Repetitive Sequence Elements Research Articles

Genetic diversity, antimicrobial resistance, toxin gene profiles, and toxin production ability of Bacillus cereus isolates from doenjang, a Korean fermented soybean paste

AbstractThe objectives of this study were to determine the toxin gene profile, toxin production, and antibiotic resistance of Bacillus cereus isolates from doenjang, a Korean fermented soybean paste. Repetitive element sequence polymorphism‐PCR (rep‐PCR) was also used to assess the genetic diversity between these isolates. Thirty‐five B. cereus strains were isolated from 67 commercial doenjang samples, and all 35 isolates carried the nheABC and entFM genes and produced NHE enterotoxin. Of them, 12 strains harbored the emetic toxin gene (ces) and tested positive for the HEp‐2 cell emetic toxin assay. The B. cereus isolates were highly resistant to ß‐lactam antibiotics including ampicillin, cefepime, penicillin, and oxacillin. The rep‐PCR fingerprints of the B. cereus isolates were not influenced by the presence of toxin genes or antibiotic resistance profiles. However, rep‐PCR assay can be used to discriminate emetic B. cereus isolates from nonemetic isolates.Practical applicationsDoenjang has been the focus of recent concern in Korea because of its potential to cause foodborne illness via Bacillus cereus. The results of the present study suggest that B. cereus isolates from doenjang could cause either diarrheal or emetic types of foodborne illness because all isolates contained at least one toxin and its gene, although B. cereus levels in doenjang meet the Korean Food and Drug Administration guidelines (<104 cfu/g). Thus, it is necessary to constantly monitor the levels of B. cereus contamination and its toxins in doenjang.

Determination of Isolated Salmonella Bacteria from Infected Animal Husbandry of Malayer City with using Molecular Methods

Salmonella species are gram negative bacteria and members of Enterobacteriaceae family. It has a rod-shaped appearance; it is catalase positive, oxidase negative, non-spore. Salmonella classified into two species, Salmonella Enterica and Salmonella Bangori. Salmonella is now one of the main reasons of diarrhea and vomiting in humans in many countries and especially in industrialized. In a study in Japan 164 Salmonella digestions were collected during 2006 to 2008 which 81 digestions were Salmonella Infantis. Salmonella-specific characteristics are studied in the two phenotype and genotype methods. In this research, with using genotype methods based on PCR, genetic diversity was evaluated; this PCR includes rep-PCR based on repetitive sequence elements (method was done by the use of three primers ERIC, REP and BOX). Studied showed most isolated strains were relevant to Salmonella Enteritidis and dendorogram study showed that the bacteria were grouped in one cluster in dendrogram that all 37 strains were put in a large cluster of Salmonella’s type which is divided into two clusters: Salmonella Enterica and Bongori. The results also in this experiment reflect the efficiency of rep-PCR method by using three ERIC, REP and BOX primers.

High-Throughput Deep Sequencing for Mapping Mammalian DNA Methylation.

This protocol describes methylation mapping analysis by paired-end sequencing (Methyl-MAPS). In addition to the sequence information, paired-end sequencing provides information about the physical distance between the two reads in the genome. Methyl-MAPS typically samples ~80% of the CpG dinucleotides in the genome and is also able to report the methylation status of individual genomic loci harboring repetitive elements. This is achieved by enzymatic fractionation of the genome into methylated and unmethylated compartments. Because the method avoids the use of bisulfite modification, DNA fragments of relatively large size are preserved, permitting the generation of paired-end libraries with DNA inserts of known size in the ranges of 0.8-1.5, 1.5-3, and 3-6 kb. The paired-end configurations can be uniquely mapped to the genome in most instances, because the paired reads will span most repetitive element sequences.

