Showing papers in "Cold Spring Harbor Monograph Archive in 1999"

20 Splicing of Precursors to mRNAs by the Spliceosomes

Abstract: The splicing of precursors to mRNAs occurs in two steps, both involving single transesterification reactions (Fig. 1). The first step generates a 2′ – 5′ bond at the branch site upstream of the 3′ splice site and a free 3′ hydroxyl group on the 5′ exon. The resulting lariat RNA intermediate, with its slow migration in gels, is the most common assay for splicing in vitro. In the second step, attack of the 3′ hydroxyl on the phosphodiester bond at the 3′ splice site displaces the lariat intron with a 3′ hydroxyl group and results in joining of the two exons. The bimolecular nature of the intermediate in splicing indicated that the reaction must occur within a stable splicing body or spliceosome. Surprisingly, assembly and functioning of the spliceosome requires approximately 100 polypeptides and five small nuclear RNAs (snRNAs), not considering gene-specific RNA-binding factors. There are two distinct types of spliceosomes in most cells. The major class or U2-type spliceosome is universal in eukaryotes, whereas the minor class or U12-type spliceosome may not be present in some organisms. The evolutionary relationship of these two spliceosomes is uncertain. The sequence specificity for the splicing of introns must be encoded within the gene. In vertebrate genes, particularly for U2-type introns, the sequence specificity for splicing is not determined solely by the consensus sequences at the intron boundaries but is more broadly distributed within the gene. In contrast, the consensus sequences of the introns in the yeast Saccharomyces cerevisiae are generally adequate to specify...

18 Group I and Group II Ribozymes as RNPs: Clues to the Past and Guides to the Future

Abstract: Group I and group II introns are not only catalytic RNAs, but also mobile genetic elements. The success of these introns as mobile elements almost certainly relates to their innate self-splicing capability, which enables them to propagate by inserting into host genes while only minimally impairing gene expression. Nevertheless, both types of introns have become dependent on proteins for efficient splicing in vivo to help fold the intron RNA into the catalytically active structure. Although group I and group II introns have very different structures and splicing mechanisms (Chapter 13), there are striking parallels in the evolution of their protein-assisted splicing reactions. For example, the splicing factors for both types of introns include intron-encoded as well as cellular proteins, and the intron-encoded proteins, DNA endonucleases for group I introns and reverse transcriptases (RTs) for group II introns, also function in intron mobility. In addition, excised group I and group II intron RNAs remain associated with splicing factors in RNP particles, which can then cleave and insert into cellular RNA or DNA target sites by reverse splicing. The need to control this deleterious ribozyme activity may have been an evolutionary driving force favoring mutations that impaired self-splicing activity and resulted in dependence on protein factors (Nikolcheva and Woodson 1997). In this chapter, we review protein-assisted reactions of group I and group II introns. These studies illustrate how proteins facilitate RNA folding and catalysis and provide unique insights into how splicing mechanisms evolve. A recurring theme, first developed in a previous review...

12 The Ever-Growing World of Small Nuclear Ribonucleoproteins

Abstract: Small r ibo n ucleo p roteins (RNPs)—defined as tight complexes of one or more proteins with a short RNA molecule (usually 60–300 nucleotides)—inhabit every compartment of eukaryotic cells. Those that reside in the nucleus, the s mall n uclear RNPs (snRNPs), can themselves be divided into two families. There are snRNPs of the nucleoplasm, whose major business is the generation of messenger RNAs for export to the cytoplasm. A different set of snRNPs, called snoRNPs, reside in the cell nucleolus, the subnuclear locale responsible for the synthesis, maturation, and assembly of rRNAs into ribosomal subunits, which are then exported to function in cytoplasmic protein synthesis. The last two years have produced the extraordinary realization that vertebrate cells contain about 200 distinct kinds of snRNPs with abundances between 10 4 (for snoRNPs directing rRNA modification) to over 10 6 (for snRNPs of the major spliceosome). All of those whose functions have been assigned play roles in gene expression, underscoring the pivotal participation of RNA molecules in the evolution of the gene expression apparatus. The one exception is the telomerase snRNP, essential for genome maintenance (see Chapter 23). Curiously, snRNPs are often the target of autoantibodies present in the sera of patients suffering from rheumatic disease (as well as in other mammals afflicted with autoimmunity). These autoantibodies almost always target epitopes on the protein component(s) of the particles, and since an antigenic protein often associates with multiple snRNAs (small nuclear RNAs), they define families of snRNPs related in structure (and usually also in function) (see Table 1).

2 Prospects for Understanding the Origin of the RNA World

Abstract: The general idea that, in the development of life on the earth, evolution based on RNA replication preceded the appearance of protein synthesis was first proposed about 30 years ago (Woese 1967; Crick 1968; Orgel 1968). It was suggested that catalysts made entirely of RNA are likely to have been important at this early stage in the evolution of life, but the possibility that RNA catalysts might still be present in contemporary organisms was overlooked. The unanticipated discovery of ribozymes (Kruger et al. 1982; Guerrier-Takada et al. 1983) initiated extensive discussion of the role of RNA in the origins of life (Pace and Marsh 1985; Sharp 1985; Lewin 1986) and led to the coining of the phrase “the RNA World” (Gilbert 1986). The RNA World means different things to different authors, so it would be futile to attempt a restrictive definition. All RNA World hypotheses include three basic assumptions: (1) At some time in the evolution of life, genetic continuity was assured by the replication of RNA; (2) Watson-Crick base-pairing was the key to replication; (3) genetically encoded proteins were not involved as catalysts. RNA World hypotheses differ in what they assume about life that may have preceded the RNA World, about the metabolic complexity of the RNA World, and about the role of low-molecular-weight cofactors, possibly including peptides, in the chemistry of the RNA World. It should be emphasized that the existence of an RNA World as a precursor of our DNA/protein world is a hypothesis. We find it...

