCAT1. Molecular Phylogeny analyses showed that wheat TdCAT1 exhibited high amino acids sequence identity to other plant catalases. Sequence homology analysis showed that TdCAT1 protein contained the putative calmodulin binding domain and a putative conserved internal peroxisomal targeting signal PTS1 motif around its C-terminus. Predicted three-dimensional structural model revealed the presence of four putative distinct structural regions which are the N-terminal arm, the β-barrel, the wrapping and the α-helical domains. TdCAT1 protein had the heme pocket that was composed by five essential residues. TdCAT1 gene expression analysis showed that this gene was induced by various abiotic stresses in durum wheat. The expression of TdCAT1 in yeast cells and Arabidopsis plants conferred tolerance to several abiotic stresses. Compared with the non-transformed plants, the transgenic lines maintained their growth and accumulated more proline under stress treatments. Furthermore, the amount of H2O2 was lower in transgenic lines, which was due to the high CAT and POD activities. Taken together, these data provide the evidence for the involvement of durum wheat catalase TdCAT1 in tolerance to multiple abiotic stresses in crop plants. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection

PubMed Central

Mesquita, Rafael D.; Vionette-Amaral, Raquel J.; Lowenberger, Carl; Rivera-Pomar, Rolando; Monteiro, Fernando A.; Minx, Patrick; Spieth, John; Carvalho, A. Bernardo; Panzera, Francisco; Lawson, Daniel; Torres, André Q.; Ribeiro, Jose M. C.; Sorgine, Marcos H. F.; Waterhouse, Robert M.; Abad-Franch, Fernando; Alves-Bezerra, Michele; Amaral, Laurence R.; Araujo, Helena M.; Aravind, L.; Atella, Georgia C.; Azambuja, Patricia; Berni, Mateus; Bittencourt-Cunha, Paula R.; Braz, Gloria R. C.; Calderón-Fernández, Gustavo; Carareto, Claudia M. A.; Christensen, Mikkel B.; Costa, Igor R.; Costa, Samara G.; Dansa, Marilvia; Daumas-Filho, Carlos R. O.; De-Paula, Iron F.; Dias, Felipe A.; Dimopoulos, George; Emrich, Scott J.; Esponda-Behrens, Natalia; Fampa, Patricia; Fernandez-Medina, Rita D.; da Fonseca, Rodrigo N.; Fontenele, Marcio; Fronick, Catrina; Fulton, Lucinda A.; Gandara, Ana Caroline; Garcia, Eloi S.; Genta, Fernando A.; Giraldo-Calderón, Gloria I.; Gomes, Bruno; Gondim, Katia C.; Granzotto, Adriana; Guarneri, Alessandra A.; Guigó, Roderic; Harry, Myriam; Hughes, Daniel S. T.; Jablonka, Willy; Jacquin-Joly, Emmanuelle; Juárez, M. Patricia; Koerich, Leonardo B.; Lange, Angela B.; Latorre-Estivalis, José Manuel; Lavore, Andrés; Lawrence, Gena G.; Lazoski, Cristiano; Lazzari, Claudio R.; Lopes, Raphael R.; Lorenzo, Marcelo G.; Lugon, Magda D.; Marcet, Paula L.; Mariotti, Marco; Masuda, Hatisaburo; Megy, Karine; Missirlis, Fanis; Mota, Theo; Noriega, Fernando G.; Nouzova, Marcela; Nunes, Rodrigo D.; Oliveira, Raquel L. L.; Oliveira-Silveira, Gilbert; Ons, Sheila; Orchard, Ian; Pagola, Lucia; Paiva-Silva, Gabriela O.; Pascual, Agustina; Pavan, Marcio G.; Pedrini, Nicolás; Peixoto, Alexandre A.; Pereira, Marcos H.; Pike, Andrew; Polycarpo, Carla; Prosdocimi, Francisco; Ribeiro-Rodrigues, Rodrigo; Robertson, Hugh M.; Salerno, Ana Paula; Salmon, Didier; Santesmasses, Didac; Schama, Renata; Seabra-Junior, Eloy S.; Silva-Cardoso, Livia; Silva-Neto, Mario A. C.; Souza-Gomes, Matheus; Sterkel, Marcos; Taracena, Mabel L.; Tojo, Marta; Tu, Zhijian Jake; Tubio, Jose M. C.; Ursic-Bedoya, Raul; Venancio, Thiago M.; Walter-Nuno, Ana Beatriz; Wilson, Derek; Warren, Wesley C.; Wilson, Richard K.; Huebner, Erwin; Dotson, Ellen M.; Oliveira, Pedro L.

2015-01-01

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods. PMID:26627243

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

A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi

PubMed Central

2013-01-01

Background Fungal pathogens cause devastating losses in economically important cereal crops by utilising pathogen proteins to infect host plants. Secreted pathogen proteins are referred to as effectors and have thus far been identified by selecting small, cysteine-rich peptides from the secretome despite increasing evidence that not all effectors share these attributes. Results We take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov model analyses followed by unsupervised protein clustering. Our method returns experimentally validated fungal effectors in Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen. Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens. Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing. Conclusions Our pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms. PMID:24252298

Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection.

PubMed

Mesquita, Rafael D; Vionette-Amaral, Raquel J; Lowenberger, Carl; Rivera-Pomar, Rolando; Monteiro, Fernando A; Minx, Patrick; Spieth, John; Carvalho, A Bernardo; Panzera, Francisco; Lawson, Daniel; Torres, André Q; Ribeiro, Jose M C; Sorgine, Marcos H F; Waterhouse, Robert M; Montague, Michael J; Abad-Franch, Fernando; Alves-Bezerra, Michele; Amaral, Laurence R; Araujo, Helena M; Araujo, Ricardo N; Aravind, L; Atella, Georgia C; Azambuja, Patricia; Berni, Mateus; Bittencourt-Cunha, Paula R; Braz, Gloria R C; Calderón-Fernández, Gustavo; Carareto, Claudia M A; Christensen, Mikkel B; Costa, Igor R; Costa, Samara G; Dansa, Marilvia; Daumas-Filho, Carlos R O; De-Paula, Iron F; Dias, Felipe A; Dimopoulos, George; Emrich, Scott J; Esponda-Behrens, Natalia; Fampa, Patricia; Fernandez-Medina, Rita D; da Fonseca, Rodrigo N; Fontenele, Marcio; Fronick, Catrina; Fulton, Lucinda A; Gandara, Ana Caroline; Garcia, Eloi S; Genta, Fernando A; Giraldo-Calderón, Gloria I; Gomes, Bruno; Gondim, Katia C; Granzotto, Adriana; Guarneri, Alessandra A; Guigó, Roderic; Harry, Myriam; Hughes, Daniel S T; Jablonka, Willy; Jacquin-Joly, Emmanuelle; Juárez, M Patricia; Koerich, Leonardo B; Lange, Angela B; Latorre-Estivalis, José Manuel; Lavore, Andrés; Lawrence, Gena G; Lazoski, Cristiano; Lazzari, Claudio R; Lopes, Raphael R; Lorenzo, Marcelo G; Lugon, Magda D; Majerowicz, David; Marcet, Paula L; Mariotti, Marco; Masuda, Hatisaburo; Megy, Karine; Melo, Ana C A; Missirlis, Fanis; Mota, Theo; Noriega, Fernando G; Nouzova, Marcela; Nunes, Rodrigo D; Oliveira, Raquel L L; Oliveira-Silveira, Gilbert; Ons, Sheila; Orchard, Ian; Pagola, Lucia; Paiva-Silva, Gabriela O; Pascual, Agustina; Pavan, Marcio G; Pedrini, Nicolás; Peixoto, Alexandre A; Pereira, Marcos H; Pike, Andrew; Polycarpo, Carla; Prosdocimi, Francisco; Ribeiro-Rodrigues, Rodrigo; Robertson, Hugh M; Salerno, Ana Paula; Salmon, Didier; Santesmasses, Didac; Schama, Renata; Seabra-Junior, Eloy S; Silva-Cardoso, Livia; Silva-Neto, Mario A C; Souza-Gomes, Matheus; Sterkel, Marcos; Taracena, Mabel L; Tojo, Marta; Tu, Zhijian Jake; Tubio, Jose M C; Ursic-Bedoya, Raul; Venancio, Thiago M; Walter-Nuno, Ana Beatriz; Wilson, Derek; Warren, Wesley C; Wilson, Richard K; Huebner, Erwin; Dotson, Ellen M; Oliveira, Pedro L

2015-12-01

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.

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

Uncovering the defence responses of Eucalyptus to pests and pathogens in the genomics age.

PubMed

Naidoo, Sanushka; Külheim, Carsten; Zwart, Lizahn; Mangwanda, Ronishree; Oates, Caryn N; Visser, Erik A; Wilken, Febé E; Mamni, Thandekile B; Myburg, Alexander A

2014-09-01

Long-lived tree species are subject to attack by various pests and pathogens during their lifetime. This problem is exacerbated by climate change, which may increase the host range for pathogens and extend the period of infestation by pests. Plant defences may involve preformed barriers or induced resistance mechanisms based on recognition of the invader, complex signalling cascades, hormone signalling, activation of transcription factors and production of pathogenesis-related (PR) proteins with direct antimicrobial or anti-insect activity. Trees have evolved some unique defence mechanisms compared with well-studied model plants, which are mostly herbaceous annuals. The genome sequence of Eucalyptus grandis W. Hill ex Maiden has recently become available and provides a resource to extend our understanding of defence in large woody perennials. This review synthesizes existing knowledge of defence mechanisms in model plants and tree species and features mechanisms that may be important for defence in Eucalyptus, such as anatomical variants and the role of chemicals and proteins. Based on the E. grandis genome sequence, we have identified putative PR proteins based on sequence identity to the previously described plant PR proteins. Putative orthologues for PR-1, PR-2, PR-4, PR-5, PR-6, PR-7, PR-8, PR-9, PR-10, PR-12, PR-14, PR-15 and PR-17 have been identified and compared with their orthologues in Populus trichocarpa Torr. & A. Gray ex Hook and Arabidopsis thaliana (L.) Heynh. The survey of PR genes in Eucalyptus provides a first step in identifying defence gene targets that may be employed for protection of the species in future. Genomic resources available for Eucalyptus are discussed and approaches for improving resistance in these hardwood trees, earmarked as a bioenergy source in future, are considered. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The genomic landscape of pediatric and young adult T-lineage acute lymphoblastic leukemia | Office of Cancer Genomics

Cancer.gov

Genetic alterations that activate NOTCH1 signaling and T cell transcription factors, coupled with inactivation of the INK4/ARF tumor suppressors, are hallmarks of T-lineage acute lymphoblastic leukemia (T-ALL), but detailed genome-wide sequencing of large T-ALL cohorts has not been carried out. Using integrated genomic analysis of 264 T-ALL cases, we identified 106 putative driver genes, half of which had not previously been described in childhood T-ALL (for example, CCND3, CTCF, MYB, SMARCA4, ZFP36L2 and MYCN).

Signaling coupled epigenomic regulation of gene expression.

PubMed

Kumar, R; Deivendran, S; Santhoshkumar, T R; Pillai, M R

2017-10-26

Inheritance of genomic information independent of the DNA sequence, the epigenetics, as well as gene transcription are profoundly shaped by serine/threonine and tyrosine signaling kinases and components of the chromatin remodeling complexes. To precisely respond to a changing external milieu, human cells efficiently translate upstream signals into post-translational modifications (PTMs) on histones and coregulators such as corepressors, coactivators, DNA-binding factors and PTM modifying enzymes. Because a protein with multiple residues for putative PTMs is expected to undergo more than one PTM in cells stimulated with growth factors, the outcome of combinational PTM codes on histones and coregulators is profoundly shaped by regulatory interplays between PTMs. The genomic functions of signaling kinases in cancer cells are manifested by the downstream effectors of cytoplasmic signaling cascades as well as translocation of the cytoplasmic signaling kinases to the nucleus. Signaling-mediated phosphorylation of histones serves as a regulatory switch for other PTMs, and connects chromatin remodeling complexes into gene transcription and gene activity. Here, we will discuss the recent advances in signaling-dependent epigenomic regulation of gene transcription using a few representative cancer-relevant serine/threonine and tyrosine kinases and their interplay with chromatin remodeling factors in cancer cells.

Exome sequencing identifies putative drivers of progression of transient myeloproliferative disorder to AMKL in infants with Down syndrome.

PubMed

Nikolaev, Sergey I; Santoni, Federico; Vannier, Anne; Falconnet, Emilie; Giarin, Emanuela; Basso, Giuseppe; Hoischen, Alexander; Veltman, Joris A; Groet, Jurgen; Nizetic, Dean; Antonarakis, Stylianos E

2013-07-25

Some neonates with Down syndrome (DS) are diagnosed with self-regressing transient myeloproliferative disorder (TMD), and 20% to 30% of those progress to acute megakaryoblastic leukemia (AMKL). We performed exome sequencing in 7 TMD/AMKL cases and copy-number analysis in these and 10 additional cases. All TMD/AMKL samples contained GATA1 mutations. No exome-sequenced TMD/AMKL sample had other recurrently mutated genes. However, 2 of 5 TMD cases, and all AMKL cases, showed mutations/deletions other than GATA1, in genes proven as transformation drivers in non-DS leukemia (EZH2, APC, FLT3, JAK1, PARK2-PACRG, EXT1, DLEC1, and SMC3). One patient at the TMD stage revealed 2 clonal expansions with different GATA1 mutations, of which 1 clone had an additional driver mutation. Interestingly, it was the other clone that gave rise to AMKL after accumulating mutations in 7 other genes. Data suggest that GATA1 mutations alone are sufficient for clonal expansions, and additional driver mutations at the TMD stage do not necessarily predict AMKL progression. Later in infancy, leukemic progression requires "third-hit driver" mutations/somatic copy-number alterations found in non-DS leukemias. Putative driver mutations affecting WNT (wingless-related integration site), JAK-STAT (Janus kinase/signal transducer and activator of transcription), or MAPK/PI3K (mitogen-activated kinase/phosphatidylinositol-3 kinase) pathways were found in all cases, aberrant activation of which converges on overexpression of MYC.

Identification of Putative Cardiovascular System Developmental Toxicants using a Classification Model based on Signaling Pathway-Adverse Outcome Pathways

EPA Science Inventory

An important challenge for an integrative approach to developmental systems toxicology is associating putative molecular initiating events (MIEs), cell signaling pathways, cell function and modeled fetal exposure kinetics. We have developed a chemical classification model based o...

Genetic differentiation and hybrid identification using microsatellite markers in closely related wild species

PubMed Central

Turchetto, Caroline; Segatto, Ana Lúcia A.; Beduschi, Júlia; Bonatto, Sandro L.; Freitas, Loreta B.

2015-01-01

Identifying the genetic basis of speciation is critical for understanding the evolutionary history of closely related wild species. Recently diverged species facilitate the study of speciation because many genetic and morphological characteristics are still shared by the organisms under study. The Petunia genus grows in South American grasslands and comprises both recently diverged wild species and commercial species. In this work, we analysed two closely related species: Petunia exserta, which has a narrow endemic range and grows exclusively in rocky shelters, and Petunia axillaris, which is widely distributed and comprises three allopatric subspecies. Petunia axillaris ssp. axillaris and P. exserta occur in sympatry, and putative hybrids between them have been identified. Here, we analysed 14 expressed sequence tag-simple sequence repeats (EST-SSRs) in 126 wild individuals and 13 putative morphological hybrids with the goals of identifying differentially encoded alleles to characterize their natural genetic diversity, establishing a genetic profile for each taxon and to verify the presence of hybridization signal. Overall, 143 alleles were identified and all taxa contained private alleles. Four major groups were identified in clustering analyses, which indicated that there are genetic distinctions among the groups. The markers evaluated here will be useful in evolutionary studies involving these species and may help categorize individuals by species, thus enabling the identification of hybrids between both their putative taxa. The individuals with intermediate morphology presented private alleles of their both putative parental species, although they showed a level of genetic mixing that was comparable with some of the individuals with typical P. exserta morphology. The EST-SSR markers scattered throughout the Petunia genome are very efficient tools for characterizing the genetic diversity in wild taxa of this genus and aid in identifying interspecific hybrids based on the presence of private alleles. These properties indicate that these markers will be helpful tools in evolutionary studies. PMID:26187606

Identification of miRNA from Bouteloua gracilis, a drought tolerant grass, by deep sequencing and their in silico analysis.

PubMed

Ordóñez-Baquera, Perla Lucía; González-Rodríguez, Everardo; Aguado-Santacruz, Gerardo Armando; Rascón-Cruz, Quintín; Conesa, Ana; Moreno-Brito, Verónica; Echavarria, Raquel; Dominguez-Viveros, Joel

2017-02-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate signal transduction, development, metabolism, and stress responses in plants through post-transcriptional degradation and/or translational repression of target mRNAs. Several studies have addressed the role of miRNAs in model plant species, but miRNA expression and function in economically important forage crops, such as Bouteloua gracilis (Poaceae), a high-quality and drought-resistant grass distributed in semiarid regions of the United States and northern Mexico remain unknown. We applied high-throughput sequencing technology and bioinformatics analysis and identified 31 conserved miRNA families and 53 novel putative miRNAs with different abundance of reads in chlorophyllic cell cultures derived from B. gracilis. Some conserved miRNA families were highly abundant and possessed predicted targets involved in metabolism, plant growth and development, and stress responses. We also predicted additional identified novel miRNAs with specific targets, including B. gracilis ESTs, which were detected under drought stress conditions. Here we report 31 conserved miRNA families and 53 putative novel miRNAs in B. gracilis. Our results suggested the presence of regulatory miRNAs involved in modulating physiological and stress responses in this grass species. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome and Transcriptome Analysis of the Fungal Pathogen Fusarium oxysporum f. sp. cubense Causing Banana Vascular Wilt Disease

PubMed Central

Zeng, Huicai; Fan, Dingding; Zhu, Yabin; Feng, Yue; Wang, Guofen; Peng, Chunfang; Jiang, Xuanting; Zhou, Dajie; Ni, Peixiang; Liang, Changcong; Liu, Lei; Wang, Jun; Mao, Chao

2014-01-01

Background The asexual fungus Fusarium oxysporum f. sp. cubense (Foc) causing vascular wilt disease is one of the most devastating pathogens of banana (Musa spp.). To understand the molecular underpinning of pathogenicity in Foc, the genomes and transcriptomes of two Foc isolates were sequenced. Methodology/Principal Findings Genome analysis revealed that the genome structures of race 1 and race 4 isolates were highly syntenic with those of F. oxysporum f. sp. lycopersici strain Fol4287. A large number of putative virulence associated genes were identified in both Foc genomes, including genes putatively involved in root attachment, cell degradation, detoxification of toxin, transport, secondary metabolites biosynthesis and signal transductions. Importantly, relative to the Foc race 1 isolate (Foc1), the Foc race 4 isolate (Foc4) has evolved with some expanded gene families of transporters and transcription factors for transport of toxins and nutrients that may facilitate its ability to adapt to host environments and contribute to pathogenicity to banana. Transcriptome analysis disclosed a significant difference in transcriptional responses between Foc1 and Foc4 at 48 h post inoculation to the banana ‘Brazil’ in comparison with the vegetative growth stage. Of particular note, more virulence-associated genes were up regulated in Foc4 than in Foc1. Several signaling pathways like the mitogen-activated protein kinase Fmk1 mediated invasion growth pathway, the FGA1-mediated G protein signaling pathway and a pathogenicity associated two-component system were activated in Foc4 rather than in Foc1. Together, these differences in gene content and transcription response between Foc1 and Foc4 might account for variation in their virulence during infection of the banana variety ‘Brazil’. Conclusions/Significance Foc genome sequences will facilitate us to identify pathogenicity mechanism involved in the banana vascular wilt disease development. These will thus advance us develop effective methods for managing the banana vascular wilt disease, including improvement of disease resistance in banana. PMID:24743270

Insights into molecular profiles and genomic evolution of an IRAK4 homolog from rock bream (Oplegnathus fasciatus): immunogen- and pathogen-induced transcriptional expression.

PubMed

Umasuthan, Navaneethaiyer; Bathige, S D N K; Whang, Ilson; Lim, Bong-Soo; Choi, Cheol Young; Lee, Jehee

2015-04-01

As a pivotal signaling mediator of toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling cascades, the IL-1R-associated kinase 4 (IRAK4) is engaged in the activation of host immunity. This study investigates the molecular and expressional profiles of an IRAK4-like homolog from Oplegnathus fasciatus (OfIRAK4). The OfIRAK4 gene (8.2 kb) was structured with eleven exons and ten introns. A putative coding sequence (1395bp) was translated to the OfIRAK protein of 464 amino acids. The deduced OfIRAK4 protein featured a bipartite domain structure composed of a death domain (DD) and a kinase domain (PKc). Teleost IRAK4 appears to be distinct and divergent from that of tetrapods in terms of its exon-intron structure and evolutionary relatedness. Analysis of the sequence upstream of translation initiation site revealed the presence of putative regulatory elements, including NF-κB-binding sites, which are possibly involved in transcriptional control of OfIRAK4. Quantitative real-time PCR (qPCR) was employed to assess the transcriptional expression of OfIRAK4 in different juvenile tissues and post-injection of different immunogens and pathogens. Ubiquitous basal mRNA expression was widely detected with highest level in liver. In vivo flagellin (FLA) challenge significantly intensified its mRNA levels in intestine, liver and head kidney indicating its role in FLA-induced signaling. Meanwhile, up-regulated expression was also determined in liver and head kidney of animals challenged with potent immunogens (LPS and poly I:C) and pathogens (Edwardsiella tarda and Streptococcus iniae and rock bream iridovirus (RBIV)). Taken together, these data implicate that OfIRAK4 might be engaged in antibacterial and antiviral immunity in rock bream. Copyright © 2014 Elsevier Ltd. All rights reserved.

Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues

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

Prody, C.A.; Zevin-Sonkin, D.; Gnatt, A.

1987-06-01

To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase and Torpedo electric organ true acetylcholinesterase. Using these probes, the authors isolated several cDNA clones from lambdagt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. Inmore » RNA blots of poly(A)/sup +/ RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These finding demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species.« less

The complete genome structure and phylogenetic relationship of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Morzunov , Sergey P.; Winton, James R.; Nichol, Stuart T.

1995-01-01

Infectious hematopoietic necrosis virus (IHNV), a member of the family Rhabdoviridae, causes a severe disease with high mortality in salmonid fish. The nucleotide sequence (11, 131 bases) of the entire genome was determined for the pathogenic WRAC strain of IHNV from southern Idaho. This allowed detailed analysis of all 6 genes, the deduced amino acid sequences of their encoded proteins, and important control motifs including leader, trailer and gene junction regions. Sequence analysis revealed that the 6 virus genes are located along the genome in the 3′ to 5′ order: nucleocapsid (N), polymerase-associated phosphoprotein (P or M1), matrix protein (M or M2), surface glycoprotein (G), a unique non-virion protein (NV) and virus polymerase (L). The IHNV genome RNA was found to have highly complementary termini (15 of 16 nucleotides). The gene junction regions display the highly conserved sequence UCURUC(U)7RCCGUG(N)4CACR (in the vRNA sense), which includes the typical rhabdovirus transcription termination/polyadenylation signal and a novel putative transcription initiation signal. Phylogenetic analysis of M, G and L protein sequences allowed insights into the evolutionary and taxonomic relationship of rhabdoviruses of fish relative to those of insects or mammals, and a broader sense of the relationship of non-segmented negative-strand RNA viruses. Based on these data, a new genus, piscivirus, is proposed which will initially contain IHNV, viral hemorrhagic septicemia virus and Hirame rhabdovirus.

Signatures of selection in tilapia revealed by whole genome resequencing.

PubMed

Xia, Jun Hong; Bai, Zhiyi; Meng, Zining; Zhang, Yong; Wang, Le; Liu, Feng; Jing, Wu; Wan, Zi Yi; Li, Jiale; Lin, Haoran; Yue, Gen Hua

2015-09-16

Natural selection and selective breeding for genetic improvement have left detectable signatures within the genome of a species. Identification of selection signatures is important in evolutionary biology and for detecting genes that facilitate to accelerate genetic improvement. However, selection signatures, including artificial selection and natural selection, have only been identified at the whole genome level in several genetically improved fish species. Tilapia is one of the most important genetically improved fish species in the world. Using next-generation sequencing, we sequenced the genomes of 47 tilapia individuals. We identified a total of 1.43 million high-quality SNPs and found that the LD block sizes ranged from 10-100 kb in tilapia. We detected over a hundred putative selective sweep regions in each line of tilapia. Most selection signatures were located in non-coding regions of the tilapia genome. The Wnt signaling, gonadotropin-releasing hormone receptor and integrin signaling pathways were under positive selection in all improved tilapia lines. Our study provides a genome-wide map of genetic variation and selection footprints in tilapia, which could be important for genetic studies and accelerating genetic improvement of tilapia.

Characterization of Urtica dioica agglutinin isolectins and the encoding gene family.

PubMed

Does, M P; Ng, D K; Dekker, H L; Peumans, W J; Houterman, P M; Van Damme, E J; Cornelissen, B J

1999-01-01

Urtica dioica agglutinin (UDA) has previously been found in roots and rhizomes of stinging nettles as a mixture of UDA-isolectins. Protein and cDNA sequencing have shown that mature UDA is composed of two hevein domains and is processed from a precursor protein. The precursor contains a signal peptide, two in-tandem hevein domains, a hinge region and a carboxyl-terminal chitinase domain. Genomic fragments encoding precursors for UDA-isolectins have been amplified by five independent polymerase chain reactions on genomic DNA from stinging nettle ecotype Weerselo. One amplified gene was completely sequenced. As compared to the published cDNA sequence, the genomic sequence contains, besides two basepair substitutions, two introns located at the same positions as in other plant chitinases. By partial sequence analysis of 40 amplified genes, 16 different genes were identified which encode seven putative UDA-isolectins. The deduced amino acid sequences share 78.9-98.9% identity. In extracts of roots and rhizomes of stinging nettle ecotype Weerselo six out of these seven isolectins were detected by mass spectrometry. One of them is an acidic form, which has not been identified before. Our results demonstrate that UDA is encoded by a large gene family.

Deep Sequencing Analysis of RNAs from Citrus Plants Grown in a Citrus Sudden Death-Affected Area Reveals Diverse Known and Putative Novel Viruses.

PubMed

Matsumura, Emilyn E; Coletta-Filho, Helvecio D; Nouri, Shahideh; Falk, Bryce W; Nerva, Luca; Oliveira, Tiago S; Dorta, Silvia O; Machado, Marcos A

2017-04-24

Citrus sudden death (CSD) has caused the death of approximately four million orange trees in a very important citrus region in Brazil. Although its etiology is still not completely clear, symptoms and distribution of affected plants indicate a viral disease. In a search for viruses associated with CSD, we have performed a comparative high-throughput sequencing analysis of the transcriptome and small RNAs from CSD-symptomatic and -asymptomatic plants using the Illumina platform. The data revealed mixed infections that included Citrus tristeza virus (CTV) as the most predominant virus, followed by the Citrus sudden death-associated virus (CSDaV), Citrus endogenous pararetrovirus (CitPRV) and two putative novel viruses tentatively named Citrus jingmen-like virus (CJLV), and Citrus virga-like virus (CVLV). The deep sequencing analyses were sensitive enough to differentiate two genotypes of both viruses previously associated with CSD-affected plants: CTV and CSDaV. Our data also showed a putative association of the CSD-symptomatic plants with a specific CSDaV genotype and a likely association with CitPRV as well, whereas the two putative novel viruses showed to be more associated with CSD-asymptomatic plants. This is the first high-throughput sequencing-based study of the viral sequences present in CSD-affected citrus plants, and generated valuable information for further CSD studies.

Structural analysis of the 5{prime} region of mouse and human Huntington disease genes reveals conservation of putative promoter region and Di- and trinucleotide polymorphisms

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

Lin, Biaoyang; Nasir, J.; Kalchman, M.A.

1995-02-10

We have previously cloned and characterized the murine homologue of the Huntington disease (HD) gene and shown that it maps to mouse chromosome 5 within a region of conserved synteny with human chromosome 4p16.3. Here we present a detailed comparison of the sequence of the putative promoter and the organization of the 5{prime} genomic region of the murine (Hdh) and human HD genes encompassing the first five exons. We show that in this region these two genes share identical exon boundaries, but have different-size introns. Two dinucleotide (CT) and one trinucleotide intronic polymorphism in Hdh and an intronic CA polymorphismmore » in the HD gene were identified. Comparison of 940-bp sequence 5{prime} to the putative translation start site reveals a highly conserved region (78.8% nucleotide identity) between Hdh and the HD gene from nucleotide -56 to -206 (of Hdh). Neither Hdh nor the HD gene have typical TATA or CCAAT elements, but both show one putative AP2 binding site and numerous potential Sp1 binding sites. The high sequence identity between Hdh and the HD gene for approximately 200 bp 5{prime} to the putative translation start site indicates that these sequences may play a role in regulating expression of the Huntington disease gene. 30 refs., 4 figs., 2 tabs.« less

Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune.

PubMed

Yano, Shigekazu; Wakayama, Mamoru; Tachiki, Takashi

2006-07-01

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases. The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.

Molecular Cloning and Characterization of G Alpha Proteins from the Western Tarnished Plant Bug, Lygus hesperus

PubMed Central

Hull, J. Joe; Wang, Meixian

2014-01-01

The Gα subunits of heterotrimeric G proteins play critical roles in the activation of diverse signal transduction cascades. However, the role of these genes in chemosensation remains to be fully elucidated. To initiate a comprehensive survey of signal transduction genes, we used homology-based cloning methods and transcriptome data mining to identity Gα subunits in the western tarnished plant bug (Lygus hesperus Knight). Among the nine sequences identified were single variants of the Gαi, Gαo, Gαs, and Gα12 subfamilies and five alternative splice variants of the Gαq subfamily. Sequence alignment and phylogenetic analyses of the putative L. hesperus Gα subunits support initial classifications and are consistent with established evolutionary relationships. End-point PCR-based profiling of the transcripts indicated head specific expression for LhGαq4, and largely ubiquitous expression, albeit at varying levels, for the other LhGα transcripts. All subfamilies were amplified from L. hesperus chemosensory tissues, suggesting potential roles in olfaction and/or gustation. Immunohistochemical staining of cultured insect cells transiently expressing recombinant His-tagged LhGαi, LhGαs, and LhGαq1 revealed plasma membrane targeting, suggesting the respective sequences encode functional G protein subunits. PMID:26463065

NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

PubMed Central

de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge

2013-01-01

NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1–33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species. PMID:23516598

Comparative analyses of putative toxin gene homologs from an Old World viper, Daboia russelii

PubMed Central

Krishnan, Neeraja M.

2017-01-01

Availability of snake genome sequences has opened up exciting areas of research on comparative genomics and gene diversity. One of the challenges in studying snake genomes is the acquisition of biological material from live animals, especially from the venomous ones, making the process cumbersome and time-consuming. Here, we report comparative sequence analyses of putative toxin gene homologs from Russell’s viper (Daboia russelii) using whole-genome sequencing data obtained from shed skin. When compared with the major venom proteins in Russell’s viper studied previously, we found 45–100% sequence similarity between the venom proteins and their putative homologs in the skin. Additionally, comparative analyses of 20 putative toxin gene family homologs provided evidence of unique sequence motifs in nerve growth factor (NGF), platelet derived growth factor (PDGF), Kunitz/Bovine pancreatic trypsin inhibitor (Kunitz BPTI), cysteine-rich secretory proteins, antigen 5, andpathogenesis-related1 proteins (CAP) and cysteine-rich secretory protein (CRISP). In those derived proteins, we identified V11 and T35 in the NGF domain; F23 and A29 in the PDGF domain; N69, K2 and A5 in the CAP domain; and Q17 in the CRISP domain to be responsible for differences in the largest pockets across the protein domain structures in crotalines, viperines and elapids from the in silico structure-based analysis. Similarly, residues F10, Y11 and E20 appear to play an important role in the protein structures across the kunitz protein domain of viperids and elapids. Our study highlights the usefulness of shed skin in obtaining good quality high-molecular weight DNA for comparative genomic studies, and provides evidence towards the unique features and evolution of putative venom gene homologs in vipers. PMID:29230357

Defense Against Cannibalism: The SdpI Family of Bacterial Immunity/Signal Transduction Proteins

PubMed Central

Povolotsky, Tatyana Leonidovna; Orlova, Ekaterina; Tamang, Dorjee G.

2010-01-01

The SdpI family consists of putative bacterial toxin immunity and signal transduction proteins. One member of the family in Bacillus subtilis, SdpI, provides immunity to cells from cannibalism in times of nutrient limitation. SdpI family members are transmembrane proteins with 3, 4, 5, 6, 7, 8, or 12 putative transmembrane α-helical segments (TMSs). These varied topologies appear to be genuine rather than artifacts due to sequencing or annotation errors. The basic and most frequently occurring element of the SdpI family has 6 TMSs. Homologues of all topological types were aligned to determine the homologous TMSs and loop regions, and the positive-inside rule was used to determine sidedness. The two most conserved motifs were identified between TMSs 1 and 2 and TMSs 4 and 5 of the 6 TMS proteins. These showed significant sequence similarity, leading us to suggest that the primordial precursor of these proteins was a 3 TMS–encoding genetic element that underwent intragenic duplication. Various deletional and fusional events, as well as intragenic duplications and inversions, may have yielded SdpI homologues with topologies of varying numbers and positions of TMSs. We propose a specific evolutionary pathway that could have given rise to these distantly related bacterial immunity proteins. We further show that genes encoding SdpI homologues often appear in operons with genes for homologues of SdpR, SdpI’s autorepressor. Our analyses allow us to propose structure–function relationships that may be applicable to most family members. Electronic supplementary material The online version of this article (doi:10.1007/s00232-010-9260-7) contains supplementary material, which is available to authorized users. PMID:20563570

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.

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.

Allopolyploid Origin of Chenopodium album s. str. (Chenopodiaceae): A Molecular and Cytogenetic Insight

PubMed Central

Krak, Karol; Vít, Petr; Belyayev, Alexander; Douda, Jan; Hreusová, Lucia; Mandák, Bohumil

2016-01-01

Reticulate evolution is characterized by occasional hybridization between two species, creating a network of closely related taxa below and at the species level. In the present research, we aimed to verify the hypothesis of the allopolyploid origin of hexaploid C. album s. str., identify its putative parents and estimate the frequency of allopolyploidization events. We sampled 122 individuals of the C. album aggregate, covering most of its distribution range in Eurasia. Our samples included putative progenitors of C. album s. str. of both ploidy levels, i.e. diploids (C. ficifolium, C. suecicum) and tetraploids (C. striatiforme, C. strictum). To fulfil these objectives, we analysed sequence variation in the nrDNA ITS region and the rpl32-trnL intergenic spacer of cpDNA and performed genomic in-situ hybridization (GISH). Our study confirms the allohexaploid origin of C. album s. str. Analysis of cpDNA revealed tetraploids as the maternal species. In most accessions of hexaploid C. album s. str., ITS sequences were completely or nearly completely homogenized towards the tetraploid maternal ribotype; a tetraploid species therefore served as one genome donor. GISH revealed a strong hybridization signal on the same eighteen chromosomes of C. album s. str. with both diploid species C. ficifolium and C. suecicum. The second genome donor was therefore a diploid species. Moreover, some individuals with completely unhomogenized ITS sequences were found. Thus, hexaploid individuals of C. album s. str. with ITS sequences homogenized to different degrees may represent hybrids of different ages. This proves the existence of at least two different allopolyploid lineages, indicating a polyphyletic origin of C. album s. str. PMID:27513342

Unveiling the complete genome sequence of clerodendrum chlorotic spot virus, a putative dichorhavirus infecting ornamental plants.

PubMed

Ramos-González, Pedro Luis; Chabi-Jesus, Camila; Banguela-Castillo, Alexander; Tassi, Aline Daniele; Rodrigues, Mariane da Costa; Kitajima, Elliot Watanabe; Harakava, Ricardo; Freitas-Astúa, Juliana

2018-06-04

The genus Dichorhavirus includes plant-infecting rhabdoviruses with bisegmented genomes that are horizontally transmitted by false spider mites of the genus Brevipalpus. The complete genome sequences of three isolates of the putative dichorhavirus clerodendrum chlorotic spot virus were determined using next-generation sequencing (Illumina) and traditional RT-PCR. Their genome organization, sequence similarity and phylogenetic relationship to other viruses, and transmissibility by Brevipalpus yothersi mites support the assignment of these viruses to a new species of dichorhavirus, as suggested previously. New data are discussed stressing the reliability of the current rules for species demarcation and taxonomic status criteria within the genus Dichorhavirus.

Cloning, characterization and sequence comparison of the gene coding for IMP dehydrogenase from Pyrococcus furiosus.

PubMed

Collart, F R; Osipiuk, J; Trent, J; Olsen, G J; Huberman, E

1996-10-03

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Pyrococcus furiosus (Pf), a hyperthermophillic archeon. Sequence analysis of the Pf gene indicated an open reading frame specifying a protein of 485 amino acids (aa) with a calculated M(r) of 52900. Canonical Archaea promoter elements, Box A and Box B, are located -49 and -17 nucleotides (nt), respectively, upstream of the putative start codon. The sequence of the putative active-site region conforms to the IMPDH signature motif and contains a putative active-site cysteine. Phylogenetic relationships derived by using all available IMPDH sequences are consistent with trees developed for other molecules; they do not precisely resolve the history of Pf IMPDH but indicate a close similarity to bacterial IMPDH proteins. The phylogenetic analysis indicates that a gene duplication occurred prior to the division between rodents and humans, accounting for the Type I and II isoforms identified in mice and humans.

Molecular Cloning and Expression of Three Polygalacturonase cDNAs from the Tarnished Plant Bug, Lygus lineolaris

PubMed Central

Allen, Margaret L.; Mertens, Jeffrey A.

2008-01-01

Three unique cDNAs encoding putative polygalacturonase enzymes were isolated from the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae). The three nucleotide sequences were dissimilar to one another, but the deduced amino acid sequences were similar to each other and to other polygalacturonases from insects, fungi, plants, and bacteria. Four conserved segments characteristic of polygalacturonases were present, but with some notable semiconservative substitutions. Two of four expected disulfide bridge—forming cysteine pairs were present. All three inferred protein translations included predicted signal sequences of 17 to 20 amino acids. Amplification of genomic DNA identified an intron in one of the genes, Llpg1, in the 5′ untranslated region. Semiquantitative RT-PCR revealed expression in all stages of the insect except the eggs. Expression in adults, male and female, was highly variable, indicating a family of highly inducible and diverse enzymes adapted to the generalist polyphagous nature of this important pest. PMID:20233096

Cloning and characterization of the nagA gene that encodes beta-n-acetylglucosaminidase from Aspergillus nidulans and its expression in Aspergillus oryzae.

PubMed

Kim, Sunhwa; Matsuo, Ichiro; Ajisaka, Katsumi; Nakajima, Harushi; Kitamoto, Katsuhiko

2002-10-01

We isolated a beta-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed beta-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of beta-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture.

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.

Export requirements of pneumolysin in Streptococcus pneumoniae.

PubMed

Price, Katherine E; Greene, Neil G; Camilli, Andrew

2012-07-01

Streptococcus pneumoniae is a major causative agent of otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolysins (CDCs), is produced by virtually all clinical isolates of S. pneumoniae, and ply mutant strains are severely attenuated in mouse models of colonization and infection. In contrast to all other known members of the CDC family, Ply lacks a signal peptide for export outside the cell. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains undefined. We show that an exogenously added signal sequence is not sufficient for Sec-dependent Ply secretion in S. pneumoniae but is sufficient in the surrogate host Bacillus subtilis. Previously, we showed that Ply is localized primarily to the cell wall compartment in the absence of detectable cell lysis. Here we show that Ply released by autolysis cannot reassociate with intact cells, suggesting that there is a Ply export mechanism that is coupled to cell wall localization of the protein. This putative export mechanism is capable of secreting a related CDC without its signal sequence. We show that B. subtilis can export Ply, suggesting that the export pathway is conserved. Finally, through truncation and domain swapping analyses, we show that export is dependent on domain 2 of Ply.

Export Requirements of Pneumolysin in Streptococcus pneumoniae

PubMed Central

Price, Katherine E.; Greene, Neil G.

2012-01-01

Streptococcus pneumoniae is a major causative agent of otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolysins (CDCs), is produced by virtually all clinical isolates of S. pneumoniae, and ply mutant strains are severely attenuated in mouse models of colonization and infection. In contrast to all other known members of the CDC family, Ply lacks a signal peptide for export outside the cell. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains undefined. We show that an exogenously added signal sequence is not sufficient for Sec-dependent Ply secretion in S. pneumoniae but is sufficient in the surrogate host Bacillus subtilis. Previously, we showed that Ply is localized primarily to the cell wall compartment in the absence of detectable cell lysis. Here we show that Ply released by autolysis cannot reassociate with intact cells, suggesting that there is a Ply export mechanism that is coupled to cell wall localization of the protein. This putative export mechanism is capable of secreting a related CDC without its signal sequence. We show that B. subtilis can export Ply, suggesting that the export pathway is conserved. Finally, through truncation and domain swapping analyses, we show that export is dependent on domain 2 of Ply. PMID:22563048

Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew.

PubMed Central

Freeman, R M; Plutzky, J; Neel, B G

1992-01-01

src homology 2 (SH2) domains direct binding to specific phosphotyrosyl proteins. Recently, SH2-containing protein-tyrosine-phosphatases (PTPs) were identified. Using degenerate oligonucleotides and the PCR, we have cloned a cDNA for an additional PTP, SH-PTP2, which contains two SH2 domains and is expressed ubiquitously. When expressed in Escherichia coli, SH-PTP2 displays tyrosine-specific phosphatase activity. Strong sequence similarity between SH-PTP2 and the Drosophila gene corkscrew (csw) and their similar patterns of expression suggest that SH-PTP2 is the human corkscrew homolog. Sequence comparisons between SH-PTP2, SH-PTP1, corkscrew, and other SH2-containing proteins suggest the existence of a subfamily of SH2 domains found specifically in PTPs, whereas comparison of the PTP domains of the SH2-containing PTPs with other tyrosine phosphatases suggests the existence of a subfamily of PTPs containing SH2 domains. Since corkscrew, a member of the terminal class signal transduction pathway, acts in concert with D-raf to positively transduce the signal generated by the receptor tyrosine kinase torso, these findings suggest several mechanisms by which SH-PTP2 may participate in mammalian signal transduction. Images PMID:1280823

Deep RNA-Seq to unlock the gene bank of floral development in Sinapis arvensis.

PubMed

Liu, Jia; Mei, Desheng; Li, Yunchang; Huang, Shunmou; Hu, Qiong

2014-01-01

Sinapis arvensis is a weed with strong biological activity. Despite being a problematic annual weed that contaminates agricultural crop yield, it is a valuable alien germplasm resource. It can be utilized for broadening the genetic background of Brassica crops with desirable agricultural traits like resistance to blackleg (Leptosphaeria maculans), stem rot (Sclerotinia sclerotium) and pod shatter (caused by FRUITFULL gene). However, few genetic studies of S. arvensis were reported because of the lack of genomic resources. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive dataset for S. arvensis for the first time. We used Illumina paired-end sequencing technology to sequence the S. arvensis flower transcriptome and generated 40,981,443 reads that were assembled into 131,278 transcripts. We de novo assembled 96,562 high quality unigenes with an average length of 832 bp. A total of 33,662 full-length ORF complete sequences were identified, and 41,415 unigenes were mapped onto 128 pathways using the KEGG Pathway database. The annotated unigenes were compared against Brassica rapa, B. oleracea, B. napus and Arabidopsis thaliana. Among these unigenes, 76,324 were identified as putative homologs of annotated sequences in the public protein databases, of which 1194 were associated with plant hormone signal transduction and 113 were related to gibberellin homeostasis/signaling. Unigenes that did not match any of those sequence datasets were considered to be unique to S. arvensis. Furthermore, 21,321 simple sequence repeats were found. Our study will enhance the currently available resources for Brassicaceae and will provide a platform for future genomic studies for genetic improvement of Brassica crops.

Deep RNA-Seq to Unlock the Gene Bank of Floral Development in Sinapis arvensis

PubMed Central

Liu, Jia; Mei, Desheng; Li, Yunchang; Huang, Shunmou; Hu, Qiong

2014-01-01

Sinapis arvensis is a weed with strong biological activity. Despite being a problematic annual weed that contaminates agricultural crop yield, it is a valuable alien germplasm resource. It can be utilized for broadening the genetic background of Brassica crops with desirable agricultural traits like resistance to blackleg (Leptosphaeria maculans), stem rot (Sclerotinia sclerotium) and pod shatter (caused by FRUITFULL gene). However, few genetic studies of S. arvensis were reported because of the lack of genomic resources. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive dataset for S. arvensis for the first time. We used Illumina paired-end sequencing technology to sequence the S. arvensis flower transcriptome and generated 40,981,443 reads that were assembled into 131,278 transcripts. We de novo assembled 96,562 high quality unigenes with an average length of 832 bp. A total of 33,662 full-length ORF complete sequences were identified, and 41,415 unigenes were mapped onto 128 pathways using the KEGG Pathway database. The annotated unigenes were compared against Brassica rapa, B. oleracea, B. napus and Arabidopsis thaliana. Among these unigenes, 76,324 were identified as putative homologs of annotated sequences in the public protein databases, of which 1194 were associated with plant hormone signal transduction and 113 were related to gibberellin homeostasis/signaling. Unigenes that did not match any of those sequence datasets were considered to be unique to S. arvensis. Furthermore, 21,321 simple sequence repeats were found. Our study will enhance the currently available resources for Brassicaceae and will provide a platform for future genomic studies for genetic improvement of Brassica crops. PMID:25192023

Complete Genome Sequence of an Avian Paramyxovirus Representative of Putative New Serotype 13

PubMed Central

Goraichuk, Iryna; Sharma, Poonam; Stegniy, Borys; Muzyka, Denys; Pantin-Jackwood, Mary J.; Gerilovych, Anton; Solodiankin, Olexii; Bolotin, Vitaliy; Miller, Patti J.; Dimitrov, Kiril M.

2016-01-01

Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13. PMID:27469958

Strigolactone-Induced Putative Secreted Protein 1 Is Required for the Establishment of Symbiosis by the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis.

