Sequence Heteroplasmy of D-Loop and rRNA Coding Regions in Mitochondrial DNA from Holstein Cows of Independent Maternal Lineages

A mitochondrial DNA (mtDNA) fragment containing the D-loop, phenylalanine tRNA, valine tRNA, and 12S and 16 rRNA genes was cloned and sequenced from 36 cows of 18 maternal lineages to identify the polymorphic sites within those regions and to detect the existence of heteroplasmic mtDNA in cows. Seventeen variable sites were observed within the D-loop and rRNA coding regions of bovine mtDNA within a 2.5-kb span. The hypervariable sites in the D-loop and rRNA coding regions were identified at nucleotide positions 169, 216, and 1594. Heteroplasmic mtDNA (variable mtDNA within a tissue) existed extensively in cows and was detected within the above regions in 11 of 36 cows sequenced. The insertion, deletion, and nucleotide transversion polymorphisms were found only in homopolymer regions. Heteroplasmy was observed frequently and seemingly is persistent in cattle. Though heteroplasmy was demonstrated, most lineages and mtDNA sites showed no heteroplasmy.     

Nonneutral Mitochondrial DNA Variation in Humans and Chimpanzees

We sequenced the NADH dehydrogenase subunit 3 (ND3) gene from a sample of 61 humans, five common chimpanzees, and one gorilla to test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution. Within humans and within chimpanzees, the ratio of replacement to silent nucleotide substitutions was higher than observed in comparisons between species, contrary to neutral expectations. To test the generality of this result, we reanalyzed published human RFLP data from the entire mitochondrial genome. Gains of restriction sites relative to a known human mtDNA sequence were used to infer unambiguous nucleotide substitutions. We also compared the complete mtDNA sequences of three humans. Both the RFLP data and the sequence data reveal a higher ratio of replacement to silent nucleotide substitutions within humans than is seen between species. This pattern is observed at most or all human mitochondrial genes and is inconsistent with a strictly neutral model. These data suggest that many mitochondrial protein polymorphisms are slightly deleterious, consistent with studies of human mitochondrial diseases.     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

High frequency of mitochondrial DNA D-loop mutations in Ewing’s sarcoma

Somatic mutations and polymorphisms in the noncoding displacement (D)-loop of mitochondrial DNA (mtDNA) are present in a variety of human cancers. To investigate whether Ewing’s sarcoma (EWS) harbors genetic alterations within the D-loop region and their potential association with EWS carcinogenesis, we analyzed and compared the complete mtDNA D-loop sequences from 17 pairs of tumor tissues and corresponding peripheral blood samples using the direct DNA sequencing method. Our results revealed that 12 of the 17 EWS tumor specimens (70.6%) carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletions. Among the tested 17 patients, we screened a total of 40 germline polymorphisms including one novel sequence variant in the D-loop fragment. Most of these identified mutations and germline variations were clustered within two hypervariable segments (HVS1 and HVS2) as well as the homopolymeric C stretch between nucleotide position 303 and 309. In addition, there was no significant correlation between mtDNA D-loop mutations and various clinicopathological factors of EWS. In conclusion, our study reports for the first time that mtDNA D-loop mutations occur at a high frequency in EWS. These data provide evidence of mtDNA alterations’ possible involvement in the initiation and/or progression of this rare malignancy.     

三种小型猪线粒体DNA控制区的比较研究

目的分析五指山小型猪、巴马小型猪和贵州香猪线粒体DNA控制区碱基序列,比较研究不同猪种的遗传标志。方法应用PCR技术分别对这三种小型猪的血液总DNA样品中线粒体DNA D-loop区进行扩增,测序比对。结果猪的线粒体DNA D-loop区分三个区域。I区（靠近5’端区域）704bp,五指山小型猪在此区共有6个变异位点,通过6个变异位点中归纳出3个单倍体,而巴马小型猪在此区有9个变异位点,通过9个变异位点归纳出4个单倍体,贵州香猪在此区共有6个变异位点,通过6个变异位点归纳出3个单倍体。Ⅱ区（串联重复序列区）,五指山小型猪、巴马小型猪和贵州香猪序列相同。Ⅲ区（靠近3’端区域）三种小型猪的序列几乎相同。结论五指山小型猪、巴马小型猪和贵州香猪三种小型猪之间线粒体DNA碱基序列变异位点较少,五指山小型猪和巴马小型猪亲缘关系较近。     

Differences Between two Subspecies of Dolly Varden, Salvelinus malma, Revealed by RFLP—PCR Analysis of Mitochondrial DNA

