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The interspecific hybrid Arbacia lixula ... × Paracentrotus lividus ... which stops developing at the onset of gastrulation, was previously found to contain much more DNA of maternal origin (P. lividus) than DNA of paternal origin. In spite of this, the RNA synthesized by the blocked gastrula hybridizes better with DNA of paternal type than with DNA of maternal type. An explanation of thru observation was sought in the present paper by means of saturation and competition experiments. The higher affinity of RNA from the hybrid gastrula for paternal DNA could be due to the fact that RNA of paternal type is complementary to a longer portion of the corresponding DNA than RNA of maternal type. This explanation was not satisfactory since the number of genes of paternal type which are active in the hybrid is not very different from the number of genes of maternal type. In normal embryos of A. lixula and P. lividus, RNA from unfertilized eggs and from plutei was found to be less competitive against labeled RNA from gastrulae than gastrula RNA itself. Transcription of the genome is therefore partially stage-specific. This stage-specificity is maintained in the hybrid as far as transcription of the maternal genome is concerned, but is lost as far as transcription of the paternal genome is concerned. The hybrid gastrula was shown to synthesize more RNA molecules of A. lixula type than does the normal gastrula of this species, whereas RNA of P. lividus type is produced in equal amounts m normal and in hybrid embryos. These observations explain the hybridization properties of the RNA synthesized by the blocked gastrula. The results suggest that the genes of paternal type that are very active in the hybrid are those genes which m normal embryos are transcribed at all stages of development and probably also in the adult. This class of genes is much more active in a foreign cytoplasm than in the cytoplasm of its own species. On the other hand, the genes whose activity is restricted to the gastrula stage m normal embryos are not active in the hybrid. This class of genes does not seem to function in a foreign cytoplasm. [ABSTRACT FROM AUTHOR]

An endo-type deoxyribonuclease isolated from autolysate of Aspergillus sojae was purified 175-fold. The purified enzyme migrated as a single band during electrophoresis on polyacrylamide gel and hydrolysed only deoxyhbonucleic acids. Optimal hydrolysis of DNA by the enzyme was observed at pH 9.4. The enzyme was strongly activated by Mg2+. Mn2+ and Zn2+ also enhanced the enzymatic activity, but to a lesser extent, Heat-denatured DNA was found to be a better substrate than native DNA. The molecular weight of the enzyme determined by gel filtration on Sephadex G-100 was 15 600. A limited amount of the enzyme preferentially splits the phosphodiester bonds of purine nucleotides in DNA. The mononucleotides liberated are P-5'-dGuo (major) and P-5'-dAdo (minor). The oligonucleotides are terminated mostly either with P-5'-dGuo or P-5'-dAdo and partly with P,5'-dCyd at the 5'-phosphate termini, and with P-5'-dGuo and P-5'-dAdo at the 3'-hydroxyl termini, respectively. [ABSTRACT FROM AUTHOR]

Highly purified RNA polymerase from Escherichia coli terminates RNA synthesis in vitro at defined sites on the DNA of coliphage TT. One such site maps at the end of the major ‘early’ transcription region of the genome. A second, distal site is most readily studied in certain mutant strains from which the first site has been deleted. At both sites, termination in vitro is simple (independent of rho or other factors) and both appear to be functional in vivo: We have applied to these sites a technique designed to study base sequences at and beyond RNA termini. The RNA terminated at the first site has cytosine at its 3′ end, and the first base coded by the DNA template beyond the RNA terminus is guanine. The specificity of this sequence is at least 92%. At the second terminator, which is found to be less efficient in its action, the corresponding bases are cytosine and adenine. [ABSTRACT FROM AUTHOR]

1. Comparison of DNA-DNA filter hybridization using either denaturated duplex DNA or artificial mixtures of the two complementary strands of T7 DNA shows that the competing renaturation reaction has two effects: (a) sequestering of 20-30 % of the input DNA as duplex, unavailable for hybridization, and (b) hybridization of partially renatured DNA as a concatenane to the filter-bound DNA. 2. Experiments using artificial mixtures of 32P-labelled and ³H-labelled L and H strands of T7 show that I0-20% of the total hybrid bound results from concatenation. With a 1: 1 input ratio of complementary strands in solution, 10% of the input bound to filter DNA of a single strand (e.g. L) is of the homologous strand (i.e. L). This effect is relatively unaffected by the concentration of DNA in solution. 3. Variation of the L:H ratio in solution shows that the strand in excess hybridizes and renatures more efficiently. This effect gives an underestimation of the amount of the minor strand present in solution. The numerical values obtained here are likely to be strongly dependent on the complexity of the DNA and the precise hybridization conditions. 4. The implication of the results for homology experiments and determination of strand specificity in transcription is discussed. [ABSTRACT FROM AUTHOR]

