Restriction endonuclease analysis of mitochondrial DNA from virus-transformed, tumor and control cells of human, hamster and avian origin. Sequence conservation and intraspecific variation

This study compares over 70 recognition sites for restriction endonucleases on mtDNAs from various control versus malignant cells, derived from Syrian hamster, chick embryo, viper and human cells, exhibiting a wide spectrum of cellular transformation and tumor histories. Agents for transformation in vitro and in vivo include Rous sarcoma viruses, simian virus 40, polyoma virus and adenovirus. The results show a striking intraspecific sequence homogeneity of different mtDNAs regardless of tissue origin and oncogenic history. mtDNA from human biopsy specimens of tumor versus pathologically normal areas yielded indistinguishable restriction cleavage patterns reflecting either the "wild-type' form (with seven restriction endonucleases) or, in one individual, a variant pattern detected with HpaI. The precise position of the HpaI variant site was determined on the physical map of human mtDNA. Additional cleavage sites in the previously reported restriction map of Syrian hamster mtDNA are also presented. It is concluded that (1) mtDNA sequence in higher animal cells are highly conserved in malignant transformation; (2) no evidence for integration of viral sequences in mtDNA is apparent; (3) variant patterns in mtDNA are likely to be intraspecific polymorphisms that pre-exist neoplastic transformation. The possibility is discussed that altered regulatory interaction with the mitochondrial genome, rather than evident changes in mtDNA primary structure, determine anomalous mitochondrial functions in malignant transformation.     

Genetic and physical analysis of the mitochondrial gene for subunit II of yeast cytochrome c oxidase.

The mitochondrial genetic locus oxi 1 contains the structural gene for subunit II of Cytochrome c oxidase. In this study, the oxi 1 locus, or at least a major portion of it, has been localized to a 2·4 kb2 HpaII fragment of mitochondrial DNA, by examining the mtDNA of oxi 1 mutants, and rho^? yeast strains that selectively retained in amplified form, this region of the mitochondria) genome. The 2·4 kb fragment is missing from the mtDNA of an oxi 1 locus deletion mutant, but is present in the mtDNAs retained by two rho^? strains that genetically recombine with all 16 oxi 1 mutants tested, to produce respiring progeny. Two other rho^? strains, that retained different but overlapping portions of the oxi 1 locus as determined genetically, contained mtDNAs consisting of “cloned” segments derived from within the 2·4 kb fragment: these rho^? mtDNAs hybridized only to the 2·4 kb HpaII fragment of wild-type mtDNA and could not be cleaved with HpaII. Furthermore, these two rho^? mtDNAs were found to correspond to sequences from opposite sides of the 2·4 kb fragment that overlap for 100 to 300 base-pairs near the middle of the fragment. Thus, five oxi 1 mutations that recombine with both of these rho^? strains could be further localized to this relatively short region of overlap. One such mutation, of particular interest because it produces an altered form of subunit II, was shown to lie on a 75-base-pair fragment that maps in this region of the overlap. The 75-base-pair fragment from the mutant migrates slightly faster during electrophoresis than the corresponding wild-type fragment. In contrast, the mobility of the fragment from a spontaneous revertant was indistinguishable from wild type.     

Pulse-label analysis and mapping of the two terminal regions of asynchronous complementary strand replication of mitochondrial DNA in transformed hamster cells

In vivo pulse-chase radioactive labeling studies were performed to localize within the physical map of $\text{C}{}_{13}\text{B}{}_4$ hamster mtDNA² the two terminal regions of heavy and light complementary strand synthesis. These terminal segments have been defined operationally as that region on the H- and L-strand that is synthesized last. mtDNA of monolayer cultures was pulsed with [³H]thymidine for a minimum period of 10 minutes, which is about one-tenth of one round of mtDNA synthesis, followed by chase periods of up to 120 minutes. The properties of the labeled closed circular replicative intermediates E-mtDNA, C-mtDNA and D-mtDNA were analyzed in $\text{CsCl}\text{PrI}{}_2$ gradients and in neutral sucrose velocity and alkaline CsCl gradients. Both terminally labeled α and β daughter molecules were found to pass through the E-mtDNA stage. Sensitivity of $\text{C}{}_{13}\text{B}{}_4$ mtDNA to alkali and ribonuclease A indicated the presence of covalently linked ribonucleotides. The distribution (specific activity) of pulse-chase radioactivity relative to uniform label was followed in electrophoretically separated HpaII + HinIII and HpaI restriction fragments (freed of 7 S initiation sequences) and corrected for thymine content. The strand specificity of the pulse-label was determined by hybridization of restriction fragments with H- and L-strands of mtDNA. The kinetic data agree precisely with electron microscopic determinations of H- and L-strand origins at respective genome positions of 0 and 67 ± 3, which are located on HpaII + HindIII fragments 9 and 6, respectively (Nass, 1980). The two terminal regions are within the predicted genome sector between about 67 and 100/0 map units; the highest terminal pulse-chase radioactivity extends within 5 to 15% of the genome's length behind each origin. The kinetics of early labeling events were found to differ at the two termini. The evidence indicates that the majority of L-strand initiation/termination sites are in the region near map position 67 ± 3, and confirms the highly asynchronous replication mechanism of this DNA.     

