Mirror Repeat Research Articles

Identification of Mirror Repeats in Viral Genomes Using FPCB Analysis

The majority of living domains consist of DNA as genetic material with the minor exception of viruses. The uniquenature of every species determines by its unique pattern of genome or gene products. The genomic features become an evidentexample of evolutionary study also. Different types of repeat patterns are observed in genomes of living domains including humanbeings whose two third portion of the genome is repetitive. Among the varied type of repeat sequences Mirror Repeats (MR) playcrucial roles at the genetic level in every species. The major focus of our research is on identification & to check the distributionof mirror repeats in some selected infectious viruses of three different domains (Animal, Plant & Human). For this, we employeda bioinformatics-based approach refer as FASTA PARALLEL COMPLEMENT BLAST (FPCB) to identify unique mirror repeat (MR)sequences. The identified repeats vary in their length as well as found to be distributed throughout the selected viral genomes.The maximum no of MR were reported in the case of Dengue virus (229) & minimum is in the case of TMV (97). In the remainingselected viruses - HCV, HPV, HTLV-1, PVY, Rabies virus 178, 156, 175, 203 & 204 MR sequences were reported. These sequencescan be utilized in many ways like in molecular diagnosis, drug delivery target as well as evolutionary study, etc. The present researchalso helps in the development of novel tools of bioinformatics to study mirror repeats and their functional perspective in thecontext of their occurrence in all domains.

A Systematic Study on Mirror Repeats in CTLA4 and KCNJ11 Genes of Diabetes

All cellular functions are controlled by the regulatory material which is constituted majorly by variety type of repeat elements. Among that types a unique pattern of bases form a Mirror repeat (MR) which thought to plays variety of roles at the genomic level that include regulation of gene expression, genomic integrity, triplex structure formation etc. The present research focused on identification of Mirror repeats in different genes types involved in both diabetes types. Two different genes CTLA4 (T1D) & KCNJ11 (T2D) were targeted to identify these sequence type. A manual method refers as FPCB is utilized to extract out MR sequences. The targeted gene sequences were divided into short regions so that it can be handled out easily while bioinformatics based analysis. Among the both types it was reported that both genes have different pattern of distribution of MR sequences. CTLA4 have 346 MR sequences in total in which MR235 have highest length of 50bps. In case of KCNJ11 a total no of 235 MR sequences were reported in which MR105 having highest length of 58bps. The present study will be helpful to elucidate the roles of MR sequences in diseases development & progression. It will be helpful for sequence based protein finding studies using bioinformatics tools as well as wet lab experiments.

In silico Approach for the Identification of Mirror Repeats within ced-3 Gene of Caenorhabditis elegans

Objectives: The repetitive elements within the genome of eukaryotes form a significant portion and are associated with various molecular functions within a cell. The goal of our study is to determine a special type of repeat i.e., mirror repeat within complete ced-3 apoptotic gene and its exons. Methods: A simple computational approach was used to search mirror repeats within the eight exons and complete ced-3 gene of Caenorhabditis elegans (C. elegans). C. elegans is a model organism widely used to study genetics, and developmental biology and ced-3 is the main apoptotic gene responsible for cell death. Findings: We identified 140 mirror repeats within the different regions of the ced-3 gene of C. elegans. These identified mirror repeats are not restricted to the genome of C. elegans but are scattered among the genome of C. vulgaris, Xenopus tropicalis and Drosophila melanogaster. Novelty: This research work is the very first endeavor to characterize mirror repeats within the complete ced-3 gene and its exons. Nobody has been studied mirror repeats within ced-3 apoptotic gene of C. elegans. Mirror repeat has the potential to form triplex DNA and is also associated with several neurological disorders. Forthcoming studies will help us to explore the functions and nature of these identified mirror repeats. Keywords: Repetitive elements, mirror repeats, Caenorhabditis elegans and ced-3 gene

Non-B-form DNA is associated with centromere stability in newly-formed polyploid wheat.

