Subcellular reorganization of mitochondria producing heavy DNA in aging wheat coleoptiles.

Unusual closed membrane vesicles containing one or more mitochondria were isolated from homogenates of aging wheat coleoptiles. Very similar (or the same) bodies were shown to exist in situ in vacuoles of undividing cells in the apical part of intact senescent coleoptiles. Vesicles isolated from coleoptile homogenate free of nuclei by 10 min centrifugation at 1700 x g and traditional mitochondria (sedimented at between 4300 x g and 17,400 x g) are similar in respiration rate, composition and content of cytochromes and sensitivity to respiration inhibitors. However, vesicles contain about 2-fold more Ca2+ ions than free mitochondria do. The specific feature of vesicles containing mitochondria in aging coleoptiles is an intensive synthesis of heavy (rho = 1.718 g/cm3) mitochondrial DNA (H-mtDNA). Thus, aging in plants is accompanied by an increased selective H-mtDNA production and change in subcellular organization of mitochondria.

In vitro DNA methylation by wheat nuclear cytosine DNA methyltransferase: effect of phytohormones.

Cytosine DNA methyltransferases (MTases) were isolated from nuclei of wheat seedlings and germinating embryos. The MTases isolated from both sources were able to perform de novo and maintenance DNA methylations. The most purified MTase fraction showed the presence of one main 67-kDa protein (embryos) and of a 85-kDa protein (in seedlings) in SDS-PAGE. Some plant growth regulators (gibberellic acid A3, 6-benzylaminopurine and fusicoccin) elevate by 30-65% the extent of in vitro DNA methylation by nuclear extracts with a maximal effect at 10(-6) M phytohormone concentration. The same phytohormones do not increase the extent of in vitro DNA methylation by purified wheat MTase; rather they inhibit it at concentrations of 10(-4)-10(-5) M. Thus, DNA methylation in the plant nucleus is controlled by phytohormones. The phytohormone effect may be mediated by other proteins in nuclear extracts.

Superproduction of heavy minicircular mitochondrial DNA in aging wheat coleoptiles.

On incubation of 7-day-old wheat seedlings in the presence of [3H]thymidine, the radioactivity incorporated into coleoptile DNA is found to be localized mainly (greater than 95%) in the fraction of heavy mitochondrial DNA (H-mt DNA; rho = 1.716 gm/cm3). Upon long (48-72 h) incubation of cut-off seedlings in water, the amount of this DNA shows a dramatic increase and corresponds to about 10% of the total coleoptile DNA. H-mtDNA is represented by open circular molecules with a contour length varying from 0.12 to 0.6 microns. The functional role of this DNA is still unknown.

DNA methylation in plants.

Both replicative and postreplicative nuclear DNA (nDNA) methylation, with the formation of 5-methylcytosine (mC) residues, occurs in plants. These two types of enzymatic DNA modification are different in amount and nucleotide sequence of methylatable sites, as well as in sensitivity to phytohormones, temperature and various inhibitors of DNA methylation, transcription and replication. The role of DNA methylation in regulation of replication, gene expression and cell differentiation is discussed.

[Determination of the adenine-specific methylase recognition site of Shigella sonnei 47, using hydrazinolysis with subsequent separation of purine oligonucleotides by thin layer chromatography on DEAE- cellulose]. / Opredelenie saita uznavaniia adeninspetsificheskoi metilazy Shigellasonnei 47 s pomoshch'iu gidrazinolizadnk i posleduiushchego pazdeleniia purinovykh oligonukleotidov tonkosloinoi khromatografiei na déaé-tselliuloze.

A procedure for separation of oligopurine blocks of different length and composition by two-dimensional thin layer chromatography on DEAE-cellulose plates has been developed. This method allows a comparative analysis of the purine isostich content in the DNAs of various origin. In case of methylated DNA, the method permits to compare the substrate specificity of different enzymes responsible for the adenine residue methylation in the DNA. In combination with enzymatic treatment of labeled methylated isostichs, the method described can be used for the deciphering of the methylated sequences as well as for constructing, in a number of cases, the recognition site of adenine-specific methylases. Thus, it was demonstrated that methylase SsoI recognizes the 5...G-A-A-T-T-C ... 3' sequence and methylates its adenine residue nearest to the 5'-end.

[Synchronous synthesis of DNA in coleoptiles and the initial leaf of developing etiolated wheat shoots: the nature and correlation of nuclear and mitochondrial DNA syntheses]. / Sinkhronnyi sintez DNK v koleoptile i pervom liste razvivaiushchikhsia étiolirovannykh prorostkov pshenitsy: priroda i sootnoshenie sinteza iadernoi i mitokhondrial'noi DNK.

