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.
Fluorescence correlation spectroscopy (FCS) is a quantitative technique where temporal fluctuations in fluorescence intensity are analyzed to yield information about physical processes that contribute to the fluctuations, These include diffusion through the observation volume, conformational dynamics, and chemical or photophysical reactions. To date, FCS applications in biochemistry and biophysics have been mostly focused on the investigation of molecular diffusion. In this chapter, we present a look into the recent applications of FCS to the study of conformational dynamics of nucleic acids. We first survey the basic theoretical and experimental aspects of the technique and then focus oil the results obtained in studies with DNA hairpins, nucleosomes, and single-stranded and duplex DNA.
Recent development of solution-phase molecular-scale Boolean calculations using deoxyribozymes is potentially an important step toward the development of autonomous therapeutic and diagnostic devices. Here, the construction of basic YES, AND, ANDNOT, and ANDANDNOT deoxyribozyme-based logic gates is described. Protocols for testing gate activity using fluorescent oligonucleotide probes have been provided, and pointers for gate optimization are included.
Eukaryotic initiation factor (eIF)2B is a guanine nucleotide exchange factor (GEF) for a second initiation factor, eIF2. During the initiation of messenger RNA (mRNA) translation, eIF2 binds Guanosine-5'-triphosphate (GTP) and initiator methionyl- transfer RNA (tRNAi) (met-tRNAi) and the resulting ternary complex subsequently binds to the 40S ribosomal subunit. Alterations in ternary complex formation due to changes in eIF2B GEF activity not only produce alterations in global rates of protein synthesis but also cause specific changes in the translation of mRNAs encoding certain proteins, such as the transcription factors general control nonrepressed 2 (GCN2) and activating transcription factor 4 (ATF4). Studies have implicated changes in eIF2B GEF activity in alterations in protein synthesis in a number of pathophysiological and physiological conditions, such as diabetes, cancer, sepsis, vanishing white matter disease, and resistance exercise. The chapter discusses the state of knowledge concerning the function of eIF2B and its role in regulating mRNA translation.
This chapter discusses progress on dissecting mechanisms underlying the establishment and regulation of the erythroid cell-specific β-globin chromatin domain while emphasizing general principles that have emerged from the work. ‘‘Chromatin domain’’ refers to a broad chromosomal region from approximately several thousand to several hundred thousand base pairs in which a gene or gene cluster resides rather than the classically defined ∼100-kb topologically constrained chromosomal loop identified by microscopy. The chapter reviews predominantly mechanistic studies conducted in mammalian systems, including genetic complementation analysis of trans-acting factor function in biologically relevant murine erythroid cell lines and analysis of cis-element function via targeted deletions in mice. Further work on the compelling problem of how cell type-specific chromatin domains are established and regulated in normal cells and how the underlying mechanisms go awry in disease states promises to be highly rewarding and to yield a continuous stream of focused and serendipitious discoveries for many years to come.
One of the major areas of emphasis has understood the role of selenium in health. Selenium is an essential micronutrient in the diet of mammals, and this element has numerous health benefits. It has roles in cancer and heart disease prevention, inhibiting viral expression, and delaying the progression of AIDS in HIV positive patients. Selenium has been reported to have roles in immune function, male reproduction, mammalian development, and slowing the aging process. This chapter discusses the means by which amino acid selenocysteine (Sec) is biosynthesized and incorporated into protein; the generation of mouse models for elucidating the role of selenoproteins in development and health; the identity and functions of selenoproteins; and the distribution and evolution of the amino acid Sec insertion machinery among eukaryotes. Sec is biosynthesized, unlike the common biosynthetic pathways of the other 20 protein amino acids, on its transfer RNA (tRNA). The machinery for inserting Sec into protein is novel and unique to this amino acid. It is apparent that tremendous effort has been expended in evolution for inserting selenium into protein in the form of Sec as discussed in the chapter.
