Electron microscopy of supercoiled pEJ4 DNA containing homopurine.homopyrimidine sequences

Supercoiled pEJ4 DNA (a derivative of pUC19 containing an insert with 60-bp-long homopurine.homopyrimidine tract from the sea urchin P. miliaris histone gene spacer) was investigated by electron microscopy using three different spreading techniques i.e., formamide and aqueous variants of the Kleinschmidt technique and protein-free benzyldimethyl-alkyl ammonium chloride (BAC) technique at different pHs. If the specimens for electron microscopy were prepared at pH 5.6 and pH 4.0 (i.e., under conditions where the homopurine.homopyrimidine tract assumes an unusual conformation) a single thick "stem" or a "denaturation bubble" in a large number of DNA molecules were observed. No such changes were found in samples prepared at neutral pH and in linearized pEJ4 DNA prepared at pH 5.6. In specimens of a control supercoiled pUC19 DNA prepared at pH 5.6 and 4.0 practically no local changes were detected. The "denaturation bubbles" were observed by BAC techniques (probably due to secondary local DNA denaturation during the specimen preparation) while the more gentle formamide technique revealed only "stems". The "stems" were almost always positioned at the sites where the curvature of supercoiled DNA molecules occurred. The results are in agreement with presence of a protonated triplex H-form in homopurine.homopyrimidine tract bringing the first evidence of curvature or kinking of the DNA molecule connected with the occurrence of the H-form in supercoiled DNA.     

Electron Microscopy of Supercoiled pEJ4 DNA Containing Homopurine · Homopyrimidine Sequences

Abstract

Supercoiled pEJ4 DNA (a derivative of pUC19 containing an insert with 60-bp-long homopurine · homopyrimidine tract from the sea urchin P. miliaris histone gene spacer) was investigated by electron microscopy using three different spreading techniques i.e., formamide and aqueous variants of the Kleinschmidt technique and protein-free benzyldimethyl-alkyl ammonium chloride (BAC) technique at different pHs. If the specimens for electron microscopy were prepared at pH 5.6 and pH 4.0 (i.e., under conditions where the homopurine · homopyridine tract assumes an unusual conformation) a single thick “stem” or a “denaturation bubble” in a large number of DNA molecules were observed. No such changes were found in samples prepared at neutral pH and in linearized pEJ4 DNA prepared at pH 5.6. In specimens of a control supercoiled pUC 19 DNA prepared at pH 5.6 and 4.0 practically no local changes were detected. The “denaturation bubbles” were observed by BAC techniques (probably due to secondary local DNA denaturation during the specimen preparation) while the more gentle formamide technique revealed only “stems”. The “stems” were almost always positioned at the sites where the curvature of supercoiled DNA molecules occurred. The results are in agreement with presence of a protonated triplex H-form in homopurine · homopyrimidine tract bringing the first evidence of curvature or kinking of the DNA molecule connected with the occurrence of the H-form in supercoiled DNA.     

Effect of DNA structure on the formation of collagen-DNA complex

Using various types of DNAs prepared from plasmid DNA, complete double-stranded DNA (ds.DNA) with linear and cyclic forms and double-stranded DNA coexisting with single-stranded DNA (ss.DNA), the structure and fibrillogenesis of the collagen-DNA complex were investigated by means of turbidity, transmission electron microscopy, and confocal laser-scanning microscopy. The rate of fibrillogenesis of the collagen-DNA complex significantly depends on the DNA structure. The structure of the fibrils formed in the complexes showed a marked difference between the ds.DNA and ss.DNA complexes with collagen. Spatial distribution of the DNA and collagen in the complexes suggests that the characteristic collagen-DNA interaction depends on the DNA forms.     

The observation of the local ordering characteristics of spermidine-condensed DNA: atomic force microscopy and polarizing microscopy studies.

Condensation of DNA by multivalent cations can provide useful insights into the physical factors governing the folding and packaging of DNA in vivo. In this work, local ordered structures of spermidine-DNA complexes prepared from different DNA concentrations have been examined by using atomic force microscopy (AFM) and polarizing microscopy (PM). Two types (I and II) of DNA condensates, significantly different in sizes, were observed. It was found that for extremely dilute solutions (DNA concentrations around 1 ng/microl or below), the DNA molecules would collapse into toroidal structures with a volume equivalent to a single lambda-DNA (type I). In relatively dilute solutions (DNA concentrations between 1 and 10 ng/microll), a significantly larger structure of multimolecular toroids (circular and elliptical, type II) were formed, which were constructed by many fine particles. Measurements show that the average diameter of these fine particles was similar to the outer diameter of the monomolecular toroids observed in extremely dilute solutions, and the thickness of the multimolecular toroids had a distribution of multi-layers with height increments of 11 nm, indicating that the multimolecular toroidal structures have lamellar characteristics. Moreover, by enriching the DNA-spermidine complexes in very diluted solution, branch-like structures constructed by subunits were observed by using AFM. The analysis of the pellets in polarizing microscopy reveals a liquid-crystal-like pattern. These observations suggest that DNA-spermidine condensation could have multiple stages, which are very sensitive to the DNA and spermidine concentrations.     