Non-B-form DNA differs from the classic B-DNA double helix structure and plays a crucial regulatory role in replication and transcription. However, the role of non-B-form DNA in centromeres, especially in polyploid wheat, remains elusive. Here, we systematically analyzed seven non-B-form DNA motif profiles (A-phased DNA repeat, direct repeat, G-quadruplex, inverted repeat, mirror repeat, short tandem repeat, and Z-DNA) in hexaploid wheat. We found that three of these non-B-form DNA motifs were enriched at centromeric regions, especially at the CENH3-binding sites, suggesting that non-B-form DNA may create a favorable loading environment for the CENH3 nucleosome. To investigate the dynamics of centromeric non-B form DNA during the alloploidization process, we analyzed DNA secondary structure using CENH3 ChIP-seq data from newly formed allotetraploid wheat and its two diploid ancestors. We found that newly formed allotetraploid wheat formed more non-B-form DNA in centromeric regions compared with their parents, suggesting that non-B-form DNA is related to the localization of the centromeric regions in newly formed wheat. Furthermore, non-B-form DNA enriched in the centromeric regions was found to preferentially form on young LTR retrotransposons, explaining CENH3's tendency to bind to younger LTR. Collectively, our study describes the landscape of non-B-form DNA in the wheat genome, and sheds light on its potential role in the evolution of polyploid centromeres.

In-silico Evaluation of Mirror Repeats in Some Selected Genes of Candida albicans

All cellular processes in a living cell are controlled by its genetic material. DNA in majority of the domains acts as a regulatory molecule by controlling various vital functions. The genetic makeup of DNA or RNA (in viruses) is unique in all the domains. Their uniqueness is determined by the presence of various types of repetitive patterns of bases. These includes inverted repeats, tandem repeats, VNTR’s, palindromes etc. Among many repetitive pattern types, Mirror repeats (MR) found to be dispersed throughout the genes or genomes. These sequences are associated with various functional features like their involvement in H-DNA formation, in replication & transcription, nervous system related diseases development etc. The major focus of this investigation is to identify MR sequences from some selected genes of Candida albicans using a bioinformatics based pipeline. The approach refers to as FPCB which utilized some manual steps to extract out MR sequence from any targeted gene or genome. The current study find out that the identified Mirror repeats found to be dispersed throughout the selected genes along with variable length. Among them the maximum & minimum MR sequences were reported in the gene FAS2 (108) & HIS1 (15) respectively. The present study will be helpful to provide a new insight for molecular as well as computational based studies of Candida albicans as well as other related fungal groups.

Mutation-site localized non-B DNA burden and survival heterogeneity in early-stage pancreatic cancer.

