The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations

Summary Restriction endonucleases and agarose gel electrophoresis have been used to demonstrate extensive nucleotide sequence diversity in mitochondrial DNA (mtDNA) within and between conspecific populations of rodents and other mammals. Cleavage of mtDNA samples with a relatively small number of endonucleases provides information concerning the phylogenetic relatedness of individual organisms which cannot now be readily obtained by any other type of molecular analysis. This information is qualitatively different from that available from the study of nuclear genes or gene products because the mitochondrial genome is inherited intact from the female parent and is not altered by recombination or meiotic segregation.The requirements for large tissue samples and laborious DNA purification procedures have imposed severe limitations on the kinds of population surveys in which this technique could be utilized. Here, we show that these difficulties can be overcome by using DNA-DNA hybridization to detect minute amounts of mtDNA in crude tissue fractions which can be more easily and rapidly prepared from very small amounts of tissue without the use of expensive and immobile laboratory equipment. The techniques are described in detail in an effort to make restriction analysis of mtDNA available to biologists who may be unfamiliar with current DNA technology.     

A restriction endonuclease cleavage map of mouse mitochondrial DNA.

A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized PstI and four sites recognized by BamI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by KpnI or SalI. The migration of linear DNA with a molecular weight greater than 1 X 10(6) was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm.     

Structural organization of interspersed repetitive elements present in the DNA of Mus musculus

Two-dimensional displays of the restriction fragments from the DNA of Mus musculus revealed a complex species-specific pattern produced from nonsatellite repetitive sequences. The patterns have been used as a guide in the direct purification of a group of broadly interspersed repeated DNA sequences (characterized by a 1350-bp Eco-Bam fragment) that have been studied by molecular cloning, restriction mapping and genomic Southern blotting. These studies show that the cloned representatives originate from an abundant group of sequences that share homology with about 2% of the mouse genome. The sequences do not appear to share homology with mouse-interspersed-family-1 (MIF-1) nor with the major AT-rich satellite sequences of mouse. They appear to be part of a group of larger repetitive elements that is both broadly interspersed and heavily methylated in normal mouse tissue.     

The myoglobin of rodents Proechimys guairae (casiragua) and Mus musculus (house mouse)

The amino acid sequences of the myoglobins of two rodents, the casiragua and the house mouse, have been determined. The myoglobin of casiragua differs from that of viscacha (another hystricomorph) at 6 positions. Mouse myoglobin differs from that of mole-rat (another myomorph) at 17 positions, whereas casiragua and mouse differ at 22 positions. Mouse myoglobin possesses several features unique among all known myoglobins (Gly 31, Cys 66, Thr 74 and Glu 113) and one substitution unique among known mammalian myoglobins (Glu 53).

Amino acid sequence Myoglobin Rodent Casiragua Mouse Phylogeny     

A restriction endonuclease cleavage map of mitochondrial DNA from transformed hamster cells.

Mitochondrial DNA from cultured C13/B4 hamster cells was cleaved by the restriction endonucleases Hpa II, Hind III, Eco RI and Bam HI into 7, 5, 3 and 2 unique fragments, respectively. The summed molecular weights of fragments obtained from electrophoretic mobilities in agarose-ethidium bromide gels (with Hpa I-cleaved T7 DNA as standard) and electron microscopic analysis of fragment classes isolated from gels (with SV40 DNA as standard) were in good agreement with the size of 10.37 +/- 0.22 x 10(6) daltons (15,700 +/- 330 nucleotide pairs) determined for the intact circular mitochondrial genome. Cyclization of all Hind III, Eco RI and Bam HI fragments was observed. A cleavage map containing the 17 restriction sites (+/- 1% s.d.) was constructed by electrophoretic analysis of 32P-labeled single- and double-enzyme digestion products and reciprocal redigestion of isolated fragments. The 7 Hpa II sites were located in one half of the genome. The total distribution of the 17 cleavages around the genome was relatively uniform. The position of the D-loop was determined from its location and expansion on 3 overlapping restriction fragments.     

