Rns Gene Research Articles

Assembly and comparative analysis of the complete mitochondrial and chloroplast genome of Cyperus stoloniferus (Cyperaceae), a coastal plant possessing saline-alkali tolerance

BackgroundCyperus stoloniferus is an important species in coastal ecosystems and possesses economic and ecological value. To elucidate the structural characteristics, variation, and evolution of the organelle genome of C. stoloniferus, we sequenced, assembled, and compared its mitochondrial and chloroplast genomes.ResultsWe assembled the mitochondrial and chloroplast genomes of C. stoloniferus. The total length of the mitochondrial genome (mtDNA) was 927,413 bp, with a GC content of 40.59%. It consists of two circular DNAs, including 37 protein-coding genes (PCGs), 22 tRNAs, and five rRNAs. The length of the chloroplast genome (cpDNA) was 186,204 bp, containing 93 PCGs, 40 tRNAs, and 8 rRNAs. The mtDNA and cpDNA contained 81 and 129 tandem repeats, respectively, and 346 and 1,170 dispersed repeats, respectively, both of which have 270 simple sequence repeats. The third high-frequency codon (RSCU > 1) in the organellar genome tended to end at A or U, whereas the low-frequency codon (RSCU < 1) tended to end at G or C. The RNA editing sites of the PCGs were relatively few, with only 9 and 23 sites in the mtDNA and cpDNA, respectively. A total of 28 mitochondrial plastid DNAs (MTPTs) in the mtDNA were derived from cpDNA, including three complete trnT-GGU, trnH-GUG, and trnS-GCU. Phylogeny and collinearity indicated that the relationship between C. stoloniferus and C. rotundus are closest. The mitochondrial rns gene exhibited the greatest nucleotide variability, whereas the chloroplast gene with the greatest nucleotide variability was infA. Most PCGs in the organellar genome are negatively selected and highly evolutionarily conserved. Only six mitochondrial genes and two chloroplast genes exhibited Ka/Ks > 1; in particular, atp9, atp6, and rps7 may have undergone potential positive selection.ConclusionWe assembled and validated the mtDNA of C. stoloniferus, which contains a 15,034 bp reverse complementary sequence. The organelle genome sequence of C. stoloniferus provides valuable genomic resources for species identification, evolution, and comparative genomic research in Cyperaceae.

The diversity of mtDNA rns introns among strains of Ophiostoma piliferum, Ophiostoma pluriannulatum and related species.

BackgroundBased on previous studies, it was suspected that the mitochondrial rns gene within the Ophiostomatales is rich in introns. This study focused on a collection of strains representing Ophiostoma piliferum, Ophiostoma pluriannulatum and related species that cause blue-stain; these fungi colonize the sapwood of trees and impart a dark stain. This reduces the value of the lumber. The goal was to examine the mtDNA rns intron landscape for these important blue stain fungi in order to facilitate future annotation of mitochondrial genomes (mtDNA) and to potentially identify mtDNA introns that can encode homing endonucleases which may have applications in biotechnology.ResultsComparative sequence analysis identified five intron insertion sites among the ophiostomatoid fungi examined. Positions mS379 and mS952 harbor group II introns, the mS379 intron encodes a reverse transcriptase, and the mS952 intron encodes a potential homing endonuclease. Positions mS569, mS1224, and mS1247 have group I introns inserted and these encode intact or eroded homing endonuclease open reading frames (ORF). Phylogenetic analysis of the intron ORFs showed that they can be found in the same insertion site in closely and distantly related species.ConclusionsBased on the molecular markers examined (rDNA internal transcribed spacers and rns introns), strains representing O. pilifera, O. pluriannulatum and Ophiostoma novae-zelandiae could not be resolved. Phylogenetic studies suggest that introns are gained and lost and that horizontal transfer could explain the presence of related intron in distantly related fungi. With regard to the mS379 group II intron, this study shows that mitochondrial group II introns and their reverse transcriptases may also follow the life cycle previously proposed for group I introns and their homing endonucleases. This consists of intron invasion, decay of intron ORF, loss of intron, and possible reinvasion.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3076-6) contains supplementary material, which is available to authorized users.

