In vitro culture of Aloe barbadensis Mill.: Morphogenetic ability and nuclear DNA content

In vitro culture of Aloe barbadensis is very difficult for both callus Induction and plant regeneration. By contrast, micropropagation from shoot meristems is easily achieved. A DNA microdensitometric study was performed on different organs of A. barbadensis and during in vitro culture of different explants. It was shown that morphogenetic ability is correlated to nuclear DNA content in the explant and/or in the callus. In particular, plant regeneration (or micropropagation) is obtained only from calli (or from explants) in which the amount of DNA per nucleus falls strictly within the diploid limits (2C and 4C) of the species.     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

In vitro and in vivo synthesis of dolichol and other main mevalonate products in various organs of the rat

The relative rate of biosynthesis of dolichol from [3H]mevalonate in nine rat organs was studied in slices and in the whole animal. This biosynthesis was also compared to that of cholesterol and ubiquinone. All tissues examined are able to synthesize dolichol, as well as ubiquinone and cholesterol. Comparison of the data from slices in vitro with the in vivo studies demonstrated relatively good agreement for dolichol and ubiquinone synthesis. Although dolichol of high specific radioactivity was recovered in the blood, redistribution between organs, such as occurs with cholesterol, appears to be insignificant. The highest rates of dolichol biosynthesis were found in kidney, spleen and liver. On the other hand, muscle makes the largest contribution to total body dolichol synthesis. Newly synthesized dolichol also appears in the bile, but excretion by this route is far from sufficient to account for dolichol turnover. Incorporation of mevalonate into the final products is mainly dependent on biosynthetic activity. For comparison of the biosynthetic rates in different organs, possible sources of errors (such as variations in the size of the precursor pool, limitation by the rate of precursor uptake or non-linear incorporation) were investigated the size of the mevalonate pool in various organs. Equilibration of this pool with exogenous mevalonate is a rapid and passive process. The size of the mevalonate pool does not determine the rates of cholesterol and dolichol biosynthesis, indicating the presence of regulatory steps in the terminal portion of these biosynthetic pathways.     

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo.     

DNA and protein components of nuclear acceptor sites for androgen receptors in the rat prostate

Androgen receptor-acceptor complexes in nuclei from rat ventral prostates were cross-linked in situ with formaldehyde and partially purified using affinity chromatography. To isolate acceptor DNA, the cross-linked receptor-acceptor complexes in formaldehyde-treated chromatin samples were adsorbed to dihydrotestosterone-17 beta-succinyl agarose, eluted with 75 microM dihydrotestosterone-1% SDS, digested with proteinase K and extracted with phenol-chloroform. After 32P end-labelling and PAGE, this DNA contained two distinct bands of DNA (about 300 and 400 base pairs respectively) which were unique relative to the total prostatic DNA. As an alternative approach for characterizing acceptor DNA, the DNA in prostatic nuclei and cross-linked chromatin was labelled with 32P by nick translation and analysed in glycerol density gradients for associations with cross-linked androgen receptors. A symmetrical 7s peak of 32P-DNA with a small amount of coincident receptor was observed in the gradients after mild trypsin treatment. In the absence of trypsin treatment, both the cross-linked receptors and the labelled DNA sedimented to the bottom of the gradients. Isolation of acceptor proteins involved iodination of cross-linked chromatin with 125I and androgen affinity chromatography. A comparison of the relative efficiency of retention and elution of 125I-proteins from different affinity columns revealed that testosterone-17 beta-succinyl agarose was potentially most suitable for purification of acceptor proteins. After electrophoresis on SDS-polyacrylamide gels, the eluates from this type of affinity matrix were found to contain two major peaks of 125I-labelled proteins--one corresponding to a protein with a similar molecular weight as the nuclear androgen receptor (33,000 Da); the other having a molecular weight of 20,000 Da. While the precise identity of this latter entity is unknown, its enrichment and retention by the affinity gel implies that it is closely associated with the androgen receptor and may be a component of the acceptor sites.     

