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The KCNN4 gene encoding a potassium channel protein whose expression has been correlated with tumor progression was found to comprise a guanine-rich minisatellite region with the ability to form a ...

Ubiquitin (Ub) is one of the proteins that are highly conserved from yeast to humans. It is an essential core unit of the welldefined post-translational modification, called ubiquitination, which is involved in a variety of biological processes. In metazoans, Ub is encoded by two monoubiquitin genes and two polyubiquitin genes, in which a single Ub is fused to a ribosomal protein or Ub coding units are arranged in tandem repeats. In mice, polyubiquitin genes (Ubb and Ubc) play a pivotal role to meet the requirement of cellular Ub pools during embryonic development. In addition, expression levels of polyubiquitin genes are increased to adapt to environmental stimuli such as oxidative, heat-shock, and proteotoxic stress. Several researchers have reported about the perturbation of Ub pools through genetic alteration or exogenous Ub delivery using diverse model systems. To study Ub pool changes in a physiologically relevant manner, changing Ub pools via the regulation of endogenous polyubiquitin gene expression has recently been introduced. Furthermore, to understand the regulation of polyubiquitin gene expression more precisely, cis-acting elements and trans-acting factors, which are regulatory components of polyubiquitin genes, have been analyzed. In this review, we discuss how the role of polyubiquitin genes has been studied during the past decade, especially focusing on their regulation. [BMB Reports 2021; 54(4): 189-195].

Background Heat shock factors (Hsfs) and Heat shock proteins (Hsps) belong to an essential group of molecular regulators involved in controlling cellular processes under normal and stress conditions. The role of Hsfs and Hsps is well known in model plant species under diverse stress conditions. While plants Hsfs are vital components of the signal transduction response to maintain cellular homeostasis, Hsps function as chaperones helping to maintain folding of damaged and newly formed proteins during stress conditions. In lettuce (Lactuca sativa), a highly consumed vegetable crop grown in the field and in hydroponic systems, the role of these gene families in response to artificial light is not well characterized. Results Using a genome-wide analysis approach, we identified 32 Hsfs and 22 small heat shock proteins (LsHsps) in lettuce, some of which do not have orthologs in Arabidopsis, poplar, and rice. LsHsp60s, LsHsp90s, and LsHsp100s are highly conserved among dicot and monocot species. Surprisingly, LsHsp70s have three times more members than Arabidopsis and two times more than rice. Interestingly, the lettuce genome triplication did not contribute to the increased number of LsHsp70s genes. The large number of LsHsp70s was the result of genome tandem duplication. Chromosomal distribution analysis shows larger tandem repeats of LsHsp70s genes in Chr1, Chr7, Chr8, and Chr9. At the transcriptional level, some genes of the LsHsfs, LsHsps, LsHsp60s, and LsHsp70s families were highly responsive to UV and high intensity light stress, in contrast to LsHsp90s and LsHsp100s which did not respond to a light stimulus. Conclusions Our genome-wide analysis provides a detailed identification of Hsfs and Hsps in lettuce. Chromosomal location and syntenic region analysis together with our transcriptional analysis under different light conditions provide candidate genes for breeding programs aiming to produce lettuce varieties able to grow healthy under hydroponic systems that use artificial light.

Variable Number Tandem Repeats (VNTRs) are tandem repeat (TR) loci that vary in copy number across a population. Using our program, VNTRseek, we analyzed human whole genome sequencing datasets from 2,770 individuals in order to detect minisatellite VNTRs, i.e., those with pattern sizes ≥7 bp. We detected 35,638 VNTR loci and classified 5,676 as commonly polymorphic (i.e., with non-reference alleles occurring in >5% of the population). Commonly polymorphic VNTR loci were found to be enriched in genomic regions with regulatory function, i.e., transcription start sites and enhancers. Investigation of the commonly polymorphic VNTRs in the context of population ancestry revealed that 1,096 loci contained population-specific alleles and that those could be used to classify individuals into super-populations with near-perfect accuracy. Search for quantitative trait loci (eQTLs), among the VNTRs proximal to genes, indicated that in 187 genes expression differences correlated with VNTR genotype. We validated our predictions in several ways, including experimentally, through the identification of predicted alleles in long reads, and by comparisons showing consistency between sequencing platforms. This study is the most comprehensive analysis of minisatellite VNTRs in the human population to date.

Patchouli, Pogostemon cablin (Blanco) Benth., is a traditional Chinese medicinal plant from the order Lamiales. It is considered a valuable herb due to its essential oil content and range of therapeutic effects. This study aimed to explore the evolutionary history of repetitive sequences in the patchouli genome by analyzing tandem repeats and transposable elements (TEs). We first retrieved genomic data for patchouli and four other Lamiales species from the GenBank database. Next, the content of tandem repeats with different period sizes was identified. Long terminal repeats (LTRs) were then identified with LTR_STRUC. Finally, the evolutionary landscape of TEs was explored using an in-house PERL program. The analysis of repetitive sequences revealed that tandem repeats constitute a higher proportion of the patchouli genome compared to the four other species. Analyses of TE families showed that most of the repetitive sequences in the patchouli genome are TEs, and that recently inserted TEs make up a comparatively larger proportion than older ones. Our analyses of LTR retrotransposons in their host genome indicated the existence of ancient LTR retrotransposon expansion, and the escape of these elements from natural selection revealed their ages. Our identification and analyses of repetitive sequences should provide new insights for further investigation of patchouli evolution.

