Molecular weight of DNA from four entomopoxviruses determined by electron microscopy.

DNA was isolated from entomopoxviruses infected Amsacta moorei and Euxoa auxiliaris (Lepidoptera), Goeldichironomus holoprasinus (Diptera), and Othnonius batesi (Coleoptera) and compared with vertebrate virus DNA (vaccinia). After incubation in Pronase, sodium lauryl sulfate, and deoxycholate, poxvirus preparations shadowed with platinum and palladium revealed subcore particles 45 to 60 nm in diameter. Continued incubation in Pronase resulted in the gradual release of DNA from the particles. Metal-shadowed DNA molecules were photographed in the electron microscope and measured, and the average molecular weights were calculated. Lepidopteran poxvirus DNA (135 X 10(6)) was approximately equal to vaccinia DNA (131.7 X 10(6)) in molecular weight. The molecular weight of dipteran and coleopteran poxvirus DNA (200 X 10(6) to 251 X 10(6)) was approximately 50% greater than vaccinia DNA. Based on the concentration of DNA and protein per virion, Amsacta entomopoxvirus contained 5.7 to 7.7% DNA.     

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA

The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10⁶. Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.     

Determination of the sedimentation coefficient of vaccinia virus DNA by analytical ultracentrifugation

By releasing intact molecules of vaccinia virus DNA, it has been possible to determine the sedimentation coefficient by analytical ultracentrifugation. The value found was 92S, which corresponds to a molecular weight of 161×10⁶.     

Strand-Length Measurements of Normal and 5-Iodo-2′-Deoxyuridine-treated Vaccinia Virus Deoxyribonucleic Acid Released by the Kleinschmidt Method

Purified vaccinia virus, which had been grown on chick-embryo chorioallantoic membranes in the presence or in the absence of 5-iodo-2′-deoxyuridine (IUdR), was suspended in 5 m ammonium acetate and subjected to the one-step Kleinschmidt procedure on surfaces of distilled water or salt solutions. Deoxyribonucleic acid (DNA) molecules were clearly revealed, and in many instances accurate length measurements could be made. The longest continuous molecules from normal virus measured 78, 77, and 65 μ. The most frequent length was approximately 30 μ, which corresponds to only one-third to one-half of the total DNA per virus particle predicted from various chemical analyses. These data provide direct evidence that normal vaccinia DNA may occur as a linear molecule of approximately 150 × 10⁶ molecular weight units, but, for reasons still unknown, the majority of these molecules appears to break into segments of equal length during release from the virion. There is no evidence for the presence of cyclic DNA. The DNA molecules are typically double-stranded. DNA from IUdR-treated vaccinia presents a markedly different picture: the molecules are mostly fragmented into small pieces, and rosettes or tangled masses equivalent to even one-quarter the length of normal molecules occur very rarely. The possibility is discussed that at least part of the virus-inhibitory effect of IUdR on vaccinia is due to extensive fragmentation of the DNA molecules into which IUdR has been incorporated in place of thymidine.     

HindIII and Sst I restriction sites mapped on rabbit poxvirus and vaccinia virus DNA.

The DNAs of two closely related orthopoxviruses, rabbit poxvirus (RPV) and vaccinia virus (VV), were mapped by overlapping-fragment analysis using restriction endonucleases HindIII and Sst I. The exact arrangement of these fragments was accomplished by total digestion of isolated partial restriction products and by end-fragment determination. RPV and VV DNAs showed identical restriction patterns in an internal region comprising approximately 60% of the genome. The size, by electrophoretical analysis of the RPV DNA, was 118 X 10(6) daltons, some 6 X 10(6) daltons less than VV DNA. The two opposite terminal restriction fragments of RPV DNA cross-hybridized to each other.     

Simple preparation of parapoxvirus genome DNA for endonuclease analysis

We compared five methods for improved extraction of very-large parapoxvirus DNA from infected cells: (i) alkaline-lysis procedure followed by phenol extraction; (ii) modified Hirt procedure, which was a neutral lysis procedure followed by phenol extraction; (iii) Hirt procedure; (iv) method used for extraction of vaccinia virus DNA; and (v) standard procedure using virus purification with an ultracentrifuge and protease-sodium dodecyl sulfate-phenol treatment. The alkaline-lysis procedure was more rapid, inexpensive and simpler than the other methods. Moreover, with this method it is not necessary to prepare any special facilities, reagents and kits. Although the extracted DNA was still crude, we could reproducibly prepare viral DNA from 2 X 10(6) infected cells in less than 2 hr and it could be readily digested by restriction endonuclease. This method will aid rapid genetic classification of parapoxvirus.     

