Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae

Abstract

A bacterial restriction endonuclease has been used to produce specific fragments of SV40 DNA. Digestion of DNA from plaque-purified stocks of SV40 with the restriction endonuclease from Hemophilus influenzae gave 11 fragments resolvable by polyacrylamide gel electrophoresis, eight of which were equimolar with the original DNA. The fragments ranged from about 6.5 X 106 to 7.4 X 104 daltons, as determined by electron microscopy, DNA content, or electrophoretic mobility.The small oncogenic virus, SV40, contains double-stranded, covalently closed-circular DNA (1) of molecular weight about 3 X 106 (2). Because of the small size of its genome, and because of its ability to transform cells in culture to a state resembling that of cancer cells, SV40 is currently under intensive study. We have been interested in preparing unique linear fragments of SV40 DNA that might be used to establish reference points on this circular genome for the mapping of such functions as initiation of DNA replication and transcription, and for the localization of genes. Unique fragments might also be helpful in nucleotide sequence determination.Our general strategy for obtaining specific fragments has been to cleave the DNA with bacterial restriction enzymes, a group of endonucleases that make double-strand breaks in DNA at specific sites (3), and to separate the products by gel electrophoresis. Although this group of enzymes has been studied primarily with natural DNA substrates, we have found that most of those tested also cleave SV40 DNA (4). In the present paper, we present our results with the restriction endonuclease from Hemophilus influenzae (5, 6). MATERIALS AND METHODSCell Lines and Virus. Two lines of African green monkey kidney cells, CV-1 and BSC-1, were obtained from M. A. Martin and Microbiological Associates, respectively. They were grown in Eagle's minimal essential medium containing 10% fetal-bovine serum, penicillin, and streptomycin. Smallplaque SV40 (from strain 776), kindly supplied by K. K. Takemoto, was plaque purified on CV-1 cells and propagated in BSC-1 cells to obtain stocks of about 108 plaque-forming units (PFU)/ml.Abbreviations: SSC, 0.15 M NaCl-0.015 M sodium citrate; TMSH, 6.6 mM Tris-HCl (pH 7.45)-6.6 mM MgC1-6.6 mM 2-mercaptoethanol. * This is paper no. 1 in a series: "Studies of SV40 DNA." 2913 Preparation of Radioactive SV40 DNA from Virions. Confluent BSC-1 monolayers in 100-mm plastic Petri dishes were infected with SV40 at a multiplicity of about 10 PFU/cell. For 32P-labeling, 0.15 mCi of carriei-free [32P]orthophosphate was added to each dish, at 24 hr after infection, in 6 ml of phosphate-free medium containing 4% dialyzed fetal-bovine serum. For labeling with [3H]thymidine of high specificactivity the medium was made 10-4M in FdU 24 hr after infection. 3 hr later, 6 ml of fresh medium, which contained 4% dialyzed serum, 50 ,gM FdU, and 0.1 mCi of [8H]thymidine (7 Ci/mmol), was added. At about 90 hr, the cells and medium were frozen, the virus was purified, and DNA was extracted from the CsCl-p...

Subunit Structure of Simian‐Virus‐40 Minichromosome

“…On the other hand, this value is in disagreement with the estimate of Griffith [17], who has reported that an SV4O minichromosome is formed of 21 nucleosomes, each containing 170 base pairs, connected by bridges of about 40 base pairs. It is likely that due to some electron microscopic artifact the measurements of Grifjith have led to an underestimation, since 21 nucleosomes, each containing 170 base pairs, connected by 40 base pair bridges, cannot account for the total SV40 genome which contains about 5000 base pairs [21,36,37].…”

Comparison of the G4 and φX174 phage genomes by electron microscopy

“…The spreading conditions used were 50% formamide, 0.1 M buffer solution (Keegstra et al, 1977) for the specimens used for mapping and 30% formamide 0.1 M buffer solution for the annealing control experiments. $1X174 replicative form DNA with at least one nick in one of the strands (RFII) was used as an internal length reference (Lang et al, 1967;Brown and Vinograd, 1974;Danna and Nathans, 1971;Stiiber and Bujard, 1977). The specimens were examined and photographed in a Philips EM 301 at a (film) magnification of 26000 x on Kodalith LR film.…”

DNA cloning: A personal view after 40 years

“…In 1970, Hamilton Smith and his colleagues at Johns Hopkins University reported that a restriction enzyme they named HindII-a protein isolated from the bacterial pathogen, Haemophilus influenzae-recognizes particular nucleotide sequences in DNA and cuts duplex DNA site-specifically at these sequences (36). The following year, Karen Danna and Daniel Nathans found that the HindII endonuclease cleaves DNA of the mammalian tumor virus SV40 into 11 fragments that can be separated by acrylamide gel electrophoresis, demonstrating the utility of restriction endonucleases for DNA analysis (37). Arber, Nathans, and Smith received the 1978 Nobel Prize in Physiology or Medicine for these accomplishments.…”