Recombinational repair is critical for survival of Escherichia coli exposed to nitric oxide

Nitric oxide (NO(.)) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO(.)-induced DNA damage on Escherichia coli. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO(.)-induced toxicity was determined. E. coli strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [alkA tag strain], oxidative damage [fpg nei nth strain], and deaminated cytosine [ung strain]) showed essentially wild-type levels of NO(.) resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (xth nfo strain) were very sensitive to killing by NO(.), which indicates that normal processing of abasic sites is critical for defense against NO(.). In addition, recA mutant cells were exquisitely sensitive to NO(.)-induced killing. Both SOS-deficient (lexA3) and Holliday junction resolvase-deficient (ruvC) cells were very sensitive to NO(.), indicating that both SOS and recombinational repair play important roles in defense against NO(.). Furthermore, strains specifically lacking double-strand end repair (recBCD strains) were very sensitive to NO(.), which suggests that NO(.) exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO(.) induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO(.), it is also important to consider recombination events among the spectrum of genetic changes that NO(. ) can induce. Furthermore, the importance of recombinational repair for cellular survival of NO(.) exposure reveals a potential susceptibility factor for invading microbes.     

Escherichia coli mutator genes

The isolation and characterization of Escherichia coli mutator genes have led to a better understanding of DNA replication fidelity mechanisms and to the discovery of important DNA repair pathways and their relationship to spontaneous mutagenesis. Mutator strains in a population of cells can be beneficial in that they allow rapid selection of variants during periods of stress, such as drug exposure.     

Methane emissions from wet grasslands on peat soil in a nature preserve

The area of wet grasslands on peat soil in the Netherlands is slowly increasing at the expense of drained, agriculturally used grasslands. This study aimed (i) to assess the contribution of wet grasslands on peat soil to methane (CH₄) emissions, and (ii) to explain differences among sites and between years in order to improve our understanding of controlling factors. For these purposes, a field study was conducted in the period 1994–1996 in the nature preserve Nieuwkoopse Plassen, which is a former peat mining and agricultural area. Net CH₄ emissions were measured weekly to monthly with vented closed flux chambers at three representative sites, and at ditches near these sites. Three-years average of CH₄ emissions was 7.9 g CH₄ m^–2 yr^–1 for Drie Berken Zudde, 13.3 for Koole, and 20.4 for Brampjesgat. Ditches near the sites emitted 4.2–22.5 g CH₄ m^–2 yr^–1. The time-course of CH₄ emissions for all experimental sites and years was fit with a multiple linear regression model with ground water level and soil temperature as independent variables. Lowering or raising the ground water level by 5 cm could decrease or increase CH₄ emissions by 30–50%. Therefore, ground water level management of these grasslands should be done with care.     

Efficient targeting of plant disease resistance loci using NBS profiling

The conserved sequences in the nucleotide-binding sites of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance (R) genes have been used for PCR-based R-gene isolation and subsequent development of molecular markers. Here we present a PCR-based approach (NBS profiling) that efficiently targets R genes and R-gene analogs (RGAs) and, at the same time, produces polymorphic markers in these genes. In NBS profiling, genomic DNA is digested with a restriction enzyme, and an NBS-specific (degenerate) primer is used in a PCR reaction towards an adapter linked to the resulting DNA fragments. The NBS profiling protocol generates a reproducible polymorphic multilocus marker profile on a sequencing gel that is highly enriched for R genes and RGAs. NBS profiling was successfully used in potato with several restriction enzymes, and several primers targeted to different conserved motifs in the NBS. Across primers and enzymes, the NBS profiles contained 50–90% fragments that were significantly similar to known R-gene and RGA sequences. The protocol was similarly successful in other crops (including tomato, barley, and lettuce) without modifications. NBS profiling can thus be used to produce markers tightly linked to R genes and R-gene clusters for genomic mapping and positional cloning and to mine for new alleles and new sources of disease resistance in available germplasm.Communicated by H.F. Linskens     

Glucocorticoid inhibition of C2C12 proliferation rate and differentiation capacity in relation to mRNA levels of the MRF gene family

