Evidence for SV40 Specific RNA containing Virus and Host Specific Sequences

AFTER infection of monkey kidney cells with simian virus 40 (SV40), several species of SV40 specific RNA are synthesized¹. Most SV40 RNA have a molecular weight of about 6×10⁵ and 8×10⁵ as measured by polyacrylamide gel electrophoresis¹. In addition to these classes of RNA, a large heterogeneous SV40 specific RNA species of up to three times the length of the monomeric SV40 DNA molecule has been observed^1–4. Nothing is known about the structure of this large heterogeneous virus specific RNA.     

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.     

Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures.

Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10⁻² μm) is smaller than the random coil segment value b (7·17 × 10⁻² μm) was observed, while circular structures of the dimer of the same molecule (12 × 10⁻² μm) were detected.     

Fractionation and molecular weight determination of deoxyribonucleic acid fragments using agarose column chromatography

DNA fragments of several sizes have been produced by shearing E. coli DNA under different pressures. These fragments have been used to demonstrate that column chromatography on agarose Bio-Gel A-15M can provide a rapid, inexpensive fractionation and sizing method for single-stranded nucleic acids having masses between 10⁵ and 10⁶ daltons. Both chromatographic and electrophoretic analysis of the sheared DNA indicated that discrete fragment populations were produced at each shearing pressure and that these fragments were distributed essentially symmetrically around a mean piece size. The average molecular weight of the several DNA fragment distributions was determined electrophoretically by comparison with standard DNA fragments obtained from restriction endonuclease cleavage of SV40 viral DNA. The molecular weights of the denatured, sheared fragments (single-stranded) ranged from 1.25 × 10⁵ to 7.4 × 10⁵. The single-stranded DNA fragments were chromatographed over agarose Bio-Gel A-15M and a linear relationship was found to exist between the mobilities and logarithms of the molecular weights. Readily available tRNA, 5s RNA, and φX174 single-stranded circular DNA chromatographed at the extremes of the linear relationship and could be used to calibrate the column chromatography.     

Homology of plasmids in strains of unicellular Cyanobacteria.

Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron microscopy and agarose gel electrophoresis. The sizes of the various plasmid species were determined; in each of the Synechococcus strains 6301, 6707, and 6908 two plasmid species were found with molecular weights of 5.3 × 10⁶ and 32.7 × 10⁶. Synechococcus strain 7425 had two plasmids of molecular weight 5.4 × 10⁶ and 24 × 10⁶. Synechococcus strain 6312 and Synechocystis strain 7005 each contained one plasmid species with molecular weight of 15.9 × 10⁶ and 2.0 × 10⁶, respectively. Restriction enzyme analysis revealed identical cleavage patterns for the plasmids of identical molecular weight.     

Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardii

The chloroplast DNA of Chlamydomonas reinhardii has been examined by restriction endonuclease analysis. EcoRI, BamHI and BglII produce 30, 17 and 12 fragments, respectively, whose sites have been determined by electron microscopy and by comparative gel electrophoresis. These fragments have been ordered into a circular map which corresponds to a genome size of M_r = 126 × 10⁶. The map was established by comparing the double digests of individual restriction fragments and by hybridizing purified labelled fragments to restriction enzyme digests of chloroplast DNA. The restriction fragments were isolated by molecular cloning or by preparative agarose gel electrophoresis.The two sets of chloroplast ribosomal RNA genes are contained within two inverted repeats of 13 × 10⁶ molecular weight, which are located nearly at opposite sides of the map. In addition, the mapping studies have revealed the presence of short repeated base sequences which are interspersed throughout the chloroplast genome.     

