Atomic force microscopy of oriented linear DNA molecules labeled with 5nm gold spheres.

The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2-7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are absorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.     

Cationic metals promote sequence-directed DNA bending

A DNA segment of approximately 200 base pairs (bp) from Crithidia fasciculata kinetoplast minicircles was previously shown by electron microscopy (EM) to bend into a small circle due to its unique nucleotide sequence containing repeated blocks of 4-6 A's. When this segment was flanked by 207 bp of plasmid DNA on one side and 460 bp on the other, the resulting 890-bp DNA was found to appear either relatively straight or extremely bent as visualized by EM. The bend was located one-third the distance from one end. The fraction of molecules with the most extreme bend increased from approximately 2% to 50-60% following incubation of the DNA with increasing concentrations of Zn2+, Co2+, Ba2+, and Mn2+. These observations suggest that sequence-directed bending in DNA is an inducible and not a static phenomenon. Possible roles of transitions between the bent and straight conformations in the control of gene expression are discussed.     

Early stages in RecA protein-catalyzed pairing. Analysis of coaggregate formation and non-homologous DNA contacts.

RecA protein will catalyze the in vitro pairing of homologous DNA molecules. To further explore the events involved in the search for homology, we have applied a nitrocellulose filter binding assay to follow pairing, and a sedimentation assay to follow the generation of aggregates (termed coaggregates) formed between RecA-complexed single-stranded (ss) DNA and double stranded (ds) DNA. Electron microscopy (EM) was used to visualize the structures involved. RecA protein promoted the pairing of circular M13 ssDNA and linear M13mp7 dsDNA efficiently in the absence of coaggregates. Indeed, pairing of homologous ss- and dsDNAs involved coaggregate formation only if the dsDNA was circular. For DNAs containing only a few hundred base-pairs of homology, for example pUC7 dsDNA and M13mp7 ssDNA, pairing and joint formation was observed if the dsDNA was superhelical but not if it was topologically relaxed or linear with the homology internal to an end of the dsDNA. The effect of non-covalently attached heterologous dsDNA on the RecA-promoted joining of M13 ssDNA and linear M13mp7 dsDNA (with non-M13 sequences at both ends) was found to depend on the topology and concentration of the heterologous DNA. A tenfold excess of superhelical pBR322 DNA strongly inhibited pairing. However, addition of relaxed or linear pBR322 DNA to the pairing reaction had little effect. As seen by EM, superhelical pBR322 DNA inhibited joint formation by excluding the homologous dsDNA form the coaggregates. EM also revealed heterologous DNA interactions presumably involved in the search for homology. Here the use of EM has provided a direct visualization of the form and architecture of coaggregates revealing a dense interweaving of presynaptic filaments and dsDNA.     

Orientation of the DNA in the filamentous bacteriophage f1

The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al., 1981a). By hybridizing the DNA isolated from either end of ³²P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.     

红外光谱法在基因探测中的应用研究

应用干涉型傅里叶变换红外分光光度计及聚乙烯膜作载体,得到了单链、双链DNA、生物素-dUTP及经过缺口翻译参入生物素的DNA探针的红外光谱,观察到Bio-dUTP分子内化学键振动产生的特征吸收谱带。利用差谱技术比较了DNA与生物素标记DNA的差异。Bio-dUTP的特征吸收探测灵敏度可达0.1ng。同时分析了硝酸纤维膜与genescreen的红外透过性质。     

Phased adenine tracts in double-stranded RNA do not induce sequence-directed bending

Tracts of four to six adenines phased with the DNA helix produce a sequence-directed bending of the helix axis. Here, using gel electrophoresis and electron microscopy (EM), we have asked whether a similar motif will induce bending in a duplex RNA helix. Single-stranded RNAs were transcribed either from short synthetic DNA templates or from Crithidia fasciculata kinetoplast bent DNA, and the complementary single-stranded RNAs were annealed to produce duplex RNA molecules containing blocks of four to six adenines. Electrophoresis on polyacrylamide gels revealed no retardation of the RNAs containing phased blocks of adenines relative to duplex RNAs lacking such blocks. Examination by EM showed most of the molecules to be straight or only slightly bent. Thus, in contrast to DNA duplexes, phased adenine tracts do not induce sequence-directed bending in double-stranded RNA. Analysis of the distribution of molecule shapes for the highly bent C. fasciculata DNA showed that the adenine blocks do not act cooperatively to induce DNA bending and that the molecules must equilibrate between a spectrum of bent shapes.     

