A new procedure for determining thymine residues in DNA sequencing. Photoinduced cleavage of DNA fragments in the presence of spermine

A new procedure for T specific cleavage of DNA fragments utilizing photoreaction with spermine has been described. Irradiation of 3'-[32P]-end-labeled DNA fragments for 10-20 min with a germicidal lamp emitting mainly 254-nm light in the presence of 1 M spermine in distilled water resulted in a T specific cleavage of the DNA chains. This method does not require piperidine treatment. By contrast, when the DNA fragments were irradiated in the presence of methylamine under similar conditions, both G and T bands with the intensity of G greater than T have appeared. A similar but less selective T cleavage has also been observed in the irradiation of 5'-[32P]-end-labeled DNA fragments in the presence of spermine followed by brief heating of the photolysate in a loading buffer for gel electrophoresis. The T specific photoreaction with spermine and the G greater than T reaction with methylamine described here may be conveniently used in combination with the standard Maxam-Gilbert's reactions to provide independent confirmatory readings.     

A photoinduced cleavage of DNA useful for determining T residues.

Irradiation of 5'-[32P]-phosphate labeled DNA fragments with ultraviolet light in the presence of primary amines followed by piperidine treatment resulted in base-specific cleavage of the DNA chain at T residues, accompanied by a less intensive G reaction. This simple, T greater than G cleavage offers an alternative method for determining T residues in chemical DNA sequencing.     

Interaction between polyamines and nucleic acids or phospholipids

The binding of polyamines to DNA, RNA, and phospholipids has been studied by gel filtration and sucrose density gradient centrifugation. Spermine was found to bind more to a GC-rich DNA. Among RNAs containing double-stranded region [poly(AU), poly(GC), and ribosomal RNA], the binding of spermine was nearly equal. Among the single-stranded RNAs, the binding of spermine was in the order poly(U) > poly(C) > poly(A). An increase in K⁺ or Mg²⁺ concentration resulted in a great decrease in spermine binding to DNA and in a slight decrease in spermine binding to RNA. Therefore, in the presence of more than 2 mm Mg²⁺ and 100 mm K⁺, the binding of spermine to RNA was greater than that to DNA. No significant difference in spermine binding was observed between 16 S ribosomal RNA and 30 S ribosomal subunits, suggesting that ribosomal proteins did not affect significantly the binding of spermine to ribosomal RNA. The binding of spermine to microsomes was dependent on phospholipids. The binding strength was in the order phosphatidylinositol > phosphatidylethanolamine > phosphatidylcholine.     

Polyamines stimulate DNA-directed DNA synthesis catalyzed by mammalian type C retroviral DNA polymerases

In the presence of optimal concentrations of Mg2+, rates of activated (gapped) DNA-directed DNA synthesis by purified mammalian type C retroviral DNA polymerases are stimulated greater than 10-fold by the polyamines spermine and spermidine. Such stimulation was not observed using either similar concentrations of the polyamines cadaverine or putrescine or exogenously provided salt or ammonium ions. Avian type C as well as mammalian type B and type D retroviral DNA polymerases, in contrast to the mammalian type C enzyme, were found to be relatively insensitive to spermine and spermidine stimulation. Kinetic analysis of the polyamine stimulation of activated DNA-directed DNA synthesis carried out using spermine and purified Rauscher leukemia virus DNA polymerase revealed at least two distinct mechanisms of activation of DNA synthesis. 1) At DNA concentrations below 2.5 micrograms/ml, spermine appears to interact with the enzyme-DNA complex in order to stimulate synthesis. 2) At DNA concentrations above 2.5 micrograms/ml, increased spermine stimulation is observed which appears to be due to its direct interaction with the activated DNA template resulting in either selective limitation of the formation of "dead-end" enzyme-DNA complexes or its ability to convert such nonproductive enzyme binding sites into productive sites for the initiation of synthetic activity. The addition of spermine to reaction mixtures was found to increase both the apparent Km and Vmax of the activated (gapped) DNA-directed reaction with regard to template concentration.     

The deoxyribonuclease induced after infection of KB cells by herpes simplex virus type 1 or type 2. I. Purification and characterization of the enzyme.

