Activation of DNA strand exchange by cationic comb-type copolymers: effect of cationic moieties of the copolymers

We have previously reported that poly(l-lysine)-graft-dextran cationic comb-type copolymers accelerate strand exchange reaction between duplex DNA and its complementary single strand by >4 orders of magnitude, while stabilizing duplex. However, the stabilization of the duplex is considered principally unfavourable for the accelerating activity since the strand exchange reaction requires, at least, partial melting of the initial duplex. Here we report the effects of different cationic moieties of cationic comb-type copolymers on the accelerating activity. The copolymer having guanidino groups exhibited markedly higher accelerating effect on strand exchange reactions than that having primary amino groups. The high accelerating effect of the former is considered to be due to its lower stabilizing effect on duplex DNA, resulting from its increased affinity to single-stranded DNA. The difference in affinity was clearly demonstrated by a fluorescence correlation spectroscopy study; the interaction of the former with single-stranded DNA still remained high even at 1 M NaCl, while that of the latter completely disappeared. These results suggest that some modes of interactions, such as hydrogen bonding, other than electrostatic interactions between the copolymers having guanidino groups and DNAs may be involved in strand exchange activation.     

Acceleration of DNA strand exchange by polycation comb-type copolymer.

Polycation comb-type copolymers which are composed of poly(L-lysine) backbone and dextran graft chain (PLL-graft-Dex) accelerated DNA duplex and triplex formation and stabilized under physiologically relevant condition remarkably. In this study, we have examined the ability of polycation copolymer in promoting strand exchange between duplex DNA and its complementary single-stranded DNA. It was demonstrated that the strand exchange rate was considerably accelerated by the polycation comb-type copolymer.     

Effect of Cationic Comb-Type Copolymer on Quadruplex Folding of Human Telomeric DNA

Cationic comb-type copolymer (CCC) consisting of a polycationic backbone and abundant graft water-soluble chains exhibited considerable stabilization effect on DNA hybrids, such as double- and triple-stranded DNAs. Here, we describe the effect of CCC on antiparallel G-quadruplex folding of human telomeric DNA, d(GGGTTA) _n in the presence of sodium ions. CCC did not significantly alter the circular dichroism (CD) spectra of d((GGGTTA) ₃ GGG) and d((GGGTTA)₇GGG) indicating that the CCC did not influence the antiparallel folding of the telomeric repeats. Hence, the ionic interaction of CCC with the DNA sequence did not interfere with specific interaction of the DNA with sodium ions to form G-quartets. Interestingly, CCC did not change the melting temperature of the d((GGGTTA) ₃ GGG) suggesting negligible stabilizing effect of CCC on the antiparallel quadruplex structure.     

Triplex formation using ODN conjugates with polycation comb-type copolymer.

Polycation comb-type copolymer that is composed of polylysine backbone and dextran side chains (PLL-g-Dex) has previously been shown to stabilize duplex and triplex DNAs quite effectively. In this study, we have conjugated PLL-g-Dex with oligonucleotides (ODN) aiming to increase the triplex stabilizing efficiency of the copolymer. Here we have demonstrated that the copolymer-TFO conjugates selectively stabilize triplex DNA. Also its potential to form triplex DNA was found to be greater than PLL-g-Dex/ODN mixture.     

Synthesis of 3-hydroxybutyrate-CO-4-hydroxybutyrate copolymers by hydrogen-oxidizing bacteria

Synthesis of 3- and 4-hydroxybutyrate copolymer (3HB-co-4HB), the most promising member of the biodegradable polyhydroxyalkanoate (PHA) family, has been studied. Cultivation conditions of naturally occurring strains of hydrogen-oxidizing bacteria Ralstonia eutropha B5786 and Cupriavidus eutrophus B10646 have been optimized to ensure efficient synthesis of the 3HB-co-4HB copolymer. A set of highly pure samples of the 3HB-co-4HB copolymer with 4HB content varying from 8.7 to 24.3 mol% has been obtained. Incorporation of 4HB into the copolymer was shown to cause a more pronounced decrease in polymer crystallinity than the incorporation of 3-hydroxyvalerate or 3-hydroxyhexanoate; samples with a degree of crystallinity below 30% have been obtained. The weight average molecular mass of the 3HB-co-4HB copolymers was shown to be independent on the monomer ratio and to vary broadly (from 540 to 1110 kDa).     

