Showing papers in "Methods in Enzymology in 1968"

[105] Determination of DNA concentration with diphenylamine

Abstract: Publisher Summary This chapter discusses the determination of DNA concentration with diphenylaraine and describes several other color reactions for DNA. The reaction between deoxyribose and diphenylamine is probably the most frequently used color reaction for the determination of DNA. The chapter focuses on the later modifications for the determination of DNA especially in microorganisms and animal tissues and presents the modifications described by Burton, Croft and Lubran, and Giles and Myers. Croft and Lubran were measuring the DNA in saline washes of human stomachs. The material has a high content of sialic acid, which seriously interferes in several color reactions for DNA, including Button's modified diphenylamine reaction. Sialic acid reacts in this method to give a color with a maximum absorption at 550 mμ. Giles and Myers found that the blank readings could be reduced by omitting the sulfuric acid and adding the acetaldehyde to the individual tubes. They also obtained a worthwhile increase of sensitivity by increasing the concentration of diphenylamine and altering the relative volumes of sample and reagent. Giles and Myers encountered turbidities in their reaction mixtures due to impurities in DNA extracts from plant tissues.

[109] Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA

Abstract: The GC content of highly purified DNA samples can be estimated from their thermal denaturation temperatures, employing small amounts of DNA (10–40 μg). The method is simple, convenient and utilizes readily available laboratory equipment; the thermal denaturation profile also gives a good picture of the quality of the DNA preparation. Denatured DNA present in native preparations, or single stranded DNA obtained by denaturing native DNA, or isolated from natural sources such as bacteriophage φX174, give broad thermal transition profiles. Denatured DNA which has been renatured by annealing, or which renatures spontaneously because it is cross-linked, yields thermal denaturation profiles which are almost similar to that of native DNA. A recent interesting observation38 is that the supercoiled component of polyoma DNA has a Tm which is 17° higher (in SSC) than the circular, nonsupercoiled or linear polyoma components. The authors would like to emphasize a point which is troublesome to many investigators, namely, the degree of hyperchromicity resulting from the denaturation of DNA. When the denaturing agent (e.g., heat, alkali) is still present, the increase in absorbance in approximately 40% at 260 mμ. Upon removal of the denaturing agent the difference in absorbance of native and denatured DNA is approximately 12%, reflecting the re-formation of intra- and intermolecular short helical structures. It should also be pointed out that irreversible denaturation is accomplished only after the last hydrogen bonds present in the GC rich regions of the DNA have been disassociated. Until this point is reached, the melting of the double-helical molecule is completely reversible, even if the heated solution is rapidly cooled. When equipment is available, analytical CsCl density gradient centrifugation is the preferred technique for estimating the GC content of DNA, especially if only small quantities (in the range of 1 μg) and/or only partially purified DNA are available.39 If possible, base compositions should be determined by two independent methods. Discrepancies in the base composition by the two methods are indicative of the presence of unusual bases.36 When large numbers of samples are to be compared, melting profiles and buoyant density determinations used together provide a great deal of information as to the composition and quality of the samples.

[103a] The use of guanidinium chloride in the isolation of nucleic acids

Abstract: Publisher Summary This chapter discusses and investigates the use of guanidinium chloride in the isolation of nucleic acids and explains that the nucleoprotein is dissociated into RNA and protein by 4 M-guanidinium chloride, which also inhibits the action of ribonuclease. RNA is precipitated from solution in the cold, by acidification to pH 5 at 0° or by the addition of alcohol (0.5 volume) at 0°. The precipitate is purified by redissolution in, and precipitation from, guanidinium chloride. In a preliminary experiment, the sedimentation pattern of the RNA component of ribosomes (100 μg/ml) in guanidinium chloride (4 M) is obtained by means of an analytical ultracentrifuge fitted with ultraviolet optics to establish that the RNA component is undegraded. The omission of the purification procedure leads to RNA contaminated with ribonuclease. In one experiment, a ribosome suspension was added to guanidinium chloride. The solution was divided into two parts. Alcohol (0.5 volume) was added to each to precipitate RNA. The precipitates were separated by low speed centrifuging. In one case, the precipitate was dissolved in guanidinium chloride, and ultimately a product was obtained that had the sedimentation pattern. In the other case, the precipitate was immediately extracted with water and, once dissolution was achieved at 0°, an aliquot was removed, bentonite was added to inhibit ribonuclease, and the sedimentation pattern was obtained. The use of 4 M guanidinium chloride for the isolation nucleic acids (principally DNA) from the nuclear fraction is also described in the chapter, in which the nucleic acid is purified by dissolution in, and precipitation from, 4 M guanidinium chloride twice more. The precipitate of purified nucleic acid is redissolved in 4 M guanidinium chloride at 25° and dialyzed overnight at 0° against standard saline citrate solution. DNA may then be freed from RNA by standard methods.

