Short Direct Repeats Research Articles

Intra-Host Citrus Tristeza Virus Populations during Prolonged Infection Initiated by a Well-Defined Sequence Variant in Nicotiana benthamiana.

Due to the error-prone nature of viral RNA-dependent RNA polymerases, the replication of RNA viruses results in a diversity of viral genomes harboring point mutations, deletions, insertions, and genome rearrangements. Citrus tristeza virus (CTV), a causal agent of diseases of economically important citrus species, shows intrinsic genetic stability. While the virus appears to have some mechanism that limits the accumulation of single-nucleotide variants, the production of defective viral genomes (DVGs) during virus infection has been reported for certain variants of CTV. The intra-host diversity generated during plant infection with variant T36 (CTV-T36) remains unclear. To address this, we analyzed the RNA species accumulated in the initially infected and systemic leaves of Nicotiana benthamiana plants inoculated with an infectious cDNA clone of CTV-T36, which warranted that infection was initiated by a known, well-defined sequence variant of the virus. CTV-T36 limited the accumulation of single-nucleotide mutants during infection. With that, four types of DVGs-deletions, insertions, and copy- and snap-backs-were found in all the samples, with deletions and insertions being the most common types. Hot-spots across the genome for DVG recombination and short direct sequence repeats suggest that sequence complementarity could mediate DVG formation. In conclusion, our study illustrates the formation of diverse DVGs during CTV-T36 infection. To the best of our knowledge, this is the first study that has analyzed the genetic variability and recombination of a well-defined sequence variant of CTV in an herbaceous host.

Horizontally transferred mitochondrial DNA tracts become circular by microhomology-mediated repair pathways.

The holoparasitic plant Lophophytum mirabile exhibits remarkable levels of mitochondrial horizontal gene transfer (HGT). Gathering comparative data from other individuals and host plants can provide insights into the HGT process. We sequenced the mitochondrial genome (mtDNA) from individuals of two species of Lophophytum and from mimosoid hosts. We applied a stringent phylogenomic approach to elucidate the origin of the whole mtDNAs, estimate the timing of the transfers, and understand the molecular mechanisms involved. Ancestral and recent HGT events replaced and enlarged the multichromosomal mtDNA of Lophophytum spp., with the foreign DNA ascending to 74%. A total of 14 foreign mitochondrial chromosomes originated from continuous regions in the host mtDNA flanked by short direct repeats. These foreign tracts are circularized by microhomology-mediated repair pathways and replicate independently until they are lost or they eventually recombine with other chromosomes. The foreign noncoding chromosomes are variably present in the population and likely evolve by genetic drift. We present the 'circle-mediated HGT' model in which foreign mitochondrial DNA tracts become circular and are maintained as plasmid-like molecules. This model challenges the conventional belief that foreign DNA must be integrated into the recipient genome for successful HGT.

Structural Differences between the Genomes of Deinococcus radiodurans Strains from Different Laboratories.

The bacterium Deinococcus radiodurans is known to efficiently and accurately reassemble its genome after hundreds of DNA double-strand breaks (DSBs). Only at very large amounts of radiation-induced DSBs is this accuracy affected in the wild-type D. radiodurans, causing rearrangements in its genome structure. However, changes in its genome structure may also be possible during the propagation and storage of cell cultures. We investigate this possibility by listing structural differences between three completely sequenced genomes of D. radiodurans strains with a recent common ancestor-the type strain stored and sequenced in two different laboratories (of the ATCC 13939 lineage) and the first sequenced strain historically used as the reference (ATCC BAA-816). We detected a number of structural differences and found the most likely mechanisms behind them: (i) transposition/copy number change in mobile interspersed repeats-insertion sequences and small non-coding repeats, (ii) variable number of monomers within tandem repeats, (iii) deletions between long direct DNA repeats, and (iv) deletions between short (4-10 bp) direct DNA repeats. The most surprising finding was the deletions between short repeats because it indicates the utilization of a less accurate DSB repair mechanism in conditions in which a more accurate one should be both available and preferred. The detected structural differences, as well as SNPs and short indels, while being important footprints of deinococcal DNA metabolism and repair, are also a valuable resource for researchers using these D. radiodurans strains.

