Organization of mitochondrial DNA in normal and texas male sterile cytoplasms of maize

Recombinant DNA and hybridization techniques have been used to compare the organization of mitochondrial DNA (mtDNA) from normal (N) and Texas male sterile (T) cytoplasms of maize. Bam H1 restriction fragments of normal mtDNA were cloned and used in molecular hybridizations against Southern blots of Bam H1 digested N and T mtDNA. Fifteen of the 35 fragments were conserved in both N and T as indicated by hybridization to comigrating bands in their restriction patterns. Only three fragments produced autoradiographs whose differences could reasonably be attributed to single changes in the cleavage site of the enzyme while approximately half (17/35) of the clones resulted in more complicated differences between N and T. The autoradiographs produced by these 17 clones indicated multiple cleavage site changes and/or sequence rearrangements of the mtDNA. Patterns of six of these 17 clones indicated partial duplication of the sequence and two showed variation in the intensity of hybridization between N and T, which may be related to the molecular heterogeneity phenomenon found in maize mitochondrial genomes. The large proportion of changes observed between N and T mtDNA indicates that rearrangements may have played an important role in the evolution of the maize mitochondrial genome.     

Structural homogeneity of mitochondrial DNA in the mitochondrial nucleoid of Physarum polycephalum

Mitochondrial DNA (mtDNA) of Physarum polycephalum was isolated gently by CsCl centrifugation. The mtDNA was linear with molecular weights ranging from 25·10⁶ to 45·10⁶ and heterogeneous in size. Nevertheless, thermal transition profiles of the mtDNA suggested that this DNA fraction was more homogeneous than nuclear DNA. Exhaustive digestions of this DNA with restriction endonucleases yielded unique fragments, and then the total of their molecular weights of each digest was around 45·10⁶. This value is equivalent to the maximum molecular weight estimated using electron microscopy and electrophoresis. Moreover, EcoRI digests of the mtDNA fractionated by the sucrose gradient showed unequimolar quantities of large fragments and a high background between bands. These results suggest that the mtDNA of Physarum has a homogeneous base sequence, and that the size heterogeneity of the mtDNA is attributable to degradation of the DNA under isolation procedures. The mtDNA was cleaved by EcoRI and XhoI to yield 16 and 7 fragments, respectively. A physical map of these fragments was constructed using the routine mapping procedures. The physical map showed that the mitochondrial genome of Physarum was linear with a molecular weight of 45·10⁶. We concluded therefore that the mitochondrial nucleoid is a structure in which the homogeneous mtDNA is highly amplified.     

Molecular analysis of cytoplasmic male sterility in chives (Allium schoenoprasum L.)

The mitochondria of chive plants with normal N or male-sterile S cytoplasms have been examined by restriction fragment analysis and Southern hybridizations of mitochondrial DNA (mtDNA) and in organello protein biosynthesis. Restriction fragment patterns of the mtDNA differed extensively between N-and S-cytoplasms. The percentage of fragments with different mobility varied between 44–48% depending on the restriction enzyme used. In contrast to mtDNA, the restriction fragment patterns of the chloropolast DNA from N- and S-cytoplasms were identical. The organization of the analyzed mitochondrial genes coxII, coxIII, nad1 and nad3 was different in N- and S-cytoplasms. Comparison of mitochondrial proteins analyzed by in organello translation revealed an 18-kDa protein present only in S-cytoplasm. The restorer gene X suppressed the synthesis of that protein in S-cytoplasm. Thus, the 18-kDa protein seems to be associated with the cytoplasmic male-sterile phenotype.     

Isolation and characteristics of DNA fragments bound to mitochondrial membrane proteins

Fragments of rat liver mitochondrial DNA were isolated. In vivo these fragments were able to form the complexes with the proteins of inner mitochondrial membrane. The fragments represent unique DNA regions with the secondary structure, their A-T content being equal to 82%. With the aid of phosphomonoesterase, polynucleotidkinase and gamma-(32P)-ATP mtDNA fragments were labeled and analyzed for oligopyrimidine composition. It was shown that they were enriched in di- and tri-oligo-pyrimidine blocks. The fragments are shown to form in vitro a complex with the membrane proteins. A single protein m. wt. 40,000) was reisolated from the complex.     

Construction and analysis of recombinant lambda phages containing mitochondrial DNA fragments.