[125] Monolayer techniques in electron microscopy of nucleic acid molecules

Abstract: Publisher Summary This chapter discusses the monolayer techniques in electron microscopy of nucleic acid molecules and describes a method in which DNA or RNA of a bulk solution is transformed to a monomolecular layer. To do this, a film of protein floated onto an aqueous solution is used. Many globular proteins in solution, and similar polymers, are capable of producing surface films on an aqueous solution in a fiat trough. As the protein is surface-denatured, it forms an insoluble film. This protein film can be considered as a monomolecular layer and assumed to exist as a molecular net of unfolded polypeptide chains. The DNA or RNA is adsorbed to it by basic side groups of amino acid residues. This adsorption effectively brings a nucleic acid molecule from a three-dimensional position in an aqueous solution (the subphase) to a two-dimensional position—that is, adsorbed to the polypeptide net. After adsorption, the monolayer, consisting of the protein net and the adsorbed nucleic acid, is itself adsorbed to a solid support and then dried. The preparation can be contrasted for electron microscopy, and electron micrographs are taken. The three procedures are employed and discussed in the chapter—a spreading procedure, a diffusion procedure, and a one-step release procedure. These three procedures explains that most globular proteins have sufficient spreading activity and these proteins become surface-denatured on top of the subphase; more information is needed as to whether DNA or RNA is enveloped as a complex of protein and nucleic acid, or more likely, is nakedly adsorbed to polypeptide chains by a few adsorption side groups per unit length; and the final concentration of nucleic acids adsorbed in the diffusion procedure depends on concentration in the subphase, period of adsorption, salt concentrations, and size of the nucleic acid.

[96] Isolation and characterization of chromosomal nucleoproteins

Abstract: Publisher Summary Chromosomes are ordinarily obtained from cells during interphase and are, therefore, in the extended form known as chromatin. The advancement in the understanding of chromosomal structure and function has been made possible by the development of new methods for the handling of chromatin and chromosomal constituents. The isolation of chromatin is based upon differential centrifugation followed by sucrose density gradient centrifugation. Chromatin is among the most pelletable components of a tissue homogenate. The tissue is, therefore, ground in a suitable medium, freed of unruptured cells and membrane fragments by filtration, and sedimented at 1000–4000 g, conditions that do not bring down mitochondria. The pellet is then washed by repeated suspension and pelleting, finally layered on sucrose solution, and centrifuged for an appropriate period. By these methods, 60–75% or more of the DNA present in the original tissue is recovered as purified chromatin. The basic steps for the isolation of the highly contracted metaphase chromosomes are (1) accumulation of a large proportion of cells in metaphase by treatment with colchieine or other mitotic poison, (2) homogenization of the cells without damage to the chromosomes, (3) separation of the released chromosomes from cell debris. The separation of chromosomal nucleoprotein into its component entities can now be accomplished by the methods that are relatively mild and nondestructive as compared to those used in the past. An excellent example is the separation of histones from DNA by banding in a cesium chloride density gradient.

[124] Use of cesium sulfate for equilibrium density gradient centrifugation☆

Abstract: Publisher Summary This chapter discusses the use of cesium sulfate for equilibrium density gradient centrifugation Equilibrium density gradient centrifugation is one of the most useful tools for fractionation and characterization of DNA The two cesium salts most commonly employed in this procedure are CsC1 and Cs 2 SO 4 The properties of density gradients prepared with these salts are rather different At similar rotor speeds, Cs 2 SO 4 , which forms approximately twice as steep a gradient as CsCl, is preferable for fractionation of DNA's with widely different buoyant densities, for example, dAT versus dABU or normal unsubstituted versus bromoor iodouracil-labeled DNA On the other hand, CsCl is better suited for routine determination of the guanine + cytosine (G + C) content of DNA, because in this solvent, there is an almost linear relationship between the percentage of G + C (20–80%) and the buoyant density of DNA In Cs 2 SO 4 , there is a much less and nonlinear dependence of density on G + C content DNA is much more heavily hydrated in the Cs 2 SO 4 gradient, with the density averaging 14 g/cm 3 as compared with 17 g/cm 3 in CsCl The chapter also explains that RNA can be banded in the Cs 2 SO 4 gradient at a concentration of about 16 g/cm and discusses the other specific advantages of Cs 2 SO 4 over CsC1

[98] Isolation of nucleic acids with phenolic solvents

Abstract: Publisher Summary Phenol was first used to extract RNA from mammalian tissues and tobacco mosaic virus, but it became clear that although phenol extracted and inhibited ribonuclease, subsequent purification resulted in degradation of the RNA. The ethylenediamine tetraacetate may inhibit ribonuclease, but this is not generally the case. Naphthalene 1,5-disulfonate is a hydrophilic salt, does not release DNA at 0.015 M concentration, and was found to lessen the amount of degradation in RNA from chicken liver. The residual protein was 1.5–2.0%. The m -Cresol is added to the phenol to serve two purposes—(1) the mixture can be cooled to 5° without the phenol crystallizing and (2) the phenol–cresol mixture is a better deproteinizing agent than phenol alone. Ribosomal RNA can be isolated without associated rapidly labeled RNA if the original homogenization is carried out in the presence of naphthalene 1,5-disulfonate and with the rapidly labeled RNA if 4-aminosalicylate is used. A method, which also yields the rapidly labeled RNA associated with the ribosomal RNA, is described in this chapter. DNA is released with ribosomal RNA when the tissues are extracted with 4-aminosalicylate, NaC1, and the phenol-cresol mixture, and it can be separated by extraction with 3 M sodium acetate (pH 6).

