Construction and analysis of viable deletion mutants of simian virus 40.

Viable mutants of simian virus 40 (SV40), with deletions ranging in size from 15 to 200 base pairs, have been obtained by infecting CV-1P cells with circularly permuted linear SV40 DNA. The linear DNA was produced by cleavage of closed circular DNA with DNase I in the presence of Mn2+, followed, in some cases, by mild digestion with lambda 5'-exonuclease. The SV40 map location and the size of each deletion were determined by using the S1 nuclease mapping procedure (Shenk et al., 1975) and the change in size of fragments produced by Hind II + III endonuclease cleavage. Deletions in at least three regions of the SV40 chromosome have slight or no effect on the rate or yield of viral multiplication and on vira-induced cellular transformation. These regions are located at the following coordinates on the SV40 physical map: 0.17 to 0.18; 0.54 to 0.59; and 0.68 to 0.74.     

A restriction endonuclease cleavage map of mitochondrial DNA from transformed hamster cells.

Mitochondrial DNA from cultured C13/B4 hamster cells was cleaved by the restriction endonucleases Hpa II, Hind III, Eco RI and Bam HI into 7, 5, 3 and 2 unique fragments, respectively. The summed molecular weights of fragments obtained from electrophoretic mobilities in agarose-ethidium bromide gels (with Hpa I-cleaved T7 DNA as standard) and electron microscopic analysis of fragment classes isolated from gels (with SV40 DNA as standard) were in good agreement with the size of 10.37 +/- 0.22 x 10(6) daltons (15,700 +/- 330 nucleotide pairs) determined for the intact circular mitochondrial genome. Cyclization of all Hind III, Eco RI and Bam HI fragments was observed. A cleavage map containing the 17 restriction sites (+/- 1% s.d.) was constructed by electrophoretic analysis of 32P-labeled single- and double-enzyme digestion products and reciprocal redigestion of isolated fragments. The 7 Hpa II sites were located in one half of the genome. The total distribution of the 17 cleavages around the genome was relatively uniform. The position of the D-loop was determined from its location and expansion on 3 overlapping restriction fragments.     

The nucleotide sequence of repetitive monkey DNA found in defective simian virus 40.

DNA fragments containing monkey DNA sequences have been isolated from defective SV40 genomes that carry host sequences in place of portions of the SV40 genome. The fragments were isolated by restriction endonuclease cleavage and contain segments homologous to sequences in both the highly repetitive and unique (or less repetitive) classes of monkey DNA. The complete nucleotide sequence of one such fragment [151 base pairs (bp)] predominantly homologous to the highly reiterated class of monkey DNA was determined using both RNA and DNA sequencing methods. The nucleotide sequence of this homogeneous DNA segment does not contain discernible multiple internal repeating units but only a few short oligonucleotide repeats. The reiteration frequency of the sequence in the monkey genome is >10⁶. Digestion of total monkey DNA (from uninfected cells) with endonuclease R Hind III produces relatively large amounts of discrete DNA fragments that contain extensive regions homologous to the fragment isolated from the defective SV40 DNA.A second fragment, also containing monkey sequences, was isolated from the same defective substituted SV40 genome. The nucleotide sequence of the 33 bp of this second fragment that are contiguous to the 151 bp fragment has also been determined.The sequences in both fragments are also present in other, independently derived, defective substituted SV40 genomes.     

Cleavage map of the simian-virus-40 genome by the restriction endonuclease III of Haemopholus aegyptius.

Enzymic digestion of Simian virus 40 (SV40) DNA with Haemophilus aegyptius restriction endonuclease Hae III results in 10 major and eight minor fragments. These were resolved by electrophoresis on graduated polyacrylamide slab gels. All fragments have been characterized with respect to the size relative to the Haemophilus influenzae Rd fragments (Hind). They were ordered on the SV40 DNA map by means of overlap analysis of the double cleavage products derived from sequential digestion of Hind fragments with Hae III endonuclease and Hae fragments with Hind II + III enzyme, as well as by other reciprocal cleavage experiments, including those involving Haemophilus para-influenzae fragments. In this way the 18 Hae III cleavage sites and the 13 Hind sites have been localized on the circular SV40 DNA map.     

Extracellular nucleases of pseudomonas BAL 31. III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes.

