Delayed DNA methylation is an integral feature of DNA replication in mammalian cells

In the majority of sites of methylation in the DNA of mammalian cells, the symmetry of methylation is restored within a few minutes of the passage of a replication fork. However, it has been shown that daughter strand methylation in immortalised cell lines is delayed in a substantial minority of sites for up to several hours after replication. We report here the results of two new approaches to the determination of the functional significance of delayed DNA methylation in mammalian cells. Firstly, we demonstrate that normal, nontransformed cells (human peripheral lymphocytes in short-term primary culture) have comparable proportions of delayed DNA methylation to many immortalised cell lines, showing that delayed DNA methylation is not just a secondary consequence of abnormally high methionine requirements commonly observed in transformed cells and that delayed DNA methylation would be unlikely not to occur in vivo. Secondly, we have used 5-aza-2'-deoxycytidine (5azadCyd) to derive subclones of cells from the Chinese hamster ovary cell line which have stably hypomethylated DNA. In three of these subclones which had lost on average one fourth of the methylation sites from their genomes, the proportion of daughter strand methylation which was delayed after replication was reduced by less than 10%. If delayed DNA methylation were site-specific, this implies that of the order of twice the number of "immediate" methylation sites than delayed methylation sites had been lost from the genomes of these hypomethylated subclones. Thus, delayed DNA methylation is an integral part of the process whereby replicating mammalian cells maintain the pattern of methylation in their genomes. These observations are discussed in relation to the significance of delayed DNA methylation for the accurate maintenance of methylation patterns in the genome and the consequent implications for the possible role of methylated deoxycytidines in mammalian gene control.     

Determination of hepatitis delta virus ribozyme N(–1) nucleobase and functional group specificity using internal competition kinetics

Biological catalysis involves interactions distant from the site of chemistry that can position the substrate for reaction. Catalysis of RNA 2′-O-transphosphorylation by the hepatitis delta virus (HDV) ribozyme is sensitive to the identity of the N(–1) nucleotide flanking the reactive phosphoryl group. However, the interactions that affect the conformation of this position, and in turn the 2′O nucleophile, are unclear. Here, we describe the application of multiple substrate internal competition kinetic analyses to understand how the N(–1) nucleobase contributes to HDV catalysis and test the utility of this approach for RNA structure–function studies. Internal competition reactions containing all four substrate sequence variants at the N(–1) position in reactions using ribozyme active site mutations at A77 and A78 were used to test a proposed base-pairing interaction. Mutants A78U, A78G, and A79G retain significant catalytic activity but do not alter the specificity for the N(–1) nucleobase. Effects of nucleobase analog substitutions at N(–1) indicate that U is preferred due to the ability to donate an H-bond in the Watson–Crick face and avoid minor groove steric clash. The results provide information essential for evaluating models of the HDV active site and illustrate multiple substrate kinetic analyses as a practical approach for characterizing structure–function relationships in RNA reactions.     

A deeper meaning for shallow-level phylogenomic studies: nested anchored hybrid enrichment offers great promise for resolving the tiger moth tree of life (Lepidoptera: Erebidae: Arctiinae)

Anchored hybrid enrichment (AHE) has emerged as a powerful tool for uncovering the evolutionary relationships within many taxonomic groups. AHE probe sets have been developed for a variety of insect groups, though none have yet been shown to be capable of simultaneously resolving deep and very shallow (e.g., intraspecific) divergences. In this study, we present NOC1, a new AHE probe set (730 loci) for Lepidoptera specialized for tiger moths and assess its ability to deliver phylogenetic utility at all taxonomic levels. We test the NOC1 probe set with 142 individuals from 116 species sampled from all the major lineages of Arctiinae (Erebidae), one of the most diverse groups of noctuoids (>11 000 species) for which no well-resolved, strongly supported phylogenetic hypothesis exists. Compared to previous methods, we generally recover much higher branch support (BS), resulting in the most well-supported, well-resolved phylogeny of Arctiinae to date. At the most shallow-levels, NOC1 confidently resolves species-level and intraspecific relationships and potentially uncovers cryptic species diversity within the genus Hypoprepia. We also implement a ‘sensitivity analysis’ to explore different loci combinations and site sampling strategies to determine whether a reduced probe set can yield results similar to those of the full probe set. At both deep and shallow levels, only 50–175 of the 730 loci included in the complete NOC1 probe set were necessary to resolve most relationships with high confidence, though only when the more rapidly evolving sites within each locus are included. This demonstrates that AHE probe sets can be tailored to target fewer loci without a significant reduction in BS, allowing future studies to incorporate more taxa at a lower per-sample sequencing cost. NOC1 shows great promise for resolving long-standing taxonomic issues and evolutionary questions within arctiine lineages, one of the most speciose clades within Lepidoptera.     

