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Results for “DNA (hypo)methylation”

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PubMed2024

Sulfonamide-induced DNA hypomethylation disturbed sugar metabolism in rice (Oryza sativa L.).

DNA methylation is well-accepted as a bridge to unravel the complex interplay between genome and environmental exposures, and its alteration regulated the cellular metabolic responses towards pollutants. However, the mechanism underlying site-specific aberrant DNA methylation and metabolic disorders under pollutant stresses remained elusive. Herein, the multilevel omics interferences of sulfonamides (i.e., sulfadiazine and sulfamerazine), a group of antibiotics pervasive in farmland soils, towards rice in 14 days of 1 mg/L hydroponic exposure were systematically evaluated. Metabolome and transcriptome analyses showed that 57.1-71.4 % of mono- and disaccharides were accumulated, and the differentially expressed genes were involved in the promotion of sugar hydrolysis, as well as the detoxification of sulfonamides. Most differentially methylated regions (DMRs) were hypomethylated ones (accounting for 87-95 %), and 92 % of which were located in the CHH context (H = A, C, or T base). KEGG enrichment analysis revealed that CHH-DMRs in the promoter regions were enriched in sugar metabolism. To reveal the significant hypomethylation of CHH, multi-spectroscopic and thermodynamic approaches, combined with molecular simulation were conducted to investigate the molecular interaction between sulfonamides and DNA in different sequence contexts, and the result demonstrated that sulfonamides would insert into the minor grooves of DNA, and exhibited a stronger affinity with the CHH contexts of DNA compared to CG or CHG contexts. Computational modeling of DNA 3D structures further confirmed that the binding led to a pitch increase of 0.1 Å and a 3.8° decrease in the twist angle of DNA in the CHH context. This specific interaction and the downregulation of methyltransferase CMT2 (log2FC = -4.04) inhibited the DNA methylation. These results indicated that DNA methylation-based assessment was useful for metabolic toxicity prediction and health risk assessment.

DNA Methylation↗

PubMed2026

Mutation in THO2, a component of THO/TREX complex, causes transcriptional gene silencing and genome-wide DNA methylation changes.

DNA methylation plays important roles in silencing of transgenes, endogenous genes, and transposable elements (TEs). To identify genes involved in antagonizing transcriptional or DNA hypermethylation-induced gene silencing, a genetic screening was conducted and thus a tho2-8 mutant was recovered. THO2 is a major component of the THO/TREX (Transcription-Export) complex, which plays essential roles in mRNA export. The tho2-8 mutation caused overaccumulation of DNA methylation on a d35S promoter ahead of LUC, suggesting its roles in antisilencing of transgenes. This mutation also resulted in significant genome-wide alterations in DNA methylation in a locus-specific manner, including 2513 hyper-DMRs and 1717 hypo-DMRs. The hyper-DMRs in the tho2-8 mutant not only exhibited a considerable overlap with those in DNA demethylation mutants (like ros1-7), but also with hypo-DMRs from nrpd1-3 and nrpe1-11 mutants, demonstrating that THO2 is able to protect those loci targeted by DNA demethylation and/or RdDM pathways from hypermethylation. The tho2-8 mutant also contained a plethora of CHH hypo-DMRs, which overlapped in large numbers with those from the nrpd1-3 and nrpe1-11 mutants, indicating that THO2 is required for the establishment/maintenance of DNA methylation at many loci. Additionally, the tho2-8 mutation caused an increase in overall 24-nt siRNA levels and many upregulated and downregulated DEGs/DETEs. The effects of THO2 on DNA methylation patterns appeared to be associated with the functioning of Pol IV and Pol V because THO2 physically interacted with NRPD7 and was necessary for normal accumulation levels of several Pol V-dependent IGNs' transcripts. Thus, this study provided valuable insights into new roles of THO2 in DNA methylation patterning.

DNA Methylation↗

PubMedDate not supplied

Mutations and epimutations in mammalian cells.