Disentangling identity of species of the genus Taphrina parasitizing herbaceous Rosaceae, with proposal of Taphrina gei-montani sp. nov.

Five strains (CCY 058-007-001T, CCY 058-007-002, CCY 058-007-003, CCY 058-007-004 and CCY 058-007-005) of a novel parasitic yeast belonging to the genus Taphrina were isolated from leaf tissues of Geum montanum L. (Rosaceae), collected from the Vysoké Tatry Mountains, Slovakia. Genetic analyses revealed that these isolates differ by 15 unique substitutions in the ITS region and by six substitutions in the rns gene from all other species of the genus Taphrina analysed hitherto. The novel strains are also distinguished from all other species of the genus Taphrina by their morphology, biochemical properties and ecology. These strains represent a novel species, for which the name Taphrina gei-montani sp. nov. is proposed. The type strain is CCY 058-007-001T (=CBS 14159=BU001). The MycoBank number is MB815677. The present study also demonstrates that two distinct species of the genus Taphrina parasitize the herbaceous Rosaceae: Taphrina gei-montani sp. nov. on Geum montanum and Taphrina tormentillae on Potentilla species.

Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease.

In Chaetomium thermophilum (DSM 1495) within the mitochondrial DNA (mtDNA) small ribosomal subunit (rns) gene a group IIA1 intron interrupts an open reading frame (ORF) encoded within a group I intron (mS1247). This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the homing endonuclease (HEase). Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 intron or a group IIB type intron. This study showed that the expression of the HEase (in vivo) in Escherichia coli can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. Exogenous magnesium chloride (MgCl2) stimulated the expression of a functional HEase but the addition of cobalt chloride (CoCl2) to growth media antagonized the expression of HEase activity. Ultimately the ability to attenuate HEase activity might be useful in precision genome engineering, minimizing off target activities, or where pathways have to be altered during a specific growth phase.

Phylogenetic analysis and the evolution of the 18S rRNA gene typing system of Acanthamoeba.

Species of Acanthamoeba were first described using morphological characters including cyst structure and cytology of nuclear division. More than 20 nominal species were proposed using these methods. Morphology, especially cyst shape and size, has proven to be plastic and dependent upon culture conditions. The DNA sequence of the nuclear small-subunit (18S) rRNA, the Rns gene, has become the most widely accepted method for rapid diagnosis and classification of Acanthamoeba. The Byers-Fuerst lab first proposed an Rns typing system in 1996. Subsequent refinements, with an increasing DNA database and analysis of diagnostic fragments within the gene, have become widely accepted by the Acanthamoeba research community. The development of the typing system, including its current state of implementation is illustrated by three cases: (i) the division between sequence types T13 and T16; (ii) the diversity within sequence supertype T2/T6, and (iii) verification of a new sequence type, designated T20. Molecular studies make clear the disconnection between phylogenetic relatedness and species names, as applied for the genus Acanthamoeba. Future reconciliation of genetic types with species names must become a priority, but the possible shortcomings of the use of a single gene when reconstructing the evolutionary history of the acanthamoebidae must also be resolved.

The mtDNA rns gene landscape in the Ophiostomatales and other fungal taxa: Twintrons, introns, and intron-encoded proteins

Comparative sequence analysis of the mitochondrial small subunit ribosomal RNA (rns) gene among species of Ophiostoma, Grosmannia, Ceratocystiopsis and related taxa provides an overview of the types of introns that have invaded this gene within the ophiostomatoid fungi. The rns gene appears to be a reservoir for a number of group I and group II introns along with intron-associated open reading frames such as homing endonucleases and reverse transcriptases. This study uncovered two twintrons, one at position mS917 where a group ID intron encoding a LAGLIDADG ORF invaded another ORF-less group ID intron. Another twintron complex was detected at position mS1247 here a group IIA1 intron invaded the open reading frame embedded within a group IC2 intron. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent losses. Results of this study will make a significant contribution to the understanding of the complexity of the mitochondrial intron landscape, and offer a resource to those annotating mitochondrial genomes. It will also serve as a resource to those that bioprospect for ribozymes and homing endonucleases.

