Stephen J. Keller

RNA synthesis by isolated mammalian mitochondria and nuclei: Effects of ethidium bromide and acriflavin

Abstract The effects of ethidium bromide and acriflavin on RNA synthesis by isolated rat liver mitochondria and nuclei were studied. Mitochondrial RNA synthesis was found to be much more sensitive to these dyes than is nuclear RNA synthesis. At low concentrations both drugs actually stimulated RNA synthesis by isolated nuclei, but inhibited at higher concentrations. Thus, it was suggested that stimulation may be due to removal of repressors since RNA synthesis using solubilized rat liver RNA polymerase and naked DNA as template failed to show such stimulation and was inhibited by much lower concentrations of dyes. Acriflavin was less effective as an inhibitor than ethidium bromide at any given concentration. Results with calf liver organelles were identical to those obtained with rat liver.

DNA biosynthesis in nuclei: I. Characterization of DNA synthesis by isolated rat liver nuclei using endogenous DNA as primer

Abstract 1. A DNA biosynthetic system consisting of isolated nuclei using endogenous DNA as primer has been characterized. The nuclei are capable of incorporating [ 3 H] dTTP into DNA linearly for 2 h, and incorporation is proportional to protein concentration up to 2 mg/ml. 2. Preincubation at 37 °C greatly enhances incorporation, but heating for 4 min at 65 °C destroys activity. The pH optimum is 8.0 with Tris-HCl and 9.0 with glycine-NaOH buffer. The system requires Mg 2+ and is stimulated by ATP, KCl, sucrose and glycerol; but deoxyribonuclease, polyamines, Na 4 P 2 O 7 , actinomycin D, ethidium bromide and acriflavin inhibit incorporation. 3. Addition of native DNA to the reaction stimulates incorporation 5-fold. DNA synthesis by endogenously and exogenously primed nuclei has been compared. CsCl density gradient centrifugation indicates that endogenously primed reactions synthesize rat liver DNA, but addition of exogenous DNA causes inhibition of endogenous DNA synthesis in favor of the exogenous DNA.

Partial purification of a chloroplast DNA polymerase from Euglena gracilis

Abstract A single DNA polymerase has been purified 965 fold from isolated chloroplasts of Euglena gracilis with a yield of 53%. The isolation methods include solubilization of the enzyme with 1M NaCl, ammonium sulfate precipitation, DNA affinity and DEAE-cellulose chromatography. The enzyme requires all four deoxynucleotide triphosphates, magnesium and denatured DNA for maximal activity. The chloroplast DNA polymerase is free of contaminating nucleases and phosphatases, has a sharp pH optimum at pH 7.2 and magnesium optimum of 6mM.

Animal virus screens for potential teratogens. I. Poxvirus morphogenesis.

The growth of poxvirions in cell culture is considered a teratogen screening test, since this virus has a rapid, simple morphogenetic pathway that is dependent upon cell proliferation. Vaccinia WR-infected BSC 40 monolayers were exposed to 42 known teratogens and 9 nonteratogens at dosages from 1 microM to 100 mM. After 24 h of infection, the number of functional virions was determined by plaque assay. Thirty-three of the 42 teratogens inhibited the virus, 3 teratogens stimulated the virus, and 6 teratogens were false-negatives. Eight of the 9 nonteratogens had no effect on virus proliferation at dosages as high as 600 times the lowest reported teratogenic dosage. The number of new virions could be directly related to the concentration of the teratogen in vitro, thus allowing each compound to be characterized by an RD50. The RD50 dosage in milligrams per liter was 98% correlated with the lowest reported teratogenic dose in vivo in milligrams per kilogram. In sum, vaccinia-infected cells have an easily identifiable endpoint, plaque-forming units, which may be an accurate prognosticator of teratogenesis.

Chloroplast DNA Replication in Chlamydomonas reinhardtii

Publisher Summary Chlamydomonas reinhardtii occupies a central position in cytoplasmic inheritance because it is the simplest organism that contains a chloroplast and non-Mendelian inheritance. Being unicellular and heterothallic, Chlamydomonas can be handled in the laboratory with the same ease as bacteria and yeast. The vegetative, haploid life cycle and the diploid meiotic sexual cycle can be physiologically controlled in large synchronous populations to facilitate either cell cycle biochemistry or genetic analysis. This chapter discusses the replication of the chloroplast DNA in vivo. Chloroplast replication usually is initiated 1–3 hours after the onset of the light period and continues for six hours, whereas nuclear DNA synthesis is initiated 2–3 hours into the dark period and continues for six hours. If cell division results in the production of four daughters, then two rounds of replication of chloroplast DNA synthesis precede two rounds of nuclear DNA synthesis. Both DNAs replicate by a semiconservative mechanism. Chlamydomonas does not appear to have dark repair reactions, so the chloroplast chromosome is stable over the entire cell cycle

DNA Replication in Simple Eukaryons: Identification of Replisomal Complexes in Cell Extracts of Chlamydomonas reinhartii

The identification of replication proteins in eukaryons has generally been hampered by the complexity of eukaryon chromosomes, the unavailability of appropriate conditional lethal mutations, and an inability to define DNA replication in Vitro as opposed to repair or recombination reactions. As a result, we have initiated a biochemical study on the unicellular green alga, Chlamydomonas reinhartii, since this organism is ideal for cell genetics, biochemical studies, and physiological manipulation in the laboratory (1). Density transfer experiments in Vivo suggest that DNA replication is restricted to a limited portion of either the mitotic or meiotic cell cycles, that DNA repair occurs during normal replication, and that recombination events are limited to meiotic gametes (2–4). Vegetatively grown cells thus appear to contain primarily the enzymes necessary for replication. We have attempted to study the DNA replication proteins by preparing extracts from the cell-wallless mutant, CW-15, of Chlamydomonas and by selecting experimental conditions which favor the formation of high molecular weight complexes which can interact with single stranded DNA. Our approach therefore relies upon observations which have been made on bacterial extracts which contain multienzymatic protein complexes that efficiently duplicate phage chromosomes (5,6).