ABSTRACT
BACKGROUND: Epigenetic clocks using DNA methylation (DNAm) to estimate biological age have become popular tools in the study of neurodegenerative diseases. Notably, several recent reports have shown a strikingly similar inverse relationship between accelerated biological aging, as measured by DNAm, and the age of onset of several neurodegenerative disorders, including Parkinson's disease (PD). Common to all of these studies is that they were performed without control subjects and using the exact same measure of accelerated aging: DNAm age minus chronological age. OBJECTIVE: We aimed to assess the validity of these findings in PD, using the same dataset as in the original study, blood DNAm data from the Parkinson's Progression Markers Initiative cohort, but also including control samples in the analyses. METHODS: We replicated the analyses and findings of the previous study and then reanalyzed the dataset incorporating control samples to account for underlying age-related biases. RESULTS: Our reanalysis shows that there is no correlation between age of onset and DNAm age acceleration. Conversely, there is a pattern of overestimating DNAm age in younger and underestimating DNAm age in older individuals in the dataset that entirely explains the previously reported association. CONCLUSIONS: Our findings refute the previously reported inverse relationship between DNAm age acceleration and age of onset in PD. We show that these findings are fully accounted for by an expected over/underestimation of DNAm age in younger/older individuals. Furthermore, this effect is likely to be responsible for nearly identical findings reported in other neurodegenerative diseases. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Subject(s)
DNA Methylation , Parkinson Disease , Humans , Aged , Parkinson Disease/epidemiology , Parkinson Disease/genetics , Epigenesis, Genetic , Age of Onset , Aging/geneticsABSTRACT
Replenishing nicotinamide adenine dinucleotide (NAD) via supplementation of nicotinamide riboside (NR) has been shown to confer neuroprotective effects in models of aging and neurodegenerative diseases, including Parkinson's disease (PD). Although generally considered safe, concerns have been raised that NR supplementation could impact methylation dependent reactions, including DNA methylation, because of increased production and methylation dependent breakdown of nicotinamide (NAM). We investigated the effect of NR supplementation on DNA methylation in a double blinded, placebo-controlled trial of 29 human subjects with PD, in blood cells and muscle tissue. Our results show that NR had no impact on DNA methylation homeostasis, including individuals with common pathogenic mutations in the MTHFR gene known to affect one-carbon metabolism. Pathway and methylation variance analyses indicate that there might be minor regulatory responses to NR. We conclude that short-term therapy with high-dose NR for up to 30 days has no deleterious impact on methylation homeostasis.
ABSTRACT
Each year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non-self-antigens is an unavoidable adverse side effect of transfusion. We describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.
Subject(s)
Blood Donors , Blood Transfusion , Genotype , Humans , Isoantibodies , Prospective StudiesABSTRACT
Next-generation sequencing (NGS) is replacing other molecular techniques to become the de facto gene diagnostics approach, transforming the speed of diagnosis for patients and expanding opportunities for precision medicine. Consequently, for accredited laboratories as well as those seeking accreditation, both objective measures of quality and external review of laboratory processes are required. External quality assessment (EQA), or Proficiency Testing (PT), can assess a laboratory's service through an independent external agency, the EQA provider. The analysis of a growing number of genes and whole exome and genomes is now routine; therefore, an EQA must be delivered to enable all testing laboratories to participate. In this paper, we describe the development of a unique platform and gene target independent EQA scheme for NGS, designed to scale from current to future requirements of clinical diagnostic laboratories testing for germline and somatic variants. The EQA results from three annual rounds indicate that clinical diagnostic laboratories are providing an increasingly high-quality NGS service and variant calling abilities are improving. From an EQA provider perspective, challenges remain regarding delivery and performance criteria, as well as in analysing similar NGS approaches between cohorts with meaningful metrics, sample sourcing and data formats.
