Studies of DNA bound RNA molecules isolated from nucleoids of Escherichia coli.

Methods are developed for studying RNA molecules bound directly to DNA in bacterial nucleoids. It is found that among the 1000-3000 nascent RNA chains that normally are attached to the DNA via their associated RNA polymerase molecules, 74 +/- 14 chains per nucleoid can be bound differently. These chains unlike the other nascent RNAs remained bound to the DNA after the chromosome was deproteinized and sheared. Sensitive assays using radioactive labels detected no RNA polymerase involved in the RNA-DNA linkage. The linkage was stable at low temperatures, but the RNA separated from the DNA at high temperature. The bound RNA molecules were heterodisperse (weight average length 1200 bases). Pulse-chase experiments and studies of the fate of these RNA molecules in rifampicin treated cells demonstrated that they are nascent RNAs, degraded or released from the DNA in vivo with kinetics similar to that of the total nascent RNA. Hybridization analyses showed that the chains are composed at least in part of nascent rRNA and known mRNA molecules. Some, but not more than 5% of the bound chains, contained sequences of about 300 nucleotides in length, bound to the DNA in an RNase resistant form.     

Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells

A new biochemical method for estimating the virtual number of mitochondria (mt) per cell was developed and used together with a plasmid probe to measure mt DNA/mitochondrion and mt DNA/cell. These methods were used in five cell types from four mammalian species. Mt DNA/mitochondrion was essentially constant in all cell types (mean 2.6 +/- 0.30 SE mitochondrial DNA molecules/mt). Mt DNA molecules/cell encompassed an eight-fold range between various cell types (low 220 +/- 6.2; high 1,720 +/- 162 mt DNA molecules/cell). Virtual mt number/cell ranged from 83 +/- 17 to 677 +/- 80 (SE) mt/cell in various cell types. All five mammalian virtual mitochondria contained the same genomic mass. The number of virtual mitochondria per cell and amount of mt DNA per cell appear to be closely regulated within a given cell type but differ widely from cell type to cell type.     

Sedimentation velocity properties of catenated DNA molecules from HeLa cell mitochondria

Catenated molecules of closed circular DNA have been isolated from the mitochondrial DNA of HeLs cells. The sedimentation coefficients of several purified species have been investigated. The catenated dimer, made up of two interlocked duplex circles, sediments at 51 S in its superhelical (closed) form. Treatment with pancreatic DNase to relax the duplex circles converts the 51 S doubly closed dimer to a 42 S singly open species, then to a 36 S doubly open catenated dimer. The triply closed trimer sediments at 63 S and is converted to a 45 S triply open form by DNase. Electron microscopy of the DNA samples before and after DNase treatment shows that under the conditions used DNase does not change the catenated nature of the DNA. The measured sedimentation coefficients, have been compared with those estimated from previously proposed correlations of sedimentation coefficient and molecular weight, and with the sedimentation coefficients for catenated DNA presented by Wang. When all the interlocked circles in a catenane are relaxed, the DNA sediments about 5–10% faster than a relaxed multiple-length circular molecule of the same molecular weight. The sedimentation coefficient, 36 S, of the fully relaxed catenated dimer is 1.4 times that of the relaxed monomer.     

Organization, dynamics and transmission of mitochondrial DNA: focus on vertebrate nucleoids

Eukaryotic cells contain numerous copies of the mitochondrial genome (from 50 to 100 copies in the budding yeast to some thousands in humans) that localize to numerous intramitochondrial nucleoprotein complexes called nucleoids. The transmission of mitochondrial DNA differs significantly from that of nuclear genomes and depends on the number, molecular composition and dynamic properties of nucleoids and on the organization and dynamics of the mitochondrial compartment. While the localization, dynamics and protein composition of mitochondrial DNA nucleoids begin to be described, we are far from knowing all mechanisms and molecules mediating and/or regulating these processes. Here, we review our current knowledge on vertebrate nucleoids and discuss similarities and differences to nucleoids of other eukaryots.     

Isolation and characterization of a DNA primase from human mitochondria

A family of enzymatic activities isolated from human mitochondria is capable of initiating DNA replication on single-stranded templates. The principal enzymes include at least a primase and DNA polymerase gamma and require that rNTPs as well as dNTPs be present in the reaction mixture. Poly(dC) and poly(dT), as well as M13 phage DNA, are excellent templates for the primase activity. A single-stranded DNA containing the cloned origin of mitochondrial light-strand synthesis can be a more efficient template than M13 phage DNA alone. Primase and DNA polymerase activities were separated from each other by sedimentation in a glycerol density gradient. Using M13 phage DNA as template, these mitochondrial enzymes synthesize RNA primers that are 9 to 12 nucleotides in size and are covalently linked to nascent DNA. The formation of primers appears to be the rate-limiting step in the replication process. Replication of M13 DNA is sensitive to N-ethylmaleimide and dideoxynucleoside triphosphates, but insensitive to rifampicin, alpha-amanitin, and aphidicolin.     