Isolation and preliminary characterization of the small circular DNA present in African green monkey kidney (BSC-1) cells

The small polydisperse circular DNA (spc-DNA) previously identified in SV40-infected African green monkey kidney (BSC-1) cells (M. G. Rush, R. Eason, and J. Vinograd, 1971, Biochim. Biophys. Acta 228, 585–594.) has been isolated in pure form from uninfected cells. This double-stranded, covalently closed circular DNA contains species ranging in molecular weight from about 0.1 to 4 × 10⁶, although most of the molecules are distributed in an apparently polydisperse population with molecular weights of less than 1 × 10⁶. There are approximately 1000 to 2000 covalently closed small DNA molecules per cell, and their average buoyant density does not appear to differ significantly from that of chromosomal and mitochondrial DNAs. This spc-DNA was resolved by polyacrylamide gel electrophoresis into three distinct bands containing comparatively homogeneous circular DNAs with molecular weights of 200,000, 520,000, and 780,000. However, the reassociation rate of in vitro labeled, denatured spc-DNA suggested a molecular complexity in the range of 1 × 10⁸, and the ability of BSC-1 chromosomal DNA to accelerate greatly the reassociation of about one third of this material indicated the presence of some repetitive chromosomal DNA sequences in spc-DNA.     

Isolation and characterization of kinetoplast DNA and RNA of Phytomonas davidi

Kinetoplast DNA networks were isolated from stationary-phase culture forms of Phytomonas davidi. The networks banded in CsCl at a density of 1.699 g/ml and consisted of covalently closed circular molecules. The networks were sensitive to shear forces and exhibited several discrete sedimenting components in neutral and alkaline sucrose. Closed monomeric minicircles were isolated from sonicated networks by alkaline band sedimentation. Closed monomers showed a heterogeneous banding pattern on electrophoresis in acrylamide-agarose gels and had sedimentation coefficients of 20.5 S in alkaline sucrose and 11 S in neutral sucrose. The mean minicircle molecular weight as measured by cospreading with φXRF II was 0.70 × 10⁶ or 1064 nucleotide pairs. Minicircles exhibited a sequence microheterogeneity as evidenced by restriction enzyme analysis, melting analysis, and renaturation kinetics. Network maxicircles were evidenced by the appearance of high molecular weight fragments after restriction with several enzymes and by the existence of supertwisted “edge loops” extending out from the periphery of networks. The maxicircle molecular weight was estimated to be approximately 24 × 10⁶. A purified kinetoplast-mitochondrion fraction was found to contain 9 and 12 S RNA species that comigrated with L. tarentolae 9 and 12 S kinetoplast RNAs.     

ATP Hydrolysis by Restriction Endonuclease from <Emphasis Type="Italic">E. coli</Emphasis> K

We wish to report a puzzling ATPase activity associated with the DNA restriction endonuclease from E. coli strain K^1–3. This enzyme makes a limited number of double chain breaks in DNA molecules lacking the host-controlled modification imparted by strain K. Unmodified DNA molecules from bacteriophage λ, which serve as a convenient substrate, are broken into fragments with a weight average molecular weight of approximately 7 × 10⁶, about one-fifth the size of the intact λ chromosome. The reaction requires Mg2⁺, ATP and S-adenosylmethionine (SAM).     

A freeze-squeeze method for recovering long DNA from agarose gels

Long DNA can be recovered from agarose gels after electrophoresis by freezing the gel slices and manually squeezing out liquid containing the DNA. With this method the recoveries of phage T₇ DNA (molecular weight 25 × 10⁶) and the open and closed forms of circular phage PM2 DNA (molecular weight 6 × 10⁶) were about 70%. Sedimentation analysis shows that the extruded DNA has not sustained double- or single-stranded breaks. The extruded DNA can be used without further purification as substrate for the restriction endonuclease Hind_II,III, from Hemophilus influenzae, for DNA·DNA hybridization and for electron microscopy.     

A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels

Electrophoresis on slab gels containing a linear gradient of polyacrylamide concentration has been used to separate DNA fragments obtained by restriction of viral DNAs. A simple method of preparing gradient gels using a sucrose density-gradient mixer and preexisting slab gel apparatus is described. DNA fragments of molecular weights 7 × 10⁴–14 × 10⁶ have been fractionated on gels of 3.5–7.5% and 2.5–7.5% acrylamide concentration. In addition to the wide range of fragment sizes which may be run on a single gel, a further advantage of the system is that much sharper bands are obtained compared to conventional constant concentration gels, thus improving resolution.In the molecular-weight range below 5 × 10⁶, for bands whose terminal velocities in the polyacrylamide concentration gradient approach zero, an approximately linear relationship holds between the logarithms of the molecular weights of the fragments and the logarithms of the distances they have migrated in the gel. Thus, by choosing a suitable upper limit to the concentration gradient, the gel system provides a method for estimating approximate molecular weights of unknown DNA fragments, by comparing their mobilities to known standards.     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA III. Host Sequences

