BACKGROUND: The insertion sequence elements (IS elements) represent the smallest and the most abundant mobile elements in prokaryotic genomes. It has been shown that they play a significant role in genome organization and evolution. To better understand their function in the host genome, it is desirable to have an effective detection and annotation tool. This need becomes even more crucial when considering rapid-growing genomic and metagenomic data. The existing tools for IS elements detection and annotation are usually based on comparing sequence similarity with a database of known IS families. Thus, they have limited ability to discover distant and putative novel IS elements. RESULTS: In this paper, we present digIS, a software tool based on profile hidden Markov models assembled from catalytic domains of transposases. It shows a very good performance in detecting known IS elements when tested on datasets with manually curated annotation. The main contribution of digIS is in its ability to detect distant and putative novel IS elements while maintaining a moderate level of false positives. In this category it outperforms existing tools, especially when tested on large datasets of archaeal and bacterial genomes. CONCLUSION: We provide digIS, a software tool using a novel approach based on manually curated profile hidden Markov models, which is able to detect distant and putative novel IS elements. Although digIS can find known IS elements as well, we expect it to be used primarily by scientists interested in finding novel IS elements. The tool is available at https://github.com/janka2012/digIS.

• MeSH
• genom bakteriální genetika   MeSH
• genomika MeSH
• lidé MeSH
• prokaryotické buňky * MeSH
• software MeSH
• transpozibilní elementy DNA * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

L1 elements are mammalian retrotransposons contributing to genome evolution and causing rare mutations in human. We describe a de novo insertion of an L1 element into the dystrophin gene resulting in skipping of exon 44 and causing Duchenne muscular dystrophy in a boy. The L1 element was rearranged due to the twin-priming mechanism, but contrary to all described L1 rearrangements the 5' region of the inverted L1 sequence ended within the poly(A) tail of the element. Furthermore, the target site for the insertion was located only 87 bp from the insertion site in another patient described previously. These findings can contribute to the understanding of the mechanisms of L1 element rearrangement, and may support the notion that some subregions of the human genome could be preferred targets for retroelements using the L1 enzymatic machinery.

• MeSH
• dítě MeSH
• Duchennova muskulární dystrofie genetika   MeSH
• dystrofin genetika   MeSH
• exony genetika   MeSH
• financování organizované MeSH
• genetická predispozice k nemoci genetika   MeSH
• genetická variace imunologie   MeSH
• lidé MeSH
• mapování chromozomů MeSH
• medicína založená na důkazech MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• retroelementy genetika   MeSH
• sekvence nukleotidů MeSH
• transpozibilní elementy DNA genetika   MeSH
• zkreslení výsledků (epidemiologie) MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

• MeSH
• Enterococcaceae * MeSH
• larva mikrobiologie   MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• Paenibacillus larvae * genetika   MeSH
• Paenibacillus * genetika   MeSH
• plazmidy genetika   MeSH
• transpozibilní elementy DNA MeSH
• včely genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The recent revision of the Acidithiobacillia class using genomic taxonomy methods has shown that, in addition to the existence of previously unrecognized genera and species, some species of the class harbor levels of divergence that are congruent with ongoing differentiation processes. In this study, we have performed a subspecies-level analysis of sequenced strains of Acidithiobacillus ferrooxidans to prove the existence of distinct sublineages and identify the discriminant genomic/genetic characteristics linked to these sublineages, and to shed light on the processes driving such differentiation. Differences in the genomic relatedness metrics, levels of synteny, gene content, and both integrated and episomal mobile genetic elements (MGE) repertoires support the existence of two subspecies-level taxa within A. ferrooxidans. While sublineage 2A harbors a small plasmid related to pTF5, this episomal MGE is absent in sublineage 2B strains. Likewise, clear differences in the occurrence, coverage and conservation of integrated MGEs are apparent between sublineages. Differential MGE-associated gene cargo pertained to the functional categories of energy metabolism, ion transport, cell surface modification, and defense mechanisms. Inferred functional differences have the potential to impact long-term adaptive processes and may underpin the basis of the subspecies-level differentiation uncovered within A. ferrooxidans. Genome resequencing of iron- and sulfur-adapted cultures of a selected 2A sublineage strain (CCM 4253) showed that both episomal and large integrated MGEs are conserved over twenty generations in either growth condition. In turn, active insertion sequences profoundly impact short-term adaptive processes. The ISAfe1 element was found to be highly active in sublineage 2A strain CCM 4253. Phenotypic mutations caused by the transposition of ISAfe1 into the pstC2 encoding phosphate-transport system permease protein were detected in sulfur-adapted cultures and shown to impair growth on ferrous iron upon the switch of electron donor. The phenotypic manifestation of the △pstC2 mutation, such as a loss of the ability to oxidize ferrous iron, is likely related to the inability of the mutant to secure the phosphorous availability for electron transport-linked phosphorylation coupled to iron oxidation. Depletion of the transpositional △pstC2 mutation occurred concomitantly with a shortening of the iron-oxidation lag phase at later transfers on a ferrous iron-containing medium. Therefore, the pstII operon appears to play an essential role in A. ferrooxidans when cells oxidize ferrous iron. Results highlight the influence of insertion sequences and both integrated and episomal mobile genetic elements in the short- and long-term adaptive processes of A. ferrooxidans strains under changing growth conditions.

