Role of polyadenylic acid in a deoxyribonucleic acid-membrane fraction extracted from pneumococci.

After the addition of radioactive polyadenylic acid to cell suspensions of pneumocci, part of the radioactivity becomes associated with a deoxyribonucleic acid (DNA)-membrane fraction extracted from the cells. A variety of techniques show that a portion of this associated radioactivity may represent oligoadenylates complexed to DNA, probaby as part of a ribonucleic acid (RNA) component. Polyadenylic acid, which had previously been shown to enhance DNA synthesis in cell suspensions (Firshein and Benson, 1968), also enhances the extent of DNA synthesis by the DNA-membrane fraction in vitro under specific conditions of concentration and conformation. The mechanism of action of this enhancement may be related to the ability of oligoadenylates to increase the number of initiation sites for DNA replication by stimulating the production of an RNA primer, thus providing additional 3'-OH groups with which DNA polymerase can react.     

Role for deoxyribonucleic acid ligase in deoxyribonucleic acid polymerase i-dependent repair synthesis in toluene-treated escherichia coli.

In a toluene-treated mutant of Escherichia coli K-12 having a temperature-sensitive, conditionally lethal mutation in the structural gene for deoxyribonucleic acid (DNA) ligase, an extensive DNA repair synthesis occurred in X-irradiated cells at the nonpermissive temperature, 42 C. At the permissive temperature, 30 C, nearly normal semiconservative synthesis and limited repair synthesis were observed when DNA ligase was activated by the addition of nicotinamide adenine dinucleotide.     

Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.

A cell-free system for the study of viral DNA replications was developed by the isolation of a nuclear membrane fraction "DNA replication complex" from adenovirus 2-infected human KB cells late after infection. This complex which possesses both DNA polymerase activity and a virus-specific endonuclease synthesizes exclusively virus-specific DNA sequences in vitro by a semiconservative mechanism. Analysis by rate zonal sedimentation in alkaline sucrose gradients showed that the products of the reaction are small DNA chains approximately 6 to 9 S, presumably "Okazaki intermediates," that are not sealed under our in vitro conditions. Analysis by rate zonal sedimentation in neutral sucrose gradients showed that labeled viral DNA fragments are hydrogen bonded to viral 18 S DNA segments, 0.25 the size of the linear, viral 31 S DNA genome. The 18 S DNA is probably a specific cleavage product of the viral endonuclease found in the replication complex and could represent intermediates in viral DNA replication or degradation products.     

Cell surface-located deoxyribonucleic acid receptors in transformable pneumococci.

We studied deoxyribonucleic acid (DNA) binding in transformable pneumococci. The relevant findings are as follows. (i) At least half of the DNA Molecules adsorbed to competent cells in the growth medium are attached to sites on the protoplast membrane. (ii) Most of the DNA bound to live competent cells in the presence of glucose is not released by moderate shear or by autolysin treatment. In contrast, most of the DNA adsorbed to competent cells in the absence of glucose is shear and autolysin sensitive. (iii) The presence of binding sites resembling in properties the sites in live competent cells can be demonstrated in wall-membrane complexes. Most of these sites are lost during preparation of cell walls and protoplasts. It is suggested that the DNA-binding site is a membrane component (protein?) Stabilized by polysaccharide (cell Wall) material. (IV) Mechanical or enzymatic damage to the cell wall or change in the ionic conditions can induce DNA binding (and surface-nuclease activity) in the incompetent pneumococci. However, such cells still show neither genetic transformation nor extensive nuclease-resistant binding of DNA. It is suggested that both competent and incompetent cells contain a large number of sequestered DNA-binding sites that can be unmasked by several experimental conditions. Induction of the competent state by the competence activator protein may involve an endogenous unmasking process.     

Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12.

The biosynthesis of ribosomal ribonucleic acid (rRNA) In wild-type Neurospora crassa growing at 25 degrees C was investigated by continuous-labeling and pulsechase experiments using [5-3H]uridine. The results of these experiments suggest the following precursor-product relationships: the first RNA molecule to be synthesized in significant quantities is the 2.4 X 10(6)-dalton (2.4-Mdal) ribosomal precursor RNA. This RNA is cleaved to produce two species of RNA with weights of 0.7 and 1.4-Mdal. The former is the mature 17S rRNA of the 37S ribosomal subunit. The 1.4-Mdal RNA is subsequently cleaved to produce the mature 1.27-Mdal (25S) and 61,000-dalton (5.8S) rRNA's of the 60S ribosomal subunit. In the maturation process, approximately 15 to 20% of the 2.4-Mdal ribosomal precursor rRNA molecule is lost. As in other eukaryotes that have been examined, 5S rRNA is not derived from this precursor molecule.     