Subject(s)
Genetic Testing/standards , Germ-Line Mutation , High-Throughput Nucleotide Sequencing/standards , Neoplasms/genetics , Quality Assurance, Health Care/methods , Sequence Analysis, DNA/standards , Algorithms , Humans , Neoplasms/diagnosis , Reproducibility of ResultsABSTRACT
SUMMARY: Genome-wide association study (GWAS) analyses, at sufficient sample sizes and power, have successfully revealed biological insights for several complex traits. RICOPILI, an open-sourced Perl-based pipeline was developed to address the challenges of rapidly processing large-scale multi-cohort GWAS studies including quality control (QC), imputation and downstream analyses. The pipeline is computationally efficient with portability to a wide range of high-performance computing environments. RICOPILI was created as the Psychiatric Genomics Consortium pipeline for GWAS and adopted by other users. The pipeline features (i) technical and genomic QC in case-control and trio cohorts, (ii) genome-wide phasing and imputation, (iv) association analysis, (v) meta-analysis, (vi) polygenic risk scoring and (vii) replication analysis. Notably, a major differentiator from other GWAS pipelines, RICOPILI leverages on automated parallelization and cluster job management approaches for rapid production of imputed genome-wide data. A comprehensive meta-analysis of simulated GWAS data has been incorporated demonstrating each step of the pipeline. This includes all the associated visualization plots, to allow ease of data interpretation and manuscript preparation. Simulated GWAS datasets are also packaged with the pipeline for user training tutorials and developer work. AVAILABILITY AND IMPLEMENTATION: RICOPILI has a flexible architecture to allow for ongoing development and incorporation of newer available algorithms and is adaptable to various HPC environments (QSUB, BSUB, SLURM and others). Specific links for genomic resources are either directly provided in this paper or via tutorials and external links. The central location hosting scripts and tutorials is found at this URL: https://sites.google.com/a/broadinstitute.org/RICOPILI/home. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Genome-Wide Association Study , Software , Algorithms , Genome , GenomicsABSTRACT
BACKGROUND: Heterozygous mutations in the gene encoding renin (REN) cause autosomal dominant tubulointerstitial kidney disease (ADTKD), early-onset anaemia and hyperuricaemia; only four different mutations have been described in the published literature to date. We report a novel dominant REN mutation discovered in an individual after forty years of renal disease. CASE PRESENTATION: A 57 year old Caucasian woman with chronic kidney disease stage five was reviewed in a regional joint renal genetics clinic. She had initially been diagnosed with chronic pyelonephritis in adolescence, around the same time that she was investigated for anaemia out of keeping with her degree of renal impairment. Hyperuricaemia was identified in her twenties following an episode of gout. A diagnosis of ADTKD was not made until the age of 37 years, when her mother was also found to have kidney disease and commenced haemodialysis. The patient's renal function continued to slowly deteriorate and, twenty years later, her sister was worked up as a potential donor for kidney transplantation. Revisiting the maternal family history during the transplant work up prompted a referral to clinical genetics and urgent REN genetic testing was requested for the patient, leading to discovery of a heterozygous mutation in the REN gene: c.49 T > C, p.(Trp17Arg). This variant was not identified in her otherwise healthy sister, allowing pre-emptive live renal transplantation to take place shortly afterwards. CONCLUSIONS: In an era where genetic testing is becoming much more readily available, this case highlights the importance of considering a genetic aetiology in all patients with long-standing renal disease and a relevant family history. Establishing a genetic diagnosis of ADTKD-REN in this individual with chronic anaemia, hyperuricaemia and slowly progressive renal impairment helped to identify a suitable live kidney donor and allowed successful pre-emptive transplantation to take place.
Subject(s)
Mutation/genetics , Nephritis, Interstitial/diagnosis , Nephritis, Interstitial/genetics , Renin/genetics , Amino Acid Sequence , Female , Humans , Kidney Transplantation , Middle Aged , Nephritis, Interstitial/surgery , Pedigree , Time FactorsABSTRACT
Osteopathia striata with cranial sclerosis (OSCS; OMIM #300373) is a rare X-linked dominant condition caused by mutations in the AMER1 gene (also known as WTX or FAM123B). It is a condition which usually affects females in whom the clinical phenotype can be extremely variable. Conversely affected males typically die in utero or during the neonatal period [Perdu et al. (); Clinical Genetics 80: 383-388; Vasiljevic et al. (); Prenatal Diagnosis 35: 302-304]. There have been a small number of reported cases of surviving males, including three patients who are somatic mosaic for the condition [Chénier, Noor, Dupuis, Stavropoulos, & Mendoza-Londono, (); American Journal of Medical Genetics Part A 158A: 2946-2952; Holman et al. (); American Journal of Medical Genetics Part A 155A: 2397-2408; Joseph, Shoji, & Econs, (); The Journal of Clinical Endocrinology and Metabolism 95: 1506-1507]. We report a case of a male child who has proven somatic mosaicism for OSCS associated with a novel pathogenic frameshift mutation, c.607_611delAGGCC (p.Arg203 fs) in AMER1. We describe the multisystemic clinical features which include macrocephaly with ventriculomegaly and requirement for ventriculoperitoneal shunt, cleft palate, and respiratory difficulties after birth requiring tracheostomy insertion, persistent patent ductus arteriosus, failure to thrive and gastrostomy insertion, growth retardation, ophthalmoplegia, kidney malformation, cryptorchidism, and developmental delay. The use of new technologies with next generation sequencing (NGS) may improve the detection rate of mosaicism in rare conditions.
