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Background – Green algae are a diverse group of photosynthetic eukaryotes, yet are still vastly understudied compared to land plants. For many years, green algae were characterized based on their morphology and life cycles. More recently, phylogenetic and genomic analyses have been added to the phycological toolkit for a better understanding of algal biodiversity and evolutionary history. Material and methods – A desert strain of green algae was isolated from Joshua Tree National Park (JTNP) in southern California as part of a larger biodiversity survey. The alga’s nuclear rRNA genes as well as the chloroplast genome were sequenced, annotated, and analysed in addition to a morphological assessment. Results – Morphologically this strain is especially similar to Pseudomuriella and Rotundella, and its lipid profile resembles that of other soil algae, but phylogenomic analyses demonstrate that it is a distinct evolutionary lineage in Chlorophyceae. The alga exhibits several unusual genomic features, the most remarkable being its highly derived yet apparently functional nuclear rRNA genes, 18S and 28S. Both genes are GC-rich and bear many compensatory base changes to maintain a similar secondary structure to that of other green algae. The chloroplast genome has a distinct gene order and repeat arrangement from other published green algal plastomes, but contains the expected genes and also provides phylogenetically informative data. Conclusion – We conclude that the strain be placed into a new species and genus in the class Chlorophyceae, and propose the name Johansenicoccus eremophilus for this new taxon. Johansenicoccus eremophilus exemplifies science’s insufficient understanding of the range of genomic variations among inconspicuous soil algae. [ABSTRACT FROM AUTHOR]

Abstract Background Clustered regularly interspaced short palindromic repeats (CRISPR) and their spacers are important components of prokaryotic CRISPR-Cas systems. In order to analyze the CRISPR loci of multiple genomes more intuitively and comparatively, here we propose a visualization analysis tool named CrisprVi. Results CrisprVi is a Python package consisting of a graphic user interface (GUI) for visualization, a module for commands parsing and data transmission, local SQLite and BLAST databases for data storage and a functions layer for data processing. CrisprVi can not only visually present information of CRISPR direct repeats (DRs) and spacers, such as their orders on the genome, IDs, start and end coordinates, but also provide interactive operation for users to display, label and align the CRISPR sequences, which help researchers investigate the locations, orders and components of the CRISPR sequences in a global view. In comparison to other CRISPR visualization tools such as CRISPRviz and CRISPRStudio, CrisprVi not only improves the interactivity and effects of the visualization, but also provides basic statistics of the CRISPR sequences, and the consensus sequences of DRs/spacers across the input strains can be inspected from a clustering heatmap based on the BLAST results of the CRISPR sequences hitting against the genomes. Conclusions CrisprVi is a convenient tool for visualizing and analyzing the CRISPR sequences and it would be helpful for users to inspect novel CRISPR-Cas systems of prokaryotes.

Background: Clustered regularly interspaced short palindromic repeats (CRISPR) and their spacers are important components of prokaryotic CRISPR-Cas systems. In order to analyze the CRISPR loci of multiple genomes more intuitively and comparatively, here we propose a visualization analysis tool named CrisprVi. Results: CrisprVi is a Python package consisting of a graphic user interface (GUI) for visualization, a module for commands parsing and data transmission, local SQLite and BLAST databases for data storage and a functions layer for data processing. CrisprVi can not only visually present information of CRISPR direct repeats (DRs) and spacers, such as their orders on the genome, IDs, start and end coordinates, but also provide interactive operation for users to display, label and align the CRISPR sequences, which help researchers investigate the locations, orders and components of the CRISPR sequences in a global view. In comparison to other CRISPR visualization tools such as CRISPRviz and CRISPRStudio, CrisprVi not only improves the interactivity and effects of the visualization, but also provides basic statistics of the CRISPR sequences, and the consensus sequences of DRs/spacers across the input strains can be inspected from a clustering heatmap based on the BLAST results of the CRISPR sequences hitting against the genomes. Conclusions: CrisprVi is a convenient tool for visualizing and analyzing the CRISPR sequences and it would be helpful for users to inspect novel CRISPR-Cas systems of prokaryotes. [ABSTRACT FROM AUTHOR]

Homologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens in Saccharomyces cerevisiae for genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionarily conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a prokaryotic adaptive immune system that checks invasion by mobile genetic elements through nuclease targeting. In this study, we investigated the occurrence, diversity, and features of the CRISPR system in the genus Aeromonas using bioinformatics tools. Only 13 out of 122 complete genomes (10.66%) of the genus Aeromonas from the NCBI GenBank database harbored the CRISPR system. The Type I–F system was the most prevalent CRISPR system among the Aeromonads, followed by the Type I–E system. Only one strain harbored a Type I–C CRISPR system. Among the Aeromonads, Aeromonas caviae (22.7%) and Aeromonas veronii (20%) had a higher prevalence rate of the complete CRISPR system. The analysis of direct repeat (DR) sequences showed that all could form stable RNA secondary structures. A phylogenetic tree generated for the Cas1 protein classified CRISPR subtypes into three distinct clusters. Among the 748 spacers investigated, 41.98% and 17.25% showed perfect homology to phage and plasmid sequences, respectively. Some arrays had duplicated spacers. The CRISPR loci are closely linked to antibiotic resistance genes in most strains. Collectively, our results would contribute to research on antibiotic resistance in the Aeromonas group, and provide new insights into the diversity and evolution of the CRISPR–Cas system. [ABSTRACT FROM AUTHOR]

Main conclusion: We assembled the complete mitochondrial genome of Scutellaria tsinyunensis in this study. Repeat-mediated recombination resulted in the formation of two conformations of the mitochondrial genome in S. tsinyunensis. Scutellaria tsinyunensis belongs to the family Lamiaceae, distributed only in the Jinyun Mountain, Chongqing, China. As a valuable endemic and small population species, it is regarded as a natural resource potentially with significant economic and ecological importance. In this study, we assembled a complete and gap-free mitochondrial genome of S. tsinyunensis. This genome had a length of 354,073 bp and the base composition of the genome was A (27.44%), T (27.30%), C (22.58%), and G (22.68%). This genome encodes 59 genes, including 32 protein-coding genes, 24 tRNA genes, and 3 rRNA genes. The Sanger sequencing and Oxford Nanopore sequencing confirmed a pair of direct repeats had mediated genome recombination, resulting in the formation of two conformations. The gene conversation between plastome and mitochondrial genome was also observed in S. tsinyunensis by detecting gene migration, including six tRNA genes (namely, trnW-CCA, trnI-CAU, trnH-UUU, trnD-GUC, trnN-GUU, and trnM-CAU), five protein-coding gene fragments, and the fragments from 2 rRNA genes. Moreover, the dN/dS analysis revealed the atp9 gene had undergone strong negative selection, and four genes (atp4, mttB, ccmFc, and ccmB) probably had undergone positive selection during evolution in Lamiales. This work reported the first mitochondrial genome of S. tsinyunensis, which could be used as a reference genome for the important medicinal plants of the genus Scutellaria, and also provide much-desired information for molecular breeding. [ABSTRACT FROM AUTHOR]

Abstract Background It is hypothesized that the highly conserved inverted repeats (IR) structure of land plant plastid genomes (plastomes) is beneficial for stabilizing plastome organization, whereas the mechanism of the occurrence and stability maintenance of the recently reported direct repeats (DR) structure is yet awaiting further exploration. Here we describe the DR structure of the Selaginella vardei (Selaginellaceae) plastome, to elucidate the mechanism of DR occurrence and stability maintenance. Results The plastome of S. vardei is 121,254 bp in length and encodes 76 genes, of which 62 encode proteins, 10 encode tRNAs, and four encode rRNAs. Unexpectedly, the two identical rRNA gene regions (13,893 bp) are arranged in a direct orientation (DR), rather than inverted. Comparing to the IR organization in Isoetes flaccida (Isoetaceae, Lycopodiopsida) plastome, a ca. 50-kb trnN-trnF inversion that spans one DR copy was found in the plastome of S. vardei, which might cause the orientation change. In addition, we find extremely rare short dispersed repeats (SDRs) in the plastomes of S. vardei and its closely related species S. indica. Conclusions We suggest that the ca. 50-kb inversion resulted in the DR structure, and the reduction in SDRs plays a key role in maintaining the stability of plastomes with DR structure by avoiding potential secondary recombination. We further confirmed the presence of homologous recombination between DR regions, which are able to generate subgenomes and form diverse multimers. Our study deepens the understanding of Selaginella plastomes and provides new insights into the diverse plastome structures in land plants.

Homologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens in Saccharomyces cerevisiae for genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionarily conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans. [ABSTRACT FROM AUTHOR]

Miniature inverted repeat transposable element (MITE) is a short transposable element, carrying no protein-coding regions. However, its high proliferation rate and sequence-specific insertion preference renders it as a good genetic tool for both natural evolution and experimental insertion mutagenesis. Recently active MITE copies are those with clear signals of Terminal Inverted Repeats (TIRs) and Direct Repeats (DRs), and are recently translocated into their current sites. Their proliferation ability renders them good candidates for the investigation of genomic evolution.

