Showing papers in "Progress in Nucleic Acid Research and Molecular Biology in 1963"
TL;DR: The chapter describes that phenol deproteinization involves the treatment of biological material with water-saturated phenol, provides immediate and practically complete inactivation of nucleases, causes no degradation of polyribonucleotide chains during the isolation procedure, completely removes the proteins, and thus secures a high purity of the RNA preparation.
Abstract: Publisher Summary This chapter discusses that the study of high-polymer RNA's is the discovery of biological activity (infectivity) of the virus RNA. Investigations first permitted the statement that the isolated RNA preparations were actually native, and that the methods of RNA isolation used, particularly phenol deproteinization, did not destroy the nativity of the RNA obtained. The chapter describes that phenol deproteinization involves the treatment of biological material with water-saturated phenol, provides immediate and practically complete inactivation of nucleases, causes no degradation of polyribonucleotide chains during the isolation procedure, completely removes the proteins, and thus secures a high purity of the RNA preparation. Apparently the methods involving phenol deproteinization are at present the best for the isolation of RNA from various biological materials. It is evident that lack of such a criterion as biological activity would be a great obstacle in any serious attempts to study the macromolecular structure of RNA. Thus the discovery made in the laboratories has greatly stimulated the investigations of macromolecular structure of native RNA from tobacco mosaic virus (TMV), at first, and of high-polymer RNA from cellular organisms later. When the “soluble” RNA was discovered and its specific biological activity established, the question of physical properties and native structure of this type of cellular RNA also arises.
208 citations
TL;DR: This chapter discusses that the main interest in the denaturation and renaturation of isolated, purified nucleic acids is in their applications and whether the short sequences that can be assayed show a complementary relationship between the twoucleic acids of the DNA-RNA hybrid region.
Abstract: Publisher Summary This chapter discusses that the main interest in the denaturation and renaturation of isolated, purified nucleic acids is in their applications. Renaturation of denatured DNA, isolated from closely related organisms, can be used to investigate their taxonomic and evolutionary relationships. Renaturation can also be employed to prepare hybrid molecules between closely related strains or species that differ in a few or many genetic markers and then used in conjunction with transformation experiments to ascertain the genetic and phenotypic potential of each of the strands of the renatured hybrid. Thus, renaturation cannot only be used to detect base sequence homologies, but also makes it possible, by adding a nucleic acid “reactant,” to isolate from a population of molecules those showing complementarity to it. The renaturation of DNA and RNA makes it possible to examine how the two nucleic acids are related structurally as well as metabolically. The chemical and enzymatic methods that are thus far available for studying the sequences of the bases in DNA and RNA are in the very early stages of development, but it would be of great interest to see whether the short sequences that can be assayed show a complementary relationship between the two nucleic acids of the DNA-RNA hybrid region. A clearer understanding of denaturation and renaturation and the factors influencing these processes would contribute significantly to a rational approach in designing experiments for gaining an insight into the interactions of nucleic acids during the growth of cells and viruses.
156 citations
TL;DR: The chapter describes that the different behaviors of bacteria under UV irradiation or X irradiation demonstrate that both kinds of radiation act fundamentally differently, although the point of attack in the cell is the same: the nucleic acids.
Abstract: Publisher Summary This chapter reviews the photochemistry of nucleic acids and discusses the difference in the reaction of thymine to ultraviolet (UV) irradiation in aqueous solution or frozen in ice. It demonstrates that thymine forms a dimer when UV irradiated in ice. If dimerization occurs within an individual DNA strand, the biological consequence may be an altered tendency to pair with the adenine of the complementary strand. The altered strength of the sugar-base linkage of deoxyribose in thymidine dimer is an indication of the changed electron density in the pyrimidine structure and this changes the ability to form hydrogen bonds. But if dimerization occurs between the two individual strands of DNA, the dividing mechanism of the cell may be completely blocked. Although an intermolecular dimerization in native DNA appears improbable, during cell duplication there are phases with cleaved DNA strands in which intermolecular dimerization is more likely. The model experiments show that both forms of dimerization are possible. The chapter describes that the different behaviors of bacteria under UV irradiation or X irradiation demonstrate that both kinds of radiation act fundamentally differently, although the point of attack in the cell is the same: the nucleic acids. The great specificity of UV radiation permitted the rapid discovery of the chemical basis of biological UV inactivation.
140 citations
TL;DR: Some of the chemical evidence bearing on the mutagenic action of some of the agents utilized on this biological system is explained, and some specific theories of mutagenesis are discussed.