The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway

Mitochondrial DNA (mtDNA) is inherited as a protein-DNA complex (the nucleoid). We show that activation of the general amino acid response pathway in rho(+) and rho(-) petite cells results in an increased number of nucleoids without an increase in mtDNA copy number. In rho(-) cells, activation of the general amino acid response pathway results in increased intramolecular recombination between tandemly repeated sequences of rho(-) mtDNA to produce small, circular oligomers that are packaged into individual nucleoids, resulting in an approximately 10-fold increase in nucleoid number. The parsing of mtDNA into nucleoids due to general amino acid control requires Ilv5p, a mitochondrial protein that also functions in branched chain amino acid biosynthesis, and one or more factors required for mtDNA recombination. Two additional proteins known to function in mtDNA recombination, Abf2p and Mgt1p, are also required for parsing mtDNA into a larger number of nucleoids, although expression of these proteins is not under general amino acid control. Increased nucleoid number leads to increased mtDNA transmission, suggesting a mechanism to enhance mtDNA inheritance under amino acid starvation conditions.     

DNA segregation in Escherichia coli cells with 5-bromodeoxyuridine-substituted nucleoids.

The pattern of segregation of DNA in Escherichia coli K-12 was analyzed by labeling replicating DNA with 5-bromodeoxyuridine followed by differential staining of nucleoids. Three types of visible arrangement were found in four-nucleoid groups derived from a native nucleoid after two replication rounds. Type A, segregation of both old strands toward cell poles, appeared with the highest frequency (0.6 to 0.8). Type B, segregation of one old strand toward the cell pole and the other toward the cell center, was twice as frequent as type C, segregation of both old strands toward the cell center. These results confirm previous data showing that DNA segregation in E. coli is nonrandom while presenting a certain degree of randomness. The proportions of the three indicated types of arrangement suggest a new probabilistic model to explain the observed segregation pattern. It is proposed that DNA strands segregate either nonrandomly, with a probability of between 0 and 1, or randomly. In nonrandom segregation, both old strands are always directed toward cell poles. Experimental data reported here or by other authors fit better with the predictions of this model than with those of other previously proposed proposed deterministic or probabilistic models.     

Presence of deletion molecules in human wart virus DNA.

Deletion mutants of human wart virus were found to occur occasionally in the virus population transmitted in vivo, although the majority of virion DNA molecules were homogeneous in length and base sequence. Human wart virus DNA was 1.48 times as long as nondefective simian virus 40 DNA.     

Differential regulation of full-length genome and a single-stranded 7S DNA along the cell cycle in human mitochondria

Mammalian mitochondria contain full-length genome and a single-stranded 7S DNA. Although the copy number of mitochondrial DNA (mtDNA) varies depending on the cell type and also in response to diverse environmental stresses, our understanding of how mtDNA and 7S DNA are maintained and regulated is limited, partly due to lack of reliable in vitro assay systems that reflect the in vivo functionality of mitochondria. Here we report an in vitro assay system to measure synthesis of both mtDNA and 7S DNA under a controllable in vitro condition. With this assay system, we demonstrate that the replication capacity of mitochondria correlates with endogenous copy numbers of mtDNA and 7S DNA. Our study also shows that higher nucleotide concentrations increasingly promote 7S DNA synthesis but not mtDNA synthesis. Consistently, the mitochondrial capacity to synthesize 7S DNA but not mtDNA noticeably varied along the cell cycle, reaching its highest level in S phase. These findings suggest that syntheses of mtDNA and 7S DNA proceed independently and that the mitochondrial capacity to synthesize 7S DNA dynamically changes not only with cell-cycle progression but also in response to varying nucleotide concentrations.     

Mitochondrial DNA nucleoids determine mitochondrial genetics and dysfunction

Mitochondrial DNA plays a crucial role in cellular homeostasis; however, the molecular mechanisms underlying mitochondrial DNA inheritance and propagation are only beginning to be understood. To ensure the distribution and propagation of the mitochondrial genome, mitochondrial DNA is packaged into macromolecular assemblies called nucleoids, composed of one or more copies of mitochondrial DNA and associated proteins. We review current research on the mitochondrial nucleoid, including nucleoid-associated proteins, nucleoid dynamics within the cell, potential mechanisms to ensure proper distribution of nucleoids, and the impact of nucleoid organization on mitochondrial dysfunction. The nucleoid is the molecular organizing unit of mitochondrial genetics, and is the site of interactions that ultimately determine the bioenergetic state of the cell as a whole. Current and future research will provide essential insights into the molecular and cellular interactions that cause bioenergetic crisis, and yield clues for therapeutic rescue of mitochondrial dysfunction.     