We have previously characterized an extracellular nuclease from Pseudomonas BAL 31 which, in addition to other activities, displays a double-strand exonuclease activity which progressively shortens both strands of linear duplex DNA molecules from both termini. This degradation is accomplished without the introduction of detectable scissions away from the ends of the duplexes. When this nuclease is used to produce a series of progressively shortened samples from a linear duplex DNA, subsequent digestion of these samples with a site-specific restriction endonuclease and analysis of the resulting fragments by gel electrophoresis permits the rapid establishment of the order of the restriction enzyme fragments through the entire genome. This is accomplished by noting from the electropherograms the order in which the various restriction enzyme fragments become noticeably shortened or disappear. Using this method, the five cleavage sites for the endonuclease Hpa I and the single cleavage sites for the nucleases Hpa II and Pst I have been mapped in PM2 bacteriophage DNA. In a more stringent test of the method, 18 of the 24 fragments produced by cleavage of coliphage lambdab2b5c DNA with the Pst I nuclease have been mapped, and five of the six remaining fragments have been assigned to small regions of the genome.     

Genome organization of RNA tumor viruses II. Physical maps of in vitro-synthesized Moloney murine leukemia virus double-stranded DNA by restriction endonucleases.

Physical maps of the genome of Moloney murine leukemia virus (M-MLV) DNA were constructed by using bacterial restriction endonucleases. The in vitro-synthesized M-MLV double-stranded DNA was used as the source of the viral DNA. Restriction endonucleases Sal I and Hind III cleave viral DNA at only one site and, thus, generate two DNA fragments. The two DNA fragments generated by Sal I are Sal IA (molecular weight, 3.5 x 10(6)) and Sal IB (molecular weight, 2.4 x 10(6)) and by Hind III are Hind IIIA (molecular weight, 3.6 x 10(6) and Hind IIIB (molecular weight, 2.3 x 10(6)). Restriction endonuclease Bam I generates four fragments of molecular weights of 2.1 x 10(6) (Bam IA), 2 X 10(6) (Bam IB), 1.25 X 10(6) (Bam IC), and 0.24 x 10(6) (Bam ID), whereas restriction endonuclease Hpa I cleaves the M-MLV double-stranded DNA twice to give three fragments of molecular weights of 4.4 x 10(6) (Hpa IA), 0.84 X 10(6) (Hpa IB), and 0.74 x 10(6) (Hpa IC). Digestion of M-MLV double-stranded DNA with restriction endonuclease Sma I produces four fragments of molecular weights of 3.9 x 10(6) (Sma IA), 1.3 X 10(6) (Sma IB), 0.28 X 10(6) (Sma IC), and 0.21 x 10(6) (Sma ID). A mixture of restriction endonucleases Bgl I and Bgl II (Bgl I + II) cleaves the viral DNA at four sites generating five fragments of approximate molecular weights of 2 x 10(6) (Bgl + IIA), 1.75 X 10(6) (Bgl I + IIB), 1.25 X 10(6) (Bgl I + IIC), 0.40 X 10(6) (Bgl I + IID), and 0.31 x 10(6) (Bgl I + IIE). The order of the fragments in relation to the 5' end and 3' end of the genome was determined either by using fractional-length M-MLV double-stranded DNA for digestion by restriction endonucleases or by redigestion of Sal IA, Sal IB, Hind IIIA, and Hind IIIB fragments with other restriction endonucleases. In addition, a number of other restriction endonucleases that cleave in vitro-synthesized M-MLV double-stranded DNA have also been listed.     

Specific cleavage and physical mapping of simian-virus-40 DNA by the restriction endonuclease of Arthrobacter luteus.

Simian virus 40 (SV40) DNA (strain 776) is cleaved by the restriction endonuclease from Arthrobacter luteus into 32 specific fragments including 20 large pieces designated Alu-A through T as well as 12 minor products named Alu m1 through m8. These were mapped on the SV40 genome by double digestion experiments. Alu fragments were treated with Hind enzymes and vice versa. Similar reciprocal digestions were also carried out with Hae III enzyme. In this way a detailed cleavage map of the SV40 genome could be constructed.     

Structural changes in simian virus 40 chromatin as probed by restriction endonucleases.