12 The Role of Metal Ions in RNA Biochemistry

Abstract: Approximately two-thirds of the elements in the periodic table can be categorized as metals. Besides luster, malleability, and conductivity, one of the fundamental characteristics of metals is their low ionization potential. As a result, the ionic forms of these elements predominate in the biosphere. Considering the diverse properties of these ions, it is not surprising that through the process of evolution, metal ions have been co-opted into numerous roles in biology. Metal ions are required for so many biochemical reactions that it is likely that they also had an important role in the RNA world. To understand both modern and prebiotic RNA biochemistry, it is therefore essential to have a basic understanding of these inorganic elements. Metal ions were abundant in the primordial soup. It is believed that 3.8 × 10 9 years ago, the ocean was between 80°C and 100°C with a pH possibly as low as 6 (Bengston 1994). Table 1 shows the concentrations of the most common metal ions in today’s seas and in blood plasma. Although the concentrations of most of these ions in the prebiotic ocean are not known, the higher temperature and lower pH relative to the current ocean would have solvated a variety of ions and leached metal ions from the mineral-rich ocean beds. Therefore, the concentrations would have been significantly higher than the current values. One important additional difference is the extremely low concentration of easily oxidized metal ions such as Fe(II). Ferrous ion has been predicted to have been very abundant in...

12 Infectious and Sporadic Prion Diseases

Abstract: Creutzfeldt-Jakob disease (CJD) was first identified in 1921 by Jakob (Jakob 1921), who referred to a previous case described by Creutzfeldt in 1920 (Creutzfeldt 1920). These original cases were clinically heterogeneous, and recent review of pathologic material provided confirmation of the diagnosis of CJD in only two of the original five cases (Masters and Gajdusek 1982). Over subsequent decades the nosology of CJD was confused and confusing. Wilson regarded CJD as a “dumping ground for several rare cases of presenile dementia” (Wilson 1940). Although an important monograph by Kirschbaum (1968) listed the clinical and pathologic features of all 150 cases identified before 1965, it included cases such as Creutzfeldt’s original case, which would not now fulfil clinical or pathologic criteria for the diagnosis of CJD. In 1954 (Jones and Nevin 1954) and 1960 (Nevin et al. 1960) Nevin and Jones described the typical clinical course, the “characteristic” electroencephalogram (EEG) and neuropathologic changes, including spongiform change, which in combination are now recognized as the paradigm features of sporadic CJD. In the late 1950s and early 1960s with remarkable perseverance in difficult conditions, Gajdusek and colleagues investigated kuru, a fatal ataxic syndrome restricted to the Okapa area of the highlands of Papua New Guinea (Gajdusek and Zigas 1957). The similarity of the neuropathologic findings in scrapie and kuru was recognized in 1959 by Hadlow (Hadlow 1959) with the implication that kuru, like scrapie, might be transmissible in the laboratory. Successful laboratory transmission of kuru in 1966 (Gajdusek et al. 1966) was followed...

1 Before RNA and After: Geophysical and Geochemical Constraints on Molecular Evolution

Abstract: 1. INTRODUCTION This chapter offers a description of some of the physical and chemical settings for the origin of life on Earth. Considering the topic of this book, particular attention is given to the conditions for a precursor RNA World; an ab initio system based on phosphate-sugar backbone structures in linear polymers and currently with nitrogen bases as recognition molecules. Both the appeal and the uncertainties in the assumption of an RNA World are obvious. The biochemical advantage of this model has geochemical and cosmochemical complements such as the abundance in the universe of simple aldehydes, “the sugar of space.” With their relatively high oxidation state, the aldehydes are compatible with plausible models of early terrestrial atmospheres dominated by CO 2 , H 2 O, N 2 , and small mixing fractions of CO, CH 4 and other reductants. Further suggestions of an RNA World come from observed and inferred sources of active oligophosphates and from concentration mechanisms based on the molecular charge conferred by phosphate esters. Finally, the facile formation of ribose phosphate has been successfully modeled under mild aqueous conditions in the laboratory. Obstacles to progress in prebiotic synthesis leading to the inception of an RNA World yet remain. The concentration of neutral molecules such as formaldehyde and glycolaldehyde required to permit the phosphorylation and sugar phosphate formation found in the laboratory remains an impediment to modeling molecular evolution. Furthermore, the oligomerization of nucleosides or nucleotides without the aid of artificial activating groups, and the synthesis and attachment of nitrogen bases in a formaldehyde environment...