Histone mRNA from HeLa cells synchronized by a double thymidine block has been identified and characterized. Its synthesis starts immediately after the cells are released from the thymidine block. Pulse labelling for 60 min with [5-³H]uridine at different times of the S-phase showed that radioactively labelled histone mRNA enters polyribosomes with a maximum during the first hour of S-phase, i.e., before the maximum of DNA synthesis Histone mRNA sediments on sucrose gradients in the 8-10-S region and separates on poly-acrylamide gels into three RNA species with molecular weights of about 1.5 × 105, 1.8 × 105, and 2.1 × 105 respectively. Polyribosomal 8-10-S RNA from synchronized cells in S-phase directs the synthesis of histones in a rabbit reticulocyte cell-free system. Histones synthesized in vitro were identified by polyacrylamide gel electrophoresis and by high-voltage paper electrophoresis of tryptic peptides. Polyribosomal 8-10-S RNA from cells in winch DNA synthesis was blocked by thymidine or hydroxyurea had only a marginal histone mRNA activity. [ABSTRACT FROM AUTHOR]

The activity of a highly purified nucleotidase from rat liver cytosol which splits certain 3'- anti 5'-nucleotides, Was studied in the presence of each of 36 (different nucleic acid nucleic acid constituents including14C, labeled and chemically modified nucleotides. It was found that the compounds either inhibited or had no effect, on dephosphorylation of thymidine 5'-phosphate, which was used as substrate for measuring 5'nucleotidase activity. On the other hand , the 3'-nucleotidase activity, which was measured with uridine 3'-phosphate as substrate, was stimulated 2.6 times by deoxy- guanosine and, furthermore, by dexyguanosine and, furthermore, by deoxyinosine, thymidine, deoxyuridine and inosine in decreasing order of effectiveness. Experiments with various phosphorylated derivatives of thymidine indicated that a 5'-phosphoryl group increase the stimulating effect of the nucleoside whereas a 3'-phosphoryl substitution reduces its ability to activate the enzyme. Di-and triphosphates were less stimulatory than the mono phosphate. The results are interpreted to indicate that the same catalytic site is responsible for the hydrolysis of the 3'-and 5'-nucleotides and that the enzyme possesses a regularly site, topographically different from the catalytic site, at which the deoxyribonucleic acid constituents act as stimulators of enzyme activity. [ABSTRACT FROM AUTHOR]

The four structural proteins of bacteriophage PM2 have been related to thc morphological structures in the virion. Protein I can be selectively removed from the virus by degradation with bromelain, a proteolytic enzyme. From electron microscopic studies and from experiments showing that the virus cannot attach to its host cell after bromelain treatment, we conclude that protein I forms the outer spikes at the vertices of the icosahedron. Protein II can be labeled with [35S]sulfanilic acid diazonium salt. As shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, proteins I and II can be removed if the virus is treated with 1 M urea. From the above studies and from packing considerations, we conclude that protein II forms the outer shell of the virus. The dissociation of the virus in 1 M urea leads to: (a) a nucleocapsid core containing viral lipid, DNA, protein III and IV, (b) a second fraction which consists of the same elements obtained by treatment of the virus with bromelain. Both panicles have been identified by electron microcopy. The dissociation of the virus in 1 M urea plus 0.5% Triton X-100, a nonionic detergent, leads to a third fraction representing an empty shell which contains all of the lipid, proteins II and III, and about 50% of protein IV, but no DNA. We have also been able to isolate a second type of nucleocapsid core by dissociation of the virus in the presence of 4.5 M urea. This core, which has an approximate s20.w = 133 S and which has been identified in the electron microscope as a core with icosahcdral shape, contains proteins III and IV, as well as the DNA, but not phospholipid. In the presence of 4.5M urea this core is resistant to Triton X-100 concentrations up to 0.1%. Higher concentrations of the nonionic detergent dissociate the core into DNA and the solubilized proteins HI and IV. In the absence or in the presence of Iow concentrations of urea (1M) the core is resistant to TritonX-100 concentrations up to 0.5%. The DNA in the core is digestible by pancreatic DNAase I and the core structure is broken down after DNAase treatment. Protein IV interacts with the DNA as shown by transformation of non-covalent interactions between DNA and protein into covalent bonds induced by a nucleophilic reagent. In conclusion, protein I forms the spikes, protein II the outer icosahedral shell and protein III the inner icosahedral shell around the DNA, which is closely associated with protein IV. The phospholipid bilayer is sandwiched between these two shells. Protein III may also form the protein bridges to protein I which connect the inner and outer shells through the bilayer. [ABSTRACT FROM AUTHOR]

1. DNA of DNase-treated mitochondria of Acanthamoeba castellanii bands at a buoyant density of 1.690 g/cm³. Up to 80% of the molecules were circular, with a circumference of 12.7 μm. Two circular conformations, the closed twisted and the open form, were separated in preparative CsCIethidium bromide gradients. 2. When mtDNA was renatured at a temperature Tm; - 23 °C, deviations from second-order kinetics were observed. At Tm; -10 °C, however, it renatured as a single kinetic class without indications of sequence repetition. The coincidence of the kinetic complexity (2.57 x 107) and the circle contour length within the limits of error of the methods suggests that each circular molecule represents a chondriome. 3. The main DNA component, which is of nuclear origin and has a peak density of 1.716 g/cm³ in CsC1, displays heterophasic renaturation kinetics. Its slow-reassociating fraction shows a kinetic complexity corresponding to a molecular weight of 2.1 x 1010. Approximately 22% of the main component appears to be repetitive at a renaturation temperature of Tm - 23 °C. [ABSTRACT FROM AUTHOR]

