Presence of displacement loops in the covalently closed circular chloroplast deoxyribonucleic acid from higher plants.

Chloroplast DNAs (ctDNA) from pea and corn plants were examined in the electron microscope for the presence of replicative intermediates. Pea and corn ctDNAs were each found to contain two displacement loops (D-loops). The D-loops were 820 (+/- 90) base pairs long in pea ctDNA and 860 (+/- 125) base pairs long in corn ctDNA. In each ctDNA, the two D-loops were located at positions that were 7100 +/- 240) base pairs apart. The displacing strands of the two D-loops were located on opposite strands of the parental DNA molecule and they were seen to expand toward each other. The D-loops in the ctDNA from pea and corn exhibited branch migration and thus were easily distinguished from the denatured regions that were also present in these closed circular ctDNAs. In addition, the positions of the D-loops were found to be distinct from the positions of the denaturation loops (Den-loops). The Den-loops were also shown to be located at AT-rich regions in these ctDNA molecules. D-loops and Den-loops were also found in the circular and catenated ctDNA oligomers from pea and corn plants. Mapping the positions of the D-loops relative to the positions of the Den-loops showed that the structure of the D-loop-containing region in the pea and corn ctDNAs has been conserved to a greater extent than the structure of the rest of the two ctDNA molecules.     

The molecular size and conformation of the chloroplast DNA from higher plants.

Covalently closed circular choloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium choloride density-gradient entrifugation. As much as 30-40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules. The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (phiX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 - 10(6) for corn ctDNA to 96.7 - 10(6) for lettuce ctDNA. Each of these ctDNAs contained 3-4% of the circular molecules as circular dimers and 1-2% of the circular molecules as catenated dimes. The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequences of the ctDNAs ranged from 83.7 - 10(6) for oat ctDNA to 93.1 - 10(6) for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules...     

Localization of replication origins in pea chloroplast DNA.

The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.     

Equilibrium melting of plasmid ColE1 DNA: electron-microscopic visualization.

The fine structure of the melting curve for the linear colE1 DNA has been obtained. To find the ColE1 DNA regions corresponding to peaks in the melting curve's fine structure, we fixed the melted DNA regions with glyoxal /12/. Electron-microscopic denaturation maps were obtained for nine temperature points within the melting range. Thereby the whole process of colE1 DNA melting was reconstructed in detail. Spectrophotometric and electron microscopic data were used for mapping the distribution of Gc-pairs over the DNA molecule. The most AT-rich DNA regions (28 and 37% of GC-pairs), 380 and 660 bp long resp., are located on both sides of the site of ColE1 DNA's cleavage by EcoR1 endonuclease. The equilibrium denaturation maps are compared with maps obtained by the method of Inman /20/ for eight points of the kinetic curve of ColE1 DNA unwinding by formaldehyde.     

Comparison of partial denaturation maps with the known sequence of simian virus 40 and phi X174 replicative form DNA.

Electron microscope partial denaturation maps of two viral DNAs, simian virus 40 and φX174 replicative form, have been obtained. A simple computer program has been developed to predict denaturation maps from any given DNA sequence, based on the percentage of A · T base-pairs along the molecule. Maps constructed from the SV40 DNA and φX174 replicative form DNA base sequence show a good correlation with the experimental maps. The results show that the regions of a DNA molecule that denature first are, in fact, those regions with the highest content of adenine and thymine base-pairs.     

The presence of covalently linked ribonucleotides in the closed circular deoxyribonucleic acid from higher plants.

Single-stranded scissions are induced in the covalently closed circular chloroplast (ct-) DNAs from peas, spinach, and lettuce plants by treatment with alkali or by incubation with a mixture of ribonucleases A and T1. These scissions are due to the presence of covalently linked ribonucleotides in these closed circular DNAs. By comparing the scission rates of these ctDNAs to the scission rate of RNA, it has been estimated that pea and spinach ctDNAs contain a maximum of 18 +/- 2 ribonucleotides/molecule, while lettuce ctDNA contains a maximum of 12 +/- 2 ribonucleotides/molecule. Further studies with pea ctDNA by electron microscopic methods have shown that pea ctDNA contains 19 alkali-labile sites at specific locations. A map of the relative positions of the alkali-labile sites has been constructed. These alkali-labile sites are presumably due to the insertion of individual ribonucleotides.     