4 Structure and Genetic Organization of Adenovirus Vectors

Abstract: Adenovirus (Ad) was first isolated in 1953 (Rowe et al. 1953) and not long after was recognized as an invaluable tool for the investigation of various aspects of mammalian molecular biology from oncogenesis to DNA replication, transcriptional regulation, and protein synthesis. Some of the features that made Ad preferable over other viruses for such studies include the ease with which the Ad genome can be experimentally manipulated, the ability to propagate Ad to high titer, and the efficiency with which Ad infects a wide variety of both quiescent and proliferating cells of various species and cell types. These properties have also made Ad an attractive vehicle for gene transfer and transgene expression in mammalian cells with numerous recent applications of Ad as a vector for human gene therapy (for review, see Bramson et al. 1995; Hitt et al. 1996; and this volume). This chapter overviews the structure and lytic cycle of wild-type Ad as well as the structure of first generation, second generation, and helper-dependent Ad vectors designed for gene therapy. GENOMIC ORGANIZATION AND VIRION STRUCTURE Adenoviruses have been isolated from many different avian and mammalian species, although the human Ads have been the most extensively studied. The approximately 50 known serotypes of human Ad have been divided into 6 subgroups based on immunological, biological, and DNA sequence similarities. The best characterized serotypes are adenovirus type 2 (Ad2) and Ad5 of subgroup C, Ad12 of subgroup A and Ad7 of subgroup B. However, all human Ads examined thus far share...

2 Development of the Prion Concept

Abstract: The prion concept developed in the aftermath of many unsuccessful attempts to decipher the nature of the scrapie agent (Prusiner 1998). In some respects, the early development of the prion concept mirrors the story of DNA (Avery et al. 1944; Stanley 1970; McCarty 1985). Prior to the acceptance of DNA as the genetic material (Hershey and Chase 1952; Watson and Crick 1953), many scientists asserted that the DNA preparations were contaminated with protein, the true genetic material (Mirsky and Pollister 1946). For more than half a century, many biologists had thought that genes were made of protein and that proteins were reproduced as replicas of themselves (Haurowitz 1950; Stanley 1935). The prejudices of these scientists were similar in some ways to those of investigators who have disputed the prion concept. However, the scientists who attacked the hypothesis that genes are composed of DNA had no well-proven alternative; they had only a set of feelings derived from poorly substantiated data sets that genes are made of protein. In contrast, those who attacked the hypothesis that the prion is composed only of protein had more than 30 years of cumulative evidence showing that genetic information in all organisms on our planet is encoded in DNA. Studies of viruses and eventually viroids extended this concept to these small infectious pathogens (Diener 1979) and showed that genes could also be composed of RNA (Fraenkel-Conrat and Williams 1955; Gierer and Schramm 1956). It is with this background that investigators working on scrapie began to unravel...

13 Building a Catalytic Active Site Using Only RNA

Abstract: WHERE DO RIBOZYMES FIT IN THE RNA WORLD HYPOTHESIS? Two of the most fundamental requirements for life are information storage and catalytic function. Without storage, transfer, and replication of information, a system cannot learn from its past and improve its viability; that is, there can be no natural selection. Equally essential to life is catalytic (enzymatic) function. At the very least, there must be machinery to catalyze the copying of the informational molecules. This process must proceed with considerable fidelity, yet some frequency of errors is also necessary to provide the diversity that allows adaptation and evolution. Beyond replication of the genome, additional catalytic functions would be highly advantageous to provide basic metabolism for even a primitive self-reproducing system. In contemporary organisms information is stored in the form of DNA. However, the persistence of RNA genomes in many viruses shows us that this sister nucleic acid is competent for information storage, at least for small genomes. Biocatalytic function in the modern world is mostly the domain of protein enzymes, although ribonucleoproteins (RNPs) still catalyze the essential cellular reactions of protein synthesis and RNA splicing. By what evolutionary pathway did this DNA-RNA-protein solution to the problem of life come about? The finding that RNA, an informational molecule, can by itself catalyze biochemical reactions has rekindled enthusiasm for the possibility that a key intermediate stage was an RNA World, with RNA providing both information and function, genotype and phenotype. One version of this RNA World hypothesis is diagrammed in Figure 1. RNA...

19 The Logic of Anti-angiogenic Gene Therapy

Abstract: Virtually all current strategies for gene therapy of cancer target the cancer cell. The goal is the destruction of cancer cells in the primary tumor and in its metastases. These approaches include, among others, the introduction of genes that (1) permit tumor cells to express toxic molecules; (2) prevent or correct genetic defects; (3) increase the immunogenicity of tumor cells; or (4) increase the sensitivity of tumor cells to drugs (Friedmann 1996; Blaese 1997; Lowenstein 1997; Roth and Cristiano 1997). Although gene therapy of cancer may be inherently less toxic than conventional chemotherapy, it may still have to overcome at least three other fundamental obstacles that hinder conventional chemotherapy, i.e., limited access to tumor cells, heterogeneity of tumor cells, and emergence of resistant tumor cells. Whereas conventional chemotherapy is directed mainly against tumor cells, anti-angiogenic therapy is directed specifically against microvascular endothelial cells that have been recruited into the tumor bed. In contrast to chemotherapy, specific anti-angiogenic therapy has little or no toxicity, does not require that the therapeutic agent enter any tumor cells nor cross the blood brain barrier, acts independent of tumor cell heterogeneity, and does not induce acquired drug resistance (Boehm et al. 1997). In addition, the effectiveness of anti-angiogenic therapy is independent of tumor type and growth fraction. Therefore, it may be fruitful to consider gene therapy of cancer that is optimized to the endothelial cell population of the tumor bed, e.g., anti-angiogenic gene therapy. We do not view anti-angiogenic gene therapy and anticancer cell gene...