Juvenile hormones (JH) regulate wide-ranging physiological and developmental processes in insects. However, molecular mechanisms underlying JH signaling remain to be determined. Vitellogenin (Vg) is primarily an egg-yolk protein, but recently proposed to serve many functions in insects. In the female American cockroach (Periplaneta americana), vitellogenin (Vg) genes are activated by JH III and suppressed by 20-hydroxyecdysone (20E) via cis-regulatory elements in a dose-dependent manner. In the present study, the upstream promoter region (935 bp) of Vg1 was cloned to elucidate the action of these hormones. A luciferase reporter assay identified an 81 bp region in the promoter region of Vg1 (−120 to −39 bp) that we found to be critical for JH III activation and 20E suppression. This 81 bp region contains a direct repeat separated by a 2-nucleotide spacer—designated Vg1 HRE— that is similar to the Drosophila ecdysone response element direct repeat 4. Moreover, nuclear proteins isolated from nymphs, males, females, and Sf9 cells successfully bound to Vg1 HRE, while binding was outcompeted by a 100-fold excess of cold probe or dephosphorylated nuclear protein extracts. In addition, binding was outcompeted by other ecdysone and JH response elements with similar half-site sequences (direct repeats) but to varying extents. Ultimately, we postulate that JH III indirectly activates Vg expression by interfering with or inhibiting the phosphorylation of nuclear proteins bound to Vg1 HRE. Involvement of JH III in both induction of Vg1 and control of nuclear proteins binding t o Vg1 HRE suggest the latter to play an important role in JH signaling. Unlabelled Image • The Vg1 promoter in the American cockroach has been cloned and characterized. • A putative JH regulatory site (Vg1 HRE) has been determined. • In vitro luciferase reporter assay conferred JH responsiveness to Vg1 HRE. • Nuclear extracts bounded to Vg1 HRE in EMSA. • Vg1 HRE plays a dual role in the induction and repression of Vg1. [ABSTRACT FROM AUTHOR]

Background: It is hypothesized that the highly conserved inverted repeats (IR) structure of land plant plastid genomes (plastomes) is beneficial for stabilizing plastome organization, whereas the mechanism of the occurrence and stability maintenance of the recently reported direct repeats (DR) structure is yet awaiting further exploration. Here we describe the DR structure of the Selaginella vardei (Selaginellaceae) plastome, to elucidate the mechanism of DR occurrence and stability maintenance. Results: The plastome of S. vardei is 121,254 bp in length and encodes 76 genes, of which 62 encode proteins, 10 encode tRNAs, and four encode rRNAs. Unexpectedly, the two identical rRNA gene regions (13,893 bp) are arranged in a direct orientation (DR), rather than inverted. Comparing to the IR organization in Isoetes flaccida (Isoetaceae, Lycopodiopsida) plastome, a ca. 50-kb trnN-trnF inversion that spans one DR copy was found in the plastome of S. vardei, which might cause the orientation change. In addition, we find extremely rare short dispersed repeats (SDRs) in the plastomes of S. vardei and its closely related species S. indica. Conclusions: We suggest that the ca. 50-kb inversion resulted in the DR structure, and the reduction in SDRs plays a key role in maintaining the stability of plastomes with DR structure by avoiding potential secondary recombination. We further confirmed the presence of homologous recombination between DR regions, which are able to generate subgenomes and form diverse multimers. Our study deepens the understanding of Selaginella plastomes and provides new insights into the diverse plastome structures in land plants. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus, a ubiquitous bacterium of the marine environment is an important food-borne pathogen responsible for gastroenteritis worldwide. In this study, we aimed to investigate the occurrence and diversity of the CRISPR-Cas system in V. parahaemolyticus genomes using a bioinformatics approach. The CRISPR-Cas system functions as an adaptive immune system in prokaryotes that provides immunity against foreign genetic elements. In total, 570 genomes V. parahaemolyticus genomes were analyzed of which 200 confirmed for the presence of CRISPR-Cas system. The CRISPR-Cas loci were further analyzed for their repeats, spacers and associated Cas proteins. Among the 200 V. parahaemolyticus strains analyzed, 16 (8%) strains possessed the CRISPR-Cas system of complete subtype I-F, while the remaining 184 (92%) harbored the minimalistic type, a subtype I-F variant. Orphan CRISPR repeats and Cas genes were found in one strain each. The CRISPR-associated direct repeat had an unit length of 28 bases. The number of repeat units in each array ranged from 3 to 5 or 5-41 depending on whether they belonged to the minimalistic or complete subtype-IF CRISPR-Cas system, respectively. Of the 768 spacers analyzed in this study, 295 were found to be unique to V. parahaemolyticus. Homology analysis of the conserved spacers revealed matches to plasmids, phages and gut viruses and self chromosomes. Among the CRISPR-associated proteins, Cas5 and Cas7 proteins were found to be conserved. However, variations were seen in the Cas6 protein, which could be grouped into four different types based on their protein length as well as amino acid composition. We present here the diversity and main features of the CRISPR-Cas system in V. parahaemolyticus, which could provide valuable insights in elucidating the role and mechanism of CRISPR/Cas elements in this pathogen. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a foodborne pathogen that causes food contamination and food poisoning, which poses great harm to health, agriculture and other hosts. Clustered regularly interspaced short palindromic repeats (CRISPR) are a recently discovered bacterial immune system that resists foreign genes such as phage DNA. This system inhibits the transfer of specific movable genetic elements that match the CRISPR spacer sequences, thereby preventing the spread of drug-resistant genes between pathogens. In this study, 57 CRISPR loci were screened from 38 strains of S. aureus based on the CRISPR database, and bioinformatics tools were used to investigate the structural features and potential functions of S. aureus CRISPR loci. The results showed that most strains contained only one CRISPR locus, a few strains contained multiple loci with sparsely distributed sites. These loci mainly included highly conserved direct repeat sequences and highly variable spacer sequences, as well as polymorphic cas genes. In addition, the analysis of secondary structure of direct repeat RNA showed that all sites can form stable RNA secondary structure. The results of constructing phylogenetic tree based on spacer sequence showed that some strains contained a high degree of phylogenetic relationship, while the differences among other strains in evolutionary processes were quite obvious. Of the 57 CRISPR loci identified, only the cas gene was found near the 4 CRISPR loci.