A preparation of serially passaged simian virus 40 (SV40) DNA, in which at least 66% of the molecules contain covalently linked cellular DNA sequences, was digested to completion with the Hemophilus influenzae restriction endonuclease. Polyacrylamide gel electrophoresis of the digest showed that the majority of the cleavage products migrated as nine classes of fragments, each class defined by a particular molecular weight. These classes of fragments differ in molecular weight from the fragments produced by the action of the same enzyme on plaque-purified virus DNA. Three classes of fragments were present in less than equimolar amounts relative to the original DNA. The remaining six classes of fragments each contain more than one fragment per original DNA molecule. DNA-DNA hybridization analysis (using the filter method) of the isolated cleavage products demonstrated the presence of highly reiterated cell DNA sequences in two of the nine classes of fragments. A third class of fragments hybridized with high efficiency only to serially passaged SV40 DNA; the level of hybridization to plaque-purified virus DNA was low and there was essentially no hybridization with cell DNA immobilized on filters. It is suggested that this class of fragments contains unique host sequences. It was estimated that at least 27% of the sequences in the substituted SV40 DNA molecules studied are host sequences. The majority of these are probably of the nonreiterated type.     

Physical and functional mapping of rat-liver mitochondrial DNA

Summary Rat-liver mitochondrial DNA (mtDNA) contains 2 cleavage sites of the restriction endonuclease XbaI. The molecular sizes of restriction fragments are 6.6×10⁶ and 3.7×10⁶ D. The results of partial cleavage of mtDNA with EcoRI allow the fragment F (0.32×10⁶ D) to be localized in the sequence ABCEGFHDA. The functional map of mtDNA is constructed for two genes of the ATP-ase mRNAs from rat-liver mitochondria. Molecular hybridization shows that the ATPase genes are located in fragment B and in the GEHD area of mtDNA EcoRI cleavage.     

Structural organization of the genome of Dictyostelium discoideum: Analysis by EcoR1 restriction endonuclease

The genome of the cellular slime mold Dictyostelium discoideum has been analyzed by limit digestion with EcoR1 restriction endonuclease. Approximately 15% of the nuclear genome is cleaved into nine discrete fragments as analyzed by agarose gel electrophoresis. These fragments appear to be derived from two nuclear buoyant density satellites, one of which contains sequences coding for ribosomal RNA. The bulk of the nuclear DNA is digested into approximately 7000 fragments with a mean molecular weight of 4 × 10⁶ to 5 × 10⁶. The mitochondrial DNA is digested into four fragments. One of the nuclear bands has been cloned in Escherichia coli using plasmid pSC101 carrying tetracyline resistance. Analysis by renaturation kinetics indicates that it is repeated approximately 200 times per haploid genome and that it is not internally repeated.     

Electric birefringence of restriction enzyme fragments of DNA: optical factor and electric polarizability as a function of molecular weight

The electric birefringence of restriction enzyme fragments of DNA has been investigated as a function of DNA concentration, buffer concentration, and molecular weight, covering a molecular weight range from 80 to 4364 base pairs (bp) (6 × 10⁴–3 × 10⁶ daltons). The specific birefringence of the DNA fragments is independent of DNA concentration below 20 μg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent. At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field at which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law region the rise of the birefringence is accurately symmetrical with the decay for fragments ≤ 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated from a 1/E extrapolation of the high field birefringence data is ?0.028, independent of molecular weight; if a 1/E² extrapolation is used, the optical factor is ?0.023. The induced polarizability, calculated from the Kerr constant and the optical factor, is proportional to the square of the length of the DNA fragments, and inversely proportional to temperature. Saturation curves for DNA fragments ≤ 161 bp can be described by theoretical saturation curves for induced dipole orientation. The saturation curves of larger fragments are broadened, because of a polarization term which is approximately linear in E, possibly related to the saturation of the induced dipole in high electric fields. This “saturated induced dipole” is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp sample is decreased 6°C in an electric field of 8 kV/cm, because the lower charge density of the coil form of DNA makes it more stable in an electric field than the helix form.     

