scholarly journals Gene Cassette PCR: Sequence-Independent Recovery of Entire Genes from Environmental DNA

2001 ◽  
Vol 67 (11) ◽  
pp. 5240-5246 ◽  
Author(s):  
H. W. Stokes ◽  
Andrew J. Holmes ◽  
Blair S. Nield ◽  
Marita P. Holley ◽  
K. M. Helena Nevalainen ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Sequence Data ◽  
Environmental Dna ◽  
Gene Families ◽  
Soil Samples ◽  
Open Reading Frames ◽  
Sequence Information ◽  
Gene Cassettes ◽  
Conserved Sequence ◽  
Reading Frames

ABSTRACT The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.

scholarly journals Sequence analysis of the equid herpesvirus 2 chemokine receptor homologues E1, ORF74 and E6 demonstrates high sequence divergence between field isolates

2007 ◽  
Vol 88 (9) ◽  
pp. 2450-2462 ◽  
Author(s):  
Emma L. Sharp ◽  
Helen E. Farrell ◽  
Kerstin Borchers ◽  
Edward C. Holmes ◽  
Nicholas J. Davis-Poynter
Keyword(s):  
Chemokine Receptors ◽  
Sequence Data ◽  
Sequence Divergence ◽  
Gene Families ◽  
Open Reading Frames ◽  
Amino Acid Difference ◽  
Transmembrane Receptors ◽  
Equid Herpesvirus ◽  
High Sequence Divergence ◽  
Reading Frames

Equid herpesvirus 2 (EHV-2), in common with other members of the subfamily Gammaherpesvirinae, encodes homologues of cellular seven-transmembrane receptors (7TMR), namely open reading frames (ORFs) E1, 74 and E6, which each show some similarity to cellular chemokine receptors. Whereas ORF74 and E6 are members of gammaherpesvirus-conserved 7TMR gene families, E1 is currently unique to EHV-2. To investigate their genetic variability, EHV-2 7TMRs from a panel of equine gammaherpesvirus isolates were sequenced. A region of gB was sequenced to provide comparative sequence data. Phylogenetic analysis revealed six ‘genogroups’ for E1 and four for ORF74, which exhibited approximately 10–38 and 11–27 % amino acid difference between groups, respectively. In contrast, E6 was highly conserved, with two genogroups identified. The greatest variation was observed within the N-terminal domains and other extracellular regions. Nevertheless, analysis of the number of non-synonymous (d N) and synonymous (d S) substitutions per site generally supported the hypothesis that the 7TMRs are under negative selective pressure to retain functionally important residues, although some site-specific positive selection (d N>d S) was also observed. Collectively, these data are consistent with transmembrane and cytoplasmic domains being less tolerant of mutations with adverse effects upon function. Finally, there was no evidence for genetic linkage between the different gB, E1, ORF74 and E6 genotypes, suggesting frequent intergenic recombination between different EHV-2 strains.

scholarly journals Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, Belonging to the “phiKMV-Like Viruses”

2011 ◽  
Vol 77 (10) ◽  
pp. 3443-3450 ◽  
Author(s):  
Evelien M. Adriaenssens ◽  
Pieter-Jan Ceyssens ◽  
Vincent Dunon ◽  
Hans-Wolfgang Ackermann ◽  
Johan Van Vaerenbergh ◽  
...  
Keyword(s):  
Soil Samples ◽  
Pantoea Agglomerans ◽  
Open Reading Frames ◽  
Human Pathogen ◽  
Content Type ◽  
Host Diversity ◽  
Double Stranded Dna ◽  
Opportunistic Human Pathogen ◽  
Reading Frames

ABSTRACTPantoea agglomeransis a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight ofP. agglomeransare lytic phages, isolated from soil samples, belonging to thePodoviridaeand are the firstPantoeaphages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of theAutographivirinae, within the genus of the “phiKMV-like viruses.” Phylogenetic analysis of all the sequenced members of theAutographivirinaesupports the classification of phages LIMElight and LIMEzero as members of the “phiKMV-like viruses” and corroborates the subdivision into the different genera. These data expand the knowledge ofPantoeaphages and illustrate the wide host diversity of phages within the “phiKMV-like viruses.”

scholarly journals Genome Sequences of Bacteriophages KaiHaiDragon and OneinaGillian, Isolated from Microbacterium foliorum in Riverside, California

