Low-level inversion of the L component of pseudorabies virus is not dependent on sequence homology.

Pseudorabies virus has a class 2 genome in which the S component is found in two orientations relative to the L component. The L component is bracketed by sequences that are partially homologous; it is found mainly in one orientation, but a small proportion is inverted (J. M. DeMarchi, Z. Lu, G. Rall, S. Kuperschmidt, and T. Ben-Porat, J. Virol. 64:4968-4977, 1990). We have ascertained the role of the patchy homologous sequences bracketing the L component in its inversion. A viral mutant, vYa, from which the sequences at the right end of the L component were deleted was constructed. Despite the absence of homologous sequences bracketing the L component in vYa, its L component inverted to an extent similar to that of the L component in the wild-type virus. These results show the following. (i) The low-frequency inversion of the L component of PrV is not mediated by homologous sequences bracketing this component. (ii) Cleavage of concatemeric DNA at the internal junction between the S and L components is responsible for the appearance of the minority of genomes with an inverted L component in populations of pseudorabies virus. (iii) The signals present near or at the end of the S component are sufficient to allow low-frequency cleavage of concatemeric DNA; the sequences at the end of the L component are not essential for cleavage, although they enhance it considerably.     

Effect of medicating ingredients and other additives on microflora present on swine and chicken feeds from a Manitoba mill

The occurrence of microfloral components on medicated and non-medicated swine and chicken feed pellets and crumbles, produced in a Manitoba feed mill between June 1991 and October 1992, was determined. Addition of medicates to feeds generally decreased bacterial incidence and increased that of Eurotium spp. fungi. The effect was less pronounced when copper sulphate was added to medicated swine feeds.Contribution No. 1662.     

Functional Analysis of the Intrathalline and Intracellular Chlorophyll Concentrations in the Lichen Family Umbilicariaceae

Intrathalline and intracellular chlorophyll concentrations togetherwith thallus structure were studied in 12 species of the lichenfamily Umbilicariaceae in order to explore pigment limitationsfor light absorption and for maximum rates of net photosynthesis(A_max). Species from high light environments tended to haverelatively low chlorophyll concentrations and vice versa. Chlorophyllconcentration on a surface area basis of all species studiedwas sufficient to absorb 80–99% of the incident light,which led us to believe that chlorophyll concentration was nota limiting factor for A_max. A_maxof Umbilicariaceae was smallerthan A_maxof leaves of vascular plants, both on a dry weightand on a surface area basis. These differences slightly decreasedbut did not disappear when referred to the chlorophyll concentrationof the photosynthetic tissues, but reference units had a confoundingeffect in the comparisons. Differences in A_maxon a dry weightbasis between Umbilicariaceae and vascular plants disappearedwhen comparisons were made with the whole plant and not simplywith the leaf. We found a significant, inverse relationshipbetween amount of alga and chlorophyll concentration withinthe algal cells in the lichens studied. This was interpretedas a mechanism to avoid pigment self-shading within the algallayer, distributing more efficiently the photosynthetic pigmentsover a given surface area, and is the first indication of sucha mechanism in lichens. It is suggested that thallus structureand physiology have a larger influence on the observed low A_maxofthe lichens studied than chlorophyll content. Algal cells; chlorophyll concentration; lichens; light harvesting; net photosynthesis; stereology; thallus structure; Umbilicariaceae     

Identifying differences in mRNA expression by representational difference analysis of cDNA.

Detection of differentially regulated genes has been severely hampered by technical limitations. In an effort to overcome these problems, the PCR-coupled subtractive process of representational difference analysis (RDA) [Lisitsyn, N. et al. (1993) Science 259, 946-951] has been adapted for use with cDNA. In a model system, RAG-1 and RAG-2, the genes responsible for activating V(D)J recombination, were identified in a genomic transfectant by cDNA RDA in a small fraction of the time taken by conventional means. The system was also modified to eliminate expected difference products to facilitate the identification of novel genes. Additional alterations to the conditions allowed isolation of differentially expressed fragments. Several caffeine up-regulated clones were obtained from the pre-B cell line 1-8, including IGF-1B, and a predicted homologue of the natural killer cell antigen, NKR-P1. The approach was found to be fast, extremely sensitive, reproducible, and predominantly lacked false positives. cDNA RDA has the capacity and adaptability to be applied to a wide range of biological problems, including the study of single gene disorders, characterization of mutant and complemented cell types, developmental or post-event expression time courses, and examination of pathogen-host interactions.     

Theory, design, and characterization of a microdialysis flow cell for Raman spectroscopy.

The theory, design, and application of a dialysis flow cell for Raman spectroscopy are described. The flow cell permits rapid collection of Raman spectra concurrent with the efflux of small solute molecules or ions into a solution of macromolecules and is well suited to acquisition of data during hydrogen-isotope exchange reactions of biological molecules. Kinetic parameters of the device are described by a diffusion model, which accounts satisfactorily for the observed rates of efflux of deuterium oxide (K2H = 0.30 min-1), calcium ions (KCa = 0.10 min-1) and EGTA (KEGTA = 0.07 min-1). Application to the kinetics of glutamate protonation in a peptide copolymer [poly(Glu, Lys, Tyr)] shows that pH-titration rates as high as 3.3 pH units/min can be monitored. It is also shown that one can extract first-order hydrogen-isotope exchange rate constants from measured second-order exchanges by taking into account the rate of entry of 2H2O effluent into the bulk H2O solution. Deuterium exchanges of the single-stranded polyribonucleotides poly(rA) and poly(rU) and of the double-stranded RNA genome from bacteriophage phi 6 have been investigated. The measured nucleotide base exchange rates are comparable with those determined previously by other methods. The results indicate that base exchanges as fast as approximately 2 min-1 can be determined reliably with the present design. Application of the Raman flow cell to hydrogen-isotope exchange of the basic pancreatic trypsin inhibitor confirms consistency with results obtained previously on this protein by tritiation and NMR techniques.     

Effects of nitrogen limitation on water relations of jack pine (Pinus banksiana Lamb.) seedlings

The water relations of shoots of young jack pine (Pinus banksiana Lamb.) seedlings were examined 6 and 15 weeks after the initiation of four different dynamic nitrogen (N) treatments using a pressure-volume analysis. The N treatments produced a wide range of needle N concentrations from 12 to 32 mg g^?1 dry mass and a 10-fold difference in total dry mass at 15 weeks. Osmotic potential at full turgor did not change over the range of needle N concentrations observed. Osmotic potential at turgor-loss point, however, declined as N concentrations decreased, indicating an increased ability of N-deficient jack pine plants to maintain turgor. The increase could be attributed largely to an increase in cell wall elasticity, suggesting that elasticity changes may be a common, significant adaptation of plants to environmental stresses. Dry mass per unit saturated water almost doubled as needle N level dropped from 32 to 12 mg g^?1 and was inversely correlated to the bulk modulus of elasticity. This suggests that cell wall elasticity is determined more by the nature of its cross-linking matrix than by the total amount of cell wall material present. Developmental change was evident in the response of some water relation variables to N limitation.