Early studies on heritable variation in cultured mammalian cells suggested that both mutation and epigenetic events might be involved. The importance of mutations has subsequently been fully documented, but only recently has an alternative form of inheritance been uncovered. This is based on the post-synthetic methylation of cytosine in regulatory regions of genes. The pattern of methylation is heritable, and in almost all cases studied, methylation of a region is associated with lack of gene expression. Such silent genes can be reactivated by the powerful demethylating agent 5-azacytidine (5-aza-CR). Changes in heritable DNA methylation which alter phenotype are referred to as epimutations. It now seems very likely that the well known 'functional hemizygosity' in CHO cells and other near diploid cell lines is due to the existence of one active and one silent gene at many autosomal loci. It is clear that permanent cell lines inactivate genes by de novo methylation, whereas normal diploid cells do not have this activity. This has important implications for our understanding of cellular transformation, tumor progression, and the increase in chromosome number frequently associated with these cellular changes. It is likely that both mutations and epimutations are important in the emergence of fully transformed tumorigenic cells. Agents which increase or reduce DNA methylation in cells can be regarded as epimutagens, although in many cases the mechanisms of inducing hypo- or hyper-methylation are not understood. Two exceptions are 5-aza-CR which inhibits the normal DNA maintenance methylase activity, and 5-methyldeoxycytidine triphosphate which is incorporated into cellular DNA following electroporation and has been shown to silence genes.

Animals↗

PubMed1994

DNA methylation and cancer.

Changes in the pattern of DNA methylation have been a consistent finding in cancer cells. The mostly descriptive nature of these studies and the fact that both hypo- and hypermethylation have been observed at various loci have made it difficult to assess whether these changes are causally involved in the transformation process or whether they reflect the altered physiology of rapidly dividing cancer cells. It is clear, however, that DNA methylation plays an important role in the generation of mutations in human tumors. The high incidence of C-to-T transitions found in the p53 tumor-suppressor gene is attributed to the spontaneous deamination of 5-methylcytosine residues. The multiple observations linking DNA methylation to cancer can be resolved in a model proposing that the high rate of mutation at CpG dinucleotides is due in part to methyltransferase-facilitated deamination. Support for a role of DNA methyltransferase as a mutator enzyme is provided by work with a prokaryotic DNA methyltransferase under S-adenosyl-methionine methyl-donor limiting conditions. Methyl-donor limiting conditions might arise in early stages of tumor development, leading to high rates of methyltransferase-mediated CpG mutagenesis, as seen in human tumors. Such a mechanism is consistent with the frequently reported methionine auxotrophy of cancer cells and with the tumorigenic effects of methyl-deficient diets. Methyl deficiency in tumor cells is also consistent with the commonly observed global hypomethylation of tumor cell DNA, despite normal or even high levels of DNA methyltransferase expression.

Animals↗

PubMed1987

Chromatin structure and methylation state of a thyroid hormone-responsive gene in rat liver.