M-13 replication was investigated in Escherichia coli mutants deficient in DNA polymerase I (polA) and DNA polymerase III (dnaEts). The following results were obtained; 1. M-13 infection is abortive in both polA- and dnaEts mutants at 42 °C. 2. The conversion of the parental single-stranded DNA to double-stranded replicative forms (RF) is only slightly affected by the dnaE mutation. However, no parental double strands are formed in the polA- dnaEts double mutant at the restrictive temperature, indicating a possible involvement of DNA polymerase 1 in this process. 3. The replication of the replicative-form molecules as well as the synthesis of progeny single strands are inhibited by thermal inactivation of the dnaE function implying a requirement of DNA polymerase 111 in both types of DNA synthesis. 4. The presence of the polA mutation leads to an accumulation of open-circular replicative forms. DNA polymerase I might, therefore, also be involved in the filling of gaps in the newly synthesized DNA molecules. [ABSTRACT FROM AUTHOR]

Low concentration (up to 20% v/v) of glycerol and dimethylsulphoxide stimulate RNA synthesis in vitro, the extent of stimulation being dependent on the nature of the DNA template. This stimulation results mainly from an increase in initiation. The target of these compounds is the DNA template rather than RNA polymerase. Ribosomal RNA synthesis is affected in a temperature-dependent manner, 20% glycerol lowering the transition temperature between the open and closed forms of the promoter by 4-5°C. Thus in a manner analogous to its effect on gal mRNA synthesis in vitro glycerol partially releases rRNA synthesis from the constraints essential for control. [ABSTRACT FROM AUTHOR]

1. The interactions of a purified calf thymus chromosomal non-histone protein (designated protein HMG1) with bacteriophage T7 DNA and calf thymus DNA have been investigated by sedimentation analysis in the ultracentrifuge. The results obtained show that (a) the non-histone protein HMG1 binds to DNA in an ionic-strength-dependent manner, (b) the DNA can bind up to approximately four to five times its weight of protein HMG1, the protein distributing itself evenly along the DNA chains, and (c) the interaction is a rapid reversible equilibrium. These results are interpreted as indicating that protein HMG1 binds to DNA through ionic bonding between the basic amino acids of the protein and the phosphate groups of the DNA. 2. Equilibrium sedimentation studies were carried out on mixtures of protein HMG1 and histone F1. The results demonstrate that protein HMG1 combines with histone F1. 3. The molecular weight of protein HMG1 has been determined by equilibrium sedimentation and a mean value of 26500 was obtained. [ABSTRACT FROM AUTHOR]

A study has been made of the action of DNAase I and of micrococcal nuclease on chromatin, on the complex of chromatin with polylysine and of DNAase I on DNA ˙ protamine; a comparison of different assay methods of DNAase I action on chromatin is included here. Also, the nature of the polylysine-binding zones in chromatin has been investigated. Measurements of acid-solubility and salt-solubility during digestion show that DNAases I and II degrade almost all of the DNA in chromatin (from various tissues), unimpeded by the chromatin proteins which are released concurrently (and which eventually aggregate on to the degraded DNA). With micrococcal nuclease concurrent release of DNA and of protein occurs similarly; however, full degradation by this nuclease is impeded apparently by aggregation of released protein on to acid-insoluble DNA. In the case of chromatin ˙ polylysine and also DNA ˙ protamine, about half of the DNA is degradable. The results indicate that discrepancies between the findings of previous workers are due to differences in assay of degradation, in conditions of digestion, in enzyme used and probably in methods of preparing chromatin resulting in products varying widely in composition. Also, the results support the previous suggestion that virtually all the DNA in chromatin is associated with protein yet is highly accessible [1], unlike DNA complexed with protamine or polylysine. The polylysine-binding zones in chromatin are discrete, double-stranded, protein-associated regions of DNA which are probably heterogenous from one molecule to another. [ABSTRACT FROM AUTHOR]

1. The activities of DNA-dependent RNA polymerases A and B purified from ovaries of Xenopus laevis were examined on various templates. 2. When bulk Xenopus DNA was centrifuged in caesium chloride gradients the ratio of incorporation of UMP and GMP into RNA by the two enzymes different in the region where the ribosomal cistrons banded when the fractionated DNAs were used as templates. The A enzyme selectively synthesised G-rich RNA under conditions where the enzyme: rDNA ratio was high, whereas no conditions were found under which the B enzyme would synthesise G-rich RNA. 3. Molecular hybridisation of the cRNA synthesised by A and B enzymes from DNA enriched with ribosomal cistrons and main-band DNA demonstrated that only transcripts of rDNA by the form A RNA polymerase were competed out by unlabelled 18-S and 28-S Xenopus ribosomal RNAs. 4. Both forms A and B RNA polymerases were capable of forming rifamycin AF/0-13 resistant complexes on either main-band or ribosomal DNA. However, whereas the amount of resistance by the B enzyme was similar on both templates, RNA polymerase A became much more resistant on rDNA than on main-band DNA. 5. Sensitivity to actinomycin D and the exotoxin from Bacillus thuringiensis, both of which inhibit ribosomal RNA synthesis preferentially in vivo, was apparently independent of the type of template used in these experiments. [ABSTRACT FROM AUTHOR]