Electron microscope heteroduplex study of Drosophila mitochondrial DNAs: evolution of A+T-rich region.

Mitochondrial DNAs (mtDNA) from three species of the genus Drosophila (D. melanogaster, D. simulons, and D. virilis) were compared by electron microscope heteroduplex mapping. Analysis of heteroduplex molecules revealed that the A + T-rich region of these mtDNAs has undergone quite extensive base sequence divergence, whereas the remainder of the molecule was found to share apparently complete base sequence homology in all three species. The differences in the sizes of the A + T-rich regions, as determined from the heteroduplex measurements, completely account for the differences in the total sizes of these mtDNAs. A segment of approximately 0.1 kb is conserved within the A + T-rich regions of D. simulans and D. virilis mtDNAs, but not within the A + T-rich region of D. melanogaster mtDNA. HaeIII restriction endonuclease analysis of the heteroduplex molecules has further shown that the unique HaeIII site of D. virilis mtDNA molecule is apparently conserved in both D. melanogaster and D. simulans mtDNA molecules. Finally, electrophoretic patterns of HaeIII-digested mtDNAs from all three species were found to be different and distinguishable from each other suggesting that single base substitutions have probably taken place throughout the entire mitochondrial genome.     

Comparison of mitochondrial components from human and hamster cell lines

Mitochondrial DNA (mtDNA) of human (KB) cells has been separated from that of Syrian hamster (BHK) cells on neutral CsCl density gradients, the density difference being 0.008 g/cm³. For three mitochondrially-located enzymes NAD-malate dehydrogenase, NADP-isocitrate dehydrogenase, and glutamate-oxaloacetate transaminase, the human enzyme has been separated from the hamster enzyme by starch gel electrophoresis. These results will facilitate the analysis of human-hamster hybrid cells for the persistence of parental mtDNA and for the presence of parental mitochondrial enzymes.     

Structural variations and optional introns in the mitochondrial DNAs of Neurospora strains isolated from nature

Mitochondrial DNAs from ten wild-type Neurospora crassa, Neurospora intermedia, and Neurospora sitophila strains collected from different geographical areas were screened for structural variations by restriction enzyme analysis. The different mtDNAs show much greater structural diversity, both within and among species, than had been apparent from previous studies of mtDNA from laboratory N. crassa strains. The mtDNAs range in size from 60 to 73 kb, and both the smallest and largest mtDNAs are found in N. crassa strains. In addition, four strains contain intramitochondrial plasmid DNAs that do not hybridize with the standard mtDNA. All of the mtDNA species have a basically similar organization. A 25-kb region that includes the rRNA genes and most tRNA genes shows very strong conservation of restriction sites in all strains. The 2.3-kb intron found in the large rRNA gene in standard N. crassa mtDNAs is present in all strains examined, including N. intermedia and N. sitophila strains. The size differences between the different mtDNAs are due to insertions or deletions that occur outside of the rRNA-tRNA region. Restriction enzyme and heteroduplex mapping suggest that four of these insertions are optional introns in the gene encoding cytochrome oxidase subunit I. Mitochondrial DNAs from different wild-type strains contain zero, one, three, or four of these introns.     