Abstract: Publisher Summary The chapter discusses the specificity of chemical mutagenesis. The chapter discusses the genetic studies with the organic chemistry, physical chemistry, and biochemistry of mutagens, bases, and polynucleotides. The chemical evidence for base analogs concerns their incorporation into the DNA, and the manner in which the analogs may hydrogen-bond with natural bases. For mutagens active on extracellular phage and nonmetabolizing DNA the chemical evidence concerns the DNA bases, with which they react and the properties of the products. It deals, primarily, with the genetic studies, using the rII mutants of phage T4. The chapter explains some of the chemical evidence bearing on the mutagenic action of some of the agents utilized on this biological system. The chapter also discusses of the some specific theories of mutagenesis, and follows this with the evidence and its interpretation. A quantitative survey of the mutation data is described, followed by its consideration in the light of the theories, and further the features of particular mutagens are dealt with, as the mutagens are discussed individually.
84 citations
TL;DR: The chapter concludes that there have been dramatic developments in this field and it now seems possible that the code of the genetic code will be found within a comparatively short time.
Abstract: Publisher Summary The Sequence Hypothesis states that the amino acid sequence of a protein is determined by the sequence of nucleotides in some particular piece of nucleic acid. The evidence in favor of this is now very considerable, and hopes that this relationship may be a simple one and that the sequence of the four bases in the nucleic acid can be thought of as a simple code for the amino acid sequence. The exact sequence of bases that determines each of the twenty amino acids found in proteins is known as the “coding problem.” The amount of degeneracy may be small andthe evidence from the cell-free system, the amino acid replacement data, and the fractionation of sRNA, is compatible with this. The amount of degeneracy may be very much higher and this is suggested by the wide range of DNA composition and by the genetic studies. Also,it is not contradicted by the more direct evidence, though this suggests that if the code is highly degenerate it is unlikely to be degenerate at random. The chapter concludes that there have been dramatic developments in this field and it now seems possible that the code will be found within a comparatively short time. The chapter deals with the recent progress and discusses the general nature of the genetic code.
78 citations
TL;DR: The chapter elaborates the over-all mechanism of gene action and concludes that the product of the RNA polymerase reaction is involved in protein synthesis; the details of this relationship are still vague.
Abstract: Publisher Summary This chapter discusses that the role of DNA in RNA synthesis is implicit in the widely accepted hypothesis that DNA is the genetic carrier of information and that RNA is an essential component in the expression of this information in polypeptide synthesis. Cellular phenotypic modification following bacteriophage infection, transformation with free DNA, mutation, or conjugation and genetic recombination would thus result from a mechanism of a DNA-directed change in RNA metabolism. Excluded from this generalization are the varieties of plant, animal, and bacterial viruses that infect suitable hosts with RNA alone. The chapter elaborates the over-all mechanism of gene action. It deals with specific demonstrations of DNA control of RNA synthesis and, most particularly, with the enzyme, RNA polymerase. Finally, it concludes that the product of the RNA polymerase reaction is involved in protein synthesis; the details of this relationship are still vague. These, and other problems, will be clarified by future studies.
75 citations
TL;DR: It seems much more likely that quantitative differences in this respect between the nuclear and ribosomal RNA are a reflection of the relative degree of disorganization of the two fractions rather than of a fundamental difference in their biological function.
Abstract: Publisher Summary The chapter discusses of the ribonucleic acid. The rapidly labeled nuclear RNA is involved, in some way, in the function of the DNA and it is not difficult to suggest either that its synthesis and breakdown is a part of the mechanism, by which the activity of the DNA, is controlled or is part of the mechanism, by which proteins are synthesized in the nucleus. Direct experimental evidence, about either of these two notions, is lacking. It seems much more likely that quantitative differences in this respect between the nuclear and ribosomal RNA are a reflection of the relative degree of disorganization of the two fractions rather than of a fundamental difference in their biological function. The mechanism of the genetic determination is a continuous flow of the RNA molecules from nucleus to cytoplasm, a passage of RNA to the cytoplasm at one particular stage of the cell cycle, or a synthesis of the cytoplasmic RNA in association with the nuclear membrane.
72 citations
TL;DR: The function of the pyrimidine base can now be seen as an alternate base-acid catalysis for the formation and the decay of the intermediate electromagnetic hypersensitivity (EHS)a, corresponding to much stronger bases and acids.
