ABSTRACT
A procedure is described for the extraction of isosorbide-5-mononitrate from serum with diethyl ether-n-butyl acetate (15:1, v/v). After extraction, the diethyl ether is evaporated at room temperature and the nitrate remains dissolved in the relatively non-volatile n-butyl acetate phase. Glassware must be used throughout as the recovery of isosorbide-5-mononitrate is not reproducible when plastic tubes or plastic pipettes are used. The extraction recovery of isosorbide-5-mononitrate from serum is 67 +/- 7%, measured relative to external standards.
Subject(s)
Isosorbide Dinitrate/analogs & derivatives , Vasodilator Agents/blood , Chromatography, Gas , Humans , Isosorbide Dinitrate/blood , Reference Standards , Reproducibility of ResultsABSTRACT
Four Petunia hybrida mitochondrial (mt) DNA fragments have been isolated, sequenced, localized on the physical map and analyzed for their ability to initiate specific DNA synthesis. When all four mtDNA fragments were tested as templates in an in vitro DNA synthesizing lysate system, developed from purified P. hybrida mitochondria, specific initiation of DNA synthesis could only be observed starting within two fragments, oriA and oriB. When DNA synthesis incubations were performed with DNA templates consisting of both the A and B origins in the same plasmid in complementary strands, DNA synthesis first initiates in the A-origin, proceeds in the direction of the B-origin after which replication is also initiated in the B-origin. Based on these observations, a replication model for the P. hybrida mitochondrial genome is presented.
Subject(s)
DNA Replication , DNA, Mitochondrial/genetics , Animals , Base Sequence , Cloning, Molecular , DNA/ultrastructure , Humans , Microscopy, Electron , Molecular Sequence Data , Organelles , Plants , Plasmids , Restriction Mapping , Sequence Homology, Nucleic AcidABSTRACT
Mitochondrial DNA ofPetunia hybrida was purified from cell suspension cultures. Up to 50% of the DNA could be isolated as supercoiled DNA molecules by CsCl-ethidium bromide density gradient centrifugation. The DNA purified from DNase-treated mitochondria bands at a single buoyant density of 1.760 gcm(-3) in neutral density gradients and runs on agarose gels as a single band with an apparent molecular weight exceeding 30 megadaltons (Md). Summing of the restriction endonuclease fragment lengths indicates a mitochondrial genome size of at least 190 Md. Electron microscopic analysis reveals the presence of a heterogeneous population of circular DNA molecules, up to 60 Md in size. Small circular DNA molecules, ranging in size from 2-30 Md are present, but unlike in cultured cells of other plant species they do not form discrete size classes and furthermore, they constitute less than 5% of the total DNA content of the mitochondria. The restriction endonuclease patterns of mitochondrial DNA do not qualitatively alter upon prolonged culture periods (up to at least two years).
