Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase

Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays--which do not interfere with biological activity--to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.     

Structure-based activity prediction for an enzyme of unknown function

With many genomes sequenced, a pressing challenge in biology is predicting the function of the proteins that the genes encode. When proteins are unrelated to others of known activity, bioinformatics inference for function becomes problematic. It would thus be useful to interrogate protein structures for function directly. Here, we predict the function of an enzyme of unknown activity, Tm0936 from Thermotoga maritima, by docking high-energy intermediate forms of thousands of candidate metabolites. The docking hit list was dominated by adenine analogues, which appeared to undergo C6-deamination. Four of these, including 5-methylthioadenosine and S-adenosylhomocysteine (SAH), were tested as substrates, and three had substantial catalytic rate constants (10(5) M(-1 )s(-1)). The X-ray crystal structure of the complex between Tm0936 and the product resulting from the deamination of SAH, S-inosylhomocysteine, was determined, and it corresponded closely to the predicted structure. The deaminated products can be further metabolized by T. maritima in a previously uncharacterized SAH degradation pathway. Structure-based docking with high-energy forms of potential substrates may be a useful tool to annotate enzymes for function.     

The Uses of Reason in Times of Technical Mediation

The art of living idiom suits well a practice-oriented approach in ethics of technology. But what remains or becomes of the functioning and use of reason in ethics? In reaction to the comments by Huijer this reply elaborates in more detail how Foucault’s art of living can be adapted for a critical contemporary ethics of technology. And the aesthetic-political rationality in Foucault’s ethics is compared with Wellner’s suggestions of holding on to the notion of code but with a new meaning. Foucault’s fourfold scheme of subjectivation and a distinction of “below and above reason” structure the argument.     

Historical variations in δ 13 C leaf of herbarium specimens in the southwestern U.S.

The uncontrolled, global increase in atmospheric CO 2 concentration (ca 80 ppmv) and decline in δ 13 C air (ca 1.5%) since industrialization provide experimental boundary conditions by which to assess physiological response of vegetation. To examine consequences of these global atmospheric changes in the southwestern U.S., 350 specimens of Atriplex confertifolia, A. canescens, Ephedra viridis, Pinus edulis, P. flexilis, Juniperus scopulorum, and Quercus turbinella of precisely known age spanning the last 150 years were acquired from 9 herbaria. Cellulose analysis of δ 13 C plant and estimation of isotopic discrimination (Δ) permitted calculation of water-use efficiency (A/g). The δ 13 C plant chronologies of C 4 Atriplex spp. show some promise as a reliable proxy for δ 13 C air because their mean trends approximate the known δ 13 C air chronology. However, the high variability would necessitate multiple samples at any time period to accurately represent the mean. The generally increasing A/g trends of the 5 C 3 species are particularly pronounced for P. edulis and, after the 1950s, for J. scopulorum, but less evident for P. flexilis, E. viridis, and Q. turbinella, evidencing possible differences in species response to rising CO 2 concentrations. The trends are statistically noisy, however, possibly due to complex microclimates, extreme seasonality, and great interannual variability typical of the southwestern U.S. Herbarium specimens, at least in the Southwest, may be less useful for precise detection of direct CO 2 effects on plant physiology than tree rings, where the variability can be constrained to a single individual over time.     

Effect of soil microtopography on seed bank distribution in the shrub-steppe

The relationship between soil surface cryptogamic crusts and seed banks was investigated in the shrubsteppe in the Lower Columbia Basin. Seventy-four percent of the seeds in a disturbed bunchgrass community were found in crevices bordering cryptogamic crust polygons. In a sagebrush/bunchgrass community, 89% of the seeds were found in crevices. In a disturbed bunchgrass community, Bromus tectorum seeds were found in both the seed bank and aboveground vegetation communities. Bromus tectorum seeds were located in the seed bank of a sagebrush/bunchgrass community, although it had a minor presence in the aboveground community. Seeds of Artemisia tridentata Nutt. were not found in either the bunchgrass or sagebrush/bunchgrass communities. The high number of seeds found in crevices bordering the cryptogamic crust suggests that crevices play a role in determining the small-scale distributional pattern of shrub-steppe plants at the Fitzner-Eberhardt Arid Lands Ecology Reserve.     

