Analysis of a 120-Kilobase Mitochondrial Chromosome in Maize

The organization of the mitochondrial genome in plants is not well understood. In maize mitochondrial DNA (mtDNA) several subgenomic circular molecules as well as an abundant fraction of linear molecules have been seen by electron microscopy. It has been hypothesized that the circular molecules are the genetic entities of the mitochondrial genome while the linear molecules correspond to randomly sheared mtDNA. A model has been proposed that explains the mechanism of generation of subgenomic circles (of a predictable size) by homologous recombination between pairs of large direct repeats found on a large (approximately 570 kb for the fertile (N) cytoplasm) master circle. So far the physical entities of the mitochondrial genome, as they exist in vivo, and the genes they carry, have not been identified. For this purpose, we used two gel systems (pulsed field gel electrophoresis and Eckhardt gels) designed to resolve large DNA. Large DNA was prepared from the Black Mexican Sweet (BMS) cultivar. We resolved several size classes of mtDNA circles and designate these as chromosomes. A 120 kb chromosome was mapped in detail. It is shown to contain the three ribosomal genes (rrn26, rrn18 and rrn5) plus two genes encoding subunits of cytochrome oxidase (Cox1 and Cox3); it appears to be colinear with the 570-kb master circle map of another fertile cytoplasm (B37N) except at the "breakpoints" required to form the 120-kb circle. The presence of the 120-kb chromosome could not have been predicted by homologous recombination through any of the known repetitive sequences nor is it a universal feature of normal maize mitochondria. It is present in mitochondria of BMS suspension cultures and seedlings, but is not detectable in seedlings of B37N. No master genome was detected in BMS.     

Analysis of rice mitochondrial genome organization using pulsed-field gel electrophoresis

Most of the plant mitochondrial (mt) genomes that have been mapped are believed to be organized as master circle molecules from which sub-genomic molecules arise through homologous recombination. We have evidence to suggest that a major part of the rice mt genome is organized as independent, sub-genomic molecules or mt chromosomes, one of which has already been mapped. This study is aimed at the identification of the other molecular entities that comprise the genome. Pulsed-field gel electrophoresis of the native rice mt DNA and Southern analysis with different mt gene probes have shown that in addition to the 117 kb mt chromosome, at least four more such molecules of sizes 130 kb, 95 kb, 70 kb and 56 kb account for most of the rice mt genome. A majority of the rice mt genes that encode products involved in oxidative phosphorylation are distributed among these five chromosomes. Partial restriction map of the 95 kborf 25/cox 3 chromosome, indicating the sites for the enzymesBglII andHindIII has also been determined.     

Genetic and physical maps and a clone bank of mitochondrial DNA from rice

Summary Mitochondrial DNA (mtDNA) was isolated from young green leaves of rice plants. DNA fragments were cloned into lambda DNA, and clones that hybridized to mitochondrial genes from other plants were selected. Distal restriction fragments of these clones were used as probes for the selection of overlapping clones. A genetic map was finally created from the library by walking along the genome. The mitochondrial genome consists of five basic circles, with each circle sharing homologous sequences with one or two other circles. A master circle was constructed from the results of recombination across repeated sequences, and its size was estimated to be 492 kb. A physical map and a bank of overlapping clones were also constructed.     

Organizational differences between cytoplasmic male sterile and male fertile Brassica mitochondrial genomes are confined to a single transposed locus.

Comparison of the physical maps of male fertile (cam) and male sterile (pol) mitochondrial genomes of Brassica napus indicates that structural differences between the two mtDNAs are confined to a region immediately upstream of the atp6 gene. Relative to cam mtDNA, pol mtDNA possesses a 4.5 kb segment at this locus that includes a chimeric gene that is cotranscribed with atp6 and lacks an approximately 1kb region located upstream of the cam atp6 gene. The 4.5 kb pol segment is present and similarly organized in the mitochondrial genome of the common nap B.napus cytoplasm; however, the nap and pol DNA regions flanking this segment are different and the nap sequences are not expressed. The 4.5 kb CMS-associated pol segment has thus apparently undergone transposition during the evolution of the nap and pol cytoplasms and has been lost in the cam genome subsequent to the pol-cam divergence. This 4.5 kb segment comprises the single DNA region that is expressed differently in fertile, pol CMS and fertility restored pol cytoplasm plants. The finding that this locus is part of the single mtDNA region organized differently in the fertile and male sterile mitochondrial genomes provides strong support for the view that it specifies the pol CMS trait.     

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.     

