Academic literature on the topic 'Slipped strand mispairing'

Flagella-mediated motility is recognized to be one of the major factors contributing to virulence in Campylobacter jejuni. Motility of this bacterium is known to be phase variable, although the mechanism of such variation remains unknown. C. jejuni genome sequencing revealed a number of genes prone to phase variation via a slipped-strand mispairing mechanism. Many of these genes are hypothetical and are clustered in the regions involved in formation of three major cell surface structures: capsular polysaccharide, lipooligosaccharide and flagella. Among the genes of unknown function, the flagellar biosynthesis and modification region contains seven hypothetical paralogous genes designated as the motility accessory factor (maf) family. Remarkably, two of these genes (maf1 and maf4) were found to be identical and both contain homopolymeric G tracts. Using insertional mutagenesis it was demonstrated that one of the genes, maf5, is involved in formation of flagella. Phase variation of the maf1 gene via slipped-strand mispairing partially restored motility of the maf5 mutant. The maffamily represents a new class of bacterial genes related to flagellar biosynthesis and phase variation. Reversible expression of flagella may be advantageous for the adaptation of C. jejunito the varied in vivo and ex vivo environments encountered during its life cycle, as well in evasion of the host immune response.

ABSTRACT Slipped-strand mispairing (SSM) has not been identified as a mechanism of phase variation in Escherichia coli. Using a reporter gene, we show that sequences that cause phase variation by SSM in Haemophilus influenzae also lead to phase variation when introduced onto the chromosome of E. coli, and the frequencies of switching are in the biologically relevant range. Thus, the absence of SSM-mediated phase variation in E. coli does not appear to be due to a mechanistic constraint.

Abstract The standard slipped-strand mispairing (SSM) model for the formation of variable number tandem repeats (VNTRs) proposes that a few tandem repeats, produced by chance mutations, provide the “raw material” for VNTR expansion. However, this model is unlikely to explain the formation of VNTRs with long motifs (e.g., minisatellites), because the likelihood of a tandem repeat forming by chance decreases rapidly as the length of the repeat motif increases. Phylogenetic reconstruction of the birth of a mitochondrial (mt) DNA minisatellite in guppies suggests that VNTRs with long motifs can form as a consequence of SSM at noncontiguous repeats. VNTRs formed in this manner have motifs longer than the noncontiguous repeat originally formed by chance and are flanked by one unit of the original, noncontiguous repeat. SSM at noncontiguous repeats can therefore explain the birth of VNTRs with long motifs and the “imperfect” or “short direct” repeats frequently observed adjacent to both mtDNA and nuclear VNTRs.

NontypeableHaemophilus influenzae(NTHi) exclusively colonize and infect humans and are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). In vitro and animal models do not accurately capture the complex environments encountered by NTHi during human infection. We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD. Genome sequences were used to elucidate the phylogeny of NTHi isolates, identify genomic changes that occur with persistence in the human airways, and evaluate the effect of selective pressure on 12 candidate vaccine antigens. Strains persisted in individuals with COPD for as long as 1,422 d. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, regulates expression of critical virulence functions, including adherence, nutrient uptake, and modification of surface molecules, and is a major mechanism for survival in the hostile environment of the human airways. A subset of strains underwent a large 400-kb inversion during persistence. NTHi does not undergo significant gene gain or loss during persistence, in contrast to other persistent respiratory tract pathogens. Amino acid sequence changes occurred in 8 of 12 candidate vaccine antigens during persistence, an observation with important implications for vaccine development. These results indicate that NTHi alters its genome during persistence by regulation of critical virulence functions primarily by slipped-strand mispairing, advancing our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract.

Objective Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events. Design and methods Observational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP-region (8.3 Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to most recent common ancestor (tMRCA) of the derived allele; forward population simulations to estimate current number of allele carriers; proposal of mutation mechanism; protein structure predictions; co-immunoprecipitation and cycloheximide chase experiments. Results Nine European-origin, unrelated c.805_825dup-positive pedigrees (four familial, five sporadic from the UK, USA and France) included 16 affected (nine gigantism/four acromegaly/two non-functioning pituitary adenoma patients and one prospectively diagnosed acromegaly patient) and nine unaffected carriers. All pedigrees shared a 2.79 Mbp haploblock around AIP with additional haploblocks privately shared between subsets of the pedigrees, indicating the existence of an evolutionarily recent common ancestor, the ‘English founder’, with an estimated median tMRCA of 47 generations (corresponding to 1175 years) with a confidence interval (9–113 generations, equivalent to 225–2825 years). The mutation occurred in a small tandem repeat region predisposed to slipped strand mispairing. The resulting seven amino-acid duplication disrupts interaction with HSP90 and leads to a marked reduction in protein stability. Conclusions The c.805_825dup allele, originating from a common ancestor, associates with a severe clinical phenotype and a high frequency of gigantism. The mutation is likely to be the result of slipped strand mispairing and affects protein–protein interactions and AIP protein stability.