Chironomidae species are universally used for studying the impact of pollutants in aquatic systems. The nonbiting midge Propsilocerus akamusi is often found in urban streams and is suitable for use as a toxicological bioindicator. However, few studies have previously examined metal stress in this species. We sequenced the genome of this urban midge to address this question. Here, we present the first chromosome-level genome of P. akamusi, obtained from Illumina short-read and PacBio long-read sequences with Hi-C technology. The size of the very small assembled genome was 85.83 Mb with a contig N50 of 6.2 Mb and a scaffold N50 of 26.1 Mb. This assembly revealed significant expansion of haemoglobin (Hb) genes, some of which formed large tandem repeats. Transcriptomic studies for copper tolerance identified four genes in the tandem array that were highly expressed, all of which presented intron loss. This characteristic might highlight the potential role of Hb genes in copper tolerance. Additionally, detoxification genes, chemosensory genes and heat shock protein genes of this midge were identified, some of which are associated with metal stress. The high-quality assembled genome of P. akamusi and the transcriptomic analyses provide new insight into the molecular mechanisms of heavy metal stress. Our comparison of the P. akamusi genome with other dipteran genomes provides valuable resources for understanding the evolutionary history, genetics, and ecology of this species as well as those of other midges.

Heptageniidae is one of the most species-rich families within Ephemeroptera, whose evolution pattern of mitogenomes and internal relationships remain unclear. In this study, the complete mitochondrial genomes (mitogenomes) of Afronurus rubromaculata, A. yixingensis and A. obliquistrita were firstly sequenced and analyzed. Three mitogenomes share similar gene arrangement with an extra trnM gene compared with the ancestral order for insects. Comprehensive analysis of tRNA secondary structures showed that the extra trnM had a high level of nucleotide variation. The conservative intergenic spacer (IGS) region in the mitogenomes was found between the trnA and trnR genes with a size of 39 nucleotides and all formed a loop-stem structure. Furthermore, tandem repeats were identified in the control region of all three mitogenomes. The phylogenetic analyses using both Bayesian inference (BI) and maximum likelihood (ML) methods based on three datasets all supported that Heptageniidae was a monophyletic family. Our results supported the relationships ((A. rubromaculata + A. obliquistrita) + A. yixingensis) within Afronurus. The analysis will improve our understanding of the mitogenome features of heptageniid mayflies and lay the foundation for further phylogenetic study of Ephemeroptera.

Background The prevalence of human brucellosis in Qinghai Province of China has been increasing rapidly, with confirmed cases distributed across 31 counties. However, the epidemiology of brucellosis transmission has not been fully elucidated. To characterize the infecting strains isolated from humans, multiple-locus variable-number tandem repeats analysis (MLVA) and whole-genome single-nucleotide polymorphism (SNP)-based approaches were employed. Methods Strains were isolated from two males blood cultures that were confirmed Brucella melitensis positive following biotyping and MLVA. Genomic DNA was extracted from these two strains, and whole-genome sequencing was performed. Next, SNP-based phylogenetic analysis was performed to compare the two strains to 94 B. melitensis strains (complete genome and draft genome) retrieved from online databases. Results The two Brucella isolates were identified as B. melitensis biovar 3 (QH2019001 and QH2019005) following conventional biotyping and were found to have differences in their variable number tandem repeats (VNTRs) using MLVA-16. Phylogenetic examination assigned the 96 strains to five genotype groups, with QH2019001 and QH2019005 assigned to the same group, but different subgroups. Moreover, the QH2019005 strain was assigned to a new subgenotype, IIj, within genotype II. These findings were then combined to determine the geographic origin of the two Brucella strains. Conclusions Utilizing a whole-genome SNP-based approach enabled differences between the two B. melitensis strains to be more clearly resolved, and facilitated the elucidation of their different evolutionary histories. This approach also revealed that QH2019005 is a member of a new subgenotype (IIj) with an ancient origin in the eastern Mediterranean Sea.

We analyzed the kinetoplast (mitochondrial genome) of Trypanosoma vivax strains from America and Africa to determine their precise architecture and to understand their adaptive response to mechanical transmission. The use of long-read based assemblies that retain individuality of tandem repeats, without erasing inter-copy variability, allowed us to investigate the evolutionary dynamics of repetitive kinetoplast-DNA. This analysis revealed that repeat elements located in edges of repeat clusters are less active in terms of renewal, whereas internal copies appear to undergo a permanent process of birth-and-death. Comparing different American strains with the African Y486 strain, we found that in the former, protein coding genes from the maxicircle contain several function disrupting mutations that with very few exceptions are present in one or the other American strain but not in both, suggesting the absence of common ancestry for most of the genomic changes that led to their loss of oxidative phosphorylation capacity. Analysis of another component of kinetoplast, the minicircles, revealed great loss of diversity, and loss of their encoded guideRNAs. Both groups of American strains retain minimal sets required to edit the still functional A6-APTase and RPS12 genes. The extensive maxi- and minicircle divergence suggests a history of multiple introduction events in America of strains that probably started to degrade their kinetoplast in Africa. The notion that kinetoplast degradation began after incursion in America would imply a pace of accumulation of genetic changes considerably faster than other trypanosomatids.

