Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (～45%) of the 1,101 essential yeast genes, with ～30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.     

Use of digital webcam images to track spring green-up in a deciduous broadleaf forest

Understanding relationships between canopy structure and the seasonal dynamics of photosynthetic uptake of CO₂ by forest canopies requires improved knowledge of canopy phenology at eddy covariance flux tower sites. We investigated whether digital webcam images could be used to monitor the trajectory of spring green-up in a deciduous northern hardwood forest. A standard, commercially available webcam was mounted at the top of the eddy covariance tower at the Bartlett AmeriFlux site. Images were collected each day around midday. Red, green, and blue color channel brightness data for a 640 × 100-pixel region-of-interest were extracted from each image. We evaluated the green-up signal extracted from webcam images against changes in the fraction of incident photosynthetically active radiation that is absorbed by the canopy (f _APAR), a broadband normalized difference vegetation index (NDVI), and the light-saturated rate of canopy photosynthesis (A _max), inferred from eddy flux measurements. The relative brightness of the green channel (green %) was relatively stable through the winter months. A steady rising trend in green % began around day 120 and continued through day 160, at which point a stable plateau was reached. The relative brightness of the blue channel (blue %) also responded to spring green-up, although there was more day-to-day variation in the signal because blue % was more sensitive to changes in the quality (spectral distribution) of incident radiation. Seasonal changes in blue % were most similar to those in f _APAR and broadband NDVI, whereas changes in green % proceeded more slowly, and were drawn out over a longer period of time. Changes in A _max lagged green-up by at least a week. We conclude that webcams offer an inexpensive means by which phenological changes in the canopy state can be quantified. A network of cameras could offer a novel opportunity to implement a regional or national phenology monitoring program.     

Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

Spatially modelling native vegetation condition

Summary   The assessment of vegetation condition is seen as an increasingly important requirement for effective biodiversity conservation in Australia. Condition assessments that operate at the scale of the site are well established. However, there is a need for mapped representations of vegetation condition at regional scales to: (i) assist with regional planning and target setting; (ii) provide regional context for site-based assessment; and (iii) monitor the change in vegetation condition at multiple scales. This paper describes a methodology for converting site condition data collected in plots into maps of vegetation condition across entire regions using a predictive statistical modelling framework (Generalized Additive Modelling) combined with a GIS. The research demonstrates how explanatory variables including topographic position, terrain roughness, landscape connectivity and remote sensing derived indices can be used to map the condition of native vegetation at the scale of a subcatchment. The inclusion of indices derived from remotely sensed imagery (SPOT4) as explanatory variables in the modelling is a novel component of this research. Although the methodology generates statistically and ecologically plausible models of vegetation condition, there are nevertheless limitations associated with the way plot data were collected and some of the explanatory variables, which impacts upon model utility. We discuss how these problems can be minimized when embarking upon studies of this type. We demonstrate how maps produced from exercises such as this could be used for conservation planning and discuss the limitations of these data for monitoring.     

Advantages of a Biomimetic Stiffness Profile in Pitching Flexible Fin Propulsion

<正> The use of oscillating flexible fins in propulsion has been the subject of several studies in recent years, but attention israrely paid to the specific role of stiffness profile in thrust production.Stiffness profile is defined as the variation in localchordwise bending stiffness (EI) of a fin, from leading to trailing edge.In this study, flexible fins with a standard NACA0012shape were tested alongside fins with a stiffness profile mimicking that of a Pumpkinseed Sunfish (Lepomis gibbosus).The finswere oscillated with a pitching sinusoidal motion over a range of frequencies and amplitudes, while torque, lateral force andstatic thrust were measured.Over the range of oscillation parameters tested, it was shown that the fin with a biomimetic stiffness profile offered a significantimprovement in static thrust, compared to a fin of similar dimensions with a standard NACA0012 aerofoil profile.Thebiomimetic fin also produced thrust more consistently over each oscillation cycle.A comparison of fin materials of different stiffness showed that the improvement was due to the stiffness profile itself, andwas not simply an effect of altering the overall stiffness of the fin.Fins of the same stiffness profile were observed to follow thesame thrust-power curve, independent of the stiffness of the moulding material.Biomimetic fins were shown to produce up to26% greater thrust per watt of input power, within the experimental range.     

Sensitive Detection of Gene Expression in Mycobacteria under Replicating and Non-Replicating Conditions Using Optimized Far-Red Reporters

Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the P_mtbB is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.     

Collagen and mature elastic fibre organisation as a function of depth in the human cornea and limbus

A network of circumferentially oriented collagen fibrils exists in the periphery of the human cornea, and is thought to be pivotal in maintaining corneal biomechanical stability and curvature. However, it is unknown whether or not this key structural arrangement predominates throughout the entire corneal thickness or exists as a discrete feature at a particular tissue depth; or if it incorporates any elastic fibres and how, with respect to tissue depth, the circumcorneal annulus integrates with the orthogonally arranged collagen of the central cornea. To address these issues we performed a three-dimensional investigation of fibrous collagen and elastin architecture in the peripheral and central human cornea using synchrotron X-ray scattering and non-linear microscopy. This showed that the network of collagen fibrils circumscribing the human cornea is located in the posterior one-third of the tissue and is interlaced with significant numbers of mature elastic fibres which mirror the alignment of the collagen. The orthogonal arrangement of collagen in the central cornea is also mainly restricted to the posterior stromal layers. This information will aid the development of corneal biomechanical models aimed at explaining how normal corneal curvature is sustained and further predicting the outcome of surgical procedures.