Structure and transcription of integrated HPV DNA in vulvar carcinomas

HPV infections are associated with a fraction of vulvar cancers. Through hybridization capture and DNA sequencing, HPV DNA was detected in five of thirteen vulvar cancers. HPV16 DNA was integrated into human DNA in three of the five. The insertions were in introns of human NCKAP1, C5orf67, and LRP1B. Integrations in NCKAP1 and C5orf67 were flanked by short direct repeats in the human DNA, consistent with HPV DNA insertions at sites of abortive, staggered, endonucleolytic incisions. The insertion in C5orf67 was present as a 36 kbp, human-HPV-hetero-catemeric DNA as either an extrachromosomal circle or a tandem repeat within the human genome. The human circularization/repeat junction was defined at single nucleotide resolution. The integrated viral DNA segments all retained an intact upstream regulatory region and the adjacent viral E6 and E7 oncogenes. RNA sequencing revealed that the only HPV genes consistently transcribed from the integrated viral DNAs were E7 and E6*I. The other two HPV DNA+ tumors had coinfections, but no evidence for integration. HPV-positive and HPV-negative vulvar cancers exhibited contrasting human, global gene expression patterns partially overlapping with previously observed differences between HPV-positive and HPV-negative cervical and oropharyngeal cancers. A substantial fraction of the differentially expressed genes involved immune system function. Thus, transcription and HPV DNA integration in vulvar cancers resemble those in other HPV-positive cancers. This study emphasizes the power of hybridization capture coupled with DNA and RNA sequencing to identify a broad spectrum of HPV types, determine human genome integration status of viral DNAs, and elucidate their structures.

Mitochondrial DNA editing in potato through mitoTALEN and mitoTALECD: molecular characterization and stability of editing events

BackgroundThe aim of this study was to evaluate and characterize the mutations induced by two TALE-based approaches, double-strand break (DSB) induction by the FokI nuclease (mitoTALEN) and targeted base editing by the DddA cytidine deaminase (mitoTALECD), to edit, for the first time, the mitochondrial genome of potato, a vegetatively propagated crop. The two methods were used to knock out the same mitochondrial target sequence (orf125).ResultsTargeted chondriome deletions of different sizes (236–1066 bp) were induced by mitoTALEN due to DSB repair through ectopic homologous recombination of short direct repeats (11–12 bp) present in the target region. Furthermore, in one case, the induced DSB and subsequent repair resulted in the amplification of an already present substoichiometric molecule showing a 4288 bp deletion spanning the target sequence. With the mitoTALECD approach, both nonsense and missense mutations could be induced by base substitution. The deletions and single nucleotide mutations were either homoplasmic or heteroplasmic. The former were stably inherited in vegetative offspring.ConclusionsBoth editing approaches allowed us to obtain plants with precisely modified mitochondrial genomes at high frequency. The use of the same plant genotype and mtDNA region allowed us to compare the two methods for efficiency, accuracy, type of modifications induced and stability after vegetative propagation.

The minor chicken class I gene BF1 is deleted between short imperfect direct repeats in the B14 and typical B15 major histocompatibility complex (MHC) haplotypes

The chicken major histocompatibility complex (MHC, also known as the BF-BL region of the B locus) is notably small and simple with few genes, most of which are involved in antigen processing and presentation. There are two classical class I genes, of which only BF2 is well and systemically expressed as the major ligand for cytotoxic T lymphocytes (CTLs). The other class I gene, BF1, is believed to be primarily a natural killer (NK) cell ligand. Among most standard chicken MHC haplotypes examined in detail, BF1 is expressed tenfold less than BF2 at the RNA level due to defects in the promoter or in a splice site. However, in the B14 and typical B15 haplotypes, BF1 RNA was not detected, and here, we show that a deletion between imperfect 32 nucleotide direct repeats has removed the BF1 gene entirely. The phenotypic effects of not having a BF1 gene (particularly on resistance to infectious pathogens) have not been systematically explored, but such deletions between short direct repeats are also found in some BF1 promoters and in the 5′ untranslated region (5′UTR) of some BG genes found in the BG region of the B locus. Despite the opposite transcriptional orientation of homologous genes in the chicken MHC, which might prevent the loss of key genes from a minimal essential MHC, it appears that small direct repeats can still lead to deletion.

Ribosomal intergenic spacers are filled with transposon remnants.

Eukaryotic ribosomal DNA (rDNA) comprises tandem units of highly-conserved coding genes separated by rapidly-evolving spacer DNA. The spacers of all 12 species examined were filled with short direct repeats (DRs) and multiple long tandem repeats (TRs), completing the rDNA maps that previously contained unannotated and inadequately studied sequences. The external transcribed spacers also were filled with DRs and some contained TRs. We infer that the spacers arose from transposon insertion, followed by their imprecise excision, leaving short DRs characteristic of transposon visitation. The spacers provided a favored location for transposon insertion because they occupy loci containing hundreds to thousands of gene repeats. The spacers' primary cellular function may be to link one rRNA transcription unit to the next, whereas transposons flourish here because they have colonized the most frequently-used part of the genome.

