Characterization of a rearrangement in viral DNA: mapping of the circular simian virus 40-like DNA containing a triplication of a specific one-third of the viral genome

Serial passage of the non-defective form of a simian virus 40-like virus (DAR) isolated from human brain results in the appearance of three distinct classes of supercoiled DNAs: R_I resistant, R_I sensitive (one cleavage site) and R_I “supersensitive” (three cleavage sites). The R_I cleavage product of the “super sensitive” form is one-third the physical size of simian virus 40 DNA (10.4 S) and reassociates about three times more rapidly than “standard” viral DNA. To identify the portions of the DAR genome present in the 10.4 S segment, the plus strand of each of the 11 fragments of ³²P-labeled simian virus 40 DNA, produced by cleavage with the Hemophilus influenzae restriction endonuclease, was hybridized in solution with the sheared R_I cleavage product of the “supersensitive” class of viral DNA. Reaction was observed with fragments located in two distinct regions of the simian virus 40 genome: (1) Hin-A and C; (2) Hin-G, J, F and K.Further studies indicated that simian virus 40 complementary RNA transcribed in vitro with Escherichia coli RNA polymerase from one strand of simian virus 40 DNA reacts with both strands of the denatured 10.4 S cleavage product when hybridization is monitored with hydroxyapatite. Treatment of the 10.4 S DNA-simian virus 40 cRNA hybrid with the single-strand spcific nuclease, S₁, converted approximately 50% of the radioactive counts to an acid-soluble product. These results indicate that the 10.4 S product contains a transposition of sequences originally present on one of the DAR DNA strands to the other strand. Examination of heteroduplexes formed between the 10.4 S segment and unique linear forms of DAR DNA produced with the R · Eco RI restriction endonuclease have confirmed these observations. Thus it appears that a molecular rearrangement(s) has resulted in the recombination and inversion of viral DNA sequences from two separate loci on the parental DAR genome. This 1.1 × 10⁶ dalton segment is reiterated three times in a supercoiled molecule equivalent in physical size to parental DAR DNA.     

An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs.

A procedure for investigating the possibility of small amounts of partial DNA sequence homology between two defined DNA molecules has been developed and used to test for sequence homology between simian virus 40 and polyoma DNAs. This procedure, which does not necessitate the use of separated viral DNA strands, involves the construction of hybrid DNA molecules containing a simian virus 40 DNA molecule covalently joined to a polyoma DNA molecule, using the sequential action of EcoRI restriction endonuclease and Escherichia coli DNA ligase. Denaturation of such hybrid DNA molecules then makes it possible to examine intramolecularly rather than intermolecularly renatured molecules. Visualization of these intramolecularly renatured “snapback” molecules with duplex regions of homology by electron microscopy reveals a 15% region of weak sequence homology. This region is denatured at about 35 °C below the melting temperature of simian virus 40 DNA and therefore corresponds to about 75% homology. This region was mapped on both the simian virus 40 and polyoma genomes by the use of Hemophilus parainfluenzae II restriction endonuclease cleavage of the simian virus 40 DNA prior to EcoRI cleavage and construction of the hybrid molecule. The 15% region of weak homology maps immediately to the left of the EcoRI restriction endonuclease cleavage site in the simian virus 40 genome and halfway around from the EcoRI restriction endonuclease cleavage site in the polyoma genome.     

