Nucleotide sequence of the D-loop region of the sperm whale (Physeter macrocephalus) mitochondrial genome

We have amplified, by the polymerase chain reaction, and have sequenced the D-loop region of the mitochondrial DNA from the sperm whale (Physeter macrocephalus). The sperm whale D-loop was aligned with D- loop sequences from four other cetaceans (Commerson's dolphin, orca, fin whale, and minke whale) and an out-group (cow). This alignment showed the sperm whale sequence to be larger than that of other cetaceans. In addition, some sequence blocks were highly conserved among all six species, suggesting roles in the functioning of mitochondrial DNA. Other blocks that were previously reported to be well conserved among cetaceans showed little sequence conservation with the sperm whale D-loop, which argues against the functional importance of these sequence blocks in cetaceans.      

Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA.

The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA (mtDNA) from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA. Both D-loop regions are bounded by the genes for tRNAPhe and tRNAPro. This region contains the most highly divergent sequences in mtDNA with the exceptions of three small conserved sequence blocks near the 5' ends of D-loop strands, a 225 nucleotide conserved sequence block in the center of the D-loop strand template region, and a short sequence associated with the 3' ends of D-loop strands. A sequence similar to that associated with the 3' termini of D-loop strands overlaps one of the conserved sequence blocks near the 5' ends of D-loop strands. The large, central conserved sequence probably does not code for a protein since no open reading frames are discretely conserved. Numerous symmetric sequences and potential secondary structures exist in these sequences, but none appear to be clearly conserved between species.     

Mapping at the nucleotide level of Xenopus laevis mitochondrial D-loop H strand: structural features of the 3' region

The D-loop resulting from limited synthesis of the newly replicated heavy (H) strand of mitochondrial DNA provides a good opportunity to examine both the origin and termination of DNA synthesis. We report here the precise determination of the 3' and 5' termini of nascent Xenopus laevis D-loop H strand. We observe two major classes of newly synthesized D-loop H strands, 1641 and 1675 nucleotides long. A stable putative secondary structure located around its 3' end is described. Analogous secondary structures are also found in the same region of the mammalian D-loop mitochondrial DNAs. Moreover a pentanucleotide (5' TACAT 3'), base-paired in these secondary structures and most often present in two copies, is conserved in all vertebrate species so far studied. The termination associated sequence previously described in mammals is part of the putative stop signal represented by the secondary structure except in man. These results show that the mechanism of arrest of H strand synthesis is common to vertebrates.     

Polymorphic sequence in the D-loop region of equine mitochondrial DNA

The D-loop regions in equine mitochondrial DNA were cloned from three thoroughbred horses by polymerase chain reaction (PCR). The total number of bases in the D-loop region were 1114bp, 1115bp and 1146bp. The equine D-loop region is A/T rich like many other mammalian D-loops. The large central conserved sequence block and small conserved sequence blocks 1, 2 and 3, that are common to other mammals, were observed. Between conserved sequence blocks 1 and 2 there were tandem repeats of an 8bp equine-specific sequence TGTGCACC, and the number of tandem repeats differed among individual horses. The base composition in the unit of these repeats is G/C rich as are the short repeats in the D-loops of rabbit and pig. Comparing DNA sequences between horse and other mammals, the difference in the D-loop region length is mostly due to the difference in the number of DNA sequences at both extremities. The similarities of the DNA sequences are in the middle part of the D-loop. In comparison of the sequences among three thoroughbred horses, it was determined that the region between tRNA^Pro and the large central conserved sequence block was the richest in variation. PCR primers in the D-loop region were designed and the expected maternal inheritance was confirmed by PCR-RFLP (restriction fragment length polymorphism).     

Structural conservation and variation in the D-loop-containing region of vertebrate mitochondrial DNA

The nucleotide sequences of the D-loop-containing regions of three rat mitochondrial DNAs (mtDNAs), two from the species Rattus norvegicus and one from R. rattus, were determined. Comparisons made among these sequences and with the mouse sequence showed that, on the basis of both base composition and frequency of nucleotide alterations, three domains could be defined within the D-loop-containing region: a central conserved segment, poor in L-strand adenine, flanked by two divergent, adenine-rich regions. Deletions and insertions were found to occur at an unexpectedly high frequency in these sequences and the conserved sequence block called CSB-1 was found not to be intact in the R. rattus sequence. Although in comparisons of more distantly related mtDNAs the D-loop region is the most divergent on the molecule, it does not diverge more than typical protein genes between R. norvegicus and R. rattus, and its central conserved domain appears to be one of the molecule's most conserved regions. The most variable domain borders the tRNAPhe gene and contains the L and H-strand promoters and the 5' terminus for H-strand DNA synthesis. Within this region we have found sequences in all the mtDNAs we have examined, including those of human, two artiodactyls and Xenopus, that are capable of folding into cloverleaf structures. In the other divergent domain of the same mtDNAs, we find sequences capable of assuming similar secondary structural configurations at or near the sites for the termination of D-loop DNA synthesis. The evolutionary preservation of the potential to form such structures despite the high primary-structural divergence of the regions they occur in, suggests the structures are of principal importance for some processes occurring in the D-loop-containing region.     

