Molecular characterization of a stable Flac plasmid

F$\text{lac}$S is a thermostable extrachromosomal element isolated in $\text{Salmonella typhimurium}$ which is altered in its replication as compared to its precursor F_ts114$\text{lac}$. Sedimentation of both these plasmids in alkaline sucrose gradients has indicated a difference in their sizes. Contour length measurements of open circular plasmid DNA molecules photographed in the electron microscope have revealed the estimated molecular weight of F_ts114$\text{lac}$ to be 81 × 10⁶ daltons while that of F$\text{lac}$S is 109 × 10⁶ daltons. F$\text{lac}$S may carry a segment of $\text{S. typhimurium}$ chromosomal or cryptic plasmid DNA.     

In vivo chain elongation of hepatic DNA: 1-beta-D-arabinofuranosyl cytosine sensitive steps.

The $\text{in vivo}$ chain elongation of rat liver DNA following partial hepatectomy was studied using alkaline sucrose gradients. DNA made in 5 min was less than 4 × 10⁷ daltons and that made in 30 min was heterodisperse and by 4 hr 75% of the DNA became larger than 1 × 10⁹ daltons. Administration of 1-β-D-arabinofuranosyl cytosine (ara-C) 5 min after thymidine-³H injection inhibited the chain elongation, whereas if given 30 minutes after thymidine-³H pulse did not inhibit the chain elongation. Thus the $\text{in vivo}$ chain elongation of rat liver DNA consists of at least two steps 1) a step sensitive to ara-C involving nucleotides addition and 2) the other insensitive to ara-C and probably involving ligation of polynucleotide chains.     

The Ribonucleic Acids of Crithidia fasciculata*

SYNOPSIS. Crithidia fasciculata ribosomes were found to be 80S and to dissociate into 58 and 41S subunits; on 5 to 50% sucrose gradients, rRNA was separated into 25, 18, and 5S components. The molecular sizes of the heavier rRNA species, estimated by polyacrylamide gel electrophoresis were 1.24 and 0.84 M (×10⁶ daltons). The 25S RNA has a tendency to interact with the 18S RNA to give a complex that is difficult to separate by sucrose gradient centrifugation. The 25S RNA is also unstable and dissociates into 0.73 and 0.57 M components. The 18S RNA has molecular size (0.84 M) higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba. Ribosomal RNA hybridized 0.29% of the nuclear DNA. Mitochondrial RNA, extracted by a rapid procedure was resolved into 16 and 5S components in sucrose gradients.     

DNA strand scission by the antitumor protein neocarzinostatin

The antibiotic protein, neocarzinostatin, induces the scission of DNA strands $\text{in vivo}$ and $\text{in vitro}$. HeLa cell DNA prelabelled with [¹⁴C] thymidine is cut into large pieces with a peak at 80–90S when cells are incubated with 0.5 to 5.0 μg/ml of highly purified neocarzinostatin. Incubation of the antibiotic (0.5 μg/ml) with [³H] SV40 DNA in the presence of 2-mercaptoethanol results in the conversion of superhelical DNA I to nicked circular duplex DNA II. At high levels of drug, smaller fragments of linear DNA are produced. Strand breaks are detected in both neutral and alkaline sucrose gradients, indicating that drug susceptibility is not due to alkali-labile bonds.     

Specific fragmentation of mitochondrial DNA from Neurospora crassa by restriction endonuclease Eco R I.

Linear, size-heterogenous mitochondrial DNA from $\text{Neurospora crassa}$ was cleaved by the restriction endonuclease Eco R I into eleven specific fragments. According to their contour lengths the fragments have molecular weights between 1.1 and 14 × 10⁶. The sum of the fragments lengths is identical with the contour length (19.8 μm, 41 × 10⁶ daltons) of the few circular molecules detectable in purified DNA preparations.The results suggest sequence homogeneity of mitochondrial DNA and further demonstrate that restriction enzymes can be used to establish a physical map of an unspecifically-fragmented DNA molecule.     

Characterization of mycoplasmatales virus DNA

The DNA of the group L1 $\text{Mycoplasmatales}$ virus, MVL51, was analyzed using alkaline sucrose velocity sedimentation, neutral and alkaline CsCl isopycnic sedimentation, and treatment of the DNA with nucleases. These treatments show that the viral chromosome is a covalently linked single-stranded DNA circle of molecular weight 2×10⁶ daltons.     