Physical studies on the size and structure of the covalently closed circular chloroplast DNA from higher plants.

The size and structure of the covalently closed circular chloroplast DNAs (ctDNA) from pea, lettuce, and spinach plants, have been studied by analytical ultracentrifugation. The values of so20,w,Na+ of the native and denatured forms of the open and closed circular DNAs from these plants have been determined. The absolute molecular weight of purified closed circular pea ctDNA monomers has been determined by buoyant equilibrium sedimentation to be 89.1 (S.D. +/- 0.7)-10(6). The value of the so20,w,Na+ of open circular pea ctDNA and its molecular weight, in conjunction with corresponding values for other sizes of circular DNA, has been used to derive an empirical relationship between so20,w,Na+ and molecular weight for open circular DNAs. Using this relationship, the molecular weights of lettuce and spinach ctDNAs have been determined to be 98.2 (S.D. +/- 1.5)-10(6) and 97.2 (S.D. +/- 1.5)-10(6), respectively. At pH values 12.7 and 13, closed circular lettuce and pea ctDNAs have been found to exist as mixtures of reversibly and irreversibly denatured closed circular DNAs.     

Restriction fragment map of sugar beet (Beta vulgaris L.) chloroplast DNA

Summary A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes SmaI, PstI and PvuII. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In common with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S+16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.     

Changes in mitochondrial DNA levels during development of pea (Pisum sativum L.)

The percentage of mitochondrial DNA (mtDNA) present in total DNA isolated from pea tissues was determined using labeled mtDNA in reassociation kinetics reactions. Embryos contained the highest level of mtDNA, equal to 1.5% of total DNA. This value decreased in light- and dark-grown shoots and leaves, and roots. The lowest value found was in dark-grown shoots; their total DNA contained only 0.3% mtDNA. This may be a reflection of increased nuclear ploidy levels without concomitant mtDNA synthesis. It was possible to compare the mtDNA values directly with previous estimates of the amount of chloroplast DNA (ctDNA) per cell because the same preparations of total DNA were used for both analyses. The embryo contained 1.5% of both mtDNA and ctDNA; this equals 410 copies of mtDNA and 1200 copies of ctDNA per diploid cell. Whereas mtDNA levels decreased to 260 copies in leaf cells of pea, the number of copies of ctDNA increased to 10300. In addition, the levels of ctDNA in first leaves of dark-grown and light-transferred pea were determined, and it was found that leaves of plants maintained in the dark had the same percentage of ctDNA as those transferred to the light.Abbreviations ctDNA chloroplast DNA - mtDNA mitochondrial DNA     

Thermodynamic and structural study of phenanthroline derivative ruthenium complex/DNA interactions: probing partial intercalation and binding properties

The binding of [Ru(PDTA-H₂)(phen)]Cl (PDTA = propylene-1,2-diaminetetra-acetic acid; phen = 1,10 phenanthroline) with ctDNA (=calf thymus DNA) has been investigated through intrinsic and induced circular dichroism, UV-visible absorption and fluorescence spectroscopies, steady-state fluorescence, thermal denaturation technique, viscosity and electrochemical measurements. The latter indicate that the cathodic and anodic peak potentials of the ruthenium complex shift to more positive values on increasing the DNA concentration, this behavior being a direct consequence of the interaction of both the reduced and oxidized form with DNA binding. From spectrophotometric titration experiments, the equilibrium binding constant and the number of monomer units of the polymer involved in the binding of one ruthenium molecule (site size) have been quantified. The intrinsic circular dichroism (CD) spectra show an unwinding and a conformational change of the DNA helix upon interaction of the ruthenium complex. Quenching process, thermal denaturation experiments and induced circular dichroism (ICD) are consistent with a partial intercalative binding mode.     

