Assays are described that permit one to distinguish the reverse transcriptase of RNA tumor viruses from known normal cellular DNA-instructed DNA polymerases. Template responses of purified reverse transcriptase were compared with those of similar preparations of the DNA polymerase I of Escherichia coli and of calf-thymus polymerase. All three enzymes responded well to the synthetic duplexes poly(dT)·poly(A), poly(U)·poly(A), and poly(dT)·poly(dA). Hence, these duplexes can detect, but cannot distinguish reverse, transcriptase from the known normal DNA polymerases. However, certain oligomer-homopolymer complexes serve as excellent distinguishing agents. The reverse transcriptase responds very well to (dT)10·poly(A) and very poorly to (dT)10·poly(dA), whereas both cellular DNA polymerases do not exhibit this behavior.
Purified single-stranded RNA also serves as a diagnostic device, since only reverse transcriptase gives a detectable response. To be definitive, a positive response to RNA must be accompanied by a demonstration via molecular hybridization that the DNA product is complementary to the RNA and not to some minor DNA contaminant.
Adenovirus deoxyribonucleic acid (DNA) was used as template for the in vitro synthesis of viral-specific ribonucleic acid (RNA). When the kinetics of the reaction were compared by using native and heat-denatured DNA templates, the latter synthesized RNA at a slower rate. The fate of the DNA after acting as template and physical characteritstics of the RNA product were studied. The DNA template, according to its sedimentation rate, was not significantly degraded by the Micrococcus lysodeicticus RNA polymerase. The products of the RNA polymerase reaction had the following properties. (i) Hybridization experiments revealed a high degree of complementarity (50 to 70%) for its homologous DNA. (ii) A very low complementarity (6 to 7%) was found for its heterologous DNA. (iii) The sedimentation rate of the synthetic RNA in a sucrose gradient was 5 to 10S when native DNA was used as the template. When heat-denatured DNA was used, the resulting RNA product, free of the template, sedimented at a rate of 3 to 16S. A rapidly sedimenting (>30S) DNA-RNA complex resulted when denatured DNA was the template. The DNA moiety of the complex was sensitive to 125 mug of deoxyribonuclease per ml. The RNA of the complex, however, was fully refractory to 50 mug of ribonuclease per ml. When the adenovirus DNA was sonically treated and then used as template, the RNA product sedimented at 3 to 9S. The heat-denatured sonically treated DNA template yielded a DNA-RNA complex that also sedimented at an unusually fast rate (>18S).
This paper describes studies on the physical and chemical properties of the helical structures obtained when polydeoxycytidylic acid (dC) or its polyribo-analogue (rC) interacts with polydeoxyinosinic acid (dI) or its polyribo-analogue (rI). At moderate concentrations of a monovalent cation (less than 0·5 M) the four complexes dI :dC, dI :rC, rI:dC and rI :rC are formed. These appear to be bihelical structures with perfect base pairing between the hypoxanthine residues of one strand and the cytosine residues of the other. The hybrid structures differ markedly from one another and from the dI:dC and rI:rC helices with respect to several properties.
Normal adult rat liver contains a high level of a synthetic RNA-dependent DNA polymerase activity, which is distinct from cellular DNA-dependent polymerases. It uses poly(dT).poly(rA) and poly(rA).poly(rU) as templates but has little or no response to DNA or several single-stranded RNAs.
The ability of Escherichia coli DNA polymerase I to retrotranscribe an RNA template was examined under steady-state conditions, using a primer extension assay which allows determination of kinetic constants on well-defined heterogeneous sequences. Equilibrium and rate constants for the initial binding step of the enzyme to two homologous DNA and RNA templates do not show striking differences. In both cases, under steady-state conditions, processivity limits the maximal velocity of the translocation process. The lower catalytic efficiency of the enzyme when it operates on RNA is then reflected by a 100-fold greater apparent average Michaelis constant for the deoxynucleotide substrates. We conclude that E.coli DNA polymerase I effectively transcribes both templates, its performances being limited in both cases by its intrinsically low processivity. Furthermore, DNA polymerase I is a strikingly accurate enzyme when operating on RNA. Magnesium has to be substituted by manganese so that a pattern of errors could be detected. This great accuracy results from a combination of factors. The 3' to 5' exonuclease activity is still operating but in a non-discriminative manner. Elongation of a mismatched primer terminus is markedly impaired. The forward polymerization rate of incorporation of an incorrect deoxynucleotide must be extremely low, when Mg2+ is present. In summary E.coli DNA polymerase I preserves its main characteristics when retrotranscribing RNA.