Genetic differences between two subspecies of Dolly Varden, northern Salvelinus malma malma and southern Salvelinus malma krascheninnikovi, from rivers of eastern Russia were studied. Mitochondrial DNA was analyzed by restriction fragment length polymorphisms (RFLP) performed on products amplified with polymerase chain reaction. Three adjacent segments (approximately 7670 bp), comprising 47% of the mitochondrial genome were used: two encoding the five complete NADH dehydrogenase subunits and the other the cytochrome b gene and the control region (D-loop). Total composite haplotypes 46 were found among 136 fishes using RFLP analysis with 14 restriction enzymes. The amount of nucleotide divergence between haplotypes of two subspecies of Dolly Varden was estimated to be approximately 4%. The differences in the level of nucleotide diversity, mismatch distribution between haplotypes, and population-genetic structure of two subspecies of Dolly Varden suggest that these two forms have existed separately for a long time.     

Screening for human mitochondrial DNA polymorphisms with denaturing gradient gel electrophoresis

A method involving denaturing gradient gel electrophoresis (DGGE) was developed to detect mitochondrial DNA (mtDNA) polymorphisms in human peripheral T-lymphocytes. DGGE analysis of 100- to 200-bp sequences of low melting temperature domains within the origin/membrane attachment site, NADH dehydrogenase subunit I, cytochrome c oxidase subunit I and two overlapping regions of the tRNA glycine/NADH dehydrogenase subunit III sequences was performed to identify sequence variants at these sites in a human B-cell line TK6 and T-cells from four individuals. A T → C transition at position 16519 in the origin/membrane attachment site in the TK6 cell line and the T-cells from one individual was found. A sequence variant resulting in a G → A transition at position 9966 in the tRNA glycine/NADH dehydrogenase III was identified in another individual. This method should be useful for the rapid screening of polymorphisms in a large number of samples. Received: 19 October 1995 / Revised: 26 March 1996     

Differentiation of Dolly Varden char Salvelinus malma from Asia and North America inferred from PCR-RFLP analysis of mitochondrial DNA

Genetic differentiation of Dolly Varden char Salvelinus malma Walbaum from the Asian and North American Pacific coasts was studied. We examined restriction fragment length polymorphism of three mitochondrial DNA (mtDNA) fragments amplified in polymerase chain reaction, which encoded four NADH dehydrogenase subunits, the cytochrome b gene, and a D-loop segment. The mtDNA haplotypes were shown to form three phylogenetic groups, whose geographic distribution corresponded to three Dolly Varden subspecies: S. malma malma, S. malma krascheninnikovi, and S. malma lordi. The nucleotide sequence divergence between S. malma malma and S. malma krascheninnikovi was 3.8%; between S. malma malma and S. malma lordi, 3.1%; and between S. malma krascheninnikovi and S. malma lordi, 2.5%. The northern Dolly Varden S. malma malma from Asia was shown to be genetically identical to that from North America.     

Mitochondrial and nuclear genes present conflicting portraits of human origins

Human mitochondrial DNA (mtDNA) sequences reveal an abundance of polymorphic sites in which the frequencies of the segregating bases are very different. A typical polymorphism involves one base at low frequency and the other base at high frequency. In contrast, nuclear gene data sets tend to show an excess of polymorphisms in which both segregating bases are at intermediate frequencies. A new statistical test of this difference finds significant differences between mtDNA and nuclear gene data sets reported in the literature. However, differences in the polymorphism patterns could be caused by different sample origins for the different data sets. To examine the mtDNA-nuclear difference more closely, DNA sequences were generated from a portion of the X-linked pyruvate dehydrogenase E1 alpha subunit (PDHA1) locus and from a portion of mitochondrial control region I (CRI) from each of eight individuals, four from sub-Saharan Africa. The two genes revealed a significant difference in the site frequency distribution of polymorphic sites. PDHA1 revealed an excess of intermediate-frequency polymorphisms, while CRI showed an excess of sites with the low-high frequency pattern. The discrepancy suggests that mitochondrial variation has been shaped by natural selection, and may not be ideal for some questions on human origins.      

Mitochondrial restriction fragment length polymorphisms in wild Phaseolus vulgaris L.: insights on the domestication of the common bean

Summary Previous examination of intraspecific mitochondrial DNA (mtDNA) diversity in common bean, Phaseolus vulgaris, showed that five restriction fragment length polymorphisms (RFLPs) distinguish the mitochondrial genomes of the two major gene pools of cultivated beans, the Mesoamerican and the Andean. In the study presented here, mtDNA was used to compare the amount of diversity in cultivated beans to that in collections of wild beans to gain an understanding of how and when the mitochondrial genomes of the gene pools became distinct. The mtDNA of six wild bean accessions from Central and South America were digested with nine restriction endonucleases and analyzed by Southern hybridization. A total of twenty RFLPs were detected demonstrating a significantly higher amount of mtDNA variability in wild beans than in cultivated ones. All of the wild beans had the same mtDNA pattern for four out of the five inter-gene pool RFLPs, indicating that the polymorphism arose soon after domestication: two in the gene pool of the cultivated Mesoamerican beans and two in the gene pool of the cultivated Andean beans. The fifth RFLP must have occurred before domestication since the locus was also polymorphic in the wild beans. Wild beans from the south Andes were distinct and less variable than wild accessions of the north Andes and Mesoamerica. The distribution of mtDNA RFLPs among the wild beans supports the concept of two distinct domestication events for P. vulgaris.     