Replitrons: A major group of eukaryotic transposons encoding HUH endonuclease

HUH endonucleases of the Rep (replication protein) class mediate the replication of highly diverse plasmids and viral genomes across all domains of life. HUH transposases have independently evolved from Reps, giving rise to three major transposable element groups: the prokaryotic insertion sequences IS200/IS605 and IS91/ISCR, and the eukaryotic Helitrons. Here, I present Replitrons, a second group of eukaryotic transposons encoding Rep HUH endonuclease. Replitron transposases feature a Rep domain with one catalytic Tyr (Y1) and an adjacent domain that may function in oligomerization, contrasting with Helitron transposases that feature Rep with two Tyr (Y2) and a fused helicase domain (i.e., RepHel). Protein clustering found no link between Replitron transposases and described HUH transposases, and instead recovered a weak association with Reps of circular Rep-encoding single-stranded (CRESS) DNA viruses and their related plasmids (pCRESS). The predicted tertiary structure of the transposase of Replitron-1, the founding member of the group that is active in the green alga Chlamydomonas reinhardtii, closely resembles that of CRESS-DNA viruses and other HUH endonucleases. Replitrons are present in at least three eukaryotic supergroups and reach high copy numbers in nonseed plant genomes. Replitron DNA sequences feature short direct repeats at, or potentially near, their termini. Finally, I characterize copy-and-paste de novo insertions of Replitron-1 using long-read sequencing of C. reinhardtii experimental lines. These results support an ancient and evolutionarily independent origin of Replitrons, in line with other major groups of eukaryotic transposons. This work expands the known diversity of both transposons and HUH endonucleases in eukaryotes.

Reovirus RNA recombination is sequence directed and generates internally deleted defective genome segments during passage.

For viruses with segmented genomes, genetic diversity is generated by genetic drift, reassortment, and recombination. Recombination produces RNA populations distinct from full-length gene segments and can influence viral population dynamics, persistence, and host immune responses. Viruses in the Reoviridae family, including rotavirus and mammalian orthoreovirus (reovirus), have been reported to package segments containing rearrangements or internal deletions. Rotaviruses with RNA segments containing rearrangements have been isolated from immunocompromised and immunocompetent children and in vitro following serial passage at relatively high multiplicity. Reoviruses that package small, defective RNA segments have established chronic infections in cells and in mice. However, the mechanism and extent of Reoviridae RNA recombination are undefined. Towards filling this gap in knowledge, we determined the titers and RNA segment profiles for reovirus and rotavirus following serial passage in cultured cells. The viruses exhibited occasional titer reductions characteristic of interference. Reovirus strains frequently accumulated segments that retained 5' and 3' terminal sequences and featured large internal deletions, while similarly fragmented segments were rarely detected in rotavirus populations. Using next-generation RNA-sequencing to analyze RNA molecules packaged in purified reovirus particles, we identified distinct recombination sites within individual viral genome segments. Recombination junctions were frequently but not always characterized by short direct sequence repeats upstream and downstream that spanned junction sites. Taken together, these findings suggest that reovirus accumulates defective gene segments featuring internal deletions during passage and undergoes sequence-directed recombination at distinct sites.IMPORTANCE Viruses in the Reoviridae family include important pathogens of humans and other animals and have segmented RNA genomes. Recombination in RNA virus populations can facilitate novel host exploration and increased disease severity. The extent, patterns, and mechanisms of Reoviridae recombination and the functions and effects of recombined RNA products are poorly understood. Here, we provide evidence that mammalian orthoreovirus regularly synthesizes RNA recombination products that retain terminal sequences but contain internal deletions, while rotavirus rarely synthesizes such products. Recombination occurs more frequently at specific sites in the mammalian orthoreovirus genome, and short regions of identical sequence are often detected at junction sites. These findings suggest that mammalian orthoreovirus recombination events are directed in part by RNA sequences. An improved understanding of recombined viral RNA synthesis may enhance our capacity to engineer improved vaccines and virotherapies in the future.