Histone mRNA was isolated from polyribosomes and other cell fractions of synchronized HeLa cells and quantitated by its translational capacity in a rabbit reticulocyte lysate. Inhibition of DNA synthesis with hydroxyurea (5 mM) results within 30 to 60 mm in a loss of 80-85% of biologically active histone mRNA from polyribosomes. In contrast, actinomycin D (5 μg/ml) during the first hour of its action, only stops further accumulation of histone mRNA on polyribosomes in early S-phase cells. Under conditions of interruption of DNA replication biologically active histone mRNA does not appear in other cell fractions suggesting that it is immediately destroyed in the cytoplasm. The possible translational control of histone synthesis in HeLa cells was investigated with special consideration of the existence of messenger-specific initiation factors. Cell-free extracts from synchronized HeLa cells in which DNA and histone synthesis were inhibited with hydroxyurea, fully retain theft capacity to translate added homologous histone mRNA as compared with extracts from synchronized S-phase cells. Using a partially purified protein-synthesizing system from rat liver supplemented with initiation factors from synchronized HeLa cells differing in their histone-synthesizing capacity, it was shown that factors from hydroxyurea-treated cells are as active in stimulating the translation of histone mRNA, globin mRNA and tobacco mosaic virus RNA as are initiation factors from S-phase cells. The results suggest that messenger discriminating translation factors are not involved in the regulation of histone synthesis in HeLa cells or they are very labile and inactivated during preparation.

1. Baker's yeast histones have been isolated either by acid extraction of purified chromatin or by salt-dissociation of crude chromatin. These two methods gave similar results as judged by the amino-acid composition and electrophoretic behaviour of histones. 2. Chemical fractionation methods have been used to isolate both, moderately lysine-rich and arginine-rich histones. Neither F1 nor F3 histones could be detected in yeast chromatin throughout these fractionation procedures. Results obtained in experiments of interactions between yeast whole histones ans calf thymus DNA also confirm the absence of a very lysine-rich histone in yeast. 3. The fractionation of arginine-rich histones was achieved by selective precipitation with acetone. F2A1 and F2A2 histones were obtained, purified and characterised by electrophoresis and amino acid analyses. The similarities between yeast and other organisms histone fractions are discussed.

When rifampicin, together with ribonucleoside triphosphates, is added at 25 °C to a mixture of RNA polymerase and DNA which had been preincubated for a few minutes, RNA is synthesized despite the presence of the inhibitor. With 0.8 mM Mg2+ present during the preincubation an optimum of RNA synthesis is observed which equals the synthesis in the absence of rifampicin. The extent of RNA synthesis at 25 °C is dependent on tile temperature at which the enzyme has been preincubated with DNA. RNA synthesis in the presence of the inhibitor increases almost linearly with preincubation temperatures from 5 to 25 °C. The same results are obtained by applying either the chain initiation assay or the chain elongation assay for measuring RNA synthesis. The effect of temperature during preincubation of enzyme and template on subsequent RNA synthesis at 25 °C proved to be reversible. Experiments with poly[d(A-T)] as competing template are consistent with the notion that during preincubation RNA polymerase is bound to the template at low as well as at higher temperatures under the conditions used. Essentially the same results concerning the initiation of RNA synthesis in the presence of rifampicin are obtained with T4 DNA, T2 DNA and poly[d(A-T)] as templates, These results support the hypothesis that RNA polymerase bound to DNA exists in at least two different states which differ in their susceptibility to rifampicin. These different states are in a rather rapid and temperature-dependent equilibrium. According to this hypothesis rifampicin prevents the conversion of the sensitive state of the complex of RNA polymerase and DNA to the resistant form. Subsequent initiation of RNA synthesis by addition of substrate is consequently blocked. [ABSTRACT FROM AUTHOR]

The action of 9-bromomethylanthracene on poly(A) either single stranded or complexed with poly(U) has been investigated. Reaction of the polycyclic hydrocarbon occurs specifically on the amino group of aclenylic residues in the polymer whereas the N¹ position is also conjugated in the monomer using the same experimental conditions. Poly(G) reacts to a lower extent than poly(A) with the formation of one conjugate instead of two for the monomer. Cross linking conjugation occurs between the two strands of DNA. The different factors governing the specificity and the extent of the reaction of this reagent with polynucleotides are discussed, the different geometry of double-stranded structure inducing various reactions of conjugation. The extent of reaction was tested for several polynucleotides and their complexes. The B form of DNA was found to be the most sensitive one. The conjugates provide convenient labels for conformational studies of polynucleotides using fluorescence spectra and polarisation data. [ABSTRACT FROM AUTHOR]