ABSTRACT
Charcot-Marie-Tooth disease (CMT) refers to a genetically heterogeneous group of disorders which cause a peripheral motor and sensory neuropathy. The overall prevalence is 1 in 2500 individuals. Mutations in the MFN2 gene are the commonest cause for the axonal (CMT2) type. We describe a Caucasian 5-year old girl affected by CMT2A since the age of 2 years. She presented with unsteady gait, in-turning of the feet and progressive foot deformities. Nerve conduction studies suggested an axonal neuropathy and molecular testing identified a previously reported pathogenic variant c.1090C > T, p.(Arg364Trp) in the MFN2 gene. This variant was also detected in a mosaic state in blood and saliva by Sanger sequencing in her subjectively healthy father. Next generation sequencing showed that the level of mosaicism was 21% in blood and 24% in saliva. A high recurrence risk was given because the father had proven somatic mosaicism and an affected child implying gonadal mosaicism. The parents were referred for pre-implantation genetic diagnosis. To the best of our knowledge, this is the first reported case of somatic mosaicism for MFN2. This study has important implications for genetic counselling in families with CMT2A.
Subject(s)
Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/pathology , GTP Phosphohydrolases/genetics , Mitochondrial Proteins/genetics , Mosaicism , Mutation, Missense , Adult , Child, Preschool , Father-Child Relations , Female , Humans , Male , Nuclear Family , Parents , Pedigree , Phenotype , Severity of Illness IndexABSTRACT
CONTEXT: Lower TSH screening cutoffs have doubled the ascertainment of congenital hypothyroidism (CH), particularly cases with a eutopically located gland-in-situ (GIS). Although mutations in known dyshormonogenesis genes or TSHR underlie some cases of CH with GIS, systematic screening of these eight genes has not previously been undertaken. OBJECTIVE: Our objective was to evaluate the contribution and molecular spectrum of mutations in eight known causative genes (TG, TPO, DUOX2, DUOXA2, SLC5A5, SLC26A4, IYD, and TSHR) in CH cases with GIS. Patients, Design, and Setting: We screened 49 CH cases with GIS from 34 ethnically diverse families, using next-generation sequencing. Pathogenicity of novel mutations was assessed in silico. PATIENTS, DESIGN, AND SETTING: We screened 49 CH cases with GIS from 34 ethnically diverse families, using next-generation sequencing. Pathogenicity of novel mutations was assessed in silico. RESULTS: Twenty-nine cases harbored likely disease-causing mutations. Monogenic defects (19 cases) most commonly involved TG (12), TPO (four), DUOX2 (two), and TSHR (one). Ten cases harbored triallelic (digenic) mutations: TG and TPO (one); SLC26A4 and TPO (three), and DUOX2 and TG (six cases). Novel variants overall included 15 TG, six TPO, and three DUOX2 mutations. Genetic basis was not ascertained in 20 patients, including 14 familial cases. CONCLUSIONS: The etiology of CH with GIS remains elusive, with only 59% attributable to mutations in TSHR or known dyshormonogenesis-associated genes in a cohort enriched for familial cases. Biallelic TG or TPO mutations most commonly underlie severe CH. Triallelic defects are frequent, mandating future segregation studies in larger kindreds to assess their contribution to variable phenotype. A high proportion (â¼41%) of unsolved or ambiguous cases suggests novel genetic etiologies that remain to be elucidated.