Mapping Z-DNA tracts in plasmid DNA using electron microscopy and gold-labeled antibodies

Using alternating poly(dG-dC).poly(dG-dC) and electron microscopy (EM), a method has been developed for detecting regions of Z conformation in DNA preparations. The procedure was developed with poly(dG-dC).poly(dG-dC) which had been converted to the Z conformation with MnCl2 and mild heat treatment. Conditions were found for reaction of this DNA with polyclonal anti-Z antibodies from rabbit, and further reaction of this mixture with gold-labelled anti-rabbit antibodies from mouse. Spreading of these samples onto air-water interfaces and examination by EM revealed gold particles aligned along strands of poly(dG-dC).poly(dG-dC). The method was refined and simplified using monoclonal antibodies and tested with the 2.2-kb plasmid, pDHg16, carrying a single tract of alternating d(G-C)23. Treatment with MnCl2 and mild heat was not necessary, as the superhelicity of this molecule ensured that the d(G-C) tract was in the Z conformation. Conditions were found for successful conjugation of mouse monoclonal anti-Z antibodies with colloidal gold (G10), 10.7-nm average diameter. The conjugate was then reacted with superhelical pDHg16, stabilized in polyethylene glycol and cross-linked with glutaraldehyde. Examination by EM showed gold particles at one site on the negatively superhelical circular DNA molecule. When these molecules were linearized with PstI, gold particles were found to occur at an average position 35% +/- 3% from one end. This location agrees well with the known position of the center of the alternating d(G-C) tract with respect to the PstI restriction site (36.8%).     

The terminase subunits pUL56 and pUL89 of human cytomegalovirus are DNA-metabolizing proteins with toroidal structure

Herpesvirus DNA packaging involves binding and cleavage of DNA containing the specific DNA-packaging motifs. Here we report a first characterization of the terminase subunits pUL56 and pUL89 of human cytomegalovirus (HCMV). Both gene products were shown to have comparable nuclease activities in vitro. Under limiting protein concentrations the nuclease activity is enhanced by interaction of pUL56 and pUL89. High amounts of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole partially inhibited the pUL89-associated nuclease activity. It was demonstrated that pUL56 is able to bind to nucleocapsids in vivo. Electron microscopy (EM) and image analysis of purified pUL56 revealed that the molecules occurred as a distinct ring-shaped structure with a pronounced cleft. EM analysis of purified pUL89 demonstrated that this protein is also a toroidal DNA-metabolizing protein. Upon interaction of pUL56 with linearized DNA, the DNA remains uncut while the cutting event itself is mediated by pUL89. Using biochemical assays in conjunction with EM pUL56 was shown to (i) bind to DNA and (ii) associate with the capsid. In contrast to this, EM analysis implied that pUL89 is required to effect DNA cleavage. The data provide the first insights into the terminase-dependent viral DNA-packaging mechanism of HCMV.     

Visualization of the bent helix in kinetoplast DNA by electron microscopy

Kinetoplast DNA minicircles from the trypanosomatid Crithidia fasciculata contain a segment of approximately 200 bp which is probably more highly bent than any other DNA previously studied. Electron microscopy (EM) of relaxed minicircles (2.5 kb) revealed 200-300 bp loops within the larger circles, and the loops could also be detected on full-length linear molecules. Examination by EM of a 219 bp cloned fragment which contains the bent helix revealed that up to 70% of the molecules appeared circular whether or not the ends were cohesive. In contrast, a 207 bp fragment from pBR322 showed no circles and the fragments in general appeared much straighter than the kinetoplast fragments. Treatment of the 219 bp bent kinetoplast fragment with the drug distamycin caused a striking reduction in curvature.     