Isolation and characterization of a 15-kilobase genomic sequence coding for part of the Pro alpha 2 chain of sheep type I collagen

DNA fragments, prepared by partial Eco RI digestion of fetal sheep liver genomic DNA, were used to prepare a "library" of amplified genomic sequences with the lambda vector Charon 4A. Several recombinant plaques were identified by their ability to hybridize to 32P-labeled cDNA prepared from fetal sheep tendon type I procollagen mRNA. Two of these recombinant DNA bacteriophages (SpC3 and SpC7) were identified as containing procollagen pro alpha 2 gene sequences by their ability to specifically anneal to procollagen pro alpha 2 mRNA. Restriction endonuclease and hybridization to a cloned pro alpha 2 cDNA demonstrated that approximately half (2.5 kilobases) of the pro alpha 2 mRNA sequence is distributed over 15 kilobases of genomic DNA. Restriction maps of SpC3 and SpC7 demonstrated that these two DNA fragments contain overlapping sequences of the pro alpha 2 gene. Electron microscopy and R-loop analysis of SpC3 revealed that at least 12 to 16 intervening sequences are distributed throughout the length of this gene fragment.     

Stable denaturation of chromosomal DNA from Saccharomyces cerevisiae during meiosis.

Partial denaturation of Saccharomyces cerevisiae chromosomal DNA was found to occur spontaneously during meiosis. Short regions of strand separation (300 base pairs long) were seen in DNA molecules prepared for electron microscopy by the aqueous spreading technique. These regions were clustered along the DNA. The time course of their appearance indicated that the denatured regions were present during the periods of premeiotic DNA replication and recombination. A similar pattern of denaturation was also detected in the DNA from vegetatively grown cells of a conditional cdc8 mutant, which is defective in DNA replication.     

DNA probes on chip surfaces studied by scanning force microscopy using specific binding of colloidal gold

Single-stranded DNA was covalently bound on chip surfaces using two different silanization procedures. The resulting surfaces were characterized by fluorescence and scanning force microscopy using sequence-complementary DNA molecules with labels. Colloidal gold (30 nm) was used as the topographic label. Scanning force microscopy revealed the individual labels on the surface and their distribution. Steps of silane layers or DNA-modified surfaces prepared using an elastomeric mask provided internal controls for comparison of modified with unmodified surfaces.     

Streptavidin conjugates with gold nanoparticles for visualization of single DNA interactions on the silicon surface

Applicability of scanning electron microscopy (SEM) for visualization of individual acts of DNA hybridization with oligonucleotide probes has been investigated using gold nanoparticles as a label. DNA or oligonucleotides were labeled with biotin molecules, which were then detected in DNA duplexes using a streptavidin conjugate with gold nanoparticles. Effective imaging of DNA duplexes was possible using the conjugate prepared by covalent binding. The detection limit of the model oligonucleotide of 19 bases was 20 pg.     

Visualization of Z sequences in form V of pBR322 by immuno-electron microscopy.

Form V DNA has been prepared from pBR322 DNA by annealing covalently closed complementary single strands. Specific rabbit antibodies to Z-DNA were shown by radioimmunoassay and electron microscopy to react with form V DNA of pBR322. The bound antibodies were visualized either directly (on synthetic polynucleotides in Z-form), or after reaction with goat anti-rabbit immunoglobulin labeled with ferritin (on form V DNA).     

The glucocorticoid receptor in homodimeric and monomeric form visualised by electron microscopy.

The purified glucocorticoid receptor (GR) from rat liver has been visualised by electron microscopy. The specimens were prepared by spreading on thin carbon support and negatively stained using uranyl acetate. Two forms of GR, the monomeric and the dimeric forms, were identified based on size, chromatographic distribution, and DNA binding properties. The GR monomer consists of two globular domains of slightly different size with a thinner connecting domain in between. In the absence of DNA the dimeric GR has a characteristic four-leaf clover structure. The size and appearance of this structure is consistent with two GR subunits arranged in a side-by-side fashion. Monomeric and dimeric GR specifically bound to DNA are also shown.     