The routine procedures used to prepare DNA from eucaryotic cells and nuclei give rise to material which is considerably degraded. This degradation takes the form of haplotomic (single-stranded) breaks arising from deoxyribonuclease action and diplotomic cleavage (double-stranded scission) of the DNA duplex due to mechanical shearing during the isolation procedure. Attempts to study the template specificities of the eucaryotic DNA-dependent RNA polymerases must take into account how these modifications to the structure of the DNA affect its template properties for these enzymes. Techniques are described which permit an accurate assessment of the state of integrity of the DNA. Treatment of the DNA with pancreatic deoxyribonuclease or sonication is shown to give rise to single-stranded interruptions in the duplex which are competent sites for the initiation of RNA synthesis by both forms AI and B rat liver RNA polymerases. However both these treatments also give rise to diplotomic cleavage of the DNA. This results in an inhibition of RNA synthesis which may be correlated with the formation of non-productive complexes by the RNA polymerases at DNA ends. Therefore the degree of template activation by pancreatic deoxyribonuclease and sonication for RNA synthesis as seen in the assay in vitro is a delicate balance between two opposing effects and is highly dependent upon the polymerase: DNA ratio. This data serves to emphasise that experiments designed to study the template specificity of eucaryotic RNA polymerases must make use of new, sophisticated techniques for the preparation of a template which is both intact and of high molecular weight. [ABSTRACT FROM AUTHOR]

It is shown that preinitiator or storage stretches on T5-DNA are not a consequence of the existence of natural nicks in this template. Nicks do not contribute to the formation of heparin-labile and stable starters. Analysis of the number of pppA and pppG starters and their products on T7-DNA in enzyme saturation in the presence of heparin yielded 8 stable pppA starters, all synthesizing one product from the r-strand sedimenting with 32 S and with a molecular weight of 1.9 x 106 (from sedimentation rate) or 2.2x105 (from chain length). Six labile pppG starters, yielding products from the l-strand and, in addition, two very labile pppG starters seen only in the absence of heparin, also on the 1-strand, were demonstrated. These results give further evidence for the existence of preinitiator (or storage) stretches, with another template for which no assumption about the number of reading units have to be made. The polyanion heparin is well suited for the elimination of artificial starts in vitro. [ABSTRACT FROM AUTHOR]

A small-angle X-ray scattering study was performed with solutions of the alternating polydeoxynucleotide poly[d(A-T) · d(A-T)] and its analogs poly[d(A-s4T) · d(A-s4T)] and poly[d(A-s4U) · d(A-s4U)]. At low concentration of NaCl and neutral pH almost identical results were obtained for these polydeoxynucleotides. At small scattering angles the same cross section radius of gyration Re = 0.92 ± 0.03 nm for all three polydeoxynucleotides was derived from the scattering curves. At larger scattering angles a second cross-section radius of gyration Rc2 could be determined which amounts to 0.855 ± 0.02 nm for poly[d(A-T) · d(A-T)], 0.86 ± 0.01 nm for poly-[d(A-s4T) · d(A-s4T)] and 0.82 ± 0.01 nm for poly[d(A-s4U) · d(A-s4U)], respectively. The length per nucleotide pair, 0.38 ± 0.03 nm, was found to be the same for all three polydeoxynucleotides. These results are similar to those for natural DNA. The above results for poly[d(A-s4T) · d(A-s4T)] and poly[d(A-s4U) · d(A-s4U)] refer to the modification of these pulydeoxynucleotides which prevails at low concentrations of 1 : 1 electrolytes and was designated as helix I. The length per nucleotide pair obtained in this study appears to be consistent with the presence of strong 0 0 8 and 0 0 16 reflections in the high-humidity X-ray fibre diffraction patterns for the sodium salt of poy[d(A-s4T) · d(A-s4T)]. The values for the cross-section radii of gyration, on the other hand, seem to be inconsistent with the reversed Hoogsteen base-pairing scheme proposed for poly[d(A-s4T) · d(A-s4T)] and poly[d(A-s4U) · d(A-s4U)], because the similarity of these values to those for poly[d(A-T) · d(A-T)] and natural DNA is contrary to the results of theoretical calculations on models built up from reversed Hoogsteen base-pairs. According to these calculations, the cross-section radius of gyration of poly [d(A-s4T) · d(A-s4T)] ought to be smaller than that of natural DNA by about 0.16–0.2 mn if this polydeoxynucleotide really were of the reversed Hoogsteen type. [ABSTRACT FROM AUTHOR]

Nalidixic acid, a very specific inhibitor of bacterial DNA synthesis, has been studied for its action on purified enzymes acting on DNA and in subcellular DNA-synthesizing systems. The thug does not inhibit the following enzymes: DNA polymerase I, endonuclease I, exonuclease I, II and III from Escherichia coli, polynucleotide-ligase and DNA methyl-transferase from T4- infected E. coli, DNA polymerase from Bacillus subtilis. A significant inhibition of ATP-dependent DNA synthesis is observed in cells of a strain of E coil lacking DNA polymerase I after short treatments with toluene (2 min); longer treatments reduce the rate of synthesis and abolish Its sensitivity to nalidixie acid. Equally insensitive are the ATP-dependent DNA synthesizing systems bound to membrane fractions of E. coli and B. subtilis. The results suggest a mode of action through a still unidentified physiological component of the growing point apparatus, and supply a new criterion of physiological integrity of subcellular DNA-synthesizing systems, namely their sensitivity to nalidixic acid. [ABSTRACT FROM AUTHOR]

