Computer-aided gene design.

A computer program, which runs on MS-DOS personal computers, is described that assists in the design of synthetic genes coding for proteins. The goal of the program is the design of a gene which (i) contains as many unique restriction sites as possible and (ii) uses a specific codon usage. The gene designed according to the criteria above is (i) suitable for 'modular mutagenesis' experiments and (ii) optimized for expression. The program 'reverse-translates' protein sequences into degenerated DNA sequences, generates a map of potential restriction sites and locates sequence positions where unique restriction sites can be accommodated. The nucleic acid sequence is then 'refined' according to a specific codon usage to remove any degeneration. Unique restriction sites, if potentially present, can be 'forced' into the degenerated nucleic acid sequence by using 'priority codes' assigned to different restriction sequences.     

Electrostatic coupling of ion pumps.

In this paper the electrostatic interactions between membrane-embedded ion-pumps and their consequences for the kinetics of pump-mediated transport processes have been examined. We show that the time course of an intrinsically monomolecular transport reaction can become distinctly nonexponential, if the reaction is associated with charge translocation and takes place in an aggregate of pump molecules. First we consider the electrostatic coupling of a single dimer of ion-pumps embedded in the membrane. Then we apply the treatment to the kinetic analysis of light-driven proton transport by bacteriorhodopsin which forms two-dimensional hexagonal lattices. Finally, for the case of nonordered molecules, we also consider a model in which the pumps are randomly distributed over the nodes of a lattice. Here the average distance is equal to that deduced experimentally and the elemental size of the lattice is the effective diameter of one single pump. This latter model is applied to an aggregate of membrane-embedded Na, K- and Ca-pumps. In all these cases the electrostatic potential considered is the exact solution calculated from the method of electrical images for a plane membrane of finite thickness immersed in an infinite aqueous solution environment. The distributions of charges (ions or charged binding sites) are considered homogeneous or discrete in the membrane and/or in the external solution. In the case of discrete distributions we compare the results from a mean field approximation and a stochastic simulation.     

Life history patterns of river invertebrates

The initiation of invertebrate distribution patterns in rivers occurs by choice of oviposition sites and is influenced by the evolved reproductive strategies of the individual species. Subsequent redistribution by migration or drifting establishes patterns which are then modified by environmental influences on growth and mortality. Continuity of life cycles is sustained by variations on a number of defined life history strategies combined with evolved behavioural responses.     

Phytoplankton and physical-chemical features of Tavropos Reservoir,Greece

Phytoplankton biomass values in Tavropos Reservoir, ranging from 92 to 1071 mg m^–3, are within a range characteristic of oligotrophic waters. The seasonal sequence of biomass shows three annual peaks, differing from the monoacmic pattern seen in oligotrophic lakes. This sequence was profoundly affected by changes in water withdrawal and inflow rates. Diatoms, cryptophytes, chrysophytes and dinoflagellates, in that order, were the major constituents of the reservoir phytoplankton. The succession, from diatoms and chrysophytes in late winter-spring, to centric diatoms in late spring-summer and again to diatom-chrysophytes in late autumn was similar to that in oligotrophic lakes.     

The relationship between water velocity,energetic costs,and microhabitat use in four North American stream fishes

Freshwater crayfish are key members of aquatic communities due to their large size and abundance. Although most commonly regarded as herbivores and detritivores, they are also selective predators. The crayfish plague fungus Aphanomyces astaci (Schikora) led to the elimination of a stock of white-clawed crayfish, Austropotamobius pallipes (Lereboullet) from Lough Lene, Co. Westmeath, in 1987. Samples taken of the flora and benthic communities of two Irish lakes, one (Lough Bane) formerly containing crayfish and the other (Lough Lene) immediately following a plague outbreak, were compared to similar samples taken a year later and ecological shifts were noted and compared to laboratory feeding results. Over time, Chara strands increased in mean length, and molluscs became more abundant.     

Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump.

In order to understand the molecular mechanism of ouabain resistance in the toad Bufo marinus, Na,K-ATPase alpha and beta subunits have been cloned and their functional properties tested in the Xenopus laevis oocyte expression system. According to sequence comparison between species, alpha 1, beta 1, and beta 3 isoforms were identified in a clonal toad urinary bladder cell line (TBM 18-23). The sequence of the alpha 1 isoform is characterized by two positively charged amino acids (Arg, Lys) at the N-terminal border of the H1-H2 extracellular loop and no charged amino acid at the C terminus, a pattern distinct from the ouabain-resistant rat alpha 1 isoform. The coexpression of alpha 1 beta 1 or alpha 1 beta 3 TBM subunits in the Xenopus oocyte resulted in the expression of identical maximum Na,K-pump currents with identical inhibition constant for ouabain (Ki) (alpha 1 beta 1: 53 +/- 3 microM; n = 7 vs. alpha 1 beta 3: 57 +/- 3.0 microM; n = 8) but distinct potassium half activation constant (K1/2) (alpha 1 beta 1: 0.87 +/- 0.08 mM, n = 16; alpha 1 beta 3: 1.29 +/- 0.07 mM, n = 17; p less than 0.005). We conclude that (i) the TBM alpha 1 isoform is necessary and sufficient to confer the ouabain resistant phenotype; (ii) the beta 3 or beta 1 subunit can associate with the alpha 1 equally well without affecting the ouabain-resistant phenotype; (iii) some specific sequence of the beta subunit can modulate the activation of the Na,K-pump by extracellular potassium ions.     

The P2 element of the td intron is dispensable despite its normal role in splicing.

The P2 region of group I introns has been proposed to be involved in the correct positioning of the P1 5'-splice site duplex in the catalytic core (Michel, F., and Westhof, E. (1990) J. Mol. Biol. 216, 585-610). The behavior of delta P2 deletion mutants of the td intron is consistent with this hypothesis. The delta P2 mutants are capable of site-specific hydrolysis, indicating that the conformation of the ribozyme is not grossly altered, but they are incapable of transesterification reactions at the splice sites, as would be predicted if P1 is not appropriately aligned within the catalytic core. Nevertheless, the function of the P2 element can be bypassed in specific pseudorevertants isolated by genetic selection from the delta P2 mutants. These results, together with phylogenetic data, support the existence of alternate strategies to create a functional P1-core interaction.