The structure of simian virus 40 (SV40) chromatin was probed by treatment with single- and multiple-site bacterial restriction endonucleases. Approximately the same fraction of the chromatin DNA was cleaved by each of three different single-site endonucleases, indicating that the nucleosomes do not have unique positions with regard to specific nucleotide sequences within the population of chromatin molecules. However, the extent of digestion was found to be strongly influenced by salt concentration. At 100 mM NaCl-5 mM MgCl2, only about 20% of the simian virus 40 (SV40) DNA I in chromatin was converted to linear SV40 DNA III. In contrast, at lower concentrations of NaCl (0.05 or 0.01 M), an additional 20 to 30% of the DNA was cleaved. These results suggest that at 100 mM NaCl only the DNA between nucleosomes was accessible to the restriction enzymes, whereas at the lower salt concentrations, DNA within the nucleosome regions became available for cleavage. Surprisingly, when SV40 chromatin was digested with multiple-site restriction enzymes, less than 2% of the DNA was digested to limit digest fragment, whereas only a small fraction (9 to 15%) received two or more cuts. Instead, the principal digest fragment was full-length linear SV40 DNA III. The failure to generate limit digest fragments was not a consequence of reduced enzyme activity in the reaction mixtures or of histone exchange. When the position of the principal cleavage site was mapped after HpaI digestion, it was found that this site was not unique. Nevertheless, all sites wree not cleaved with equal probability. An additional finding was that SV40 chromatin containing nicked-circular DNA II produced by random nicking of DNA I was also resistant to digestion by restriction enzymes. These results suggest that the initial cut which causes relaxation of topological constraint in SV40 chromatin DNA imparts resistance to further digestion by restriction enzymes. We propose that this may be accomplished by either "winding" of the internucleosomal DNA into the body of the nucleosome, or as suggested by others, by successive right-hand rotation of nucleosomes.     

P1 nuclease defines a subpopulation of active SV40 chromatin--a new nuclease hypersensitivity assay.

Under exhaustive digestion conditions P1 nuclease was found to cleave a subpopulation of intracellular SV40 chromatin only once. The major P1 cleavage site in SV40 DNA was mapped at the origin of DNA replication, and the two minor sites at the SV40 enhancers. The P1-sensitive SV40 chromatin subpopulation was found to have higher superhelical density than the bulk of the intracellular SV40 chromatin. Furthermore, pulse labeled SV40 DNA which had higher superhelical density than that of the steady state viral DNA (S.S.Chen and M.T.Hsu, J.Virol 51:14-19, 1984) was also found to be preferentially cleaved by P1 nuclease. These results are consistent with a supercoil-dependent alteration of chromatin conformation near the regulatory region of the viral genome that can be recognized by P1 nuclease. Since P1 nuclease cleaves the subpopulation of SV40 chromatin only once without further degradation, this nuclease can be used as a general tool to define viral or cellular chromatin fraction with altered chromatin conformation and to map nuclease hypersensitive sites. Preliminary studies indicate that P1 makes limited double stranded cleavages in cellular chromatin to generate large DNA fragments.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

Simian virus 40 minichromosomes contain torsionally strained DNA molecules.

Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.     

Isolation of DNA fragment containing phoS gene of Escherichia coli K-12

The DNA fragment containing the phoS gene, a regulatory gene for alkaline phosphatase, has been isolated from Escherichia coli K-12 chromosomal DNA by cutting off the DNA with Hind III restriction enzyme and by cloning the gene with plasmid vector pTP 4 which was constructed in this study. The isolated fragment was of about 12.3 kbp and seemed to contain the phoT, glmS, and bgl genes. The 12.3 kbp Hind III fragment was subjected to restriction enzymes EcoR I, BamH I, Sal I, and Pst I, and was found to possess two EcoR I, no BamH I, a Sal I, and four Pst I sites. Partial deletion using these restriction enzymes suggested that the about 6 kbp Hind III-Pst I fragment contained the phoS and phoT genes. Further analysis with other restriction enzymes revealed that the 6 kbp Hind III-Pst I fragment contained a BstE II, two Mlu I and four Hpa I sites. The deletion of these restriction sites using single-strand-specific nuclease S1 suggested that the BstE II and one of Mlu I sites were in the phoT gene, and the BstE II and two Mlu I sites were not in the phoS gene.     