The gene for S14 in the rat codes for an mRNA which in lipogenic tissues (liver, fat, mammary gland) responds both to L-triiodothyronine and a high-carbohydrate, fat-free diet. In an effort to understand the molecular basis for the tissue-specific regulation of mRNA-S14 expression, we have examined the organization of this gene in chromatin. Specifically, we examined the distribution of DNase I-hypersensitive sites and DNA methylation sites associated with a 25-kilobase chromosomal domain containing the S14 gene. Our results show that DNase I preferentially digests four regions of the DNA flanking the 5' end of the hepatic S14 gene which have characteristics of DNase I-hypersensitive sites. The sites, identified as HS-1, HS-2, HS-3, and HS-4, are located at or adjacent to the site of transcription initiation and at 1.2, 7, and 8 kilobases upstream from this site, respectively. In lactating mammary gland where the S14 gene is also highly expressed and regulated by L-triiodothyronine, sites HS-1, HS-2, and HS-4 are present, but HS-3 is absent. No hypersensitive sites were detected either within the gene or flanking the 3' end of the gene. In brain, kidney, and spleen, tissues in which mRNA-S14 is expressed at levels less than or equal to 10% of that found in euthyroid liver, sites HS-1 and HS-3 were absent. Despite the marked effect of thyroid state on the abundance of hepatic mRNA-S14, no significant alterations were observed in the DNase I sensitivity of hepatic chromatin containing the S14 gene. Analysis of the DNA methylation pattern at HpaII and HhaI sites showed a positive correlation of hypomethylation of the gene and the contiguous flanking regions with S14 gene expression. All HpaII/MspI sites and most HhaI sites either within or flanking the S14 gene were undermethylated in liver and lactating mammary gland. Although thyroid status had no generalized effect on the site-specific DNA methylation state of the hepatic S14 chromosomal domain, one site (H3) situated in the second exon close to the 3' terminus of the S14 gene appeared to undergo demethylation in the transition from hypo- to euthyroidism. In essence, our results show that the 5' DNA flanking region of the S14 gene contains a tissue-specific DNase I-hypersensitive site which, although not influenced by thyroid status, appears essential for the expression of S14 and its regulation by L-triiodothyronine.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

The effect of E. coli host strain on the consensus sequence of regions of the human L1 transposon.

We have used highly methylation tolerant host strains to clone hyper- and hypo-methylated genomic elements from different regions of the same family of long interspersed repetitive elements from human DNA, specifically the 1.8 kilobase (kb) and 1.2kb KpnI fragments from members of the L1 family of transposable elements in which respectively some 18% and 2.7% of cytosines are methylated in vivo in human spleen DNA. The consensus of the DNA sequences of the ends of 13 clones from the hypomethylated region of human L1 agreed exactly with the consensus derived previously from clones made using conventional host strains. However the sequences of 18 of our clones from the 5' end of the hypermethylated region differed significantly from the sequences of clones made using conventional hosts (P less than 0.0001). The 5' region of the 1.8kb L1 region is a CpG island which, in human somatic tissue, appears to be maintained in a highly methylated state, including methylation at sites other than CpG dinucleotides. The consensus sequence of this region also has features suggestive of a previously unrecognized open reading frame.

Base Sequence↗

PubMed2025

Effect of extracellular vesicles in remodeling the tumor microenvironment by DNMT1 downregulation for enhanced cancer immunotherapy.

BACKGROUND: The efficacy of immunotherapy is often hindered by the suppression of immune responses via the tumor microenvironment (TME). The presence of cancer cells forces other proximal non-cancerous cells to support tumor growth and persistence. A clear example of this cancerous-to-non-cancerous communication is represented by the accumulation of myeloid-derived suppressor cells (MDSCs) within the TME. Several studies have convergently shown that the overexpression of DNA-methyl-transferase-1 (DNMT1) in these cells results in protection from necroptosis and enhanced accumulation in vivo. Conversely, targeting DNMT1 through hypo-methylating agents has shown promising therapeutic potential by not only reducing the levels of MDSCs but also enhancing cancer immunogenicity and the efficacy of immune checkpoint inhibitors (ICI). METHODS: Murine 4T1 (triple-negative breast cancer (TNBC)) and CT26 (colon carcinoma) cell lines were cultured under standard conditions and used to generate tumor models in BALB/c mice. An oncolytic adenovirus expressing a DNMT1-targeting short hairpin RNA (OAd.shDNMT1) was engineered and validated for DNMT1 knockdown and genome-wide methylation reduction. Small extracellular vesicles (sEVs) were isolated from virus-infected cancer cells and characterized for RNA content and uptake by MDSCs. MDSC differentiation and suppressive function were assessed in vitro using flow cytometry and co-culture assays with murine splenocytes. In vivo, tumor-bearing mice received intratumoral OAd.shDNMT1, systemic decitabine, or immune checkpoint inhibitors (anti-Programmed cell Death protein-1), and tumor growth, immune infiltration, and systemic MDSC levels were evaluated. RESULTS: In this study, we report that, by using virally infected TNBC murine cells as a source for shDNMT1-loaded sEVs, OAd.shDNMT1 successfully reduced MDSC levels in vitro and in vivo. Furthermore, the co-administration with ICI resulted in a significant tumor growth reduction in mice bearing poorly immunogenic TNBC 4T1 cells. Also, our treatment promoted antitumor immunity, prolonged survival, and complete tumor eradication in modestly immunogenic colon CT26 cancer cells. CONCLUSION: This multifaceted strategy, based on OV-mediated immune stimulation and reduction of MDSC levels via sEVs, may improve clinical outcomes and the success of immuno-based regimens for patients facing MDSC-rich and highly aggressive cancer subtypes.