PubMed

Tsuzuki, Syusaku; Handa, Yoshihiro; Takeda, Naoya; Kawaguchi, Masayoshi

2016-04-01

Arbuscular mycorrhizal (AM) symbiosis is the most widespread association between plants and fungi. To provide novel insights into the molecular mechanisms of AM symbiosis, we screened and investigated genes of the AM fungus Rhizophagus irregularis that contribute to the infection of host plants. R. irregularis genes involved in the infection were explored by RNA-sequencing (RNA-seq) analysis. One of the identified genes was then characterized by a reverse genetic approach using host-induced gene silencing (HIGS), which causes RNA interference in the fungus via the host plant. The RNA-seq analysis revealed that 19 genes are up-regulated by both treatment with strigolactone (SL) (a plant symbiotic signal) and symbiosis. Eleven of the 19 genes were predicted to encode secreted proteins and, of these, SL-induced putative secreted protein 1 (SIS1) showed the largest induction under both conditions. In hairy roots of Medicago truncatula, SIS1 expression is knocked down by HIGS, resulting in significant suppression of colonization and formation of stunted arbuscules. These results suggest that SIS1 is a putative secreted protein that is induced in a wide spatiotemporal range including both the presymbiotic and symbiotic stages and that SIS1 positively regulates colonization of host plants by R. irregularis.

Preferential Representation of Past Outcome Information and Future Choice Behavior by Putative Inhibitory Interneurons Rather Than Putative Pyramidal Neurons in the Primate Dorsal Anterior Cingulate Cortex.

PubMed

Kawai, Takashi; Yamada, Hiroshi; Sato, Nobuya; Takada, Masahiko; Matsumoto, Masayuki

2018-05-02

The dorsal anterior cingulate cortex (dACC) plays crucial roles in monitoring the outcome of a choice and adjusting a subsequent choice behavior based on the outcome information. In the present study, we investigated how different types of dACC neurons, that is, putative pyramidal neurons and putative inhibitory interneurons, contribute to these processes. We analyzed single-unit database obtained from the dACC in monkeys performing a reversal learning task. The monkey was required to adjust choice behavior from past outcome experiences. Depending on their action potential waveforms, the recorded neurons were classified into putative pyramidal neurons and putative inhibitory interneurons. We found that these neurons do not equally contribute to outcome monitoring and behavioral adjustment. Although both neuron types evenly responded to the current outcome, a larger proportion of putative inhibitory interneurons than putative pyramidal neurons stored the information about the past outcome. The putative inhibitory interneurons further represented choice-related signals more frequently, such as whether the monkey would shift the last choice to an alternative at the next choice opportunity. Our findings suggest that putative inhibitory interneurons, which are thought not to project to brain areas outside the dACC, preferentially transmit signals that would adjust choice behavior based on past outcome experiences.

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

Sequence variability of Campylobacter temperate bacteriophages

PubMed Central

Clark, Clifford G; Ng, Lai-King

2008-01-01

Background Prophages integrated within the chromosomes of Campylobacter jejuni isolates have been demonstrated very recently. Prior work with Campylobacter temperate bacteriophages, as well as evidence from prophages in other enteric bacteria, suggests these prophages might have a role in the biology and virulence of the organism. However, very little is known about the genetic variability of Campylobacter prophages which, if present, could lead to differential phenotypes in isolates carrying the phages versus those that do not. As a first step in the characterization of C. jejuni prophages, we investigated the distribution of prophage DNA within a C. jejuni population assessed the DNA and protein sequence variability within a subset of the putative prophages found. Results Southern blotting of C. jejuni DNA using probes from genes within the three putative prophages of the C. jejuni sequenced strain RM 1221 demonstrated the presence of at least one prophage gene in a large proportion (27/35) of isolates tested. Of these, 15 were positive for 5 or more of the 7 Campylobacter Mu-like phage 1 (CMLP 1, also designated Campylobacter jejuni integrated element 1, or CJIE 1) genes tested. Twelve of these putative prophages were chosen for further analysis. DNA sequencing of a 9,000 to 11,000 nucleotide region of each prophage demonstrated a close homology with CMLP 1 in both gene order and nucleotide sequence. Structural and sequence variability, including short insertions, deletions, and allele replacements, were found within the prophage genomes, some of which would alter the protein products of the ORFs involved. No insertions of novel genes were detected within the sequenced regions. The 12 prophages and RM 1221 had a % G+C very similar to C. jejuni sequenced strains, as well as promoter regions characteristic of C. jejuni. None of the putative prophages were successfully induced and propagated, so it is not known if they were functional or if they represented remnant prophage DNA in the bacterial chromosomes. Conclusion These putative prophages form a family of phages with conserved sequences, and appear to be adapted to Campylobacter. There was evidence for recombination among groups of prophages, suggesting that the prophages had a mosaic structure. In many of these properties, the Mu-like CMLP 1 homologs characterized in this study resemble temperate bacteriophages of enteric bacteria that are responsible for contributions to virulence and host adaptation. PMID:18366706

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence.

PubMed

Lahr, Roni M; Mack, Seshat M; Héroux, Annie; Blagden, Sarah P; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc; Berman, Andrea J

2015-09-18

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

DOE PAGES

Lahr, Roni M.; Mack, Seshat M.; Heroux, Annie; ...

2015-07-22

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. Amore » putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.« less

Complete Genome Sequence of an Avian Paramyxovirus Representative of Putative New Serotype 13.

PubMed

Goraichuk, Iryna; Sharma, Poonam; Stegniy, Borys; Muzyka, Denys; Pantin-Jackwood, Mary J; Gerilovych, Anton; Solodiankin, Olexii; Bolotin, Vitaliy; Miller, Patti J; Dimitrov, Kiril M; Afonso, Claudio L

2016-07-28

Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13. Copyright © 2016 Goraichuk et al.

Identification and Characterization of Putative Integron-Like Elements of the Heavy-Metal-Hypertolerant Strains of Pseudomonas spp.

PubMed

Ciok, Anna; Adamczuk, Marcin; Bartosik, Dariusz; Dziewit, Lukasz

2016-11-28

Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas .

The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

PubMed Central

Urano, Y; Kominami, R; Mishima, Y; Muramatsu, M

1980-01-01

Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Images PMID:6162156

Molecular detection of a putatively novel cyprinid herpesvirus in sichel (Pelecus cultratus) during a mass mortality event in Hungary.

PubMed

Doszpoly, Andor; Papp, Melitta; Deákné, Petra P; Glávits, Róbert; Ursu, Krisztina; Dán, Ádám

2015-05-01

In the early summer of 2014, mass mortality of sichel (Pelecus cultratus) was observed in Lake Balaton, Hungary. Histological examination revealed degenerative changes within the tubular epithelium, mainly in the distal tubules and collecting ducts in the kidneys and multifocal vacuolisation in the brain stem and cerebellum. Routine molecular investigations showed the presence of the DNA of an unknown alloherpesvirus in some specimens. Subsequently, three genes of the putative herpesviral genome (DNA polymerase, terminase, and helicase) were amplified and partially sequenced. A phylogenetic tree reconstruction based on the concatenated sequence of these three conserved genes implied that the virus belongs to the genus Cyprinivirus within the family Alloherpesviridae. The sequences of the sichel herpesvirus differ markedly from those of the cypriniviruses CyHV-1, CyHV-2 and CyHV-3, putatively representing a fifth species in the genus.

Genome-wide identification and expression analysis of MAPK and MAPKK gene family in Malus domestica.

PubMed

Zhang, Shizhong; Xu, Ruirui; Luo, Xiaocui; Jiang, Zesheng; Shu, Huairui

2013-12-01

MAPK signal transduction modules play crucial roles in regulating many biological processes in plants, which are composed of three classes of hierarchically organized protein kinases, namely MAPKKKs, MAPKKs, and MAPKs. Although genome-wide analysis of this family has been carried out in some species, little is known about MAPK and MAPKK genes in apple (Malus domestica). In this study, a total of 26 putative apple MAPK genes (MdMPKs) and 9 putative apple MAPKK genes (MdMKKs) have been identified and located within the apple genome. Phylogenetic analysis revealed that MdMAPKs and MdMAPKKs could be divided into 4 subfamilies (groups A, B, C and D), respectively. The predicted MdMAPKs and MdMAPKKs were distributed across 13 out of 17 chromosomes with different densities. In addition, analysis of exon-intron junctions and of intron phase inside the predicted coding region of each candidate gene has revealed high levels of conservation within and between phylogenetic groups. According to the microarray and expressed sequence tag (EST) analysis, the different expression patterns indicate that they may play different roles during fruit development and rootstock-scion interaction process. Moreover, MAPK and MAPKK genes were performed expression profile analyses in different tissues (root, stem, leaf, flower and fruit), and all of the selected genes were expressed in at least one of the tissues tested, indicating that the MAPKs and MAPKKs are involved in various aspects of physiological and developmental processes of apple. To our knowledge, this is the first report of a genome-wide analysis of the apple MAPK and MAPKK gene family. This study provides valuable information for understanding the classification and putative functions of the MAPK signal in apple. © 2013.

Molecular cloning and characterization of a new basic peroxidase cDNA from soybean hypocotyls infected with Phytophthora sojae f.sp. glycines.

PubMed

Yi, S Y; Hwang, B K

1998-10-31

Differential display techniques were used to isolate cDNA clones corresponding to genes which were expressed in soybean hypocotyls by Phytophthora sojae f.sp. glycines infection. With a partial cDNA clone C20CI4 from the differential display PCR as a probe, a new basic peroxidase cDNA clone, designated GMIPER1, was isolated from a cDNA library of soybean hypocotyls infected with P. sojae f.sp. glycines. Sequence analysis revealed that the peroxidase clone encodes a mature protein of 35,813 Da with a putative signal peptide of 27 amino acids in its N-terminus. The amino acid sequence of the soybean peroxidase GMIPER1 is between 54-75% identical to other plant peroxidases including a soybean seed coat peroxidase. Southern blot analysis indicated that multiple copies of sequences related to GMIPER1 exist in the soybean genome. The mRNAs corresponding to the GMIPER1 cDNA accumulated predominantly in the soybean hypocotyls infected with the incompatible race of P. sojae f.sp. glycines, but were expressed at low levels in the compatible interaction. Soybean GMIPER1 mRNAs were not expressed in hypocotyls, leaves, stems, and roots of soybean seedlings. However, treatments with ethephon, salicylic acid or methyl jasmonate induced the accumulation of the GMIPER1 mRNAs in the different organs of soybean. These results suggest that the GMIPER1 gene encoding a putative pathogen-induced peroxidase may play an important role in induced resistance of soybean to P. sojae f.sp. glycines and in response to various external stresses.

A putative carbohydrate-binding domain of the lactose-binding Cytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of the L-fucose-binding Ulex europaeus anti-H(O) lectin.

PubMed

Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

1995-04-01

The complete amino acid sequence of a lactose-binding Cytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of the L-fucose-binding Ulex europaeus lectin I (UEA-I).

In vivo functional mapping of the conserved protein domains within murine Themis1.

PubMed

Zvezdova, Ekaterina; Lee, Jan; El-Khoury, Dalal; Barr, Valarie; Akpan, Itoro; Samelson, Lawrence; Love, Paul E

2014-09-01

Thymocyte development requires the coordinated input of signals that originate from numerous cell surface molecules. Although the majority of thymocyte signal-initiating receptors are lineage-specific, most trigger 'ubiquitous' downstream signaling pathways. T-lineage-specific receptors are coupled to these signaling pathways by lymphocyte-restricted adapter molecules. We and others recently identified a new putative adapter protein, Themis1, whose expression is largely restricted to the T lineage. Mice lacking Themis1 exhibit a severe block in thymocyte development and a striking paucity of mature T cells revealing a critical role for Themis1 in T-cell maturation. Themis1 orthologs contain three conserved domains: a proline-rich region (PRR) that binds to the ubiquitous cytosolic adapter Grb2, a nuclear localization sequence (NLS), and two copies of a novel cysteine-containing globular (CABIT) domain. In the present study, we evaluated the functional importance of each of these motifs by retroviral reconstitution of Themis1(-/-) progenitor cells. The results demonstrate an essential requirement for the PRR and NLS motifs but not the conserved CABIT cysteines for Themis1 function.

BASH, a novel signaling molecule preferentially expressed in B cells of the bursa of Fabricius.

PubMed

Goitsuka, R; Fujimura, Y; Mamada, H; Umeda, A; Morimura, T; Uetsuka, K; Doi, K; Tsuji, S; Kitamura, D

1998-12-01

The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.

Combinatorial Discovery of Defined Substrates That Promote a Stem Cell State in Malignant Melanoma

PubMed Central

2017-01-01

The tumor microenvironment is implicated in orchestrating cancer cell transformation and metastasis. However, specific cell–ligand interactions between cancer cells and the extracellular matrix are difficult to decipher due to a dynamic and multivariate presentation of many signaling molecules. Here we report a versatile peptide microarray platform that is capable of screening for cancer cell phenotypic changes in response to ligand–receptor interactions. Using a screen of 78 peptide combinations derived from proteins present in the melanoma microenvironment, we identify a proteoglycan binding and bone morphogenic protein 7 (BMP7) derived sequence that selectively promotes the expression of several putative melanoma initiating cell markers. We characterize signaling associated with each of these peptides in the activation of melanoma pro-tumorigenic signaling and reveal a role for proteoglycan mediated adhesion and signaling through Smad 2/3. A defined substratum that controls the state of malignant melanoma may prove useful in spatially normalizing a heterogeneous population of tumor cells for discovery of therapeutics that target a specific state and for identifying new drug targets and reagents for intervention. PMID:28573199

Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands

PubMed Central

Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G.

2016-01-01

The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands. PMID:27576083

Molecular Diagnosis of Putative Stargardt Disease by Capture Next Generation Sequencing

PubMed Central

Shi, Wei; Huang, Ping; Min, Qingjie; Li, Minghan; Yu, Xinping; Wu, Yaming; Zhao, Guangyu; Tong, Yi; Jin, Zi-Bing; Qu, Jia; Gu, Feng

2014-01-01

Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in ABCA4 and two in PROM1 were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in PROM1 gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis. PMID:24763286

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

Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues.

PubMed Central

Prody, C A; Zevin-Sonkin, D; Gnatt, A; Goldberg, O; Soreq, H

1987-01-01

To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase (BtChoEase; EC 3.1.1.8) and Torpedo electric organ "true" acetylcholinesterase (AcChoEase; EC 3.1.1.7). Using these probes, we isolated several cDNA clones from lambda gt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A)+ RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These findings demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species. Images PMID:3035536

The Nostoc punctiforme Genome

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

John C. Meeks

2001-12-31

Nostoc punctiforme is a filamentous cyanobacterium with extensive phenotypic characteristics and a relatively large genome, approaching 10 Mb. The phenotypic characteristics include a photoautotrophic, diazotrophic mode of growth, but N. punctiforme is also facultatively heterotrophic; its vegetative cells have multiple development alternatives, including terminal differentiation into nitrogen-fixing heterocysts and transient differentiation into spore-like akinetes or motile filaments called hormogonia; and N. punctiforme has broad symbiotic competence with fungi and terrestrial plants, including bryophytes, gymnosperms and an angiosperm. The shotgun-sequencing phase of the N. punctiforme strain ATCC 29133 genome has been completed by the Joint Genome Institute. Annotation of an 8.9more » Mb database yielded 7432 open reading frames, 45% of which encode proteins with known or probable known function and 29% of which are unique to N. punctiforme. Comparative analysis of the sequence indicates a genome that is highly plastic and in a state of flux, with numerous insertion sequences and multilocus repeats, as well as genes encoding transposases and DNA modification enzymes. The sequence also reveals the presence of genes encoding putative proteins that collectively define almost all characteristics of cyanobacteria as a group. N. punctiforme has an extensive potential to sense and respond to environmental signals as reflected by the presence of more than 400 genes encoding sensor protein kinases, response regulators and other transcriptional factors. The signal transduction systems and any of the large number of unique genes may play essential roles in the cell differentiation and symbiotic interaction properties of N. punctiforme.« less

Envelope-like retrotransposons in the plant kingdom: evidence of their presence in gymnosperms (Pinus pinaster).

PubMed

Miguel, Célia; Simões, Marta; Oliveira, Maria Margarida; Rocheta, Margarida

2008-11-01

Retroviruses differ from retrotransposons due to their infective capacity, which depends critically on the encoded envelope. Some plant retroelements contain domains reminiscent of the env of animal retroviruses but the number of such elements described to date is restricted to angiosperms. We show here the first evidence of the presence of putative env-like gene sequences in a gymnosperm species, Pinus pinaster (maritime pine). Using a degenerate primer approach for conserved domains of RNaseH gene, three clones from putative envelope-like retrotransposons (PpRT2, PpRT3, and PpRT4) were identified. The env-like sequences of P. pinaster clones are predicted to encode proteins with transmembrane domains. These sequences showed identity scores of up to 30% with env-like sequences belonging to different organisms. A phylogenetic analysis based on protein alignment of deduced aminoacid sequences revealed that these clones clustered with env-containing plant retrotransposons, as well as with retrotransposons from invertebrate organisms. The differences found among the sequences of maritime pine clones isolated here suggest the existence of different putative classes of env-like retroelements. The identification for the first time of env-like genes in a gymnosperm species may support the ancestrality of retroviruses among plants shedding light on their role in plant evolution.

The use of high-throughput small RNA sequencing reveals differentially expressed microRNAs in response to aster yellows phytoplasma-infection in Vitis vinifera cv. ‘Chardonnay’

PubMed Central

Solofoharivelo, Marie-Chrystine; Souza-Richards, Rose; Stephan, Dirk; Murray, Shane; Burger, Johan T.

2017-01-01

Phytoplasmas are cell wall-less plant pathogenic bacteria responsible for major crop losses throughout the world. In grapevine they cause grapevine yellows, a detrimental disease associated with a variety of symptoms. The high economic impact of this disease has sparked considerable interest among researchers to understand molecular mechanisms related to pathogenesis. Increasing evidence exist that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation during plant development and responses to biotic and abiotic stresses. Thus, we aimed to dissect complex high-throughput small RNA sequencing data for the genome-wide identification of known and novel differentially expressed miRNAs, using read libraries constructed from healthy and phytoplasma-infected Chardonnay leaf material. Furthermore, we utilised computational resources to predict putative miRNA targets to explore the involvement of possible pathogen response pathways. We identified multiple known miRNA sequence variants (isomiRs), likely generated through post-transcriptional modifications. Sequences of 13 known, canonical miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a unique genomic location, were predicted, of which 23 were differentially expressed. A homology search revealed that some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of randomly selected known and novel miRNAs was determined with real-time RT-qPCR analysis, thereby validating the trend of expression seen in the normalised small RNA sequencing read count data. Among the putative miRNA targets, we identified genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. Our results may assist in understanding the role that miRNA pathways play during plant pathogenesis, and may be crucial in understanding disease symptom development in aster yellows phytoplasma-infected grapevines. PMID:28813447

EphB4 localises to the nucleus of prostate cancer cells

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

Mertens-Walker, Inga, E-mail: inga.mertenswalker@qut.edu.au; Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD; Lisle, Jessica E.

2015-04-10

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of bothmore » NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor. - Highlights: • The EphB4 protein can be found in the nucleus of prostate cancer cell lines. • EphB4 contains two functional nuclear localisation signals. • Chromatin immunoprecipitation has identified potential genome sequences to which EphB4 binds. • Lef1 is a confirmed target for EphB4-mediated gene regulation.« less

Cloning and sequencing of the Thermoanaerobacterium saccharolyticum B6A-RI apu gene and purification and characterization of the amylopullulanase from Escherichia coli.

PubMed

Ramesh, M V; Podkovyrov, S M; Lowe, S E; Zeikus, J G

1994-01-01

The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an M(r) of 142,000 +/- 2,000 and had same the catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase.

Cloning and sequencing of the Thermoanaerobacterium saccharolyticum B6A-RI apu gene and purification and characterization of the amylopullulanase from Escherichia coli.

PubMed Central

Ramesh, M V; Podkovyrov, S M; Lowe, S E; Zeikus, J G

1994-01-01

The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an M(r) of 142,000 +/- 2,000 and had same the catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase. Images PMID:8117096

A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS).

PubMed

Unwin, Richard D; Griffiths, John R; Whetton, Anthony D

2009-01-01

The application of a targeted mass spectrometric workflow to the sensitive identification of post-translational modifications is described. This protocol employs multiple reaction monitoring (MRM) to search for all putative peptides specifically modified in a target protein. Positive MRMs trigger an MS/MS experiment to confirm the nature and site of the modification. This approach, termed MIDAS (MRM-initiated detection and sequencing), is more sensitive than approaches using neutral loss scanning or precursor ion scanning methodologies, due to a more efficient use of duty cycle along with a decreased background signal associated with MRM. We describe the use of MIDAS for the identification of phosphorylation, with a typical experiment taking just a couple of hours from obtaining a peptide sample. With minor modifications, the MIDAS method can be applied to other protein modifications or unmodified peptides can be used as a MIDAS target.

Large-scale oscillation of structure-related DNA sequence features in human chromosome 21

NASA Astrophysics Data System (ADS)

Li, Wentian; Miramontes, Pedro

2006-08-01

Human chromosome 21 is the only chromosome in the human genome that exhibits oscillation of the (G+C) content of a cycle length of hundreds kilobases (kb) ( 500kb near the right telomere). We aim at establishing the existence of a similar periodicity in structure-related sequence features in order to relate this (G+C)% oscillation to other biological phenomena. The following quantities are shown to oscillate with the same 500kb periodicity in human chromosome 21: binding energy calculated by two sets of dinucleotide-based thermodynamic parameters, AA/TT and AAA/TTT bi- and tri-nucleotide density, 5'-TA-3' dinucleotide density, and signal for 10- or 11-base periodicity of AA/TT or AAA/TTT. These intrinsic quantities are related to structural features of the double helix of DNA molecules, such as base-pair binding, untwisting or unwinding, stiffness, and a putative tendency for nucleosome formation.

Sequence of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Nicotiana plumbaginifolia and phylogenetic origin of the gene family.

PubMed

Habenicht, A; Quesada, A; Cerff, R

1997-10-01

A cDNA-library has been constructed from Nicotiana plumbaginifolia seedlings, and the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GapN, EC 1.2.1.9) was isolated by plaque hybridization using the cDNA from pea as a heterologous probe. The cDNA comprises the entire GapN coding region. A putative polyadenylation signal is identified. Phylogenetic analysis based on the deduced amino acid sequences revealed that the GapN gene family represents a separate ancient branch within the aldehyde dehydrogenase superfamily. It can be shown that the GapN gene family and other distinct branches of the superfamily have its phylogenetic origin before the separation of primary life-forms. This further demonstrates that already very early in evolution, a broad diversification of the aldehyde dehydrogenases led to the formation of the superfamily.

In Silico Prediction and Validation of Novel RNA Binding Proteins and Residues in the Human Proteome.

PubMed

Chowdhury, Shomeek; Zhang, Jian; Kurgan, Lukasz

2018-05-28

Deciphering a complete landscape of protein-RNA interactions in the human proteome remains an elusive challenge. We computationally elucidate RNA binding proteins (RBPs) using an approach that complements previous efforts. We employ two modern complementary sequence-based methods that provide accurate predictions from the structured and the intrinsically disordered sequences, even in the absence of sequence similarity to the known RBPs. We generate and analyze putative RNA binding residues on the whole proteome scale. Using a conservative setting that ensures low, 5% false positive rate, we identify 1511 putative RBPs that include 281 known RBPs and 166 RBPs that were previously predicted. We empirically demonstrate that these overlaps are statistically significant. We also validate the putative RBPs based on two major hallmarks of their RNA binding residues: high levels of evolutionary conservation and enrichment in charged amino acids. Moreover, we show that the novel RBPs are significantly under-annotated functionally which coincides with the fact that they were not yet found to interact with RNAs. We provide two examples of our novel putative RBPs for which there is recent evidence of their interactions with RNAs. The dataset of novel putative RBPs and RNA binding residues for the future hypothesis generation is provided in the Supporting Information. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genes under positive selection in a model plant pathogenic fungus, Botrytis.

PubMed

Aguileta, Gabriela; Lengelle, Juliette; Chiapello, Hélène; Giraud, Tatiana; Viaud, Muriel; Fournier, Elisabeth; Rodolphe, François; Marthey, Sylvain; Ducasse, Aurélie; Gendrault, Annie; Poulain, Julie; Wincker, Patrick; Gout, Lilian

2012-07-01

The rapid evolution of particular genes is essential for the adaptation of pathogens to new hosts and new environments. Powerful methods have been developed for detecting targets of selection in the genome. Here we used divergence data to compare genes among four closely related fungal pathogens adapted to different hosts to elucidate the functions putatively involved in adaptive processes. For this goal, ESTs were sequenced in the specialist fungal pathogens Botrytis tulipae and Botrytis ficariarum, and compared with genome sequences of Botrytis cinerea and Sclerotinia sclerotiorum, responsible for diseases on over 200 plant species. A maximum likelihood-based analysis of 642 predicted orthologs detected 21 genes showing footprints of positive selection. These results were validated by resequencing nine of these genes in additional Botrytis species, showing they have also been rapidly evolving in other related species. Twenty of the 21 genes had not previously been identified as pathogenicity factors in B. cinerea, but some had functions related to plant-fungus interactions. The putative functions were involved in respiratory and energy metabolism, protein and RNA metabolism, signal transduction or virulence, similarly to what was detected in previous studies using the same approach in other pathogens. Mutants of B. cinerea were generated for four of these genes as a first attempt to elucidate their functions. Copyright © 2012 Elsevier B.V. All rights reserved.

Genomic investigation of a suspected outbreak of Legionella pneumophila ST82 reveals undetected heterogeneity by the present gold-standard methods, Denmark, July to November 2014

PubMed Central

Schjørring, Susanne; Stegger, Marc; Kjelsø, Charlotte; Lilje, Berit; Bangsborg, Jette M; Petersen, Randi F; David, Sophia; Uldum, Søren A

2017-01-01

Between July and November 2014, 15 community-acquired cases of Legionnaires´ disease (LD), including four with Legionella pneumophila serogroup 1 sequence type (ST) 82, were diagnosed in Northern Zealand, Denmark. An outbreak was suspected. No ST82 isolates were found in environmental samples and no external source was established. Four putative-outbreak ST82 isolates were retrospectively subjected to whole genome sequencing (WGS) followed by phylogenetic analyses with epidemiologically unrelated ST82 sequences. The four putative-outbreak ST82 sequences fell into two clades, the two clades were separated by ca 1,700 single nt polymorphisms (SNP)s when recombination regions were included but only by 12 to 21 SNPs when these were removed. A single putative-outbreak ST82 isolate sequence segregated in the first clade. The other three clustered in the second clade, where all included sequences had < 5 SNP differences between them. Intriguingly, this clade also comprised epidemiologically unrelated isolate sequences from the UK and Denmark dating back as early as 2011. The study confirms that recombination plays a major role in L. pneumophila evolution. On the other hand, strains belonging to the same ST can have only few SNP differences despite being sampled over both large timespans and geographic distances. These are two important factors to consider in outbreak investigations. PMID:28662761

Identification of Putative Nuclear Receptors and Steroidogenic Enzymes in Murray-Darling Rainbowfish (Melanotaenia fluviatilis) Using RNA-Seq and De Novo Transcriptome Assembly.

PubMed

Bain, Peter A; Papanicolaou, Alexie; Kumar, Anupama

2015-01-01

Murray-Darling rainbowfish (Melanotaenia fluviatilis [Castelnau, 1878]; Atheriniformes: Melanotaeniidae) is a small-bodied teleost currently under development in Australasia as a test species for aquatic toxicological studies. To date, efforts towards the development of molecular biomarkers of contaminant exposure have been hindered by the lack of available sequence data. To address this, we sequenced messenger RNA from brain, liver and gonads of mature male and female fish and generated a high-quality draft transcriptome using a de novo assembly approach. 149,742 clusters of putative transcripts were obtained, encompassing 43,841 non-redundant protein-coding regions. Deduced amino acid sequences were annotated by functional inference based on similarity with sequences from manually curated protein sequence databases. The draft assembly contained protein-coding regions homologous to 95.7% of the complete cohort of predicted proteins from the taxonomically related species, Oryzias latipes (Japanese medaka). The mean length of rainbowfish protein-coding sequences relative to their medaka homologues was 92.1%, indicating that despite the limited number of tissues sampled a large proportion of the total expected number of protein-coding genes was captured in the study. Because of our interest in the effects of environmental contaminants on endocrine pathways, we manually curated subsets of coding regions for putative nuclear receptors and steroidogenic enzymes in the rainbowfish transcriptome, revealing 61 candidate nuclear receptors encompassing all known subfamilies, and 41 putative steroidogenic enzymes representing all major steroidogenic enzymes occurring in teleosts. The transcriptome presented here will be a valuable resource for researchers interested in biomarker development, protein structure and function, and contaminant-response genomics in Murray-Darling rainbowfish.

Genome sequence of Plasmopara viticola and insight into the pathogenic mechanism

PubMed Central

Yin, Ling; An, Yunhe; Qu, Junjie; Li, Xinlong; Zhang, Yali; Dry, Ian; Wu, Huijuan; Lu, Jiang

2017-01-01

Plasmopara viticola causes downy mildew disease of grapevine which is one of the most devastating diseases of viticulture worldwide. Here we report a 101.3 Mb whole genome sequence of P. viticola isolate ‘JL-7-2’ obtained by a combination of Illumina and PacBio sequencing technologies. The P. viticola genome contains 17,014 putative protein-coding genes and has ~26% repetitive sequences. A total of 1,301 putative secreted proteins, including 100 putative RXLR effectors and 90 CRN effectors were identified in this genome. In the secretome, 261 potential pathogenicity genes and 95 carbohydrate-active enzymes were predicted. Transcriptional analysis revealed that most of the RXLR effectors, pathogenicity genes and carbohydrate-active enzymes were significantly up-regulated during infection. Comparative genomic analysis revealed that P. viticola evolved independently from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis. The availability of the P. viticola genome provides a valuable resource not only for comparative genomic analysis and evolutionary studies among oomycetes, but also enhance our knowledge on the mechanism of interactions between this biotrophic pathogen and its host. PMID:28417959

Complete nucleotide sequence and annotation of the temperate corynephage ϕ16 genome.

PubMed

Lobanova, Juliya S; Gak, Evgueni R; Andreeva, Irina G; Rybak, Konstantin V; Krylov, Alexander A; Mashko, Sergey V

2017-08-01

The complete genome of ϕ16, a temperate corynephage from Corynebacterium glutamicum ATCC 21792, was sequenced and annotated (GenBank: KY250482). The electron microscopy study of ϕ16 virion confirmed that it belongs to the family Siphoviridae. The ϕ16 genome consists of a linear double-stranded DNA molecule of 58,200 bp (G+C = 52.2%) with protruding cohesive 3'-ends of 14 nt. Four major structural proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting technique. Using bioinformatics analysis, 101 putative ORFs and 5 tRNA genes were predicted. Only 27 putative gene products could be assigned to known biological functions. The ϕ16 genome was divided into functional modules. Seven putative promoters and eight putative unidirectional intrinsic terminators were predicted. One site of putative «-1» programmed ribosomal frameshifting was proposed in the phage tail assembly genome region. C. glutamicum genetic tools could be broadened by exploiting the known integrase gene (gp33) and the newly identified excisionase gene (gp47), participating in site-specific recombination between ϕ16-attP/attB.

The unique genomic landscape surrounding the EPSPS gene in glyphosate resistant Amaranthus palmeri: a repetitive path to resistance.

PubMed

Molin, William T; Wright, Alice A; Lawton-Rauh, Amy; Saski, Christopher A

2017-01-17

The expanding number and global distributions of herbicide resistant weedy species threaten food, fuel, fiber and bioproduct sustainability and agroecosystem longevity. Amongst the most competitive weeds, Amaranthus palmeri S. Wats has rapidly evolved resistance to glyphosate primarily through massive amplification and insertion of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene across the genome. Increased EPSPS gene copy numbers results in higher titers of the EPSPS enzyme, the target of glyphosate, and confers resistance to glyphosate treatment. To understand the genomic unit and mechanism of EPSPS gene copy number proliferation, we developed and used a bacterial artificial chromosome (BAC) library from a highly resistant biotype to sequence the local genomic landscape flanking the EPSPS gene. By sequencing overlapping BACs, a 297 kb sequence was generated, hereafter referred to as the "EPSPS cassette." This region included several putative genes, dense clusters of tandem and inverted repeats, putative helitron and autonomous replication sequences, and regulatory elements. Whole genome shotgun sequencing (WGS) of two biotypes exhibiting high and no resistance to glyphosate was performed to compare genomic representation across the EPSPS cassette. Mapping of sequences for both biotypes to the reference EPSPS cassette revealed significant differences in upstream and downstream sequences relative to EPSPS with regard to both repetitive units and coding content between these biotypes. The differences in sequence may have resulted from a compounded-building mechanism such as repetitive transpositional events. The association of putative helitron sequences with the cassette suggests a possible amplification and distribution mechanism. Flow cytometry revealed that the EPSPS cassette added measurable genomic content. The adoption of glyphosate resistant cropping systems in major crops such as corn, soybean, cotton and canola coupled with excessive use of glyphosate herbicide has led to evolved glyphosate resistance in several important weeds. In Amaranthus palmeri, the amplification of the EPSPS cassette, characterized by a complex array of repetitive elements and putative helitron sequences, suggests an adaptive structural genomic mechanism that drives amplification and distribution around the genome. The added genomic content not found in glyphosate sensitive plants may be driving evolution through genome expansion.

Signatures of selection in tilapia revealed by whole genome resequencing

PubMed Central

Hong Xia, Jun; Bai, Zhiyi; Meng, Zining; Zhang, Yong; Wang, Le; Liu, Feng; Jing, Wu; Yi Wan, Zi; Li, Jiale; Lin, Haoran; Hua Yue, Gen

2015-01-01

Natural selection and selective breeding for genetic improvement have left detectable signatures within the genome of a species. Identification of selection signatures is important in evolutionary biology and for detecting genes that facilitate to accelerate genetic improvement. However, selection signatures, including artificial selection and natural selection, have only been identified at the whole genome level in several genetically improved fish species. Tilapia is one of the most important genetically improved fish species in the world. Using next-generation sequencing, we sequenced the genomes of 47 tilapia individuals. We identified a total of 1.43 million high-quality SNPs and found that the LD block sizes ranged from 10–100 kb in tilapia. We detected over a hundred putative selective sweep regions in each line of tilapia. Most selection signatures were located in non-coding regions of the tilapia genome. The Wnt signaling, gonadotropin-releasing hormone receptor and integrin signaling pathways were under positive selection in all improved tilapia lines. Our study provides a genome-wide map of genetic variation and selection footprints in tilapia, which could be important for genetic studies and accelerating genetic improvement of tilapia. PMID:26373374

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

Olfactory Ionotropic Receptors in Mosquito Aedes albopictus (Diptera: Culicidae).

PubMed

Chen, Qian; Man, Yahui; Li, Jianyong; Pei, Di; Wu, Wenjian

2017-09-01

Ionotropic glutamate receptors (iGluRs) are a conserved family of ligand-gated ion channels that primarily function to mediate neuronal communication at synapses. A variant subfamily of iGluRs, the ionotropic receptors (IRs), was recently identified in insects and proved with the function in odorant recognition. Ionotropic receptors participate in a distinct olfactory signaling pathway that is independent of olfactory receptors activity. In the present study, we identify 102 putative IR genes, dubbed as AalbIr genes, in mosquito Aedes albopictus (Skuse) by in silico comparative sequence analysis. Among AalbIr genes, 19 show expression in the female antenna by RT-PCR. These putative olfactory AalbIRs share four conservative hydrophobic domains of amino acids, similar to the transmembrane and ion channel pore regions found in conventional iGluRs. To determine the potential function of these olfactory AalbIRs in host-seeking, we compared their transcript expression levels in the antennae of blood-fed females with that of non-blood-fed females by quantitative real-time RT-PCR. Three AalbIr genes showed downregulation when the mosquito finished a bloodmeal. These results may help to improve our understanding of the IR-mediated olfactory signaling in mosquitoes. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Structure prediction of Fe(II) 2-oxoglutarate dioxygenase from a psychrophilic yeast Glaciozyma antarctica PI12

NASA Astrophysics Data System (ADS)

Yusof, Nik Yusnoraini; Bakar, Farah Diba Abu; Mahadi, Nor Muhammad; Raih, Mohd Firdaus; Murad, Abdul Munir Abdul

2015-09-01

A cDNA encoding Fe(II) 2-oxoglutarate (2OG) dependent dioxygenases was isolated from psychrophilic yeast, Glaciozyma antarctica PI12. We have successfully amplified 1,029 bp cDNA sequence that encodes 342 amino acid with predicted molecular weight 38 kDa. The prediction protein was analysed using various bioinformatics tools to explore the properties of the protein. Based on a BLAST search analysis, the Fe2OX amino acid sequence showed 61% identity to the sequence of oxoglutarate/iron-dependent oxygenase from Rhodosporidium toruloides NP11. SignalP prediction showed that the Fe2OX protein contains no putative signal peptide, which suggests that this enzyme most probably localised intracellularly.The structure of Fe2OX was predicted by homology modelling using MODELLER9v11. The model with the lowest objective function was selected from hundred models generated using MODELLER9v11. Analysis of the structure revealed the longer loop at Fe2OX from G.antarctica that might be responsible for the flexibility of the structure, which contributes to its adaptation to low temperatures. Fe2OX hold a highly conserved Fe(II) binding HXD/E…H triad motif. The binding site for 2-oxoglutarate was found conserved for Arg280 among reported studies, however the Phe268 was found to be different in Fe2OX.

Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

PubMed

Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

2009-02-01

Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

Systematic variation in mRNA 3′-processing signals during mouse spermatogenesis

PubMed Central

Liu, Donglin; Brockman, J. Michael; Dass, Brinda; Hutchins, Lucie N.; Singh, Priyam; McCarrey, John R.; MacDonald, Clinton C.; Graber, Joel H.

2007-01-01

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis. PMID:17158511

Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro-β-1,3-glucanase

PubMed Central

Shinshi, H.; Wenzler, H.; Neuhaus, J.-M.; Felix, G.; Hofsteenge, J.; Meins, F.

1988-01-01

Tobacco glucan endo-1,3-β-glucosidase (β-1,3-glucanase; 1,3-β-D-glucan glucanohydrolase; EC 3.2.1.39) exhibits complex hormonal and developmental regulation and is induced when plants are infected with pathogens. We determined the primary structure of this enzyme from the nucleotide sequence of five partial cDNA clones and the amino acid sequence of five peptides covering a total of 70 residues. β-1,3-Glucanase is produced as a 359-residue preproenzyme with an N-terminal hydrophobic signal peptide of 21 residues and a C-terminal extension of 22 residues containing a putative N-glycosylation site. The results of pulse-chase experiments with tunicamycin provide evidence that the first step in processing is loss of the signal peptide and addition of an oligosaccharide side chain. The glycosylated intermediate is further processed with the loss of the oligosaccharide side chain and C-terminal extension to give the mature enzyme. Heterogeneity in the sequences of cDNA clones and of mature protein and in Southern blot analysis of restriction endonuclease fragments indicates that tobacco β-1,3-glucanase is encoded by a small gene family. Two or three members of this family appear to have their evolutionary origin in each of the progenitors of tobacco, Nicotiana sylvestris and Nicotiana tomentosiformis. Images PMID:16593965

Template Based Design of Anti-Metastatic Drugs from the Active Conformation of Laminin Peptide II

DTIC Science & Technology

2001-01-01

p40 (LBP/p40) gene Maeda, M., Kawasaki, K., Mu, Y., Kamada, H., during sea urchin development. Exp. Cell Res. 221, Tsutsumi, Y., Smith, T. J. & Mayumi...represents the average of six replicates + SEM . minance of putative heparin-binding phage recov- ered from elution with peptide 11. Putative heparin...scrambled sequence peptide, WAQADSTPE, was used as a sequence specificity control. The data shown is the average of six replicate wells ± SEM . Statistics were

Single nucleotide polymorphism discovery in rainbow trout by deep sequencing of a reduced representation library.

PubMed

Sánchez, Cecilia Castaño; Smith, Timothy P L; Wiedmann, Ralph T; Vallejo, Roger L; Salem, Mohamed; Yao, Jianbo; Rexroad, Caird E

2009-11-25

To enhance capabilities for genomic analyses in rainbow trout, such as genomic selection, a large suite of polymorphic markers that are amenable to high-throughput genotyping protocols must be identified. Expressed Sequence Tags (ESTs) have been used for single nucleotide polymorphism (SNP) discovery in salmonids. In those strategies, the salmonid semi-tetraploid genomes often led to assemblies of paralogous sequences and therefore resulted in a high rate of false positive SNP identification. Sequencing genomic DNA using primers identified from ESTs proved to be an effective but time consuming methodology of SNP identification in rainbow trout, therefore not suitable for high throughput SNP discovery. In this study, we employed a high-throughput strategy that used pyrosequencing technology to generate data from a reduced representation library constructed with genomic DNA pooled from 96 unrelated rainbow trout that represent the National Center for Cool and Cold Water Aquaculture (NCCCWA) broodstock population. The reduced representation library consisted of 440 bp fragments resulting from complete digestion with the restriction enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the estimated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 independent contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually represent paralogous loci in the tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Approximately 48% (183) of the putative SNPs were validated; 167 markers were successfully incorporated into the rainbow trout linkage map. In addition, 2% of the sequences from the validated markers were associated with rainbow trout transcripts. The use of reduced representation libraries and pyrosequencing technology proved to be an effective strategy for the discovery of a high number of putative SNPs in rainbow trout; however, modifications to the technique to decrease the false discovery rate resulting from the evolutionary recent genome duplication would be desirable.

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.

Domain fusion analysis by applying relational algebra to protein sequence and domain databases

PubMed Central

Truong, Kevin; Ikura, Mitsuhiko

2003-01-01

Background Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. Results This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at . Conclusion As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time. PMID:12734020

The Rose (Rosa hybrida) NAC Transcription Factor 3 Gene, RhNAC3, Involved in ABA Signaling Pathway Both in Rose and Arabidopsis

PubMed Central

Lü, Peitao; Liu, Jitao; Gao, Junping; Zhang, Changqing

2014-01-01

Plant transcription factors involved in stress responses are generally classified by their involvement in either the abscisic acid (ABA)-dependent or the ABA-independent regulatory pathways. A stress-associated NAC gene from rose (Rosa hybrida), RhNAC3, was previously found to increase dehydration tolerance in both rose and Arabidopsis. However, the regulatory mechanism involved in RhNAC3 action is still not fully understood. In this study, we isolated and analyzed the upstream regulatory sequence of RhNAC3 and found many stress-related cis-elements to be present in the promoter, with five ABA-responsive element (ABRE) motifs being of particular interest. Characterization of Arabidopsis thaliana plants transformed with the putative RhNAC3 promoter sequence fused to the β-glucuronidase (GUS) reporter gene revealed that RhNAC3 is expressed at high basal levels in leaf guard cells and in vascular tissues. Moreover, the ABRE motifs in the RhNAC3 promoter were observed to have a cumulative effect on the transcriptional activity of this gene both in the presence and absence of exogenous ABA. Overexpression of RhNAC3 in A. thaliana resulted in ABA hypersensitivity during seed germination and promoted leaf closure after ABA or drought treatments. Additionally, the expression of 11 ABA-responsive genes was induced to a greater degree by dehydration in the transgenic plants overexpressing RhNAC3 than control lines transformed with the vector alone. Further analysis revealed that all these genes contain NAC binding cis-elements in their promoter regions, and RhNAC3 was found to partially bind to these putative NAC recognition sites. We further found that of 219 A. thaliana genes previously shown by microarray analysis to be regulated by heterologous overexpression RhNAC3, 85 are responsive to ABA. In rose, the expression of genes downstream of the ABA-signaling pathways was also repressed in RhNAC3-silenced petals. Taken together, we propose that the rose RhNAC3 protein could mediate ABA signaling both in rose and in A. thaliana. PMID:25290154

The rose (Rosa hybrida) NAC transcription factor 3 gene, RhNAC3, involved in ABA signaling pathway both in rose and Arabidopsis.

PubMed

Jiang, Guimei; Jiang, Xinqiang; Lü, Peitao; Liu, Jitao; Gao, Junping; Zhang, Changqing

2014-01-01

Plant transcription factors involved in stress responses are generally classified by their involvement in either the abscisic acid (ABA)-dependent or the ABA-independent regulatory pathways. A stress-associated NAC gene from rose (Rosa hybrida), RhNAC3, was previously found to increase dehydration tolerance in both rose and Arabidopsis. However, the regulatory mechanism involved in RhNAC3 action is still not fully understood. In this study, we isolated and analyzed the upstream regulatory sequence of RhNAC3 and found many stress-related cis-elements to be present in the promoter, with five ABA-responsive element (ABRE) motifs being of particular interest. Characterization of Arabidopsis thaliana plants transformed with the putative RhNAC3 promoter sequence fused to the β-glucuronidase (GUS) reporter gene revealed that RhNAC3 is expressed at high basal levels in leaf guard cells and in vascular tissues. Moreover, the ABRE motifs in the RhNAC3 promoter were observed to have a cumulative effect on the transcriptional activity of this gene both in the presence and absence of exogenous ABA. Overexpression of RhNAC3 in A. thaliana resulted in ABA hypersensitivity during seed germination and promoted leaf closure after ABA or drought treatments. Additionally, the expression of 11 ABA-responsive genes was induced to a greater degree by dehydration in the transgenic plants overexpressing RhNAC3 than control lines transformed with the vector alone. Further analysis revealed that all these genes contain NAC binding cis-elements in their promoter regions, and RhNAC3 was found to partially bind to these putative NAC recognition sites. We further found that of 219 A. thaliana genes previously shown by microarray analysis to be regulated by heterologous overexpression RhNAC3, 85 are responsive to ABA. In rose, the expression of genes downstream of the ABA-signaling pathways was also repressed in RhNAC3-silenced petals. Taken together, we propose that the rose RhNAC3 protein could mediate ABA signaling both in rose and in A. thaliana.

Characterization by Suppression Subtractive Hybridization of Transcripts That Are Differentially Expressed in Leaves of Anthracnose-Resistant Ramie Cultivar.

