A NON-CLEAVABLE UmuD VARIANT THAT ACTS AS A UmuD' MIMIC

UmuD{sub 2} cleaves and removes its N-terminal 24 amino acids to form UmuD'{sub 2}, which activates UmuC for its role in UV-induced mutagenesis in E. coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD has been shown to bind to the beta processivity clamp (''beta'') of the replicative DNA polymerase, pol III. A possible beta-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with beta. We performed alanine-scanning mutagenesis of this motif (14-TFPLF-18) in UmuD and showed that it has a moderate influence on UV-induced mutagenesis but is required for the cold sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the beta-binding motif variant bind to beta with similar K{sub d} values as determined by changes in tryptophan fluorescence. However, this data also implies that the single tryptophan in beta is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is noncleavable, we find that cells harboring it exhibit phenotypes more consistent with the cleaved form UmuD', such as resistance to killing by UV light and failure to exhibit the cold sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD' mimic

Selective disruption of the DNA polymerase III α–β complex by the umuD gene products

DNA polymerase III (DNA pol III) efficiently replicates the Escherichia coli genome, but it cannot bypass DNA damage. Instead, translesion synthesis (TLS) DNA polymerases are employed to replicate past damaged DNA; however, the exchange of replicative for TLS polymerases is not understood. The umuD gene products, which are up-regulated during the SOS response, were previously shown to bind to the α, β and ε subunits of DNA pol III. Full-length UmuD inhibits DNA replication and prevents mutagenic TLS, while the cleaved form UmuD′ facilitates mutagenesis. We show that α possesses two UmuD binding sites: at the N-terminus (residues 1–280) and the C-terminus (residues 956–975). The C-terminal site favors UmuD over UmuD′. We also find that UmuD, but not UmuD′, disrupts the α–β complex. We propose that the interaction between α and UmuD contributes to the transition between replicative and TLS polymerases by removing α from the β clamp

Asymmetric response of forest and grassy biomes to climate variability across the African Humid Period : influenced by anthropogenic disturbance?

A comprehensive understanding of the relationship between land cover, climate change and disturbance dynamics is needed to inform scenarios of vegetation change on the African continent. Although significant advances have been made, large uncertainties exist in projections of future biodiversity and ecosystem change for the world's largest tropical landmass. To better illustrate the effects of climate–disturbance–ecosystem interactions on continental‐scale vegetation change, we apply a novel statistical multivariate envelope approach to subfossil pollen data and climate model outputs (TraCE‐21ka). We target paleoenvironmental records across continental Africa, from the African Humid Period (AHP: ca 14 700–5500 yr BP) – an interval of spatially and temporally variable hydroclimatic conditions – until recent times, to improve our understanding of overarching vegetation trends and to compare changes between forest and grassy biomes (savanna and grassland). Our results suggest that although climate variability was the dominant driver of change, forest and grassy biomes responded asymmetrically: 1) the climatic envelope of grassy biomes expanded, or persisted in increasingly diverse climatic conditions, during the second half of the AHP whilst that of forest did not; 2) forest retreat occurred much more slowly during the mid to late Holocene compared to the early AHP forest expansion; and 3) as forest and grassy biomes diverged during the second half of the AHP, their ecological relationship (envelope overlap) fundamentally changed. Based on these asymmetries and associated changes in human land use, we propose and discuss three hypotheses about the influence of anthropogenic disturbance on continental‐scale vegetation change

Genomic organisation of the Mal d 1 gene cluster on linkage group 16 in apple

European populations exhibit progressive sensitisation to food allergens, and apples are one of the foods for which sensitisation is observed most frequently. Apple cultivars vary greatly in their allergenic characteristics, and a better understanding of the genetic basis of low allergenicity may therefore allow allergic individuals to increase their fruit intake. Mal d 1 is considered to be a major apple allergen, and this protein is encoded by the most complex allergen gene family. Not all Mal d 1 members are likely to be involved in allergenicity. Therefore, additional knowledge about the existence and characteristics of the different Mal d 1 genes is required. In the present study, we investigated the genomic organisation of the Mal d 1 gene cluster in linkage group 16 of apple through the sequencing of two bacterial artificial chromosome clones. The results provided new information on the composition of this family with respect to the number and orientation of functional and pseudogenes and their physical distances. The results were compared with the apple and peach genome sequences that have recently been made available. A broad analysis of the whole apple genome revealed the presence of new genes in this family, and a complete list of the observed Mal d 1 genes is supplied. Thus, this study provides an important contribution towards a better understanding of the genetics of the Mal d 1 family and establishes the basis for further research on allelic diversity among cultivars in relation to variation in allergenicity