Abstract Spontaneous deletion mutations often occur at short direct repeats that flank inverted repeat sequences. Inverted repeats may initiate genetic rearrangements by formation of hairpin secondary structures that block DNA polymerases or are processed by structure-specific endonucleases. We have investigated the ability of inverted repeat sequences to stimulate deletion of flanking direct repeats in Escherichia coli. Propensity for cruciform extrusion in duplex DNA correlated with stimulation of flanking deletion, which was partially sbcD dependent. We propose two mechanisms for palindrome-stimulated deletion, SbcCD dependent and SbcCD independent. The SbcCD-dependent mechanism is initiated by SbcCD cleavage of cruciforms in duplex DNA followed by RecA-independent single-strand annealing at the flanking direct repeats, generating a deletion. Analysis of deletion endpoints is consistent with this model. We propose that the SbcCD-independent pathway involves replication slipped mispairing, evoked from stalling at hairpin structures formed on the single-stranded lagging-strand template. The skew of SbcCD-independent deletion endpoints with respect to the direction of replication supports this hypothesis. Surprisingly, even in the absence of palindromes, SbcD affected the location of deletion endpoints, suggesting that SbcCD-mediated strand processing may also accompany deletion unassociated with secondary structures.

We have identified two individuals from Glasgow in Scotland who have a deletion of one of two copies of the cytochrome oxidise II (COII) intergenic 9–bp sequence motif CCCCCTCTA, located between the COII and tRNA Lys genes of the human mitochondrial genome. Although this polymorphism is common in Africa and Asia, it has not been reported in Northern Europe. Analysis of the mitochondrial DNA control region sequences of these two individuals suggests that they belong to a lineage that originated independently of the previously characterized African and Asian 9–bp deleted lineages. Among the Scottish population we have also identified a maternal lineage of three generations exhibiting heteroplasmy for two, three and four copies of the CCCCCTCTA motif. Polymerase chain reaction amplification across the COII – tRNA Lys intergenic region of these individuals gives different ratios of the three product lengths that are dependent on the concentration of the DNA–binding dye crystal violet. To investigate whether changes in repeat number were generated de novo , we constructed clones containing known numbers of the CCCCCTCTA motif. In the presence of high concentrations of crystal violet we obtained two, three and four copies of this motif when the amplification template contained only four copies. Various DNA–binding drugs are known to stabilize bulged structures in DNA and contribute to the process of slipped–strand–mispairing during DNA replication. These results suggest that the COII – tRNA Lys intergenic region is unstable owing to slipped–strand mispairing. Although sequences containing four copies of the CCCCCTCTA motif are less stable in vitro , we observed an increase in the proportion of mitochondrial genomes with four repeats between a mother and a daughter in the heteroplasmic lineage. From this we conclude that drift in the germ–line lineage is a main factor in the maintenance or loss of heteroplasmy.

ABSTRACTA nonhemolyticListeria monocytogenesstrain isolated from a fish processing plant was avirulent in a plaque-forming assay and in a subcutaneous mouse virulence assay. However, it showed 60% lethality (9/15 mice) when 109CFU were intraperitoneally injected into mice. HemolyticL. monocytogenesbacteria were recovered from liver and spleen of the deceased mice, and the pulsed-field gel electrophoresis patterns were indistinguishable for the nonhemolytic and the hemolytic isolates. Sequencing ofprfAfrom the nonhemolytic strain revealed a duplication of 7 bp in the helix-turn-helix region, resulting in a truncated PrfA protein. We propose that the direct repeat of 7 bp causes a reversible inactivation ofprfAand that slipped-strand mispairing regulates the phase variation in hemolytic activity and virulence. NonhemolyticL. monocytogenesstrains with identical duplications inprfAwere isolated from several sources in France, as well as in Norway, suggesting that the reversible inactivation described in this study is not an isolated event.