EVOLUTIONARY INFERENCES FROM RESTRICTION MAPS OF MITOCHONDRIAL DNA FROM NINE TAXA OF XENOPUS FROGS

Restriction endonuclease cleavage maps were prepared by the double digestion method for mitochondrial DNAs (mtDNAs) purified from Xenopus borealis, X. clivii, X. fraseri, X. muelleri, X. ruwenzoriensis, X. vestitus, X. laevis victorianus, X. l. laevis, and a variant of X. laevis designated X. laevis “davis.” An average of 21 cleavage sites per genome were mapped with 11 restriction endonucleases. Among the four invariant sites found are three conserved not only among the Xenopus mtDNAs tested but also among nearly all vertebrate mtDNAs examined to date. Two of these are Sac II sites in the 12S and 16S ribosomal RNA genes, and one is a Hpa I site in the gene for asparagine transfer RNA. These three sites permit the alignment and comparison of mtDNAs from different vertebrate classes. Although most of the differences observed among the Xenopus maps are attributable to point mutations causing gain or loss of restriction sites, the maps also differ by three large length mutations in or near the displacement loop. Phylogenetic analysis of 30 informative sites suggests that those members of the laevis species-group that have 36 chromosomes per somatic cell can be divided into three subgroups: 1) X. borealis, X. clivii, and perhaps X. fraseri (the “borealis” subgroup), 2) X. muelleri, and 3) the subspecies of X, laevis. The mtDNA of the hexaploid (2n = 108) species, X. ruwenzoriensis, is most similar to that of taxa in the latter two subgroups, which contrasts with the morphological similarity of this species to X. fraseri. X. ruwenzoriensis may be an allopolyploid with a mother (the contributor of the cytoplasmic mtDNA genome) on the X. laevis or X. muelleri lineage and a father on the X. fraseri lineage. We present a model showing how mtDNA and nuclear genomes can yield contrasting phytogenies for species-groups that have undergone several rounds of interspecific hybridization. Comparison of mitochondrial and nuclear sequence divergences suggests that Xenopus mtDNA, like that of mammals and birds, evolves faster than nuclear DNA. Genetic distances among mtDNAs of Xenopus species are very large, generally approaching or exceeding one substitution per nucleotide.     

Identification of cytoplasmically transferred mitochondrial DNA in female germlines of Drosophila and its propagation in the progeny

Summary The composition of mitochondrial DNA (mtDNA) was analyzed in single female flies that developed from fertilized Drosophila melanogaster eggs, into which germ plasm of D. simulans had been introduced. HpaII cleavage patterns showed that all 12 individual female flies examined had developed from eggs in which 37%–71% of the total mtDNA was D. simulans mtDNA (Ds mtDNA) and the rest was D. melanogaster mtDNA (Dm mtDNA). The stability of this heteroplasmic state in these isofemale lines was monitored for seven generations at both individual and population levels. Results showed that the heteroplasmy of Dm and Ds mtDNAs was stably transmitted for at least three generations at the population level, but showed stochastic segregation at the individual level. After 4–6 generations, all individuals lost Ds mtDNA. The mechanisms of preferential loss of Ds mtDNA and of transmission of heteroplasmic mtDNA to descendants are discussed.     

Length polymorphisms, restriction site variation, and maternal inheritance of mitochondrial DNA of Drosophila melanogaster

We have studied the mitochondrial DNA in three wild type laboratory strains of Drosophila melanogaster, ry⁺⁵ and two Oregon R-substrains, called here R and E. Lengths of the restriction bands for EcoRI, BglII, HpaII, MspI, HaeIII, and HindIII were compared. The number of restriction sites was identical in all strains, with the exception of an extra HaeIII site in ry⁺⁵. Careful comparison of restriction fragment lengths showed that bands containing the AT-rich region were different in length among all strains. The laboratory strains, ry⁺⁵, proved to be a mixture of strains carrying different mtDNAs; these separated into substrains G1 and G2 in the progeny of single pair matings. Adult progeny of reciprocal crosses of G1 and R were analyzed by HaeIII restriction digestion. The results demonstrated maternal inheritance for both the extra restriction site and band containing the AT-rich region.     

Chondriome analysis in sexual progenies of Nicotiana cybrids

Summary We studied the chondriomes (the mitochondrial genomes) of sexual-progeny plants derived from eleven Nicotiana cybrids which resulted from donor-recipient protoplast fusions. The recipients were either N. tabacum or N. sylvestris and the donor (of the cytoplasm) was N. bigelovii. The chondriomes were characterized by the mitochondrial DNA (mtDNA) restriction-patterns. The differences in mtDNA restriction patterns were revealed after Sal I digestions and probing the respective Southern-blots with three mtDNA fragments. The hybridization patterns of mtDNAs from 35 second-generation plants (i.e. the sexual progeny derived from the cybrid plants) indicated only minor variations between plants derived from the same cybrid but pronounced variations among sibs derived from different cybrids. The mtDNA of 32 second-generation plants varied from both original fusion partners but the mtDNA of one (male-sterile) plant was apparently identical with the mtDNA of one of the original donor (N. bigelovii) and the mtDNA of two other (male-fertile) plants was apparently identical to the mtDNA of an original recipient (N. sylvestris). Generally, the mtDNAs of male-fertile, second-generation plants were similar to the mtDNAs of the original recipients while the mtDNAs of the male-sterile second-generation plants were similar to the mtDNA of the donor (N. begelovii). The analyses of mtDNAs from the thirdgeneration plants indicated stabilization of the chondriomes; no variations were detected between the mtDNAs of plants derived from a given second-generation plant.     