The deoxyribonuclease induced in KB cells by herpes simplex virus (HSV) type 1 and type 2 has been purified. Both enzymes are able to completely degrade single- and double-stranded DNA yielding 5'-monophosphonucleotides as the sole products. A divalent cation, either Mg2+ or Mn2+, is an absolute requirement for catalysis and a reducing agent is necessary for enzyme stability. The maximum rate of reaction is achieved with 5 mM MgCl2 for both HSV-1 and HSV-2 DNase. The optimum concentration for Mn2+ is 0.1 to 0.2 mM and no exonuclease activity is observed when the concentration of Mn2+ is greater than 1 mM. The rate of reaction at the optimal Mg2+ concentration is 3- to 5-fold greater than that at the optimal Mn2+ concentration. In the presence of Mg2+, the enzymes are inhibited upon the addition of Mn2+, Ca2+, and Zn2+. The enzymatic reaction is also inhibited by spermine and spermidine, but not by putrescine. Crude and purified HSV-1 and HSV-2 DNase can degrade both HSV-1 and HSV-2 DNA, but native HSV-1 DNA is hydrolyzed at only 22% of the rate and HSV-2 DNA at only 32% of the rate of Escherichia coli DNA. Although HSV-1 and HSV-2 DNase were similar, minor differences were observed in most other properties such as pH optimum, inhibition by high ionic strength, activation energy, and sedimentation coefficient. However, the enzymes differ immunologically.     

The binding of polyamines and magnesium to DNA.

1. The binding of spermine and Mg2+ to DNA has been investigated using the dye arsenazo III to measure unbound cations. 2. The apparent dissociation constant, Kd, of DNA for spermine has been found to be 7.4 +/- 3.9 x 10(-8) M and that for Mg2+, 6.5 +/- 3.3 x 10(-7) M. 3. Binding of spermine in the presence of 1 mM Mg2+ has been shown to have a Kd of about 4 x 10(-6) M. 4. Magnesium ion (2 mM) halves the concentration of spermine needed to cause DNA aggregation. 5. Spermidine binds to DNA with a similar affinity to spermine but 3,3'-iminobispropylamine and 1,5,9,13-tetra-azatridecane bind with a lower affinity. The naturally occurring polyamines thus have a higher affinity for DNA than the related polyamines which do not occur naturally. 6. Binding of spermine or spermidine to DNA alters the spectrophotometric absorbance of DNA at 260 nm.     

High-performance liquid chromatographic determination of N-[2- (hydroxyethyl)-N-(2-(7-guaninyl)ethyl)]methylamine, a reaction product between nitrogen mustard and DNA and its application to biological samples

Nitrogen mustard (HN2) is a bifunctional alkylating agent which is thought to cause cytotoxicity by covalently binding to DNA. Most studies to date have looked at qualitatively determining the presence of DNA–HN2 adducts from reactions with native DNA. The adduct which is predominately formed in these reactions is N-[2-(hydroxyethyl)-N-(2-(7-guaninyl)ethyl]methylamine (N7G). A simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of N7G from DNA using ultraviolet detection is described. DNA samples having been exposed to HN2 treatment were hydrolyzed and preseparated from high-molecular-mass material by filtration using a molecular mass cut-off of 3000. The mobile phase consisted of methanol–26 mM ammonium formate, pH 6.5 (24:76, v/v). N7G, as well as the internal standard, methoxyphenol, were separated within 30 min. The recovery of N7G after hydrolysis of the DNA reaction product was quantitative and limits of detection and quantification of 10 and 20 ng/ml, respectively, were calculated. The method was validated in DNA–HN2 dose response experiments. The N7G reaction product appears to be the first reaction product formed at lower ratios of HN2/DNA but its production plateaus at higher ratios of HN2/DNA probably due to increased formation of hitherto unknown adducts. The method is simple and sensitive and for this reason, may be suited for the determination of DNA/HN2 reaction products.     

Repair of base-base mismatches in simian and human cells

Mismatched heteroduplexes arise as intermediates of several dissimilar genetic processes. The outcome of these genetic events will therefore be influenced by the efficiency and specificity of mismatch repair. We have studied the correction of base-base mispairs in simian and human fibroblasts by transfecting the cells with derivatives of SV40 DNA, each harboring a single mispair in a defined orientation. Analysis of plaques revealed that correction efficiencies for homomispairs followed the pattern G.G greater than C.C greater than or equal to A.A greater than T.T. Repair bias was influenced by flanking sequences. Correction efficiences for heteromispairs followed the pattern of G.T greater than A.C greater than C.T greater than A.G and repair favored the retention of G + C by a substantial margin. This repair specificity could lead to a gene conversion bias favoring the accumulation of G + C in sequences subject to high levels of recombination or unequal exchange.     