Thermodynamics of the interaction of daunomycin with DNA

The association constant for the interaction of daunomycin with DNA was determined as a function of temperature (using [³H] daunomycin in conventional equilibrium dialysis cells) and ionic strength (using a spectrophotometric titration method). The association constant varied between 3.1 × 10⁶ M^?1 (4°C) and 3.9 × 10⁵ M^?1 (65°C). The free energy change was ?8.2 to ?8.8 $\text{kcal}\text{mol}$, the enthalpy change ?5.3 $\text{kcal}\text{mol}$ and the entropy change +10 to +11 eu, all values being consistent with that expected of an intercalation process. The apparent number of intercalation sites detected (0.15 to 0.16 per nucleotide) was independent of temperature. The large positive entropy change accompanying the interaction appeals to be due to extensive release of water from the DNA and daunomycin. The apparent number of binding sites increased dramatically with decrease of ionic strength, although the apparent association constant remained largely unaffected by ionic strength.     

The early synthesis and possible function of a 0.5 X 10(6) Mr RNA after transfer of dark-grown Spirodela plants to light.

A 0.5 × 10⁶$\text{M}$_r RNA found in plastids of the aquatic angiosperm $\text{Spirodela}$, is synthesized at a much higher rate than any other rapidly labeling RNA species about $\text{3–3}\text{1}\text{2}$ h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 10⁶$\text{M}$_r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in $\text{Spirodela}$, poly(A) sequences are not detected in the 0.5 × 10⁶$\text{M}$_r RNA; yet a sucrose gradient fraction which includes RNA of this $\text{M}$_r stimulates amino acid incorporation by an $\text{E. coli}$ cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 10⁶$\text{M}$_r RNA as a chloroplast messenger is discussed.     

Dimers of escherichia coli F' factors

Covalently closed circular DNA dimers of several $\text{E.}$$\text{coli}$ sex factors have been isolated. One of these, F′451, a dimer of F′450, has a molecular weight of $\text{ca.}$ 230 × 10⁶ daltons. F′451 (λ) containing a λ prophage has a molecular weight of 260 × 10⁶ and is probably the largest covalent closed circle of DNA yet reported. These dimers arise spontaneously and are of unknown origin and significance.     

A manganese-stimulated endonuclease from Bacillus subtilis

An endonuclease activity has been identified in extracts of $\text{Bacillus subtilis}$. This activity is stimulated by Mn⁺⁺ or Ca⁺⁺ ions but not by Mg⁺⁺ ions. The enzyme catalyzes the breakdown of native DNA of high molecular weight to fragments of molecular weights ranging from 3 × 10⁶ to 20 × 10⁶. A variety of DNA's from sources such as $\text{B. subtilis, Salmonella}$ and T7 phage are attacked. About 61% of the activity of the cells is released into the medium during protoplast formation under conditions where 98% of the glucose 6-P dehydrogenase activity is retained by the cells.     

Evidence for the attachment of RNA to pulse-labeled DNA in the slime mold, Physarum polycephalum

When $\text{Physarum}\text{polycephalum}$ is pulse-labeled for up to 20 minutes with ³H-thymidine and the shortest labeled DNA strands are partially purified by sedimentation through a neutral aqueous sucrose gradient and then through a formamide-sucrose gradient, these short strands band in Cs₂SO₄ isopycnic density gradients at a density greater than that of bulk single-stranded DNA. Their density is brought partially or nearly completely back to that of single-stranded DNA by hydrolysis with pancreatic RNase A or alkali, respectively. Therefore the dense material attached to the short pulse-labeled DNA strands consists at least partially of RNA.     

Messenger RNA of mouse immune interferon (MuIFN-gamma)

A lysate of $\text{Staphylococcus aureus}$ ($\text{S. aureus}$) induced high-titered (10^3.6 ～ 10^4.6 units/ml) mouse immune interferon (MuIFN-γ) in spleen cell cultures. mRNA was extracted from such cells, purified by oligo(dT) cellulose chromatography and sucrose gradient centrifugation, and injected in $\text{Xenopus laevis}$ oocytes. Fractions containing RNA of 15 to 16 S gave rise to IFN activity, that was characterized as IFN-γ by virtue of its acid lability, neutralization by antiserum to partially purified MuIFN-γ, and lack of neutralization by antiserum to NDV-induced L-cell IFN.     