Publisher Summary Interferons were discovered as antiviral agents. These cytokines possess multiple activities that include the ability to affect cell growth, differentiation, and death, in addition to their hallmark ability to interfere with virus multiplication. This chapter focuses on the organization and regulated expression of the ADAR1 and PKR genes, the biochemical and biophysical properties of the ADAR1 and PKR proteins, and the mechanisms by which ADAR1 and PKR modulate the physiology of cultured cells and intact animals. Two important genes regulated by interferons are ADAR1 and PKR. ADAR1 and PKR encode double-stranded RNA (dsRNA)-binding proteins that are responsible, in part, for the biochemical and mechanistic actions of interferons. Both forms of ADAR1, p150 and p110, function to modify the expression of genetic information by changing cellular and viral RNAs through substitution of an inosine, which is recognized as guanine, for adenine. PKR is an RNA-dependent protein kinase that controls the translational pattern in cells through phosphorylation of the α subunit of protein synthesis initiation factor eIF-2; PKR also modulates signal transduction processes. The chapter discusses the roles that these proteins might play in genetic and infectious human diseases.
This chapter discusses evidence and hypotheses concerning the place of RNA circularization during messenger RNA (mRNA) translation. It provides a short list that is representative of the conundrums facing the researcher in the field and presents different RNAs according to the chemical nature of the interactions involved in mRNA 5'-3' end-to-end communication: RNA–RNA, RNA–protein, and protein–protein interactions, where it focuses on the potential biological consequences of mRNA circularization. This field of research is an exciting, young, dynamic one with richness of choice as to the problem facing the researcher. The chapter underlines the cases that would seem to the nonspecialist to be resolved are in fact far from being cut-and-dried, and much work remains to be done in all cases considered. Therefore, the choice comes down to the specialities of individual laboratories, and it seems highly likely that significant progress can now only be made by collaborative efforts in many areas.
This chapter provides a detailed summary about human Natural killer (NK) cells and their sophisticated competencies to discriminate between autologous healthy cells and autologous unhealthy cells that have been rendered ‘‘unusual’’ by malignant transformation, virus infection, or other means. In this chapter, heterogeneity of the NK cell population is a major topic, because this population heterogeneity provides the basis of broad specificity. This heterogeneity results mainly from a highly variable and at least in part stochastic expression of numerous inhibitory and eventually also activating receptors at the cell surface, and the surface expression of these receptors and the subsequent signal integration process are part of a regulatory network. The fine tuning of this network, probably by a nontarget-specific educational process, provides not only the basis for self-tolerance of NK cells to normal tissues but also allows fast and sensitive detection of the unusual properties of diseased cells, leading subsequently to their elimination.
This chapter discusses the use of inhibitors of tyrosyl-DNA phosphodiesterase (Tdp1) and Chk1/2 in combination with Topoisomerase I (TopI) inhibitors. TopI is an abundant and essential enzyme. It is the selective target of camptothecins, which are effective anticancer agents. TopI–DNA cleavage complexes can also be trapped by various endogenous and exogenous DNA lesions, including mismatches, abasic sites, and carcinogenic adducts. Tdp1 is one of the repair enzymes for Top1–DNA covalent complexes. It forms a multiprotein complex that includes poly (Adenosine diphosphate (ADP)–ribose) polymerase (PARP). PARP-deficient cells are hypersensitive to camptothecins and functionally deficient for Tdp1. This chapter reviews the developments in several pathways involved in the repair of Top1 cleavage complexes and the role of Chk1 and Chk2 checkpoint kinases in the cellular responses to Top1 inhibitors. The genes conferring camptothecin hypersensitivity are compiled for humans, budding yeast, and fission yeast.
