Comparison of the requirements for ribonucleic acid synthesis with the requirements for deoxyribonucleic acid synthesis in animal tissues

Ribonucleic acid polymerase and deoxyribonucleic acid polymerase have been partially purified from bovine lymphosarcoma, lymph node, and thymus. An examination of the deoxyribonucleic acid requirements of the two enzymes indicates that “native” deoxyribonucleic acid is the preferred template for ribonucleic acid synthesis; heat-denatured deoxyribonucleic acid is considerably less active. The primer requirements for deoxyribonucleic acid synthesis differ: “native” deoxyribonucleic acid is usually inactive, while denatured deoxyribonucleic acid is active. The two enzymes also differ in pH optima and in their requirements for metal cofactors.     

Nitrous acid damage to duplex deoxyribonucleic acid: distinction between deamination of cytosine residues and a novel mutational lesion.

The rate of nitrous acid deamination of labeled cytosine residues in native Escherichia coli deoxyribonucleic acid was monitored in vitro by release of acid-soluble counts after treatment with uracil deoxyribonucleic acid glycosylase. The reaction exhibited a lag and was not stimulate by several agents previously shown to enhance base substitution mutagenesis during nitrous acid treatment of duplex deoxyribonucleic acid. We conclude that a significant proportion of nitrous acid induced mutagenic lesions are novel lesions and not cytosine deaminations.     

Incorporation and excision of 5-fluorouracil from deoxyribonucleic acid in Escherichia coli.

When Escherichia coli are grown in the presence of 5-fluorouracil, the 5-fluorouracil is incorporated almost exclusively into ribonucleic acid as fluorouridylate. In this study, small but detectable amounts were incorporated into ribonucleic acid as fluorocytidylate and into deoxyribonucleic acid as fluorodeoxyuridylate and fluorodeoxycytidylate. The amount of 5-fluorouracil found in deoxyribonucleic acid as fluorodeoxyuridylate increased 50-fold when the cells were deficient in both deoxyuridine triphosphatase and uracil-deoxyribonucleic acid glycosylase activities. Therefore, the same mechanisms which excluded uracil from deoxyribonucleic acid in vivo also excluded 5-fluorouracil. Even though purified uracil-deoxyribonucleic acid glycosylase excised 5-fluorouracil from deoxyribonucleic acid at only 5% the rate with which it excised uracil, most of the 5-fluorouracil excised from deoxyribonucleic acid in vivo was apparently excised directly by uracil-deoxyribonucleic acid glycosylase rather than by repair initiated by excision of uracil.     

Entry of double-stranded deoxyribonucleic acid during transformation of Neisseria gonorrhoeae.

The state of donor deoxyribonucleic acid after entry into competent cells was examined by assaying the transformed cell lysates for donor-marker transforming activity and density of donor deoxyribonucleic acid in CsCl gradients. The experiments showed that deoxyribonucleic acid entered in native, double-stranded form.     

Transformation in pneumococcus: nuclease resistance of deoxyribonucleic acid in the eclipse complex.

Donor deoxyribonucleic acid strands in the eclipse phase of genetic transformation of pnuemococcus (Streptococcus pneumoniae) are purified as a complex with a cf the deoxyribonucleic acid strand in this complex to digestion by nucleases was shown to be 50- to 1,000-fold less than that of uncomplexed single strands of deoxyribonucleic acid. Deoxyribonuclease I, micrococcal nuclease, Neurospora endonuclease, nuclease P1, and the major endogenous nuclease of cell-free extracts were studied. Sensitivity to nuclease attack was not uniform along the deoxyribonucleic acid strand; sequences of strongly protected bases were separated by more sensitive regions. The minimum size of protected fragments was about 70 bases. A complex of protein with the protected deoxyribonucleic acid segments was obtained after partial digestion. The sizes of these complexes, of the protected deoxyribonucleic acid segments, and of the protein subunit released by complete nuclease digestion, are all approximately identical, as determined by gel exclusion chromatography. Deoxyribonucleic acid strands of eclipse complex were also shown to be particularly well protected from attack by the major pneumococcal endonuclease in cell extracts.     

Plasmid deoxyribonucleic acid in Rhizobium trifolii.

In deoxyribonucleic acid of Rhizobium trifolii centrifuged in cesium chloride-ethidium bromide equilibrium was found a sattelite peak containing covalently closed circular deoxyribonucleic acid. The plasmid had a molecular weight of about 64 x 10(6) shown by sedimentation in sucrose gradients and electron microscopy.     

Scanning Feulgen-deoxyribonucleic acid cytophotometry of Papanicolaou destained preparations.

Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou destained specimens revealed a differential loss in Feulgen reactivity among human buccal and cervical smears, cultured embryonic lung fibroblasts and invasive cervical carcinoma cells. Loss in Feulgen reactivity in Papanicolaou destained fibroblasts and polyploid nuclei of malignant lesions was observed to result in underestimates of relative Feulgen deoxyribonucleic acid and nuclear area values using scanning integrating microdensitometry. Thus, Papanicolaou stained preparations may not be suitable for deoxyribonucleic acid quantification of high ploidy lesions since distributional absorption error is unpredictably influenced by such factors as ploidy level, nuclear size, chromatin dispersion and differential aldehyde loss during destaining. Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou stained preparations can be useful for differentiating benign from malignant lesions if extent of aneuploidy (as reflected in abnormal deoxyribonucleic acid frequency distribution profile) is used as a diagnostic indicator.     

Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors.

A mouse complementary deoxyribonucleic acid segment coding for the enzyme dihydrofolate reductase has been cloned in two general classes of vectors containing simian virus 40 deoxyribonucleic acid: (i) those that can be propagated as virions in permissive cells and (ii) those that can be introduced into and maintained stably in various mammalian cells. Both types of vectors express the mouse dihydrofolate reductase by using signals supplied by simian virus 40 deoxyribonucleic acid sequences. Moreover, plasmid vectors carrying the complementary deoxyribonucleic acid segment can complement Chinese hamster ovary cells lacking dihydrofolate reductase.     

Excretion of glutamic acid in Citrobacter intermedius C3 associated with plasmid deoxyribonucleic acid.

Several mutants of Citrobacter intermedius C3 lacking both the ability to synthesize proline and the ability to excrete glutamic acid were isolated by treatment with nitrosoguanidine. No revertants for either characteristic were obtained from these mutants. The ability to excrete glutamic acid was transferred to those mutants with very high frequencies in mating experience by using auxotropic excreting strains as donors. Moreover, the ability to synthesize proline was transferred together with the ability to excrete glutamic acid when an excreting strain was used as donor. The transconjugants showed a rapid spontaneous curing of both genetic markers. It was shown by two different methods that a band of covalently closed circular deoxyribonucleic acid is present in the cesium chloride gradients corresponding to the wild type and excretor mutants. Nonexcretor mutants described herein lacked such a band. Pro + transformants that were also excretors were obtained with plasmid deoxyribonucleic acid isolated either from wild type or from an excretor mutant. These data strongly indicate that glutamic acid excretion in C. intermedius C3 is related to the presence of extrachromosomal deoxyribonucleic acid.     

Quantitation of cellular deoxyribonucleic acid by flow microfluorometry.

This report characterizes for the first time an easy, reproducible means of standardizing the relative fluorescent units normally reported for flow microfluorometry. Absolute values for deoxyribonucleic acid/cell are obtained by using nucleated red blood cells as references. Cell were selected and characterized for the quantitative analysis of deoxyribonucleic acid per cell over a range from 2 pg/cell to 93 pg/cell using literature values for species having nucleated erythrocytes. Fluorescence staining by either acridine-orange (green wavelength) or propidium iodide (red wavelength) gave linear curves over the entire range investigated only when "gain controls" and current are optimized. The range was equivalent to mammalian cell values from 1 N (=3.5 pg deoxyribonucleic acid/cell) to 28 N (=91 pg deoxyribonucleic acid/cell). The standard curves obtained with nonmammalian erythrocytes were compared to mammalian free-cell preparations of bovine thymus and liver cells which fell at 6.8 and 6.9 pg deoxyribonucleic acid/cell, respectively. The routine use of these easily obtainable red blood cells will allow ready comparisons on the basis of absolute values for deoxyribonucleic acid per cell for work between experiments, work between staining procedures and dye types and work between laboratories.     

Structure of Caulobacter deoxyribonucleic acid.

The deoxyribonucleic acid of the dimorphic bacterium Caulobacter crescentus contains a component that renatures with rapid, unimolecular kinetics. This component was present in both swarmer and stalked cells and exhibited the sensitivity to endonuclease S1 expected for hairpin loops. Double-stranded side branches between 100 and 600 nucleotide pairs in length were visible in electron micrographs of rapidly reassociating deoxyribonucleic acid isolated by hydroxyapatite chromatography. No extrachromosomal elements were found in spite of systematic attempts to detect their presence. These results indicate that the rapidly reassociating fraction derives from inverted repeat sequences within the chromosome and not from cross-links or plasmids. We estimate that there are approximately 350 inverted repeat regions per Caulobacter genome. The kinetic complexity of Caulobacter deoxyribonucleic acid, however, is no greater than that of other bacteria.     

Reinitiation of deoxyribonucleic acid synthesis by deoxyribonucleic acid initiation mutants of Escherichia coli: role of ribonucleic acid synthesis, protein synthesis, and cell division.

The dnaA and dnaC genes are thought to code for two proteins required for the initiation of chromosomal deoxyribonucleic acid replication in Escherichia coli. When a strain carrying a mutation in either of these genes is shifted from a permissive to a restrictive temperature, chromosome replication ceases after a period of residual synthesis. When the strains are reincubated at the permissive temperature, replication again resumes after a short lag. This reinitiation does not require either protein synthesis (as measured by resistance to chloramphenicol) or ribonucleic acid synthesis (as measured by resistance to rifampin). Thus, if there is a requirement for the synthesis of a specific ribonucleic acid to initiate deoxyribonucleic acid replication, this ribonucleic acid can be synthesized prior to the time of initiation and is relatively stable. Furthermore, the synthesis of this hypothetical ribonucleic acid does not require either the dnaA of dnaC gene products. The buildup at the restrictive temperature of the potential to reinitiate deoxyribonucleic acid synthesis at the permissive temperature shows rather complex kinetics the buildup roughly parallels the rate of mass increase of the culture for at least the first mass doubling at the restrictive temperature. At later times there appears to be a gradual loss of initiation potential despite a continued increase in mass. Under optimal conditions the increase in initiation potential can equal, but not exceed, the increase in cell division at the restrictive temperature. These results are most easily interpreted according to models that postulate a relationship between the initiation of deoxyribonucleic acid synthesis and the processes leading to cell division.     