Pig liver fatty acid synthetase: purification and physicochemical properties.

This paper reports the first detailed study of the physicochemical properties of a fatty acid synthetase multienzyme complex from a mammalian liver. Fatty acid synthetase from pig liver was purified by a procedure including the following main steps: (i) preparation of a clarified supernatant solution (50,000 g), (ii) ammonium sulfate fractionation, (iii) DEAE-cellulose chromatography to separate 11 S catalase from the 13 S fatty acid synthetase, (iv) a preparative sucrose density gradient step to remove a 7 S impurity, and (v) a calcium phosphate gel step to remove an unusual yellow 16 S heme protein to yield a colorless preparation. The purified fatty acid synthetase was colorless and showed a single symmetrical peak in sucrose density gradient and conventional sedimentation velocity experiments. Fatty acid synthetase was very stable at 4 °C in the presence of 1 mm dithiothreitol and 25% sucrose. Extrapolation to zero protein concentration yielded values of S^o_20,w = 13.3 S and D^o_20,w = 2.60 × 10^?7cm²/s for the sedimentation and diffusion coefficients of the enzyme. Frictional coefficient values of 1.55 and 1.56 × 10^?7 cm, respectively, were calculated from the values for the sedimentation and diffusion coefficients. Based on these frictional coefficient values, the Stokes radius of the enzyme was calculated to be 82.4 Å. Sedimentation and diffusion coefficient data yielded a molecular weight value of $\text{M}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{s}\text{D}\text{) = 478,000}$ and sedimentation equilibrium data yielded a value of M_w = 476,000. Preliminary intrinsic viscosity measurements at 20 °C gave a value of 7.3 ml/g, indicating that the enzyme is somewhat asymmetric. This is supported by the value of 1.58 calculated for the frictional ratio and by the fact that the values for the sedimentation and diffusion coefficients are both slightly lower than expected for a globular protein of molecular weight 478,000. The enzyme possesses about 90 SH groups per molecule, assuming a molecular weight of 478,000. The ultraviolet absorption spectrum of the enzyme shows a maximum at 280 nm and an unusual shoulder at 290 nm. The fluorescence spectrum of the enzyme is dominated by tryptophan fluorescence and, over the excitation range of 260–300 nm, there is a single emission maximum at 344 nm.     

An Escherichia coli K12 mutant carrying altered ribosomal protein (S10).

An $\text{Escherichia coli}$ mutant (JE14373) carrying decreased stability of stable RNA species was found to have altered electrophoretic mobility of a 30S ribosomal protein (S10). Recombinants covering $\text{str}$ gene (76 min on $\text{E. coli}$ linkage map by Bachmann, Low and Taylor, 1976 (ref. 1)) obtained from a cross of CSH64 × JE14373, restored normal S10 protein. The size analysis of RNAs labeled for 15 min with [³H]uridine showed 50 to 60 % decrease of 16S RNA in this mutant strain, but almost no decrease of 23S RNA at 10 or 40 min after addition of rifampicin. On the other hand, no change was observed in the stability of both rRNA pieces in its parental PA3092 strain even at 40 min after addition of rifampicin.     

Organization of the nuclear ribosomal DNA of Chlamydomonas reinhardii

Summary Hybridization of cytoplasmic ribosomal RNA (rRNA) to restriction endonuclease digests of nuclear DNA of Chlamydomonas reinhardii reveals two BamHI ribosomal fragments of 2.95 and 2.35×10⁶ d and two SalI ribosomal fragments of 3.8 and 1.5×10⁶ d. The ribosomal DNA (rDNA) units, 5.3×10⁶ d in size, appear to be homogeneous since no hybridization of rDNA to other nuclear DNA fragments can be detected. The two BamHI and SalI ribosomal fragments have been cloned and a restriction map of the ribosomal unit has been established. The location of the 25S, 18S and 5.8S rRNA genes has been determined by hibridizing the rRNAs to digests of the ribosomal fragments and by observing RNA/DNA duplexes in the electron microscope. The data also indicate that the rDNA units are arranged in tandem arrays. The 5S rRNA genes are not closely located to the 25S and 18S rRNA genes since they are not contained within the nuclear rDNA unit. In addition no sequence homology is detectable between the nuclear and chloroplast rDNA units of C. reinhardii.Abbreviations used rRNA ribosomal RNA - rDNA ribosomal DNA d, dalton     

