S1 Endonuclease Research Articles

A novel mRNA and a low molecular weight polypeptide encoded in the transforming region of adenovirus DNA.

Immunoprecipitation was used to identify adenovirus type 2 (ad2) tumor antigens synthesized in vivo. The antisera, prepared from tumor-bearing animals, reacted with a wide spectrum of ad2 early proteins including a 11 000-dalton (11 K) polypeptide. The gene for this polypeptide was mapped to the transforming region of the viral genome by hybridization selection followed by in vitro translation and immunoprecipitation. Hybrid arrest translation revealed that the 11 K RNA was transcribed from the leftward reading strand (1-strand) in contrast to other mRNAs from this region. Sucrose gradient analysis of the selected 11 K mRNA revealed that the size of the mRNA was 20S corresponding to approximately 2000 nucleotides. Novel 1-strand transcripts of this length from the transforming region were identified by S1 endonuclease analysis. Taken together, these results suggest that both strands of the transforming region of ad2 DNA are actively transcribed into functional mRNA early after viral infection.

Factors affecting the kinetics of DNA reassociation in phenol-water emulsion at high DNA concentrations.

AbstractDNA reassociation kinetics using the phenol emulsion reassociation technique (PERT) [Kohne, D. E., Levison, S. A. & Byers, M. J. (1977) Biochemistry 16, 5329–5341] has been investigated at high DNA concentrations using an endonuclease S1 assay of reaction progress. Apparent second‐order rate constants fall on two intersecting straight lines when presented as a function of DNA concentrations on a log–log plot. In the low DNA concentration range, the rate constants drop about 10‐fold when concentration increases 1000‐fold. In the high DNA concentration range, the rate constants drop more than 10‐fold when concentration increases 10‐fold. The slopes of these lines are the same in different solvents and at different temperatures. The intersection between the lines occurs when the available catalytic surface is saturated. At high DNA concentrations, high‐complexity heterologous denatured DNA apparently competes 2–4 times better for the surface than homologous DNA because it does not participate in a reassociation reaction. Native and partially native DNA molecules cannot compete with single‐stranded DNA for a saturated surface. At high DNA concentrations, reactions using PERT become dependent on the single‐strand DNA length. Increasing length lowers reassociation rates.

Partially deficient methylation of cytosine in DNA at CC TAGG sites stimulates genetic recombination of bacteriophage lambda

Lambda bacteriophages grown on arl mutants of Escherichia coil (“Arl −” phages) display intermediate levels of cytosine methylation: less 5-methylcytosine (m 5C) than phages grown on wild-type bacteria (“Arl +” phages) but more than phages grown on dcm mutants, and thus lacking the methylated sequences (Cm 5C T AGG) characteristic of E. coil K-12 bacteria (“Dcm −” phages). “Arl −” phages are one twelfth as resistant to Eco RII restriction (recognition site CC T AGG) as “Arl +” phages, but 40-fold more resistant than “Dcm −” phages. Chromatographic analyses show the 5-methylcytosine content of “Arl −” DNA to be one third that of “Arl +” DNA. Altered cytosine methylation frequency correlates with two previously described properties of “Arl −” phages, increased genetic recombination and unusual sensitivity of phage DNA to endonuclease S1, which are absent in phages grown on dcm or dcm arl bacteria. Methylated/unmethylated heteroduplex DNA prepared in vitro (one strand from Eco RII-modified phages/one from “Dcm −” phages) is highly recombinogenic but not S1-sensitive. We hypothesize that hemimethylated CC T AGG sites in “Arl −” DNA are necessary and sufficient for enhanced recombination, and necessary but not sufficient for S1 sensitivity.

Identification of thymidine-5′-aldehyde at DNA strand breaks induced by neocarzinostatin chromophore

Snake venom phosphodiesterase or endonuclease S1 digestion of neocarzinostatin chromophore-treated DNA, labeled in its thymidine residues, liberates an unusual labeled nucleoside from the 5' end of a drug-induced break. This substance, isolated by reverse-phase HPLC, possesses carbons from both the thymine and the deoxyribose moieties of thymidine in the DNA but, unlike thymidine, is readily degraded at pH 12 to thymine and a sugar fragment. The altered nucleoside was shown to contain a carbonyl group by its reduction with NaBH(4) to form a substance that has the chromatographic properties of thymidine and by its reaction with various hydrazines to form the respective hydrazone derivatives; the carbonyl exists as the 5' aldehyde as shown by its mild chemical oxidation to the carboxylic acid with simultaneous loss of the 5' (3)H. Mass spectral analysis showed a fragmentation pattern compatible with the structure thymidine-5'-aldehyde. These data indicate that the nonprotein chromophore of neocarzinostatin, in the presence of a reducing substance (2-mercaptoethanol) and molecular oxygen, selectively oxidizes the 5' carbon of nucleosides in DNA to the aldehyde, resulting in a strand break and a DNA fragment bearing nucleoside-5'-aldehyde at its 5' end.

