Structures and stability of simple DNA repeats from bacteria.

DNA is a fundamentally important molecule for all cellular organisms due to its biological role as the store of hereditary, genetic information. On the one hand, genomic DNA is very stable, both in chemical and biological contexts, and this assists its genetic functions. On the other hand, it is also a dynamic molecule, and constant changes in its structure and sequence drive many biological processes, including adaptation and evolution of organisms. DNA genomes contain significant amounts of repetitive sequences, which have divergent functions in the complex processes that involve DNA, including replication, recombination, repair, and transcription. Through their involvement in these processes, repetitive DNA sequences influence the genetic instability and evolution of DNA molecules and they are located non-randomly in all genomes. Mechanisms that influence such genetic instability have been studied in many organisms, including within human genomes where they are linked to various human diseases. Here, we review our understanding of short, simple DNA repeats across a diverse range of bacteria, comparing the prevalence of repetitive DNA sequences in different genomes. We describe the range of DNA structures that have been observed in such repeats, focusing on their propensity to form local, non-B-DNA structures. Finally, we discuss the biological significance of such unusual DNA structures and relate this to studies where the impacts of DNA metabolism on genetic stability are linked to human diseases. Overall, we show that simple DNA repeats in bacteria serve as excellent and tractable experimental models for biochemical studies of their cellular functions and influences.

Telomeric-Like Repeats Flanked by Sequences Retrotranscribed from the Telomerase RNA Inserted at DNA Double-Strand Break Sites during Vertebrate Genome Evolution.

Interstitial telomeric sequences (ITSs) are stretches of telomeric-like repeats located at internal chromosomal sites. We previously demonstrated that ITSs have been inserted during the repair of DNA double-strand breaks in the course of evolution and that some rodent ITSs, called TERC-ITSs, are flanked by fragments retrotranscribed from the telomerase RNA component (TERC). In this work, we carried out an extensive search of TERC-ITSs in 30 vertebrate genomes and identified 41 such loci in 22 species, including in humans and other primates. The fragment retrotranscribed from the TERC RNA varies in different lineages and its sequence seems to be related to the organization of TERC. Through comparative analysis of TERC-ITSs with orthologous empty loci, we demonstrated that, at each locus, the TERC-like sequence and the ITS have been inserted in one step in the course of evolution. Our findings suggest that telomerase participated in a peculiar pathway of DNA double-strand break repair involving retrotranscription of its RNA component and that this mechanism may be active in all vertebrate species. These results add new evidence to the hypothesis that RNA-templated DNA repair mechanisms are active in vertebrate cells.

Identification of passion fruit (Passiflora edulis) chromosomes using BAC-FISH.

Passiflora edulis, the yellow passion fruit, is the main crop from the Passiflora genus, which comprises 525 species with its diversity center in South America. Genetic maps and a BAC (bacterial artificial chromosome) genomic library are available, but the nine chromosome pairs of similar size and morphology (2n = 18) hamper chromosome identification, leading to different proposed karyotypes. Thus, the aim of this study was to establish chromosome-specific markers for the yellow passion fruit using single-copy and repetitive sequences as probes in fluorescent in situ hybridizations (FISH) to allow chromosome identification and future integration with whole genome data. Thirty-six BAC clones harboring genes and three retrotransposons (Ty1-copy, Ty3-gypsy, and LINE) were selected. Twelve BACs exhibited a dispersed pattern similar to that revealed by retroelements, and one exhibited subtelomeric distribution. Twelve clones showed unique signals in terminal or subterminal regions of the chromosomes, allowing their genes to be anchored to six chromosome pairs that can be identified with single-copy markers. The markers developed herein will provide an important tool for genomic and evolutionary studies in the Passiflora genus.

Molecular cytogenetic analysis of genome-specific repetitive elements in Citrus clementina Hort. Ex Tan. and its taxonomic implications.