DNA interaction and antitumor activity of a Pt(III) derivative of 2-mercaptopyridine

The complex [Pt2(Spy-)4Cl2], where Spy- is deprotonated 2-mercaptopyridine, was prepared and analyzed spectroscopically. A single signal in the 195Pt NMR spectrum indicates the equivalence of the two Pt(III) ions. The interaction of this complex with DNA was studied by circular dichroism and the modifications caused by the complex in plasmid pBR322 DNA were imaged by atomic force microscopy. Preliminary results showed higher activity against HeLa and U937 tumor lines for the Pt-2-mercaptopyridine complex in comparison with cisplatin. The values of LC50 were lower than those obtained for cisplatin. Promising perspectives for this compound are expected due to its similarity with the analogous Pt and 2-mercaptopyrimidine antitumor compound.     

Three-dimensional approaches to the residual structure of histone-depleted HeLa cell nuclei

Histone-depleted nuclei were prepared by high-salt extraction of interphase HeLa cell nuclei. A large amount of the nuclear DNA remained associated with a rapidly sedimenting residual nuclear structure including cytoplasmic (intermediate filament) and nuclear (matrix and lamina) proteins. Electron microscopy allowed detection in the insoluble structure of a residual nuclear envelope, nucleolar residues, and an intranuclear network whose correspondence with components of in situ fixed nuclei is discussed. Using three-dimensional electron microscopy, it is further demonstrated that the salt-insoluble structure remaining after histone depletion in 2 M NaCl is highly ordered. This is of the utmost importance when considering the roles reportedly ascribed to this structure in nuclear functions.     

Physicochemical characterization and purification of cationic lipoplexes.

Cationic lipid-nucleic acid complexes (lipoplexes) consisting of dioleoyltrimethylammoniumpropane (DOTAP) liposomes and plasmid DNA were prepared at various charge ratios (cationic group to nucleotide phosphate), and the excess component was separated from the lipoplex. We measured the stoichiometry of the lipoplex, noted its colloidal properties, and observed its morphology and structure by electron microscopy. The colloidal properties of the lipoplexes were principally determined by the cationic lipid/DNA charge ratio and were independent of the lipid composition. In lipoplexes, the lipid membranes as observed in freeze-fracture electron microscopy were deformed into high-radius-of-curvature features whose characteristics depended on the lipid composition. Lipoplexes prepared at a threefold or greater excess of either DOTAP or DNA could be resolved into complexes with a defined stoichiometry and the excess component by sedimentation to equilibrium on sucrose gradients. The separated, positively charged complex retained high transfection activity and had reduced toxicity. The negatively charged lipoplex showed increased transfection activity compared to the starting mixture. In cryoelectron micrographs the positively charged complex was spherical and contained a condensed but indistinct interior structure. In contrast, the separated negatively charged lipoplexes had a prominent internal 5.9 +/- 0.1-nm periodic feature with material projecting as spikes from the spherical structure into the solution. It is likely that these two lipoplexes represent structures with different lipid and DNA packing.     

Comparison of JC and BK Human Papovaviruses with Simian Virus 40: Restriction Endonuclease Digestion and Gel Electrophoresis of Resultant Fragments

JC virus was found to have a buoyant density of 1.20 g/cm(3) in linear sucrose-D(2)O and 1.35 g/cm(3) in cesium chloride isopycnic gradients. DNA extracted either from JC-infected cultures or from gradient-purified virions occupied a dense position relative to linear DNA in cesium chloride/ethidium bromide gradients, and the circular configuration of the extracted DNA was confirmed by electron microscopy, with a measured molecular weight of 2.93 x 10(6). DNA from BK virus was similarly prepared and compared to JC and to an SV40 DNA standard by digestion with restriction endonuclease preparations from Haemophilus influenzae, Haemophilus parainfluenzae, and Escherichia coli. Digests were electrophoretically analyzed on gradient polyacrylamide slab gels or agarose gels, and the three viruses were found to have distinctly different cleavage patterns by this form of analysis: JC and BK viruses were almost entirely different from SV40 and significantly different from each other. Thus, JC and BK human papovaviruses appear to be discrete new members of the papovavirus group, rather than SV40 variants.     

Immobilization of proteins on magnetic nanoparticles

Magnetic nanoparticles prepared from an alkaline solution of divalent and trivalent iron ions could covalently bind protein via the activation ofN-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC). Trypsin and avidin were taken as the model proteins for the formation of protein-nanoparticle conjugates. The immobilized yield of protein increased with molar ratio of EDC/nanoparticle. Higher concentrations of added protein could yield higher immobilized protein densities on the particles. In contrast to EDC, the yields of protein immobilization via the activation of cyanamide were relatively lower. Nanoparticles bound with avidin could attach a single-stranded DNA through the avidin-biotin interaction and hybridize with a DNA probe. The DNA hybridization was confirmed by fluorescence microscopy observations. Immobilized DNA on nanoparticles by this technique may have widespread applicability to the detection of specific nucleic acid sequence and targeting of DNA to particular cells.     

