Comparison of the Restriction Endonuclease Digestion Patterns of Mitochondrial DNA from Normal and Male Sterile Cytoplasms of ZEA MAYS L

High resolution gel electrophoresis has allowed the assignment of fragment number and molecular weight to EcoRI, SalI and PstI restriction fragments of mitochondrial DNA from B37 normal (N) and B37 T, C and S male sterile cytoplasmic types of maize. A minimum complexity of 450-475 kb has been established. Hybridization of cloned EcoRI fragments to restriction digests of total mitochondrial DNA suggests that at least 80% of the genome is composed of unique sequences. Restriction fragments of identical size in N, T, C and S contain similar sequence information as evidenced by their hybridization behavior.—The total SalI digest and the larger PstI fragments representing 80% of the total complexity were used to calculate the fraction of shared fragments of each pairwise combination of cytoplasmic types. The C type mtDNA is most closely allied with the other mtDNAs and shares 67% of fragments with S, 65% with N, and 60% with T. The S type mtDNA is quite divergent from N (53% shared fragments) and T (56% shared fragments). N and T share 59% of the fragments. These results are discussed in terms of the origin of mtDNA diversity in maize.     

Cloning of human mitochondrial DNA in Escherichia coli

In order to determine its nucleotide sequence, human mitochondrial DNA (mtDNA) purified from term placentae was cloned in Escherichia coli using the plasmid vector pBR322. The products of an mtDNA MboI digestion (23 fragments ranging in size from 2800 to 25 base-pairs (bp)) were ligated with BamHI-cut pBR322. The ampicillin-resistant tetracycline-sensitive colonies obtained upon transformation of E. coli χ1776 were screened by agarose gel electrophoresis of colony lysates, colony hybridization and restriction analysis. All but MboI fragment 2 were obtained in this way. MboI fragments 5 and 8 were each found only once among the 705 clones screened. All other MboI fragments were approximately equally represented in the population of clones except for a slight bias towards smaller fragments. MboI fragment 2 overlaps with the mtDNA BamHI/EcoRI (1.7 kb³) and the 0.9 kb HinIII fragments. These were cloned in similarly restricted pBR322 to provide a set of clones covering most of the mtDNA molecule. Clones representative of each MboI fragment were shown to be complementary to mtDNA by hybridization to Southern blots of mtDNA digests and were thereby partially mapped. Further mapping was obtained by restriction analysis of mtDNA sequentially degraded by exonuclease III. A collection of recombinant clones has thus been obtained using the mtDNA isolated from a single placenta and is now being used to obtain a complete nucleotide sequence of human mtDNA.     

A detailed physical map of HeLa cell mitochondria DNA and its alignment with the positions of known genetic markers

Twenty-one fragments have been identified among the products of digestion of HeLa cell mtDNA with the restriction enzyme Hpa II. The sum of the molecular sizes of these fragments, estimated from their mobility relative to that of known markers, accounts, within experimental error, for the total length of HeLa cell mtDNA. The 21 fragments have been ordered in a physical map by two approaches: (1) sequential digestion with Hpa II of the fragments produced by Eco RI, Hind III, andHpa I enzymes, and (2) fragment-primed DNA synthesis. The Hpa II map has been aligned with the maps constructed with the other three enzymes and with the unique cutting site produced by Bam I. The combined map thus obtained has resolved HeLa cell mtDNA into 27 recognizable segments in the molecular size range between 75 and 1950 base pairs. This physical map has been aligned with the known positions of the rRNA and 4 S RNA genes on the two mtDNA strands by RNA-DNA hybridization experiments utilizing purified ³²P-labeled 12 and 16 S rRNA.     

Organization and expression of the mitochondrial genome of plants I. The genes for wheat mitochondrial ribosomal and transfer RNA: evidence for an unusual arrangement.

