Buoyant density constancy of Schizosaccharomyces pombe cells.

Buoyant densities of cells from exponentially growing cultures of the fission yeast Schizosaccharomyces pombe 972h- with division rates from 0.14 to 0.5 per h were determined by equilibrium centrifugation in Percoll gradients. Buoyant densities were independent of growth rate, with an average value (+/- standard error) of 1.0945 (+/- 0.00037) g/ml. When cells from these cultures were separated by size, mean cell volumes were independent of buoyant density, indicating that buoyant densities also were independent of cell age during the division cycle. These results support the suggestion that most or all kinds of cells that divide by equatorial fission may have similar, evolutionarily conserved mechanisms for regulation of buoyant density.     

Renaturation and Hybridization Studies of Mitochondrial DNA

The products of the renaturation reaction of mitochondrial DNA from oocytes of Xenopus laevis have been studied by electron microscopy and CsCl equilibrium density gradient centrifugation. The reaction leads to the formation of intermediates containing single-stranded and double-stranded regions. Further reactions of these intermediates result in large complexes of interlinking double-stranded filaments. The formation of circular molecules of the same length as native circles of mitochondrial DNA was also observed. The formation of common high molecular weight complexes during joint reannealing of two DNA's with complementary sequences was used as a method to detect sequence homology in different DNA samples. Although this method does not produce quantitative data it offers several advantages in the present study. No homologies could be detected between the nuclear DNA and the mitochondrial DNA of X. laevis or of Rana pipiens. In interspecies comparisons homologies were found between the nuclear DNA's of X. laevis and the mouse and between the mitochondrial DNA's of X. laevis and the chick, but none between the mitochondrial DNA's of X. laevis and yeast. These results are interpreted as indicating the continuity of mitochondrial DNA during evolution.     

Satellite Dnas in the Embryos of Various Species of the Genus Drosophila

A tentative evolutionary pattern has been found for two classes of the multiple satellite DNA's found in the genus Drosophila. The satellite DNA's from five Drosophila species (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) were analyzed and found to fall into three arbitrary CsCl buoyant density classes: Class I, rho = 1.661-1.669 g cm(-3), DNA molecules composed of primarily dA and dT moieties; Class II, rho = 1.685 and rho = 1.692, DNA molecules of low GC content; and Class III, rho = 1.711, a DNA of high GC composition. The dAT satellite DNA's appear in all the species studied except D. hydei, the species of most recent evolutionary divergence, whereas the heavy satellite appears only in the two species of most recent divergence, D. virilis and D. hydei.     

Sedimentation equilibrium of proteins in density gradients.

The technique of sedimentation equilibrium in density gradients in the analytical ultracentrifuge has been applied to the study of proteins. A variety of effects and procedures including the use of density marker beads, the effects of pressure on buoyant density and pH, and the calculation of compositional density gradient proportionality constants and density--refractive index relations have been developed. The buoyant densities of twenty-four proteins have been measured and hydration values computed. The buoyant titrations of six proteins have been measured. These data have been interpreted in terms of the buoyant titrations which have been obtained for six ionizable homopolypeptides, five copolypeptides, two non-ionizable homopolypeptides and three chemically modified proteins. Spectropolarimetry and potentiometric titrations were employed to further interpret these data. Approximate values for dissociation constants, numbers of ionizable residues, and the nature of ions bound or dissociated upon ionization have been obtained. The relation between potentiometric and buoyant titrations and the use of density gradient centrifugation as a probe for protein structure have been explored.     

The isolation of IS1 and IS2 DNA

Summary DNA of the IS-elements IS1 and IS2 was prepared by digestion of appropriate heteroduplex molecules with endonuclease S1, followed by sucrose gradient centrifugation or gel electrophoresis. The material obtained is homogeneous with regard to size. The length of IS1 DNA is 820±65 nucleotides, the length of IS2 DNA is 1.350±70 nucleotides. IS1 DNA is not cleaved by the restriction endonucleases Eco R1, Hind II or Hind III. IS2 DNA is cleaved once by each of the two latter enzymes. The buoyant density determined by equilibrium centrifugation of Hg-complexes in Cs₂SO₄ corresponds to a GC content of approximately 50%. Labelling with polynucleotide kinase indicates that both IS DNA's have a guanosyl residue at both of their 5-termini.     

