scholarly journals Genome of a novel circovirus of starlings, amplified by multiply primed rolling-circle amplification

2006 ◽  
Vol 87 (5) ◽  
pp. 1189-1195 ◽  
Author(s):  
Reimar Johne ◽  
Daniel Fernández-de-Luco ◽  
Ursula Höfle ◽  
Hermann Müller
Keyword(s):  
Rolling Circle Amplification ◽  
Open Reading Frames ◽  
Degenerate Primers ◽  
Rolling Circle ◽  
Specific Pcr ◽  
Sturnus Unicolor ◽  
Evolutionarily Conserved ◽  
Degree Of Similarity ◽  
Reading Frames ◽  
Apparently Healthy

The genus Circovirus comprises small non-enveloped viruses with a circular single-stranded DNA genome. By using PCR with degenerate primers, a novel circovirus (starling circovirus, StCV) was detected in spleen samples of wild starlings (Sturnus vulgaris and Sturnus unicolor) found dead during an epidemic outbreak of septicaemic salmonellosis in northeastern Spain. Using a specific PCR, StCV was also detected in apparently healthy birds from the same population. The genome was amplified using multiply primed rolling-circle amplification and cloned. Open reading frames (ORFs) with similarities to the replication-associated protein and the capsid protein of circoviruses as well as an additional ORF encoding a protein of 106 aa were evident from the sequence. Phylogenetic analysis of circovirus genomes revealed the highest degree of similarity (67·1 %) between StCV and canary circovirus. A similar analysis of the evolutionarily conserved cytochrome b gene of the circovirus host species revealed a strict co-evolution of circoviruses with their hosts; however, the circoviruses showed about a threefold higher genetic divergence than their hosts.

scholarly journals Overexpression-based detection of translatable circular RNAs is vulnerable to coexistent linear RNA byproducts

2021 ◽  
Author(s):  
Yanyi Jiang ◽  
Xiaofan Chen ◽  
Wei Zhang
Keyword(s):  
Open Reading Frames ◽  
Systematic Evaluation ◽  
Circular Rnas ◽  
Protein Coding ◽  
Rolling Circle ◽  
Functional Investigation ◽  
Overexpression System ◽  
Translation Signals ◽  
Coding Potential ◽  
Reading Frames

AbstractIn RNA field, the demarcation between coding and non-coding has been negotiated by the recent discovery of occasionally translated circular RNAs (circRNAs). Although absent of 5’ cap structure, circRNAs can be translated cap-independently. Complementary intron-mediated overexpression is one of the most utilized methodologies for circRNA research but not without bearing echoing skepticism for its poorly defined mechanism and latent coexistent side products. In this study, leveraging such circRNA overexpression system, we have interrogated the protein-coding potential of 30 human circRNAs containing infinite open reading frames in HEK293T cells. Surprisingly, pervasive translation signals are detected by immunoblotting. However, intensive mutagenesis reveals that numerous translation signals are generated independently of circRNA synthesis. We have developed a dual tag strategy to isolate translation noise and directly demonstrate that the fallacious translation signals originate from cryptically spliced linear transcripts. The concomitant linear RNA byproducts, presumably concatemers, can be translated to allow pseudo rolling circle translation signals, and can involve backsplicing junction (BSJ) to disqualify the BSJ-based evidence for circRNA translation. We also find non-AUG start codons may engage in the translation initiation of circRNAs. Taken together, our systematic evaluation sheds light on heterogeneous translational outputs from circRNA overexpression vector and comes with a caveat that ectopic overexpression technique necessitates extremely rigorous control setup in circRNA translation and functional investigation.

scholarly journals Sequence and Organization of pXO1, the Large Bacillus anthracis Plasmid Harboring the Anthrax Toxin Genes

1999 ◽  
Vol 181 (20) ◽  
pp. 6509-6515 ◽  
Author(s):  
R. T. Okinaka ◽  
K. Cloud ◽  
O. Hampton ◽  
A. R. Hoffmaster ◽  
K. K. Hill ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Regulatory Elements ◽  
Open Reading Frames ◽  
Significant Similarity ◽  
Toxin Genes ◽  
Theta Replication ◽  
Large Plasmids ◽  
High Degree ◽  
Degree Of Similarity ◽  
Reading Frames