蛹虫草线粒体DNA与细胞核DNA进化关系的比较

【目的】分析蛹虫草是否存在核内线粒体DNA片段,比较蛹虫草线粒体DNA与细胞核DNA的碱基变异程度及所反映的菌株间的系统发育关系。【方法】通过本地BLAST或LAST对蛹虫草线粒体基因组和核基因组进行序列相似性搜索;从10个已知线粒体基因组的蛹虫草菌株中分别扩增7个细胞核蛋白编码基因片段,并与其在14个线粒体蛋白编码基因上的碱基变异情况进行比较。【结果】蛹虫草核基因组中存在5处较短的核内线粒体DNA片段,总长只有278bp。蛹虫草核DNA的变异频率整体上高于线粒体DNA。核DNA和线粒体DNA所反映的蛹虫草菌株间的系统发育关系存在显著差异。【结论】蛹虫草线粒体DNA与核DNA间不存在长片段的基因交流,二者变异频率不同,所反映的蛹虫草菌株间的系统发育关系也有差异。本研究增加了对蛹虫草线粒体与细胞核DNA进化关系的认识。     

Single-stranded DNA versus tailed duplex in sequence conversion of lacZα DNA

Abstract

Targeted DNA editing has great potential to cure some genetic diseases; however, the use of artificial nucleases such as CRISPR-Cas9 and TALEN in gene therapy can potentially cause severe side effects due to off-target DNA cleavages. Single-stranded (ss) DNAs and 5'-tailed duplexes (TDs) can achieve target base substitutions when introduced without artificial nucleases into cultured cells and mouse liver. In this study, ss DNA and TD were separately co-introduced into human U2OS cells, together with a target plasmid DNA bearing an inactivated lacZα gene, and the gene correction efficiencies were compared. Unlike the genes examined in previous studies, ss DNA and TD showed similar efficiencies. Therefore, ss DNAs might be as useful as TD for gene correction, depending on the target sequence.     

Preparation and evaluation of ODN conjugates with polycation comb-type copolymer.

Oligodeoxynucleotide (ODN) conjugates with the polylysine comb-type copolymer having an ability to promote and stabilize duplex and triplex DNA formation were prepared. 5'-Aminated ODN was succinylated with succinic anhydride. The resulting ODNs having carboxyl terminus were coupled with epsilon-amino groups of the comb-type copolymer using water soluble carbodiimide. The conjugate free from unconjugated ODNs was obtained by gel permeation chromatography. The resulting conjugate maintains ability to form duplex and triplex DNA as estimated by melting curve analysis. Both specificity and stability of the triplex DNA formation were increased by employing the ODN-copolymer conjugates compared to those with their mixture.     

Basic polypeptides as histone models. Synthesis, conformation, and interaction with DNA of statistical copolymers (Lys x,Ala y)n.

Statistical copolymers (Lys^x,Ala^y)_n were synthesized by copolymerization of N-carboxyanhydrides of L -amino acids. The conformation of copolymers in aqueous solutions was investigated using circular dichroism (CD). Calculations based on the CD data showed that polymers (Lys^x,Ala^y)_n can exhibit a random conformation, an α-helix, and a β-structure in various ratios. CD spectra of complexes of copolymers with DNA prepared by gradual dialysis from a high ionic strength to 0.15 M NaCl can be correlated with the copolymer conformation in medium and high ionic strength. For copolymers forming an α-helix and β-structure, these spectra show resemblance with similar spectra of complexes of those histones that are able to exhibit ordered conformations.     

Relative Effects of Graft Copolymer and Polyamines on Triplex Stabilization Under Physiological Conditions

Abstract

Triplex-stabilizing effect of a graft copolymer under physiologically relevant conditions has been evaluated and compared with other polyamines. Here we show that the graft copolymer significantly stabilizes triplex DNAs with amazingly higher efficicacy than that of physiological concentrations of spermine and spermidine.     