Interaction of saffron carotenoids as anticancer compounds with ctDNA, Oligo (dG.dC)15, and Oligo (dA.dT)15

Crocin and crocetin are two important natural saffron carotenoids, which, along with dimethylcrocetin (DMC) as a semi-synthetic product, are responsible for its color. Many biological properties of saffron have been reported, among which the anticancer property is the most important. Some anticancer drugs have direct interaction with DNA, and thus the present study attempted to investigate the interaction of three major saffron carotenoids-crocin, crocetin, and DMC--with calf thymus DNA (ctDNA) and oligonucleotides. The spectrophotometric data showed some changes in ctDNA absorption spectra due to the formation of complex with saffron extract and each of these three components. Also, all the three components caused the quenching of the fluorescence emission of ctDNA-ethidium bromide complex. The Scatchard analysis of these data indicated a noncompetitive manner for quenching, which is accompanied by the outside groove-binding pattern. The circular dichroism (CD) spectra also indicated the nonintercalative binding and induction of the conformational changes, and B to C transition in ctDNA structure and then unstacking of ctDNA bases at higher concentrations of the carotenoids. The CD spectra of G.C and A.T oligonucleotides after addition of these carotenoids indicated the transition from B- to C-DNA, which is very similar to the ctDNA spectral changes. The DeltaG(H(2)O), the best parameter for the estimation of macromolecule stability, was determined for ctDNA denaturation using dodecyl trimethylammonium bromide in the absence and presence of crocin, crocetin, or DMC. Our results showed a decrease in the Delta G(H(2)O), indicating the ctDNA destabilization due to its interaction with the mentioned ligands. In conclusion, the results show that saffron and its carotenoids interact with DNA and induce some conformational changes in it. Of these carotenoids, the order of potential of interaction with DNA is crocetin > DMC > crocin.     

Capecitabine as a minor groove binder of DNA: molecular docking,molecular dynamics,and multi-spectroscopic studies

The interaction mechanism and binding mode of capecitabine with ctDNA was extensively investigated using docking and molecular dynamics simulations, fluorescence and circular dichroism (CD) spectroscopy, DNA thermal denaturation studies, and viscosity measurements. The possible binding mode and acting forces on the combination between capecitabine and DNA had been predicted through molecular simulation. Results indicated that capecitabine could relatively locate stably in the G-C base-pairs-rich DNA minor groove by hydrogen bond and several weaker nonbonding forces. Fluorescence spectroscopy and fluorescence lifetime measurements confirmed that the quenching was static caused by ground state complex formation. This phenomenon indicated the formation of a complex between capecitabine and ctDNA. Fluorescence data showed that the binding constants of the complex were approximately 2 × 10⁴ M^?1. Calculated thermodynamic parameters suggested that hydrogen bond was the main force during binding, which were consistent with theoretical results. Moreover, CD spectroscopy, DNA melting studies, and viscosity measurements corroborated a groove binding mode of capecitabine with ctDNA. This binding had no effect on B-DNA conformation.     

Structure of hepatitis B Dane particle DNA before and after the Dane particle DNA polymerase reaction.

DNA isolated from the hepatitis B antigen form known as the Dane particle was examined by electron microscopy before and after the endogenous Dane particle DNA polymerase reaction. The most frequently occurring form was an untwisted circular double-stranded DNA molecule approximately 1 mum in length. Less frequently occurring forms included circular DNA of approximately unit length and having one or more small single-stranded regions, similar circular molecules with one or more tails either shorter or longer than 1 mum in length, and very small circular molecules with tails. There was no increase in frequency or length of tails after a DNA polymerase reaction, suggesting that tails were not formed during this reaction. The mean length of circular molecules increased by 23% when DNA was spread in formamide compared with aqueous spreading, suggesting that single-stranded regions are present in most of the molecules. The mean length of circular molecules obtained from aqueous spreading increased by 27% after a Dane particle DNA polymerase reaction. This indicates that single-stranded regions were converted to double-stranded DNA during the reaction.     