Assembly of the durian chloroplast genome using long PacBio reads

We have assembled the complete sequence of the Durio zibethinus chloroplast genome using long PacBio reads. Durian is a valuable commercial tree that produces durian fruit, which is popular in Southeast Asia. The chloroplast genome assembled into a single 143 kb cyclic contig that contained 111 genes. There were 46 short direct repeats (45 to 586 bp) and five short inverted repeats (63 to 169 bp). The long reads that were used for the assembly span the entire chloroplast with > 10 kb overlaps and multiple long reads join the start of the contig to the end of the contig. The durian chloroplast was found to lack the large inverted repeat that is common in chloroplast genomes. An additional 24 durian varieties were sequenced and compared to the assembly and found to also lack the large inverted repeat. There were nine SNPs among the varieties.

Short Direct Repeats in the 3' Untranslated Region Are Involved in Subgenomic Flaviviral RNA Production.

Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation.IMPORTANCE Flaviviruses include many important human pathogens. The production of subgenomic flaviviral RNAs (sfRNAs) is important for viral pathogenicity as a common feature of flaviviruses. sfRNAs are formed through the incomplete degradation of viral genomic RNA by the cytoplasmic 5'-3' exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3'-UTR. The 3'-UTRs of the flavivirus genome also contain distinct short direct repeats (DRs), such as RCS3, CS3, RCS2, and CS2. However, the biological functions of these ancient primary DR sequences remain largely unknown. Here, we found that DR sequences are involved in sfRNA formation and viral virulence and provide novel targets for the rational design of live attenuated flavivirus vaccine.

Omp2b Porin Alteration in the Course of Evolution of Brucella spp.

The genus Brucella comprises major pathogenic species causing disease in livestock and humans, e.g. B. melitensis. In the past few years, the genus has been significantly expanded by the discovery of phylogenetically more distant lineages comprising strains from diverse wildlife animal species, including amphibians and fish. The strains represent several potential new species, with B. inopinata as solely named representative. Being genetically more distant between each other, relative to the “classical” Brucella species, they present distinct atypical phenotypes and surface antigens. Among surface protein antigens, the Omp2a and Omp2b porins display the highest diversity in the classical Brucella species. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. They share between 85 and 100% sequence identity depending on the Brucella species, biovar, or genotype. Only the omp2b gene copy has been shown to be expressed and genetic variation is extensively generated by gene conversion between the two copies. In this study, we analyzed the omp2 loci of the non-classical Brucella spp. Starting from two distinct ancestral genes, represented by Australian rodent strains and B. inopinata, a stepwise nucleotide reduction was observed in the omp2b gene copy. It consisted of a first reduction affecting the region encoding the surface L5 loop of the porin, previously shown to be critical in sugar permeability, followed by a nucleotide reduction in the surface L8 loop-encoding region. It resulted in a final omp2b gene size shared between two distinct clades of non-classical Brucella spp. (African bullfrog isolates) and the group of classical Brucella species. Further evolution led to complete homogenization of both omp2 gene copies in some Brucella species such as B. vulpis or B. papionis. The stepwise omp2b deletions seemed to be generated through recombination with the respective omp2a gene copy, presenting a conserved size among Brucella spp., and may involve short direct DNA repeats. Successive Omp2b porin alteration correlated with increasing porin permeability in the course of evolution of Brucella spp. They possibly have adapted their porin to survive environmental conditions encountered and to reach their final status as intracellular pathogen.

Detection of CRISPR cassettes and cas genes in the Arabidopsis thaliana genome

The state of the art in the evolution of plant viruses allows the genetic foundations of antiviral immunity in higher (including the most important crops) plants to be categorized as one of the most pressing issues of genetics and selection. According to the endosymbiotic theory, mitochondria descended from alphaproteobacteria that had been absorbed but not degraded by the host cell. The discovery of CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins), which implement the adaptive immunity function in prokaryotes, raises the question whether such a mechanism of antiviral protection could be caught up by evolution and used by representatives of eukaryotes (in particular, plants). The purpose of this work was to analyze the complete sequences of nuclear, mitochondrial, and chloroplast genomes of Arabidopsis thaliana in order to search for genetic elements similar to those in CRISPR-Cas systems of bacteria and archaea. As a result, in silico methods helped us to detect a locus of regularly intermittent short direct repeats in the mitochondrial genome of A. thaliana ecotypes. The structure of this locus corresponds to the CRISPR locus of the prokaryotic adaptive antiviral immune system. The probable connection between the locus found in the mitochondrial genome of the higher plant and the function of adaptive immunity is indicated by a similarity between the spacer sequences in the CRISPR cassette found and the genome of Cauliflower mosaic virus affecting Arabidopsis plants. Sequences of repeats and spacers of CRISPR cassettes in Arabidopsis C24 and Ler lines are perfectly identical. However, the locations of the CRISPR locus in the mitochondrial genomes of these lines differ significantly. The CRISPR cassette in the Col-0 line was found to be completely broken as a result of four deletions and one insertion. Although cas genes were not detected in the mitochondrial genome of the studied Arabidopsis ecotypes, their presence was detected in the nuclear genome. Both cas genes and numerous CRISPR cassettes were found on all the five chromosomes in the nuclear genome of the Col-0 ecotype. The results suggest the existence of a system of adaptive immunity in plants, which is similar to the CRISPR immunity of bacteria and archaea.