PubMed

Xuxia, Wang; Jie, Chen; Bo, Wang; Lijun, Liu; Hui, Jiang; Diluo, Tang; Dingxiang, Peng

2012-01-01

For the purpose of screening putative anthracnose resistance-related genes of ramie ( Boehmeria nivea L. Gaud), a cDNA library was constructed by suppression subtractive hybridization using anthracnose-resistant cultivar Huazhu no. 4. The cDNAs from Huazhu no. 4, which were infected with Colletotrichum gloeosporioides , were used as the tester and cDNAs from uninfected Huazhu no. 4 as the driver. Sequencing analysis and homology searching showed that these clones represented 132 single genes, which were assigned to functional categories, including 14 putative cellular functions, according to categories established for Arabidopsis . These 132 genes included 35 disease resistance and stress tolerance-related genes including putative heat-shock protein 90, metallothionein, PR-1.2 protein, catalase gene, WRKY family genes, and proteinase inhibitor-like protein. Partial disease-related genes were further analyzed by reverse transcription PCR and RNA gel blot. These expressed sequence tags are the first anthracnose resistance-related expressed sequence tags reported in ramie.

Putative Porin of Bradyrhizobium sp. (Lupinus) Bacteroids Induced by Glyphosate▿

PubMed Central

de María, Nuria; Guevara, Ángeles; Serra, M. Teresa; García-Luque, Isabel; González-Sama, Alfonso; de Lacoba, Mario García; de Felipe, M. Rosario; Fernández-Pascual, Mercedes

2007-01-01

Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed. PMID:17557843

Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

PubMed Central

Niskanen, Einari A; Hytönen, Vesa P; Grapputo, Alessandro; Nordlund, Henri R; Kulomaa, Markku S; Laitinen, Olli H

2005-01-01

Background A chicken egg contains several biotin-binding proteins (BBPs), whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins. PMID:15777476

DArT Markers Effectively Target Gene Space in the Rye Genome

PubMed Central

Gawroński, Piotr; Pawełkowicz, Magdalena; Tofil, Katarzyna; Uszyński, Grzegorz; Sharifova, Saida; Ahluwalia, Shivaksh; Tyrka, Mirosław; Wędzony, Maria; Kilian, Andrzej; Bolibok-Brągoszewska, Hanna

2016-01-01

Large genome size and complexity hamper considerably the genomics research in relevant species. Rye (Secale cereale L.) has one of the largest genomes among cereal crops and repetitive sequences account for over 90% of its length. Diversity Arrays Technology is a high-throughput genotyping method, in which a preferential sampling of gene-rich regions is achieved through the use of methylation sensitive restriction enzymes. We obtained sequences of 6,177 rye DArT markers and following a redundancy analysis assembled them into 3,737 non-redundant sequences, which were then used in homology searches against five Pooideae sequence sets. In total 515 DArT sequences could be incorporated into publicly available rye genome zippers providing a starting point for the integration of DArT- and transcript-based genomics resources in rye. Using Blast2Go pipeline we attributed putative gene functions to 1101 (29.4%) of the non-redundant DArT marker sequences, including 132 sequences with putative disease resistance-related functions, which were found to be preferentially located in the 4RL and 6RL chromosomes. Comparative analysis based on the DArT sequences revealed obvious inconsistencies between two recently published high density consensus maps of rye. Furthermore we demonstrated that DArT marker sequences can be a source of SSR polymorphisms. Obtained data demonstrate that DArT markers effectively target gene space in the large, complex, and repetitive rye genome. Through the annotation of putative gene functions and the alignment of DArT sequences relative to reference genomes we obtained information, that will complement the results of the studies, where DArT genotyping was deployed, by simplifying the gene ontology and microcolinearity based identification of candidate genes. PMID:27833625

DArT Markers Effectively Target Gene Space in the Rye Genome.

PubMed

Gawroński, Piotr; Pawełkowicz, Magdalena; Tofil, Katarzyna; Uszyński, Grzegorz; Sharifova, Saida; Ahluwalia, Shivaksh; Tyrka, Mirosław; Wędzony, Maria; Kilian, Andrzej; Bolibok-Brągoszewska, Hanna

2016-01-01

Large genome size and complexity hamper considerably the genomics research in relevant species. Rye ( Secale cereale L.) has one of the largest genomes among cereal crops and repetitive sequences account for over 90% of its length. Diversity Arrays Technology is a high-throughput genotyping method, in which a preferential sampling of gene-rich regions is achieved through the use of methylation sensitive restriction enzymes. We obtained sequences of 6,177 rye DArT markers and following a redundancy analysis assembled them into 3,737 non-redundant sequences, which were then used in homology searches against five Pooideae sequence sets. In total 515 DArT sequences could be incorporated into publicly available rye genome zippers providing a starting point for the integration of DArT- and transcript-based genomics resources in rye. Using Blast2Go pipeline we attributed putative gene functions to 1101 (29.4%) of the non-redundant DArT marker sequences, including 132 sequences with putative disease resistance-related functions, which were found to be preferentially located in the 4RL and 6RL chromosomes. Comparative analysis based on the DArT sequences revealed obvious inconsistencies between two recently published high density consensus maps of rye. Furthermore we demonstrated that DArT marker sequences can be a source of SSR polymorphisms. Obtained data demonstrate that DArT markers effectively target gene space in the large, complex, and repetitive rye genome. Through the annotation of putative gene functions and the alignment of DArT sequences relative to reference genomes we obtained information, that will complement the results of the studies, where DArT genotyping was deployed, by simplifying the gene ontology and microcolinearity based identification of candidate genes.

Five putative nucleoside triphosphate diphosphohydrolase genes are expressed in Trichomonas vaginalis.

PubMed

Frasson, Amanda Piccoli; Dos Santos, Odelta; Meirelles, Lúcia Collares; Macedo, Alexandre José; Tasca, Tiana

2016-01-01

Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1-5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Isolation of pheromone precursor genes of Magnaporthe grisea.

PubMed

Shen, W C; Bobrowicz, P; Ebbole, D J

1999-01-01

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia. Copyright 1999 Academic Press.

Molecular cloning of a cDNA encoding the precursor of adenoregulin from frog skin. Relationships with the vertebrate defensive peptides, dermaseptins.

PubMed

Amiche, M; Ducancel, F; Lajeunesse, E; Boulain, J C; Ménez, A; Nicolas, P

1993-03-31

Adenoregulin has recently been isolated from Phyllomedusa skin as a 33 amino acid residues peptide which enhanced binding of agonists to the A1 adenosine receptor. In order to study the structure of the precursor of adenoregulin we constructed a cDNA library from mRNAs extracted from the skin of Phyllomedusa bicolor. We detected the complete nucleotide sequence of a cDNA encoding the adenoregulin biosynthetic precursor. The deduced sequence of the precursor is 81 amino acids long, exhibits a putative signal sequence at the NH2 terminus and contains a single copy of the biologically active peptide at the COOH terminus. Structural and conformational homologies that are observed between adenoregulin and the dermaseptins, antimicrobial peptides exhibiting strong membranolytic activities against various pathogenic agents, suggest that adenoregulin is an additional member of the growing family of cytotropic antimicrobial peptides that allow vertebrate animals to defend themselves against microorganisms. As such, the adenosine receptor regulating activity of adenoregulin could be due to its ability to interact with and disrupt membranes lipid bilayers.

The Genome of the Obligately Intracellular Bacterium Ehrlichia canis Reveals Themes of Complex Membrane Structure and Immune Evasion Strategies

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

Mavromatis, K; Doyle, C Kuyler; Lykidis, A

2006-01-01

Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, {alpha}-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associatedmore » with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (n = 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (n = 7).« less

Prevalence of transcription promoters within archaeal operons and coding sequences

PubMed Central

Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

2009-01-01

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements. PMID:19536208

Prevalence of transcription promoters within archaeal operons and coding sequences.

PubMed

Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

2009-01-01

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of approximately 64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes-events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

The genome of obligately intracellular Ehrlichia canis revealsthemes of complex membrane structure and immune evasion strategies

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

Mavromatis, K.; Kuyler Doyle, C.; Lykidis, A.

2005-09-01

Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, a-proteobacterium is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, and 17 putative pseudogenes, and a substantial proportion of non-coding sequence (27 percent). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences, and a unique serine-threonine bias associated with the potential for O-glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein familiesmore » associated with immune evasion were identified, one of which contains poly G:C tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Proteins associated with pathogen-host interactions were identified including a small group of proteins (12) with tandem repeats and another with eukaryotic-like ankyrin domains (7).« less

Deep mRNA Sequencing of the Tritonia diomedea Brain Transcriptome Provides Access to Gene Homologues for Neuronal Excitability, Synaptic Transmission and Peptidergic Signalling

PubMed Central

Senatore, Adriano; Edirisinghe, Neranjan; Katz, Paul S.

2015-01-01

Background The sea slug Tritonia diomedea (Mollusca, Gastropoda, Nudibranchia), has a simple and highly accessible nervous system, making it useful for studying neuronal and synaptic mechanisms underlying behavior. Although many important contributions have been made using Tritonia, until now, a lack of genetic information has impeded exploration at the molecular level. Results We performed Illumina sequencing of central nervous system mRNAs from Tritonia, generating 133.1 million 100 base pair, paired-end reads. De novo reconstruction of the RNA-Seq data yielded a total of 185,546 contigs, which partitioned into 123,154 non-redundant gene clusters (unigenes). BLAST comparison with RefSeq and Swiss-Prot protein databases, as well as mRNA data from other invertebrates (gastropod molluscs: Aplysia californica, Lymnaea stagnalis and Biomphalaria glabrata; cnidarian: Nematostella vectensis) revealed that up to 76,292 unigenes in the Tritonia transcriptome have putative homologues in other databases, 18,246 of which are below a more stringent E-value cut-off of 1x10-6. In silico prediction of secreted proteins from the Tritonia transcriptome shotgun assembly (TSA) produced a database of 579 unique sequences of secreted proteins, which also exhibited markedly higher expression levels compared to other genes in the TSA. Conclusions Our efforts greatly expand the availability of gene sequences available for Tritonia diomedea. We were able to extract full length protein sequences for most queried genes, including those involved in electrical excitability, synaptic vesicle release and neurotransmission, thus confirming that the transcriptome will serve as a useful tool for probing the molecular correlates of behavior in this species. We also generated a neurosecretome database that will serve as a useful tool for probing peptidergic signalling systems in the Tritonia brain. PMID:25719197

A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plasticity among diplomonads and significant lateral gene transfer in eukaryote genome evolution

PubMed Central

Andersson, Jan O; Sjögren, Åsa M; Horner, David S; Murphy, Colleen A; Dyal, Patricia L; Svärd, Staffan G; Logsdon, John M; Ragan, Mark A; Hirt, Robert P; Roger, Andrew J

2007-01-01

Background Comparative genomic studies of the mitochondrion-lacking protist group Diplomonadida (diplomonads) has been lacking, although Giardia lamblia has been intensively studied. We have performed a sequence survey project resulting in 2341 expressed sequence tags (EST) corresponding to 853 unique clones, 5275 genome survey sequences (GSS), and eleven finished contigs from the diplomonad fish parasite Spironucleus salmonicida (previously described as S. barkhanus). Results The analyses revealed a compact genome with few, if any, introns and very short 3' untranslated regions. Strikingly different patterns of codon usage were observed in genes corresponding to frequently sampled ESTs versus genes poorly sampled, indicating that translational selection is influencing the codon usage of highly expressed genes. Rigorous phylogenomic analyses identified 84 genes – mostly encoding metabolic proteins – that have been acquired by diplomonads or their relatively close ancestors via lateral gene transfer (LGT). Although most acquisitions were from prokaryotes, more than a dozen represent likely transfers of genes between eukaryotic lineages. Many genes that provide novel insights into the genetic basis of the biology and pathogenicity of this parasitic protist were identified including 149 that putatively encode variant-surface cysteine-rich proteins which are candidate virulence factors. A number of genomic properties that distinguish S. salmonicida from its human parasitic relative G. lamblia were identified such as nineteen putative lineage-specific gene acquisitions, distinct mutational biases and codon usage and distinct polyadenylation signals. Conclusion Our results highlight the power of comparative genomic studies to yield insights into the biology of parasitic protists and the evolution of their genomes, and suggest that genetic exchange between distantly-related protist lineages may be occurring at an appreciable rate in eukaryote genome evolution. PMID:17298675

Comparative genomics of Roseobacter clade bacteria isolated from the accessory nidamental gland of Euprymna scolopes

PubMed Central

Collins, Andrew J.; Fullmer, Matthew S.; Gogarten, Johann P.; Nyholm, Spencer V.

2015-01-01

The accessory nidamental gland (ANG) of the female Hawaiian bobtail squid, Euprymna scolopes, houses a consortium of bacteria including members of the Flavobacteriales, Rhizobiales, and Verrucomicrobia but is dominated by members of the Roseobacter clade (Rhodobacterales) within the Alphaproteobacteria. These bacteria are deposited into the jelly coat of the squid’s eggs, however, the function of the ANG and its bacterial symbionts has yet to be elucidated. In order to gain insight into this consortium and its potential role in host reproduction, we cultured 12 Rhodobacterales isolates from ANGs of sexually mature female squid and sequenced their genomes with Illumina sequencing technology. For taxonomic analyses, the ribosomal proteins of 79 genomes representing both roseobacters and non-roseobacters along with a separate MLSA analysis of 33 housekeeping genes from Roseobacter organisms placed all 12 isolates from the ANG within two groups of a single Roseobacter clade. Average nucelotide identity analysis suggests the ANG isolates represent three genera (Leisingera, Ruegeria, and Tateyamaria) comprised of seven putative species groups. All but one of the isolates contains a predicted Type VI secretion system, which has been shown to be important in secreting signaling and/or effector molecules in host–microbe associations and in bacteria–bacteria interactions. All sequenced genomes also show potential for secondary metabolite production, and are predicted to be involved with the production of acyl homoserine lactones (AHLs) and/or siderophores. An AHL bioassay confirmed AHL production in three tested isolates and from whole ANG homogenates. The dominant symbiont, Leisingera sp. ANG1, showed greater viability in iron-limiting conditions compared to other roseobacters, possibly due to higher levels of siderophore production. Future comparisons will try to elucidate novel metabolic pathways of the ANG symbionts to understand their putative role in host development. PMID:25755651

Complete genome sequence of two tomato-infecting begomoviruses in Venezuela: evidence of a putative novel species and a novel recombinant strain.

PubMed

Romay, Gustavo; Chirinos, Dorys T; Geraud-Pouey, Francis; Gillis, Annika; Mahillon, Jacques; Bragard, Claude

2018-02-01

At least six begomovirus species have been reported infecting tomato in Venezuela. In this study the complete genomes of two tomato-infecting begomovirus isolates (referred to as Trujillo-427 and Zulia-1084) were cloned and sequenced. Both isolates showed the typical genome organization of New World bipartite begomoviruses, with DNA-A genomic components displaying 88.8% and 90.3% similarity with established begomoviruses, for isolates Trujillo-427 and Zulia-1084, respectively. In accordance to the guidelines for begomovirus species demarcation, the Trujillo-427 isolate represents a putative new species and the name "Tomato wrinkled mosaic virus" is proposed. Meanwhile, Zulia-1084 represents a putative new strain classifiable within species Tomato chlorotic leaf distortion virus, for which a recombinant origin is suggested.

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.

Analysis of the DNA sequence of a 15,500 bp fragment near the left telomere of chromosome XV from Saccharomyces cerevisiae reveals a putative sugar transporter, a carboxypeptidase homologue and two new open reading frames.

PubMed

Gamo, F J; Lafuente, M J; Casamayor, A; Ariño, J; Aldea, M; Casas, C; Herrero, E; Gancedo, C

1996-06-15

We report the sequence of a 15.5 kb DNA segment located near the left telomere of chromosome XV of Saccharomyces cerevisiae. The sequence contains nine open reading frames (ORFs) longer than 300 bp. Three of them are internal to other ones. One corresponds to the gene LGT3 that encodes a putative sugar transporter. Three adjacent ORFs were separated by two stop codons in frame. These ORFs presented homology with the gene CPS1 that encodes carboxypeptidase S. The stop codons were not found in the same sequence derived from another yeast strain. Two other ORFs without significant homology in databases were also found. One of them, O0420, is very rich in serine and threonine and presents a series of repeated or similar amino acid stretches along the sequence.

Mapping of aldose reductase gene sequences to human chromosomes 1, 3, 7, 9, 11, and 13

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

Bateman, J.B.; Kojis, T.; Heinzmann, C.

1993-09-01

Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase; EC 1.1.1.21) (AR) catalyzes the reduction of several aldehydes, including that of glucose, to the corresponding sugar alcohol. Using a complementary DNA clone encoding human AR, the authors mapped the gene sequences to human chromosomes 1, 3, 7, 9, 11, 13, 14, and 18 by somatic cell hybridization. By in situ hybridization analysis, sequences were localized to human chromosomes 1q32-q43, 3p12, 7q31-q35, 9q22, 11p14-p15, and 13q14-q21. As a putative functional AR gene has been mapped to chromosome 7 and a putative pseudogene to chromosome 3, the sequences on the other seven chromosomes may represent other activemore » genes, non-aldose reductase homologous sequences, or pseudogenes. 24 refs., 3 figs., 2 tabs.« less

In Vivo Ligands of MDA5 and RIG-I in Measles Virus-Infected Cells

PubMed Central

Hembach, Katharina; Baum, Alina; García-Sastre, Adolfo; Söding, Johannes; Conzelmann, Karl-Klaus

2014-01-01

RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5′-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling. PMID:24743923

Characterisation of the genomes of four putative vesiculoviruses: tench rhabdovirus, grass carp rhabdovirus, perch rhabdovirus and eel rhabdovirus European X.

PubMed

Stone, David M; Kerr, Rose C; Hughes, Margaret; Radford, Alan D; Darby, Alistair C

2013-11-01

The complete coding sequences were determined for four putative vesiculoviruses isolated from fish. Sequence alignment and phylogenetic analysis based on the predicted amino acid sequences of the five main proteins assigned tench rhabdovirus and grass carp rhabdovirus together with spring viraemia of carp and pike fry rhabdovirus to a lineage that was distinct from the mammalian vesiculoviruses. Perch rhabdovirus, eel virus European X, lake trout rhabdovirus 903/87 and sea trout virus were placed in a second lineage that was also distinct from the recognised genera in the family Rhabdoviridae. Establishment of two new rhabdovirus genera, "Perhabdovirus" and "Sprivivirus", is discussed.

Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins.

PubMed

Amiche, M; Ducancel, F; Mor, A; Boulain, J C; Menez, A; Nicolas, P

1994-07-08

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.

De Novo Sequencing and Characterization of the Floral Transcriptome of Dendrocalamus latiflorus (Poaceae: Bambusoideae)

PubMed Central

Li, De-Zhu; Guo, Zhen-Hua

2012-01-01

Background Transcriptome sequencing can be used to determine gene sequences and transcript abundance in non-model species, and the advent of next-generation sequencing (NGS) technologies has greatly decreased the cost and time required for this process. Transcriptome data are especially desirable in bamboo species, as certain members constitute an economically and culturally important group of mostly semelparous plants with remarkable flowering features, yet little bamboo genomic research has been performed. Here we present, for the first time, extensive sequence and transcript abundance data for the floral transcriptome of a key bamboo species, Dendrocalamus latiflorus, obtained using the Illumina GAII sequencing platform. Our further goal was to identify patterns of gene expression during bamboo flower development. Results Approximately 96 million sequencing reads were generated and assembled de novo, yielding 146,395 high quality unigenes with an average length of 461 bp. Of these, 80,418 were identified as putative homologs of annotated sequences in the public protein databases, of which 290 were associated with the floral transition and 47 were related to flower development. Digital abundance analysis identified 26,529 transcripts differentially enriched between two developmental stages, young flower buds and older developing flowers. Unigenes found at each stage were categorized according to their putative functional categories. These sequence and putative function data comprise a resource for future investigation of the floral transition and flower development in bamboo species. Conclusions Our results present the first broad survey of a bamboo floral transcriptome. Although it will be necessary to validate the functions carried out by these genes, these results represent a starting point for future functional research on D. latiflorus and related species. PMID:22916120

smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data. Results To identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. Conclusions This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to understand their roles in key stevia traits. PMID:23116282

smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni.

PubMed

Mandhan, Vibha; Kaur, Jagdeep; Singh, Kashmir

2012-11-01

MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data. To identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to understand their roles in key stevia traits.

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.

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity

PubMed Central

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-01-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity. PMID:26646790

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

PubMed

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-12-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity.

Nucleotide sequence of the gene encoding the nitrogenase iron protein of Thiobacillus ferrooxidans

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

Pretorius, I.M.; Rawlings, D.E.; O'Neill, E.G.

1987-01-01

The DNA sequence was determined for the cloned Thiobacillus ferrooxidans nifH and part of the nifD genes. The DNA chains were radiolabeled with (..cap alpha..-/sup 32/P)dCTP (3000 Ci/mmol) or (..cap alpha..-/sup 35/S)dCTP (400 Ci/mmol). A putative T. ferrooxidans nifH promoter was identified whose sequences showed perfect consensus with those of the Klebsiella pneumoniae nif promoter. Two putative consensus upstream activator sequences were also identified. The amino acid sequence was deduced from the DNA sequence. In a comparison of nifH DNA sequences from T. ferrooxidans and eight other nitrogen-fixing microbes, a Rhizobium sp. isolated from Parasponia andersonii showed the greatest homologymore » (74%) and Clostridium pasteurianum (nifH1) showed the least homology (54%). In the comparison of the amino acid sequences of the Fe proteins, the Rhizobium sp. and Rhizobium japonicum showed the greatest homology (both 86%) and C. pasteurianum (nifH1 gene product) demonstrated the least homology (56%) to the T. ferrooxidans Fe protein.« less

Transcriptome sequencing and de novo analysis of the copepod Calanus sinicus using 454 GS FLX.

PubMed

Ning, Juan; Wang, Minxiao; Li, Chaolun; Sun, Song

2013-01-01

Despite their species abundance and primary economic importance, genomic information about copepods is still limited. In particular, genomic resources are lacking for the copepod Calanus sinicus, which is a dominant species in the coastal waters of East Asia. In this study, we performed de novo transcriptome sequencing to produce a large number of expressed sequence tags for the copepod C. sinicus. Copepodid larvae and adults were used as the basic material for transcriptome sequencing. Using 454 pyrosequencing, a total of 1,470,799 reads were obtained, which were assembled into 56,809 high quality expressed sequence tags. Based on their sequence similarity to known proteins, about 14,000 different genes were identified, including members of all major conserved signaling pathways. Transcripts that were putatively involved with growth, lipid metabolism, molting, and diapause were also identified among these genes. Differentially expressed genes related to several processes were found in C. sinicus copepodid larvae and adults. We detected 284,154 single nucleotide polymorphisms (SNPs) that provide a resource for gene function studies. Our data provide the most comprehensive transcriptome resource available for C. sinicus. This resource allowed us to identify genes associated with primary physiological processes and SNPs in coding regions, which facilitated the quantitative analysis of differential gene expression. These data should provide foundation for future genetic and genomic studies of this and related species.

A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay.

PubMed

Perego, M

1997-08-05

The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export-import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase-prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

A peptide export–import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay

PubMed Central

Perego, Marta

1997-01-01

The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export–import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase–prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction. PMID:9238025

Characterization of sequences in human TWIST required for nuclear localization

PubMed Central

Singh, Shalini; Gramolini, Anthony O

2009-01-01

Background Twist is a transcription factor that plays an important role in proliferation and tumorigenesis. Twist is a nuclear protein that regulates a variety of cellular functions controlled by protein-protein interactions and gene transcription events. The focus of this study was to characterize putative nuclear localization signals (NLSs) 37RKRR40 and 73KRGKK77 in the human TWIST (H-TWIST) protein. Results Using site-specific mutagenesis and immunofluorescences, we observed that altered TWISTNLS1 K38R, TWISTNLS2 K73R and K77R constructs inhibit nuclear accumulation of H-TWIST in mammalian cells, while TWISTNLS2 K76R expression was un-affected and retained to the nucleus. Subsequently, co-transfection of TWIST mutants K38R, K73R and K77R with E12 formed heterodimers and restored nuclear localization despite the NLSs mutations. Using a yeast-two-hybrid assay, we identified a novel TWIST-interacting candidate TCF-4, a basic helix-loop-helix transcription factor. The interaction of TWIST with TCF-4 confirmed using NLS rescue assays, where nuclear expression of mutant TWISTNLS1 with co-transfixed TCF-4 was observed. The interaction of TWIST with TCF-4 was also seen using standard immunoprecipitation assays. Conclusion Our study demonstrates the presence of two putative NLS motifs in H-TWIST and suggests that these NLS sequences are functional. Furthermore, we identified and confirmed the interaction of TWIST with a novel protein candidate TCF-4. PMID:19534813

Mitogen-activated protein kinase 1 from disk abalone (Haliotis discus discus): Roles in early development and immunity-related transcriptional responses.

PubMed

Perera, N C N; Godahewa, G I; Lee, Jehee

2016-12-01

Mitogen-activated protein kinase (MAPK) is involved in the regulation of cellular events by mediating signal transduction pathways. MAPK1 is a member of the extracellular-signal regulated kinases (ERKs), playing roles in cell proliferation, differentiation, and development. This is mainly in response to growth factors, mitogens, and many environmental stresses. In the current study, we have characterized the structural features of a homolog of MAPK1 from disk abalone (AbMAPK1). Further, we have unraveled its expressional kinetics against different experimental pathogenic infections or related chemical stimulants. AbMAPK1 harbors a 5' untranslated region (UTR) of 23 bps, a coding sequence of 1104 bps, and a 3' UTR of 448 bp. The putative peptide comprises a predicted molecular mass of 42.2 kDa, with a theoretical pI of 6.28. Based on the in silico analysis, AbMAPK1 possesses two N-glycosylation sites, one S_TK catalytic domain, and a conserved His-Arg-Asp domain (HRD). In addition, a conservative glycine rich ATP-phosphate-binding loop and a threonine-x-tyrosine motif (TEY) important for the autophosphorylation were also identified in the protein. Homology assessment of AbMAPK1 showed several conserved regions, and ark clam (Aplysia californica) showed the highest sequence identity (87.9%). The phylogenetic analysis supported close evolutionary kinship with molluscan orthologs. Constitutive expression of AbMAPK1 was observed in six different tissues of disk abalone, with the highest expression in the digestive tract, followed by the gills and hemocytes. Highest AbMAPK1 mRNA expression level was detected at the trochophore developmental stage, suggesting its role in abalone cell differentiation and proliferation. Significant modulation of AbMAPK1 expression under pathogenic stress suggested its putative involvement in the immune defense mechanism. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a novel salt-inducible gene encoding an acidic isoform of PR-5 protein in soybean (Glycine max [L.] Merr.).

PubMed

Onishi, M; Tachi, H; Kojima, T; Shiraiwa, M; Takahara, H

2006-10-01

We identified a novel salt-inducible soybean gene encoding an acidic-isoform of pathogenesis-related protein group 5 (PR-5 protein). The soybean PR-5-homologous gene, designated as Glycine max osmotin-like protein, acidic isoform (GmOLPa)), encodes a putative polypeptide having an N-terminal signal peptide. The mature GmOLPa protein without the signal peptide has a calculated molecular mass of 21.5 kDa and a pI value of 4.4, and was distinguishable from a known PR-5-homologous gene of soybean (namely P21 protein) through examination of the structural features. A comparison with two intracellular salt-inducible PR-5 proteins, tobacco osmotin and tomato NP24, revealed that GmOLPa did not have a C-terminal extension sequence functioning as a vacuole-targeting motif. The GmOLPa gene was transcribed constitutively in the soybean root and was induced almost exclusively in the root during 24 h of high-salt stress (300 mM NaCl). Interestingly, GmOLPa gene expression in the stem and leaf, not observed until 24 h, was markedly induced at 48 and 72 h after commencement of the high-salt stress. Abscisic acid (ABA) and dehydration also induced expression of the GmOLPa gene in the root; additionally, dehydration slightly induced expression in the stem and leaf. In fact, the 5'-upstream sequence of the GmOLPa gene contained several putative cis-elements known to be involved in responsiveness to ABA and dehydration, e.g. ABA-responsive element (ABRE), MYB/MYC, and low temperature-responsive element (LTRE). These results suggested that GmOLPa may function as a protective PR-5 protein in the extracellular space of the soybean root in response to high-salt stress and dehydration.

Genome-Wide Survey and Expression Profile Analysis of the Mitogen-Activated Protein Kinase (MAPK) Gene Family in Brassica rapa

PubMed Central

Yu, Hao; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Chai, Yourong; Liang, Ying

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in plants, controlling cell division, development, hormone signaling, and biotic and abiotic stress responses. Although MAPKs have been investigated in several plant species, a comprehensive analysis of the MAPK gene family has hitherto not been performed in Brassica rapa. In this study, we identified 32 MAPKs in the B. rapa genome by conducting BLASTP and syntenic block analyses, and screening for the essential signature motif (TDY or TEY) of plant MAPK proteins. Of the 32 BraMAPK genes retrieved from the Brassica Database, 13 exhibited exon splicing errors, excessive splicing of the 5' sequence, excessive retention of the 5' sequence, and sequencing errors of the 3' end. Phylogenetic trees of the 32 corrected MAPKs from B. rapa and of MAPKs from other plants generated by the neighbor-joining and maximum likelihood methods suggested that BraMAPKs could be divided into four groups (groups A, B, C, and D). Gene number expansion was observed for BraMAPK genes in groups A and D, which may have been caused by the tandem duplication and genome triplication of the ancestral genome of the Brassica progenitor. Except for five members of the BraMAPK10 subfamily, the identified BraMAPKs were expressed in most of the tissues examined, including callus, root, stem, leaf, flower, and silique. Quantitative real-time PCR demonstrated that at least six and five BraMAPKs were induced or repressed by various abiotic stresses and hormone treatments, respectively, suggesting their potential roles in the abiotic stress response and various hormone signal transduction pathways in B. rapa. This study provides valuable insight into the putative physiological and biochemical functions of MAPK genes in B. rapa. PMID:26173020

Molecular characterization of putative Hepatozoon sp. from the sedge warbler (Acrocephalus schoenobaenus).

PubMed

Biedrzycka, Aleksandra; Kloch, Agnieszka; Migalska, Magdalena; Bielański, Wojciech

2013-05-01

We characterized partial sequences of 18S rDNA from sedge warblers infected with a parasite described previously as Hepatozoon kabeeni. Prevalence was 47% in sampled birds.We detected 3 parasite haplotypes in 62 sequenced samples from infected animals. In phylogenetic analyses, 2 of the putative Hepatozoon haplotypes closely resembled Lankesterella minima and L. valsainensis. The third haplotype grouped in a wider clade composed of Caryospora and Eimeria. None of the haplotypes showed resemblance to sequences of Hepatozoon from reptiles and mammals. Molecular detection results were consistent with those from microscopy of stained blood smears, confirming that the primers indeed amplified the parasite sequences. Here we provide evidence that the avian Hepatozoon-like parasites are most likely Lankesterella, supporting the suggestion that the systematic position of avian Hepatozoon-like species needs to be revised.

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.

The presence of an insulin-like androgenic gland factor (IAG) and insulin-like peptide binding protein (ILPBP) in the ovary of the blue crab, Callinectes sapidus and their roles in ovarian development.

PubMed

Huang, Xiaoshuai; Ye, Haihui; Chung, J Sook

2017-08-01

Insulin-like androgenic gland factor (IAG) that is produced by the male androgenic gland (AG), plays a role in sexual differentiation and maintenance of male secondary sex characteristics in decapod crustaceans. With an earlier finding of IAG expression in a female Callinectes sapidus ovary, we aimed to examine a putative role of IAG during the ovarian development of this species. To this end, the full-length cDNA sequence of the ovarian CasIAG (termed CasIAG-ova) has been isolated. The predicted mature peptide sequence of CasIAG-ova is identical to that of the IAG from the AG, except in their signal peptide regions. The CasIAG-ova contains an alternative initiation codon (UUG) as the start codon, which suggests that the translational regulation of CasIAG-ova may differ from that of the IAG from AG. To define the function of CasIAG-ova, the expressions of CasIAG-ova as well as its putative binding protein, insulin-like peptide binding protein (ILPBP), are measured in the ovaries at various developmental stages obtained from different seasons. Season affects both CasIAG and ILPBP expression in the ovary. Overall, summer females at earlier ovarian stages contain high levels of CasIAG and ILPBP than spring or fall females. These findings indicate that CasIAG-ova and CasILPBP may be involved in the ovarian development. When comparing the levels of CasIAG and CasILPBP in the ovary, the latter are much higher (∼10-10000 fold) than the former. Expression patterns of CasILPBP differ from those of CasIAG-ova during ovarian development and by season, suggesting that ILPBP may have an additional role in ovarian development rather than a function of a putative binding protein of IAG. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification and Phylogenetic Analysis of Tityus pachyurus and Tityus obscurus Novel Putative Na+-Channel Scorpion Toxins

PubMed Central

Guerrero-Vargas, Jimmy A.; Mourão, Caroline B. F.; Quintero-Hernández, Verónica; Possani, Lourival D.; Schwartz, Elisabeth F.

2012-01-01

Background Colombia and Brazil are affected by severe cases of scorpionism. In Colombia the most dangerous accidents are caused by Tityus pachyurus that is widely distributed around this country. In the Brazilian Amazonian region scorpion stings are a common event caused by Tityus obscurus. The main objective of this work was to perform the molecular cloning of the putative Na+-channel scorpion toxins (NaScTxs) from T. pachyurus and T. obscurus venom glands and to analyze their phylogenetic relationship with other known NaScTxs from Tityus species. Methodology/Principal Findings cDNA libraries from venom glands of these two species were constructed and five nucleotide sequences from T. pachyurus were identified as putative modulators of Na+-channels, and were named Tpa4, Tpa5, Tpa6, Tpa7 and Tpa8; the latter being the first anti-insect excitatory β-class NaScTx in Tityus scorpion venom to be described. Fifteen sequences from T. obscurus were identified as putative NaScTxs, among which three had been previously described, and the others were named To4 to To15. The peptides Tpa4, Tpa5, Tpa6, To6, To7, To9, To10 and To14 are closely related to the α-class NaScTxs, whereas Tpa7, Tpa8, To4, To8, To12 and To15 sequences are more related to the β-class NaScTxs. To5 is possibly an arthropod specific toxin. To11 and To13 share sequence similarities with both α and β NaScTxs. By means of phylogenetic analysis using the Maximum Parsimony method and the known NaScTxs from Tityus species, these toxins were clustered into 14 distinct groups. Conclusions/Significance This communication describes new putative NaScTxs from T. pachyurus and T. obscurus and their phylogenetic analysis. The results indicate clear geographic separation between scorpions of Tityus genus inhabiting the Amazonian and Mountain Andes regions and those distributed over the Southern of the Amazonian rainforest. Based on the consensus sequences for the different clusters, a new nomenclature for the NaScTxs is proposed. PMID:22355312

Cloning, sequencing and characterization of lipase genes from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

USDA-ARS?s Scientific Manuscript database

Lipase (lip) and lipase-specific foldase (lif) genes of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans NRRL B-2649 were cloned using primers based on consensus sequences, followed by PCR-based genome walking. Sequence analyses showed a putative Lip gene-product (...

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.

Identification of (R)-selective ω-aminotransferases by exploring evolutionary sequence space.

PubMed

Kim, Eun-Mi; Park, Joon Ho; Kim, Byung-Gee; Seo, Joo-Hyun

2018-03-01

Several (R)-selective ω-aminotransferases (R-ωATs) have been reported. The existence of additional R-ωATs having different sequence characteristics from previous ones is highly expected. In addition, it is generally accepted that R-ωATs are variants of aminotransferase group III. Based on these backgrounds, sequences in RefSeq database were scored using family profiles of branched-chain amino acid aminotransferase (BCAT) and d-alanine aminotransferase (DAT) to predict and identify putative R-ωATs. Sequences with two profile analysis scores were plotted on two-dimensional score space. Candidates with relatively similar scores in both BCAT and DAT profiles (i.e., profile analysis score using BCAT profile was similar to profile analysis score using DAT profile) were selected. Experimental results for selected candidates showed that putative R-ωATs from Saccharopolyspora erythraea (R-ωAT_Sery), Bacillus cellulosilyticus (R-ωAT_Bcel), and Bacillus thuringiensis (R-ωAT_Bthu) had R-ωAT activity. Additional experiments revealed that R-ωAT_Sery also possessed DAT activity while R-ωAT_Bcel and R-ωAT_Bthu had BCAT activity. Selecting putative R-ωATs from regions with similar profile analysis scores identified potential R-ωATs. Therefore, R-ωATs could be efficiently identified by using simple family profile analysis and exploring evolutionary sequence space. Copyright © 2017 Elsevier Inc. All rights reserved.

Wnt/β-catenin signaling stimulates the expression and synaptic clustering of the autism-associated Neuroligin 3 gene.

PubMed

Medina, Matías A; Andrade, Víctor M; Caracci, Mario O; Avila, Miguel E; Verdugo, Daniela A; Vargas, Macarena F; Ugarte, Giorgia D; Reyes, Ariel E; Opazo, Carlos; De Ferrari, Giancarlo V

2018-03-05

Synaptic abnormalities have been described in individuals with autism spectrum disorders (ASD). The cell-adhesion molecule Neuroligin-3 (Nlgn3) has an essential role in the function and maturation of synapses and NLGN3 ASD-associated mutations disrupt hippocampal and cortical function. Here we show that Wnt/β-catenin signaling increases Nlgn3 mRNA and protein levels in HT22 mouse hippocampal cells and primary cultures of rat hippocampal neurons. We characterized the activity of mouse and rat Nlgn3 promoter constructs containing conserved putative T-cell factor/lymphoid enhancing factor (TCF/LEF)-binding elements (TBE) and found that their activity is significantly augmented in Wnt/β-catenin cell reporter assays. Chromatin immunoprecipitation (ChIP) assays and site-directed mutagenesis experiments revealed that endogenous β-catenin binds to novel TBE consensus sequences in the Nlgn3 promoter. Moreover, activation of the signaling cascade increased Nlgn3 clustering and co- localization with the scaffold PSD-95 protein in dendritic processes of primary neurons. Our results directly link Wnt/β-catenin signaling to the transcription of the Nlgn3 gene and support a functional role for the signaling pathway in the dysregulation of excitatory/inhibitory neuronal activity, as is observed in animal models of ASD.

RNA-Seq of the Caribbean reef-building coral Orbicella faveolata (Scleractinia-Merulinidae) under bleaching and disease stress expands models of coral innate immunity.

PubMed

Anderson, David A; Walz, Marcus E; Weil, Ernesto; Tonellato, Peter; Smith, Matthew C

2016-01-01

Climate change-driven coral disease outbreaks have led to widespread declines in coral populations. Early work on coral genomics established that corals have a complex innate immune system, and whole-transcriptome gene expression studies have revealed mechanisms by which the coral immune system responds to stress and disease. The present investigation expands bioinformatic data available to study coral molecular physiology through the assembly and annotation of a reference transcriptome of the Caribbean reef-building coral, Orbicella faveolata. Samples were collected during a warm water thermal anomaly, coral bleaching event and Caribbean yellow band disease outbreak in 2010 in Puerto Rico. Multiplex sequencing of RNA on the Illumina GAIIx platform and de novo transcriptome assembly by Trinity produced 70,745,177 raw short-sequence reads and 32,463 O. faveolata transcripts, respectively. The reference transcriptome was annotated with gene ontologies, mapped to KEGG pathways, and a predicted proteome of 20,488 sequences was generated. Protein families and signaling pathways that are essential in the regulation of innate immunity across Phyla were investigated in-depth. Results were used to develop models of evolutionarily conserved Wnt, Notch, Rig-like receptor, Nod-like receptor, and Dicer signaling. O. faveolata is a coral species that has been studied widely under climate-driven stress and disease, and the present investigation provides new data on the genes that putatively regulate its immune system.

RNA-Seq of the Caribbean reef-building coral Orbicella faveolata (Scleractinia-Merulinidae) under bleaching and disease stress expands models of coral innate immunity

PubMed Central

Walz, Marcus E.; Weil, Ernesto; Smith, Matthew C.

2016-01-01

Climate change-driven coral disease outbreaks have led to widespread declines in coral populations. Early work on coral genomics established that corals have a complex innate immune system, and whole-transcriptome gene expression studies have revealed mechanisms by which the coral immune system responds to stress and disease. The present investigation expands bioinformatic data available to study coral molecular physiology through the assembly and annotation of a reference transcriptome of the Caribbean reef-building coral, Orbicella faveolata. Samples were collected during a warm water thermal anomaly, coral bleaching event and Caribbean yellow band disease outbreak in 2010 in Puerto Rico. Multiplex sequencing of RNA on the Illumina GAIIx platform and de novo transcriptome assembly by Trinity produced 70,745,177 raw short-sequence reads and 32,463 O. faveolata transcripts, respectively. The reference transcriptome was annotated with gene ontologies, mapped to KEGG pathways, and a predicted proteome of 20,488 sequences was generated. Protein families and signaling pathways that are essential in the regulation of innate immunity across Phyla were investigated in-depth. Results were used to develop models of evolutionarily conserved Wnt, Notch, Rig-like receptor, Nod-like receptor, and Dicer signaling. O. faveolata is a coral species that has been studied widely under climate-driven stress and disease, and the present investigation provides new data on the genes that putatively regulate its immune system. PMID:26925311

Localization, cloning, and sequence determination of the conjugative plasmid ColB2 pilin gene.

PubMed Central

Finlay, B B; Frost, L S; Paranchych, W

1984-01-01

ColB2 is a colicin-producing, 96-kilobase plasmid which encodes a conjugative system that is similar, but not identical, to F. A restriction map of this plasmid was generated, and DNA homology studies between F and ColB2 plasmids revealed homology only between their transfer operons. The locations of the ColB2 transfer operon and ColB2 pilin gene were localized on this restriction map. The gene encoding ColB2 pilin, traA, was cloned and sequenced. The pilin protein of ColB2 is identical to F, except at the amino terminus, where ala-gln of ColB2 pilin corresponds to Ala-Gly-Ser-Ser of F pilin. This is due to a 6-base-pair deletion in the ColB2 pilin gene. Biochemical studies on tryptic peptides derived from ColB2 pilin demonstrate the location of this gene to be correct. There is a putative signal peptidase cleavage site after the sequence Ala-Met-Ala, giving a signal peptide of 51 amino acids and a mature pilin protein of 68 amino acids (7,000 daltons). The amino terminus is blocked, probably with an acetyl group. A chimera containing the ColB2 pilin gene was able to complement an F traA mutant, demonstrating that the pilus assembly proteins of F can utilize the ColB2 pilin protein to form a pilus. Images PMID:6090427

Begin at the beginning: A BAC-end view of the passion fruit (Passiflora) genome.

PubMed

Santos, Anselmo Azevedo; Penha, Helen Alves; Bellec, Arnaud; Munhoz, Carla de Freitas; Pedrosa-Harand, Andrea; Bergès, Hélène; Vieira, Maria Lucia Carneiro

2014-09-26

The passion fruit (Passiflora edulis) is a tropical crop of economic importance both for juice production and consumption as fresh fruit. The juice is also used in concentrate blends that are consumed worldwide. However, very little is known about the genome of the species. Therefore, improving our understanding of passion fruit genomics is essential and to some degree a pre-requisite if its genetic resources are to be used more efficiently. In this study, we have constructed a large-insert BAC library and provided the first view on the structure and content of the passion fruit genome, using BAC-end sequence (BES) data as a major resource. The library consisted of 82,944 clones and its levels of organellar DNA were very low. The library represents six haploid genome equivalents, and the average insert size was 108 kb. To check its utility for gene isolation, successful macroarray screening experiments were carried out with probes complementary to eight Passiflora gene sequences available in public databases. BACs harbouring those genes were used in fluorescent in situ hybridizations and unique signals were detected for four BACs in three chromosomes (n=9). Then, we explored 10,000 BES and we identified reads likely to contain repetitive mobile elements (19.6% of all BES), simple sequence repeats and putative proteins, and to estimate the GC content (~42%) of the reads. Around 9.6% of all BES were found to have high levels of similarity to plant genes and ontological terms were assigned to more than half of the sequences analysed (940). The vast majority of the top-hits made by our sequences were to Populus trichocarpa (24.8% of the total occurrences), Theobroma cacao (21.6%), Ricinus communis (14.3%), Vitis vinifera (6.5%) and Prunus persica (3.8%). We generated the first large-insert library for a member of Passifloraceae. This BAC library provides a new resource for genetic and genomic studies, as well as it represents a valuable tool for future whole genome study. Remarkably, a number of BAC-end pair sequences could be mapped to intervals of the sequenced Arabidopsis thaliana, V. vinifera and P. trichocarpa chromosomes, and putative collinear microsyntenic regions were identified.

Partial DNA sequencing of Douglas-fir cDNAs used in RFLP mapping

Treesearch

K.D. Jermstad; D.L. Bassoni; C.S. Kinlaw; D.B. Neale

1998-01-01

DNA sequences from 87 Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) cDNA RFLP probes were determined. Sequences were submitted to the GenBank dbEST database and searched for similarity against nucleotide and protein databases using the BLASTn and BLASTx programs. Twenty-one sequences (24%) were assigned putative functions; 18 of which...

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.

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

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.

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.

Accelerated Evolution of the Pituitary Adenylate Cyclase-Activating Polypeptide Precursor Gene During Human Origin

PubMed Central

Wang, Yin-qiu; Qian, Ya-ping; Yang, Su; Shi, Hong; Liao, Cheng-hong; Zheng, Hong-Kun; Wang, Jun; Lin, Alice A.; Cavalli-Sforza, L. Luca; Underhill, Peter A.; Chakraborty, Ranajit; Jin, Li; Su, Bing

2005-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition. PMID:15834139

Characterization of a Theta-Type Plasmid from Lactobacillus sakei: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli

PubMed Central

Alpert, Carl-Alfred; Crutz-Le Coq, Anne-Marie; Malleret, Christine; Zagorec, Monique

2003-01-01

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed. PMID:12957947

Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs

PubMed Central

Yang, Mingyu; Du, Lianming; Li, Wujiao; Shen, Fujun; Fan, Zhenxin; Jian, Zuoyi; Hou, Rong; Shen, Yongmei; Yue, Bisong; Zhang, Xiuyue

2015-01-01

The giant panda (Ailuropoda melanoleuca) is one of the world’s most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs) or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity. PMID:26599861

Profile of microRNA in Giant Panda Blood: A Resource for Immune-Related and Novel microRNAs.