During plant infection, fungi secrete effector proteins in coordination with distinct infection stages. Thus, the success of plant infection is determined by precise control of effector gene expression. We analysed the PWL2 effector gene of the rice blast fungus Magnaporthe oryzae to understand how effector genes are activated specifically during the early biotrophic stages of rice infection. Here, we used confocal live‐cell imaging of M. oryzae transformants with various PWL2 promoter fragments fused to sensitive green fluorescent protein reporter genes to determine the expression patterns of PWL2 at the cellular level, together with quantitative reverse transcription PCR analyses at the tissue level. We found PWL2 expression was coupled with sequential biotrophic invasion of rice cells. PWL2 expression was induced in the appressorium upon penetration into a living rice cell but greatly declined in the highly branched hyphae when the first‐invaded rice cell was dead. PWL2 expression then increased again as the hyphae penetrate into living adjacent cells. The expression of PWL2 required fungal penetration into living plant cells of either host rice or nonhost onion. Deletion and mutagenesis experiments further revealed that the tandem repeats in the PWL2 promoter contain 12‐base pair sequences required for expression. We conclude that PWL2 expression is (a) activated by an unknown signal commonly present in living plant cells, (b) specific to biotrophic stages of fungal infection, and (c) requires 12‐base pair cis‐regulatory sequences in the promoter., Expression of the effector gene PWL2 of the rice blast fungus is controlled by tandem repeat cis‐regulatory sequences in response to unknown signals from living cells of host (rice) or non‐host (onion).

Background Chromosomal variants play important roles in crop breeding and genetic research. The development of single-stranded oligonucleotide (oligo) probes simplifies the process of fluorescence in situ hybridization (FISH) and facilitates chromosomal identification in many species. Genome sequencing provides rich resources for the development of oligo probes. However, little progress has been made in peanut due to the lack of efficient chromosomal markers. Until now, the identification of chromosomal variants in peanut has remained a challenge. Results A total of 114 new oligo probes were developed based on the genome-wide tandem repeats (TRs) identified from the reference sequences of the peanut variety Tifrunner (AABB, 2n = 4x = 40) and the diploid species Arachis ipaensis (BB, 2n = 2x = 20). These oligo probes were classified into 28 types based on their positions and overlapping signals in chromosomes. For each type, a representative oligo was selected and modified with green fluorescein 6-carboxyfluorescein (FAM) or red fluorescein 6-carboxytetramethylrhodamine (TAMRA). Two cocktails, Multiplex #3 and Multiplex #4, were developed by pooling the fluorophore conjugated probes. Multiplex #3 included FAM-modified oligo TIF-439, oligo TIF-185-1, oligo TIF-134-3 and oligo TIF-165. Multiplex #4 included TAMRA-modified oligo Ipa-1162, oligo Ipa-1137, oligo DP-1 and oligo DP-5. Each cocktail enabled the establishment of a genome map-based karyotype after sequential FISH/genomic in situ hybridization (GISH) and in silico mapping. Furthermore, we identified 14 chromosomal variants of the peanut induced by radiation exposure. A total of 28 representative probes were further chromosomally mapped onto the new karyotype. Among the probes, eight were mapped in the secondary constrictions, intercalary and terminal regions; four were B genome-specific; one was chromosome-specific; and the remaining 15 were extensively mapped in the pericentric regions of the chromosomes. Conclusions The development of new oligo probes provides an effective set of tools which can be used to distinguish the various chromosomes of the peanut. Physical mapping by FISH reveals the genomic organization of repetitive oligos in peanut chromosomes. A genome map-based karyotype was established and used for the identification of chromosome variations in peanut following comparisons with their reference sequence positions.

During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51–DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.

Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ∼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.

Developing a mass spectrometry-based assay for the ovarian cancer biomarker CA125 (MUC16) is a desirable goal, because it may enable detection of molecular regions that are not recognized by antibodies and are therefore analytically silent in the current immunoassay. Additionally, the ability to characterize the CA125 proteoforms expressed by individuals may offer clinical insight. Enrichment of CA125 from malignant ascites may provide a high-quality source of this important ovarian cancer biomarker, but a reliable strategy for such enrichment is currently lacking. Beginning with crude ascites isolated from three individual patients with high grade serous ovarian cancer, we enriched for MUC16 using filtration, ion exchange, and size exclusion chromatography and then performed bottom-up proteomics on the isolated proteins. This approach of enrichment and analysis reveals that the peptides detected via mass spectrometry map to the SEA domain and C-loop regions within the tandem repeat domains of CA125 and that peptide abundance correlates with clinical CA125 counts.

Telomeres are protective cap on the ends of DNA of non-coding tandem repeats of TTAGGG. Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase that maintains the structure of telomeres. Type 2 diabetes (T2D) affects multi-organ and telomere length by altering telomerase activity. We aimed to evaluate the relative telomere length (RTL) and risk association of rs2853669 with T2D in Bangladeshi population. RTL was measured in 408 unrelated Bangladeshi (224 T2D and 184 healthy) using primers for target gene and reference gene albumin. Genotypic frequencies for rs2853669 were determined using TaqMan® probes. The mean level of age adjusted RTL (AARTL) varied significantly between the healthy and individuals with T2D for all the genotypes with respect to rs2853669. Moreover, healthy individuals had significantly higher AARTL than T2D. Similar findings were observed when study participants were stratified based on their gender. Association studies revealed that under codominant model of inheritance, TC genotype showed protective role against development of type 2 diabetes. This study suggests a possible role of telomere biology in T2DM, but their association needs to be evaluated further with a larger series and matched healthy controls.