[108] Use of CsCl density gradient analysis for determining the guanine plus cytosine content of DNA

Abstract: Publisher Summary This chapter investigates the use of CsCl density gradient analysis for determining the guanine plus cytosine content of DNA. The buoyant density of DNA in CsC1 increases linearly with its content of guanine plus cytosine (GC). Thermally denatured DNA and naturally occurring single-stranded DNA behave in a similar manner but are uniformly higher in density. The ease and reproducibility of buoyant density determinations provide a standard method for evaluating the composition and other attributes, for example, strandedness, molecular weight, and degree of homogeneity of DNA samples. As each centrifuge cell passes directly over the light beam, the light passes through the solution and out through the upper window, where it is slightly deflected in the ease of the three wedge windows. Thus, each beam emerges from a different cell. The beams can be observed by holding a white card between the camera lens and the 4-cell mask and looking at the card from the direction of the mirror when green light is used in the optical system. Alternatively, a photograph can be taken by exposing to ultraviolet (UV) light a film at this position. Light passing through the air spaces of the cells produces the smaller images, which are toward the back of the ultracentrifuge. The light passing through the CsCl gradient is refracted toward the front of the ultracentrifuge. At this position, these images are not in focus and do not yet correspond to the final photographs taken at the plate holder.

[106b] Filter paper disk techniques for assaying radioactive macromolecules

Abstract: Publisher Summary This chapter discusses the filter paper disk techniques for assaying radioactive macromolecules. The method consists of numbering a series of Whatman No. 1 (or No. 3) cellulose disks and then applying the impure radioactive sample, such as an aliquot of a reaction mixture, to the properly numbered disk. The enzyme reaction is stopped by placing the filter paper disk in a large beaker of trichloroacetic acid. A large number of disks, for example, all the time points in a multitube kinetic experiment or density gradient run, can be accumulated and subsequently processed simultaneously through several reagents necessary for extraction of extraneous material. After radioactive precursors have been removed from the radioactive product, the washing reagents that may interfere with the analysis of the radioactive product are removed by suitable solvents and residual water is removed by drying either with solvents, air, or heat. The radioactivity of the sample can then be analyzed for the various types of isotopes that it may contain, either by scintillation techniques or by a straightforward Geiger counting procedure, under standard counting conditions. Descriptions of several specific modifications of this disk procedure are outlined in the chapter, which are useful for a number of enzymatic polymerizations and depolymerizations that use radioactive substrates to measure the radioactive product.

[148] The formation and detection of DNA-RNA hybrids

Abstract: Publisher Summary Hybridizations between RNA and denatured (single stranded) DNA from a variety of sources have been widely used as a test for complementarity between the two participating nucleic acid molecules. The technique is based on the notion that the synthesis of RNA on a native DNA template utilizes the transient formation of complementary hydrogen bonds (in the Watson–Crick sense) between a part of the growing RNA molecule and one strand of the DNA template. When completed RNA molecules are isolated and mixed with denatured DNA in vitro under conditions that favor the formation of hydrogen bonds, structures arise that have the predicted characteristics of RNA–DNA hybrids. These structures involve the entire RNA molecule and a part of one strand of the DNA molecule. Hybrids are formed in aqueous solutions buffered at neutral pH, containing salt, and held at high temperatures. The temperature of hybridization must be carefully selected for each experiment. A detailed description of annealing conditions and general analysis of such conditions are provided in the chapter. When RNA and single-stranded DNA are mixed in solution, two reactions may occur—(1) the hybridization reaction involving RNA and one strand of DNA and (2) the DNA renaturation reaction. Renaturation introduces serious errors into quantitative interpretation of hybridization experiments, because renatured DNA is not able to accept RNA. Estimates of the amount of DNA that can anneal with a given type of RNA will be low if the renaturation reaction occurs. The chapter explains the two major complications inherent in DNA–RNA hybridizations.

[104] Recovery and purification of nucleic acids by means of cetyltrimethylammonium bromide

Abstract: Publisher Summary High molecular weight impurities and contaminating traces of nucleases in phenol-prepared nucleic acids are usually precipitated together with the nucleic acids by two volumes of ethanol. Many of these impurities may be reduced or eliminated by precipitating the nucleic acids with cetyltrimethylammonium (CTA) bromide. The CTA nucleic acids are converted into sodium salts without solution in water, and the opportunity for enzymatic degradation is minimized. The CTA nucleic acid precipitates do not appear to retain water so do nucleic acids precipitated from aqueous ethanol, and after conversion to sodium salts, the nucleic acids may be easily dried by washing with ethanol and acetone. DNA appears to be recovered unchanged following CTA bromide precipitation. Nucleic acids isolated by phenol extraction of purified viruses, tobacco leaf, Chinese cabbage leaf, or rat liver, and recovered by CTA bromide precipitation, contain intact, high molecular weight RNA as judged by sedimentation properties. Purification is often necessary to ensure complete removal of polysaccharides, polyhydroxybutyric acids, sugar phosphates, or nucleoside mono-, di-, and triphozphates. To eliminate these materials from phenol-prepared nucleic acids, Kirby method is discussed, which is a two-phase separation technique that removes glycogen, starch, and sugar phosphates from phenol prepared nucleic acids.