Control of transcriptional pausing by biased thermal fluctuations on repetitive genomic sequences

In the process of transcription elongation, RNA polymerase (RNAP) pauses at highly nonrandom positions across genomic DNA, broadly regulating transcription; however, molecular mechanisms responsible for the recognition of such pausing positions remain poorly understood. Here, using a combination of statistical mechanical modeling and high-throughput sequencing and biochemical data, we evaluate the effect of thermal fluctuations on the regulation of RNAP pausing. We demonstrate that diffusive backtracking of RNAP, which is biased by repetitive DNA sequence elements, causes transcriptional pausing. This effect stems from the increased microscopic heterogeneity of an elongation complex, and thus is entropy-dominated. This report shows a linkage between repetitive sequence elements encoded in the genome and regulation of RNAP pausing driven by thermal fluctuations.

Emergence of multidrug-resistant Proteus mirabilis in along-term care facility in Croatia.

An increased frequency of Proteus mirabilis isolates resistant to expanded-spectrum cephalosporins was observed recently in along-term care facility in Zagreb (Godan). The aim of this study was the molecular characterization of resistance mechanisms to new cephalosporins in P.mirabilis isolates from this nursing home. Thirty-eight isolates collected from 2013-2015 showing reduced susceptibility to ceftazidime were investigated. Antibiotic susceptibilities were determined by broth microdilution method. Inhibitor-based tests were performed to detect extended-spectrum (ESBLs) and AmpC β-lactamases. AmpC β-lactamases were characterized by polymerase chain reaction (PCR) followed by sequencing of bla ampC genes. Quinolone resistance determinants (qnr genes) were characterized by PCR. Genotyping of the isolates was performed by repetitive element sequence (rep)-PCR and pulsed-field gel electrophoresis (PFGE). Presence of an AmpC β-lactamase was confirmed in all isolates by combined-disk test with phenylboronic acid. All isolates were resistant to amoxicillin alone and combined with clavulanate, cefotaxime, ceftriaxone, cefoxitin, and ciprofloxacin; but susceptible to cefepime, imipenem, and meropenem. PCR followed by sequencing using primers targeting bla ampc genes revealed CMY-16 β-lactamase in all but one strain. Bla cmy-16 was carried by anon-conjugative plasmid which did not belong to any known plasmid-based replicon typing (PBRT) group. Rep-PCR identified one large clone consisting of 15isolates, three pairs or related isolates, one triplet, and four singletons. PFGE confirmed the clonality of the isolates. This is the first report of multidrug resistant P.mirabilis in anursing home in Croatia. Cephalosporin resistance was due to plasmid-mediated AmpC β-lactamase CMY-16.

Identification of the Long-Sought Leptin in Chicken and Duck: Expression Pattern of the Highly GC-Rich Avian leptin Fits an Autocrine/Paracrine Rather Than Endocrine Function.

More than 20 years after characterization of the key regulator of mammalian energy balance, leptin, we identified the leptin (LEP) genes of chicken (Gallus gallus) and duck (Anas platyrhynchos). The extreme guanine-cytosine content (∼70%), the location in a genomic region with low-complexity repetitive and palindromic sequence elements, the relatively low sequence conservation, and low level of expression have hampered the identification of these genes until now. In vitro-expressed chicken and duck leptins specifically activated signaling through the chicken leptin receptor in cell culture. In situ hybridization demonstrated expression of LEP mRNA in granular and Purkinje cells of the cerebellum, anterior pituitary, and in embryonic limb buds, somites, and branchial arches, suggesting roles in adult brain control of energy balance and during embryonic development. The expression patterns of LEP and the leptin receptor (LEPR) were explored in chicken, duck, and quail (Coturnix japonica) using RNA-sequencing experiments available in the Short Read Archive and by quantitative RT-PCR. In adipose tissue, LEP and LEPR were scarcely transcribed, and the expression level was not correlated to adiposity. Our identification of the leptin genes in chicken and duck genomes resolves a long lasting controversy regarding the existence of leptin genes in these species. This identification was confirmed by sequence and structural similarity, conserved exon-intron boundaries, detection in numerous genomic, and transcriptomic datasets and characterization by PCR, quantitative RT-PCR, in situ hybridization, and bioassays. Our results point to an autocrine/paracrine mode of action for bird leptin instead of being a circulating hormone as in mammals.

