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.     

DNA of a Human Hepatitis B Virus Candidate

Particles containing DNA polymerase (Dane particles) were purified from the plasma of chronic carriers of hepatitis B antigen. After a DNA polymerase reaction with purified Dane particle preparations treated with Nonidet P-40 detergent, Dane particle core structures containing radioactive DNA product were isolated by sedimentation in a sucrose density gradient. The radioactive DNA was extracted with sodium dodecyl sulfate and isolated by band sedimentation in a preformed CsCl gradient. Examination of the radioactive DNA band by electron microscopy revealed exclusively circular double-stranded DNA molecules approximately 0.78 mum in length. Identical circular molecules were observed when DNA was isolated by a similar procedure from particles that had not undergone a DNA polymerase reaction. The molecules were completely degraded by DNase 1. When Dane particle core structures were treated with DNase 1 before DNA extraction, only 0.78-mum circular DNA molecules were detected. Without DNase treatment of core structures, linear molecules with lengths between 0.5 and 12 mum, in addition to the 0.78-mum circles were found. These results suggest that the 0.78-mum circular molecules were in a protected position within Dane particle cores and the linear molecules were not within core structures. Length measurements on 225 circular molecules revealed a mean length of 0.78 +/- 0.09 mum which would correspond to a molecular weight of around 1.6 x 10(6). The circular molecules probably serve as primer-template for the DNA polymerase reaction carried out by Dane particle cores. Thermal denaturation and buoyant density measurements on the Dane particle DNA polymerase reaction product revealed a guanosine plus cytosine content of 48 to 49%.     

Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum Dane particles: possible implications in persistent HBV infection

In chimpanzee hepatitis B virus (HBV) carriers, the mechanism of viral persistence has been examined by analyzing viral DNA molecules in liver and serum. Chimpanzee liver DNA contained two extrachromosomal HBV DNA molecules migrating on hybridization blots at 4.0 kb and 2.3 kb. There was no evidence for integration of HBV DNA into the host genome. The extrachromosomal molecules were distinct from Dane particle DNA and were converted to linear 3.25 kb full-length double-stranded HBV DNA on digestion with Eco RI. Nucleases S1 and Bal 31 converted "2.3 kb" HBV DNA to 3.25 kb via an intermediate of "4.0 kb" apparent length. The HBV DNA molecule that migrated at 2.3 kb represents a supercoiled form I of the HBV genome, and the molecule that migrated at 4.0 kb represents a full-length "nicked," relaxed circular form II. Evidence for supercoiled HBV DNA in serum Dane particles was obtained by production of form II molecules upon digestion with nuclease S1 or Bal 31. It is proposed that most Dane particles represent interfering noninfectious virus containing partially double-stranded DNA circles and that particles containing supercoiled HBV DNA may represent infectious hepatitis B virus.     

DNA synthesized in the hepatitis B Dane particle DNA polymerase reaction.

Radioactive DNA was prepared in extensive (4 h) Dane particle DNA polymerase reactions. In different experiments the amount of new DNA, determined by the amount of nucleotide incorporation into an acid-insoluble form, was between 29 and 45% of the total circular DNA isolated from Dane particle preparations after the reaction. DNA reassociation kinetics were used to determine the complexity of the newly synthesized DNA. In different experiments COt1/2 values, corresponding to between 625 and 1,250 nucleotide pairs, were obtained for the radioactive Dane particle DNA. These results suggest that a unique region (or regions), corresponsing to approximately one-fourth to one-half of the circular Dane particle DNA template, was copied one time during the reaction. DNA and RNA extracted from hepatitis B virus-infected liver but not from uninfected liver accelerated the rate of reassociation of radioactive DNA from Dane particles. These Dane particle DNA base sequences were found in alkali-stable, rapidly sedimenting DNA from infected liver as well as in DNA sedimenting at a rate similar to the DNA extracted from Dane particles. These findings are consistent with Dane particle DNA being hepatitis B virus DNA that is integrated into high-molecular-weight cellular DNA and transcribed into RNA in infected liver.     

Very efficient template/primer-independent DNA synthesis by thermophilic DNA polymerase in the presence of a thermophilic restriction endonuclease

We have found that, in the presence of a thermophilic restriction endonuclease, thermophilic DNA polymerase efficiently synthesizes and amplifies DNA in the absence of any added template and primer nucleic acid under isothermal conditions. More than 10 microg of DNA can be synthesized by 1 unit of DNA polymerase in 1 h, and the reaction proceeds until available dNTPs are consumed. We used mostly the Tsp509I restriction endonuclease (recognition sequence: decreasing AATT), the TspRI restriction endonuclease (recognition sequence: NNCA(G/C)TGNN decreasing), and Vent (exo(-)) and Vent DNA polymerase. The synthesized double-stranded DNA has a highly repetitive palindromic sequence, e.g. (AAAAATTTTT)(n) and (ATACACTGTATATACAGTGTAT)(n). In every repeating unit, there are one or two recognition sites for the restriction enzyme. Our data show that the high efficiency of the restriction-endonuclease-DNA-polymerase (RE-pol) DNA synthesis results from an efficient exponential amplification involving digestion-elongation cycles: a longer DNA with numerous recognition sites for the restriction enzyme is digested to short fragments, and the short fragments are used as seeds for elongation to synthesize longer DNA. A possible role of RE-pol DNA synthesis in the evolutionary development of genetic materials is briefly discussed.     