In etiolated coleoptiles and initial leaf of developing wheat shoots the DNA synthesis is periodical and synchronous. In the initial leaf each step of DNA synthesis results in a stepwise increase of DNA content and is doubled at the first three steps. During the leaf plane formation the synthesis of nuclear DNA (nDNA) is decreased, while that of mitochondrial DNA (mitDNA) continues in synchronous cycles. This is the cause of relative stabilization of DNA content per unit of leaf plane length. The DNA increase in this organ occurs due to synchronous synthesis of nDNA and mitDNA in intercalary meristem cells. In coleoptiles a marked replication of nDNA is observed at the first three steps of the synthesis; in each cycle nDNA synthesis precedes that of mitDNA. With completion of coleoptile formation the nDNA synthesis in it practically ceases, whereas that of mitDNA continues in synchronous cycles. MitDNA is non-methylated and its composition (56 mol.% GC) differs significantly from that of the newly synthesized nDNA (44 mol.% GC; 100 X m5C/(C + m5C) = 16-17%). It may be concluded that in various organs of wheat shoots the composition and methylation of newly synthesized DNA depend on the age of the shoot and on the ratio of nDNA/mitDNA syntheses.

[Mitochondrial nature of newly synthesized DNA in aging coleoptiles of etiolated wheat shoots]. / Mitokhondrial'naia priroda novoobrazovannoi DNK v stareiushchikh koleoptiliakh étiolirovannykh prorostkov pshenitsy.

In mature ageing coleoptiles as well as in cut-off etiolated wheat shoots exhausted by incubation in H2O nuclear DNA (nDNA) synthesis does not occur. However, the synthesis of another DNA is continued under these conditions. The parameters of this DNA (e. g. buoyant density, rho = 1.716 g/cm3, and composition - 56 mol.% GC) differs from those of nDNA (rho = 1.700 g/cm3, 44 mol.% GC). The newly synthesized DNA is non-methylated and metabolically stable. It is not sensitive to cycloheximide (20 micrograms/ml) but is markedly inhibited by ethidium bromide (2-10 micrograms/ml). The synthesis of this DNA is localized in the mitochondria, thus indicating that in ageing coleoptiles and exhausted wheat shoots all the newly synthesized DNA is of mitochondrial origin.

[Methylation of newly formed DNA in developing wheat seedlings]. / Metilirovanie novoobrazovannoi DNK v razvivaiushchikhsia prorostkakh pshenitsy.

The base composition (45 divided by 56 mol.% GC) and methylation level (100 . m5C/(m5C + C) = = 20% divided by 0.4%) of pulse-labelled newly formed DNA in etiolated wheat seedlings and their individual organs are markedly changed with age. The base composition of total leaf and coleoptile DNA (45 mol.% GC) remains unchanged; the changes in the methylation level are not significant (1). In the course of development of the plant organs (e. g. leaf) the level of synthesis of DNA whose composition is identical to that of nuclear DNA, is decreased and the incorporation of labelled precursors into the GC-rich (56 mol.% GC) practically unmethylated DNA is increased. In ageing coleoptiles only the synthesis of unmethylated DNA of the GC-type takes place. It is assumed that this DNA may correspond to mitochondrial DNA.

[Replicative methylation of DNA in L-cells: effect of S-isobutyladenosine and cycloheximide and possible existence of two DNA-methylases]. / Replikativnoe metilirovanie DNK v L-kletkakh: deistvie S-izobutiladenozina i tsiklogeksimida i vosmozhnoe sushchestvovanie dvukh DNK-metilaz.

Methylation of DNA in cultured cells of mouse fibroblasts (L-cells) occurs at least in two steps, i. e. methylation of Okazaki fragments (up to 100 . m5C/C + m5C = 2.8-2.9) and methylation of linkage sites of DNA formed by ligation of the fragments (up to 6.0). The synthesized Okazaki fragments are not subjected to further methylation, since about one half of their methylation sites (CG) remains non-modified. The transmethylation inhibitor S-isobutyladenosine (SiBA) inhibits the methylation of the "linkage" sites of the newly synthesized DNA without affecting that of the Okazaki fragments. The repression of protein synthesis (including that of histones) by cycloheximide in the course of replication reveals some additional methylation sites. The level of methylation of the newly synthesized polymeric DNA reaches thereby 6.0, which corresponds to modification of all its CG-dinucleotides. A model for replicative methylation of DNA based on the existence of two DNA-methylases differing in their specificity is proposed. It is assumed that one of the DNA methylases is highly specific and functions within the complex with DNA-polymerase, while the other possesses a restricted specificity and functions in a free form (i. e. apart from the replicative complex).