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.     

Glycolipids stimulate DNA polymerase activity in a DNA-membrane fraction and in a partially purified polymerase system extracted from pneumococci.

We have assayed the ability of various lipids to affect DNA polymerases activity in a DNA-membrane complex extracted from Streptococcus pneumoniae by the Sarkosyl-M-band technique. In addition, to determine which DNA polymerases were affected by the lipids, we partially purified three DNA polymerase activities from cell lysates, the first such demonstration outside of Escherichia coli and Bacillus subtilis. Glycolipids are unique among polar lipids in stimulating the rate and extent of DNA polymerase activity in M-bands and in Sarkosyl lysates from which the M-band is derived. It appears that they exert this stimulatory effect, in part, by removing (neutralizing) detergent molecules which act as inhibitors, as well as by substituting for the detergent, thereby creating a favorable environment for the polymerases involved in DNA synthesis. That the stimulatory effect is not simply a detoxification of the detergent was shown by two observations. One, phospholipids, although interacting with Sarkosyl and therefore "potentially" capable of detoxifying the system, did not stimulate DNA polymerase activity in vitro. Two, glycolipids were capable of stimulating the activity of at least two DNA polymerases partially purified from cell lysates in the absence of any Sarkosyl. The stimulatory effect was greater for a polymerase that had four characteristics similar to those observed with polymerase III in other organisms.     

Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli.

A new Escherichia coli deoxyribonucleic acid (DNA) ligase mutant has been identified among a collection of temperature-sensitive DNA replication mutants isolated recently (Sevastopoulos, Wehr, and Glaser, Proc. Natl. Acad. Sci. U.S.A. 74:3485-3489, 1977). At the nonpermissive temperature DNA synthesis in the mutant stops rapidly, the DNA is degraded to acid-soluble material, and cell death ensures. This suggests that the mutant may be among the most ligase-deficient strains yet characterized.     

A deoxyribonucleic acid ligase from nuclei of rat liver. Purification and properties.

A DNA ligase has been extensively purified from nuclei of rat livers. The ligase seals single strand nicks in DNA with any of the four usual bases on either the 3 or 5 sides. It requires ATP and a divalent cation (Mg-2plus or Mn-2plus) for activity. At low Mg-2plus concentrations the activity is greatly stimulated by a variety of monovalent cations. Relatively small excesses of either monovalent or divalent cation above the amounts which give maximal activity lead to inhibition of activity. Poly(G) and poly(I) inhibit ligase activity; several other polyribonucleotides are not inhibitory. Low concentrations of inorganic pyrophosphate are inhibitory. The molecular weight of the ligase is estimated from gel filtration to be about 10 times 10-4.     

Selective release of a deoxyribonucleic acid-binding factor from the surface of competent pneumococci.

Methods are described that resulted in the selective release of deoxyribonucleic acid (DNA)-binding factor from the surface of competent pneumococci. The same methods caused a parallel inactivation of the DNA-binding capacity of the extracted bacteria. Genetically or physiologically incompetent pneumococci did not yield binding factor upon exposure to the same methods. The solubilized binding factor appeared to be a protein; it could be assayed by a membrane filter binding procedure. The binding factor had properties reminiscent of those of the DNA receptors of transformable pneumococci (Seto et al., 1975).     

Calcium-requiring step in the uptake of deoxyribonucleic acid molecules through the surface of competent pneumococci.

The conversion of surface-adsorbed deoxyribonucleic acid (DNA) molecules to a state in which they are inaccessible to exogenous deoxyribonuclease requires specifically calcium ions; magnesium ions cannot replace calcium ions. Virtually maximal levels of nuclease-resistant DNA binding and genetic transformation can be obtained in media free from magnesium and containing only calcium ions. It is suggested that the calcium-requiring process is the transport of DNA molecules across the plasma membrane. Magnesium ions stimulate both the loss of surface-adsorbed DNA to the medium and the extracellular degradation of DNA.     

Phosphatidic acid releases calcium from a platelet membrane fraction in vitro

A platelet membrane fraction which actively sequesters calcium in the presence of ATP was prepared and the influence of phosphatidic acid evaluated. At 10–60 μg/ml phosphatidic acid caused a concentration dependent release of calcium from the membrane fraction. The calcium was released from inside the vesicles, since release occurred in the presence of EGTA used to bind calcium outside the membrane vesicles. Aspirin failed to inhibit release of calcium by phosphatidic acid. Our results may explain, in part, the prostaglandin and thromboxane independent calcium release which occurs in response to certain aggregating agents. Thus, phosphatidic acid, or a metabolite, may have an important role intracellularly in platelets in promoting calcium movement.