The refractory responses of the White Swiss strain of Mus musculus to infection with Taenia taeniaeformis

Penetration of intestinal mucosa by oncospheres of Taenia taeniaeformis occurred among three age groups of White Swiss Mus musculus. Highest frequency of penetration was demonstrated among the 10–30 day-old group with the other two older groups, 40–60 and 70–100 days, each having succeedingly less penetration. The invasion process was more cytolytic than ground substance lysis; no difference in this process, or the reaction of the villi to it, was detected among the various age groups. Liver reaction, displayed by the same three age groups against larvae of T. taeniaeformis, was age dependent. Disorganization, loss of larval tissue integrity, and formation of a non-cellular capsule were demonstrated within 48 h in the 10–30 day group, while a rapid cellular response of inflammation and walling-off of larvae was seen in the 40–60 day group. No larvae were observed in the 70–100 day group.     

鼠源Filia N端蛋白在GSH条件中的一种全新三维结构

母性效应因子复合体SCMC(subcorticalmaternalcomplex)中的Filia蛋白,其N端具有Ⅰ型KH结构域的特征,通过结合RNA参与卵子发生及胚胎发育过程中的RNA转录调控.此外,Filia蛋白对染色体整倍体化的维持起重要作用.缺乏Filia的雌鼠体内早期胚胎发育进程延迟,致使雌鼠生殖力降低,但不会不育.在卵母细胞成熟过程中,还原型谷胱甘肽(glutathione,GSH)浓度发生变化以维持胞内的氧化还原环境.本文成功解析了在含有GSH的条件中生长的Filia(N1-124)蛋白晶体结构.与在不含GSH的条件中生长的Filia(N1-124)蛋白晶体相比,在含有GSH的条件中,Filia(N1-124)蛋白晶体的一个不对称单位中含有5对二聚体,Filia(N1-124)单体分子在α3螺旋区与邻近分子发生了结构域交换.Filia(N1-124)晶体内凭借链间的离子键、氢键和疏水相互作用,形成了独特的十聚体组装方式.本文为进一步研究在不同生理条件下Filia的结构与功能的关系提供了结构依据.     

The effects of testosterone, estradiol, and dihydrotestosterone on male mouse (Mus musculus) ultrasonic vocalizations

Two experiments examined the effects of the free forms of testosterone (T), dihydrotestosterone (DHT), and estradiol (E₂) upon male mouse (Mus musculus) courtship vocalizations and seminal vesicle weight. In the first experiment, administration of T to castrated males was associated with a large number of vocalizations and large seminal vesicle weights, DHT was associated with large seminal vesicle weights but very few vocalizations, whereas E₂ was associated with low measures of both vocalization and seminal vesicle weight. In the second experiment, T and DHT had effects highly similar to those of the previous experiment; however, in contrast to the previous experiment, both low and high dosages of E₂ were associated with large numbers of ultrasonic vocalizations but small seminal vesicles. A mixture of E₂ and DHT was very similar to T in its effect upon both measures. We suggest from these results that hormonal mechanisms similar to those reported for rat sex behavior may interact with situational variables to determine the expression of male mouse courtship.     

Spontaneous parthenogenesis in Mus musculus: Comparison of protein synthesis in parthenogenetic and normal preimplantation embryos

Summary In preimplantation stages of normal and spontaneously activated parthenogenetic embryos of the LT/Sv mouse strain, protein synthesis was analyzed by using two-dimensional polyacrylamide gel electrophoresis. Fertilization and parthenogenetic activation cause similar changes of polypeptide synthesis when compared with those of unfertilized eggs. The overt developmental delay of early parthenotes, which is probably due to an initial retarded activation in comparison with normal fertilization, is documented molecularly by a similar delay in their protein synthesis pattern. These differences are clearly visible at the two-cell stage but gradually disappear during further cleavage. The basic protein patterns of normal and parthenogenetic embryos are remarkably similar up to the blastocyst stage. However, quantitative differences occur in all preimplantation embryos analyzed and become more distinct at the blastocyst stage. In addition, only minor qualitative changes appear during late preimplantation. These alterations in protein synthesis may reflect at the molecular level early events in abnormal development of parthenotes. Our biochemical results are discussed in context with biological experiments rescuing parthenogenetic LT/ Sv embryos by chimera formation.     

Peculiarities of recognition of CCA/TGG sequences in DNA by restriction endonucleases MvaI and EcoRII.