High-fidelity PCR amplification of infectious copies of the complete simian virus 40 genome from plasmids and virus-infected cell lysates

We describe here a long-polymerase chain reaction (PCR) method that can be used to amplify complete simian virus 40 (SV40) DNA with high fidelity, and we show that authentic, viable virus can be produced from molecular clones of the PCR-amplified viral DNAs. A commercial long-PCR kit that employed a combination of Taq and GB-D polymerases was used, together with a pair of overlapping primers that recognized a unique EcoRI site in the SV40 genome. Efficient amplification required linearization of the circular SV40 genomic DNAs with EcoRI. Entire SV40 genomes were successfully PCR-amplified from an SV40 plasmid and from two different SV40-infected cell lysates and were cloned into pUC-19. Three separate segments of the cloned viral genomes were DNA sequenced, and no nucleotide changes relative to the parental virus were detected, suggesting that the viral DNAs had been amplified with high fidelity. Each PCR clone was infectious, and no differences were detected in the growth characteristics of viruses derived from these clones as compared to the original viral strain. The procedure we utilized shortens and simplifies the molecular cloning of small double-stranded DNA viruses and will be useful for viral diagnostic tests and for recovery of virus from clinical samples. The results of these experiments have broad implications, as the methodology is applicable to many systems.     

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.     

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.     

Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: indications of a phase relation between restriction sites and chromatin subunits in African green monkey and calf nuclei.

The periodicities of the restriction enzyme cleavage sites in highly repetitive DNAs of six mammalian species (monkey, mouse, sheep, human, calf and rat) appear related to the length of DNA contained in the nucleosome subunit of chromatin. We suggest that the nucleosome structure is an essential element in the generation and evolution of repeated DNA sequences in mammals (Brown et al., 1978; Maio et al., 1977). The possibility of a phase relation between DNA repeat sequences and associated nucleosome proteins is consistent with this hypothesis and has been tested by restriction enzyme and micrococcal nuclease digestions of repetitive DNA sequences in isolated, intact nuclei.Sites for four different restriction enzyme activities, EcoRI, EcoRI¹, HindIII and HaeIII have been mapped within the repeat unit of component α DNA, a highly repetitive DNA fraction of the African green monkey. The periodicity of cleavage sites for each of the enzymes (176 ± 4 nucleotide base-pairs) corresponds closely to the periodicity (about 185 nucleotide base-pairs) of the sites attacked in the initial stages of micrococcal nuclease digestion of nuclear chromatin. In intact monkey nuclei, EcoRI-RI¹ sites are accessible to restriction enzyme cleavage; the HindIII and HaeIII sites are not. The results suggest (1) that, in component α chromatin, the EcoRI-RI¹ sites are found at the interstices of adjacent nucleosomes and (2) the HindIII and HaeIII sites are protected from cleavage by their location on the protein core of the nucleosome. This interpretation was confirmed by experiments in which DNA segments of mononucleosomes and nucleosome cores released from CV-1 nuclei by micrococcal nuclease were subsequently treated with EcoRI, EcoRI¹ and HindIII. A major secondary segment of component α, about 140 nucleotide base-pairs in length, was released only by treatment with HindIII, in keeping with the location of the HindIII sites in the restriction map and their resistance to cleavage in intact nuclei.EcoRI reduces calf satellite I DNA to a segment of about 1408 nucleotide basepairs. In contrast, restriction of calf satellite I DNA with EcoRI¹ produces six prominent segments ranging in size from 176 to 1408 nucleotide base-pairs. Treatment of isolated calf nuclei with either EcoRI or EcoRI¹ did not produce segments shorter than 1408 base-pairs, indicating that while canonical EcoRI sites are accessible to attack, the irregularly spaced EcoRI¹ sites are specifically blocked. The results are consistent with a phase relation between the repeat sequence of calf satellite I DNA and an octameric array of nucleosomes.     

Methylation and cleavage sequences of the EcoP1 restriction-modification enzyme.

EcoP1 is a restriction modification enzyme encoded by bacteriophage P1. It requires ATP for cleavage and S-adenosyl methionine for methylation of DNA. We have mapped the sites of both cleavage and methylation in simian virus 40 DNA and determined their sequences. The enzyme methylates the sequence A-G-mA-C-C and cuts the DNA 25 to 27 base-pairs from the site of methylation in the 3′ direction, with a two to four base-pair stagger between cuts. Consistent with the fact that the methylation sequence is asymmetric, the enzyme methylates only one strand in vitro. One variant of simian virus 40 has acquired an additional EcoP1 methylation and cleavage site by changing a A-G-A-A-C sequence to A-G-A-C-C.     