2019 ◽  
Vol 8 (15) ◽  
Author(s):  
Gillian Miller ◽  
Steven Tran ◽  
Rhiannon Abrahams ◽  
Daniel Bazan ◽  
Ethan Blaylock ◽  
...  
Keyword(s):  
Soil Samples ◽  
Open Reading Frames ◽  
Genome Sequences ◽  
Bacterial Host ◽  
Content Type ◽  
Reading Frames

KaiHaiDragon and OneinaGillian are two bacteriophages which have been recovered from soil samples using the bacterial host Microbacterium foliorum. Their genome lengths are 52,992 bp and 61,703 bp, with 91 and 104 predicted open reading frames, respectively.

scholarly journals Tranosema rostrale ichnovirus repeat element genes display distinct transcriptional patterns in caterpillar and wasp hosts

2009 ◽  
Vol 90 (6) ◽  
pp. 1505-1514 ◽  
Author(s):  
Asieh Rasoolizadeh ◽  
Catherine Béliveau ◽  
Don Stewart ◽  
Conrad Cloutier ◽  
Michel Cusson
Keyword(s):  
Fat Body ◽  
Gene Dosage ◽  
Choristoneura Fumiferana ◽  
Gene Families ◽  
Transcript Abundance ◽  
Repeat Element ◽  
Open Reading Frames ◽  
Regulation Of Transcription ◽  
Lepidopteran Host ◽  
Reading Frames

The endoparasitic wasp Tranosema rostrale transmits an ichnovirus to its lepidopteran host, Choristoneura fumiferana, during parasitization. As shown for other ichnoviruses, the segmented dsDNA genome of the T. rostrale ichnovirus (TrIV) features several multi-gene families, including the repeat element (rep) family, whose products display no known similarity to non-ichnovirus proteins, except for a homologue encoded by the genome of the Helicoverpa armigera granulovirus; their functions remain unknown. This study applied linear regression of efficiency analysis to real-time PCR quantification of transcript abundance for all 17 TrIV rep open reading frames (ORFs) in parasitized and virus-injected C. fumiferana larvae, as well as in T. rostrale ovaries and head–thorax complexes. Although transcripts were detected for most rep ORFs in infected caterpillars, two of them clearly outnumbered the others in whole larvae, with a tendency for levels to drop over time after infection. The genome segments bearing the three most highly expressed rep genes in parasitized caterpillars were present in higher proportions than other rep-bearing genome segments in TrIV DNA, suggesting a possible role for gene dosage in the regulation of transcription level. TrIV rep genes also showed important differences in the relative abundance of their transcripts in specific tissues (cuticular epithelium, the fat body, haemocytes and the midgut), implying tissue-specific roles for individual members of this gene family. Significantly, no rep transcripts were detected in T. rostrale head–thorax complexes, whereas some were abundant in ovaries. There, the transcription pattern was completely different from that observed in infected caterpillars, suggesting that some rep genes have wasp-specific functions.

scholarly journals Analysis of Expressed Sequence Tags from Uromyces appendiculatus Hyphae and Haustoria and Their Comparison to Sequences from Other Rust Fungi

2008 ◽  
Vol 98 (10) ◽  
pp. 1126-1135 ◽  
Author(s):  
D. P. Puthoff ◽  
A. Neelam ◽  
M. L. Ehrenfried ◽  
B. E. Scheffler ◽  
L. Ballard ◽  
...  
Keyword(s):  
Expressed Sequence Tags ◽  
Sequence Data ◽  
Cellular Function ◽  
Rust Fungi ◽  
Open Reading Frames ◽  
Uromyces Appendiculatus ◽  
Data Set ◽  
Uromyces Fabae ◽  
Expressed Sequence ◽  
Reading Frames

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

scholarly journals Nitrogen or Sulfur Starvation Differentially Affects Phycobilisome Degradation and Expression of the nblA Gene in Synechocystis Strain PCC 6803

2001 ◽  
Vol 183 (10) ◽  
pp. 2989-2994 ◽  
Author(s):  
Catherine Richaud ◽  
Gérald Zabulon ◽  
Annette Joder ◽  
Jean-Claude Thomas
Keyword(s):  
Sequence Data ◽  
Open Reading Frames ◽  
Northern Hybridization ◽  
Sulfur Starvation ◽  
P Limitation ◽  
Kanamycin Cassette ◽  
N Starvation ◽  
Pcc 7942 ◽  
Reading Frames ◽  
Pcc 6803