BACKGROUND: Clementine mandarin (Citrus clementina Hort. ex Tan.) is one of the most famous and widely grown citrus cultivars worldwide. Variations in relation to the composition and distribution of repetitive DNA sequences that dominate greatly in eukaryote genomes are considered to be species-, genome-, or even chromosome-specific. Repetitive DNA-based fluorescence in situ hybridization (FISH) is a powerful tool for molecular cytogenetic study. However, to date few studies have involved in the repetitive elements and cytogenetic karyotype of Clementine. RESULTS: A graph-based similarity sequence read clustering methodology was performed to analyze the repetitive DNA families in the Clementine genome. The bioinformatics analysis showed that repetitive DNAs constitute 41.95% of the Clementine genome, and the majority of repetitive elements are retrotransposons and satellite DNAs. Sequential multicolor FISH using a probe mix that contained CL17, four satellite DNAs, two rDNAs and an oligonucleotide of (TTTAGGG)3 was performed with Clementine somatic metaphase chromosomes. An integrated karyotype of Clementine was established based on unequivocal and reproducible chromosome discriminations. The distribution patterns of these probes in several Citrus, Poncirus and Fortunella species were summarized through extensive FISH analyses. Polymorphism and heterozygosity were commonly observed in the three genera. Some asymmetrical FISH loci in Clementine were in agreement with its hybrid origin. CONCLUSIONS: The composition and abundance of repetitive elements in the Clementine genome were reanalyzed. Multicolor FISH-based karyotyping provided direct visual proof of the heterozygous nature of Clementine chromosomes with conspicuous asymmetrical FISH hybridization signals. We detected some similar and variable distribution patterns of repetitive DNAs in Citrus, Poncirus, and Fortunella, which revealed notable conservation among these genera, as well as obvious polymorphism and heterozygosity, indicating the potential utility of these repetitive element markers for the study of taxonomic, phylogenetic and evolutionary relationships in the future.

Structure, clustering and functional insights of repeats configurations in the upstream promoter region of the human coding genes.

BACKGROUND: Repetitive DNA sequences (Repeats) are significant regions in the human genome that have a specific genomic distribution, structure, and several binding sites for genome architecture and function. In consequence, the possible configurations of Repeats in specific and dynamic regions like the gene promoters could define footprints for molecular mechanisms, pathways, and cell function beyond their density in the genome. Here we explored the distribution of Repeats in the upstream promoter region of the human coding genes with the aim to identify specific configurations, clusters and functional meaning of those elements. Our method includes structural descriptions, hierarchical clustering, pathway association, and functional enrichment analysis. RESULTS: We report here several configurations of Repeats in the upstream promoter region (UPR), which define 2729 patterns for the 80% of the human coding genes. There are 47 types of Repeats in these configurations, where the most frequent were Alu, Low_complexity, MIR, Simple_repeat, LINE/L2, LINE/L1, hAT-Charlie, and ERV1. The distribution, length, and the high frequency of Repeats in the UPR defines several patterns and clusters, where the minimum frequency of configuration among Repeats was higher than 0.7. We found those clusters associated with cellular pathways and ontologies; thus, it was plausible to determine groups of Repeats to specific functional insights, for example, pathways for Genetic Information Processing or Metabolism shows particular groups of Repeats with specific configurations. CONCLUSION: Based on these findings, we propose that specific configurations of repetitive elements describe frequent patterns in the upstream promoter for sets of human coding genes, which those correlated to specific and essential cell pathways and functions.

Chromosomal Mapping of the Histone Multigene Family and U2 snRNA in Hypancistrus Species (Siluriformes, Loricariidae) from the Brazilian Amazon.

Loricariidae (Siluriformes) comprises ∼1026 species of neotropical fish, being considered the most diverse among the Siluriformes. Studies on repetitive DNA sequences have provided important data on the evolution of the genomes of members of this family, especially of the Hypostominae subfamily. In this study, the chromosomal mapping of the histone multigene family and U2 snRNA was performed in two species belonging to the Hypancistrus genus, Hypancistrus sp. "pão" (2n = 52, 22m + 18sm +12st) and Hypancistrus zebra (2n = 52, 16m + 20sm +16st). The presence of dispersed signals of histones H2A, H2B, H3, and H4 in the karyotype of both species, with each sequence displaying a varied level of accumulation and dispersion of these sequences between them was observed; in addition, U2 snDNA probe only showed positive results in H. zebra, which present this multigene in the terminal region of three chromosomal pairs. The obtained results resemble data already analyzed in the literature, in which the action of transposable elements interfere in the organization of these multigene families, in addition to other evolutionary processes that shape the evolution of the genome, such as circular or ectopic recombination. This study also shows that the dispersion of the multigene histone family is quite complex, and from this, these data serve as a point of discussion for the evolutionary processes that occur in the Hypancistrus karyotype.

