Studies of simian virus 40 DNA. VII. A cleavage map of the SV40 genome

A physical map of the Simian virus 40 genome has been constructed on the basis of specific cleavage of Simian virus 40 DNA by bacterial restriction endonucleases. The 11 fragments produced by enzyme from Hemophilus influenzae have been ordered by analysis of partial digest products and by analysis of an overlapping set of fragments produced by enzyme from Hemophilus parainfluenzae. In addition, the single site in SV40 DNA cleaved by the Escherichia coli R_I restriction endonuclease has been located. With this site as a reference point, the H. influenzae cleavage sites and the H. parainfluenzae cleavage sites have been localized on the map.     

Characterization and sequence analysis of a recombination site in the hybrid virus Ad2+ND.

Restriction endonuclease mapping of an adenovirus-simian virus 40 hybrid virus and adenovirus-2 DNA allowed the characterization of fragments of Ad2⁺ND₁² which contain the two junctions between simian virus (SV40) sequences and adenovirus-2 sequences. The corresponding fragments of Ad2⁺⁺ DNA were also characterized. One fragment of Ad2⁺ND₁ containing a recombination site, and the corresponding fragment of Ad2⁺⁺ were analyzed by direct DNA sequence analysis. Comparison of nucleotide sequences in Ad2⁺⁺, Ad2⁺ ND₁, and SV40 DNAs precisely localized those sequences involved in the final recombination event which produced the stable hybrid virus Ad2⁺ND₁. No sequence homology was detected between the two parent DNAs.     

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.     

Structure of two adenovirus-simian virus 40 hybrids which contain the entire SV40 genome

Two defective adenovirus-simian virus 40 hybrids which contain the entire SV40 genome (Ad2⁺⁺HEY and Ad2⁺⁺LEY)² have been isolated. Upon infection of cells permissive for SV40 both hybrids give rise to infectious SV40 virions, but with markedly different efficiencies. In the case of Ad2⁺⁺HEY nearly all cells infected with a hybrid particle yield SV40 progeny, whereas in the case of Ad2⁺⁺LEY infectious SV40 is produced in only about one in 10⁴ cells infected with hybrid particles. The structures of the DNA molecules in the Ad2⁺⁺HEY and Ad2⁺⁺LEY populations were examined using electron microscope heteroduplex methods. Both populations were found to be heterogeneous. Ad2⁺⁺HEY contained three hybrids (HEY-I, HEY-II, and HEY-III) whose genomes differed only in their content of SV40 DNA (0.45 ± 0.02, 1.43 ± 0.04, and 2.39 ± 0.09 SV40 genomes, respectively). Ad2⁺⁺LEY contained two hybrids (LEY-I and LEY-II), which also differed only in their content of SV40 DNA (0.03 ± 0.01 and 1.05 ± 0.01 SV40 genomes, respectively). In those hybrids which contained more than one complete SV40 genome (HEY-II, HEY-III, LEY-II) the excess SV40 DNA was shown to be organized as a tandem repetition. These data suggest that the various hybrid genomes within each population are interconvertible by recombination events, which insert or excise an SV40 genome. It is proposed that HEY-II and HEY-III yield infectious SV40 with higher efficiency than LEY-II because their SV40 DNA segments contain longer tandem repetitions; thus, the probability of an intramolecular recombination event which results in excision of an SV40 genome is greater.     

Nucleotide recognition seouence at the cleavage site of Haemophilus aegyptius II (Hae II) restriction endonuclease

We have determined the nucleotide sequence recognized by the restriction endonuclease Hae II from Haemophilus aegyptius which cleaves the simian virus 40 (SV40) DNA at a single specific site. By using terminal radioactive labeling of the cleavage site at both the 5′ and 3′-ends we have deduced the recognition sequence,

with elements of a two-fold rotational symmetry. The endonuclease produces staggered ends with protruding 3′-terminated single-strands, four nucleotides in length. In plasmid RSF 2124 DNA, which contains multiple Hae II cleavage sites, it was observed that the 5th nucleotide from the 3′ terminus is either a pdA or a pdG, indicating alternating recognition sequences.     

