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 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.     

Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease

According to the current paradigm type IIE restriction endonucleases are homodimeric proteins that simultaneously bind to two recognition sites but cleave DNA at only one site per turnover: the other site acts as an allosteric locus, activating the enzyme to cleave DNA at the first. Structural and biochemical analysis of the archetypal type IIE restriction enzyme EcoRII suggests that it has three possible DNA binding interfaces enabling simultaneous binding of three recognition sites. To test if putative synapsis of three binding sites has any functional significance, we have studied EcoRII cleavage of plasmids containing a single, two and three recognition sites under both single turnover and steady state conditions. EcoRII displays distinct reaction patterns on different substrates: (i) it shows virtually no activity on a single site plasmid; (ii) it yields open-circular DNA form nicked at one strand as an obligatory intermediate acting on a two-site plasmid; (iii) it cleaves concertedly both DNA strands at a single site during a single turnover on a three site plasmid to yield linear DNA. Cognate oligonucleotide added in trans increases the reaction velocity and changes the reaction pattern for the EcoRII cleavage of one and two-site plasmids but has little effect on the three-site plasmid. Taken together the data indicate that EcoRII requires simultaneous binding of three rather than two recognition sites in cis to achieve concerted DNA cleavage at a single site. We show that the orthodox type IIP enzyme PspGI which is an isoschisomer of EcoRII, cleaves different plasmid substrates with equal rates. Data provided here indicate that type IIE restriction enzymes EcoRII and NaeI follow different mechanisms. We propose that other type IIE restriction enzymes may employ the mechanism suggested here for EcoRII.     

A new estimate of sequence divergence of mitochondrial DNA using restriction endonuclease mappings

Summary A new estimate of the sequence divergence of mitochondrial DNA in related species using restriction enzyme maps is constructed. The estimate is derived assuming a simple Posisson-like model for the evolutionary process and is chosen to maximize an expression which is a reasonable approximation to the true likelihood of the restriction map data. Using this estimate, four sets of mitochondrial DNA data are analyzed and discussed.     

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg²⁺ ions, EcoRV is able to cleave the DNA but not in presence of Ca²⁺, although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg²⁺(A)-EcoRV-DNA complex compared to Ca²⁺(S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca²⁺(S)-EcoRV-DNA compared to Mg²⁺(A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca²⁺(S) bound complex than in Mg²⁺(A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.     

Subunit assembly for DNA cleavage by restriction endonuclease SgrAI

The SgrAI endonuclease usually cleaves DNA with two recognition sites more rapidly than DNA with one site, often converting the former directly to the products cut at both sites. In this respect, SgrAI acts like the tetrameric restriction enzymes that bind two copies of their target sites before cleaving both sites concertedly. However, by analytical ultracentrifugation, SgrAI is a dimer in solution though it aggregates to high molecular mass species when bound to its specific DNA sequence. Its reaction kinetics indicate that it uses different mechanisms to cleave DNA with one and with two SgrAI sites. It cleaves the one-site DNA in the style of a dimeric restriction enzyme acting at an individual site, mediating neither interactions in trans, as seen with the tetrameric enzymes, nor subunit associations, as seen with the monomeric enzymes. In contrast, its optimal reaction on DNA with two sites involves an association of protein subunits: two dimers bound to sites in cis may associate to form a tetramer that has enhanced activity, which then cleaves both sites concurrently. The mode of action of SgrAI differs from all restriction enzymes characterised previously, so this study extends the range of mechanisms known for restriction endonucleases.     

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.     

Reduction of nuclear DNA contamination in the analysis of chloroplast DNA with restriction endonucleases

If chloroplasts purified on sucrose step gradients are treated for 10 min at 4°C with 2 M NaCl, followed by a 1000-g centrifugation, nuclear DNA contamination is reduced 1.5 to 3 fold as estimated by densitometry.     

A variant in the restriction endonuclease cleavage pattern of mitochondrial DNA in the domestic fowl, Gallus gallus domesticus

Summary. The cleavage patterns of mitochondrial DNAs (mtDNAs) were investigated from 15 lines of domestic fowls, Gallus gallus domesticus . using 11 restriction endonucleases. The cleavage patterns with 10 restriction endonucleases were identical in all the lines. A variant was found in a line of White Leghorn in the pattern with Mspl digestions. Cleavage patterns of the red jungle fowl, Gallus gallus gallus , were identical to the common patterns shown by the 14 lines of domestic fowls.     

Polymorphism of Mitochondrial DNAs of Yunnan Domestic Water Buffaloes, Bubalus bubalis, in China, Based on Restriction Endonuclease Cleavage Patterns

Restriction endonuclease cleavage patterns of mitochondrial DNA(mtDNA) of three local types of Yunnan native water buffalo were analyzed using 18 enzymes which recognize six nucleotides. Among the 12 animals analyzed, 3 of 18 enzymes, BamHI, EcoRI, and ScaI, revealed polymorphisms. Three mtDNA types were identified. The results indicate that a relatively low level of mtDNA variation exists in Yunnan domestic water buffaloes. The origin of Chinese buffalo derived from Yunnan province of China is discussed.     