Submicromolar free calcium modulates dexamethasone binding to the glucocorticoid receptor

The relative distribution of bound cis- and trans-(NH₃)₂PtCl₂ at specific sites in SV40 DNA is evaluated by monitoring the extent to which five restriction endonucleases, each of which cleave at a single, unique site, are inhibited as a result of the DNA modification. The order of cleavage inhibition is Bgl 1 ? Bam HI > Hpa II, Kpn I > Eco RI. Both isomers produce a comparable effect for any particular endonuclease. Inhibition correlates with the % (G+C) content within and about the recognition sequences. That modified sequences immediately adjacent to the recognition sequence influence cleavage is further supported by differential cleavage observed with the multicut Hind III endonuclease. The binding of cis-(NH₃)₂PtCl₂ at the hyper-reactive Bgl 1 site may well be directly responsible for inhibiting SV40 replication.     

Iodination as a probe for small regions of disrupted secondary structure in double-stranded DNAs.

Conditions were established where the thallium-catalyzed iodination of random coil DNA proceeded 100-200 times faster than for native DNA. This reaction was explored as a probe for localized regions of disrupted base pairs in duplex DNA. A heteroduplex was constructed between DNA fragments produced by Hind II + III cleavage of phi80 plac DNA and phi80 plac DNA containing the Ll deletion (73 nucleotides in length). This heteroduplex incorporated twelve times as much iodine as the parent homoduplex fragments. Hence the technique could reveal the presence of a few (two or more) nonpaired cytosines, if they existed within an otherwise helical DNA fragment 789 base pairs long. Iodination studies were performed on superhelical SV40 DNA and on linear lambdaplac DNA. Analysis of the relative amount of iodine in restriction endonuclease fragments of these DNA's revealed the absence of localized single-stranded regions.     

Nuclease-sensitive sites in the two major intracellular simian virus 40 nucleoproteins

Simian virus 40 nucleoprotein isolated from the nuclei of infected cells contains a nuclease-sensitive site adjacent to the viral origin of replication (between 0.66 and 0.73 map unit). Nuclear extracts were subfractionated by sucrose gradient centrifugation to yield provirions (200S) and simian virus 40 chromatin (80S). The 80S fraction was cleaved either by DNase I or by an endonuclease endogenous to BSC-1 cells with high preference for the 0.66 to 0.73 region. The 200S fraction was treated to release core particles that were sensitive to nuclease cleavage; however, DNase I showed little or no preference for the 0.66 to 0.73 region of the provirion core nucleoprotein.     

Structure of eukaryotic chromatin. Evaluation of periodicity using endogenous and exogenous nucleases.

DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endonuclease, (b) prostate chromatin digested in situ with micrococcal nuclease or pancreatic DNAase I, and (c) isolated liver chromatin digested with micrococcal nuclease or pancreatic DNAase I has been analyzed electrophoretically on polyacrylamide gels. The electrophoretic patterns of DNA prepared from chromatin digested in situ with either endogenous endonuclease (liver nuclei) or micrococcal nuclease (prostate nuclei) are virtually identical. Each pattern consists of a series of discrete bands representing multiples of the smallest fragment of DNA 200 +/- 20 base pairs in length. The smallest DNA fragment (monomer) accumulates during prolonged digestion of chromatin in situ until it accounts for nearly all of the DNA on the gel; approx. 20% of the DNA of chromatin is rendered acid soluble during this period. Digestion of liver chromatin in situ in the presence of micrococcal nuclease results initially in the reduction of the size of the monomer from 200 to 170 base pairs of DNA and subsequently results in its conversion to as many as eight smaller fragments. The electrophoretic pattern obtained with DNA prepared from micrococcal nuclease digests of isolated liver chromatin is similar, but not identical, to that obtained with liver chromatin in situ. These preparations are more heterogeneous and contain DNA fragments smaller than 200 base pairs in length. These results suggest that not all of the chromatin isolated from liver nuclei retains its native structure. In contrast to endogenous endonuclease and micrococcal nuclease digests of chromatin, pancreatic DNAase I digests of isolated chromatin and of chromatin in situ consist of an extremely heterogeneous population of DNA fragments which migrates as a continuum on gels. A similar electrophoretic pattern is obtained with purified DNA digested by micrococcal nuclease. The presence of spermine (0.15 mM) and spermidine (0.5 mM) in preparative and incubation buffers decreases the rate of digestion of chromatin by endogenous endonuclease in situ approx. 10-fold, without affecting the size of the resulting DNA fragments. The rates of production of the smallest DNA fragments, monomer, dimer, and trimer, are nearly identical when high molecular weight DNA is present in excess, indicating that all of the chromatin multimers are equally susceptible to endogenous endonuclease. These observations points out the effects of various experimental conditions on the digestion of chromatin by nucleases.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. II. Biochemical Evidence for Genetic Exchange

