Nuclear Fraction of Bacillus subtilis as a Template for Ribonucleic Acid Synthesis

A "nuclear fraction" prepared from Bacillus subtilis was a more efficient template than purified deoxyribonucleic acid for the synthesis of ribonucleic acid by exogenously added ribonucleic acid polymerase isolated from B. subtilis. The initial rate of synthesis with the nuclear fraction was higher and synthesis continued for several hours, yielding an amount of ribonucleic acid greater than the amount of deoxyribonucleic acid used as the template. The product was heterogenous in size, with a large portion exceeding 23S. When purified deoxyribonucleic acid was the template, a more limited synthesis was observed with a predominantly 7S product. However, the ribonucleic acids produced in vitro from these templates were very similar to each other and to in vivo synthesized ribonucleic acid as determined by the competition of ribonucleic acid from whole cells in the annealing of in vitro synthesized ribonucleic acids to deoxyribonucleic acid. Treatment of the nuclear fraction with heat (60 C for 15 min) or trypsin reduced the capacity of the nuclear fraction to synthesize ribonucleic acid to the level observed with purified deoxyribonucleic acid.     

Biochemical characterization of nonintegrated plasmid-folded chromosome complexes: sex factor F and the Escherichia coli nucleoid.

The existence of nonintegrated plasmid-chromosome complexes has been deduced in previous work from the cosedimentation of covalently closed, circular plasmids with host folded chromosomes. In the present work, it is shown that about 70 to 90% of the covalently closed, circular F deoxyribonucleic acid could be released in vitro from chromosome complexes by ribonuclease treatment but not by protease, Sarkosyl, or ethidium bromide. Consistent with the in vitro studies, Escherichia coli cells treated for 5 min with rifampin, an inhibitor of ribonucleic acid initiation, released upon lysis 90% of their plasmid deoxyribonucleic acid as freely sedimenting molecules.     

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.     

Isolation and characterization of thermosensitive Escherichia coli mutants defective in deoxyribonucleic acid replication

Thermosensitive deoxyribonucleic acid replication-defective mutants have been isolated by using an autoradiographic selection method. The mutants have been analyzed genetically and biochemically. Some of the mutants show thermosensitivity of in vitro deoxyribonucleic acid replication. These can be classified into three groups according to their behavior in in vitro complementation assays. This classification is congruent with that obtained by genetic mapping by using cotransduction frequencies with selected markers in P1 transduction analysis.     

Cyanobacterial ribonucleic acid polymerases recognize lambda promoters.

We compared the initiation specificities in vitro of deoxyribonucleic acid-dependent ribonucleic acid polymerases purified from two cyanobacteria, Fremyella diplosiphon and Anacystis nidulans, and from Escherichia coli. A restriction fragment made from lambda deoxyribonucleic acid was used as a template. The cyanobacterial and E. coli ribonucleic acid polymerases recognized the same lambda promoters but exhibited different sensitivities to the inhibitor heparin, suggsesting differences in the structure of the initiation complexes.     

Spontaneous transformation of a clonal population of dermis-derived multipotent cells in culture

It is reported that adult multipotent stem cells can undergo spontaneous transformation after long-term in vitro culture. Understanding the molecular mechanisms involved in this spontaneous transformation process can help in the design of future therapeutic applications. By far, the transformation process of adult multipotent stem cell is not well understood. In this study, a tumorigenic cell line nominated TDMC1 was established from a clonal population of rat dermis-derived multipotent cells (DMCs) following spontaneous transformation in culture. The transformed cells could produce tumors with characteristics of fibrous histocytoma when they are inoculated subcutaneously into nude mice. The molecular profiles of the nontransformed DMCs and transformed cells were analyzed by a deoxyribonucleic acid microarray. Our results showed that the overactivation of the K-ras/mitogen-activated protein kinase kinase signaling pathway played an important role in the transformation process. These data may be helpful to explain, at least in part, the possible mechanism for the malignant transformation of adult multipotent cells.     

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.     

