Single-copy insertion of transgenes in Caenorhabditis elegans

At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.     

Anion sensitivity and spectral tuning of middle- and long-wavelength-sensitive (MWS/LWS) visual pigments

The long-wavelength-sensitive (LWS) opsins form one of four classes of vertebrate cone visual pigment and exhibit peak spectral sensitivities (λ(max)) that generally range from 525 to 560 nm for rhodopsin/vitamin-A(1) photopigments. Unique amongst the opsin classes, many LWS pigments show anion sensitivity through the interaction of chloride ions with a histidine residue at site 197 (H197) to give a long-wavelength spectral shift in peak sensitivity. Although it has been shown that amino acid substitutions at five sites (180, 197, 277, 285 and 308) are useful in predicting the λ(max) values of the LWS pigment class, some species, such as the elephant shark and most marine mammals, express LWS opsins that possess λ(max) values that are not consistent with this 'five-site' rule, indicating that other interactions may be involved. This study has taken advantage of the natural mutation at the chloride-binding site in the mouse LWS pigment. Through the use of a number of mutant pigments generated by site-directed mutagenesis, a new model has been formulated that takes into account the role of charge and steric properties of the side chains of residues at sites 197 and 308 in the function of the chloride-binding site in determining the peak sensitivity of LWS photopigments.     

A latent class procedure for the structural analysis of two-way compositional data

This paper develops a new procedure for simultaneously performing multidimensional scaling and cluster analysis on two-way compositional data of proportions. The objective of the proposed procedure is to delineate patterns of variability in compositions across subjects by simultaneously clustering subjects into latent classes or groups and estimating a joint space of stimulus coordinates and class-specific vectors in a multidimensional space. We use a conditional mixture, maximum likelihood framework with an E-M algorithm for parameter estimation. The proposed procedure is illustrated using a compositional data set reflecting proportions of viewing time across television networks for an area sample of households.     

Observations on the reproduction, sources of mortality, and diet of the Kendall Warm Spring dace

The life history of the endangered Kendall Warm Springs dace ( Rhinichthys osculus thermalis ) is largely unknown. Our study of its reproduction, sources of mortality, and diet indicated that Kendall Warm Springs dace seem to be reproductively active throughout the year, but the rate of reproduction appears to decrease during the winter. Males become sexually mature at 34 mm total length and females at 40 mm total length. We observed 2 sources of mortality: (1) emigration from the warm spring over a waterfall into the Green River and (2) predation on larvae by dragonfly ( Libellula saturata ) nymphs. Stomachs of Kendall Warm Springs dace contained small (≤ 1.5 mm total length) benthic invertebrates, primarily dipterans and mollusks.     

Science,Politics and Networks: Shibukawa Harumi and the Birth of the New Almanac in Seventeenth-century Japan

In 1684, during the Edo period (1603–1868), the imperial court of Japan passed a reform act that resulted in a new almanac called the Jôkyô almanac. This was the first reform in more than eight hundred years, and marked a departure from the past practice of adopting almanacs from China. Yet, the reform was complicated, and it was achieved after decades through the efforts of Shibukawa Harumi (1639–1715). How was the reform accomplished, and why was it significant? In this study, I will focus on Harumi's role in the reform. I propose to examine the reform as the culmination of a process deeply linked to Harumi's political connections and his intellectual growth especially in the field of calendrical sciences. I will show that this approach of looking at the reform by analyzing different aspects of Harumi's life has implications for understanding trends in society in seventeenth-century Japan. First, despite the ban on Christianity, intellectuals like Harumi assimilated European sciences in unique ways. Second, Harumi's activities exemplified how intellectual ties were increasingly embedded within political networks. Overall, I will argue that the reform of the almanac, viewed as a process surrounding the core figure of Harumi, was driven by scientific goals and personal ambitions.     

A stress response pathway regulates DNA damage through β2-adrenoreceptors and β-arrestin-1

The human mind and body respond to stress, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the 'fight-or-flight' response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders, and epidemiological studies strongly indicate that chronic stress leads to DNA damage. This stress-induced DNA damage may promote ageing, tumorigenesis, neuropsychiatric conditions and miscarriages. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates β(2)-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos. Activated β(2)-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of β-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right. Here we elucidate a molecular mechanism by which β-adrenergic catecholamines, acting through both Gs-PKA and β-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, β-arrestin-1 (ARRB1), activated via β(2)-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1(-/-)) mice, which show preserved p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress, and in the testes, in which paternal stress may affect the offspring's genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.     

The auroral footprint of Enceladus on Saturn

Although there are substantial differences between the magnetospheres of Jupiter and Saturn, it has been suggested that cryovolcanic activity at Enceladus could lead to electrodynamic coupling between Enceladus and Saturn like that which links Jupiter with Io, Europa and Ganymede. Powerful field-aligned electron beams associated with the Io-Jupiter coupling, for example, create an auroral footprint in Jupiter's ionosphere. Auroral ultraviolet emission associated with Enceladus-Saturn coupling is anticipated to be just a few tenths of a kilorayleigh (ref. 12), about an order of magnitude dimmer than Io's footprint and below the observable threshold, consistent with its non-detection. Here we report the detection of magnetic-field-aligned ion and electron beams (offset several moon radii downstream from Enceladus) with sufficient power to stimulate detectable aurora, and the subsequent discovery of Enceladus-associated aurora in a few per cent of the scans of the moon's footprint. The footprint varies in emission magnitude more than can plausibly be explained by changes in magnetospheric parameters--and as such is probably indicative of variable plume activity.     

The representation of three-way proximity data by single and multiple tree structure models

Models for the representation of proximity data (similarities/dissimilarities) can be categorized into one of three groups of models: continuous spatial models, discrete nonspatial models, and hybrid models (which combine aspects of both spatial and discrete models). Multidimensional scaling models and associated methods, used for thespatial representation of such proximity data, have been devised to accommodate two, three, and higher-way arrays. At least one model/method for overlapping (but generally non-hierarchical) clustering called INDCLUS (Carroll and Arabie 1983) has been devised for the case of three-way arrays of proximity data. Tree-fitting methods, used for thediscrete network representation of such proximity data, have only thus far been devised to handle two-way arrays. This paper develops a new methodology called INDTREES (for INdividual Differences in TREE Structures) for fitting various(discrete) tree structures to three-way proximity data. This individual differences generalization is one in which different individuals, for example, are assumed to base their judgments on the same family of trees, but are allowed to have different node heights and/or branch lengths.We initially present an introductory overview focussing on existing two-way models. The INDTREES model and algorithm are then described in detail. Monte Carlo results for the INDTREES fitting of four different three-way data sets are presented. In the application, a single ultrametric tree is fitted to three-way proximity data derived from intention-to-buy-data for various brands of over-the-counter pain relievers for relieving three common types of maladies. Finally, we briefly describe how the INDTREES procedure can be extended to accommodate hybrid modelling, as well as to handle other types of applications.