Analysis of the Vicia faba genome by use of restriction endonucleases.

DNA of the broad bean, Vicia faba, was cleaved by the restriction endonucleases endoR . EcoRI, endoR . HindIII, endoR . HincII, endoR . BamI, and endoR . BspRI. Separation in agarose gels of the resulting fragments revealed, in addition to the bulk DNA, an enzyme-specific pattern of bands composed of restriction fragments of 300 to more than 30,000 base pairs in length. Bulk DNA was characterized by an unusual size distribution which significantly deviated from that expected according to the random fragmentation theory. It is argued that the observed distribution is due to the high proportion of repetitive DNA within this species (approximately equal to 75%). In all digests, a class of high-molecular-weight restriction fragments of more than 30,000 base pairs in length was observed which comprised 5-8% of the genome. It showed hybridization with highly repetitive DNA (c0t less than or equal to 2 x 10(-2) M . s) and included a fraction (2-3% of the genome) highly resistant to the activity of all the enzymes tested. The buoyant density in CsCl of this resistant DNA was not different from that of the total DNA (36% dG + dC). In endoR . EcoRI digests, the high-molecular-weight fragment class contained, in addition to the resistant DNA, a fraction of relatively high buoyant density (calculated dG + dC content: 61%) containing cleavage sites for the other enzymes used.     

The mitochondrial genome of wild-type yeast cells. IV. Genes and spacers

The organization of the mitochondrial genome of wild-type Saccharomyces cerevisiae cells has been investigated further, by degrading mitochondrial DNA with micrococcal nuclease. Under the conditions used, this enzyme very strongly degrades the A + T-rich stretches (spacers) whereas it only inflicts a limited number of breaks into the G + C-rich stretches (genes). The macromolecular fragments derived from the “genes” have been separated from the oligonucleotides originating from the “spacers” by gel filtration, and both sorts of products have been investigated. It has been shown (a) that the spacers are very homogeneous in base composition and have a G + C content lower than 5% (mitochondrial DNA has a G + C content of 18%); (b) that the genes are very heterogeneous in base composition, the G + C content ranging from about 25% to 50%, when the average size of the fragments is 1·2 × 10⁵; smaller fragments, molecular weight 4 × 10⁴, having a G + C level as high as 65%, have been isolated in a yield of 10%; the average G + C content of genes is about 32%; (c) that genes and spacers are present in about equal amounts in the mitochondrial genome and that they have comparable average sizes.     

Organization of nucleotide sequences in the chicken genome

The four major components of chicken DNA were prepared by density gradient centrifugation and characterized in several basic properties: relative amounts, dG + dC content, buoyant densities, compositional heterogeneity, and reassociation kinetics. While the relative amounts and the compositions of the major components of chicken DNA were similar to those found in mammalian genomes, their compositional heterogeneities were found to be narrower. The relative amounts of interspersed repeated and unique sequences were strikingly different in different components and also different from those found in the corresponding major components of mouse and human DNAs. If one takes into consideration that major DNA components (a) account for practically all of main-band DNA and (b) derive by preparative breakage from very long DNA segments of fairly homogeneous composition, the isochores, our findings indicate that the distribution of interspersed repeats is different in different chromosomal regions and is species-specific.     

Characterization of the genome of the basidiomycete Schizophyllum commune

DNA of Schizophyllum commune was isolated both from mycelial cells and from protoplasts. Nuclear DNA was isolated after solubilization of the mitochondria with the detergent Nonidet. The G + C content of the nuclear DNA was 57%, calculated from its buoyant density (1.7165 g/ml) and from the Tm (77.4 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). The buoyant density of the ribosomal cistrons was 1.707 g/ml. DNA isolated from purified mitochondria had a very low G + C content: 22% (rho = 1.6845 g/ml, Tm = 61.8 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). Analysis of CsCl profiles and melting patterns suggested that mitochondrial DNA contains interspersed (A + T)-rich sequences. From reassociation analysis of sheared nuclear DNA the genome size of S. commune was determined to be 22.8 . 10(9) daltons. A small amount of DNA (0.5 . 10(9) daltons) bound to hydroxyapatite at zero time Cot. 7% of the genome (1.6 . 10(9) daltons) represented repetitive DNA.     