Cleavage of Circular, Superhelical Simian Virus 40 DNA to a Linear Duplex by S1 Nuclease

S(1) nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S(1) nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S(1) nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR(I) restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.     

RNA primers in Simian virus 40 DNA replication. II. Distribution of 5' terminal oligoribonucleotides in nascent DNA.

A cell-free simian virus 40 (SV40) DNA replication system served to study the role of RNA in the initiation of nascent DNA chains of less than 200 nucleotides (Okazaki pieces). RNA-DNA covalent linkages were found to copurify with SV40 replicating DNA. These linkages were identified by transfer of a fraction of the ³²P from the 5′ position of a deoxyribonucleotide to 2′(3′)rNMPs upon either alkaline hydrolysis or RNAase T₂ digestion of SV40 replicating [³²P]DNA. Alkaline hydrolysis also exposed 5′ terminal hydroxyl groups in the nascent DNA which were detected as nucleosides after digestion with P1 nuclease. The RNA-DNA covalent linkages resulted from a population of Okazaki pieces containing uniquely sized oligoribonucleotides covalently attached to their 5′ termini (RNA primers). The density of a portion of the Okazaki pieces in potassium iodide gradients corresponded to a content of 90% DNA and 10% RNA, while the remaining Okazaki pieces appeared to contain only DNA. Incubation of Okazaki pieces with a defined length in the presence of either RNAase T₂ or potassium hydroxide converted about one-third to one-half of them intto a second well defined group of DNA chains of greater electrophoretic mobili y in polyacrylamide gels. The increased mobility corresponded to the removalof at least seven-residues. Since alkaline hydrolysis of similar Okazaki pieces revealed that one-third to one-half of them contained rN-³²P-dN linkages, the oligoribonucleotides must be covalently attached to the 5′ ends of nascent DNA chains. Although the significance of two populations of Okazaki pieces, one with and one without RNA primers, is imperfectly understood, a sizable fraction of nascent DNA chains clearly contained RNA primers.Neither the length of the RNA primer nor the number of RNA primers per DNA chain changed significantly with increasing length of Okazaki pieces. Since the frequency of RNA-DNA junctions found in nascent DNA chains greater than 400 nucleotides was similar to that of Okazaki pieces, the complete excision of RNA primers appears to occur after Okazaki pieces are joined to the 5′ end of growing daughter strands.³²P-label transfer analysis of Okazaki pieces recovered from hybrids with isolated HindII + III restriction fragments of SV40 DNA revealed a uniform distribution of rN-P-dN sequences around the replicating DNA molecule. Therefore, most, if not all, RNA primers serve to initiate Okazaki pieces rather than to initiate DNA replication at the origin of the genome. Moreover, the positions of RNA primers are not determined by a specific set of nucleotide sequences.     

Fine structure of DNA melting curves.

Theoretical calculations predict that the differential melting curves for random polynucleotide sequences having lengths up to several tens of thousands of base pairs have a clear-cut fine structure. This structure appears in the form of multiple narrow peaks 0.3–0.4°C wide on the bell shaped main curve. The differential melting curves have different shapes for different specific sequences. The theory also predicts the disappearance of the fine structure when the length of the sequence increases and when circular, covalently closed DNA is considered instead of the open structure. The predictions of the theory were confirmed by the measurements of differential melting curves for open and covalently closed circular forms of DNA for PM2 phage (N = 10⁴ base pairs) and also for other phage DNA's of different length: T7 (N = 3.8 × 10⁴); S_D (N = 9.2 × 10⁴); T2 (N = 17 × 10⁴). It was shown that the effect of fine structure results mainly from the cooperative melting out of DNA regions 300–500 base pairs long.     

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40². For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.