The SELEX method and oligonucleotide combinatorial chemistry discovery process yields highaffinity͞high-specificity ligands for virtually any molecular target. Typically, the enormous starting libraries used in the SELEX process contain 10 14 -10 15 sequences. We now ask if the smaller sequences, complexity of extant organisms, and evolutionary history provide useful interactions between oligonucleotides and at least some unexpected targets. That is, do organisms contain a robust "linkage map" between their oligonucleotides and proteins and͞or small molecules that enriches life?We have accepted the challenge of writing an inaugural article, hoping to stimulate and provoke other scientists to wonder with us about a chance to discover surprising molecular interactions in biology between nucleic acids and other intracellular components. We will chart the conceptual triangle surrounding in vitro evolution [the SELEX (for systematic evolution of ligands by exponential enrichment) methodology]: insights into the prebiotic earth, applied medical research, and novel regulatory elements and circuits in cells (identified through the genomic SELEX process). We came to this set of ideas through the study of bacteriophage T4 development, with a focus on translational regulation. It was that work and the extraordinary environment for RNA studies that exists at the University of Colorado in Boulder that led us to our present interests.
Translational Regulation in Bacteriophage T4-Infected Escherichia coliAmong the first experiments on coupled transcription and translation in vitro were those done in Lipmann's laboratory by one of us (1). For the first time, the synthesis of active, full-length enzymes from a DNA template was achieved. The templates used for most of those experiments were from T-even bacteriophage, which ultimately led to our interest in T4 translation and its regulation.What a time to study a simple developing system-Peter Geiduschek's and Dick Epstein's laboratories (40, 41) had made clear the phage transcriptional pattern, while the invention of high-resolution SDS gels by Uli Laemmli (42) made it possible to detect a corresponding translational pattern. We thought that the development of this organism could be understood, given the power of T4 genetics (2) and the availability of mutations in most of the essential genes.The early work we did at the University of Colorado was so simple. We received T4 mutant strains from people (who for the most part still shared things) and ran gels to see what happened to phage gene expression after infections of nonpermissive E. coli with those T4
The alpha‐proteobacteria of the genus Wolbachia is a widespread group of maternally inherited endosymbionts of arthropod and nematode hosts. Wolbachia infection induces a range of host phenotypes, including cytoplasmic incompatibility, male killing, feminization, and induction of thelytokous parthenogenesis. Heterogony (cyclical parthenogenesis) is a remarkable characteristic of oak gallwasps, Cynipini, the largest tribe of the Cynipidae. A few species of Cynipini are exceptional in that they are univoltine and exhibit thelytokous parthenogenesis, probably because they lost the arrhenotokous generation of their heterogonic ancestor species due to Wolbachia infection. In this study, the presence of Wolbachia was detected using polymerase chain reaction primers for the wsp genes in a thelytokous parthenogenetic species [Dryocosmus kuriphilus (Yasumatsu)] (Hymenoptera: Cynipidae: Cynipini). Approximately 29.8 and 87.1% of adults of the Zhuzhou and Fuzhou strains, respectively, were infected with Wolbachia while all females of the remaining four strains collected from other localities in China were Wolbachia free. The length of the wsp fragment of Zhuzhou and Fuzhou strains was found to be 573 and 561 bp, respectively. The nucleotide sequence of the bacterial wsp fragment indicated that the endosymbiotic bacteria of the Zhuzhou and Fuzhou strains are members of supergroup A, but belong to different clades; they probably originated from two independent infection events. In conclusion, thelytokous parthenogenesis of D. kuriphilus is not caused by Wolbachia infection and the deletion of the arrhenotokous generation is thus not associated with such an infection.
The three-dimensional crystal structure of the Escherichia coli methionine repressor, MetJ, complexed with a DNA operator fragment is described in an accompanying article. The complex exhibits several novel features of DNA-protein interaction. DNA sequence recognition is achieved largely by hydrogen-bond contacts between the bases and amino-acid side chains located on a beta-ribbon, a mode of recognition previously hypothesized on the basis of modelling of idealized beta-strands and DNA, and mutagenesis of the Salmonella phage P22 repressors Arc and Mnt. The complex comprises a pair of MetJ repressor dimers which bind to adjacent met-box sites on the DNA, and contact each other by means of a pair of antiparallel alpha-helices. Here we assess the importance of these contacts, and also of contacts that would be made between the C-helices of the protein and DNA in a previous model of the complex, by studying mutations aimed at disrupting them. The role of the carboxy-terminal helix face in operator binding was unclear, but we demonstrate that recognition of operator sequences occurs through side chains in the beta-strand motif and that dimer-dimer interactions are required for effective repression.
Herein, the excellent Na+ selectivity of a few RNA-cleaving DNAzymes was exploited, where Na+ can be around 3000-fold more effective than K+ for promoting catalysis. By using a double mutant based on the Ce13d DNAzyme, and by lowering the temperature, increased 2-aminopurine (2AP) fluorescence was observed with addition of both Na+ and K+. The fluorescence increase was similar for these two metals at below 10 mM, after which K+ took a different pathway. Since 2AP probes its local base stacking environment, K+ can be considered to induce misfolding. Binding of both Na+ and K+ was specific, since single base mutations could fully inhibit 2AP fluorescence for both metals. The binding thermodynamics was measured by temperature-dependent experiments revealing enthalpy-driven binding for both metals and less coordination sites compared to G-quadruplex DNA. Cleavage activity assays indicated a moderate cleavage activity with 10 mM K+, while further increase of K+ inhibited the activity, also supporting its misfolding of the DNAzyme. For comparison, a G-quadruplex DNA was also studied using the same system, where Na+ and K+ led to the same final state with only around 8-fold difference in Kd. This study provides interesting insights into strategies for discriminating Na+ and K+.
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