Phylogenetic relationships of Northeast Asian cattle to other cattle populations determined using mitochondrial DNA D-loop sequence polymorphism

Phylogenetic relationships of Northeast Asian cattle to various other cattle breeds including Bos taurus, Bos indicus, and Bison bison were assessed using mtDNA D-loop sequences. A neighbor-joining tree was constructed using sequences determined for 4 Cheju Black, 4 Cheju Yellow, 4 Korean Yellow cattle (Bos taurus), and 2 American Brahman cattle (Bos indicus), and also published sequences for 31 Japanese Black cattle, 45 European breed cattle, 6 African zebus, 2 African taurines, and 6 Indian zebus. Five American bisons (Bison bison) were used as an outgroup. The neighbor-joining tree showed that American bisons and Indian zebus are clearly separate from other cattle breeds, respectively, and African cattle clustered together, although with a low bootstrap probability (<50%). Results indicate that cattle in Northeast Asia, Europe, and Africa are closely related to each other–suggesting their recent divergence, but are separate from Indian zebus.     

Nickase and a protein encoded by an open reading frame downstream from the nickase BspD6I gene form a restriction endonuclease complex

We are the first to have isolated a protein (186 amino acid residues) encoded by the open reading frame adjacent to the end of the BspD6I nickase (N.BspD6I) gene. Cleavage of both DNA strands near the sequence recognized by nickase (5 -GAGTC/5 -GACTC) occurs when this protein is added to the reaction mixture containing N.BspD6I. The protein encoded by the open reading frame and the nickase are suggested to be subunits of heterodimeric restriction endonuclease R.BspD6I.     

DNA recognition by the EcoRV restriction endonuclease probed using base analogues

The EcoRV restriction endonuclease recognises palindromic GATATC sequences and cuts between the central T and dA bases in a reaction that has an absolute requirement for a divalent metal ion, physiologically Mg(2+). Use has been made of base analogues, which delete hydrogen bonds between the protein and DNA (or hydrophobic interactions in the case of the 5-CH(3) group of thymine), to evaluate the roles of the outer two base-pairs (GATATC) in DNA recognition. Selectivity arises at both the binding steps leading to the formation of the enzyme-DNA-metal ion ternary complex (assayed by measuring the dissociation constant in the presence of the non-reactive metal Ca(2+)) and the catalytic step (evaluated using single-turnover hydrolysis in the presence of Mg(2+)), with each protein-DNA contact contributing to recognition. With the A:T base-pair, binding was reduced by the amount expected for the simple loss of a single contact; much more severe effects were observed with the G:C base-pair, suggesting additional conformational perturbation. Most of the modified bases lowered the rate of hydrolysis; furthermore, the presence of an analogue in one strand of the duplex diminished cutting at the second, unmodified strand, indicative of communication between DNA binding and the active site. The essential metal ion Mg(2+) plays a key role in mediating interactions between the DNA binding site and active centre and in many instances rescue of hydrolysis was seen with Mn(2+). It is suggested that contacts between the GATATC site are required for tight binding and for the correct assembly of metal ions and bound water at the catalytic site, functions important in providing acid/base catalysis and transition state stabilisation.     

Polymorphism of mitochondrial DNA (mtDNA) in cattle and buffaloes

Mitochondrial DNA (mtDNA) from two breeds of cattle, viz., [Hariana (Bos indicus), Holstein (Bos taurus)] and Indian water buffalo (Bubalis bubalus), was analyzed using 13 restriction endonucleases which recognized an average of about 40 six-base sites. Polymorphism among cattle was detected with six of these enzymes. The two Holstein differed at six sites, whereas the Hariana breed (Bos indicus) did not show any site polymorphism. Surprisingly, the Hariana type differed by only one site from one of the Holstein types. The total size of buffalo mtDNA was estimated to be 16.4 kb. Polymorphism within the Murrah buffalo breed was observed with respect to aBglI site. Scarcely any of the restriction fragments of buffalo mtDNA matched those of cattle mtDNA.     