Structure of varicella-zoster virus DNA

Varicella-zoster virus (VZV) DNA was prepared from nucleocapsids and from enveloped virions of a laboratory strain (Ellen) and directly from the vesicle fluids of patients with zoster infections. VZV Ellen nucleocapsid DNA was cleaved with 11 different restriction endonucleases and electrophoresed in agarose gels. The restriction profiles of the nucleocapsid DNA were identical to those of the DNA recovered from purified virions, but differed from those of another VZV strain (KM). In vitro-labeled VZV K.M. DNA purified directly from vesicle fluid yielded a distinct restriction pattern which appeared to be unchanged after several tissue culture passages of the isolate from that fluid. Restriction endonuclease analysis (EcoRI or BglII) of VZV DNA revealed the presence of four cleavage fragments with a molar ratio of approximately 0.5. No individual fragments with molar ratios of 0.25 were noted. This observation suggests that the VZV genome may contain one invertible segment. Comparison of the electrophoretic migrations of VZV DNA fragments relative to those of DNAs of known size permitted calculation of the VZV genome size to be 72 X 10(6) to 80 X 10(6) daltons. These results were confirmed by electron microscopy which demonstrated a genome size of about 76 X 10(6) daltons for passaged and unpassaged VZV DNA. Electron microscopy also revealed that some of the DNA molecules recovered from nucleocapsids or directly from vesicle fluids were superhelical circles.     

DNA Polymerase Associated with Human Hepatitis B Antigen

DNA polymerase activity was detected in each of eight preparations of concentrated human hepatitis B antigen (HBAg) rich in Dane particles prepared by high-speed centrifugation of antigen-positive human plasma and in none of seven control preparations prepared in the same way from HBAg-negative plasma. The incorporation of (3)H-thymidine-methyl-5'-triphosphate into DNA was dependent on four deoxyribonucleoside triphosphates and MgCl(2). Treatment of the concentrated HBAg preparations with the nonionic detergent Nonidet P-40 (NP40) more than doubled the enzyme activity. Fractionation of the concentrated HBAg preparation in sucrose density gradients after treatment with NP40 revealed that the enzyme activity appeared within the density range of Dane core antigen but at a slightly higher density than the average for core antigen. The only particles observed by electron microscopy in this region of the gradient were typical 28-nm cores, suggesting that the DNA polymerase activity was associated with a subpopulation of cores. No DNA polymerase activity was found in purified 20-nm HBAg particles. The DNA product of the reaction remained associated with the 110S core and was not susceptible to DNase digestion when associated with the core. Inhibition of the reaction by actinomycin D and daunomycin suggested that the reaction was dependent on a DNA template associated with the core.     

Electron microscopy of membrane-free folded chromosomes from Escherichia coli

Membrane-free folded chromosomes were purified from log-phase cultures of Escherichia coli and prepared for electron microscopy by aqueous (Kleinschmidt and Zahn) spreading. The appearance of the chromosomes depended on the salt concentrations in spreading. At certain salt concentration, the chromosomes resembled rosettes, with supercoiled loops of DNA radiating from a central core containing RNA. The rosettes support previous models deduced from physical studies of folded chromosomes. Apparently, cores contain must of the visible RNA, and the organization of the core is linked to the organization of the DNA loops.Submitted in celebration of Julius Marmur's birthday—his teachings made this study possible     

The diagnosis of malaria and identification of plasmodium species by polymerase chain reaction in Turkey

More than half of the world's population is exposed to malaria in approximately 100 countries. Rapid diagnosis and correct treatment of cases are the main objectives of control programs in malaria endemic areas. We have developed a PCR method to determine the presence of plasmodium DNA in blood. The method can also identify the species of the plasmodium by restriction enzyme analysis of the amplified product. We evaluated the performance of this method in the diagnosis of malaria suspected cases in Turkey by comparing to microscopy of the blood smears: blood samples were obtained from 114 patients with malaria symptoms, including fever and/or chills lasting for several days, before starting treatment. Thin and thick blood smears were prepared immediately in the region of specimen collection. After isolation of DNA from blood samples, DNA was amplified by PCR and digested by restriction enzyme AluI. The obtained fragments were analyzed by agarose gel electrophoresis. The number of parasites in the thick and thin smears of the blood samples was evaluated microscopically after staining by Giemsa and results were compared by PCR results. Among 114 plasmodium positive cases detected by microscopy, 100 were also detected by PCR. There were 14 false negatives and no false positive by PCR. Compared to microscopy, the sensitivity, specificity and Positive Predictive Value (PPV) of PCR were determined as 76%, 100% and 100%, respectively.