Publisher Summary This chapter reviews important findings relating to the regulation of histidine decarboxylase (HDC) at the transcriptional and posttranslational levels and discusses the role of HDC or histamine in carcinogenesis. Histamine is a bioamine whose roles in allergy, inflammation, neurotransmission, and gastric acid secretion have been well described. Animal studies using mice deficient in histamine production have confirmed these important functions and identified a number of new ones. A single enzyme, L-histidine decarboxylase, is responsible for histamine biosynthesis in mammals. It is expressed in the liver of the developing fetus and in the stomach, brain, thymus, spleen, and bones of adults. At the transcription level, HDC gene expression is regulated by several stimuli, including gastrin (a stomach peptide hormone), lipopolysaccharide (LPS), phorbol 12-myristate-13-acetate (PMA), oxidative stress, and Helicobacter pylori infection. At the posttranslational level, processing into multiple truncated isoforms provides a cellular mechanism for controlling the activity of the actual enzyme. While these aspects of regulation are important for normal physiological function, histamine is increasingly being recognized as a contributory factor in the development of some cancer types.
There are several scientific reviews available that deal with the multitude of molecular processes involved in repairing various DNA lesions. The chapter discusses prokaryotic DNA mismatch repair pathway. Repair of base mismatches in Escherichia coli and related bacteria is performed by two molecular yet overlapping processes: the long-patch mismatch repair and very-short-patch mismatch repair pathways. DNA mismatch repair is inevitable for maintaining genomic stability and is highly conserved from prokaryotes to eukaryotes. Availability of several completely sequenced bacterial genomes has helped in the identification of proteins, which are involved in the DNA mismatch repair process and their subsequent biochemical characterization. Comparative studies of the activities of these proteins have helped in elucidating molecular pathway involved in the complex process of DNA mismatch repair. The characteristic features of the prokaryotic DNA mismatch repair proteins and their biochemical activities are reviewed in the chapter.
This chapter discusses the alterations of protein tyrosine phosphatases (PTPs) and the implications of growth, proliferation, and apoptosis phenotypes attributable to the altered function of this family of phosphatases in cancer. Protein phosphorylation and dephosphorylation are complex enzymatic reactions that are performed by the concerted action of protein kinases and phosphatases, respectively. Deregulation of such coordination due to the loss or gain of a single component of the process can result in disease conditions that include, but are not limited to, neoplastic transformation, developmental, autoimmune, and metabolic disorders. Unlike many protein tyrosine kinases that function as oncoproteins, PTPs could impart positive or negative effect on cell proliferation. The potential applications of different therapeutic approaches to rectify the adverse effects of alterations in expression of the phosphatase genes and of the phosphatase activity in cancer are described in the chapter.
Virus‐like particles (VLPs), formed by the structural elements of viruses, have received considerable attention over the past two decades. The number of reports on newly obtained VLPs has grown proportionally with the systems developed for the expression of these particles. The chapter outlines the recent achievements in two important fields of research brought about by the availability of VLPs produced in a foreign host. These are: (1) The requirements for VLP assembly and (2) the use of VLPs as carriers for foreign epitopes. VLP technology is a rapidly advancing domain of molecular and structural biology. Extensive progress in VLP studies was achieved as the insect cell based protein production system was developed. This baculovirus expression system has many advantages for the synthesis of viral structural proteins resulting in the formation of VLPs. It allows production of large amounts of correctly folded proteins while also providing cell membranes that can serve as structural elements for enveloped viruses. These features give us the opportunity to gain insights into the interactions and requirements accompanying VLP formation that are similar to the assembly events occurring in mammalian cells. Other encouraging elements are the ability to easily scale up the system and the simplicity of purification of the assembled VLPs. The growing number of VLPs carrying foreign protein fragments on their surface and studies on the successful assembly of these chimeric molecules is a promising avenue towards the development of a new technology, in which the newly designed VLPs will be directed to particular mammalian cell types by exposing specific binding domains. The progress made in modeling the surface of VLPs makes them to date the best candidates for the design of delivery systems that can efficiently reach their targets.