[5-Methylcytosine in pyrimidine sequences of plant and animal DNA: specificity of methylation]. / 5-Metiltsitozin v pirimidinovykh posledovatel'nostiakh DNK rastenii i zhivotnykh: spetsifichnost' metilirovaniia.

A method for two-dimensional DEAE-TLC fractionation and a subsequent quantitative spectrophotometric determination of the 5-methylcytosine containing pyrimidine oligodeoxyribonucleotides isolated from plant and animal DNA is proposed. Using this method, the distribution of 5-methylcytosine among pyrimidine oligodeoxyribonucleotides in various plant and animal DNA was studied. It was found that the methylation patterns of DNA in higher plants and animals are quite different, but they are similar among various plants or among different animal DNAs. More than half of the 5-methylcytosine residues in animal DNAs are localized in the monopyrimidine fraction (Pu-C-Pu); however, in plant DNAs there are two types of heavily methylated sequences (Pu-C-Pu and Pu-(CT)-Pu). It is suggested that methylation of plant DNAs occurs in two different sites possessing the second order symmetry with axis lined across the dinucleotide CpG or complementary AT base pair. Possible involvement of eucaryotic DNA-methylases in replicative and reparative DNA synthesis and in hormonal regulation of DNA methylation at and after DNA synthesis in plants and animals as well as a possible role of DNA methylation in regulation of gene activity in tissues and cells are discussed.

[Replication and methylation of DNA in cells of tobacco suspension culture and the effect of auxin]. / Replikatsiia i metilirovanie DNK v kletkakh suspenzionnoi kul'tury tabaka i vliianie auksina.

After 2 min of incubation of tobacco cell culture in a medium with [3H] -- thymidine the bulk of radioactivity of newly synthesized DNA is found in short (about 5S) fragments, whereas after a prolonged incubation of the cells, i. e. 5--60 min--in long replication fragments as well. Hence DNA replication in tobacco cells occurs discretely via formation and cross-linking of Okazaki fragments. At high cell concentrations in the medium the linking of 5S fragments is suppressed. It was shown that the Okazaki fragments and other fragments of DNA replication are subjected to methylation, the DNA methylation occurring immediately after the onset of replication. The level of methylation of the 4--6S fragments is two times less than that of the linked ones; therefore replicative methylation occurs in at least two steps: at first the Okazaki fragments undergo methylation and once they are linked, an additional methylation of DNA takes place. Auxin (2,4-dichlorophenoxyacetic acid) at concentration of 5 mg per 1 of medium does not affect the ratio of the replication fragments and methylation of the Okazaki fragments, but completely inhibits the second step of replicative methylation of DNA, i. e. methylation of the linked fragments. Phytohormones can probably control the transcription of newly synthesized DNA via regulation of methylation.

[Quantitative spectrophotometric determination of non-identified nucleotides in DNA]. / Kolichestvennoe spektrofotometricheskoi opredelenie neizvestnykh nukleotidov v DNK.

A method for calculation of molar extinction coefficients (epsilon) and for quantitative spectrophotometric determination of non-identified nucleotide analogs from nucleic acids of some bacteriophages is proposed. The method is based on spectral properties of known and unknown nucleotides and of their dinucleotide isolated from the DNA studied.

Structure of animal mitochondrial DNA (base composition, pyrimidine clusters, character of methylation).

Base composition, content of pyrimidine isopliths and methylation degree of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from various vertebrates and protozoon Crithidia oncopelti have been studied. mtDNAs from mammals (ox, rat) do not differ in fact in the G + C content from the respective nDNA. The G + C content in mtDNA from fishes (sheat-fish) and birds (duck, chicken) is 1.5--2.5 mol % higher than in the respective nDNA. Kinetoplast DNA (kDNA) from Crithidia oncopelti (G + C = 42.9 mol %) differs significantly in base composition from nDNA (G + C 51.3 mol%). All the mtDNA and kDNA studied differ from the respective nDNA by a lower degree of pyrimidine clustering. The amount of mono- and dipyrimidine fragments in mtDNA is more than 30 mol %, whereas in nDNA it does not exceed 23 mol %. The quantity of long pyrimidine clusters (hexa- and others) is 2--4 times lower in mtDNA than in nDNA. The lower degree of clustering of pyrimidine nucleotides seems to be a specific feature of all the mtDNA studied. This may be indicative of common traits in the organization and origin of mtDNA. All mtDNA of vertebrates contain 5-methylcytosine as "minor" base (1.5--3.15 mol %) and surpass by 1.5--2 times the respective nDNA in the methylation degree. It has been found that in animals mtDNA is species specific as far as the 5-methylcytosine content is concerned. mtDNA of beef heart differs significantly from nDNA in the mode of 5-methylcytosine distribution in pyrimidine isopliths, which may indicate that methylation specificity of nuclear and mitochondrial DNA is not the same. In mitochondria and nuclei of rat liver certain DNA-methylase activity has been detected, which provides in vitro the methylation of cytosine residues both in homologbous DNA and various heterologous DNAs. Specificity of methylation in vitro of cytosine residues in one and the same heterologous DNA from Escherichia coli B with nuclear and mitochondrial enzymes is different. Mitochondrial enzyme methylates cytosine residues chiefly in mono-, whereas nuclear enzyme, in di- and tripyrimidine fragments.

