Mitochondrial DNA polymorphism in Japanese

Summary Mitochondrial DNA (mtDNA) from 116 Japanese was analyzed with nine restriction enzymes that recognize a four or five base pair sequence. The sizes of the mtDNA fragments produced by digestion by each enzyme were compared after gel electrophoresis. Double digestion experiments indicated that, in the coding region from URF2 (unidentified reading frame) to tRNA^Asn (bp 5274–5691), there is an insertion of about 60 base pairs (bp) compared with the published mtDNA sequence, which is common to all individuals in the present sample. A total of 95 different morphs were detected with the nine enzymes, 60 of which have not been documented previously. Based on a comparison of the cleavage maps of all individuals, 62 different combinations of restriction types were observed. By pairwise comparison of each restriction type, the average number of nucleotide substitutions per nucleotide site () was estimated to be 0.0026. Phylogenetic analysis of the present data indicates that at least two distinct lineages exist in the Japanese population.     

Polymorphism of mitochondrial DNA in pigs based on restriction endonuclease cleavage patterns

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of pigs and Japanese wild boars were analyzed using 17 enzymes which recognize six nucleotides. The map of cleavage sites was made by double-digestion methods. Polymophism of mtDNA was detected in the digestion by BglII, EcoRV, ScaI, and StuI. The restriction cleavage patterns were identical among the breeds of Landrace, Hampshire, Duroc I, and Large White I (A type). The patterns of Large White II were the same as those of Japanese wild boars (B type). A difference between the A type and the B type of mtDNA was found in the case of three restriction enzymes, BglII, ScaI, and StuI, and the nucleotide alterations between them were estimated as more than six. On the other hand, a difference between mtDNA from almost all pigs and mtDNA from Duroc II was detected using EcoRV. We suggest that the difference of mtDNA between the A type and the B type of mtDNA could result from the different origin of boars, that is, whether they were of European or Asian origin.     

Genetic diversity in the Chinese pangolin (Manis pentadactyla): Inferred from restriction enzyme analysis of mitochondrial DNAs

Two different forms of Chinese pangolins can be recognized according to the color of their scales, i.e., brown and dusky. We analyzed mitochondrial DNA (mtDNA) purified from the livers of seven dusky and six brown Chinese pangolins from the same locality, using cleavage patterns from 19 restriction enzymes. From the 19 6-bp recognition enzymes used, 51–56 sites were observed. By combining the cleavage patterns for each enzyme, the 13 samples were classified into four restriction types: two in dusky and two in brown Chinese pangolins. The estimated number of nucleotide substitutions per site in dusky and brown types is 0.002, and that between dusky and brown types is 0.012. Divergence between brown and dusky forms began 0.6 Myr ago, provided the mean rate of sequence divergence is 0.02 per Myr in mtDNA. Our results suggest that there is considerable divergence in Chinese pangolins, and brown and dusky Chinese pangolins may be quite different forms or, at least, belong to different maternal groups.     

Ovine mitochondrial DNA: restriction enzyme analysis, mapping and sequencing data

Restriction endonuclease fragment patterns of mitochondrial DNA (mtDNA) in sheep were analysed with 11 enzymes. Four breeds (Merinolandschaf, Rhoenschaf, Schwarzkoepfiges Fleischschaf and Skudde) of domestic sheep and European Mouflon were examined. A restriction map with 28 cleavage sites of seven enzymes was established. KpnI and PstI do not cut ovine mtDNA. Two EcoRI fragments of Merinolandschaf, Rhoenschaf and Mouflon each were cloned and partially sequenced. Intraspecific nucleotide sequence differences within 1.101 kb ranged from 0.09 to 0.27%. Hybridization analysis with a fragment of porcine mtDNA along with sequencing data from cloned fragments was used for orientation of the restriction map along the bovine sequence. Ovine mtDNA sequences encompassing parts of the Cyt.b-, ND5-, CoIII- and ATPase6 genes were compared with the corresponding sequences of the bovine mtDNA.     

Pig mitochondrial DNA: Polymorphism,restriction map orientation,and sequence data

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) in pigs were analyzed using 18 enzymes which recognize six nucleotides and 1 four-nucleotide-recognizing enzyme. Pigs including Taiwan native breeds and miniature strains maintained in Japan were examined in this study; four commercial breeds of pigs and Japanese wild boars have been investigated earlier [Watanabe, T., et al. (1985). Biochem. Genet. 23:105]. mtDNA polymorphisms were observed in the cleavage patterns of five restriction enzymes, Bg1II, EcoRV, ScaI, StuI, and TaqI. The results support the previous hypothesis that pigs must be derived from two different maternal origins, European and Asian wild boars, and that a breed, Large White, arises from both European and Asian pigs. Two HindIII cleavage fragments were cloned into the HindIII site of M13mp10 and were partially sequenced by the dideoxynucleotide-chain termination method. Furthermore, DraI and StuI cleavage sites were newly determined on the restriction endonuclease map. On the basis of these results, the restriction endonuclease cleavage map of pig mtDNA was rewritten. Comparing sequence data of pig mtDNA at 237 positions with those of cow, human, mouse, and rat mtDNA, the sequence difference, silent and replacement changes, and transitions and transversions among mammalian species were estimated. The relationships among them are discussed.     