To elucidate the mechanism of action of restriction endonucleases MvaI and EcoRII a study was made of their interaction with a set of synthetic substrates in which the heterocyclic bases or the sugar-phosphate backbone had been modified; individual nucleotide residues had been removed or replaced with hydrocarbon bridges, and mismatched base pairs had been introduced. The groups of atoms in the heterocyclic bases and the phosphates in the recognition site that produce the most significant influence on the functioning of endonucleases MvaI and EcoRII were discerned. Profound differences were found in the functioning of the MvaI and EcoRII neoschizomers. The catalytic activity of EcoRII is significantly affected by any alteration in the recognition site structure and conformation, with a modification in one strand of the substrate causing the same decrease in the hydrolysis rate of both strands. Endonuclease MvaI is tolerant to a number of structural abnormalities; the latter sometimes affect only hydrolysis of one strand of the recognition site. The enzyme can preferentially cleave one of the substrate strands. Mismatched base pairs retard and sometimes block the hydrolysis. The effect depends on the particular enzyme, mismatch and its location.     

Use of the antibiotics actinomycin D and distamycin A to limit the action of restriction endonucleases and to map DNA

It is shown that distamycin A and actinomycin D protect the recognition sites of certain restriction endonucleases from the attack by these nucleases due to specific interaction of these antibiotics with double-stranded DNA. Distamycin A protects A-T containing sites and actinomycin G-C rich sites. Among Hind II recognition sites which have alternative structure (GTPyPuAC) distamycin A protects only Hpa I similar sites (GTTAAC). It is shown with several restriction endonucleases that antibiotic action depends on the nucleotide sequences in the recognition sites and in their closest environment. Proper concentrations of antibiotic give rise to larger fragments. Use of both distamycin A and actinomycin D allows to obtain a set of overlapping fragments. The data obtained with various DNAs and restriction endonucleases allow to conclude that these antibiotics may be useful for DNA mapping and for preparation of large functional fragments of DNA.     

Analysis of mitochondrial DNA species in interspecific hybrid somatic cells using restriction endonucleases. Identification of recombinant mtDNA molecules

Interspecific hybrid cells were isolated by fusion between thymidine kinase-deficient (TK⁻) mouse B82 cells and hypoxanthine-guanine-phosphoribosyl-transferase-deficient (HGPRT⁻) rat L6TG cells, and cultivating them in selective medium with hypoxanthine-aminopterin-thymidine (HAT). Karyo-type analysis revealed that they contained both mouse and rat chromosomes. Mitochondrial DNA (mtDNA) species of the hybrid cells were identified by digesting them with three kinds of restriction endonucleases, Hae II, EcoR I and Hpa II. Their restriction endonuclease cleavage patterns indicated that a portion of the mtDNAs was of mouse parent cell origin, while the remainings were recombinant molecules, i.e., part of the rat mtDNA sequence could be detected, but not whole rat mtDNA. The molecular weights of hybrid cell mtDNAs were calculated to be almost the same as that of the parent cells (˜10⁷ D).     

4-Quinolones cause a selective loss of mitochondrial DNA from mouse L1210 leukemia cells

The 4-quinolone antibiotics nalidixic acid and ciprofloxacin and potent inhibitors of the bacterial type II topoisomerase DNA gyrase. Treatment of mouse L1210 leukemia cells with these drugs resulted in a delayed inhibition of cell proliferation. Prior to inhibition of cell proliferation, there was a time-dependent decrease in the cellular content of mitochondrial DNA (mtDNA). The decrease in mtDNA was associated with a decrease in the rate of mitochondrial respiration and an increase in the concentration of lactate in the growth medium. Inhibition of cell proliferation by 4-quinolones was reversible upon drug washout. However, there was a 2- to 4-day lag before the growth rate returned to normal levels. This was preceeded by an increase in mtDNA content and mitochondrial respiration. These studies suggest that inhibition of mammalian cell proliferation by 4-quinolone drugs is related to the selective depletion of mtDNA. © 1993 Wiley-Liss, Inc.     

Molecular characterization of Fasciola gigantica from Mauritania based on mitochondrial and nuclear ribosomal DNA sequences