A simple autoradiographic method for investigating long range chromosome substructure: size and number of DNA molecules in isolated nucleoids of Escherichia coli.

A method is described for gently dissociating large DNA-protein complexes and for visualizing and quantitating the substructures by autoradiography. Using this technique, it is shown that nucleoids isolated from exponentially growing Escherichia coli (mean generation time = 35 min) contain on average 2.8 genome equivalents of DNA and that this nucleoid can be dissociated by deproteinization into two substructures having on average 1.4 genome equivalents. This result is correlated with previous sedimentation studies on the unfolded nucleoid DNA to explain prior inconsistencies. Scanning electron microscopy studies demonstrate that the shape and size of the isolated nucleoid is consistent with the proposed subunit structure of the in vivo nucleoid.     

In vitro replication of heavy strand DNA in permeabilized human mitochondria.

We have characterized some of the experimental conditions that are essential for initiation of human mitochondrial DNA synthesis. Mitochondria were purified from HeLa cells and were permeabilized with Triton X-100. When supplied with rNTPs and dNTPs, the permeabilized mitochondria synthesized nucleic acids that ranged in size from about 600 to 2000 nucleotides. In vitro DNA synthesis occurred on endogenous DNA templates and required a continuous supply of ATP. Analyses of the synthetic products revealed that almost all of them were of heavy-strand sequence and included authentic 7S DNA. Most of the synthetic products had 5' ends that mapped to similar locations as those previously identified for nascent heavy-strand DNA. Identification of these parameters should facilitate our efforts to achieve in vitro replication of heavy-strand mitochondrial DNA.     

DNA polymerase of mitochondria is a gamma-polymerase.

Mitochondria isolated from rat liver cells or mycoplasma-free HeLa cells contain a single DNA polymerase activity which is closely related to, or identical to, the DNA polymerase gamma activity found in the homologous cell. In rat liver cells, about 16% of the total cytoplasmic gamma-polymerase activity is found associated with mitochondria and in HeLa cells about 20% of the total cellular gamma-polymerase is mitochondria associated. Since mitochondria possess no unique DNA polymerase activity, the number of DNA polymerases now known in mammalian cells is reduced, from the previously proposed four enzymes, to three--DNA polymerases alpha, beta, and gamma.     

Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis

Intact and fast-sedimenting nucleoids of Bacillus licheniformis were isolated under low-salt conditions and without addition of detergents, polyamines or Mg2+. These nucleoids were partially unfolded by treatment with RNase and completely unfolded by treatments that disrupt protein-DNA interactions, like incubation with proteinase K, 0.1% sodium dodecyl sulphate and high ionic strength. Ethidium bromide intercalation studies on RNase-treated, proteinase-K-treated and non-treated nucleoids in combination with sedimentation analysis of DNase-I-treated nucleoids revealed that DNA is organized in independent, negatively supertwisted domains. In contrast to the DNA organization in bacterial nucleoids, isolated under high-salt conditions and in the presence of detergents (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972), the domains of supertwisted DNA in the low-salt-isolated nucleoids studied here are restrained by protein-DNA interactions. A major role for nascent RNA in restraining supertwisted DNA was not observed. The superhelix density of B. licheniformis nucleoids calculated from the change of the sedimentation coefficient upon ethidium bromide intercalation, was of the same order of magnitude as that of other bacterial nucleoids and eukaryotic chromosomes, isolated under high-salt conditions: namely, -0.150 (corrected to standard conditions: 0.2 M-NaCl, 37 degrees C; Bauer, 1978). Electron microscopy of spread nucleoids showed relaxed DNA and regions of condensed DNA. Spreading in the presence of 100 micrograms ethidium bromide per ml revealed only condensed structures, indicating that nucleoids are intact. From spreadings of proteinase-K-treated nucleoids we infer that supertwisted DNA and the protein-DNA interactions, responsible for restraining the superhelical DNA conformation, are localized in the regions of condensed DNA.     

Size of the intracellular circular Epstein-Barr virus DNA molecules in infectious mononucleosis-derived human lymphoid cell lines.

The size of non-integrated circular Epstein-Barr virus (EBV) DNA molecules isolated from seven different human lymphoblastoid cell lines of infectious mononucleosis origin has been determined by sedimentation analysis and by direct contour length measurements on electron micrographs. Six lines had intracellular circular EBV genomes of the same size as linear virion DNA molecules. The seventh line, established with the B95-8 strain of EBV, was the only one found to have circular EBV DNA molecules significantly smaller than virion DNA. The data show that intracellular EBV DNA circles of reduced size do not generally occur in infectious mononucleosis-derived cell lines.