Identification of novel high-frequency DNA methylation changes in breast cancer

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.     

Localization of<Emphasis Type="Italic"> jointless-2</Emphasis> gene in the centromeric region of tomato chromosome 12 based on high resolution genetic and physical mapping

Abstract Abscission is a universal process whereby plants shed their organs, such as flowers, fruit and leaves. In tomato, the non-allelic mutations jointless and jointless-2 have been discovered as recessive mutations that completely suppress the formation of pedicel abscission zones. A high resolution genetic map of jointless-2 was constructed using 1,122 jointless F₂ plants. Restriction fragment length polymorphism (RFLP) marker RPD140 completely co-segregated with the jointless-2 locus and mapped in a 2.4 cM interval between RFLP markers CD22 and TG618. To chromosome walk to jointless-2, all three markers were used to screen a bacterial artificial chromosome (BAC) library and contigs were developed. Intensive efforts to expand and merge the BAC contigs were unsuccessful because of the highly repetitive sequence content on the distal ends of each contig. To determine the physical distance between and the orientation of the three contigs, we used high resolution pachytene fluorescence in situ hybridization (FISH) mapping. The RPD140 contig was positioned in the centromeric region of chromosome 12 between two large pericentric heterochromatin blocks, about 50 Mb from the TG618 contig on the short arm and 10 Mb from the CD22 contig on the long arm, respectively. Based on high resolution genetic and physical mapping, we conclude that the jointless-2 gene is located within or near the chromosome 12 centromere where 1 cM is approximately 25 Mb in length.Communicated by Q. ZhangM.A. Budiman, S-B. Chang and S. Lee contributed equally to the work.     

Mechanism of electrogenic cation transport by the cloned organic cation transporter 2 from rat

The organic cation transporter 2 (OCT2) is expressed in plasma membranes of kidney and brain. Its transport mechanism and substrates are debated. We studied substrate-induced changes of electrical current with the patch clamp technique after expression of rat OCT2 in oocytes. Activation of current, corresponding to efflux, was observed for small organic cations, e.g. choline. In contrast, the bigger cations quinine and tetrabutylammonium elicited no change in current. However, transport of choline could be inhibited by applying quinine or tetrabutylammonium to the cytoplasmic side. Inhibition of organic cation efflux by quinine was competitive with substrates. Quinine at the inside also inhibited substrate influx from the outside. Current-voltage analysis showed that both maximal turnover and apparent affinity to substrates are voltage-dependent. Substrate-induced currents with organic cations on both membrane sides reversed as predicted from the Nernst potential. Our results clearly identify the electrochemical potential as driving force for transport at neutral pH and exclude an electroneutral H(+)/organic cation(+) exchange. We suggest the existence of an electroneutral organic cation(+) exchange and propose a model for a carrier-type transport mechanism.     

Sorghum genome sequencing by methylation filtration

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.     

Digital Mapping of Soil Organic Carbon Contents and Stocks in Denmark

Estimation of carbon contents and stocks are important for carbon sequestration, greenhouse gas emissions and national carbon balance inventories. For Denmark, we modeled the vertical distribution of soil organic carbon (SOC) and bulk density, and mapped its spatial distribution at five standard soil depth intervals (0−5, 5−15, 15−30, 30−60 and 60−100 cm) using 18 environmental variables as predictors. SOC distribution was influenced by precipitation, land use, soil type, wetland, elevation, wetness index, and multi-resolution index of valley bottom flatness. The highest average SOC content of 20 g kg⁻¹ was reported for 0−5 cm soil, whereas there was on average 2.2 g SOC kg⁻¹ at 60−100 cm depth. For SOC and bulk density prediction precision decreased with soil depth, and a standard error of 2.8 g kg⁻¹ was found at 60−100 cm soil depth. Average SOC stock for 0−30 cm was 72 t ha⁻¹ and in the top 1 m there was 120 t SOC ha⁻¹. In total, the soils stored approximately 570 Tg C within the top 1 m. The soils under agriculture had the highest amount of carbon (444 Tg) followed by forest and semi-natural vegetation that contributed 11% of the total SOC stock. More than 60% of the total SOC stock was present in Podzols and Luvisols. Compared to previous estimates, our approach is more reliable as we adopted a robust quantification technique and mapped the spatial distribution of SOC stock and prediction uncertainty. The estimation was validated using common statistical indices and the data and high-resolution maps could be used for future soil carbon assessment and inventories.     

The role of tryptophan 272 in the Paracoccus denitrificans cytochrome c oxidase

The mechanism of electron coupled proton transfer in cytochrome c oxidase (CcO) is still poorly understood. The P(M)-intermediate of the catalytic cycle is an oxoferryl state whose generation requires one additional electron, which cannot be provided by the two metal centres. The missing electron has been suggested to be donated to this binuclear site by a tyrosine residue. A tyrosine radical species has been detected in the P(M) and F* intermediates (formed by addition of H2O2) of the Paraccocus denitrificans CcO using electron paramagnetic resonance (EPR) spectroscopy. From the study of conserved variants its origin was determined to be Y167 which is surprising as this residue is not part of the active site. Upon inspection of the active site it becomes evident that W272 could be the actual donor of the missing electron, which can then be replenished from Y167 or from the Y280-H276 cross link in the natural cycle. To address the question, whether such a direct electron transfer pathway to the binuclear centre exists two tryptophan 272 variants in subunit I have been generated. These variants are characterised by their turnover rates as well as using EPR and optical spectroscopy. From these experiments it is concluded, that W272 is an important intermediate in the formation of the radical species appearing in P(M) and F* intermediates produced with hydrogen peroxide. The significance of this finding for the catalytic function of the enzyme is discussed.     

Eco-floristic sectors and deforestation threats in Sumatra: identifying new conservation area network priorities for ecosystem-based land use planning

Biogeographical studies are a necessary step in establishing conservation area networks. Determining the ecological factors influencing vegetation is also a basic principle for hierarchical ecological classifications and a necessary prerequisite for ecosystem-based land use planning. Eco-floristic sectors (EFS) have already been identified for the Indonesian island of Sumatra, combining both approaches, dividing it into 38 EFSs representing unique ecosystems in terms of tree flora and environment (Laumonier 1997). The impact of deforestation on individual EFSs has been highly varied and in some cases extreme. We assigned one of five ‘extinction risk categories’ to each EFS based on the percentage of forest lost between 1985 and 2007. Eighty-five percent of all forest loss (10.2 million ha) occurred in the eastern peneplain, western lowland regions and swamps. In 2007, only 29% of forests were protected by conservation areas, only nine of the 38 EFS had more than 50% of their remaining forest cover protected. 38% of remaining forest was “critically endangered”, “endangered” or “vulnerable” EFSs (5 million ha) but only 1 million ha (20%) were protected. Sumatra’s existing network of conservation areas does not adequately represent the island’s ecosystems. Priorities for a new conservation area network can be formulated for integration into Sumatra’s new land use plans at provincial and district level. Decision makers can now use EFSs to locate new conservation areas so they represent and maintain the whole range of the island’s diversity.