The genome of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 rescued by passage on transformed permissive monkey lines (see accompanying paper [Y. Gluzman et al., J. Virol. 24:534-540, 1977]) was analyzed by restriction endonuclease cleavage mapping to obtain biochemical evidence that the rescue of the ts phenotype results from recombination with the resident SV40 genome of the transformed cell. It was demonstrated that the endonuclease R. HaeIII cleavage site, which is located at 0.9 map unit in the standard viral genome (and which is in the proximity of the known map position of the tsD lesion), is missing in the DNAs of the parental tsD202 virus and of three independent revertants of tsD202. In contrast, this cleavage site was shown to be present in the DNAs of four out of five independently derived rescued D202 populations and in the DNA of the SV40 strain, 777, used to transform the monkey cells. Comparison of the endonuclease R. Hin(II + III) cleavage patterns of SV40 strain 777 DNA and tsD202 DNA revealed differences in the electrophoretic mobilities of Hin fragments A, B, and F. However, the corresponding Hin fragments from all four rescued D202 genomes were identical in their mobilities to those of tsD202 DNA, indicating that these regions of the rescued D202 genome are characteristic of the tsD202 parent. We conclude, therefore, that the genome of the rescued D202 virus is a true recombinant, since it contains restriction endonuclease cleavage sites characteristic of both parents, the endogenous resident SV40 genome of the transformed monkey cells and the exogenous tsD202 mutant.     

Location of the cleavage sites on the SV 40 DNA map produced by the restriction endonucleases Pst1 and Bam1

Restriction endonucleases from Providencia stuartii (Pst 1) and Bacillus amyloliquefaciens H (Bam 1) cleave SV 40 DNA at two and one specific sites, respectively. Using EcoRI and Hind III endonuclease restriction sites as reference, the two Pst I sites were mapped at 0.050; 0.265 and the Bam I site was mapped at 0.170 of the genome length, clockwise, from the single EcoRI cleavage site.     

A stretch of "late" SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes.

Examination of DNA fragments produced from either formaldehyde-fixed or unfixed SV40 minichromosomes by multiple-cut restriction endonucleases has led to the following major results: Exhaustive digestion of unfixed minichromosomes with Hae III generated all ten major limit-digest DNA fragments as well as partial cleavage products. In striking contrast to this result, Hae III acted on formaldehyde-fixed minichromosomes to yield only one of the limit-digest fragments, F, which is located in the immediate vicinity of the origin of replication, spanning nucleotides 5169 and 250 on the DNA sequence map of Reddy et al. (1978). This 300 base pair (bp) fragment was released as naked DNA from formaldehyde-fixed, Hae III-digested minichromosomes following treatment either by pronase-SDS or by SDS alone. In the latter case, the remainder of the minichromosome retained its compact configuration as assayed by both sedimentational and electrophoretic methods. In minichromosomes, the F fragment is therefore not only accessible to Hae III at its ends, but is also neither formaldehyde cross-linked into any SDS-resistant nucleoprotein structure nor topologically "locked" within the compact minichromosomal particle. This same fragment was preferentially produced during the early stages of digestion of unfixed minichromosomes with Hae III, and its final yield in the exhaustive Hae III digest was significantly higher than that of other limit-digest fragments. Similar results were obtained upon digestion of either unfixed or formaldehyde-fixed minichromosomes with Alu I. In particular, of approximately twenty major limit-digest DNA fragments, only two fragments (F and P, encompassing nucleotides 5146 to 190, and 190 to 325, respectively) were produced by Alu I from the formaldehyde-fixed minichromosomes. All other restriction endonucleases tested (Mbo I, Mbo II, Hind III, Hin II+III and Hinf I), for which there are no closely spaced recognition sequences in the above mentioned regions of the SV40 genome, did not produce any significant amount of limit-digest DNA fragments from formaldehyde-fixed minichromosomes. These findings, taken together with our earlier data on the preferential exposure of the origin of replication in SV40 minichromosomes (Varshavsky, Sundin and Bohn, 1978), strongly suggest that a specific region of the "late" SV40 DNA approximately 400 bp long is uniquely exposed in the compact minichromosome. It is of interest that, in addition to the origin of replication, this region contains binding sites for T antigen (Tjian, 1977), specific tandem repeated sequences and apparently also the promoters for synthesis of late SV40 mRNAs (Fiers et al., 1978; Reddy et al., 1978).