Resolution of α, β and γ DNA of Saccharomyces cerevisiae with the antitumor drug cis-Pt(NH3)2Cl2. Evidence for preferential drug binding by GpG sequences of DNA

This paper reports further studies on the separation of DNAs with the antitumor drug cis-Pt(NH₃)₂Cl₂. cis-Pt(NH₃)₂Cl₂ permits resolution of the three DNA components from whole Saccharomyces cerevisiae in CsCl gradients, avoids pelleting of mitochondrial (β) DNA and does not require a critical molar ratio of platinum drug to DNA-P. However, the difficulty in removing all of the DNA-bound platinum may limit its preparative use. The linear relationship between the increase in buoyant density of platinized double-stranded DNA and its G + C content is employed to calculate a G + C content of 41.2% and 45.8% for α and γ DNA, respectively, using a value of 20% G + C for β DNA. In parallel experiments, we find that poly(dG)·poly(dC), which contains sequential guanine bases, exhibits an unexpectedly large buoyant density increase with cis-Pt(NH₃)₂ Cl₂, while the buoyant density increase of poly[d(G-C)]is markedly retarded, indicating an effect of nucleotide base sequence on DNA separation. The trans platinum compound, which has no antitumor properties, separates DNAs on the basis of G + C content in a similar fashion, but does not preferentially increase the buoyant density of poly(dG)·poly(dC).     

Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.     

Effect of mercuric and silver ions on cesium sulfate equilibrium buoyant densities of synthetic polydeoxyribonucleotides.

The effect of Hg2+ and Ag+ on the buoyant density (rho) of four synthetic DNA polymers, poly[d(A-T)]; poly(dA) - poly(dT); oikt[d(G-C)]; and poly(dG) - poly(dC), was investigated. The buoyant density of poly[d(A-T)] in Cs2SO4 increased dramatically after complexing with Hg2+, but little change in the buoyant density of other polymers resulted except at very high molar ratios of Hg2+/DNA-P (rf). Hg2+ raised the thermal transition temperature (Tm) of alternating polymers and lowered the Tm of homopolymers. Measurements in the preparative ultracentrifuge indicated that lowered Tm correlated with Hg2+-induced strand separation of one homopolymer [poly(dA) - poly(dT)], but strand separation was not observed with another homopolymer [poly(dG) - poly(dC)] complexed with Hg2+. When Ag+ was mixed with the polymers, the buoyant density of poly(dG) - poly(dC) increased most markedly. A substantial increase in the buoyant density of poly[d(A-T)] and a small increase in the buoyant density of poly[d(G-C)] were also observed. The Tm changes induced by Ag+ were not related in any obvious way to buoyant density changes. These findings indicate that nucleotide sequence as well as overall base composition is of importance in understanding the buoyant density changes induced by metal ions. Although these data do not allow construction of a detailed molecular model of polymer-metal ion interactions, they may be used to explain much of the behavior of naturally occurring DNA sequences, such as heterochromatic satellite sequences and 5 S and rRNA sequences, in Hg2+/Cs2SO4 and Ag+/Cs2SO4 gradients.     

Detection of triple-helix related structures adopted by poly(dG)-poly(dC) sequences in supercoiled plasmid DNA.

Negative superhelical strain induces the poly(dG)-poly(dC) sequence to adopt two totally different types of triple-helices, either a dG.dG.dC triplex in the presence of Mg(+)+ at both neutral and acidic pHs or a protonated dC+.dG.dC triplex in the absence of Mg(+)+ ions at acidic pH (1). To examine whether there are still other types of non-B DNA structures formed by the same sequence, we constructed supercoiled plasmid DNAs harboring varying lengths of the poly(dG) tract, and the structures adopted by each supercoiled plasmid DNA were studied with a chemical probe, chloroacetaldehyde. The potential of a poly(dG)-poly(dC) sequence to adopt non-B DNA structures depends critically on the length of the tract. Furthermore, in the presence of Mg(+)+ and at a mildly acidic pH, in addition to the expected dG.dG.dC triplex detected for the poly(dG) tracts of 14 to 30 base pairs (bp), new structures were also detected for the tracts longer than 35 bp. The structure formed by a poly(dG) tract of 45 bp revealed chemical reaction patterns consistent with a dG.dG.dC triplex and protonated dC+.dG.dC triple-helices fused together. This structure lacks single-stranded stretches typical of intramolecular triplexes.     