The EcoRI site of simian virus 40 deoxyribonucleic acid: nucleotide sequences of the minus and plus strands.

The nucleotide sequences of the minus and plus strands of simian virus 40 DNA in a 17-base-pair segment spanning the EcoRI restriction site have been identified. The minus-strand sequence is (5′) T-G-G-C-G-A-G-A-A-T-T-C-C-T-T-T-G and the plus-strand sequence is its complement: (5′) C-A-A-A-G-G-A-A-T-T-C-T-C-G-C-C-A.     

Preparation of large quantities of separated strands from simian virus 40 DNA restriction fragments by low-temperature low-salt agarose gel electrophoresis.

We have used agarose gel electrophoresis to separate complementary DNA strands obtained from simian virus 40 DNA restriction fragments produced by HindII and III or by EcoRI and HpaII digestion. By modifying existing methods we have virtually eliminated the problematic renaturation of DNA during electrophoresis. This has allowed us to recover large quantities of separated DNA strands (approximately 20 μg of DNA per 12-mm-diameter preparative tube gel). By using a combination of low temperature and low buffer concentration during electrophoresis, we have also significantly improved the resolution of DNA strands.     

Loss of Polyoma Virus Infectivity as a Result of a Single Amino Acid Change in a Region of Polyoma Virus Large T-Antigen Which Has Extensive Amino Acid Homology with Simian Virus 40 Large T-Antigen

The polyoma virus (Py) transformed cell line 7axB, selected by in vivo passage of an in vitro transformed cell, contains an integrated tandem array of 2.4 genomes and produces the large, middle, and small Py T-antigen species, with molecular weights of 100,000, 55,000, and 22,000, respectively (Hayday et al., J. Virol. 44:67-77, 1982; Lania et al., Cold Spring Harbor Symp. Quant. Biol. 44:597-603, 1980). The integrated viral and adjacent host DNA sequences have been molecularly cloned as three EcoRI fragments (Hayday et al.). One of these fragments (7B-M), derived from within the tandem viral sequences, is equivalent to an EcoRI viral linear molecule. Fragment 7B-M has been found to be transformation competent but incapable of producing infectious virus after DNA transfection (Hayday et al.). By constructing chimerae between 7B-M and Py DNA and by direct DNA sequencing, the mutation responsible for the loss of infectivity has been located to a single base change (adenine to guanine) at nucleotide 2503. This results in a conversion of an aspartic acid to a glycine in the C-terminal region of the Py large T-antigen but does not appear to affect the binding of the Py large T-antigen to Py DNA at the putative DNA replication and autoregulation binding sites. The mutation is located within a 21-amino acid homology region shared by the simian virus 40 large T-antigen (Friedmann et al., Cell 17:715-724, 1979). These results suggest that the mutation in the 7axB large T-antigen may be involved in the active site of the protein for DNA replication.     

G4 DNA replication: I. Origin of synthesis of the viral and complementary DNA strands

The break in the complementary DNA strand of early G4 replicative form II DNA (RFII) and in the viral DNA strand of late RFII DNA was located using two single cleavage restriction enzymes (EcoRI and PstI) and by limited nick translation of the break using DNA polymerase I and ³²P-labelled deoxyribonucleotides followed by digestion with the restriction enzymes HaeIII and HindII. The break in the complementary DNA strand was unique and in HaeIII Z5 close to the EcoRI cleavage site whereas the break in the viral DNA strand was on the other side of the molecule in HaeIII Z2 approxiately 50% away from the EcoRI cleavage site. Distribution of a short ³H pulse in early G4 replicating intermediates that were synthesising both DNA strands at the same time showed that synthesis of the strands started on opposite sides of the molecule and proceeded in opposite convergent directions, suggesting that initiation of synthesis of the two strands was independent and not unified in a single growing fork.     