Transposable elements: multifunctional players in the plant genome.

Transposable elements (TEs) are indispensable components of eukaryotic genomes that play diverse roles in gene regulation, recombination, and environmental adaptation. Their ability to mobilize within the genome leads to gene expression and DNA structure changes. TEs serve as valuable markers for genetic and evolutionary studies and facilitate genetic mapping and phylogenetic analysis. They also provide insight into how organisms adapt to a changing environment by promoting gene rearrangements that lead to new gene combinations. These repetitive sequences significantly impact genome structure, function and evolution. This review takes a comprehensive look at TEs and their applications in biotechnology, particularly in the context of plant biology, where they are now considered "genomic gold" due to their extensive functionalities. The article addresses various aspects of TEs in plant development, including their structure, epigenetic regulation, evolutionary patterns, and their use in gene editing and plant molecular markers. The goal is to systematically understand TEs and shed light on their diverse roles in plant biology.

Differential Spreading of Microsatellites in Holocentric Chromosomes of Chagas Disease Vectors: Genomic and Evolutionary Implications.

This study focused on analyzing the distribution of microsatellites in holocentric chromosomes of the Triatominae subfamily, insect vectors of Chagas disease. We employed a non-denaturing FISH technique to determine the chromosomal distribution of sixteen microsatellites across twenty-five triatomine species, involving five genera from the two principal tribes: Triatomini and Rhodniini. Three main hybridization patterns were identified: strong signals in specific chromosomal regions, dispersed signals dependent on microsatellite abundance and the absence of signals in certain chromosomal regions or entire chromosomes. Significant variations in hybridization patterns were observed between Rhodniini and Triatomini species. Rhodniini species displayed weak and scattered hybridization signals, indicating a low abundance of microsatellites in their genomes. In contrast, Triatomini species exhibited diverse and abundant hybridization patterns, suggesting that microsatellites are a significant repetitive component in their genomes. One particularly interesting finding was the high abundance of GATA repeats, and to a lesser extent AG repeats, in the Y chromosome of all analyzed Triatomini species. In contrast, the Y chromosome of Rhodniini species did not show enrichment in GATA and AG repeats. This suggests that the richness of GATA repeats on the Y chromosome likely represents an ancestral trait specific to the Triatomini tribe. Furthermore, this information can be used to elucidate the evolutionary relationships between Triatomini and other groups of reduviids, contributing to the understanding of the subfamily's origin. Overall, this study provides a comprehensive understanding of the composition and distribution of microsatellites within Triatominae genomes, shedding light on their significance in the evolutionary processes of these species.

The nature and organization of satellite DNAs in Petunia hybrida, related, and ancestral genomes.