High-frequency genetic recombination and reactivation of orthopoxviruses from DNA fragments transfected into leporipoxvirus-infected cells

Poxvirus DNA is not infectious because establishing an infection requires the activities of enzymes packaged in the virion. This barrier can be overcome by transfecting virus DNA into cells previously infected with another poxvirus, since the resident virus can provide the trans-acting systems needed to reactivate transfected DNA. In this study we show that cells infected with a leporipoxvirus, Shope fibroma virus (SFV), can reactivate vaccinia virus DNA. Similar heterologous packaging systems which used fowlpox-infected cells to reactivate vaccinia virus have been described, but SFV-infected cells promoted a far more efficient reaction that can produce virus titers exceeding 10(6) PFU/ micro g of transfected DNA. SFV-promoted reactions also exploit the hyperrecombinogenic systems previously characterized in SFV-infected cells, and these coupled recombination and reactivation reactions could be used to delete nonessential regions of the vaccinia virus genome and to reconstruct vaccinia virus from overlapping DNA fragments. SFV-catalyzed recombination reactions need only two 18- to 20-bp homologies to target PCR amplicons to restriction enzyme-cut vaccinia virus vectors, and this reaction feature was used to rapidly clone and express a gene encoding fluorescent green protein without the need for plaque purification or selectable markers. The ability of SFV-infected cells to reactivate fragments of vaccinia virus was ultimately limited by the number of recombinational exchanges required and one cannot reconstruct vaccinia virus from multiple PCR fragments spanning essential portions of the genome. These observations suggest that recombination is an integral part of poxvirus reactivation reactions and provide a useful new technique for altering the structure of poxvirus genomes.     

Cells of pea (Pisum sativum) that differentiate from G2 phase have extrachromosomal DNA.

Velocity sedimentation in an alkaline sucrose gradient of newly replicated chromosomal DNA revealed the presence of extrachromosomal DNA that was not replicated by differentiating cells in the elongation zone. The extrachromosomal DNA had a number average molecular weight of 12 X 10(6) to 15 X 10(6) and a weight average molecular weight of 25 X 10(6), corresponding to about 26 X 10(6) and 50 X 10(6) daltons, respectively, of double-stranded DNA. The molecules were stable, lasting at least 72 h after being formed. Concurrent measurements by velocity sedimentation, autoradiography, and cytophotometry of isolated nuclei indicated that the extrachromosomal molecules were associated with root-tip cells that stopped dividing and differentiated from G2 phase but not with those that stopped dividing and differentiated from G1 phase.     

Interferon-mediated, double-stranded RNA-dependent protein kinase is inhibited in extracts from vaccinia virus-infected cells

The interferon-inducible, double-stranded RNA (dsRNA)-dependent protein kinase which phosphorylates an endogenous HeLa 69 kilodalton polypeptide or exogenous initiation factor eIF2 was inhibited during vaccinia virus infection. High interferon doses (20,000 reference units per ml) did not prevent this inhibition. The inhibition required protein synthesis but not viral DNA synthesis during infection, suggesting that an early vaccinia virus gene function was responsible. An active dsRNA-dependent protein kinase could be recovered from an inactive extract by purification on polyinosinate X polycytidylate-cellulose. An inhibitor of the protein kinase, therefore, must be present in the inactive extract. Similar results have been obtained with mouse L929 cells. At early time points of infection, the protein kinase in cell extracts required exogenous dsRNA for activity. This argues against endogenous viral dsRNA and activation of the kinase in the intact cell. At late time points of infection (when vaccinia virus dsRNA was almost certainly formed), the inhibitor of the kinase is present. Accordingly, it seems unlikely that the kinase played any role in the interferon-mediated inhibition of virus growth observed in these cells under these particular conditions.     

Epstein-Barr virus DNA. IX. Variation among viral DNAs from producer and nonproducer infected cells.