Staphylococcal nuclease makes a single non-random cut in the simian virus 40 viral minichromosome.

When compact simian virus 40 (SV40) minichromosomes are treated with staphylococcal nuclease at 0 °C under limit-digest conditions, about one-third of the minichromosomes remain resistant to nuclease, a third of them are nicked, while the remaining third suffer one and only one double-stranded cut. Results show that each cleaved minichromosome is cut only once and afterwards becomes resistant to further fragmentation. This is in marked contrast to the action of staphylococcal nuclease at 37 °C, which leads to a rapid fragmentation of all minichromosomes to oligo- and mononucleosomes.The SV40 linear DNA III produced by low-temperature nuclease digestion of minichromosomes was redigested with single-cut restriction endonucleases. By this mapping procedure it was determined that the location of the staphylococcal nuclease cut is neither unique nor random; it occurs at a number of discrete sites on the DNA, half of all cuts being concentrated at the origin of replication and nearby in the “late” portion of the SV40 genome. Control experiments have shown that when staphylococcal nuclease digests naked SV40 DNA at 0 °C it does not “hesitate” after the first cut. Although initial cuts in the purified DNA are non-random in location, their distribution is quite different from that generated by a low-temperature nuclease digestion of compact SV40 minichromosomes. Possible interpretations of these results are discussed in view of the recent finding that a specific region of the SV40 genome is uniquely exposed in the minichromosome (Varshavsky et al., 1978, 1979; Scott &; Wigmore, 1978).     

Studies on the organization of the α-satellite DNA from African green monkey cells using restriction nucleases and molecular cloning

α-Satellite DNA from African green monkey cells was analysed with restriction nucleases in some detail confirming and complementing our earlier results. With EcoRI and HaeIII (or BsuRI isoschizomer), about 25 and 10%, respectively, of the satellite DNA were cleaved into a series of fragments of the 172 bp repeat length and multiples thereof. To allow studies with fragments of homogeneous sequence unit length, HindIII fragments were covalently joined with the plasmid pBR313. After transformation 19 clones were obtained, containing up to three monomer fragments. Nine of the clones were characterized by digestion with EcoRI. Three of these had cleavage sites for this nuclease in the satellite DNA portion. In the six clones tested with HaeIII no cleavage site was detected in the cloned DNA. The results are discussed in relation to the nucleotide sequence data recently published by Rosenberg et al. (1978) and in the context of random and nonrandom processes in satellite DNA evolution.     

Physical map of the BK virus genome.

Two new human papovavirus isolates (JMV and MMV) from the urines of patients with Wiskott-Aldrich syndrome were morphologically and serologically identical to BK virus (BKV). The genomes of these two new isolates were found to be indistinguishable from prototype BKV DNA in a variety of nucleic acid hybridization experiments. Like BKV DNA, JMV and MMV DNAs share approximately 20% of their polynucleotide sequences with simian virus 40 DNA. The genome of JMV was indistinguishable from that of BKV by restriction endonuclease analysis; MMV DNA contained three instead of four R-Hind cleavage sites and one rather than no R-HpaII cleavage sites. Physical maps of the BKV and MMV genomes were constructed using restriction endonucleases, and these maps were oriented to the map of simian virus 40 DNA.     

Detection of sequence heterology by use of the N. Crassa nucleases

We have used the single-strand specific nucleases of $\text{Neurospora crassa}$ to detect sequence divergencies between two similar DNA molecules: restriction endonuc lease $\text{Eco}$RI produced linears from Simian Virus 40 and a variant of human origin, DAR. Enzyme treatment of the heteroduplex DNA resulted in specific cleavage into two fragments of one-third and two-thirds genome length. These two viral DNAs therefore have at least one region of heterology located about 0.35 map units from the $\text{Eco}$RI site. Due to the known specificities of the $\text{N.crassa}$ nucleases, this technique is applicable to detect mutations in RNA or DNA genomes.     