Screening of recreational areas of rivers for potentially pathogenic free-living amoebae in the suburbs of Tehran, Iran

A survey was conducted to determine the presence of free-living amoebae (FLA), especially Acanthamoeba and Naegleria, in river recreation areas in Tehran Province, Iran. All rivers surveyed were associated with human activity, and two were also a source of municipal tap water. Fifty-five water samples from 10 major rivers were screened for FLA and identified by morphological characters, PCR amplification targeting specific genes for Acanthamoeba (DF3 region of Rns gene) and other FLA (ITS PCR), and homology analysis. The percentage of positive FLA isolates was 27.3%, of which 80% were Acanthamoeba, assigned to the T4 and T15 genotype, and 20% were Naegleria. Isolation of Acanthamoeba T4 genotype (91.7%) from recreation areas could be a health threat and a sanitary risk associated with human activity where young people and tourists congregate in summer. Posting of warning signs and education of high-risk individuals are important for disease prevention. To the best of our knowledge this is the first report of genotype T15 (clustered with A. jacobsi) identified in Iran and the first report of the distribution of FLA such as Naegleria (N. pagei, N. clarki and N. fultoni) in recreation areas in rivers of Tehran Province using molecular methods.

The highly variable mitochondrial small-subunit ribosomal RNA gene of Ophiostoma minus

Mitochondrial genomes in the true fungi are highly variable both in size and organization. Most of this size variation is due to the presence of introns and intron-encoded open reading frames (ORFs). The objectives for this work were to examine the mitochondrial small-subunit ribosomal RNA ( rns) gene of strains of Ophiostoma minus for the presence of introns and to characterize such introns and their encoded ORFs. DNA sequence analysis showed that among different strains of O. minus various rns gene exon/intron configurations can be observed. Based on comparative sequence analysis and RNA secondary structure modeling group I introns with LAGLIDADG ORFs were uncovered at positions mS569 and mS1224 and group II introns were present at positions mS379 and mS952. The mS379 group II intron encoded a fragmented reverse transcriptase (RT)-like ORF and the mS952 group II intron encoded a LAGLIDADG-type ORF. Examples of intron ORF degeneration due to frameshift mutations were observed. The mS379 group II intron is the first mitochondrial group II intron to have an ORF inserted within domain II, typically RT-like ORFs are inserted in domain IV. The evolutionary dynamics of the intron-encoded ORFs have also been examined.

A 971-bp insertion in the rns gene is associated with mitochondrial hypovirulence in a strain of Cryphonectria parasitica isolated from nature

In the chestnut-blight fungus Cryphonectria parasitica, cytoplasmically transmissible hypovirulence phenotypes frequently are elicited by double-stranded RNA (dsRNA) virus infections. However, some strains manifest cytoplasmically transmissible hypovirulence traits without containing any mycovirus. In this study, we describe an altered form of mtDNA that is associated with hypovirulence and senescence in a virus-free strain of C. parasitica, KFC9, which was obtained from nature and has an elevated level of cyanide-resistant respiration. In this strain, a 971-bp DNA element, named InC9, has been inserted into the first exon of the mitochondrial small-subunit ribosomal RNA (rns) gene. Sequence analysis indicates that InC9 is a type A1 group II intron that lacks a maturase-encoding ORF. RT-PCR analyses showed that the InC9 sequence is spliced inefficiently from the rRNA precursor. The KFC9 strain had very low amounts of mitochondrial ribosomes relative to virulent strains, thus most likely is deficient in mitochondrial protein synthesis and lacks at least some of the components of the cyanide-sensitive, cytochrome-mediated respiratory pathway. The attenuated-virulence trait and the splicing-defective intron are transferred asexually and concordantly by hyphal contact from hypovirulent donor strains to virulent recipients, confirming that InC9 causes hypovirulence.