DNA sequences in regulatory regions and in telomers at the ends of chromosomes frequently contain tandem repeats of guanine nucleotides that can form stacked structures stabilized by Hoogsten pairing and centrally bound monovalent cations. The replication and elongation of telomeres requires the disruption of these G-quadruplex structures. Hence, drug molecules such as gold (Au)-carbene that stabilize G-quadruplexes may also interfere with the elongation of telomeres and, in turn, could be used to control cell replication and growth. To better understand the molecular mechanism of Au-carbene binding to G-quadruplexes, we employed molecular dynamics simulations and free energy simulations. Whereas very restricted mobility of two Au-carbene ligands was found upon binding as a doublet to one side of the G-quadruplex, much larger translational and orientational mobility was observed for a single Au-carbene binding at the second G-quadruplex surface. Comparative simulations on duplex DNA in the presence of Au-carbene ligands indicates a preference for the minor groove and weaker unspecific and more salt-dependent binding than to the G-quadruplex surface. Analysis of energetic contributions reveals a dominance of nonpolar and van der Waals interactions to drive binding. The simulations can also be helpful for proposing possible modifications that could improve Au-carbene affinity and specificity for G-quadruplex binding.

Although centromeres have conserved function, centromere-specific histone H3 (CenH3) and centromeric DNA evolve rapidly. The centromere drive model explains this phenomenon as a consequence of the conflict between fast-evolving DNA and CenH3, suggesting asymmetry in female meiosis as a crucial factor. We characterized evolution of the CenH3 protein in three closely related, polyploid mitotic parthenogenetic species of the Meloidogyne incognita group, and in the distantly related meiotic parthenogen Meloidogyne hapla. We identified duplication of the CenH3 gene in a putative sexual ancestral Meloidogyne. We found that one CenH3 (αCenH3) remained conserved in all extant species, including in distant Meloidogyne hapla, whereas the other evolved rapidly and under positive selection into four different CenH3 variants. This pattern of CenH3 evolution in Meloidogyne species suggests the subspecialization of CenH3s in ancestral sexual species. Immunofluorescence performed on mitotic Meloidogyne incognita revealed a dominant role of αCenH3 on its centromere, whereas the other CenH3s have lost their function in mitosis. The observed αCenH3 chromosome distribution disclosed cluster-like centromeric organization. The ChIP-Seq analysis revealed that in M. incognita αCenH3-associated DNA dominantly comprises tandem repeats, composed of divergent monomers which share a completely conserved 19-bp long box. Conserved αCenH3-associated DNA is also confirmed in the related mitotic Meloidogyne incognita group species suggesting preservation of both centromere protein and DNA constituents. We hypothesize that the absence of centromere drive in mitosis might allow for CenH3 and its associated DNA to achieve an equilibrium in which they can persist for long periods of time.

Lipoid proteinosis (LP) is a rare autosomal recessive disorder caused by pathological mutations in the glycoprotein extracellular matrix protein 1 gene (ECM1). In this study, we examined two sibling patients who were suspected of LP in a consanguineous Chinese family for clinical manifestations and sequenced the all coding exonic regions of ECM1 in the proband. Both siblings were detected a homozygous three-nucleotide duplication, c.506_508dupCTG in the exon 6 of ECM1. This mutation introduces an alanine addition between two highly conserved amino acids (Pro169 and Gly170), designated as p.169_170insA, within one of the two tandem repeat domains which are functional important for protein-protein interactions. Their parents were unaffected and heterozygous for this mutation. This mutation wasn't found in one hundred normal Chinese individuals screened and wasn't previously reported elsewhere, excluding it as a common neutral polymorphism. These evidences supported this duplication as the causative mutation of LP. Our finding expanded the spectrum of disease-causing mutations in LP and provides further evidence for the importance of ECM1 gene in the development of this rare genodermatosis.

