USE OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACERS FOR PHYLOGENETIC STUDIES IN DIATOMS1

Sequence variation of ribosomal DNA internal transcribed spacers (ITS) among populations, species, and genera of the diatom genus Stephanodiscus was investigated. ITS 1 and ITS 2, including the 5.8S gene, were sequenced from geographically distant and nearby populations of S. niagarae Ehrenberg. In addition, repeats from S. hantzschii Grunow and Cyclotella meneghiniana Kützing were sequenced to determine the taxonomic range over which the ITS region could be used for diatom systematics. The morphologically distinct S. yellowstonensis Theriot & Stoermer, thought to have evolved from S. niagarae in Yellowstone Lake between 12,000 and 8000 years ago, also was sequenced to assess its relationship to nearby S. niagarae populations. The organization and relative sizes of ITS 1 and ITS 2 in Stephanodiscus species were similar to those reported for other eukaryotes. In general, ITS 2 was slightly larger and more variable than ITS 1. Cladistic analysis of ITS sequences did not resolve relationships of nearby S. niagarae and S. yellowstonensis populations. However, central North American S. niagarae populations were in a clade supported by two nucleotide changes. For Cyclotella, much of the ITS region was not alignable with that for Stephanodiscus species; therefore, generic-level comparison within the Thalassiosiraceae may not be possible. The variation (95–96% similarity) between S. hantzschii and other Stephanodiscus species suggests that interspecific relationships could be assessed with ITS sequences. Although S. yellowstonensis is morphologically distinct from S. niagarae, no autapomorphic nucleotide sites were identified. Two S. niagarae populations (Heart and Lewis Lakes), however, did possess autapomorphic ITS sites.     

Bi-directional inflorescence development inArabidopsis thaliana: Acropetal initiation of flowers and basipetal initiation of paraclades

In this study we investigated Arabidopsis thaliana (L.) Heynh. inflorescence development by characterizing morphological changes at the shoot apex during the transition to flowering. Sixteen-hour photoperiods were used to synchronously induce flowering in vegetative plants grown for 30 d in non-inductive 8-h photoperiods. During the first inductive cycle, the shoot apical meristem ceased producing leaf primordia and began to produce flower primordia. The differentiation of paraclades (axillary flowering shoots), however, did not occur until after the initiation of multiple flower primordia from the shoot apical meristem. Paraclades were produced by the basipetal activation of buds from the axils of leaf primordia which had been initiated prior to photoperiodic induction. Concurrent with the activation of paraclades was the partial suppression of paraclade-associated leaf primordia, which became bract leaves. The suppression of bract-leaf primordia and the abrupt initiation of flower primordia during the first inductive photoperiod is indicative of a single phase change during the transition to flowering in photoperiodically induced Arabidopsis. Morphogenetic changes characteristic of the transition to flowering in plants grown continuously in 16-h photoperiods were qualitatively equivalent to the changes observed in plants which were photoperiodically induced after 30 d. These results suggest that Arabidopsis has only two phases of development, a vegetative phase and a reproductive phase; and that the production of flower primordia, the differentiation of paraclades from the axils of pre-existing leaf primordia and the elongation of internodes all occur during the reproductive phase.     

Effects of Ca(II) ions on Mn(II) dynamics in chick glia and rat astrocytes: Potential regulation of glutamine synthetase

Previous studies have demonstrated that in glia and astrocytes Mn(II) is distributed with ca. 30–40% in the cytoplasm, 60–70% in mitochondria. Ca(II) ions were observed to alter both the flux rates and distribution of Mn(II) ions in primary cultues of chick glia and rat astrocytes. External (influxing) Ca(II) ions had the greatest effect on Mn(II) uptake and efflux, compared to internal (effluxing) or internal-external equilibrated Ca(II) ions. External (influxing) Ca(II) ions inhibited the net rate and extent of Mn(II) uptake but enhanced Mn(II) efflux from mitochondria. These observations differ from Ca(II)–Mn(II) effects previously reported with brain (neuronal) mitochondria. Overall, increased cytoplasmic Ca(II) acts to block Mn(II) uptake and enhance Mn(II) release by mitochondria, which serve to increase the cytoplasmic concentration of free Mn(II). A hypothesis is presented involving external L-glutamate acting through membrane receptors to mobilize cell Ca(II), which in turn causes mitochondrial Mn(II) to be released. Because the concentration of free cytoplasmic Mn(II) is poised near the K_d for Mn(II) with glutamine synthetase, a slight increase in cytoplasmic Mn(II) will directly enhance the activity of glutamine synthetase, which catalyzes removal of neurotoxic glutamate and ammonia.     