A comparative analysis of three Epstein-Barr virus DNAs from American patients with infectious mononucleosis (B95-8, Cherry, and Lamont) and four Epstein-Barr virus DNAs from African patients with Burkitt lymphoma (AG876, W91, Raji, and P3HR-1) indicated that the usual format of Epstein-Barr virus DNA includes a variable number of direct repeats of a 0.35 X 10(6)-dalton sequence (TR) at both ends of the DNA, a 9 X 10(6)-dalton sequence of largely unique DNA (Us), a variable number of repeats of a 2 X 10(6)-dalton sequence (IR), and a 89 X 10(6)-dalton sequence of largely unique DNA (UL). Within UL there was homology between DNA at 26 X 10(6) to 28 X 10(6) daltons and DNA at 93 X 10(6) to 95 X 10(6) daltons. The relative sequence order (TR, US, IR, UL, TR) did not vary among "standard" Epstein-Barr virus DNA molecules of each isolate. B95-8 DNA had an unusual deletion extending from 91 X 10(6) to 100 X 10(6) daltons, and P3HR-1 DNA had an unusual deletion extending from 23.5 X 10(6) to 26 X 10(6) daltons. There was sufficient variability among the EcoRI and BamHI fragments of the DNAs to identify each isolate specifically. However, we discerned no distinguishing features for the two geographic or pathogenic origins of the seven isolates. Three intracellular DNAs (Raji, Lamont, and Cherry) and one virion DNA (P3HR-1) were heterogenous in molecular organization and had subpopulations of rearranged or defective molecules. Some regions, particularly 59 X 10(6) to 63 X 10(6) daltons and sequences around TR, frequently participated in rearrangements. Restriction endonuclease maps of the standard and rearranged DNAs of the seven isolates are presented.     

Excision and replication of extrachromosomal DNA of pea (Pisum sativum).

Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80% of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 X 10(6) to 140 X 10(6) daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 X 10(6) daltons and another of 7 X 10(6) daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 X 10(6) and 7 X 10(6) daltons.     

Use of a restriction endonuclease in analyzing the genomes from two different strains of vaccinia virus.

A restriction endonuclease from Haemophilus influenzae (Hind III) specifically cleaved vaccinia DNA into 14 fragments. The molecular weights of these fragments were determined by gel electrophoresis and ranged from 0.5 x 10(6) to 30 x 10(6). Hind III digestion of the DNA from the WR and CV-1 strains of vaccinia revealed a small molecular difference in one of the resulting fragments. The average molecular weight of the entire vaccinia genome was calculated to be 125 x 10(6).     

Characterization of two types of yeast ribosomal DNA genes.

The intragenic organization of ribosomal DNA from a diploid strain of Saccharomyces cerevisiae was analyzed by using recombinant DNA molecules constructed in vitro. Restriction analysis of the yeast ribosomal DNA with the EcoRI restriction enzyme indicated that eight restriction fragments were present in the ribosomal DNA of this strain: X' (1.87 X 10(6) daltons), A (1.77 X 10(6) daltons), B (1.48 X 10(6) daltons), C (1.22 X 10(6) daltons), D (0.39 X 10(6) daltons), E (0.36 X 10(6) daltons), F (0.22 X 10(6) daltons), and G (0.17 X 10(6) daltons). These fragments were distributed between two different types of ribosomal DNA genes, which had the restriction maps: (formula: see text) in which the underlined region shows the repeating unit. The diploid yeast strain contained approximately equal amounts of each of these two types of genes. The analysis of the recombinant DNA molecules also indicated that the yeast ribosomal genes are homogeneous and extensively clustered.     

Restriction enzyme digests of rapidly renaturing fragments of vaccinia virus DNA.

Vaccinia virus DNA fragments that have been denatured by alkali and then neutralized contain a fraction that rapidly reforms duplex structures. The fraction is enriched by fractionating on hydroxyapatite columns and serves as as substrate for digestion by two restriction endonucleases isolated from Hemophilus parainfluenzae, Hpa I and HPa II. The patterns obtained by gel electrophoresis of the digested fragments show the presence of three major bands after Hpa I digestion and four major bands after Hpa II digestion. The DNA that is isolated from some of these bands quickly reforms duplex regions after alkaline denaturation. The size of the DNA segments in the major bands has been estimated to be in the range of 0.44 X 10(6) to 3.2 X 10(6) daltons. The fragments which rapidly reform duplex chains after denaturation are sensitive to single-strand-specific nucleases. These results are consistent with a model of vaccinia virus DNA which has a covalent link connecting complementary chains.     