Fasciolosis caused by Fasciola hepatica and Fasciola gigantica (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. From Africa, F. gigantica has been previously characterized from Burkina Faso, Senegal, Kenya, Zambia and Mali, while F. hepatica has been reported from Morocco and Tunisia, and both species have been observed from Ethiopia and Egypt on the basis of morphometric differences, while the use of molecular markers is necessary to distinguish exactly between species. Samples identified morphologically as F. gigantica (n = 60) from sheep and cattle from different geographical localities of Mauritania were genetically characterized by sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes and the mitochondrial Cytochrome c Oxidase I (COI) gene.Comparison of the sequences of the Mauritanian samples with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. gigantica. The nucleotide sequencing of ITS rDNA of F. gigantica showed no nucleotide variation in the ITS-1, 5.8S, and ITS-2 rDNA sequences among all samples examined and those from Burkina Faso, Kenya, Egypt and Iran. The phylogenetic trees based on the ITS-1 and ITS-2 sequences showed a close relationship of the Mauritanian samples with isolates of F. gigantica from different localities of Africa and Asia. The COI genotypes of the Mauritanian specimens of F. gigantica had a high level of diversity, and they belonged to the F. gigantica phylogenically distinguishable clade. The present study is the first molecular characterization of F. gigantica in sheep and cattle from Mauritania, allowing a reliable approach for the genetic differentiation of Fasciola spp. and providing basis for further studies on liver flukes in the African countries.     

DNA sequences homologous to mitochondrial genes in nuclei from normal rat tissues and from rat hepatoma cells

Using specific probes we show that sequences homologous to NADH dehydrogenase Subunit 6, and Cytochrome oxidase Subunits I, II, and III mitochondrial genes are present in nuclear DNA from various tissues. These mitochondrial-like sequences are also present in rat hepatoma nuclear DNA but with an abnormal organization and a higher copy number than in normal hepatocytes.     

Genetic characterization of common carp (Cyprinus carpio) populations from Greece using mitochondrial DNA sequences

Wild common carp from two lakes and two rivers in Greece were genetically characterized with sequencing analysis of two mitochondrial DNA segments: cytochrome b (1119 bp) and D-loop (646 bp). A total of 9 variable singleton sites and 7 unique haplotypes were detected. A common haplotype was found in three out of the four populations examined, which seems to be the ancestral one and represents the European origin of common carp from Greece. This haplotype could be also justified by the introductions reported with individuals belonging to the Central European race, into many natural habitats in Greece. Limited genetic variation — in Evros and Aliakmonas populations — could be due to bottleneck effects and small effective population sizes, whereas the different haplotypes found in Lake Volvi could represent different common carp stocks. Values of sequence divergence among Greek haplotypes ranged from 0.0006 to 0.0023. The Neighbour-Joining (NJ) phylogenetic tree constructed based on the combined sequences, reveals that the populations of common carp from Greece belong to the European group of populations — which is highly divergent from the South East-Asia cluster — and to the subspecies Cyprinus carpio carpio.     

The determination of complete human mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations

Studies of single cells have previously shown intracellular clonal expansion of mitochondrial DNA (mtDNA) mutations to levels that can cause a focal cytochrome c oxidase (COX) defect. Whilst techniques are available to study mtDNA rearrangements at the level of the single cell, recent interest has focused on the possible role of somatic mtDNA point mutations in ageing, neurodegenerative disease and cancer. We have therefore developed a method that permits the reliable determination of the entire mtDNA sequence from single cells without amplifying contaminating, nuclear-embedded pseudogenes. Sequencing and PCR–RFLP analyses of individual COX-negative muscle fibres from a patient with a previously described heteroplasmic COX II (T7587C) mutation indicate that mutant loads as low as 30% can be reliably detected by sequencing. This technique will be particularly useful in identifying the mtDNA mutational spectra in age-related COX-negative cells and will increase our understanding of the pathogenetic mechanisms by which they occur.     

The extent of self-MHC restriction of cytotoxic T cells in nude mice varies from mouse to mouse

There are conflicting results as to whether the response of athymic nude mice to TNP-modified self determinants is or is not H-2 restricted. We cultured spleen cells from 29 individual RNC (H-2k) nude mice with TNP-modified self determinants and tested the cultures for their ability to lyse TNP-modified self (RNC-TNP) and TNP-modified allogeneic (BALB/c-TNP) target cells. Each mouse was stimulated by two different protocols: either by the addition of TNP-modified irradiated nu/+ spleen cells or by TNP modification of the nude responder cells without addition of other cells. All mice could lyse RNC-TNP targets and about one-half could also lyse BALB/c-TNP targets, i.e., there was a 50:50 division between restricted and unrestricted responses. The magnitude of the response against RNC-TNP and whether the response was restricted were both independent of the method of stimulation. We conclude that H-2 restriction in these mice is imposed by an as yet unidentified environmental influence that can vary from one nude mouse to the next. The influence appears to act through negative selection because the modified self response is, if anything, higher in mice showing an unrestricted response.