Evaluation Of Eight Fluorochrome Combinations For Simultaneous Dna-Protein Flow Analyses

Eight fluorescent dye combinations for simultaneous DNA-protein staining have been evaluated spectroscopically and flow microfluoromctrically: propidium iodide (PI) with fluorescein-isothiocyanate (FITC), fluorescamine (FC), and dansylchloride (DANS); diamidinophenylindole (DAPI) with sulphorhodamin (SR101), tetramethylrhodamin isothiocyanate (TRITC), and nitroben-zodiazole (NBD); acriflavine (AF) with stilbene isothiocyanate sulphonic acid (SITS), and DAPI. Three different experimental tumor cell lines have been employed in the investigations. Simultaneous DNA-protein analyses have been carried out with the newly developed HEIFAS instrument. Spectroscopically two groups of dyes were distinguishable according to their excitation maximum below 400 nm and above 450 nm respectively. DANS and NBD were found to be unsatisfactory with respect to their protein distributions obtained by flow analysis. The remaining stains involved in the dye combinations revealed comparable flow distributions of the cellular DNA and protein content. With respect to preparation time and number of centrifugal steps involved in the staining protocols, and in connection with the stability of the dye used, the DAPI-SR101 method proved to be fastest and easiest With this combination DNA and protein flow analysis can be performed simultaneously within 30 min.     

The chromosome fiber: Evidence for an ordered superstructure of nucleosomes

Chromosome fibers isolated from lymphocyte nuclei and prepared for electron microscopy by techniques designed to preserve their native structure have a distinctly knobby appearance, suggesting that DNA and protein are not distributed evenly along the fiber axis. Individual knobs (superbeads) are arranged in tandem and have an average diameter of about 200 Å. Mild nuclease digestion of isolated nuclei releases apparent monomer superbeads that are composed of nucleohistone particles with the properties of nucleosomes. The kinetics of digestion indicate that the superbead is a discrete structural unit containing, on the average, about eight nucleosomes.     

Repair rate in human fibroblasts measured by thymine dimer excorporation

Summary The UV photoproduct, thymine dimer ( ), is excorporated with a remarkably low rate from the DNA of human fibroblasts grown in cell culture. An UV dose of 18 J/m² creates 0.045% (related to thymine). Within the first two days of repair logarithmically growing and quiescent fibroblasts exhibit the same repair rates; thereafter, the proportion of is lower in growing cells due to recovery of DNA replication. Only about 50% of the lesions are excised within 24 h. In quiescent cells, 13% of the thymine dimers originally present can be detected as late as a week after UV-irradiation. Two distinct first-order rate constants indicate that approximately half of the dimers are less accessible to repair. Repair measured by the nucleoid decondensation technique corresponds to the faster repair rate, whereas the slow repair rate cannot be detected by this method. Saturation of repair is found beyond 27 J/m². The remarkably slow rate of excision indicates that thymine dimers are not lethal lesions in human fibroblasts.     

Genome organization in Xiphophorus (Poeciliidae; Telostei)

Summary Xiphophorus represents a valuable model for studying genomic contributions to neoplasia. For analyzing these contributions at the molecular level, basic information about the genome organization is a prerequisite. This study presents data on the organization and complexity of the genomes of three species of Xiphophorus, maculatus, variatus and helleri, representative of the problem. Their diploid nuclei, as measured in the erythrocyte, contain 1.19 pg, 1.23 pg, and 1.27 pg DNA, these values representing approximately 50% of that of birds, 20% of that of mammals. The melting curves of native, high molecular weight DNA are homogeneous, the T_m was determined for maculatus as 85.0° C (corresponding to a mean GC-content of 38.3%) for variatus as 86.0° C (GC=40.7%), for helleri as 85.0° C (GC=39.3%). Reassociation of sheared denatured DNA indicated approximately 90% single copy sequences, the remaining 10% are predominantly multiple copy sequences. The complexity of single copy DNA was determined from reassociation kinetics for maculatus as 3.97×10⁸ base pairs, for variatus as 4.31×10⁸ base pairs, and for helleri as 4.49×10⁸ base pairs. The DNA of the three species upon isopycnic density gradient centrifugation in the presence of the fluorescence dye Hoechst 33258 shows in addition to the main band, two heavy (GC-rich) satellites, denoted in the order of increasing density, components I and II. Analytical centrifugation reveals for the main band DNA a buoyant density of 1.6980 gcm^-3 (GC=38.7%), for component I 1.7080 gcm^-3 (GC=48.9%), for component II 1.7150 gcm^-3 (GC=56.1%). Each of the components comprises approximately 0.38% of the total DNA. Complete digestion of components I and II with restriction enzymes EcoRI and BamHI yields a complex banding pattern upon agarose gel-electrophoresis. A 2.4 kb fragment of component I and a 5.3 kb fragment of component II of helleri, cloned and amplified in the pBR322/E. coli RR1 system, hybridize efficiently to purified nuclei of liver. Furthermore, restriction fragments of component II DNA, transferred to nitrocellulose by Southern-blotting, hybridize with 18S and 28S ribosomal DNA.     

The carbonyl reactivity of 3-bromopyruvate and related compounds

The covalent hydration of β-halopyruvic acids and β-halopyruvamides has been investigated in the uv region by stopped-flow techniques. In aqueous solutions of β-bromopyruvate and related compounds, the geminal diol is the predominant form at all pH values. Kinetic investigations have also been carried out on these hydrations. General bases enhance the rate of approaching the equilibrium. Specific acid catalysis was not detected. In the water-catalyzed reaction of pyruvamides, the relationship between the equilibrium constants K and the rate constants of the forward reaction shows that the transition state is more productlike.     

Rhythmic changes in transcriptional activity during the development of potato tubers

Chromatin-bound, DNA-dependent RNA polymerase (EC 2.7.7.6) activity and chromatin template availability, as measured with saturating amounts of E. coli RNA polymerase, changes rhythmically during the formation, dormancy, and sprouting of potato tubers. Active growth processes coincide with the highest RNA polymerase activity as well as the greatest template accessibility, during tuberization and sprouting. Consequently, chromatin-associated RNA and protein content is highest in young developing tubers and in old tubers at the onset of sprouting. Ribosomal RNA content, in turn, is maximal in small tubers, remains constant during dormancy, and decreases when sprouting begins, probably due to the translocation of rRNA into the sprouts. The nucleolus changes its shape and size concomitantly with the process of tuberization.     

Chemical carcinogenesis by the two-stage protocol in the skin ofMastomys natalensis (Muridae) using topical initiation with 7,12-dimethylbenz(a)anthracene and topical promotion with 12-0-tetradecanoylphorbol-13-acetate

Virchows Archiv B Cell Pathology - The long-term (34 weeks) topical administration of 7,12-dimethylbenz(a)anthracene (DMBA) to the skin of male and femaleMastomys induced a broad spectrum of benign...