Rat mtDNA has a molecular length of about 16 kilobase (kb) pairs and is cleaved into seven fragments by restriction endonuclease EcoRI. These fragments were cloned in Escherichia coli K-12 host using lambda gtWES.lambda B' (lambda gtWES.lambda B, for short, in this paper) as a vector. Recombinant DNAs containing one or a few fragments of the mtDNA were transfected to CaCl2-treated E. coli, and the plaques containing specific recombinant phages were selected. DNA amplified in the recombinanat phage lambda gt.mt was shown to contain the same restriction endonuclease cleavage sites as those found in the mtDNA. Present results permitted the DNA sequencing of any portion of the mitochondrial genome.     

Repair of lesions induced by bruneomycin in DNA of isolated mitochondria from the mature eggs of the teleost fish Misgurnus fossilis

Addition of a radiomimetic antibiotic bruneomycin (Streptonigrin) to isolated mitochondria from mature quiescent oocytes of the teleost fish loach Misgurnus fossilis leads to the induction of unscheduled synthesis of mitochondrial DNA. Most of the newly synthesized DNA has the sedimentation properties of open circles and up to 15% of the label is present in the fraction of the covalently closed-circular molecules. The size of the newly synthesized DNA stretches determined from the bouyant shift of DNA labeled with 5-bromouracil and [3H]dAMP and sonicated to fragments of different molecular weight, was found to be equal to about 1000 nucleotides for the labeled covalently closed circles and to about 2000 nucleotides for the labeled open-circular DNA. Experiments with the centrifugation of non-sheared and sonicated 5-bromouracil and [3H]dAMP-labeled mitochondrial DNA (mtDNA) in alkaline CsCl density gradients provided evidence of a covalent linkage between newly-synthesized stretches and the parental DNA strands. It is concluded from these data that the unscheduled mtDNA synthesis induced by bruneomycin does at least in part represent mtDNA repair synthesis.     

Computer prediction of peptide maps: assignment of polypeptides to human and mouse mitochondrial DNA genes by analysis of two-dimensional-proteolytic digest gels.

We have prepared a computer program that predicts complete and partial peptide maps from amino acid sequences. The program fragments amino acid sequences at designated cleavage sites and calculates the molecular weight and relative labeling of each peptide. These data are graphed as log molecular weight of the original protein (X-axis) vs. log molecular weight of the component peptides (Y-axis). The program is interactive, permitting adjustment of a number of graphic parameters and alteration of the position of proteins in the first dimension to accommodate aberrations in protein mobility. The program has been used to predict the V8 protease peptide maps of the 13 open reading frames (ORFs) identified in the human and the mouse mitochondrial DNA (mtDNA) sequences. The results were compared to the V8 protease peptide maps obtained for mouse and human mitochondrially synthesized proteins by two-dimensional proteolytic digest gels. A high correlation was observed between the predicted and observed peptide maps. These results suggest the assignment of several proteins to mtDNA genes.     

DNA Replication by a membrane-DNA complex from rat liver mitochondria

The results presented here indicate that mitochondrial DNA (mtDNA) synthesis occurs on the inner mitochondrial membrane and that a membrane-DNA complex, enriched in newly synthesized DNA, can be isolated. The complex is able to synthesize DNA in vitro. Enrichment studies demonstrated that mtDNA synthesis occurs on an intact membrane-DNA complex in vitro and that pulse-labeled mtDNA could be chased from the membrane-DNA complex to the top fraction of the discontinuous sucrose gradient. The membrane-DNA complex was also shown to carry out replicative synthesis of mtDNA in vitro. Replication was shown to be asynchronous with heavy-strand synthesis preceding light-strand synthesis. The progression of mtDNA replication by the membrane-DNA complex was shown to be from small fragments (<13 S) to larger fragments (14–24 S) liberated from closed circular molecules, to a heat-stable 27 S molecule, and finally to a 38 S heat-stable molecule. The time estimated to progress from small fragments to the 38 S molecule is 120 min.     

Analysis of Schizosaccharomyces pombe mitochondrial DNA replication by two dimensional gel electrophoresis

The entire mitochondrial genome of Schizosaccharomyces pombe ura4-294h ^-was analyzed by the 2D pulsed field gel electrophoresis technique developed by Brewer and Fangman. The genome consists of multimers with an average size of 100 kb and analysis of the overlapping restriction fragments of the complete mitochondrial DNA (mtDNA) genome resulted in simply Y 2D gel patterns. Large single-stranded DNA molecules or double-stranded DNA molecules containing large or numerous single-stranded regions were found in the S. pombe mtDNA preparation. The replication of mtDNA monomers was found to occur in either direction. On the basis of these results, a replication mechanism for S. pombe mtDNA that is most consistent with a rolling circle model is suggested.     