Mitochondrial acetyl-CoA acetyltransferase in various organs from rat: form patterns and coenzyme-A-mediated modification

The mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9), which is involved in the biosynthesis or degradation of ketone bodies, was directly demonstrated in organ extracts applying a two-step chromatography-immunoelectrophoresis method. In liver, the enzyme can be shown in at least three forms: in an unmodified state, designated as AAT, and in the CoASH-modified forms A1 and A2, in amounts of 51.5 +/- 5.0%, 39.4 +/- 4.8% and 9.1 +/- 2.7% (areas of immunoprecipitation), respectively. This pattern, which could not be altered by a treatment with glutathione, resembles that of mitochondrial acetyl-CoA acetyltransferase in extrahepatic tissues. However, the proportion of the unmodified enzyme (AAT) is lower as compared to those in other tissues such as brain (81.5 +/- 4.4%). CoASH-modification and transformation into modified forms, which equal naturally occurring forms, can be demonstrated in vitro with acetyl-CoA acetyltransferase from both liver and brain. Thus CoASH-modification of mitochondrial acetyl-CoA acetyltransferase seems to be a process of general importance.     

Sequence organisation in nuclear DNA from Physarum polycephalum: methylation of repetitive sequences.

Nuclear DNA from the slime mould Physarum polycephalum is digested by the restriction endonuclease HpaII to generate a high molecular weight and a low molecular weight component. These are referred to as the M+ and the M- compartment, respectively. Sequences that are present in the M+ compartment are cleaved by MspI, the restriction enzyme isoschizomer of HpaII, thus showing that the recognition sequences for these enzymes in M+ DNA contain methylated CpG doublets. The distribution of repetitive sequences in the M+ and M- DNA compartments was investigated by comparison of the 'fingerprint' patterns of total Physarum DNA and isolated M+ DNA after digestion using different restriction endonucleases, and by probing for the presence of specific repetitive sequences in Southern blots of M+ and M- DNA by the use of cloned DNA segments. Both types of experiment indicate that many repetitive sequences are shared by both compartments, though some repetitive sequences appear to be considerably enriched, or are present exclusively, either in M+ DNA or in M- DNA.     

In vitro photomorphogenesis,plant growth regulators,melatonin content,and DNA methylation under various wavelengths of light in Phalaenopsis amabilis

Plant Cell, Tissue and Organ Culture (PCTOC) - We tested the feasibility to promote growth and shoot proliferation of Phalaenopsis through different wavelengths of LED and fluorescent. Therefore,...     

Mitochondrial DNA sequences are present inside nuclear DNA in rat tissues and increase with age

Mitochondrial DNA (mtDNA) mutations increase with age. However, the number of cells with predominantly mutated mtDNA is small in old animals. Here a new hypothesis is proposed: mtDNA fragments may insert into nuclear DNA contributing to aging and related diseases by alterations in the nucleus. Real-time PCR quantification shows that sequences of cytochrome oxidase III and 16S rRNA from mtDNA are present in highly purified nuclei from liver and brain in young and old rats. The sequences of these insertions revealed that they contain single nucleotide polymorphisms identical to those present in mtDNA of the same animal. Interestingly, the amount of mitochondrial sequences in nuclear DNA increases with age in both tissues. In situ hybridization of mtDNA to nuclear DNA confirms the presence of mtDNA sequences inside nuclear DNA in rat hepatocytes. Bone marrow metaphase cells from both young and old rats show mtDNA at centromeric regions in 20 out of the 2n = 40 chromosomes. Consequently, mitochondria can be a major trigger of aging but the final target could also be the nucleus.     

In vitro synthesis of double-stranded DNA from the Kilham rat virus single-stranded DNA genome.

Double-stranded, full-length linear DNA was synthesized in vitro by using single-stranded linear DNA as a self-priming template from the parvovirus Kilham rat virus and Escherichia coli DNA polymerase "large fragment" as the polymerizing enzyme. To ascertain the order of the synthesis of the cleavage fragments and to assess the accuracy of the in vitro synthesis, restriction endonuclease cleavage sites with known recognition sequences were mapped on the DNA. Comparing the cleavage pattern of the synthesized DNA with that of double-stranded viral DNA isolated from infected cells confirms that the in vitro synthesis produces a faithful copy of the viral single-stranded genome. Electron micrographs of the in vitro product reveal it to be a double-stranded linear molecule.     

In vitro studies on the methylation of histones in rat brain nuclei.

