We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

The investigation of the three-dimensional structure of the DNA aptamer d(G1G2T3-T4G5G6T7G8T9G10G11T12T13G14G15) which binds to and inhibits thrombin has been carried out by NMR methods. This DNA exhibits a number of long-range NOEs between residues which are not adjacent in sequence, which allowed the determination of the novel tertiary structure adopted. This DNA adopts a highly compact, highly symmetrical structure which consists of two tetrads of guanosine base pairs and three loops. The residues of the tetrads alternate anti-syn-anti-syn. This novel structural motif for DNA may also be relevant to the structure of telomere DNA.

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Tissue factor (TF) is a membrane-bound glycoprotein that functions as a cofactor for coagulation factor VIIa (VIIa) and initiates blood coagulation at sites of vascular injury. On the basis of sequence alignments, TF was predicted to be a member of the cytokine receptor superfamily. Utilizing the structural information available for the cytokine receptor superfamily, we have used site-directed mutagenesis to identify the binding site on TF for VIIa. The predicted loop regions in TF were systematically replaced with the homologous loops from the gamma-interferon receptor (gamma-IFN-R), the protein most related to TF in the superfamily of cytokine receptors. Six discontinuous regions (residues 16-20, 40-46, 60-69, 101-111, 129-151, 193-207) were identified that are required for interaction with VIIa and enhancement of activity. Individual substitution of 68 residues within these loops with alanine revealed eight residues (K20, D44, W45, K46, Q110, R135, F140, V207) that are required for cofactor activity. These residues fall into two groups, those that are required only for interactions with VIIa (K46, Q110, R135, F140, V207) and those that are also required to induce the conformational change in VIIa required for enhanced activity (K20, D44, W45). The discontinuous regions of TF required for interactions with VIIa form a single binding surface for VIIa that is analogous to the interface defined by the crystal structure of the complex between growth hormone and its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Despite the global importance of As in rice, research has primarily focused on Bangladesh, India, China, and the United States with limited attention given to other countries. Owing to both indigenous As within the soil and the possible increases arising from the onset of irrigation with groundwater, an assessment of As in rice within Cambodia is needed, which offers a "base-case" comparison against sediments of similar origin that comprise rice paddy soils where As-contaminated water is used for irrigation (e.g., Bangladesh). Here, we evaluated the As content of rice from five provinces (Kandal, Prey Veng, Battambang, Banteay Meanchey, and Kampong Thom) in the rice-growing regions of Cambodia and coupled that data to soil-chemical factors based on extractions of paddy soil collected and processed under anoxic conditions. At total soil As concentrations ranging 0.8 to 18 μg g(-1), total grain As concentrations averaged 0.2 μg g(-1) and ranged from 0.1 to 0.37 with Banteay Meanchey rice having significantly higher values than Prey Veng rice. Overall, soil-extractable concentrations of As, Fe, P, and Si and total As were poor predictors of grain As concentrations. While biogeochemical factors leading to reduction of As(V)-bearing Fe(III) oxides are likely most important for predicting plant-available As, husk and straw As concentrations were the most significant predictors of grain-As levels among our measured parameters.