PCR-based cloning of the complete mouse mitochondrial genome and stable engineering in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

We have devised a method for cloning an entire mammalian mitochondrial genome (mtDNA) in Escherichia coli using PCR-based amplification and sequential ligation. Here we test this approach by cloning the complete mouse mtDNA. The mtDNA was divided into four to five fragments based on unique restriction enzyme sites and amplified by high-fidelity long-range DNA polymerase. The synthesized fragments were cloned individually to test their toxicity in the E. coli host and then combined sequentially into a vector containing the E. coli R6K origin of DNA replication. The synthetic complete mouse mtDNA clones were replicated stably and faithfully in E. coli when maintained at moderately low copy numbers per cell. The sequence integrity of the synthetic mouse mtDNA clones was confirmed by nucleotide sequencing; no mutations or rearrangements in the genome were found. This approach can facilitate the cloning of entire mammalian mitochondrial genomes in E. coli and assist in the introduction of desired modifications into the mitochondrial genome.     

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high‐energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA. However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La‐related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI‐Larp complex promotes the synthesis of a subset of nuclear‐encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI‐Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron‐transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI‐Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis.     

The use of restriction endonucleases to compare mitochondrial dna sequences in Mus musculus: A detailed restriction map of mitochondrial DNA from mouse L cells

The complete mitochondrial genome of a chloramphenicol-resistant, oligomycin-resistant mouse L cell has been cloned in E. coli. Using fragments of this DNA, purified by preparative agarose gel electrophoresis, we have mapped 139 restriction sites cleaved by 15 endonucleases. We have then compared the positions of these sites with those found in mtDNA purified from several other L-cell lines, from the tissue of several strains of laboratory mice, and from a plasmid containing mtDNA of a single, wild Mus musculus sample. The results indicate that all of the L-cell mtDNAs are identical except at a single EcoRI site, and that the L-cell sequence is identical to that found in mtDNA from normal tissue. They also suggest that mtDNA sequences found in North American Mus musculus populations may be much more homogeneous than expected from analysis of other rodents.     

滑鼠蛇肝线粒体DNA的限制酶图谱

用六种限制性内切酶BamHⅠ、EcoRⅠ、PstⅠ、BglⅠ、BglⅡ和SalⅠ对滑鼠蛇肝脏线粒体DNA(mtDNA)进行酶解。发现BglⅡ、PstⅠ、BamHⅠ、BglⅠ和EcoRⅠ在滑鼠蛇肝mtDNA上分别有1、2、3、3和4个切点。SalⅠ不能切割滑鼠蛇肝mtDNA。根据滑鼠蛇肝mtDNA的单酶、双酶完全酶解及部分酶解片段的数目和分子量,建立了滑鼠蛇肝mtDNA的限制酶图谱。     

Genetic differentiation and hybridization in two naturally occurring sympatric trout Salmo spp. forms from a small karstic lake

In this study, multiple molecular markers [genotyping of 12 nuclear microsatellite loci and the protein‐coding gene ldh‐c1* plus sequencing of the mitochondrial DNA (mtDNA) control region] were employed to investigate the genetic structure of the two trout forms, Salmo cettii and Salmo fibreni, inhabiting Lake Posta Fibreno, central Italy. The two forms were found to share a unique mtDNA haplotype, belonging to a widespread Mediterranean haplogroup (AD). Bayesian clustering analyses showed that these two forms correspond to well‐defined autochthonous gene pools. Genetic introgression between the two gene pools, however, was observed, whose frequency appears to correlate with the environmental features of the spawning sites. The interplay of selection for the spawning sites, philopatry and natural selection can be argued to maintain genetic differentiation despite the lack of complete reproductive isolation.     