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.     

东方田鼠四个种群线粒体D-loop区遗传多态性分析

东方田鼠(Microtus fortis)主要分布在我国和俄罗斯、朝鲜、蒙古等地,我国是东方田鼠主要分布区,在东北、西北、黄河流域、长江流域和浙江、福建等省都有分布.     

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.     

Variation of mitochondrial DNA in Far Eastern mullet pilengas, Liza haematocheilus Temminck and Schlegel, acclimatized in the Azov-Black Sea basin

This study continues the investigation of genetic variation in the populations of native and acclimatized in the Azov-Black Sea basin pilengas from the Sea of Japan. The previous comparison based on allozyme analysis was supplemented by analysis of restriction polymorphism of a mitochondrial DNA fragment containing the cytochrome b gene and the D-loop. Five out of fifteen tested endonucleases detected polymorphic sites. In the samples of native and acclimatized pilengas, five common haplotypes were found; ten and three “unique” haplotypes were detected in the Far Eastern and the Azov populations, respectively. The differences in haplotype distributions between these populations were highly significant (P < 0.001). The mtDNA variation was lower in the Azov than in the Far Eastern population (haplotype diversity μ respectively 6.35 ± 0.27 and 9.14 ± 0.55), which is in good agreement with the decrease in the number of polymorphic loci and the mean number of alleles per locus, found earlier for allozyme markers in this population. The reasons for these differences in the acclimatized population are discussed.     

Asian affinities and continental radiation of the four founding Native American mtDNAs.

The mtDNA variation of 321 individuals from 17 Native American populations was examined by high-resolution restriction endonuclease analysis. All mtDNAs were amplified from a variety of sources by using PCR. The mtDNA of a subset of 38 of these individuals was also analyzed by D-loop sequencing. The resulting data were combined with previous mtDNA data from five other Native American tribes, as well as with data from a variety of Asian populations, and were used to deduce the phylogenetic relationships between mtDNAs and to estimate sequence divergences. This analysis revealed the presence of four haplotype groups (haplogroups A, B, C, and D) in the Amerind, but only one haplogroup (A) in the Na-Dene, and confirmed the independent origins of the Amerinds and the Na-Dene. Further, each haplogroup appeared to have been founded by a single mtDNA haplotype, a result which is consistent with a hypothesized founder effect. Most of the variation within haplogroups was tribal specific, that is, it occurred as tribal private polymorphisms. These observations suggest that the process of tribalization began early in the history of the Amerinds, with relatively little intertribal genetic exchange occurring subsequently. The sequencing of 341 nucleotides in the mtDNA D-loop revealed that the D-loop sequence variation correlated strongly with the four haplogroups defined by restriction analysis, and it indicated that the D-loop variation, like the haplotype variation, arose predominantly after the migration of the ancestral Amerinds across the Bering land bridge.     

Variation in mitochondrial DNA in Far Eastern mullet pilengas, Liza haematocheilus Temminck and Schlegel, acclimatized in the Azov-Black Sea basin

This study continues the investigation of genetic variation in the populations of native and acclimatized in the Azov-Black Sea basin pilengas from the Sea of Japan. The previous comparison based on allozyme analysis was supplemented by analysis of restriction polymorphism of a mitochondrial DNA fragment containing the cytochrome b gene and the D-loop. Five out of fifteen endonucleases tested detected polymorphic sites. In the samples of native and acclimatized pilengas, five common haplotypes were found; ten and three "population-specific" haplotypes were detected in the Far Eastern and the Azov populations, respectively. The differences in haplotype distributions between these populations were highly significant (P < 0.001). The mtDNA variation was lower in the Azov than in the Far Eastern population (haplotype diversity mu respectively 6.35 +/- 0.27 and 9.14 +/- 0.55), which is in good agreement with the decrease in the number of polymorphic loci and the mean number of alleles per locus, found earlier for allozyme markers in this population. The reasons for these differences in the acclimatized population are discussed.     

