Alkaline Sucrose Gradient Sedimentation of Chromosomal Deoxyribonucleic Acid from Escherichia coli PolA+ and PolA− Strains During Thymine Starvation

Single-strand breaks were not detected in the deoxyribonucleic acid of Escherichia coli after thymine starvation for up to 180 min, even in a sensitive PolA(-) strain.     

Single-Strand Breaks in Deoxyribonucleic Acid and Viability Loss During Deoxyribonucleic Acid Synthesis Inhibition in Escherichia coli

The effects of deoxyribonucleic acid (DNA) synthesis inhibition brought about in four different ways-thymidine starvation, nalidixic acid, hydroxyurea, and dnaB mutation-were examined in isogenic strains of Escherichia coli K-12. Three parameters were examined to determine whether there are strict correlations among them: (i) the extent of DNA synthesis inhibition; (ii) cell survival; and (iii) the rate of breakage of DNA molecules. There was no significant correlation between the extent of DNA synthesis inhibition and the rate of viability loss caused by the four DNA synthesis inhibitors, nor was there a strict correlation between the rate of occurrence of single-strand breaks in DNA and loss of viability. During treatment with hydroxyurea (0.1 M), no viability loss was observed and little, if any, single-strand breakage of DNA occurred. Both thymidine starvation and nalidixic-acid (20 mug/ml) treatment resulted in viability loss and breakage of DNA. For these latter two inhibitors, the two events appeared to be associated because greater rates of both viability loss and DNA breakage were observed for nalidixic acid compared with thymidine starvation. However, viability loss need not be associated with extensive breakage of DNA as demonstrated with a temperature-sensitive DNA synthesis mutant; at 39 C, viability loss occurred at a high rate without significant DNA breakage. With the other agents, the amount of DNA breakage accumulated when a cell population has sustained an average of one lethal hit was estimated to be about 30 single-strand breaks per genome. Differences in chromosomal and episomal breakage rates were observed.     

Separation of the Herpesvirus Deoxyribonucleic Acid Duplex into Unique Fragments and Intact Strand on Sedimentation in Alkaline Gradients

Deoxyribonucleic acid (DNA) extracted from herpes simplex virions forms multiple partially overlapping bands upon denaturation and centrifugation in alkaline sucrose density gradients. The most rapidly sedimenting DNA corresponds to an intact strand 48 x 10(6) daltons in molecular weight. In this study, we analyzed the DNA fragments generated in alkaline sucrose gradients with respect to size and uniqueness of base sequences. The distribution of sedimentation constants of the various fragments obtained in numerous gradients showed that the fragments smaller than the whole strand fall into six distinct classes ranging in molecular weight from 10 x 10(6) to 39 x 10(6) daltons. Four types of DNA strands can be reconstructed from the whole strand and six fragments on the basis of their molecular weights. DNA from each of the bands self-hybridizes to a lower extent than unfractionated viral DNA, indicating that each of the bands preferentially contains sequences from one unique strand. The data permit reconstruction of four possible types of DNA duplexes differing in the positions of the strand interruptions. Analysis of viral DNA extracted from nuclei of cells labeled with (3)H-thymidine for intervals from 3 to 120 min showed that nascent DNA is invariably attached to small fragments and that the fragments become elongated only upon prolonged incubation of cells. The experiments suggest that viral DNA replication begins at numerous initiation sites along each strand and that the elongation beyond the size of the replication unit involves repair or ligation, or both. Since newly made DNA yields more fragments than viral DNA extracted from mature virions, it is suggested that the fragmentation of mature DNA on denaturation with alkali arises from incomplete processing of specific initiation sites. Comparison of viral DNA extracted from nuclei with that extracted from mature cytoplasmic virions in cells labeled for 120 min indicates that packaged DNA is not randomly selected from among the nuclear DNA population but rather represents DNA molecules which in alkaline gradients yield a minimal number of fragments.     

Methylation Pattern of Lambda Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from phage lambda grown on Escherichia coli K-12 strain W4032 had 113 +/- 10 5-methylcytosine residues and 215 +/- 20 6-methyl adenine residues per genome, as determined by three independent methods. These methylated nucleotides were distributed equally among the two strands of lambda DNA. Shearing of double-stranded DNA to half-length fragments revealed a slight deficiency of 5-methyl cytosine in the 55% guanine plus cytosine half. Shearing the DNA to fragments of smaller length showed that the distribution of methylated nucleotides along the double helix was uniform with the exception of an undermethylated fragment arising from the center of the lambda DNA molecule. The implication of these results for the function of methylated nucleotides in the lambda DNA molecule is discussed.     