Hexahydrohippuric Acid,a Newly Isolated Compound from the Cattle’s Urine

A cosmid library and physical maps of mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, were constructed using the cosmid clones. Electrophoresis profile and the physical maps indicated that the liverwort mtDNA was approximately 183 kb long, the smallest among plant mtDNAs, and that it consisted of a single circular molecule. Southern hybridization analysis showed that genes typical to the mitochondrial genome existed in a single copy, and also that there was no incorporation of chloroplast DNA fragments into the mitochondrial genome.     

Mitochondrial DNA recombination in Brassica campestris

The structure of the mitochondrial genome in plants is unclear, but appears to consist of mostly linear DNA with some other structures, including branched molecules and subgenomic circles. Mitochondrial DNA (mtDNA) recombination was analyzed in Brassica campestris, which has one of the smallest mitochondrial genomes (218 kb) in higher plants. Field-inversion gel electrophoresis (FIGE) separated mtDNA into discrete populations that each represents the entire genome. Electron microscopy revealed large, mostly linear molecules trapped in the wells, slower migrating populations with mostly linear DNA and a low level of circular and networked mtDNA molecules of 10–140 kbp, and a fast migrating population of 10–50 kbp linear mtDNA. Some smaller than genome size circular molecules and circles with tails were observed, and may represent recombination or rolling circle replication intermediates. Hybridization of end-labeled mtDNA suggests there may be specific ends (or recombination hotspots) for some linear molecules. Analysis of mtDNA enriched by BND-cellulose and separated by two-dimensional agarose gel electrophoresis shows the presence of complex recombination structures and the presence of significant single-stranded regions in mtDNA. These findings provide further evidence that DNA recombination contributes to the complex structure of mtDNA in plants.     

The linear 20 kb mitochondrial genome of Pandorina morum (Volvocaceae,Chlorophyta)

A physical restriction map of the mitochondrial genome from one clone (TCC 854) of the sexually isolated populations (syngens) of the morphologically uniform species Pandorina morum Bory has been constructed using restriction endonucleases Ava I, Bam HI, Bgl II, Eco RI, Kpn I, and Pst I. The 20 kb linear genome can easily be separated from plastid DNA, nuclear satellite rDNA, and main band (nuclear) DNA on a Hoechst/CsCl buoyant density gradient. The Pandorina mitochondrial DNA shows sufficient similarity to the 16 kb mitochondrial genome of Chlamydomonas reinhardtii to cross-hybridize, and also hybridizes with a probe containing maize mitochondrial 18S rRNA genes. Double digests, self-probing, and Bal31 exonuclease experiments suggest that 1.8 to 3.3 kb of sequence is repeated at each end of the genome as an inverted repeat. Mitochondrial genome sizes of other P. morum syngens were found to range from ca. 20 to ca. 38 kb. The mitochondrial genome should be valuable for taxonomic studies; it can be used for comparative organellar studies; and it should be of interest to compare with that of other plant and animal mitochondrial genomes.     

Three copies of a single recombination repeat occur on the 443 kb master circle of the Petunia hybrida 3704 mitochondrial genome.

At 443 kb, the map of Petunia hybrida line 3704 mitochondrial DNA is the largest yet produced from a dicot plant. Regions of similarity to known plant mitochondrial genes and to the chloroplast genome have been placed on a master circle. One long repeated sequence, apparently active in recombination, is present in three copies. Two copies of 6.6 kb occur in a direct orientation and are separated by 199 kb. A third truncated copy of 3.5 kb is inverted relative to the other two and is separated from the others by 99 and 145 kb. The presence of the recombination repeats predicts a multipartite molecular organization, consisting of four master circles and three subgenomic circles. Two other repeated regions were found not to be substrates for, or products of recombination. The absence of recombination at certain reiterated regions indicates that there is specificity of recombination at the recombination repeats.     

