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.     

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...     

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.     

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.     

Isolation and physical studies of the intact supercoiled, the open circular and the linear forms of ColE1-plasmid DNA.

For the study of DNA conformations, conformational transitions, and DNA-protein interactions, covalently closed supercoiled ColE1-plasmid DNA has been purified from cultures of Escherichia coli harboring this plasmid and grown in the presence of chloramphenicol according to the method of D.B. Clewell [J. Bact. 110 (1972)667]. The open circular and linear forms of the plasmid were prepared by digestion of the covalently closed, supercoiled form with pancreatic deoxyribonuclease and EcoRI-restriction endonuclease, respectively. The linear form was found to be very homogeneous by electron microscopy and sedimenting boundary analysis. Its physical properties (s0 20,w=16.3 S,D0 20,W=1.98 X 10(-8) cm2 s-1 and [eta]=2605 ml g-1) have been carefully determined in 0.2 M NaCl, 0.002 M NaPO4 pH 7.0,0.002 M EDTA, at 23 degrees C. Combination of s0 20, w (obtained by quasielastic laser light scattering) gave Ms,D=4.39 x 10(6). This value is in reasonable agreement with the molecular weight from total intensity laser light scattering M=4.30 x 10(6). The covalently closed and open circular forms of the ColE1-plasmid are less homogeneous due to slight cross-contamination and the presence of small amounts of dimers in these preparations. The weight fractions of the various components as determined by boundary analysis or electron microscopy are given together with the average quantities obtained in the same solvent for the supercoiled form ((s0 20,w)w=25.4 S, (D0 20,w)z=2.89 x 10(-8) cm2 s-1, [eta]= 788 ML G-1,Ms,D=4.69 x 10(6) and Mw=4.59 x 10(6)) and the open circular form (s0 20, w)w=20.1 S, (D0 20,w)z=2.45 x 10(-8) cm2 s-1, [eta]=1421 ml g-1,Ms,D=4.37 x 10(6) and Mw=4.15 x 10(6)). Midpoint analysis of the sedimenting boundaries allows unambiguous determination of the sedimentation coefficients of these two forms: s0 20,w=24.5 S and s0 20,w=18.8 S, respectively. Also deduced from total intensity light scattering were radii of gyration Rg (103.5, 134.2 and 186 nm) and second virial coefficients A2 (0.7, 4.8 AND 5.4 x 10(-4) mole ml/g2) for the supercoiled, the open circular and linear forms, respectively. The data presented are discussed in relation to the conformational parameters for the three forms in solution.     

Ethidium bromide-mediated renaturation of denatured closed circular DNAs: mechanistic aspects and fractionation of DNA on a molecular weight basis

When closed circular duplex DNAs are exposed to alkali in the presence of ethidium bromide, from 0 to 100% of the DNA can be recovered as the fully base-paired duplex (native) form upon neutralization of the solutions. The fraction of native DNA depends on the concentration of ethidium bromide, time of incubation, ionic strength and temperature of the solutions before neutralization as well as the molecular weight and superhelix density of the DNA. Limiting ethidium concentrations exist below and above which 0 and 100% of the DNA, respectively, is recovered as native material under a given set of incubation conditions regardless of the length of time of incubation before neutralization. The strong molecular weight dependence of the fraction of DNA recovered in the native form after a given time of pre-neutralization incubation at ethidium concentrations between the limiting values noted above allows larger DNAs to remain fully denatured upon neutralization while smaller DNAs in the same mixture are fully renatured. This permits the rapid fractionation of mixtures of closed duplex DNAs on the basis of molecular weight when a technique for the separation of denatured from fully base-paired DNA is applied to such mixtures. Such a separation has been demonstrated through the marked enrichment of plasmid cloning vector DNA containing cloned inserts in the fractions that remain denatured after neutralization of alkaline solutions of these DNAs containing ethidium bromide.     