Polyamines inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis. Studies with permeabilized GH3 cells.

[3H]Inositol-labelled GH3 rat anterior pituitary tumour cells were permeabilized with digitonin and were incubated at 37 degrees C in the presence of ATP and Mg2+. [3H]Polyphosphoinositide breakdown and [3H]inositol phosphate production were stimulated by hydrolysis-resistant GTP analogues and by Ca2+. Of the nucleotides tested, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) was the most effective stimulus. Activation by GTP gamma S appeared to be mediated by a guanine nucleotide-binding (G) protein as GTP gamma S-stimulated [3H]inositol phosphate production was inhibited by other nucleotides with a potency order of GTP = GDP = guanosine 5'-[beta-thio]diphosphate greater than ITP greater than GMP greater than UTP = CTP = adenosine 5'-[gamma-thio]triphosphate. The stimulatory effects of 10 microM-GTP gamma S on [3H]inositol phosphate levels were reversed by spermine and spermidine with IC50 values of approx. 0.25 and 2 mM respectively. Putrescine was inhibitory only at higher concentrations. Similarly, GTP gamma S-induced decreases in [3H]polyphosphoinositide levels were reversed by 2.5 mM-spermine. The inhibitory effects of spermine were not overcome by supramaximal concentrations of GTP gamma S. In contrast, [3H]inositol phosphate production stimulated by addition of 0.3-0.6 mM-Ca2+ to incubation media was only partially inhibited by spermine (5 mM), and spermine was not inhibitory when added Ca2+ was increased to 1 mM. These data show that polyamines, particularly spermine, inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis with a marked selectivity towards the stimulatory effects of GTP gamma S.     

The effects of spermine and Mg2+ on the catalytic mechanism of isoleucine: tRNA ligase.

Isoleucyl-tRNA formation catalysed by isoleucine: tRNA ligase is stimulated by both Mg2+ and spermine in the pH-range 7.0 to 8.0 at 310 K. At low [Mg2+] the acceleration caused by both cations together exceeds the sum of their individual effects. 2. The spermine-stimulated reaction has a steeper temperature-dependence than reaction in the presence of Mg2+. Two phases in the kinetics of isoleucyl-tRNA formation are detected in the presence of Mg2+ plus or minus spermine, but only a single step is observed in the presence of spermine alone. Thus the rate-limiting steps under normal assay conditions are different for the two cations. 3. Enzyme-bound isoleucyl-AMP can be formed in the absence of Mg-2+ and plus or minus spermine. 4. It is concluded that there is no evidence for cation-dependent differences in the reaction mechanism of isoleucine: tRNA ligase, though there are certainly differences in the relative rates of some of the individual steps.     

The regulation of G0-S transition in mouse T lymphocytes by polyamines

While the role of polyamines in DNA synthesis during the S phase of the cell cycle has been repeatedly postulated, recent studies point also to polyamine involvement in the early phase of the G0-S transition. In order to determine polyamine-dependent steps in the cell cycle we have studied the effects of inhibitors of polyamine biosynthesis and exogenous polyamines on the proliferation of T lymphocytes as well as on the expression of some growth-regulated genes. The ability of Con A-stimulated mouse T lymphocytes to enter DNA synthesis was markedly inhibited by methylglyoxal bis(guanylhydrazone) in a dose-dependent manner. This inhibitory effect was stronger in the presence of fetal calf serum containing a high level of activities of polyamine oxidases than in the presence of horse serum. Putrescine and spermine added to T splenocyte culture instead of mitogen-Con A stimulated [3H]thymidine incorporation with kinetics similar to that observed with Con A. The growth-stimulating effects of polyamines were concentration-dependent. Polyamines at optimal growth-stimulating concentrations (10 microM spermine and 80 microM putrescine) induced the expression of genes encoding the cytoskeletal proteins beta-actin, vimentin, and alpha-tubulin to an extent and with kinetics similar to those of Con A. The results presented herein suggest that polyamines are capable of stimulating the transition of G0 cells to the S phase and that this effect may be mediated by their influence on the gene expression.     