Toxin Profile, Biofilm Formation, and Molecular Characterization of Emetic Toxin-Producing Bacillus cereus Group Isolates from Human Stools.

Emetic toxin-producing Bacillus cereus group species are an important problem, because the staple food for Korean is grains such as rice. In this study, we determined the prevalence (24 of 129 isolates) of emetic B. cereus in 36,745 stool samples from sporadic food-poisoning cases in Korea between 2007 and 2008. The toxin gene profile, toxin production, and biofilm-forming ability of the emetic B. cereus isolates were investigated. Repetitive element sequence polymorphism polymerase chain reaction fingerprints (rep-PCR) were also used to assess the intraspecific biodiversity of these isolates. Emetic B. cereus was present in 0.07% of the sporadic food-poisoning cases. The 24 emetic isolates identified all carried the nheABC and entFM genes and produced NHE enterotoxin. However, they did not have hemolysin BL toxin or related genes. A relationship between biofilm formation and toxin production was not observed in this study. The rep-PCR fingerprints of the B. cereus isolates were not influenced by the presence of toxin genes, or biofilm-forming ability. The rep-PCR assay discriminated emetic B. cereus isolates from nonemetic isolates, even if this assay did not perfectly discriminate these isolates. Further study on emetic isolates possessing a high degree of diversity may be necessary to evaluate the performance of the subtyping assay to discriminate emetic and nonemetic B. cereus isolates and could provide a more accurate indication of the risk from B. cereus strains.

Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using in vitro TF-DNA preferences obtained from the universal protein binding microarrays (PBM) for ~90 eukaryotic TFs belonging to 22 different DNA-binding domain types. As a result of this new analysis, we conclude that nonconsensus protein-DNA binding is a widespread phenomenon that significantly affects protein-DNA binding preferences and need not require the presence of consensus (specific) TFBSs in order to achieve genome-wide TF-DNA binding specificity.

Molecular and phenotypic characteristics of methicillin-resistant Staphylococcus aureus isolated from hospitalized patients.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of infections acquired in both community and hospital settings. In this study, MRSA isolated from different sources of hospitalized patients was characterized by molecular and phenotypic methods. A total of 123 S. aureus isolates were characterized according to their genetic relatedness by repetitive element sequence based-PCR (REP-PCR), in vitro antimicrobial susceptibility profile, SCCmec typing and presence of seven virulence factor-encoding genes. REP-PCR fingerprinting showed low relatedness between the isolates, and the predominance of one specific lineage or clonal group was not observed. All isolates were susceptible to teicoplanin and linezolide. All isolates were resistant to cefoxitin and penicillin, and the majority were also resistant to one or more other antimicrobials. Fifty isolates (41.7%) were intermediately resistant to vancomycin. Most isolates harbored SCCmec type II (53.7%), followed by type I (22.8%), type IV (8.1%) and type III (1.6%). All isolates harbored at least two virulence factor-encoding genes, and the prevalence was as follows: coa, 100%; icaA, 100%; hla, 13.0%; hlb, 91.1%, hld, 91.1%; lukS-PV and lukF-PV, 2.4%; and tst, 34.1%. A positive association with the presence of hla and SCCmec type II, and tst and SCCmec type I was observed. This study showed the high virulence potential of multidrug-resistant MRSA circulating in a teaching hospital. A high prevalence of MRSA showing intermediate vancomycin resistance was also observed, indicating the urgent need to improve strategies for controlling the use of antimicrobials for appropriate management of S. aureus infections.