The muscle regulatory factors (MRF) gene family regulate muscle fibre development. Several hormones and drugs also affect muscle development. Glucocorticoids are the only drugs reported to have a beneficial effect on muscle degenerative disorders. We investigated the glucocorticoid-related effects on C2C12 myoblast proliferation rate, morphological differentiation, and subsequent mRNA expression patterns of the MRF genes. C2C12 cells were incubated with the glucocorticoids dexamethasone or alpha-methyl-prednisolone. Both glucocorticoids showed comparable effects. Glucocorticoid treatment of C2C12 cells during the proliferative phase reduced the proliferation rate of the cells dose dependently, especially during the third and fourth day of culture, increased MyoD1, myf-5, and MRF4 mRNA levels, and reduced myogenin mRNA level, compared to untreated control cells. Thus, the mRNA level of proliferation-specific MyoD1 and myf-5 expression does not seem to associate with C2C12 myoblast proliferation rate. Glucocorticoid treatment of C2C12 cells during differentiation reduced the differentiation capacity dose dependently, which is accompanied by a dose dependent reduction of myogenin mRNA level, and increased MyoD1, myf-5, and MRF4 mRNA levels compared to untreated control cells. Therefore, we conclude that glucocorticoid treatment reduces differentiation of C2C12 myoblasts probably through reduction of differentiation-specific myogenin mRNA level, while inducing higher mRNA levels of proliferation-associated MRF genes.     

Domains of a Transit Sequence Required for in Vivo Import in Arabidopsis Chloroplasts

Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

R.M. Cowling,D.M. Richardson & S.M. Pierce (eds.) 1977. Vegetation of Southern Africa.

We determined the role of bird dispersal in seed and seedling dynamics of the tree Kalopanax pictus from 7 years of observing seed rain and seedling emergence in a broad-leaved deciduous forest in central Japan. We also performed an experiment on the influence of seed pulp on germination of seeds of K. pictus. Seeds of this species can lie dormant for several years, and this causes rather constant yearly seedling emergence in spite of irregular seed production. The spatial distribution of the seedlings that emerged each year (maximum distance from nearest conspecific seed-bearing tree of 90 m) was wider than that of gravity-dispersed seeds (max. distance of 37 m), suggesting seed dispersal by birds in winter. Emerged seedling densities at sites over 20 m from the nearest conspecific seed-bearing tree were highest in the spring of 1991, about half a year after the largest seed fall of the observation period. However, emerged seedling densities within 20 m from seed-bearing trees were highest in 1992, 1.5 years after the largest seed fall. These field observations may be explained by the experimental results on the effects of seed pulp on germination. Intact seeds germinate slowly at low germination rates, while seeds without seed pulp germinate quickly at high germination rates. Fallen seeds with seed pulp thus appear to form a seed bank near seed sources (temporal dispersal), while seeds scattered by birds appear to increase the possibility of reaching the present safe sites in distant areas with quick germination (spatial dispersal).     

Nitrogen distribution and leaf area indices in relation to photosynthetic nitrogen use efficiency in savanna grasses

Leaf photosynthetic characteristics, distribution patterns of nitrogen content per unit leaf area (n_L) and leaf area production per unit n_Lwere measured in natural stands of a C₄ grass (Hyparrhenia rufa) from the seasonal savannas and of a C₄grass (Paspalum fasciculatum) and two C₃grasses (Leersia hexandra and Hymenachne amplexicaulis) from the flooded savannas in central Venezuela. Daily rates of canopy photosynthesis (P_cD) as well as the optimal leaf area production per unit n_Lat which P_cDfor a given total amount of nitrogen in the canopy (i.e., canopy-PNUE) is maximized were also calculated. The C₃and C₄species from the flooded savannas had similar light saturated rates of photosynthesis per unit n_L(i.e. leaf-PNUE) and similar canopy-PNUEs which was in strong contrast with previous studies. Especially H. rufa but also L. hexandra and H. amplexicaulis had leaf- and canopy-PNUEs which were considerably higher than the values calculated for most other species with the same photosynthetic pathway (i.e., C₃or C₄). In contrast to previous studies, differences in the light gradient in the canopy between stands only partially explained differences in N distribution. Measured leaf area indices were greater and the average n_L values were consequently smaller than the calculated optima. There was, however, a very strong linear correlation between the optimal and actual average n_Lindicating that even though the model overestimated average n_L, it did predict the differences in leaf area production per unit nitrogen – the inverse average n_L– very well. This result strongly indicates that leaf area production per unit of leaf nitrogen increases with leaf-PNUE and decreases with the extinction coefficient for light. Grass species from seasonal savannas have extremely high leaf-PNUEs and thus optimally produce large amounts of leaf area per unit n_L. This helps explain how stands of these species may have high leaf area indices and achieve high photosynthetic productivity despite the very low nutrient availability at which they grow.