EVIDENCE which has accumulated over the past decade from mammalian and non-mammalian sources about the enzyme(s) involved in DNA synthesis has indicated the possibility of a multiunit structure for this enzyme. Mammalian DNA polymerase DNA nucleotidyl-transferase (E.G.184.108.40.206) was first isolated from a high speed supernatant fraction of cell homogenates, instead of a nuclear fraction where DNA synthesis takes place1. Later it was shown that cytoplasmic fractions, obtained by non-aqueous techniques2 and also by the use of a homogeniza-tion medium containing Ca++ (ref. 3) to retain morphological integrity of nuclei, contained substantial amounts of this enzyme.
This paper presents a preliminary survey of the nucleic acid polymerases of the developing chicken embryo, especially of the 4-day stage. The predominant activity is that of a DNA polymerase preferring a DNA-RNA hybrid as the template. The enzyme, which is activated by Mn(2+) ions and inhibited by p-chloromercuriphenylsulfonate, copies preferentially the ribo strand of a hybrid, such as poly(rA).poly(dT), but is relatively inactive with all-ribo duplexes. DNA polymerase I of Escherichia coli was also found to use the hybrid template with high efficiency, copying preferentially the ribo strand. With the chicken enzyme, the template activity of denatured DNA was increased tenfold by simultaneous transcription with RNA polymerase. DNA polymerase activity reaches a maximum in the 6- to 8-day chicken embryo and then declines progressively to about one-third of the maximal value in the adult chicken.
DNA has been synthesized by direction of 28S ribosomal RNA (rRNA) or by tobacco mosaic virus RNA using homogeneous Escherichia coli DNA polymerase I. The reaction requires all four deoxynucleoside triphosphates and Mg2+.
A FORMALDEHYDE-induced methylene-bridged dimer of either adenosine or adenylic acid has been implicated as the mediator of mutagenic activity following the addition of formaldehyde to the medium on which Drosophila larvae develop1-7. RNA is also effective3, and it has been shown that adenylate is the component of RNA uniquely concerned, as none of the other RNA nucleotides is effective when supplied singly or in combination4'5.Fig. 1As the chemical binding of formaldehyde by RNA nucleotides is a specific function of the amino groups of the bases8-10, it was suggested4 that methylene bis-adenylic acid was formed in the culture medium by formaldehyde linkage of two adenylate molecules (Fig. 1).
A comparative study was performed on the template specificities of the highly purified DNA polymerases from Escherichia coli and Micrococcus luteus and of a partially purified DNA polymerase from virions of avian myeloblastosis virus (AMV). The three DNA polymerases show approximately the same capacity to utilize twenty different high molecular weight templates. Thus, when tested with polymer templates (primers), the two bacterial DNA polymerases are at least as effective "reverse transcriptases" as the tumor virus associated DNA polymerase. However, (rA)n·oligo(dT) is a markedly better template than (dA)n· oligo(dT) for the AMV DNA polymerase, as reported previously. For the M. luteus DNA polymerase, the two templates are approximately equally effective. The AMV DNA polymerase provides faithful DNA synthesis when either DNAs or RNAs serve as templates (primers). DNA synthesis is dependent on the presence of a suitable primer strand and the newly synthesized DNA strand is covalently attached to the primer strand through a phosphodiester linkage. Thus, when a polyribonucleotide serves as a primer, the new DNA strand is joined to an RNA molecule. The AMV DNA polymerase apparently cannot initiate the synthesis of a new DNA strand. This behavior is identical with that observed for the two bacterial DNA polymerases.
Particles apparently similar to type B mouse mammary tumour virus, which
are found in human milk and may be associated with breast cancer,
contain reverse transcriptase activity. Milks lacking the particles do
not have this activity. This finding will help to establish the
relationship between these particles and tumour viruses.
The Central Dogma of molecular biology which postulates the unidirectional transmission of genetic specifications for protein biosynthesis was enunciated by Crick (1958) who proposed explicitly that “once ‘information’ has passed into protein it cannot get out again. In more detail, the transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein may be possible, but transfer from protein to protein or from protein to nucleic acid is impossible. Information means here the precise determination of sequence either of bases in the nucleic acids or of amino acids in the protein.”
Six RNA viruses have now been shown to contain DNA polymerase activities directed by single-stranded RNA, double-stranded RNA and double-stranded DNA. It is further demonstrated that DNA-RNA hybrids, such as synthetic dC.rG, act as even more effective templates.