Summary Background Neisseria meningitidis is the causative agent of invasive meningococcal disease and the polysaccharide capsule is one of its major virulence factors. Biosynthesis of the meningococcal capsule is controlled by an RNA thermosensor (RNAT) in the 5′-untranslated region (5′-UTR) of the cssA gene. The function of the RNAT depends on an 8-bp tandem repeat configuration. We aimed to identify and characterise novel RNATs in meningococcal isolates responsible for regulating capsule production. Methods We investigated the allele igr_up_NEIS0055, containing the 5′-UTR of the cssA gene, in clinical meningococcal isolates for which whole-genome sequences are available on the Neisseria PubMLST database and that were isolated in Europe between Jan 1, 2010, and Dec 31, 2018. Eight isolates with different RNAT tandem repeat configurations were selected for genetic and phenotypic studies. The thermosensing capability of the RNAT and capsule production was tested with immunoblots. Bacterial survival by capsule protection was assessed with a human serum stress assay and capsule interference with bacterial cell adhesion was evaluated with a bacterial adhesion assay. The dataset of RNAT configurations was analysed for an association with invasive meningococcal disease, and was stratified to visualise the distribution of RNAT configurations within the meningococcal population. Findings Our search of PubMLST identified 112 alleles for the igr_up_NEIS0055 locus and 7013 N meningitidis isolates. Five novel RNAT tandem repeat configurations were identified and eight RNAT tandem repeat configurations, ranging from 1 × 8-bp up to 8 × 8-bp, were characterised. The disrupted RNATs (1 × 8-bp and 3 × 8-bp to 8 × 8-bp) confer upregulated CssA expression and increased capsule production compared with the native 2 × 8-bp configuration, resulting in a hypercapsulation phenotype. Increased capsule production was associated with higher survival rates in up to 25% human serum. The prevalence of a disrupted RNAT resulting in hypercapsulation was almost twice as high in invasive meningococcal disease isolates compared with carrier isolates. Disrupted RNATs were especially attributed to isolates of capsule group B and C, and clonal complexes 23, 32, 213, and 269. Hypercapsulation in one isolate led to lower adhesion onto pharyngeal cells compared with a similar isolate with low capsule production. Interpretation Six non-canonical RNAT tandem repeat variants (3 × 8-bp to 8 × 8-bp) were identified in the igr_up_NEIS0055 locus of N meningitidis that induce a hypercapsulation phenotype, thus providing the meningococci with better protection against host complement-mediated killing than does the native RNAT (2 × 8-bp). Further research is warranted to strengthen the association between hypercapsulation and the progression of invasive meningococcal disease, and to investigate the role of regulatory RNAs in meningococcal virulence and as potential markers for disease progression. Funding Swedish Foundation for Strategic Research, Knut and Alice Wallenberg Foundation, and Swedish Research Council.

Y. pestis MLVA typing is used both to seek for similarities and differences between individual isolates upon conducting epidemiological investigations as well as for clonal clustering of intraspecies phylogenetic groups while analyzing microevolution and taxonomy issues. It cannot be ruled out that the most variable loci may be indicators allowing to approximate the unique strain-related properties circulating in certain natural plague foci. The Central Caucasian Highland Natural Plague Focus distinguished by heterogeneity of the circulating strains therein, including proline pro- and auxo-trophy, may represent a convenient model for testing this hypothesis. The purpose of our work was to assess the frequencies of the VNTR alleles associated with proline dependence among the Y. pestis strain VNTR loci, determined during previous MLVA-25 typing in the Central Caucasian Highland Natural Plague Focus. The main task was to identify the most informative sets of VNTR loci suitable for predicting proline pro— and auxotrophy (pro+, pro—). It was found that the loci ms45, ms56, ms46, ms07, ms69, ms62 displayed peak variability by allele frequencies and/or exhibited significant differences of mean allele frequencies in the pro— and pro+ strains. In particular, it was showed that the alleles of the ms45 locus contained 6 tandem repeats suggesting probability for pro+ reaching 0.944, whereas the alleles of the ms45 locus contained 7 tandem repeats with expected probability for pro— reaching 0.783. Moreover, the ms56 and ms46 contained 9 and more than 18 tandem repeats, respectively, thereby pointing at probability for pro+ equal to 0.933 and 0.818, respectively. Diagnostics for pro+/pro— phenotype by using specific statistical methods demonstrated statistical error 13.33% and 26.67% for the pro— and pro+ strains, respectively. All pro+ strains bearing a 6 tandem repeat complex from the ms45 locus, 9 tandem repeats derived from the ms56 locus and ms46 locus-derived 29—30 tandem repeats were accurately diagnosed solely based on these 3 loci. Thus, it is possible to predict some properties of Y. pestis strains based on determining the allele frequencies. While the number of MLVA typed plaque strains isolated in such natural focus has been progressively increased, it may be expected that opportunities for prognosing their properties based on determining locus tandem repeat composition having diagnostic value would be elevated.

Although bacteriophages are more numerous and have smaller genomes than their bacterial hosts, relatively few have their genomes sequenced. Here, we isolated the Pseudomonas fluorescens bacteriophage from Napahai plateau wetland and performed de novo genome sequencing. Based on the previous biological characteristics and bioinformatics analysis, it was determined that VW-6B was a linear double-stranded DNA (dsDNA) phage with 35,306 bp, with 56.76% G+C content and 197 bp tandem repeats. The VW-6B genome contained 46 open-reading frames (ORFs), and no tRNA genes were found. Based on phage genome structure, sequence comparison, and collinear analysis, VW-6B should be classified into the family Siphoviridae and be considered as a member of a new species in the Mu-like phage. The newly isolated bacteriophage can specifically infect P. fluorescens, which further enriches the diversity of known bacteriophages and provides a basis for the subsequent research and application of bacteriophages.

This study presented 96 cox1 and 76 cox3 genes of Amphioctopus neglectus populations. Three distinct lineages were formed from phylogenetic trees and networks constructed using haplotypes. Mitogenomes of A. neglectus-a and A. neglectus-b as the representatives of two lineages separated from population genetics were sequenced to compare with A. neglectus at the genome-level. Amphioctopus neglectus-a showed significant differences with A. neglectus, mainly reflected in gene length, intergenic regions and the secondary structure of tandem repeat motifs. Notably, two sequence deletions in mitogenomes of the two representative species were detected in different positions of major non-coding regions, which were the most distinct differences with A. neglectus. Pairwise genetic distances and the phylogenetic analysis supported the relationship of (A. neglectus-a + (A. neglectus + A. neglectus-b)). This study suggested that A. neglectus-a should be considered as a potential cryptic species of this complex, while A. neglectus-b needed further verification to be defined.