Genetic differentiation between laboratory lines of the musk shrew (Suncus murinus,insectivora) based on restriction endonuclease cleavage patterns of mitochondrial DNA

The cleavage patterns of mitochondrial DNA (mtDNA) by 17 restriction endonucleases were compared between eight lines of musk shrews derived from different wild-caught stocks. Enzymatic digestion byBamHI,PvuII,XbaI, andXhoI showed a cleavage pattern common to all lines that were from five Japanese islands (Nag, Ize, OKI, TKU, and Tr), Bangladesh (BAN), Sri Lanka (SRI), and Java (Bog), and every line lacked cleavage sites forSalI andSmaI. Different cleavage patterns were detected by the remaining 11 enzymes. Within the BAN line, the presence of at least two types of mtDNAs was proved by six enzymes and was not contradictory to the maternal pedigrees going up to the wild ancestors of the stock. More than 30 cleavage sites of the shrew mtDNA were mapped by double-digestion methods. Nucleotide diversities of mtDNA were calculated from these maps and were estimated to be less than 0.5% among Japanese and Bog lines but to be 3.8% between BAN and the other seven lines and 2.3% within the BAN line. These results indicate that BAN shrews differentiate from the other lines to the intersubspecific extent reported in mice previously.     

Bovine mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites

Cleavage patterns of mitochondrial DNA (mtDNA) by restriction endonuclease analysis were examined in four Japanese Black cows, three Japanese Shorthorn cows, and six Holstein cows. Seventeen restriction enzymes which recognize six base pairs and two restriction enzymes which recognize four base pairs were used in this study. Polymorphism was observed with three restriction enzymes, HindIII, TaqI, and MspI, and was detected within the breeds. Nucleotide substitution was determined in the HindIII polymorphic site by DNA cloning and sequencing; this is C----T at position 10126 of the URF-3 region. Furthermore, the MspI and TaqI polymorphic sites were located on the physical map.     

Human mitochondrial DNA types in Finland

Summary Variation in mitochondrial DNA (mtDNA) in a sample of 110 Finns was analyzed with six restriction enzymes, AvaII, BamHI, HaeII, HinII, HpaI, and MspI, by using total blood cell DNA probed with mouse mtDNA. Two new enzyme morphs were observed, one for HaeII and one for HindII. Double-digestion experiments indicated that the BamHI morphs 2 and 3 result from base changes leading to AvaII morphs 3 and 9, respectively. Of the ten different mtDNA types observed, defined by restriction fragment patterns, seven have been previously described in Caucasoid populations. The three new Finnish mtDNA types can be derived from Caucasoid lineages by single restriction site changes. The results were used to reconstruct a phylogenetic tree for Caucasoid mtDNA types defined by the enzymes used. The frequencies of mtDNA types were used to compute genetic distances between Finns, Italians, and Israeli Jews. The frequencies of both enzyme morphs and mtDNA types show that the Finnish population is highly homogeneous.     

Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine population of native cattle, indicating the existence of two separate types of mtDNA. These two types of mtDNA are very different from each other, at the level of subspecies. Since the native Philippine cattle are considered to represent an admixture of European and Indian cattle, the two types of mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and the nucleotide substitutions at some of the sites have also been estimated.     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.     

Genetic relationship among the musk shrews,Suncus murinus insectivora,inhabiting islands and the continent based on mitochondrial DNA types

Cleavage patterns of mitochondrial DNA (mtDNA) by restriction endonucleases were examined in musk shrews collected at six trapping sites on two Japanese and two Indonesian islands, on Sri Lanka, situated close to the Indian subcontinent, and on the mainland of East Bengal in Bangladesh. No variation of mtDNAs was found among the Japanese and Indonesian shrews, despite their geographical isolation by the sea. In contrast, at least six mtDNA types were present in the Sri Lanka and the Bangladesh populations (three types for each), and these two populations seemed to be differentiated to the extent, which could be compared to the mice-intersubspecific differences. These populations were also differentiated from the Japanese-Indonesian type. Furthermore, a similar level of differentiation was also estimated between two mtDNA types within these respective populations. This feral species might be considered unique because of its high emigration rate caused by human movements and its high rate of subspecific hybridization.     