PubMed

Yang, Mingyu; Du, Lianming; Li, Wujiao; Shen, Fujun; Fan, Zhenxin; Jian, Zuoyi; Hou, Rong; Shen, Yongmei; Yue, Bisong; Zhang, Xiuyue

2015-01-01

The giant panda (Ailuropoda melanoleuca) is one of the world's most beloved endangered mammals. Although the draft genome of this species had been assembled, little was known about the composition of its microRNAs (miRNAs) or their functional profiles. Recent studies demonstrated that changes in the expression of miRNAs are associated with immunity. In this study, miRNAs were extracted from the blood of four healthy giant pandas and sequenced by Illumina next generation sequencing technology. As determined by miRNA screening, a total of 276 conserved miRNAs and 51 novel putative miRNAs candidates were detected. After differential expression analysis, we noticed that the expressions of 7 miRNAs were significantly up-regulated in young giant pandas compared with that of adults. Moreover, 2 miRNAs were up-regulated in female giant pandas and 1 in the male individuals. Target gene prediction suggested that the miRNAs of giant panda might be relevant to the expressions of 4,602 downstream genes. Subseuqently, the predicted target genes were conducted to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and we found that these genes were mainly involved in host immunity, including the Ras signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In conclusion, our results provide the first miRNA profiles of giant panda blood, and the predicted functional analyses may open an avenue for further study of giant panda immunity.

The acoustic repertoire and behavioural context of the vocalisations of a nocturnal dasyurid, the eastern quoll (Dasyurus viverrinus)

PubMed Central

Dorph, Annalie

2017-01-01

Defining an acoustic repertoire is essential to understanding vocal signalling and communicative interactions within a species. Currently, quantitative and statistical definition is lacking for the vocalisations of many dasyurids, an important group of small to medium-sized marsupials from Australasia that includes the eastern quoll (Dasyurus viverrinus), a species of conservation concern. Beyond generating a better understanding of this species' social interactions, determining an acoustic repertoire will further improve detection rates and inference of vocalisations gathered by automated bioacoustic recorders. Hence, this study investigated eastern quoll vocalisations using objective signal processing techniques to quantitatively analyse spectrograms recorded from 15 different individuals. Recordings were collected in conjunction with observations of the behaviours associated with each vocalisation to develop an acoustic-based behavioural repertoire for the species. Analysis of recordings produced a putative classification of five vocalisation types: Bark, Growl, Hiss, Cp-cp, and Chuck. These were most frequently observed during agonistic encounters between conspecifics, most likely as a graded sequence from Hisses occurring in a warning context through to Growls and finally Barks being given prior to, or during, physical confrontations between individuals. Quantitative and statistical methods were used to objectively establish the accuracy of these five putative call types. A multinomial logistic regression indicated a 97.27% correlation with the perceptual classification, demonstrating support for the five different vocalisation types. This putative classification was further supported by hierarchical cluster analysis and silhouette information that determined the optimal number of clusters to be five. Minor disparity between the objective and perceptual classifications was potentially the result of gradation between vocalisations, or subtle differences present within vocalisations not discernible to the human ear. The implication of these different vocalisations and their given context is discussed in relation to the ecology of the species and the potential application of passive acoustic monitoring techniques. PMID:28686679

Genome-wide digital transcript analysis of putative fruitlet abscission related genes regulated by ethephon in litchi

PubMed Central

Li, Caiqin; Wang, Yan; Ying, Peiyuan; Ma, Wuqiang; Li, Jianguo

2015-01-01

The high level of physiological fruitlet abscission in litchi (Litchi chinensis Sonn.) causes severe yield loss. Cell separation occurs at the fruit abscission zone (FAZ) and can be triggered by ethylene. However, a deep knowledge of the molecular events occurring in the FAZ is still unknown. Here, genome-wide digital transcript abundance (DTA) analysis of putative fruit abscission related genes regulated by ethephon in litchi were studied. More than 81 million high quality reads from seven ethephon treated and untreated control libraries were obtained by high-throughput sequencing. Through DTA profile analysis in combination with Gene Ontology and KEGG pathway enrichment analyses, a total of 2730 statistically significant candidate genes were involved in the ethephon-promoted litchi fruitlet abscission. Of these, there were 1867 early-responsive genes whose expressions were up- or down-regulated from 0 to 1 d after treatment. The most affected genes included those related to ethylene biosynthesis and signaling, auxin transport and signaling, transcription factors (TFs), protein ubiquitination, ROS response, calcium signal transduction, and cell wall modification. These genes could be clustered into four groups and 13 subgroups according to their similar expression patterns. qRT-PCR displayed the expression pattern of 41 selected candidate genes, which proved the accuracy of our DTA data. Ethephon treatment significantly increased fruit abscission and ethylene production of fruitlet. The possible molecular events to control the ethephon-promoted litchi fruitlet abscission were prompted out. The increased ethylene evolution in fruitlet would suppress the synthesis and polar transport of auxin and trigger abscission signaling. To the best of our knowledge, it is the first time to monitor the gene expression profile occurring in the FAZ-enriched pedicel during litchi fruit abscission induced by ethephon on the genome-wide level. This study will contribute to a better understanding for the molecular regulatory mechanism of fruit abscission in litchi. PMID:26217356

Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

DOE PAGES

Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica; ...

2016-04-28

Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three proteins are indeed lipid-binding and act in the vasculature possibly in a function related to long-distance signaling, the three proteins do not act in the same but rather in distinct pathways. Furthermore, it points toward PLAFP as a prime candidate to investigate long-distance lipid signaling in the plant drought response.« less

Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

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

Barbaglia, Allison M.; Tamot, Banita; Greve, Veronica

Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho-) lipids could act as long-distance developmental signals in response to abiotic stress, and thatmore » they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012). Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I) a putative GDSL-motif lipase (II) a PIG-P-like protein, with a possible receptor-like function; (III) and PLAFP (phloem lipid-associated family protein), a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH), which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while all three proteins are indeed lipid-binding and act in the vasculature possibly in a function related to long-distance signaling, the three proteins do not act in the same but rather in distinct pathways. Furthermore, it points toward PLAFP as a prime candidate to investigate long-distance lipid signaling in the plant drought response.« less

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

Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower.

PubMed

Tamborindeguy, C; Ben, C; Liboz, T; Gentzbittel, L

2004-04-01

Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs represented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previously reported for sunflower. Several potentially interesting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The libraries described here represent an original and valuable resource for the discovery of yet unknown genes putatively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos.

Sequence analysis and gene expression of putative exo- and endo-glucanases from oil palm (Elaeis guineensis) during fungal infection.

PubMed

Yeoh, Keat-Ai; Othman, Abrizah; Meon, Sariah; Abdullah, Faridah; Ho, Chai-Ling

2012-10-15

Glucanases are enzymes that hydrolyze a variety β-d-glucosidic linkages. Plant β-1,3-glucanases are able to degrade fungal cell walls; and promote the release of cell-wall derived fungal elicitors. In this study, three full-length cDNA sequences encoding oil palm (Elaeis guineensis) glucanases were analyzed. Sequence analyses of the cDNA sequences suggested that EgGlc1-1 is a putative β-d-glucan exohydolase belonging to glycosyl hydrolase (GH) family 3 while EgGlc5-1 and EgGlc5-2 are putative glucan endo-1,3-β-glucosidases belonging to GH family 17. The transcript abundance of these genes in the roots and leaves of oil palm seedlings treated with Ganoderma boninense and Trichoderma harzianum was profiled to investigate the involvement of these glucanases in oil palm during fungal infection. The gene expression of EgGlc1-1 in the root of oil palm seedlings was increased by T. harzianum but suppressed by G. boninense; while the gene expression of both EgGlc5-1 and EgGlc5-2 in the roots of oil palm seedlings was suppressed by G. boninense or/and T. harzianum. Copyright © 2012 Elsevier GmbH. All rights reserved.

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.

Analysis of the beak and feather disease viral genome indicates the existence of several genotypes which have a complex psittacine host specificity.

PubMed

de Kloet, E; de Kloet, S R

2004-12-01

A study was made of the phylogenetic relationships between fifteen complete nucleotide sequences as well as 43 nucleotide sequences of the putative coat protein gene of different strains belonging to the virus species Beak and feather disease virus obtained from 39 individuals of 16 psittacine species. The species included among others, cockatoos ( Cacatuini), African grey parrots ( Psittacus erithacus) and peach-faced lovebirds ( Agapornis roseicollis), which were infected at different geographical locations, within and outside Australia, the native origin of the virus. The derived amino acid sequences of the putative coat protein were highly diverse, with differences between some strains amounting to 50 of the 250 amino acids. Phylogenetic analysis demonstrated that the putative coat gene sequences form six clusters which show a varying degree of psittacine species specificity. Most, but not all strains infecting African grey parrots formed a single cluster as did the strains infecting the cockatoos. Strains infecting the lovebirds clustered with those infecting such Australasian species as Eclectus roratus, Psittacula kramerii and Psephotus haematogaster. Although individual birds included in this study were, where studied, often infected by closely related strains, infection by highly diverged trains was also detected. The possible relationship between BFD viral strains and clinical disease signs is discussed.

A new cryptic virus belonging to the family Partitiviridae was found in watermelon co-infected with Melon necrotic spot virus.

PubMed

Sela, Noa; Lachman, Oded; Reingold, Victoria; Dombrovsky, Aviv

2013-10-01

A novel virus was detected in watermelon plants (Citrullus lanatus Thunb.) infected with Melon necrotic spot virus (MNSV) using SOLiD next-generation sequence analysis. In addition to the expected MSNV genome, two double-stranded RNA (dsRNA) segments of 1,312 and 1,118 bp were also identified and sequenced from the purified virus preparations. These two dsRNA segments encode two putative partitivirus-related proteins, an RNA-dependent RNA polymerase (RdRP) and a capsid protein, which were sequenced. Genomic-sequence analysis and analysis of phylogenetic relationships indicate that these two dsRNAs together make up the genome of a novel Partitivirus. This virus was found to be closely related to the Pepper cryptic virus 1 and Raphanus sativus cryptic virus. It is suggested that this novel virus putatively named Citrullus lanatus cryptic virus be considered as a new member of the family Partitiviridae.

The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion.

PubMed Central

Ringwald, M; Schuh, R; Vestweber, D; Eistetter, H; Lottspeich, F; Engel, J; Dölz, R; Jähnig, F; Epplen, J; Mayer, S

1987-01-01

We have determined the amino acid sequence of the Ca2+-dependent cell adhesion molecule uvomorulin as it appears on the cell surface. The extracellular part of the molecule exhibits three internally repeated domains of 112 residues which are most likely generated by gene duplication. Each of the repeated domains contains two highly conserved units which could represent putative Ca2+-binding sites. Secondary structure predictions suggest that the putative Ca2+-binding units are located in external loops at the surface of the protein. The protein sequence exhibits a single membrane-spanning region and a cytoplasmic domain. Sequence comparison reveals extensive homology to the chicken L-CAM. Both uvomorulin and L-CAM are identical in 65% of their entire amino acid sequence suggesting a common origin for both CAMs. Images Fig. 1. Fig. 4. Fig. 7. PMID:3501370

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

Domain fusion analysis by applying relational algebra to protein sequence and domain databases.

PubMed

Truong, Kevin; Ikura, Mitsuhiko

2003-05-06

Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at http://calcium.uhnres.utoronto.ca/pi. As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time.

A high-resolution genetic, physical, and comparative gene map of the doublefoot (Dbf) region of mouse chromosome 1 and the region of conserved synteny on human chromosome 2q35.

PubMed

Hayes, C; Rump, A; Cadman, M R; Harrison, M; Evans, E P; Lyon, M F; Morriss-Kay, G M; Rosenthal, A; Brown, S D

2001-12-01

The mouse doublefoot (Dbf) mutant exhibits preaxial polydactyly in association with craniofacial defects. This mutation has previously been mapped to mouse chromosome 1. We have used a positional cloning strategy, coupled with a comparative sequencing approach using available human draft sequence, to identify putative candidates for the Dbf gene in the mouse and in homologous human region. We have constructed a high-resolution genetic map of the region, localizing the mutation to a 0.4-cM (+/-0.0061) interval on mouse chromosome 1. Furthermore, we have constructed contiguous BAC/PAC clone maps across the mouse and human Dbf region. Using existing markers and additional sequence tagged sites, which we have generated, we have anchored the physical map to the genetic map. Through the comparative sequencing of these clones we have identified 35 genes within this interval, indicating that the region is gene-rich. From this we have identified several genes that are known to be differentially expressed in the developing mid-gestation mouse embryo, some in the developing embryonic limb buds. These genes include those encoding known developmental signaling molecules such as WNT proteins and IHH, and we provide evidence that these genes are candidates for the Dbf mutation.

The rDNA ITS region in the lessepsian marine angiosperm Halophila stipulacea (Forssk.) Aschers. (Hydrocharitaceae): intragenomic variability and putative pseudogenic sequences.

PubMed

Ruggiero, Maria Valeria; Procaccini, Gabriele

2004-01-01

Halophila stipulacea is a dioecious marine angiosperm, widely distributed along the western coasts of the Indian Ocean and the Red Sea. This species is thought to be a Lessepsian immigrant that entered the Mediterranean Sea from the Red Sea after the opening of the Suez Canal (1869). Previous studies have revealed both high phenotypic and genetic variability in Halophila stipulacea populations from the western Mediterranean basin. In order to test the hypothesis of a Lessepsian introduction, we compare genetic polymorphism between putative native (Red Sea) and introduced (Mediterranean) populations through rDNA ITS region (ITS1-5.8S-ITS2) sequence analysis. A high degree of intraindividual variability of ITS sequences was found. Most of the intragenomic polymorphism was due to pseudogenic sequences, present in almost all individuals. Features of ITS functional sequences and pseudogenes are described. Possible causes for the lack of homogenization of ITS paralogues within individuals are discussed.

Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

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

Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki

2006-05-12

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLSmore » might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.« less

Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis

DOE PAGES

Plett, Jonathan M.; Yin, Hengfu; Mewalal, Ritesh; ...

2017-03-23

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as ‘effectors’, i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa andmore » 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. Finally, we conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.« less

Cyclophilin B enhances HIV-1 Infection

PubMed Central

DeBoer, Jason; Madson, Christian J.; Belshan, Michael

2016-01-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. PMID:26774171

Cyclophilin B enhances HIV-1 infection.

PubMed

DeBoer, Jason; Madson, Christian J; Belshan, Michael

2016-02-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of novel nuclear localization signals of Drosophila myeloid leukemia factor.

PubMed

Sugano, Wakana; Yamaguchi, Masamitsu

2007-01-01

Myeloid leukemia factor 1 (MLF1) was first identified as part of a leukemic fusion protein produced by a chromosomal translocation, and MLF family proteins are present in many animals. In mammalian cells, MLF1 has been described as mainly cytoplasmic, but in Drosophila, one of the dMLF isoforms (dMLFA) localized mainly in the nucleus while the other isoform (dMLFB), that appears to be produced by the alternative splicing, displays both nuclear and cytoplasmic localization. To investigate the difference in subcellular localization between MLF family members, we examined the subcellular localization of deletion mutants of dMLFA isoform. The analyses showed that the C-terminal 40 amino acid region of dMLFA is necessary and sufficient for nuclear localization. Based on amino acid sequences, we hypothesized that two nuclear localization signals (NLSs) are present within the region. Site-directed mutagenesis of critical residues within the two putative NLSs leads to loss of nuclear localization, suggesting that both NLS motifs are necessary for nuclear localization.

Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis

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

Plett, Jonathan M.; Yin, Hengfu; Mewalal, Ritesh

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as ‘effectors’, i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa andmore » 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. Finally, we conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.« less

The SH2 domain interaction landscape.

PubMed

Tinti, Michele; Kiemer, Lars; Costa, Stefano; Miller, Martin L; Sacco, Francesca; Olsen, Jesper V; Carducci, Martina; Paoluzi, Serena; Langone, Francesca; Workman, Christopher T; Blom, Nikolaj; Machida, Kazuya; Thompson, Christopher M; Schutkowski, Mike; Brunak, Søren; Mann, Matthias; Mayer, Bruce J; Castagnoli, Luisa; Cesareni, Gianni

2013-04-25

Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Serotype IV Sequence Type 468 Group B Streptococcus Neonatal Invasive Disease, Minnesota, USA.

PubMed

Teatero, Sarah; Ferrieri, Patricia; Fittipaldi, Nahuel

2016-11-01

To further understand the emergence of serotype IV group B Streptococcus (GBS) invasive disease, we used whole-genome sequencing to characterize 3 sequence type 468 strains isolated from neonates in Minnesota, USA. We found that strains of tetracycline-resistant sequence type 468 GBS have acquired virulence genes from a putative clonal complex 17 GBS donor by recombination.

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed Central

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-01-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis. PMID:8892842

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-11-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.

Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

PubMed

Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng

2013-11-01

Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

Isolation, cloning, and characterization of the 2S albumin: a new allergen from hazelnut.

PubMed

Garino, Cristiano; Zuidmeer, Laurian; Marsh, Justin; Lovegrove, Alison; Morati, Maria; Versteeg, Serge; Schilte, Piet; Shewry, Peter; Arlorio, Marco; van Ree, Ronald

2010-09-01

2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP-HPLC. After N-terminal sequencing, degenerated and poly-d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut-allergic patients. N-terminal sequencing of a approximately 10 kDa peak from size exclusion chromatography/RP-HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N-terminal and a 10 aa internal peptide. The obtained clone (441 bp) encoded a 147 aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.

RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

PubMed

Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

2016-02-01

During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks. Copyright © 2015 Elsevier B.V. All rights reserved.

Functional Genomics Analysis of Singapore Grouper Iridovirus: Complete Sequence Determination and Proteomic Analysis

PubMed Central

Song, Wen Jun; Qin, Qi Wei; Qiu, Jin; Huang, Can Hua; Wang, Fan; Hew, Choy Leong

2004-01-01

Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV). Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp. One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified. Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins. Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database. An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified. Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation. The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins. Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products. PMID:15507645

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection

PubMed Central

De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain

2016-01-01

ABSTRACT Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae. Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum. IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete’s foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae. Comparing gene expression during infection on guinea pigs with keratin degradation in vitro, which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo, encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates. PMID:27822542

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection.

PubMed

Tran, Van Du T; De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain; Pagni, Marco; Monod, Michel

2016-01-01

Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae . Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum . IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete's foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae . Comparing gene expression during infection on guinea pigs with keratin degradation in vitro , which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo , encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates.

Detecting false positive sequence homology: a machine learning approach.

PubMed

Fujimoto, M Stanley; Suvorov, Anton; Jensen, Nicholas O; Clement, Mark J; Bybee, Seth M

2016-02-24

Accurate detection of homologous relationships of biological sequences (DNA or amino acid) amongst organisms is an important and often difficult task that is essential to various evolutionary studies, ranging from building phylogenies to predicting functional gene annotations. There are many existing heuristic tools, most commonly based on bidirectional BLAST searches that are used to identify homologous genes and combine them into two fundamentally distinct classes: orthologs and paralogs. Due to only using heuristic filtering based on significance score cutoffs and having no cluster post-processing tools available, these methods can often produce multiple clusters constituting unrelated (non-homologous) sequences. Therefore sequencing data extracted from incomplete genome/transcriptome assemblies originated from low coverage sequencing or produced by de novo processes without a reference genome are susceptible to high false positive rates of homology detection. In this paper we develop biologically informative features that can be extracted from multiple sequence alignments of putative homologous genes (orthologs and paralogs) and further utilized in context of guided experimentation to verify false positive outcomes. We demonstrate that our machine learning method trained on both known homology clusters obtained from OrthoDB and randomly generated sequence alignments (non-homologs), successfully determines apparent false positives inferred by heuristic algorithms especially among proteomes recovered from low-coverage RNA-seq data. Almost ~42 % and ~25 % of predicted putative homologies by InParanoid and HaMStR respectively were classified as false positives on experimental data set. Our process increases the quality of output from other clustering algorithms by providing a novel post-processing method that is both fast and efficient at removing low quality clusters of putative homologous genes recovered by heuristic-based approaches.

Transcriptomics of the Bed Bug (Cimex lectularius)

PubMed Central

Rajarapu, Swapna P.; Jones, Susan C.; Mittapalli, Omprakash

2011-01-01

Background Bed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance. Methodology and Principal Findings Using 454 pyrosequencing, we obtained a total of 216,419 reads with 79,596,412 bp, which were assembled into 35,646 expressed sequence tags (3902 contigs and 31744 singletons). Nearly 85.9% of the C. lectularius sequences showed similarity to insect sequences, but 44.8% of the deduced proteins of C. lectularius did not show similarity with sequences in the GenBank non-redundant database. KEGG analysis revealed putative members of several detoxification pathways involved in pesticide resistance. Lamprin domains, Protein Kinase domains, Protein Tyrosine Kinase domains and cytochrome P450 domains were among the top Pfam domains predicted for the C. lectularius sequences. An initial assessment of putative defense genes, including a cytochrome P450 and a glutathione-S-transferase (GST), revealed high transcript levels for the cytochrome P450 (CYP9) in pesticide-exposed versus pesticide-susceptible C. lectularius populations. A significant number of single nucleotide polymorphisms (296) and microsatellite loci (370) were predicted in the C. lectularius sequences. Furthermore, 59 putative sequences of Wolbachia were retrieved from the database. Conclusions To our knowledge this is the first study to elucidate the genetic makeup of C. lectularius. This pyrosequencing effort provides clues to the identification of potential detoxification genes involved in pesticide resistance of C. lectularius and lays the foundation for future functional genomics studies. PMID:21283830

Establishing the role of rare coding variants in known Parkinson's disease risk loci.

PubMed

Jansen, Iris E; Gibbs, J Raphael; Nalls, Mike A; Price, T Ryan; Lubbe, Steven; van Rooij, Jeroen; Uitterlinden, André G; Kraaij, Robert; Williams, Nigel M; Brice, Alexis; Hardy, John; Wood, Nicholas W; Morris, Huw R; Gasser, Thomas; Singleton, Andrew B; Heutink, Peter; Sharma, Manu

2017-11-01

Many common genetic factors have been identified to contribute to Parkinson's disease (PD) susceptibility, improving our understanding of the related underlying biological mechanisms. The involvement of rarer variants in these loci has been poorly studied. Using International Parkinson's Disease Genomics Consortium data sets, we performed a comprehensive study to determine the impact of rare variants in 23 previously published genome-wide association studies (GWAS) loci in PD. We applied Prix fixe to select the putative causal genes underneath the GWAS peaks, which was based on underlying functional similarities. The Sequence Kernel Association Test was used to analyze the joint effect of rare, common, or both types of variants on PD susceptibility. All genes were tested simultaneously as a gene set and each gene individually. We observed a moderate association of common variants, confirming the involvement of the known PD risk loci within our genetic data sets. Focusing on rare variants, we identified additional association signals for LRRK2, STBD1, and SPATA19. Our study suggests an involvement of rare variants within several putatively causal genes underneath previously identified PD GWAS peaks. Copyright © 2017 Elsevier Inc. All rights reserved.

Post-genomic insights into the plant polysaccharide degradation potential of Aspergillus nidulans and comparison to Aspergillus niger and Aspergillus oryzae.

PubMed

Coutinho, Pedro M; Andersen, Mikael R; Kolenova, Katarina; vanKuyk, Patricia A; Benoit, Isabelle; Gruben, Birgit S; Trejo-Aguilar, Blanca; Visser, Hans; van Solingen, Piet; Pakula, Tiina; Seiboth, Bernard; Battaglia, Evy; Aguilar-Osorio, Guillermo; de Jong, Jan F; Ohm, Robin A; Aguilar, Mariana; Henrissat, Bernard; Nielsen, Jens; Stålbrand, Henrik; de Vries, Ronald P

2009-03-01

The plant polysaccharide degradative potential of Aspergillus nidulans was analysed in detail and compared to that of Aspergillus niger and Aspergillus oryzae using a combination of bioinformatics, physiology and transcriptomics. Manual verification indicated that 28.4% of the A. nidulans ORFs analysed in this study do not contain a secretion signal, of which 40% may be secreted through a non-classical method.While significant differences were found between the species in the numbers of ORFs assigned to the relevant CAZy families, no significant difference was observed in growth on polysaccharides. Growth differences were observed between the Aspergilli and Podospora anserina, which has a more different genomic potential for polysaccharide degradation, suggesting that large genomic differences are required to cause growth differences on polysaccharides. Differences were also detected between the Aspergilli in the presence of putative regulatory sequences in the promoters of the ORFs of this study and correlation of the presence of putative XlnR binding sites to induction by xylose was detected for A. niger. These data demonstrate differences at genome content, substrate specificity of the enzymes and gene regulation in these three Aspergilli, which likely reflect their individual adaptation to their natural biotope.

Draft Genome Sequence of Aeromonas caviae Strain 429865 INP, Isolated from a Mexican Patient

PubMed Central

Padilla, Juan Carlos A.; Bustos, Patricia; Sánchez-Varela, Alejandro; Palma-Martinez, Ingrid; Arzate-Barbosa, Patricia; García-Pérez, Carlos A.; López-López, María de Jesús; González, Víctor

2015-01-01

Aeromonas caviae is an emerging human pathogen. Here, we report the draft genome sequence of Aeromonas caviae strain 429865 INP which shows the presence of various putative virulence-related genes. PMID:26494682

A resource for characterizing genome-wide binding and putative target genes of transcription factors expressed during secondary growth and wood formation in Populus

Treesearch

Lijun Liu; Trevor Ramsay; Matthew S. Zinkgraf; David Sundell; Nathaniel Robert Street; Vladimir Filkov; Andrew Groover

2015-01-01

Identifying transcription factor target genes is essential for modeling the transcriptional networks underlying developmental processes. Here we report a chromatin immunoprecipitation sequencing (ChIP-seq) resource consisting of genome-wide binding regions and associated putative target genes for four Populus homeodomain transcription factors...

Genome-based insights into the resistome and mobilome of multidrug-resistant Aeromonas sp. ARM81 isolated from wastewater.

PubMed

Adamczuk, Marcin; Dziewit, Lukasz

2017-01-01

The draft genome of multidrug-resistant Aeromonas sp. ARM81 isolated from a wastewater treatment plant in Warsaw (Poland) was obtained. Sequence analysis revealed multiple genes conferring resistance to aminoglycosides, β-lactams or tetracycline. Three different β-lactamase genes were identified, including an extended-spectrum β-lactamase gene bla PER-1 . The antibiotic susceptibility was experimentally tested. Genome sequencing also allowed us to investigate the plasmidome and transposable mobilome of ARM81. Four plasmids, of which two carry phenotypic modules (i.e., genes encoding a zinc transporter ZitB and a putative glucosyltransferase), and 28 putative transposase genes were identified. The mobility of three insertion sequences (isoforms of previously identified elements ISAs12, ISKpn9 and ISAs26) was confirmed using trap plasmids.

Fungal Genes in Context: Genome Architecture Reflects Regulatory Complexity and Function

PubMed Central

Noble, Luke M.; Andrianopoulos, Alex

2013-01-01

Gene context determines gene expression, with local chromosomal environment most influential. Comparative genomic analysis is often limited in scope to conserved or divergent gene and protein families, and fungi are well suited to this approach with low functional redundancy and relatively streamlined genomes. We show here that one aspect of gene context, the amount of potential upstream regulatory sequence maintained through evolution, is highly predictive of both molecular function and biological process in diverse fungi. Orthologs with large upstream intergenic regions (UIRs) are strongly enriched in information processing functions, such as signal transduction and sequence-specific DNA binding, and, in the genus Aspergillus, include the majority of experimentally studied, high-level developmental and metabolic transcriptional regulators. Many uncharacterized genes are also present in this class and, by implication, may be of similar importance. Large intergenic regions also share two novel sequence characteristics, currently of unknown significance: they are enriched for plus-strand polypyrimidine tracts and an information-rich, putative regulatory motif that was present in the last common ancestor of the Pezizomycotina. Systematic consideration of gene UIR in comparative genomics, particularly for poorly characterized species, could help reveal organisms’ regulatory priorities. PMID:23699226

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.

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.

A single base substitution in the coding region for neurophysin II associated with familial central diabetes insipidus.

PubMed Central

Ito, M; Mori, Y; Oiso, Y; Saito, H

1991-01-01

To elucidate the molecular mechanism of familial central diabetes insipidus (FDI), we sequenced the arginine vasopressin-neurophysin II (AVP-NPII) gene in 2 patients belonging to a pedigree that is consistent with an autosomal dominant mode of inheritance. 10 patients with idiopathic central diabetes insipidus (IDI) and 5 normals were also studied. The AVP-NPII gene, locating on chromosome 20, consists of three exons that encode putative signal peptide, AVP, NPII, and glycoprotein. Using polymerase chain reaction, fragments including the promoter region and all coding regions were amplified from genomic DNA and subjected to direct sequencing. Sequences of 10 patients with IDI were identical with those of normals, while in 2 patients with FDI, a single base substitution was detected in one of two alleles of the AVP-NPII gene, indicating they were heterozygotes for this mutation. It was a G----A transition at nucleotide position 1859 in the second exon, resulting in a substitution of Gly for Ser at amino acid position 57 in the NPII moiety. It was speculated that the mutated AVP-NPII precursor or the mutated NPII molecule, through their conformational changes, might be responsible for AVP deficiency. Images PMID:1840604

De Novo Assembly, Gene Annotation, and Marker Discovery in Stored-Product Pest Liposcelis entomophila (Enderlein) Using Transcriptome Sequences

PubMed Central

Wei, Dan-Dan; Chen, Er-Hu; Ding, Tian-Bo; Chen, Shi-Chun; Dou, Wei; Wang, Jin-Jun

2013-01-01

Background As a major stored-product pest insect, Liposcelis entomophila has developed high levels of resistance to various insecticides in grain storage systems. However, the molecular mechanisms underlying resistance and environmental stress have not been characterized. To date, there is a lack of genomic information for this species. Therefore, studies aimed at profiling the L. entomophila transcriptome would provide a better understanding of the biological functions at the molecular levels. Methodology/Principal Findings We applied Illumina sequencing technology to sequence the transcriptome of L. entomophila. A total of 54,406,328 clean reads were obtained and that de novo assembled into 54,220 unigenes, with an average length of 571 bp. Through a similarity search, 33,404 (61.61%) unigenes were matched to known proteins in the NCBI non-redundant (Nr) protein database. These unigenes were further functionally annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A large number of genes potentially involved in insecticide resistance were manually curated, including 68 putative cytochrome P450 genes, 37 putative glutathione S-transferase (GST) genes, 19 putative carboxyl/cholinesterase (CCE) genes, and other 126 transcripts to contain target site sequences or encoding detoxification genes representing eight types of resistance enzymes. Furthermore, to gain insight into the molecular basis of the L. entomophila toward thermal stresses, 25 heat shock protein (Hsp) genes were identified. In addition, 1,100 SSRs and 57,757 SNPs were detected and 231 pairs of SSR primes were designed for investigating the genetic diversity in future. Conclusions/Significance We developed a comprehensive transcriptomic database for L. entomophila. These sequences and putative molecular markers would further promote our understanding of the molecular mechanisms underlying insecticide resistance or environmental stress, and will facilitate studies on population genetics for psocids, as well as providing useful information for functional genomic research in the future. PMID:24244605

Sequence of a cDNA and expression of the gene encoding a putative epidermal chitin synthase of Manduca sexta.

PubMed

Zhu, Yu-Cheng; Specht, Charles A; Dittmer, Neal T; Muthukrishnan, Subbaratnam; Kanost, Michael R; Kramer, Karl J

2002-11-01

Glycosyltransferases are enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. One type of glycosyltransferase is chitin synthase, a very important enzyme in biology, which is utilized by insects, fungi, and other invertebrates to produce chitin, a polysaccharide of beta-1,4-linked N-acetylglucosamine. Chitin is an important component of the insect's exoskeletal cuticle and gut lining. To identify and characterize a chitin synthase gene of the tobacco hornworm, Manduca sexta, degenerate primers were designed from two highly conserved regions in fungal and nematode chitin synthase protein sequences and then used to amplify a similar region from Manduca cDNA. A full-length cDNA of 5152 nucleotides was assembled for the putative Manduca chitin synthase gene, MsCHS1, and sequencing of genomic DNA verified the contiguity of the sequence. The MsCHS1 cDNA has an ORF of 4692 nucleotides that encodes a transmembrane protein of 1564 amino acid residues with a mass of approximately 179 kDa (GenBank no. AY062175). It is most similar, over its entire length of protein sequence, to putative chitin synthases from other insects and nematodes, with 68% identity to enzymes from both the blow fly, Lucilia cuprina, and the fruit fly, Drosophila melanogaster. The similarity with fungal chitin synthases is restricted to the putative catalytic domain, and the MsCHS1 protein has, at equivalent positions, several amino acids that are essential for activity as revealed by mutagenesis of the fungal enzymes. A 5.3-kb transcript of MsCHS1 was identified by northern blot hybridization of RNA from larval epidermis, suggesting that the enzyme functions to make chitin deposited in the cuticle. Further examination by RT-PCR showed that MsCHS1 expression is regulated in the epidermis, with the amount of transcript increasing during phases of cuticle deposition.

In silico analysis of Mn transporters (NRAMP1) in various plant species.

PubMed

Vatansever, Recep; Filiz, Ertugrul; Ozyigit, Ibrahim Ilker

2016-03-01

Manganese (Mn) is an essential micronutrient in plant life cycle. It may be involved in photosynthesis, carbohydrate and lipid biosynthesis, and oxidative stress protection. Mn deficiency inhibits the plant growth and development, and causes the various plant symptoms such as interveinal chlorosis and tissue necrosis. Despite its importance in plant life cycle, we still have limited knowledge about Mn transporters in many plant species. Therefore, this study aimed to identify and characterize high affinity Arabidopsis Mn root transporter NRAMP1 orthologs in 17 different plant species. Various in silico methods and digital gene expression data were used in identification and characterization of NRAMP1 homologs; physico-chemical properties of sequences were calculated, putative transmembrane domains (TMDs) and conserved motif signatures were determined, phylogenetic tree was constructed, 3D models and interactome map were generated, and gene expression data was analyzed. 49 NRAMP1 homologs were identified from proteome datasets of 17 plant species using AtNRAMP1 as query. Identified sequences were characterized with a NRAMP domain structure, 10-12 putative TMDs with cytosolic N- and C-terminuses, and 10-14 exons encoding a protein of 500-588 amino acids and 53.8-64.3 kDa molecular weight with basic characteristics. Consensus transport residues, GQSSTITGTYAGQY(/F)V(/I)MQGFLD(/E/N) between TMD-8 and 9 were identified in all sequences but putative N-linked glycosylation sites were not highly conserved. In phylogeny, NRAMP1 sequences demonstrated divergence in lower and higher plants as well as in monocots and dicots. Despite divergence of lower plant Physcomitrella patens in phylogeny, it showed similarity in superposed 3D models. Phylogenetic distribution of AtNRAMP1 and 6 homologs inferred a functional relationship to NRAMP6 sequences in Mn transport, while distribution of OsNRAMP1 and 5 homologs implicated an involvement of NRAMP1 sequences in Mn transport or a cross-talk between in Fe-Mn homeostasis. Interactome analysis further confirmed this cross-talk between Mn and Fe pathways. Gene expression profile of AtNRAMP1 under Fe-, K-, P- and S-deficiencies, and cold, drought, heat and salt stresses revealed various proteins involving in transcription regulation, cofactor biosynthesis, diverse developmental roles, carbohydrate metabolism, oxidation-reduction reactions, cellular signaling and protein degradation pathways. Mn deficiency or toxicity could cause serious adverse effects in plants as well as in humans. To reduce these adversities mainly rely on understanding the molecular mechanisms underlying Mn uptake from the soil. However, we still have limited knowledge regarding the structural and functional roles of Mn transporters in many plant species. Therefore, identification and characterization of Mn root uptake transporter, NRAMP1 orthologs in various plant species will provide valuable theoretical knowledge to better understand Mn transporters as well as it may become an insight for future studies aiming to develop genetically engineered and biofortified plants.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

PubMed

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-02-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

PubMed Central

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-01-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators. Images PMID:3257578

Pseudomonas aeruginosa Possesses Two Putative Type I Signal Peptidases, LepB and PA1303, Each with Distinct Roles in Physiology and Virulence

PubMed Central

Rose, Ruth S.; Rangarajan, Minnie; Aduse-Opoku, Joseph; Hashim, Ahmed; Curtis, Michael A.

2012-01-01

Type I signal peptidases (SPases) cleave signal peptides from proteins during translocation across biological membranes and hence play a vital role in cellular physiology. SPase activity is also of fundamental importance to the pathogenesis of infection for many bacteria, including Pseudomonas aeruginosa, which utilizes a variety of secreted virulence factors, such as proteases and toxins. P. aeruginosa possesses two noncontiguous SPase homologues, LepB (PA0768) and PA1303, which share 43% amino acid identity. Reverse transcription (RT)-PCR showed that both proteases were expressed, while a FRET-based assay using a peptide based on the signal sequence cleavage region of the secreted LasB elastase showed that recombinant LepB and PA1303 enzymes were both active. LepB is positioned within a genetic locus that resembles the locus containing the extensively characterized SPase of E. coli and is of similar size and topology. It was also shown to be essential for viability and to have high sequence identity with SPases from other pseudomonads (≥78%). In contrast, PA1303, which is small for a Gram-negative SPase (20 kDa), was found to be dispensable. Mutation of PA1303 resulted in an altered protein secretion profile and increased N-butanoyl homoserine lactone production and influenced several quorum-sensing-controlled phenotypic traits, including swarming motility and the production of rhamnolipid and elastinolytic activity. The data indicate different cellular roles for these P. aeruginosa SPase paralogues; the role of PA1303 is integrated with the quorum-sensing cascade and includes the suppression of virulence factor secretion and virulence-associated phenotypes, while LepB is the primary SPase. PMID:22730125

DNA sequence similarity recognition by hybridization to short oligomers

DOEpatents

Milosavljevic, Aleksandar

1999-01-01

Methods are disclosed for the comparison of nucleic acid sequences. Data is generated by hybridizing sets of oligomers with target nucleic acids. The data thus generated is manipulated simultaneously with respect to both (i) matching between oligomers and (ii) matching between oligomers and putative reference sequences available in databases. Using data compression methods to manipulate this mutual information, sequences for the target can be constructed.

Prokaryotic Caspase Homologs: Phylogenetic Patterns and Functional Characteristics Reveal Considerable Diversity

PubMed Central

Asplund-Samuelsson, Johannes; Bergman, Birgitta; Larsson, John

2012-01-01

Caspases accomplish initiation and execution of apoptosis, a programmed cell death process specific to metazoans. The existence of prokaryotic caspase homologs, termed metacaspases, has been known for slightly more than a decade. Despite their potential connection to the evolution of programmed cell death in eukaryotes, the phylogenetic distribution and functions of these prokaryotic metacaspase sequences are largely uncharted, while a few experiments imply involvement in programmed cell death. Aiming at providing a more detailed picture of prokaryotic caspase homologs, we applied a computational approach based on Hidden Markov Model search profiles to identify and functionally characterize putative metacaspases in bacterial and archaeal genomes. Out of the total of 1463 analyzed genomes, merely 267 (18%) were identified to contain putative metacaspases, but their taxonomic distribution included most prokaryotic phyla and a few archaea (Euryarchaeota). Metacaspases were particularly abundant in Alphaproteobacteria, Deltaproteobacteria and Cyanobacteria, which harbor many morphologically and developmentally complex organisms, and a distinct correlation was found between abundance and phenotypic complexity in Cyanobacteria. Notably, Bacillus subtilis and Escherichia coli, known to undergo genetically regulated autolysis, lacked metacaspases. Pfam domain architecture analysis combined with operon identification revealed rich and varied configurations among the metacaspase sequences. These imply roles in programmed cell death, but also e.g. in signaling, various enzymatic activities and protein modification. Together our data show a wide and scattered distribution of caspase homologs in prokaryotes with structurally and functionally diverse sub-groups, and with a potentially intriguing evolutionary role. These features will help delineate future characterizations of death pathways in prokaryotes. PMID:23185476

Genome-wide identification of Hami melon miRNAs with putative roles during fruit development

PubMed Central

Wang, Guangzhi; Ma, Xinli; Li, Meihua; Wu, Haibo; Fu, Qiushi; Zhang, Yi; Yi, Hongping

2017-01-01

MicroRNAs represent a family of small endogenous, non-coding RNAs that play critical regulatory roles in plant growth, development, and environmental stress responses. Hami melon is famous for its attractive flavor and excellent nutritional value, however, the mechanisms underlying the fruit development and ripening remains largely unknown. Here, we performed small RNA sequencing to investigate the roles of miRNAs during Hami melon fruit development. Two batches of flesh samples were collected at four fruit development stages. Small RNA sequencing yielded a total of 54,553,424 raw reads from eight libraries. 113 conserved miRNAs belonging to 30 miRNA families and nine novel miRNAs comprising nine miRNA families were identified. The expression of 42 conserved miRNAs and three Hami melon-specific miRNAs significantly changed during fruit development. Furthermore, 484 and 124 melon genes were predicted as putative targets of 29 conserved and nine Hami melon-specific miRNA families, respectively. GO enrichment analysis were performed on target genes, “transcription, DNA-dependent”, “rRNA processing”, “oxidation reduction”, “signal transduction”, “regulation of transcription, DNA-dependent”, and “metabolic process” were the over-represented biological process terms. Cleavage sites of six target genes were validated using 5’ RACE. Our results present a comprehensive set of identification and characterization of Hami melon fruit miRNAs and their potential targets, which provide valuable basis towards understanding the regulatory mechanisms in programmed process of normal Hami fruit development and ripening. Specific miRNAs could be selected for further research and applications in breeding practices. PMID:28742088

DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains.

PubMed

Johnson, Timothy J; Siek, Kylie E; Johnson, Sara J; Nolan, Lisa K

2006-01-01

ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains.

DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains

PubMed Central

Johnson, Timothy J.; Siek, Kylie E.; Johnson, Sara J.; Nolan, Lisa K.

2006-01-01

ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains. PMID:16385064

Regulation of the alpha-glucuronidase-encoding gene ( aguA) from Aspergillus niger.

PubMed

de Vries, R P; van de Vondervoort, P J I; Hendriks, L; van de Belt, M; Visser, J

2002-09-01

The alpha-glucuronidase gene aguA from Aspergillus niger was cloned and characterised. Analysis of the promoter region of aguA revealed the presence of four putative binding sites for the major carbon catabolite repressor protein CREA and one putative binding site for the transcriptional activator XLNR. In addition, a sequence motif was detected which differed only in the last nucleotide from the XLNR consensus site. A construct in which part of the aguA coding region was deleted still resulted in production of a stable mRNA upon transformation of A. niger. The putative XLNR binding sites and two of the putative CREA binding sites were mutated individually in this construct and the effects on expression were examined in A. niger transformants. Northern analysis of the transformants revealed that the consensus XLNR site is not actually functional in the aguA promoter, whereas the sequence that diverges from the consensus at a single position is functional. This indicates that XLNR is also able to bind to the sequence GGCTAG, and the XLNR binding site consensus should therefore be changed to GGCTAR. Both CREA sites are functional, indicating that CREA has a strong influence on aguA expression. A detailed expression analysis of aguA in four genetic backgrounds revealed a second regulatory system involved in activation of aguA gene expression. This system responds to the presence of glucuronic and galacturonic acids, and is not dependent on XLNR.

Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia.

PubMed

Ficarelli, A; Tassi, F; Restivo, F M

1999-03-01

We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

Mitogen-activated protein kinase cascades in Vitis vinifera

PubMed Central

Çakır, Birsen; Kılıçkaya, Ozan

2015-01-01

Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

Excessive burden of lysosomal storage disorder gene variants in Parkinson's disease.

PubMed

Robak, Laurie A; Jansen, Iris E; van Rooij, Jeroen; Uitterlinden, André G; Kraaij, Robert; Jankovic, Joseph; Heutink, Peter; Shulman, Joshua M

2017-12-01

Mutations in the glucocerebrosidase gene (GBA), which cause Gaucher disease, are also potent risk factors for Parkinson's disease. We examined whether a genetic burden of variants in other lysosomal storage disorder genes is more broadly associated with Parkinson's disease susceptibility. The sequence kernel association test was used to interrogate variant burden among 54 lysosomal storage disorder genes, leveraging whole exome sequencing data from 1156 Parkinson's disease cases and 1679 control subjects. We discovered a significant burden of rare, likely damaging lysosomal storage disorder gene variants in association with Parkinson's disease risk. The association signal was robust to the exclusion of GBA, and consistent results were obtained in two independent replication cohorts, including 436 cases and 169 controls with whole exome sequencing and an additional 6713 cases and 5964 controls with exome-wide genotyping. In secondary analyses designed to highlight the specific genes driving the aggregate signal, we confirmed associations at the GBA and SMPD1 loci and newly implicate CTSD, SLC17A5, and ASAH1 as candidate Parkinson's disease susceptibility genes. In our discovery cohort, the majority of Parkinson's disease cases (56%) have at least one putative damaging variant in a lysosomal storage disorder gene, and 21% carry multiple alleles. Our results highlight several promising new susceptibility loci and reinforce the importance of lysosomal mechanisms in Parkinson's disease pathogenesis. We suggest that multiple genetic hits may act in combination to degrade lysosomal function, enhancing Parkinson's disease susceptibility. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Transcriptome Sequencing and Developmental Regulation of Gene Expression in Anopheles aquasalis

PubMed Central

Silva, Maria C. P.; Lopes, Adriana R.; Barros, Michele S.; Sá-Nunes, Anderson; Kojin, Bianca B.; Carvalho, Eneas; Suesdek, Lincoln; Silva-Neto, Mário Alberto C.; James, Anthony A.; Capurro, Margareth L.