4166 Background: Pancreatic cancer is a highly aggressive disease with a poor prognosis, and one in which most measure of genomic instability (e.g., tumor mutation burden (TMB), fraction of genome altered (FGA)) have thus far, not proven informative in differentiating survival. Non-B DNA are alternative DNA forms that deviate from the canonical B-DNA structure with potential to increase susceptibility to mutations, leading to the development of cancer. The role of non-B in pancreatic cancer has not been fully explored. Herein, we investigate the relationship between gene mutation sites co-localized with non-B DNA motifs in terms of survival and B-DNA features. Methods: Using TCGA data, we derived a genome-wide mutation signature on 104 early-stage pancreatic patients. We introduced a metric, mutation-localized non-B burden (MLNB) by the number of non-b motifs containing mutation sites, normalized to the number of mutations and motif library size. We applied MLNB to patient-specific mutation signatures to derive an MLNB burden for non-B DNA structure motifs: G-quadruplexes (G4), Z-DNA, inverted repeats (IR), mirror repeats (MR), direct repeats (DR), and short tandem repeats (STR). We performed a cluster analysis on MLNB and compared groups based on overall survival (OS) in months (mos) using a log-rank test. Comparisons of B-DNA-derived molecular features among clusters were performed using a Kruskal-Wallis and Fisher Exact test. Results: Among the 104 early-stage pancreatic patients with a mutation signature, MLNB clustering resulted in six patient clusters that differentiated by non-B DNA structure, with DR burden the longest OS cluster (n=23, median OS=30mos), significantly (p < 0.05) differing as compared to IR (n=23, median OS=15 mos), STR (n=20, median OS=16mos), MR with lack of IR (n=14, median OS=13mos) and MR with IR (n=16, median OS=8mos). A mix of Z-DNA and G4 burden defined a cluster with shorter OS (n=22, median OS=14mos) as compared to DR, though not significant. Patients with the longest OS, MLNB-DR burden cluster had mutation signatures enriched in MAPK and Notch signaling pathways, as compared to the other clusters enriched with double-stranded break and mismatch repair (IR), hedgehog and WNT signaling (STR), and interleukin-4 signaling (MR) pathways. Among all clusters, CDKN2A deletion was most prevalent except for MLNB-DR burden cluster. No Significant differences were found between the MLNB clusters with age, race gender, KRAS and TP53 mutations, FGA, TMB, tumor purity, Bailey and Moffit subtypes. Conclusions: Our results point towards the potential use of MLNB as an emerging marker of site-structure genomic instability. Considering that triplex DNA is emerging as a marker genomic instability, and that we distinguish between two MR groups based on IR burden, our results may provide insights into various instability mechanisms associated with poor survival in pancreatic cancer.

Identification of mirror repeats within the <i>Maleless</i> (<i>MLE</i>) gene of <i>Drosophila melanogaster</i> Meigen

DNA repeats present in prokaryotes and eukaryotes genomes that include simple tandem repeats, satellite DNA, or palindromic sequences are classified as inverted, direct, and mirror repeats (MRs). Out of these, MRs are not well studied in Drosophila melanogaster Meigen. In this study, manual bioinformatics approach was used to find MRs in D. melanogaster maleless (mle) gene. In this analysis, 123 MRs were found within the complete mle gene, while 78 MRs were found in the exonic region of the mle gene. MegaBLAST was performed to elucidate the presence of identified MRs across the genomes of D. melanogaster, D. nasuta Lamb and D. bipectinate Duda. These demonstrated the conserved characteristics of specific MRs in Drosophila genome and the evolutionary and functional significance of MRs in diverse genomes. This study establishes a link between the presence of MRs in mle gene of D. melanogaster and MRs in human genome.

Mirror repeats in the <i>intersex</i> gene of <i>Drosophila melanogaster</i> Meigen

Genomes of many organisms, including both prokaryotes and eukaryotes, contain numerous repeat elements. Among these DNA repeats, mirror repeats have been studied rarely. Hence, this study on the mirror repeats within the intersex gene of Drosophila melanogaster, which reveal 11 mirror repeats. Out of these mirror repeats, ten mirror repeats are perfect, and six are within a single exon. The presence of mirror repeats within the intersex gene raises further questions concerning their origin, evolution, and biological function.

In silico Evaluation of mirror repeats within ced-9 Gene of Caenorhabditis elegans

In silico Evaluation of mirror repeats within ced-9 Gene of Caenorhabditis elegans

S1-END-seq reveals DNA secondary structures in human cells

DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing. This approach revealed two predominant non-B DNA structures: cruciform DNA formed by expanded (TA)n repeats that accumulate in microsatellite unstable human cancer cell lines and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine mirror repeats common across a variety of cell lines. We show that H-DNA is enriched during replication, that its genomic location is highly conserved, and that H-DNA formed by (GAA)n repeats can be disrupted by treatment with a (GAA)n-binding polyamide. Finally, we show that triplex-forming repeats are hotspots for mutagenesis. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.