Evolutionary Relationships among Five Subspecies of MUS MUSCULUS Based on Restriction Enzyme Cleavage Patterns of Mitochondrial DNA

The intra- and intersubspecific genetic distances between five subspecies of Mus musculus were estimated from restriction enzyme cleavage patterns or maps of mitochondrial DNA (mtDNA). The European subspecies, M. m. domesticus and Asian subspecies, M. m. bactrianus, M. m. castaneus, M. m. molossinus and M. m. urbanus were examined. For each subspecies, except M. m. urbanus, at least two local races from widely separated localities were examined. Intrasubspecific heterogeneity was found in the mtDNA cleavage patterns of M. m. bactrianus and M. m. castaneus. M. m. molossinus and M. m. domesticus, however, revealed no intrasubspecific heterogeneity. Four of the subspecies had distinct cleavage patterns. The fifth, M. m. urbanus, had cleavage patterns identical to those of M. m. castaneus with several enzymes. Estimates of genetic distances between the various races and subspecies were obtained by comparing cleavage maps of the mtDNAs with various restriction enzymes. Nucleotide sequence divergences of mtDNA between local races were estimated to be less than 0.4% in M. m. bactrianus and less than 0.3% in M. m. castaneus. The times of divergence of both subspecies were calculated to be 0.1–0.2 x 10⁶ years. These values suggest that the intrasubspecific divergence began some 0.1–0.2 x 10⁶ years ago. On the other hand, nucleotide sequence divergences between European subspecies M. m. domesticus and Asian subspecies M. m. bactrianus and M. m. castaneus were 7.1% and 5.8%, respectively. The times of divergence were calculated to be 2.1–2.6 x 10⁶ years. Further, the nucleotide sequence divergence and time of divergence between M. m. molossinus and the other two Asian subspecies were comparable to those between M. m. molossinus and M. m. domesticus (about 3% and 1 x 10⁶ years, respectively). These results suggest that M. m. molossinus is situated in a unique evolutionary position among Asian subspecies.     

Amino acid change associated with the major polymorphic Hinc II site of Oriental and Caucasian mitochondrial DNAs.

The mitochondrial DNAs (mtDNAs) from 116 Oriental and Caucasian blood samples were analyzed for their Hinc II restriction endonuclease cleavage patterns using Southern analysis and 32P human mtDNA probes. Seven distinct patterns were found, all of which could be interrelated by single nucleotide changes. The predominant pattern (mtHinc II-2) was found in 97% of the Caucasians and 73% of the Orientals. This mtDNA morph had one more Hinc II site than did the second most common morph (mtHinc II-1), which was found only in 20% of the Orientals. Three additional patterns were in a single Oriental sample, a fourth in a single Caucasian sample, and a fifth in one member of each population. The polymorphic site that differentiated mtHinc II-1 and mtHinc II-2 was cloned and sequenced. A single nucleotide change was found that created an Hinc II site and changed the amino acid sequence of the URF5 gene. Comparison of these sequences with those of other primates [15] revealed that the Asian mtHinc II-1 and mtHinc II-4 mtDNAs were identical in this region with those of chimpanzees and orangutans. These results suggest that the Asian mtHinc II-1 mtDNA may have been ancestral to other human mtDNAs.     

Nucleotide recognition seouence at the cleavage site of Haemophilus aegyptius II (Hae II) restriction endonuclease

We have determined the nucleotide sequence recognized by the restriction endonuclease Hae II from Haemophilus aegyptius which cleaves the simian virus 40 (SV40) DNA at a single specific site. By using terminal radioactive labeling of the cleavage site at both the 5′ and 3′-ends we have deduced the recognition sequence,

with elements of a two-fold rotational symmetry. The endonuclease produces staggered ends with protruding 3′-terminated single-strands, four nucleotides in length. In plasmid RSF 2124 DNA, which contains multiple Hae II cleavage sites, it was observed that the 5th nucleotide from the 3′ terminus is either a pdA or a pdG, indicating alternating recognition sequences.     