Effect of cationic comb-type copolymer on quadruplex folding of human telomeric DNA

Cationic comb-type copolymer (CCC) consisting of a polycationic backbone and abundant graft water-soluble chains exhibited considerable stabilization effect on DNA hybrids, such as double- and triple-stranded DNAs. Here, we describe the effect of CCC on antiparallel G-quadruplex folding of human telomeric DNA, d(GGGTTA)(n) in the presence of sodium ions. CCC did not significantly alter the circular dichroism (CD) spectra of d((GGGTTA)(3)GGG) and d((GGGTTA)(7)GGG) indicating that the CCC did not influence the antiparallel folding of the telomeric repeats. Hence, the ionic interaction of CCC with the DNA sequence did not interfere with specific interaction of the DNA with sodium ions to form G-quartets. Interestingly, CCC did not change the melting temperature of the d((GGGTTA)(3)GGG) suggesting negligible stabilizing effect of CCC on the antiparallel quadruplex structure.     

Molecular encapsulation of berberine by a modified β-cyclodextrin and binding of host: guest complex to G-quadruplex DNA

Abstract

The capacity to control quadruplex formation, especially in cancer cells, is captivating and entails a reasonable comprehension of the ligand-G-quadruplex binding. Herein, we report an iminopyrenyl-β-cyclodextrin conjugate interacting with duplex and G-quadrulex DNAs. In addition, the host: guest association of the established G-quadruplex binder, berberine, with the β-cyclodextrin derivative is studied employing 2-D ROESY. NMR, UV-visible, and fluorescence spectroscopic techniques are utilized to explore the β-cyclodextrin conjugate's interaction with the quadruplexes. The Binding constants are accounted for the association of the ligands to each of the DNAs viz., calf thymus DNA (duplex), kit22, telo24, and myc22 (quadruplexes). The modulation of the iminopyrenyl-β-cyclodextrin binding to the DNAs are observed when berberine is loaded in the host molecule. A vivid distinction between the interactions of the ligands with duplex and quadruplex structures is inferred. Berberine-loaded iminopyrenyl-β-cyclodextrin shows a higher affinity for binding to kit22.     

No enhancing effects of plasmid-specific histone acetyltransferase recruitment system on transgene expression in vivo

Abstract

Altered levels of histone acetylation are associated with changes in chromosomal gene expression. Thus, the specific acetylation of histones bound to plasmid DNA might increase transgene expression. Previously, the expression of the histone acetyltransferase domain of CREB-binding protein fused to the sequence-dependent DNA binding domain of GAL4 (GAL4-HAT) successfully improved reporter gene expression in cultured cells [J. Biosci. Bioengng. 123, 277–280 (2017)]. In this study, the same approach was applied for transgene expression in mice. The activator and reporter plasmid DNAs bearing the genes for GAL4-HAT and Gaussia princeps luciferase, respectively, were co-administered into the mouse liver by hydrodynamics-based tail vein injection, and the Gaussia luciferase activity in serum was measured for two weeks. Unexpectedly, the co-injection of the GAL4-HAT and luciferase plasmid DNAs seemed to decrease, rather than increase, luciferase expression. Moreover, the co-injection apparently reduced the amount of luciferase DNA in the liver. These results indicated that this system is ineffective in vivo and suggested the exclusion of hepatic cells expressing GAL4-HAT.     

