Transposition Of Mobile Elements Research Articles

Resistance of Klebsiella pneumoniae Strains Carrying bla NDM-1 Gene and the Genetic Environment of bla NDM-1.

ObjectiveRegional dissemination is the major cause of the widespread prevalence of a plasmid-encoding NDM-1 enzyme. We investigated the drug resistance, joint efficiency, and gene environment of a Klebsiella pneumoniae strain carrying blaNDM–1 gene.Materials and MethodsCarbapenem-non-susceptible strains were analyzed using the VITEK 2 Compact. Strains carrying blaNDM–1 were identified using polymerase chain reaction and sequencing. Antimicrobial susceptibility testing and plasmid conjugation experiments were then conducted. Strains carrying blaNDM–1 were subjected to Southern blot analysis. After the gene mapping of blaNDM–1, library construction, and sequencing, plasmids were subsequently spliced and genotyped using the software Glimmer 3.0, and then analyzed using Mauve software.ResultsAmong 1735 carbapenem-non-susceptible strains, 54 strains of blaNDM–1-positive bacteria were identified, which consisted of 44 strains of K. pneumoniae, 8 strains of Acinetobacter baumannii and 2 strains of Escherichia coli. Strains carrying blaNDM–1 had a resistance rate of more than 50% in most antibiotics. Plasmid conjugation between strains carrying blaNDM–1 and E. coli strain J53 had a success rate of 50%. Southern blot analysis indicated that each strain had multiple plasmids containing blaNDM–1. Among the five plasmids containing blaNDM–1 in K. pneumoniae for sequencing, two plasmids with complete sequences were obtained. The findings were as follows: (i) The p11106 and p12 plasmids were highly similar to pNDM-BTR; (ii) the p11106 and p12 plasmids showed differences in the 20–30 kb region (orf00032–orf00043) from the other six plasmids; and (iii) blaNDM–1 was located at orf00037, while ble was found at orf00038. Two tnpA genes were located in the upstream region, and orf00052 (tnpA) in the 36 kb region was in the downstream sequence.ConclusionblaNDM–1-containing bacteria exhibit multidrug resistance, which rapidly spreads and is transferred through efficient plasmid conjugation; the multidrug resistance of these bacteria may be determined by analyzing their drug-resistant plasmids. The presence of ble and tnpA genes suggests a possible hypothesis that blaNDM–1 originates from A. baumannii, which is retained in K. pneumoniae over a long period by transposition of mobile elements.

Gene duplication and transposition of mobile elements drive evolution of the Rpv3 resistance locus in grapevine.

A wild grape haplotype (Rpv3-1) confers resistance to Plasmopara viticola. We mapped the causal factor for resistance to an interval containing a TIR-NB-LRR (TNL) gene pair that originated 1.6-2.6 million years ago by a tandem segmental duplication. Transient coexpression of the TNL pair in Vitis vinifera leaves activated pathogen-induced necrosis and reduced sporulation compared with control leaves. Even though transcripts of the TNL pair from the wild haplotype appear to be partially subject to nonsense-mediated mRNA decay, mature mRNA levels in a homozygous resistant genotype were individually higher than the mRNA trace levels observed for the orthologous single-copy TNL in sensitive genotypes. Allelic expression imbalance in a resistant heterozygote confirmed that cis-acting regulatory variation promotes expression in the wild haplotype. The movement of transposable elements had a major impact on the generation of haplotype diversity, altering the DNA context around similar TNL coding sequences and the GC-content in their proximal 5'-intergenic regions. The wild and domesticated haplotypes also diverged in conserved single-copy intergenic DNA, but the highest divergence was observed in intraspecific and not in interspecific comparisons. In this case, introgression breeding did not transgress the genetic boundaries of the domesticated species, because haplotypes present in modern varieties sometimes predate speciation events between wild and cultivated species.

Genome analysis of Rubritalea profundi SAORIC-165T, the first deep-sea verrucomicrobial isolate, from the northwestern Pacific Ocean.

