Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping

Atomic force microscopy (AFM) allows the study of single protein–DNA interactions such as those observed with the Type I Restriction–Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPγS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein–DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping

Assessing the Factorial Validity of the Attitudes and Belief Scale 2-Abbreviated Version: A Call for the Development a Gold Standard Method of Measuring Rational and Irrational Beliefs

Rational Emotive Behaviour Therapy (REBT) does not possess a measure of rational and irrational beliefs that meets internationally recognised standards for acceptable psychometric properties. Without such a measure the theory/practice of REBT cannot be rigorously evaluated, thus undermining its scientific veracity. The current study investigates the validity and reliability of a recently developed measure of rational and irrational beliefs: the Attitudes and Belief Scale 2-Abbreviated Version (ABS-2-AV). University students from three countries completed the ABS-2-AV (N = 397). An alternative models framework using confirmatory factor analysis indicated that a theoretically consistent eight-factor model of the ABS-2-AV provided the best fit of the data. A number of post-hoc modifications were required in order to achieve acceptable model fit results, and these modifications revealed important methodological limitations with the ABS-2-AV. Results indicated that the validity of the ABS-2-AV was undermined due to items measuring both the psychological process of interest (rational and irrational beliefs) and the context in which these beliefs processes are presented. This is a serious methodological limitation of the ABS-2 and all questionnaires derived from it, including the ABS-2-AV. This methodological limitation resulted in the ABS-2-AV possessing poor internal reliability. These limitations are discussed in relation to the broader REBT literature and the impact such problems have on research and practice. A call is made for REBT researchers to come together to develop a “gold standard” method of assessing rational and irrational beliefs that meets international standard for psychometric excellence

Antimicrobial Resistance among Campylobacter Strains, United States, 1997–2001

We summarize antimicrobial resistance surveillance data in human and chicken isolates of Campylobacter. Isolates were from a sentinel county study from 1989 through 1990 and from nine state health departments participating in National Antimicrobial Resistance Monitoring System for enteric bacteria (NARMS) from 1997 through 2001. None of the 297 C. jejuni or C. coli isolates tested from 1989 through 1990 was ciprofloxacin-resistant. From 1997 through 2001, a total of 1,553 human Campylobacter isolates were characterized: 1,471 (95%) were C. jejuni, 63 (4%) were C. coli, and 19 (1%) were other Campylobacter species. The prevalence of ciprofloxacin-resistant Campylobacter was 13% (28 of 217) in 1997 and 19% (75 of 384) in 2001; erythromycin resistance was 2% (4 of 217) in 1997 and 2% (8 of 384) in 2001. Ciprofloxacin-resistant Campylobacter was isolated from 10% of 180 chicken products purchased from grocery stores in three states in 1999. Ciprofloxacin resistance has emerged among Campylobacter since 1990 and has increased in prevalence since 1997

Shape - but Not Size - Codivergence between Male and Female Copulatory Structures in Onthophagus Beetles

Genitalia are among the fastest evolving morphological traits in arthropods. Among the many hypotheses aimed at explaining this observation, some explicitly or implicitly predict concomitant male and female changes of genital traits that interact during copulation (i.e., lock and key, sexual conflict, cryptic female choice and pleiotropy). Testing these hypotheses requires insights into whether male and female copulatory structures that physically interact during mating also affect each other's evolution and patterns of diversification. Here we compare and contrast size and shape evolution of male and female structures that are known to interact tightly during copulation using two model systems: (a) the sister species O. taurus (1 native, 3 recently established populations) and O. illyricus, and (b) the species-complex O. fracticornis-similis-opacicollis. Partial Least Squares analyses indicated very little to no correlation between size and shape of copulatory structures, both in males and females. Accordingly, comparing shape and size diversification patterns of genitalia within each sex showed that the two components diversify readily - though largely independently of each other - within and between species. Similarly, comparing patterns of divergence across sexes showed that relative sizes of male and female copulatory organs diversify largely independent of each other. However, performing this analysis for genital shape revealed a signature of parallel divergence. Our results therefore suggest that male and female copulatory structures that are linked mechanically during copulation may diverge in concert with respect to their shapes. Furthermore, our results suggest that genital divergence in general, and co-divergence of male and female genital shape in particular, can evolve over an extraordinarily short time frame. Results are discussed in the framework of the hypotheses that assume or predict concomitant evolutionary changes in male and female copulatory organs

Contribution of CgPDR1-Regulated Genes in Enhanced Virulence of Azole-Resistant Candida glabrata

In Candida glabrata, the transcription factor CgPdr1 is involved in resistance to azole antifungals via upregulation of ATP binding cassette (ABC)-transporter genes including at least CgCDR1, CgCDR2 and CgSNQ2. A high diversity of GOF (gain-of-function) mutations in CgPDR1 exists for the upregulation of ABC-transporters. These mutations enhance C. glabrata virulence in animal models, thus indicating that CgPDR1 might regulate the expression of yet unidentified virulence factors. We hypothesized that CgPdr1-dependent virulence factor(s) should be commonly regulated by all GOF mutations in CgPDR1. As deduced from transcript profiling with microarrays, a high number of genes (up to 385) were differentially regulated by a selected number (7) of GOF mutations expressed in the same genetic background. Surprisingly, the transcriptional profiles resulting from expression of GOF mutations showed minimal overlap in co-regulated genes. Only two genes, CgCDR1 and PUP1 (for PDR1 upregulated and encoding a mitochondrial protein), were commonly upregulated by all tested GOFs. While both genes mediated azole resistance, although to different extents, their deletions in an azole-resistant isolate led to a reduction of virulence and decreased tissue burden as compared to clinical parents. As expected from their role in C. glabrata virulence, the two genes were expressed as well in vitro and in vivo. The individual overexpression of these two genes in a CgPDR1-independent manner could partially restore phenotypes obtained in clinical isolates. These data therefore demonstrate that at least these two CgPDR1-dependent and -upregulated genes contribute to the enhanced virulence of C. glabrata that acquired azole resistance

GeoOrigins: A new method and R package for trait mapping and geographic provenancing of specimens without categorical constraints

Biologists often seek to geographically provenance organisms using their traits. This is typically achieved by defining spatial groups using distinct patterns of trait variation. Here, we present a new spatial provenancing and trait boundary identification methodology, based on correlations between geographic and trait distances that require no a priori group assumptions. We apply this to three datasets where spatial provenance is sought: morphological rat and vole dentition data (human commensal translocation datasets); and birdsong data (cultural transmission dataset). We also present the results of cross‐validation testing. Spatial provenancing is possible with differing degrees of accuracy for each dataset, with birdsong providing the most accurate geographic origin (identifying an average spatial region of 0.22 km2 as the area of origin with 99.9% confidence). Our method has a wide range of potential applications to diverse data types—including phenotypic, genetic and cultural—to identify trait boundaries and spatially provenance the origin of unknown or translocated specimens where trait differences are geographically structured and correlated with spatial separation

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genome