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...

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Two of seven sucrose-fermenting Salmonella strains obtained from clinical sources were found capable of conjugal transfer of the sucrose fermentation (Scr+) property to the Escherichia coli K-12 strain WR3026. The genetic elements conferring this Scr+ property, designated scr-53 and scr-94, were then conjugally transmissible from E. coli WR3026 Scr+ exconjugants to other strains of E. coli at frequencies of 5 x 10-I to 5 x 10-I for the scr-53 element and 10 6 to 10-s for the scr-94 element. In E. coli hosts, both of these elements were compatible with F-lac and with each of six previously characterized transmissible lac elements. No antibiotic resistance characteristics or colicin production were discovered to be associated with either scr-53 or scr-94. Neither scr element generated a male host response to the female-specific phage XII, but the scr-53 element rendered its E. coli host sensitive to the male-specific phage R-17. E. coli hosts containing scr-53 were susceptible to lysis by Plvir, and transduction of the scr-53 element was accomplished with this phage. The scr-53 element was isolated from E. coli WR3026, Scr+ transductants, and E. coli WR2036 Scr+ exconjugants as a covalently closed circular deoxyribonucleic acid molecule with a molecular weight (determined by electron microscopy) of approximately 52 x 106. Receipt of the scr-94 element rendered E. coli hosts of this element unsusceptible to lysis by Plvir, although adsorption of the phage by an E. coli WR3026 exconjugant containing scr-94 occurred as efficiently as it did on WR3026 itself. Repeated examination of E. coli strains harboring scr-94, as well as of the Salmonella strain which initially contained it, did not reveal the presence of circular deoxyribonucleic acid. The synthesis of the sucrose cleaving enzyme was inducible in E. coli exconjugants containing either scr-53 or scr-94.

The transcription and processing of mitochondrial 21S rRNA in a petite strain of Saccharomyces cerevisiae has been examined by electron microscopic analysis of R-loop hybrids and by hybridization of labeled mitochondrial DNA probes to RNA transferred to diazobenzyloxymethyl paper. We have shown the presence of a large [5.1-to 5.4-kilobase (kb)J transcript that appears to be a precursor of mitochondrial 21S rRNA. This transcript contains sequences homologous to those of the mature 21S rRNA, to the intervening sequence present in the gene, and to additional sequences at the 3' end of the molecule. Our data suggest that this precursor of 21S rRNA is processed in two steps. The intron sequence is usually excised first, followed by removal of the extra 3' sequences. In some cases, however, the 3' extension is first removed and the intron sequence is then excised. Both pathways appear to lead to formation of the 3.1-kb mature 21S rRNA and a stable 1.2-kb intron transcript. Similar results were obtained with grande MH41-7B mitochondrial RNA by RNA transfer hybridization. We have also observed a number of additional transcripts that may be normal processing intermediates or may result from faulty cleavage-ligation during excision of the intervening sequence. The 70--to 75-kilobase (kb) yeast mitochondrial genome and its products have been analyzed extensively. Detailed genetic (e.g., refs. 1 and 2) and restriction endonuclease maps have been derived (e.g., refs. 3-6), regions of the DNA sequence have been determined (e.g., refs. 7-9), and a number of the transcripts have been characterized and mapped (10-15). Yeast mitochondrial DNA (mtDNA) specifies mitochondrial 14S and 21S rRNA, about 25 tRNAs, and seven to nine polypeptides (for review, see ref. 16). Although these gene products account for at most 20-30% of a single-strand DNA equivalent, more than 60% of the mitochondrial genome is transcribed (17). Analysis of mitochondrial transcripts from grande and petite strains by gel electrophoresis has shown that their aggregate molecular weight exceeds the coding capacity of the genome (11,12). Similarly, transcript mapping with petite yeast strains indicates that multiple RNA species are specified by individual regions of the mitochondrial genome (11,13). Therefore, large regions of the genome appear to be transcribed, and the products are then processed into mature RNA species. Furthermore, intervening sequences have been demonstrated in the cytochrome b (18, 19) and the 21S rRNA (20-22) cistrons and possibly the OXI 3 region (unpublished results). Transcripts derived from these regions thus require extensive processing. Characterization of RNA processing pathways would appear to be essential for an understanding of the biogenesis of mitochondria.In this study, we have focused on the analysis of transcripts derived from the 21S rRNA cistron. This gene contains a 1.2-kb intervening sequence (20)(21)(22) (11,13).The Fl1 mitochondrial genome has been extensively characterized both genetically and physically (23,25...