3 The Genomic Tag Hypothesis: What Molecular Fossils Tell Us about the Evolution of tRNA

Abstract: INTRODUCTION Holley’s realization that tRNA could be folded into a two-dimensional cloverleaf posed more questions than it answered (Dudock et al 1969) One of the most perplexing was whether the three-dimensional structure of tRNA would turn out to be an “integral fold” in which all parts were essential for the correct structure, or whether tRNA could be decomposed into smaller, structurally independent units The crystal structure of tRNA immediately revealed that tRNA is composed of two perpendicular coaxial stacks (Quigley and Rich 1976): a stack of the acceptor stem on the di-hydrouridine stem/loop (the “top half”) and a stack of the T ψ C stem/loop on the anticodon stem/loop (the “bottom half”) (see Fig 1) Remarkably, the covalent connections between the middle of one helical stack and the middle of the other hardly distorted either helical stack: The top and bottom halves of tRNA appeared to be inserted into each other with surgical precision A great deal of evidence has subsequently shown that the top and bottom halves of tRNA are indeed structurally and functionally independent units This suggests that the two halves of tRNA could have evolved independently Here we review the experimental evidence bearing on our hypothesis (Weiner and Maizels 1987) that the top half of tRNA evolved first as a 3′ terminal “genomic tag” that marked single-stranded RNA genomes for replication in what Gilbert was first to call the “RNA World” (Gilbert 1986) The bottom half of tRNA would then have evolved separately as replication in the

6 Did the RNA World Exploit an Expanded Genetic Alphabet

Abstract: SINGLE BIOPOLYMER LIFE FORMS BASED ON RNA In terms of its macromolecular chemistry, life on Earth can be classified as a “two-biopolymer” system. Nucleic acid is the encoding biopolymer, storing information within an organism and passing it to its descendants. Nucleic acids also direct the biosynthesis of the second biopolymer, proteins. Proteins generate most of the selectable traits in contemporary organisms, from structure to motion to catalysis. The two-biopolymer strategy evidently works rather well. It has lasted on Earth for several billion years, adapting in this time to a remarkable range of environments, surviving formidable geobiological (and perhaps cosmic) events that threatened its extinction, and generating intelligence capable of exploring beyond Earth. The terrestrial version of two-biopolymer life contains a well recognized paradox, however, one relating to its origins. It is difficult enough to envision a nonbiological mechanism that would allow either proteins or nucleic acids to emerge spontaneously from nonliving precursors. But it seems astronomically improbable that both biopolymers arose simultaneously and spontaneously, and even more improbable (if that can be imagined) that both biopolymers so arose with an encoder-encoded relationship. Accordingly, a variety of “single-biopolymer” models have been proposed as forms of life that antedated the two-biopolymer system. These (presumably) could have emerged more easily than a two-biopolymer system. Such models postulate that a single biopolymer can perform the catalytic and information repository roles and undergo the Darwinian evolution that defines life (Joyce 1994). For example, Rich (1962), Woese (1967), Orgel (1968), and Crick (1968) proposed that the...

3 Lentiviral Vectors

Abstract: Somatic gene therapy requires the efficient delivery and sustained expression of a therapeutic gene into the tissues of a human body. Such an approach has tremendous therapeutic potential for several inherited and acquired diseases. However, major obstacles must be overcome to fulfill these high expectations. Among them, the development of more effective gene delivery systems is widely viewed as a critical challenge to gene therapy investigators (Verma and Somia 1997). Retroviral vectors have long been favored as a gene transfer tool for several reasons. First, they integrate efficiently into the genome of the target cell. Second, they do not transfer any viral gene, thus alleviating the risk of immune response against the transduced cells. Both of these properties are likely to be crucial for achieving sustained expression of the transgene. Third, the genome and the life cycle of retroviruses are relatively simple and well studied, which has allowed a continuous improvement in the vector design and the generation of stable producer systems amenable to characterization and scaleup. Fourth, retroviral vectors can transfer a sizeable amount of DNA, up to 7.0 kb of foreign genetic material (Miller et al. 1993). Until now, the retroviral vectors used in clinical trials have been derived from onco-retroviruses such as the Moloney murine leukemia virus (MLV). A major drawback of these vectors is that they can only transduce cells that divide shortly after infection (Miller et al. 1990). While actively dividing cells are found in several body tissues, they are short-lived and continuously replaced. The...

21 tRNA Splicing: An RNA World Add-on or an Ancient Reaction?