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.     

Mitochondrial DNA associations with East Asian metabolic syndrome

Mitochondrial dysfunction has repeatedly been reported associated with type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS), as have mitochondrial DNA (mtDNA) tRNA and duplication mutations and mtDNA haplogroup lineages. We identified 19 Taiwanese T2DM and MS pedigrees from Taiwan, with putative matrilineal transmission, one of which harbored the pathogenic mtDNA tRNA^Leu(UUR) nucleotide (nt) 3243A>G mutation on the N9a3 haplogroup background. We then recruited three independent Taiwanese cohorts, two from Taipei (N?=?498, mean age 52 and N?=?1002, mean age 44) and one from a non-urban environment (N?=?501, mean age 57). All three cohorts were assessed for an array of metabolic parameters, their mtDNA haplogroups determined, and the haplogroups correlated with T2DM/MS phenotypes. Logistic regression analysis revealed that mtDNA haplogroups D5, F4, and N9a conferred T2DM protection, while haplogroups F4 and N9a were risk factors for hypertension (HTN), and F4 was a risk factor for obesity (OB). Additionally, the 5263C>T (ND2 A165V) variant commonly associated with F4 was associated with hypertension (HTN). Cybrids were prepared with macro-haplogroup N (defined by variants m.ND3 10398A (114T) and m.ATP6 8701A (59T)) haplogroups B4 and F1 mtDNAs and from macro-haplogroup M (variants m.ND3 10398G (114A) and m.ATP6 8701G (59A)) haplogroup M9 mtDNAs. Additionally, haplogroup B4 and F1 cybrids were prepared with and without the mtDNA variant in ND1 3394T>C (Y30H) reported to be associated with T2DM. Assay of mitochondria complex I in these cybrids revealed that macro-haplogroup N cybrids had lower activity than M cybrids, that haplogroup F cybrids had lower activity than B4 cybrids, and that the ND1 3394T>C (Y30H) variant reduced complex I on both the B4 and F1 background but with very different cumulative effects. These data support the hypothesis that functional mtDNA variants may contribute to the risk of developing T2DM and MS.     

Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA. II. The organization of sequences in petite mitochondrial DNAs carrying genetic markers from the 21S region.

We have investigated the organization of sequences in ten rho- petite mtDNAs by restriction enzyme analysis and electron microscopy. From the comparison of the physical maps of the petite mtDNAs with the physical map of the mtDNA of the parental rho+ strain we conclude that there are at least three different classes of petite mtDNAs: I. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA. II. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA with a terminal inverted duplication. III. Mixed repeats of an (almost) continuous rho+ mtDNA segment. In out petite mtDNAs of the second type, the inverted duplications do not cover the entire conserved rho+ mtDNA segment. We have found that the petite mtDNAs of the third type contain a local inverted duplication at the site where repeating units can insert in two orientations. At least in one case this local inverted duplication must have arisen by mutation. The rearrangements that we have found in the petite mtDNAs do not cluster at specific sites on the rho+ mtDNA map. Large rearrangements or deletions within the conserved rho+ mtDNA segment seem to contribute to the suppressiveness of a petite strain. There is also a positive correlation between the retention of certain segments of the rho+ mtDNA and the suppressiveness of a petite strain. We found no correlation between the suppressiveness of a petite strain and its genetic complexity. The relevance of these findings for the mechanism of petite induction and the usefulness of petite strains for the physical mapping of mitochondrial genetic markers and for DNA sequence analysis are discussed.     