Fine tuning the level of the Cdc13 telomere-capping protein for maximal chromosome stability performance.

Chromosome stability relies on an adequate length and complete replication of telomeres, the physical ends of chromosomes. Telomeres are composed of short direct repeat DNA and the associated nucleoprotein complex is essential for providing end-stability. In addition, the so-called end-replication problem of the conventional replication requires that telomeres be elongated by a special mechanism which, in virtually all organisms, is based by a reverse transcriptase, called telomerase. Although, at the conceptual level, telomere functions are highly similar in most organisms, the telomeric nucleoprotein composition appears to diverge significantly, in particular if it is compared between mammalian and budding yeast cells. However, over the last years, the CST complex has emerged as a central hub for telomere replication in most systems. Composed of three proteins, it is related to the highly conserved replication protein A complex, and in all systems studied, it coordinates telomerase-based telomere elongation with lagging-strand DNA synthesis. In budding yeast, the Cdc13 protein of this complex also is essential for telomerase recruitment and this specialisation is accompanied by additional regulatory adaptations. Based on recent results obtained in yeast, here, we review these issues and present an updated telomere replication hypothesis. We speculate that the similarities between systems far outweigh the differences, once we detach ourselves from the historic descriptions of the mechanisms in the various organisms.

GC content elevates mutation and recombination rates in the yeast Saccharomyces cerevisiae.

The chromosomes of many eukaryotes have regions of high GC content interspersed with regions of low GC content. In the yeast Saccharomyces cerevisiae, high-GC regions are often associated with high levels of meiotic recombination. In this study, we constructed URA3 genes that differ substantially in their base composition [URA3-AT (31% GC), URA3-WT (43% GC), and URA3-GC (63% GC)] but encode proteins with the same amino acid sequence. The strain with URA3-GC had an approximately sevenfold elevated rate of ura3 mutations compared with the strains with URA3-WT or URA3-AT About half of these mutations were single-base substitutions and were dependent on the error-prone DNA polymerase ζ. About 30% were deletions or duplications between short (5-10 base) direct repeats resulting from DNA polymerase slippage. The URA3-GC gene also had elevated rates of meiotic and mitotic recombination relative to the URA3-AT or URA3-WT genes. Thus, base composition has a substantial effect on the basic parameters of genome stability and evolution.

Identification of novel MITEs (miniature inverted-repeat transposable elements) in Coxiella burnetii: implications for protein and small RNA evolution

BackgroundCoxiella burnetii is a Gram-negative gammaproteobacterium and zoonotic agent of Q fever. C. burnetii’s genome contains an abundance of pseudogenes and numerous selfish genetic elements. MITEs (miniature inverted-repeat transposable elements) are non-autonomous transposons that occur in all domains of life and are thought to be insertion sequences (ISs) that have lost their transposase function. Like most transposable elements (TEs), MITEs are thought to play an active role in evolution by altering gene function and expression through insertion and deletion activities. However, information regarding bacterial MITEs is limited.ResultsWe describe two MITE families discovered during research on small non-coding RNAs (sRNAs) of C. burnetii. Two sRNAs, Cbsr3 and Cbsr13, were found to originate from a novel MITE family, termed QMITE1. Another sRNA, CbsR16, was found to originate from a separate and novel MITE family, termed QMITE2. Members of each family occur ~ 50 times within the strains evaluated. QMITE1 is a typical MITE of 300-400 bp with short (2-3 nt) direct repeats (DRs) of variable sequence and is often found overlapping annotated open reading frames (ORFs). Additionally, QMITE1 elements possess sigma-70 promoters and are transcriptionally active at several loci, potentially influencing expression of nearby genes. QMITE2 is smaller (150-190 bps), but has longer (7-11 nt) DRs of variable sequences and is mainly found in the 3′ untranslated region of annotated ORFs and intergenic regions. QMITE2 contains a GTAG repetitive extragenic palindrome (REP) that serves as a target for IS1111 TE insertion. Both QMITE1 and QMITE2 display inter-strain linkage and sequence conservation, suggesting that they are adaptive and existed before divergence of C. burnetii strains.ConclusionsWe have discovered two novel MITE families of C. burnetii. Our finding that MITEs serve as a source for sRNAs is novel. QMITE2 has a unique structure and occurs in large or small versions with unique DRs that display linkage and sequence conservation between strains, allowing for tracking of genomic rearrangements. QMITE1 and QMITE2 copies are hypothesized to influence expression of neighboring genes involved in DNA repair and virulence through transcriptional interference and ribonuclease processing.