Characterization of Rabensburg virus, a flavivirus closely related to West Nile virus of the Japanese encephalitis antigenic group

Rabensburg virus (RABV), a Flavivirus with ～76% nucleotide and 90% amino acid identity with representative members of lineage one and two West Nile virus (WNV), previously was isolated from Culex pipiens and Aedes rossicus mosquitoes in the Czech Republic, and phylogenetic and serologic analyses demonstrated that it was likely a new lineage of WNV. However, no direct link between RABV and human disease has been definitively established and the extent to which RABV utilizes the typical WNV transmission cycle is unknown. Herein, we evaluated vector competence and capacity for vertical transmission (VT) in Cx. pipiens; in vitro growth on avian, mammalian, and mosquito cells; and infectivity and viremia production in birds. RABV infection and replication only were detected on mosquito cells. Experimentally inoculated birds did not become infected. Cx. pipiens had poor peroral vector competence and a higher VT rate as compared to US-WNV in Cx. pipiens. As a result, we postulate that RABV is an intermediate between the mosquito-specific and horizontally transmitted flaviviruses.     

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.     

Transport of DNA from Lymphocytes to Fibroblasts in Culture

The exchange of radioactivity between lymphocytes, labelled with (³H) thymidine after stimulation with Concanavalin A, and recipient V79 fibroblasts in culture was studied. The radioactive material involved in this exchange was macromolecular deoxyribonucleic acid as well as its breakdown products. This deoxyribonucleic acid from lymphocytes localised in the nuclei of the host cells soon after contact between donor and recipients. This occurred even when the V79 fibroblasts were confluent at high cell density, and thus in a steady, non-growing state with respect to cell numbers.
The fate of the radioactive donor lymphocyte deoxyribonucleic acid, substituted with bromodeoxyuridine, was followed in the recipient cells by analysing its buoyant density in caesium chloride gradients. This deoxyribonucleic acid was found to become associated with the nuclear deoxyribonucleic acid of the host cells, involving both retention of relatively intact donor deoxyribonucleic acid as well as its breakdown and re-utilisation for host cell deoxyribonucleic acid synthesis. Nongrowing recipient cells were found to retain the donor deoxyribonucleic acid in relatively intact form for much longer periods than when the same cells were in logarithmic growth phase.     

Template requirement of maize RNA polymerase

Maize RNA polymerase utilizes heated deoxyribonucleic acid more effectively than native deoxyribonucleic acid as a template for ribonucleic acid synthesis. A ribonucleic acid-deoxyribonucleic acid hybrid accumulates in the presence of heated deoxyribonucleic acid. The amount of product formed with either native or heat-denatured deoxyribonucleic acid does not exceed the amount of deoxyribonucleic acid added as template.     

Changing proportions of DNA components in Euglena gracilis

The ratios of satellite deoxyribonucleic acid components to chromosomal deoxyribonucleic acid in Euglena gracilis Z were measured by analytical density gradient ultracentrifugation. Chloroplast deoxyribonucleic acid with a buoyant density of 1.685 g/cm³ exhibited a constant ratio to chromosomal deoxyribonucleic acid during exponential growth and increased twofold as the culture reached the end of the exponential growth phase. The quantity of a satellite deoxyribonucleic acid with a buoyant density of 1.691 g/cm³ was not sufficient to measure the ratio to chromosomal deoxyribonucleic acid during exponential growth but increased to approximately equal the quantity of chloroplast deoxyribonucleic acid as the culture approached the end of the exponential growth phase. The quantity of a deoxyribonucleic acid component with a buoyant density of 1.700 g/cm³ was not sufficient to measure the ratio to chromosomal deoxyribonucleic acid during exponential growth but represented approximately one-third of the total deoxyribonucleic acid as the culture entered the stationary phase of growth.     

Plasmid-mediated transformation in Bacillus megaterium.