In-silico evaluation of ‘Mirror Repeats’ In HIV Genome

The repetitive sequences played an important role in the characterization of both prokaryotic & eukaryotic organisms. Various different patterns of repetitive sequences have also been identified in organisms. Among all the repeat sequences. Mirror Repeats (MR`s) play an important role in various types of neurological disorders. These MR`s have also been reported for structure determination of genomes, triplex DNA formation & various other genome functions. We have followed a distinguished method referred to as FPCB (FASTA PARALLEL COMPLEMENT BLAST) for the identification of MR`s. The above said method used to identify MR’s in both types of HIV viruses (HIV-1 & HIV-2). Present investigation reported that MR’s are frequently distributed in all the regions of the genomes of both types. As a result, 232 & 248 total numbers of MR`s identified in both the HIV-1 & HIV-2 genome respectively. In addition, it was also revealed that the majority of the identified sequences are imperfect. The maximum length of MR`s in HIV-1 is of 47 nucleotides (NTD`s), however in case of HIV-2, it is of 49 nucleotides (NTD`s). Present investigation will be helpful for further development of a link between mirror repeats and host genome, which will be a new trend to block the viral integration as well as pathogenicity.

APOBEC mutagenesis is low in most types of non-B DNA structures

SummaryWhile somatic mutations are known to be enriched in genome regions with non-canonical DNA secondary structure, the impact of particular mutagens still needs to be elucidated. Here, we demonstrate that in human cancers, the APOBEC mutagenesis is not enriched in direct repeats, mirror repeats, short tandem repeats, and G-quadruplexes, and even decreased below its level in B-DNA for cancer samples with very high APOBEC activity. In contrast, we observe that the APOBEC-induced mutational density is positively associated with APOBEC activity in inverted repeats (cruciform structures), where the impact of cytosine at the 3’-end of the hairpin loop is substantial. Surprisingly, the APOBEC-signature mutation density per TC motif in the single-stranded DNA of a G-quadruplex (G4) is lower than in the four-stranded part of G4 and in B-DNA. The APOBEC mutagenesis, as well as the UV-mutagenesis in melanoma samples, are absent in Z-DNA regions, owing to the depletion of their mutational signature motifs.

In Silico Analysis of Structural Photosynthetic Genes of Arabidopsis thaliana for Unique Mirror Repeats

Objectives: The underlying work explores mirror sequences in the photosynthetic genes of Arabidopsis thaliana. At present, these sequences are standing at the forefront to be explored for their origin, distribution and function in plants. Methods: FPCB, a recently developed bioinformatics approach was utilized for identification of mirror sequences. It is a three step strategy based on pattern matching of alignments, produced after aligning gene sequence and its complement using mega-BLAST. This algorithm was quick and efficient enough to characterize a range of mirror sequences. Findings: All the analyzed genes were reported to harbor great variety of mirror sequences at quite high frequencies. LHCA1 gene have the highest total count of these sequences and ATPB gene have lowest of all. A total of 401 unique mirror sequences of different lengths and compositions were reported in the twelve selected genes. Promoter motifs were found to be greatly enriched with these repeats. Eleven mirror sequences of significant lengths were also reported using the above approach. Novelty: This work is the very first attempt to characterize photosynthetic genes of Arabidopsis thaliana for mirror repeats. This will further aggravate efforts to develop fingerprinting techniques based on these unique mirror sequences, which are very powerful tools to study taxonomic and evolutionary relationships. Mirror sequences are also potential candidates as drug delivery systems and in molecular medicine. Keywords: Mirror repeats; HDNA; FPCB; photosynthetic genes; Arabidopsis thaliana

In silico approach for the identification of mirror repeats in selected operon genes of Escherichia coli strain K-12 substrain MG1655