In vitro termination of adenovirus DNA synthesis by a soluble replication complex.

The double-stranded DNA from a soluble DNA replication complex that was labeled with deoxyribonucleoside triphosphates and completed in vitro was digested with EcoRI, Sma I, and Hpa I restriction endonucleases. All regions of the adenovirus type 2 genome were labeled in vitro, but restriction fragments derived from the ends of the DNA molecules were relatively more highly labeled than those derived from internal regions. The in vitro endogenous DNA polymerase reaction also exhibited strand-specific labeling near the molecular ends, in that restriciton fragments from the left end were labeled predominantly in the r strand and fragments from the right end were labeled predominantly in the l strand.     

Association of transformation of xenosomes from nonkiller to killer with extrachromosomal DNA.

Extrachromosomal DNA in the form of covalently closed circular DNA molecules was isolated from killer and nonkiller xenosomes, bacterial endosymbionts of the marine protozoan Parauronema acutum. Restriction endonuclease digests of these molecules derived from 12 isolates revealed consistent, readily identifiable, differences in the pattern of fragments of the killer as compared with those present in the nonkiller. Transformation of the nonkiller to killer by infection is also accompanied by a change from the nonkiller to killer pattern. Based on analysis of fragments resulting from restriction endonuclease digests, two circular duplex DNA molecules, each 63 kilobase pairs (kbp) in length, were identified in the 263-20 nonkiller stock and mapped. The maps revealed that each possesses a single BamHI site and multiple BglI, BstIIE, PstI, and SalI sites. A distinguishing feature of these maps is that the two molecules share a region about 17 kbp in length in which multiple restriction sites are in register with each other. Allowing for a 0.5-kbp insertion or deletion and the introduction or removal of only a few restriction sites, an additional stretch extending approximately 31 kbp beyond this sequence could also be considered to be homologous. The structure of the killer plasmid appears to be more complex, and we have been unable, as yet, to construct physical maps for this DNA. We postulate that the killer plasmid DNA is composed of three, perhaps four, circular 63-kbp duplexes, at least one which contains a single BamHI site and another which contains two BamHI sites. The remaining molecules may represent copies of either or both of the other two, modified to contain additional restriction sites. Transformation from the nonkiller to the killer is visualized as the insertion of restriction sites at various points along parent nonkiller plasmid DNA molecules. The mechanism by which these sites are introduced is unknown.     

Action of Escherichia coli P1 restriction endonuclease on simian virus 40 DNA

The P1 restriction endonuclease (EcoP1) prepared from a P1 lysogen of Escherichia coli makes one double-strand break in simian virus (SV40) DNA. In the presence of cofactors S-adenosylmethionine and ATP the enzyme cleaves 70% of the closed circular SV40 DNA molecules once to produce unit-length linear molecules and renders the remaining 30% resistant to further cleavage. No molecules were found by electron microscopy or by gel electrophoresis that were cleaved more than once. It would appear that the double-strand break is made by two nearly simultaneous single-strand breaks, since no circular DNA molecules containing one single-strand break were found as intermediates during the cleavage reaction. The EcoP1 endonuclease-cleaved linear SV40 DNA molecules are not cleaved at a unique site, as shown by the generation of about 65% circular molecules after denaturation and renaturation. These EcoP1 endonuclease-cleaved, renatured circular molecules are resistant to further cleavage by EcoP1 endonuclease.The EcoP1 endonuclease cleavage sites on SV40 DNA were mapped relative to the partial denaturation map and to the EcoRI and HpaII restriction endonuclease cleavage sites. These maps suggest there are a minimum of four unique but widely spaced cleavage sites at 0.09, 0.19, 0.52, and 0.66 SV40 units relative to the EcoRI site. The frequency of cleavage at any particular site differs from that at another site. If S-adenosylmethionine is omitted from the enzyme reaction mix, SV40 DNA is cleaved into several fragments.An average of 4.6 ± 1 methyl groups are transferred to SV40 DNA from S-adenosylmethionine during the course of a normal reaction containing the cofactors. Under conditions which optimize this methylation, 7 ± 1 methyl groups can be transferred to DNA. This methylation protects most of the molecules from further cleavage. The methyl groups were mapped relative to the Hemophilus influenzae restriction endonuclease fragments. The A fragment receives three to four methyl groups and the B and G fragments each receive one to two methyl groups. These fragments correspond to those in which cleavage sites are located.     

