Physicochemical Characterization of Mitochondrial DNA from Pea Leaves

Abstract

The mitochondrial DNA from pea leaves exists in a circular conformation. 25% of the circular molecules exist as supercoils, and 10% of the molecules are dimers. The molecular weight of mitochondrial DNA is about 66 to 70 X 106 by electron microscopy, and 74 X 106 from its renaturation kinetics. No evidence for inter-and intramolecular heterogeneity is found.The mitochondrial (mt-) DNA from animal tissues exists in the form of closed circular duplex molecules of 10 X 106 molecular weight (1). Luck and Reich (2, 3) reported the isolation of linear molecules of molecular weight 13 X 106 from Neurospora mitochondria, and calculated that the molecular weight of mt-DNA might be about 66 X 106 from its renaturation kinetics. Recent studies (4) have shown that mt-DNA from Neurospora can be obtained in a linear structure 26 jum in length. Similarly, mt-DNA from slime mold Physarium has been isolated in linear forms, with molecular weights ranging from 20 to 30 X 106 (5). Extensive studies in osmotically shocked preparations of yeast have demonstrated the existence of mt-DNA in circular form (6). However, all attempts to isolate these circular molecules -were unsuccessful. The molecular weight of yeast mt-DNA was calculated to be 50 X 106 (6). Mt-DNA from protozoa, Tetrahymena, and Paramecium, are linear structures of 17.6 jim in length (7). Mt-DNA from higher plants has always been isolated in linear molecules with mean lengths ranging from 10 to 20 ,um (7,8). We have studied mt-DNA from pea leaves; it has a molecular weight of 74 X 106 from its renaturation rate. The DNA released from osmotically shocked mitochondria, as well as that isolated by deproteinization, has a circular form with an average contour length of 30 jum under experimental conditions in which replicative form (RF) II DNA from bacteriophage OX-174 has a length of 1.45 Mum. This is the largest circular mt-DNA isolated to date. MATERIALS AND METHODSI8olation of Mitochondrial DNA. 12-to 15-day-old pea leaves were homogenized with 4 volumes of buffer containing 0.3 M mannitol-0.05 M Tris HCl (pH 8.0)-3 mM EDTA-0.1% bovine-serum albumin-1 mM 2-mercaptoethanol.Homogenates were filtered through cheese cloth and centrifuged at 1020 X g for 15 min; the supernatant was then centrifuged at 12,000 X g for 20 min. The mitochondrial fraction (contaminated with broken nuclei and chloroplasts) was suspended in the buffer (200 ml/kg of leaves) and centrifuged twice at 1020 X g for 15 min; the final mitochondrial pellet was obtained after centrifugation at 12,000 X g for 20 min. This pellet was suspended in the buffer (40 ml/kg of leaves) and treated with 50 MLg/ml of DNase for 1 hr at 4°.All subsequent operations to obtain DNA free from RNA and protein were those reported elsewhere (9).Isolation of Mitochondrial DNA for Electron Microscopy. 100 g of leaves were chopped with razor blades in 250 ml of buffer as reported (10). The mitochondrial pellet obtained as described above was suspended in 10 ml of buffer, treated with DNase, and washed -three times by suspe...

Physicochemical Characterization of Mitochondrial DNA from Soybean

“…Mitochondrial DNA from a number of higher plant sources (14) has been isolated. The buoyant density in neutral CsCl of plant mtDNA is approximately 1.706 g/cm:', suggesting a conserved base ratio for mtDNA of higher plants (22,26).…”

“…In class 4, there appear to be at least three distinct denaturation patterns, indicating heterogeneity within this class. It is proposed that the mitochondrial genome of soybean is distributed among the different size circular molecules, several copies of the genome are contained within these classes and that the majority of the various size molecules may be a result of recombination events between circular molecules.Mitochondrial DNA from a number of higher plant sources (14) has been isolated. The buoyant density in neutral CsCl of plant mtDNA is approximately 1.706 g/cm:', suggesting a conserved base ratio for mtDNA of higher plants (22,26).…”

Identification of mitochondrial rRNA from plant cells

“…The position of higher plants has been recently reported; they display long mtDNA molecules: 19.5 tam for red bean [4], 45 tam for pea, [5], 30/am for potato tubers [6]. The plant mt rRNA have not been evidenced with certainty [7,8].…”

Characterization of Mitochondrial DNA in Citrus

“…However, striking differences have been reported in the size of mt DNA in different plant species, ranging from 3 to 30,um. Plant species with a single or several discrete size classes have been described (2,4,8), but in other cases the occurrence of discrete classes is less obvious (6, 7).In addition, linear molecules of varying length are consistently observed in electron microscopic preparations of plant mt DNA. In fact, this form has been proposed as characteristic of the mt DNA of several species (5, 9, 11) with a wide range of variability in length.…”

“…However, striking differences have been reported in the size of mt DNA in different plant species, ranging from 3 to 30,um. Plant species with a single or several discrete size classes have been described (2,4,8), but in other cases the occurrence of discrete classes is less obvious (6, 7).…”