Sequence-specific fragmentation of macronuclear DNA in a holotrichous ciliate

Macronuclear DNA from the protozoan G. chattoni, a holotrichous ciliate, was analyzed. Most, if not all, of the macronuclear DNA is subchromosomal, ranging in size from above 100 kb down to 2.1 kb, with molecules in the lower molecular weight range being resolvable by gel electrophoresis into reproducible, specific, discrete size classes. A prominent class of linear 9.3 kb molecules consists of single free rRNA genes. Upon denaturation and partial renaturation, a high percentage of total macronuclear DNA was found as single-stranded circles. Sequence analysis showed that a minimum of 38 tandem repeats of the sequence CCCCAA is present in inverted orientation at each end of most or all Glaucoma macronuclear DNA molecules, including the rDNA. This sequence must therefore be recognized during site-specific fragmentation of chromosomes in macronuclear development.     

Electron microscopy mapping of oligopurine tracts in duplex DNA by peptide nucleic acid targeting.

Biotinylated homopyrimidine decamer peptide nucleic acids (PNAs) are shown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The noncovalent complexes are visualized by electron microscopy (EM) without chemical fixation using streptavidin as an EM marker. The triplex stoichiometry of the PNA-DNA complexes (two PNA molecules presumably binding by Watson-Crick and Hoogsteen pairing with one of the strands of the duplex DNA) is indicated by the appearance of two streptavidin 'beads' per target site in some micrographs, and is also supported by the formation of two retardation bands in a gel shift assay. Quantitative analysis of the positions of the streptavidin 'beads' revealed that under optimized conditions PNA-DNA complexes are preferably formed with the fully complementary target. An increase in either the PNA concentration or the incubation time leads to binding at sites containing one or two mismatches. Our results demonstrate that biotinylated PNAs can be used for EM mapping of short targets in duplex DNA.     

Effect of pulsed and reversing electric fields on the orientation of linear and supercoiled DNA molecules in agarose gels

When linear or supercoiled DNA molecules are imbedded in agarose gels and subjected to electric fields, they become oriented in the gel matrix and give rise to an electric birefringence signal. The sign of the birefringence is negative, indicating that the DNA molecules are oriented parallel to the electric field lines. If the DNA molecules are larger than about 1.5 kilobase pairs, a delay is observed before the birefringence signal appears. This time lag, which is roughly independent of DNA molecular weight, decreases with increasing electric field strength. The field-free decay of the birefringence is much slower for the DNA molecules imbedded in agarose gels than observed in free solution, indicating that orientation in the gel is accompanied by stretching. Both linear and supercoiled molecules become stretched, although the apparent change in conformation is much less pronounced for supercoiled molecules. When the electric field is rapidly reversed in polarity, very little change in the birefringence signal is observed for linear or supercoiled DNAs if the equilibrium orientation (i.e., birefringence) had been reached before field reversal. Apparently, completely stretched, oriented DNA molecules are able to reverse their direction of migration with little or no loss of orientation. If the steady-state birefringence had not been reached before the field reversal, complicated orientation patterns are observed after field reversal. Very large, partially stretched DNA molecules exhibit a rapid decrease in orientation at field reversal. The rate of decrease of the birefringence signal in the reversing field is faster than the field-free decay of the birefringence and is approximately equal to the rate of orientation in the field (after the lag period).(ABSTRACT TRUNCATED AT 250 WORDS)     

Visualization of DNA and RNA molecules, and protein-DNA complexes for electron microscopy

This article provides step-by step instructions for the preparation of double- and single-stranded DNA and RNA molecules and protein-DNA complexes for electron microscopy (EM). Absorption, spreading, staining, dark-field imaging, and metal shadowing techniques are described in detail. A number of examples are illustrated on analysis of DNA replication, DNA repair and DNA recombination to demonstrate the usefulness of the technique for EM visualisation. Application of immunogold labeling of specific protein in DNA-protein complexes is also covered.     