The Male Sterility-Associated Pcf Gene and the Normal Atp9-1 Gene in Petunia Are Located on Different Mitochondrial DNA Molecules

A mitochondrial DNA (mtDNA) region termed the S-pcf locus has previously been correlated with cytoplasmic male sterility (CMS) in Petunia. In order to understand the relationship of the S-pcf locus to homologous sequences found elsewhere in mtDNAs of both CMS and fertile lines, the structure of the mitochondrial genome of CMS Petunia line 3688 was determined by cosmid walking. The S-pcf locus, which includes the only copies of genes for NADH dehydrogenase subunit 3 (nad3) and small ribosomal subunit protein 12 (rps12) was found to be located on a circular map of 396 kb, while a second almost identical circular map of 407 kb carries the only copies of the genes for 18S and 5S rRNA (rrn18 and rrn5), the only copy of a conserved unidentified gene (orf25), and the only known functional copy of atp9. Three different copies of a recombination repeat were found in six genomic environments, predicting sub-genomic circles of 277, 266 and 130 kb. The ratio of atp9 to S-pcf mtDNA sequences was approximately 1.5 to 1, indicating that sub-genomic molecules carrying these genes differ in abundance. Comparison of the mtDNA organization of the CMS line with that of the master circle of fertile Petunia line 3704 reveals numerous changes in order and orientation of ten different sectors.     

Analysis of Schizosaccharomyces pombe mitochondrial DNA replication by two dimensional gel electrophoresis

The entire mitochondrial genome of Schizosaccharomyces pombe ura4-294h ^-was analyzed by the 2D pulsed field gel electrophoresis technique developed by Brewer and Fangman. The genome consists of multimers with an average size of 100 kb and analysis of the overlapping restriction fragments of the complete mitochondrial DNA (mtDNA) genome resulted in simply Y 2D gel patterns. Large single-stranded DNA molecules or double-stranded DNA molecules containing large or numerous single-stranded regions were found in the S. pombe mtDNA preparation. The replication of mtDNA monomers was found to occur in either direction. On the basis of these results, a replication mechanism for S. pombe mtDNA that is most consistent with a rolling circle model is suggested.     

Mitochondrial DNA of Marchantia polymorpha as a single circular form with no incorporation of foreign DNA.

A cosmid library and physical maps of mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, were constructed using the cosmid clones. Electrophoresis profile and the physical maps indicated that the liverwort mtDNA was approximately 183 kb long, the smallest among plant mtDNAs, and that it consisted of a single circular molecule. Southern hybridization analysis showed that genes typical to the mitochondrial genome existed in a single copy, and also that there was no incorporation of chloroplast DNA fragments into the mitochondrial genome.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^– mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

Cloning and characterization of the Chlamydomonas moewusii mitochondrial genome.

Summary We report that the mitochondrial genome of Chlamydomonas moewusii has a 22 kb circular map and thus contrasts with the mitochondrial genome of Chlamydomonas reinhardtii, which is linear and about 6 kb shorter. Overlapping restriction fragments spanning over 90% of the C. moewusii mitochondrial DNA (mtDNA) were identified in a clone bank constructed using a Sau3AI partial digest of a C. moewusii DNA fraction enriched for mtDNA by preparative CsCI density gradient centrifugation. Overlapping Sau3AI clones were identified by a chromosome walk initiated with a clone of C. moewusii mtDNA. The mtDNA map was completed by Southern blot analysis of the C. moewusii mtDNA fraction using isolated mtDNA clones. Regions that hybridized to C. reinhardtii or wheat mitochondrial gene probes for subunit I of cytochrome oxidase (cox1), apocytochrome b (cob), three subunits of NADH dehydrogenase (nadl, nad2 and nad5) and the small and the large ribosomal RNAs (rrnS and rrnL, respectively) were localized on the C. moewusii mtDNA map by Southern blot analysis. The results show that the order of genes in the mitochondrial genome of C. moewusii is completely rearranged relative to that of C. reinhardtii.     

MITOCHONDRIAL GENOME CONFORMATION AMONG CW‐GROUP CHLOROPHYCEAN ALGAE1

Most green algal taxa have circular‐mapping mitochondrial genomes, whereas some have linear genome‐ or subgenomic‐sized mitochondrial DNAs (mtDNA). It is not clear, however, if the circular‐mapping genomes represent genome‐sized circular molecules, if such circular molecules and the linear forms are the predominant in vivo mtDNA structures, or if the linear forms arose only once or multiple times among extant green algal lineages. We therefore examined the DNA components detected with homologous mtDNA probes after pulsed‐field gel electrophoresis of total cellular DNA from the chlorophycean basal bodies displaced clockwise(CW)‐group taxa Chlamydomonas reinhardtii and Chlamydomonas moewusii. For C. reinhardtii, the 15.8‐kb linear mtDNA was the only DNA component detected, and there was no evidence of circular or large linear precursors of this DNA. In the case of C. moewusii, which is known to have a circular‐mapping 22.9‐kb mitochondrial genome, three DNA components were detected; these appeared to be circular (relaxed and supercoiled) and genome‐sized linear DNA molecules, the latter of which likely resulted from random double‐strand breaks in the circular forms during DNA isolation. In further studies, DNA from additional CW‐group taxa was examined using conventional gel electrophoresis and DNA‐filter blot analysis with C. reinhardtii and C. moewusii mtDNA probes. We conclude that all taxa from the “Volvox clade” (sensu Nakayama et al. 1996 of the CW‐group have genome‐ or subgenomic‐sized linear mtDNAs as their predominant mtDNA form and that these arose from a genome‐sized circular form in an ancestor that existed near the base of this clade.     