A transformation system was developed for Bacillus megaterium by using antibiotic resistance plasmid deoxyribonucleic acid molecules derived from Staphylococcus aureus and Bacillus cereus. Lysozyme-generated protoplasts of B. megaterium allowed uptake of plasmid deoxyribonucleic acid in the presence of polyethylene glycol. Transformants expressed the antibiotic resistance determinants present on the plasmid deoxyribonucleic acid, and reisolated plasmid deoxyribonucleic acid yielded restriction endonuclease digestion patterns identical to those of the donor deoxyribonucleic acid.     

Synchronous germination and outgrowth of fractionated Bacillus subtilis spores: tool for the analysis of differentiation and division of bacterial cells.

A fraction of Bacillus subtilis 168 spores, purified by Urografin isopycnic density centrifugation, heat activated, and inoculated in nutrient broth plus glucose, germinated and outgrew very synchronously. Synchrony was documented by nuclear staining and fluorescence microscopy, and by determining the variation of the buoyant density of the cells during outgrowth. Cell mass increased at constant rates and the passage from one rate to the next was dependent upon deoxyribonucleic acid (DNA) synthesis. DNA synthesis inhibitors induced the formation of anucleated sister cells in a vast majority of the population, indicating that septation was programmed and became independent from DNA synthesis very early in the cell cycle.     

Transformation in Group H Streptococci: Blackburn Strain Transformable Mutants

Transformable mutants of the nontransformable Blackburn strain group H streptococci have been isolated. In contrast with the wild strain, the cell surface extracts of these mutants contain the factor inactivating deoxyribonucleic acid in vitro.     

Concurrent changes in transducing efficiency and content of transforming deoxyribonucleic acid in Bacillus subtilis bacteriophage SP-10

Taylor, Martha J. (Fort Detrick, Frederick, Md.), and Curtis B. Thorne. Concurrent changes in transducing efficiency and content of transforming deoxyribonucleic acid in Bacillus subtilis bacteriophage SP-10. J. Bacteriol. 91:81-88. 1966.-Spores of Bacillus subtilis W-23-S(r) infected with transducing phage SP-10 served as convenient inocula for broth cultures from which transducing phage was harvested. Methods are described for producing highly infected spores. The inoculum level of infected spores in nutrient broth-yeast extract-glucose medium affected the transducing efficiency of SP-10 in lysates of these cultures. Phage in lysates of cultures inoculated with about 10(5) or fewer spores per milliliter transduced 20- to 350-fold more efficiently than did phage in lysates from cultures inoculated with 10(6) to 10(7) spores per milliliter. Transduction frequencies in the order of 10(-5) per plaque-forming unit were obtained routinely, and some infected-spore preparations yielded phage that gave frequencies as high as 10(-4). The combination of inoculum level and incubation time required to produce the best transducing phage had to be determined empirically for each batch of infected spores. Several possible explanations for the difference between lysates having high (HTE) and those having low (LTE) transducing efficiency were ruled out by special experiments. The hypothesis is presented that some cultural condition resulting from a relatively low inoculum of phage-infected spores favors the incorporation by phage particles of bacterial deoxyribonucleic acid (DNA) in the manner required for the production of transducing phage. Support for this hypothesis is a demonstration, through transformation experiments with DNA extracted from HTE and LTE phage particles, that populations of HTE phage particles yielded significantly more (7 to 27 times) transforming activity per microgram of DNA than did populations of LTE phage.     

Experimental avian PMV-2 infection in a domesticated wild host: daily behavior and effect on activity levels

Paramyxovirus type 2 (PMV-2) isolated from wild birds is often considered non-pathogenic, but nothing is known about its effects on overall behavior and fitness of free-flying birds. Domestically bred, African cut-throat finches (Amadina fasciata), a species from which PMV-2 has been isolated in the wild, were inoculated with a Central American field strain of PMV-2. Patterns of behavior were examined before and after viral challenge to quantify inapparent, sublethal effects of the disease. Infected birds demonstrated a significant decrease in activity (P = 0.01) followed by an apparent recovery period. Antibody titers confirmed infection in inoculated birds and indicated that sentinel birds did not become infected.