A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation.

The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists. Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids. To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively. The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine. In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 273, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. However, the mutated protein is instead activated specifically by succinate. Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them.     

Use of EDTA to prevent spontaneous cell-to-cell transformation provides a more accurate estimation of gene transfer frequencies

Summary Spontaneous cell-to-cell transformation between naturally competent bacteria on selective media resulted in an overestimation of the transferability of genetic information. EDTA effectively prevented transformation on selective media whereas DNaseI did not reliably inhibit cell-to-cell transformation. An improved method to estimate gene transfer frequencies is described.     

The effect of behavioural and morphological plasticity on foraging efficiency in the threespine stickleback (Gasterosteus sp.)

We conducted an experiment to assess the change in foraging efficiency resulting from diet-induced morphological and behavioural plasticity in a species of freshwater, threespine stickleback (Gasterosteus sp.). Different degrees of morphological and behavioural change were induced using two prey items commonly found in the diet of this species, allowing us to estimate the relative importance of each type of plasticity. The purpose of the experiment was twofold. First, earlier work had suggested that diet variability might be an important factor in the evolution of trophic morphological plasticity in sticklebacks. The present results extend this work by revealing the adaptive significance of morphological plasticity. The current experiment also qualitatively assessed the compatibility of the time scale of morphological change with that of the natural resource variability experienced by this species. The results indicate that diet-induced plasticity improves foraging efficiency continuously for up to 72 days of prey exposure. This is probably due in part to plasticity of the external trophic morphology but our results also suggest a complex interplay between morphology and behaviour. The time scale appears to be matched to that of natural diet variability although it is possible that some traits exhibit non-labile plasticity. Our discussion highlights the important distinction between conditions favouring the evolution of labile versus non-labile plasticity. The second objective of the experiment was to determine the relative importance of morphological and behavioural plasticity. Few studies have attempted to quantify the adaptive significance of morphological plasticity and no study to our knowledge has separated the effects of morphological and behavioural plasticity. Our experiment reveals that both behavioural and morphological plasticity are important and it also suggests a dichotomy between the two: behavioural plasticity predominately affects searching efficiency whereas morphological plasticity predominately affects handling efficiency.     

Conservation in structure of TOC1 transposons from Chlamydomonas reinhardtii.

TOC1 transposons from Chlamydomonas reinhardtii have an unusual arrangement of long terminal repeats. Polymorphic regions between TOC1 transposons were identified by restriction mapping on Southern blots. The variation in size of an internal MluI fragment defines two subclasses of TOC1 elements. Full-length cloned members of each subclass of TOC1 element were compared by electron microscope heteroduplex analysis. The cloned elements were co-linear over their entire length with no large sequence discontinuities. Base substitutions and small insertion/deletion events of less than 50 bp are responsible for forming the two subclasses of TOC1 elements.     

Comparison of different methods of determining cell viability after exposure to cytotoxic compounds

Cell-survival (of DON and L1210 cells) after treatment with cytotoxic compounds was assessed by measuring cloning efficiency, exclusion of trypan blue and erythrosin B, [⁵¹Cr] release, and attachment of DON cells to glass. Cell survival as measured by cloning efficiency did not correlate with survival measured by any of the other methods. We found that the stainability of cells after drug exposure depended on the cell line used. For example, after 3 h exposure to tubercidin although 100% of both DON and L1210 cells were killed (on basis of cloning efficiency), only 11% of DON cells and 68% of L1210 cells were dead as indicated by staining with erythrosin B. The stainability of cells also depended on the particular drug used. For example, after 24 h exposure of L1210 cells to adriamycin and tubercidin (both killed >99% of cells on basis of cloning efficiency) 21% of cells exposed to adriamycin and 99% of cells exposed to tubercidin were stained. The results obtained with several other cytotoxic compounds are discussed.     

Exogenous O6-methylguanine inhibits adduct removal and sensitizes human cells to killing by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine

We partially depleted the O6-methylguanine-DNA methyltransferase activity in four O6-methylguanine (O6-mGua) repair-proficient (Mer+) human cell strains with exogenous O6-mGua (2 mM for 3 h, a non-toxic regimen) and then challenged them with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT-partially depleted HT29 cells removed O6-mGua from DNA at about half the rate of control cells, while removal of 3-methyladenine was unaffected. In spite of partial depletion of MT, however, cell killing by MNNG in a colony-forming assay with HT29, A549, A498 or KD cells was not greatly affected. (This is in contrast to the dramatic potentiation of CNU cytotoxicity observed previously.) In an attempt to sensitize Mer+ strains to killing by MNNG, we treated cells with O6-mGua following MNNG exposure (0.4 mM for 4 days), in addition to the pre-MNNG treatment of 2 mM O6-mGua for 3 h. This sensitized KD and HT29 cells 2-fold to killing by MNNG, based on the dose at 10% survival, but did not sensitive Mer- A1336. However, post-treatment alone was as effective as combined pre- and post-treatment in sensitizing KD cells to killing. Thus, when the O6-mGua post-treatment was begun, greater than 50% of O6-mGua was already removed from cell DNA. Our findings may be accounted for by at least two schemes, one in which nonlethal O6-mGua are removed from DNA rapidly, while potentially lethal O6-mGua are repaired later. The other scheme proposes that exogenous O6-mGua increases the lethality of a non-O6-mGua lesion by reducing its repair both in Mer+ and Mer- cells. Both schemes are consistent with the hypothesis that O6-mGua may be a lethal DNA lesion in human cells.