Independence of buoyant cell density and growth rate in Escherichia coli

The relationship between growth rate and buoyant density was determined for cells from exponential-phase cultures of Escherichia coli B/r NC32 by equilibrium centrifugation in Percoll gradients at growth rates ranging from 0.15 to 2.3 doublings per h. The mean buoyant density did not change significantly with growth rate in any of three sets of experiments in which different gradient conditions were used. In addition, when cultures were allowed to enter the stationary phase of growth, mean cell volumes and buoyant densities usually remained unchanged for extended periods. These and earlier results support the existence of a highly regulated, discrete state of buoyant density during steady-state growth of E. coli and other cells that divide by equatorial fission.     

Characterization of the DNA in DROSOPHILA MELANOGASTER

DNA has been quantitatively extracted from Drosophila melanogaster at various stages of embryonic development and analyzed by isopycnic centrifugation in CsCl and by fractionation on methylated albumin columns. The DNA is composed of three main classes of DNA, as defined by their buoyant density, rho, in CsCl: a bulk DNA, rho = 1.699 g cm(-3), and two satellite DNAs, rho = 1.685 g cm(-3) and rho = 1.669 g cm(-3). These three types of DNA persist throughout the development of the insect. In the unfertilized egg, 80% of the total DNA consists of the satellite DNAs; this amount decreases to 18% during the first three hours after fertilization and then remains constant through embryogenesis. There is a concomitant increase of the satellite DNA's with the bulk DNA after blastoderm formation.     

Different forms of a lipopolysaccharide-protein complex from Yersinia pseudotuberculosis

Two forms of lipopolysaccharide-protein complex with buoyant densities of 1,43 and 1,40 g/cm3 were found in the Yersinia pseudotuberculosis cell wall. These forms have the similar monosaccharide, fatty acid and polypeptide compositions, but differ in the length of O-specific chains. The differences in density are stipulated by the different contents of the main components of the complex. Both forms contain the related antigenic determinants but have some differences in the antigenic structure. The ability of the two forms to produce a hybrid form with the intermediate density of 1,41 g/cm3 has been shown.     

Properties of satellite DNA of three plant species of the subtribe Citrinae

The properties of stDNA of three species of the subtribe Citrinae have been investigated. The buoyant density of the main component is 1.693 g/cm3, that of satellite component is 1.712 g/cm3 and 1.715 g/cm3. The differential melting curves of satellite components reveal two melting zones. Some of stDNAs are melted within a broad temperature range, while others--at higher temperatures as a narrow peak. The reassociation kinetics suggest that 50-54% of stDNA are a fast reassociating fraction with the length of repeating sequences of 0.8-1.1 x 10(2) base pairs. Based on the values of Tm and buoyant density the 5-methylcytosine content in stDNA was calculated and was found equal to 20-35%. Using equilibrium ultracentrifugation in the actinomycin D--CsCl density gradient the stDNAs of the subtribe Citrinae were separated into constituent components.     

Characterization of HCV-like particles produced in a human hepatoma cell line by a recombinant baculovirus

Although processing of the hepatitis C virus (HCV) polyprotein and characterization of each of its viral proteins have been described in detail, analysis of the structure and assembly of HCV particles has been hampered by the lack of a robust cell culture system to support efficient replication of HCV. In this study, we generated HCV-like particles (HCV-LP) using a recombinant baculovirus encoding structural and a part of non-structural proteins in a human hepatoma cell line. The HCV-LP exhibited a buoyant density of 1.17 g/ml in CsCl equilibrium gradient and particles of 40 to 50 nm in diameter. Binding of the HCV-LP to human hepatoma cells was partially inhibited by the treatment with anti-hCD81 antibody, in contrast to the hCD81-independent binding of HCV-LP produced in insect cells. These results indicate that HCV-LP generated in different types of cells exhibit different cellular tropism for binding to target cells.     