We show here that mitochondrial-specific ribosomal and transfer RNAs of wheat (Triticum vulgare Vill. [Triticum aestivum L.] var. Thatcher) are encoded by the mitochondrial DNA (mtDNA). Individual wheat mitochondrial rRNA species (26S, 18S, 5S) each hybridized with several mtDNA fragments in a particular restriction digest (Eco RI, Xho I, or Sal I). In each case, the DNA fragments to which 18S and 5S rRNAs hybridized were the same, but different from those to which 26S rRNA hybridized. From these results, we conclude that the structural genes for wheat mitochondrial 18S and 5S rRNAs are closely linked, but are physically distant from the genes for wheat mitochondrial 26S rRNA. This arrangement of rRNA genes is clearly different from that in prokaryotes and chloroplasts, where 23S, 16S and 5S rRNA genes are closely linked, even though wheat mitochondrial 18S rRNA has previously been shown to be prokaryotic in nature. The mixed population of wheat mitochondrial 4S RNAs (tRNAs) hybridized with many large restriction fragments, indicating that the tRNA genes are broadly distributed throughout the mitochondrial genome, with some apparent clustering in regions containing 18S and 5S rRNA genes.     

The structures and fidelity of replication of mouse mitochondrial DNA-pSC101 EcoRI recombinant plasmids grown in E. coli K12

Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (LA9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoRI endonuclease digests and were used to transform E. coli K12. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the E. coli mtDNA lacked the alkali lability characteristic of animal mtDNAs.Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single-base replication errors occur at a frequency of fewer than 1 nucleotide in 300. Electron microscopic analyses of plasmid-LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSC101 and mtDNA fragments.A third EcoRI fragment in LA9 mtDNA, not found in an earlier study (Brown and Vinograd, 1974), has been positioned in the LA9, EcoRI map. This fragment contains 165±10 nucleotide pairs.     

Different methylation pattern of melon satellite DNA sequences in hypocotyl and callus tissues

Satellite DNA sequences from Cucumis melo have been examined with respect to modification at CCGG sequences in hypocotyls and in callus tissues. For this purpose, restriction fragments given by HpaII and MspI were compared (both enzymes recognize CCGG sequences but have different sensitivity to methylation at this site). Whereas the methylation level of satellite DNA sequences is on average higher in hypocotyls than in callus tissues, the comparison of partially methylated repeat units of satellite DNA reveals that in callus tissues, all methylated restriction sites are doubly methylated.     

Rearrangements in sugar beet mitochondrial DNA induced by cell suspension,callus cultures and regeneration

Structural alterations in mitochondrial DNAs (mtDNAs) from a plant of a sterile sugar beet line, callus derived from it, suspension-cultured cells and plants regenerated from the callus were studied. BamHI restriction analysis revealed that structural alterations between the mtDNAs of the callus and the control plant had occurred. Multiple rearrangements were also demonstrated in the mtDNA from the suspension culture, of which some were similar to those appearing in the callus, and others had arisen de novo. Rearrangements were also identified by means of blot hybridization of BamHI-digested mtDNA from suspension-cultured cells with the genes encoding subunit II of cytochrome oxidase (cox II) and subunit 1 of NADH-dehydrogenase (Nd1). No alterations were observed in the mitochondrial genome of the callus and regenerants. The location of the genes for the -subunit of F1-ATPase (atpA) and apocytochrome b (cob) in the mtDNA remained unchanged.Our salient finding was of a plant with an altered mitochondrial genome as judged by EcoRI and BamHI restriction analysis. This exceptional plant had retained the sterile phenotype like all of the other regenerants and the parent. The set of plasmid-like molecules of mtDNA remained the same as that in the control plant and in all of the regenerants, callus and suspension-cultured cells. The only type of plasmid-like molecule found in all of the DNAs was the 1.6-kbp minicircle, which is a feature of sterile cytoplasms. These structural changes in mtDNA were obviously a consequence of somaclonal variation during the in vitro cultivation of the sugar beet cells.     