Divergence of tRNA genes in chloroplast DNA of higher plants

The saturation hybridization between spinach chloroplast (ct) DNA and spinach ¹²⁵I-labelled chloroplast tRNA has shown that about 1.1% of the spinach ctDNA codes for tRNAs. The observed hybridization is a result of specific base-pairing as shown by competition hybridization experiments and thermal stability of the ctDNA-tRNA hybrids. The amount of hybridization shows that spinach ctDNA contains about 40 tRNA genes. Similar hybridization studies have shown that corn ctDNA contains about 28 tRNA genes. The cross-hybridizations between ctDNA and tRNAs of corn, spinach and pea have shown that tRNAs in chloroplasts of higher plants have undergone significant divergence. The pea and spinach tRNAs have been found to have 50% of the base sequences in common. The corn tRNAs have been found to have only about 30% of the base sequences in common with pea and spinach. These data have been confirmed by extensive heterologous competition experiments and thermal stability of the heterologous DNA-tRNA hybrids. The experiments have also shown that the base sequences of tRNAs common in all three plants are the same.     

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.     

Isciatica and Observation of Chloroplast DNA from the Rape and Tobacco

Chloroplast DNA (ctDNA)from the rape (Brassica napus) and tobacco (Nicotiana tabacum) has been isolated using a intact pure chloroplast lysis methed followed by discontinute sucrose gradient centrifugation and DNase treatment. Our electron microscopic observation on the chloroplast DNAs revealed clearly flowerlike configurations made up of many “petal-loops” linked together in the middle to forming one “central-loop”. The number of petal-loops per molecule varies from 5 to 50, usually about 7 or 8. The size of the petal-loops is not much different, being 1.52 ± 0.48μm and 1.28 ± 0.37μm in average length for rape and tobacco respactively. Some petal-loops appear attached to membrane protein, indicating the possibility that ctDNAs may have a similar organization as chromosome. Besides, some configurations are quite large with a total of over 50 petal-loops including 2 or 3 molecules linked together by petal-loops, and some rather small ones with a single circular molecule, the size of which is about to 1–2 petal-loops in length. Such variation in the size of ctDNA may suggest the possibility of hithly organized internal molecular arragement of the ctDNA and occurrence of intramolecular of intermolecular recombination.     

Denaturation mapping studies on the circular chloroplast deoxyribonucleic acid from pea leaves.

The structure of circular pea chloroplast DNA (ctDNA) has been analyzed by denaturation mapping. All of the pea ctDNA molecules that were examined had identical gross base sequences. Denaturation maps were constructed at denaturation levels of 2.5%, 22%, and 44%. These denaturation maps showed that the circular pea ctDNA contained six small AT-rich regions on one-half of the DNA molecule, and two small GC-rich regions on the other half of the DNA molecule. The structure of pea ctDNA circular dimers was also examined. The results showed that the pea ctDNA circular dimers consisted of two monomer length units integrated in tandem repeat.     

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.     

Nuclear and chloroplast DNA differentiation in Andean potatoes.

Over 3500 accessions of Andean landraces have been known in potato, classified into 7 cultivated species ranging from 2x to 5x (Hawkes 1990). Chloroplast DNA (ctDNA), distinguished into T, W, C, S, and A types, showed extensive overlaps in their frequencies among cultivated species and between cultivated and putative ancestral wild species. In this study, 76 accessions of cultivated and 19 accessions of wild species were evaluated for ctDNA types and examined by ctDNA high-resolution markers (ctDNA microsatellites and H3 marker) and nuclear DNA restriction fragment length polymorphisms (RFLPs). ctDNA high-resolution markers identified 25 different ctDNA haplotypes. The S- and A-type ctDNAs were discriminated as unique haplotypes from 12 haplotypes having C-type ctDNA and T-type ctDNA from 10 haplotypes having W-type ctDNA. Differences among ctDNA types were strongly correlated with those of ctDNA high-resolution markers (r = 0.822). Differentiation between W-type ctDNA and C-, S-, and A-type ctDNAs was supported by nDNA RFLPs in most species except for those of recent or immediate hybrid origin. However, differentiation among C-, S-, and A-type ctDNAs was not clearly supported by nDNA RFLPs, suggesting that frequent genetic exchange occurred among them and (or) they shared the same gene pool owing to common ancestry.     