[123] Ultraviolet circular dichroism in nucleic acid structural analysis

Abstract: Publisher Summary This chapter describes the ultraviolet circular dichroism in nucleic acid structural analysis and investigates whether nucleic acids, due to their conformational dissymmetry, give rise to Cotton effects in the ultraviolet and whether these can be used for structural investigation. Ordinary spectrophotometry is a necessary supplementation of circular dichroism (CD) work for the determination of hypochromicity or other forms of difference spectra as additional criteria of structural features in case of proteins and nucleic acids. The α-helix is considered as an exciton, thought to give rise to a splitting of the major absorption band of the peptide structure (around or below 200 mμ) into two spectral components, which are polarized parallel and perpendicular to the longitudinal axis of the helix. The CD spectrum of peptides known to be helically structured reveals a positive CD band at about 194 mμ and a negative band at 207 mμ, with rotational strengths of about 80×10–40 and –30×10–40, respectively, ascribed to the exciton structure. These features have been found in few proteins that have been investigated such as apomyoglobin, myosin, actin, and rhodopsin. One can anticipate a fruitful further development of this field, which is of interest also to nucleic acid research inasmuch as the structural aspects of protein–polynucleotide interactions will be revealing.

[103b] The use of sodium and lithium dodecyl sulfate in nucleic acid isolation

Abstract: Publisher Summary Dodecyl sulfate (lauryl sulfate) used either as the sodium salt—sodium dodecyl sulfate (SDS)—or the more soluble lithium salt (LDS) is a powerful detergent that has found wide application in the analytical biochemistry of nucleic acids because of its ability to release nucleic acids from their associations with protein or lipoprotein structures in which they normally occur inside the cell Recently, the amide derivative sodium dodecyl sarcosinate has been introduced because of its solubility in high concentrations of cesium chloride The precise mechanism of action of these detergents is not known, but it is likely that the hydrocarbon chain of SDS competes for hydrophobic bonds This tendency in combination with the strongly hydrophilic sulfate group presumably causes the formation of water-soluble complexes or micelles Two additional properties make dodecyl sulfate particularly suitable for use in nucleic acid isolation;the anionic detergent appears to be an inhibitor of nucleases and its negative charge prevents it from interacting with nucleic acids SDS has often been used in combination with phenol for the preparative isolation of nucleic acids free of protein This chapter confines to the examples of the use of dodecyl sulfate alone and illustrates the great potential of the method in the cases where optimal analytical resolution is of paramount importance The chapter presents detailed directions for the release and characterization of nucleic acids from a number of representative sources such as (1) viral RNA, (2) bacterial DNA, (3) ribosomal RNA (rRNA), (4) messenger RNA (mRNA), (5) and transfer RNA

[143] Deoxynucleotide polymerizing enzymes from calf thymus gland☆☆☆

Abstract: Publisher Summary This chapter discusses the deoxynucleotide polymerizing enzymes from calf thymus gland. Calf thymus gland is a source of two separate deoxynucleotidyl transferases—DNA polymerase and a terminal deoxynucleotidyl transferase. Both enzymes are conveniently isolated from the same preparative process because separation of the two polymerizing activities is the final purification step. The assays measure the conversion of 14C-deoxynucleoside triphosphate (acid soluble) to l4 C-polydeoxynucleotide (acid insoluble) and are applicable at any stage of purification. The DNA polymerase reaction mixtures contain only one labeled triphosphate, and all the four are incorporated in proportion to the base composition of the denatured DNA template used. To obtain total nucleotide incorporation for calf thymus DNA template, multiply 14 C-dATP incorporation by 3.4. Other methods of assay, useful after partial purification, are the measurement of pyrophosphate formation and hypochromicity due to polymer formation. Terminal deoxynucleotidyl transferase, isolated from calf thymus gland, may be used to prepare a series of a single-chain polydeoxynucleotides. Replicative deoxynueleotidyl transferase (DNA polymerase), isolated from calf thymus gland, may be used to prepare a series of double-chain polydeoxynucleotides. The availability of procedures for the synthesis of single-chain polydeoxynucleotides with terminal deoxynucleotidyl transferase and the homopolymer complexes with calf thymus DNA polymerase or Escherichia coli DNA polymerase provides material for the degradative synthesis of oligodeoxynueleotides. The chapter also discusses the degradation of single-chain polydeoxynucleotides and double-chain polydeoxynucleotides.