Subcellular localization of minicircle DNA in the dinoflagellate Amphidinium massartii

Peridinin‐containing dinoflagellates have small circular DNA molecules called minicircle DNAs, each of which encodes one, or occasionally a few, plastid proteins or ribosomal RNA. Dinoflagellate minicircle DNA is composed of two parts: a gene‐coding sequence and a non‐coding sequence that consists of several variable and core regions. The core regions are identical among the minicircle DNAs with different genes within a species or strain. Because such structure is very different from those of well known plastid DNAs, many functional and evolutionary questions have been raised for the minicircle DNAs, and several studies that focus on answering those questions are underway. However, the localization of minicircle DNA is still controversial: several lines of indirect evidence have implied plastid localization, whereas the nuclear localization of minicircle DNA has also been suggested in a species. In order to understand the evolution and function of minicircle DNA, it is important to know its precise localization. In this study, we sequenced two typical minicircle DNAs, one encodes psbA and the other encodes 23S rRNA genes, from an Amphidinium massartii strain (TM16). To determine the subcellular localization of these minicircle DNAs, we performed DNA‐targeted whole cell fluorescence in situ hybridization with A. massartii minicircle DNA‐specific probes and demonstrated that minicircle DNAs were present in plastids. This study provides the first direct evidence for the plastid localization of dinoflagellate minicircle DNAs.     

Enhanced production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) copolymer with manipulated variables and its properties

Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M _n) of copolymers ranged from 260 × 10³ to 590 × 10³Da, and the polydispersities (M _w/M _n) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T _m), glass transition temperature (T _g), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.     

The Affinity of DNA-Microtubule Protein Complexes and their Disruption by Tubulin Binding Drugs

Abstract

Using the gel shift assay system, we have measured the apparent affinity constant for the interaction of two different DNAs with MAP proteins found in both total calf brain microtubules and heat stable brain preparations. Both DNAs studied contained centromere/kinetochore sequences- one was enriched in the calf satellite DNA; the other was a large restriction fragment containing the yeast CEN11 DNA sequence. Complexes formed using both DNAs had similar K_app values in the range of 2.1×10⁷ M^?1 to 2.0×10⁸ M^?1. CEN11 DNA-MTP complexes had by far the highest K_app value of 2.0×10⁸ M^?1. The CEN11 DNA sequence is where the yeast kinetochore of chromosome 11 is formed and where the single yeast microtubule is bound in vivo. The CEN11 conserved region II known binding sites -(dA/dT)n runs- for mammalian MAP2 protein, are in good agreement with this higher K_app value. The effects of the classical tubulin binding drugs colchicine, podophyllotoxin and vinblastine on the DNA-MAP protein complex stability were investigated by determining the drug concentrations where the complexes were destabilized. Only the complexes formed from total microtubule protein (tubulin containing) were destabilized over a wide drug concentration range. Heat stable brain protein complexes (no tubulin) were largely unaffected. Furthermore, it took 10–100 fold higher drug concentrations to disrupt the CEN 11 DNA complexes compared to the calf thymus satellite DNA enriched complexes. These data support our previous results suggesting that there is a DNA sequence dependent interaction with MAP proteins that appears to be conserved in evolution (Marx et. al., Biochim. Biophys. Acta. 783, 383–392,1984; Marx and Denial, Molecular Basis of Cancer 172B,65-15 1985). In addition, these results imply that the classical tubulin binding drugs may exert their biological effects in cells at least in part by disrupting DNA-Protein complexes of the type we have studied here.     

Plasmid-like DNAs associated with mitochondria of cytoplasmic male-sterile Sorghum

Summary Plasmid-like, linear DNAs were detected in preparations of mitochondrial DNA from cytoplasmic male-sterile sorghum. Designated N-1 and N-2, the DNAs exhibited molecular sizes of ca. 5,700 and 5,300 bp, respectively. The DNAs occurred in only the IS1112C entry among 24 entries examined. Electron microscopy of the DNAs indicated that the molecules were linear as isolated. Nick translation of N-1 and N-2 followed by membrane hybridization indicated substantial homology between the two DNAs, and some homology to the S-1 and S-2 maize plasmid-like DNAs. At least four additional DNA species, ranging from ca. 1,000–4,000 bp if linear, were also detected in sorghum mitochondrial DNA. The detection of plasmid-like DNAs in sorghum, with homology to the plasmid-like DNAs of maize, suggests an etiological relationship of the molecules to the inheritance and expression of cytoplasmic male sterility in sorghum, perhaps in a manner analogous to the postulated role of these elements in maize.Cooperative Investigations of Agricultural Research Service, U.S. Department of Agriculture, Institute of Food and Agricultural Sciences, University of Florida, Texas Agricultural Experiment Station, and Department of Genetics, North Carolina State University. Florida Experiment Stations Journal Series No. 3578     