The structure of animal mitochondrial DNA (base composition, pyrimidine clusters, character of methylation).

Base composition, content of pyrimidine isopliths and the degree of methylation of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from various vertebrates and protozoon Crithidia oncopelti have been studied. MtDNAs from mammals (ox, rat) do not differ in fact in the GC content from the respective nDNA. The GC content in mtDNA from fishes (sheat fish) and birds (duck, chicken) is 1.5-2.5 mole % higher than in the respective nDNA. Kinetoplast DNA (kDNA) from Crithidia oncopelti (GC = 42.9 mole %) differs significantly in base composition from nDNA (GC = 51.3 mole %). All the mtDNA and kDNA studied differ from the respective nDNA by a lower degree of pyrimidine clustering. The amount of mono and dipyrimidine fragments in mtDNA is more than 30 mole %, whereas in nDNA it does not exceed 23 mole %. The quantity of long pyrimidine clusters (hexa and others) is 2-4 times lower in mtDNA than in nDNA. The lower degree of clustering of pyrimidine nucleotides seems to be a specific feature of all the mtDNA studied. This may be indicative of common traits in the organization and origin of mtDNA. All mtDNA of vertebrates contain 5-methylcytosine as a 'minor' base (1.5- 3.15 mole %) and surpass by 1.5-2 times the respective nDNA in the methylation degree. It has been found that in animals mtDNA is species specific as far as the 5-methyl-cytosine content is concerned. In mitochondria and nuclei of rat liver certain DNA methylase activity has been detected, which provides in vitro the methylation of cytosine residues both in homologous DNA and various heterologous DNAs. The specificity of methylation in vitro of cytosine residues in the same heterologous DNA from E. coli B varies with the source of enzymes. The mitochondrial enzyme methylates cytosine as the lone monopyrimidine residue, whereas the nuclear enzyme methylase cytosine in the di- and tripyrimidine fragments.

[DNA-methylase activities from animal mitochondria and nuclei: different specificity of DNA methylation]. / DNK iad DNK-metilaznye aktivnosti iz mitokhondrii i iader zhivotnykh. Razlichnaia spetsifichnost' metilirovaniia DNK

DNA-methylase activities which methylate cytosine residues in homo- and heterologous DNA were detected in mitochondria and nuclei from rat liver and beef heart. Adenine modifying DNA-methylases in mitochondria and nuclei were not found. DNA from mitochondria and nuclei differ significantly in the methylation degree and in the pattern of the 5-methyl-cytosine distribution by pyrimidine isostichs as DNA in vivo and in vitro being methylated. Mitochondrial DNA methylase has the maximum activity at 30 degrees and pH 7.8 this enzyme(s) differ(s) from the nuclear one(s) in the pH dependence of its activity. After exhaustive in vitro methylation of various DNA by the nuclear enzyme DNA-methylase from mitochondria additionally introduces CH3 groups from S-adenosylmethionine into these DNA (about 3 times more CH3 groups than nuclear enzyme). Nuclear DNA-methylase also methylates DNA which is previously fully-methylated by the mitochondrial enzyme, but to a lesser degree. In conditions of exhaustive DNA methylation mitochondrial enzyme introduces into E. coli B DNA about four times more methyl groups as compared to the nuclear one. After the methylation of E. coli B DNA by mitochondrial enzyme the label (3H-methyl) was detected predominantly in mono-, and in case of nuclear enzyme--in di- and tripyrimidine fragments. Mitochondrial DNA-methylase differs from the nuclear one in the nature of recognized DNA sequences; these enzymes seems to be represented by different proteins. The mitochondrial enzyme methylates shorter nucleotide sequences in DNA as compared to the nuclear DNA-methylase. All these data suggest there exist organoid specificity of genome methylation in animal cell and the modification-restriction systems in animal nucleus and mitochondria are different in character.