[118] Stepwise degradation of RNA: Periodate followed by aniline cleavage

Abstract: Publisher Summary RNA, which carries no phosphate at the 2 ' or 3 ' positions of the (right) terminus, is susceptible to periodate oxidation at this glycol group. If the RNA is terminally phosphorylated, pretreatment with the Escherichia coli phosphatase renders it reactive toward periodate. The transformation of the (2 ' and) 3 ' carbon group to an aldehyde weakens the 5′-phosphate ester bond. Various primary aliphatic amines condense with the aldehyde groups and catalyze cleavage of this 5 ' ester bond at about pH 8, but only the very weakly basic amine, aniline, is found to achieve this quantitatively at 25° with an optimum at pH 5, under conditions that are acceptable for long-chain polynucleotides such as viral RNA. The eliminated base and degraded ribose moiety is separated from the macromolecule by alcohol precipitation of the latter. The RNA is subsequently treated with phosphatase to remove the terminal phosphate, subjected to the cycle—oxidation, ester bond cleavage, dephospherylation to remove one nucleotide at a time. The released base is chromatographically identified. The difficulties in assuring quantitative cleavage of the terminal phosphate ester bond, without incipient random ester bond hydrolysis, represent the weak link in this procedure. This chapter describes the stepwise degradation of RNA by periodate oxidation followed by aniline cleavage. The other method of monitoring the progress of the stepwise degradation on the microscale is by means of 32 P-labeled RNA used in a parallel experiment. This permits the analysis for inorganic phosphate in the 70% alcohol supernatant after each step.

[100] Isolation and purification of plant nucleic acids from whole tissues and from isolated nuclei

Abstract: Publisher Summary This chapter discusses the isolation and purification of plant nucleic acids from whole tissues and isolated nuclei. Three factors are taken into account—(1) Hardness of tissue, (2) pH, and (3) Nucleic acid content. Tissues that are soft pose no problems in mechanical breakage. In a number of plant species, vacuolar pH is low and in such cases, macromolecules may be denatured upon cell injury. Nucleic acids may be isolated and purified from most types of plant tissues, but special procedures are required for tissues that contain a great deal of storage material and/or a great deal of secondary wall growth. A procedure is described in the chapter that separates cytoplasmic and nuclear materials from these interfering components. In the case of plants, tissue bulk is usually a deceptive indicator of the amount of nucleic acid present, and the nucleic acid content should first be determined analytically. The chapter also discusses the preparation of tissues, the fractionation of tissues by nonaqueous procedures and aqueous procedures, preparation of RNA, and preparation of DNA from plant tissues.

[146] Measurement of DNA synthesis in bacterial cells

Abstract: Publisher Summary DNA determinations based on the well-known diphenylamine reaction are useful when an absolute measure of the total amount, or variations in the total amount, of DNA in a bacterial culture is required Radioisotopes are ideally suited to experiments in which the kinetics of DNA synthesis is to be measured They are not so convenient for determining absolute amounts of DNA per cell but can be used for this purpose providing (1) the specific activity of the labeled precursor is accurately known, (2) the base composition of the DNA is known, and (3) the specific activity of the intracellular precursor pool is the same as that of the medium The advantages of using radiophosphorus ( 32 P) to label DNA lie in the ready availability of high specific activity material; the fact that it can be assayed with an ordinary thin-window Geiger counter commonly present in most laboratories and the fact that it is readily incorporated into bacterial cells Adenine, guanine, or cytosine can be used in conjunction with a suitable fractionation procedure to measure DNA synthesis Possibly the most convenient way to measure DNA synthesis is by the incorporation of radioactively labeled precursors that are specific for DNA alone Thymine and thymidine are ideal for this purpose and are discussed briefly

[116b] Borohydride reduction of periodate-oxidized chain ends

Abstract: Publisher Summary This chapter discusses the borohydride reduction of periodate-oxidized chain ends. Periodate-oxidized chain ends of RNA by reaction with 14C- or 3H-isoniazid or reduction with tritium borohydride. The borohydride method has the advantage that the label is introduced with minimal change in structure, thereby allowing the use of separation techniques commonly employed for nucleic acids. The derivative is stable to alkaline hydrolysis and thus better suited to end group determination. Disadvantages are the low counting efficiency for tritium and nonspecific labeling, which occurs to the extent of about one tritium atom per 1000 nucleotides. With tritiated reagents of the highest specific activity available, the borohydride and isoniazid methods lead to specific activities of 1.5 and 2.0 mC/micromole of RNA, respectively. Periodate oxidation cleaves the bond between carbons 2' and 3' with formation of a dialdehyde. Reduction by tritiated sodium borohydride yields primary alcohol groups at carbons 2' and 3' with one non-exchangeable tritium atom at each. Alkaline hydrolysis releases the terminal residue as a nucleoside trialcohol, which can be identified after chromatography. Digestion with specific endonucleases and analysis of the labeled product in a chain-length selective system indicates the sequence of adjacent residues. The optimum reaction conditions depend on the chain length of the material to be labeled and whether one seeks to determine the end group or obtain an intact labeled polynucleotide. Adjustment to higher pH after periodate treatment is more economical of borohydride (decomposition half-life in minutes given by log T1/2 = pH– 8) but enhances the base-catalyzed side reactions of phosphodiester hydrolysis and flelimination of the terminal residue.