Preferential binding of cisplatin to mitochondrial DNA of Chinese hamster ovary cells

Some chemical carcinogens localize preferentially in mitochondrial DNA (mtDNA) when compared with genomic DNA (gDNA). Here we compare the ability of cisplatin (cis-diamminedichloroplatimum[II]) to induce DNA adducts in both genomic and mtDNA of Chinese hamster ovary (CHO) cells in culture. Cytotoxicity was examined by cell survival 4, 8 and 24 h afer exposure to 50 μM cisplatin. Cisplatin-DNA adducts were measured in DNA from nuclear and mitochondrial fractions by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), a sensitive competitive microtiter-based immunoassay utilizing antiserum elicited against cisplatin-modified DNA. An additional comparison of cisplatin-DNA binding in both compartments was performed by immunoelectron microscopy using the cisplatin-DNA antiserum and colloidal gold. DELFIA analysis of cisplatin-DNA adducts in gDNA and mtDNA showed a six-fold higher incorporation of drug into mtDNA as compared to gDNA. Morphometric studies of colloidal gold distribution in photomicrographs of CHO cells showed mtDNA to contain a four-fold higher concentration of cisplatin as compared to nuclear DNA. Therefore, both methods demonstrated a preferential binding of cisplatin to mtDNA versus gDNA.     

Population structure of mitochondrial genomes in Saccharomyces cerevisiae

Background

Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences.

Results

To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae.

Conclusions

Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1664-4) contains supplementary material, which is available to authorized users.     

Nucleotide sequence of Rattus norvegiens mitochondrial DNA that includes the genes for tRNAile, tRNAgln and tRNAf-met

The nucleotide sequence of a segment of mtDNA from Rattus norvegiens (rat) which contains the genes for tRNA^ile, tRNA^gl and tRNA^f-met has been determined. A detailed comparison has been made between this sequence and the corresponding sequences of mouse, human and bovine mtDNAs with regard to the primary and secondary structure of the tRNA genes, the regions connecting the tRNA genes, and the regions flanking the tRNA genes which code for the carboxyl terminus of URF-1 and the amino terminus of URF-2. No differences were found in the nucleotide sequences of the genes for tRNA^ile, tRNA^gln and tRNA^f-met in mtDNAs from three different female lines of rats (SASCO-1, SASCO-2 and Wild-UT) that differ by substitutions of 0.8% to 1.8% of their total nucleotides.     

Characterization of multiple Chinese hamster carbonyl reductases

Carbonyl reductase (CR) is an enzyme which can catalyze the oxidoreduction of various carbonyl compounds in the presence of NAD(P)H. With the PCR method, using primers carrying the conserved nucleotide sequence among mammalian CRs, we isolated three different cDNAs (CHCR1, CHCR2 and CHCR3) which encode a unique carbonyl reductase from the Chinese hamster. The PCR products of CHCR1 and CHCR2 were clearly isolated with Bpu1102I, BspEI and XmaI restriction enzymes. The nucleotide-sequence of CHCR3 was completely different from those of CHCR1 and CHCR2. The predicted double-wound βαβα-structures of the CHCRs suggests the presence of a typical NADP⁺-binding motif and is similar to the corresponding region of 3α,20β-hydroxysteroid dehydrogenase and mouse lung tetrameric carbonyl reductase. The deduced amino acid sequence of CHCR1 showed a high homology to CHCR2 (>96%) and the other mammalian CRs (>81%). However, CHCR3 showed a high homology to human CBR3 (>86%) and a relatively lower homology to the other CHCRs (<76%). Bacterial recombinant CHCRs showed typical carbonyl reductase activities towards 4-benzoylpyridine, 4-nitrobenzaldehyde and pyridine 4-carboxyaldehyde. These three CRs showed not only 3-keto reductase of steroids, but also 20-keto reductase. However, these CRs did not show any activity of 17-keto reductase activity. Both CHCR1 and CHCR2 have prostaglandin 9-keto reductase and 15-hydroxyprostaglandin dehydrogenase activities towards PGE₂ and PGF_2α from the analyses of enzymatic reaction products. The results of Western blotting and RT-PCR suggest these CHCRs have a tissue-dependent-distribution in the Chinese hamster.     

Restriction mapping and molecular cloning of adenovirus type 4 (subgroup E) DNA

The locations of thirty restriction endonuclease cleavage sites were determined on the genome of adenovirus type 4 (Ad4), the sole member of the subgroup E adenovirions. The restriction endonucleases BglII, EcoRI, HindIII, HpaI, KpnI, SalI, and XbaI cut Ad4 DNA 10, 3, 2, 3, 5, 5 and 3 times, respectively. Orientation of the linear Ad4 map with respect to left and right molecular ends was accomplished by taking advantage of the limited sequence homology between Ad2 and Ad4. Ten non-overlapping fragments of Ad4 DNA representing 98% of the genome, map units 1.6 to 99.6, have been cloned into the plasmid vector pKC7.