ABSTRACT Nitrogen (N) limitation in cyanobacteria is well documented: a reduced growth rate is observed, accompanied by a cessation of phycobiliprotein synthesis and an ordered degradation of phycobilisomes (PBS). This leads to a dramatic bleaching phenomenon known as chlorosis. In Synechococcus strain PCC 7942, bleaching due to PBS degradation is also observed under sulfur (S) or phosphorus (P) limitation, and all three are under the control of the nblAgene product, a 59-amino-acid polypeptide which is overexpressed under N, S, and P starvation (J. L. Collier, and A. R. Grossman, EMBO J. 13:1039–1047, 1994). Cyanobase sequence data forSynechocystis strain PCC 6803 indicate the presence of two tandem open reading frames (sll0452 and sll0453) homologous tonblA. We cloned the two genes, identified a unique 5′ mRNA end suggestive of a single transcription start site, and studiednblA expression under conditions of N or S starvation by Northern hybridization: transcripts were detected only under N starvation (no signal is detected in replete medium or with S starvation), whether nblA1 or nblA2 was used as a probe. Mutations in nblA1 and nblA2 were constructed by insertion of a kanamycin cassette; both mutations were nonbleaching under N starvation. Synechocystis strain PCC 6803 does not bleach under S starvation, consistent with the absence ofnblA induction in these conditions. These results were confirmed by analysis of the PBS components: sequential degradation of phycocyanin and associated linkers was observed only under conditions of N starvation. This indicates differences betweenSynechocystis strain PCC 6803 and Synechococcusstrain PCC 7942 in their regulatory and signaling pathways leading to N- and S-starved phenotypes.

scholarly journals Cloning and Genetic Analyses of the Bacteriocin 41 Determinant Encoded on the Enterococcus faecalis Pheromone-Responsive Conjugative Plasmid pYI14: a Novel Bacteriocin Complemented by Two Extracellular Components (Lysin and Activator)

2008 ◽  
Vol 190 (6) ◽  
pp. 2075-2085 ◽  
Author(s):  
Haruyoshi Tomita ◽  
Elizabeth Kamei ◽  
Yasuyoshi Ike
Keyword(s):  
Enterococcus Faecalis ◽  
Sequence Data ◽  
Open Reading Frames ◽  
Conjugative Plasmid ◽  
Cell Surface Receptor ◽  
Bacteriocin Activity ◽  
Lytic Enzymes ◽  
Repeat Structure ◽  
Surface Receptor ◽  
Reading Frames

ABSTRACT The conjugative plasmid pYI14 (61 kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E. faecalis. Physical mapping of pYI14 showed that it consisted of EcoRI fragments A to P. The clone pHT1100, containing EcoRI fragments A (12.6 kbp) and H (3.5 kbp), conferred the bacteriocin activity on E. faecalis strains. Genetic analysis showed that the determinant was located in a 6.6-kbp region within the EcoRI AH fragments. Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL1 ) ORF8 (bacL2 ), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL1 , bacL2 , bacA, and bacI were essential for expression of the bacteriocin in E. faecalis. Extracellular complementation of bacteriocin expression was possible for bacL1 and -L2 and bacA mutants. bacL1 and -L2 and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, providing the host with resistance to its own bacteriocin activity. The bacL1 -encoded protein had significant homology with lytic enzymes that attack the gram-positive bacterial cell wall. Sequence data for the deduced bacL1 -encoded protein suggested that it has a domain structure consisting of an N-terminal signal peptide, a second domain with the enzymatic activity, and a third domain with a three-repeat structure directing the proenzyme to its cell surface receptor.

scholarly journals Identification and analysis of proton-translocating pyrophosphatases in the methanogenic archaeonMethanosarcina mazei

Archaea ◽  
2002 ◽  
Vol 1 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Sebastian Bäumer ◽  
Sabine Lentes ◽  
Gerhard Gottschalk ◽  
Uwe Deppenmeier
Keyword(s):  
Specific Activity ◽  
Sequence Data ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Inorganic Pyrophosphatase ◽  
Amino Acid Sequence Similarity ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Inorganic Pyrophosphate ◽  
Reading Frames