Subject(s)
Autoantigens/genetics , Congenital Hypothyroidism/genetics , Iodide Peroxidase/genetics , Iron-Binding Proteins/genetics , Receptors, Thyrotropin/genetics , Thyroglobulin/genetics , Humans , Mutation , Pedigree , PhenotypeABSTRACT
MOTIVATION: For most research approaches, genome analyses are dependent on the existence of a high quality genome reference assembly. However, the local accuracy of an assembly remains difficult to assess and improve. The gEVAL browser allows the user to interrogate an assembly in any region of the genome by comparing it to different datasets and evaluating the concordance. These analyses include: a wide variety of sequence alignments, comparative analyses of multiple genome assemblies, and consistency with optical and other physical maps. gEVAL highlights allelic variations, regions of low complexity, abnormal coverage, and potential sequence and assembly errors, and offers strategies for improvement. Although gEVAL focuses primarily on sequence integrity, it can also display arbitrary annotation including from Ensembl or TrackHub sources. We provide gEVAL web sites for many human, mouse, zebrafish and chicken assemblies to support the Genome Reference Consortium, and gEVAL is also downloadable to enable its use for any organism and assembly. AVAILABILITY AND IMPLEMENTATION: Web Browser: http://geval.sanger.ac.uk, Plugin: http://wchow.github.io/wtsi-geval-plugin CONTACT: kj2@sanger.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Genomics , Web Browser , Animals , Genome , Humans , Internet , Mice , Sequence AlignmentABSTRACT
We report a case of a female child who has classical Freeman-Sheldon syndrome (FSS) associated with a previously reported recurrent pathogenic heterozygous missense mutation, c.2015G > A, p. (Arg672His), in MYH3 where the phenotypically normal mother is a molecularly confirmed mosaic. To the best of our knowledge, this is the first report in the medical literature of molecularly confirmed parental mosaicism for a MYH3 mutation causing FSS. Since proven somatic mosaicism after having an affected child is consistent with gonadal mosaicism, a significantly increased recurrence risk is advised. Parental testing is thus essential for accurate risk assessment for future pregnancies and the use of new technologies with next generation sequencing (NGS) may improve the detection rate of mosaicism. © 2016 Wiley Periodicals, Inc.
Subject(s)
Craniofacial Dysostosis/diagnosis , Craniofacial Dysostosis/genetics , Cytoskeletal Proteins/genetics , Genetic Association Studies , Mosaicism , Mutation , Phenotype , Alleles , Amino Acid Substitution , Audiometry , Comparative Genomic Hybridization , DNA Mutational Analysis , Echocardiography , Female , Genotype , Humans , Infant, Newborn , Physical ExaminationSubject(s)
Epigenesis, Genetic , High-Throughput Nucleotide Sequencing/methods , T-Lymphocytes, Regulatory/metabolism , DNA Methylation/genetics , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Humans , Male , Polymerase Chain Reaction/methods , SulfitesABSTRACT
High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues.
Subject(s)
Formaldehyde/chemistry , Genetic Testing/methods , High-Throughput Nucleotide Sequencing/methods , Multiplex Polymerase Chain Reaction , Paraffin Embedding , Algorithms , Amino Acid Substitution , DNA Mutational Analysis/instrumentation , DNA Mutational Analysis/methods , False Positive Reactions , Genetic Testing/instrumentation , High-Throughput Nucleotide Sequencing/instrumentation , Humans , INDEL Mutation , Lymphoma/genetics , Lymphoma/pathology , Multiplex Polymerase Chain Reaction/instrumentation , Multiplex Polymerase Chain Reaction/methods , Paraffin Embedding/methods , Tissue Fixation/methodsABSTRACT
BACKGROUND: Familial juvenile hyperuricaemic nephropathy is a rare inherited nephropathy with genetic heterogeneity. Categorised by genetic defect, mutations in uromodulin (UMOD), renin (REN) and hepatocyte nuclear factor-1ß (HNF-1ß) genes as well as linkage to chromosome 2p22.1-21 have previously been identified. Knowledge of the genetics of this phenotype has provided important clues to developmental pathways in the kidney. CASE PRESENTATION: We report a novel phenotype, with the typical features of hyperuricemia and renal deterioration, but with the additional unexpected feature of unilateral renal hypoplasia. Mutation analyses of the existing known genes and genetic loci were negative indicating a new monogenic cause. Interestingly two cousins of the index case did not share the latter feature, suggesting a modifier gene effect. CONCLUSION: Unilateral renal hypo/aplasia is usually sporadic and relatively common, with no genetic cause to date identified. This reported pedigree reveals the possibility that a new, unknown renal developmental gene may be implicated in the FJHN phenotype.