Direct Enzymatic Repair of Deoxyribonucleic Acid Single-Strand Breaks in Dormant Spores

With the alkaline sucrose gradient centrifugation method, it was found that dormant spores of Clostridium botulinum subjected to 300 krads of gamma radiation showed a distinct decrease in deoxyribonucleic acid (DNA) fragment size, indicating induction of single-strand breaks (SSB). A two- to threefold difference in radiation resistance of spores of two strains of C. botulinum, 33A (37% survival dose [D(37)] = 110 krads) and 51B (D(37) = 47 krads), was accompanied by relatively larger DNA fragments (molecular weight 7.9 x 10(7)) obtained during extraction from the radiation-resistant strain 33A and smaller DNA fragments (molecular weight 1.8 x 10(7)) obtained under identical conditions from radiation-sensitive strain 51B. The apparent number of DNA SSB produced by 300 krads in strains 33A and 51B was 0.37 and 3.50, respectively, per 10(8) daltons of DNA. Addition of 0.02 M ethylenediaminetetraacetic acid (EDTA) to spore suspensions during irradiation doubled the apparent number of SSB in strain 33A but had no effect on strain 51B. In vivo, 0.02 M EDTA present during irradiation to 100 to 300 krads decreased survival of spores of 33A by about 30% but had little or no effect on 51B. Survival of 33A was also reduced by about 45% when the spores were irradiated while frozen in dry ice (-75 C) and, after irradiation, immediately exposed to 0.03 M EDTA for 1 h to inhibit repair in the dormant spores. These results suggest that the highly radiation-resistant strain 33A may be able to accomplish repair of SSB during irradiation or after irradiation under nonphysiological conditions, i.e., in the dormant state. This repair can be inhibited by EDTA. Sedimentation patterns show that DNA from spores of both strains 33A and 51B did not show any postirradiation repair during the first 6 h of germination, as opposed to Bacillus subtilis spores, which exhibit repair immediately after germination. These observations suggest the existence of direct repair in physiological dormant spores of strain 33A in the cryptobiotic resting state in the absence of germination. The repair seems to be similar to that of polynucleotide ligase activity shown to be operative in some vegetative cells. Apparently radiation-sensitive strains such as 51B and B. subtilis are generally poor in DNA repair enzyme activity under conditions of spore dormancy, which may account for the approximately threefold difference in radiation sensitivity or DNA fragility of different strains, or both.     

Repair of Single-Strand Deoxyribonucleic Acid Breaks in Ultraviolet Light-Irradiated Haemophilus influenzae

The wild-type strain and mutants of Haemophilus influenzae, sensitive or resistant to ultraviolet light (UV) as defined by colony-forming ability, were examined for their ability to perform the incision and rejoining steps of the deoxyribonucleic acid (DNA) dark repair process. Although UV-induced pyrimidine dimers are excised by the wild-type Rd and a resistant mutant BC200, the expected single-strand DNA breaks could not be detected on alkaline sucrose gradients. Repair of the gap resulting from excision must be rapid when experimental conditions described by us are employed. Single-strand DNA breaks were not detected in a UV-irradiated sensitive mutant (BC100) incapable of excising pyrimidine dimers, indicating that this mutant may be defective in a dimer-recognizing endonuclease. No single-strand DNA breaks were detected in a lysogen BC100(HP1c1) irradiated with a UV dose large enough to induce phage development in 80% of the cells.     

Denaturation Pattern of the Deoxyribonucleic Acid from Chicken Embryo Lethal Orphan Virus

The denaturation pattern of chicken embryo lethal orphan virus deoxyribonucleic acid confirms that the sequences are unique. The molecular weight of the deoxyribonucleic acid was determined by length measurements to be 29 x 10(6).     

Activity of Deoxyribonucleic Acid Fragments of Defined Size in Bacillus subtilis Transformation

The transforming activity of Bacillus subtilis deoxyribonucleic acid (DNA) that had been sheared and purified with respect to size by sucrose gradient sedimentation is given as a function of the DNA molecular weight. It is shown (i) that fragments of median molecular weight 1.2 x 10(6) have finite activity (10(-4)), (ii) that the shape of the activity-versus-molecular weight function is qualitatively similar to that observed previously for Diplococcus pneumoniae, and (iii) that this shape precludes interpretation in terms of critical size models.     