Small, repetitive DNAs contribute significantly to the expanded mitochondrial genome of cucumber

Closely related cucurbit species possess eightfold differences in the sizes of their mitochondrial genomes. We cloned mitochondrial DNA (mtDNA) fragments showing strong hybridization signals to cucumber mtDNA and little or no signal to watermelon mtDNA. The cucumber mtDNA clones carried short (30-53 bp), repetitive DNA motifs that were often degenerate, overlapping, and showed no homology to any sequences currently in the databases. On the basis of dot-blot hybridizations, seven repetitive DNA motifs accounted for >13% (194 kb) of the cucumber mitochondrial genome, equaling >50% of the size of the Arabidopsis mitochondrial genome. Sequence analysis of 136 kb of cucumber mtDNA revealed only 11.2% with significant homology to previously characterized mitochondrial sequences, 2.4% to chloroplast DNA, and 15% to the seven repetitive DNA motifs. The remaining 71.4% of the sequence was unique to the cucumber mitochondrial genome. There was <4% sequence colinearity surrounding the watermelon and cucumber atp9 coding regions, and the much smaller watermelon mitochondrial genome possessed no significant amounts of cucumber repetitive DNAs. Our results demonstrate that the expanded cucumber mitochondrial genome is in part due to extensive duplication of short repetitive sequences, possibly by recombination and/or replication slippage.     

The Mitochondrial Genome Organization of a Maize Fertile Cmst Revertant Line Is Generated through Recombination between Two Sets of Repeats

The mitochondrial genome (mtDNA) organization from a fertile revertant line (V3) derived from the maize cytoplasmic male sterile type T (cmsT) callus tissue culture has been determined. We report that the sequence complexity can be mapped on to a circular ``master chromosome' of 705 kb which includes a duplication of 165 kb of DNA when compared to its male sterile progenitor. Associated with this event is also a 0.423-kb deletion, which removed the cmsT-associated urf13 gene. As found for the maize normal type (N) and cmsT mitochondrial genomes, the V3 master chromosome also exists as a multipartite structure generated by recombination through repeated sequences.     

Complex organization of the mitochondrial genome of petaloid CMS carrot

The petaloid trait in carrot is a mitochondrially associated homeotic-like conversion of stamens into petals which results in cytoplasmic male sterility (cms), but little is understood about the phenomenon at the molecular level. Identification of region(s) of the mitochndrial (mt) genome that are causally implicated in cms may be aided by a physical map of cms-associated mtDNA. The mt genome of petaloid cms carrot is 255 kb in length and contains three pairs of repeated sequences, two in direct orientation and one in inverted orientation. All regions of the genome are present in equal stoichiometries, but the arrangement of the repeated sequences prevented the representation of the entire genome as a single master circle or multiple subgenomes. An alternative model of mtDNA replication that accounts for our data is discussed.     

Tricircular mitochondrial genomes of Brassica and Raphanus: reversal of repeat configurations by inversion.

We constructed complete physical maps of the tripartite mitochondrial genomes of two Crucifers, Brassica nigra (black mustard) and Raphanus sativa (radish). Both genomes contain two copies of a direct repeat engaged in intragenomic recombination. The outcome of this recombination in black mustard is to interconvert a 231 kb master chromosome with two subgenomic circles of 135 kb and 96 kb. In radish, a 242 kb master chromosome interconverts with subgenomic circles of 139 kb and 103 kb. The recombination repeats are 7 kb in size in black mustard and 10 kb in radish, and are nearly identical except for two insertions in the radish repeat relative to the black mustard one. The two repeat configurations present on the master chromosome of black mustard are located on the subgenomes of radish and vice-versa. To explain this, we postulate the existence of an evolutionarily intermediate mitochondrial genome in which the recombination repeats were (are) present in an inverted orientation. The recombination repeats described for these two species are completely different from those previously found in the closely related species B. campestris, implying that such repeats are created and lost frequently in plant mitochondrial DNAs and making it less than likely that recombination occurs in a site-specific manner.