Evolutionary Dynamics of the mS952 Intron: A Novel Mitochondrial Group II Intron Encoding a LAGLIDADG Homing Endonuclease Gene

Examination of the mitochondrial small subunit ribosomal RNA (rns) gene of five species of the fungal genus Leptographium revealed that the gene has been invaded at least once at position 952 by a group II intron encoding a LAGLIDADG homing endonuclease gene. Phylogenetic analyses of the intron and homing endonuclease sequences indicated that each element in Leptographium species forms a single clade and is closely related to the group II intron/homing endonuclease gene composite element previously reported at position 952 of the mitochondrial rns gene of Cordyceps species and of Cryphonectria parasitica. The results of an intron survey of the mt rns gene of Leptographium species superimposed onto the phylogenetic analysis of the host organisms suggest that the composite element was transmitted vertically in Leptographium lundbergii. However, its stochastic distribution among strains of L. wingfieldii, L. terebrantis, and L. truncatum suggests that it has been horizontally transmitted by lateral gene transfer among these species, although the random presence of the intron may reflect multiple random loss events. A model is proposed describing the initial invasion of the group II intron in the rns gene of L. lundbergii by a LAGLIDADG homing endonuclease gene and subsequent evolution of this gene to recognize a novel DNA target site, which may now promote the mobility of the intron and homing endonuclease gene as a composite element.

A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions

A group II intron encoding a protein belonging to the LAGLIDADG family of homing endonucleases was identified in the mitochondrial rns gene of the filamentous fungus Leptographium truncatum, and the catalytic activities of both the intron and its encoded protein were characterized. A model of the RNA secondary structure indicates that the intron is a member of the IIB1 subclass and the open reading frame is inserted in ribozyme domain III. In vitro assays carried out with two versions of the intron, one in which the open reading frame was removed and the other in which it was present, demonstrate that both versions of the intron readily self-splice at 37 degrees C and at a concentration of MgCl(2) as low as 6 mM. The open reading frame encodes a functional LAGLIDADG homing endonuclease that cleaves 2 (top strand) and 6 (bottom strand) nucleotides (nt) upstream of the intron insertion site, generating 4 nt 3' OH overhangs. In vitro splicing assays carried out in the absence and presence of the intron-encoded protein indicate that the protein does not enhance intron splicing, and RNA-binding assays show that the protein does not appear to bind to the intron RNA precursor transcript. These findings raise intriguing questions concerning the functional and evolutionary relationships of the two components of this unique composite element.

The Cryphonectria parasitica mitochondrial rns gene: Plasmid-like elements, introns and homing endonucleases

The mt- rns gene of Cryphonectria parasitica is 9872 bp long and includes two group I and two group II introns. An analysis of intronic protein-encoding sequences revealed that LAGLIDADG ORFs, which usually are associated with group I introns, were transferred at least twice into group II introns. A plasmid-like mitochondrial element (plME) that appears in high amounts in previously mutagen-induced mit1 and mit2 hypovirulent mutants of the Ep155 standard virulent strain of C. parasitica was found to be derived from a short region of the mt- rns gene, including the exon 1 and most of the first intron. The plME is a 4.2-kb circular, multimeric DNA and an autonomously-replicating mtDNA fragment. Although sexual transmission experiments indicate that the plME does not directly cause hypovirulence, its emergence is one manifestation of the many complex molecular and genetic events that appear to underlie this phenotype.

Identification of the 18S-ribosomal-DNA genotypes ofAcanthamoebaisolates from the Philippines

Cyst morphology has been commonly used to identify the free-living amoeba Acanthamoeba to subgenus level. A more accurate and consistent method, based on the sequence analysis of the gene coding for the amoeba's small-subunit ribosomal RNA (Rns), has, however, been developed. There have been no attempts to identify the Acanthamoeba genotypes circulating in the Philippines. In this study, therefore, the ASA.S1 region of the Rns gene from 17 Acanthamoeba isolates, collected from soil, water and contact-lens storage cases in different regions of the Philippines, was sequenced. After the isolates were genotyped, using the BLAST program, their phylogenetic positions relative to known Acanthamoeba isolates were determined. For this, the model-based (GTR + Gamma) neighbour-joining, maximum-likelihood and Bayesian-inference analyses and the non-model-based maximum-parsimony analysis were used. All but two of the isolates were identified as the T5 or T4 genotypes, which are probably common in soil, water and contact-lens cases across the Philippines. The only other genotypes identified were T15 (as a single isolate from a contact-lens case) and T3 (as a single soil isolate).

