Site-directed spin labeling (SDSL) uses electron paramagnetic resonance (EPR) spectroscopy to monitor the behavior of a stable nitroxide radical attached at specific locations within a macromolecule such as protein, DNA, or RNA. Parameters obtained front EPR measurements, such as internitroxide distances and descriptions of the rotational motion of a nitroxide, provide unique information on features near the labeling site. With recent advances in solid-phase synthesis of nucleic acids and developments in EPR methodologies, particularly pulsed EPR technologies, SDSL has been increasingly used to study the structure and dynamics of DNA and RNA at the level of the individual nucleotides. This chapter summarizes the current SDSL studies oil nucleic acids, with discussions focusing oil literature from the last decade.
This chapter discusses various scientific and practical applications of molecular colony technique (MCT) developed to date in different laboratories in Russia, United States, and China. In addition to already mentioned applications, these include in vitro gene cloning and molecular diagnostics. MCT comprises the amplification of nucleic acids in immobilized media, such as in a gel. Because the gel matrix prevents the convection of the entrapped liquid and restricts diffusion of nucleic acids, the amplification products do not spread throughout the medium. Rather, they form more or less compact molecular colonies, which are reminiscent of bacterial colonies growing on the surface of a nutrient agar. If amplification is carried out in a thin gel layer, a 2-D pattern of molecular colonies is generated, each colony comprising many copies (a clone) of a single starting RNA or DNA template molecule. The ability of MCT to detect single molecules makes it a unique tool for studying extremely rare reactions, such as recombinations between RNA molecules. After the discovery of genetic recombination, at the level of DNA, indications began to accumulate that a similar process may occur at the level of RNA as well.
This chapter presents a broader view of the state of human disease modeling in Drosophila melanogaster and discusses new directions in the study of the genetic basis of human disorders in flies. The Drosophila classical genetics powerhouse, in combination with rapidly developing genomic and postgenomic tools, accelerates the identification and characterization of gene networks. Because the molecular mechanisms controlling a variety of physiological pathways are largely conserved between flies and humans, flies are quite useful in modeling a variety of human diseases. These include nervous system disorders, cancer, immune responses, elements of the cardiovascular system, and many more. Possibly the most successful area of human neurological disease modeling in Drosophila is the models of polyglutamine tract repeat disorders. The fly eye is an excellent readout for polyglutamine tract repeat disorders, like such as Huntington's disease and the spinocerebellar ataxias. In both conditions, there is a critical threshold of polyglutamine repeats that must be reached before a clinical presentation is observed. In flies, the expression of the human Huntingtin protein or the SCA3/MJD protein, containing the clinically relevant number of repeats, leads to the degeneration of photoreceptor neurons.
RNA modeling has become an increasingly attractive field for researchers as new functions for RNA are identified and characterized. However, our progress in determining three-dimensional structures is still behind our discovery of functional RNA molecules. Continuous development of experimental methods has enabled us to characterize biochemical and physical properties of the RNA molecules. Advancement in computer simulation and modeling is bringing us closer to the all-atomic-detail modeling. But there is still a big gap between our current achievement and our goal of predicting the three-dimensional structure based on their sequence information. In this chapter, we will go over the important progresses and techniques of structure characterization of nucleic acids, as an introduction for readers to wider range of approaches.
DNA polymerase epsilon (Pol epsilon) is a large, multi-subunit polymerase that is conserved throughout all eukaryotes. In addition to its role as one of the three DNA polymerases responsible for bulk chromosomal replication, Pot epsilon is implicated in a wide variety of important cellular processes, including the repair of damaged DNA, DNA recombination and the regulation of proper cell cycle progression. Additionally, recent work has suggested that Pol epsilon is linked to chromatin remodeling and the regulation of epigenetic inheritance. Though much has been learned in the past two decades about the various functions of Pol epsilon, a great deal remains unknown due in large Part to the complexities of both the various implicated pathways and the Pol epsilon holoenzyme itself. While most of the progress in understanding Pol epsilon has come from work using the human and bakers yeast systems, the Xenopus and fission yeast systems have provided valuable insights. Here we discuss what is currently known about this unique DNA polymerase, from its initial identification through the present.