Abstract: Publisher Summary The chapter discusses the function of the pyrimidine base in the ribonuclease reaction. The analysis of the chemical course of the enzymatic reaction was concluded that the pyrimidine base acts specifically in the catalysis and not in the binding, and that the increase of electrophilicity at the phosphorus required for reaction is furnished, by the enzyme, through a protonation. The chapter discusses the various mechanism of the nonenzymatic cleavage of 3' ribonucleoside di- and tri-esters. The function of the pyrimidine base can now be seen as an alternate base-acid catalysis for the formation and the decay of the intermediate electromagnetic hypersensitivity (EHS)a. This role depends first on a steric requirement that the base holds the proton of the 2' OH group, between the attacking and the leaving oxygen. This cannot be done by the purine bases. Secondly, it depends on the easily polarizable system that allows a rapid change of the electron density at the C-2 oxygen. This is required to dissipate the positive charge, by interaction, with the solvent, thus stabilizing the intermediate and producing actual basicities and acidities, corresponding to much stronger bases and acids. These actual values are responsible for the reaction rates. The chapter concludes with the mechanism of the enzymatic reaction of ribonuclease reaction.
70 citations
TL;DR: It is possible to affect the pH of neoplastic tissues and to intensify the inhibitory effects of the azapyrimidine derivatives, by preparing compounds that yield the active form of the antimetabolite, when enzymatically decomposed in the cell, and at the same time shift the pH toward the optimum for the action of the antibacterial substance.
Abstract: Publisher Summary The chapter discusses the mechanism of action and application of azapyrimidines. The mechanism of action of the most important azapyrimidines, such as (a) 6-azathymine, (b) 6-azauracil, (c) 5-assuracil and 5-azaorotic acid, and (d) 6-azacytidine is discussed in this chapter. Among azapyrimidines and their derivatives, greatest attention has been paid to the cancerostatic effects of 6-azauridine. It followed from the very first tests that the antineoplastic effect of 6-azauracil is associated with the formation of riboside derivatives. The biological effects of 6-azauridine, such as cancerostatic effects, the virostatic effect, and other effects are also discussed in this chapter. Some of the inhibitory effects of azapyrimidines and of their derivatives, mentioned in this chapter, probably, arise from the different degree of dissociation of the azapyrimidine derivatives as compared with the dissociation of the corresponding natural analogs at equal pH values. The chapter discusses several experimental findings supporting this concept. It is possible to affect the pH of neoplastic tissues and to intensify the inhibitory effects of the azapyrimidine derivatives. The aim is attained, by preparing compounds that yield the active form of the antimetabolite, when enzymatically decomposed in the cell, and at the same time shift the pH toward the optimum for the action of the antimetabolite.
61 citations
TL;DR: The chapter discusses that certain aspects of primer function in DNA synthesis are susceptible to experiment, and an evaluation of the kinetics of DNA synthesis utilizing a variety of the chemical and physical agents that can affect primer activity may also be fruitful in evaluating replication mechanisms.
Abstract: Publisher Summary This chapter reviews two degrees of primer activation. The first concerns the secondary structure of the DNA molecule, and for the calf thymus and phage-induced E. coli polymerase this type of activation, typically produced by heat, acid, or alkaline denaturation, results in rate increases of one to two orders of magnitude. The second type of primer activation results from changes in the primary structure of the DNA chain and is typically seen in sonicated or enzymatically degraded primers. A uniform interpretation of the second type of activation based on the improved kinetic situation resulting from chain shortening seems a most useful generalization. The validity of this interpretation in detail still depends, however, upon a careful and thorough study of the kinetic constants in the sonicated primer systems. Extension of this interpretation to enzymatically degraded primers is probably a gross oversimplification, and in this instance adequate characterization of the degraded primers is an impossible task. The chapter discusses that certain aspects of primer function in DNA synthesis are susceptible to experiment. The final disposition of primer molecules is amenable to centrifugal experiments with various isotopic labels, and, in enzymes utilizing denatured primers, to separation on methylated albumin columns followed by physical analysis. An evaluation of the kinetics of DNA synthesis utilizing a variety of the chemical and physical agents that can affect primer activity may also be fruitful in evaluating replication mechanisms, although the complete analysis of a system with such complexity seems, at times, presumptuous.
56 citations
TL;DR: The authors reviewed the relatively new appearance of a variety of RNA's that are functionally distinct are closely interrelated Ribosomal RNA seems to stand apart with only a passive participation in information transfer Its relationship to messenger ribonucleic acid (mRNA) is unsettled, one group favoring a melting of mRNA into the ribosome, while others favor separate paths of synthesis from a common pool of mononucleotides.