Chimeric classical swine fever viruses containing envelope protein E RNS or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response

Three chimeric classical swine fever virus (CSFV)/bovine viral diarrhoea virus (BVDV) full-length DNA copies were constructed, based on the infectious DNA copy of the CSFV vaccine strain C. The antigenic region of E2 and/or the complete E RNS gene were replaced by the analogous sequence of BVDV II strain 5250. Viable chimeric virus Flc11, in which E RNS was replaced, was directly recovered from supernatant of SK6.T7 cells transfected with full-length DNA. Viable chimeric virus Flc9, in which E2 was replaced, resulted in recovery of virus only when SK6.T7 transfected cells were maintained for several passages. However, no virus could be recovered after replacement of both E RNS and E2, even after 10 cell passages. Both Flc9 and Flc11 grow in swine kidney cells (SK6), stably maintain their heterologous BVDV sequences and, as assessed by monoclonal antibody typing and radio-immunoprecipitation assays, express their heterologous proteins. Flc9 showed a slower growth rate on SK6 cells than Flc11 and wild-type Flc2 virus. Replacement of E RNS or E2 of C-strain-based chimeric viruses did not alter cell tropism compared to wild-type C-strain virus for SK6 and FBE cells. Both Flc9 and Flc11 induced E2 or E RNS antibodies, which could be discriminated from those induced after wild-type virus infection, even after repeated vaccination. Furthermore, pigs were completely protected against a lethal CSFV challenge. These results indicate the feasibility of introduction of marker antigens in a live-attenuated marker C-strain vaccine for CSFV.

Association of rns homologs with colonization factor antigens in clinical Escherichia coli isolates

rns is a trans-acting positive regulatory factor required for expression of the colonization factor antigen II (CFA/II) antigens CS1 and CS2 (J. Caron, L. M. Coffield, and J. R. Scott, Proc. Natl. Acad. Sci. USA 86:963-967, 1989). All 35 CFA/II-positive strains hybridized with a rns gene probe, as did all 10 CFA/I strains and all 4 CS4 strains. Hybridization with rns was detected in 25% of non-enterotoxigenic Escherichia coli strains and was not detected in enteric pathogens with low G + C content.

Expression of CFA/I fimbriae is positively regulated

Production of the plasmid-coded fimbrial antigen CFA/I of Escherichia coli requires both CFA/I region 1 and CFA/I region 2, which are separated by about 40 kb on the wildtype plasmid. The nucleotide sequence of region 2 was determined and contains an open reading frame ( cfa d), encoding a protein of 265 amino acids. The protein has no signal sequence and upon sequence analysis appeared to be a DNA-binding protein. A plasmid was constituted, with a promoterless β-galactosidase gene preceded by the promoter of region 1. Introduction of a plasmid, carrying the cfa d gene, into a strain containing this construct enhanced expression of β-galactosidase by at least five-fold indicating that the cfa d protein was enhancing expression from the promoter of region 1. The cfa d gene sequence differed at 28 positions from the Rns gene, which encodes a protein that is a positive regulator of the expression of CS1 or CS2 fimbriae. It was shown that the cfa d gene and the Rns gene can functionally substitute each other in regulating fimbrial synthesis.

A plasmid-encoded regulatory gene, rns, required for expression of the CS1 and CS2 adhesins of enterotoxigenic Escherichia coli.

To be virulent, enterotoxigenic Escherichia coli (ETEC) must produce a toxin and a pilus-like structure that mediates specific attachment to host tissue. Expression of two of these specific adherence structures, CS1 and CS2, requires the presence of a plasmid in an ETEC strain of a particular serotype and biotype. We show here that this plasmid does not contain the structural gene for a pilin protein, as previously believed. Instead we have identified a plasmid-encoded gene called rns that is required for expression of CS1 or CS2 colonization factor antigens and for adhesion. The rns gene, defined by two separately isolated insertion mutations, produces a 26-kDa protein when transcribed and translated in vitro. At the protein level the rns gene product is homologous to AraC, a positive regulator of the arabinose operon of enteric bacteria, and to RhaR and RhaS, which regulate the rhamnose operon of E. coli. The homology of the Rns protein to AraC is localized to regions that are believed to bind to DNA. Moreover, the sequence of one of these homologous regions is consistent with a DNA binding helix-turn-helix motif. The average G + C content of E. coli DNA is 50%; yet the rns gene contains only 28% G + C, suggesting that it was acquired from some other organism.