Organization of mitochondrial DNA in normal and texas male sterile cytoplasms of maize

Recombinant DNA and hybridization techniques have been used to compare the organization of mitochondrial DNA (mtDNA) from normal (N) and Texas male sterile (T) cytoplasms of maize. Bam H1 restriction fragments of normal mtDNA were cloned and used in molecular hybridizations against Southern blots of Bam H1 digested N and T mtDNA. Fifteen of the 35 fragments were conserved in both N and T as indicated by hybridization to comigrating bands in their restriction patterns. Only three fragments produced autoradiographs whose differences could reasonably be attributed to single changes in the cleavage site of the enzyme while approximately half (17/35) of the clones resulted in more complicated differences between N and T. The autoradiographs produced by these 17 clones indicated multiple cleavage site changes and/or sequence rearrangements of the mtDNA. Patterns of six of these 17 clones indicated partial duplication of the sequence and two showed variation in the intensity of hybridization between N and T, which may be related to the molecular heterogeneity phenomenon found in maize mitochondrial genomes. The large proportion of changes observed between N and T mtDNA indicates that rearrangements may have played an important role in the evolution of the maize mitochondrial genome.     

Cytochrome Oxidase Subunit II Gene from Carrot Contains an Intron

Introns in the cytochrome oxidase subunit II (COXII) gene of plant mitochondrial DNA (mtDNA) have been observed only in monocots. The COXII genes in dicots investigated to date do not contain introns. This is the first report of an intron in the COXII gene of a dicot. The presence of an intron in the carrot COXII intron was verified by restriction mapping and hybridization using specific maize and wheat COXII probes. Regions of the carrot COXII intron are homologous to the maize COXII intron and homologous to the wheat COXII intron-insert as demonstrated by hybridization. Homology of these regions was confirmed by sequencing portions of the gene. A comparison of the restriction map of the carrot COXII gene with the restriction maps of the COXII genes from pea, Oenothera, maize, wheat, and rice revealed that the carrot map coincides with the rice restriction map.     

Organization of the large mitochondrial genome in the isopod Armadillidium vulgare.

The mitochondrial DNA (mtDNA) in animals is generally a circular molecule of approximately 15 kb, but there are many exceptions such as linear molecules and larger ones. RFLP studies indicated that the mtDNA in the terrestrial isopod Armadillidium vulgare varied from 20 to 42 kb. This variation depended on the restriction enzyme used, and on the restriction profile generated by a given enzyme. The DNA fragments had characteristic electrophoretic behaviors. Digestions with two endonucleases always generated fewer fragments than expected; denaturation of restriction profiles reduced the size of two bands by half; densitometry indicated that a number of small fragments were present in stoichiometry, which has approximately twice the expected concentration. Finally, hybridization to a 550-bp 16S rDNA probe often revealed two copies of this gene. These results cannot be due to the genetic rearrangements generally invoked to explain large mtDNA. We propose that the large A. vulgare mtDNA is produced by the tripling of a 14-kb monomer with a singular rearrangement: one monomer is linear and the other two form a circular dimer. Densitometry suggested that these two molecular structures were present in different proportions within a single individual. The absence of mutations within the dimers also suggests that replication occurs during the monomer phase.     

Stable heterogeneity of mitochondrial DNA in grande and petite strains of S. cerevisiae.

Several instances of mitochondrial DNA heterogeneity in grande and petite strains of Saccharomyces cerevisiae were examined. We have detected heterogeneity in the mtDNA from some of the progeny strains of a cross between two grande strains (D273-10B, MH41-7B) which differ in genome size and restriction cleavage pattern of their mtDNA. The progeny strains transmit restriction fragments characteristic of both parental strains from homologous regions of the mitochondrial genome, and this sequence heterogeneity is not eliminated by additional subcloning. Sequence diversity is more common in the mtDNA of petite than of grande strains of yeast. We have examined subclones of one petite strain to identify the origin of this variability. Many of the submolar restriction fragments persist in independent subclones of this petite after 15 and 30 cell divisions; some submolar fragments disappear, and some new fragments appear. We conclude that the observed sequence heterogeneity is due to molecular heterogeneity, i.e., to differences in the multiple copies of the petite mitochondrial genome, as well as to clonal heterogeneity. It is likely that tandem repeats on the same mtDNA molecule also differ, i.e., that there is intramolecular heterogeneity, and that this accounts for the stability of the heterogeneity. Continuing deletion is probably responsible for the appearance of “new” fragments in petite subclones.     