Genetic and geographical differentiation of <Emphasis Type="Italic">Pandaka</Emphasis> gobies in Japan

The mitochondrial DNA (mtDNA) phylogeny of Japanese Pandaka species (Perciformes: Gobiidae) was inferred from partial nucleotide sequences of the mitochondrial 12S and 16S rRNA genes (1083bp). The resultant mtDNA tree showed two major clades (clade I and clade II), which were inconsistent with the present taxonomic classification. One of the major clades was further divided into two geographical groups, distributed on the Japanese Major Islands (clade I-A) and from Amami-oshima Island to Iriomote Island (clade I-B). The mtDNA haplotypes in clade II were found only on Iriomote Island. The mtDNA divergences in clade I indicated that the Japanese Major Island (clade I-A) and Ryukyu (clade I-B) groups have been geographically isolated from each other for millions of years, based on the putative molecular divergence rate. The geographical distributions of mtDNA haplotypes in clade I-A and clade I-B also suggested that Pandaka gobies had not dispersed to distant offshore islands, indicating that their geographical differentiation may be closely associated with the geological history of the Japanese and Ryukyu Archipelagos.This revised version was published online in January 2005 with corrections to the repetition of the 1st authors name.     

Segregation of mitochondrial DNA in human somatic cell hybrids

Summary The maintenance of mtDNA has been examined in human intraspecific hybrid cells constructed from the fusion of HEB7A, a HeLa tumor cell line carrying the mitochondrially coded chloramphenical (CAP) resistance mutation, and GM 2291, a limited lifespan human diploid fibroblast which is CAP sensitive. These two cells can be distinguished by a polymorphism in a site for the restriction endonuclease, HaeIII. Independently isolated clones of hybrid cells were characterized for their growth properties (either normal limited lifespan or transformed and immortal). Whole cell DNA preparations were made from each hybrid, digested with HaeIII, and the resultant fragments were detected by hybridization to ³²P labelled mouse mtDNA as probe. Experiments with mixtures of HEB7A and GM2291 DNA reveal that HEB7A mtDNA can be detected when it constitutes as little as 5% of the total cell mtDNA.The results indicate that the HEB7A mtDNA is lost from most hybrids, and when it does persist it is usually a minor component of total mtDNA. The addition of CAP at the time of fusion slightly increases the quantity of HEB7A mtDNA, but not enough to confer CAP resistance. Furthermore, five limited lifespan hybrids contained no detectable HEB7A mtDNA, while three transformed hybrids contained varying quantities of HEB7A mtDNA, suggesting that retention of this tumor form of mtDNA is associated with tumor growth behavior. These results suggest that cytoplasmic genetic incompatibility occurs in intraspecific hybrids.     

Restriction patterns of adult chicken globin genes at early and late stages of embryonic development

Globin mRNA isolated from anemic chicken was transcribed into cDNA and integrated into the Pst I cleavage site of plasmid pBR 322. After cloning in E. coli strain HB 101 and colony hybridization with¹²⁵I-labelled globin mRNA the plasmids of individual clones were characterized by hybrid arrested cell-free translation. Thus we could isolate clones containing or globin chain nucleotide sequences.DNA was isolated from chicken blastoderms incubated for 18–20 h and from 11 d chicken embryos. A comparison of the restriction maps of the DNA from the two developmental stages with labelled nick translated plasmids and labelled cDNA did not indicate any globin gene rearrangements between these two stages of embryonic development. We conclude, that the adult chicken globin genes show a constant genomic organization during embryonic development. However, the restriction patterns of the globin gene family of the chicken strain investigated revealed some differences after 2 generations of propagation.     

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.     

Mitochondrial DNA polymorphism in Russians from west Siberia.

The mitochondrial DNA (mtDNA) of 60 Russians from West Siberia was analyzed with the following restriction enzymes: BamHI, HindIII, PstI, PvuII and SacI that recognize 6 bp. The observed restriction fragment length polymorphisms (morphs) were classified into 13 types of distinct cleavage patterns (mitotypes). The distributions of the mtDNA morphs were compared with those characteristic of some other human populations.     

Variation of mitochondrial DNA in chinook salmon <Emphasis Type="Italic">Oncorhynchus tschawytscha</Emphasis> Walbaum populations from Kamchatka

The variation in mitochondrial DNA (mtDNA) structure among chinook salmon Oncorhynchus tschawytscha Walbaum populations from Kamchatka was inferred from restriction length polymorphism analysis using eight restriction endonucleases. The nucleotide sequence variation in three amplified mtDNA regions was examined at seven polymorphic restriction sites in 579 fish from 13 localities. Based on the frequencies of 11 combined haplotypes and the number of nucleotide substitutions, the among-and within-population variation was estimated. The heterogeneity test showed highly significant differences among all the populations. The estimated maximum time of independent divergence of the asian chinook salmon populations, whose differences was about 0.02% nucleotide substitutions, did not exceed 10 000–20 000 years. Apparently, the retreat of the late Pleistocene glacier triggered spreading, recolonization, and formation of the present-day pattern of the species subdivision into structural components.