Microsporidia are a group of obligate intracellular parasites causing significant disease in human beings and economically important animals. Though a few spore wall proteins (SWPs) have now been identified in these intriguing species, the information on SWPs remains too little to elucidate the spore wall formation mechanisms of microsporidia. It has been well described that numerous proteins with tandem repeats tend to be localized on the cell wall of fungi and parasites. Previously, by scanning the proteins with tandem repeats in microsporidian Nosema bombycis, we obtained 83 candidate SWPs based on whether those proteins possess a signal peptide and/or transmembrane domain. Here, we further characterized a candidate protein (EOB13250) with three tandem repeats in the N-terminal region and a transmembrane domain in C-terminus of N. bombycis. Sequence analysis showed that the tandem repeat domain of EOB13250 was species-specific for this parasite. RT-PCR indicated that the expression of the gene encoding this protein started on the fourth day postinfection. After cloned and expressed in Escherichia coli, a polyclone antibody against the recombinant EOB13250 protein was prepared. Western blotting demonstrated this protein exist in N. bombycis. Immunofluorescence analysis (IFA) and immunoelectron microscopy analysis (IEM) further provided evidence that EOB13250 was an endospore wall protein. These results together suggested that EOB13250 was a novel spore wall protein of N. bombycis. This study provides a further enrichment of the number of identified spore wall proteins in microsporidia and advances our understanding of the spore wall formation mechanism in these obligate unicellular parasites.

The RepeatsDB database (URL: https://repeatsdb.org/) provides annotations and classification for protein tandem repeat structures from the Protein Data Bank (PDB). Protein tandem repeats are ubiquitous in all branches of the tree of life. The accumulation of solved repeat structures provides new possibilities for classification and detection, but also increasing the need for annotation. Here we present RepeatsDB 3.0, which addresses these challenges and presents an extended classification scheme. The major conceptual change compared to the previous version is the hierarchical classification combining top levels based solely on structural similarity (Class > Topology > Fold) with two new levels (Clan > Family) requiring sequence similarity and describing repeat motifs in collaboration with Pfam. Data growth has been addressed with improved mechanisms for browsing the classification hierarchy. A new UniProt-centric view unifies the increasingly frequent annotation of structures from identical or similar sequences. This update of RepeatsDB aligns with our commitment to develop a resource that extracts, organizes and distributes specialized information on tandem repeat protein structures., Facultad de Ciencias Exactas, Instituto de Biotecnologia y Biologia Molecular

Motivation Long tandem repeat expansions of more than 1000 nt have been suggested to be associated with diseases, but remain largely unexplored in individual human genomes because read lengths have been too short. However, new long-read sequencing technologies can produce single reads of 10 000 nt or more that can span such repeat expansions, although these long reads have high error rates, of 10–20%, which complicates the detection of repetitive elements. Moreover, most traditional algorithms for finding tandem repeats are designed to find short tandem repeats ( Results Here, we report an efficient algorithm for solving this problem that takes advantage of the length of the repeat. Namely, a long tandem repeat has hundreds or thousands of approximate copies of the repeated unit, so despite the error rate, many short k-mers will be error-free in many copies of the unit. We exploited this characteristic to develop a method for first estimating regions that could contain a tandem repeat, by analyzing the k-mer frequency distributions of fixed-size windows across the target read, followed by an algorithm that assembles the k-mers of a putative region into the consensus repeat unit by greedily traversing a de Bruijn graph. Experimental results indicated that the proposed algorithm largely outperformed Tandem Repeats Finder, a widely used program for finding tandem repeats, in terms of sensitivity. Availability and implementation https://github.com/morisUtokyo/mTR.

Chromosome level assemblies are accumulating in various taxonomic groups including mosquitoes. However, even in the few reference-quality mosquito assemblies, a significant portion of the heterochromatic regions including telomeres remain unresolved. Here we produce a de novo assembly of the New World malaria mosquito, Anopheles albimanus by integrating Oxford Nanopore sequencing, Illumina, Hi-C and optical mapping. This 172.6 Mbps female assembly, which we call AalbS3, is obtained by scaffolding polished large contigs (contig N50=13.7 Mbps) into three chromosomes. All chromosome arms end with telomeric repeats, which is the first in mosquito assemblies and represents a significant step towards the completion of a genome assembly. These telomeres consist of tandem repeats of a novel 30-32 bp telomeric repeat unit (TRU) and are confirmed by analysing the termini of long reads and through both chromosomal in situ hybridization and a Bal31 sensitivity assay. The AalbS3 assembly included previously uncharacterized centromeric and rDNA clusters and more than doubled the content of transposable elements and other repetitive sequences. This telomere-to-telomere assembly, although still containing gaps, represents a significant step towards resolving biologically important but previously hidden genomic components. The comparison of different scaffolding methods will also inform future efforts to obtain reference-quality genomes for other mosquito species.100-word Article SummaryWe report AalbS3, a telomere-to-telomere assembly of the Anopheles albimanus genome produced by integrating advancing technologies including Oxford Nanopore and Bionano optical mapping. AalbS3 features much of the difficult-to-assemble genomic ‘dark matters’ including previously missed transposons, centromeres and rDNA clusters. We describe novel telomeric repeats that are confirmed by analysis of long reads and by telomere hybridization assays. This reference-quality assembly represents a significant step towards completing the genomic puzzle pieces and informs efforts to improve the assembly of other mosquito species. Future research into the relationship between telomere and mosquito life span may have significant implications to disease control.

