Isolation and physicochemical characterization of mitochondrial DNA from cultured cells ofPetunia hybrida

Abstract

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).

Characterization of cytoplasmic male sterility in Petunia hybrida and Zea mays. Localization and activity of cytochrome c oxidase

“…Currently, it is still uncertain which mitochondrially encoded protein is responsible for the developmental abnormalities in the ems petunia anthers. Deviations in mitochondrial DNA arrangement are found in organelles purified from suspension cultured cells (KOOL et al, 1985;BOESHORE et al, 1985). Certainly, a ems-specific variation in gene expression may be expressed in these vegetative cells, as well as in reproductive tissues.…”

“…In ems Petunia hybrida, KOOL et al (1985) demonstrated that the ems plasmatype is correlated with changes in mitochondrial DNA composition and its translational products. The ems-associated DNA arrangement and a homologous mitochondrial DNA region from a male fertile line are subjected to sequence and transcription analysis (BOESHORE et al, 1985).…”

Two potential Petunia hybrida mitochondrial DNA replication origins show structural and in vitro functional homology with the animal mitochondrial DNA heavy and light strand replication origins

“…Isolation, characterization and sequencing of P. hybrida mtDNA potential replication origin regions. In order to isolate P. hybrida mtDNA replication origins, purified P. hybrida mtDNA (Kool et al 1985) was digested with BamHI or BamHI/EeoRI, ligated in the appropriate linearized yeast-E, coli shuttle vector YIp5 and analyzed for autonomous replication in yeast by transforming the mtDNA bank to the ura3 yeast strain DLlc~. Transformants were selected for uracil-independent growth; because the YIp5 vector itself cannot replicate in yeast, the observed high frequency transformation should be the result of recombinant plasmids carrying mtDNA fragments which promote autonomous replication in yeast (so-called mtDNA ARS fragments).…”

“…The P. hybrida liquid cell suspenison culture AK5000 was grown as described (Kool et al (1985) with the exception that the homogenization buffer H consisted of 330 mM sorbitol, 30 mM Tris-HC1 pH 8.0, 10 mM NaC1, 10 mM MgC12 and 2 mM 2-mercaptoethanol. The final mitochondrial pellet was resuspended in 1.5 ml of H-buffer and lysed by adding two volumes of lysis buffer L [0.75 M NaC1, 50 mM Tris-HC1 pH 8.0, 2 mM DTT, 20% (v/v) glycerol, 50 p.g/ml PMSF and 50 ~tg/ml TOSF; the latter two components were dissolved at 10 mg/ml in iso-propanol].…”

Mechanism of Male Sterility in Nicotiana tabacum L.

“…When male sterility is governed by cytoplasmic factors (plasmagenes), in association with nuclear gene (s), it has been termed as cytoplasmic male sterility (CMS). Biochemical studies indicate that the CMS trait is encoded by the mitochondrial genome (Levings 1983, Kool et al 1985, Mann et al 1989.…”