A chemical and physical method for determining the complete base composition of plant DNA.

Two physical methods are routinely used to determine the base composition of DNA. One measures the temperature corresponding to the midpoint of the absorbance rise (TM) and relates it to base composition with the equation, TM = 41 (dG + dC) + 69, the other measures buoyant density (rho) and relates it to base composition rho = 0.098(dG + dC) + 1.6535. The base composition of DNA from various sources was first determined by a chemical method and these values compared to those determined by the physical methods. Higher plants contained up to 7 mol% 5-methyldeoxycytidine in their DNA and in all cases tested deoxyguanosine = deoxycytidine + 5-methyldeoxycytidine. After determining that TM was unaffected by the amount of 5-methyldeoxycytidine in DNA, the mol% of dA, dT, dG, and the total of dC plus 5-methyldeoxycytidine for any DNA could be calculated. Buoyant density on the other hand, was lowered 0.004 g . cm-3 for every 6.3 mol% 5-methyldeoxycytidine. Therefore, both physical parameters were related to the mole fraction of 5-methyldeoxycytidine by the following equation: (see article). With a value of r 5-methyldeoxycytidine an estimation of deoxycytidine was made. The resultant values agreed with the chromatographic determinations.     

Factor C from rabbit liver. A new poly(dC) and poly[d(G-C)] template-selective stimulatory protein of DNA polymerases

We have undertaken a search for mammalian DNA-binding proteins that enhance the activity of DNA polymerases in a template sequence-specific fashion. In this paper, we report the extensive purification and characterization of a new DNA-binding protein from rabbit liver that selectively stimulates DNA polymerases to copy synthetic poly[d(G-C)] and the poly(dC) strand of poly(dC).poly(dG) as well as single-stranded natural DNA that contains stretches of oligo(dC). The enhancing protein, a polypeptide of 65 kDa designated factor C, stimulates the copying of the two synthetic templates by Escherichia coli DNA polymerase I, Micrococcus luteus polymerase, and eukaryotic DNA polymerases alpha and beta, but not by avian myeloblastosis virus polymerase. Factor C, however, does not affect utilization by these polymerases of the poly(dG) strand of poly(dC).poly(dG), of poly(dC) primed by oligo(dG), or of poly(dA).poly(dT) and poly[d(A-T)]. With polymerase I, Michaelis constants (Km) of poly[d(G-C)] and of the poly(dC) strand of poly(dC).poly(dG) are decreased by factor C 37- and 4.7-fold, respectively, whereas maximum velocity (Vmax) remains unchanged. By contrast, neither the Km value of the poly(dG) strand of poly(dC).poly(dG) nor the Vmax value with this template is altered by factor C. Rates of copying of activated DNA, denatured DNA, or singly primed M13 DNA are not affected significantly by factor C. However, primer extension analysis of the copying of recombinant M13N4 DNA that contains runs of oligo(dC) within an inserted thymidine kinase gene shows that factor C increases processivity by specifically augmenting the efficiency at which polymerase I traverses the oligo(dC) stretches. Direct binding of factor C to denatured DNA is indicated by retention of the protein-DNA complex on columns of DEAE-cellulose. Binding of factor C to poly[d(G-C)] is demonstrated by the specific adsorption of the enhancing protein to columns of poly[d(G-C)]-Sepharose. We propose that by binding to poly[d(G-C)] and to poly(dC).poly(dG), factor C enables tighter binding of some DNA polymerases to these templates and facilitates enzymatic activity.     

The isolation of duplex DNA fragments containing (dG.dC) clusters by chromatography on poly(rC)-Sephadex.