• MeSH
• Acidithiobacillus * genetika   metabolismus   MeSH
• oxidace-redukce MeSH
• síra metabolismus   MeSH
• transpozibilní elementy DNA * genetika   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Polyploidization is considered one of the main mechanisms of plant genome evolution. The presence of multiple copies of the same gene reduces selection pressure and permits sub-functionalization and neo-functionalization leading to plant diversification, adaptation and speciation. In bread wheat, polyploidization and the prevalence of transposable elements resulted in massive gene duplication and movement. As a result, the number of genes which are non-collinear to genomes of related species seems markedly increased in wheat. RESULTS: We used new-generation sequencing (NGS) to generate sequence of a Mb-sized region from wheat chromosome arm 3DS. Sequence assembly of 24 BAC clones resulted in two scaffolds of 1,264,820 and 333,768 bases. The sequence was annotated and compared to the homoeologous region on wheat chromosome 3B and orthologous loci of Brachypodium distachyon and rice. Among 39 coding sequences in the 3DS scaffolds, 32 have a homoeolog on chromosome 3B. In contrast, only fifteen and fourteen orthologs were identified in the corresponding regions in rice and Brachypodium, respectively. Interestingly, five pseudogenes were identified among the non-collinear coding sequences at the 3B locus, while none was found at the 3DS locus. CONCLUSION: Direct comparison of two Mb-sized regions of the B and D genomes of bread wheat revealed similar rates of non-collinear gene insertion in both genomes with a majority of gene duplications occurring before their divergence. Relatively low proportion of pseudogenes was identified among non-collinear coding sequences. Our data suggest that the pseudogenes did not originate from insertion of non-functional copies, but were formed later during the evolution of hexaploid wheat. Some evidence was found for gene erosion along the B genome locus.

• MeSH
• Brachypodium genetika   MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná genetika   MeSH
• duplikace genu MeSH
• fylogeneze MeSH
• genetické lokusy genetika   MeSH
• genom rostlinný genetika   MeSH
• inzerční mutageneze MeSH
• kontigové mapování MeSH
• molekulární evoluce * MeSH
• polyploidie MeSH
• pšenice genetika   MeSH
• pseudogeny genetika   MeSH
• rýže (rod) genetika   MeSH
• sekvenční analýza DNA MeSH
• umělé bakteriální chromozomy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

The genomic shock hypothesis suggests that allopolyploidy is associated with genome changes driven by transposable elements, as a response to imbalances between parental insertion loads. To explore this hypothesis, we compared three allotetraploids, Nicotiana arentsii, N. rustica and N. tabacum, which arose over comparable time frames from hybridisation between increasingly divergent diploid species. We used sequence-specific amplification polymorphism (SSAP) to compare the dynamics of six transposable elements in these allopolyploids, their diploid progenitors and in corresponding synthetic hybrids. We show that element-specific dynamics in young Nicotiana allopolyploids reflect their dynamics in diploid progenitors. Transposable element mobilisation is not concomitant with immediate genome merger, but occurs within the first generations of allopolyploid formation. In natural allopolyploids, such mobilisations correlate with imbalances in the repeat profile of the parental species, which increases with their genetic divergence. Other restructuring leading to locus loss is immediate, nonrandom and targeted at specific subgenomes, independently of cross orientation. The correlation between transposable element mobilisation in allopolyploids and quantitative imbalances in parental transposable element loads supports the genome shock hypothesis proposed by McClintock.

• MeSH
• fylogeneze MeSH
• genetická variace MeSH
• genetické lokusy MeSH
• hybridizace genetická * MeSH
• polyploidie * MeSH
• sekvence nukleotidů MeSH
• tabák genetika   MeSH
• transpozibilní elementy DNA genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Burkholderia cenocepacia can cause serious infections and epidemics in patients with cystic fibrosis (CF). A CF population in the Czech Republic experienced an epidemic outbreak caused by a B. cenocepacia ST-32 strain. The clonality of the isolates was evident by multilocus sequence typing; however, fingerprinting profiles obtained by pulsed-field gel electrophoresis (PFGE) showed substantial band variability. We investigated whether the PFGE pattern diversity resulted from genomic rearrangements mediated by insertion sequences (IS); in addition, we determined whether stressful growth conditions altered the transposition activity of these IS. DNA probes for IS commonly found in B. cenocepacia were designed using the B. cenocepacia J2315 genome. Southern hybridization analysis of ST-32 isolates demonstrated diversity in both the copy number and the insertion site for a homologue of ISBcen20. Movement of the ISBcen20 homologue was detected when the ST-32 isolate CZ1238 was exposed to oxidative stress (growth in the presence of H(2)O(2)). PFGE analysis of CZ1238 derivatives exposed to oxidative stress demonstrated genomic rearrangements. Interestingly, when the closely related B. cenocepacia strain J2315 was exposed to oxidative stress, no movement of ISBcen20 was detected. Since frameshift mutations are present within the transposases of all copies of this IS in J2315, our data suggest that the transposase is inactive. In summary, we have demonstrated for the first time that IS movement can be mediated by oxidative stress and can lead to genomic rearrangements in the CF pathogen B. cenocepacia. These IS movements may alter the PFGE fingerprints of isolates that are clonal by other typing methods.