Abstract: Introns are encoded in the genes for tRNA in organisms from all three kingdoms of life. Their removal is an essential step in the maturation of tRNA precursors. In Bacteria, introns are self-splicing and are removed by a group 1 splicing mechanism (Kuhsel et al. 1990; Reinhold-Hurek and Shub 1992; Biniszkiewicz et al. 1994). In Eukaryotes and Archaea, intron removal is mediated enzymatically by proteins. Recent progress in understanding both eukaryotic and archaeal tRNA splicing has revealed that the two processes, previously thought to be unrelated, are in fact similar. Insight gained from the comparison has provided a clearer understanding of intron recognition, the catalysis of intron removal, and has given new insight into the evolution of the tRNA splicing process. INTRON-CONTAINING tRNA tRNA Introns in Eukaryotes Intervening sequences (introns) were discovered 20 years ago in the yeast genes for the tyrosine-inserting non-sense suppressor tRNA (Goodman et al. 1977) and for tRNA Phe (Valenzuela et al. 1978). With the completion of the yeast genome, it is known that of the 274 yeast tRNA genes 61, or 20%, contain introns. Table 1 lists the tRNAs that contain introns. PCR cloning of tRNAs from higher eukaryotes has revealed a similar distribution of intron-containing tRNA (Stange and Beier 1986; Green et al. 1990; Schneider et al. 1993). The introns in all of the genes are small (14–60 bases), and they are all located in the same position, one base to the 3′ side of the anticodon (Fig. 1A). Structure probing revealed that the...

3 Bioassays of Prions

Abstract: Prior to the experimental transmission of scrapie to goats inoculated with tissue extracts prepared from sheep with scrapie (Cuille and Chelle 1939), no experimental studies of the disease were possible. Despite the cumbersome bioassays in sheep and goats, some limited information was obtained. For example, the susceptibility of 24 different breeds of sheep was measured after subcutaneous inoculation with brain extract prepared from a sheep with scrapie (Gordon 1946). The resistance of the scrapie agent to inactivation by formalin and heat was also shown using bioassays performed in sheep (Pattison and Millson 1960). The first evidence for strains of prions accumulated with goats that presented with two different clinical syndromes (Pattison and Millson 1961b). One set of prion-infected goats was described as “drowsy” due to the lethargy manifest during the clinical phase of scrapie, and the other was called “hyper” because these animals were highly irritable and easily aroused. One of the most distinctive and remarkable features of slow infections, such as those caused by prions, is their prolonged incubation periods, during which the host is free of recognizable clinical dysfunction. The onset of clinical symptoms marks the end of the incubation period and the beginning of a relatively short, progressive course of illness that ends in death. Although prolonged incubation periods are a fascinating phenomenon, they have been the biggest impediment to prion research. Because animals remain healthy throughout the incubation period, investigators must wait until signs of clinical illness appear before assigning a positive score. In early studies...

7 Adeno-associated Viral Vectors

Abstract: The discipline of gene therapy is relatively young when compared to other fields of study. However, from the start, there has been a clear vision of the expectations required to achieve successful clinical results. These have included (1) efficient transduction of the target cell, (2) long-term expression of the therapeutic gene, (3) lack of immune response to the vector or transduced cell, and (4) absence of toxicity to the patient following delivery. Until recently, this mandate has been met only in part by the numerous viral and nonviral delivery systems. Now, definitive in vivo results meeting all of the above criteria have been established by using the parvovirus adeno-associated virus (AAV) vector system supporting a thorough testing of this vector in the clinical arena. This chapter describes in detail the AAV vector system, production methods, successful in vivo models, and questions related to AAV biology that, when understood, may further enhance the existing vector system. Research employing AAV as a vector for gene therapy has been driven by the desire to exploit the unique biology and life cycle of this virus. This human parvovirus exhibits many natural features that are absent from alternative vectors. AAV’s most prominent feature, which suits it to applications for long-term gene therapy, is the tendency to establish latent infections through integration into the chromosomal DNA. The fact that AAV lytic cycle generally depends on the presence of a coinfecting helper virus means that, essentially, any uninfected helper cell can potentially serve as a host for...

9 Introns and the RNA World

Abstract: The RNA World is a hypothesis about the origin of life based on the view that the most critical event is the emergence of a self-replicating molecule, a molecule that can both copy itself and mutate and, hence, evolve to more efficient copying (Gilbert 1986). Evolution works on variation and selection, and selection is always measured in terms of more efficient multiplication, the ability to make more of the entity in question. The concept of an RNA World is a way of answering the basic problem of what was the molecular biology involved at the beginning of life. Our understanding of the molecular basis of biology today is in terms of a genetic material, commonly DNA, translated through an apparatus involving RNA and the mechanism of protein synthesis to specify the positions of 20 amino acids in protein enzymes. That picture of life, in which the genetic material is of one chemical kind, DNA, made up of four bases, a second chemical, RNA, is used for structural and transfer purposes, and the enzymatic activities in the cell are a third chemical kind made up of 20 ingredient amino acids, creates a complex paradox in trying to formulate how life could have begun. This paradox was resolved by two realizations. One was that RNA is likely to be more primary than DNA, but the picture of an RNA–protein world, in which RNA is the genetic material specifying the positions of amino acids in proteins, still left one with a complex...