Efficiency of one- and two-stage selection indexes for improvement of net economic merit in White Leghorns

Summary Mitochondrial (mt) and chloroplast (ct) DNAs from sugar beet lines carrying normal and introduced sources of male sterile cytoplasms have been characterized and compared on the basis of restriction enzyme analysis. Normal cytoplasm was shown to contain mt and ctDNAs which differed from those of the male sterile cytoplasms examined in the present investigation. On the other hand, four groups of male sterile cytoplasms could be differentiated by their own characteristic mtDNA digest patterns, while two were separated by ctDNA comparisons. In addition, a greater degree of variability of the mitochondrial genome is suggested. Our results also imply strict maternal inheritance of mt and ctDNAs. Thus, the organelle DNA assay provides a positive and alternative means of identifying various male sterile cytoplasms.     

Variations of chloroplast DNAs in the genus Pelargonium and their biparental inheritance

Summary The comparison of EcoRI patterns of chloroplast DNAs (ctDNAs) from five species of the genus Pelargonium and from 16 cultivars and varieties of Pelargonium zonale hort. demonstrates a remarkable inter- and intraspecific ctDNA (plastome) variation. The plastome of the P. zonale varieties could be differentiated into groups I, II and III. Reasons for this variation seem to be: occurrence of numerous spontaneous plastome mutations, intense hybridisation by gardeners and breeders, and biparental plastid inheritance.Crosses of P. zonale varieties with different ctDNA types lead to the direct evidence on the molecular level of biparental plastid inheritance and plastid sorting-out in F₁-hybrids.     

An adenosine triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Improved purification, subunit structure and substrate specificity

The ATP-dependent deoxyribonuclease from Bacillus laterosporus has been purified to near homogeneity by a procedure involving ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex A-25 chromatography and DNA-cellulose affinity chromatography. The purified enzyme has a molecular weight of 210,000 +/- 8,000 as determined by sucrose gradient sedimentation. It is composed of two nonidentical polypeptide chains with close molecular weights of around 110,000. The substrate preference of the pure enzyme is essentially identical with the previous result obtained with the partially purified enzyme preparation (Anai, M., Mihara, T., Yamanaka, M., Shibata, T., & Takagi, Y. (1975) J. Biochem. 78, 105-114). Thus, the enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of ATP. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of ATP. Furthermore, no endonuclease activity is observed on covalently closed circular duplex DNA and open circular duplex DNA.     

Chloroplast DNA variation between the common cultivated potato (Solanum tuberosum ssp. tuberosum) and several South American relatives

Summary Chloroplast DNA (ctDNA) from the tuberbearing Solanum species tuberosum, vernei, phureja, and chacoense has been compared by restriction endonuclease analysis. Digestion by Hind III or Xba I reveal no differences, but digestion with Bam HI and Eco RI reveals minor differences in the ctDNA among these species. The ctDNA restriction patterns of the tetraploid common cultivated potato of North America and Europe, S. tuberosum ssp. tuberosum and the South American tetraploid, S. tuberosum ssp. andigena are identical for all four restriction endonucleases. These data suggest that ssp. tuberosum and ssp. andigena contain similar ctDNA and therefore may share a common ancestor, or direct lineage. The ctDNA restriction patterns of S. vernei and S. chacoense are identical for all four restriction endonucleases, and S. phureja ctDNA, can be distinguished from the other diploid ctDNAs by digestion with Bam HI. None of the diploids analyzed contain ctDNA identical to the tetraploids and therefore either did not contribute their chloroplast genomes to the evolution of the tetraploids, or the ctDNA has diverged since this evolutionary event. The ctDNAs studied did not contain restriction polymorphisms which could be correlated to cytoplasmic male sterility in Solanum. This is the first demonstration of ctDNA diversity in the tuber-bearing Solanum species.     