Subject(s)
Genetic Predisposition to Disease/genetics , Gout/diagnosis , Gout/genetics , Hyperuricemia/diagnosis , Hyperuricemia/genetics , Kidney Diseases/diagnosis , Kidney Diseases/genetics , Kidney/abnormalities , Penetrance , Renal Insufficiency, Chronic/diagnosis , Renal Insufficiency, Chronic/genetics , Adolescent , Humans , Kidney/pathology , Male , PedigreeABSTRACT
Serratia sp. strain ATCC 39006 is a Gram-negative bacterium and a member of the Enterobacteriaceae that produces various bioactive secondary metabolites, including the tripyrrole red pigment prodigiosin and the ß-lactam antibiotic 1-carbapenen-2-em-3-carboxylic acid (a carbapenem). This strain is the only member of the Enterobacteriaceae known to naturally produce gas vesicles, as flotation organelles. Here we present the genome sequence of this strain, which has served as a model for analysis of the biosynthesis and regulation of antibiotic production.
ABSTRACT
The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse "payload" of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5' sequence in the crRNA, but not on the presence of a protospacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.
Subject(s)
Archaeal Proteins/chemistry , Inverted Repeat Sequences , RNA, Archaeal/chemistry , Sulfolobus solfataricus/metabolism , Archaeal Proteins/isolation & purification , Archaeal Viruses/immunology , Base Sequence , Crystallography, X-Ray , Macromolecular Substances/chemistry , Macromolecular Substances/isolation & purification , Microscopy, Electron , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/isolation & purification , RNA Cleavage , RNA, Archaeal/genetics , RNA, Archaeal/isolation & purification , Sulfolobus solfataricus/genetics , Sulfolobus solfataricus/immunology , Sulfolobus solfataricus/virologyABSTRACT
The genomes of two Sulfolobus islandicus strains obtained from Icelandic solfataras were sequenced and analyzed. Strain REY15A is a host for a versatile genetic toolbox. It exhibits a genome of minimal size, is stable genetically, and is easy to grow and manipulate. Strain HVE10/4 shows a broad host range for exceptional crenarchaeal viruses and conjugative plasmids and was selected for studying their life cycles and host interactions. The genomes of strains REY15A and HVE10/4 are 2.5 and 2.7 Mb, respectively, and each genome carries a variable region of 0.5 to 0.7 Mb where major differences in gene content and gene order occur. These include gene clusters involved in specific metabolic pathways, multiple copies of VapBC antitoxin-toxin gene pairs, and in strain HVE10/4, a 50-kb region rich in glycosyl transferase genes. The variable region also contains most of the insertion sequence (IS) elements and high proportions of the orphan orfB elements and SMN1 miniature inverted-repeat transposable elements (MITEs), as well as the clustered regular interspaced short palindromic repeat (CRISPR)-based immune systems, which are complex and diverse in both strains, consistent with them having been mobilized both intra- and intercellularly. In contrast, the remainder of the genomes are highly conserved in their protein and RNA gene syntenies, closely resembling those of other S. islandicus and Sulfolobus solfataricus strains, and they exhibit only minor remnants of a few genetic elements, mainly conjugative plasmids, which have integrated at a few tRNA genes lacking introns. This provides a possible rationale for the presence of the introns.
Subject(s)
Genome, Archaeal , Sulfolobus/genetics , Sulfolobus/metabolism , Attachment Sites, Microbiological , DNA Transposable Elements , Gene Expression Regulation, Archaeal/physiology , Genetic Variation , Iceland , Open Reading Frames , Phylogeny , RNA, Archaeal/genetics , RNA, Archaeal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolismABSTRACT
Spindle-shaped virus-like particles are abundant in extreme geothermal environments, from which five spindle-shaped viral species have been isolated to date. They infect members of the hyperthermophilic archaeal genus Sulfolobus, and constitute the Fuselloviridae, a family of double-stranded DNA viruses. Here we present four new members of this family, all from terrestrial acidic hot springs. Two of the new viruses exhibit a novel morphotype for their proposed attachment structures, and specific features of their genome sequences strongly suggest the identity of the host-attachment protein. All fuselloviral genomes are highly conserved at the nucleotide level, although the regions of conservation differ between virus-pairs, consistent with a high frequency of homologous recombination having occurred between them. We propose a fuselloviral specific mechanism for interviral recombination, and show that the spacers of the Sulfolobus CRISPR antiviral system are not biased to the highly similar regions of the fusellovirus genomes.