ABSTRACT The Bacillus anthracis Sterne plasmid pXO1 was sequenced by random, “shotgun” cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a “pathogenicity island,” defined by a 44.8-kb region that is bordered by inverted IS1627 elements at each end. This region contains the three toxin genes (cya, lef, and pagA), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in Bacillus spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.

scholarly journals A Novel Virus Detected in Papillomas and Carcinomas of the Endangered Western Barred Bandicoot (Perameles bougainville) Exhibits Genomic Features of both the Papillomaviridae and Polyomaviridae

2007 ◽  
Vol 81 (24) ◽  
pp. 13280-13290 ◽  
Author(s):  
Lucy Woolford ◽  
Annabel Rector ◽  
Marc Van Ranst ◽  
Andrea Ducki ◽  
Mark D. Bennett ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Open Reading Frames ◽  
Nonstructural Proteins ◽  
T Antigens ◽  
Virus Family ◽  
Rolling Circle ◽  
Large Genome Size ◽  
Novel Virus ◽  
L1 And L2

ABSTRACT Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size (∼7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families.

scholarly journals Robust Utilization of Phospholipase-Generated Metabolites, Glycerophosphodiesters, by Candida albicans: Role of the CaGit1 Permease

2011 ◽  
Vol 10 (12) ◽  
pp. 1618-1627 ◽  
Author(s):  
Andrew C. Bishop ◽  
Tao Sun ◽  
Mitchell E. Johnson ◽  
Vincent M. Bruno ◽  
Jana Patton-Vogt
Keyword(s):  
Candida Albicans ◽  
Insertional Mutagenesis ◽  
Single Gene ◽  
Open Reading Frames ◽  
Content Type ◽  
Phosphate Source ◽  
High Degree ◽  
Degree Of Similarity ◽  
Reading Frames

ABSTRACTGlycerophosphodiesters are the products of phospholipase-mediated deacylation of phospholipids. InSaccharomyces cerevisiae, a single gene,GIT1, encodes a permease responsible for importing glycerophosphodiesters, such as glycerophosphoinositol and glycerophosphocholine, into the cell. In contrast, theCandida albicansgenome contains four open reading frames (ORFs) with a high degree of similarity toS. cerevisiaeGIT1(ScGIT1) Here, we report thatC. albicansutilizes glycerophosphoinositol (GroPIns) and glycerophosphocholine (GroPCho) as sources of phosphate at both mildly acidic and physiological pHs. Insertional mutagenesis ofC. albicansGIT1(CaGIT1) (orf19.34), the ORF most similar toScGit1, abolished the ability of cells to use GroPIns as a phosphate source at acidic pH and to transport [3H]GroPIns at acidic and physiological pHs, while reintegration of aGIT1allele into the genome restored those functions. Several lines of evidence, including the detection of internal [3H]GroPIns, indicated that GroPIns is transported intact through CaGit1. GroPIns transport was shown to conform to Michaelis-Menten kinetics, with an apparentKmof 28 ± 6 μM. Notably, uptake of label from [3H]GroPCho was found to be roughly 50-fold greater than uptake of label from [3H]GroPIns and roughly 500-fold greater than the equivalent activity inS. cerevisiae.Insertional mutagenesis ofCaGIT1had no effect on the utilization of GroPCho as a phosphate source or on the uptake of label from [3H]GroPCho. Growth under low-phosphate conditions was shown to increase label uptake from both [3H]GroPIns and [3H]GroPCho. Screening of a transcription factor deletion set identifiedCaPHO4as required for the utilization of GroPIns, but not GroPCho, as a phosphate source.

scholarly journals Nucleotide Sequence and Analysis of Conjugative Plasmid pVT745

2001 ◽  
Vol 183 (5) ◽  
pp. 1585-1594 ◽  
Author(s):  
Dominique M. Galli ◽  
Jinbiao Chen ◽  
Karen F. Novak ◽  
Donald J. Leblanc
Keyword(s):  
Nucleotide Sequence ◽  
Open Reading Frames ◽  
Conjugative Plasmid ◽  
Periodontal Pathogen ◽  
Gene Products ◽  
E Coli ◽  
Replication Region ◽  
High Degree ◽  
Degree Of Similarity ◽  
Reading Frames