When isolated nuclei from 12-day-old rat brains were incubated with S-adenosyl-L-[methyl-3H]methionine, significant amounts of 3H-methyl were incorporated into lysyl residues in histones H3 and H4. About 0.024% of the total methylation sites on histone H3 and 0.013% of the sites on histone H4 were unmethylated at the time the nuclei were isolated. Methylation of these sites proceeded stepwise, progressing to a stable ratio of 0.93:1.0:0.17 for N epsilon-mono-, N epsilon-di-, and N epsilon-trimethyllysine in histone H3 and 0.19:1.0 for N epsilon-mono- and N epsilon-dimethyllysine in histone H4. The Km values of the enzyme for S-adenosyl-L-methionine were 11.5 +/- 1.1 micron and 12.5 +/- 1.3 micron with histones H3 and H4 as methyl acceptors, respectively. The Vmax values were 11.1 and 5.3 pmol of 3H-methyl incorporated/min/mg of histone H3 and H4, respectively. Since histone H3 contains 2 mol of N epsilon-methyllysine/mol and histone H4 contains 1 mol/mol, no difference in the overall rates of methylation can be deduced from the data. S-Adenosyl-L-homocysteine, one of the products of the reaction, was a competitive inhibitor with respect to S-adenosyl-L-methionine. The Ki values for S-adenosyl-L-homocysteine were 5.5 +/- 0.4 micron and 5.9 +/- 0.5 micron with histones H3 and H4 as methyl acceptors, respectively.     

In vivo and in vitro phosphoarylation of nuclear proteins in rat liver.

The incorporation of 32P into nuclear nonhistone proteins was compared in rat liver in vivo, in liver slices incubated in vitro, and in isolated nuclei incubated with gamma-[32P]ATP. The highest specific activities of nuclear phosphorproteins were obtained by incubating isolated nuclei. However, the Radioactivity profiles of polyacrylamide gel electrophoretograms of these proteins differed from those obtained in vivo or in liver slice experiments. A group of low molecular weight nonhistone proteins exhibited a very high incporation of labelled phosphate. These proteins could be obtained from the interface when the phosphoproteins were isolated by the buffered phenol extraction procedure. Phosphorylated proteins were also obtained from three cytoplasmic fractions (mitochondria, microsomes, and cytosol). The specific activities of these proteins were much lower than of the nuclear phosphoproteins.     

Effect of hydrocortisone on methylation and molecular population of DNA in rat liver]

The content of 5-methyl cytosine in rat liver DNA increases 1,7-fold 8 hours after intraperitoneal injection of hydrocortisone (5 mg per 100 g animal weight). The content of GC, physicochemical parameters (Tm, delta T, etc.) and DNA renaturation pattern did not show any changes. No changes were observed in the pattern of H3-thymidine incorporation into rat liver DNA: after hydrocortisone injection the radioactivity was found to be equally distributed in all isolated sequences of DNA, differing in the degree of reiteration (specific radioactivities of these DNA, fractions are very similar). Thus, the molecular population of DNA in liver cells remains unchanged, which suggests that the hormone-induced increase in the 5-methyl cytosine content is due to a change in the DNA methylation level. The methyation level of unique sequences (COt greater than 600), i. e. that of structural genes, does not undergo any essential changes. The reversible methylation of DNA regulated by hormones seems to be one of the mechanisms controlling gene activity.     

Gender specific differences in levels of DNA methylation at selected loci from human total blood: a tendency toward higher methylation levels in males

Abnormal patterns of DNA methylation are observed in many diseases such as tumors and imprinting disorders. Little is known about inter-individual and gender specific variations. Here, we report on accurate and sensitive quantitative measurements of methylation in DNA from total blood in 96 healthy human males and 96 healthy human females. Global methylation was estimated by studying two repetitive DNA elements, namely Line-1 and Alu repeats, while single loci were investigated for three differentially methylated regions (DMRs) at PEG3, NESP55 and H19 imprinted genes and two additional loci at Xq28 (F8 gene) and at 19q13.4 (locus between PEG3 and ubiquitin specific protease 29). We observed inter-individual correlations in the degree of methylation between Alu and Line-1 repeats. Moreover, all studied CpGs showed slightly higher methylation in males (P < 0.0003–0.0381), with the exception of DMRs at imprinted genes (P = 0.0342–0.9616) which were almost equally methylated in both sexes with only a small tendency towards higher methylation in males. This observed difference could be due to the process of X chromosome inactivation or merely to the presence of an additional X chromosome in female cells or could be a result of downstream effects of sex determination. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.