MIR retrotransposon sequences provide insulators to the human genome

Insulators are regulatory elements that help to organize eukaryotic chromatin via enhancer-blocking and chromatin barrier activity. Although there are several examples of transposable element (TE)-derived insulators, the contribution of TEs to human insulators has not been systematically explored. Mammalian-wide interspersed repeats (MIRs) are a conserved family of TEs that have substantial regulatory capacity and share sequence characteristics with tRNA-related insulators. We sought to evaluate whether MIRs can serve as insulators in the human genome. We applied a bioinformatic screen using genome sequence and functional genomic data from CD4(+) T cells to identify a set of 1,178 predicted MIR insulators genome-wide. These predicted MIR insulators were computationally tested to serve as chromatin barriers and regulators of gene expression in CD4(+) T cells. The activity of predicted MIR insulators was experimentally validated using in vitro and in vivo enhancer-blocking assays. MIR insulators are enriched around genes of the T-cell receptor pathway and reside at T-cell-specific boundaries of repressive and active chromatin. A total of 58% of the MIR insulators predicted here show evidence of T-cell-specific chromatin barrier and gene regulatory activity. MIR insulators appear to be CCCTC-binding factor (CTCF) independent and show a distinct local chromatin environment with marked peaks for RNA Pol III and a number of histone modifications, suggesting that MIR insulators recruit transcriptional complexes and chromatin modifying enzymes in situ to help establish chromatin and regulatory domains in the human genome. The provisioning of insulators by MIRs across the human genome suggests a specific mechanism by which TE sequences can be used to modulate gene regulatory networks.

ProGeRF: proteome and genome repeat finder utilizing a fast parallel hash function.

Repetitive element sequences are adjacent, repeating patterns, also called motifs, and can be of different lengths; repetitions can involve their exact or approximate copies. They have been widely used as molecular markers in population biology. Given the sizes of sequenced genomes, various bioinformatics tools have been developed for the extraction of repetitive elements from DNA sequences. However, currently available tools do not provide options for identifying repetitive elements in the genome or proteome, displaying a user-friendly web interface, and performing-exhaustive searches. ProGeRF is a web site for extracting repetitive regions from genome and proteome sequences. It was designed to be efficient, fast, and accurate and primarily user-friendly web tool allowing many ways to view and analyse the results. ProGeRF (Proteome and Genome Repeat Finder) is freely available as a stand-alone program, from which the users can download the source code, and as a web tool. It was developed using the hash table approach to extract perfect and imperfect repetitive regions in a (multi)FASTA file, while allowing a linear time complexity.

Rhizobia Indigenous to the Okavango Region in Sub-Saharan Africa: Diversity, Adaptations, and Host Specificity

The rhizobial community indigenous to the Okavango region has not yet been characterized. The isolation of indigenous rhizobia can provide a basis for the formulation of a rhizobial inoculant. Moreover, their identification and characterization contribute to the general understanding of species distribution and ecology. Isolates were obtained from nodules of local varieties of the pulses cowpea, Bambara groundnut, peanut, hyacinth bean, and common bean. Ninety-one of them were identified by BOX repetitive element PCR (BOX-PCR) and sequence analyses of the 16S-23S rRNA internally transcribed spacer (ITS) and the recA, glnII, rpoB, and nifH genes. A striking geographical distribution was observed. Bradyrhizobium pachyrhizi dominated at sampling sites in Angola which were characterized by acid soils and a semihumid climate. Isolates from the semiarid sampling sites in Namibia were more diverse, with most of them being related to Bradyrhizobium yuanmingense and Bradyrhizobium daqingense. Host plant specificity was observed only for hyacinth bean, which was nodulated by rhizobia presumably representing yet-undescribed species. Furthermore, the isolates were characterized with respect to their adaptation to high temperatures, drought, and local host plants. The adaptation experiments revealed that the Namibian isolates shared an exceptionally high temperature tolerance, but none of the isolates showed considerable adaptation to drought. Moreover, the isolates' performance on different local hosts showed variable results, with most Namibian isolates inducing better nodulation on peanut and hyacinth bean than the Angolan strains. The local predominance of distinct genotypes implies that indigenous strains may exhibit a better performance in inoculant formulations.

Molecular diversity and functional variability of environmental isolates of Bacillus species.