Clutch size, offspring performance, and intergenerational fitness

It is now generally recognized that clutch size affects morethan offspring number. In particular, clutch size affects asuite of traits associated with offspring reproductive performance.Optimal clutch size is therefore determined not by the numericallymost productive clutch but by the clutch that maximizes collectiveoffspring reproductive success. Calculation of optimal clutchsize thus requires a consideration of ecological factors operatingduring an intergenerational time frame, spanning the lifetimeof the egglaying adult and the lifetimes of her offspring. Theoptimal clutch cannot define reproductive values in advance,but instead requires that the strategy chosen is the best responseto the set of reproductive values that it itself generates.In this article, we introduce methods for solving this problem,based on an iterative solution of the equation characterizingexpected lifetime reproductive success. We begin by consideringa semelparous organism, in which case lifetime reproductivesuccess is a function only of the state of the organism. Foran iteroparous organism, lifetime reproductive success dependsupon both state and time, so that our methods extend the usualstochastic dynamic programming approach to the evaluation oflifetime reproductive success. The methods are intuitive andeasily used. We consider both semelparous and iteroparous organisms,stable and varying environments, and describe how our methodscan be employed empirically.     

16S rDNA analysis reveals phylogenetic diversity among the polysaccharolytic clostridia

Abstract Small subunit rDNA sequences were determined for 13 mesophilic, polysaccharolytic, mainly cellulolytic species of the genus Clostridium and one cellulolytic Eubacterium specues. Sequences were compared to those of 36 representatives of mesophilic and thermophilic clostridia, including those of nine thermophilic polysaccharolytic species published previously. The majority of strains group with 23S rRNA clusters I and III, while the others group with the thermophilic polysaccharolytic clostridia, i.e. C. stercorarium, C. thermolacticum and C. thermocellum . Lack of close genetic relationships between the various polysaccharolytic species is unexpected and may indicate that these biotechnologically important organisms differ with respect to the enzymology of polysaccharolytic degradation as well.     

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

PHYLOGENY OF THE ULVOPHYCEAE (CHLOROPHYTA): CLADISTIC ANALYSIS OF NUCLEAR-ENCODED rRNA SEQUENCE DATA1

Cladistic analysis of nuclear-encoded rRNA sequence data provided us with the basis for some new hypotheses of relationships within the green algal class Ulvophyceae. The orders Ulotrichales and Ulvales are separated from the clade formed by the remaining orders of siphonous and siphonocladous Ulvophyceae (Caulerpales, Siphonocladales /Cladophorales [S/C] complex, and the Dasycladales), by the Chlorophyceae and Pleurastrophyceae. Our results suggest that the Ulvophyceae is not a monophyletic group. Examination of inter- and intra-ordinal relationships within the siphonous and siphonocladous ulvophycean algae revealed that Cladophora, Chaetomorpha, Anadyomene, Microdictyon, Cladophoropsis and Dictyosphaeria form a clade. Thus the hypothesis, based on ultrastructural features, that the Siphonocladales and Cladophorales are closely related is supported. Also, the Caulerpales is a monophyletic group with two lineages; Caulerpa, Halimeda, and Udotea comprise one, and Bryopsis and Codium comprise the other. The Dasycladales (Cymopolia and Batophora) also forms a clade, but this clade is not inferred to be the sister group to the S/C complex as has been proposed. Instead, it is either the sister taxon to the Caulerpales or basal to the Caulerpales and S/C clade The Trentepohliales is also included at the base of the siphonous and siphonocladous ulvophycean clade. The Pleurastrophyceae, which, like the Ulvophyceae, posses a counter-clockwise arrangement of flagellar basal bodies, are more closely related to the Chlorophyceae than to the Ulvophyceae based on rRNA sequences. Thus, the arrangement of basal bodies does not diagnose a monophyletic group. Previously reported hypotheses of phylogenetic relationships of ulvophycean algae were tested. In each case, additional evolutionary steps were required to obtain the proposed relationships. Relationships of ulvophycean algae to other classes of green algae are discussed.     