Although culture-independent studies have shown the presence of Verrucomicrobia in the deep sea, verrucomicrobial strains from deep-sea environments have been rarely cultured and characterized. Recently, Rubritalea profundi SAORIC-165T, a psychrophilic bacterium of the phylum Verrucomicrobia, was isolated from a depth of 2,000 m in the northwestern Pacific Ocean. In this study, the genome sequence of R. profundi SAORIC-165T, the first deep-sea verrucomicrobial isolate, is reported with description of the genome properties and comparison to surface-borne Rubritalea genomes. The draft genome consisted of four contigs with an entire size of 4,167,407 bp and G+C content of 47.5%. The SAORIC-165T genome was predicted to have 3,844 proteincoding genes and 45 non-coding RNA genes. The genome contained a repertoire of metabolic pathways, including the Embden-Meyerhof-Parnas pathway, pentose phosphate pathway, tricarboxylic acid cycle, assimilatory sulfate reduction, and biosynthesis of nicotinate/nicotinamide, pantothenate/coenzyme A, folate, and lycopene. The comparative genomic analyses with two surface-derived Rubritalea genomes showed that the SAORIC-165T genome was enriched in genes involved in transposition of mobile elements, signal transduction, and carbohydrate metabolism, some of which might be related to bacterial enhancement of ecological fitness in the deep-sea environment. Amplicon sequencing of 16S rRNA genes from the water column revealed that R. profundi-related phylotypes were relatively abundant at 2,000 m and preferred a particle-associated life style in the deep sea. These findings suggest that R. profundi represents a genetically unique and ecologically relevant verrucomicrobial group well adapted to the deep-sea environment.

Genomic Structural Variations Affecting Virulence During Clonal Expansion of Pseudomonas syringae pv. actinidiae Biovar 3 in Europe.

Pseudomonas syringae pv. actinidiae (Psa) biovar 3 caused pandemic bacterial canker of Actinidia chinensis and Actinidia deliciosa since 2008. In Europe, the disease spread rapidly in the kiwifruit cultivation areas from a single introduction. In this study, we investigated the genomic diversity of Psa biovar 3 strains during the primary clonal expansion in Europe using single molecule real-time (SMRT), Illumina and Sanger sequencing technologies. We recorded evidences of frequent mobilization and loss of transposon Tn6212, large chromosome inversions, and ectopic integration of IS sequences (remarkably ISPsy31, ISPsy36, and ISPsy37). While no phenotype change associated with Tn6212 mobilization could be detected, strains CRAFRU 12.29 and CRAFRU 12.50 did not elicit the hypersensitivity response (HR) on tobacco and eggplant leaves and were limited in their growth in kiwifruit leaves due to insertion of ISPsy31 and ISPsy36 in the hrpS and hrpR genes, respectively, interrupting the hrp cluster. Both strains had been isolated from symptomatic plants, suggesting coexistence of variant strains with reduced virulence together with virulent strains in mixed populations. The structural differences caused by rearrangements of self-genetic elements within European and New Zealand strains were comparable in number and type to those occurring among the European strains, in contrast with the significant difference in terms of nucleotide polymorphisms. We hypothesize a relaxation, during clonal expansion, of the selection limiting the accumulation of deleterious mutations associated with genome structural variation due to transposition of mobile elements. This consideration may be relevant when evaluating strategies to be adopted for epidemics management.

The Role of Transposable Elements in Emergence of Metazoa.