S-adenosyl methionine prevents promiscuous DNA cleavage by the EcoP1I type III restriction enzyme

DNA cleavage by the type III restriction endonuclease EcoP1I was analysed on circular and catenane DNA in a variety of buffers with different salts. In the presence of the cofactor S-adenosyl methionine (AdoMet), and irrespective of buffer, only substrates with two EcoP1I sites in inverted repeat were susceptible to cleavage. Maximal activity was achieved at a Res2Mod2 to site ratio of approximately 1:1 yet resulted in cleavage at only one of the two sites. In contrast, the outcome of reactions in the absence of AdoMet was dependent upon the identity of the monovalent buffer components, in particular the identity of the cation. With Na+, cleavage was observed only on substrates with two sites in inverted repeat at elevated enzyme to site ratios (>15:1). However, with K+ every substrate tested was susceptible to cleavage above an enzyme to site ratio of approximately 3:1, including a DNA molecule with two directly repeated sites and even a DNA molecule with a single site. Above an enzyme to site ratio of 2:1, substrates with two sites in inverted repeat were cleaved at both cognate sites. The rates of cleavage suggested two separate events: a fast primary reaction for the first cleavage of a pair of inverted sites; and an order-of-magnitude slower secondary reaction for the second cleavage of the pair or for the first cleavage of all other site combinations. EcoP1I enzymes mutated in either the ATPase or nuclease motifs did not produce the secondary cleavage reactions. Thus, AdoMet appears to play a dual role in type III endonuclease reactions: Firstly, as an allosteric activator, promoting DNA association; and secondly, as a "specificity factor", ensuring that cleavage occurs only when two endonucleases bind two recognition sites in a designated orientation. However, given the right conditions, AdoMet is not strictly required for DNA cleavage by a type III enzyme.     

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.     

Mitochondrial genetic variation in European, African and Indian cattle populations

Mitochondrial DNA from representative animals of 13 different cattle breeds was assayed for restriction fragment length polymorphisms (RFLP) to determine phylogenetic relationships and levels of variation among breeds; 16 different mitotypes were found, described by 20 polymorphisms. Within these 16 mitotypes two major lineages were apparent: an Afro-European and an Asian type. These were found to differ at over 2.3% of sites surveyed. None of the mitotypes found in the Asian lineage was detectable in the Afro-European lineage and vice versa. Within each of the major mitotypes there were no further significant differences within or among breeds. Using rates of mitochondrial evolution estimated from other species, the two lineages were estimated to have diverged between 575000 and 1150000 years ago; well outside the 10000 years bp timeframe postulated by a single domestication hypothesis. The results presented are concordant with those generated in other studies and provide strong evidence for an independent domestication of Asian Bos indicus. Furthermore, the grouping of all African indicine populations within the clade containing all Bos taurus lineages points to the hybrid origins of the humped cattle of that continent.     

Monomeric restriction endonuclease BcnI in the apo form and in an asymmetric complex with target DNA

Restriction endonuclease BcnI cleaves duplex DNA containing the sequence CC/SGG (S stands for C or G, / designates a cleavage position) to generate staggered products with single nucleotide 5'-overhangs. Here, we show that BcnI functions as a monomer that interacts with its target DNA in 1:1 molar ratio and report crystal structures of BcnI in the absence and in the presence of DNA. In the complex with DNA, BcnI makes specific contacts with all five bases of the target sequence and not just with a half-site, as the protomer of a typical dimeric restriction endonuclease. Our data are inconsistent with BcnI dimerization and suggest that the enzyme introduces double-strand breaks by sequentially nicking individual DNA strands, although this remains to be confirmed by kinetic experiments. BcnI is remotely similar to the DNA repair protein MutH and shares approximately 20% sequence identity with the restriction endonuclease MvaI, which is specific for the related sequence CC/WGG (W stands for A or T). As expected, BcnI is structurally similar to MvaI and recognizes conserved bases in the target sequence similarly but not identically. BcnI has a unique machinery for the recognition of the central base-pair.     

Mitochondrial DNA polymorphism and phylogenetic relationships inHevea brasiliensis

Using fourteen random mitochondrial DNA probes, we have examined restriction fragment length polymorphism (RFLP) in wild and cultivatedHevea brasiliensis. A total of 395 accessions, including 345 from various prospectings collected in Brazil, Colombia and Peru and 50 cultivated clones, were analyzed. Two other species (H. benthamiana andH. pauciflora) were also included in the study for comparison. The high level of mitochondrial polymorphism allowed us to divide all the accessions analyzed into 212 distinct genotypes. The genetic variability of cultivated clones was limited to four genotypes forming two clusters. In contrast, considerable genetic variation was found in the wild collections. In almost all cases, accessions displaying the same RFLP profile were restricted to the same geographical area (same or neighbor administrative districts). In addition, accessions whose genetic closeness was predicted by RFLP profiles were also clustered according to geographical origin. In a few cases, however, similar RFLP profiles were found for accessions originating from geographically distant districts. This discrepancy can be explained either by seed dispersion (by river) or possibly by similar genetic events occurring independently in different geographical locations. Chloroplast DNA RFLP was also analyzed in 217 accessions, representative of 126 distinct mitochondrial genotypes. Very few differences were found, indicating that the chloroplast genome is more highly conserved than the mitochondrial genome.