The transcription of rat-liver chromatin has been studied using partially purified form AI and highly purified form B DNA-dependent RNA polymerases isolated from rat liver. Chromatin contains endogenous RNA polymerase activity. This activity is evident only when the RNA polymerase assays are carried out at high ionic strength and its appearance as the ionic-strength increases is thought to be due to the removal of chromatin-associated proteins which block further transcription by the bound enzyme. This activity is insensitive to the rifampicin derivative AF/0-13 which is shown to inhibit initiation of RNA synthesis by mammalian RNA polymerases. Form AI polymerase is virtually inactive in the transcription of chromatin whereas the form B RNA polymerase (α-amanitin sensitive) actively transcribes chromatin. This activity has two salt concentration optima: (a) 0.01 M (NH4)2SO4 or 0.03 M KCl; (b) 0.16 M (NH4)2SO4 or 0.25 M KCl. Both these activities are inhibited by rifampicin AF/0-13. A comparison of the activity of the rat-liver form B polymerase and the Micrococcus lysodeikticus RNA polymerase demonstrates that chromatin is a more efficient template for the rat-liver enzyme. Evidence is presented that the rat-liver form B and the Micrococcus lysodeikticus RNA polymerases bind to and transcribe from different sites on the chromatin DNA. [ABSTRACT FROM AUTHOR]

1. After infection of Escherichia coli K12 strains with the single stranded DNA bacteriophage M13, between 85-95% of the intracellular parental DNA molecules are found attached to the host membrane. 2. This attachment is not irreversible. Even in singly infected cells up to 30% of the membrane bound parental DNA molecules are released from the membrane site and transferred to progeny phages during the first hour of infection. 3. The turnover of the membrane-bound parental DNA depends strongly on the host system studied, indicating that this process may reflect primarily a property of the host. [ABSTRACT FROM AUTHOR]

The addition of tRNA to chromatin gel (initial insoluble deoxyribonucleoprotein preparation) in the presence of 1 mM MgCl2or of 40 mM NaCl results in the transfer of the very lysine-rich histone (F1) and of some nonhistone proteins to the tRNA, whereas all other histones remain in the deoxyribonucleoprotein. This result provides a new general method for the mild removal of histone F1 from the chromatin gel. The addition of tRNA to chromatin gel in the absence of Mg2+ and Na+ results not only in the rapid removal of histone F1 and of some nonhistone proteins from the ehromatin gel, but also in the slow transfer of histones F2a2 and F2b to the tRNA. A similar pattern of histone redistribution is observed when the tRNA (or DNA) is added to the soluble deoxyribonucleoprotein preparation obtained by mechanical disruption of the chromatin gel. There is no protein redistribution in the above-mentioned soluble deoxyribonucleoprotein preparation (in the absence of exogenous free polynucleotides). The addition of free DNA or tRNA to the soluble deoxyribonucleoprotein preparation, obtained by treatment of the chromatin gel with 2 M urea, allows one to remove quantitatively histones F1, F2a2 and F2b from the deoxyribonucleoprotein, leaving the initial distribution of the arginine­rich histones (F2al and F3) on the endogenous DNA unchanged. The redistribution of proteins (and in particular of histones) occurs in the soluble deoxyribonucleoprotein preparation obtained by urea treatment even in the absence of any exogenous polynucleotides suggesting that conservation of the initial distribution of proteins in this soluble deoxyribonueleoprotein preparation is not complete. [ABSTRACT FROM AUTHOR]

Cells made permeable to nucleotides by treatment with ether synthesize DNA from exogenous deoxynucleoside triphosphates in a semi-conservative fashion, The reaction proceeds from sites of previous synthesis on the chromosome, and not detectably from new sites. Short chains appear as precursors of long units and have a mean sedimentation coefficient of about 10 S in alkali, like Okazaki-pieces when synthesized in the presence of 10 to 20 μM deoxynucleoside tri- phosphates, and of only 5 S when synthesized in the presence of 1μM dNTP, Under the former conditions short chains contain not more than half of the newly synthesized DNA; under the latter conditions they can contain more than half, the remainder of the newly synthesized DNA being in long units, Nascent short chains labeled after several minutes of synthesis in the presence of 5-bromodeoxyuridine 5'-triphosphate at high deoxynucleoside triphosphate concentrations show bimodal density distribution in an alkaline Cs2SO4 equilibrium gradient with peaks of material corresponding to the density of heavy single strands and almost light single strands, respectively. The implications of these findings are discussed. Judged from controls, repair synthesis was not a source of error in these experiments. In contrast with results reported for intact cells, ether-treated polA1- (Kornberg-polymerase deficient) cells join nascent short chains as efficiently as do polA+ cells. [ABSTRACT FROM AUTHOR]

The action of netropsin and distamycin A on the DNA secondary structure and on tile template properties of DNA in the DNA- and RNA-polymerase systems have been studied. Ultraviolet absorption measurements indicate characteristic hypochromic effects for the DNA-antibiotic complexes due to binding of netropsin and distamycin A to DNA. These are accompanied by the appearance of an absorption peak beyond 320 nm. This ultraviolet absorption maximum depends on the (A + T) content of DNA and disappears upon heat denaturation. These results together with earlier observations indicate an A and T specificity of the binding of the oligopeptides netropsin and distamycin A. As demonstrated by absorbance-temperature measurements netropsin increases considerably the thermal stability of DNA. Drastic changes can even be obtained with ratios below 5 moles netropsin per 100 DNA phosphate groups. The antibiotic dependent increase in tile melting temperature is more pronounced for the DNA netropsin than for the DNA distamycin A complex. The structure of denatured DNA is also affected markedly by both antibiotics. From the results it is concluded that binding of netropsin and distamycin A to DNA is the result of their affinity to double-helical and base stacked regions. Comparison of the effect of these antibiotics on DNA- and RNA-synthesis in vitro shows that both netropsin and distamycin. A inhibit the DNA- and RNA-polymerase reactions In both systems the template activity of native as well as of denatured DNA is affected by these antibiotics, Using ehromatin of Ehrlich ascites tumor cells as template it could be shown, that the nucleoprotein complex is as sensitive to the action of distamycin A as pure DNA. [ABSTRACT FROM AUTHOR]