The chapter discusses various deoxyribonucleic acid (DNA) polymerases from mammalian cells. Progress in the study of these mammalian systems has been hampered, by the fact that they do not lend themselves to the purification to the same level, as achieved, with the preparations from bacterial sources. However, perseverance with mammalian systems is to be welcomed, as it is a reasonable expectation that the answers to the unsolved problems of DNA biosynthesis (certainly the more complex aspects of it, with the concomitant clinical as well as theoretical implications) are more likely to emerge from continued investigation on a broad front than from the intense concentration on one or a very few aspects. There is a wide variety of mammalian systems available for study and, as these have already yielded much information, the time is propitious for a general appraisal of the existing data together with an attempt to relate them to the observations reported for nonmammalian systems.
As part of a biochemical search for a virus in melanomas preliminary kinetic studies with RNA directed DNA polymerase from avian myeloblastosis virus showed that thymidine labeled triphosphate (TTP) stimulates the enzyme. This stimulatory effect was used as an additional probe in detecting RNA directed DNA polymerase activity in Greene hamster melanoma in vivo. This enzyme activity, however, was not found in the Greene hamster melanoma cells growing in tissue culture under ordinary conditions. When the cells were treated with BUDR the enzyme could be demonstrated and the stimulating effect of TTP was observed.
The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. It is transcribed at different stages of Drosophila ontogenesis. The Drosophila LINE family includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. As demonstrated here, a 2.23 kb DNA fragment from the region of jockey encoding the putative reverse transcriptase was stably introduced into an expression system under inducible control of the Escherichia coli lac regulatory elements. We describe the expression of the 92 kDa protein and identify this polypeptide alone as the authentic jockey reverse transcriptase based on some of its physical and enzymic properties. The jockey polymerase demonstrates RNA and DNA-directed DNA polymerase activities but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulphydryl reagent. The enzyme prefers poly(rC) and poly(rA) as template and 'activated' DNA is not effective.
The kinetic properties of Escherichia coli DNA polymerase I were simplified to those of a 1 deoxynucleotide substrate reaction by the use of polynucleotide templates. With poly(dA)-oligo(dT) as the template-primer complex, Mg2+ decreases the Km of the substrate dTTP but has little or no effect on the Km of the substrate Mg-dTTP, suggesting that multiple pathways involving the binding of Mg2+, dTTP, and Mg-dTTP are operative in forming the active complex. The Km of free Mg2+, extrapolated to zero concentration of substrate (830 = 62 muM), agrees within a factor of 2 with the dissociation constant of magnesium from 4 +/- 1 sites on the enzyme determined previously by binding studies (Slater, J.P., Tamir, I., Loeb, L.A., and Mildvan, A.S. (1972) J. Biol. Chem. 247, 6784-6794). The maximal turnover number with poly(dA) as template is 5.7 +/- 0.7 s-1. Changing the nature of the base in the polydeoxynucleotide template alters the maximal rate of polydeoxynucleotide synthesis by an overall factor of 31 with poly(dC) is greater than poly(dT) is greater than poly(dA) is greater than poly(dG), indicating that pyrimidine templates are copied faster than purine templates. Changing the sugar structure from poly(dA) to poly(rA) causes a 3-fold increase in the rate of template copying. A study of the kinetic effects of all noncomplementary deoxynucleotides with all deoxynucleotide templates, as well as with poly(rA)-oligo(dT), yields complex patterns of activation and inhibition requiring from 1 to 2 additional binding sites for the noncomplementary nucleotides. The kinetically determined affinities of the active site of the enzyme-template-primer complex for the complementary free nucleotide (as measured by Km) generally exceed those for the noncomplementary neuclotides (as measured by KI slope) by 1 or more than 3 orders of magnitude.
RNA-dependent DNA-polymerase activity is present in specimens of freshly excised human medulloblastoma. This activity is demonstrated in the cell homogenates and activity remains in the supernatant fluid at 30,000g after 30 minutes and pellets at 100,000g in 2 hrs. Detergent is necessary to elicit significant activity. The enzyme activity was determined using a poly(A)·oligo(dT) template. Retinoblastoma, neuroblastoma and ocular malignant melanoma also showed activity but in much lower amounts than medulloblastoma. Normal human orbital connective tissue, cerebellum and retina, similarly studied, failed to show activity with this template. Thus, by these methods a RNA-dependent DNA-polymerase activity is demonstrated in human tumors, but not in the corresponding normal tissues.