Tandem repeats are proposed to contribute to human-specific traits, and more than 40 tandem repeat expansions are known to cause neurological disease. Here, we characterize a human-specific 69 bp variable number tandem repeat (VNTR) in the last intron of WDR7, which exhibits striking variability in both copy number and nucleotide composition, as revealed by long-read sequencing. In addition, greater repeat copy number is significantly enriched in three independent cohorts of individuals with sporadic amyotrophic lateral sclerosis (ALS). Each unit of the repeat forms a stem-loop structure with the potential to produce microRNAs, and the repeat RNA can aggregate when expressed in cells. We leveraged its remarkable sequence variability to align the repeat in 288 samples and uncover its mechanism of expansion. We found that the repeat expands in the 3′-5′ direction, in groups of repeat units divisible by two. The expansion patterns we observed were consistent with duplication events, and a replication error called template switching. We also observed that the VNTR is expanded in both Denisovan and Neanderthal genomes but is fixed at one copy or fewer in non-human primates. Evaluating the repeat in 1000 Genomes Project samples reveals that some repeat segments are solely present or absent in certain geographic populations. The large size of the repeat unit in this VNTR, along with our multiplexed sequencing strategy, provides an unprecedented opportunity to study mechanisms of repeat expansion, and a framework for evaluating the roles of VNTRs in human evolution and disease.

Immunomolecular characterization of Ehrlichia chaffeensis (E. ch.) and E. canis (E. ca.) has defined protein orthologs, including tandem repeat proteins (TRPs) that have immunodominant linear antibody epitopes. In this study, we combined bioinformatic analysis and cell-free protein expression to identify undiscovered immunoreactive E. ch. and E. ca. hypothetical proteins. Antigenicity of the E. ch. and E. ca. ORFeomes (n = 1105 and n = 925, respectively) was analyzed by the sequence-based prediction model ANTIGENpro, and we identified ~250 ORFs in each respective ORFeome as highly antigenic. The hypothetical proteins (E. ch. n = 93 and E. ca. n = 98) present in the top 250 antigenic ORFs were further investigated in this study. By ELISA, 46 E. ch. and 30 E. ca. IVTT-expressed hypothetical proteins reacted with antibodies in sera from naturally E. ch.-infected patients or E. ca.-infected dogs. Moreover, 15 E. ch. and 16 E. ca. proteins consistently reacted with a panel of sera from patients or dogs, including many that revealed the immunoreactivity of “gold standard” TRPs. Antibody epitopes in most (>70%) of these proteins exhibited partial or complete conformation-dependence. The majority (23/31; 74%) of the major immunoreactive proteins identified were small (≤250 aa), and 20/31 (65%) were predicted to be secreted effectors. Unlike the strong linear antibody epitopes previously identified in TRP and OMP orthologs, there were contrasting differences in the E. ch. and E. ca. antigenic repertoires, epitopes and ortholog immunoreactivity. This study reveals numerous previously undefined immunodominant and subdominant antigens, and illustrates the breadth, complexity, and diversity of immunoreactive proteins/epitopes in Ehrlichia.

Short tandem repeats (STRs) contribute to structural variation in plant mitochondrial genomes, but the mechanisms underlying their formation and expansion are unclear. In this study, we detected high polymorphism in the nad7-1 region of the Pinus tabuliformis mitogenome caused by the rapid accumulation of STRs and rearrangements over a few million years ago. The STRs in nad7-1 have a 7-bp microhomology (TAG7) flanking the repeat array. We then scanned the mitogenomes of 136 seed plants to understand the role of microhomology in the formation of STR and mitogenome evolution. A total of 13,170 STRs were identified, and almost half of them were associated with microhomologies. A substantial amount (1,197) of microhomologies was long enough to mediate structural variation, and the length of microhomology is positively correlated with the length of tandem repeat unit. These results suggest that microhomology may be involved in the formation of tandem repeat via microhomology-mediated pathway, and the formation of longer duplicates required greater length of microhomology. We examined the abundance of these 1,197 microhomologies, and found 75% of them were enriched in the plant mitogenomes. Further analyses of the 400 prevalent microhomologies revealed that 175 of them showed differential enrichment between angiosperms and gymnosperms and 186 differed between angiosperms and conifers, indicating lineage-specific usage and expansion of microhomologies. Our study sheds light on the sources of structural variation in plant mitochondrial genomes and highlights the importance of microhomology in mitochondrial genome evolution.