In vitro enzymatic recognition of DNA modified by O,O'-diacetyl or O-acetyl derivatives of the carcinogen 4-hydroxyaminoquinoline-1-oxide.

Purified DNA was modified in vitro by 3H-labelled O-acetyl or O,O'-diacetyl-4-hydroxyaminoquinoline-1-oxide (Ac4HAQO or di Ac-4HAQO). It was then subjected to the action of the single-stranded DNA specific nuclease S1 and the digested fractions were analysed. For both types of modified DNA, the release of non-modified nucleotides was faster than the release of modified nucleotides. This result is at variance with that obtained with acetoxy-acetylaminofluorene-modified DNA: in the latter case, the modified nucleotides were preferentially released. The results suggest that the S1 endonuclease can recognize different conformational changes in DNA, which depend on the carcinogen used. The enzymatic activity (or activities) present in Micrococcus luteus cell extracts released ethanol-soluble products from Ac-4HAQO modified DNA.

Genetic relationships among clinical and environmental isolates ofVibrio cholerae from Australia

DNA-DNA homology among twenty-nine isolates having the phenotypic properties ofVibrio cholerae was studied using the S1 endonuclease method. Ten strains ofV. cholerae O1 isolated from patients and from the environment in Australia showed greater than 88% homology with the neotype strain ofV. cholerae NCTC 8021. Strains of the non-O1 serotype isolated from a variety of clinical and environmental sources also showed a high level of relatedness, including four luminescent strains and a reference strain of the biotypealbensis. A group of sucrose-negative strains showed a low level of homology (40 to 43%) withV. cholerae, but 75% and 82% homology within the group.

Covalent adducts of DNA and the nonprotein chromophore of neocarzinostatin contain a modified deoxyribose.

When the nonprotein chromophore of neocarzinostatin was allowed to react with either calf thymus DNA or poly(dA-dT) . poly(dA-dT) in the presence of 2-mercaptoethanol and the DNA was precipitated with ethanol, 5% of the fluorescence attributable to the naphthalene rings of the chromophore coprecipitated with the DNA. Most of this fluorescence remained attached to DNA through successive reprecipitations, suggesting formation of covalent adducts between chromophore and DNA. Enzymatically digested poly(dA-dT) . poly(dA-dT)-chromophore adduct contained, in addition to deoxyadenosine and thymidine, several highly fluorescent hydrophobic products, separable by reverse-phase chromatography, all of which contained both adenine and thymine radiolabel, as well as chromophore radiolabel. One such product consistently had twice as much thymine as adenine, suggesting a structure chromophore-d(TpApT), in which the attached chromophore rendered both phosphodiester bonds refractory to endonuclease S1. This adduct fragment was completely hydrolyzed at pH 12, releasing adenine, 3'-dTMP, and 5'-dTMP. At pH 7, the adduct fragment slowly released chromophore and 3'-dTMP with parallel kinetics, leaving a modified d(ApT), which was cleaved by snake venom phosphodiesterase to yield 5'-dTMP and a modified deoxyadenosine. These hydrolysis patterns are unlike those of any previously characterized base or phosphotriester DNA adduct but rather indicate an altered deoxyadenosine sugar. The formation of adducts containing a modified deoxyribose suggests that deoxyribose may be the site of covalent chromophore attachment. Alteration of this same site, possibly the 5'-carbon of the sugar moiety, may account for the extreme lability of the phosphodiester bond.

Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease.