Studies on T4-head maturation. 2. Substrate specificity of gene-49-controlled endonuclease

The substrate specificity of 49+-enzyme was investigated in vitro. The enzyme showed a marked preference for rapidly sedimenting T4 DNA (greater than 1000 S) when helix-destabilizing proteins from Escherichia coli or phage T4 were added to the reaction. Regular replicative T4 DNA (200-S DNA) or denatured T4 DNA was not cleaved by the enzyme in the presence of these proteins but if they were omitted from the reaction both DNAs become good substrates for the enzyme. 200-S DNA was cleaved at its natural sites of single strandedness which occur at one-genome intervals. Gaps in T4 DNA which were constructed by treatment of a nicked DNA with exonuclease III were also cleaved by 49+-enzyme in the absence of helix-destabilizing proteins. Single-stranded T4 DNA was extensively degraded and up to 50% of the material was found to be acid-soluble in a limit digest. The degradation products were predominantly oligonucleotides of random size. No preference for a 5'-terminal nucleotide was observed in material from a limit digest with M13 DNA. Double-stranded DNA was nicked upon exposure to 49+-enzyme and double-strand breakage finally occurred by an accumulation of single-strand interruptions. No acid-soluble material was produced from native T4 DNA. The introduction of nicks in native DNA did not improve its properties as a substrate for the enzyme. Double-stranded DNA was about 100-fold less sensitive to the enzyme than single-stranded DNA.     

The effect of silver ion binding and pH on the buoyant density of DNA and its use in fractionating heterogeneous DNA.

1. The effect of pH on the buoyant density of the complexes of Ag+ with DNA has been studied using 3H-labeled human DNA and several bacterial DNAs to determine the conditions necessary for the maximum resolution of compositional heterogeneity. In neutral CS2SO4 density gradients, Ag+ complexes with (G - C)-rich components are always denser than those with (A - T)-rich components, since (G - C)rich DNAs have a larger affinity for Ag+ than (A - T)-rich DNAs and their complexes are denser than (A - T)-rich complexes. In alkaline (pH greater than 9) CS2SO4 gradients, the buoyant density of the Ag+ - DNA complex is not a simple function of base composition. The Ag+ affinity of (A - T)-rich DNA is larger than that of (G - C)-rich DNA but the density of a (G - C)-rich complex is larger. Thus the ordering of the buoyant density changes depends on the amount of added Ag+. 2. The problem of resolving the density heterogeneity within a tracer DNA, and minor components of DNA, is explored and useful fractionation techniques are developed.     

Continuous cell-free protein synthesis directed by messenger DNA and catalyzed by extract of Thermus thermophilus HB27

Polypeptide synthesis directed by DNA as the messenger in a cell-free system of Thermus thermophilus was investigated. Polypeptides were synthesized with the addition of neomycin in the presence of DNA catalyzed by the cell extract. The stability of messenger DNA was greater than that of messenger RNA. Continuous cell-free translation with messenger DNA produced polypeptides at the rate of more than 8 microg/h in the presence of spermine.     

Protecting or promoting effects of spermine on DNA strand breakage induced by iron or copper ions as a function of metal concentration

Polyamines are ubiquitous polycations that participate in cellular processes such as growth, differentiation and cell death. Among the different functions ascribed to these organic cations, the polyamine spermine is known to protect DNA from the damage produced by reactive oxygen species (ROS) generated by different agents including copper ions. We have found that spermine exerts opposite effects on DNA strand breakage induced by Fenton reaction depending on metal concentration. Whereas at low concentration of the transition metals, 10 microM copper or 50 microM Fe(II), 1 mM spermine exerted a protective role, at metal concentrations higher than 25 microM copper or 100 microM Fe(II), spermine stimulated DNA strand breakage. The promotion of the damage induced by spermine was independent of DNA sequence but decreased by increasing the ionic concentration of the media or by the presence of metal-chelating agents. Moreover, spermine did not increase the oxidation of 2-deoxyribose by metal/H2O2 when DNA was substituted by 2-deoxyribose as a target for damage. Our results corroborate that spermine may protect DNA and 2-deoxyribose from the damage induced by ROS but also demonstrate that under certain conditions spermine may promote DNA strand breakage. The fact that this promoting effect of spermine on ROS-induced damage was observed only in the presence of DNA suggests that this polyamine under certain conditions may facilitate the interaction of copper and iron ions with DNA leading to the formation of ROS in close proximity to DNA.     