Abstract: Publisher Summary This chapter reviews the relatively new appearance of a variety of RNA's that are functionally distinct are closely interrelated Ribosomal RNA seems to stand apart with only a passive participation in information transfer Its relationship to messenger ribonucleic acid (mRNA) is unsettled, one group favoring a “melting” of mRNA into the ribosome, while others favor separate paths of synthesis from a common pool of mononucleotides The general mode of biosynthesis of sequence patterns has proven to be a one-by-one addition and the differences of sequence pattern between ribosomal and the template carrying mRNA are difficult to explain The chapter discusses that the rapid turnover of mRNA was originally thought to be a characteristic of this class of material Such turnover now seems rather to be a feature of microbial mRNA during rapid growth It is related to a rapid rate of protein synthesis and the need for fast replacement in view of an apparently rapidly changing mRNA population with a probably sequential translating of DNA into protein synthesis during the growth cycle Thus mRNA may be defined, as of now, as a sequence-determining template that combines with ribosomes for catalytic functioning in protein synthesis
TL;DR: The inability of this enzyme to attack the last two nucleotide residues at the 5'-phosphoryl or 5'-hydroxyl terminus of a polydeoxyribonucleotide chain, coupled with its lack of endonuclease activity, permits the use of this enzymes as an end group reagent.
Abstract: Publisher Summary This chapter discusses the nucleases of Escherichia coli. The chapter compiles the information currently available, with respect to the nucleases characteristic of a single cellular species— namely, Escherichia COX. This chapter also describes the enzymes that catalyze the hydrolysis or phosphorolysis of the various nucleic acid constituents. The nucleic acids of E. coli, their structure and biosynthesis, have been and continue to be under intensive genetic and biochemical investigation and much of our knowledge, regarding the enzymatic synthesis of nucleic acids and the involvement of nucleic acids in protein synthesis, are being derived from the studies carried out with this particular organism. Emphasis, in the foregoing discussion, has been on the properties of the nucleases of E. coli and their place in the nucleic acid economy of this organism. Furthermore, the inability of this enzyme to attack the last two nucleotide residues at the 5'-phosphoryl or 5'-hydroxyl terminus of a polydeoxyribonucleotide chain, coupled with its lack of endonuclease activity, permits the use of this enzyme as an end group reagent.
TL;DR: Procedures, by which the transfer RNA can be separated from one or more of the other major components, are described in this chapter, and some of their limitations discussed.
Abstract: Publisher Summary The chapter discusses the preparation, fractionation, and properties of sRNA. Many of the physical and chemical properties of the transfer RNA differ in greater or lesser degree from those of the other components of cells, such as proteins, polysaccharides, DNA, and ribosomal RNA. These differences provide the bases of the methods used for the separation of the transfer RNA in a more or less purified state. Procedures, by which the transfer RNA can be separated from one or more of the other major components, are described in this chapter, and some of their limitations discussed. A large number of fractionation procedures have been devised in attempts to achieve some degree of purity, with respect to specific amino acid acceptor capacities. These procedures fall into two groups, the physical methods and the chemical methods. The chapter also discusses some of the major properties of sRNA, such as the nucleotide compositions of the transfer sRNA's, the secondary structure of sRNA's, and the nucleotide sequences of sRNA.
TL;DR: The chapter describes that double-stranded molecules of DNA show a greater stability to such mutagenic agents as heat, radioactive decay, and nitrous acid than to those not possessing such a structure.
Abstract: Publisher Summary This chapter discusses that the twin-stranded helical structure of DNA has had a tremendous impact on contemporary research and thinking in biology and biochemistry The most provoking aspect of the proposed structure is that it furnishes a persuasive insight into the chemical mode of biological replication and the transcription of genetic information The chapter describes that double-stranded molecules of DNA show a greater stability to such mutagenic agents as heat, radioactive decay, and nitrous acid than to those not possessing such a structure Twin-stranded forms might have a superior evolutionary survival value, and natural selection may be based, in part, on the differential stabilities of the two forms The hereditary determinants of higher organisms may well be associated with DNA containing singly, doubly, or multiply stranded forms The analytical data on chromatographically separated fractions of DNA from calf thymus also indicate such possibilities The fact that deoxyribonucleases with specificities for single-stranded DNA exist may imply the occurrence of this type of nucleic acid in nature
TL;DR: The separation of oligonucleotide and polynucleotide material on certain other adsorbents that have been introduced more recently, such as diethylaminoethyl cellulose (DEAE-cellulose) and methylated serum albumin, is described in this chapter.