[128] Molecular weight and conformation of DNA

Abstract: Of the variety of molecular weight methods applicable to DNA, the sedimentation and/or viscosity method, and the electron microscopic-length method have proved to be the most satisfactory and the most widely accepted These two techniques are additionally useful because of the information about conformation and topology which they can provide However, in view of the pitfalls of sedimentation-viscosity measurements and the empirical nature of their relation to molecular weight, and because of the presence of an unknown source of variability in the electron microscopic method,109 absolute molecular weights with less than 10% uncertainty cannot be claimed, and errors as large as 20% are probably not uncommon, as inspection of Table I will suggest The situation today is nevertheless infinitely improved over what it was a few years ago when errors by factors of two and more were common-place in reported molecular weights of DNA In addition, there now exist several other independent techniques which provide molecular weight results consistent with those obtained by the two popular methods Most importantly, “standard” DNA's are now available from the dozen DNA-containing viruses listed in Table I With further study the molecular weights of the nucleic acids of these and other DNA viruses will be more accurately established The methodology of DNA molecular weight determinations therefore appears to be capable of meeting the demands put upon it by current experimentation with intact viral chromosomes Eventually the molecular weights of bacterial chromosomes may also be routinely determined, a possibility suggested by the last three entries in Table I

[154] Isolation and properties of the Escherichia coli amino acid polymerizing enzymes

Abstract: Publisher Summary This chapter discusses the isolation and properties of the Escherichia Coli amino acid polymerizing enzymes. The result of a continued study of the components of ribosome-linked polypeptide synthesis from activated amino acids in extracts of homogenates of Escherichia coli is considered. In order to test for the supernatant factors, the extract was fractionated and ribosomes were freed of adsorbed components. The principle of fractionation was to prepurify a complex fraction, from which two complementary components were separated by chromatography on DEAE-Sephadex. The KC1 gradient procedure is described by which the two complementary fractions, T and G, were obtained. Cell growth, disruption, extraction (step 1), protamine sulfate treatment (step 2), and ammonium sulfate fractionation (step 3) are carried out for G; T is separated from G and purified by following steps—alumina Cγ gel fractionation, DEAE-sephadex column chromatography, and hydroxylapatite column chromatography. The Escherichia coli supernatant fractions have been found to fully satisfy Pseudomonas and Bacillus subtilis ribosomal systems but animal, and in particular mammalian, ribosomes (reticulocyte, liver) appear not to interact with these bacterial factors.

[129] Molecular weight and conformation of RNA

Abstract: Publisher Summary The RNAs, whose molecular weight can vary from 26,000 to about 2,000,000 depending on the type, assumes a quite compact conformation in dilute salt solutions. It thus has a size and structure readily accessible to the physical chemical techniques developed to characterize macromolecules. However, only a few reliable molecular weight measurements of RNA have been reported. The chapter briefly describes some of the difficulties encountered in physical measurements of RNA. These include the problem of obtaining sufficient amounts of reasonably pure material, the effects of trace amounts of nucleases, and the tendency of RNA molecules to aggregate, both with its own and with other RNA species. After a consideration how these factors add to the complexity of properly characterizing the size and shape of RNA, some of the methods, which have been successfully applied to this problem, are described in the chapter with special emphasis on light scattering and sedimentation-viscosity measurements. The measurements concern only the conformation of RNA in vitro, and any extension of the results obtained to the in vivo conformation is purely conjectural. Requirements for purity of RNA preparations could conceal, at least in some cases, the presence of a native structure. Repeated phenol extractions to remove the last trace of protein, prolonged dialysis against chelating agents to remove metal ions, and brief exposure to high temperature to dissociate possible aggregates could certainly denature a native structure that might have been present initially.

[157b] Methods of identification of peptides during hemoglobin biosynthesis and measurement of their sequential synthesis

Abstract: Publisher Summary This chapter describes the methods for the identification of peptides during hemoglobin biosynthesis and measurement of their sequential synthesis. A model of protein synthesis by sequential growth whereby amino acids are joined one after the other down a polypeptide chain is postulated. Taking hemoglobin as an example, it is demonstrated that peptide chains grow by serial addition of amino acids, starting at the free amino terminal and going toward the free carboxyl terminal. A method of preparative analysis chromatography of amino acids is described in the chapter that allows the determination of specific radioactivity on any number of amino acids at once in protein biosynthesis studies. The general technical procedure presents an adaptation of paper chromatography and paper electrophoresis procedures for the isolation and characterization of tryptic peptides of hemoglobin. Another new technique is ion-exchange chromatography with volatile organic developers, which appears to have a considerably higher resolving power than paper electrophoresis. Electrophoresis on filter paper strips provides a method of separating peptides in an electric field.

[137] Phage Qβ RNA polymerase☆

Abstract: Publisher Summary The procedure described in this chapter yields a 2000–5000 fold purified Qβ RNA polymerase with an activity of 4000–5000 millimicromoles GMP incorporated per milligram of protein per 20 minutes. The procedure for purification of the Qβ polymerase has been described in the chapter. The phage is banded by centrifugation in the Spinco 40 rotor at 37,000 rpm for 48 hours. After centrifugation, the phage layer is removed and dialyzed against SSC for 24 hours. A suspension of purified phage is mixed with an equal volume of phenol in a glass-stoppered test tube, and the tube is rolled gently at room temperature for about 10 minutes. The phases are separated by centrifugation at 15,000 g for 5 minutes. The size of the RNA should be determined because the Qβ RNA polymerase reaction requires intact Qβ RNA. Isolation of an RNA phage RNA polymerase has been impeded in large measure by the lability of the phage polymerase activity in extracts of cells infected with f2, MS2, or R-17 phages. The extensive purification described in the chapter has been possible apparently because the Qβ RNA polymerase has greater stability.