2014-01-01

Background Anopheles aquasalis is a major malaria vector in coastal areas of South and Central America where it breeds preferentially in brackish water. This species is very susceptible to Plasmodium vivax and it has been already incriminated as responsible vector in malaria outbreaks. There has been no high-throughput investigation into the sequencing of An. aquasalis genes, transcripts and proteins despite its epidemiological relevance. Here we describe the sequencing, assembly and annotation of the An. aquasalis transcriptome. Methodology/Principal Findings A total of 419 thousand cDNA sequence reads, encompassing 164 million nucleotides, were assembled in 7544 contigs of ≥2 sequences, and 1999 singletons. The majority of the An. aquasalis transcripts encode proteins with their closest counterparts in another neotropical malaria vector, An. darlingi. Several analyses in different protein databases were used to annotate and predict the putative functions of the deduced An. aquasalis proteins. Larval and adult-specific transcripts were represented by 121 and 424 contig sequences, respectively. Fifty-one transcripts were only detected in blood-fed females. The data also reveal a list of transcripts up- or down-regulated in adult females after a blood meal. Transcripts associated with immunity, signaling networks and blood feeding and digestion are discussed. Conclusions/Significance This study represents the first large-scale effort to sequence the transcriptome of An. aquasalis. It provides valuable information that will facilitate studies on the biology of this species and may lead to novel strategies to reduce malaria transmission on the South American continent. The An. aquasalis transcriptome is accessible at http://exon.niaid.nih.gov/transcriptome/An_aquasalis/Anaquexcel.xlsx. PMID:25033462

Genome-Wide Analysis Reveals Novel Regulators of Growth in Drosophila melanogaster

PubMed Central

Vonesch, Sibylle Chantal; Lamparter, David; Mackay, Trudy F. C.; Bergmann, Sven; Hafen, Ernst

2016-01-01

Organismal size depends on the interplay between genetic and environmental factors. Genome-wide association (GWA) analyses in humans have implied many genes in the control of height but suffer from the inability to control the environment. Genetic analyses in Drosophila have identified conserved signaling pathways controlling size; however, how these pathways control phenotypic diversity is unclear. We performed GWA of size traits using the Drosophila Genetic Reference Panel of inbred, sequenced lines. We find that the top associated variants differ between traits and sexes; do not map to canonical growth pathway genes, but can be linked to these by epistasis analysis; and are enriched for genes and putative enhancers. Performing GWA on well-studied developmental traits under controlled conditions expands our understanding of developmental processes underlying phenotypic diversity. PMID:26751788

Dose- and time-dependent expression of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) in PCB-, B[a]P-, and TBT-exposed intertidal copepod Tigriopus japonicus.

PubMed

Kim, Bo-Mi; Rhee, Jae-Sung; Hwang, Un-Ki; Seo, Jung Soo; Shin, Kyung-Hoon; Lee, Jae-Seong

2015-02-01

The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon nuclear translocator (ARNT) genes from the copepod Tigriopus japonicus (Tj) were cloned to examine their potential functions in the invertebrate putative AhR-CYP signaling pathway. The amino acid sequences encoded by the Tj-AhR and Tj-ARNT genes showed high similarity to homologs of Daphnia and Drosophila, ranging from 68% and 70% similarity for the AhR genes to 56% for the ARNT genes. To determine whether Tj-AhR and Tj-ARNT are modulated by environmental pollutants, transcriptional expression of Tj-AhR and Tj-ARNT was analyzed in response to exposure to five concentrations of polychlorinated biphenyl (PCB 126) (control, 10, 50, 100, 500 μg L(-1)), benzo[a]pyrene (B[a]P) (control, 5, 10, 50, 100 μg L(-1)), and tributyltin (TBT) (control, 1, 5, 10, 20 μg L(-1)) 24h after exposure. A time-course experiment (0, 3, 6, 12, 24h) was performed to analyze mRNA expression patterns after exposure to PCB, B[a]P, and TBT. T. japonicus exhibited dose-dependent and time-dependent upregulation of Tj-AhR and Tj-ARNT in response to pollutant exposure, and the degree of expression was dependent on the pollutant, suggesting that pollutants such as PCB, B[a]P, and TBT modulate expression of Tj-AhR and Tj-ARNT genes in the putative AhR-CYP signaling pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

An insight into the sialotranscriptome of the seed-feeding bug, Oncopeltus fasciatus.

PubMed

Francischetti, Ivo M B; Lopes, Angela H; Dias, Felipe A; Pham, Van M; Ribeiro, José M C

2007-09-01

The salivary transcriptome of the seed-feeding hemipteran, Oncopeltus fasciatus (milkweed bug), is described following assembly of 1025 expressed sequence tags (ESTs) into 305 clusters of related sequences. Inspection of these sequences reveals abundance of low complexity, putative secreted products rich in the amino acids (aa) glycine, serine or threonine, which might function as silk or mucins and assist food canal lubrication and sealing of the feeding site around the mouthparts. Several protease inhibitors were found, including abundant expression of cystatin transcripts that may inhibit cysteine proteases common in seeds that might injure the insect or induce plant apoptosis. Serine proteases and lipases are described that might assist digestion and liquefaction of seed proteins and oils. Finally, several novel putative proteins are described with no known function that might affect plant physiology or act as antimicrobials.

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

PubMed

Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Transposon variation by order during allopolyploidisation between Brassica oleracea and Brassica rapa.

PubMed

An, Z; Tang, Z; Ma, B; Mason, A S; Guo, Y; Yin, J; Gao, C; Wei, L; Li, J; Fu, D

2014-07-01

Although many studies have shown that transposable element (TE) activation is induced by hybridisation and polyploidisation in plants, much less is known on how different types of TE respond to hybridisation, and the impact of TE-associated sequences on gene function. We investigated the frequency and regularity of putative transposon activation for different types of TE, and determined the impact of TE-associated sequence variation on the genome during allopolyploidisation. We designed different types of TE primers and adopted the Inter-Retrotransposon Amplified Polymorphism (IRAP) method to detect variation in TE-associated sequences during the process of allopolyploidisation between Brassica rapa (AA) and Brassica oleracea (CC), and in successive generations of self-pollinated progeny. In addition, fragments with TE insertions were used to perform Blast2GO analysis to characterise the putative functions of the fragments with TE insertions. Ninety-two primers amplifying 548 loci were used to detect variation in sequences associated with four different orders of TE sequences. TEs could be classed in ascending frequency into LTR-REs, TIRs, LINEs, SINEs and unknown TEs. The frequency of novel variation (putative activation) detected for the four orders of TEs was highest from the F1 to F2 generations, and lowest from the F2 to F3 generations. Functional annotation of sequences with TE insertions showed that genes with TE insertions were mainly involved in metabolic processes and binding, and preferentially functioned in organelles. TE variation in our study severely disturbed the genetic compositions of the different generations, resulting in inconsistencies in genetic clustering. Different types of TE showed different patterns of variation during the process of allopolyploidisation. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Multi-Virulence-Locus Sequence Typing of Staphylococcus lugdunensis Generates Results Consistent with a Clonal Population Structure and Is Reliable for Epidemiological Typing

PubMed Central

Didi, Jennifer; Lemée, Ludovic; Gibert, Laure; Pons, Jean-Louis

2014-01-01

Staphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlLR2, atlLR3, hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (dN/dS) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlLR2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the dN/dS ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlLR3, isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis. PMID:25078912

Identification and analysis of pig chimeric mRNAs using RNA sequencing data

PubMed Central

2012-01-01

Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs. PMID:22925561

Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis

USDA-ARS?s Scientific Manuscript database

Technical Abstract: Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered...

Identification and characterization of cell-specific enhancer elements for the mouse ETF/Tead2 gene.

PubMed

Tanoue, Y; Yasunami, M; Suzuki, K; Ohkubo, H

2001-12-21

We have identified and characterized by transient transfection assays the cell-specific 117-bp enhancer sequence in the first intron of the mouse ETF (Embryonic TEA domain-containing factor)/Tead2 gene required for transcriptional activation in ETF/Tead2 gene-expressing cells, such as P19 cells. The 117-bp enhancer contains one GC-rich sequence (5'-GGGGCGGGG-3'), termed the GC box, and two tandemly repeated GA-rich sequences (5'-GGGGGAGGGG-3'), termed the proximal and distal GA elements. Further analyses, including transfection studies and electrophoretic mobility shift assays using a series of deletion and mutation constructs, indicated that Sp1, a putative activator, may be required to predominate over its competition with another unknown putative repressor, termed the GA element-binding factor, for binding to both the GC box, which overlapped with the proximal GA element, and the distal GA element in the 117-bp sequence in order to achieve a full enhancer activity. We also discuss a possible mechanism underlying the cell-specific enhancer activity of the 117-bp sequence.

Application of Targeted Functional Assays to Assess a Putative Vascular Disruption Developmental Toxicity Pathway Informed By ToxCast High-Throughput Screening Data

EPA Science Inventory

Chemical perturbation of vascular development is a putative toxicity pathway which may result in developmental toxicity. EPA’s high-throughput screening (HTS) ToxCast program contains assays which measure cellular signals and biological processes critical for blood vessel develop...

Complete genome sequence of Enterobacter sp. IIT-BT 08: A potential microbial strain for high rate hydrogen production.

PubMed

Khanna, Namita; Ghosh, Ananta Kumar; Huntemann, Marcel; Deshpande, Shweta; Han, James; Chen, Amy; Kyrpides, Nikos; Mavrommatis, Kostas; Szeto, Ernest; Markowitz, Victor; Ivanova, Natalia; Pagani, Ioanna; Pati, Amrita; Pitluck, Sam; Nolan, Matt; Woyke, Tanja; Teshima, Hazuki; Chertkov, Olga; Daligault, Hajnalka; Davenport, Karen; Gu, Wei; Munk, Christine; Zhang, Xiaojing; Bruce, David; Detter, Chris; Xu, Yan; Quintana, Beverly; Reitenga, Krista; Kunde, Yulia; Green, Lance; Erkkila, Tracy; Han, Cliff; Brambilla, Evelyne-Marie; Lang, Elke; Klenk, Hans-Peter; Goodwin, Lynne; Chain, Patrick; Das, Debabrata

2013-12-20

Enterobacter sp. IIT-BT 08 belongs to Phylum: Proteobacteria, Class: Gammaproteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae. The organism was isolated from the leaves of a local plant near the Kharagpur railway station, Kharagpur, West Bengal, India. It has been extensively studied for fermentative hydrogen production because of its high hydrogen yield. For further enhancement of hydrogen production by strain development, complete genome sequence analysis was carried out. Sequence analysis revealed that the genome was linear, 4.67 Mbp long and had a GC content of 56.01%. The genome properties encode 4,393 protein-coding and 179 RNA genes. Additionally, a putative pathway of hydrogen production was suggested based on the presence of formate hydrogen lyase complex and other related genes identified in the genome. Thus, in the present study we describe the specific properties of the organism and the generation, annotation and analysis of its genome sequence as well as discuss the putative pathway of hydrogen production by this organism.

Molecular characterization of Helja, an extracellular jacalin-related protein from Helianthus annuus: Insights into the relationship of this protein with unconventionally secreted lectins.

PubMed

Pinedo, Marcela; Orts, Facundo; Carvalho, André de Oliveira; Regente, Mariana; Soares, Julia Ribeiro; Gomes, Valdirene Moreira; de la Canal, Laura

2015-07-01

Jacalin-related lectins (JRLs) encompass cytosolic, nuclear and vacuolar members displaying the jacalin domain in one or more copies or in combination with unrelated domains. Helianthus annuus jacalin (Helja) is a mannose-specific JRL previously identified in the apoplast of Helianthus annuus seedlings, and this protein has been proposed to follow unconventional secretion. Here, we describe the full-length Helja cDNA sequence, which presents a unique jacalin domain (merolectin) and the absence of a signal peptide, confirming that the protein cannot follow the classical ER-dependent secretory pathway. Helja mRNA is present in seeds, cotyledons, roots and hypocotyls, but no transcripts were detected in the leaves. Searches for sequence similarity showed that Helja is barely similar to other JRLs present in H. annuus databases and less than 45% identical to other monocot or dicot JRLs. Strikingly, most of the merolectins recovered through data mining using Helja as a query were predicted as apoplastic, although most of these proteins lack the signal peptide required for classical secretion. Thus, Helja is the first bait identified to recover putative unconventionally secreted lectins. Because the recovered JRLs are widely distributed among the plant kingdom, an as yet unknown role for jacalin lectins in the apoplast is emerging. Copyright © 2015 Elsevier GmbH. All rights reserved.

An Aspergillus oryzae acetyl xylan esterase: molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris.

PubMed

Koseki, Takuya; Miwa, Yozo; Akao, Takeshi; Akita, Osamu; Hashizume, Katsumi

2006-02-10

We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae (http://www.nrib.go.jp/ken/EST/db/index.html) and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused alpha-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.

Two novel genes, fanA and fanB, involved in the biogenesis of K99 fimbriae.

PubMed

Roosendaal, E; Boots, M; de Graaf, F K

1987-08-11

The nucleotide sequence of the region located transcriptionally upstream of the K99 fimbrial subunit gene (fanC) was determined. Several putative transcription signals and two open reading frames, designated fanA and fanB, became apparent. Frameshift mutations in fanA and fanB reduced K99 fimbriae expression 8-fold and 16-fold, respectively. Complementation of the mutants in trans restored the K99 expression to about 75% of the wild type level, indicating that fanA and fanB code for transacting polypeptides involved in the biogenesis of K99 fimbriae. The fanA and fanB gene products FanA and FanB were not detectable in minicell preparations, indicating that both polypeptides are synthesized in very small amounts. However, in an in vitro DNA directed translation system FanA and FanB could be identified. The deduced amino acid sequences of FanA and FanB showed that both polypeptides contain no signal peptides, indicating a cytoplasmic location. Furthermore, the polypeptides are very hydrophilic, mainly basic, and exhibit remarkable homology to each other and to a regulatory protein (papB) encoded by the pap-operon (1). Some of these features are characteristics of nucleic acid binding proteins, which suggests that FanA and FanB have a regulatory function in the synthesis of FanC and the auxiliary polypeptides FanD-H.

Experimental Evidence and In Silico Identification of Tryptophan Decarboxylase in Citrus Genus.

PubMed

De Masi, Luigi; Castaldo, Domenico; Pignone, Domenico; Servillo, Luigi; Facchiano, Angelo

2017-02-11

Plant tryptophan decarboxylase (TDC) converts tryptophan into tryptamine, precursor of indolealkylamine alkaloids. The recent finding of tryptamine metabolites in Citrus plants leads to hypothesize the existence of TDC activity in this genus. Here, we report for the first time that, in Citrus x limon seedlings, deuterium labeled tryptophan is decarboxylated into tryptamine, from which successively deuterated N , N , N -trimethyltryptamine is formed. These results give an evidence of the occurrence of the TDC activity and the successive methylation pathway of the tryptamine produced from the tryptophan decarboxylation. In addition, with the aim to identify the genetic basis for the presence of TDC, we carried out a sequence similarity search for TDC in the Citrus genomes using as a probe the TDC sequence reported for the plant Catharanthus roseus . We analyzed the genomes of both Citrus clementina and Citrus sinensis , available in public database, and identified putative protein sequences of aromatic l-amino acid decarboxylase. Similarly, 42 aromatic l-amino acid decarboxylase sequences from 23 plant species were extracted from public databases. Potential sequence signatures for functional TDC were then identified. With this research, we propose for the first time a putative protein sequence for TDC in the genus Citrus .

Honey bee (Apis mellifera) transferrin-gene structure and the role of ecdysteroids in the developmental regulation of its expression.

PubMed

do Nascimento, Adriana Mendes; Cuvillier-Hot, Virginie; Barchuk, Angel Roberto; Simões, Zilá Luz Paulino; Hartfelder, Klaus

2004-05-01

Social life is prone to invasion by microorganisms, and binding of ferric ions by transferrin is an efficient strategy to restrict their access to iron. In this study, we isolated cDNA and genomic clones encoding an Apis mellifera transferrin (AmTRF) gene. It has an open reading frame (ORF) of 2136 bp spread over nine exons. The deduced protein sequence comprises 686 amino acid residues plus a 26 residues signal sequence, giving a predicted molecular mass of 76 kDa. Comparison of the deduced AmTRF amino acid sequence with known insect transferrins revealed significant similarity extending over the entire sequence. It clusters with monoferric transferrins, with which it shares putative iron-binding residues in the N-terminal lobe. In a functional analysis of AmTRF expression in honey bee development, we monitored its expression profile in the larval and pupal stages. The negative regulation of AmTRF by ecdysteroids deduced from the developmental expression profile was confirmed by experimental treatment of spinning-stage honey bee larvae with 20-hydroxyecdysone, and of fourth instar-larvae with juvenile hormone. A juvenile hormone application to spinning-stage larvae, in contrast, had only a minor effect on AmTRF transcript levels. This is the first study implicating ecdysteroids in the developmental regulation of transferrin expression in an insect species.

Characterization and mapping of cDNA encoding aspartate aminotransferase in rice, Oryza sativa L.

PubMed

Song, J; Yamamoto, K; Shomura, A; Yano, M; Minobe, Y; Sasaki, T

1996-10-31

Fifteen cDNA clones, putatively identified as encoding aspartate aminotransferase (AST, EC 2.6.1.1.), were isolated and partially sequenced. Together with six previously isolated clones putatively identified to encode ASTs (Sasaki, et al. 1994, Plant Journal 6, 615-624), their sequences were characterized and classified into 4 cDNA species. Two of the isolated clones, C60213 and C2079, were full-length cDNAs, and their complete nucleotide sequences were determined. C60213 was 1612 bp long and its deduced amino acid sequence showed 88% homology with that of Panicum miliaceum L. mitochondrial AST. The C60213-encoded protein had an N-terminal amino acid sequence that was characteristic of a mitochondrial transit peptide. On the other hand, C2079 was 1546 bp long and had 91% amino acid sequence homology with P. miliaceum L. cytosolic AST but lacked in the transit peptide sequence. The homologies of nucleotide sequences and deduced amino acid sequences of C2079 and C60213 were 54% and 52%, respectively. C2079 and C60213 were mapped on chromosomes 1 and 6, respectively, by restriction fragment length polymorphism linkage analysis. Northern blot analysis using C2079 as a probe revealed much higher transcript levels in callus and root than in green and etiolated shoots, suggesting tissue-specific variations of AST gene expression.

Evolutionary profiles from the QR factorization of multiple sequence alignments

PubMed Central

Sethi, Anurag; O'Donoghue, Patrick; Luthey-Schulten, Zaida

2005-01-01

We present an algorithm to generate complete evolutionary profiles that represent the topology of the molecular phylogenetic tree of the homologous group. The method, based on the multidimensional QR factorization of numerically encoded multiple sequence alignments, removes redundancy from the alignments and orders the protein sequences by increasing linear dependence, resulting in the identification of a minimal basis set of sequences that spans the evolutionary space of the homologous group of proteins. We observe a general trend that these smaller, more evolutionarily balanced profiles have comparable and, in many cases, better performance in database searches than conventional profiles containing hundreds of sequences, constructed in an iterative and computationally intensive procedure. For more diverse families or superfamilies, with sequence identity <30%, structural alignments, based purely on the geometry of the protein structures, provide better alignments than pure sequence-based methods. Merging the structure and sequence information allows the construction of accurate profiles for distantly related groups. These structure-based profiles outperformed other sequence-based methods for finding distant homologs and were used to identify a putative class II cysteinyl-tRNA synthetase (CysRS) in several archaea that eluded previous annotation studies. Phylogenetic analysis showed the putative class II CysRSs to be a monophyletic group and homology modeling revealed a constellation of active site residues similar to that in the known class I CysRS. PMID:15741270

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

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.

Molecular cloning of trypsin cDNAs and trypsin gene expression in the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae).

PubMed

Johnson, S C; Ewart, K V; Osborne, J A; Delage, D; Ross, N W; Murray, H M

2002-09-01

The salmon louse, Lepeophtheirus salmonis, is a marine ectoparasitic copepod that infects salmonid fishes. We are studying the interactions between this parasite and its salmonid hosts, as it is a common cause of disease in both wild and farmed stocks of salmon. In this paper, we report on the cloning and sequencing of seven trypsin-like enzymes from a cDNA library prepared from whole body preadult female and male L. salmonis. The predicted trypsin activation peptides are 23 or 24 residues in length, considerably longer than previously reported activation peptides of other animals. Differences in the putative signal and activation peptide sequences of the trypsin isoforms suggest that these forms differ in their regulation and function. The calculated molecular weights of the trypsins range from 23.6 to 23.7 kDa. There are eight cysteine residues, which suggest the presence of four disulfide bridges. These trypsins are very similar (>or=46% aa identity) to other crustacean trypsins and insect hypodermins. Using in situ hybridization techniques trypsinogen expression could be identified in all three cell types of the midgut.

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.

Dynamic Redox Regulation of IL-4 Signaling.

PubMed

Dwivedi, Gaurav; Gran, Margaret A; Bagchi, Pritha; Kemp, Melissa L

2015-11-01

Quantifying the magnitude and dynamics of protein oxidation during cell signaling is technically challenging. Computational modeling provides tractable, quantitative methods to test hypotheses of redox mechanisms that may be simultaneously operative during signal transduction. The interleukin-4 (IL-4) pathway, which has previously been reported to induce reactive oxygen species and oxidation of PTP1B, may be controlled by several other putative mechanisms of redox regulation; widespread proteomic thiol oxidation observed via 2D redox differential gel electrophoresis upon IL-4 treatment suggests more than one redox-sensitive protein implicated in this pathway. Through computational modeling and a model selection strategy that relied on characteristic STAT6 phosphorylation dynamics of IL-4 signaling, we identified reversible protein tyrosine phosphatase (PTP) oxidation as the primary redox regulatory mechanism in the pathway. A systems-level model of IL-4 signaling was developed that integrates synchronous pan-PTP oxidation with ROS-independent mechanisms. The model quantitatively predicts the dynamics of IL-4 signaling over a broad range of new redox conditions, offers novel hypotheses about regulation of JAK/STAT signaling, and provides a framework for interrogating putative mechanisms involving receptor-initiated oxidation.

Dynamic Redox Regulation of IL-4 Signaling

PubMed Central

Dwivedi, Gaurav; Gran, Margaret A.; Bagchi, Pritha; Kemp, Melissa L.

2015-01-01

Quantifying the magnitude and dynamics of protein oxidation during cell signaling is technically challenging. Computational modeling provides tractable, quantitative methods to test hypotheses of redox mechanisms that may be simultaneously operative during signal transduction. The interleukin-4 (IL-4) pathway, which has previously been reported to induce reactive oxygen species and oxidation of PTP1B, may be controlled by several other putative mechanisms of redox regulation; widespread proteomic thiol oxidation observed via 2D redox differential gel electrophoresis upon IL-4 treatment suggests more than one redox-sensitive protein implicated in this pathway. Through computational modeling and a model selection strategy that relied on characteristic STAT6 phosphorylation dynamics of IL-4 signaling, we identified reversible protein tyrosine phosphatase (PTP) oxidation as the primary redox regulatory mechanism in the pathway. A systems-level model of IL-4 signaling was developed that integrates synchronous pan-PTP oxidation with ROS-independent mechanisms. The model quantitatively predicts the dynamics of IL-4 signaling over a broad range of new redox conditions, offers novel hypotheses about regulation of JAK/STAT signaling, and provides a framework for interrogating putative mechanisms involving receptor-initiated oxidation. PMID:26562652

Generation and Analysis of Expressed Sequence Tags from Olea europaea L.

PubMed Central

Ozdemir Ozgenturk, Nehir; Oruç, Fatma; Sezerman, Ugur; Kuçukural, Alper; Vural Korkut, Senay; Toksoz, Feriha; Un, Cemal

2010-01-01

Olive (Olea europaea L.) is an important source of edible oil which was originated in Near-East region. In this study, two cDNA libraries were constructed from young olive leaves and immature olive fruits for generation of ESTs to discover the novel genes and search the function of unknown genes of olive. The randomly selected 3840 colonies were sequenced for EST collection from both libraries. Readable 2228 sequences for olive leaf and 1506 sequences for olive fruit were assembled into 205 and 69 contigs, respectively, whereas 2478 were singletons. Putative functions of all 2752 differentially expressed unique sequences were designated by gene homology based on BLAST and annotated using BLAST2GO. While 1339 ESTs show no homology to the database, 2024 ESTs have homology (under 80%) with hypothetical proteins, putative proteins, expressed proteins, and unknown proteins in NCBI-GenBank. 635 EST's unique genes sequence have been identified by over 80% homology to known function in other species which were not previously described in Olea family. Only 3.1% of total EST's was shown similarity with olive database existing in NCBI. This generated EST's data and consensus sequences were submitted to NCBI as valuable source for functional genome studies of olive. PMID:21197085

Comprehensive analysis of CLE polypeptide signaling gene expression and over-expression activity in Arabidopsis

USDA-ARS?s Scientific Manuscript database

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis thaliana, only a few families of extracellular signaling molecules have been discovered and their biological roles are lar...

Genome mining of ascomycetous fungi reveals their genetic potential for ergot alkaloid production.

PubMed

Gerhards, Nina; Matuschek, Marco; Wallwey, Christiane; Li, Shu-Ming

2015-06-01

Ergot alkaloids are important as mycotoxins or as drugs. Naturally occurring ergot alkaloids as well as their semisynthetic derivatives have been used as pharmaceuticals in modern medicine for decades. We identified 196 putative ergot alkaloid biosynthetic genes belonging to at least 31 putative gene clusters in 31 fungal species by genome mining of the 360 available genome sequences of ascomycetous fungi with known proteins. Detailed analysis showed that these fungi belong to the families Aspergillaceae, Clavicipitaceae, Arthrodermataceae, Helotiaceae and Thermoascaceae. Within the identified families, only a small number of taxa are represented. Literature search revealed a large diversity of ergot alkaloid structures in different fungi of the phylum Ascomycota. However, ergot alkaloid accumulation was only observed in 15 of the sequenced species. Therefore, this study provides genetic basis for further study on ergot alkaloid production in the sequenced strains.

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.

Diversity of the P2 protein among nontypeable Haemophilus influenzae isolates.

PubMed Central

Bell, J; Grass, S; Jeanteur, D; Munson, R S

1994-01-01

The genes for outer membrane protein P2 of four nontypeable Haemophilus influenzae strains were cloned and sequenced. The derived amino acid sequences were compared with the outer membrane protein P2 sequence from H. influenzae type b MinnA and the sequences of P2 from three additional nontypeable H. influenzae strains. The sequences were 76 to 94% identical. The sequences had regions with considerable variability separated by regions which were highly conserved. The variable regions mapped to putative surface-exposed loops of the protein. PMID:8188390

Expressed sequence tags (ESTs) and phylogenetic analysis of floral genes from a paleoherb species, Asarum caudigerum.

PubMed

Zhao, Yinhe; Wang, Guoying; Zhang, Jinpeng; Yang, Junbo; Peng, Shang; Gao, Lianming; Li, Chengyun; Hu, Jinyong; Li, Dezhu; Gao, Lizhi

2006-07-01

Asarum caudigerum (Aristolochiaceae) is an important species of paleoherb in relation to understanding the origin and evolution of angiosperm flowers, due to its basal position in the angiosperms. The aim of this study was to isolate floral-related genes from A. caudigerum, and to infer evolutionary relationships among florally expression-related genes, to further illustrate the origin and diversification of flowers in angiosperms. A subtracted floral cDNA library was constructed from floral buds using suppression subtractive hybridization (SSH). The cDNA of floral buds and leaves at the seedling stage were used as a tester and a driver, respectively. To further identify the function of putative MADS-box transcription factors, phylogenetic trees were reconstructed in order to infer evolutionary relationships within the MADS-box gene family. In the forward-subtracted floral cDNA library, 1920 clones were randomly sequenced, from which 567 unique expressed sequence tags (ESTs) were obtained. Among them, 127 genes failed to show significant similarity to any published sequences in GenBank and thus are putatively novel genes. Phylogenetic analysis indicated that a total of 29 MADS-box transcription factors were members of the APETALA3(AP3) subfamily, while nine others were putative MADS-box transcription factors that formed a cluster with MADS-box genes isolated from Amborella, the basal-most angiosperm, and those from the gymnosperms. This suggests that the origin of A. caudigerum is intermediate between the angiosperms and gymnosperms.

Putative Monofunctional Type I Polyketide Synthase Units: A Dinoflagellate-Specific Feature?

PubMed Central

Eichholz, Karsten; Beszteri, Bánk; John, Uwe

2012-01-01

Marine dinoflagellates (alveolata) are microalgae of which some cause harmful algal blooms and produce a broad variety of most likely polyketide synthesis derived phycotoxins. Recently, novel polyketide synthesase (PKS) transcripts have been described from the Florida red tide dinoflagellate Karenia brevis (gymnodiniales) which are evolutionarily related to Type I PKS but were apparently expressed as monofunctional proteins, a feature typical of Type II PKS. Here, we investigated expression units of PKS I-like sequences in Alexandrium ostenfeldii (gonyaulacales) and Heterocapsa triquetra (peridiniales) at the transcript and protein level. The five full length transcripts we obtained were all characterized by polyadenylation, a 3′ UTR and the dinoflagellate specific spliced leader sequence at the 5′end. Each of the five transcripts encoded a single ketoacylsynthase (KS) domain showing high similarity to K. brevis KS sequences. The monofunctional structure was also confirmed using dinoflagellate specific KS antibodies in Western Blots. In a maximum likelihood phylogenetic analysis of KS domains from diverse PKSs, dinoflagellate KSs formed a clade placed well within the protist Type I PKS clade between apicomplexa, haptophytes and chlorophytes. These findings indicate that the atypical PKS I structure, i.e., expression as putative monofunctional units, might be a dinoflagellate specific feature. In addition, the sequenced transcripts harbored a previously unknown, apparently dinoflagellate specific conserved N-terminal domain. We discuss the implications of this novel region with regard to the putative monofunctional organization of Type I PKS in dinoflagellates. PMID:23139807

Discovery and Annotation of Plant Endogenous Target Mimicry Sequences from Public Transcriptome Libraries: A Case Study of Prunus persica.

PubMed

Karakülah, Gökhan

2017-06-28

Novel transcript discovery through RNA sequencing has substantially improved our understanding of the transcriptome dynamics of biological systems. Endogenous target mimicry (eTM) transcripts, a novel class of regulatory molecules, bind to their target microRNAs (miRNAs) by base pairing and block their biological activity. The objective of this study was to provide a computational analysis framework for the prediction of putative eTM sequences in plants, and as an example, to discover previously un-annotated eTMs in Prunus persica (peach) transcriptome. Therefore, two public peach transcriptome libraries downloaded from Sequence Read Archive (SRA) and a previously published set of long non-coding RNAs (lncRNAs) were investigated with multi-step analysis pipeline, and 44 putative eTMs were found. Additionally, an eTM-miRNA-mRNA regulatory network module associated with peach fruit organ development was built via integration of the miRNA target information and predicted eTM-miRNA interactions. My findings suggest that one of the most widely expressed miRNA families among diverse plant species, miR156, might be potentially sponged by seven putative eTMs. Besides, the study indicates eTMs potentially play roles in the regulation of development processes in peach fruit via targeting specific miRNAs. In conclusion, by following the step-by step instructions provided in this study, novel eTMs can be identified and annotated effectively in public plant transcriptome libraries.

Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response

PubMed Central

Sakurai, Tetsuya; Plata, Germán; Rodríguez-Zapata, Fausto; Seki, Motoaki; Salcedo, Andrés; Toyoda, Atsushi; Ishiwata, Atsushi; Tohme, Joe; Sakaki, Yoshiyuki; Shinozaki, Kazuo; Ishitani, Manabu

2007-01-01

Background Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs). Results The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. Conclusion The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome. PMID:18096061

Comparative Analyses of the β-Tubulin Gene and Molecular Modeling Reveal Molecular Insight into the Colchicine Resistance in Kinetoplastids Organisms

PubMed Central

Luis, Luis; Serrano, María Luisa; Hidalgo, Mariana; Mendoza-León, Alexis

2013-01-01

Differential susceptibility to microtubule agents has been demonstrated between mammalian cells and kinetoplastid organisms such as Leishmania spp. and Trypanosoma spp. The aims of this study were to identify and characterize the architecture of the putative colchicine binding site of Leishmania spp. and investigate the molecular basis of colchicine resistance. We cloned and sequenced the β-tubulin gene of Leishmania (Viannia) guyanensis and established the theoretical 3D model of the protein, using the crystallographic structure of the bovine protein as template. We identified mutations on the Leishmania   β-tubulin gene sequences on regions related to the putative colchicine-binding pocket, which generate amino acid substitutions and changes in the topology of this region, blocking the access of colchicine. The same mutations were found in the β-tubulin sequence of kinetoplastid organisms such as Trypanosoma cruzi, T. brucei, and T. evansi. Using molecular modelling approaches, we demonstrated that conformational changes include an elongation and torsion of an α-helix structure and displacement to the inside of the pocket of one β-sheet that hinders access of colchicine. We propose that kinetoplastid organisms show resistance to colchicine due to amino acids substitutions that generate structural changes in the putative colchicine-binding domain, which prevent colchicine access. PMID:24083244

Isolation of a polyphenol oxidase (PPO) cDNA from artichoke and expression analysis in wounded artichoke heads.

PubMed

Quarta, Angela; Mita, Giovanni; Durante, Miriana; Arlorio, Marco; De Paolis, Angelo

2013-07-01

The polyphenol oxidase (PPO) enzyme, which can catalyze the oxidation of phenolics to quinones, has been reported to be involved in undesirable browning in many plant foods. This phenomenon is particularly severe in artichoke heads wounded during the manufacturing process. A full-length cDNA encoding for a putative polyphenol oxidase (designated as CsPPO) along with a 1432 bp sequence upstream of the starting ATG codon was characterized for the first time from [Cynara cardunculus var. scolymus (L.) Fiori]. The 1764 bp CsPPO sequence encodes a putative protein of 587 amino acids with a calculated molecular mass of 65,327 Da and an isoelectric point of 5.50. Analysis of the promoter region revealed the presence of cis-acting elements, some of which are putatively involved in the response to light and wounds. Expression analysis of the gene in wounded capitula indicated that CsPPO was significantly induced after 48 h, even though the browning process had started earlier. This suggests that the early browning event observed in artichoke heads was not directly related to de novo mRNA synthesis. Finally, we provide the complete gene sequence encoding for polyphenol oxidase and the upstream regulative region in artichoke. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Temporal specificity of reward prediction errors signaled by putative dopamine neurons in rat VTA depends on ventral striatum

PubMed Central

Takahashi, Yuji K.; Langdon, Angela J.; Niv, Yael; Schoenbaum, Geoffrey

2016-01-01

Summary Dopamine neurons signal reward prediction errors. This requires accurate reward predictions. It has been suggested that the ventral striatum provides these predictions. Here we tested this hypothesis by recording from putative dopamine neurons in the VTA of rats performing a task in which prediction errors were induced by shifting reward timing or number. In controls, the neurons exhibited error signals in response to both manipulations. However, dopamine neurons in rats with ipsilateral ventral striatal lesions exhibited errors only to changes in number and failed to respond to changes in timing of reward. These results, supported by computational modeling, indicate that predictions about the temporal specificity and the number of expected rewards are dissociable, and that dopaminergic prediction-error signals rely on the ventral striatum for the former but not the latter. PMID:27292535

Cloning of Russian sturgeon (Acipenser gueldenstaedtii) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth.

PubMed

Yom Din, S; Hurvitz, A; Goldberg, D; Jackson, K; Levavi-Sivan, B; Degani, G

2008-03-01

In this study, the GH and IGF-I of the Russian sturgeon (rs), Acipenser gueldenstaedtii, were cloned and sequenced, and their mRNA gene expression determined. In addition, to improve our understanding of the GH function, the expression of this hormone was assessed in young males and females. Moreover, IGF-I expression was quantified in young males and compared to that in older ones. The nucleotide sequence of the rsGH cDNA was 980 bp long and had an open reading frame of 642 bp, beginning with the first ATG codon at position 39 and ending with the stop codon at position 683. A putative polyadenylation signal, AATAAA, was recognized 42 bp upstream of the poly (A) tail. The position of the signal- peptide cleavage site was predicted to be at position 111, yielding a signal peptide of 24 amino-acids (aa) and a mature peptide of 190 aa. When the rsGH aa sequence was compared with other species, the highest degree of identity was found to be with mammalians (66-70% identity), followed by anguilliformes and amphibia (61%) and other fish (39-47%). The level of rsGH mRNA was discovered to be similar in pituitaries of females and males of 5 age groups (1, 2, 3, 4, and 5- yr-old). In females and males, the levels did not change dramatically during the first 5 yr of growth. The partial nucleotide sequence of the rsIGF-I was 445 bp long and had an open reading frame of 396 bp, beginning with the ATG codon at position 50. The position of the signal-peptide cleavage site was predicted to be at position 187, yielding a signal peptide of 44 aa. The highest level of IGF-I mRNA expression was recorded in the kidney of adult sturgeons. The IGF-I mRNA expression levels in the intestine, pituitary gland, and liver were not significantly different. Low levels of expression were found in the brain, heart, and muscle. In most tissues, there was no significant difference between mRNA levels of one and 5-yr-old fish. In conclusion, based on the GH-sequence analysis, A. gueldenstaedtii is genetically distant from other teleosts. The expression of the GH mRNA was similar in males and females, and its level remained constant during the first 5 yr of growth. While the IGF-I mRNA expression differed amongst various tissues, the level in each tissue was similar in 1 and 5-yr-old fish.

Cloning, sequencing and characterization of lipase from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

USDA-ARS?s Scientific Manuscript database

Lipase gene (lip) of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing bacterium P. resinovorans NRRL B-2649 was cloned, sequenced and characterized by using consensus primers and PCR-based genome walking method. The ORF of the putative Lip (314 amino acids) and its active site (Ser111, Asp...

Neuropeptidomics of the Mosquito Aedes Aegypti

DTIC Science & Technology

2010-01-01

translational processing ( pyroglutamate formation) was detected for AST-C and CAPA-PVK-2. For the first time in insects, we succeeded in the direct...hormones, trace DNA sequences generated by TIGR and the Broad Institute were first searched by TBLASTN24 using amino acid sequences of candidate peptides...previously described.1 TBLASTN searches, using the amino acid sequences of putative Ae. aegypti neuropeptide and peptide hormone orthologs identified in

Draft genome sequence of Therminicola potens strain JR

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

Byrne-Bailey, K.G.; Wrighton, K.C.; Melnyk, R.A.

'Thermincola potens' strain JR is one of the first Gram-positive dissimilatory metal-reducing bacteria (DMRB) for which there is a complete genome sequence. Consistent with the physiology of this organism, preliminary annotation revealed an abundance of multiheme c-type cytochromes that are putatively associated with the periplasm and cell surface in a Gram-positive bacterium. Here we report the complete genome sequence of strain JR.

Complete Genome Sequence of a Putative New Bacterial Strain, I507, Isolated from the Indian Ocean

PubMed Central

Wang, Shu-yan; Wei, Jia-qiang

2018-01-01

ABSTRACT Bacterial strain I507 was isolated from the central Indian Ocean and may be a potential novel species, according to the 16S rRNA gene sequence. Here, we present its complete genome sequence and expect that it will provide researchers with valuable information to further understand its classification and function in the future. PMID:29674539

Identification and expression analysis of a putative fatty acidbinding protein gene in the Asian honeybee, Apis cerana cerana.

PubMed

Yu, Xiaoli; Kang, Mingjiang; Liu, Li; Guo, Xingqi; Xu, Baohua

2013-01-01

Fatty acid-binding proteins (FABPs) play pivotal roles in cellular signaling, gene transcription, and lipid metabolism in vertebrates and invertebrates. In this study, a putative FABP gene, referred to as AccFABP, was isolated from the Asian honeybee, Apis cerana cerana Fabricius (Hymenoptera: Apidae). The full-length cDNA consisted of 725 bp, and encoded a protein of 204 amino acids. Homology and phylogenetic analysis indicated that AccFABP was a member of the FABP multifamily. The genomic structure of this gene, which was common among FABP multifamily members, spanned 1,900 bp, and included four exons and three introns. Gene expression analysis revealed that AccFABP was highly expressed in the dark-pigmented phase of pupal development, with peak expression observed in the fat bodies of the dark-pigmented phase pupae. The AccFABP transcripts in the fat body were upregulated by exposure to dietary fatty acids such as conjugated linoleic acid, docosahexaenoic acid, and arachidonic acid. Transcription factor binding sites for Caudal-Related Homeobox and functional CCAAT/enhancer binding site, which were respectively associated with tissue expression and lipid metabolism, were detected in the 5' promoter sequence. The evidence provided in the present study suggests that AccFABP may regulate insect growth and development, and lipid metabolism.

Gamarada debralockiae gen. nov. sp. nov.-the genome of the most widespread Australian ericoid mycorrhizal fungus.

PubMed

Midgley, David J; Sutcliffe, Brodie; Greenfield, Paul; Tran-Dinh, Nai

2018-05-01

This study describes a novel ericoid mycorrhizal fungus (ErMF), Gamarada debralockiae Midgley and Tran-Dinh gen. nov. sp. nov. Additionally, catabolism was explored from a genomic perspective. The nuclear and mitochondrial genomes of G. debralockiae were sequenced. Morphological characteristics were assessed on various media. Catabolic genes of G. debralockiae were explored using SignalP and dbCAN. Phylogenetic comparisons were undertaken using Phylogeny.fr. The 58.5-Mbp draft genome of G. debralockiae contained 17,075 putative genes. The complete mitochondrial genome was 28,168 bp in length. In culture, G. debralockiae produces slow-growing non-sporulating colonies. Gamarada debralockiae has many putative secreted catabolic enzymes. Phylogeny indicated G. debralockiae was distinct from known ascomycetous ErMF: Pezoloma ericae, Meliniomyces spp., Oidiodendron spp., and Cairneyella variabilis. It is closely related to many undescribed plant root-associated fungi and its nearest described relative is Hyphodiscus brevicollaris. Gamarada debralockiae has been recovered from virtually all Australian ericoid mycorrhizal studies and biogeographic data suggests the taxon is widespread in Australia. Gamarada debralockiae has similar catabolic potential to C. variabilis and co-occurs with C. variabilis at Australian sites. Plants that host multiple ErMF may benefit from subtle differences in catabolism that improve access to nitrogen and phosphorus from within recalcitrant organic matter.

RNA-Seq Analysis of Human Trigeminal and Dorsal Root Ganglia with a Focus on Chemoreceptors

PubMed Central

Flegel, Caroline; Schöbel, Nicole; Altmüller, Janine; Becker, Christian; Tannapfel, Andrea; Hatt, Hanns; Gisselmann, Günter

2015-01-01

The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq) to conduct the first expression analysis of human trigeminal ganglia (TG) and dorsal root ganglia (DRG). We analyzed the data with a focus on G-protein coupled receptors (GPCRs) and ion channels, which are (potentially) involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP) channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs) with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues. PMID:26070209

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

Transcriptional Profiles of Mating-Responsive Genes from Testes and Male Accessory Glands of the Mediterranean Fruit Fly, Ceratitis capitata

PubMed Central

Scolari, Francesca; Gomulski, Ludvik M.; Ribeiro, José M. C.; Siciliano, Paolo; Meraldi, Alice; Falchetto, Marco; Bonomi, Angelica; Manni, Mosè; Gabrieli, Paolo; Malovini, Alberto; Bellazzi, Riccardo; Aksoy, Serap; Gasperi, Giuliano; Malacrida, Anna R.

2012-01-01

Background Insect seminal fluid is a complex mixture of proteins, carbohydrates and lipids, produced in the male reproductive tract. This seminal fluid is transferred together with the spermatozoa during mating and induces post-mating changes in the female. Molecular characterization of seminal fluid proteins in the Mediterranean fruit fly, Ceratitis capitata, is limited, although studies suggest that some of these proteins are biologically active. Methodology/Principal Findings We report on the functional annotation of 5914 high quality expressed sequence tags (ESTs) from the testes and male accessory glands, to identify transcripts encoding putative secreted peptides that might elicit post-mating responses in females. The ESTs were assembled into 3344 contigs, of which over 33% produced no hits against the nr database, and thus may represent novel or rapidly evolving sequences. Extraction of the coding sequences resulted in a total of 3371 putative peptides. The annotated dataset is available as a hyperlinked spreadsheet. Four hundred peptides were identified with putative secretory activity, including odorant binding proteins, protease inhibitor domain-containing peptides, antigen 5 proteins, mucins, and immunity-related sequences. Quantitative RT-PCR-based analyses of a subset of putative secretory protein-encoding transcripts from accessory glands indicated changes in their abundance after one or more copulations when compared to virgin males of the same age. These changes in abundance, particularly evident after the third mating, may be related to the requirement to replenish proteins to be transferred to the female. Conclusions/Significance We have developed the first large-scale dataset for novel studies on functions and processes associated with the reproductive biology of Ceratitis capitata. The identified genes may help study genome evolution, in light of the high adaptive potential of the medfly. In addition, studies of male recovery dynamics in terms of accessory gland gene expression profiles and correlated remating inhibition mechanisms may permit the improvement of pest management approaches. PMID:23071645

The prediction of a pathogenesis-related secretome of Puccinia helianthi through high-throughput transcriptome analysis.

PubMed

Jing, Lan; Guo, Dandan; Hu, Wenjie; Niu, Xiaofan

2017-03-11

Many plant pathogen secretory proteins are known to be elicitors or pathogenic factors,which play an important role in the host-pathogen interaction process. Bioinformatics approaches make possible the large scale prediction and analysis of secretory proteins from the Puccinia helianthi transcriptome. The internet-based software SignalP v4.1, TargetP v1.01, Big-PI predictor, TMHMM v2.0 and ProtComp v9.0 were utilized to predict the signal peptides and the signal peptide-dependent secreted proteins among the 35,286 ORFs of the P. helianthi transcriptome. 908 ORFs (accounting for 2.6% of the total proteins) were identified as putative secretory proteins containing signal peptides. The length of the majority of proteins ranged from 51 to 300 amino acids (aa), while the signal peptides were from 18 to 20 aa long. Signal peptidase I (SpI) cleavage sites were found in 463 of these putative secretory signal peptides. 55 proteins contained the lipoprotein signal peptide recognition site of signal peptidase II (SpII). Out of 908 secretory proteins, 581 (63.8%) have functions related to signal recognition and transduction, metabolism, transport and catabolism. Additionally, 143 putative secretory proteins were categorized into 27 functional groups based on Gene Ontology terms, including 14 groups in biological process, seven in cellular component, and six in molecular function. Gene ontology analysis of the secretory proteins revealed an enrichment of hydrolase activity. Pathway associations were established for 82 (9.0%) secretory proteins. A number of cell wall degrading enzymes and three homologous proteins specific to Phytophthora sojae effectors were also identified, which may be involved in the pathogenicity of the sunflower rust pathogen. This investigation proposes a new approach for identifying elicitors and pathogenic factors. The eventual identification and characterization of 908 extracellularly secreted proteins will advance our understanding of the molecular mechanisms of interactions between sunflower and rust pathogen and will enhance our ability to intervene in disease states.