Mitochondrial D-loop sequence variation among the 16 maternal lines of the Lipizzan horse breed

Mitochondrial DNA from 49 Lipizzan horses representing 16 maternal lines from the original stud at Lipica was used for SSCP analysis and DNA sequencing. The SSCP analysis of the 444 bp long fragment of the D-loop region extending from the tRNA(Pro) gene to the central conserved sequence block revealed three distinct groups of SSCP patterns. Both ends of the D-loop region (378 bp and 310 bp), which are considered as the most variable regions within the mammalian mitochondrial DNA, were sequenced. According to 49 polymorphic sites identified within the both parts of the D-loop region, the 16 maternal lines were grouped into 13 distinct mitochondrial haplotypes. The minimal difference between two different haplotype DNA sequences was one nucleotide and the maximal 24 nucleotides. The inheritance of mitochondrial haplotypes was stable and no sequence variation potentially attributable to mutation within maternal line was observed. Considerable DNA sequence similarity of Lipizzan mitochondrial haplotypes with the haplotypes from other breeds was observed. Phylogenetic analysis of the sequence data revealed a dendrogram with three separated branches, supporting the historical data about the multiple origin of the Lipizzan breed.     

The main regulatory region of mammalian mitochondrial DNA: Structure-function model and evolutionary pattern

Summary The evolution of the main regulatory region (D-loop) of the mammalian mitochondrial genome was analyzed by comparing the sequences of eight mammalian species: human, common chimpanzee, pygmy chimpanzee, dolphin, cow, rat, mouse, and rabbit. The best alignment of the sequences was obtained by optimization of the sequence similarities common to all these species.The two peripheral left and right D-loop domains, which contain the main regulatory elements so far discovered, evolved rapidly in a species-specific manner generating heterogeneity in both length and base composition. They are prone to the insertion and deletion of elements and to the generation of short repeats by replication slippage. However, the preservation of some sequence blocks and similar cloverleaf-like structures in these regions, indicates a basic similarity in the regulatory mechanisms of the mitochondrial genome in all mammalian species.We found, particularly in the right domain, significant similarities to the telomeric sequences of the mitochondrial (mt) and nuclear DNA ofTetrahymena thermophila. These sequences may be interpreted as relics of telomeres present in ancestral linear forms of mtDNA or may simply represent efficient templates of RNA primase-like enzymes.Due to their peculiar evolution, the two peripheral domains cannot be used to estimate in a quantitative way the genetic distances between mammalian species. On the other hand the central domain, highly conserved during evolution, behaves as a good molecular clock.Reliable estimates of the times of divergence between closely and distantly related species were obtained from the central domain using a Markov model and assuming nonhomogeneous evolution of nucleotide sites.     

Structure and evolution of teleost mitochondrial control regions

We amplified and sequenced the mitochondrial control region from 23 species representing six families of teleost fish. The length of this segment is highly variable among even closely related species due to the presence of tandemly repeated sequences and large insertions. The position of the repetitive sequences suggests that they arise during replication both near the origin of replication and at the site of termination of the D-loop strand. Many of the conserved sequence blocks (CSBs) observed in mammals are also found among fish. In particular, the mammalian CSB-D is present in all of the fish species studied. Study of potential secondary structures of RNAs from the conserved regions provides little insight into the functional constraints on these regions. The variable structure of these control regions suggests that particular care should be taken to identify the most appropriate segment for studies of intraspecific variation. Correspondence to: T.D. Kocher     

Sequence length and variation in the mitochondrial control region of two freshwater gobiid fishes belonging to Rhinogobius (Teleostei: Gobioidei)

The control region (D-loop) of mitochondrial DNA (mtDNA) was amplified and sequenced for eight samples of the rhinogobies Rhinogobius maculafasciatus and R. giurinus from Taiwan and southern China. The control regions of both species are of 841–842 bp; the length of these sequences being the most compact among all known sequences in teleost fishes. Three conserved sequence blocks (CSB) were observed. The full D-loop and tRNA Phe gene sequences were determined and compared with other fishes. The interspecific sequence divergence between the two species is 11.3–11.7%; and the intraspecific variation in R. guirinus 0.8–1.8%. Results suggest that the control region of Rhinogobius is informative for phylogenetic reconstruction at both intraspecific and interspecific levels in this gobiid genus.     