Forensic genotyping uses a multiplex short tandem repeat (STR) assay to amplify deoxyribonucleic acid (DNA) samples. One of the artifacts mostly commonly encountered in forensic DNA analysis is stutter, which are non-specific products from the polymerase chain reaction (PCR) that are typically one repeat unit shorter in length than the allelic amplicon. While stutter peaks are typically no taller than 10% of the parent peak on electropherograms, their peak heights can fall into similar ranges as minor contributor alleles in mixtures, creating a problem of how to distinguish artifacts from true allele peaks in these situations. One way to potentially address this issue is to find a PCR method that produces a much lower amount of stutter than the method currently used, which involves amplifying samples with commercial PCR kits designed for forensic applications. These kits all use some form of Taq DNA polymerase (derived from Thermus aquaticus). In an effort to examine whether the type of enzyme used in an assay affects the resulting stutter rates observed, the existing GlobalFiler™ PCR Amplification Kit (Applied Biosystems) protocol for forensic multiplex STR assays was modified to test different types of enzymes. This was done by amplifying the same DNA sample with GlobalFiler primers and different commercial proofreading enzymes and their accompanying reaction buffer using manufacturer-recommended PCR parameters. The DNA sample originated from a buccal swab that was extracted on the EZ1® Advanced (Qiagen). The DNA solution was quantified using the Quantifiler™ Duo DNA Quantification Kit (Applied Biosystems) on the 7500 Real-Time PCR System (Applied Biosystems). In order to first establish the validity of switching out enzymes in an established protocol, a DNA sample was amplified with the Type-it® Microsatellite Kit (Qiagen), another Taq-based kit that is also marketed for use in multiplex STR assays. After a complete profile was successfully generated, research proceeded with testing various high-fidelity DNA polymerases. Some of the enzymes tested were known to be Pyrococcus-like while others were fused to a DNA-binding domain to enhance processivity. Taq polymerases tend to produce products with 3’adenine-overhangs while proofreading enzymes produce blunt-ends. This change caused a one base pair difference in the resulting amplicon lengths, which was accommodated by manually assigning genotypes after results from fragment analysis by capillary electrophoresis using a 3130 Genetic Analyzer (Applied Biosystems) were interpreted by the GeneMapper™ software (Applied Biosystems). Additional amplification kits tested were: the UCP HiFidelity PCR Kit (Qiagen), Phusion™ Hot Start II High-Fidelity DNA Polymerase (Thermo Scientific), Platinum™ SuperFi™ II DNA Polymerase (Invitrogen), iProof™ High-Fidelity DNA Polymerase (Bio-Rad), Q5® High-Fidelity DNA Polymerase (New England Biolabs), and TruFi™ DNA Polymerase (Azura Genomics). Most of the kits produced profiles exhibiting a high degree of uneven amplification and varying levels of allelic dropout. In addition, all of the kits tested had much shorter peak heights compared to using GlobalFiler. Changing the type of enzyme used in an established protocol was found to be less straightforward than anticipated. Due to the poor quality results obtained in the first pass of trials, a few kits were selected to undergo optimization in the hopes of achieving higher quality results from which further analyses, such as comparing stutter rates, could be more reliably conducted. Both altered reagent amounts (higher enzyme concentration, higher DNA input mass) and different PCR parameters (decreased denaturation temperature, varying annealing temperature, decreased extension temperature, longer extension cycles, and longer final extension stage) were assessed. Only an increase in extension cycling time was found to produce better peak heights while maintaining balanced amplification of most of the targeted loci. Initial samples amplified with the Phusion enzyme exhibited multiple non-specific artifacts that were not stutter. Raising the annealing temperature for that enzyme’s protocol eliminated this issue. Therefore, higher annealing temperatures were pre-emptively used for several of the other enzymes tested. One of the explanations proposed for the uneven amplification observed is the presence of inhibitors in the commercial buffers used affecting downstream capillary electrophoresis. The Q5 High-Fidelity and TruFi DNA polymerases produced the best quality profiles; the UCP HiFidelity PCR Kit had the poorest results. Preliminary results indicated that none of the protocol alterations implemented significantly decreased stutter rates, nor was any one commercial enzyme found to have consistently lower stutter rates than the GlobalFiler kit. Due to the low number of trials carried out, the findings from this study require more replications with a wider variety of DNA polymerases to confirm that the type of enzyme used in an assay does not affect stutter rates.