Introduction: The garden petunia, Petunia hybrida (Solanaceae) is a fertile, diploid, annual hybrid species (2n=14) originating from P. axillaris and P. inflata 200 years ago. To understand the recent evolution of the P. hybrida genome, we examined tandemly repeated or satellite sequences using bioinformatic and molecular cytogenetic analysis. Methods: Raw reads from available genomic assemblies and survey sequences of P. axillaris N (PaxiN), P. inflata S6, (PinfS6), P. hybrida (PhybR27) and the here sequenced P. parodii S7 (PparS7) were used for graph and k-mer based cluster analysis of TAREAN and RepeatExplorer. Analysis of repeat specific monomer lengths and sequence heterogeneity of the major tandem repeat families with more than 0.01% genome proportion were complemented by fluorescent in situ hybridization (FISH) using consensus sequences as probes to chromosomes of all four species. Results: Seven repeat families, PSAT1, PSAT3, PSAT4, PSAT5 PSAT6, PSAT7 and PSAT8, shared high consensus sequence similarity and organisation between the four genomes. Additionally, many degenerate copies were present. FISH in P. hybrida and in the three wild petunias confirmed the bioinformatics data and gave corresponding signals on all or some chromosomes. PSAT1 is located at the ends of all chromosomes except the 45S rDNA bearing short arms of chromosomes II and III, and we classify it as a telomere associated sequence (TAS). It is the most abundant satellite repeat with over 300,000 copies, 0.2% of the genomes. PSAT3 and the variant PSAT7 are located adjacent to the centromere or mid-arm of one to three chromosome pairs. PSAT5 has a strong signal at the end of the short arm of chromosome III in P. axillaris and P.inflata, while in P. hybrida additional interstitial sites were present. PSAT6 is located at the centromeres of chromosomes II and III. PSAT4 and PSAT8 were found with only short arrays. Discussion: These results demonstrate that (i) repeat families occupy distinct niches within chromosomes, (ii) they differ in the copy number, cluster organization and homogenization events, and that (iii) the recent genome hybridization in breeding P. hybrida preserved the chromosomal position of repeats but affected the copy number of repetitive DNA.

Repetitive DNA Sequences in the Human Y Chromosome and Male Infertility.

The male-specific Y chromosome, which is well known for its diverse and complex repetitive sequences, has different sizes, genome structures, contents and evolutionary trajectories from other chromosomes and is of great significance for testis development and function. The large number of repetitive sequences and palindrome structure of the Y chromosome play an important role in maintaining the stability of male sex determining genes, although they can also cause non-allelic homologous recombination within the chromosome. Deletion of certain Y chromosome sequences will lead to spermatogenesis disorders and male infertility. And Y chromosome genes are also involved in the occurrence of reproductive system cancers and can increase the susceptibility of other tumors. In addition, the Y chromosome has very special value in the personal identification and parentage testing of male-related cases in forensic medicine because of its unique paternal genetic characteristics. In view of the extremely high frequency and complexity of gene rearrangements and the limitations of sequencing technology, the analysis of Y chromosome sequences and the study of Y-gene function still have many unsolved problems. This article will introduce the structure and repetitive sequence of the Y chromosome, summarize the correlation between Y chromosome various sequence deletions and male infertility for understanding the repetitive sequence of Y chromosome more systematically, in order to provide research motivation for further explore of the molecules mechanism of Y-deletion and male infertility and theoretical foundations for the transformation of basic research into applications in clinical medicine and forensic medicine.

Genome-Wide Analysis of Transposable Elements and Satellite DNAs in Spinacia Species to Shed Light on Their Roles in Sex Chromosome Evolution.

Sex chromosome evolution has mostly been studied in species with heteromorphic sex chromosomes. The Spinacia genus serves as an ideal model for investigating evolutionary mechanisms underlying the transition from homomorphic to heteromorphic sex chromosomes. Among evolutionary factors, repetitive sequences play multiple roles in sex chromosome evolution while their forces have not been fully explored in Spinacia species. Here, we identified major repetitive sequence classes in male and female genomes of Spinacia species and their ancestral relative sugar beet to elucidate the evolutionary processes of sex chromosome evolution using next-generation sequencing (NGS) data. Comparative analysis revealed that the repeat elements of Spinacia species are considerably higher than of sugar beet, especially the Ty3/Gypsy and Ty1/Copia retrotransposons. The long terminal repeat retroelements (LTR) Angela, Athila, and Ogre may be accounted for the higher proportion of repeats in the spinach genome. Comparison of the repeats proportion between female and male genomes of three Spinacia species indicated the different representation in Spinacia tetrandra samples but not in the S. oleracea or S. turkestanica samples. From these results, we speculated that emergence of repetitive DNA sequences may correlate the formation of sex chromosome and the transition from homomorphic sex chromosomes to heteromorphic sex chromosomes as heteromorphic sex chromosomes exclusively existed in Spinacia tetrandra. Three novel sugar beet-specific satellites were identified and confirmed by fluorescence in situ hybridization (FISH); six out of eight new spinach-specific satellites were mapped to the short arm of sex chromosomes. A total of 141 copies of SolSat01-171-s were found in the sex determination region (SDR). Thus, the accumulation of satellite DNA on the short arm of chromosome 1 may be involved in the sex chromosome evolution in Spinacia species. Our study provides a fundamental resource for understanding repeat sequences in Spinacia species and their roles in sex chromosome evolution.