Isolation,physical map and gene map of mitochondrial DNA from the cryptomonad Pyrenomonas salina

Mitochondrial DNA (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant density centrifugation of whole-cell DNA in the presence of Hoechst dye 33258. mtDNA consists of circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sac I) has been constructed. Genes coding for the small subunit of rRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for 5S rRNA and NADH dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic and phylogenetic studies.     

Differentiation of Dolly Varden Char <Emphasis Type="Italic">Salvelinus malma</Emphasis> from Asia and North America Inferred from PCR-RFLP Analysis of Mitochondrial DNA

Genetic differentiation of Dolly Varden char Salvelinus malma Walbaum from the Asian and North American Pacific coasts was studied. We examined restriction fragment length polymorphism of three mitochondrial DNA (mtDNA) fragments amplified in polymerase chain reaction, which encoded four NADH dehydrogenase subunits, the cytochrome b gene, and a D-loop segment. The mtDNA haplotypes were shown to form three phylogenetic groups, whose geographic distribution corresponded to three Dolly Varden subspecies: S. malma malma, S. malma krascheninnikovi, and S. malma lordi. The nucleotide sequence divergence between S. malma malma and S. malma krascheninnikovi was 3.8%; between S. malma malma and S. malma lordi, 3.1%; and between S. malma krascheninnikovi and S. malma lordi, 2.5%. The northern Dolly Varden S. malma malma from Asia was shown to be genetically identical to that from North America.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 626–634.Original Russian Text Copyright © 2005 by Oleinik, Skurikhina, Brykov, Crane, Wenburg.     

Polymorphic Sites and the Mechanism of Evolution in Human Mitochondrial DNA

Twelve restriction enzymes were used to screen for the presence or absence of cleavage sites at 441 locations in the mitochondrial DNA of 112 humans from four continents. Cleavage maps were constructed by comparison of DNA fragment sizes with those expected from the published sequence for one human mtDNA. One hundred and sixty-three of the sites were polymorphic, i.e., present in some individuals but absent from others, 278 sites being invariant. These polymorphisms probably result from single base substitutions and occur in all functional regions of the genome.--In 77 cases, it was possible to specify the exact nature and location (within a restriction site) of the mutation responsible for the absence of a restriction site in a known human mtDNA sequence and its presence in another human mtDNA. Fifty-two of these 77 gain mutations occur in genes coding for proteins, 34 being silent and 18 causing amino acid replacements; moreover, nine of the replacements are radical.--Notable also is the anomalous ratio of transitions to transversions required to account for these 77 restriction site differences between the known human mtDNA sequences and other human mtDNAs. This ratio is lower for most groups of restriction sites than has been reported from sequence comparisons of limited parts of the mtDNA genome in closely related mammals, perhaps indicating a special functional role or sensitivity to mutagenesis for palindromic regions containing high levels of guanine and cytosine.--From the genomic distribution of the 163 polymorphic sites, it is inferred that the level of point mutational variability in tRNA and rRNA genes is nearly as high as in protein-coding genes but lower than in noncoding mtDNA. Thus, the functional constraints operating on components of the protein-synthetic apparatus may be lower for mitochondria than for other systems. Furthermore, the mitochondrial genes for tRNAs that recognize four codons are more variable than those recognizing only two codons.--Among the more variable of the human mitochondrial genes coding for proteins is that for subunit 2 of cytochrome oxidase; this polypeptide appears to have been evolving about five times faster in primates than in other mammals. Cytochrome c, a nuclearly encoded protein that interacts directly with the oxidase 2 subunit in electron transport, has also evolved faster in primates than in rodents or ungulates. This example, along with that for the mitochondrial rRNA genes and the nuclear genes coding for mitochondrial ribosomal proteins, provides evidence for coevolution between specific nuclear and mitochondrial genes.     

Polymorphism of the Noncoding Region of the Mitochondrial Genome in Three Kazakh Populations Inhabiting Different Areas of Kazakhstan and in DNA Samples from Ancient Populations of the Kazakhstani Altai

The polymorphism of the major noncoding region of mitochondrial DNA (mtDNA D loop, 528 bp) has been studied in samples from three modern Kazakh populations (from Almaty, the Semipalatinsk Region, and the Altai Mountains) and in DNA samples of ancient human populations of the Kazakhstani Altai. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis for 13 restriction sites, including BamHI, EcoRV, Sau3AI (one site each), KpnI (two sites), HaeIII (three sites), and RsaI (five sites) were used. The frequency distributions of all sites have been determined. The gene diversity (h) and the genetic distances between different Kazakh populations and other populations of the world have been calculated. The RFLP analysis of the mtDNA control region of fossil samples has been performed similarly to the analysis of modern mtDNA samples. Two fossil mtDNA samples from burial mound 11 are monomorphic with respect to all restriction sites analyzed.