Separation of T-even Bacteriophage Deoxyribonucleic Acid from Host Deoxyribonucleic Acid by Hydroxyapatite Chromatography

A simple and rapid method is described for separation of T-even bacteriophage deoxyribonucleic acid (DNA) from host (Escherichia coli) DNA by hydroxyapatite column chromatography with a shallow gradient of phosphate buffer at neutral pH. By this method, bacteriophage T2, T4, and T6 DNA (but not T5, T7, or lambda DNA) could be separated from host E. coli DNA. It was found that glucosylation of the T-even phage DNA is an important factor in separation.     

Deoxyribonucleic Acid Breaks in Heated Salmonella typhimurium LT-2 After Exposure to Nutritionally Complex Media

Minimal medium recovery of heat-treated Salmonella typhimurium LT-2 has been expressed as the reduced viability on trypticase soy agar supplemented with 0.5% yeast extract (TSY) relative to a glucose-salts (M-9) agar. Incubation of S. typhimurium LT-2 in water at 50 C for 15 min did not change the sedimentation patterns of deoxyribonucleic acid (DNA) in alkaline sucrose gradients. The same pattern was obtained if the heated cells were further incubated for 15 min at 37 C in M-9 broth. However, a marked increase in DNA single-strand breakage accompanied by a loss of viability was observed after a similar incubation of heated bacteria in TSY broth. If heated bacteria were incubated in M-9 broth before TSY broth, there was a decrease in the single-strand breakage occurring in the TSY broth. This decrease is believed to be the result of repair of heat-induced damage. We conclude that minimal medium recovery after heat treatment is due to DNA damage caused by sequential exposure to heat and TSY medium, such damage not occurring after sequential exposure to heat and M-9 medium.     

Deoxyribonucleic Acid polymerase from wheat embryos

A soluble DNA-dependent DNA polymerase was extracted from wheat embryos. In vitro, the incorporation of radioactive thymidine triphosphate into acid-insoluble material is dependent upon the presence of the enzyme, all four deoxyribonucleotide triphosphates, Mg²⁺, and a DNA template. Incorporation occurs on native, alkali-denatured, and strictly double-stranded DNA. The in vitro synthesized product is a polydeoxynucleotide with a chain length shorter than the template; it has the same buoyant density as wheat embryo DNA when this DNA is used as template; and it forms a double-stranded complex with the DNA template. These data suggest that the in vitro DNA synthesis catalyzed by proteins extracted from wheat embryos occurs in a semiconservative way.     

Ultraviolet-Induced Cross-Links in the Deoxyribonucleic Acid of Single-Stranded Deoxyribonucleic Acid Viruses as a Probe of Deoxyribonucleic Acid Packaging

Deoxyribonucleic acid (DNA) from ultraviolet (UV)-irradiated phiX174 sediments in alkali at rates up to 1.7 times that of unirradiated phiX174 DNA and is observed as a condensed, cross-linked structure when examined in the electron microscope by the formamide spreading technique. This structure appears to result from multiple cross-links induced in the tightly coiled DNA contained within the spherical phiX174 capsid. In contrast, the DNA extracted after UV irradiation of the filamentous bacteriophage M13 is not strikingly altered in its sedimentation properties and appears by electron microscopy to be rod-shaped as a result of side-to-side association of the circular DNA. The differences in these UV-induced structures reflect the differences in the packaging of the single-stranded DNA in the two virions.     

Disruption of Adenovirus Type 7 by Lithium Iodide Resulting in the Release of Viral Deoxyribonucleic Acid

Adenovirus type 7 exposed to solutions of LiI was progressively converted into slower sedimenting deoxyribonucleic acid (DNA)-containing particles, and, ultimately, under proper conditions, DNA free or almost free from protein was released from the virus. The degree of viral degradation was dependent on the time of treatment, on the temperature, and on the concentration of the reagent.     

Method for Isolation of Deoxyribonucleic Acid from Mycobacteria

A relatively gentle and efficient procedure for obtaining deoxyribonucleic acid from mycobacteria by the use of glycine and lysozyme is described.     