• MeSH
• Burkholderia klasifikace   genetika   izolace a purifikace   MeSH
• cystická fibróza komplikace   MeSH
• DNA bakterií genetika   MeSH
• DNA fingerprinting metody   MeSH
• epidemický výskyt choroby MeSH
• genetická variace * MeSH
• genotyp MeSH
• infekce bakteriemi rodu Burkholderia epidemiologie   mikrobiologie   MeSH
• lidé MeSH
• molekulární epidemiologie metody   MeSH
• oxidační stres * MeSH
• pulzní gelová elektroforéza metody   MeSH
• rekombinace genetická * MeSH
• shluková analýza MeSH
• techniky typizace bakterií metody   MeSH
• transpozibilní elementy DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

We describe the molecular etiology of β(+)-thalassemia that is caused by the insertion of the full-length transposable element LINE-1 (L1) into the intron-2 of the β-globin gene (HBB). The transcript level of the affected β-globin gene was severely reduced. The remaining transcripts consisted of full-length, correctly processed β-globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half-life due to activation of the nonsense-mediated decay pathway. The lower steady-state amount of mRNA produced by the β-globin(L1) allele also resulted from a reduced rate of transcription and decreased production of full-length β-globin primary transcripts. The promoter and enhancer sequences of the β-globin(L1) allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the β-globin(L1) transcription despite permanent β-globin(L1) promoter CpG methylation. This result indicates that the decreased rate of transcription from the β-globin(L1) allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the β-globin gene represents a novel etiology of β-thalassemia.

• MeSH
• alely MeSH
• alternativní sestřih MeSH
• beta-globiny genetika   MeSH
• beta-talasemie genetika   MeSH
• CpG ostrůvky MeSH
• dlouhé rozptýlené jaderné elementy * MeSH
• dospělí MeSH
• genetická transkripce MeSH
• introny * MeSH
• inzerční mutageneze * MeSH
• lidé MeSH
• metylace DNA MeSH
• pořadí genů MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese MeSH
• stabilita RNA MeSH
• umlčování genů MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

Analysis of plants bearing a T-DNA insertion is a potent tool of modern molecular biology, providing valuable information about the function and involvement of genes in metabolic pathways. A collection of 12 Arabidopsis thaliana lines with T-DNA insertions in the gene coding for the catalytic subunit of telomerase (AtTERT) and in adjacent regions was screened for telomerase activity [telomere repeat amplification protocol (TRAP) assay], telomere length (terminal restriction fragments), and AtTERT transcription (quantitative reverse transcription-PCR). Lines with the insertion located upstream of the start codon displayed unchanged telomere stability and telomerase activity, defining a putative minimal AtTERT promoter and the presence of a regulatory element linked to increased transcription in the line SALK_048471. Lines bearing a T-DNA insertion inside the protein-coding region showed telomere shortening and lack of telomerase activity. Transcription in most of these lines was unchanged upstream of the T-DNA insertion, while it was notably decreased downstream. The expression profile varied markedly in mutant lines harbouring insertions at the 5' end of AtTERT which showed increased transcription and abolished tissue specificity. Moreover, the line FLAG_385G01 (T-DNA insertion inside intron 1) revealed the presence of a highly abundant downstream transcript with normal splicing but without active telomerase. The role of regulatory elements found along the AtTERT gene is discussed in respect to natural telomerase expression and putative intron-mediated enhancement.

• MeSH
• Arabidopsis genetika   MeSH
• DNA bakterií genetika   MeSH
• genotyp MeSH
• inzerční mutageneze MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny huseníčku genetika   MeSH
• regulace genové exprese u rostlin MeSH
• regulační oblasti nukleových kyselin genetika   MeSH
• telomerasa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The current studies on human and veterinary epidemiology have highly contributed to the detection of specific, small and mobile elements of the DNA - insertion sequences (IS). These sequences were: IS900 in Mycobacterium avium subspecies paratuberculosis, IS6110 in M. tuberculosis, and IS901 in M. avium complex. It has been ten years since the discovery of mycobacterial insertion Sequences. Accordingly, the aim of this abstract was to present a summary of various research aspects and utilization of insertion sequences in mycobacteriology. The discovery, composition and function of IS are described in the introduction part. Priority has been given to the insertion sequences found in M. avium complex: IS902, IS1245, IS1311, IS1110, IS1141, IS1613.

• MeSH
• lidé MeSH
• molekulární typizace MeSH
• Mycobacterium tuberculosis MeSH
• Mycobacterium MeSH
• mykobakteriózy epidemiologie   MeSH
• transpozibilní elementy DNA MeSH
• tuberkulóza epidemiologie   přenos   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Geografické názvy
• Česká republika MeSH