Radiation-induced double-strand scission of the DNA of mammalian metaphase chromosomes

The molecular weight distribution of double-stranded mammalian (hamster) DNA was determined by ultracentrifugation of isolated metaphase chromosomes previously layered onto sucrose gradients containing high salt concentrations to dissociate the protein and nucleic acid components. In untreated controls the distribution (as determined by counting the incorporated radioactivity in the resultant fractions) exhibited a peak at 225 X 10(6) daltons. Inclusion of mercaptoethanol and hydroxylamine into the gradients produced no significant change of these sedimentation patterns. Gamma-radiation-induced reduction in the number and weight average molecular weights was used to calculate a value of 1.05 X 10(11) double-strand breaks/gram rad, equivalent to about 600 eV/break. No significant difference was observed for chromosomes irradiated either before or after isolation from intact mitotic cells. Irradiation in the presence of cystamine resulted in at least a sevenfold reduction in the apparent double-strand scission. The observed sedimentation patterns were compared with those generated by a theoretical computer simulation of radiation-induced degradation which assumed random selection and breakage of molecules. These results suggested that at least 80 to 90 % of the isolated DNA was distributed approximately normally with a mean molecular weight of about 200 X 10(6) daltons and a standard deviation of about 50 X 10(6) daltons.     

DNA of Epstein-Barr virus. VI. Mapping of the internal tandem reiteration.

Epstein-Barr virus (B95-8) DNA consists of short (10 X 10(6)) and long (87 X 10(6)) unique DNA sequences joined by 10 tandem reiterations of a 1.85 X 10(6) DNA segment. The reiterated sequence contains BamI and BglII sites separated by 4 X 10(5). The 4.5 X 10(5) and 14.0 X 10(5) segments generated by cleavage of the reiterated DNA with BamI and BglII contain sequences which hybridize to each other, suggesting that the internal tandemly reiterated sequence has a direct or inverted repeat within it. The opposite ends of the linear, nicked, double-stranded DNA molecule (R. F. Pritchett, S. D. Hayward, and E. D. Kieff, J. Virol. 15:556--569, 1975) consist of from 1 to 12 direct repeats of another 3 X 10(5) sequence (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; D. Given, D. Yee, K. Griem, and E. Kieff, J. Virol. 30:852--862, 1979). There is no homology between the internal reiterated sequence and either terminus. However, part of the internal reiteration (less than 5 X 10(5) is reiterated at two separate locations in the long unique region. The internal reiterations are a source of variation within EBV (B95-8) DNA preparations. Thus, although the majority of molecules contain 10 tandem reiterations, some molecules have 9, 8, 7, 6, 5, 4, or fewer tandem reiterations. A consequence of this variability is that the KpnI A fragment and the EcoRI/Hsul A fragment consist of a family of seven or more fragments differing in the number of tandem internal reiterations. The EcoRI/HsuI A fragment of EBV (W91) DNA is approximately 6 X 10(6) smaller than the largest and dominant EcoRI/HsuI A fragment of EBV (B95-8) DNA. EBV (W91 DNA also differs from EBV (B95-8) DNA by an additional 7 X 10(6) to 8 X 10(6) of DNA in the long unique DNA region (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388--398, 1978). These data suggest the possibility that the smaller number of internal reiterations in EBV (W91) DNA may be a consequence of the additional unique DNA and a restriction in the overall size of EBV DNA.     

Naturally occurring cross-links in yeast chromosomal DNA.

Chromosome-size yeast DNA molecules with a number average molecular weight (Mn) of 3-4 X 10(8) were isolated from sucrose gradients after sedimentation of lysed yeast spheroplasts. Resedimentation showed that the molecules were isolated without introducing appreciable single-strand or double-strand breaks. The presence of cross-links in these molecules was suggested by the observation that the apparent Mn in alkali was greater than expected for separated single strands. Since cross-linked molecules would have strands which fail to separate upon denaturation, this was tested more directly. Neutralization of alkaline denaturing conditions resulted in up to 70% of the intact molecules rapidly reforming duplex structures, as shown by equilibrium banding in CsCI. Experiments with larger E. coli DNA molecules (Mn = 5.2 X 10(8)) indicated that the conditions used were sufficient to denature completely molecules of this size. Results of enzyme treatments suggest that the cross-links are not RNA or protein. Experiments with density-labeled yeast DNA molecules showed that the rapid reformation of duplex DNA is not the consequence either of a bimolecular reaction between separated DNA strands or of intrastrand renaturation. The data indicate that when the yeast DNA molecules are completely denatured, the strands fail to separate. Hence they must be cross-linked. Experiments with sheared DNA show that there are small number of cross-links, one to four, permolecule.     