Duplex DNA containing oligo(dG.dC)-rich clusters can be isolated by specific binding to poly(rC)-Sephadex. This binding, probably mediated by the formation of an oligo(dG.dC)rC+ triple helix, is optimal at pH 5 in 50% formamide, 2 M LiCl; the bound DNA is recovered by elution at pH 7.5. Using this method we find that the viral DNAs PM2, lambda and SV40 contain at least 1, 1 and 2 sites for binding to poly(rC)-Sephadex, respectively. These binding sites have been mapped in the case of SV40; the binding sites can in turn be used for physical mapping studies of DNAs containing (dG.dC) clusters. Inspection of the sequence of the bound fragments of SV40 DNA shows that a (dG.dC)6-7 tract is required for the binding of duplex DNA to poly(rC)-Sephadex. Although about 60% of rabbit DNA cleaved with restriction endonuclease KpnI binds to poly(rC)-Sephadex, no binding is observed for the 5.1 kb DNA fragment generated by KpnI digestion, which contains the rabbit beta-globin gene. This indicates that oligo(dG.dC) clusters are not found close to the rabbit beta-globin gene.     

Magnesium-dependent supercoiling-induced transition in (dG)n.(dC)n stretches and formation of a new G-structure by (dG)n strand.

Plasmids containing (dG)27.(dC)27 inserts (pPG27), (dG)37.(dC)37 inserts (pPG37), and (dG)24C(dG)21.(dC)24G(dC)21 inserts (pPG46C) were constructed for the study of structural transitions within (dG)n.(dC)n stretches. Two-dimensional gel electrophoresis has shown that a Mg2+-dependent supercoiling-induced structural transition takes place at pH 8 in plasmid pPG46C. The transition occurs at -0=0.06 and involves a supercoiling release corresponding to 5 superhelical turns. After denaturation of the restriction fragments containing (dG)n.(dC)n inserts, the strands do not renature completely and (dG)n-containing strand migrates in PAGE much faster than the (dC)n-containing one. Chemical modification experiments with the (dG)n-strand have revealed the periodic nature of the protection of guanines against dimethyl sulfate methylation. The (dG)n strand in the presence of Mg2+ forms complexes with the complementary (dC)n strand, which differ from the native duplex in mobility. We believe these effects to be due to the formation of an intrastrand structure within the (dG)n strand stabilized by G.G interactions (we called it G-structure), which in the presence of Mg2+ forms an interstrand complex. with the (dC)n strand.     

Nucleosome cores reconstituted from poly (dA-dT) and the octamer of histones.

In this paper we describe a detailed investigation of the reconstitution of nucleosome cores from poly (dA-dT) and the octamer of histones. We also attempted the reconstitution from the copolymers poly dA.poly dT, poly dG.poly dC and poly (dG-dC). The repeat of the reconstituted chromatin fibre is discussed. The micrococcal nuclease released poly (dA-dT) core particle is found to contain a considerably narrower DNA size distribution that of the native random DNA nucleosome core (12). In addition we have succeeded in obtaining small crystals of the poly (dA-dT) nucleosome core. The DNAase I digestion pattern of the poly (dA-dT) containing nucleosome core is presented. The periodicity of DNAase I cutting sites is found to be about 10.5 bases and is similar to that of the native nucleosome core (12, 13).     

Sequence specific cleavage of DNA by micrococcal nuclease.

Micrococcal nuclease is shown to cleave DNA under conditions of partial digestion in a specific manner. Sequences of the type 5'CATA and 5'CTA are attacked preferentially, followed by exonucleolytic degradation at the newly generated DNA termini. GC-rich flanking sequences further increase the probability of initial attack. Unexpectedly, long stretches containing only A and T are spared by the nuclease. These results, which were obtained with spared by the nuclease. These results, which were obtained with mouse satellite DNA and two fragments from the plasmid pBR22, do not support the previous contention that it is the regions of high At-content which are initially cleaved by micrococcal nuclease. This specificity of micrococcal nuclease complicates its use in experiments intended to monitor the nucleoprotein structure of a DNA sequence in chromatin.     

Comparison of nuclease digestion of polyoma virus nucleoprotein complex and mouse chromatin.

We digested polyoma virus nucleoprotein complex, isolated from disrupted virions, with micrococcal nuclease and DNase I. The results were compared with digestions of chromatin from mouse nuclei. The nucleosome "core" structures were similar, but the spacing of the nucleosomes in the isolated polymoma nucleoprotein complexes was irregular, whereas in mouse chromatin it was regular. The average nucleosome repeat length in each case was 190 to 200 base pairs. This figure suggests that, unless there are substantial stretches of free DNA, the polyoma nucleoprotein complex contains about 26 nucleosomes. The commonly used method of preparing the nucleoprotein complex by disruption of virions at pH 10.2 may lead to significant damage to the structure. Such damage may be more clearly revealed by the susceptibility of the DNA to nuclease digestion than by the usual criteria of sedimentation velocity and buoyant density.     

