Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Fungal populations of the haulm base of wheat

Abstract In a 2-year field survey, the phylloplane mycoflora on the haulm base of wheat was studied by the dilution plate technique. No significant colonisation was registered before the end of May. Cladosporium spp., 'white' and 'red' yeasts were found to be the most abundant fungi.     

Effects of grazing intensity and grazing exclusion on litter decomposition in the temperate steppe of Nei Mongol,China

Aims Grazing intensity and grazing exclusion affect ecosystem carbon cycling by changing the plant community and soil micro-environment in grassland ecosystems. The aims of this study were: 1) to determine the effects of grazing intensity and grazing exclusion on litter decomposition in the temperate grasslands of Nei Mongol; 2) to compare the difference between above-ground and below-ground litter decomposition; 3) to identify the effects of precipitation on litter production and decomposition. Methods We measured litter production, quality, decomposition rates and soil nutrient contents during the growing season in 2011 and 2012 in four plots, i.e. light grazing, heavy grazing, light grazing exclusion and heavy grazing exclusion. Quadrate surveys and litter bags were used to measure litter production and decomposition rates. All data were analyzed with ANOVA and Pearson’s correlation procedures in SPSS. Important findings Litter production and decomposition rates differed greatly among four plots. During the two years of our study, above-ground litter production and decomposition in heavy-grazing plots were faster than those in light-grazing plots. In the dry year, below-ground litter production and decomposition in light-grazing plots were faster than those in heavy-grazing plots, which is opposite to the findings in the wet year. Short-term grazing exclusion could promote litter production, and the exclusion of light-grazing could increase litter decomposition and nutrient cycling. In contrast, heavy-grazing exclusion decreased litter decomposition. Thus, grazing exclusion is beneficial to the restoration of the light-grazing grasslands, and more human management measures are needed during the restoration of heavy-grazing grasslands. Precipitation increased litter production and decomposition, and below-ground litter was more vulnerable to the inter-annual change of precipitation than above-ground litter. Compared to the light-grazing grasslands, heavy-grazing grasslands had higher sensitivity to precipitation. The above-ground litter decomposition was strongly positively correlated with the litter N content (R² = 0.489, p < 0.01) and strongly negatively correlated with the soil total N content (R² = 0.450, p < 0.01), but it was not significantly correlated with C:N and lignin:N. Below-ground litter decomposition was negatively correlated with the litter C (R² = 0.263, p < 0.01), C:N (R² = 0.349, p < 0.01) and cellulose content (R² = 0.460, p < 0.01). Our results will provide a theoretical basis for ecosystem restoration and the research of carbon cycling.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Aggregating land use quantity and intensity to link water quality in upper catchment of Miyun Reservoir

Eutrophication resulting from nutrient enrichment decreases water quality and harms ecosystem structure and function, and its degree is significantly affected by land use in the catchment. Quantifying the relationship between eutrophication and land use can help effectively manage land use to improve water quality. Previous studies principally utilized land use quantity as an indicator to link water quality parameters, but these studies lacked insight into the impact of land use intensity. Taking the upper catchment of Miyun Reservoir as a case study, we developed a method of aggregating land use quantity and intensity to build a new land use indicator and tested its explanatory power on water quality. Six nutrient concentrations from 52 sub-watersheds covering the whole catchment were used to characterize the spatial distribution of eutrophication. Based on spatial techniques, empirical conversion coefficients, remote sensing data, and socio-economic statistical data, land use intensity was measured and mapped visually. The new land use indicator was calculated and linked to nutrient concentrations by Pearson correlation coefficients. Results demonstrated that our new indicator incorporating intensity information can quantify the different nutrient-exporting abilities of different land use areas. Compared to traditional indicators that only incorporate land use quantity, most Pearson correlation coefficients between the new indicator and water nutrient concentrations increased. This new information enhanced the explanatory power of land use on water nutrient concentrations, and so will be able to help us understand the impact of land use on water quality and guide decision making for better land use management.     

Competitive and facilitative relationships among three shrub species,and the role of browsing intensity and rooting depth in the Succulent Karoo,South Africa

Abstract. The nearest‐neighbour technique is used to infer competition and facilitation between the three most abundant species in a semi‐arid region of western South Africa. Relationships among the shrubs Leipoldtia schultzei and Ruschia robusta, which are leaf‐succulent members of the Mesembryanthemaceae (‘mesembs’) and Hirpicium alienatum a non‐succulent Asteraceae, were compared on two adjacent sites with different histories of browsing intensity. Competition was more prevalent and more important than facilitation. The only evidence for facilitation was found at the heavily‐browsed site where the palatable Hirpicium was larger under the unpalatable Leipoldtia. Generally the prevalence and importance of competition was reduced at the heavily‐browsed site. Strong evidence was obtained for intraspecific competition in each of the three species; also, competition was evident between the two mesembs, where Leipoldtia was competitively dominant over Ruschia, although neither species inhibited Hirpicium. Minimal competition between the mesembs and the asteraceous shrub was interpreted in terms of differentiation in rooting depth, and competition within the mesembs, in terms of overlap in rooting depth. The mesembs had the bulk of their roots in the top 5 cm of soil, while the asteraceous shrub had the bulk of its roots, and all its fine roots, at greater depths. The shallow‐rooted morphology of the mesembs is well adapted to utilize small rainfall events, which occur frequently in the Succulent Karoo, and do not penetrate the soil deeply. Modifications of existing methods are applied for analysing nearest‐neighbour interactions.