Systems initially emerged for protecting genomes against insertions of transposable elements and represented by mechanisms of splicing regulation, RNA-interference, and epigenetic factors have played a key role in the evolution of animals. Many studies have shown inherited transpositions of mobile elements in embryogenesis and preservation of their activities in certain tissues of adult organisms. It was supposed that on the emergence of Metazoa the self-regulation mechanisms of transposons related with the gene networks controlling their activity could be involved in intercellular cell coordination in the cascade of successive divisions with differentiated gene expression for generation of tissues and organs. It was supposed that during evolution species-specific features of transposons in the genomes of eukaryotes could form the basis for creation of dynamically related complexes of systems for epigenetic regulation of gene expression. These complexes could be produced due to the influence of noncoding transposon-derived RNAs on DNA methylation, histone modifications, and processing of alternative splicing variants, whereas the mobile elements themselves could be directly involved in the regulation of gene expression in cis and in trans. Transposons are widely distributed in the genomes of eukaryotes; therefore, their activation can change the expression of specific genes. In turn, this can play an important role in cell differentiation during ontogenesis. It is supposed that transposons can form a species-specific pattern for control of gene expression, and that some variants of this pattern can be favorable for adaptation. The presented data indicate the possible influence of transposons in karyotype formation. It is supposed that transposon localization relative to one another and to protein-coding genes can influence the species-specific epigenetic regulation of ontogenesis.

Asking the Right Question about the Genetic Basis of Domestication: What is the Source of Genetic Diversity of Domesticated Species?

In this paper we investigate source of increased genetic diversity - one of the most obvious characteristics of domesticated species, leading to a wide range of phenotypic variability (breeds). The hypothesis that we put forward during the course of our studies focuses on transposons and retro-viral elements in the domesticated genomes. The inherent tolerance for transposition of mobile elements could lead to enhanced genetic diversity in the domesticated species.

Stress-induced transcriptional activation of retrotransposon-like sequences in the Scots pine (Pinus sylvestris L.) genome

Transposition of mobile elements has been implicated in genome instability, rearrangements and therefore also adaptation to changing environmental conditions. Transposons could influence gene activity directly by transposition inside or close to coding regions by their disruption or by addition of regulative sequences. Further, class I transposable elements, which are the most abundant in plant genomes, utilize a RNA intermediate in their life cycle, therefore retrotransposons could act by producing non-coding RNAs that could affect other transcripts by RNA interference. Transposition activity is suppressed by chromatin modifications, and both classes of transposons have been shown to be activated in plants under various stress conditions and developmental stages. Using a nonspecific amplification approach, we demonstrate the differential transcriptional activation of sequences with homology to transposable elements and other associated sequences in the complex genome of Scots pine (Pinus sylvestris L.) after exposure to heat stress, infestation with pine woolly aphids, and salicylic acid and abscisic acid treatment. Sequences with homology to several retrotransposon classes and families were identified, as well as several chimeric transcript types. Some of them represent chloroplast sequence insertions into the pine nuclear genome and these sequences are highly represented in EST databases of a wide range of species. In this study, we identified several retrotransposon classes and families with differing levels of similarity with known transposable elements from other plant species, and which are differentially expressed under various stress conditions in Scots pine.

A Deterministic Analysis of Genome Integrity during Neoplastic Growth in Drosophila

The development of cancer has been associated with the gradual acquisition of genetic alterations leading to a progressive increase in malignancy. In various cancer types this process is enabled and accelerated by genome instability. While genome sequencing-based analysis of tumor genomes becomes increasingly a standard procedure in human cancer research, the potential necessity of genome instability for tumorigenesis in Drosophila melanogaster has, to our knowledge, never been determined at DNA sequence level. Therefore, we induced formation of tumors by depletion of the Drosophila tumor suppressor Polyhomeotic and subjected them to genome sequencing. To achieve a highly resolved delineation of the genome structure we developed the Deterministic Structural Variation Detection (DSVD) algorithm, which identifies structural variations (SVs) with high accuracy and at single base resolution. The employment of long overlapping paired-end reads enables DSVD to perform a deterministic, i.e. fragment size distribution independent, identification of a large size spectrum of SVs. Application of DSVD and other algorithms to our sequencing data reveals substantial genetic variation with respect to the reference genome reflecting temporal separation of the reference and laboratory strains. The majority of SVs, constituted by small insertions/deletions, is potentially caused by erroneous replication or transposition of mobile elements. Nevertheless, the tumor did not depict a loss of genome integrity compared to the control. Altogether, our results demonstrate that genome stability is not affected inevitably during sustained tumor growth in Drosophila implying that tumorigenesis, in this model organism, can occur irrespective of genome instability and the accumulation of specific genetic alterations.