Organization of the large mitochondrial genome in the isopod Armadillidium vulgare.

The mitochondrial DNA (mtDNA) in animals is generally a circular molecule of approximately 15 kb, but there are many exceptions such as linear molecules and larger ones. RFLP studies indicated that the mtDNA in the terrestrial isopod Armadillidium vulgare varied from 20 to 42 kb. This variation depended on the restriction enzyme used, and on the restriction profile generated by a given enzyme. The DNA fragments had characteristic electrophoretic behaviors. Digestions with two endonucleases always generated fewer fragments than expected; denaturation of restriction profiles reduced the size of two bands by half; densitometry indicated that a number of small fragments were present in stoichiometry, which has approximately twice the expected concentration. Finally, hybridization to a 550-bp 16S rDNA probe often revealed two copies of this gene. These results cannot be due to the genetic rearrangements generally invoked to explain large mtDNA. We propose that the large A. vulgare mtDNA is produced by the tripling of a 14-kb monomer with a singular rearrangement: one monomer is linear and the other two form a circular dimer. Densitometry suggested that these two molecular structures were present in different proportions within a single individual. The absence of mutations within the dimers also suggests that replication occurs during the monomer phase.     

Denaturation map of the circular mitochondrial genome of Neurospora crassa.

A denaturation map of mitochondrial DNA from the wild type strain 5256 of Neurospora crassa was constructed by computer analysis of the contour length distribution of single- and double-stranded regions of nineteen circular and three full length linear molecules after partial denaturation. The data suggest that mitochondrial DNA in this strain is a homogeneous population of a circular molecule of molecular weight 41 - 10(6) with an asymmetric distribution of AT-rich regions, and that linear molecules derive from this genome by random breaks during isolation.     

Physical map of the DNA of bacteriophage phiB

A physical map of DNA phi B bacteriophage was constructed. Using the data of denaturation maps and heteroduplex analysis the positions of 10 (from 11) peaks were estimated. These peaks belong to DNA regions obtained after hydrolysis of phi B DNA by EcoRI endonuclease. This map is orientated to the end of the DNA molecule, which first entered the head, during phage maturation. The disposition of AT-enriched regions of the DNA molecule were shown.     

Physical mapping of the pea chloroplast DNA and localization of the ribosomal RNA genes

The closed circular DNA of pea chloroplast has been digested with restriction endonucleases SalI, SmaI, BamHI, XbaI, XhoI, HindIII, and EcoRI. A physical restriction map of pea ctDNA has been constructed by mapping the SalI and SmaI sites. The pea ctDNA has been found to contain one set of ribosomal RNA genes by Southern hybridization of restriction endonuclease digest, R-loop studies, and DNA-DNA heteroduplex mapping. The 23 S and 16 S RNA genes are confined to a DNA region of 3.0 and 1.5 kbp, respectively. The two rRNA chains are separated by a spacer region of 2.2 kbp.     

Locating Interrupted Hydrogen Bonding in the Secondary Structure of PM2 Circular DNA by Comparative Denaturation Mapping