The Landscape of Repetitive Elements in the Refined Genome of Chilli Anthracnose Fungus Colletotrichum truncatum.

The ascomycete fungus Colletotrichum truncatum is a major phytopathogen with a broad host range which causes anthracnose disease of chilli. The genome sequencing of this fungus led to the discovery of functional categories of genes that may play important roles in fungal pathogenicity. However, the presence of gaps in C. truncatum draft assembly prevented the accurate prediction of repetitive elements, which are the key players to determine the genome architecture and drive evolution and host adaptation. We re-sequenced its genome using single-molecule real-time (SMRT) sequencing technology to obtain a refined assembly with lesser and smaller gaps and ambiguities. This enabled us to study its genome architecture by characterising the repetitive sequences like transposable elements (TEs) and simple sequence repeats (SSRs), which constituted 4.9 and 0.38% of the assembled genome, respectively. The comparative analysis among different Colletotrichum species revealed the extensive repeat rich regions, dominated by Gypsy superfamily of long terminal repeats (LTRs), and the differential composition of SSRs in their genomes. Our study revealed a recent burst of LTR amplification in C. truncatum, C. higginsianum, and C. scovillei. TEs in C. truncatum were significantly associated with secretome, effectors and genes in secondary metabolism clusters. Some of the TE families in C. truncatum showed cytosine to thymine transitions indicative of repeat-induced point mutation (RIP). C. orbiculare and C. graminicola showed strong signatures of RIP across their genomes and "two-speed" genomes with extensive AT-rich and gene-sparse regions. Comparative genomic analyses of Colletotrichum species provided an insight into the species-specific SSR profiles. The SSRs in the coding and non-coding regions of the genome revealed the composition of trinucleotide repeat motifs in exons with potential to alter the translated protein structure through amino acid repeats. This is the first genome-wide study of TEs and SSRs in C. truncatum and their comparative analysis with six other Colletotrichum species, which would serve as a useful resource for future research to get insights into the potential role of TEs in genome expansion and evolution of Colletotrichum fungi and for development of SSR-based molecular markers for population genomic studies.

Effects of genome structure variation, homeologous genes and repetitive DNA on polyploid crop research in the age of genomics.

Compared to diploid species, allopolyploid crop species possess more complex genomes, higher productivity, and greater adaptability to changing environments. Next generation sequencing techniques have produced high-density genetic maps, whole genome sequences, transcriptomes and epigenomes for important polyploid crops. However, several problems interfere with the full application of next generation sequencing techniques to these crops. Firstly, different types of genomic variation affect sequence assembly and QTL mapping. Secondly, duplicated or homoeologous genes can diverge in function and then lead to emergence of many minor QTL, which increases difficulties in fine mapping, cloning and marker assisted selection. Thirdly, repetitive DNA sequences arising in polyploid crop genomes also impact sequence assembly, and are increasingly being shown to produce small RNAs to regulate gene expression and hence phenotypic traits. We propose that these three key features should be considered together when analyzing polyploid crop genomes. It is apparent that dissection of genomic structural variation, elucidation of the function and mechanism of interaction of homoeologous genes, and investigation of the de novo roles of repeat sequences in agronomic traits are necessary for genomics-based crop breeding in polyploids.

Chromatin organization and cytological features of carnivorous Genlisea species with large genome size differences.