Comparison of the Restriction Endonuclease Digestion Patterns of Mitochondrial DNA from Normal and Male Sterile Cytoplasms of ZEA MAYS L

High resolution gel electrophoresis has allowed the assignment of fragment number and molecular weight to EcoRI, SalI and PstI restriction fragments of mitochondrial DNA from B37 normal (N) and B37 T, C and S male sterile cytoplasmic types of maize. A minimum complexity of 450-475 kb has been established. Hybridization of cloned EcoRI fragments to restriction digests of total mitochondrial DNA suggests that at least 80% of the genome is composed of unique sequences. Restriction fragments of identical size in N, T, C and S contain similar sequence information as evidenced by their hybridization behavior.—The total SalI digest and the larger PstI fragments representing 80% of the total complexity were used to calculate the fraction of shared fragments of each pairwise combination of cytoplasmic types. The C type mtDNA is most closely allied with the other mtDNAs and shares 67% of fragments with S, 65% with N, and 60% with T. The S type mtDNA is quite divergent from N (53% shared fragments) and T (56% shared fragments). N and T share 59% of the fragments. These results are discussed in terms of the origin of mtDNA diversity in maize.     

Cloning and characterization of a linear 2.3 kb mitochondrial plasmid of maize

Summary A linear 2.3 kb DNA molecule found in maize mitochondria was cloned into pUC8. A natural deletion of this plasmid, found in cmsT and some N (fertile) types of maize plants, was mapped to one end of the plasmid. A minor sequence homology to S-2, another linear mitochondrial plasmid, was detected, as well as more significant sequence homology with chloroplast and maize nuclear DNA. Hybridization to teosinte mitochondrial DNA (mtDNA) revealed the presence of part of the maize plasmid in the high molecular weight mtDNA of the maize relatives. RNA dot hybridization indicates that the plasmid is transcribed in mitochondria. The termini of the 2.3 kb linear plasmid contain inverted repeated sequences; of the first 17 nucleotides of the termini, 16 are identical to the terminal inverted repeats of the linear S plasmids found in the mitochondria of cmsS maize plants.     

DNA synthesis in isolated mitochondrial nucleoids from plasmodia ofPhysarum polycephalum

Summary A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mtnucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia ofPhysarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl₂ for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.Abbreviations BrdU 5-bromodeoxyuridine - BrdUTP 5-bromo-deoxyuridine triphosphate - DAPI 4,6-diamidino-2-phenylindole - dNTP deoxyribonucleoside triphosphate - ddCTP dideoxycytidine triphosphate - NEM N-ethylmaleimide - MRC mitochondrial replicon cluster; mt mitochondrial - NP-40 Nonidet P-40 - PBS phosphatebuffered saline - PMSF phenylmethanesulfonyl fluoride - rNTP ribonucleoside triphosphate - VIMPCS video-intensified microscope photon-counting system     

Some physicochemical properties of rice mitochondrial DNA

Summary Certain physicochemical properties of rice mitochondrial DNA (mtDNA) were determined. Certain low-molecular-weight mtDNA bands were found in addition to the major mtDNA band. Rice mtDNA appeared in the electron microscope as a collection of linear molecules with heterogeneous length in the range of 1–156 kb. The major distribution area was 60–105 kb. A small fraction (less than 5%) of rice mtDNA was found in the form of a circular molecule. Some molecules had the appearance of being supercoiled. Replication fork structures were found in both circular and linear mtDNA molecules. In one rice species, Jin Nante, 15 different circular molecules were found. Rice mtDNA was digested with different restriction enzymes. The total molecular weight of rice mtDNA was calculated to be about 300 kb according to the data of restriction enzyme digestion and electron microscopy.     

A Novel Mitochondrial Heteroplasmic C13806A Point Mutation Associated with Iranian Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by decreased expression of the protein Frataxin. Frataxin deficiency leads to excessive free radical production and dysfunction of chain complexes. Mitochondrial DNA (mtDNA) could be considered a candidate modifier factor for FRDA disease, since mitochondrial oxidative stress is thought to be involved in the pathogenesis of this disease. It prompted us to focus on the mtDNA and monitor the nucleotide changes of genome which are probably the cause of respiratory chain defects and reduced ATP generation. We searched about 46% of the entire mitochondrial genome by temporal temperature gradient gel electrophoresis (TTGE) and DNA fragments showing abnormal banding patterns were sequenced for the identification of exact mutations. In 18 patients, for the first time, we detected 26 mtDNA mutations; of which 5 (19.2%) was novel and 21 (80.8%) have been reported in other diseases. Heteroplasmic C13806A polymorphisms were associated with Iranian FRDA patients (55.5%). Our results showed that NADH dehydrogenase (ND) genes mutations in FRDA samples were higher than normal controls (P < 0.001) and we found statistically significant inverse correlation (r = −0.8) between number of mutation in ND genes and age of onset in FRDA patients. It is possible that mutations in ND genes could constitute a predisposing factor which in combination with environmental risk factors affects age of onset and disease progression.