Previous studies with HCHO have revealed a reaction with superhelical DNA that strongly suggests that this DNA consists of small regions of interrupted secondary structure. To map these sites in PM2 DNA, the following set of experiments was performed using electron microscopy. (i) A denaturation map of nicked form II was obtained using Inman's alkaline-HCHO conditions. (ii) The superhelical form I was reacted with HCHO at 30 C until equilibrium was achieved at the interrupted sites (3.6% reactivity). The excess HCHO was removed rapidly and X-ray treatment was employed to nick these prereacted molecules. These form II molecules containing HCHO (form II HCHO) were also subjected to denaturation mapping. It would be expected that the HCHO-unpaired regions would serve as induction sites for the propagation of melting. Hence, depending on the location of the induction sites; we would anticipate either the creation of new regions of melting or a normal denaturation map shifted to lower pH values. Comparison of the development of progressive denaturation of form II and form II HCHO reveals that the latter is the case. The denaturation maps of form II are highly organized patterns of adenine-thymine (AT)-rich regions, with a total of five regions at extreme pH conditions. There are six highly organized regions for form II HCHO, i.e., smaller adjacent loops, at low denaturation conditions where no denaturation is seen for form II. These coalesce into the pattern for form II containing four of five A-T-rich regions observed for form II. Hence we conclude that the regions of altered hydrogen bonding in superhelical PM2 DNA are four to six in number and they map in the A-T-rich regions of the DNA.     

Structure of nonintegrated, circular Herpesvirus saimiri and Herpesvirus ateles genomes in tumor cell lines and in vitro-transformed cells.

Nonintegrated, circular DNA molecules of Herpesvirus saimiri and Herpesvirus ateles were found in five lymphoid cell lines originating from tumor tissues or established by in vitro immortalization of T lymphocytes. The arrangement of unique (L) and repetitive (H) DNA sequences in circular viral genomes was analyzed by partial denaturation mapping followed by visualization with an electron microscope. Three types of circular viral DNA structures were found. (i) The virus-producing cell line RLC, which is derived from an H. ateles-induced rabbit lymphoma, contains circular viral genomes which consist of a single L-DNA and a single H-DNA region, both the same length as in virion DNA. (ii) The circular viral genomes of the nonproducer cell lines H1591 and A1601, in vitro transformed by H. saimiri and H. ateles, respectively, have deletions in the unique L-DNA region and larger H-DNA regions. Cell line A1601 lacks about 8% of virion L-DNA, and H1591 cells lack about 40% of viral L-DNA information. (iii) The nonproducing H. saimiri tumor cell lines 1670 and 70N2 harbor viral genomes with two L-DNA and two H-DNA regions, respectively. Both types of circular molecules have a long and a short L-segment. The sequence arrangements of circular DNA molecules from H. saimiri-transformed cell lines were compared with those of linear virion DNA by computer alignment of partial denaturation histograms. The L-DNA deletion in cell line H1591 was found to map in the right half of the virion DNA. Comparison of the denaturation patterns of both L regions of cell lines 1670 and 70N2 identified the short L regions as subsets of the long L regions. Thus, circular viral DNA molecules of all four nonproducer cell lines represent defective genomes.     

Characterization of the Pea Chloroplast DNA OriA Region

One of the two origins of replication in pea chloroplast DNA (oriA) maps in the rRNA spacer region downstream of the 16S rRNA gene, and further characterization of this origin is presented here. End-labeling of nascent DNA strands from in vivo replicating ctDNA was used to generate probes for Southern hybridization. Hybridization data identified the same region that was previously mapped to contain D-loops by electron microscopy. Subclones of the ori A region were tested for their ability to support in vitro DNA replication using a partially purified pea ctDNA replication system. Two-dimensional agarose gel electrophoresis identified replication intermediates for clones from the region just downstream of the 16S rRNA gene, with a 450-bp SacI-EcoRI clone showing the strongest activity. The experiments presented in this paper identify the 940 base pair region in the rRNA spacer between the 3′ end of the 16S rRNA gene and the Eco RI site as containing oriA. Previous studies by electron microscopy localized the D-loop in the spacer region just to the right of the Bam HI site, but the experiments presented here show that sequences to the left of the BamHI site are required for replication initiation from ori A. DNA sequence analysis of this region of pea ctDNA shows the presence of characteristic elements of DNA replication origins, including several direct and inverted repeat sequences, an A + T rich region, and dna A-like binding sites, most of which are unique to the pea ctDNA ori A region when compared with published rRNA spacer sequences from other chloroplast genomes.