10 Cell Biology of Prions

Abstract: The cellular prion protein (PrP C ) is a cell-surface glycoprotein anchored by a glycosylphosphatidylinositol (GPI) moiety (Stahl et al. 1987). PrP C is expressed throughout the brain, particularly in neurons (Kretzschmar et al. 1986;Moser et al. 1995), and to a lesser extent in extraneural tissues (Bendheim et al. 1992; Ford et al. 2002). In the prion diseases, PrP C is converted to an abnormal, conformationally altered isoform (PrP Sc ), which subsequently accumulates in the brain and results in extensive neurodegeneration with an inevitably fatal outcome (Prusiner 1996). Therefore, localizing PrP C in the brain is an important step in understanding the biology of the normal protein and in mapping changes in models of experimental prion diseases. EXPRESSION AND LOCALIZATION OF PrP C IN BRAIN The precise localization of PrP C remains enigmatic, due to conflicting data obtained using different techniques. Immunohistochemical studies have described a somatic expression of PrP C in neurons with no or only a minor signal in the neuropil (DeArmond et al. 1987; Piccardo et al. 1990; Safar et al. 1990; Bendheim et al. 1992; Verghese-Nikolakaki et al. 1999; Ford et al. 2002). Contradictory findings probably reflect the peculiarities inherently associated with pre-embedding techniques. Many immunoelectron microscopic procedures may result in a destruction of cellular membranes, possibly leading to an artificial redistribution of GPI-anchored proteins within the membrane. These uncertainties with regard to the precise subcellular localization of PrP C therefore encouraged two of us (P.J.P. and S.B.P.) to perform a quantitative study of the ultrastructural localization of PrP C in the mouse brain. We used a...

14 Neuropathology of Prion Diseases

Abstract: The discovery of mechanisms of nerve cell dysfunction, degeneration, and death in prion diseases acquired by infection, in dominantly inherited forms, and in sporadic (idiopathic) forms has gone hand in hand with the discovery of the prion and how prions are propagated. The generation of PrP-specific antibodies led to our first immunohistochemical study, in which we discovered that the amyloid plaques in experimental scrapie in Syrian hamsters contain protease-resistant PrP (Fig. 1A) (Bendheim et al. 1984; DeArmond et al. 1985). That finding convinced us that we had a unique opportunity to obtain a better understanding of the pathogenesis of scrapie in animals and the related human disorders that include sporadic, iatrogenic, and familial Creutzfeldt-Jakob disease (CJD), and the rare familial disorder Gerstmann-Straussler-Scheinker syndrome (GSS). The overall objective was to test the hypothesis that PrP Sc accumulation in the brain causes the clinically relevant neuronal dysfunction, vacuolation, and death that are the characteristics of prion diseases. The results of many studies have led to the unifying hypothesis that neuronal degeneration in spontaneous, infectious, and genetic prion diseases and the propagation of prions in those diseases are both related exclusively to abnormalities of PrP. Multiple investigators in Great Britain, Europe, Japan, and the US have contributed to our understanding of the mechanisms of CNS degeneration peculiar to prion diseases. We have attempted to identify and acknowledge their many contributions; however, the number of prion disease investigators is increasing logarithmically and, therefore, inevitably we will have missed some and apologize to them for that...

15 The Hematopoietic System as a Target for Gene Therapy

Abstract: The ability to transfer a gene into repopulating stem cells and to achieve lineage-specific expression in differentiating hematopoietic cells would create many therapeutic opportunities. The hemoglobin disorders, severe β-thalassemia and sickle cell anemia, because of their frequency worldwide and their severe morbidity and mortality, were early targets for human gene therapy. Molecular cloning of the globin genes in the late 1970s (Efstratiadis et al. 1980; Lawn et al. 1980) fueled interest in these diseases as targets for genetic intervention. Demonstration that DNA fragments could be incorporated into the genome of eukaryotic cells by calcium-phosphate-mediated DNA uptake (Pellicer et al. 1978) provided the methodology for attempted gene transfer into stem cells. Reported success in a murine model (Cline et al. 1980) prompted an attempt to treat patients with severe β-thalassemia by introduction of a globin gene into hematopoietic cells using calcium-phosphate-mediated transfection. In retrospect, this attempt carried little, if any, risk to the patients, but it was widely discredited because the very low efficiency of transduction and gene integration achieved by this methodology rendered success very unlikely. Furthermore, the investigators failed to obtain prior approval from the appropriate regulatory bodies before undertaking clinical trials (Anderson and Fletcher 1980). The field of human gene therapy has evolved substantially from this uncertain beginning. Studies in the murine model provided a sound experimental basis for subsequent work in larger animal models and ultimately for the initiation of human clinical trials. Many additional diseases that are potentially amenable to gene-therapy intervention have been characterized. The...

13 Inherited Prion Diseases

Abstract: HISTORICAL OUTLINE The first report of an inherited prion disease can be traced to an affected member of the “H” family carrying a neurologic disorder through multiple generations. It was presented at a meeting of the Viennese Neurological and Psychiatric Association in 1912 (Dimitz 1913). Two decades passed before Gerstmann in 1928 and Gerstmann, Straussler, and Scheinker in 1936 reported clinical presentations and neuropathologic findings for several affected members of the “H” family. These reports establish the disease that is currently referred to as Gerstmann-Straussler- Scheinker disease or GSS (Gerstmann 1928; Gerstmann et al. 1936). The subject reported by Creutzfeldt in 1920 and 1921, which had a positive family history (Creutzfeldt 1920Creutzfeldt 1921), is unlikely to have been affected by the condition now called Creutzfeldt-Jakob disease (CJD). The first authentic familial case of CJD was recorded in 1924 by Kirschbaum (1924). However, it was Meggendorfer in 1930 who showed that the subject described by Kirschbaum was a member of a large kindred that became known as the “Backer” family and has been proven in subsequent publications to be affected by an inherited form of CJD (Meggendorfer 1930; Stender 1930; Jakob et al. 1950). In 1973, Gajdusek, Gibbs, and their colleagues first demonstrated the transmissibility of inherited prion diseases to nonhuman primates (Roos et al. 1973). This finding followed earlier studies that reported the transmissibility of the sporadic form of CJD and kuru, a prion disease of the Fore tribe of New Guinea propagated through endocannibalism (Gajdusek et al. 1966; Gibbs...