Trends in the 5' vs. 3' flanks of oligonucleotides in eukaryotic and prokaryotic genomes: the asymmetric roles played by cytosine and guanine.

Studies of sequence context preferences of oligonucleotides composed of (G/C)n and (A/T)m blocks (n + m = 3,4,5) unravel strong patterns. Comparisons of the 5' and 3' nearest neighbor doublets flanking these oligomers reveal the preference of (G/C)2 to be positioned immediately next to the (A/T)m block, enclosing it by (G/C) nucleotides rather than extending the (G/C)n block. That is, for a (G/C)n(A/T)m oligomer and a (G/C)2 doublet, (G/C)n(A/T)m(G/C)2 greater than (G/C)n + 2 (A/T)m. Similarly for an (A/T)m(G/C)n oligomer, (G/C)2(A/T)m(G/C)n greater than (A/T)m(G/C)n + 2. In an analogous manner, (A/T)2 flanking doublets prefer enclosing the (G/C)n blocks, although these patterns are weaker. Here we show a strong, direct relationship between the magnitude of the trends and the presence of Cs in the (G/C)n block in the (G/C)n(A/T)m oligomer, and the presence of Gs in the complementary (A/T)m(G/C)n oligomers. The trends are stronger in eukaryotic than in prokaryotic sequences. They are stronger for longer (G/C)n and shorter (A/T)m blocks. We suggest that the preference for (A/T)m to be enclosed by (G/C) rather than be flanked by them on only one side is related to DNA structure and DNA-protein interaction. Sequences of the (G/C)(A/T)(G/C) type may have more homogeneous minor groove geometry. In particular, the strong G vs. C asymmetry in the trends may be related to pyrimidine-purine junctions, possibly to CG sequences.     

Polyamines stimulate natural RNA-directed DNA synthesis by Rauscher murine leukemia virus DNA polymerase

In the presence of optimal concentrations of Mg²⁺, spermine and spermidine were found to stimulate rabbit globin mRNA-directed cDNA synthesis by Rauscher murine leukemia virus (R-MuLV) DNA polymerase. Stimulation of DNA synthesis did not occur with the polyamines putrescine or cadaverine, nor could exogenously provided salt or ammonium ions duplicate the stimulation. Analysis of the mechanism of stimulation showed that inclusion of spermine in reaction mixtures a) increased Vmax and decreased apparent Km with respect to the globin mRNA-oligo(dT) tem?late-primer complex, and b) decreased the quantity of oligo (dT) required for optimal rates of cDNA synthesis on a fixed quantity of mRNA template. Genomic 70S RNA-directed cDNA synthesis was also stimulated by spermine addition to reaction mixtures, but only at supra-optimal RNA concentrations. Our results suggest that stimulation of R-MuLV DNA polymerase activity by polyamines is primarily due to stabilization of the enzyme-templateprimer initiation complex resulting in increased efficiency of initiation of cDNA synthesis.     

Taxonomy of alkaliphilic Bacillus strains

The DNA base compositions of 78 alkaliphilic Bacillus strains were determined. These strains were grouped as follows: DNA group A, guanine-plus-cytosine (G+C) content of 34.0 to 37.5 mol% (17 strains); DNA group B, G+C content of 38.2 to 40.8 mol% (33 strains); and DNA group C, G+C content of 42.1 to 43.9 mol% (28 strains). DNA group A includes the type strain of Bacillus alcalophilus Vedder 1934. DNA-DNA hybridization studies with DNA group A strains revealed that only one strain, strain DSM 2526, exhibited a high level of DNA homology with B. alcalophilus DSM 485T (T = type strain). Neither strain DSM 485T nor any other DNA group A strain is homologous to any of the Bacillus type strains with comparable base compositions. Six strains formed a distinct group containing three highly homologous strains and three strains exhibiting greater than 50% DNA homology.