Structure of the KcsA potassium channel from Streptomyces lividans: a site-directed spin labeling study of the second transmembrane segment.

KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure. Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale. Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O(2) or nickel(II) ethylenediaminediacetate is low. This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements. However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature. These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.     

Hydrocarbon-metabolizing activity of various mammalian cells in culture

Summary Two methods for determining the hydrocarbon-metabolizing enzyme activity of cultured mammalian cells were compared. The method designed to measure benzo[a]an-thracene-induced aryl hydrocarbon hydroxylase activity could detect and quantify enzyme activities in low passage rodent cells, but could not reproducibly detect levels in intermediate or high passage mouse, rat, or human cells. The method designed to measure the ability of a cell to convert benzo[a]pyrene from an organic-soluble to an aqueous acetone-soluble form proved to be more reproducible. This technique, when modified, was demonstrated to be an effective screening test for the detection of those lines with higher levels of hydrocarbon-metabolizing enzymes. Supported by the Council for Tobacco Research and Contract NIH 70-2068 within the Virus Cancer Program, National Cancer Institute, National Institutes of Health.     

The Growth Response of the Stems of Genetically Modified Tobacco Plants (Nicotiana tabacum'Samsun') to Flexural Stimulation

Genetically modified tobacco plants (Nicotiana tabacum‘Samsun’)with antisense cinnamyl alcohol dehydrogenase DNA, produce secondaryxylem of a reduced tensile stiffness. These plants were grownalongside control plants. The stems of the plants were flexedor protected from flexing over a period of several weeks. Thetensile moduli and second moments of areas of the differenttissues inside the stems were measured and used to calculatethe bending stiffness of the plants. In tobacco, the cylinderof xylem was found to be the most important tissue in determiningthe bending stiffness of the plants. The thickness of the xylemtissue cylinder increased when plants were subjected to flexuralstimulation. This increased the bending stiffness of the stems.The response to mechanical stimulation was found to be correlatedwith tissue strain and the genetically modified plants wereable to exactly compensate for the reduced modulus of theirxylem tissue by increasing the thickness of the xylem tissuecylinder more than in control plants.Copyright 1999 Annals ofBotany Company. Tobacco plants, stem bending, xylem tissue, second moment of area, thigmomorphogenesis, mechanical strain.     

Sexual receptivity and post-emergence ovarian development in females of Coproica vagans (Diptera: Sphaeroceridae)

Abstract .Males and females of the dung fly species Coproica vagans Haliday 1833 (Diptera: Sphaeroceridae) mate soon after emergence from the puparium. At this time females still have immature ovaries. Therefore, mating precedes vitellogenesis in this species. Data presented here show that mating enhances oogenesis in C. vagans females. Mated females mature their first egg batch sooner and oviposit four days earlier than virgin conspecifics. Mating-related enhancement of oogenesis could be explained either through nutritional benefits to females or male chemical or stimulatory manipulation of the females. Oogenesis was divided into six arbitrary stages, with vitellogenesis beginning in stage 4. Ovarian development beyond stage 4 is rapid compared with pre-vitellogenetic development. Virgin females pause oogenesis in stage 4. The genital opening of mated females is blocked by a mating plug that persists until oviposition begins. The plug seems to ensure the paternity of the last male to mate by preventing females from remating. The operational sex ratio in C. vagans populations is presumed to be strongly male-biased.     

Levels of genetic diversity at different stages of the domestication cycle of interior spruce in British Columbia

Concerns over the reductionist nature of the domestication of forest-tree species focus on the possibility of potential genetic erosion during this process. To address these concerns, genetic diversity assessments in a breeding zone the Province of British Columbia “interior” spruce (Picea glauca×engelmanni) program was conducted using allozyme markers. Genetic-variation comparisons were made between natural and production (seed orchard) populations as well as seed and seedling crops produced from the same breeding zone’s seed orchard. The natural population sample consisted of a total of 360 trees representing three stands within each of three watersheds present in the Shuswap-Adams low-elevation zone of interior British Columbia. Small amounts of genetic differentiation were observed among the nine natural populations (4%) and this was attributable to extensive gene flow Consequently, the sum of these nine populations was considered as a baseline for the genetic variation present in the breeding zone. The comparisons between the seed orchard and the breeding zone produced a similar percentage of polymorphic loci while the expected hetrozygosity (0.207 vs 0.210) and the average number of alleles per locus (2.7 vs 2.4) were slightly lower in the seed orchard. A total of seven natural populations’ rare alleles were not present in the orchard population, while one allele was unique to the orchard. The %P increased to 70.6% in the seedlot, but dropped to the natural populations level (64.7%) in the plantation. The observed increase in %P was a result of pollen contamination in the orchard. It is suspected that the reduction in the plantation was caused by an unintentional selection in the nursery. Simulated roguing in the orchard did not drastically reduce even if up to 50% of the orchard’s clones were rogued. However, roguing was associated with a reduction in the average number of alleles per locus (i.e., sampling effect). Received: 2 January 1996 / Accepted: 24 May 1996