Simultaneous addition of EGF prolongs the increase in cytosolic free calcium seen in response to bradykinin in NRK-49F cells

The calcium-sensitive fluorescent indicator fura-2 and a microscope equipped for rapidly changing excitation wavelengths were used to look at the effects of growth factors on cytosolic free calcium ([Ca²⁺]_i,) in NRK-49F cells. In these cells bradykinin induced a rapid increase in [Ca²⁺]_i, which generally decayed to near basal [Ca²⁺]_i within 3 minutes. The initial rise in [Ca²⁺]_i in response to bradykinin was relatively independent of extracellular calcium; however, the decay to basal [Ca²⁺]_i was more rapid in the absence of extracellular calcium. Measurements made on individual cells showed a heterogeneity in the response to bradykinin. Epidermal growth factor (EGF) had no effect on [Ca²⁺]_i in NRK-49F cells when added alone in the presence of extracellular calcium. Simultaneous addition of bradykinin and EGF produced a more prolonged increase in [Ca²⁺]_i than bradykinin alone. The prolongation was dependent on the presence of extracellular calcium and did not occur in its absence. Transient increases in [Ca²⁺]_i occurring after the initial peak were occasionally seen in these cells. Our results indicate that there is rapid interaction between the signaling mechanisms for bradykinin and EGF. When this occurs, one effect is the transport of calcium into the cell from the extracellular environment, causing a more prolonged rise in [Ca²⁺]_i. This effect occurs within 1 minute after combined addition of bradykinin and EGF.     

The ecosystem approach to environmental assessment: moving from theory to practice

The ecosystem approach to environmental management is viewed by many as being fundamental to the development of appropriate management strategies. While this approach represents a major advance in the way researchers view environmental assessment, the approach in itself does not provide practical information as to what questions to ask and what tools to use in assessing and managing ecosystems. Similarly, the concept of ecosystem health, as it is usually defined, has little practical value for ecosystem managers. We suggest the next stage in environmental assessment will be the development of specific frameworks designed to assess individual ecosystems. Of primary importance is the need to consider the basic structure and function of the ecosystem itself. Such consideration, together with explicit identification of anthropogenic stresses particular to the system, serves to identify those components most at risk and those issues most deserving of attention. Researchers should explore critical linkages between environmental stressors and their observable, measurable and predictable effects on ecological parameters and use this understanding to develop a management strategy that incorporates appropriate ecological indicators. The importance of these considerations will be illustrated using examples from the Northern River Basins Study.     

Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy

Summary The growth factor receptor-bound protein-2 (Grb2) is an adaptor protein that mediates signal transduction pathways. Chemical shift assignments were obtained for the SH2 domain of Grb2 by heteronuclear NMR spectroscopy, employing the uniformly ¹³C-/¹⁵N-enriched protein as well as the protein containing selectively ¹⁵N-enriched amino acids. Using the Chemical Shift Index (CSI) method, the chemical shift indices of four nuclei, ¹H, ¹³C, ¹³C and ¹³CO, were used to derive the secondary structure of the protein. Nuclear Overhauser enhancements (NOEs) were then employed to confirm the secondary structure. The CSI results were compared to the secondary structural elements predicted for the Grb2 SH2 domain from a sequence alignment [Lee et al. (1994) Structure, 2, 423–438]. The core structure of the SH2 domain contains an antiparallel -sheet and two -helices. In general, the secondary structural elements determined from the CSI method agree well with those predicted from the sequence alignment.Abbreviations crk viral p47^gag-crk - EGF epidermal growth factor - GAP GTPase-activating protein - PI3K phosphatidylinositol-3-kinase - PLC- phospholipase-C-, shc, src homologous and collagen - src sarcoma family of nonreceptor tyrosine kinase