In the present study, out of 264 phosphate (P) solubilizing Bacillus strains isolated from apple rhizosphere, only twelve isolates were found to be efficient (showed most of the plant growth promoting activity) which were further characterized at molecular level using 16S rDNA partial gene sequencing. Out of 12 isolates, MZPSB 207 was found to be most efficient P-solubilizing (864.71 μg/ml) isolate which also showed indole acetic acid production (51.83 μg/ml), siderophore production, ammonia production, antagonistic property (against Rhizoctonia solani and Fusarium oxysporum), hydrolytic enzymes productions (protease, chitinase and cellulase), 1-aminocyclopropane-1-carboxylate (ACC) deaminase production (7.7 μm αKB mg-1 h-1). The in-vitro seed germination assay showed that Bacillus (twelve isolates) inoculated seeds showed more seed germination and seedling vigor rate as compared to uninoculated control treatment.For the genetic diversity studies of efficient 12 strains, the polyphasic approach using 16S-rDNA, Repetitive element sequence (rep) based PCR (ERIC-PCR and BOX-PCR) were used. Based on 16S rDNA partial gene sequencing the isolated Bacillus genus was divide into four groups. First group (five isolates), second group (two isolates), third group (three isolates) and fourth group (two isolates) which showed close genetic relatedness to the B. subtilis, B. pumulis, B. megaterium and B. amyloliquefaciens, respectively. The rep PCR fingerprinting showed variability between and within the species. The large variability was showed by ERIC-PCR whereas some variability was showed by BOX-PCR. The results clearly showed that 16S rRNA gene sequencing is unable to discriminate the isolates at strain level. But rep-PCR fingerprinting is excellent tool to characterize and discriminate the strains at the genomic level.

Shiga Toxin–Producing Escherichia coli Distribution and Characterization in a Pasture-Based Cow-Calf Production System

Shiga toxin–producing Escherichia coli (STEC) strains are commonly found in cattle gastrointestinal tracts. In this study, prevalence and distribution of E. coli virulence genes (stx1, stx2, hlyA, and eaeA) were assessed in a cow-calf pasture-based production system. Angus cows (n = 90) and their calves (n = 90) were kept in three on-farm locations, and fecal samples were collected at three consecutive times (July, August, and September 2011). After enrichment of samples, stx1, stx2, eaeA, and hlyA were amplified and detected with a multiplex PCR (mPCR) assay. Fecal samples positive for stx genes were obtained from 93.3% (84 of 90) of dams and 95.6% (86 of 90) of calves at one or more sampling times. Age class (dam or calf), spatial distribution of cattle (farm locations B, H, K), and sampling time influenced prevalence and distribution of virulence genes in the herd. From 293 stx-positive fecal samples, 744 E. coli colonies were isolated. Virulence patterns of isolates were determined through mPCR assay: stx1 was present in 41.9% (312 of 744) of the isolates, stx2 in 6.5% (48 of 744), eaeA in 4.2% (31 of 744), and hlyA in 2.4% (18 of 744). Prevalence of non-O157 STEC was high among the isolates: 33.8% (112 of 331) were STEC O121, 3.6% (12 of 331) were STEC O103, and 1.8% (6 of 331) were STEC O113. One isolate (0.3%) was identified as STEC O157. Repetitive element sequence–based PCR (rep-PCR) fingerprinting was used to study genetic diversity of stx-positive E. coli isolates. Overall, rep-PCR fingerprints were highly similar, supporting the hypothesis that strains are transmitted between animals but not necessarily from a dam to its calf. Highly similar STEC isolates were obtained at each sampling time, but isolates obtained from dams were more diverse than those from calves, suggesting that strain differences in transference may exist. Understanding the transfer of E. coli from environmental and animal sources to calves may aid in developing intervention strategies to reduce E. coli colonization of young cattle.