Replicating simian virus 40 (SV40) chromosomes were found to be similar to other eukaryotic chromosomes in that the rate and extent of micrococcal nuclease (MNase) digestion were greater with replicating than with nonreplicating mature SV40 chromatin. MNase digestion of replicating SV40 chromosomes, pulse labeled in either intact cells or nuclear extracts, resulted in the rapid release of nascent DNA as essentially bare fragments of duplex DNA (3-7S) that had an average length of 120 base pairs and were degraded during the course of the reaction. In addition, nucleosomal monomers, equivalent in size to those from mature chromosomes, were released. On the other hand, MNase digestion of uniformly labeled mature SV40 chromosomes resulted in the release of only nucleosomal monomers and oligomers. The small nascent DNA fragments released from replicating chromosomes represented prenucleosomal DNA (PN-DNA) from the region of replication forks that encompasses the actual sites of DNA synthesis and includes Okazaki fragments. Predigestion of replicating SV40 chromosomes with both Escherichia coli exonuclease III (3'-5') and bacteriophage T7 gene 6 exonuclease (5'-3') resulted in complete degradation of PN-DNA. This result, together with the observation that isolated PN-DNA annealed equally well to both strands of SV40 restriction fragments, demonstrated that PN-DNA originates from both sides of replication forks. Over 90% of isolated Okazaki fragments annealed only to the retrograde DNA template. The characteristics of isolated PN-DNA were assessed by examining its sensitivity to MNase and single strand specific S1 endonuclease, sedimentation behavior before and after deproteinization, buoyant density in CsCl after formaldehyde treatment, and size on agarose gels. In addition, it was observed that MNase digestion of purified SV40 DNA also resulted in the release of a transient intermediate similar in size to PN-DNA, indicating that a DNA-protein complex is not required to account for the appearance of PN-DNA. These and other data provide a model of replicating chromosomes in which DNA synthesis occurs on a region of replication forks that is free of nucleosomes and is designated as prenucleosomal DNA.

Control of adenovirus early gene expression: accumulation of viral mRNA after infection of transformed cells.

We studied the accumulation of viral mRNA in the presence of inhibitors of protein synthesis in an adenovirus type 5-transformed cell line (line 293 cells). An analysis of the endogenous viral mRNA's and proteins revealed that only early regions 1A and 1B were expressed in uninfected 293 cells. However, viral mRNA's from early regions 2, 3, and 4, as well as mRNA's from early regions 1A and 1B, accumulated in 293 cells after infection with adenovirus type 2. Cells treated with anisomycin before infection showed a drastic enhancement of mRNA from early region 4 compared with drug-free controls, This increase in viral mRNA was detected by using filter hybridization, S1 endonuclease mapping, and in vitro translation. The rate of transcription of early region 4 nuclear RNA also increased significantly in the presence of anisomycin. In contrast to the results with early region 4, the levels of cytoplasmic mRNA's from early regions 2 and 3 did not increase in cells treated with inhibitors. These results suggested that multiple virus encoded controls operate on the early regions of the adenovirus genome.

In vivo synthesis of a polycistronic messenger RNA for the ribosomal proteins L11, L1, L10 and L7/12 in Escherichia coli.

Sucrose density gradient centrifugation and DNA/RNA hybridization have been used to analyse the mRNA synthesized from the ribosomal protein - RNA polymerase subunits gene cluster rplKAJL-rpoBC in Escherichia coli. DNA/RNA hybrids obtained from total E. coli RNA and specific DNA restriction fragments from this chromosomal area were further subjected to endonuclease S1 digestion. This analysis permits the mapping of the ends of mRNA molecules for specific genes or operons by sizing the S1 resistant hybrids. Our results show that the predominant mRNA synthesized under conditions of balanced growth from the rplKAJL-rpoBC region codes for the four ribosomal proteins L11, L1, L10 and L7/12. This tetracistronic mRNA puts the transcription of the following rpoBC genes under the main control of the L11 promoter. Smaller distinct mRNA species could also be detected by this technique. They originate from intercistronic transcription termination and re-initiation as well as from processing of the larger polycistronic mRNA.

Control of adenovirus early gene expression: posttranscriptional control mediated by both viral and cellular gene products.

An adenovirus type 5 host range mutant (hr-1) located in region E1A and phenotypically defective in expressing viral messenger ribonucleic acid (RNA) from other early regions (Berk et al., Cell 17:935-944, 1979) was analyzed for accumulation of viral RNA in the presence of protein synthesis inhibitors. Nuclear RNA was transcribed from all early regions at the same rate, regardless of whether the drug was present or absent. As expected, low or undetectable levels of RNA were found in the cytoplasm of hr-1-infected cells compared with the wild-type adenovirus type 5 in the absence of drug. When anisomycin was added 30 min before hr-1 infection, cytoplasmic RNA was abundant from early regions E3 and E4 when assayed by filter hybridization. In accordance, early regions E3 and E4 viral messenger RNA species were detected by the S1 endonuclease mapping technique only in hr-1-infected cells that were treated with the drug. Similar results were obtained by in vitro translation studies. Together, these results suggest that this adenovirus type 5 mutant lacks a viral gene product necessary for accumulation of viral messenger RNA, but not for transcription. It is proposed that a cellular gene product serves as a negative regulator of viral messenger RNA accumulation at the posttranscriptional level.

Control of adenovirus early gene expression: posttranscriptional control mediated by both viral and cellular gene products.