Regulatory small RNAs

Recent years have been marked by a burst of studies on the role of various RNAs in the regulation of gene expression. These regulatory effects act on the level of both chromatin in the nuclei and the cytoplasm during translation. The review papers of this issue are mainly dedicated to different types of small RNAs of 20-30 nucleotides. The small RNAs control diverse cellular functions including genome protection against transpositions of mobile elements of the genome.

Small RNAs and transposon silencing in plants

Transposons are highly conserved in plants and have created a symbiotic relationship with the host genome. An important factor of the successful communication between transposons and host plants is epigenetic modifications including DNA methylation and the modifications of the histone tail. In plants, small interfering RNAs (siRNAs) are responsible for RNA-directed DNA methylation (RdDM) that suppresses transposon activities. Although most transposons are silent in their host plants, certain genomic shocks, such as an environmental stress or a hybridization event, might trigger transposon activation. Further, since transposons can affect the regulation mechanisms of host genes, it is possible that transposons have co-evolved as an important mechanism for plant development and adaptation. Recent new findings reveal that siRNAs control not only transcriptional activation, but also suppress transgenerational transposition of mobile elements making siRNAs critically important towards maintaining genome stability. Together these data suggest host-mediated siRNA regulation of transposons appears to have been adapted for controlling essential systems of plant development, morphogenesis, and reproduction.

Study of the activity of integrase of retrotransposone Gypsy Drosophila melanogaster in vitro

Introduction of retrotransposone gypsy in the genome of Drosophila melanogaster occurs specifically and is performed by integrase of this mobile element. Gypsy integrase is capable not only of introduction but also of precise excision of retrotransposon DNA from the target site. In the present work, we study the endonuclease activity of recombinant integrase gypsy in vitro. It is indicated that the enzyme hydrolyzes the substrate, forming single-chain and double-chain breaks. Conformation of the substrate greatly influences hydrolysis: integrase conducts double-chain breaks only in a supertwisted circular DNA molecule. The data make it possible to understanding the integration mechanism of retroviruses and transposition of mobile elements.

Conserved flanking microsatellite sequences (ReFS) differentiate between Lepidoptera species, and provide insight into microsatellite evolution

High rates of mutation and homoplasy mean that microsatellites generally are not considered to be useful molecular markers for inferring systematic relationships between species. However, an earlier pilot study suggested that conserved flanking microsatellite sequences, also known as repetitive flanking sequences (ReFS), may form a basis for a dominant marker that can differentiate between species of Lepidoptera. We present data that demonstrate that ReFS are quick and easy to use, and generate highly repeatable banding patterns from a range of Lepidoptera species. Sequence data from a subset of ReFS‐amplified bands revealed microsatellite families with flanking sequences that are more conserved within than among species: this is probably attributable to recombination‐mediated events, transposition of mobile elements or a combination of the two. Our data support the use of ReFS as dominant interspecific molecular markers, and add to the growing literature on the evolution of microsatellites in Lepidoptera.

Amplification of “defective” retrotransposon gtwin in D. melanogaster strain carrying large complex chromosomal aberration

Transposable elements (TE) are found in all eukaryotic genomes and play a significant role in their structure and functioning. The majority of mobile elements are silent in the genomes indicating the existence of cell control mechanisms of their activity. Establishment of immunity to TE is of great interest, but it cannot be studied directly and there are only few examples of present or recent active transpositions of mobile elements. G32, a Drosophila melanogaster strain, is characterized by the presence of large complex chromosomal aberration in the 3rd chromosome, active transpositions of gtwin in the past, and its stability at present. To address the question as to what had happened to the element while the cell took it under the control, we performed the detailed cytological and molecular analyses of gtwin's structure and its distribution in G32. Two variants of gtwin were found, one of which is amplified in G32 despite the alteration of tRNA-primer binding site. This element is accumulated in the aberrant chromosome and associated with the inversions breakpoints. Gtwin copies are predominantly localized in euchromatic regions and at least three of them are situated in heterochromatin. One copy was found in the piRNA cluster that might have caused silencing of the element.