8 On the Origin of the Ribosome: Coevolution of Subdomains of tRNA and rRNA

Abstract: Translation is one of the most complicated of biological processes, involving literally hundreds of specific macromolecules. Not the least of this complexity is the structure of the ribosome itself, which even in the relatively simple Escherichia coli version, consists of over 50 different proteins and three RNA molecules, comprising more than 4500 nucleotides, giving an aggregate mass of around 2.5 million daltons (Hill et al. 1990; Matheson et al. 1995). The difficulty of imagining how such a structure evolved is eased somewhat by accepting the notion that the original ribosome was made solely of RNA, as tentatively suggested by Crick (1968) more than two decades ago, and asserted with increasing force (Woese 1980) and enthusiasm (Gesteland and Atkins 1993) since then. Adherents of RNA World scenarios imagine, to varying degrees, that something resembling protein synthesis was carried out by ribozyme-like proto-ribosomes prior to the advent of ribosomal proteins and translation factors (not to mention aminoacyl-tRNA synthetases). Such scenarios solve the chicken-or-the-egg problem of the molecular evolution of ribosomes but raise some difficult new questions. The most obvious is that rRNA itself has a vast and intricate structure containing thousands of nucleotides (see, e.g., Fig. 1), and is not likely to have evolved by chance in a few simple evolutionary steps. Compounding this problem is that, prior to the existence of protein synthesis, it is difficult to imagine what selective pressures could have driven its evolution; in other words, the RNA World could not have anticipated the invention of protein synthesis.

10 The Interactions That Shape RNA Structure

Abstract: This chapter describes the effects of noncovalent interactions on RNA structure and evolution. The building blocks of RNA are well suited for taking advantage of relatively strong noncovalent interactions such as stacking and hydrogen-bonding to form ordered structures. These ordered structures are able to protect RNA from chemical degradation and to allow the specific binding and catalysis required for further evolution. The noncovalent interactions important for shaping RNA during evolution are revealed by the RNA structures that occur naturally, and by thermodynamic measurements on model systems. First, we discuss the fundamentals of the molecular interactions, then the contributions of stacking, hydrogen-bonding, and metal ions to formation of helices and other motifs. Examples are given of how these interactions shape RNA structures. Finally, some speculations are presented as to how these interactions directed evolution. Since understanding of noncovalent interactions, and knowledge of three-dimensional structures of RNA, are limited, this chapter represents an early stage in the evolution of our understanding of how the two are connected. FUNDAMENTALS The equilibrium constant, K , relating the concentrations of two conformations, C 1 and C 2 , of an RNA strand is given by(1)K=[C1]/[C2]=exp(-ΔG°/RT) For an association of two non-self-complementary strands A and B to give A·B, the relevant K = [A·B]/[A][B]. Here Δ G ° is the standard free-energy difference between the two conformations, [C 1 ]/[C 2 ] and [A·B]/[A][B] are the ratios of the equilibrium concentrations, R is the gas constant (1.987 cal K −1 mole −1 ), and T is the temperature in Kelvins. Thus, Changes in Δ G ° of 1.4 and 2.8...

6 Prions of Fungi: [URE3], [PSI], and [Het-s] Discovered as Heritable Traits

Abstract: In 1994, it was proposed that two non-Mendelian genetic elements of Saccharomyces cerevisiae , called [URE3] and [PSI], were in fact prion (infectious protein) forms of the chromosomally encoded proteins, Ure2p and Sup35p, respectively (Wickner 1994). Recently, it has been suggested that [Het-s], a non-Mendelian genetic element of the filamentous fungus Podospora anserina , is a prion of the protein encoded by het- s (Coustou et al. 1997). Here we describe the properties of these phenomena, the evidence that they are prions, and their general implications for prion biology. The concept of an infectious protein was first proposed to explain the unusual properties of the agent producing the transmissible spongiform encephalopathies (Griffith 1967). The term “prion” was coined to mean the scrapie agent, including the possibility that it may have no essential nucleic acid component, but not restricted to this case (Prusiner 1982). We shall use the term prion here to mean infectious protein (the protein-only model), regardless of the organism or protein, and making no assumptions about the mechanism involved. In general, a prion is a protein that has undergone a change such that it no longer carries out its normal function but has acquired the ability to convert the normal form of the protein into the same form as itself, the prion form. By this definition, the prion change need not be one of conformation. A protein methylase might methylate another molecule of itself by mistake and, having done so, the methylated methylase may no longer be able to modify its...