N-2-acetylaminofluorene modification of poly(dG-m5dC)·poly(dG-m5dC) induces the Z-DNA conformation

Poly(dG-m⁵dC)·poly(dG-m⁵dC) was modified by treatment with N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) and its conformation examined by circular dichroism (CD) and susceptibility to S₁ nuclease digestion. A sample with a modification level of 10% shows a CD spectrum characteristic of the Z form and is resistant to digestion by S₁ nuclease. The relative reactivity of several polymers with N-Aco-AAF was shown to follow the order of ease of formation of Z DNA: poly(dG-m⁵dC)·poly(dG-m⁵dC) > poly(dG-dC)·poly(dG-dC) > poly(dG)·poly(dC). This suggests that AAF reacts more readily with Z DNA than B DNA.     

Chromatin organization in Paracentrotus lividus eggs

After purification by buoyant density centrifugation in ethidium bromide - CsCl gradient and electrophoretic fractionation, the DNA fragments isolated from P. lividus egg nuclei incubated with micrococcal nuclease exhibit a typical oligomeric pattern. Analysis of chromatin samples digested to an increasing extent by micrococcal nuclease reveals that the structural organization of egg chromatin is heterogeneous, both in terms of repeat size and degree of sensitivity to nuclease attack. The nucleosomal repeats of P. lividus sperms and embryos up to the mesenchyme blastula stage have also been determined, for comparison.     

S1 nuclease sensitivity to cis- and trans-diamminedichloroplatinum(II) modified DNAS: influence of (G+C) content and nucleotide sequence

The sensitivity of S1 nuclease to cis- and trans-(NH3)2PtCl2 modified DNAs is examined as a function of the level of cis- and trans-(NH3)2PtCl2 bound, the % (G+C) content in DNA from different sources and the sequence dependence in poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). The extent of DNA digested increases with increasing levels of either isomer and is inversely influenced by the % (G+C) content of the DNA. However, the difference in the extent of digestion between the cis-and trans-(NH3)2PtCl2 modified DNAs at equivalent levels of bound isomer follows the order, calf-thymus greater than M. lysodeikticus greater than poly(dG-dC).poly(dG-dC). While there is virtually no difference in the digestion profiles for poly(dG-dC).poly(dG-dC) modified with the two isomers, there is a striking difference in the extent of digestion between cis- and trans-(NH3)2PtCl2 modified poly(dG).poly(dC). These results are discussed in light of the possible modes of binding for cis-(NH3)2PtCl2, previously reported findings on modified DNA and possible implications for modifications in cellular chromatin.     

Recognition of (dG)n.(dC)n sequences by endonuclease G. Characterization of the calf thymus nuclease

We report the purification of endonuclease G (Ruiz-Carrillo, A., and Renaud, J. (1987) EMBO J. 6, 401-407) from calf thymus nuclei and whole tissue. The enzyme has been enriched 29,000-fold, and the activity was unambiguously identified with a 26-kDa protein after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native nuclease behaves as a 50-kDa species by gel filtration, suggesting that it is composed of two subunits, presumably identical. In terms of absolute amounts, endonuclease G (endo G) is a nuclear enzyme although it was also detected in purified mitochondria. Endo G is highly specific for (dG)n.(dC)n tracts in DNA, nicking either strand of relaxed substrates with similar kinetics. The sensitivity of the homopolymer tracts is proportional to their length (from n = 8 to 29), insofar as the flanking sequences are constant. However, the overall rate of cleavage is influenced by the composition of the flanking DNA. Minor cleavage sites contain shorter (dG)n.(dC)n clusters (n = 3-7). Endo G efficiently cleaves (dC)n but not (dG)n runs in single-stranded DNA, suggesting that it may recognize an asymmetric strand conformation of the homopolymer tracts. Endo G does not recognize other homo(co)-polymer sequences or cruciform structures in DNA.