Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme.     

Cultivation of the sapropelic ciliate Plagiopyla nasuta Stein and isolation of the endosymbiont Methanobacterium formicicum

The sapropelic ciliate Plagiopyla nasuta was isolated and cultured in monoculture. Optimal conditions for growth were: 15–20°C, pH about 7, and about 2% of oxygen in the headspace. Cultures of P. nasuta produced methane. Epifluorescence microscopy revealed the presence of methanogenic bacteria as endosymbionts. An endosymbiont of the ciliate was isolated and identified as Methanobacterium formicicum. In the ciliate cell these methanogens were found to be closely associated with microbody-like organelles. No mitochondria could be detected.     

Influence of template primary and secondary structure on the rate and fidelity of DNA synthesis

High resolution gel electrophoresis was used to monitor the successive addition of dNMP residues onto the 3'-OH ends of discrete 5'-32P-primers, during DNA synthesis on natural templates. Resulting autoradiographic banding patterns revealed considerable variation in the relative rates of incorporation at different positions along the template. The pattern of "pause sites" along the template was unique for each of three different DNA polymerases (polymerase I (the "large fragment" form of Escherichia coli), T4 polymerase (encoded by bacteriophage T4), and AMV polymerase (DNA polymerase of avian myeloblastosis virus]. Most pause sites were not caused by attenuation of polymerization at regions of local secondary structure in the template. Assays of the accuracy of incorporation at different positions along the template (in which elongation was monitored in the presence of only 3 of the 4 2'-deoxynucleoside 5'-triphosphates) strongly suggested that the relative fidelity of DNA synthesis catalyzed by different polymerases depends on the position on the template at which the comparison is made. Primer-templates were constructed that permitted comparison of elongation during synthesis on a single-stranded template with that during polymerization through a double-stranded region (wherein elongation required concomitant displacement of a strand annealed adjacent to the 5'-32P-primer). Although strand displacement DNA synthesis catalyzed by polymerase I occurred approximately ten times more slowly than synthesis in the same region of a single-stranded viral template, most of the pause sites were the same in the presence or absence of "tandem" primer. Electrophoretic assays of the fidelity of DNA synthesis suggested that an increased tendency toward misincorporational "hotspots" occurred when elongation required concomitant strand displacement.     

Template-dependent variation in the relative fidelity of DNA polymerase I of Escherichia coli in the presence of Mg2+ versus Mn2+

The fidelity of E. coli DNA polymerase I in the presence of Mg2+ vs Mn2+ was examined at many positions along natural DNA templates, by use of an electrophoretic assay of misincorporation. Although there was an overall greater tendency for misincorporation to occur in Mn2+-activated chain elongation, some specific sites on the template were more prone to misincorporation with Mg2+ and others with Mn2+. This sequence-dependent effect was seen in spite of the finding that the relative rate of incorporation of the correct nucleotide at different positions on the template was essentially the same with Mg2+ and Mn2+. In agreement with previous studies, the fidelity of E. coli pol I was higher at activating, than at inhibiting, concentrations of Mg2+. The results reveal new complexities regarding the role of divalent cation in the control of fidelity in DNA synthesis and attest to the dynamic nature of interactions between DNA polymerase, its substrates and divalent metal activator during the course of polymerization on natural templates.     

Life‐history constraints in grassland plant species: a growth‐defence trade‐off is the norm

Plant growth can be limited by resource acquisition and defence against consumers, leading to contrasting trade‐off possibilities. The competition‐defence hypothesis posits a trade‐off between competitive ability and defence against enemies (e.g. herbivores and pathogens). The growth‐defence hypothesis suggests that strong competitors for nutrients are also defended against enemies, at a cost to growth rate. We tested these hypotheses using observations of 706 plant populations of over 500 species before and following identical fertilisation and fencing treatments at 39 grassland sites worldwide. Strong positive covariance in species responses to both treatments provided support for a growth‐defence trade‐off: populations that increased with the removal of nutrient limitation (poor competitors) also increased following removal of consumers. This result held globally across 4 years within plant life‐history groups and within the majority of individual sites. Thus, a growth‐defence trade‐off appears to be the norm, and mechanisms maintaining grassland biodiversity may operate within this constraint.     

Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster

Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5′→3′ direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences. All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 °C independent of the substrate analyzed (IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 °C) and PPN (35–40 °C). A potential role for NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform in developing embryos and in germinating seeds, as judged from an analysis of β-glucuronidase reporter gene expression under the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development rather than during stratification. Received: 13 May 2000 / Accepted: 14 August 2000     

Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems

The cycles of the key nutrient elements nitrogen (N) and phosphorus (P) have been massively altered by anthropogenic activities. Thus, it is essential to understand how photosynthetic production across diverse ecosystems is, or is not, limited by N and P. Via a large-scale meta-analysis of experimental enrichments, we show that P limitation is equally strong across these major habitats and that N and P limitation are equivalent within both terrestrial and freshwater systems. Furthermore, simultaneous N and P enrichment produces strongly positive synergistic responses in all three environments. Thus, contrary to some prevailing paradigms, freshwater, marine and terrestrial ecosystems are surprisingly similar in terms of N and P limitation.