Abstract: Publisher Summary The chapter discusses the column chromatography of oligonucleotides and polynucleotides. The separation of oligonucleotide and polynucleotide material on certain other adsorbents that have been introduced more recently, such as diethylaminoethyl cellulose (DEAE-cellulose) and methylated serum albumin, is described in this chapter. The former offers wide possibilities in the fractionation of oligonucleotides and the latter deserves special attention, because of its ability to separate nucleic acids of high molecular weight by column chromatography. The chromatographic separation of oligonucleotides, according to chain length, is discussed in this chapter. Many components can be isolated from an RNase digest, by chromatography on DEAE-cellulose, at a slightly alkaline pH. The chromatographic separation of the oligonucleotides on sephadex is also discussed in this chapter. Sephadex is a cross-linked dextran that, in principle, allows the separation of substances according to the molecular size. Chromatographic separation of nucleic acids, such as chromatographic separation on substituted cellulose anion exchangers and chromatographic separation of nucleic acids on methylated albumin columns, are also discusses in this chapter.
TL;DR: The understanding of the mechanisms of biosynthesis of RNA have become much clearer, partly as a result of the elucidation of the structure of RNA, and partly from the demonstration of the natural occurrence of the 5'-mono, di-, and triphosphates of all four ribonucleosides-adenosine, guanosine, cytidine, and uridine.
Abstract: Publisher Summary This chapter describes that RNA is synthesized in animal tissues from small molecular precursors. Much of the evidence for this is derived from isotopic experiments in which labeled precursors have been observed to be incorporated into the RNA of animal cells. Thus, P 32 -orthophosphate, N 15 -ammonium citrate, N 15 -glycine, C 13 -formate, C 13 -glycine, C 13 -bicarbonate, C 14 -formate, C 14 -glycine, and C 14 - bicarbonate, as well as labelled purine and pyrimidine nucleosides and bases, have been shown to be effective precursors of tissue RNA's. The understanding of the mechanisms of biosynthesis of RNA have become much clearer, partly as a result of the elucidation of the structure of RNA, and partly from the demonstration of the natural occurrence in mammalian and other tissues of the 5'-mono, di-, and triphosphates of all four ribonucleosides-adenosine, guanosine, cytidine, and uridine. The chapter also describes the stages of biosynthesis of RNA. It discusses that since RNA is a complex molecule composed of purine and pyrimidine ribonucleotides, it consider its biosynthesis in three main stages: (1) the formation of purine and pyrimidine nucleotides; (2) their phosphorylation to the ribonucleoside di- and triphosphates; and (3) the polymerization reactions leading to the formation of polyribonucleotides.
TL;DR: This chapter discusses that it is important to distinguish the essential features of the structure of DNA that can be taken as firmly established from those that may be restricted to some particular experimental conditions and shows that X-ray diffraction techniques provide the most direct tool for solving some of these problems.
Abstract: Publisher Summary This chapter reviews a model of DNA that provides a precise molecular basis for the interpretation of genetic phenomena. Many of the chemical and physical properties of DNA had been studied at that time, laying a firm foundation for the construction of a model, and biochemistry and genetics were sufficiently advanced to assimilate the new ideas and to test the working hypothesis that the model suggested. The chapter discusses that it is important to distinguish the essential features of the structure of DNA that can be taken as firmly established from those that may be restricted to some particular experimental conditions. Second, it is of interest to investigate the structural modifications that DNA can undergo in vitro without irreversible damage, and to try to correlate them with any polymorphism that might be related to the physiological functions of DNA. The chapter focuses on these two problems and also provides description of the X-ray diffraction study of DNA in solution with the purpose of showing that X-ray diffraction techniques provide the most direct tool for solving some of these problems.
TL;DR: The chapter discusses the various base compositions of the plant virus RNA's and of the distribution of the various polynucleotides that are liberated on enzymatic digestion and the claimed synthesis of infectious tobacco mosaic virus RNA in vitro.
Abstract: Publisher Summary This chapter discusses certain aspects of the vast amount of work being carried out on plant virus nucleic acids at present. The chapter discusses the various base compositions of the plant virus RNA's and of the distribution of the various polynucleotides that are liberated on enzymatic digestion. These measurements are particularly in virus taxonomy and it is necessary to review information particularly in relation to the protein composition of the strains of the plant viruses. The chapter explains the various amount of nucleic acid in plant viruses. The plant viruses have to direct the host cells into producing the enzyme systems in addition to the coat proteins and nucleic acids required for constituting new virus particles, so that many of the rather simple ideas about the relationship of the virus nucleic acids and proteins that have been put forward are revised. The chapter also discusses virus ribonucleic acids as “messengers” and mutations in plant viruses. The chapter concludes with the claimed synthesis of infectious tobacco mosaic virus RNA in vitro .