The monophyletic carnivorous genus Genlisea (Lentibulariaceae) is characterized by a bi-directional genome size evolution resulting in a 25-fold difference in nuclear DNA content. This is one of the largest ranges found within a genus so far and makes Genlisea an interesting subject to study mechanisms of genome and karyotype evolution. Genlisea nigrocaulis, with 86 Mbp one of the smallest plant genomes, and the 18-fold larger genome of G. hispidula (1,550 Mbp) possess identical chromosome numbers (2n = 40) but differ considerably in chromatin organization, nuclear and cell size. Interphase nuclei of G. nigrocaulis and of related species with small genomes, G. aurea (133 Mbp, 2n ≈ 104) and G. pygmaea (179 Mbp, 2n = 80), are hallmarked by intensely DAPI-stained chromocenters, carrying typical heterochromatin-associated methylation marks (5-methylcytosine, H3K9me2), while in G. hispidula and surprisingly also in the small genome of G. margaretae (184 Mbp, 2n = 38) the heterochromatin marks are more evenly distributed. Probes of tandem repetitive sequences together with rDNA allow the unequivocal discrimination of 13 out of 20 chromosome pairs of G. hispidula. One of the repetitive sequences labeled half of the chromosome set almost homogenously supporting an allopolyploid status of G. hispidula and its close relative G. subglabra (1,622 Mbp, 2n = 40). In G. nigrocaulis 11 chromosome pairs could be individualized using a combination of rDNA and unique genomic probes. The presented data provide a basis for future studies of karyotype evolution within the genus Genlisea.

Lower LINE-1 methylation in first-episode schizophrenia patients with the history of childhood trauma.

AIM: We investigated methylation of DNA repetitive sequences (LINE-1 and BAGE) in peripheral blood leukocytes from first-episode schizophrenia (FES) patients and healthy controls (HCs) with respect to childhood adversities. MATERIALS & METHODS: Patients were divided into two subgroups based on the history of childhood trauma - FES(+) and FES(-) subjects. The majority of HCs had a negative history of childhood trauma - HCs(-) subjects. RESULTS: FES(+) patients had significantly lower LINE-1 methylation in comparison with FES(-) patients or HC(-) subjects. Emotional abuse and total trauma score predicted lower LINE-1 methylation in FES patients, while general trauma score was associated with lower BAGE methylation in HCs. CONCLUSION: Childhood adversities might be associated with global DNA hypomethylation in adult FES patients.

Indole acetic acid production by fluorescent Pseudomonas spp. from the rhizosphere of Plectranthus amboinicus (Lour.) Spreng. and their variation in extragenic repetitive DNA sequences.

Fluorescent Pseudomonas (FP) is a heterogenous group of growth promoting rhizobacteria that regulate plant growth by releasing secondary metabolic compounds viz., indole acetic acid (IAA), siderophores, ammonia and hydrogen cyanide. In the present study, IAA producing FPs from the rhizosphere of Plectranthus amboinicus were characterized morphologically, biochemically and at the molecular level. Molecular identification of the isolates were carried out using Pseudomonas specific primers. The effect of varying time (24, 48, 72 and 96 h), Trp concentrations (100, 200, 300, 400 and 500 µg.ml-1), temperature (10, 26, 37 and 50±2 °C) and pH (6, 7 and 8) on IAA production by 10 best isolates were studied. Results showed higher IAA production at 72 h incubation, at 300 µg.ml-1 Trp concentration, temperature 26±2 °C and pH 7. TLC with acidified ethyl acetate extract showed that the IAA produced has a similar Rf value to that of the standard IAA. Results of TLC were confirmed by HPLC analysis. Genetic diversity of the isolates was also studied using 40 RAPD and 4 Rep primers. Genetic diversity parameters such as dominance, Shannon index and Simpson index were calculated. Out of 40 RAPD primers tested, 9 (2 OP-D series and 7 OP-E series) were shortlisted for further analysis. Studies using RAPD, ERIC, BOX, REP and GTG5 primers revealed that isolates exhibit significant diversity in repetitive DNA sequences irrespective of the rhizosphere.

Construction of Small Genes with Repetitive Elements Using Oligo Extension and Golden Gate Assembly.

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences and results in synthesis failure or delays by DNA synthesis vendors. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden Gate-compatible overhangs. Then synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate assembly.