Clustering of Genetically Defined Allele Classes in the Caenorhabditis elegans DAF-2 Insulin/IGF-1 Receptor

PubMed Central

Patel, Dhaval S.; Garza-Garcia, Acely; Nanji, Manoj; McElwee, Joshua J.; Ackerman, Daniel; Driscoll, Paul C.; Gems, David

2008-01-01

The DAF-2 insulin/IGF-1 receptor regulates development, metabolism, and aging in the nematode Caenorhabditis elegans. However, complex differences among daf-2 alleles complicate analysis of this gene. We have employed epistasis analysis, transcript profile analysis, mutant sequence analysis, and homology modeling of mutant receptors to understand this complexity. We define an allelic series of nonconditional daf-2 mutants, including nonsense and deletion alleles, and a putative null allele, m65. The most severe daf-2 alleles show incomplete suppression by daf-18(0) and daf-16(0) and have a range of effects on early development. Among weaker daf-2 alleles there exist distinct mutant classes that differ in epistatic interactions with mutations in other genes. Mutant sequence analysis (including 11 newly sequenced alleles) reveals that class 1 mutant lesions lie only in certain extracellular regions of the receptor, while class 2 (pleiotropic) and nonconditional missense mutants have lesions only in the ligand-binding pocket of the receptor ectodomain or the tyrosine kinase domain. Effects of equivalent mutations on the human insulin receptor suggest an altered balance of intracellular signaling in class 2 alleles. These studies consolidate and extend our understanding of the complex genetics of daf-2 and its underlying molecular biology. PMID:18245374

THE GENOMIC LANDSCAPE OF PEDIATRIC AND YOUNG ADULT T-LINEAGE ACUTE LYMPHOBLASTIC LEUKEMIA

PubMed Central

Liu, Yu; Easton, John; Shao, Ying; Maciaszek, Jamie; Wang, Zhaoming; Wilkinson, Mark R.; McCastlain, Kelly; Edmonson, Michael; Pounds, Stanley B.; Shi, Lei; Zhou, Xin; Ma, Xiaotu; Sioson, Edgar; Li, Yongjin; Rusch, Michael; Gupta, Pankaj; Pei, Deqing; Cheng, Cheng; Smith, Malcolm A.; Auvil, Jaime Guidry; Gerhard, Daniela S.; Relling, Mary V.; Winick, Naomi J.; Carroll, Andrew J.; Heerema, Nyla A.; Raetz, Elizabeth; Devidas, Meenakshi; Willman, Cheryl L.; Harvey, Richard C.; Carroll, William L.; Dunsmore, Kimberly P.; Winter, Stuart S.; Wood, Brent L; Sorrentino, Brian P.; Downing, James R.; Loh, Mignon L.; Hunger, Stephen P; Zhang, Jinghui; Mullighan, Charles G.

2017-01-01

Genetic alterations activating NOTCH1 signaling and T cell transcription factors, coupled with inactivation of the INK4/ARF tumor suppressors are hallmarks of T-ALL, but detailed genome-wide sequencing of large T-ALL cohorts has not been performed. Using integrated genomic analysis of 264 T-ALL cases, we identify 106 putative driver genes, half of which were not previously described in childhood T-ALL (e.g. CCND3, CTCF, MYB, SMARCA4, ZFP36L2 and MYCN). We described new mechanisms of coding and non-coding alteration, and identify 10 recurrently altered pathways, with associations between mutated genes and pathways, and stage or subtype of T-ALL. For example, NRAS/FLT3 mutations were associated with immature T-ALL, JAK3/STAT5B mutations in HOX1 deregulated ALL, PTPN2 mutations in TLX1 T-ALL, and PIK3R1/PTEN mutations in TAL1 ALL, suggesting that different signaling pathways have distinct roles according to maturational stage. This genomic landscape provides a logical framework for the development of faithful genetic models and new therapeutic approaches. PMID:28671688

Genome-Wide Identification of Medicago Peptides Involved in Macronutrient Responses and Nodulation1[OPEN

PubMed Central

Dai, Xinbin; Zhuang, Zhaohong; Torres-Jerez, Ivone; Nogales, Joaquina

2017-01-01

Growing evidence indicates that small, secreted peptides (SSPs) play critical roles in legume growth and development, yet the annotation of SSP-coding genes is far from complete. Systematic reannotation of the Medicago truncatula genome identified 1,970 homologs of established SSP gene families and an additional 2,455 genes that are potentially novel SSPs, previously unreported in the literature. The expression patterns of known and putative SSP genes based on 144 RNA sequencing data sets covering various stages of macronutrient deficiencies and symbiotic interactions with rhizobia and mycorrhiza were investigated. Focusing on those known or suspected to act via receptor-mediated signaling, 240 nutrient-responsive and 365 nodulation-responsive Signaling-SSPs were identified, greatly expanding the number of SSP gene families potentially involved in acclimation to nutrient deficiencies and nodulation. Synthetic peptide applications were shown to alter root growth and nodulation phenotypes, revealing additional regulators of legume nutrient acquisition. Our results constitute a powerful resource enabling further investigations of specific SSP functions via peptide treatment and reverse genetics. PMID:29030416

Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans

PubMed Central

2004-01-01

Numerous invertebrate species belonging to several phyla cannot synthesize sterols de novo and rely on a dietary source of the compound. SCPx (sterol carrier protein 2/3-oxoacyl-CoA thiolase) is a protein involved in the trafficking of sterols and oxidation of branched-chain fatty acids. We have isolated SCPx protein from Spodoptera littoralis (cotton leafworm) and have subjected it to limited amino acid sequencing. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.9 kb), which encodes a 57 kDa protein. Northern blotting detected two mRNA transcripts, one of 1.9 kb, encoding SCPx, and one of 0.95 kb, presumably encoding SCP2 (sterol carrier protein 2). The former mRNA was highly expressed in midgut and Malpighian tubules during the last larval instar. Furthermore, constitutive expression of the gene was detected in the prothoracic glands, which are the main tissue producing the insect moulting hormone. There was no significant change in the 1.9 kb mRNA in midgut throughout development, but slightly higher expression in the early stages. Conceptual translation of the cDNA and a database search revealed that the gene includes the SCP2 sequence and a putative peroxisomal targeting signal in the C-terminal region. Also a cysteine residue at the putative active site for the 3-oxoacyl-CoA thiolase is conserved. Southern blotting showed that SCPx is likely to be encoded by a single-copy gene. The mRNA expression pattern and the gene structure suggest that SCPx from S. littoralis (a lepidopteran) is evolutionarily closer to that of mammals than to that of dipterans. PMID:15149283

Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115.

PubMed

Oh, Ji-eun; Karlmark, Karlin Raja; Shin, Jooho; Hengstschläger, Markus; Lubec, Gert

2006-05-15

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND. (c) 2006 Wiley-Liss, Inc.

High-Throughput Sequencing Reveals Hypothalamic MicroRNAs as Novel Partners Involved in Timing the Rapid Development of Chicken (Gallus gallus) Gonads.

PubMed

Han, Wei; Zou, Jianmin; Wang, Kehua; Su, Yijun; Zhu, Yunfen; Song, Chi; Li, Guohui; Qu, Liang; Zhang, Huiyong; Liu, Honglin

2015-01-01

Onset of the rapid gonad growth is a milestone in sexual development that comprises many genes and regulatory factors. The observations in model organisms and mammals including humans have shown a potential link between miRNAs and development timing. To determine whether miRNAs play roles in this process in the chicken (Gallus gallus), the Solexa deep sequencing was performed to analyze the profiles of miRNA expression in the hypothalamus of hens from two different pubertal stages, before onset of the rapid gonad development (BO) and after onset of the rapid gonad development (AO). 374 conserved and 46 novel miRNAs were identified as hypothalamus-expressed miRNAs in the chicken. 144 conserved miRNAs were showed to be differentially expressed (reads > 10, P < 0.05) during the transition from BO to AO. Five differentially expressed miRNAs were validated by real-time quantitative RT-PCR (qRT-PCR) method. 2013 putative genes were predicted as the targets of the 15 most differentially expressed miRNAs (fold-change > 4.0, P < 0.01). Of these genes, 7 putative circadian clock genes, Per2, Bmal1/2, Clock, Cry1/2, and Star were found to be targeted multiple times by the miRNAs. qRT-PCR revealed the basic transcription levels of these clock genes were much higher (P < 0.01) in AO than in BO. Further functional analysis suggested that these 15 miRNAs play important roles in transcriptional regulation and signal transduction pathways. The results provide new insights into miRNAs functions in timing the rapid development of chicken gonads. Considering the characteristics of miRNA functional conservation, the results will contribute to the research on puberty onset in humans.

XET Activity is Found Near Sites of Growth and Cell Elongation in Bryophytes and Some Green Algae: New Insights into the Evolution of Primary Cell Wall Elongation

PubMed Central

Van Sandt, Vicky S. T.; Stieperaere, Herman; Guisez, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

2007-01-01

Background and Aims In angiosperms xyloglucan endotransglucosylase (XET)/hydrolase (XTH) is involved in reorganization of the cell wall during growth and development. The location of oligo-xyloglucan transglucosylation activity and the presence of XTH expressed sequence tags (ESTs) in the earliest diverging extant plants, i.e. in bryophytes and algae, down to the Phaeophyta was examined. The results provide information on the presence of an XET growth mechanism in bryophytes and algae and contribute to the understanding of the evolution of cell wall elongation in general. Methods Representatives of the different plant lineages were pressed onto an XET test paper and assayed. XET or XET-related activity was visualized as the incorporation of fluorescent signal. The Physcomitrella genome database was screened for the presence of XTHs. In addition, using the 3′ RACE technique searches were made for the presence of possible XTH ESTs in the Charophyta. Key Results XET activity was found in the three major divisions of bryophytes at sites corresponding to growing regions. In the Physcomitrella genome two putative XTH-encoding cDNA sequences were identified that contain all domains crucial for XET activity. Furthermore, XET activity was located at the sites of growth in Chara (Charophyta) and Ulva (Chlorophyta) and a putative XTH ancestral enzyme in Chara was identified. No XET activity was identified in the Rhodophyta or Phaeophyta. Conclusions XET activity was shown to be present in all major groups of green plants. These data suggest that an XET-related growth mechanism originated before the evolutionary divergence of the Chlorobionta and open new insights in the evolution of the mechanisms of primary cell wall expansion. PMID:17098750

ESTs Analysis Reveals Putative Genes Involved in Symbiotic Seed Germination in Dendrobium officinale

PubMed Central

Zhao, Ming-Ming; Zhang, Gang; Zhang, Da-Wei; Hsiao, Yu-Yun; Guo, Shun-Xing

2013-01-01

Dendrobium officinale (Orchidaceae) is one of the world’s most endangered plants with great medicinal value. In nature, D . officinale seeds must establish symbiotic relationships with fungi to germinate. However, the molecular events involved in the interaction between fungus and plant during this process are poorly understood. To isolate the genes involved in symbiotic germination, a suppression subtractive hybridization (SSH) cDNA library of symbiotically germinated D . officinale seeds was constructed. From this library, 1437 expressed sequence tags (ESTs) were clustered to 1074 Unigenes (including 902 singletons and 172 contigs), which were searched against the NCBI non-redundant (NR) protein database (E-value cutoff, e-5). Based on sequence similarity with known proteins, 579 differentially expressed genes in D . officinale were identified and classified into different functional categories by Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression levels of 15 selected genes emblematic of symbiotic germination were confirmed via real-time quantitative PCR. These genes were classified into various categories, including defense and stress response, metabolism, transcriptional regulation, transport process and signal transduction pathways. All transcripts were upregulated in the symbiotically germinated seeds (SGS). The functions of these genes in symbiotic germination were predicted. Furthermore, two fungus-induced calcium-dependent protein kinases (CDPKs), which were upregulated 6.76- and 26.69-fold in SGS compared with un-germinated seeds (UGS), were cloned from D . officinale and characterized for the first time. This study provides the first global overview of genes putatively involved in D . officinale symbiotic seed germination and provides a foundation for further functional research regarding symbiotic relationships in orchids. PMID:23967335

ESTs analysis reveals putative genes involved in symbiotic seed germination in Dendrobium officinale.

PubMed

Zhao, Ming-Ming; Zhang, Gang; Zhang, Da-Wei; Hsiao, Yu-Yun; Guo, Shun-Xing

2013-01-01

Dendrobiumofficinale (Orchidaceae) is one of the world's most endangered plants with great medicinal value. In nature, D. officinale seeds must establish symbiotic relationships with fungi to germinate. However, the molecular events involved in the interaction between fungus and plant during this process are poorly understood. To isolate the genes involved in symbiotic germination, a suppression subtractive hybridization (SSH) cDNA library of symbiotically germinated D. officinale seeds was constructed. From this library, 1437 expressed sequence tags (ESTs) were clustered to 1074 Unigenes (including 902 singletons and 172 contigs), which were searched against the NCBI non-redundant (NR) protein database (E-value cutoff, e(-5)). Based on sequence similarity with known proteins, 579 differentially expressed genes in D. officinale were identified and classified into different functional categories by Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression levels of 15 selected genes emblematic of symbiotic germination were confirmed via real-time quantitative PCR. These genes were classified into various categories, including defense and stress response, metabolism, transcriptional regulation, transport process and signal transduction pathways. All transcripts were upregulated in the symbiotically germinated seeds (SGS). The functions of these genes in symbiotic germination were predicted. Furthermore, two fungus-induced calcium-dependent protein kinases (CDPKs), which were upregulated 6.76- and 26.69-fold in SGS compared with un-germinated seeds (UGS), were cloned from D. officinale and characterized for the first time. This study provides the first global overview of genes putatively involved in D. officinale symbiotic seed germination and provides a foundation for further functional research regarding symbiotic relationships in orchids.

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.

Previously unknown and highly divergent ssDNA viruses populate the oceans.

PubMed

Labonté, Jessica M; Suttle, Curtis A

2013-11-01

Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton.

Characterization of a restriction modification system from the commensal Escherichia coli strain A0 34/86 (O83:K24:H31).

PubMed

Weiserová, Marie; Ryu, Junichi

2008-06-27

Type I restriction-modification (R-M) systems are the most complex restriction enzymes discovered to date. Recent years have witnessed a renaissance of interest in R-M enzymes Type I. The massive ongoing sequencing programmes leading to discovery of, so far, more than 1 000 putative enzymes in a broad range of microorganisms including pathogenic bacteria, revealed that these enzymes are widely represented in nature. The aim of this study was characterisation of a putative R-M system EcoA0ORF42P identified in the commensal Escherichia coli A0 34/86 (O83: K24: H31) strain, which is efficiently used at Czech paediatric clinics for prophylaxis and treatment of nosocomial infections and diarrhoea of preterm and newborn infants. We have characterised a restriction-modification system EcoA0ORF42P of the commensal Escherichia coli strain A0 34/86 (O83: K24: H31). This system, designated as EcoAO83I, is a new functional member of the Type IB family, whose specificity differs from those of known Type IB enzymes, as was demonstrated by an immunological cross-reactivity and a complementation assay. Using the plasmid transformation method and the RM search computer program, we identified the DNA recognition sequence of the EcoAO83I as GGA(8N)ATGC. In consistence with the amino acids alignment data, the 3' TRD component of the recognition sequence is identical to the sequence recognized by the EcoEI enzyme. The A-T (modified adenine) distance is identical to that in the EcoAI and EcoEI recognition sites, which also indicates that this system is a Type IB member. Interestingly, the recognition sequence we determined here is identical to the previously reported prototype sequence for Eco377I and its isoschizomers. Putative restriction-modification system EcoA0ORF42P in the commensal Escherichia coli strain A0 34/86 (O83: K24: H31) was found to be a member of the Type IB family and was designated as EcoAO83I. Combination of the classical biochemical and bacterial genetics approaches with comparative genomics might contribute effectively to further classification of many other putative Type-I enzymes, especially in clinical samples.

In situ localization and tissue distribution of ostreid herpesvirus 1 proteins in infected Pacific oyster, Crassostrea gigas.

PubMed

Martenot, Claire; Segarra, Amélie; Baillon, Laury; Faury, Nicole; Houssin, Maryline; Renault, Tristan

2016-05-01

Immunohistochemistry (IHC) assays were conducted on paraffin sections from experimentally infected spat and unchallenged spat produced in hatchery to determine the tissue distribution of three viral proteins within the Pacific oyster, Crassostrea gigas. Polyclonal antibodies were produced from recombinant proteins corresponding to two putative membrane proteins and one putative apoptosis inhibitor encoded by ORF 25, 72, and 87, respectively. Results were then compared to those obtained by in situ hybridization performed on the same individuals, and showed a substantial agreement according to Landis and Koch numeric scale. Positive signals were mainly observed in connective tissue of gills, mantle, adductor muscle, heart, digestive gland, labial palps, and gonads of infected spat. Positive signals were also reported in digestive epithelia. However, few positive signals were also observed in healthy appearing oysters (unchallenged spat) and could be due to virus persistence after a primary infection. Cellular localization of staining seemed to be linked to the function of the viral protein targeted. A nucleus staining was preferentially observed with antibodies targeting the putative apoptosis inhibitor protein whereas a cytoplasmic localization was obtained using antibodies recognizing putative membrane proteins. The detection of viral proteins was often associated with histopathological changes previously reported during OsHV-1 infection by histology and transmission electron microscopy. Within the 6h after viral suspension injection, positive signals were almost at the maximal level with the three antibodies and all studied organs appeared infected at 28h post viral injection. Connective tissue appeared to be a privileged site for OsHV-1 replication even if positive signals were observed in the epithelium cells of different organs which may be interpreted as a hypothetical portal of entry or release for the virus. IHC constitutes a suited method for analyzing the early infection stages of OsHV-1 infection and a useful tool to investigate interactions between OsHV-1 and its host at a protein level. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis, transport and regulation.

PubMed

Sun, Haiyue; Liu, Yushan; Gai, Yuzhuo; Geng, Jinman; Chen, Li; Liu, Hongdi; Kang, Limin; Tian, Youwen; Li, Yadong

2015-09-02

Cranberries (Vaccinium macrocarpon Ait.), renowned for their excellent health benefits, are an important berry crop. Here, we performed transcriptome sequencing of one cranberry cultivar, from fruits at two different developmental stages, on the Illumina HiSeq 2000 platform. Our main goals were to identify putative genes for major metabolic pathways of bioactive compounds and compare the expression patterns between white fruit (W) and red fruit (R) in cranberry. In this study, two cDNA libraries of W and R were constructed. Approximately 119 million raw sequencing reads were generated and assembled de novo, yielding 57,331 high quality unigenes with an average length of 739 bp. Using BLASTx, 38,460 unigenes were identified as putative homologs of annotated sequences in public protein databases, including NCBI NR, NT, Swiss-Prot, KEGG, COG and GO. Of these, 21,898 unigenes mapped to 128 KEGG pathways, with the metabolic pathways, secondary metabolites, glycerophospholipid metabolism, ether lipid metabolism, starch and sucrose metabolism, purine metabolism, and pyrimidine metabolism being well represented. Among them, many candidate genes were involved in flavonoid biosynthesis, transport and regulation. Furthermore, digital gene expression (DEG) analysis identified 3,257 unigenes that were differentially expressed between the two fruit developmental stages. In addition, 14,473 simple sequence repeats (SSRs) were detected. Our results present comprehensive gene expression information about the cranberry fruit transcriptome that could facilitate our understanding of the molecular mechanisms of fruit development in cranberries. Although it will be necessary to validate the functions carried out by these genes, these results could be used to improve the quality of breeding programs for the cranberry and related species.

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.

Environmental genomics of "Haloquadratum walsbyi" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species

PubMed Central

Legault, Boris A; Lopez-Lopez, Arantxa; Alba-Casado, Jose Carlos; Doolittle, W Ford; Bolhuis, Henk; Rodriguez-Valera, Francisco; Papke, R Thane

2006-01-01

Background Mature saturated brine (crystallizers) communities are largely dominated (>80% of cells) by the square halophilic archaeon "Haloquadratum walsbyi". The recent cultivation of the strain HBSQ001 and thesequencing of its genome allows comparison with the metagenome of this taxonomically simplified environment. Similar studies carried out in other extreme environments have revealed very little diversity in gene content among the cell lineages present. Results The metagenome of the microbial community of a crystallizer pond has been analyzed by end sequencing a 2000 clone fosmid library and comparing the sequences obtained with the genome sequence of "Haloquadratum walsbyi". The genome of the sequenced strain was retrieved nearly complete within this environmental DNA library. However, many ORF's that could be ascribed to the "Haloquadratum" metapopulation by common genome characteristics or scaffolding to the strain genome were not present in the specific sequenced isolate. Particularly, three regions of the sequenced genome were associated with multiple rearrangements and the presence of different genes from the metapopulation. Many transposition and phage related genes were found within this pool which, together with the associated atypical GC content in these areas, supports lateral gene transfer mediated by these elements as the most probable genetic cause of this variability. Additionally, these sequences were highly enriched in putative regulatory and signal transduction functions. Conclusion These results point to a large pan-genome (total gene repertoire of the genus/species) even in this highly specialized extremophile and at a single geographic location. The extensive gene repertoire is what might be expected of a population that exploits a diverse nutrient pool, resulting from the degradation of biomass produced at lower salinities. PMID:16820057

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

PubMed

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional enrichments were related to the cellular functions. The normalized number of functional enrichments of human putative transcriptional target genes changed according to the criteria of enhancer-promoter assignments and correlated with the median expression level of the target genes. These analyses and characters of human putative transcriptional target genes would be useful to examine the criteria of enhancer-promoter assignments and to predict the novel mechanisms and factors such as DNA binding proteins and DNA sequences of enhancer-promoter interactions.

Cdc6 localizes to S- and G2-phase centrosomes in a cell cycle-dependent manner

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

Kim, Gwang Su; Kang, Jeeheon; Bang, Sung Woong

2015-01-16

Highlights: • Cdc6 protein is a component of the pre-replicative complex required for chromosomal replication initiation. • Cdc6 localized to centrosomes of S and G2 phases in a cell cycle-dependent manner. • The centrosomal localization was governed by centrosomal localization signal sequences of Cdc6. • Deletions or substitution mutations on the centrosomal localization signal interfered with centrosomal localization of the Cdc6 proteins. - Abstract: The Cdc6 protein has been primarily investigated as a component of the pre-replicative complex for the initiation of chromosome replication, which contributes to maintenance of chromosomal integrity. Here, we show that Cdc6 localized to the centrosomesmore » during S and G2 phases of the cell cycle. The centrosomal localization was mediated by Cdc6 amino acid residues 311–366, which are conserved within other Cdc6 homologues and contains a putative nuclear export signal. Deletions or substitutions of the amino acid residues did not allow the proteins to localize to centrosomes. In contrast, DsRed tag fused to the amino acid residues localized to centrosomes. These results indicated that a centrosome localization signal is contained within amino acid residues 311–366. The cell cycle-dependent centrosomal localization of Cdc6 in S and G2 phases suggest a novel function of Cdc6 in centrosomes.« less

Murine c-mpl: a member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal.

PubMed Central

Skoda, R C; Seldin, D C; Chiang, M K; Peichel, C L; Vogt, T F; Leder, P

1993-01-01

The murine myeloproliferative leukemia virus has previously been shown to contain a fragment of the coding region of the c-mpl gene, a member of the cytokine receptor superfamily. We have isolated cDNA and genomic clones encoding murine c-mpl and localized the c-mpl gene to mouse chromosome 4. Since some members of this superfamily function by transducing a proliferative signal and since the putative ligand of mpl is unknown, we have generated a chimeric receptor to test the functional potential of mpl. The chimera consists of the extracellular domain of the human interleukin-4 receptor and the cytoplasmic domain of mpl. A mouse hematopoietic cell line transfected with this construct proliferates in response to human interleukin-4, thereby demonstrating that the cytoplasmic domain of mpl contains all elements necessary to transmit a growth stimulatory signal. In addition, we show that 25-40% of mpl mRNA found in the spleen corresponds to a novel truncated and potentially soluble isoform of mpl and that both full-length and truncated forms of mpl protein can be immunoprecipitated from lysates of transfected COS cells. Interestingly, however, although the truncated form of the receptor possesses a functional signal sequence and lacks a transmembrane domain, it is not detected in the culture media of transfected cells. Images PMID:8334987

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon–carbon bond cleavage during α-oxidation of 3-methyl-branched fatty acids

PubMed Central

Foulon, Veerle; Antonenkov, Vasily D.; Croes, Kathleen; Waelkens, Etienne; Mannaerts, Guy P.; Van Veldhoven, Paul P.; Casteels, Minne

1999-01-01

In the third step of the α-oxidation of 3-methyl-branched fatty acids such as phytanic acid, a 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde. The cleavage enzyme was purified from the matrix protein fraction of rat liver peroxisomes and identified as a protein made up of four identical subunits of 63 kDa. Its activity proved to depend on Mg2+ and thiamine pyrophosphate, a hitherto unrecognized cofactor of α-oxidation. Formyl-CoA and 2-methylpentadecanal were identified as reaction products when the purified enzyme was incubated with 2-hydroxy-3-methylhexadecanoyl-CoA as the substrate. Hence the enzyme catalyzes a carbon–carbon cleavage, and we propose calling it 2-hydroxyphytanoyl-CoA lyase. Sequences derived from tryptic peptides of the purified rat protein were used as queries to recover human expressed sequence tags from the databases. The composite cDNA sequence of the human lyase contained an ORF of 1,734 bases that encodes a polypeptide with a calculated molecular mass of 63,732 Da. Recombinant human protein, expressed in mammalian cells, exhibited lyase activity. The lyase displayed homology to a putative Caenorhabditis elegans protein that resembles bacterial oxalyl-CoA decarboxylases. Similarly to the decarboxylases, a thiamine pyrophosphate-binding consensus domain was present in the C-terminal part of the lyase. Although no peroxisome targeting signal, neither 1 nor 2, was apparent, transfection experiments with constructs encoding green fluorescent protein fused to the full-length lyase or its C-terminal pentapeptide indicated that the C terminus of the lyase represents a peroxisome targeting signal 1 variant. PMID:10468558

A genome-wide study of two-component signal transduction systems in eight newly sequenced mutans streptococci strains

PubMed Central

2012-01-01

Background Mutans streptococci are a group of gram-positive bacteria including the primary cariogenic dental pathogen Streptococcus mutans and closely related species. Two component systems (TCSs) composed of a signal sensing histidine kinase (HK) and a response regulator (RR) play key roles in pathogenicity, but have not been comparatively studied for these oral bacterial pathogens. Results HKs and RRs of 8 newly sequenced mutans streptococci strains, including S. sobrinus DSM20742, S. ratti DSM20564 and six S. mutans strains, were identified and compared to the TCSs of S. mutans UA159 and NN2025, two previously genome sequenced S. mutans strains. Ortholog analysis revealed 18 TCS clusters (HK-RR pairs), 2 orphan HKs and 2 orphan RRs, of which 8 TCS clusters were common to all 10 strains, 6 were absent in one or more strains, and the other 4 were exclusive to individual strains. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. While TCS complements were comparable within the six S. mutans strains, S. sobrinus DSM20742 lacked TCSs possibly involved in acid tolerance and fructan catabolism, and S. ratti DSM20564 possessed 3 unique TCSs but lacked the quorum-sensing related TCS (ComDE). Selected computational predictions were verified by PCR experiments. Conclusions Differences in the TCS repertoires of mutans streptococci strains, especially those of S. sobrinus and S. ratti in comparison to S. mutans, imply differences in their response mechanisms for survival in the dynamic oral environment. This genomic level study of TCSs should help in understanding the pathogenicity of these mutans streptococci strains. PMID:22475007

An ordered EST catalogue and gene expression profiles of cassava (Manihot esculenta) at key growth stages.

PubMed

Li, You-Zhi; Pan, Ying-Hua; Sun, Chang-Bin; Dong, Hai-Tao; Luo, Xing-Lu; Wang, Zhi-Qiang; Tang, Ji-Liang; Chen, Baoshan

2010-12-01

A cDNA library was constructed from the root tissues of cassava variety Huanan 124 at the root bulking stage. A total of 9,600 cDNA clones from the library were sequenced with single-pass from the 5'-terminus to establish a catalogue of expressed sequence tags (ESTs). Assembly of the resulting EST sequences resulted in 2,878 putative unigenes. Blastn analysis showed that 62.6% of the unigenes matched with known cassava ESTs and the rest had no 'hits' against the cassava database in the integrative PlantGDB database. Blastx analysis showed that 1,715 (59.59%) of the unigenes matched with one or more GenBank protein entries and 1,163 (40.41%) had no 'hits'. A cDNA microarray with 2,878 unigenes was developed and used to analyze gene expression profiling of Huanan 124 at key growth stages including seedling, formation of root system, root bulking, and starch maturity. Array data analysis revealed that (1) the higher ratio of up-regulated ribosome-related genes was accompanied by a high ratio of up-regulated ubiquitin, proteasome-related and protease genes in cassava roots; (2) starch formation and degradation simultaneously occur at the early stages of root development but starch degradation is declined partially due to decrease in UDP-glucose dehydrogenase activity with root maturity; (3) starch may also be synthesized in situ in roots; (4) starch synthesis, translocation, and accumulation are also associated probably with signaling pathways that parallel Wnt, LAM, TCS and ErbB signaling pathways in animals; (5) constitutive expression of stress-responsive genes may be due to the adaptation of cassava to harsh environments during long-term evolution.

Functional analysis of duplicated Symbiosis Receptor Kinase (SymRK) genes during nodulation and mycorrhizal infection in soybean (Glycine max).

PubMed

Indrasumunar, Arief; Wilde, Julia; Hayashi, Satomi; Li, Dongxue; Gresshoff, Peter M

2015-03-15

Association between legumes and rhizobia results in the formation of root nodules, where symbiotic nitrogen fixation occurs. The early stages of this association involve a complex of signalling events between the host and microsymbiont. Several genes dealing with early signal transduction have been cloned, and one of them encodes the leucine-rich repeat (LRR) receptor kinase (SymRK; also termed NORK). The Symbiosis Receptor Kinase gene is required by legumes to establish a root endosymbiosis with Rhizobium bacteria as well as mycorrhizal fungi. Using degenerate primer and BAC sequencing, we cloned duplicated SymRK homeologues in soybean called GmSymRKα and GmSymRKβ. These duplicated genes have high similarity of nucleotide (96%) and amino acid sequence (95%). Sequence analysis predicted a malectin-like domain within the extracellular domain of both genes. Several putative cis-acting elements were found in promoter regions of GmSymRKα and GmSymRKβ, suggesting a participation in lateral root development, cell division and peribacteroid membrane formation. The mutant of SymRK genes is not available in soybean; therefore, to know the functions of these genes, RNA interference (RNAi) of these duplicated genes was performed. For this purpose, RNAi construct of each gene was generated and introduced into the soybean genome by Agrobacterium rhizogenes-mediated hairy root transformation. RNAi of GmSymRKβ gene resulted in an increased reduction of nodulation and mycorrhizal infection than RNAi of GmSymRKα, suggesting it has the major activity of the duplicated gene pair. The results from the important crop legume soybean confirm the joint phenotypic action of GmSymRK genes in both mycorrhizal and rhizobial infection seen in model legumes. Copyright © 2015 Elsevier GmbH. All rights reserved.

A perchlorate sensitive iodide transporter in frogs

PubMed Central

Carr, Deborah L.; Carr, James A.; Willis, Ray E.; Pressley, Thomas A.

2008-01-01

Nucleotide sequence comparisons have identified a gene product in the genome database of African clawed frogs (Xenopus laevis) as a probable member of the solute carrier family of membrane transporters. To confirm its identity as a putative iodide transporter, we examined the function of this sequence after heterologous expression in mammalian cells. A green monkey kidney cell line transfected with the Xenopus nucleotide sequence had significantly greater 125I uptake than sham-transfected control cells. The uptake in carrier-transfected cells was significantly inhibited in the presence of perchlorate, a competitive inhibitor of mammalian Na+/iodide symporter. Tissue distributions of the sequence were also consistent with a role in iodide uptake. The mRNA encoding the carrier was found to be expressed in the thyroid gland, stomach, and kidney of tadpoles from X. laevis, as well as the bullfrog Rana catesbeiana. The ovaries of adult X. laevis also were found to express the carrier. Phylogenetic analysis suggested that the putative X. laevis iodide transporter is orthologous to vertebrate Na+-dependent iodide symporters. We conclude that the amphibian sequence encodes a protein that is indeed a functional Na+/iodide symporter in Xenopus laevis, as well as Rana catesbeiana. PMID:18275962

Annotation of differentially expressed genes in the somatic embryogenesis of musa and their location in the banana genome.

PubMed

Maldonado-Borges, Josefina Ines; Ku-Cauich, José Roberto; Escobedo-Graciamedrano, Rosa Maria

2013-01-01

Analysis of cDNA-AFLP was used to study the genes expressed in zygotic and somatic embryogenesis of Musa acuminata Colla ssp. malaccensis, and a comparison was made between their differential transcribed fragments (TDFs) and the sequenced genome of the double haploid- (DH-) Pahang of the malaccensis subspecies that is available in the network. A total of 253 transcript-derived fragments (TDFs) were detected with apparent size of 100-4000 bp using 5 pairs of AFLP primers, of which 21 were differentially expressed during the different stages of banana embryogenesis; 15 of the sequences have matched DH-Pahang chromosomes, with 7 of them being homologous to gene sequences encoding either known or putative protein domains of higher plants. Four TDF sequences were located in all Musa chromosomes, while the rest were located in one or two chromosomes. Their putative individual function is briefly reviewed based on published information, and the potential roles of these genes in embryo development are discussed. Thus the availability of the genome of Musa and the information of TDFs sequences presented here opens new possibilities for an in-depth study of the molecular and biochemical research of zygotic and somatic embryogenesis of Musa.

Spliced DNA Sequences in the Paramecium Germline: Their Properties and Evolutionary Potential

PubMed Central

Catania, Francesco; McGrath, Casey L.; Doak, Thomas G.; Lynch, Michael

2013-01-01

Despite playing a crucial role in germline-soma differentiation, the evolutionary significance of developmentally regulated genome rearrangements (DRGRs) has received scant attention. An example of DRGR is DNA splicing, a process that removes segments of DNA interrupting genic and/or intergenic sequences. Perhaps, best known for shaping immune-system genes in vertebrates, DNA splicing plays a central role in the life of ciliated protozoa, where thousands of germline DNA segments are eliminated after sexual reproduction to regenerate a functional somatic genome. Here, we identify and chronicle the properties of 5,286 sequences that putatively undergo DNA splicing (i.e., internal eliminated sequences [IESs]) across the genomes of three closely related species of the ciliate Paramecium (P. tetraurelia, P. biaurelia, and P. sexaurelia). The study reveals that these putative IESs share several physical characteristics. Although our results are consistent with excision events being largely conserved between species, episodes of differential IES retention/excision occur, may have a recent origin, and frequently involve coding regions. Our findings indicate interconversion between somatic—often coding—DNA sequences and noncoding IESs, and provide insights into the role of DNA splicing in creating potentially functional genetic innovation. PMID:23737328

Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese

PubMed Central

Wüthrich, Daniel; Bruggmann, Rémy; Berthoud, Hélène; Arias-Roth, Emmanuelle

2015-01-01

Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively. PMID:25767226

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

Hypoxia Sensing in Plants: On a Quest for Ion Channels as Putative Oxygen Sensors.

PubMed

Wang, Feifei; Chen, Zhong-Hua; Shabala, Sergey

2017-07-01

Over 17 million km2 of land is affected by soil flooding every year, resulting in substantial yield losses and jeopardizing food security across the globe. A key step in resolving this problem and creating stress-tolerant cultivars is an understanding of the mechanisms by which plants sense low-oxygen stress. In this work, we review the current knowledge about the oxygen-sensing and signaling pathway in mammalian and plant systems and postulate the potential role of ion channels as putative oxygen sensors in plant roots. We first discuss the definition and requirements for the oxygen sensor and the difference between sensing and signaling. We then summarize the literature and identify several known candidates for oxygen sensing in the mammalian literature. This includes transient receptor potential (TRP) channels; K+-permeable channels (Kv, BK and TASK); Ca2+ channels (RyR and TPC); and various chemo- and reactive oxygen species (ROS)-dependent oxygen sensors. Identified key oxygen-sensing domains (PAS, GCS, GAF and PHD) in mammalian systems are used to predict the potential plant counterparts in Arabidopsis. Finally, the sequences of known mammalian ion channels with reported roles in oxygen sensing were employed to BLAST the Arabidopsis genome for the candidate genes. Several plasma membrane and tonoplast ion channels (such as TPC, AKT and KCO) and oxygen domain-containing proteins with predicted oxygen-sensing ability were identified and discussed. We propose a testable model for potential roles of ion channels in plant hypoxia sensing. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

1-Aminocyclopropane-1-carboxylic acid oxidase reaction mechanism and putative post-translational activities of the ACCO protein

PubMed Central

Dilley, David R.; Wang, Zhenyong; Kadirjan-Kalbach, Deena K.; Ververidis, Fillipos; Beaudry, Randolph; Padmanabhan, Kallaithe

2013-01-01

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyses the final step in ethylene biosynthesis converting ACC to ethylene, cyanide, CO2, dehydroascorbate and water with inputs of Fe(II), ascorbate, bicarbonate (as activators) and oxygen. Cyanide activates ACCO. A ‘nest’ comprising several positively charged amino acid residues from the C-terminal α-helix 11 along with Lys158 and Arg299 are proposed as binding sites for ascorbate and bicarbonate to coordinately activate the ACCO reaction. The binding sites for ACC, bicarbonate and ascorbic acid for Malus domestica ACCO1 include Arg175, Arg244, Ser246, Lys158, Lys292, Arg299 and Phe300. Glutamate 297, Phe300 and Glu301 in α-helix 11 are also important for the ACCO reaction. Our proposed reaction pathway incorporates cyanide as an ACCO/Fe(II) ligand after reaction turnover. The cyanide ligand is likely displaced upon binding of ACC and ascorbate to provide a binding site for oxygen. We propose that ACCO may be involved in the ethylene signal transduction pathway not directly linked to the ACCO reaction. ACC oxidase has significant homology with Lycopersicon esculentum cysteine protease LeCp, which functions as a protease and as a regulator of 1-aminocyclopropane-1-carboxylic acid synthase (Acs2) gene expression. ACC oxidase may play a similar role in signal transduction after post-translational processing. ACC oxidase becomes inactivated by fragmentation and apparently has intrinsic protease and transpeptidase activity. ACC oxidase contains several amino acid sequence motifs for putative protein–protein interactions, phosphokinases and cysteine protease. ACC oxidase is subject to autophosphorylaton in vitro and promotes phosphorylation of some apple fruit proteins in a ripening-dependent manner. PMID:24244837

Genome-Wide Prediction and Analysis of 3D-Domain Swapped Proteins in the Human Genome from Sequence Information.

PubMed

Upadhyay, Atul Kumar; Sowdhamini, Ramanathan

2016-01-01

3D-domain swapping is one of the mechanisms of protein oligomerization and the proteins exhibiting this phenomenon have many biological functions. These proteins, which undergo domain swapping, have acquired much attention owing to their involvement in human diseases, such as conformational diseases, amyloidosis, serpinopathies, proteionopathies etc. Early realisation of proteins in the whole human genome that retain tendency to domain swap will enable many aspects of disease control management. Predictive models were developed by using machine learning approaches with an average accuracy of 78% (85.6% of sensitivity, 87.5% of specificity and an MCC value of 0.72) to predict putative domain swapping in protein sequences. These models were applied to many complete genomes with special emphasis on the human genome. Nearly 44% of the protein sequences in the human genome were predicted positive for domain swapping. Enrichment analysis was performed on the positively predicted sequences from human genome for their domain distribution, disease association and functional importance based on Gene Ontology (GO). Enrichment analysis was also performed to infer a better understanding of the functional importance of these sequences. Finally, we developed hinge region prediction, in the given putative domain swapped sequence, by using important physicochemical properties of amino acids.

Candidate causative mutation on BTA18 associated with calving and conformation traits in Holstein bulls

USDA-ARS?s Scientific Manuscript database

Complementing quantitative methods with sequence data analysis is a major goal of the post-genome era of biology. In this study, we analyzed Illumina HiSeq sequence data derived from 11 US Holstein bulls in order to identify putative causal mutations associated with calving and conformation traits. ...

A resource of single-nucleotide polymorphisms for rainbow trout generated by restriction-site associated DNA sequencing of doubled haploids

USDA-ARS?s Scientific Manuscript database

Salmonid genomes are considered to be in a pseudo-tetraploid state as a result of an evolutionarily recent genome duplication event. This situation complicates single nucleotide polymorphism (SNP) discovery in rainbow trout as many putative SNPs are actually paralogous sequence variants (PSVs) and ...

Complete genome sequence of a novel potyvirus, Callistephus mottle virus identified in Callistephus chinensis

USDA-ARS?s Scientific Manuscript database

The complete genomic sequence of a novel putative member of the genus Potyvirus was detected from Callistephus chinensis (china aster) in South Korea. The genomic RNA consists of 9,859 nucleotides excluding the 3’ poly(A) tail. The Callistephus virus genome, which contains the typical open reading f...

Taxonomic evaluation of putative Streptomyces scabiei strains held in the ARS (NRRL) Culture Collection using multi-locus sequence analysis

USDA-ARS?s Scientific Manuscript database

Multi-locus sequence analysis has been demonstrated to be a useful tool for identification of Streptomyces species and was previously applied to phylogenetically differentiate the type strains of species pathogenic on potatoes (Solanum tuberosum L.). The ARS Culture Collection (NRRL) contains 43 str...

Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing.

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

Hong, R. L., Hamaguchi, L., Busch, M. A., and Weigel, D.

2003-06-01

OAK-B135 In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3 kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae, several other motifs, but not the LFY and WUS binding sites previously identified, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally importantmore » for activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection, but also highlight that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.« less

PlantTFDB: a comprehensive plant transcription factor database

PubMed Central

Guo, An-Yuan; Chen, Xin; Gao, Ge; Zhang, He; Zhu, Qi-Hui; Liu, Xiao-Chuan; Zhong, Ying-Fu; Gu, Xiaocheng; He, Kun; Luo, Jingchu

2008-01-01

Transcription factors (TFs) play key roles in controlling gene expression. Systematic identification and annotation of TFs, followed by construction of TF databases may serve as useful resources for studying the function and evolution of transcription factors. We developed a comprehensive plant transcription factor database PlantTFDB (http://planttfdb.cbi.pku.edu.cn), which contains 26 402 TFs predicted from 22 species, including five model organisms with available whole genome sequence and 17 plants with available EST sequences. To provide comprehensive information for those putative TFs, we made extensive annotation at both family and gene levels. A brief introduction and key references were presented for each family. Functional domain information and cross-references to various well-known public databases were available for each identified TF. In addition, we predicted putative orthologs of those TFs among the 22 species. PlantTFDB has a simple interface to allow users to search the database by IDs or free texts, to make sequence similarity search against TFs of all or individual species, and to download TF sequences for local analysis. PMID:17933783

Transcriptome analysis of eyestalk and hemocytes in the ridgetail white prawn Exopalaemon carinicauda: assembly, annotation and marker discovery.

PubMed

Li, Jitao; Li, Jian; Chen, Ping; Liu, Ping; He, Yuying

2015-01-01

The ridgetail white prawn Exopalaemon carinicauda is one of major economic mariculture species in eastern China. The deficiency of genomic and transcriptomic data is becoming the bottleneck of further researches on its good traits. In the present study, 454 pyrosequencing was undertaken to investigate the transcriptome profiles of E. carinicauda. A collection of 1,028,710 sequence reads (459.59 Mb) obtained from cDNA prepared from eyestalk and hemocytes was assembled into 162,056 expressed sequence tags (ESTs). Of these, 29.88 % of 48,428 contigs and 70.12 % of 113,628 singlets possessed high similarities to sequences in the GenBank non-redundant database, with most significant (E value <1e(-10)) unigenes matches occurring with crustacean and insect sequences. KEGG analysis of unigenes identified putative members of biological pathways related to growth and immunity. In addition, we obtained a total of putative 125,112 SNPs and 13,467 microsatellites. These results will contribute to the understanding of the genome makeup and provide useful information for future functional genomic research in E. carinicauda.

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.

Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site

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

Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de

1994-04-01

The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-pointmore » sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.« less

Molecular cloning and functional analysis of tumor necrosis factor receptor-associated factor 6 (TRAF6) in Crossastrea gigas.

PubMed

Mao, Fan; Li, Jun; Zhang, Yuehuan; Xiang, Zhiming; Zhang, Yang; Yu, Ziniu

2017-09-01

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been demonstrated to be a key signaling molecule involved in adaptive and innate immunity. In this study, we obtained the full length CgTRAF6 cDNA and analyzed the characteristics of the ORF and the peptide sequence in Crassostrea gigas. The deduced protein sequence of CgTRAF6 includes a conserved C-terminal TRAF domain following the RING and the zinc finger domain. The TRAF domain is composed of coiled-coil TRAF-N and MATH (meprin and TRAF-C homology) subdomains. Furthermore, phylogenetic analysis revealed that CgTRAF6 is clustered together with other members TRAF6 family and is placed in a sub-cluster singly which had a close relationship with Drosophila melanogaster. Expression analysis of CgTRAF6 indicated its constitutive expression in all tissues including mantle, adductor muscle, digestive tract, gonads, heart, gill, and hemocyte. Immune challenge with Vibrio alginolyticus and poly I:C resulted in significant up-regulation of CgTRAF6 expression. Dual-luciferase reporter assays showed that CgTRAF6 could activate both pNF-κB-Luc and pISRE-Luc expression, suggesting CgTRAF6 is potentially involved in NF-κB and the interferon signaling pathway. Furthermore, RNAi mediated knockdown of CgTRAF6 resulted in the down-regulation of several putative anti-viral signaling (IRF) and effector (PKR & Viperin) molecules coding genes, 7 days post-injection. These results collectively indicate that CgTRAF6 is a member of TRAF6 sub-family and is potentially involved in immune defense system against invading bacteria and viruses in Crassostrea gigas. Copyright © 2017 Elsevier Ltd. All rights reserved.

The sigma factor SigD of Mycobacterium tuberculosis putatively enhances gene expression of the septum site determining protein under stressful environments.