Genome-wide analysis of DNA turnover and gene expression in stationary-phase Saccharomyces cerevisiae

Exponential-phase yeast cells readily enter stationary phase when transferred to fresh, carbon-deficient medium, and can remain fully viable for up to several months. It is known that stationary-phase prokaryotic cells may still synthesize substantial amounts of DNA. Although the basis of this phenomenon remains unclear, this DNA synthesis may be the result of DNA maintenance and repair, recombination, and stress-induced transposition of mobile elements, which may occur in the absence of DNA replication. To the best of our knowledge, the existence of DNA turnover in stationary-phase unicellular eukaryotes remains largely unstudied. By performing cDNA-spotted (i.e. ORF) microarray analysis of stationary cultures of a haploid Saccharomyces cerevisiae strain, we demonstrated on a genomic scale the localization of a DNA-turnover marker [5-bromo-2'-deoxyuridine (BrdU); an analogue of thymidine], indicative of DNA synthesis in discrete, multiple sites across the genome. Exponential-phase cells on the other hand, exhibited a uniform, total genomic DNA synthesis pattern, possibly the result of DNA replication. Interestingly, BrdU-labelled sites exhibited a significant overlap with highly expressed features. We also found that the distribution among chromosomes of BrdU-labelled and expressed features deviates from random distribution; this was also observed for the overlapping set. Ty1 retrotransposon genes were also found to be labelled with BrdU, evidence for transposition during stationary phase; however, they were not significantly expressed. We discuss the relevance and possible connection of these results to DNA repair, mutation and related phenomena in higher eukaryotes.

Role of short RNAs in regulating the expression of genes and mobile elements in germ cells

Two main types of short RNAs, 21 to 25 nucleotides long, are involved in the negative regulation of gene expression in eukaryotes: microRNAs and small interfering RNAs (siRNAs) of the RNA interference system. MicroRNAs predominantly suppress the translation of mRNA targets, while siRNAs not only prevent mRNA translation and/or lead to mRNA degradation, but are also involved in the regulation of gene expression at the transcriptional level. In germ cells translational regulation of gene expression plays a significant role and its mechanism has been extensively studied in oogenesis of Drosophila@. The role of heterochromatization and chromatin compaction, which can repress the expression of mobile elements and other repeated elements of the genome, was studied to a lesser extent. Activation and transposition of mobile elements accompanied by mutations and chromosome rearrangements are especially dangerous in germline cells. It has been proposed that a specialized class of short RNAs, repeat associated siRNAs (rasiRNAs), can be involved in repression of the expression of mobile elements in Drosophila germ cells. Here we describe the findings on subcellular ribonucleoprotein structures characteristic of germ cells: perinuclear and polar granules containing proteins of the RNA interference and microRNA maturation system. Also, we present our own results revealing the role of genes of the RNA interference system in mobile element silencing in Drosophila.

Evolutionary dynamics of multilocus microsatellite arrangements in the genome of the butterfly Bicyclus anynana, with implications for other Lepidoptera

The sequences flanking microsatellites isolated from the butterfly Bicyclus anynana display high levels of similarity among different loci. We examined sequence data for evidence of the two mechanisms most likely to generate these similarities, namely recombination mediated events, such as unequal crossing over or gene conversion and through transposition of mobile elements (MEs). Many sequences contained tandemly arranged microsatellites, lending support to recombination as the multiplication mechanism. There is, however, also support for ME-mediated multiplication of microsatellites and their flanking sequences. Homology with a known Lepidopteran ME was found in B. anynana microsatellite regions, and polymorphic microsatellite markers with partial similarities in their flanking sequences were passed on to the next generation independently, indicating that they are not linked. Therefore, the rise of these similarities appears to be mediated through both processes, either as an interaction between the two, or by each being responsible for part of the observations. A large proportion of microsatellites embedded in repetitive DNA is representative for most studied butterflies and moths, and a BLAST survey of the B. anynana sequences revealed four short microsatellite-associated sequences that were present in many species of Lepidoptera. The similarities usually start to deviate beyond these sequences, which suggests that they define the extremes of a repeated unit. Further study of these conserved sequences may help to understand the mechanism underlying the multiplication events, and answer the question of why these redundancies are predominantly found in this insect group.