Organization of the ribosomal RNA gene cluster in the yeast Saccharomyces cerevisiae

The chromosomal organization of the ribosomal RNA gene cluster from Saccharomyces cerevisiae was investigated. 18 S rRNA R-loops were formed with unfractionated high molecular weight DNA crosslinked once per 2.7 × 10³ bases with trioxsalen and observed in the electron microscope. Almost all the R-loops were found in very long continuous 9.34 ± 0.18 × 10³ base repeating units. In addition, molecules were found at a frequency of one to two per genome equivalent of rDNA where several rRNA genes were linked to long stretches of non-rDNA. These results suggest that rDNA is arranged in a single tandem repetitive cluster of 100 to 140 genes flanked on one or both sides by non-rDNA.     

Isolation and some properties of a mammalian ribosomal DNA

The DNA coding for 28 S and 18 S ribosomal RNA, including the spacer regions, has been isolated from calf (Bos taurus) thymus gland. The method used included shearing of the total DNA to a highly homogeneous size population, selective heat denaturation and S 1 nuclease treatment to remove single stranded DNA. Repeated centrifugation on density gradients yields a 140-fold purified rDNA fraction with a GC content of 61.2%. Eco RI nuclease cleaves this DNA into two fragments of 16.4 and 4.9×10⁶ daltons. Hybridization of these fragments with 28 S and 18 S rRNA shows that the 28 S coding sequence is located mostly on the 4.9×10⁶ dalton fragment, while both the 16.4 and 4.9×10⁶ dalton fragments contain the 18 S sequence. The data indicate that the ribosomal RNA gene has a repeat unit of 21.3×10⁶ daltons which includes a nontranscribed spacer of about 12.5×10⁶ daltons.     

Anomalous enhancing effect of hydroxyurea on DNA synthesis

$\text{In vitro}$ cultures of $\text{Crithidia}$ sp. were exposed to various concentrations of hydroxyurea (HU) during the logarithmic phase. In the presence of 5 × 10^?2M HU, cell division was completely blocked after an initial increase in cell numbers by about 20%. Inhibition of incorporation of ³H-thymidine into acid-insoluble material was effective within 1 hr of exposure to the drug (5 × 10^?2M) and it reached a level of 80% after 8 hr. At lower concentrations (5 × 10^?4M ? 1 × 10^?3M), however, incorporation of ³H-thymidine was remarkably increased while cell division remained unaffected indicating that the increase in incorporation was not due to increased DNA synthesis in preparation for cell division.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Nuclear and cytoplasmic binding components for vitamin D metabolites.

Specific binding of 1α,25-dihydroxyvitamin D₃ to macromolecular components of small intestinal nuclei and cytosol is demonstrated. The nuclear 1α,25-dihydroxyvitamin D₃ complex can be extracted from chromatin by 0.3 M KCl and sediments at 3.7S in sucrose density gradients. The cytoplasmic 1α,25-dihydroxyvitamin D₃-binding components also sediment at 3.7S, identically to the nuclear complex under the ultracentrifugation procedures employed.Macromolecular binding components with a high affinity for 25-hydroxyvitamin D₃ (K_d = 4.5 × 10⁻⁹ M) were also identified in intestinal cytosol which differ from the 1α,25-hydroxyvitamin D₃ receptor in that: 1) they sediment at 5–6S in sucrose gradients, 2) they are observed in organs other than the intestine, and 3) while they do bind 1α,25-dihydroxyvitamin D₃ at higher concentrations than 25-hydroxyvitamin D₃, they are not observed to transfer either 25-hydroxyvitamin D₃ or 1α,25-dihydroxyvitamin D₃ to the nucleus, $\text{in vitro}$.