Whole Genome Sequence Analysis of Mutations Accumulated in rad27Δ Yeast Strains with Defects in the Processing of Okazaki Fragments Indicates Template-Switching Events.

Okazaki fragments that are formed during lagging strand DNA synthesis include an initiating primer consisting of both RNA and DNA. The RNA fragment must be removed before the fragments are joined. In Saccharomyces cerevisiae, a key player in this process is the structure-specific flap endonuclease, Rad27p (human homolog FEN1). To obtain a genomic view of the mutational consequence of loss of RAD27, a S. cerevisiae rad27Δ strain was subcultured for 25 generations and sequenced using Illumina paired-end sequencing. Out of the 455 changes observed in 10 colonies isolated the two most common types of events were insertions or deletions (INDELs) in simple sequence repeats (SSRs) and INDELs mediated by short direct repeats. Surprisingly, we also detected a previously neglected class of 21 template-switching events. These events were presumably generated by quasi-palindrome to palindrome correction, as well as palindrome elongation. The formation of these events is best explained by folding back of the stalled nascent strand and resumption of DNA synthesis using the same nascent strand as a template. Evidence of quasi-palindrome to palindrome correction that could be generated by template switching appears also in yeast genome evolution. Out of the 455 events, 55 events appeared in multiple isolates; further analysis indicates that these loci are mutational hotspots. Since Rad27 acts on the lagging strand when the leading strand should not contain any gaps, we propose a mechanism favoring intramolecular strand switching over an intermolecular mechanism. We note that our results open new ways of understanding template switching that occurs during genome instability and evolution.

Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12.

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, also in the E. coli K12 context. However, it did not encode a site-specific recombinase as mobile genomic islands usually do. It was then deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E.coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

Short Interspersed Nuclear Element (SINE) Sequences in the Genome of the Human Pathogenic Fungus Aspergillus fumigatus Af293.

Novel families of short interspersed nuclear element (SINE) sequences in the human pathogenic fungus Aspergillus fumigatus, clinical isolate Af293, were identified and categorised into tRNA-related and 5S rRNA-related SINEs. Eight predicted tRNA-related SINE families originating from different tRNAs, and nominated as AfuSINE2 sequences, contained target site duplications of short direct repeat sequences (4–14 bp) flanking the elements, an extended tRNA-unrelated region and typical features of RNA polymerase III promoter sequences. The elements ranged in size from 140–493 bp and were present in low copy number in the genome and five out of eight were actively transcribed. One putative tRNAArg-derived sequence, AfuSINE2-1a possessed a unique feature of repeated trinucleotide ACT residues at its 3’-terminus. This element was similar in sequence to the I-4_AO element found in A. oryzae and an I-1_AF long nuclear interspersed element-like sequence identified in A. fumigatus Af293. Families of 5S rRNA-related SINE sequences, nominated as AfuSINE3, were also identified and their 5'-5S rRNA-related regions show 50–65% and 60–75% similarity to respectively A. fumigatus 5S rRNAs and SINE3-1_AO found in A. oryzae. A. fumigatus Af293 contains five copies of AfuSINE3 sequences ranging in size from 259–343 bp and two out of five AfuSINE3 sequences were actively transcribed. Investigations on AfuSINE distribution in the fungal genome revealed that the elements are enriched in pericentromeric and subtelomeric regions and inserted within gene-rich regions. We also demonstrated that some, but not all, AfuSINE sequences are targeted by host RNA silencing mechanisms. Finally, we demonstrated that infection of the fungus with mycoviruses had no apparent effects on SINE activity.