Molecular and chromosomal analysis of ribosomal cistrons in two cartilaginous fish, Taeniura lymma and Raja montagui (Chondrichthyes, Batoidea)

We used silver nitrate staining, CMA3 and FISH to study the chromosomal localization of both the major ribosomal genes and the nucleolar organizer regions as well as that of the minor ribosomal genes (5S rDNA) in two species of Batoidea, Taeniura lymma (Dasyatidae) and Raja montagui (Rajidae). In both species, all the metaphases examined showed the presence of multiple NOR-bearing sites, while the gene for 5S rRNA proved to be localized on two chromosome pairs. Furthermore, one of the two 5S rDNA sites in T. lymma was shown to be co-localized with the major ribosomal cluster. The presence of multiple nucleolar organizer regions in the two species might be interpreted as being the result of intraspecific polymorphisms, or as a phenomenon of the amplified transposition of mobile elements of the genome. We also determined the nucleotide sequence of the 5S rRNA gene, consisting of 564 bp in R. montagui and 612 bp in T. lymma. We also found TATA-like and (TGC)n trinucleotides, (CA)n dinucleotides and (GTGA)n tetranucleotides, which probably influence gene regulation.

Genetic variability of nematodes: a threat to the durability of plant resistance genes?

Plant resistance is currently the most effective and environmentally safe method to control plant parasitic nematodes (PPNs). Resistance genes generally act against sedentary PPNs by inducing a hypersensitive reaction that prevents the parasite installation and/or reproduction. However, the recent emergence of virulent biotypes able to overcome the plant resistance genes may constitute a severe limitation to this control strategy. In selection experiments conducted under controled environment, the genetic variation, specificity and inheritance of nematode virulence have been demonstrated. Moreover, the occurrence of gene-for-gene interactions has been shown in a few cases. Moleculars markers have been extensively used to investigate the genetic variability of PPNs, but so far, the genomic polymorphisms observed are largely independent of virulence. Such data suggest that, within a species, virulent isolates do not share a common origin, but are probably the result of independent mutational events. To understand the molecular mechanisms responsible for virulence in PPNs, several strategies have been developed, in relation with their mode of reproduction (parthenogenesis versus amphimixis). As an example, recent results obtained in our laboratory on the root-knot nematodes Meloidogyne spp. are presented. On a more general point of view, factors that may induce stable genome variability in PPNs, e.g. Transposition of mobile elements and chromosomal rearrangements (leading to polyploidy, aneuploidy, etc) will also be considered. Advances in knowledge in these areas should have important consequences for the management and durability of natural resistance genes, and for the engineering of new forms of resistance.

Role of double-stranded RNA in eukaryotic gene silencing

Data on RNA interference, that is, posttranscriptional gene silencing by homologous double-stranded (ds) RNA, are reviewed. Gene silencing caused by exogenous dsRNA in artificial systems and observed in transgenic organisms carrying additional gene copies is considered. Data are summarized on the mechanism that arose during evolution of the Drosophila melanogaster genome to suppress repetitive genes with the use of dsRNA and thereby to prevent male sterility. The role of dsRNA in inhibiting expression and transposition of mobile elements is discussed on the basis of authors own and published findings.

Different mechanisms of telomere length regulation

Eukaryotic chromosomes are linear and have their, ends formed by DNA-protein structures, telomeres. At present more and more facts demonstrate the diversity of telomere functions. Telomeres protect the chromosome ends from degradation, fusion, recombination, and from the repair system that recognizes nicks in DNA strands. As shown recently, shortening of the telomeres is a cause of cell aging. In most organisms, telomeres are elongated by means of a special ribonucleoprotein complex; however, in some insects this takes place by either gene conversion or transposition of mobile elements. Evolutionary relations between different types of telomeres are discussed.