Herpes simplex virus type 1 Angelotti and a defective viral genotype: analysis of genome structures and genetic relatedness by DNA-DNA reassociation kinetics.

DNA-DNA reassociation kinetics of herpes simplex virus type 1 Angelotti DNA and a class of defective viral DNA revealed that the viral standard genome has a total sequence complexity of about 93 X 10(6) daltons and that a portion of 11 X 10(6) daltons occurs twice on the viral genome. These results agree with structural features of herpes simplex virus type 1 DNA derived from electron microscopic studies and restriction enzyme analyses by several investigators. The defective viral DNA (molecular weight, about 97 X 10(6)) displays a sequence complexity of about 11 X 10(6) daltons, suggesting that the molecule is built up by repetitions of standard DNA sequences comprising about 15,000 base pairs. A 2 X 10(6)-dalton portion of these sequences maps in the redundant region and a 9 X 10(6)-dalton portion maps in the unique part of the standard herpes simplex virus type 1 Angelotti DNA, as could be shown by reassociation of viral standard DNA in the presence of defective DNA and vice versa. No cellular DNA sequences could be detected in defective DNA. A 12% molar fraction of the defective DNA consists of highly repetitive sequences of about 350 to 500 base pairs in length.     

Analysis of Mycoplasma hyorhinis genome by use of restriction endonucleases and by electron microscopy.

The chromosome of Mycoplasma hyorhinis was analyzed by using different restriction endonucleases and electron microscopy. It was found that restriction enzymes BstEII, XhoI, and SacI are the enzymes of choice for analysis and characterization of M. hyorhinis. The bands resulting from digestion of M. hyorhinis DNA with BstEII had apparent molecular weights ranging from 1.2 X 10(6) to 75 X 10(6). The apparent total molecular weight of DNA was calculated from the molecular weights of the individual bands and found to be 251 X 10(6). Electron microscopic contour length measurements of the largest DNA fragments verified the molecular weight values calculated from gel analysis. Electron microscopic contour length measurements of intact DNA of M. hyorhinis revealed a molecular weight of 5.4 +/- 5 X 10(8). The discrepancy between the values of molecular weight of M. hyorhinis DNA as determined by restriction enzyme analysis and contour length measurement is based on the fact that some of the DNA fragments which migrate as an apparent single band in the agarose gel really are double or multiple DNA fragments.     

Irradiation damage in chromatin isolated from V-79 Chinese hamster lung fibroblasts

The effect of chromatin structure on the extent of radiation damage induced by low doses of 100 KeV X rays was investigated using a fluorescent assay for DNA unwinding. Chromatin was isolated from V-79 Chinese hamster lung fibroblast nuclei by partial digestion with micrococcal nuclease. Gel electrophoresis of the isolated DNA showed the molecular weight of the chromatin preparation to be 10.6 X 10(6) with a size range of 6.6-21.7 X 10(6) Da while a size of 10.2 +/- 0.9 X 10(6) Da was found by sedimenting the DNA in alkaline sucrose gradients. The repeat length of V-79 chromatin was found to be 194 +/- 3 bp. The typical nucleosomal repeat structure of the isolated chromatin and that of intact nuclei was identical. Irradiation with 50 and 100 Gy of 100 KeV X rays and analysis by alkaline sucrose density centrifugation indicated that V-79 chromatin sustained 0.56 +/- 0.19 and 0.69 +/- 0.09 single-strand breaks per 10 Gy per 10(8) Da of DNA, respectively. Irradiation with doses of 0.5-3.0 Gy of 100 KeV X rays and analysis by the fluorometric assay showed that the radiation sensitivity of V-79 chromatin decreases sharply on compaction with MgCl2. Histone H1 depletion, which inhibits compaction and causes chromatin to expand by increasing the linker from 26 to 48 bp, results in a considerable increase in the radiation sensitivity. It is concluded that radiation damage sustained by DNA is greatly influenced by chromatin structure.