Trenimon (2,3,5-aziridine-1,4-benzoquinone) reacts with DNA by alkylation of the purine moiety, by formation of apurinic sites, and by induction of strand breaks. The presence of cross links was noted in the later stages of treatment, as was a diminished susceptibility to DNAase action. The agent causes progressive loss of priming ability in the DNA-dependent RNA polymerase system. The RNA synthesized was of shorter chain length and sedimented in the sucrose gradient for the most part with a coefficient smaller than 6 S. [ABSTRACT FROM AUTHOR]

The physical properties of DNA after reaction with 7-bromomethylbenz[a]anthracene have been examined. Covalent binding of up to one hydrocarbon residue for 10 nucleotides does not cause denaturation, even locally, of the DNA. Equal numbers of adenine and guanine residues are substituted Reactivity of DNA is greater than that of polyribonucleotide complexes. Fluorescence characteristics, particularly polarisation of fluorescence, of the carcinogen at levels of between 1 to 800 and 1 to 10 nucleotides in the DNA double helix are described and energy transfer discussed. A geometrical arrangement of the methylbenz[a]anthracene residues, in winch the axis of the molecule lies in the large groove of the DNA double helix is proposed. [ABSTRACT FROM AUTHOR]

A fast sedimenting complex of gently lysed pot A- cells (an Escherichia coli strain lacking DNA polymerase I) included fragmented cell wall structures, lipid components, about 20% of the cellular protein and most of the bacterial chromosome. A major fraction of the DNA synthesizing activity in these strains cosedimented with this complex Several biochemical properties of this complex are described. The DNA synthesizing activity can be released from the complex by ultrasonication and by various detergents. The results are interpreted to mean that the DNA synthesizing activity is attached to the bacterial chromosome. [ABSTRACT FROM AUTHOR]

Since hydroxyurea specifically inhibits the biosynthesis of deoxyribonucleotides in Escherichia coli, analysis of the deoxyribonucleoside triphosphate pools following hydroxyurea addition makes possible a determination of the turnover of these compounds in vivo. This method has been used in the present work to study the effect of thymine starvation of E. coli 15 T on the turnover of dGTP, dATP, and dCTP. From the results obtained the following conclusions may be made. In exponentially growing cultures DNA synthesis can account for the entire turnover of the deoxyribonucleoside triphosphates. Removal of thee from the growth medium results in the appearance of catabolic reactions that degrade dATP and dCTP. During the first 20 min of thymine starvation the rates of degradation of dATP and dCTP are of the same magnitude as those accounted for by DNA polymerization during exponential growth. After 75 min, however, the decay rate of dCTP has increased 5-fold while the rate of dATP decay remains constant. During the entire period of thymine starvation the rate of decay of dGTP is very low, i.e. one sixth of that observed during exponential growth. The end products of the catabolic reactions involved are to a large extent excreted into the growth medium as diphenylamine-reacting material. By determining the rate of accumulation of the individual deoxyribonucleoside triphosphates following thymine deprivation, and adding these values to the decay rates, determined in the presence of hydroxyurea, it is possible to estimate the endogenous rate of synthesis of dGTP, dATP and dCTP under these conditions. The resets show that removal of thymine from E. coli 15 T results in a significant reduction in the rate of dGTP synthesis, essentially no effect on the rate of dATP synthesis, and an increasing rate of dCTP synthesis reaching 6-fold values after 75 min of starvation. [ABSTRACT FROM AUTHOR]

Diethyl pyrocarbonate has been shown to react with poly(A), poly(C) and poly(G) but poly(U) does not react. Diethyl pyrocarbenate appears to carbethoxylate amino groups on the bases. Poly(A) can bind one carbethoxy group per base but the binding capacity of poly(G) and poly(C) under non-denaturing conditions is 0.32 and 0.23, respectively. The difference in binding between poly(A) on one hand and poly(G) and poly(C) on the other may depend on conformation of the polynucleotides. The double helix poly(A) · poly(U) is essentially not carbethoxylated and native adenovirus DNA does neither bind carbethoxy groups. Carbethoxylation of poliovirus RNA leading to a 3-fold decrease in infectivity resulted in an average of 11 carbethoxy groups per RNA molecule. The specificity of the carbethoxylation for single strands was used to estimate the helix content of transfer RNA. Denatured yeast tRNAAla was carbethoxylated on 55 out of 56 bases containing amino groups. 18 carbethoxy, groups could be introduced under native conditions in the absence of magnesium, compared with 16 carbethoxy groups in the presence of magnesium. This is in agreement with the number of A, G and C in single-stranded regions of the dover-leaf moded. Unfractionated yeast tRNA was carbethoxylated on 29 and 20 bases respectively in the absence and presence of magnesium. The results suggest that carbethoxylation could be used to determine secondary structure of RNA. [ABSTRACT FROM AUTHOR]