太湖新银鱼线粒体D-loop和Cytb片段序列结构与进化速率比较

共获得49个太湖新银鱼(Neosalanx taihuensis)个体的线粒体细胞色素b(Cyt b)全序列和控制区(D-loop)部分序列。所测线粒体D-loop部分序列长度变化范围为648～680bp,识别到位于前端的一个串联重复序列、一个终止相关序列(ETAS),3个中央保守区保守序列(CSB-F、CSB-E、CSB-D)及一个保守序列区保守序列(CSB-1),结构与其他鱼类的研究结果类似。太湖新银鱼线粒体Cyt b和D-loop片段的相对进化速率的比较研究结果表明,太湖新银鱼D-loop总的序列多态性位点的比例为0.83%,低于线粒体Cyt b部分总的序列多态性位点的比例(1.31%)。假设太湖新银鱼Cyt b基因平均进化速率相对值为1,贝叶斯(Bayes)MCMC模拟给出Cyt b基因的相对速率区间估计为1.000±0.131,而D-loop基因的相对速率为0.859±0.261,表明太湖新银鱼D-loop基因的进化速率低于Cyt b基因,同时,后验概率分布的变异方差也比较大。说明Cyt b基因比D-loop基因具有相对较高的进化速率,也相对更接近分子钟假设。因此,可以认为Cyt b基因比D-loop基因更适于太湖新银鱼种内及近缘种间相关分子生态及系统地理格局的研究。     

Sequence variation in the Tbx4 gene in marine mammals

The amino-acid sequences of the T-domain region of the Tbx4 gene, which is required for hindlimb development, are 100% identical in humans and mice. Cetaceans have lost most of their hindlimb structure, although hindlimb buds are present in very early cetacean embryos. To examine whether the Tbx4 gene has the same function in cetaceans as in other mammals, we analyzed Tbx4 sequences from cetaceans, dugong, artiodactyls and marine carnivores. A total of 39 primers were designed using human and dog Tbx4 nucleotide sequences. Exons 3, 4, 5, 6, 7, and 8 of the Tbx4 genes from cetaceans, artiodactyls, and marine carnivores were sequenced. Non-synonymous substitution sites were detected in the T-domain regions from some cetacean species, but were not detected in those from artiodactyls, the dugong, or the carnivores. The C-terminal regions contained a number of non-synonymous substitutions. Although some indels were present, they were in groups of three nucleotides and therefore did not cause frame shifts. The dN/dS values for the T-domain and C-terminal regions of the cetacean and artiodactylous Tbx4 genes were much lower than 1, indicating that the Tbx4 gene maintains it function in cetaceans, although full expression leading to hindlimb development is suppressed.     

Interfamilial characterization of a region of the ZFX and ZFY genes facilitates sex determination in cetaceans and other mammals

Sequence polymorphism of homologues ZFX and ZFY, in a 604-base pair exon region, was examined in 10 known males and 10 known females across seven cetacean families and used to design a simple, highly sensitive and widely applicable fluorescent 5' exonuclease assay for gender determination in cetaceans. Multiplex amplification, cloning, and sequencing of these previously uncharacterized regions revealed (i) eight fixed differences between ZFX and ZFY, (ii) 29 variable sites between ZFX and ZFY and (iii) very low interspecific nucleotide diversity for both ZFX and ZFY across all families examined. We developed a 5' exonuclease assay that produces a small (105 bp) polymerase chain reaction (PCR) product from both the X and the Y chromosome orthologs, and used double-labelled fluorescent probes to distinguish between the two genes in a real-time PCR assay that is highly reproducible and sensitive. We demonstrated sex specificity for 33 cetacean species in nine families. Given the availability of conserved primers and sequence information for many mammalian species, this approach to designing sexing assays for a wide range of species is both practical and efficient.     

Efficient site-specific cleavage by RNase MRP requires interaction with two evolutionarily conserved mitochondrial RNA sequences.

RNase MRP is a site-specific endonuclease that processes primer mitochondrial RNA from the leading-strand origin of mitochondrial DNA replication. Using deletional analysis and saturation mutagenesis, we have determined the substrate requirements for cleavage by mouse mitochondrial RNase MRP. Two regions of sequence homology among vertebrate mitochondrial RNA primers, conserved sequence blocks II and III, were found to be critical for both efficient and accurate cleavage; a third region of sequence homology, conserved sequence block I, was dispensable. Analysis of insertion and deletion mutations within conserved sequence block II demonstrated that the specificity of RNase MRP accommodates the natural sequence heterogeneity of conserved sequence block II in vivo. Heterologous assays with human RNase MRP and mutated mouse mitochondrial RNA substrates indicated that sequences essential for substrate recognition are conserved between mammalian species.