Circulating Tumor DNA as Biomarkers for Cancer Detection

Detection of circulating tumor DNAs (ctDNAs) in cancer patients is an important component of cancer precision medicine ctDNAs. Compared to the traditional physical and biochemical methods, blood-based ctDNA detection offers a non-invasive and easily accessible way for cancer diagnosis, prognostic determination, and guidance for treatment. While studies on this topic are currently underway, clinical translation of ctDNA detection in various types of cancers has been attracting much attention, due to the great potential of ctDNA as blood-based biomarkers for early diagnosis and treatment of cancers. ctDNAs are detected and tracked primarily based on tumor-related genetic and epigenetic alterations. In this article, we reviewed the available studies on ctDNA detection and described the representative methods. We also discussed the current understanding of ctDNAs in cancer patients and their availability as potential biomarkers for clinical purposes. Considering the progress made and challenges involved in accurate detection of specific cell-free nucleic acids, ctDNAs hold promise to serve as biomarkers for cancer patients, and further validation is needed prior to their broad clinical use.     

Isolation and preliminary characterization of the small circular DNA present in African green monkey kidney (BSC-1) cells

The small polydisperse circular DNA (spc-DNA) previously identified in SV40-infected African green monkey kidney (BSC-1) cells (M. G. Rush, R. Eason, and J. Vinograd, 1971, Biochim. Biophys. Acta 228, 585–594.) has been isolated in pure form from uninfected cells. This double-stranded, covalently closed circular DNA contains species ranging in molecular weight from about 0.1 to 4 × 10⁶, although most of the molecules are distributed in an apparently polydisperse population with molecular weights of less than 1 × 10⁶. There are approximately 1000 to 2000 covalently closed small DNA molecules per cell, and their average buoyant density does not appear to differ significantly from that of chromosomal and mitochondrial DNAs. This spc-DNA was resolved by polyacrylamide gel electrophoresis into three distinct bands containing comparatively homogeneous circular DNAs with molecular weights of 200,000, 520,000, and 780,000. However, the reassociation rate of in vitro labeled, denatured spc-DNA suggested a molecular complexity in the range of 1 × 10⁸, and the ability of BSC-1 chromosomal DNA to accelerate greatly the reassociation of about one third of this material indicated the presence of some repetitive chromosomal DNA sequences in spc-DNA.     

Clone bank and physical and genetic map of potato chloroplast DNA

Summary Clone banks of PvuII, BamHI and XhoI fragments were generated of the Solanum tuberosum cv Katahdin plastome. These clone banks, in conjunction with molecular hybridization to tobacco ctDNA probes, were used to construct a physical map of potato ctDNA. The potato plastome was found to be a circular molecule of 155–156 Kbp containing two inverted repeat regions of 23–27 Kbp. The arrangement of restriction sites is very similar to that of other Solanaceae plastomes. Heterologous hybridization to known ctDNA encoded gene probes from tobacco allowed us to establish a genetic map of the potato chloroplast genome. The arrangement of these genes on the potato plastome resembles that on most higher plant ctDNAs.     

Physical properties and gel electrophoresis behavior of R12-derived plasmid DNAs.

A series of closed circular (I) plasmid DNAs has been derived from drug resistance factor R12, and the nicked circular (II) and linear (III) derivatives of these molecules prepared by irradiation in the presence of ethidium bromide and by treatment with restriction enzyme EcoRI, respectively. These DNAs encompass the molecular weight range 3.6 to 61 megadaltons. The base compositions range from 45% to 51% (GC) as estimated by buoyant density determinations. The smaller plasmids are significantly less supercoiled (9-10%) than are the larger (12-13%). The gel electrophoretic behavior of the three DNA structural forms was determined as a function of molecular weight in agarose gels of concentrations ranging from 0.7% to 1.6% and at electrophoresis salt concentrations from 0.02 M to 0.08 M sodium acetate. The mobilities of DNAs I and III undergo a reversal relative to each other at a molecular weight which decreases with increasing agarose gel concentration. The molecular weight at which DNA II fails to enter a gel depends upon the ionic strength during electrophoresis but not upon the gel concentration.