Two DNA specific exonucleases, DNase III and DNase IV, are present in mammalian cell nuclei. The activity of the purified enzymes with ultraviolet-irradiated polydeoxynucleotides and DNA as substrates has been investigated. DNase III shows a very poor ability to release pyrimidine dimers, although excision can be demonstrated by using high concentrations of enzyme. In contrast, DNase IV liberates dinners in an effective fashion in vitro, and is a likely candidate to serve in this function also in vivo. The pyrimidine dimers are released by DNase IV as parts of oligonucleotides containing 5-8 residues. Neither of the two exonucleases is markedly inhibited by caffeine. [ABSTRACT FROM AUTHOR]

The effect of deprotonation and alkaline denaturation on the conformation of DNA from various sources has been investigated by optical rotatory dispersion and circular dichroism measurements. Upon transition to alkaline pH, changes in the optical rotatory dispersion at the characteristic peaks around 228 and 290 nm are observed. The variation of the optical rotations in case of A · T-rich DNA is more pronounced than in ease of G · C-rich DNA. For G · C-rich DNA, changes of rotations are mainly observed at pH values beyond 11.5; the transition ranging between pH 11.6 and 11.7. The transition is accompanied by a shift of the "cross-over" point towards longer wavelengths presumably due to deprotonation of guanine residues. Native DNAs show characteristic differences in their circular dichroism spectra dependent on the G · C content. Increasing the temperature (below the melting region) reveals enhancement of the circular dichroism band of native DNA around 270 nm whereas the opposite effect is found in the denatured state. As a possible explanation an increase of the chirality in the native helical DNA structure or a structure of intermediary character is considered. Titration up to pH 12 or higher produces a drastic increase of the rotational strength around 228 nm as well as changes in the other two circular dichroism-maxima which may be correlated to a diminuition of the chiral and helical structure. [ABSTRACT FROM AUTHOR]

Rat liver nucleoli were isolated from nuclear rat liver preparations. The nucleolar DNA and extranucleolar DNA, i.e. chromosomal DNA were examined. After deproteinization, nucleolar and chromosomal DNA were separated from RNA on 6 M CsCl isopycnic gradient. Nucleolar and chromosomal DNA were of the same density (1.703 g/cm³) in CsCl. The specific activity of nucleolar DNA from normal adult rat liver was significantly higher than chromosomal DNA after 32P incorporation in vivo, independent of the time after the label. Nucleolar DNA is metabolically distinct from chromosomal DNA. Results are discussed and a possible role of nucleolar DNA replication in ribosomal RNA synthesis is suggested. [ABSTRACT FROM AUTHOR]

The intracellular flow and the transfer to progeny phages of the single stranded parental DNA of the filamentous phage M13 has been studied in Escherichia coli KMBL 42 at low multiplicities of infection (<1). The cellular bound parental DNA was found to be first converted quantitatively into replicative form DNA. Then, contrary to the events in phage ΦX 174 infection the parental DNA and to progeny phage particles. The efficiency of transfer per infecting phage is approximately as high as 0.75 and 0.5, respectively. Evidence is presented that during transfer the parental DNA is incorporated into DNA strands having up to several times the length of single stranded phage DNA. [ABSTRACT FROM AUTHOR]

When chromatin from calf thymus is treated with dilute hydrochloric acid under mild conditions variable proportions of histones become bound to the DNA, thus becoming insoluble and appearing as non-histones. This is confirmed by model experiments in which artificial mixtures of pure histones and DNA were treated with acid. The acid insoluble residual proteins formed are strongly bound (presumably covalently) to the DNA, but can be separated by density gradient centrifugation at pH 11.5. Some properties of these "non-histones" are discussed. Their amino acid analyses are different from those of the histones. The differences, also recognizable in model experiments must originate from the mild acid treatment in presence of DNA and suggest modifications of amino acids. These "nonhistones" are considered to be masked histones, because after strong alkali treatment, definite histone fractions can be recognized by electrophoreses. The conclusion is drawn that mild acid treatment of chromatin artificially produces "nonhistones". [ABSTRACT FROM AUTHOR]

Dna-dependent RNA polymerase from Escherichia coli can use ATP and UTP as substrates in a template free de novo reaction when incubated with a single triphosphate. The following observations on this reaction were made: 1. The yield of polymer produced in these reactions amounts to only 3% of that in the simultaneous (ATP + UTP) de novo polymerization and is stimulated by traces of DNA contaminating the enzyme. 2. Poly G · poly C also, stimulates de novo formation of poly A and poly U up to 60-fold. This reaction depends on the concentration of monovalent cations, e.g. Na= or K=, with optimum concentrations at 0.2 M. A model for the stimulation effect is discussed. 3. In the (ATP = UTP) reaction the obligatory lag phase was eliminated by preincubation with either of the two substrates. The optimum conditions of the reaction turned out to be the same as for polyribonucleotide transcription. Therefor (ATP = UTP) polymerization is interpreted as transcription of originally de novo formed homopolymers like poly A and poly U. [ABSTRACT FROM AUTHOR]