Microarray analysis of the in vivo sequence preferences of a minor groove binding drug

<p>Abstract</p> <p>Background</p> <p>Minor groove binding drugs (MGBDs) interact with DNA in a sequence-specific manner and can cause changes in gene expression at the level of transcription. They serve as valuable models for protein interactions with DNA and form an important class of antitumor, antiviral, antitrypanosomal and antibacterial drugs. There is a need to extend knowledge of the sequence requirements for MGBDs from <it>in vitro </it>DNA binding studies to living cells.</p> <p>Results</p> <p>Here we describe the use of microarray analysis to discover yeast genes that are affected by treatment with the MGBD berenil, thereby allowing the investigation of its sequence requirements for binding <it>in vivo</it>. A novel approach to sequence analysis allowed us to address hypotheses about genes that were directly or indirectly affected by drug binding. The results show that the sequence features of A/T richness and heteropolymeric character discovered by <it>in vitro </it>berenil binding studies are found upstream of genes hypothesized to be directly affected by berenil but not upstream of those hypothesized to be indirectly affected or those shown to be unaffected.</p> <p>Conclusion</p> <p>The data support the conclusion that effects of berenil on gene expression in yeast cells can be explained by sequence patterns discovered by <it>in vitro </it>binding experiments. The results shed light on the sequence and structural rules by which berenil binds to DNA and affects the transcriptional regulation of genes and contribute generally to the development of MGBDs as tools for basic and applied research.</p

Evaluation of Three Automated Genome Annotations for Halorhabdus utahensis

Genome annotations are accumulating rapidly and depend heavily on automated annotation systems. Many genome centers offer annotation systems but no one has compared their output in a systematic way to determine accuracy and inherent errors. Errors in the annotations are routinely deposited in databases such as NCBI and used to validate subsequent annotation errors. We submitted the genome sequence of halophilic archaeon Halorhabdus utahensis to be analyzed by three genome annotation services. We have examined the output from each service in a variety of ways in order to compare the methodology and effectiveness of the annotations, as well as to explore the genes, pathways, and physiology of the previously unannotated genome. The annotation services differ considerably in gene calls, features, and ease of use. We had to manually identify the origin of replication and the species-specific consensus ribosome-binding site. Additionally, we conducted laboratory experiments to test H. utahensis growth and enzyme activity. Current annotation practices need to improve in order to more accurately reflect a genome's biological potential. We make specific recommendations that could improve the quality of microbial annotation projects

Engineering bacteria to solve the Burnt Pancake Problem

<p>Abstract</p> <p>Background</p> <p>We investigated the possibility of executing DNA-based computation in living cells by engineering <it>Escherichia coli </it>to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA.</p> <p>Results</p> <p>Inversions (or "flips") of the DNA fragment pancakes are driven by the <it>Salmonella typhimurium </it>Hin/<it>hix </it>DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in <it>E. coli</it>. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; <it>E. coli </it>cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of <it>in vivo </it>flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes.</p> <p>Conclusion</p> <p>The Hin/<it>hix </it>system is a proof-of-concept demonstration of <it>in vivo </it>computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/<it>hix </it>may provide a flexible new tool for manipulating transgenic DNA <it>in vivo</it>.</p

Finding Common Sequence and Structure Motifs in a set of RNA sequences

Abstract Ve present a computational scheme to search for the most common motif, composed of a combination of sequence and structure constraints, among a collection of RNA sequences. The method uses a simplified version of the Sankoff algorithm for simultaneous folding and alignment of RNA sequences, but maintains tractability by constructing multi-sequence alignments from pairwise comparisons. The overall method has similarities to both CLUSTAL and CONSENSUS, but the core algorithm assures that the pairwise alignments are optimized for both sequence and structure conservation. Example solutions, and comparisons with other approaches, are provided. The solutions include finding consensus structures identical to published ones