Structural homogeneity of mitochondrial DNA in the mitochondrial nucleoid of Physarum polycephalum

Mitochondrial DNA (mtDNA) of Physarum polycephalum was isolated gently by CsCl centrifugation. The mtDNA was linear with molecular weights ranging from 25·10⁶ to 45·10⁶ and heterogeneous in size. Nevertheless, thermal transition profiles of the mtDNA suggested that this DNA fraction was more homogeneous than nuclear DNA. Exhaustive digestions of this DNA with restriction endonucleases yielded unique fragments, and then the total of their molecular weights of each digest was around 45·10⁶. This value is equivalent to the maximum molecular weight estimated using electron microscopy and electrophoresis. Moreover, EcoRI digests of the mtDNA fractionated by the sucrose gradient showed unequimolar quantities of large fragments and a high background between bands. These results suggest that the mtDNA of Physarum has a homogeneous base sequence, and that the size heterogeneity of the mtDNA is attributable to degradation of the DNA under isolation procedures. The mtDNA was cleaved by EcoRI and XhoI to yield 16 and 7 fragments, respectively. A physical map of these fragments was constructed using the routine mapping procedures. The physical map showed that the mitochondrial genome of Physarum was linear with a molecular weight of 45·10⁶. We concluded therefore that the mitochondrial nucleoid is a structure in which the homogeneous mtDNA is highly amplified.     

Genetic variability in mitochondrial DNA of the screwworm,Cochliomyia hominivorax (Diptera: Calliphoridae), from Brazil

Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) was used to examine genetic variation and population structure of screwworm flies in four populations from São Paulo State, Brazil. The total DNA of 405 individuals was digested with 15 restriction endonucleases and probed with five clonedHindIII fragments representing the entire mitochondrial genome ofCochliomyia hominivorax. The survey revealed that four enzymes (HaeIII,HindIII,MspI, andPvuII) were suitable to detect mtDNA variation among all populations. Based on the fragment patterns obtained for these four enzymes, a total of 15 haplotypes in combination was detected. Heteroplasmic individuals for thePvuII pattern were obtained in one of the populations. The estimated average for nucleotide sequence divergence (δ) was 0.92%. The cladogram of the geographical distribution among the observed haplotypes suggests that the sampled screwworms probably belong to a single evolutionary lineage with populations interconnected by reduced gene flow.     

Linear mitochondrial DNAs of yeasts: frequency of occurrence and general features.

In most yeast species, the mitochondrial DNA (mtDNA) has been reported to be a circular molecule. However, two cases of linear mtDNA with specific termini have previously been described. We examined the frequency of occurrence of linear forms of mtDNA among yeasts by pulsed-field gel electrophoresis. Among the 58 species from the genera Pichia and Williopsis that we examined, linear mtDNA was found with unexpectedly high frequency. Thirteen species contained a linear mtDNA, as confirmed by restriction mapping, and labeling, and electron microscopy. The mtDNAs from Pichia pijperi, Williopsis mrakii, and P. jadinii were studied in detail. In each case, the left and right terminal fragments shared homologous sequences. Between the terminal repeats, the order of mitochondrial genes was the same in all of the linear mtDNAs examined, despite a large variation of the genome size. This constancy of gene order is in contrast with the great variation of gene arrangement in circular mitochondrial genomes of yeasts. The coding sequences determined on several genes were highly homologous to those of the circular mtDNAs, suggesting that these two forms of mtDNA are not of distant origins.     

Molecular analysis of the inheritance and stability of the mitochondrial genome of an inbred line of maize

Summary We have investigated the inheritance of the mitochondrial DNA (mtDNA) restriction endonuclease digestion patterns of maize inbred line B37N in individual plants and pooled siblings in lineages derived from five separate plants in the third generation following successive self-pollinations. The restriction fragment patterns of the different mtDNA samples were compared after digestion with five endonucleases. No differences were visible in the mobilities of the 199 fragments scored per sample. Hybridization analysis with two different cloned mtDNA probes, one of which contains homologies to a portion of the S2 plasmid characteristic of cms-S maize, failed to reveal cryptic variation. The apparent rate of genomic change in maize mtDNA from inbred plants appears to be very slow, compared with the faster rates of change seen in maize tissue cultures and with the documented rapid rate of inter- and intraspecific variation for mammalian mtDNA.     