ABSTRACT The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitansto Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerousA. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.

scholarly journals Characterization of Plasmid pRT1 from Pyrococcus sp. Strain JT1

2002 ◽  
Vol 184 (9) ◽  
pp. 2561-2566 ◽  
Author(s):  
Donald E. Ward ◽  
Ingrid M. Revet ◽  
Renu Nandakumar ◽  
Jon H. Tuttle ◽  
Willem M. de Vos ◽  
...  
Keyword(s):  
Dna Replication ◽  
Open Reading Frames ◽  
Single Stranded Dna ◽  
Rolling Circle ◽  
Hyperthermophilic Archaeon ◽  
Rolling Circle Mechanism ◽  
Replication Intermediates ◽  
Reading Frames

ABSTRACT We discovered a 3,373-bp plasmid (pRT1) in the hyperthermophilic archaeon Pyrococcus sp. strain JT1. Two major open reading frames were identified, and analysis of the sequence revealed some resemblance to motifs typically found in plasmids that replicate via a rolling-circle mechanism. The presence of single-stranded DNA replication intermediates of pRT1 was detected, confirming this mode of replication.

scholarly journals Sinorhizobium meliloti Plasmid pRm1132f Replicates by a Rolling-Circle Mechanism

2001 ◽  
Vol 183 (8) ◽  
pp. 2704-2708 ◽  
Author(s):  
L. R. Barran ◽  
N. Ritchot ◽  
E. S. P. Bromfield
Keyword(s):  
Nucleotide Sequence ◽  
Plasmid Dna ◽  
Sinorhizobium Meliloti ◽  
Open Reading Frames ◽  
Rolling Circle ◽  
Group Iii ◽  
Coding Regions ◽  
Origins Of Replication ◽  
Rolling Circle Mechanism ◽  
Reading Frames

ABSTRACT pRm1132f isolated from Sinorhizobium meliloti is a group III rolling-circle-replicating (RCR) plasmid. At least seven of eight open reading frames in the nucleotide sequence represented coding regions. The minimal replicon contained a rep gene and single- and double-stranded origins of replication. Detection of single-stranded plasmid DNA confirmed that pRm1132f replicated via an RCR mechanism.

scholarly journals Identification of a Novel Geminivirus in Fraxinus rhynchophylla in Korea

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2385
Author(s):  
Aamir Lal ◽  
Yong-Ho Kim ◽  
Thuy Thi Bich Vo ◽  
I Gusti Ngurah Prabu Wira Sanjaya ◽  
Phuong Thi Ho ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Indian Subcontinent ◽  
Chemical Constituents ◽  
Rolling Circle Amplification ◽  
Open Reading Frames ◽  
Stem Loop ◽  
Rolling Circle ◽  
Reading Frame ◽  
Infectious Clones ◽  
Ash Trees

Fraxinus rhynchophylla, common name ash, belongs to the family Oleaceae and is found in China, Korea, North America, the Indian subcontinent, and eastern Russia. It has been used as a traditional herbal medicine in Korea and various parts of the world due to its chemical constituents. During a field survey in March 2019, mild vein thickening (almost negligible) was observed in a few ash trees. High-throughput sequencing of libraries of total DNA from ash trees, rolling-circle amplification (RCA), and polymerase chain reaction (PCR) allowed the identification of a Fraxinus symptomless virus. This virus has five confirmed open reading frames along with a possible sixth open reading frame that encodes the movement protein and is almost 2.7 kb in size, with a nonanucleotide and stem loop structure identical to begomoviruses. In terms of its size and structure, this virus strongly resembles begomoviruses, but does not show any significant sequence identity with them. To confirm movement of the virus within the trees, different parts of infected trees were examined, and viral movement was successfully observed. No satellite molecules or DNA B were identified. Two-step PCR confirmed the virion and complementary strands during replication in both freshly collected infected samples of ash tree and Nicotiana benthamiana samples agro-inoculated with infectious clones. This taxon is so distantly grouped from other known geminiviruses that it likely represents a new geminivirus genus.

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