Analysis of genome sequence data from the methanogenic archaeonMethanosarcina mazeiGö1 revealed the existence of two open reading frames encoding proton-translocating pyrophosphatases (PPases). These open reading frames are linked by a 750-bp intergenic region containing TC-rich stretches and are transcribed in opposite directions. The corresponding polypeptides are referred to as Mvp1 and Mvp2 and consist of 671 and 676 amino acids, respectively. Both enzymes represent extremely hydrophobic, integral membrane proteins with 15 predicted transmembrane segments and an overall amino acid sequence similarity of 50.1%. Multiple sequence alignments revealed that Mvp1 is closely related to eukaryotic PPases, whereas Mvp2 shows highest homologies to bacterial PPases. Northern blot experiments with RNA from methanol-grown cells harvested in the mid-log growth phase indicated that only Mvp2 was produced under these conditions. Analysis of washed membranes showed that Mvp2 had a specific activity of 0.34 U mg (protein)–1. Proton translocation experiments with inverted membrane vesicles prepared from methanol-grown cells showed that hydrolysis of 1 mol of pyrophosphate was coupled to the translocation of about 1 mol of protons across the cytoplasmic membrane. Appropriate conditions formvp1 expression could not be determined yet. The pyrophosphatases ofM. mazeiGö1 represent the first examples of this enzyme class in methanogenic archaea and may be part of their energy-conserving system. Abbreviations: DCCD,N,N′-dicyclohexylcarbodiimide; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; Δp, proton motive force.

scholarly journals Thousands of novel translated open reading frames in humans inferred by ribosome footprint profiling

2015 ◽  
Author(s):  
Anil Raj ◽  
Sidney H. Wang ◽  
Heejung Shim ◽  
Arbel Harpak ◽  
Yang I. Li ◽  
...  
Keyword(s):  
Significant Negative Correlation ◽  
Selective Constraint ◽  
Open Reading Frames ◽  
Sequence Information ◽  
The Novel ◽  
Protein Coding ◽  
Coding Sequences ◽  
Coding Regions ◽  
Human Lymphoblastoid Cell ◽  
Reading Frames

AbstractAccurate annotation of protein coding regions is essential for understanding how genetic information is translated into biological functions. Here we describe riboHMM, a new method that uses ribosome footprint data along with gene expression and sequence information to accurately infer translated sequences. We applied our method to human lymphoblastoid cell lines and identified 7,273 previously unannotated coding sequences, including 2,442 translated upstream open reading frames. We observed an enrichment of harringtonine-treated ribosome footprints at the inferred initiation sites, validating many of the novel coding sequences. The novel sequences exhibit significant signatures of selective constraint in the reading frames of the inferred proteins, suggesting that many of these are functional. Nearly 40% of bicistronic transcripts showed significant negative correlation in the levels of translation of their two coding sequences, suggesting a key regulatory role for these novel translated sequences. Our work significantly expands the set of known coding regions in humans.

scholarly journals Target enrichment of long open reading frames and ultraconserved elements to link microevolution and macroevolution in non-model organisms

2021 ◽  
Author(s):  
Claudia Ortiz-Sepulveda ◽  
Mathieu Genete ◽  
Christelle Blassiau ◽  
Cécile Godé ◽  
Christian Albrecht ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Data ◽  
Early Stage ◽  
Phylogenetic Analyses ◽  
Open Reading Frames ◽  
Model Organisms ◽  
Recent Common Ancestor ◽  
Ultraconserved Elements ◽  
Most Recent Common Ancestor ◽  
Reading Frames

Despite the increasing accessibility of high-throughput sequencing, obtaining high-quality genomic data on non-model organisms without proximate well-assembled and annotated genomes remains challenging. Here we describe a workflow that takes advantage of distant genomic resources and ingroup transcriptomes to select and jointly enrich long open reading frames (ORFs) and ultraconserved elements (UCEs) from genomic samples for integrative studies of microevolutionary and macroevolutionary dynamics. This workflow is applied to samples of the African unionid bivalve tribe Coelaturini (Parreysiinae) at basin and continent-wide scales. Our results indicate that ORFs are efficiently captured without prior identification of intron-exon boundaries. The enrichment of UCEs was less successful, but nevertheless produced a substantial dataset. Exploratory continent-wide phylogenetic analyses with ORF supercontigs (>515,000 parsimony informative sites) resulted in a fully resolved phylogeny, the backbone of which was also retrieved with UCEs (>11,000 informative sites), although some branches lack support in the latter case. Variant calling on the exome of Coelaturini from the Malawi Basin produced ~2,000 SNPs per population pair. Nucleotide diversity and population differentiation was low compared to previous estimates in mollusks, but comparable to those in recently diversifying Malawi cichlids and other taxa at an early stage of speciation. Skimming non-specific sequence data obtained for Coelaturini of the Malawi Basin, we reconstructed the maternally-inherited mitogenome, which displays an identical gene order to that of the most recent common ancestor of Unionidae. Overall, our workflow and results provide exciting perspectives for the development of integrative genomic studies on micro- and macroevolutionary dynamics in non-model organisms.

Sign in / Sign up

Export Citation Format

Share Document