Jasmine [Jasminum sambac (L.) Aiton] is an important cultivated plant species that is valued for its ornamental, medicinal and edible values. However, the genomic information available for jasmine is limited. In this study, the complete chloroplast (cp) genomes of single-petal (SP) and double-petal (DP) cultivars of jasmine were investigated. An entire cp genome comparison, inverted repeat contraction and expansion were analyzed among several species, including the two jasmine cultivars, and phylogenetic analysis was performed. The results showed that the complete cp genomes were 163,315 bp (SP) and 163,281 bp (DP) long, respectively. Both genomes exhibited a single circular molecule with quadripartite structure consisting of a pair of inverted repeats separated by the large and small single-copy regions. Both SP and DP cultivars contained 114 unique genes. Sequence analysis identified 31 tandem repeats, 15 forward repeats, 14 palindrome repeats and five complement repeats in both cp genomes. A total of 118 and 117 simple sequence repeats (SSRs) were detected in the cp genomes of SP and DP cultivars, respectively. Phylogenetic analysis strongly supported that the SP cultivar was close to DP, and J. sambac belonged to the family Oleaceae, order Contortae. The complete cp genome sequences presented in this report may contribute to genetic and phylogenetic studies of this species.

Ralstonia solanacearum, a bacterial plant pathogen that cause wilt in many economically important vegetable crops including potato is called R. solanacearum species complex (RSSC) due to its enormous diversity and vast host range. The whole genome of three strains (Rs2, Rs25 and Rs75) belonging to different phylotypes that are known to cause potato wilt in India were sequenced and assembled. The genome-wide microsatellite content of these strains were studied along with four reference strains (GMI1000/I; CFBP2957/II; CMR15/III; PSI07/IV) to excavate the compositional biasness among the phylotypes. Genome wide G + C content was found to be 66.0–67.50% for all the strains with all the megaplsmids having higher than chromosomes. The highest tandem repeats were found in phylotype IV (Rs75 and PSI07) whereas, phylotype III which is not found to infect potato in India have the lowest. The dinucleotides TA and AT are underrepresented in both chromosome as well as megaplsmid whereas, GC and CG/CG were the most abundantly occurring repeats.

Haynaldia villosa is a wild relative of wheat and a valuable gene resource for wheat improvement. Owing to the limited number of probes available for fluorescence in situ hybridization (FISH), the resolution at which the karyotype of H. villosa can be characterized is poor, hampering accurate characterization of small segmental alien introgressions. We designed ten oligonucleotide probes using tandem repeats in DNA sequences derived from the short arm of H. villosa chromosome 6V (6VS). FISH with seven of them resulted in clear signals on H. villosa chromosomes. Using these, we constructed FISH karyotypes for H. villosa using oligo-6VS-1 and oligo-6VS-35 oligonucleotides and characterized the distribution of the two probes in five different H. villosa accessions. The new FISH probes can efficiently characterize H. villosa introgressions into wheat.

Antimicrobial peptides (AMPs) mainly introduced as a new generation of antibiotics, could be used for broad medical and biotechnological applications including treatment of multi drug resistant bacterial infections, anti-biofilm agents, wound healing, drug delivery and biosensors. High cost of peptide synthesis is one of the major obstacles of industrial scale production of AMPs. Recombinant expression of tandem repeats (multimeric forms) of AMPs with proper linkers is a useful approach for overcoming the high cost of peptides synthesis. In the present study, the effect of using glycine- serine [(GGGGS) 3] and aspartic acid-proline (DP) Linkers between four tandem repeats of sushi S3 antimicrobial peptide on its biological activity was investigated. S3 peptide, derived from factor C of horseshoe crab hemolymph has been known as a promising candidate as anti-Gram negative bacterial agent. Each variant of S3 tetramers with (GGGGS) 3 and DP linkers (named S3-4mer-GS and S3-4mer-DP, respectively) were expressed in E. coli. BL21 (DE3) as inclusion bodies and were purified by using immobilized metal affinity chromatography (IMAC). Antimicrobial activities and cytotoxic properties of variants were evaluated by determination of minimum inhibitory concentration assay (MIC) and MTT assay. Statistical analysis revealed that glycine serine linker improved antimicrobial activity of S3-tetramer approximately 25% and 86% in comparison to tetramer with aspartic acid - proline linker and S3 monomer respectively. According to the enhanced antibacterial activity of glycine- serine linker without any significant effects on its cytotoxicity, this linker will be a good choice for recombinant tandem expression of small antimicrobial peptides.

Recent advances in functional genomics have facilitated the identification of multiple genes and isoforms associated with the genetic risk of schizophrenia, yet the causal variations remain largely unclear. A previous study reported that the schizophrenia risk single-nucleotide polymorphism (SNP) rs7085104 at 10q24.32 was in high linkage disequilibrium (LD) with a human-specific variable number of tandem repeat (VNTR), and both were significantly associated with the brain mRNA expression of a human-unique AS3MTd2d3 isoform in Europeans and African Americans. In this study, we have shown the direct regulation of the AS3MTd2d3 mRNA expression by this VNTR through an in vitro minigene splicing assay, suggesting that it is likely a causative functional variation. Intriguingly, we have further confirmed that the VNTR and rs7085104 are significantly associated with AS3MTd2d3 mRNA expression in brains of Han Chinese donors, and rs7085104 is also associated with risk of schizophrenia in East Asians. Finally, the overexpression of AS3MTd2d3 in cultured primary hippocampal neurons results in significantly reduced densities of mushroom dendritic spines, implicating its potential functional impact. Considering the crucial roles of dendritic spines in neuroplasticity, these results reveal the potential regulatory impact of the schizophrenia risk VNTR on AS3MTd2d3 and provide insights into the underlying biological mechanisms.