Influence of water size and type on bat captures in the lower Sonoran Desert

We compared bat use by mist-netting at 4 different types of wildlife water developments in southwestern Arizona during summer 2000 and 2001. Scaling our results by netting effort, we caught bats more frequently and observed higher species diversity at tinajas (modified natural rock pools) with larger open-water area compared with ""guzzler"" type water developments that had less open water and more obstacles to bat flight. We caught the fewest bats at guzzlers with buried concrete vault drinkers, which impede bat access and have the smallest areas of open water. Water development designs that minimize evaporative water loss by reducing the amount of open water apparently reduce use by bats in this area.     

Roost sites of the silver-haired bat ( Lasionycyeris noctivagans ) in the Black Hills, South Dakota

We investigated the roosting ecology of silver-haired bats ( Lasionycteris noctivagans ) in the Black Hills, of western South Dakota. Using radiotelemetry, we located 39 roosts, 10 of which were maternity aggregations containing 6 to 55 bats. The roosts were mostly in ponderosa pine ( Pinus ponderosa ) snags that averaged 39 cm diameter at breast height. Solitary bats preferred roosting under loose bark or in crevices in trees, regularly moving among trees. All maternity aggregations were found in tree cavities, primarily those created by woodpeckers. Roost trees were located in patches of forest with relatively high snag densities, about 21 snags/ha. This study suggests that snags play an important role in maintain silver-haired bat populations in ponderosa pine ecosystems.     

Efficacy of using passive integrated transponder technology to track individual shorthead sculpins

We evaluated the feasibility of using passive integrated transponder (PIT) tags to mark and track individual shorthead sculpins ( Cottus confusus ). We implanted PIT tags in 80 shorthead sculpins in 2 size categories (60–80 mm and 81–106 mm total length) and in 1 of 2 locations (subcutaneous along the spinous dorsal fin or in the body cavity). We evaluated tag retention, sculpin survival, and net-avoidance behavior for 29 days. We observed no mortality directly attributable to PIT-tag injection at either tagging location. We observed a 38.8% loss rate of dorsal tags and a 2.5% loss rate of body-cavity tags. No change in net-avoidance behavior was observed. We released 97 PIT-tagged sculpins into an artificial stream and monitored them with 3 stationary and 1 portable antenna. Sixty sculpins were detected at least once, and 18 sculpins were detected multiple times with the portable antenna. These results indicate that PIT-tagging within the body cavity is a feasible method for marking and tracking individual shorthead sculpins.     

Fire history of Paunsaugunt Plateau in southern Utah

A fire history of the Pausaugunt Plateau in southern Utah was developed using dendrochronological methods. Fire frequencies of individual ponderosa pine trees from three sites on the plateau varied from 19.5 to 47 years. Composite fire intervals for the three sites ranged from 15.2 to 18.4 years. The last recorded fires in these study areas occurred in 1892, 1902, and 1911, corresponding to the initiation of fire suppression policies in the West. The absence of fire since 1911 may be contributing to a recently documented decrease in ponderosa pine regeneration within the high-elevation, mixed-coniferous forests of the Paunsaugunt Plateau.     

The relation between species numbers and island characteristics for habitat islands in a volcanic landscape

Within the Craters of the Moon Lava Flow in southeastern Idaho are kipukas, islands of sagebrush habitat isolated by relatively barren lava. In 1979 I counted the number of species of plants, small mammals, and reptiles for a series of kipukas. The degree of isolation of a kipuka was not related to numbers of species found there; thus, these data do not support the equilibrium theory of island biogeography. For most of the study organisms lava is not a significant barrier to dispersal. Larger kipukas support larger numbers of species. For plants this relationship likely is a result of the increased topographic variety of larger kipukas, whereas for small mammal species minimum area requirements for maintaining populations are met only by larger kipukas. More distant kipukas showed increased density of small mammals possibly as a result of reduced predation. Patterns of distribution in this ecosystem are best explained, not by any all-inclusive community mechanism, but through the agglomerative contributions of a variety of population and community functions.