Specific Origin in SV40 DNA Replication

THE oncogenic virus SV40 contains a covalently closed circular DNA molecule of 3 × 10⁶ molecular weight¹. In infected permissive cells, SV40 DNA replicates through a Cairns type intermediate² with the parental strands forming a partially twisted, covalently closed molecule³. We have used a specific bacterial restriction endonuclease⁴ to analyse SV40 DNA replication. The restriction endonuclease of H. influenzae makes double-strand breaks in DNA at specific hexanucleotide sequences^{5, 6} and splits SV40 DNA into eleven fragments, separable by Polyacrylamide gel electrophoresis, ranging in molecular weight from 6.5 × 10⁵ (about 20% of the molecule) to 7.4 × 10⁴ (about 2.5% of the molecule). The largest eight fragments are present in the digest in amounts equimolar with the starting DNA⁴. Therefore, by digesting labelled replicating SV40 DNA and newly completed DNA and measuring the relative yield of each fragment, we could determine whether a particular region of the DNA is synthesized first or last and also estimate the time needed to replicate one molecule completely.     

Separation and analysis of promoter sites in bacteriophage lambda DNA by specific endonucleases

Specific endonucleases from Hemophilus influenzae, H. parainfluenzae and H. aegyptius were used to separate fragments bearing only one of the various promoters in phage λ DNA. Fragments containing these promoters were characterized by comparative analysis on polyacrylamide gels of the digestion products from λ and a variety of deletion or deletion-substitution derivatives. A single endonuclease from H. influenzae, Hin-II, is shown to cleave the early leftward and rightward promoters, p_L and p_R, at the sites of cleavage of the operators, O_L and O_R, because the corresponding cleavage sites are specifically protected by the DNA-dependent RNA polymerase. With altered p_L (mutations sex1 and sex3), the cleavage in the corresponding promoter is abolished. With X13, a mutation that presumably inactivates p_R, the cleavage in O_R still occurs.     

Characterization of the simian virus 40-specific messenger RNAs isolated from HeLa cells infected with the non-defective adenovirus 2-simian virus 40 hybrid viruses Ad2+ND2 and Ad2+ND4

Previous work has shown that cells infected with the non-defective adenovirus 2-simian virus 40 hybrid viruses, Ad2⁺ND2 and Ad2⁺ND4 synthesize more than one SV40⁴ large T antigen-related protein. These proteins overlap in amino acid sequence and have their carboxy-terminal sequences in common (Mann et al., 1977). We have characterized the messenger RNAs coding for these SV40-specific proteins. By translating in vitro SV40-specific mRNA isolated from cells infected with these viruses we have shown that each SV40-specific protein can incorporate ³⁵S-labeled formyl methionine at its N-terminus donated by [³⁵S]-fmet-tRNA_f^met, demonstrating that each protein results from a de novo initiation event. Furthermore, analysis of the N-terminal tryptic peptides of these proteins indicates that each protein has a unique N-terminal peptide and therefore a unique initiation site for protein synthesis, with the possible exception of the 74,000 and 95,000 molecular weight proteins, which may have the same N-terminal sequence. Therefore, these proteins cannot be derived by proteolytic cleavage of a large precursor protein.The messenger activities for many of the hybrid virus proteins can be resolved by gel electrophoresis, demonstrating the presence of multiple SV40-specific mRNA species. This result is consistent with the possibility that each SV40-specific protein is coded by a distinct species of RNA.     