Background: The repeating elements in the genes or genomes of living organisms are associated with a variety of functions at the molecular level. Mirror repeats (MRs) are unique type of repeat sequences among them, which are found to be linked with H-DNA formation and they have also associated with several neurological disorders with many other functional roles are also being reported. Methods: The manual bioinformatics-based approach is used to identify the MRs in the genome. The applied approach FASTA-parallel complement-BLAST is used by following some simple steps to identify MRs. This methodology is initiated by the downloading of a sequence of interest in FASTA format followed by development of the parallel complement and final step of BLAST analysis. By using this approach, the present study identifies MRs in lac, trp, and ara operon genes of Escherichia coli str. K-12 substr. MG1655 (NC_000913.3). Results: Present investigation identified the frequent distribution MRs in all the analyzed operon genes. These identified MRs vary in their length or size. In case of lac, trp, and ara operon, maximum number of MRs reported in lacZ (61), trpE (40), and araE (41) genes, respectively. Conclusion: The frequent existence of MRs (shorter as well as larger length) in analyzed genes gives a hint about their significant roles in the genes or genomes of all bacterial species. These may be useful to study the evolutionary history of living world. These types of studies will be exploring new trends and tools of molecular biology research as well as development of new concept for MR identification.

Genome-Wide Analysis to Identify Palindromes, Mirror and Inverted Repeats in SARS-CoV-2, MERS-CoV and SARS-CoV-1

Research pertaining to SARS-CoV-2 is in full swing to understand the origin and evolution of this deadly virus that can lead to its rapid detection. To achieve this, atypical genomic sequences which may be unique to SARS-CoV-2 or Coronaviridae family in general may be investigated. Such sequences in virus genomes may be responsible for target prediction, replication, defence mechanisms and viral packaging. This fact has motivated us to explore the different types of repeats such as palindromes, mirror repeats and inverted repeats in SARS-CoV-2, MERS-CoV and SARS-CoV-1. For this purpose, the respective reference sequence of SARS-CoV-2, MERS-CoV and SARS-CoV-1 is divided into descriptors of sequences of length <inline-formula> <tex-math notation="LaTeX">${k}$ </tex-math></inline-formula> using <inline-formula> <tex-math notation="LaTeX">${k}$ </tex-math></inline-formula>-mer technique. Thereafter, these descriptors are represented as a collection of tokens which are subsequently used for the identification of palindrome, mirror repeat and inverted repeat in the respective reference sequence. The highest number of palindromes, mirror repeats and inverted repeats are identified for descriptor length 10. As a result, for palindromes such values are 38, 42 and 33 and for mirror repeats they are 52, 38 and 33 for SARS-CoV-2, MERS-CoV and SARS-CoV-1 respectively. For inverted repeats, with a descriptor length 10 and intervening length 5, the values are 59, 56 and 70 respectively. Moreover, the identified repeats are then searched for in 108246, 291 and 340 SARS-CoV-2, MERS-CoV and SARS-CoV-1 virus sequences respectively to find the population coverage of such repeats. It surpasses 99&#x0025; in most cases and even 100&#x0025; for some. Furthermore, GC contents which mostly lie between 20&#x0025;-50&#x0025; are evaluated for these repeats as well in order to understand their binding efficacy.

A novel approach for identification of mirror repeats within the Engrailed Homeobox-1 gene of Xenopus tropicalis

Background: Repetitive sequences constitute the major portion of genomic DNA in most of the organisms and are responsible for variation in DNA structure, function, etc., These sequences also have the potential to adopt various noncanonical DNA structures. Methods: By using a swift, manual approach mirror repeats has been identified within the complete engrailed homeobox-1 gene (en-1) of X. tropicalis. Another tool Non- B DNA motif search was also deployed for comparative analysis. Results: A total of 166 mirror repeats were identified within the complete en-1 gene of X. tropicalis. The similar sequences were also searched among the genome of different organisms such as Xenopus laevis, Caenorhabditis elegans, Drosophila melanogaster, etc., Conclusion: To the best of our knowledge, it was novel identification of mirror repeats in the engrailed-1 gene of X. tropicalis. Few of these sequences may adopt various noncanonical B-DNA forms and are potent sites for mutation and recombination events.

PolyPurine Reverse Hoogsteen Hairpins Work as RNA Species for Gene Silencing.

PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs.