Homologies of repetitive DNA sequences among Crustacea

The interrelationships of a number of Crustacea were measured by nucleic acid hybridization techniques, with special emphas is on the question of whether GC-rich satellite DNA contains nucleotide sequences homologous to sequences found in other Crustacea with and without similar satellite DNAs. Repetitious sequences from both main-band DNA and GC-rich satellite DNA from the land crab, Gecarcinus lateralis, were hybridized to the total DNAs of crustaceans ranging from the brine shrimp (Subclass: Branchiopoda) to the North American lobster (Homarus americanus, Subclass: Malacostraca; Suborder: Repantia; Section: Macrura) and the true crabs (Subclass: Malacostraca; Suborder: Reptantia; Section: Brachyura). Approximately half of the Gecarcinus repetitious main-band DNA sequences were found to be represented in the DNA of the other true crabs, while a lesser but still significant amount of homology (5 to 10%) to the GC-rich satellite DNA was observed. We also observed a significant amount of homology of the Gecarcinus GC-rich satellite to other crustacean DNAs, even at the level of a different taxonomic Section. This is the first observation of hybrid formation between a purified satellite and DNAs from other organisms under stringent hybridization conditions.Research sponsored by the U.S. Atomic Energy Commission under contact with the Union Carbide Corporation.Research performed while an Oak Ridge Graduate Fellow under appointment from the Oak Ridge Associated Universities in partial fulfillment of the Ph. D. degree from the University of Tennessee, Knoxville, Tennessee.     

Centromeric DNA from Saccharomyces uvarum is functional in Saccharomyces cerevisiae

Previous comparisons of centromeric DNA sequences in laboratory strains of Saccharomyces cerevisiae have revealed conserved sequences within 120 base pairs (bp) which appear to be essential for centromere function. We wanted to find out whether centromeric DNA in Saccharomyces strains with different degrees of DNA sequence divergence carry the same conserved sequences or not. Bam HI DNA fragments from two S. cerevisiae strains and one Saccharomyces uvarum strain were cloned into a centromere selection vector and tested for centromere function in a S. cerevisiae laboratory strain. Fragments having centromere function were obtained at approximately equal frequencies from all three strains. Two of the S. uvarum centromeric DNAs and two of the S. cerevisiae centromeric DNAs were sequenced and shown to carry in a 120 bp region sequences essentially like those of centromeric DNA in S. cerevisiae laboratory strains. DNA hybridization to separated chromosomal DNAs revealed that the two newly determined S. cerevisiae centromeric DNA sequences belong to chromosomes V and XIII, respectively. On leave from: Department of Cell and Tumor Biology, Roswell Park Memorial Institute, Buffalo, NY 14263, USA; On leave from: The Biological Laboratories, University of Leiden, The Netherlands     

Selective allele loss and interference between cauliflower mosaic virus DNAs

Summary Some cauliflower mosaic virus (CaMV) alleles are selectively lost during growth of the virus in mixedly infected turnip plants. Viral DNA from plants co-inoculated with DNA of the cabbage S isolate and infectious cabbage S DNA with an extra EcoRI restriciion site lacked the extra site. The EcoRI allele was also lost in most plants co-inoculated with a non-infectious mutant of cabbage S DNA while little selective allele loss was observed with two other non-infectious mutant DNAs. Plants co-inoculated with DNAs of closely-related isolates (CM4-184 and W) contained both parental viral DNAs and some DNAs with characteristics of both parents. Interference, scored as a reduced frequency of infection or a delay in symptom appearance relative to plants inoculated with wild-type DNA, occurred when plants were inoculated with wild-type and mutant DNAs covalently attached to one another in partial dimer plasmid DNAs. Similarities in the conditions leading to selective allele loss and those leading to interference suggest that both may have been due to active gene conversion between CaMV DNA molecules.