Mitochondrial DNA of the blowflyPhormia regina: restriction analysis and gene localization

A study of an invertebrate mitochondrial genome, that of the blowflyPhormia regina, has been initiated to compare its structural and functional relatedness to other metazoan mitochondrial genomes. A restriction map of mitochondrial DNA (mtDNA) isolated from sucrose gradient-purified mitochondria has been established using a combination of single and double restriction endonuclease digestions and hybridizations with isolated mtDNA fragments, revealing a genome size of 17.5 kilobases (kb). A number of mitochondrial genes including those encoding the 12 S and 16 S ribosomal RNA, the cytochromec oxidase I subunit (COI) and an unidentified open reading frame (URF2) have been located on thePhormia mtDNA by Southern blot analysis using as probes both isolated mtDNA fragments and oligonucleotides derived from the sequences of previously characterized genes from rat andDrosophila yakuba mtDNAs. These data indicate that for those regions examined, the mitochondrial genome organization of blowfly mtDNA is the same as that ofDrosophila yakuba, the order being COI-URF2-12 S-16 S. These data also report the presence of an A + T-rich region, located as a 2.5-kb region between the URF2 and the 12 S rRNA genes, and its amplification by the polymerase chain reaction is described.     

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.     

Cloning of mitochondrial DNA from the petite negative yeast Schizosaccharomyces pombe in the bacterial plasmid pBR322

Summary The entire mitochondrial (mt) genome of the yeast Schizosaccharomyces pombe (S. pombe) was cloned in the BamHI site of the Escherichia coli plasmid pBR322. Three lines of evidence demonstrate that the complete mtDNA molecule was amplified without rearrangement or partial loss. First, restriction of the hybrid plasmid with BamHI led to the recovery of two fragments corresponding to the linearized plasmid and the BamHI-cut mtDNA. Second, restriction of cloned and native mtDNA with HindIII revealed identical fragments. Third, mitochondrial ribosomal RNA hybridized to the same HindIII fragments from cloned mtDNA and from mtDNA isolated from mitochondria.     

Circular mitochondrial genome of Candida albicans contains a large inverted duplication.

The mitochondrial DNA (mtDNA) of the dimorphic fungus Candida albicans has a molecular size of 41 kilobase pairs as judged by summation of the fragment sizes produced by digestion with restriction endonucleases EcoRI, PvuII, and a combination of both enzymes. Five of the six EcoRI fragments comprising the mitochondrial genome have been cloned into the plasmid vector, pBR322. Restriction mapping revealed a circular map as predicted by previous observations with the electron microscope. The use of nick-translated, purified mtDNA to probe digests of mtDNA from other strains of C. albicans revealed a common restriction pattern. Use of nick-translated, cloned EcoRI fragments to probe digests of mtDNA revealed a large (at least 5 kilobase pairs), inverted duplication as well as a smaller (less than 0.4 kilobase pairs) region of related sequences.     

A Comprehensive Study of Genic Variation in Natural Populations of Drosophila Melanogaster. IV. Mitochondrial DNA Variation and the Role of History Vs. Selection in the Genetic Structure of Geographic Populations

Preliminary studies with restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) in natural populations of Drosophila melanogaster revealed considerable variation in terms of nucleotide sequence and overall size. In this report we present data from more isofemale lines and more restriction enzymes, and explore the utility of the data in inferring a colonization history of this species. Size variation in the noncoding A + T-rich region is particularly plentiful, with size variants occurring in all restriction site haplotypes in all populations. We report here classes of small-scale mobility polymorphisms (apparent range of 20 bp) in specific restriction fragments in the coding region. The variation in one such fragment appears to be generated even more rapidly than in the noncoding region. On the basis of the distribution of restriction site haplotypes, the species range can be divided into three major regions along longitudinal lines: Euro-African populations are the most diverse and are taken to be oldest; Far East populations have a complex distribution of haplotypes; Western Hemisphere populations are the least diverse and are interpreted to be the youngest. The history inferred from mtDNA alone is remarkably similar to one based on several nuclear markers. The mtDNA haplotype distribution is also very different from that of allozymes in these same populations. We interpret this as further evidence that natural selection is still the most parsimonious explanation for the parallel latitudinal allozyme clines in this species.