Base Composition of Deoxyribonucleic Acid Isolated from Athiorhodaceae

Guanine plus cytosine content of deoxyribonucleic acid ranged from 60.5 to 65.0% for five Rhodospirillum species and from 64.4 to 70.3% for six Rhodopseudomonas species. These values were compared to those of two Hyphomicrobiaceae and two hydrogen-oxidizing bacteria.     

Unlinking of Cell Division from Deoxyribonucleic Acid Replication in a Temperature-sensitive Deoxyribonucleic Acid Synthesis Mutant of Escherichia coli

A new type of temperature-sensitive deoxyribonucleic acid (DNA) synthesis mutant, which can divide without a completion of DNA replication, was isolated from a thymidine-requiring Escherichia coli strain by means of photo-bromouracil selection after nitrosoguanidine mutagenesis. In this mutant, in spite of the fact that DNA synthesis stopped immediately after the temperature shift from 30 to 41 C, cells could continue to divide, though at a reduced rate. This cell division without DNA synthesis at 41 C is further supported by the following results. (i) Cell division took place at high temperature without addition of thymidine but not at all at 30 C. The parent strain of the mutant did not divide at 41 C without thymidine. (ii) Smaller cells isolated from the culture grown at 41 C did not contain DNA. This was shown by chemical analysis of the smaller cells and on electron micrographs. Ability of cells to divide was examined according to sizes of cells. By using the culture at 30 C, cells of various sizes were separated by means of sucrose-density gradient centrifugation. It was found that all cell fractions, including the smallest one, could divide at high temperature. These results suggest that in this mutant the completion of DNA replication is not required for triggering cell division at high temperature. Heat sensitivity of a factor which links cell division with DNA replication appears to be responsible. Some possible mechanisms of the coordination between cell division and DNA replication are discussed.     

Turnover of Myelin Proteins of Rat Brain, Determined in Fractions Separated by Sedimentation in a Continuous Sucrose Gradient

Abstract: Rats that Received intracranial injections of [³H]leucine at 14 days of age were killed on days 17, 24, 38, 55, and 89 post-injection. Brains were homogenized and the myelin membranes separated in a sucrose density gradient. At day 17 sodium dodecylsulfate polyacrylamide gels of water-shocked, delipidated membrane fractions showed a difference in the specific activity of myelin proteins across the gradient. A decrease in specific activity was found in all of the proteins in the denser fractions, compared with the lighter fractions. As time after injection progressed, the difference became more pronounced; a two- to threefold decrease in specific activity was seen across the gradient in the various myelin proteins. The proteins of the lightest membrane fractions retained their high specific activity throughout the experiment in spite of extensive new myelin synthesis. Taking this new myelin into account, the decrease in specific activity in the denser myelin fractions could be explained by isotope dilution. Therefore, proteins present in at least some of the myelin are essentially stable.     

Binding of Deoxyribonucleic Acid-Dependent Deoxyribonucleic Acid Polymerase to Poxvirus

Poxvirus has a deoxyribonucleic acid polymerase activity that remains associated with the virus despite repeated centrifugation through sucrose gradients. Highly purified poxvirus preparation can adsorb deoxyribonucleic acid polymerase from cytoplasmic extracts of cells containing such an activity. These results indicate that caution must be used in assuming that an enzyme associated with a purified virus is necessarily an integral part of the virion.     

Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization Assay for Replication Origin Deoxyribonucleic Acid of Escherichia coli

Deoxyribonucleic acid (DNA)-DNA hybridization on nitrocellulose filters can be used to assay for replication origin DNA from Escherichia coli if the DNA attached to the filters is enriched for the replication origin sequences. Such DNA can be readily isolated from very rapidly growing cells. When low amounts of this DNA were attached to filters, radioactively labeled DNA from the replication origin hybridized 1.7 times as well as radioactive replication terminus DNA. Under identical conditions, radioactively labeled DNA from exponentially growing cells hybridized only 1.3 times as well as radioactive replication terminus DNA. The replication origin, replication terminus, and randomly labeled DNA hybridized with similar efficiencies to filters containing DNA isolated from cells incubated in the absence of required amino acids. This DNA appeared to have all sequences present at equal frequencies. The hybridization assay was used to demonstrate that the DNA synthesized shortly after the addition of amino acids to cells previously deprived of required amino acids was primarily from the replication origin and then rapidly became similar to DNA synthesized by exponentially growing cells.