PubMed

Ares, Miguel A; Rios-Sarabia, Nora; De la Cruz, Miguel A; Rivera-Gutiérrez, Sandra; García-Morales, Lázaro; León-Solís, Lizbel; Espitia, Clara; Pacheco, Sabino; Cerna-Cortés, Jorge F; Helguera-Repetto, Cecilia A; García, María Jesús; González-Y-Merchand, Jorge A

2017-07-01

This work examined the expression of the septum site determining gene (ssd) of Mycobacterium tuberculosis CDC1551 and its ∆sigD mutant under different growing conditions. The results showed an up-regulation of ssd during stationary phase and starvation conditions, but not during in vitro dormancy, suggesting a putative role for SigD in the control of ssd expression mainly under lack-of-nutrients environments. Furthermore, we elucidated a putative link between ssd expression and cell elongation of bacilli at stationary phase. In addition, a -35 sigD consensus sequence was found for the ssd promoter region, reinforcing the putative regulation of ssd by SigD, and in turn, supporting this protein role during the adaptation of M. tuberculosis to some stressful environments.

TNF induction of jagged-1 in endothelial cells is NFκB-dependent

PubMed Central

Johnston, Douglas A.; Dong, Bamboo; Hughes, Christopher C.W.

2009-01-01

TNF-α is a potent proinflammatory cytokine that induces endothelial cell (EC) adhesion molecules. In addition, TNF promotes angiogenesis by inducing an EC tip cell phenotype and the expression of jagged-1, a ligand for the notch pathway. Notch signaling is critical for vascular patterning and helps to restrict the proliferation of tip cells. Here we demonstrate that TNF induction of jagged-1 in human EC is rapid and dependent upon signaling through TNFR1, but not TNFR2. A luciferase reporter construct carrying 3.7 kb of 5′ promoter sequence from the human gene was responsive to both TNF and overexpression of NFκB pathway components. TNF-induced promoter activation was blocked by treatment with an NFκB inhibitor or co-expression of dominant-negative IKKβ. Mutations in a putative NFκB-binding site at −3.0 kb, which is conserved across multiple species, resulted in a loss of responsiveness to TNF and NFκB. Electromobility shift and chromatin immunoprecipitation assays revealed binding of both p50 and p65 to the promoter in response to TNF treatment. Full promoter activity also depends on an AP-1 site at −2.0 kb. These results indicate that canonical NFκB signaling is required for TNF induction of the notch ligand jagged-1 in EC. PMID:19393188

Within-genome evolution of REPINs: a new family of miniature mobile DNA in bacteria.

PubMed

Bertels, Frederic; Rainey, Paul B

2011-06-01

Repetitive sequences are a conserved feature of many bacterial genomes. While first reported almost thirty years ago, and frequently exploited for genotyping purposes, little is known about their origin, maintenance, or processes affecting the dynamics of within-genome evolution. Here, beginning with analysis of the diversity and abundance of short oligonucleotide sequences in the genome of Pseudomonas fluorescens SBW25, we show that over-represented short sequences define three distinct groups (GI, GII, and GIII) of repetitive extragenic palindromic (REP) sequences. Patterns of REP distribution suggest that closely linked REP sequences form a functional replicative unit: REP doublets are over-represented, randomly distributed in extragenic space, and more highly conserved than singlets. In addition, doublets are organized as inverted repeats, which together with intervening spacer sequences are predicted to form hairpin structures in ssDNA or mRNA. We refer to these newly defined entities as REPINs (REP doublets forming hairpins) and identify short reads from population sequencing that reveal putative transposition intermediates. The proximal relationship between GI, GII, and GIII REPINs and specific REP-associated tyrosine transposases (RAYTs), combined with features of the putative transposition intermediate, suggests a mechanism for within-genome dissemination. Analysis of the distribution of REPs in a range of RAYT-containing bacterial genomes, including Escherichia coli K-12 and Nostoc punctiforme, show that REPINs are a widely distributed, but hitherto unrecognized, family of miniature non-autonomous mobile DNA.

Multi-virulence-locus sequence typing of Staphylococcus lugdunensis generates results consistent with a clonal population structure and is reliable for epidemiological typing.

PubMed

Didi, Jennifer; Lemée, Ludovic; Gibert, Laure; Pons, Jean-Louis; Pestel-Caron, Martine

2014-10-01

Staphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlLR2, atlLR3, hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (dN/dS) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlLR2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the dN/dS ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlLR3, isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Characterization of two new putative adhesins of Leptospira interrogans.

PubMed

Figueredo, Jupciana M; Siqueira, Gabriela H; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Chapola, Erica G B; Nascimento, Ana L T O

2017-01-01

We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

Structural Insights into the Quadruplex-Duplex 3' Interface Formed from a Telomeric Repeat: A Potential Molecular Target.

PubMed

Russo Krauss, Irene; Ramaswamy, Sneha; Neidle, Stephen; Haider, Shozeb; Parkinson, Gary N

2016-02-03

We report here on an X-ray crystallographic and molecular modeling investigation into the complex 3' interface formed between putative parallel stranded G-quadruplexes and a duplex DNA sequence constructed from the human telomeric repeat sequence TTAGGG. Our crystallographic approach provides a detailed snapshot of a telomeric 3' quadruplex-duplex junction: a junction that appears to have the potential to form a unique molecular target for small molecule binding and interference with telomere-related functions. This unique target is particularly relevant as current high-affinity compounds that bind putative G-quadruplex forming sequences only rarely have a high degree of selectivity for a particular quadruplex. Here DNA junctions were assembled using different putative quadruplex-forming scaffolds linked at the 3' end to a telomeric duplex sequence and annealed to a complementary strand. We successfully generated a series of G-quadruplex-duplex containing crystals, both alone and in the presence of ligands. The structures demonstrate the formation of a parallel folded G-quadruplex and a B-form duplex DNA stacked coaxially. Most strikingly, structural data reveals the consistent formation of a TAT triad platform between the two motifs. This triad allows for a continuous stack of bases to link the quadruplex motif with the duplex region. For these crystal structures formed in the absence of ligands, the TAT triad interface occludes ligand binding at the 3' quadruplex-duplex interface, in agreement with in silico docking predictions. However, with the rearrangement of a single nucleotide, a stable pocket can be produced, thus providing an opportunity for the binding of selective molecules at the interface.

Increased taxon sampling reveals thousands of hidden orthologs in flatworms

PubMed Central

2017-01-01

Gains and losses shape the gene complement of animal lineages and are a fundamental aspect of genomic evolution. Acquiring a comprehensive view of the evolution of gene repertoires is limited by the intrinsic limitations of common sequence similarity searches and available databases. Thus, a subset of the gene complement of an organism consists of hidden orthologs, i.e., those with no apparent homology to sequenced animal lineages—mistakenly considered new genes—but actually representing rapidly evolving orthologs or undetected paralogs. Here, we describe Leapfrog, a simple automated BLAST pipeline that leverages increased taxon sampling to overcome long evolutionary distances and identify putative hidden orthologs in large transcriptomic databases by transitive homology. As a case study, we used 35 transcriptomes of 29 flatworm lineages to recover 3427 putative hidden orthologs, some unidentified by OrthoFinder and HaMStR, two common orthogroup inference algorithms. Unexpectedly, we do not observe a correlation between the number of putative hidden orthologs in a lineage and its “average” evolutionary rate. Hidden orthologs do not show unusual sequence composition biases that might account for systematic errors in sequence similarity searches. Instead, gene duplication with divergence of one paralog and weak positive selection appear to underlie hidden orthology in Platyhelminthes. By using Leapfrog, we identify key centrosome-related genes and homeodomain classes previously reported as absent in free-living flatworms, e.g., planarians. Altogether, our findings demonstrate that hidden orthologs comprise a significant proportion of the gene repertoire in flatworms, qualifying the impact of gene losses and gains in gene complement evolution. PMID:28400424

Integrating metagenomic and amplicon databases to resolve the phylogenetic and ecological diversity of the Chlamydiae

PubMed Central

Lagkouvardos, Ilias; Weinmaier, Thomas; Lauro, Federico M; Cavicchioli, Ricardo; Rattei, Thomas; Horn, Matthias

2014-01-01

In the era of metagenomics and amplicon sequencing, comprehensive analyses of available sequence data remain a challenge. Here we describe an approach exploiting metagenomic and amplicon data sets from public databases to elucidate phylogenetic diversity of defined microbial taxa. We investigated the phylum Chlamydiae whose known members are obligate intracellular bacteria that represent important pathogens of humans and animals, as well as symbionts of protists. Despite their medical relevance, our knowledge about chlamydial diversity is still scarce. Most of the nine known families are represented by only a few isolates, while previous clone library-based surveys suggested the existence of yet uncharacterized members of this phylum. Here we identified more than 22 000 high quality, non-redundant chlamydial 16S rRNA gene sequences in diverse databases, as well as 1900 putative chlamydial protein-encoding genes. Even when applying the most conservative approach, clustering of chlamydial 16S rRNA gene sequences into operational taxonomic units revealed an unexpectedly high species, genus and family-level diversity within the Chlamydiae, including 181 putative families. These in silico findings were verified experimentally in one Antarctic sample, which contained a high diversity of novel Chlamydiae. In our analysis, the Rhabdochlamydiaceae, whose known members infect arthropods, represents the most diverse and species-rich chlamydial family, followed by the protist-associated Parachlamydiaceae, and a putative new family (PCF8) with unknown host specificity. Available information on the origin of metagenomic samples indicated that marine environments contain the majority of the newly discovered chlamydial lineages, highlighting this environment as an important chlamydial reservoir. PMID:23949660

Report on the development of putative functional SSR and SNP markers in passion fruits.

PubMed

da Costa, Zirlane Portugal; Munhoz, Carla de Freitas; Vieira, Maria Lucia Carneiro

2017-09-06

Passionflowers Passiflora edulis and Passiflora alata are diploid, outcrossing and understudied fruit bearing species. In Brazil, passion fruit cultivation began relatively recently and has earned the country an outstanding position as the world's top producer of passion fruit. The fruit's main economic value lies in the production of juice, an essential exotic ingredient in juice blends. Currently, crop improvement strategies, including those for underexploited tropical species, tend to incorporate molecular genetic approaches. In this study, we examined a set of P. edulis transcripts expressed in response to infection by Xanthomonas axonopodis, (the passion fruit's main bacterial pathogen that attacks the vines), aiming at the development of putative functional markers, i.e. SSRs (simple sequence repeats) and SNPs (single nucleotide polymorphisms). A total of 210 microsatellites were found in 998 sequences, and trinucleotide repeats were found to be the most frequent (31.4%). Of the sequences selected for designing primers, 80.9% could be used to develop SSR markers, and 60.6% SNP markers for P. alata. SNPs were all biallelic and found within 15 gene fragments of P. alata. Overall, gene fragments generated 10,003 bp. SNP frequency was estimated as one SNP every 294 bp. Polymorphism rates revealed by SSR and SNP loci were 29.4 and 53.6%, respectively. Passiflora edulis transcripts were useful for the development of putative functional markers for P. alata, suggesting a certain level of sequence conservation between these cultivated species. The markers developed herein could be used for genetic mapping purposes and also in diversity studies.

Characterization of a human X-linked gene from the DXS732E locus in the candidate region for the anhidrotic ectodermal dysplasia (EDA) gene (Xq13.1)

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

Gault, J.; Zonana, J.; Zeltinger, J.

A conserved mouse genomic clone was used to identify a homologous human genomic clone (the DXS732E locus), which was subsequently employed to isolate cDNAs from a human fetal brain library. Nine unique overlapping cDNAs were isolated, and sequences analysis of 3.9 kb identified a putative 1 kb ORF. GRAIL analysis of the sequence supported the hypothesis that the putative ORF was coding sequence, and Prosite analysis of the putative ORF identified potential glycosylation and phosphorylation sites. The 5{prime} end of the gene maps within a CpG island, and comparison of cDNA sequences indicate the gene is alternatively spliced at itsmore » 3{prime} end. Northern analysis and RT-PCR indicate that two different sized messages appear to be expressed with the gene expressed in human fetal kidney, intestine, brain, and muscle. The gene is expressed in 77 day human skin, a time when hair follicle formation occurs. Anhidrotic ectodermal dysplasia (EDA) results in the abnormal morphogenesis of hair, teeth and eccrine sweat glands. A positional cloning strategy towards cloning the EDA gene had been used, and deletion and X-autosome translocation patients have been useful in further delimiting the EDA region. The present gene at the DXS732E locus is partially deleted in one EDA patient who does not have other apparent abnormalities. No rearrangements of the gene have been detected in two female X-autosome translocation EDA patients, nor in four additional male patients with submicroscopic molecular deletions.« less

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

Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

PubMed Central

Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

2015-01-01

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

Genome annotation provides insight into carbon monoxide and hydrogen metabolism in Rubrivivax gelatinosus

DOE PAGES

Wawrousek, Karen; Noble, Scott; Korlach, Jonas; ...

2014-12-05

In this article, we report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. This microbe is a model for studies of its carboxydotrophic life style under anaerobic condition, based on its ability to utilize carbon monoxide (CO) as the sole carbon substrate and water as the electron acceptor, yielding CO 2 and H 2 as the end products. The CO-oxidation reaction is known to be catalyzed by two enzyme complexes, the CO dehydrogenase and hydrogenase. As expected, analysis of the genome of Rx. gelatinosus CBS reveals the presence of genes encodingmore » both enzyme complexes. The CO-oxidation reaction is CO-inducible, which is consistent with the presence of two putative CO-sensing transcription factors in its genome. Genome analysis also reveals the presence of two additional hydrogenases, an uptake hydrogenase that liberates the electrons in H 2 in support of cell growth, and a regulatory hydrogenase that senses H 2 and relays the signal to a two-component system that ultimately controls synthesis of the uptake hydrogenase. The genome also contains two sets of hydrogenase maturation genes which are known to assemble the catalytic metallocluster of the hydrogenase NiFe active site. Finally and collectively, the genome sequence and analysis information reveals the blueprint of an intricate network of signal transduction pathways and its underlying regulation that enables Rx. gelatinosus CBS to thrive on CO or H 2 in support of cell growth.« less

Identification and expression analysis of BoMF25, a novel polygalacturonase gene involved in pollen development of Brassica oleracea.

PubMed

Lyu, Meiling; Liang, Ying; Yu, Youjian; Ma, Zhiming; Song, Limin; Yue, Xiaoyan; Cao, Jiashu

2015-06-01

BoMF25 acts on pollen wall. Polygalacturonase (PG) is a pectin-digesting enzyme involved in numerous plant developmental processes and is described to be of critical importance for pollen wall development. In the present study, a PG gene, BoMF25, was isolated from Brassica oleracea. BoMF25 is the homologous gene of At4g35670, a PG gene in Arabidopsis thaliana with a high expression level at the tricellular pollen stage. Collinear analysis revealed that the orthologous gene of BoMF25 in Brassica campestris (syn. B. rapa) genome was probably lost because of genome deletion and reshuffling. Sequence analysis indicated that BoMF25 contained four classical conserved domains (I, II, III, and IV) of PG protein. Homology and phylogenetic analyses showed that BoMF25 was clustered in Clade F. The putative promoter sequence, containing classical cis-acting elements and pollen-specific motifs, could drive green fluorescence protein expression in onion epidermal cells. Quantitative RT-PCR analysis suggested that BoMF25 was mainly expressed in the anther at the late stage of pollen development. In situ hybridization analysis also indicated that the strong and specific expression signal of BoMF25 existed in pollen grains at the mature pollen stage. Subcellular localization showed that the fluorescence signal was observed in the cell wall of onion epidermal cells, which suggested that BoMF25 may be a secreted protein localized in the pollen wall.

Gene Expression and Molecular Characterization of a Xylanase from Chicken Cecum Metagenome

PubMed Central

AL-Darkazali, Hind; Meevootisom, Vithaya

2017-01-01

A xylanase gene xynAMG1 with a 1,116-bp open reading frame, encoding an endo-β-1,4-xylanase, was cloned from a chicken cecum metagenome. The translated XynAMG1 protein consisted of 372 amino acids including a putative signal peptide of 23 amino acids. The calculated molecular mass of the mature XynAMG1 was 40,013 Da, with a theoretical pI value of 5.76. The amino acid sequence of XynAMG1 showed 59% identity to endo-β-1,4-xylanase from Prevotella bryantii and Prevotella ruminicola and 58% identity to that from Prevotella copri. XynAMG1 has two conserved motifs, DVVNE and TEXD, containing two active site glutamates and an invariant asparagine, characteristic of GH10 family xylanase. The xynAMG1 gene without signal peptide sequence was cloned and fused with thioredoxin protein (Trx.Tag) in pET-32a plasmid and overexpressed in Escherichia coli Tuner™(DE3)pLysS. The purified mature XynAMG1 was highly salt-tolerant and stable and displayed higher than 96% of its catalytic activity in the reaction containing 1 to 4 M NaCl. It was only slightly affected by common organic solvents added in aqueous solution to up to 5 M. This chicken cecum metagenome-derived xylanase has potential applications in animal feed additives and industrial enzymatic processes requiring exposure to high concentrations of salt and organic solvents. PMID:28751915

Immunological and biochemical characterization of processing products from the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line.

PubMed Central

Bidard, J N; de Nadai, F; Rovere, C; Moinier, D; Laur, J; Martinez, J; Cuber, J C; Kitabgi, P

1993-01-01

Neurotensin (NT) and neuromedin N (NN) are two related biologically active peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting with an N-terminal signal peptide and containing in its C-terminal region one copy each of NT and NN. NN precedes NT and is separated from it by a Lys-Arg sequence. Two other Lys-Arg sequences flank the N-terminus of NN and the C-terminus of NT. A fourth Lys-Arg sequence occurs near the middle of the precursor and is followed by an NN-like sequence. Finally, an Arg-Arg pair is present within the NT moiety. The four Lys-Arg doublets represent putative processing sites in the precursor molecule. The present study was designed to investigate the post-translational processing of the NT/NN precursor in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT upon dexamethasone treatment. Five region-specific antisera recognizing the free N- or C-termini of sequences adjacent to the basic doublets were produced, characterized and used for immunoblotting and radioimmunoassay studies in combination with gel filtration, reverse-phase h.p.l.c. and trypsin digestion of rMTC 6-23 cell extracts. Because two of the antigenic sequences, i.e. NN and the NN-like sequence, start with a lysine residue that is essential for recognition by their respective antisera, a micromethod by which trypsin specifically cleaves at arginine residues was developed. The results show that dexamethasone-treated rMTC 6-23 cells produced comparable amounts of NT, NN and a peptide corresponding to a large N-terminal precursor fragment lacking the NN and NT moieties. This large fragment was purified. N-Terminal sequencing revealed that it started at residue Ser23 of the prepro-NT/NN sequence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing respectively the NN and NT sequences at their C-termini were present in lower amounts. The NN-like sequence was internal to all the large fragments. There was no evidence for the presence of peptides with the NN-like sequence at their N-termini. This shows that, in rMTC 6-23 cells, the precursor is readily processed at the three Lys-Arg doublets that flank and separate the NT and NN sequences. In contrast, the Lys-Arg doublet that precedes the NN-like sequence is not processed in this system.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 3 PMID:8471039

Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC).

PubMed

Brzuszkiewicz, Elzbieta; Thürmer, Andrea; Schuldes, Jörg; Leimbach, Andreas; Liesegang, Heiko; Meyer, Frauke-Dorothee; Boelter, Jürgen; Petersen, Heiko; Gottschalk, Gerhard; Daniel, Rolf

2011-12-01

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).

Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo.

PubMed

Leekitcharoenphon, Pimlapas; Friis, Carsten; Zankari, Ea; Svendsen, Christina Aaby; Price, Lance B; Rahmani, Maral; Herrero-Fresno, Ana; Fashae, Kayode; Vandenberg, Olivier; Aarestrup, Frank M; Hendriksen, Rene S

2013-10-15

Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria. Eighteen S. Typhimurium ST313 isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Additionally, six of the isolates were characterized by whole genome sequence typing (WGST). The presence of a putative virulence determinant was examined in 177 Salmonella isolates belonging to 57 different serovars. All S. Typhimurium ST313 isolates harbored resistant genes encoded by blaTEM1b, catA1, strA/B, sul1, and dfrA1. Additionally, aac(6')1aa gene was detected. Phylogenetic analyses revealed close genetic relationships among Congolese and Nigerian isolates from both blood and stool. Comparative genomic analyses identified a putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and S. Dublin. We showed in a limited number of isolates that S. Typhimurium ST313 is a prevalent sequence-type causing gastrointestinal diseases and septicemia in patients from Nigeria and DRC. We found three distinct phylogenetic clusters based on the origin of isolation suggesting some spatial evolution. Comparative genomics showed an interesting putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and invasive S. Dublin.

Carbon and nitrogen nutrient balance signaling in plants.

PubMed

Zheng, Zhi-Liang

2009-07-01

Cellular carbon (C) and nitrogen (N) metabolism must be tightly coordinated to sustain optimal growth and development for plants and other cellular organisms. Furthermore, C/N balance is also critical for the ecosystem response to elevated atmospheric CO(2). Despite numerous physiological and molecular studies in C/N balance or ratio response, very few genes have been shown to play important roles in C/N balance signaling. During recent five years, exciting progress was made through genetic and genomic studies. Several DNA microarray studies have shown that more than half of the transcriptome is regulated by C, N and the C-N combination. Three genetic studies involving distinct bioassays have demonstrated that a putative nitrate transporter (NTR2.1), a putative glutamate receptor (GLR1.1) and a putative methyltransferase (OSU1) have important functions in the C/N balance response. OSU1 is identical to QUA2/TSD2 which has been implicated to act in cell wall biogenesis, indicating a link between cell wall property and the C/N balance signaling. Given that many investigations are only focused on C alone or N alone, the C/N balance bioassays and gene expression patterns are discussed to assist phenotypic characterization of C/N balance signaling. Further, re-examination of those previously reported sugar or nitrogen responsive genes in C/N balance response may be necessary to dissect the C/N signaling pathways. In addition, key components involved in C-N interactions in bacterial, yeast and animal systems and whether they are functionally conserved in plants are discussed. These rapid advances have provided the first important step towards the construction of the complex yet elegant C/N balance signaling networks in plants.

Draft Genome Sequence of a Novel Chitinophaga sp. Strain, MD30, Isolated from a Biofilm in an Air Conditioner Condensate Pipe

PubMed Central

Darris, Maxwell

2017-01-01

ABSTRACT Most of the 24 known Chitinophaga species were originally isolated from soils. We report the draft genome sequence of a putatively novel Chitinophaga sp. from a biofilm in an air conditioner condensate pipe. The genome comprises 7,661,303 bp in one scaffold, 5,694 predicted protein-coding sequences, and a G+C content of 47.6%. PMID:29051259

Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese.

PubMed

Storari, Michelangelo; Wüthrich, Daniel; Bruggmann, Rémy; Berthoud, Hélène; Arias-Roth, Emmanuelle

2015-03-12

Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively. Copyright © 2015 Storari et al.

Complete Genome Sequence of the Electricity-Producing “Thermincola potens” Strain JR▿

PubMed Central

Byrne-Bailey, Kathryne G.; Wrighton, Kelly C.; Melnyk, Ryan A.; Agbo, Peter; Hazen, Terry C.; Coates, John D.

2010-01-01

“Thermincola potens” strain JR is one of the first Gram-positive dissimilatory metal-reducing bacteria (DMRB) for which there is a complete genome sequence. Consistent with the physiology of this organism, preliminary annotation revealed an abundance of multiheme c-type cytochromes that are putatively associated with the periplasm and cell surface in a Gram-positive bacterium. Here we report the complete genome sequence of strain JR. PMID:20525829

SNPs in putative regulatory regions identified by human mouse comparative sequencing and transcription factor binding site data

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

Banerjee, Poulabi; Bahlo, Melanie; Schwartz, Jody R.

2002-01-01

Genome wide disease association analysis using SNPs is being explored as a method for dissecting complex genetic traits and a vast number of SNPs have been generated for this purpose. As there are cost and throughput limitations of genotyping large numbers of SNPs and statistical issues regarding the large number of dependent tests on the same data set, to make association analysis practical it has been proposed that SNPs should be prioritized based on likely functional importance. The most easily identifiable functional SNPs are coding SNPs (cSNPs) and accordingly cSNPs have been screened in a number of studies. SNPs inmore » gene regulatory sequences embedded in noncoding DNA are another class of SNPs suggested for prioritization due to their predicted quantitative impact on gene expression. The main challenge in evaluating these SNPs, in contrast to cSNPs is a lack of robust algorithms and databases for recognizing regulatory sequences in noncoding DNA. Approaches that have been previously used to delineate noncoding sequences with gene regulatory activity include cross-species sequence comparisons and the search for sequences recognized by transcription factors. We combined these two methods to sift through mouse human genomic sequences to identify putative gene regulatory elements and subsequently localized SNPs within these sequences in a 1 Megabase (Mb) region of human chromosome 5q31, orthologous to mouse chromosome 11 containing the Interleukin cluster.« less

Molecular cloning and characterization of rat sperm surface antigen 2B1, a glycoprotein implicated in sperm-zona binding.

PubMed

Hou, S T; Ma, A; Jones, R; Hall, L

1996-10-01

The rat sperm surface antigen, 2B1, that has been proposed to play a key role in sperm adhesion to the zona pellucida, has been cloned and its entire cDNA sequenced. Northern blot analysis indicates that 2B1 is encoded by a 2.2-kb RNA transcript that is abundantly expressed in the testis. The deduced protein sequence contains 512 amino-acid residues with a strong candidate signal sequence and C-terminal transmembrane domain. Data base searches reveal a high degree of sequence similarity to guinea pig, rabbit, monkey, and human PH20 sperm surface antigens, and a lower degree of similarity to honey bee and whiteface hornet venom hyaluronidases. Rat 2B1 antigen also possesses hyaluronidase activity, suggesting that it is a bifunctional protein with putative roles in the dispersion of cumulus oophorus cells as well as zona adhesion. However, while it would appear that 2B1 is the rat homologue of the guinea pig PH20 antigen, they differ in a number of important biochemical respects (including their mode of attachment to the sperm membrane and distribution between soluble and membrane-bound fractions), as well as in their localization on the sperm membrane. Expression of regions of the 2B1 protein in recombinant bacterial cells has allowed a preliminary mapping of the 2B1 epitope, and has provided more definitive information on the endoproteolytic processing of 2B1 during epididymal transit.

Genome of turbot rhabdovirus exhibits unusual non-coding regions and an additional ORF that could be expressed in fish cell.

PubMed

Zhu, Ruo-Lin; Lei, Xiao-Ying; Ke, Fei; Yuan, Xiu-Ping; Zhang, Qi-Ya

2011-02-01

Genomic sequence of Scophthalmus maximus rhabdovirus (SMRV) isolated from diseased turbot has been characterized. The complete genome of SMRV comprises 11,492 nucleotides and encodes five typical rhabdovirus genes N, P, M, G and L. In addition, two open reading frames (ORF) are predicted overlapping with P gene, one upstream of P and smaller than P (temporarily called Ps), and another in P gene which may encodes a protein similar to the vesicular stomatitis virus C protein. The C ORF is contained within the P ORF. The five typical proteins share the highest sequence identities (48.9%) with the corresponding proteins of rhabdoviruses in genus Vesiculovirus. Phylogenetic analysis of partial L protein sequence indicates that SMRV is close to genus Vesiculovirus. The first 13 nucleotides at the ends of the SMRV genome are absolutely inverse complementarity. The gene junctions between the five genes show conserved polyadenylation signal (CATGA(7)) and intergenic dinucleotide (CT) followed by putative transcription initiation sequence A(A/G)(C/G)A(A/G/T), which are different from known rhabdoviruses. The entire Ps ORF was cloned and expressed, and used to generate polyclonal antibody in mice. One obvious band could be detected in SMRV-infected carp leucocyte cells (CLCs) by anti-Ps/C serum via Western blot, and the subcellular localization of Ps-GFP fusion protein exhibited cytoplasm distribution as multiple punctuate or doughnut shaped foci of uneven size. Copyright © 2010 Elsevier B.V. All rights reserved.

Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli.

PubMed Central

Pi, J; Wookey, P J; Pittard, A J

1991-01-01

The phenylalanine-specific permease gene (pheP) of Escherichia coli has been cloned and sequenced. The gene was isolated on a 6-kb Sau3AI fragment from a chromosomal library, and its presence was verified by complementation of a mutant lacking the functional phenylalanine-specific permease. Subcloning from this fragment localized the pheP gene on a 2.7-kb HindIII-HindII fragment. The nucleotide sequence of this 2.7-kb region was determined. An open reading frame was identified which extends from a putative start point of translation (GTG at position 636) to a termination signal (TAA at position 2010). The assignment of the GTG as the initiation codon was verified by site-directed mutagenesis of the initiation codon and by introducing a chain termination mutation into the pheP-lacZ fusion construct. A single initiation site of transcription 30 bp upstream of the start point of translation was identified by the primer extension analysis. The pheP structural gene consists of 1,374 nucleotides specifying a protein of 458 amino acid residues. The PheP protein is very hydrophobic (71% nonpolar residues). A topological model predicted from the sequence analysis defines 12 transmembrane segments. This protein is highly homologous with the AroP (general aromatic transport) system of E. coli (59.6% identity) and to a lesser extent with the yeast permeases CAN1 (arginine), PUT4 (proline), and HIP1 (histidine) of Saccharomyces cerevisiae. Images PMID:1711024

The expression pattern of the C-terminal kinesin gene kifc1 during the spermatogenesis of Sepiella maindroni.

PubMed

Tan, Fu-Qing; Ma, Xiao-Xin; Zhu, Jun-Quan; Yang, Wan-Xi

2013-12-10

In this study, we investigated the gene sequence and characteristic of kifc1 in Sepiella maindroni through PCR and RACE technology. Our research aimed particularly at the spatio-temporal expression pattern of kifc1 in the developmental testis through in situ hybridization. The particular role of kifc1 in the spermatogenesis of S. maindroni was our particular interest. Based on multiple protein sequence alignments of KIFC1 homologues, kifc1 gene from the testis of S. maindroni was identified, which consisted of 2432bp including a 2109 in-frame ORF corresponding to 703 continuous amino acids. The encoded polypeptide shared highest similarity with Octopus tankahkeei. Through the prediction of the secondary and tertiary structures, the motor domain of KIFC1 was conserved at the C-terminal, having putative ATP-binding and microtubule-binding motifs, while the N-terminal was more specific to bind various cargoes for cellular events. The stalk domain connecting between the C-terminal and N-terminal determined the direction of movement. According to RT-PCR results, the kifc1 gene is not tissue-specific, commonly detected in different tissues, for example, the testis, liver, stomach, muscle, caecum and gills. Through an in situ hybridization method, the expression pattern of KIFC1 protein mimics in the spermatogenesis of S. maindroni. During the primary stage of the spermatogenesis, the kifc1 mRNA signal was barely detectable. At the early spermatids, the signal started to be present. With the elongation of spermatids, the signals increased substantially. It peaked and gathered around the acrosome area when the spermatids began to transform to spindle shape. As the spermatids developed into mature sperm, the signal vanished. In summary, the expression of kfic1 at specific stages during spermiogenesis and its distribution shed light on the potential functions of this motor in major cytological transformations. The KIFC1 homologue may provide a direct shaping force to the nucleus or influence the shaping process through indirect regulation. © 2013.

Acyl-Homoserine Lactone Production in Nitrifying Bacteria of the Genera Nitrosospira, Nitrobacter, and Nitrospira Identified via a Survey of Putative Quorum-Sensing Genes.

PubMed

Mellbye, Brett L; Spieck, Eva; Bottomley, Peter J; Sayavedra-Soto, Luis A

2017-11-15

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus , Nitrobacter , and Nitrospira Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N -decanoyl-l-homoserine lactone (C 10 -HSL), N -3-hydroxy-tetradecanoyl-l-homoserine lactone (3-OH-C 14 -HSL), a monounsaturated AHL (C 10:1 -HSL), and N -octanoyl-l-homoserine lactone (C 8 -HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria. IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with bacterial cell-cell signaling or quorum sensing (QS). QS is a method of bacterial communication and gene regulation that is well studied in bacterial pathogens, but less is known about QS in environmental systems. Our previous work suggested that QS might be involved in the regulation of nitrogen oxide gas production during nitrite metabolism. This study characterized putative QS signals produced by different genera and species of nitrifiers. Our work lays the foundation for future experiments investigating communication between nitrifying bacteria, the purpose of QS in these microorganisms, and the manipulation of QS during nitrification. Copyright © 2017 American Society for Microbiology.

Incorrectly predicted genes in rice?

PubMed

Cruveiller, Stéphane; Jabbari, Kamel; Clay, Oliver; Bernardi, Giorgio

2004-05-26

Between one third and one half of the proposed rice genes appear to have no homologs in other species, including Arabidopsis. Compositional considerations, and a comparison of curated rice sequences with ex novo predictions, suggest that many or most of the putative genes without homologs may be false positive predictions, i.e., sequences that are never translated into functional proteins in vivo.

Genome Sequence of an Alphaherpesvirus from a Beluga Whale (Delphinapterus leucas)

PubMed Central

Davison, Andrew J.; Nielsen, Ole; Jacob, Jessica M.; Romero, Carlos H.; Burek-Huntington, Kathy A.

2017-01-01

ABSTRACT Beluga whale alphaherpesvirus 1 was isolated from a blowhole swab taken from a juvenile beluga whale. The genome is 144,144 bp in size and contains 86 putative genes. The virus groups phylogenetically with members of the genus Varicellovirus in subfamily Alphaherpesvirinae and is the first alphaherpesvirus sequenced from a marine mammal. PMID:29051247

Genome Sequence of Actinobacillus seminis Strain ATCC 15768, a Reference Strain of Ovine Pathogens That Causes Infections in Reproductive Organs

PubMed Central

Negrete-Abascal, Erasmo; Montes-Garcia, Fernando; Vaca-Pacheco, Sergio; Leyto-Gil, Abraham M.; Fragoso-Garcia, Edgar; Carvente-Garcia, Roberto; Perez-Agueros, Sandra; Castelan-Sanchez, Hugo G.; Garcia-Molina, Alejandra; Villamar, Tomas E.; Sánchez-Alonso, Patricia

2018-01-01

ABSTRACT The draft genome sequence of Actinobacillus seminis strain ATCC 15768 is reported here. The genome comprises 22 contigs corresponding to 2.36 Mb with 40.7% G+C content and contains several genes related to virulence, including a putative RTX protein. PMID:29326222

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

PubMed Central

Moreira, Rebeca; Balseiro, Pablo; Planas, Josep V.; Fuste, Berta; Beltran, Sergi; Novoa, Beatriz; Figueras, Antonio

2012-01-01

Background The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. Methodology and Principal Findings High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. Conclusions This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum. PMID:22536348

The OGCleaner: filtering false-positive homology clusters.

PubMed

Fujimoto, M Stanley; Suvorov, Anton; Jensen, Nicholas O; Clement, Mark J; Snell, Quinn; Bybee, Seth M

2017-01-01

Detecting homologous sequences in organisms is an essential step in protein structure and function prediction, gene annotation and phylogenetic tree construction. Heuristic methods are often employed for quality control of putative homology clusters. These heuristics, however, usually only apply to pairwise sequence comparison and do not examine clusters as a whole. We present the Orthology Group Cleaner (the OGCleaner), a tool designed for filtering putative orthology groups as homology or non-homology clusters by considering all sequences in a cluster. The OGCleaner relies on high-quality orthologous groups identified in OrthoDB to train machine learning algorithms that are able to distinguish between true-positive and false-positive homology groups. This package aims to improve the quality of phylogenetic tree construction especially in instances of lower-quality transcriptome assemblies. https://github.com/byucsl/ogcleaner CONTACT: sfujimoto@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

Experimental Identification of Actinobacillus pleuropneumoniae Strains L20 and JL03 Heptosyltransferases, Evidence for a New Heptosyltransferase Signature Sequence

PubMed Central

Merino, Susana; Knirel, Yuriy A.; Regué, Miguel; Tomás, Juan M.

2013-01-01

We experimentally identified the activities of six predicted heptosyltransferases in Actinobacillus pleuropneumoniae genome serotype 5b strain L20 and serotype 3 strain JL03. The initial identification was based on a bioinformatic analysis of the amino acid similarity between these putative heptosyltrasferases with others of known function from enteric bacteria and Aeromonas. The putative functions of all the Actinobacillus pleuropneumoniae heptosyltrasferases were determined by using surrogate LPS acceptor molecules from well-defined A. hydrophyla AH-3 and A. salmonicida A450 mutants. Our results show that heptosyltransferases APL_0981 and APJL_1001 are responsible for the transfer of the terminal outer core D-glycero-D-manno-heptose (D,D-Hep) residue although they are not currently included in the CAZY glycosyltransferase 9 family. The WahF heptosyltransferase group signature sequence [S(T/S)(GA)XXH] differs from the heptosyltransferases consensus signature sequence [D(TS)(GA)XXH], because of the substitution of D261 for S261, being unique. PMID:23383222

The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution.

PubMed

Haarmann, Thomas; Machado, Caroline; Lübbe, Yvonne; Correia, Telmo; Schardl, Christopher L; Panaccione, Daniel G; Tudzynski, Paul

2005-06-01

The genomic region of Claviceps purpurea strain P1 containing the ergot alkaloid gene cluster [Tudzynski, P., Hölter, K., Correia, T., Arntz, C., Grammel, N., Keller, U., 1999. Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol. Gen. Genet. 261, 133-141] was explored by chromosome walking, and additional genes probably involved in the ergot alkaloid biosynthesis have been identified. The putative cluster sequence (extending over 68.5kb) contains 4 different nonribosomal peptide synthetase (NRPS) genes and several putative oxidases. Northern analysis showed that most of the genes were co-regulated (repressed by high phosphate), and identified probable flanking genes by lack of co-regulation. Comparison of the cluster sequences of strain P1, an ergotamine producer, with that of strain ECC93, an ergocristine producer, showed high conservation of most of the cluster genes, but significant variation in the NRPS modules, strongly suggesting that evolution of these chemical races of C. purpurea is determined by evolution of NRPS module specificity.

The complete mitochondrial genome sequence of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae) contains triplicate putative control regions.

PubMed

Yan, Dankan; Tang, Yunxia; Xue, Xiaofeng; Wang, Minghua; Liu, Fengquan; Fan, Jiaqin

2012-09-10

To investigate the features of the control region (CR) and the gene rearrangement in the mitochondrial (mt) genome of Thysanoptera insects, we sequenced the whole mt genome of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae). The mt genome is a circular molecule with 14,889 nucleotides and an A+T content of 76.6%, and it has triplicate putative CRs. We propose that tandem duplication and deletion account for the evolution of the CR and the gene translocations. Intramitochondrial recombination is a plausible model for the gene inversions. We discuss the excessive duplicate CR sequences and the transcription of the rRNA genes, which are distant from one another and from the CR. Finally, we address the significance of the complicated mt genomes in Thysanoptera for the evolution of the CR and the gene arrangement of the mt genome. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Application of Signaling Pathway-Based Adverse Outcome Pathways and High Throughput Toxicokinetic-PBPK for Developmental Cardiac Malformations

EPA Science Inventory

Associating putative molecular initiating events (MIE) with downstream cell signaling pathways and modeling fetal exposure kinetics is an important challenge for integration in developmental systems toxicology. Here, we describe an integrative systems toxicology model for develop...

Both neurons and astrocytes exhibited tetrodotoxin-resistant metabotropic glutamate receptor-dependent spontaneous slow Ca2+ oscillations in striatum.

PubMed

Tamura, Atsushi; Yamada, Naohiro; Yaguchi, Yuichi; Machida, Yoshio; Mori, Issei; Osanai, Makoto

2014-01-01

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson's disease. The group I mGluRs are known to modulate the intracellular Ca(2+) signaling. To characterize Ca(2+) signaling in striatal cells, spontaneous cytoplasmic Ca(2+) transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca(2+) transients (referred to as slow Ca(2+) oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca(2+) oscillation. Depletion of the intracellular Ca(2+) store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca(2+) oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca(2+) oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca(2+) oscillation in both putative-neurons and astrocytes. The slow Ca(2+) oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca(2+) oscillation may involve in the neuron-glia interaction in the striatum.

Exploring the Genes of Yerba Mate (Ilex paraguariensis A. St.-Hil.) by NGS and De Novo Transcriptome Assembly

PubMed Central

Aguilera, Patricia M.; Bubillo, Rosana E.; Otegui, Mónica B.; Ducasse, Daniel A.; Zapata, Pedro D.; Marti, Dardo A.

2014-01-01

Yerba mate (Ilex paraguariensis A. St.-Hil.) is an important subtropical tree crop cultivated on 326,000 ha in Argentina, Brazil and Paraguay, with a total yield production of more than 1,000,000 t. Yerba mate presents a strong limitation regarding sequence information. The NCBI GenBank lacks an EST database of yerba mate and depicts only 80 DNA sequences, mostly uncharacterized. In this scenario, in order to elucidate the yerba mate gene landscape by means of NGS, we explored and discovered a vast collection of I. paraguariensis transcripts. Total RNA from I. paraguariensis was sequenced by Illumina HiSeq-2000 obtaining 72,031,388 pair-end 100 bp sequences. High quality reads were de novo assembled into 44,907 transcripts encompassing 40 million bases with an estimated coverage of 180X. Multiple sequence analysis allowed us to predict that yerba mate contains ∼32,355 genes and 12,551 gene variants or isoforms. We identified and categorized members of more than 100 metabolic pathways. Overall, we have identified ∼1,000 putative transcription factors, genes involved in heat and oxidative stress, pathogen response, as well as disease resistance and hormone response. We have also identified, based in sequence homology searches, novel transcripts related to osmotic, drought, salinity and cold stress, senescence and early flowering. We have also pinpointed several members of the gene silencing pathway, and characterized the silencing effector Argonaute1. We predicted a diverse supply of putative microRNA precursors involved in developmental processes. We present here the first draft of the transcribed genomes of the yerba mate chloroplast and mitochondrion. The putative sequence and predicted structure of the caffeine synthase of yerba mate is presented. Moreover, we provide a collection of over 10,800 SSR accessible to the scientific community interested in yerba mate genetic improvement. This contribution broadly expands the limited knowledge of yerba mate genes, and is presented as the first genomic resource of this important crop. PMID:25330175

Development of a bioassay to screen for chemicals mimicking the anti-aging effects of calorie restriction

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

Chiba, Takuya, E-mail: takuya@nagasaki-u.ac.jp; Tsuchiya, Tomoshi; Komatsu, Toshimitsu

2010-10-15

Research highlights: {yields} We identified four sequence motifs lying upstream of putative pro-longevity genes. {yields} One of these motifs binds to HNF-4{alpha}. {yields} HNF-4{alpha}/PGC-1{alpha} could up-regulate the transcription of a reporter gene linked to this motif. {yields} The reporter system described here could be used to screen candidate anti-aging molecules. -- Abstract: Suppression of the growth hormone/insulin-like growth factor-I pathway in Ames dwarf (DF) mice, and caloric restriction (CR) in normal mice extends lifespan and delays the onset of age-related disorders. In combination, these interventions have an additive effect on lifespan in Ames DF mice. Therefore, common signaling pathways regulatedmore » by DF and CR could have additive effects on longevity. In this study, we tried to identity the signaling mechanism and develop a system to assess pro-longevity status in cells and mice. We previously identified genes up-regulated in the liver of DF and CR mice by DNA microarray analysis. Motif analysis of the upstream sequences of those genes revealed four major consensus sequence motifs, which have been named dwarfism and calorie restriction-responsive elements (DFCR-REs). One of the synthesized sequences bound to hepatocyte nuclear factor-4{alpha} (HNF-4{alpha}), an important transcription factor involved in liver metabolism. Furthermore, using this sequence information, we developed a highly sensitive bioassay to identify chemicals mimicking the anti-aging effects of CR. When the reporter construct, containing an element upstream of a secreted alkaline phosphatase (SEAP) gene, was co-transfected with HNF-4{alpha} and its regulator peroxisome proliferator-activated receptor (PPAR) {gamma} coactivator-1{alpha} (PGC-1{alpha}), SEAP activity was increased compared with untransfected controls. Moreover, transient transgenic mice established using this construct showed increased SEAP activity in CR mice compared with ad libitum-fed mice. These data suggest that because of its rapidity, ease of use, and specificity, our bioassay will be more useful than the systems currently employed to screen for CR mimetics, which mimic the beneficial effects of CR. Our system will be particularly useful for high-throughput screening of natural and synthetic candidate molecules.« less

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, characterization and leucocyte expression of Siglec-10, a novel human sialic acid-binding receptor.

PubMed Central

Munday, J; Kerr, S; Ni, J; Cornish, A L; Zhang, J Q; Nicoll, G; Floyd, H; Mattei, M G; Moore, P; Liu, D; Crocker, P R

2001-01-01

Here we characterize Siglec-10 as a new member of the Siglec family of sialic acid-binding Ig-like lectins. A full-length cDNA was isolated from a human spleen library and the corresponding gene identified. Siglec-10 is predicted to contain five extracellular Ig-like domains and a cytoplasmic tail containing three putative tyrosine-based signalling motifs. Siglec-10 exhibited a high degree of sequence similarity to CD33-related Siglecs and mapped to the same region, on chromosome 19q13.3. The expressed protein was able to mediate sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates. Using specific antibodies, Siglec-10 was detected on subsets of human leucocytes including eosinophils, monocytes and a minor population of natural killer-like cells. The molecular properties and expression pattern suggest that Siglec-10 may function as an inhibitory receptor within the innate immune system. PMID:11284738

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

Abraitiene, Asta; US Department of Agriculture, Agricultural Research Service, Molecular Plant Pathology Laboratory, Room 214 Building 004 BARC-West, 10300 Baltimore Avenue, Beltsville, MD 20705; Zhao Yan

Transient expression of engineered reporter RNAs encoding an intron-containing green fluorescent protein (GFP) from a Potato virus X-based expression vector previously demonstrated the nuclear targeting capability of the 359 nucleotide Potato spindle tuber viroid (PSTVd) RNA genome. To further delimit the putative nuclear-targeting signal, PSTVd subgenomic fragments were embedded within the intron, and recombinant reporter RNAs were inoculated onto Nicotiana benthamiana plants. Appearance of green fluorescence in leaf tissue inoculated with PSTVd-fragment-containing constructs indicated shuttling of the RNA into the nucleus by fragments as short as 80 nucleotides in length. Plant-to-plant variation in the timing of intron removal and subsequentmore » GFP fluorescence was observed; however, earliest and most abundant GFP expression was obtained with constructs containing the conserved hairpin I palindrome structure and embedded upper central conserved region. Our results suggest that this conserved sequence and/or the stem-loop structure it forms is sufficient for import of PSTVd into the nucleus.« less

Repeated divergent selection on pigmentation genes in a rapid finch radiation

PubMed Central

Campagna, Leonardo; Repenning, Márcio; Silveira, Luís Fábio; Fontana, Carla Suertegaray; Tubaro, Pablo L.; Lovette, Irby J.

