Physicochemical Characterization of Mitochondrial DNA from Soybean

Abstract

Mitochondrial DNA (mtDNA) of soybean ( Glycine max L.) was isolated and its buoyant density was contrasted with that of nuclear (nDNA) and chloroplast (ctDNA) DNA. Each of the three DNAs banded at a single, characteristic buoyant density when centrifuged to equilibrium in a CsCI gradient. Buoyant densities were 1.694 g/cm1 for nDNA and 1.706 g/cm ' for mtDNA. These values correspond to G-C contents of 34.7 and 46.9%, respectively. Covalently closed, circular mtDNA molecules were isolated from soybean hypocotyls by ethidium bromide-cesium chloride density gradient centrifugation. Considerable variation in mtDNA circle size was observed by electron microscopy. There were seven apparent size classes with mean lengths of 5.9 psm (class 1), 10 pum (class 2), 12.9 pum (class 3), 16.6 pm (class 4), 20.4 jum (class 5), 24.5 p,m (class 6), and 29.9 pm (class 7). In addition, minicircles were observed in all preparations. Partially denatured, circular mtDNA molecules with at least one representative from six of the seven observed size classes were mapped. 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). This also appears to be the case with ctDNAs from higher plants in which buoyant densities of approximately 1.697 g/cm: have consistently been found (26). Mitochondrial DNAs from animal sources appear to exist as circular molecules of 4 to 6 ,um in size (19). A circular conformation has also been observed by electron microscopy in mtDNA from yeast (17) 3)-3 mm EDTA-0. 1% BSA-I mM 2-mercaptoethanol. Homogenates were filtered through four layers of cheesecloth and centrifuged at 12 Ig for 10 min to obtain a crude nuclear pellet. This pellet was resuspended in 20 ml of 0.15 M NaCI-0. I M EDTA (pH 8.5)-2% sodium laurylsulfate and the nuclei allowed to lyse for 15 min. The mixture was placed in a 60 C bath for 10 min, phenol-extracted twice, and the nDNA alcohol-precipitated and spooled onto a glass stirring rod. The spooled nDNA was resuspended in a 0.01 M Tris-HCl (pH 8) buffer, incubated with RNase (50 ,g/ml) for I hr at 37 C, phenolextracted, dialyzed against 0.01 M Tris-HCI (pH 8) for 48 hr, and alcohol-precipitated by placing in a freezer for 48 hr. The nDNA was resuspended in 0.01 M Tris-HCl (pH 8) buffer and the UV spectrum obtained with a Beckman DB-G spectrophotometer. The ratio of the A at 260 to 280 nm always exceeded 1.8.The supernatant fraction from which the crude nuclear fraction had been isolated was first r...

Can partial methylation explain the complex fragment patterns observed when plant mitochondrial DNA is cleaved with restriction endonucleases?

“…[26]). Depending on the plant system and the methods of analysis, plant mitochondria have yielded both circular [3, 4,27] and linear [28-301 DNA forms, in varying proportions and length distributions, Maximum molecular weight estimates are 60-l 20 X I 06, based on electron microscopy [3, 27,28] or 70-140 X 106, based on renaturation kinetics [3, 30,31], whereas restriction analysis yields minimum molecular weight estimates which are usually appreciably higher than these values [S-7]. While some of these discrepancies undoubtedly have their origin in preparative artifacts and differences in the analytical techniques, the possibility remains that real differences in the organization of the mitochondrial genome may exist in different higher plants.…”

Mitochondrial DNA from Oenothera berteriana

“…Circular molecules were also found in mt-DNA from 0. berteriana, but generally associated with electron-dense particles (1). No smaller circular molecules could be observed as were found to exist in maize (15) and in soybean (21).…”

“…These data do not seem valid for the whole plant kingdom. In some species such as maize (16) and soybean (21), different size classes of circular molecules are observed, the largest being about the size of mt-DNA from the pea. These different mt-DNA molecules within the same species or even organism, as well as the mini-and maxicircles in Trypanosoma (2) …”

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).…”