Automated apparatus for quantitation of social approach behaviors in mice

Mouse models of social dysfunction, designed to investigate the complex genetics of social behaviors, require an objective methodology for scoring social interactions relevant to human disease symptoms. Here we describe an automated, three chambered apparatus designed to monitor social interaction in the mouse. Time spent in each chamber and the number of entries are scored automatically by a system detecting photocell beam breaks. When tested with the automated equipment, juvenile male C57BL/6J mice spent more time in a chamber containing a stranger mouse than in an empty chamber (sociability), similar to results obtained by the observer scored method. In addition, automated scoring detected a preference to spend more time with an unfamiliar stranger than a more familiar conspecific (preference for social novelty), similar to results obtained by the observer scored method. Sniffing directed at the wire cage containing the stranger mouse correlated significantly with time spent in that chamber, indicating that duration in a chamber represents true social approach behavior. Number of entries between chambers did not correlate with duration of time spent in the chambers; entries instead proved a useful control measure of general activity. The most significant social approach behavior took place in the first five minutes of both the sociability and preference for social novelty tests. Application of these methods to C57BL/6J, DBA/2J and FVB/NJ adult males revealed that all three strains displayed tendencies for sociability and preference for social novelty. To evaluate the importance of the strain of the stranger mouse on sociability and preference for social novelty, C57BL/6J subject mice were tested either with A/J strangers or with C57BL/6J strangers. Sociability and preference for social novelty were similar with both stranger strains. The automated equipment provides an accurate and objective approach to measuring social tendencies in mice. Its use may allow higher-throughput scoring of mouse social behaviors in mouse models of social dysfunction.     

Are human preferences for facial symmetry focused on signals of developmental instability?

Humans find symmetrical faces more attractive than are asymmetricalfaces. Evolutionary psychologists claim that our preferencefor symmetry can be explained in the context of mate choicebecause symmetry is an honest indicator of the genetic qualityof potential mates. These arguments assume that asymmetry inhuman faces is fluctuating asymmetry (FA), because this formof asymmetry can be revealing of developmental instability.However, no study has yet examined the characteristics of facialasymmetry. Here we provide the first detailed study of the patternsof asymmetry in human faces. We measured asymmetry in 35 facialtraits. Although some traits had distributions characteristicof FA, many had distributions that characterize directionalasymmetry (DA); on average, both men and women had right hemi-facedominance. For DA traits we used deviations from the mean asymmetryas a measure of developmental instability. Our measures of asymmetryaccounted for a moderate proportion of the variance in perceivedsymmetry. Importantly, only FAs and random deviations from DAcontributed to people's perception of symmetry. DA was not importantin symmetry judgments. Faces rated as symmetrical were alsorated as attractive. Random deviations from DA were weakly relatedto women's attractiveness judgments of men's faces. DAs didnot influence attractiveness judgments. Our data suggest thatpeople focus on aspects of facial asymmetry that may be revealingof developmental instability. Further studies that isolate FAfrom other forms of asymmetry are required to accurately assessthe influence of developmental instability on the quality ofindividuals and its potential role in mate preferences.     

Enzyme activities of aerobic lignocellulolytic bacteria isolated from wet tropical forest soils

Lignocellulolytic bacteria have promised to be a fruitful source of new enzymes for next-generation lignocellulosic biofuel production. Puerto Rican tropical forest soils were targeted because the resident microbes decompose biomass quickly and to near-completion. Isolates were initially screened based on growth on cellulose or lignin in minimal media. 75 Isolates were further tested for the following lignocellulolytic enzyme activities: phenol oxidase, peroxidase, β-d-glucosidase, cellobiohydrolase, β-xylopyranosidase, chitinase, CMCase, and xylanase. Cellulose-derived isolates possessed elevated β-d-glucosidase, CMCase, and cellobiohydrolase activity but depressed phenol oxidase and peroxidase activity, while the contrary was true of lignin isolates, suggesting that these bacteria are specialized to subsist on cellulose or lignin. Cellobiohydrolase and phenol oxidase activity rates could classify lignin and cellulose isolates with 61% accuracy, which demonstrates the utility of model degradation assays. Based on 16S rRNA gene sequencing, all isolates belonged to phyla dominant in the Puerto Rican soils, Proteobacteria, Firmicutes, and Actinobacteria, suggesting that many dominant taxa are capable of the rapid lignocellulose degradation characteristic of these soils. The isolated genera Aquitalea, Bacillus, Burkholderia, Cupriavidus, Gordonia, and Paenibacillus represent rarely or never before studied lignolytic or cellulolytic species and were undetected by metagenomic analysis of the soils. The study revealed a relationship between phylogeny and lignocellulose-degrading potential, supported by Kruskal–Wallis statistics which showed that enzyme activities of cultivated phyla and genera were different enough to be considered representatives of distinct populations. This can better inform future experiments and enzyme discovery efforts.     

CALLING SONGS OF FIELD CRICKETS (TELEOGRYLLUS OCEANICUS) WITH AND WITHOUT PHONOTACTIC PARASITOID INFECTION

The field cricket Teleogryllus oceanicus has been introduced to Hawaii, where it is parasitized by an acoustically orienting parasitoid fly, Ormia ochracea. Previous work showed that call parameters from parasitized populations differ from those in unparasitized populations in a direction expected if selection by flies is occurring. Here we examined songs of males collected in the field and compare calling song characters of crickets later found to harbor parasitoid larvae with those of males free of parasitoids. The two groups differ significantly in several song characteristics, particularly the trill-like long chirp given at the beginning of each song. Males with longer long chirps containing shorter interpulse intervals are more likely to be parasitized, suggesting that the flies find such males more attractive. Depending on the traits females prefer, sexual selection may oppose natural selection in altering T. oceanicus song in parasitized populations.