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
- Genome, Bacterial genetics MeSH
- Genomics MeSH
- Humans MeSH
- Prokaryotic Cells * MeSH
- Software MeSH
- DNA Transposable Elements * genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article 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 genetics MeSH
- Chromosomes, Plant genetics MeSH
- DNA, Plant genetics MeSH
- Gene Duplication MeSH
- Phylogeny MeSH
- Genetic Loci genetics MeSH
- Genome, Plant genetics MeSH
- Mutagenesis, Insertional MeSH
- Contig Mapping MeSH
- Evolution, Molecular * MeSH
- Polyploidy MeSH
- Triticum genetics MeSH
- Pseudogenes genetics MeSH
- Oryza genetics MeSH
- Sequence Analysis, DNA MeSH
- Chromosomes, Artificial, Bacterial MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study 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 microbiology MeSH
- Multiplex Polymerase Chain Reaction methods MeSH
- Paenibacillus larvae * genetics MeSH
- Paenibacillus * genetics MeSH
- Plasmids genetics MeSH
- DNA Transposable Elements MeSH
- Bees genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article 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 classification genetics isolation & purification MeSH
- Cystic Fibrosis complications MeSH
- DNA, Bacterial genetics MeSH
- DNA Fingerprinting methods MeSH
- Disease Outbreaks MeSH
- Genetic Variation * MeSH
- Genotype MeSH
- Burkholderia Infections epidemiology microbiology MeSH
- Humans MeSH
- Molecular Epidemiology methods MeSH
- Oxidative Stress * MeSH
- Electrophoresis, Gel, Pulsed-Field methods MeSH
- Recombination, Genetic * MeSH
- Cluster Analysis MeSH
- Bacterial Typing Techniques methods MeSH
- DNA Transposable Elements MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
We herein present a rare case of an EML4-ALK positive patient. A 61-year-old man was diagnosed with locoregional non-small cell lung cancer (NSCLC). No EGFR mutations were detected, and therefore the ALK rearrangement was evaluated using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and the reverse transcription PCR (RT-PCR) method for EML4-ALK. All methods showed a positive result and, therefore, the patient was treated with crizotinib with a good therapeutic response. However, a detailed RT-PCR analysis and sequencing revealed an unexpected 138 bp insertion of attractin-like 1 (ATRNL1) gene into the EML4-ALK fusion gene. In our case, the positive therapeutic response suggests that ATRNL1 insertion does not affect EML4-ALK's sensitivity to crizotinib. This case shows great EML4-ALK heterogeneity and also that basic detection methods (IHC, FISH) cannot fully specify ALK rearrangement but in many cases a full specification seems to be important for an effective TKI indication, and sequencing ALK variants might contribute to optimized patient selection.
- MeSH
- Oncogene Proteins, Fusion chemistry genetics MeSH
- Protein Kinase Inhibitors therapeutic use MeSH
- Mutagenesis, Insertional * MeSH
- Middle Aged MeSH
- Humans MeSH
- Molecular Sequence Data MeSH
- Lung Neoplasms diagnosis drug therapy genetics MeSH
- Carcinoma, Non-Small-Cell Lung diagnosis drug therapy genetics MeSH
- Pyrazoles therapeutic use MeSH
- Pyridines therapeutic use MeSH
- Radiography, Thoracic MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA MeSH
- Neoplasm Staging MeSH
- Treatment Outcome MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
- Research Support, Non-U.S. Gov't MeSH
Renal hypouricemia is a heterogeneous inherited disorder characterized by impaired uric acid handling in the renal tubules. Patients are usually asymptomatic; however, some may experience urolithiasis and/or acute kidney injury. Most of the described patients (compound heterozygous and/or homozygous) are Japanese with mutations in the SLC22A12 gene (OMIM #220150). Four patients with renal hypouricemia caused by heterozygous defects and two families with homozygous mutations in the SLC2A9 gene have been recently described (OMIM #612076). We describe the clinical history, biochemical and molecular genetics findings of a Czech family with renal hypouricemia. The concentration of serum uric acid in the proband (16-year-old Czech girl with unrelated parents) was 0.17 ± 0.05 mg/dl and expressed as an increase in the fractional excretion of uric acid (194 ± 99%). The sequencing analysis of the coding region of uric acid transporters SLC22A12, SLC2A9, SLC17A3, ABCC4 and ABCG2, was performed. Analysis of genomic DNA revealed novel one nucleotide homozygote insertion in exon 3 in the SLC2A9 gene in proband and her brother resulting in a truncated protein (p.Ile118HisfsX27). No sequence variants in other candidate uric acid transporter were found. Homozygous loss-of-function mutations cause massive renal hypouricemia via total loss of uric acid absorption; however, they do not necessarily lead to nephrolithiasis and acute kidney injury. In contrast to previously reported heterozygous patients with renal hypouricemia type 2, we did not find even slight hypouricemia and found no decrease in the FE-UA of the heterozygous parents of the reported siblings.
- MeSH
- Genetic Association Studies MeSH
- Glomerular Filtration Rate MeSH
- Homozygote MeSH
- Mutagenesis, Insertional MeSH
- Uric Acid blood MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Urinary Calculi diagnosis genetics MeSH
- Glucose Transport Proteins, Facilitative genetics MeSH
- Pedigree MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA MeSH
- Case-Control Studies MeSH
- Renal Tubular Transport, Inborn Errors diagnosis genetics MeSH
- Check Tag
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
- Research Support, Non-U.S. Gov't 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.
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
- Alleles MeSH
- Alternative Splicing MeSH
- beta-Globins genetics MeSH
- beta-Thalassemia genetics MeSH
- CpG Islands MeSH
- Long Interspersed Nucleotide Elements * MeSH
- Adult MeSH
- Transcription, Genetic MeSH
- Introns * MeSH
- Mutagenesis, Insertional * MeSH
- Humans MeSH
- DNA Methylation MeSH
- Gene Order MeSH
- Promoter Regions, Genetic MeSH
- Gene Expression Regulation MeSH
- RNA Stability MeSH
- Gene Silencing MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
- Research Support, Non-U.S. Gov't 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 genetics MeSH
- DNA, Bacterial genetics MeSH
- Genotype MeSH
- Mutagenesis, Insertional MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Arabidopsis Proteins genetics MeSH
- Gene Expression Regulation, Plant MeSH
- Regulatory Sequences, Nucleic Acid genetics MeSH
- Telomerase genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