Restriction insensitivity in bacteriophage T5. III. Characterization of EcoRI-sensitive mutants by restriction analysis.

Despite the fact that its DNA carries six EcoRI cleavage sites, bacteriophage T5 is able to grow on an EcoRI restricting host, suggesting that it specifies a restriction protection system. In the hope of identifying this protection system, mutants of T5 have been isolated which are unable to grow on an EcoRI restricting host. Analysis of the DNA of such mutants shows that they have each acquired two new EcoRI sites per molecule as a consequence of a single EcoRI site (ris) mutation located in the terminally repetitious, first step transfer (FST) region of the genome. The EcoRI sites generated by the ris mutations differ from the natural EcoRI sites in that the latter are situated on the second step transfer (SST) DNA, which suggests that the in vivo sensitivity of ris mutants is a consequence of having an EcoRI site on the FST DNA. This is understandable, if the hypothetical restriction protection genes are also located on the FST DNA. While expression of these genes would protect natural sites on the SST DNA, the ris sites would, on the contrary, enter an environment in which the protection, products had not yet been synthesized.Construction of double and triple ris mutants has allowed the ordering of the ris sites and the construction of an EcoRI restriction map of the FST region. In addition, the ris mutants allow estimation of the size of the terminal repetition of T5 DNA as 5.9 × 10⁶ to 6.0 × 10⁶ daltons. Correlation of the physical map of the FST region with the already established genetic map of this region allows orientation of the pre-early genes on the genetic and physical maps, and approximate localization of two amber mutations on the physical map.     

Restriction and modification enzymes detect no allosteric changes in DNA with bound lac repressor or RNA polymerase

A 203 base-pair fragment containing the lac operator/promoter region of Escherichia coli was inserted into the EcoRI site of the plasmid vector pKC7. Rates of restriction endonuclease cleavage of the flanking EcoRI sites and of several other restriction sites on the DNA molecule were then compared in the presence and absence of bound RNA polymerase or lac repressor. The rates were identical whether or not protein had been bound, even for sites as close as 40 base-pairs from a protein binding site. No difference was detected using supercoiled, nicked circular, or linear DNA substrates. No apparent change in the rates of methylation of EcoRI sites by EcoRI methylase was produced by binding the regulatory proteins.     

A simplified method for mapping deletion/substitution mutants of bacteriophage lambda.

A convenient and rapid method for mapping deletion/substitution mutants of phage λ is presented. The method involves: (a) forming heteroduplex DNA molecules between the wild-type and the mutant: (b) digestion of the single-stranded regions with S₁ nuclease; (c) cleavage of a portion of the remaining duplex DNA with EcoRI nuclease or any convenient restriction endonuclease; and (d) separation of the resulting DNA fragments by gel electrophoresis. Using three deletion/substitution mutants with known endpoints, we show that the values obtained by this method deviate, on the average, by ±120 base pairs from the values obtained by electron microscopy.     

Propagation of a segment of bacteriophage lamda-DNA in monkey cells after covalent linkage to a defective simian virus 40 genome.

A 520 base pair DNA segment was excised from the bacteriophage lamda-genome by cleavage with the bacterial restriction endonuclease, endo R. Hindll. This segment was covalently joined in vitro to an 880 base pair simian virus 40 (SV40) DNA segment which contains the initation site for SV40 DNA replication. The latter segment was derived from the genome of a defective reiteration mutant of SV40 also by endo R. Hindlll cleavage. When the recombinant molecule, together with wild-type SV40 DNA as helper, was introduced into monkey cells by DNA infection, replication of the lamda-DNA sequences was observed, and hybrid genomes were encapsidated into progeny SV40 virions. The structure of the lamda-DNA segment after serial passage in monkey cells was examined by use of restriction endonucleases and electron microscopic heteroduplex analysis.