Improvement in biocontrol activity of Bacillus subtilis UTB1 against Aspergillus flavus using gamma-irradiation

Bacillus subtilis UTB1, a biocontrol bacterium isolated from Iranian pistachio nuts, has revealed to have antagonistic activity against aflatoxin-producing Aspergillus flavus R5. The strain UTB1 produces lipopeptide compounds and is able to degrade aflatoxin B1. In this study, a random mutagenesis generated using different doses of gamma irradiation (0.1–3 KGy) was applied on B. subtilis UTB1 to improve its antagonistic activity against A. flavus R5. Five hundred bacterial colonies were selected randomly after irradiation, and their effects against A. flavus R5 were assessed in a plate assay. Forty-five colonies (9%) exhibited higher inhibition activity as compared to the non-irradiated wild type. Eight colonies out of the 45 were selected based on different polymorphism patterns obtained by repetitive element sequence polymorphism-PCR (ERIC and BOX) analyses; six of which could significantly inhibit the fungal growth utilizing washed cells and cell-free supernatants as compared to the parental strain. According to thin-layer chromatograms, the production of lipopeptides including surfactin, fengycin and iturin families increased in these six mutants. A considerable inhibition of the fungal growth was observed using bioautography analysis, which associated with iturins production. A. flavus sporulation and aflatoxin content decreased significantly in pistachio nuts treated with mutants M419 and M464 as compared to the strain UTB1. These results suggest that both mutants M419 and M464 could be promising biocontrol candidates against A. flavus in pistachio nuts.

Distribution and sequencing of enterobacterial repetitive intergenic consensus sequence elements in Streptococcus mutans serotype c

ObjectiveThe banding pattern of Streptococcus mutans strains generated using repetitive extragenic palindromic PCR (rep-PCR) with enterobacterial repetitive intergenic consensus (ERIC) primers has been shown previously. In the present study, the sequencing and analysis of gene sequences of the 6 most intense and commonly occurring amplicons from S. mutans serotype c was investigated. MethodsThe sequences of the amplicons from S. mutans serotype c strain by using rep-PCR with ERIC1R and ERIC2 primers were compared to the whole genome sequence of S. mutans UA159 and NN2025 strains. ResultsThe amplicons were found to contain partial gene sequences coding for proteins such as the hypothetical protein, glucan binding protein A, putative methylated-DNA-protein-cysteine S-methyltransferase, putative d-3-phosphoglycerate dehydrogenase, putative excinuclease ABC (subunit A) and putative GTP-binding protein. The locations of 5 of the 6 amplicons were found to be assembled downstream in the UA159 genome. The coding direction of the amplicons in the NN2025 genome was in reverse orientation relative to that in the UA159 strain, except in the 2170-bp amplicon. ConclusionsThe repeating sequence elements of ERIC in S. mutans serotype c are located on one side of the genome and have less frequency and similarity compared to those in enterobacteria.

Extensive chromosomal reshuffling drives evolution of virulence in an asexual pathogen

Sexual recombination drives genetic diversity in eukaryotic genomes and fosters adaptation to novel environmental challenges. Although strictly asexual microorganisms are often considered as evolutionary dead ends, they comprise many devastating plant pathogens. Presently, it remains unknown how such asexual pathogens generate the genetic variation that is required for quick adaptation and evolution in the arms race with their hosts. Here, we show that extensive chromosomal rearrangements in the strictly asexual plant pathogenic fungus Verticillium dahliae establish highly dynamic lineage-specific (LS) genomic regions that act as a source for genetic variation to mediate aggressiveness. We show that such LS regions are greatly enriched for in planta-expressed effector genes encoding secreted proteins that enable host colonization. The LS regions occur at the flanks of chromosomal breakpoints and are enriched for retrotransposons and other repetitive sequence elements. Our results suggest that asexual pathogens may evolve by prompting chromosomal rearrangements, enabling rapid development of novel effector genes. Likely, chromosomal reshuffling can act as a general mechanism for adaptation in asexually propagating organisms.