HindII, HindIII,and HpaI restriction fragment maps of the left arm of bacteriophage λ DNA

The sites on the left arm of bacteriophage λ DNA cleaved by the restriction endonucleases isolated from Hemophilus influenzae strain Rc (HincII) and Rd (HindII+III), and Hemophilus parainfluenzae (HpaI) were localized on the λ physical map, and the fragments resulting from these cleavages were identified by gel electrophoresis. The restriction sites within the b2 region of λ were mapped by analysis of the digestion profiles of deletion and substitution derivatives of λ, as well as by digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease. The restriction sites on the λ genome between the left vegetative end and the b2 region were mapped entirely by successive digestion experiments. The restriction fragment map for the right arm of λ may be found in the accompanying paper (Robinson and Landy, 1977).     

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.     

Different regions of a complex statellite DNA vary in size and sequence of the repeating unit.

The 1.688 g/cm³ satellite DNA of Drosophila melanogaster is composed primarily of 359 base-pair units repeated in tandem. Most of these units contain a single cleavage site for both HaeIII and HinfI restriction endonucleases; however, some units lack one or both sites. Previously we had shown that the distribution of HaeIII and HinfI endonuclease sites varies widely between different regions of 1.688 g/cm³ satellite DNA; for example, some regions contain HaeIII sites in every unit and other regions (>10,000 base-pairs) contain no HaeIII sites (Carlson &; Brutlag, 1977). We have now cloned molecules of 1.688 g/cm³ satellite DNA which lack HaeIII sites and have shown that the absence of sites is caused by sequence variation rather than base modification. This result indicates that regions of 1.688 g/cm³ satellite DNA with different distributions of restriction sites differ in the sequence of their repeating units. We also show that a large fraction of the satellite DNA which is not cleaved by HaeIII endonuclease still contains HinfI endonuclease sites (and AluI sites) spaced about 359 base-pairs apart. However, one cloned segment lacking HaeIII sites was found to contain 33 tandem copies of a novel 254 base-pair unit. Sequence analysis showed that this 254 base-pair unit is homologous to the 359 repeat except for a 98 base-pair deletion. These data suggest that both units have evolved from a common ancestor and that each has subsequently become amplified into separate tandem arrays.     

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.     

A map of the sites on bacteriophage PM2 DNA for the restriction endonucleases HindIII and HpaII

The map of the seven sites for the restriction endonuclease HindIII³ and the single site for endo R.HpaII on PM2 DNA was determined. This map was oriented with respect to the denaturation map of this DNA (Brack et al., 1975) by partial denaturation mapping of the fragments. A new method for localizing restriction fragments by DNA-DNA hybridization and electron microscopy is described.     

Arthrobacter luteus restriction endonuclease recognition sequence and its cleavage map of SV40 DNA.

The nucleotide sequence at the cleavage site of the restriction endonuclease isolated from Arthrobacter luteus (Alu) has been determined. The endonuclease cleaves at the center of a palindromic tetranucleotide sequence to give even-ended duplex DNA fragments phosphorylated at the 5'-end. The endonuclease cleaves SV40 form I DNA into 32 fragments. The order and sizes of these fragments have been determined to provide an Alu cleavage map of the SV40 genome.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses VIII. Association of Simian Virus 40 Transplantation Antigen with a Specific Region of the Early Viral Genome

Two of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrids induce SV40 transplantation resistance in immunized hamsters. These two hybrids, Ad2(+)ND(2) and Ad2(+)ND(4), contain 32 and 43% of the SV40 genome, respectively. The pattern of induction of SV40 transplantation antigen (TSTA) by the various hybrids differentiates TSTA from both SV40 U and T antigens. Since the SV40 RNA induced by both these hybrids is early SV40 RNA, these findings confirm that TSTA is an early SV40 function. By combining available data on SV40 antigen induction by these hybrids with electron microscopy heteroduplex mapping studies, the DNA segment responsible for the induction of SV40 TSTA can be inferred to lie in the region between 0.17 and 0.43 SV40 units from the site on the SV40 chromosome cleaved by E. coli R(1) restriction endonuclease.