The distribution of deoxyribonucleic acid in the female gametophyte of Myosurus minimus

Summary An investigation of the distribution of deoxyribonucleic acid in the female gametophyte of the angiosperm Myosurus has revealed that the egg and polar nuclei show no detectable Feulgen reaction. Deoxyribonucleic acid was nevertheless demonstrated in these nuclei by means of fluorescence microscopy and ultraviolet microspectrography.These latter techniques also showed the presence of appreciable quantities of deoxyribonucleic acid in the cytoplasm of the two-nucleate embryosac and of the egg.The significance of these results is discussed in relation to the existing knowledge of the distribution of deoxyribonucleic acid in the eggs of plants and animals.This work constitutes part of a Thesis for the Ph.D. degree of the University of London.     

Plasmid deoxyribonucleic acid replication in Streptomyces griseus

A series of electron micrographs showing the presence of different molecular forms representing various replication stages of plasmid deoxyribonucleic acid from Streptomyces griseus was obtained. Based upon an analysis of these electron micrographs, a tentative model for plasmid deoxyribonucleic acid replication in S. griseus is proposed.     

Rate stimulation of deoxyribonucleic acid synthesis after inhibition.

The degree to which the rate of deoxyribonucleic acid synthesis in thy- cultures of Escherichia coli is stimulated after a period of thymine starvation is shown to be a function of the concentration of thymine present as well as of the culture doubling time. Inhibition of deoxyribonucleic acid synthesis by nalidixic acid yields comparable results. Periods of thymine starvation exceeding one doubling time appear to cause an irreversible inactivation of a fraction of the replication forks in the culture.     

Four proteins synthesized in response to deoxyribonucleic acid damage in Micrococcus radiodurans.

Four proteins, alpha beta, gamma, and delta, preferentially synthesized in ultraviolet light-treated cells of Micrococcus radiodurans, were characterized in terms of their molecular weights and isoelectric points. Within the sublethal-dose range, the differential rate of synthesis for these proteins increased linearly with the inducing UV dose. The degree of induction reached 100-fold, and the most abundant protein beta, amounted to approximately 2% of the total newly synthesized protein after irradiation. Damage caused by ionizing radiation or by treatment with mitomycin C also provoked the synthesis of the four proteins. The proportions between the individual proteins, however, varied strikingly with the damaging agent. In contrast to treatments which introduced damage in the cellular deoxyribonucleic acid, the mere arrest of deoxyribonucleic acid replication, caused by nalidixic acid or by starvation for thymine, failed to elicit the synthesis of either protein. Repair of deoxyribonucleic acid damage requires that a number of versatile and efficient processes by employed. It is proposed that the induced proteins participate in deoxyribonucleic acid repair in M. radiodurans. Mechanisms are discussed which would allow a differentiated cellular response to damages of sufficiently distinctive nature.     

Interactions between exogenous deoxyribonucleic acid and membrane vesicles isolated from competent and noncompetent Bacillus subtilis.

Competent cultures of Bacillus subtilis 168 have been fractionated into a high-competent and a low-competent fraction by a large-scale separation technique. Membrane vesicles isolated from both cell fractions are equally active in the transport of L-glutamate. Both membrane vesicle preparations seem to have similar endo- and exonuclease activities. Also, both preparations are capable of binding deoxyribonucleic acid. However, especially at low deoxyribonucleic acid concentrations (1 mug or less per ml), vesicles obtained from competent cells bind significantly more deoxyribonucleic acid (up to sixfold) than vesicles from noncompetent cells.     

Nucleotide sequence homology between the heat-labile enterotoxin gene of Escherichia coli and Vibrio cholerae deoxyribonucleic acid.

Isolated deoxyribonucleic acid fragments encoding the heat-labile enterotoxin of Escherichia coli were used to probe for homologous sequences in restricted whole-cell deoxyribonucleic acid from Vibrio cholerae. Significant sequence homology between the heat-labile enterotoxin gene and V. cholerae deoxyribonucleic acid was demonstrated, and apparent differences were observed in the organization of the cholera toxin gene among different strains of V. cholerae.     

Nature of transforming deoxyribonucleic acid in calcium-treated Escherichia coli.

A study of the reactivation of ultraviolet-irradiated plasmid and phage deoxyribonucleic acid molecules after transformation into Escherichia coli strains indicated that, when double-stranded deoxyribonucleic acid was used as the donor species, it was taken up without conversion to the single-standed form.