Mutations Disrupting Histone Methylation Have Different Effects on Replication Timing in S. pombe Centromere

The fission yeast pericentromere comprises repetitive sequence elements packaged into heterchromatin marked by histone H3K9 methylation and Swi6 binding. Transient disruption of Swi6 during S phase allows a period of RNA synthesis which programs the RNAi machinery to maintain histone methylation. However, Swi6 is also required for early replication timing. We show that not only Swi6 but also the chromodomain protein Chp1 are delocalized during S phase. Different from loss of swi6, mutations that disrupt histone methylation in the centromere, chp1Δ and clr4Δ, undergo early DNA replication. However, timing is modestly delayed in RNAi mutants dcr1Δ or rdp1Δ, while hrr1Δ mutants resemble swi6Δ in their replication delay. Finally, we show that recruitment of RNA polymerase II in the centromere occurs independently of replication. These different effects indicate that replication timing is not simply linked to histone methylation.

Molecular Epidemiological Analysis of Methicillin-Resistant Staphylococcus aureus Isolates From a Medical Intensive Care Unit: A Comparison of Nasal and Clinical Isolates

BackgroundThe control of nosocomial transmission of methicillin-resistant Staphylococcus aureus (MRSA) represents a significant challenge to infection control professionals. Nasal carriage colonization by MRSA plays a crucial role in the epidemiology and pathogenesis of this infection. MethodsPatients in the medical intensive care unit (ICU) between November 2010 and March 2011 were swabbed when hospitalized, reswabbed 1 week later and for a third time, when they were discharged from the ICU. All swabs were examined within 2 hours of collection using ChromID MRSA-Select agar plates to detect MRSA. Positive specimens were determined to have the mecA and femB gene through amplification with duplex polymerase chain reaction. Repetitive element sequence–based polymerase chain reaction was used to investigate the epidemiological types of MRSA isolates in the third screening and clinical isolates obtained from 2007 to 2010 in West China Hospital. A comparison of molecular types was performed to investigate the genetic relationship between nasal and clinical isolates. ResultsAfter the third screening, 16 nasal MRSA isolates were identified. Epidemiological analysis revealed that 16 nasal MRSA isolates and 37 clinical MRSA isolates differentiated into 2 clusters, comprising 9 subclusters. Of the 16 nasal strains, 11 (68.8%) belonged to subcluster I of cluster I; 3 of 9 subclusters consisted of both nasal and clinical isolates, while 4 of 9 subclusters consisted of clinical isolates and only 2 of 9 consisted of nasal isolates. ConclusionsOur study indicated a high degree of genetic relatedness between nasal and clinical MRSA isolates. The molecular typing of MRSA is critical for controlling the nosocomial transmission of this pathogen in ICU setting and defining a nosocomial infection control policy.

Mechanism of Replicative DNA Polymerase Delta Pausing and a Potential Role for DNA Polymerase Kappa in Common Fragile Site Replication

Common fragile sites (CFSs) are hot spots of chromosomal breakage, and CFS breakage models involve perturbations of DNA replication. Here, we analyzed the contribution of specific repetitive DNA sequence elements within CFSs to the inhibition of DNA synthesis by replicative and specialized DNA polymerases (Pols). The efficiency of in vitro DNA synthesis was quantitated using templates corresponding to regions within FRA16D and FRA3B harboring AT-rich microsatellite and quasi-palindrome (QP) sequences. QPs were predicted to form stems of ~75-100% self-homology, separated by 3-9 bases of intervening sequences. Analysis of DNA synthesis progression by human Pol δ demonstrated significant synthesis perturbation both at [A](n) and [TA](n) repeats in a length-dependent manner and at short (<40 base pairs) QP sequences. DNA synthesis by the Y-family polymerase κ was significantly more efficient than Pol δ through both types of repetitive elements. Using DNA trap experiments, we show that Pol δ pauses within CFS sequences are sites of enzyme dissociation, and dissociation was observed in the presence of RFC-loaded PCNA. We propose that enrichment of microsatellite and QP elements at CFS regions contributes to fragility by perturbing replication through multiple mechanisms, including replicative Pol pausing and dissociation. Our finding that Pol δ dissociates at specific CFS sequences is significant, since dissociation of the replication machinery and inability to efficiently recover the replication fork can lead to fork collapse and/or formation of double-strand breaks in vivo. Our biochemical studies also extend the potential involvement of Y-family polymerases in CFS maintenance to include polymerase κ.