Subject(s)
Fuselloviridae/genetics , Fuselloviridae/ultrastructure , Hot Springs/virology , Recombination, Genetic , Virion/ultrastructure , Conserved Sequence , DNA, Viral/chemistry , DNA, Viral/genetics , Fuselloviridae/isolation & purification , Gene Order , Microscopy, Electron, Transmission , Molecular Sequence Data , Sequence Analysis, DNA , Sulfolobus/virology , Viral Structural Proteins/ultrastructureABSTRACT
Clusters of regularly interspaced short palindromic repeats (CRISPRs) of Sulfolobus fall into three main families based on their repeats, leader regions, associated cas genes and putative recognition sequences on viruses and plasmids. Spacer sequence matches to different viruses and plasmids of the Sulfolobales revealed some bias particularly for family III CRISPRs. Transcription occurs on both strands of the five repeat-clusters of Sulfolobus acidocaldarius and a repeat-cluster of the conjugative plasmid pKEF9. Leader strand transcripts cover whole repeat-clusters and are processed mainly from the 3'-end, within repeats, yielding heterogeneous 40-45 nt spacer RNAs. Processing of the pKEF9 leader transcript occurred partially in spacers, and was incomplete, probably reflecting defective repeat recognition by host enzymes. A similar level of transcripts was generated from complementary strands of each chromosomal repeat-cluster and they were processed to yield discrete approximately 55 nt spacer RNAs. Analysis of the partially identical repeat-clusters of Sulfolobus solfataricus strains P1 and P2 revealed that spacer-repeat units are added upstream only when a leader and certain cas genes are linked. Downstream ends of the repeat-clusters are conserved such that deletions and recombination events occur internally.
Subject(s)
Inverted Repeat Sequences , Multigene Family , Sulfolobus/genetics , Transcription, Genetic , 5' Untranslated Regions , Phylogeny , RNA, Archaeal/genetics , Sequence Alignment , Sequence Analysis, DNA , Transcription Initiation SiteABSTRACT
Four novel filamentous viruses with double-stranded DNA genomes, namely, Acidianus filamentous virus 3 (AFV3), AFV6, AFV7, and AFV8, have been characterized from the hyperthermophilic archaeal genus Acidianus, and they are assigned to the Betalipothrixvirus genus of the family Lipothrixviridae. The structures of the approximately 2-mum-long virions are similar, and one of them, AFV3, was studied in detail. It consists of a cylindrical envelope containing globular subunits arranged in a helical formation that is unique for any known double-stranded DNA virus. The envelope is 3.1 nm thick and encases an inner core with two parallel rows of protein subunits arranged like a zipper. Each end of the virion is tapered and carries three short filaments. Two major structural proteins were identified as being common to all betalipothrixviruses. The viral genomes were sequenced and analyzed, and they reveal a high level of conservation in both gene content and gene order over large regions, with this similarity extending partly to the earlier described betalipothrixvirus Sulfolobus islandicus filamentous virus. A few predicted gene products of each virus, in addition to the structural proteins, could be assigned specific functions, including a putative helicase involved in Holliday junction branch migration, a nuclease, a protein phosphatase, transcriptional regulators, and glycosyltransferases. The AFV7 genome appears to have undergone intergenomic recombination with a large section of an AFV2-like viral genome, apparently resulting in phenotypic changes, as revealed by the presence of AFV2-like termini in the AFV7 virions. Shared features of the genomes include (i) large inverted terminal repeats exhibiting conserved, regularly spaced direct repeats; (ii) a highly conserved operon encoding the two major structural proteins; (iii) multiple overlapping open reading frames, which may be indicative of gene recoding; (iv) putative 12-bp genetic elements; and (v) partial gene sequences corresponding closely to spacer sequences of chromosomal repeat clusters.