An adenovirus type 5 host range mutant (hr-1) located in region E1A and phenotypically defective in expressing viral messenger ribonucleic acid (RNA) from other early regions (Berk et al., Cell 17:935-944, 1979) was analyzed for accumulation of viral RNA in the presence of protein synthesis inhibitors. Nuclear RNA was transcribed from all early regions at the same rate, regardless of whether the drug was present or absent. As expected, low or undetectable levels of RNA were found in the cytoplasm of hr-1-infected cells compared with the wild-type adenovirus type 5 in the absence of drug. When anisomycin was added 30 min before hr-1 infection, cytoplasmic RNA was abundant from early regions E3 and E4 when assayed by filter hybridization. In accordance, early regions E3 and E4 viral messenger RNA species were detected by the S1 endonuclease mapping technique only in hr-1-infected cells that were treated with the drug. Similar results were obtained by in vitro translation studies. Together, these results suggest that this adenovirus type 5 mutant lacks a viral gene product necessary for accumulation of viral messenger RNA, but not for transcription. It is proposed that a cellular gene product serves as a negative regulator of viral messenger RNA accumulation at the posttranscriptional level.

Single-stranded fraction of deoxyribonucleic acid from Bacillus subtilis.

About 13% of the deoxyribonucleic acid (DNA) of various strains of Bacillus subtilis, independent of the stage of growth or competence for transformation, was rendered acid soluble by endonuclease S1. In a pH 11.2 CsCl gradient, 4% of the untreated DNA banded at the density typical for single-stranded molecules, whereas 9% of the remaining DNA (main band) was sensitive to endonuclease S1. Selective inhibition of DNA polymerase III, or of DNA-dependent ribonucleic acid polymerase, did not increase or abolish single-strandedness. The DNA purification procedure did affect the level of single-stranded DNA, indicating its binding to cell constituents containing ribonucleic acid, protein, and membranous material. The molecular weight of the single-stranded fraction resembled that of total denatured DNA, and its buoyant density in an alkaline CsCl gradient was centered partially at a density of 1.772 g/cm3 and partially at a density of 7.759 g/cm3. Incubation of DNA under conditions leading to renaturation of its single-stranded fraction led to an increase in transforming activity for the purA16+ marker (close to the origin of replication) relative to leu-8+ and metC3+ markers (located in the middle of the chromosome), indicating this region is the main source of the single-stranded fraction.

Alteration of guanine residues during proflavine mediated photosensitization of DNA.

Abstract— Mild photodynamic treatments of proflavine‐calf thymus DNA complexes induce a unique and quantitatively important alteration of the guanine residues which can be related to the lethal lesions due to the combined action of proflavine and light on phages. The ‘altered guanine’ is destroyed by HClO4 but is recovered after partial DNA depurination under the form of two photoproducts. The first product, Gox, elutes as guanine on a Sephadex column but has a modified UV absorbance spectrum. It gives rise by further irradiation to another product, X, which elutes at pH 9.7 as a pyrimidine compound and presented a maximal UV absorbance at 246 nm. Product X is also selectively released by piperidine fixation onto the photo‐damaged DNA. The guanine degradation process is markedly decreased in the presence of the singlet oxygen quencher, NaN3. The photodynamic lesion inhibits the enzymatic degradation of the DNA but generates locally denatured regions that are sensitive to S1 endonuclease.

Single-stranded poly(deoxyguanylic acid) associates into double- and triple-stranded structures.

Circular plasmid deoxyribonucleic acid (DNA), pBR322, was digested with the restriction endonuclease PstI to give full-length double-stranded DNA molecules, terminated by two self-complementary single-stranded sequences: (formula: see text). The protruding 3' termini were extended with dG by using calf thymus terminal deoxynucleotidyl transferase and dGTP, to form single-stranded tails of oligo(dG). At a length of about dG15, such tails become resistant to single strand specific endonuclease S1, and also cease to function as substrate (initiator) for the terminal deoxynucleotidyl transferase. This altered reactivity arises from association of the oligo(dG) tails into double- and triple-stranded structures, resulting in linear, circular, and branched polymers of the monomeric linear plasmid DNA. All these polymeric structures of the plasmid DNA are stable at room temperature, can be observed in the electron microscope, and can be separated from each other by agarose gel electrophoresis. At 60 degrees C or in 50% formamide, most of the oligo(dG) self-association can be reversed (melted), and the plasmid DNA is again found as the original linear monomer.

The inverted repeat as a recognizable structural feature in supercoiled DNA molecules.