The presence of RNA in a double helix inhibits its interaction with histone protein

The binding of core histones (H2A, H2B, H3, H4) to a circular plasmid DNA and to a circular DNA-RNA hybrid molecule of similar size has been compared. Circular hybrid molecules were formed from single stranded fd DNA by synthesis of the complimentary strand with ribonucleotides using wheat germ RNA polymerase II. Upon reconstitution of plasmid DNA circles with histone, the sedimentation profiles of the DNA remained sharp by increased several fold in rate. Material from the peak fractions of these sedimentations appeared to be condensed circular loops of nucleosomes when examined by electron microscopy (EM), and the mass ratio of DNA to histone (at the histone concentrations which produced the fastest sedimentations) was typical of native chromatin. In contrast, the sedimentation behavior of DNA-RNA hybrid circles after addition of histone remained unchanged except for a minor fraction which exhibited a broad and faster sedimentation rate. Examination by EM revealed that most of the molecules appeared identical to protein free hybrid circles while the minor, faster sedimenting fraction appeared to be two or more circles bound together by protein aggregates. Finally, a linear molecule consisting of about 3000 base pairs of duplex DNA covalently joined on both ends to 1500 base pairs of RNA-DNA hybrid helix was constructed. Reconstitution of this molecule with core histone showed nucleosome formation only on the central DNA duplex region. Isopycnic banding of fixed hybrid-histone mixtures showed that little or no histone had bound to the bulk of the full hybrid molecules. We suggest that the presence of RNA in a nucleic acid duplex inhibits the condensation of the duplex into a nucleosomal structure by histone.     

Analysis of DNA replication forks encountering a pyrimidine dimer in the template to the leading strand.

Electron microscopy (EM) was used to visualize intermediates of in vitro replication of closed circular DNA plasmids. Cell-free extracts were prepared from human cells that are proficient (IDH4, HeLa) or deficient (CTag) in bypass replication of pyrimidine dimers. The DNA substrate was either undamaged or contained a single cis, syn thymine dimer. This lesion was inserted 385 bp downstream from the center of the SV40 origin of replication and sited specifically in the template to the leading strand of the newly synthesized DNA. Products from 30 minute reactions were crosslinked with psoralen and UV, linearized with restriction enzymes and spread for EM visualization. Extended single-stranded DNA regions were detected in damaged molecules replicated by either bypass-proficient or deficient extracts. These regions could be coated with Escherichia coli single-stranded DNA binding protein. The length of duplex DNA from a unique restriction site to the single-stranded DNA region was that predicted from blockage of leading strand synthesis by the site-specific dimer. These results were confirmed by S1nuclease treatment of replication products linearized with single cutting restriction enzymes, followed by detection of the diagnostic fragments by gel electrophoresis. The absence of an extended single-stranded DNA region in replication forks that were clearly beyond the dimer was taken as evidence of bypass replication. These criteria were fulfilled in 17 % of the molecules replicated by the IDH4 extract.     

DNA of a new parvo-like virus isolated from the silkworm,Bombyx mori

Parvo-like virus, which was designated as “Ina-flacherie virus (Ina-FV),” was isolated from the silkworm, Bombyx mori, and the properties of its DNA were characterized. Purified Ina-FV had a diameter of 22 ± 0.5 nm and a sedimentation coefficient of 102 S. On density gradient separation in CsCl, particles were found at densities of 1.40 and 1.45 g/ml. The DNA content of Ina-FV was 28 ± 2%. The DNA in low-salt buffer possessed properties typical of a single-stranded (ss) molecule. Double-stranded (ds) DNA was extracted under conditions of appropriate high salt and elevated temperature. Electron microscopical examination revealed that the ds DNA was composed of linear molecules with an average length of 1.7 μm and other less well-defined structures. The linear ds molecule had a molecular weight of about 3.4 × 10⁶ determined by electron microscopy (EM) and agarose gel electrophoresis. When the ds DNA was alkali-denatured and examined in an EM, linear ss molecules with approximate length of 1.7 μm were observed, indicating that the linear ds molecule was formed from the annealing of the linear ss molecules of unit length. These data suggest that Ina-FV is closely related to members of the densovirus subgroup.