Mitochondrial DNA from Podospora anserina. I. Isolation and characterization.

Mitochondrial (Mt) DNA from Podospora anserina was isolated and characterized with respect to density in CsCl, contour length and endonuclease restriction enzymes. The density of Mt DNA for four races examined was 1.694 g/cm3, compared with 1.712 g/cm3 for nuclear DNA. Extraction in the presence of a nuclease inhibitor, aurintricarboxylic acid and isolation in DAPI CsCl gradients allowed us to isolate high molecular weight DNA. Mt DNA isolated by total DNA extraction contained ca. 1% of circular molecules, 31 micron in contour length; Mt DNA isolated from purified mitochondria contained 2--4% of these 31 micron circles. Analysis with Eco RI restriction endonuclease revealed that each of the four races examined, s, A, T and E had a characteristic fragment pattern. Races s and A Mt DNA differed by only one fragment after Eco RI enzymatic digestion; similarly, these two DNA differed by only one or two fragments after Hae III digestion.     

Specific fragmentation of mitochondrial DNA from Neurospora crassa by restriction endonuclease Eco R I.

Linear, size-heterogenous mitochondrial DNA from $\text{Neurospora crassa}$ was cleaved by the restriction endonuclease Eco R I into eleven specific fragments. According to their contour lengths the fragments have molecular weights between 1.1 and 14 × 10⁶. The sum of the fragments lengths is identical with the contour length (19.8 μm, 41 × 10⁶ daltons) of the few circular molecules detectable in purified DNA preparations.The results suggest sequence homogeneity of mitochondrial DNA and further demonstrate that restriction enzymes can be used to establish a physical map of an unspecifically-fragmented DNA molecule.     

Conformation-selective methylation of geminivirus DNA

Geminiviruses with small circular single-stranded DNA genomes replicate in plant cell nuclei by using various double-stranded DNA (dsDNA) intermediates: distinct open circular and covalently closed circular as well as heterogeneous linear DNA. Their DNA may be methylated partially at cytosine residues, as detected previously by bisulfite sequencing and subsequent PCR. In order to determine the methylation patterns of the circular molecules, the DNAs of tomato yellow leaf curl Sardinia virus (TYLCSV) and Abutilon mosaic virus were investigated utilizing bisulfite treatment followed by rolling circle amplification. Shotgun sequencing of the products yielded a randomly distributed 50% rate of C maintenance after the bisulfite reaction for both viruses. However, controls with unmethylated single-stranded bacteriophage DNA resulted in the same level of C maintenance. Only one short DNA stretch within the C2/C3 promoter of TYLCSV showed hyperprotection of C, with the protection rate exceeding the threshold of the mean value plus 1 standard deviation. Similarly, the use of methylation-sensitive restriction enzymes suggested that geminiviruses escape silencing by methylation very efficiently, by either a rolling circle or recombination-dependent replication mode. In contrast, attempts to detect methylated bases positively by using methylcytosine-specific antibodies detected methylated DNA only in heterogeneous linear dsDNA, and methylation-dependent restriction enzymes revealed that the viral heterogeneous linear dsDNA was methylated preferentially.     

Quantitation of heteroplasmy in mitochondrial DNA mutations by primer extension using Vent(R)(exo-) DNA polymerase and RFLP analysis

In this report we describe a simple and rapid protocol for reliable quantitation of mitochondrial DNA (mtDNA) mutations, which is basically a modification of the traditional polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis technique. Up to now, the PCR/RFLP method has been of limited use for the accurate determination of ratios of mutant and wild type molecules, largely owing to the formation of heteroduplex molecules by PCR and incompleteness of restriction digestion. In order to overcome this problem, we have introduced a single-step primer extension reaction using Vent(R)(exo-) DNA polymerase and a fluorescence-labeled primer to the standard assay. The labeled homoduplex molecules are then digested with a restriction endonuclease, and the nucleic acids fractionated on an automated DNA sequencer equipped with GENESCAN analysis software. The amount of mutant mtDNA is readily estimated from fluorescence intensities of the wild-type and mutant mtDNA fragments corrected for incomplete digestion as monitored by a homologous control fragment. The accuracy of the improved protocol was determined by constructing standard curves obtained from defined mixtures of genomic DNA containing homoplasmic wild-type and mutant mtDNA. The expected values were obtained, with an observed correlation coefficient of 0.997 and a typical variability of +/-5% between repeated measurements. Further validation of the protocol is provided by the screening of five patients and unaffected subjects carrying the guanine to adenine transition at the nucleotide 3460 of the mitochondrial genome responsible for the mitochondrial disorder of Leber's hereditary optic neuropathy.     