2017-01-01

Instances of recent and rapid speciation are suitable for associating phenotypes with their causal genotypes, especially if gene flow homogenizes areas of the genome that are not under divergent selection. We study a rapid radiation of nine sympatric bird species known as capuchino seedeaters, which are differentiated in sexually selected characters of male plumage and song. We sequenced the genomes of a phenotypically diverse set of species to search for differentiated genomic regions. Capuchinos show differences in a small proportion of their genomes, yet selection has acted independently on the same targets in different members of this radiation. Many divergent regions contain genes involved in the melanogenesis pathway, with the strongest signal originating from putative regulatory regions. Selection has acted on these same genomic regions in different lineages, likely shaping the evolution of cis-regulatory elements, which control how more conserved genes are expressed and thereby generate diversity in classically sexually selected traits. PMID:28560331

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.

Dihydroartemisinin as a Putative STAT3 Inhibitor, Suppresses the Growth of Head and Neck Squamous Cell Carcinoma by Targeting Jak2/STAT3 Signaling

PubMed Central

Jia, Lifeng; Song, Qi; Zhou, Chenyang; Li, Xiaoming; Pi, Lihong; Ma, Xiuru; Li, Hui; Lu, Xiuying; Shen, Yupeng

2016-01-01

Developing drugs that can effectively block STAT3 activation may serve as one of the most promising strategy for cancer treatment. Currently, there is no putative STAT3 inhibitor that can be safely and effectively used in clinic. In the present study, we investigated the potential of dihydroartemisinin (DHA) as a putative STAT3 inhibitor and its antitumor activities in head and neck squamous cell carcinoma (HNSCC). The inhibitory effects of DHA on STAT3 activation along with its underlying mechanisms were studied in HNSCC cells. The antitumor effects of DHA against HNSCC cells were explored both in vitro and in vivo. An investigation on cooperative effects of DHA with cisplatin in killing HNSCC cells was also implemented. DHA exhibited remarkable and specific inhibitory effects on STAT3 activation via selectively blocking Jak2/STAT3 signaling. Besides, DHA significantly inhibited HNSCC growth both in vitro and in vivo possibly through induction of apoptosis and attenuation of cell migration. DHA also synergized with cisplatin in tumor inhibition in HNSCC cells. Our findings demonstrate that DHA is a putative STAT3 inhibitor that may represent a new and effective drug for cancer treatment and therapeutic sensitization in HNSCC patients. PMID:26784960

Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

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

Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

2003-12-31

Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involvedmore » in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.« less

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.

Annotation of Differentially Expressed Genes in the Somatic Embryogenesis of Musa and Their Location in the Banana Genome

PubMed Central

Maldonado-Borges, Josefina Ines; Ku-Cauich, José Roberto; Escobedo-GraciaMedrano, Rosa Maria

2013-01-01

Analysis of cDNA-AFLP was used to study the genes expressed in zygotic and somatic embryogenesis of Musa acuminata Colla ssp. malaccensis, and a comparison was made between their differential transcribed fragments (TDFs) and the sequenced genome of the double haploid- (DH-) Pahang of the malaccensis subspecies that is available in the network. A total of 253 transcript-derived fragments (TDFs) were detected with apparent size of 100–4000 bp using 5 pairs of AFLP primers, of which 21 were differentially expressed during the different stages of banana embryogenesis; 15 of the sequences have matched DH-Pahang chromosomes, with 7 of them being homologous to gene sequences encoding either known or putative protein domains of higher plants. Four TDF sequences were located in all Musa chromosomes, while the rest were located in one or two chromosomes. Their putative individual function is briefly reviewed based on published information, and the potential roles of these genes in embryo development are discussed. Thus the availability of the genome of Musa and the information of TDFs sequences presented here opens new possibilities for an in-depth study of the molecular and biochemical research of zygotic and somatic embryogenesis of Musa. PMID:24027442

Preparing and Analyzing Expressed Sequence Tags (ESTs) Library for the Mammary Tissue of Local Turkish Kivircik Sheep

PubMed Central

Omeroglu Ulu, Zehra; Ulu, Salih; Un, Cemal; Ozdem Oztabak, Kemal; Altunatmaz, Kemal

2017-01-01

Kivircik sheep is an important local Turkish sheep according to its meat quality and milk productivity. The aim of this study was to analyze gene expression profiles of both prenatal and postnatal stages for the Kivircik sheep. Therefore, two different cDNA libraries, which were taken from the same Kivircik sheep mammary gland tissue at prenatal and postnatal stages, were constructed. Total 3072 colonies which were randomly selected from the two libraries were sequenced for developing a sheep ESTs collection. We used Phred/Phrap computer programs for analysis of the raw EST and readable EST sequences were assembled with the CAP3 software. Putative functions of all unique sequences and statistical analysis were determined by Geneious software. Total 422 ESTs have over 80% similarity to known sequences of other organisms in NCBI classified by Panther database for the Gene Ontology (GO) category. By comparing gene expression profiles, we observed some putative genes that may be relative to reproductive performance or play important roles in milk synthesis and secretion. A total of 2414 ESTs have been deposited to the NCBI GenBank database (GW996847–GW999260). EST data in this study have provided a new source of information to functional genome studies of sheep. PMID:28239610

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach.

PubMed

Musumeci, Matías A; Lozada, Mariana; Rial, Daniela V; Mac Cormack, Walter P; Jansson, Janet K; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe M

2017-04-09

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

PubMed Central

Musumeci, Matías A.; Lozada, Mariana; Rial, Daniela V.; Mac Cormack, Walter P.; Jansson, Janet K.; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe M.

2017-01-01

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer–Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments. PMID:28397770

Comparative sequence analysis of a region on human chromosome 13q14, frequently deleted in B-cell chronic lymphocytic leukemia, and its homologous region on mouse chromosome 14.

PubMed

Kapanadze, B; Makeeva, N; Corcoran, M; Jareborg, N; Hammarsund, M; Baranova, A; Zabarovsky, E; Vorontsova, O; Merup, M; Gahrton, G; Jansson, M; Yankovsky, N; Einhorn, S; Oscier, D; Grandér, D; Sangfelt, O

2000-12-15

Previous studies have indicated the presence of a putative tumor suppressor gene on human chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have recently identified a minimally deleted region encompassing parts of two adjacent genes, termed LEU1 and LEU2 (leukemia-associated genes 1 and 2), and several additional transcripts. In addition, 50 kb centromeric to this region we have identified another gene, LEU5/RFP2. To elucidate further the complex genomic organization of this region, we have identified, mapped, and sequenced the homologous region in the mouse. Fluorescence in situ hybridization analysis demonstrated that the region maps to mouse chromosome 14. The overall organization and gene order in this region were found to be highly conserved in the mouse. Sequence comparison between the human deletion hotspot region and its homologous mouse region revealed a high degree of sequence conservation with an overall score of 74%. However, our data also show that in terms of transcribed sequences, only two of those, human LEU2 and LEU5/RFP2, are clearly conserved, strengthening the case for these genes as putative candidate B-CLL tumor suppressor genes.

Draft Genome Sequence of a Novel Chitinophaga sp. Strain, MD30, Isolated from a Biofilm in an Air Conditioner Condensate Pipe.

PubMed

Wan, Xuehua; Darris, Maxwell; Hou, Shaobin; Donachie, Stuart P

2017-10-19

Most of the 24 known Chitinophaga species were originally isolated from soils. We report the draft genome sequence of a putatively novel Chitinophaga sp. from a biofilm in an air conditioner condensate pipe. The genome comprises 7,661,303 bp in one scaffold, 5,694 predicted protein-coding sequences, and a G+C content of 47.6%. Copyright © 2017 Wan et al.

Assessing the genetic diversity of Cu resistance in mine tailings through high-throughput recovery of full-length copA genes

PubMed Central

Li, Xiaofang; Zhu, Yong-Guan; Shaban, Babak; Bruxner, Timothy J. C.; Bond, Philip L.; Huang, Longbin

2015-01-01

Characterizing the genetic diversity of microbial copper (Cu) resistance at the community level remains challenging, mainly due to the polymorphism of the core functional gene copA. In this study, a local BLASTN method using a copA database built in this study was developed to recover full-length putative copA sequences from an assembled tailings metagenome; these sequences were then screened for potentially functioning CopA using conserved metal-binding motifs, inferred by evolutionary trace analysis of CopA sequences from known Cu resistant microorganisms. In total, 99 putative copA sequences were recovered from the tailings metagenome, out of which 70 were found with high potential to be functioning in Cu resistance. Phylogenetic analysis of selected copA sequences detected in the tailings metagenome showed that topology of the copA phylogeny is largely congruent with that of the 16S-based phylogeny of the tailings microbial community obtained in our previous study, indicating that the development of copA diversity in the tailings might be mainly through vertical descent with few lateral gene transfer events. The method established here can be used to explore copA (and potentially other metal resistance genes) diversity in any metagenome and has the potential to exhaust the full-length gene sequences for downstream analyses. PMID:26286020

A Third Approach to Gene Prediction Suggests Thousands of Additional Human Transcribed Regions

PubMed Central

Glusman, Gustavo; Qin, Shizhen; El-Gewely, M. Raafat; Siegel, Andrew F; Roach, Jared C; Hood, Leroy; Smit, Arian F. A

2006-01-01

The identification and characterization of the complete ensemble of genes is a main goal of deciphering the digital information stored in the human genome. Many algorithms for computational gene prediction have been described, ultimately derived from two basic concepts: (1) modeling gene structure and (2) recognizing sequence similarity. Successful hybrid methods combining these two concepts have also been developed. We present a third orthogonal approach to gene prediction, based on detecting the genomic signatures of transcription, accumulated over evolutionary time. We discuss four algorithms based on this third concept: Greens and CHOWDER, which quantify mutational strand biases caused by transcription-coupled DNA repair, and ROAST and PASTA, which are based on strand-specific selection against polyadenylation signals. We combined these algorithms into an integrated method called FEAST, which we used to predict the location and orientation of thousands of putative transcription units not overlapping known genes. Many of the newly predicted transcriptional units do not appear to code for proteins. The new algorithms are particularly apt at detecting genes with long introns and lacking sequence conservation. They therefore complement existing gene prediction methods and will help identify functional transcripts within many apparent “genomic deserts.” PMID:16543943

Genome-Wide Identification and Characterization of Novel Laccase Genes in the White-Rot Fungus Flammulina velutipes

PubMed Central

Kim, Hong-Il; Kwon, O-Chul; Kong, Won-Sik; Lee, Chang-Soo

2014-01-01

The aim of this study was to identify and characterize new Flammulina velutipes laccases from its whole-genome sequence. Of the 15 putative laccase genes detected in the F. velutipes genome, four new laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) were found to contain four complete copper-binding regions (ten histidine residues and one cysteine residue) and four cysteine residues involved in forming disulfide bridges, fvLac-1, fvLac-2, fvLac3, and fvLac-4, encoding proteins consisting of 516, 518, 515, and 533 amino acid residues, respectively. Potential N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified in the cDNA sequence of fvLac-1 (Asn-454), fvLac-2 (Asn-437 and Asn-455), fvLac-3 (Asn-111 and Asn-237), and fvLac4 (Asn-402 and Asn-457). In addition, the first 19~20 amino acid residues of these proteins were predicted to comprise signal peptides. Laccase activity assays and reverse transcription polymerase chain reaction analyses clearly reveal that CuSO4 affects the induction and the transcription level of these laccase genes. PMID:25606003

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

Rapid development of molecular markers by next-generation sequencing linked to a gene conferring phomopsis stem blight disease resistance for marker-assisted selection in lupin (Lupinus angustifolius L.) breeding.

PubMed

Yang, Huaan; Tao, Ye; Zheng, Zequn; Shao, Di; Li, Zhenzhong; Sweetingham, Mark W; Buirchell, Bevan J; Li, Chengdao

2013-02-01

Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F(8) recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F(8) population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of "false positives" (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in development of markers for molecular plant breeding.

Overexpression of a cotton gene that encodes a putative transcription factor of AP2/EREBP family in Arabidopsis affects growth and development of transgenic plants.

PubMed

Zhou, Ying; Xia, Hui; Li, Xiao-Jie; Hu, Rong; Chen, Yun; Li, Xue-Bao

2013-01-01

In the study, a gene encoding a putative ethylene response factor of AP2/EREBP family was isolated from cotton (Gossypium hirsutum) and designated as GhERF12. Sequence alignment showed that GhERF12 protein contains a central AP2/ERF domain (58 amino acids) with two functional conserved amino acid residues (ala14 and asp19). Transactivation assay indicated that GhERF12 displayed strong transcription activation activity in yeast cells, suggesting that this protein may be a transcriptional activator in cotton. Quantitative RT-PCR analysis showed that GhERF12 expression in cotton was induced by ACC and IAA. Overexpression of GhERF12 in Arabidopsis affected seedling growth and development. The GhERF12 transgenic plants grew slowly, and displayed a dwarf phenotype. The mean bolting time of the transgenic plants was delayed for about 10 days, compared with that of wild type. Further study revealed that some ethylene-related and auxin-related genes were dramatically up-regulated in the transgenic plants, compared with those of wild type. Collectively, we speculated that GhERF12, as a transcription factor, may be involved in regulation of plant growth and development by activating the constitutive ethylene response likely related to auxin biosynthesis and/or signaling.

Transcriptional Response of Virus-Infected Cassava and Identification of Putative Sources of Resistance for Cassava Brown Streak Disease

PubMed Central

Maruthi, M. N.; Bouvaine, Sophie; Tufan, Hale A.; Mohammed, Ibrahim U.; Hillocks, Rory J.

2014-01-01

Cassava (Manihot esculenta) is a major food staple in sub-Saharan Africa, which is severely affected by cassava brown streak disease (CBSD). The aim of this study was to identify resistance for CBSD as well as to understand the mechanism of putative resistance for providing effective control for the disease. Three cassava varieties; Kaleso, Kiroba and Albert were inoculated with cassava brown streak viruses by grafting and also using the natural insect vector the whitefly, Bemisia tabaci. Kaleso expressed mild or no disease symptoms and supported low concentrations of viruses, which is a characteristic of resistant plants. In comparison, Kiroba expressed severe leaf but milder root symptoms, while Albert was susceptible with severe symptoms both on leaves and roots. Real-time PCR was used to estimate virus concentrations in cassava varieties. Virus quantities were higher in Kiroba and Albert compared to Kaleso. The Illumina RNA-sequencing was used to further understand the genetic basis of resistance. More than 700 genes were uniquely overexpressed in Kaleso in response to virus infection compared to Albert. Surprisingly, none of them were similar to known resistant gene orthologs. Some of the overexpressed genes, however, belonged to the hormone signalling pathways and secondary metabolites, both of which are linked to plant resistance. These genes should be further characterised before confirming their role in resistance to CBSD. PMID:24846209

Overexpression of a Cotton Gene That Encodes a Putative Transcription Factor of AP2/EREBP Family in Arabidopsis Affects Growth and Development of Transgenic Plants

PubMed Central

Li, Xiao-Jie; Hu, Rong; Chen, Yun; Li, Xue-Bao

2013-01-01

In the study, a gene encoding a putative ethylene response factor of AP2/EREBP family was isolated from cotton (Gossypium hirsutum) and designated as GhERF12. Sequence alignment showed that GhERF12 protein contains a central AP2/ERF domain (58 amino acids) with two functional conserved amino acid residues (ala14 and asp19). Transactivation assay indicated that GhERF12 displayed strong transcription activation activity in yeast cells, suggesting that this protein may be a transcriptional activator in cotton. Quantitative RT-PCR analysis showed that GhERF12 expression in cotton was induced by ACC and IAA. Overexpression of GhERF12 in Arabidopsis affected seedling growth and development. The GhERF12 transgenic plants grew slowly, and displayed a dwarf phenotype. The mean bolting time of the transgenic plants was delayed for about 10 days, compared with that of wild type. Further study revealed that some ethylene-related and auxin-related genes were dramatically up-regulated in the transgenic plants, compared with those of wild type. Collectively, we speculated that GhERF12, as a transcription factor, may be involved in regulation of plant growth and development by activating the constitutive ethylene response likely related to auxin biosynthesis and/or signaling. PMID:24194949

Base Flipping in V(D)J Recombination: Insights into the Mechanism of Hairpin Formation, the 12/23 Rule, and the Coordination of Double-Strand Breaks▿ †

PubMed Central

Bischerour, Julien; Lu, Catherine; Roth, David B.; Chalmers, Ronald

2009-01-01

Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position −1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex. PMID:19720743

Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

PubMed

Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

2013-09-01

Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

PubMed

Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

2013-08-01

Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

Expressed sequence tag analysis of functional genes associated with adventitious rooting in Liriodendron hybrids.

PubMed

Zhong, Y D; Sun, X Y; Liu, E Y; Li, Y Q; Gao, Z; Yu, F X

2016-06-24

Liriodendron hybrids (Liriodendron chinense x L. tulipifera) are important landscaping and afforestation hardwood trees. To date, little genomic research on adventitious rooting has been reported in these hybrids, as well as in the genus Liriodendron. In the present study, we used adventitious roots to construct the first cDNA library for Liriodendron hybrids. A total of 5176 expressed sequence tags (ESTs) were generated and clustered into 2921 unigenes. Among these unigenes, 2547 had significant homology to the non-redundant protein database representing a wide variety of putative functions. Homologs of these genes regulated many aspects of adventitious rooting, including those for auxin signal transduction and root hair development. Results of quantitative real-time polymerase chain reaction showed that AUX1, IRE, and FB1 were highly expressed in adventitious roots and the expression of AUX1, ARF1, NAC1, RHD1, and IRE increased during the development of adventitious roots. Additionally, 181 simple sequence repeats were identified from 166 ESTs and more than 91.16% of these were dinucleotide and trinucleotide repeats. To the best of our knowledge, the present study reports the identification of the genes associated with adventitious rooting in the genus Liriodendron for the first time and provides a valuable resource for future genomic studies. Expression analysis of selected genes could allow us to identify regulatory genes that may be essential for adventitious rooting.

The primary and subunit structure of a novel type killer toxin produced by a halotolerant yeast, Pichia farinosa.

PubMed

Suzuki, C; Nikkuni, S

1994-01-28

A halotolerant yeast, Pichia farinosa KK1 strain, produces a unique killer toxin termed SMK toxin (salt-mediated killer toxin) which shows its maximum killer activity in the presence of 2 M NaCl. The toxin consists of two distinct subunits, alpha and beta, which are tightly linked without a disulfide bond under acidic conditions, even in the presence of 6 M urea. Under neutral conditions, however, the alpha subunit precipitates, resulting in the dissociation of the subunits and the loss of killer activity. The nucleotide sequence of the SMK1 gene predicts a 222 amino acid preprotoxin with a typical signal sequence, the hydrophobic alpha, an interstitial gamma polypeptide with a putative glycosylation site, and the hydrophilic beta. Amino acid sequence analyses of peptide fragments including the carboxyl-terminal peptides fragments including the carboxyl-terminal peptides from each subunit suggest that the alpha and beta subunits consist of amino acid residues 19-81 and 146-222 of the preprotoxin, respectively, and the molecular weight of the mature alpha beta dimer is 14,214. The KEX2-like endopeptidase and KEX1-like carboxypeptidase may be involved in the stepwise processing of the SMK preprotoxin. The maturation process and the functions of the SMK toxin are compared with the K1 toxin of Saccharomyces cerevisiae.

RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.

PubMed

Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

2016-01-01

Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

Persistence of evolutionary memory: primordial six-transmembrane helical domain mu opiate receptors selectively linked to endogenous morphine signaling.

PubMed

Kream, Richard M; Sheehan, Melinda; Cadet, Patrick; Mantione, Kirk J; Zhu, Wei; Casares, Federico; Stefano, George B

2007-12-01

Biochemical, molecular and pharmacological evidence for two unique six-transmembrane helical (TMH) domain opiate receptors expressed from the micro opioid receptor (MOR) gene have been shown. Designated micro3 and micro4 receptors, both protein species are Class A rhodopsin-like members of the superfamily of G-protein coupled receptors but are selectively tailored to mediate the cellular regulatory effects of endogenous morphine and related morphinan alkaloids via stimulation of nitric oxide (NO) production and release. Both micro3 and micro4 receptors lack an amino acid sequence of approximately 90 amino acids that constitute the extracellular N-terminal and TMH1 domains and part of the first intracellular loop of the micro1 receptor, but retain the empirically defined ligand binding pocket distributed across conserved TMH2, TMH3, and TMH7 domains of the micro1 sequence. Additionally, the receptor proteins are terminated by unique intracellular C-terminal amino acid sequences that serve as putative coupling or docking domains required for constitutive NO synthase activation. Because the recognition profile of micro3 and micro4 receptors is restricted to rigid benzylisoquinoline alkaloids typified by morphine and its extended family of chemical congeners, it is hypothesized that conformational stabilization provided by interaction of extended extracellular N-terminal protein domains and the extracellular loops is required for binding of endogenous opioid peptides as well as synthetic flexible opiate alkaloids.

Characterisation of a large family of polymorphic collagen-like proteins in the endospore-forming bacterium Pasteuria ramosa.

PubMed

McElroy, Kerensa; Mouton, Laurence; Du Pasquier, Louis; Qi, Weihong; Ebert, Dieter

2011-09-01

Collagen-like proteins containing glycine-X-Y repeats have been identified in several pathogenic bacteria potentially involved in virulence. Recently, a collagen-like surface protein, Pcl1a, was identified in Pasteuria ramosa, a spore-forming parasite of Daphnia. Here we characterise 37 novel putative P. ramosa collagen-like protein genes (PCLs). PCR amplification and sequencing across 10 P. ramosa strains showed they were polymorphic, distinguishing genotypes matching known differences in Daphnia/P. ramosa interaction specificity. Thirty PCLs could be divided into four groups based on sequence similarity, conserved N- and C-terminal regions and G-X-Y repeat structure. Group 1, Group 2 and Group 3 PCLs formed triplets within the genome, with one member from each group represented in each triplet. Maximum-likelihood trees suggested that these groups arose through multiple instances of triplet duplication. For Group 1, 2, 3 and 4 PCLs, X was typically proline and Y typically threonine, consistent with other bacterial collagen-like proteins. The amino acid composition of Pcl2 closely resembled Pcl1a, with X typically being glutamic acid or aspartic acid and Y typically being lysine or glutamine. Pcl2 also showed sequence similarity to Pcl1a and contained a predicted signal peptide, cleavage site and transmembrane domain, suggesting that it is a surface protein. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Replication of a low-pathogenic avian influenza virus is enhanced by chicken ubiquitin-specific protease 18.

PubMed

Tanikawa, Taichiro; Uchida, Yuko; Saito, Takehiko

2017-09-01

Previous research revealed the induction of chicken USP18 (chUSP18) in the lungs of chickens infected with highly pathogenic avian influenza viruses (HPAIVs). This activity was correlated with the degree of pathogenicity of the viruses to chickens. As mammalian ubiquitin-specific protease (USP18) is known to remove type I interferon (IFN I)-inducible ubiquitin-like molecules from conjugated proteins and block IFN I signalling, we explored the function of the chicken homologue of USP18 during avian influenza virus infection. With this aim, we cloned chUSP18 from cultured chicken cells and revealed that the putative chUSP18 ORF comprises 1137 bp. Comparative analysis of the predicted aa sequence of chUSP18 with those of human and mouse USP18 revealed relatively high sequence similarity among the sequences, including domains specific for the ubiquitin-specific processing protease family. Furthermore, we found that chUSP18 expression was induced by chicken IFN I, as observed for mammalian USP18. Experiments based on chUSP18 over-expression and depletion demonstrated that chUSP18 significantly enhanced the replication of a low-pathogenic avian influenza virus (LPAIV), but not an HPAIV. Our findings suggest that chUSP18, being similar to mammalian USP18, acts as a pro-viral factor during LPAIV replication in vitro.

Within-Genome Evolution of REPINs: a New Family of Miniature Mobile DNA in Bacteria

PubMed Central

Bertels, Frederic; Rainey, Paul B.

2011-01-01

Repetitive sequences are a conserved feature of many bacterial genomes. While first reported almost thirty years ago, and frequently exploited for genotyping purposes, little is known about their origin, maintenance, or processes affecting the dynamics of within-genome evolution. Here, beginning with analysis of the diversity and abundance of short oligonucleotide sequences in the genome of Pseudomonas fluorescens SBW25, we show that over-represented short sequences define three distinct groups (GI, GII, and GIII) of repetitive extragenic palindromic (REP) sequences. Patterns of REP distribution suggest that closely linked REP sequences form a functional replicative unit: REP doublets are over-represented, randomly distributed in extragenic space, and more highly conserved than singlets. In addition, doublets are organized as inverted repeats, which together with intervening spacer sequences are predicted to form hairpin structures in ssDNA or mRNA. We refer to these newly defined entities as REPINs (REP doublets forming hairpins) and identify short reads from population sequencing that reveal putative transposition intermediates. The proximal relationship between GI, GII, and GIII REPINs and specific REP-associated tyrosine transposases (RAYTs), combined with features of the putative transposition intermediate, suggests a mechanism for within-genome dissemination. Analysis of the distribution of REPs in a range of RAYT–containing bacterial genomes, including Escherichia coli K-12 and Nostoc punctiforme, show that REPINs are a widely distributed, but hitherto unrecognized, family of miniature non-autonomous mobile DNA. PMID:21698139

Identification and characterization of ARS-like sequences as putative origin(s) of replication in human malaria parasite Plasmodium falciparum.

PubMed

Agarwal, Meetu; Bhowmick, Krishanu; Shah, Kushal; Krishnamachari, Annangarachari; Dhar, Suman Kumar

2017-08-01

DNA replication is a fundamental process in genome maintenance, and initiates from several genomic sites (origins) in eukaryotes. In Saccharomyces cerevisiae, conserved sequences known as autonomously replicating sequences (ARSs) provide a landing pad for the origin recognition complex (ORC), leading to replication initiation. Although origins from higher eukaryotes share some common sequence features, the definitive genomic organization of these sites remains elusive. The human malaria parasite Plasmodium falciparum undergoes multiple rounds of DNA replication; therefore, control of initiation events is crucial to ensure proper replication. However, the sites of DNA replication initiation and the mechanism by which replication is initiated are poorly understood. Here, we have identified and characterized putative origins in P. falciparum by bioinformatics analyses and experimental approaches. An autocorrelation measure method was initially used to search for regions with marked fluctuation (dips) in the chromosome, which we hypothesized might contain potential origins. Indeed, S. cerevisiae ARS consensus sequences were found in dip regions. Several of these P. falciparum sequences were validated with chromatin immunoprecipitation-quantitative PCR, nascent strand abundance and a plasmid stability assay. Subsequently, the same sequences were used in yeast to confirm their potential as origins in vivo. Our results identify the presence of functional ARSs in P. falciparum and provide meaningful insights into replication origins in these deadly parasites. These data could be useful in designing transgenic vectors with improved stability for transfection in P. falciparum. © 2017 Federation of European Biochemical Societies.

High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

PubMed Central

2010-01-01

Background Bathymodiolus azoricus is a deep-sea hydrothermal vent mussel found in association with large faunal communities living in chemosynthetic environments at the bottom of the sea floor near the Azores Islands. Investigation of the exceptional physiological reactions that vent mussels have adopted in their habitat, including responses to environmental microbes, remains a difficult challenge for deep-sea biologists. In an attempt to reveal genes potentially involved in the deep-sea mussel innate immunity we carried out a high-throughput sequence analysis of freshly collected B. azoricus transcriptome using gills tissues as the primary source of immune transcripts given its strategic role in filtering the surrounding waterborne potentially infectious microorganisms. Additionally, a substantial EST data set was produced and from which a comprehensive collection of genes coding for putative proteins was organized in a dedicated database, "DeepSeaVent" the first deep-sea vent animal transcriptome database based on the 454 pyrosequencing technology. Results A normalized cDNA library from gills tissue was sequenced in a full 454 GS-FLX run, producing 778,996 sequencing reads. Assembly of the high quality reads resulted in 75,407 contigs of which 3,071 were singletons. A total of 39,425 transcripts were conceptually translated into amino-sequences of which 22,023 matched known proteins in the NCBI non-redundant protein database, 15,839 revealed conserved protein domains through InterPro functional classification and 9,584 were assigned with Gene Ontology terms. Queries conducted within the database enabled the identification of genes putatively involved in immune and inflammatory reactions which had not been previously evidenced in the vent mussel. Their physical counterpart was confirmed by semi-quantitative quantitative Reverse-Transcription-Polymerase Chain Reactions (RT-PCR) and their RNA transcription level by quantitative PCR (qPCR) experiments. Conclusions We have established the first tissue transcriptional analysis of a deep-sea hydrothermal vent animal and generated a searchable catalog of genes that provides a direct method of identifying and retrieving vast numbers of novel coding sequences which can be applied in gene expression profiling experiments from a non-conventional model organism. This provides the most comprehensive sequence resource for identifying novel genes currently available for a deep-sea vent organism, in particular, genes putatively involved in immune and inflammatory reactions in vent mussels. The characterization of the B. azoricus transcriptome will facilitate research into biological processes underlying physiological adaptations to hydrothermal vent environments and will provide a basis for expanding our understanding of genes putatively involved in adaptations processes during post-capture long term acclimatization experiments, at "sea-level" conditions, using B. azoricus as a model organism. PMID:20937131

Complete genome sequence of Bacillus amyloliquefaciens strain Co1-6, a plant growth-promoting rhizobacterium of Calendula officinalis

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

Köberl, Martina; White, Richard A.; Erschen, Sabine

The genome sequence of Bacillus amyloliquefaciens strain Co1-6, a plant growth-promoting rhizobacterium (PGPR) with broad-spectrum antagonistic activity against plant-pathogenic fungi, bacteria, and nematodes, consists of a single 3.9-Mb circular chromosome. The genome reveals genes putatively responsible for its promising biocontrol and PGP properties.

Complete genome sequence of Bacillus amyloliquefaciens strain Co1-6, a plant growth-promoting rhizobacterium of Calendula officinalis

DOE PAGES

Köberl, Martina; White, Richard A.; Erschen, Sabine; ...

2015-08-13

The genome sequence of Bacillus amyloliquefaciens strain Co1-6, a plant growth-promoting rhizobacterium (PGPR) with broad-spectrum antagonistic activity against plant-pathogenic fungi, bacteria, and nematodes, consists of a single 3.9-Mb circular chromosome. The genome reveals genes putatively responsible for its promising biocontrol and PGP properties.

Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar

Treesearch

John Kuzio; Margot N. Pearson; Steve H. Harwood; C. Joel Funk; Jay T. Evans; James M. Slavicek; George F. Rohrmann

1999-01-01

The genome of the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) was sequenced and analyzed. It is composed of 161,046 bases with a G + C content of 57.5% and contains 163 putative open reading frames (ORFs) of ≥150 nucleotides. Homologs were found to 95 of the 155 genes predicted for the Autographa californica...

Genome Sequence of Lactobacillus sakei LK-145 Isolated from a Japanese Sake Cellar as a High Producer of d-Amino Acids

PubMed Central

Kato, Shiro

2017-01-01

ABSTRACT This announcement reports the complete genome sequence of strain LK-145 of Lactobacillus sakei isolated from a Japanese sake cellar as a potent strain for the production of large amounts of d-amino acids. Three putative genes encoding an amino acid racemase were identified. PMID:28818888

Genome Sequence of an Alphaherpesvirus from a Beluga Whale (Delphinapterus leucas).

PubMed

Davison, Andrew J; Nielsen, Ole; Subramaniam, Kuttichantran; Jacob, Jessica M; Romero, Carlos H; Burek-Huntington, Kathy A; Waltzek, Thomas B

2017-10-19

Beluga whale alphaherpesvirus 1 was isolated from a blowhole swab taken from a juvenile beluga whale. The genome is 144,144 bp in size and contains 86 putative genes. The virus groups phylogenetically with members of the genus Varicellovirus in subfamily Alphaherpesvirinae and is the first alphaherpesvirus sequenced from a marine mammal. Copyright © 2017 Davison et al.

Genome Sequence of Actinobacillus seminis Strain ATCC 15768, a Reference Strain of Ovine Pathogens That Causes Infections in Reproductive Organs.

PubMed

Negrete-Abascal, Erasmo; Montes-Garcia, Fernando; Vaca-Pacheco, Sergio; Leyto-Gil, Abraham M; Fragoso-Garcia, Edgar; Carvente-Garcia, Roberto; Perez-Agueros, Sandra; Castelan-Sanchez, Hugo G; Garcia-Molina, Alejandra; Villamar, Tomas E; Sánchez-Alonso, Patricia; Vazquez-Cruz, Candelario

2018-01-11

The draft genome sequence of Actinobacillus seminis strain ATCC 15768 is reported here. The genome comprises 22 contigs corresponding to 2.36 Mb with 40.7% G+C content and contains several genes related to virulence, including a putative RTX protein. Copyright © 2018 Negrete-Abascal et al.

Joint Estimation of Contamination, Error and Demography for Nuclear DNA from Ancient Humans

PubMed Central

Slatkin, Montgomery

2016-01-01

When sequencing an ancient DNA sample from a hominin fossil, DNA from present-day humans involved in excavation and extraction will be sequenced along with the endogenous material. This type of contamination is problematic for downstream analyses as it will introduce a bias towards the population of the contaminating individual(s). Quantifying the extent of contamination is a crucial step as it allows researchers to account for possible biases that may arise in downstream genetic analyses. Here, we present an MCMC algorithm to co-estimate the contamination rate, sequencing error rate and demographic parameters—including drift times and admixture rates—for an ancient nuclear genome obtained from human remains, when the putative contaminating DNA comes from present-day humans. We assume we have a large panel representing the putative contaminant population (e.g. European, East Asian or African). The method is implemented in a C++ program called ‘Demographic Inference with Contamination and Error’ (DICE). We applied it to simulations and genome data from ancient Neanderthals and modern humans. With reasonable levels of genome sequence coverage (>3X), we find we can recover accurate estimates of all these parameters, even when the contamination rate is as high as 50%. PMID:27049965

Transcriptome Sequencing, and Rapid Development and Application of SNP Markers for the Legume Pod Borer Maruca vitrata (Lepidoptera: Crambidae)

PubMed Central

Margam, Venu M.; Coates, Brad S.; Bayles, Darrell O.; Hellmich, Richard L.; Agunbiade, Tolulope; Seufferheld, Manfredo J.; Sun, Weilin; Kroemer, Jeremy A.; Ba, Malick N.; Binso-Dabire, Clementine L.; Baoua, Ibrahim; Ishiyaku, Mohammad F.; Covas, Fernando G.; Srinivasan, Ramasamy; Armstrong, Joel; Murdock, Larry L.; Pittendrigh, Barry R.

2011-01-01

The legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), is an insect pest species of crops grown by subsistence farmers in tropical regions of Africa. We present the de novo assembly of 3729 contigs from 454- and Sanger-derived sequencing reads for midgut, salivary, and whole adult tissues of this non-model species. Functional annotation predicted that 1320 M. vitrata protein coding genes are present, of which 631 have orthologs within the Bombyx mori gene model. A homology-based analysis assigned M. vitrata genes into a group of paralogs, but these were subsequently partitioned into putative orthologs following phylogenetic analyses. Following sequence quality filtering, a total of 1542 putative single nucleotide polymorphisms (SNPs) were predicted within M. vitrata contig assemblies. Seventy one of 1078 designed molecular genetic markers were used to screen M. vitrata samples from five collection sites in West Africa. Population substructure may be present with significant implications in the insect resistance management recommendations pertaining to the release of biological control agents or transgenic cowpea that express Bacillus thuringiensis crystal toxins. Mutation data derived from transcriptome sequencing is an expeditious and economical source for genetic markers that allow evaluation of ecological differentiation. PMID:21754987

Nucleotide sequence of a resistance breaking mutant of southern bean mosaic virus.

PubMed

Lee, L; Anderson, E J

1998-01-01

SBMV-S is a resistance-breaking mutant of an Arkansas isolate of the bean strain of southern bean mosaic virus (SBMV-BARK) that is able to move systemically in Phaseolus vulgaris cvs. Pinto and Great Northern, whereas the wild-type SBMV-BARK causes local necrotic lesions and is restricted to the inoculated leaves of these hosts. Sequence analysis of the 4136 nucleotide genomes of SBMV-BARK and SBMV-S revealed seven nucleotide differences, but only four deduced amino acid changes. A single amino acid change occurred in the C-terminal region of the putative RNA-dependent RNA polymerase and three differences were identified in the N-terminal portion of the virus coat protein. SBMV-BARK and SBMV-S were compared with other sobemoviruses and were found to contain a high level of nucleotide sequence identity (91.3%) to SBMV-B. Unlike SBMV-B however, SBMV-BARK and SBMV-S contained four putative overlapping open reading frames, making them more similar in genome organization to the cowpea strain, SBMV-C. The possibility exists that mutations or even errors, that resulted in mis-identification of open reading frames, occurred in previously published information on nucleotide sequence and genomic organization for SBMV-B.

Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

PubMed

van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

2016-01-01

RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.

Characterization of the venom from the Australian scorpion Urodacus yaschenkoi: Molecular mass analysis of components, cDNA sequences and peptides with antimicrobial activity.

PubMed

Luna-Ramírez, Karen; Quintero-Hernández, Veronica; Vargas-Jaimes, Leonel; Batista, Cesar V F; Winkel, Kenneth D; Possani, Lourival D

2013-03-01

The Urodacidae scorpions are the most widely distributed of the four families in Australia and represent half of the species in the continent, yet their venoms remain largely unstudied. This communication reports the first results of a proteome analysis of the venom of the scorpion Urodacus yaschenkoi performed by mass fingerprinting, after high performance liquid chromatography (HPLC) separation. A total of 74 fractions were obtained by HPLC separation allowing the identification of approximately 274 different molecular masses with molecular weights varying from 287 to 43,437 Da. The most abundant peptides were those from 1 K Da and 4-5 K Da representing antimicrobial peptides and putative potassium channel toxins, respectively. Three such peptides were chemically synthesized and tested against Gram-positive and Gram-negative bacteria showing minimum inhibitory concentration in the low micromolar range, but with moderate hemolytic activity. It also reports a transcriptome analysis of the venom glands of the same scorpion species, undertaken by constructing a cDNA library and conducting random sequencing screening of the transcripts. From the resultant cDNA library 172 expressed sequence tags (ESTs) were analyzed. These transcripts were further clustered into 120 unique sequences (23 contigs and 97 singlets). The identified putative proteins can be assorted in several groups, such as those implicated in common cellular processes, putative neurotoxins and antimicrobial peptides. The scorpion U. yaschenkoi is not known to be dangerous to humans and its venom contains peptides similar to those of Opisthacanthus cayaporum (antibacterial), Scorpio maurus palmatus (maurocalcin), Opistophthalmus carinatus (opistoporines) and Hadrurus gerstchi (scorpine-like molecules), amongst others. Copyright © 2012 Elsevier Ltd. All rights reserved.

Genome of Epinotia aporema granulovirus (EpapGV), a polyorganotropic fast killing betabaculovirus with a novel thymidylate kinase gene

PubMed Central

2012-01-01

Background Epinotia aporema (Lepidoptera: Tortricidae) is an important pest of legume crops in South America. Epinotia aporema granulovirus (EpapGV) is a baculovirus that causes a polyorganotropic infection in the host larva. Its high pathogenicity and host specificity make EpapGV an excellent candidate to be used as a biological control agent. Results The genome of Epinotia aporema granulovirus (EpapGV) was sequenced and analyzed. Its circular double-stranded DNA genome is 119,082 bp in length and codes for 133 putative genes. It contains the 31 baculovirus core genes and a set of 19 genes that are GV exclusive. Seventeen ORFs were unique to EpapGV in comparison with other baculoviruses. Of these, 16 found no homologues in GenBank, and one encoded a thymidylate kinase. Analysis of nucleotide sequence repeats revealed the presence of 16 homologous regions (hrs) interspersed throughout the genome. Each hr was characterized by the presence of 1 to 3 clustered imperfect palindromes which are similar to previously described palindromes of tortricid-specific GVs. Also, one of the hrs (hr4) has flanking sequences suggestive of a putative non-hr ori. Interestingly, two more complex hrs were found in opposite loci, dividing the circular dsDNA genome in two halves. Gene synteny maps showed the great colinearity of sequenced GVs, being EpapGV the most dissimilar as it has a 20 kb-long gene block inversion. Phylogenetic study performed with 31 core genes of 58 baculoviral genomes suggests that EpapGV is the baculovirus isolate closest to the putative common ancestor of tortricid specific betabaculoviruses. Conclusions This study, along with previous characterization of EpapGV infection, is useful for the better understanding of the pathology caused by this virus and its potential utilization as a bioinsecticide. PMID:23051685

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

Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

PubMed

Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

2015-05-01

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

New generic primer system targeting mucosal/genital and cutaneous human papillomaviruses leads to the characterization of HPV 115, a novel Beta-papillomavirus species 3

PubMed Central

Chouhy, Diego; Gorosito, Mario; Sánchez, Adriana; Serra, Esteban C; Bergero, Adriana; Bussy, Ramón Fernandez; Giri, Adriana A

2009-01-01

We explored the cutaneotropic HPV genetic diversity in 71 subjects from Argentina. New generic primers (CUT) targeting 88 mucosal/cutaneous HPV were designed and compared to FAP primers. Overall, 69 different HPV types/putative types were identified, being 17 of them novel putative types. Phylogenetic analysis of partial L1 sequences grouped 2 novel putative types in the Beta-PV, 14 in the Gamma-PV and 1 in the Mu-PV genera. CUT primers showed broader capacity than FAP primers in detecting different genera/species and novel putative types (p<0.01). Using overlapping PCR, the full-length genome of a Beta-PV putative type was amplified and cloned. The new virus, designated HPV 115, encodes 5 early genes and 2 late genes. Phylogenetic analysis indicated HPV 115 as the most divergent type within the genus Beta-PV species 3. This report is the first providing data on cutaneous HPVs circulating in South America and expands our knowledge of the Papillomaviridae family. PMID:19948351

Localization of the putative precursor of Alzheimer's disease-specific amyloid at nuclear envelopes of adult human muscle.

PubMed Central

Zimmermann, K; Herget, T; Salbaum, J M; Schubert, W; Hilbich, C; Cramer, M; Masters, C L; Multhaup, G; Kang, J; Lemaire, H G

1988-01-01

Cloning and sequence analysis revealed the putative amyloid A4 precursor (pre-A4) of Alzheimer's disease to have characteristics of a membrane-spanning glycoprotein. In addition to brain, pre-A4 mRNA was found in adult human muscle and other tissues. We demonstrate by in situ hybridization that pre-A4 mRNA is present in adult human muscle, in cultured human myoblasts and myotubes. Immunofluorescence with antipeptide antibodies shows the putative pre-A4 protein to be expressed in adult human muscle and associated with some but not all nuclear envelopes. Despite high levels of a single 3.5-kb pre-A4 mRNA species in cultured myoblasts and myotubes, the presence of putative pre-A4 protein could not be detected by immunofluorescence. This suggests that putative pre-A4 protein is stabilized and therefore functioning in the innervated muscle tissue but not in developing, i.e. non-innervated cultured muscle cells. The selective localization of the protein on distinct nuclear envelopes could reflect an interaction with motor endplates. Images PMID:2896589

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

USDA-ARS?s Scientific Manuscript database

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE-receptor kinase-WOX signaling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem), and vascular cambium are tightly controlled by CLE signaling pathway...

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

Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

PubMed Central

Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Caballero, Primitivo

2014-01-01

In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 µg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins. PMID:24784323

Transcriptome analysis of molecular mechanisms responsible for light-stress response in Mythimna separata (Walker)

PubMed Central

Duan, Yun; Gong, ZhongJun; Wu, RenHai; Miao, Jin; Jiang, YueLi; Li, Tong; Wu, XiaoBo; Wu, YuQing

2017-01-01

Light is an important environmental signal for most insects. The Oriental Armyworm, Mythimna separata, is a serious pest of cereal crops worldwide, and is highly sensitive to light signals during its developmental and reproductive stages. However, molecular biological studies of its response to light stress are scarce, and related genomic information is not available. In this study, we sequenced and de novo assembled the transcriptomes of M. separata exposed to four different light conditions: dark, white light (WL), UV light (UVL) and yellow light (YL). A total of 46,327 unigenes with an average size of 571 base pairs (bp) were obtained, among which 24,344 (52.55%) matched to public databases. The numbers of genes differentially expressed between dark vs WL, dark vs UVL, dark vs YL, and UVL vs YL were 12,012, 12,950, 14,855, and 13,504, respectively. These results suggest that light exposure altered gene expression patterns in M. separata. Putative genes involved in phototransduction-fly, phototransduction, circadian rhythm-fly, olfactory transduction, and taste transduction were identified. This study thus identified a series of candidate genes and pathways potentially related to light stress in M. separata. PMID:28345615

In Search of the Neural Circuits of Intrinsic Motivation

PubMed Central

Kaplan, Frederic; Oudeyer, Pierre-Yves

2007-01-01

Children seem to acquire new know-how in a continuous and open-ended manner. In this paper, we hypothesize that an intrinsic motivation to progress in learning is at the origins of the remarkable structure of children's developmental trajectories. In this view, children engage in exploratory and playful activities for their own sake, not as steps toward other extrinsic goals. The central hypothesis of this paper is that intrinsically motivating activities correspond to expected decrease in prediction error. This motivation system pushes the infant to avoid both predictable and unpredictable situations in order to focus on the ones that are expected to maximize progress in learning. Based on a computational model and a series of robotic experiments, we show how this principle can lead to organized sequences of behavior of increasing complexity characteristic of several behavioral and developmental patterns observed in humans. We then discuss the putative circuitry underlying such an intrinsic motivation system in the brain and formulate two novel hypotheses. The first one is that tonic dopamine acts as a learning progress signal. The second is that this progress signal is directly computed through a hierarchy of microcortical circuits that act both as prediction and metaprediction systems. PMID:18982131