24 Dynamics of the Genetic Code

Abstract: Shape is crucial for catalysis. In the hypothetical RNA World, the replicative RNAs, which constituted the hereditary information, also functioned as the “shapes” for catalysis. There was no need for decoding. When decoding originated, presumably discrimination between alternate coding possibilities was initially weak, but once one mode became predominant, there would have been selection to lock it in with increasing efficiency. Nontriplet translocation and nonstandard meaning of code words presumably generally approached a minimum compatible with speed and energy use optimization. In the present day, nonstandard decoding alternatives generally just contribute to a low level of translational errors, of which frameshifting errors (Atkins et al. 1972; Kurland 1992) are a grave type. However, some unknown proportion of genes in probably all organisms has special sites where efficient decoding alternatives are programmed into the mRNA. The group of mechanisms involved in redirection of decoding is called “recoding” (Gesteland et al. 1992). Either the evolution of the ability to perform recoding was coincident with evolution of the ability of the decoding apparatus to perform standard decoding, or it is a later sophistication. Of course, the answer is unknown, but the conservation of the required mRNA signals presented below indicates that recoding has been part of the decoding repertoire for at least several hundred million years and must therefore be favored by evolution. This is clearly distinct from the error rate that is a trade-off between energy expenditure, speed, and accuracy. In the latter case, evolution has optimized the balance as a whole.

10 Advances in Synthetic Gene-delivery System Technology

Abstract: Twenty-six years ago in a Science article, Friedmann and Roblin (1972) outlined prospects for human gene therapy. This forward-looking review anticipated the development of two alternative gene-delivery systems: viral-gene therapy vectors and synthetic gene-delivery systems using purified gene sequences. As molecular biology techniques matured, the tools to package genes into nonreplicating, recombinant viral vectors became available (Mann et al. 1983), allowing the efficient introduction of recombinant genes into living cells in vitro (cultured cells) and in vivo (animals and humans). During the last several years, we have witnessed an exponential growth in preclinical research and clinical development of recombinant viral vectors for gene-therapy applications. However, the introduction of synthetic, nonviral gene-delivery systems into the clinical gene-therapy repertoire took somewhat longer to develop. Scientists at Vical, Inc. and the University of Wisconsin made a key discovery that led to increased interest in direct nonviral gene-transfer technology (Wolff et al. 1990). These investigators were the first to show that under certain conditions, muscle tissues could absorb plasmids, leading to expression of the encoded protein persisting for periods of weeks to several months. Improvements in this basic finding led to what has been referred to as “naked DNA” reagents for gene transfer. In addition, numerous laboratories and biotechnology companies are developing other technologies that allow the delivery of DNA directly into nonmuscle tissues, including the use of cationic lipid molecules that facilitate direct absorption of DNA into cells. The plasmids used for these products are chemically well defined and highly purified and can...

4 Transmission and Replication of Prions

Abstract: One of the most remarkable features of slow infections is the clockwork precision with which the replication of prions occurs. Inoculation of numerous animals with the same dose of prions results in illness at the same time months later. The molecular mechanisms controlling this extraordinarily precise process are unknown. The term infection implies that a pathogen replicates during this process. When the infectious pathogen has achieved a high titer, a disease in the host often appears. Prior to the recognition of the existence of prions, all infectious pathogens contained a nucleic acid genome that encoded their progeny. Copying of this genome by a polynucleotide polymerase provided a means of replicating the pathogen; in the case of prions, another mechanism functions. GENERAL FEATURES OF PRION TRANSMISSION AND REPLICATION As we learn more about prions, some general features and rules of prion replication are beginning to emerge. Although many of the results described here can be found in other chapters, we felt that it was important to collect information on prion transmission and replication in one place within this monograph. Prion Replication and Incubation Times When the titer of prions reaches a critical threshold level, the animals develop signs of neurologic dysfunction. The length of the interval from inoculation to reach the threshold prion concentration, at which CNS dysfunction becomes evident, is referred to as the incubation time or incubation period. The length of the incubation time can be modified by (1) the dose of prions, (2) the route of inoculation, (3)...

5 Re-creating an RNA Replicase

Abstract: The idea that certain RNA sequences can use the information from one RNA strand to accurately and efficiently produce another RNA is central to the RNA World hypothesis (Pace and Marsh 1985; Sharp 1985; Cech 1986; Orgel 1986). Such RNA-dependent RNA polymerase ribozymes would have been responsible for replicating the ribozymes of the RNA World, including themselves (via their complement sequences). The most extreme versions of the RNA World hypothesis suggest that life began with a self-replicating system seeded by a pair of replicase molecules, one serving as the template, the other as the enzyme. Less extreme versions of the RNA World hypothesis cite difficulties with prebiotic synthesis and stability of RNA on the early Earth and instead postulate that life originated in a “pre-RNA world”—an era dominated by an RNA-like polymer that could catalyze reactions and code for its own replication (Chapter 2). Because the pre-RNA polymer presumably facilitated production of RNA, issues regarding prebiotic availability of RNA are less problematic in this conservative RNA World view. However, the scenario calls for eventual transition to the RNA World, and thus still relies on some radical assumptions regarding the intrinsic catalytic capability of RNA—including the idea that there exist, somewhere among all RNA sequence possibilities, sequences that can promote the replicase reaction. The RNA replicase is one of the few assumptions common to all versions of the RNA World hypothesis. Because nearly four billion years of evolution have obscured any vestiges of the presumed RNA replicases, our only...