Properties of satellite DNA from Brassica nigra

The DNA components of B. nigra were preparatively separated by equilibrium ultracentrifugation in a CsCl density gradient, the buoyant density of the main component being 1,696 g . cm-3, that of the satellite component--1,704 g . cm-3. The properties of individual DNA fractions were investigated. Four major components could be observed on the differential melting curve of satellite DNA. Using the reassociation kinetics method it was shown that 30% of satellite DNA are presented as a fast reassociating component with a length of a repeated unit of approximately 2,5 . 10(3) nucleotide pairs. The calculated values of Tm and buoyant density suggest that the m5C content in satellite DNA is lower than that in the main component. During equilibrium ultracentrifugation in the density gradients of actinomycin D--CsCl and Hg2+--Cs2SO4 the satellite DNA is split into 4 major components.     

Buoyant density constancy during the cell cycle of Escherichia coli

Cell buoyant densities were determined in exponentially growing cultures of Escherichia coli B/r NC32 and E. coli K-12 PAT84 by equilibrium centrifugation in Percoll gradients. Distributions within density bands were measured as viable cells or total numbers of cells. At all growth rates, buoyant densities had narrow normal distributions with essentially the same value for the coefficient of variation, 0.15%. When the density distributions were determined in Ficoll gradients, they were more than twice as broad, but this increased variability was associated with the binding of Ficoll to the bacteria. Mean cell volumes and cell lengths were independent of cell densities in Percoll bands, within experimental errors, both in slowly and in rapidly growing cultures. Buoyant densities of cells separated by size, and therefore by age, in sucrose gradients also were observed to be independent of age. The results make unlikely any stepwise change in mean buoyant density of 0.1% or more during the cycle. These results also make it unlikely that signaling functions for cell division or for other cell cycle events are provided by density variations.     

Density fluctuation during the cell cycle in the defective vacuolar morphology mutants of Saccharomyces cerevisiae.

The buoyant densities of the yeast cells of defective vacuolar morphology mutants were examined by equilibrium sedimentation centrifugation in a Percoll density gradient. These vacuoleless mutants also show density fluctuation as wild-type cells during the cell cycle. This suggests that morphological changes of the vacuole are not related to cyclic density fluctuation in Saccharomyces cerevisiae.     

Interactions of non-histone proteins with immobilized components of chromatin

The specific interaction between non-histone proteins (NHP) of rat thymus and the dextran-immobilized components of chromatin has been investigated. DNA's from E. coli and rat thymus, total histone and reconstituted nucleohistone were used as the affinity sorbents. The distribution of protein fractions in the NHP groups dissociated from chromatin with different ionic strengths was studied by binding on the columns and by SDS-PAAG-electrophoresis. It is shown that NHP of chromatin include the protein components with selective affinity to nucleohistone. These results are discussed in connection with the different functions of NHP in chromatin.     

Buoyant density variation during the cell cycle of Saccharomyces cerevisiae

Cell buoyant densities of the budding yeast Saccharomyces cerevisiae were determined for rapidly growing asynchronous and synchronous cultures by equilibrium sedimentation in Percoll gradients. The average cell density in exponentially growing cultures was 1.1126 g/ml, with a range of density variation of 0.010 g/ml. Densities were highest for cells with buds about one-fourth the diameter of their mother cells and lowest when bud diameters were about the same as their mother cells. In synchronous cultures inoculated from the least-dense cells, there was no observable perturbation of cell growth: cell numbers increased without lag, and the doubling time (66 min) was the same as that for the parent culture. Starting from a low value at the beginning of the cycle, cell buoyant density oscillated between a maximum density near midcycle (0.4 generations) and a minimum near the end of the cycle (0.9 generations). The pattern of cyclic variation of buoyant density was quantitatively determined from density measurements for five cell classes, which were categorized by bud diameter. The observed variation in buoyant density during the cell cycle of S. cerevisiae contrasts sharply with the constancy in buoyant density observed for cells of Escherichia coli, Chinese hamster cells, and three murine cell lines.     