[138] The preparation of an RNA replicase capable of synthesizing biologically active viral RNA☆

Abstract: Publisher Summary This chapter discusses the preparation of an RNA replicase capable of synthesizing biologically active viral RNA. Synthesis of RNA by replicase preparations is monitored by the conversion of radioactive nucleoside triphosphates (XTP's) into acid-insoluble materials. During the early stages of the purification schedule, nuclease activity and high primer-independent incorporation of XTP make impossible an estimation of replicase content. The purification scheme for the replicase is, therefore, directed toward the removal of nucleases and cellular polymerases rather than bulk protein. Several DEAE preparations are pooled and rechromatographed on DEAE. The process of rechromatography routinely results in a two- to threefold increase in replicase specific activity, as well as removal of certain basic proteins, which may complex with and precipitate nucleic acids. After the DEAE stage, replicase preparations retain quantities of mature virus particles too large to allow direct assay of reaction mixtures for infectious nucleic acid production. The Q β replicase requires as template its homologous viral genome. Other viral RNA species, as well as cellular RNA, do not stimulate nucleotide incorporation by the replicase nor do fragments of Q β -RNA promote extensive synthesis. The use of highly purified and intact Q β -RNA is essential in any study of the reaction catalyzed by the Q β replicase. Two preparations of Q β -RNA, which cannot be distinguished by either their size or relative infectivity, can nevertheless vary widely in their ability to initiate the replicase reaction. For maximum rate of nucleotide incorporation and maximum involvement of input template strands in replicative intermediates, freshly prepared Q β -RNA must be employed. The Q β replicase carries out in vitro a reaction of biological significance. The purified replicase, therefore, allows study of the RNA replication process divorced from cellular activity.

[136] Enzymatic synthesis of polyguanylic acid and copolymers containing guanylic acid

Abstract: Publisher Summary This chapter discusses enzymatic synthesis of polyguanylic acid and copolymers containing guanylic acid. The synthesis of high molecular weight polyguanylie acid proceed with great difficulty under conditions where the other homopolymers are readily formed. Polyguanylic acid can be obtained without a primer when the concentration of substrate GDP and Mg ++ are very low but in the presence of a large amount of enzyme. Thus, with 1 m M GDP and polynucleotide phosphorylase extracted from Azotobacter vinelandii, high molecular weight poly G is formed. When polymerization of GDP, catalyzed by highly purified polynucleotide phosphorylase isolated from Escherichia coli , is made at 60° and in the presence of Mn ++ instead of Mg ++ , the reaction proceeds readily; the procedure is quite reproducible. The polyguanylic acid thus obtained is fairly homogeneous and has a high molecular weight. The only limiting factor is the enzyme. Polynucleotide phosphorylase isolated from Escherichia coli and purified to at least DEAE-Sephadex step can synthesize the polyguanylic acid under the conditions described in the chapter, as far as the yield and the properties of the polymer are concerned. At a lower temperature, polynucleotide phosphorylase isolated from Micrococcus lysodeikticus can also synthesize poly G in the presence of a primer. Enzymatic synthesis of copolymers in the presence of high ratio of GDP also presents some difficulty whether in the presence or the absence of primer. The ratio of guanylic acid to other nucleotides incorporated into the copolymers is always higher than the ratio of GDP to other nucleoside diphosphatcs in the input pool.

[152] Techniques for measuring specific sRNA binding to Escherichia coli ribosomes

Abstract: Publisher Summary This chapter describes the techniques for measuring specific soluble RNA (sRNA) binding to Escherichia coli ribosomes. Specific sRNA binds to the template ribosome complex. The binding is specific, in which only the sRNA coded for in the messenger RNA (mRNA) sequence is stimulated to bind to the ribosomes. The bound sRNA sediments faster than the rest of sRNA during the sucrose density gradient centrifugation. The faster-sedimenting bound sRNA is assayed for its amino acid acceptor ability after the sucrose density gradient centrifugation. After the sucrose density gradient centrifugation of the mixture, the amounts of the various amino acid sRNA's in each fraction are determined by measuring the incorporation of 14C-amino acid into the fraction that is insoluble in cold trichloroacetic acid but soluble in hot trichloroacetic acid. The radioactive aminoacyl sRNA formed is soluble in hot trichloroacetic acid and insoluble in cold trichloroacetic acid. Radioactivity incorporated into the protein fraction is insoluble in both hot and cold trichloroacetie acid. It is, therefore, possible to measure the amount of each aminoacyl sRNA by subtracting the radioactivity insoluble in hot trichloroacetic acid from that insoluble in cold trichloroacetic acid. The sRNA sedimenting faster than the rest of sRNA is regarded as bound sRNA. Specific aminoacyl sRNA, like sRNA, binds to the template ribosome complex. Ribosomes are absorbed by Millipore filter quantitatively, while aminoaeyl sRNA is not. Using aminoacyl-14C sRNA, the ribosome-bound aminoacyl-14C sRNA can be conveniently measured by counting the radioactivity retained on the Millipore filter.