The chapter discusses various synthetic polynucleotides. Synthetic polynucleotides are of considerable interest from the biological viewpoint of code cracking, and, also, as grossly simplified models for the study of the numerous physical properties, manifested by nucleic acids. This chapter is discusses the physical studies of the structure in synthetic polynucleotides, including homopolymers of naturally occurring nucleotides and of various analogs. The chapter very briefly reviews some technical aspects of the physical chemistry of polynucleotides. The various random coil and structured forms of polynucleotides, containing a single naturally occurring purine or pyrimidine base, are discussed in this chapter. Under suitable conditions of pH or salt concentration, all such polymers form hydrogen-bonded, multi stranded, secondary structures. The chapter discusses various polynucleotide complexes, such as complexes between Poly G and Poly C. All homopolynucleotides are derived from natural or almost natural nucleotides form multistranded secondary structures under appropriate conditions. In case of the “basic” (or 6-amino) nucleosides (A and C), slightly acidic conditions are necessary, whereas the “acidic” (or 6-keto) polymers (G, U, I, X), all give defined secondary structures at pH 7 albeit, with vast different stabilities.
RNA polymerase from Micrococcus lysodeikticus has been used to synthesize rG : rC† from rC and GTP. The rG: rC product appears to consist of one continuous rC strand and many short pieces of rG comprising the other strand. rG:rC is completely resistant to pancreatic RNase at 37°C. Attempts at thermal dissociation of the complex were unsuccessful. Dissociation can be achieved in alkali, for which the pKa is about 12·5 at 25°C. Alkaline dissociation is essentially reversible, but the complex formed by neutralization is distinguishable from the rG:rC synthesized initially. The density of rG:rC in cesium sulfate is 1·685.
The interaction with divalent cations of the synthetic polynucleotides, polyadenylic acid and polyuridylic acid, has been studied by conductiometric titration. It is shown that further binding of divalent cation by polynucleotide ceases when one equivalent of cation has been added per mole of polymer phosphate, and that the titration curve is the same for both polymers. This result is compared with that obtained previously for DNA in distilled water, and it is shown that the anomalous end-point for DNA is eliminated if sufficient points are taken in the course of the titration.The interaction of polynucleotides with polylysine has also been investigated, both spectrophotometrically and by conductiometric titration. It is shown that a stoichiometrically well defined complex is formed, and that in the course of the reaction the polylysine is capable of displacing an equivalent amount of disalent cation.
RNA polymerase converts single-stranded DNA templates to a DNA-RNA hybrid. Free RNA does not appear until the composition of the hybrid reaches a llimiting value of equal quantities of DNA and RNA. This is in contrast to the situation with double-stranded DNA templates where the double helix is conserved and only free RNA is produced. The bacteriophage φX174 DNA-RNA hybrid undergoes a relatively sharp optical density transition with increasing temperature ; the Tm of DNA-RNA hybrid is slightly less than that of the corresponding double-stranded φX DNA although the dependence of Tm on counter-ion concentration of the two helices is quite similar. After dissociation of the hybrid at high temperature, the helical structure can be reformed by annealing at high ionic strength. The RNA of the hybrid is less susceptible to low levels of pancreatic ribonuclease than the free RNA. φ hybrid can serve as template for RNA synthesis; in doing so a major portion of the RNA of the hybrid template is replaced by newly-synthesized RNA; i.e. the reaction is predominantly semi-conservative. These results have been discussed in terms of possible models of the copying process.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
J T August
P J Ortiz
August, J. T., P. J. Ortiz, and J. Hurwitz, J. Biol. Chem., 237, 3786 (1962).
Lowry, O., N. Rosebrough, A. Farr, and R. Randall, J. Biol. Chem., 193, 265 (1951).
D G Comb
Katz, S., and D. G. Comb, J. Biol. Chem., 238, 9 (1963).
There appears to be a discrepancy in the transition temperature of the OX 174 hybrid; at a lower ionic strength Sinsheimer and Lawrence14 observed a higher Tm than Chamberlin and Berg
Chamberlin, M., and P. Berg, J. Mol. Biol., 8, 297 (1964). There appears to be a discrepancy in the transition temperature of the OX 174 hybrid; at a lower ionic strength Sinsheimer
and Lawrence14 observed a higher Tm than Chamberlin and Berg.
14 Sinsheimer, R. L., and M. Lawrence, J. Mol. Biol., 8, 289 (1964).
R M Franklin
Baltimore, D., and R. M. Franklin, Biochem. Biophys. Res. Commun., 9, 388 (1962).
Lee-Huang, S., and G. Felsenfeld, unpublished data.
12 Radding, C. M., J. Josse, and A. Kornberg, J. Biol. Chem., 237, 2869 (1962).