The single-strand-specific endonuclease S1 from Aspergillus oryzae introduces highly selective cleavages into supercoiled covalently closed circular DNA molecules, but not into their previously linearized counterparts. The cleavage sites are inverted repeats of unit length between 9 and 13 base pairs, separated by a nonrepetitious 2-6 base pairs. Such regions may adopt hairpin or similar structures stabilized by the negative superhelix density and may constitute recognition sites for cellular proteins.

A taxonomic study of Gardnerella vaginalis (Haemophilus vaginalis) Gardner and Dukes 1955.

Fifty-five strains received as Haemophilus vaginalis or as catalase-negative coryneform bacteria from the vagina together with 61 marker cultures were subjected to numerical phenetic analyses using 149 unit characters. The data were examined using the simple matching (SSM), Jaccard (SJ) and pattern (DP) coefficients and clustering was achieved using the average linkage algorithm. Cluster composition was not markedly affected by the coefficient used or by test error, estimated at 6 . 5%. The H. vaginalis strains formed a tight cluster which was only distantly related to representatives of the genera arthrobacter, Cellulomonas, Corynebacterium sensu stricto, Erysipelothrix, Haemophilus, Kurthia, Lactobacillus, Listeria and Propionibacterium but shared a high overall affinity to unclassified catalase-negative coryneforms which formed a discrete taxon, cluster 9. The H. vaginalis strains could be distinguished from the related strains in cluster 9 by several unrelated phenotypic characters. Using the S1 endonuclease assay, DNA-DNA hybridizations were performed with representative strains from the numerical as well as with reference strains of Bifidobacterium and Actinomyces. Haemophilus vaginalis was found to be a genotypically legitimate group and its DNA showed little homology with DNA from the marker strains tested. The DNA base composition of H. vaginalis was 42 to 44 mol % guanine plus cytosine. A new genus should be created to incorporate strains known as H. vaginalis or Corynebacterium vaginale. The name Gardnerella vaginalis proposed by Greenwood & Pickett (1979) is supported.

Targeted deletions of sequences from closed circular DNA.

Closed circular DNA interacts with complementary sequences of single-stranded DNA to form displacement loop (D loop) structures in vitro. The site of D-loop formation can be directed by using single-stranded DNA derived from a selected restriction fragment. Circular DNA containing a D loop can then be linearized by cleavage with endonuclease S1. This cleavage appears to remove a limited number of nucleotides from each strand of the circular DNA substrate. Incubation with polynucleotide ligase followed by propagation in vivo leads to circular DNA molecules that bear small, single deletions in the region of the single-stranded DNA sequence chosen for the formation of the D loops. We have utilized these manipulations of DNA to construct tetracycline-sensitive deletion mutants of plasmid pBR322. The level of mutagenesis obtained by the procedure is sufficiently high that selective growth and screening procedures are not necessary for the isolation or identification of mutants. The frequency, variety, and small size of the deletions obtained within the selected target regions present considerable advantage for genetic and biochemical analysis. The method is quite general in rationale and should be immediately applicable to phage and viruses having infectious circular DNA genomes or recombinant DNA species propagated in circular plasmid vectors.

68] Transcription maps of polyoma virus-specific RNA: Analysis by two-dimensional nuclease S1 gel mapping

Publisher Summary This chapter describes the analysis of transcription maps of polyoma virus-specific RNA by 2-D nuclease S1 gel mapping. The chapter describes the methodology used to map polyoma virus transcripts on the physical map of the viral genome. The ultimate characterization of viral RNA is the determination of the complete nucleotide sequence of individual RNA species. A rapid procedure for the analysis of labeled viral RNA using an adaptation of the Southern transfer technique (mini-blot hybridization) has also been described. The hybrids formed between polyoma virus RNA and DNA are digested with single-strand-specific endonuclease S1 and the resulting duplexes or their component DNA strands are sized by gel electrophoresis. The 2-D variation of this basic procedure is a general diagonal assay for splicing within RNA molecules. The chapter describes the standard procedures used for the preparation of viral nucleic acids and hybridization probes.

Isolation of transposon TnA from plasmid RP4 carrying two copies of this element

Employing heteroduplex and restriction analyses, two inverted copies of a 3.2 · 10 6 dalton transposable sequence, TnA, were found in RP4::TnA, a spontaneously arisen derivative of the plasmid RP4. Integration of the second copy of TnA causes loss of the conjugative properties of RP4. Both TnA sequences in RP4:: TnA were localized and found to have opposite orientations. The DNA fragment corresponding to the individual transposon TnA was isolated after the endonuclease S1 digestion of RP4::TnA molecules annealed under conditions favoring intramolecular renaturation. The attempts to transform the cells of Escherichia coli QD5003, HB101[pCRI] and JC7623 with the isolated transposon were unsuccessful.