Animals↗

PubMed1989

Endogenous virus genomes become hypomethylated tissue--specifically during aging process of C57BL mice.

In an attempt to find out a cause for age-dependent derepression of endogenous viruses, extents of DNA methylation at the endogenous B- and C-type ecotropic viruses in brain, liver and spleen of C57BL/6NJc1 were examined at three ages, newborn, young adult and old. Both endogenous viruses showed a slight but significant tissue-specific either hypo- or hypermethylation during post-natal developmental phase in the three tissues. After maturation, however, no such change was detectable at most of the sites examined. The exceptions were C-type ecotropic virus in brain and B-type virus in spleen, where the age-dependent decreases of methylation were observed. The changes seemed to be continuations of preceding developmental hypomethylation. They indicated that the hypomethylation could be one of the causes for the age-dependent derepression of endogenous virus. It was further suggested that a mechanism to stop the developmental changes of DNA methylation at the maturation of individuals would be important in considering the reasons for the changes in senescent phase.

Aging↗

PubMed1988

RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase.

In vitro methylation of Bluescribe plasmid DNA (pBS) with human placental DNA methyltransferase to 6% 5-methylcytosine (mC) reduced transformation efficiencies in rglB+ host strains C600 and DS410 by almost 2 orders of magnitude. By contrast, the rglB- derivative of DS410 showed no reduction in transformation efficiency with methylation while the rglB- derivative of C600 was partially tolerant to methylation. Further, we show that the 1.8 kilobase (kb) and 1.2 kb KpnI fragments derived from the human L1 repeat have respectively 18.3% and 2.3% mC in vivo. Using these hyper- and hypo-methylated genomic segments ligated into the pBS plasmid, transformants with the highly methylated 1.8 kb L1 insert were recovered at 17 to 40 fold higher frequency with the rglB- host strains than with the rglB+ hosts. In addition, recombinant phage (lambda 2001) containing inserts of plant genomic DNA with 26.7% mC (from Petunia hybrida) when plated on rglB- hosts gave titres up to 222 times higher than on the rglB+ strains.

Cell Line↗

PubMed2025

Hypermethylation at 45S rDNA promoter in cancers.

The ribosomal genes (rDNA genes) encode 47S rRNA which accounts for up to 80% of all cellular RNA. At any given time, no more than 50% of rDNA genes are actively transcribed, and the other half is silent by forming heterochromatin structures through DNA methylation. In cancer cells, upregulation of ribosome biogenesis has been recognized as a hallmark feature, thus, the reduced methylation of rDNA promoter has been thought to support conformational changes of chromatin accessibility and the subsequent increase in rDNA transcription. However, an increase in the heterochromatin state through rDNA hypermethylation can be a protective mechanism teetering on the brink of a threshold where cancer cells rarely successfully proliferate. Hence, clarifying hypo- or hypermethylation of rDNA will unravel its additional cellular functions, including organization of genome architecture and regulation of gene expression, in response to growth signaling, cellular stressors, and carcinogenesis. Using the bisulfite-based quantitative real-time methylation-specific PCR (qMSP) method after ensuring unbiased amplification and complete bisulfite conversion of the minuscule DNA amount of 1 ng, we established that the rDNA promoter was significantly hypermethylated in 107 breast, 65 lung, and 135 colon tumour tissue samples (46.81%, 51.02% and 96.60%, respectively) as compared with their corresponding adjacent normal samples (26.84%, 38.26% and 77.52%, respectively; p < 0.0001). An excessive DNA input of 1 μg resulted in double-stranded rDNA remaining unconverted even after bisulfite conversion, hence the dramatic drop in the single-stranded DNA that strictly required for bisulfite conversion, and leading to an underestimation of rDNA promoter methylation, in other words, a faulty hypomethylation status of the rDNA promoter. Our results are in line with the hypothesis that an increase in rDNA methylation is a natural pathway protecting rDNA repeats that are extremely sensitive to DNA damage in cancer cells.