Long-chain triglyceride lipases of pig liver

The blood-group specific glycoproteins of human ovarian cyst fluids have been isolated by equilibrium density gradient centrifugation in CsCl; they have been characterised in terms of buoyant density, selective salvation and apparent molecular weight, both in CsCl and Cs(2)SO(4).     

Polypyrimidine segments in drosophila melanogaster DNA: I. Detection of a cryptic satellite containing polypyrimidine/polypurine DNA

Very long runs of pyrimidine nucleotides (polypyrimidines), previously detected in DNA from Drosophila melanogaster, have now been localized to a “cryptic” satellite. These polypyrimidines have an average length of 750 nucleotides and account for about 3% of the thymine residues in total DNA. The buoyant density of the DNA component which contains the polypyrimidines was detected by centrifuging native DNA to equilibrium in a CsCI gradient, and then assaying each fraction for its content of polypyrimidines. A peak was detected at a density of about 1.707 gm/cm³, distinctly heavier than the main band of DNA (1.702 gm/cm³). The buoyant density of polypyrimidine-containing molecules was little affected by differences in the molecular weight of the starting DNA in the range 10⁵-10⁷ daltons (single-stranded). Thus polypyrimidines (and their complementary polypurines) appear to form all or part of a “cryptic” satellite.Polypyrimidines have been isolated and characterized with respect to composition and buoyant density. Direct nucleoside analysis of unlabeled material indicated 34.5% deoxycytidine, 65.5% thymidine. Their banding position in neutral and alkaline CsCI gradients was consistent with a single-stranded DNA polymer of this composition.     

Antigenic and deoxyribonucleic acid base composition of aMycoplasma and associated diphtheroids

Three diphtheroids isolated from cultures ofMycoplasma arthritidis (Campo strain) were shown to cross-react serologically with theMycoplasma. Stable L-forms prepared from these bacteria were shown to have still greater antigenic similarity to theMycoplasma. But the three diphtheroids isolated on three separate occasions were shown not to be identical antigenically. The buoyant densities of the DNA's of the L-forms were similar to those of the diphtheroids from which they were derived. The caesium chloride gradient method indicated a significant difference in nucleotide base composition between the purified deoxyribonucleic acid of the diphtheroids and of theMycoplasma.Junior Research Fellow supported by USPH training grant 5 ROI AI 00232.     

Four human carcinoma cell lines with novel mutations in position 12 of c-K-ras oncogene.

We have used synthetic oligonucleotides to probe for mutations affecting amino acid 12 of the c-K-ras gene in human cell line DNA. Of seven carcinoma cell lines tested, four were found to contain a mutation at this position. In each the nucleotide G was replaced with an A resulting in a Gly to Asp substitution in three cases (cell lines A427, A1165 and A1663) and Gly to Ser in the fourth (A549). Neither of these substitutions have been previously reported in either human tumor or human tumor-derived cell line DNA's. These results indicate that association between mutations involving position 12 of the human c-K-ras oncogene and carcinomas may be stronger than previously recognized.     

Peptidase activity in the inner membrane of Pseudomonas aeruginosa

The location of peptidase activity within the cell envelope structure of Pseudomonas aeruginosa has been studied. Inner and outer membrane fractions were separated on the basis of buoyant density using two consecutive sucrose steps gradients and identified on the basis of known components. The inner membrane was shown to contain peptidase activity while the outer membrane contained none. These data support the hypothesis that P. aeruginosa transports intact peptides.