Mechanism of Replication of Single-Stranded φX174 DNA VII. Circularization of the Progeny Viral Strand

Linear phiX174 single-stranded DNA can be isolated from phiX phage particles produced under various conditions. About half of the linear strands have a dGMP residue at the 5' end, the remaining have roughly comparable amounts of dCMP, dTMP, and dAMP. The linear strands can be converted to covalently closed circular molecules by polynucleotide ligase, but only after they have been incubated with T4 DNA polymerase and deoxynucleoside triphosphates. Experiments with endonuclease R, the restriction enzyme from Haemophilus influenzae, indicated that the nucleotides incorporated into the DNA during this reaction were found predominantly in a limited region of the genome. The results suggest that the normal intermediate in single-stranded phiX174 DNA synthesis may be a single-stranded linear molecule which is shorter than unit length and is intrinsically capable of circularization.     

Rapid and reliable dideoxy sequencing of double-stranded DNA

We report a simple and reliable protocol for nucleotide sequencing using the Sanger dideoxy technique on linearized double-stranded DNA molecules with specific oligonucleotide primers. The method is demonstrated for restriction fragments cloned into the plasmid vectors pSP64 and pSP65 using two vector-specific primers, the M 13 reverse primer and a new SP6 primer, flanking the multiple cloning site. Template DNA may be prepared by a rapid alkaline lysis procedure. Mild linearization conditions with the appropriate restriction endonuclease avoid the appearance of artifact bands.     

In vitro synthesis of double-stranded DNA from the Kilham rat virus single-stranded DNA genome.

Double-stranded, full-length linear DNA was synthesized in vitro by using single-stranded linear DNA as a self-priming template from the parvovirus Kilham rat virus and Escherichia coli DNA polymerase "large fragment" as the polymerizing enzyme. To ascertain the order of the synthesis of the cleavage fragments and to assess the accuracy of the in vitro synthesis, restriction endonuclease cleavage sites with known recognition sequences were mapped on the DNA. Comparing the cleavage pattern of the synthesized DNA with that of double-stranded viral DNA isolated from infected cells confirms that the in vitro synthesis produces a faithful copy of the viral single-stranded genome. Electron micrographs of the in vitro product reveal it to be a double-stranded linear molecule.     

Comparison of the fine structure of mitochondrial DNA from Saccharomyces cerevisiae and S. carlsbergensis: electron microscopy of partially denatured molecules.

Denaturation-maps of mitochondrial DNA from Saccharomyces cerevisiae and S. carlsbergensis have been derived from electron microscopic observations of partially denatured complete circular molecules and large fragments of these circles. The mitochondrial DNA from the two species differ by 6% in total length, but seems from the maps to contain some regions of apparent close homology. The cleavage pattern of the two DNAs by the restriction endonuclease EcoRI is compared by gel electrophoresis.     

系统性红斑狼疮(SLE)患者血清免疫复合物中DNA的提取及电镜观察

从活动期SLE患者血清DNA/抗-DNA免疫复合物中分离DNA,用电镜观察结果表明:这些DNA是很不均质的双链片段。它们的分子量范围很宽,镜下可见的最小片段长553A(约150bp),最大片段长10431A(约2800bp),多数DNA片段长约200—400bp,与正常对照相比较有明显区别。另外,还观察到具有单链末端的双链DNA片段。     

Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase

The DNA polymerase induced after infection of Escherichia coli by bacteriophage T7 can exist in two forms. One distinguishing property of Form I, the elimination of nicks in double-stranded DNA templates, strongly suggests that this form of the polymerase catalyzes limited DNA synthesis at nicks, resulting in displacement of the downstream strand. In this paper, we document this reaction by a detailed characterization of the DNA product. DNA synthesis on circular, duplex DNA templates containing a single site-specific nick results in circular molecules bearing duplex branches. Analysis of newly synthesized DNA excised from the product shows that the majority of the branches are less than 500 base pairs in length and that they arise from a limited number of sites. The branches have fully base-paired termini but are attached by two noncomplementary DNA strands that have a combined length of less than 30 nucleotides. The product molecules are topologically constrained as a result of the duplex branch. DNA sequence analysis has provided an unequivocal structure of one such product molecule. We conclude that strand displacement synthesis catalyzed by Form I of T7 DNA polymerase is terminated by a template-switching reaction. We propose two distinct models for template-switching that we call primer relocation and rotational strand exchange. Strand displacement synthesis catalyzed by Form I of T7 DNA polymerase effectively converts T7 DNA circles that are held together by hydrogen bonds in their 160-nucleotide-long terminal redundancy to T7-length linear molecules. We suggest that strand displacement synthesis catalyzed by T7 DNA polymerase is essential in vivo to the processing of a T7 DNA concatemer to mature T7 genomes.