Characterization of Flowering Genes of Arabidopsis thaliana for Mirror Repeats

A variety of simple DNA repeats are enriched in the eukaryotic genomes. Recent studies have proven their importance in understanding genome organization and function, especially how genomes evolve using them as mutational hotspots during DNA replication. Mirror repeat sequences, the most underrated subset of this class of repeats, are now gaining importance because of their probable involvement in developing several genetic diseases in humans. These repeats typically adopt H-DNA conformations in both in-vitro and in-vivo conditions. On the other end, plants were still not analyzed for their presence or distribution and whether they are responsible for causing diseases in them or not. The present study aims to extract mirror repeats in the flowering genes of Arabidopsis thaliana. To this end, we have deployed FPCB (FASTA-PARALLEL COMPLEMENT-BLAST), an efficacious and quick method to extract perfect and degenerate mirror repeat sequences through pattern matching of alignments with user-defined algorithmic parameters. All the analyzed genes were reported to have quite high densities of mirror sequences. A total of 93 unique mirror repeats of significant lengths were extracted in the analyzed genes.

Characterization of G4 DNA formation in mitochondrial DNA and their potential role in mitochondrial genome instability.

Mitochondria possess their own genome which can be replicated independently of nuclear DNA. Mitochondria being the powerhouse of the cell produce reactive oxygen species, due to which the mitochondrial genome is frequently exposed to oxidative damage. Previous studies have demonstrated an association of mitochondrial deletions to aging and human disorders. Many of these deletions were present adjacent to non-B DNA structures. Thus, we investigate noncanonical structures associated with instability in mitochondrial genome. Insilico studies revealed the presence of >100 G-quadruplex motifs (of which 5 have the potential to form 3-plate G4 DNA), 23 inverted repeats, and 3 mirror repeats in the mitochondrial DNA (mtDNA). Further analysis revealed that among the deletion breakpoints from patients with mitochondrial disorders, majority are located at G4 DNA motifs. Interestingly, ~50% of the deletions were at base-pair positions 8271-8281, ~35% were due to deletion at 12362-12384, and ~12% due to deletion at 15516-15545. Formation of 3-plate G-quadruplex DNA structures at mitochondrial fragile regions was characterized using electromobility shift assay, circular dichroism (CD), and Taq polymerase stop assay. All 5 regions could fold into both intramolecular and intermolecular G-quadruplex structures in a KCl-dependent manner. G4 DNA formation was in parallel orientation, which was abolished in the presence of LiCl. The formation of G4 DNA affected both replication and transcription. Finally, immunolocalization of BG4 with MitoTracker confirmed the formation of G-quadruplex in mitochondrial genome. Thus, we characterize the formation of 5 different G-quadruplex structures in human mitochondrial region, which may contribute toward formation of mitochondrial deletions.

Cruciform Formable Sequences within Pou5f1 Enhancer Are Indispensable for Mouse ES Cell Integrity.

DNA can adopt various structures besides the B-form. Among them, cruciform structures are formed on inverted repeat (IR) sequences. While cruciform formable IRs (CFIRs) are sometimes found in regulatory regions of transcription, their function in transcription remains elusive, especially in eukaryotes. We found a cluster of CFIRs within the mouse Pou5f1 enhancer. Here, we demonstrate that this cluster or some member(s) plays an active role in the transcriptional regulation of not only Pou5f1, but also Sox2, Nanog, Klf4 and Esrrb. To clarify in vivo function of the cluster, we performed genome editing using mouse ES cells, in which each of the CFIRs was altered to the corresponding mirror repeat sequence. The alterations reduced the level of the Pou5f1 transcript in the genome-edited cell lines, and elevated those of Sox2, Nanog, Klf4 and Esrrb. Furthermore, transcription of non-coding RNAs (ncRNAs) within the enhancer was also upregulated in the genome-edited cell lines, in a similar manner to Sox2, Nanog, Klf4 and Esrrb. These ncRNAs are hypothesized to control the expression of these four pluripotency genes. The CFIRs present in the Pou5f1 enhancer seem to be important to maintain the integrity of ES cells.