Staphylococcus aureus is a foodborne pathogen that causes food contamination and food poisoning, which poses great harm to health, agriculture and other hosts. Clustered regularly interspaced short palindromic repeats (CRISPR) are a recently discovered bacterial immune system that resists foreign genes such as phage DNA. This system inhibits the transfer of specific movable genetic elements that match the CRISPR spacer sequences, thereby preventing the spread of drug-resistant genes between pathogens. In this study, 57 CRISPR loci were screened from 38 strains of S. aureus based on the CRISPR database, and bioinformatics tools were used to investigate the structural features and potential functions of S. aureus CRISPR loci. The results showed that most strains contained only one CRISPR locus, a few strains contained multiple loci with sparsely distributed sites. These loci mainly included highly conserved direct repeat sequences and highly variable spacer sequences, as well as polymorphic cas genes. In addition, the analysis of secondary structure of direct repeat RNA showed that all sites can form stable RNA secondary structure. The results of constructing phylogenetic tree based on spacer sequence showed that some strains contained a high degree of phylogenetic relationship, while the differences among other strains in evolutionary processes were quite obvious. Of the 57 CRISPR loci identified, only the cas gene was found near the 4 CRISPR loci. [ABSTRACT FROM AUTHOR]

Quantitative PCR is a diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of human herpesvirus 6A/B (HHV-6A/B) is important for detection of viral reactivation and inherited chromosomally integrated HHV-6A/B in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies Inc. Eleven of 17 (65%) HHV-6A/B candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1,254 bp and 983 bp, respectively. The average copy number measured was between 5 and 10 times the number of copies of the rest of the genome. We also report the first interspecies recombinant HHV-6A/B strain with a HHV-6A backbone and a >5.5-kb region from HHV-6B, from U41 to U43, that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at U1/U2, U87, and U89, as well as deletion in the U12-to-U24 region and the U94/U95 genes. HHV-6A/B strains derived from cord blood mononuclear cells from different laboratories on different continents with fewer passages revealed no copy number differences throughout the viral genome. These data indicate that large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae. [ABSTRACT FROM AUTHOR]

The Clustered regularly interspaced short palindromic repeats (CRISPR) systems are a type of innate immunity found in some prokaryotes, which protect them against alien genetic elements by targeting foreign nucleic acids. Some other functions are also attributed to these systems. Clostridium botulinum bacteria produce botulinum neurotoxins (BoNT), one of the deadliest known toxins for humans and some animals. Food poisoning due to these bacteria is still a challenge in food industries. On the other hand, BoNT has been widely investigated for therapeutic applications including different muscle disorders. Bont genes may be located on bacterial chromosomes, plasmids, or even prophages. Generally, the genomes of Cl . botulinum show a high level of plasticity. In order to investigate the presence and characteristics of CRISPRs in these anaerobe bacteria, an in silico study on 113 CRISPR arrays identified in 38 Cl . botulinum strains was performed. A high occurrence of CRISPR arrays (80%) were found, with a remarkable frequency on plasmids. Several (CRISPR-associated) Cas proteins from different types were recognized in the studied strains, which were mostly Cas6. The CRISPR-Cas systems were identified as type I or III, but no type II. The spacers showed more homology with bacterial plasmids than phages. Active CRISPR-Cas systems can prevent the transfer of foreign genes, which may also include bont genes. This study provides the first insight into the probable roles of CRISPR-Cas systems in Cl . botulinum strains such as toxigenicity. [ABSTRACT FROM AUTHOR]