[97] Isolation and purification of nuclear proteins☆

Abstract: Publisher Summary The nuclear proteins are divisible into histones, acidic nuclear and nucleolar proteins, and nuclear and nucleolar enzymes Nucleoproteins are deoxyribonucleoprotein complexes containing mainly DNA and histones but also some RNA and acidic proteins The problems involved in the isolation of nuclear proteins are extensions of the problems of isolation of nuclei Other problems in the isolation of these proteins are those of isolation of gels, particles, and soluble fractions from the nuclei in native states, the insolubility of acidic nuclear proteins, and the possibility that biological activities may be altered For comparison from tissue to Tissue, there is the problem of extraction of nuclear proteins in satisfactory yield sufficient to permit subfractionation into individual molecular species For the isolation of histones, the methods for purification of deoxyribonucleoproteins are of greatest importance The three methods are discussed for isolation of deoxyribonucleoproteins—(a) direct extraction of nuclei with water or dilute buffer solutions such as 07 M phosphate buffer, (b) extraction of nuclei with 014 M NaC1 containing 001 M sodium citrate to remove most of the nuclear components, and (c) extraction of nuclei with 1–2 M NaC1 containing 001 M trisodium citrate The standard method for the separation of histones from deoxyribonucleoproteins is extraction with HCl Fractionation and subfractionation methods of nuclear proteins are discussed and chemical procedure for the isolation of nuclear protein fractions is also tabulated in the chapter

[99] The isolation of nucleic acids from bacterial spores

Abstract: Publisher Summary This chapter discusses the isolation and purification of plant nucleic acids from whole tissues and from isolated nuclei. Tissues that are soft pose no problems in mechanical breakage (for example, meristematic regions). Specific methods for some types of tissue are provided in the chapter, but it is emphasized that plant cell walls are formidable barriers to the diffusion of large molecules. Incomplete cell breakage may result not only in low yields of nucleic acid but also what is more important; preferential breakage of young cells results in selective extractions. In a number of plant species, vacuolar pH is low and in such cases, macromolecules may be denatured upon cell injury. Vacuolar membranes are likely to be disrupted before cells are broken and Tris or phosphate buffers may not penetrate rapidly enough to offset vacuolar acidity. If preprocessing with organic solvents is not desired, prior infiltration of tissue pieces with dilute ammonium hydroxide (e.g., 0.001 M) should be tried. Nucleic acids may be isolated and purified from most types of plant tissues.

[174] Preparation and assay of nucleic acids as antigens

Abstract: Publisher Summary DNA-specific antibodies may be produced in individuals with lupus erythematosus (LE) and in rabbits injected with a lysate of T4 bacteriophage. Purine and pyrimidine bases are haptens when chemically coupled to bovine serum albumin or synthetic polypeptides. Rabbits injected with such protein-hapten conjugates produced purine and pyrimidine-specifie antibodies capable of reacting with DNA and RNA. Bovine serum albumin is extensively used as a carrier protein for simple haptens; methylated bovine serum albumin (MBSA)—a basic protein—is tested as a carrier for DNA. The stability of complexes formed between nucleic acids and MBSA depends upon the concentration and type of salt in the medium, as well as the pH. Owing to the greater ease in dissolving MBSA in water than in 0.15 M NaC1, MBSA is added to the hapten as a 1% aqueous solution. A relatively small volume is required because of the greater concentration of MBSA so that the concentration of salt in the final solution is essentially 0.15 M . Protein-nucleic acid conjugates are formed by mixing the solutions of nucleic acid and MBSA at room temperature in amounts such that the weight ratio of nucleic acid to MBSA is one. The chapter also discusses the preparation of MBSA, the preparation of antiserum, and assay of antiserum.

[116a] Periodate oxidation of ribonucleic acids and their derivatives

Abstract: Publisher Summary This chapter discusses the periodate oxidation of RNAs and their derivatives. When ribonucleosides or ribonueleotides are incubated with excess periodate at room temperature near neutrality during short reaction periods (up to 3 hours), the oxidation is practically specific for the glycol groups of the ribosyl components. The number of the glycol groups is obviously determined by the ring (furanose or pyranose) structure of the ribose groups and the position of the phosphoryl groups. Under the reaction conditions defined above, the amounts of periodate consumed can be considered as an accurate measure of the number of glycolic hydroxy groups of the ribose groups, whereas the nitrogenous rings are not affected. The products of the periodate oxidation of natural (ribofuranosyl) ribonucleosides and 5'-ribonucleotides are the corresponding 2′,3′-dialdehyde compounds. Neither formaldehyde nor formic acid is formed. As would be anticipated on the basis of the cis configuration of the 2′- and 3′-OH groups in ribose, the periodate oxidation proceeds rapidly to completion. The chapter also discusses the influence of periodate oxidation on the stability of the phosphoric ester linkage and the purine- and pyrimidine-ribosyl linkages of 5′-nucleotides.