DNA Methylation↗

PubMed1986

Spontaneous neurinoma in an African lungfish, Protopterus annectens, and DNA repair studies on normal and neoplastic tissues.

Neurinomas developed in an African lungfish (Protopterus annectens), living in an aquarium in Western Japan. The 2 tumors, measuring 7.5 X 9.0 X 6.5 and 13 X 4 X 6 cm, were located on the skin. As shown by light microscopy, tumor cells were composed of spindle-shaped cells with huge pleomorphic nuclei, which were arranged in parallel rows or whorls in interlacing connective tissue. Long-term culture of these tumor cells was achieved in vitro at 25 degrees C with use of conditioned medium over a period of more than 4 months. The nuclear DNA contents of erythrocytes (normal diploids, 2C) and tumor cells dispersed from the fixed tumor tissues were measured by 4',6-diamidino-2-phenylindole hydrochloride-DNA microfluorometry by using mouse cerebellar small granule cells (normal diploids, 2C) as a reference. The 2C value of the lungfish was approximately 28-fold greater than that of the mouse. Furthermore, consistent with the nuclear pleomorphism observed by light microscopy, the nuclear DNA contents of tumor cells showed a wide distribution from hypo-2C to hyper-4C. DNA repair synthesis was measured autoradiographically in organ cultures of the tumor, lung, and skin, exposed to chemical carcinogens or UV radiation. Considerable repair was observed in the tumor and skin cells exposed to 1-methyl-1-nitrosourea (CAS: 684-93-5), N-methyl-N'-nitro-N-nitrosoguanidine (CAS: 70-25-7), or 254-nm or sunlamp UV light. Only traces of repair synthesis were detected in lung exposed to 1-methyl-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. 4-Hydroxyaminoquinoline 1-oxide (CAS: 4637-56-3) did not induce repair in any of the three tissues. The observed values for repair, relative to the amount of DNA, were similar to those in other fishes.

4-Hydroxyaminoquinoline-1-oxide↗

PubMed1988

Evidence for dedifferentiation and metaplasia in amphibian limb regeneration from inheritance of DNA methylation.

Amphibian limb regeneration is a process in which it has been suggested that cells of one differentiated type may dedifferentiate and give rise to cells of another type in the regenerate. We have used two tissue-specific hypomethylations in the newt cardioskeletal myosin heavy chain gene as lineage markers to follow the fate of cells during limb regeneration. Analysis of genomic DNA from different muscle cell populations allowed the assignment of one marker to the muscle (Hypo A) lineage and the other, more tentatively, to the 'connective tissue' (Hypo B) component of muscle. The contribution to regenerated limb cartilage and limb blastemal tissue by cells carrying these markers was estimated by quantitative analysis of Southern blot hybridizations using DNA from regenerate tissues. The results are consistent with a contribution of cells from both muscle and connective tissue lineages to cartilage in regenerated limbs. In addition, removal of the humerus at the time of amputation (eliminating any contribution from pre-existing cartilage), has provided evidence for an increased representation of cells carrying the connective tissue marker in regenerate cartilage but did not affect the representation of cells carrying the muscle cell marker.

Animals↗

PubMed1997

Epigenetic variation illustrated by DNA methylation patterns of the fragile-X gene FMR1.

Genomic methylation patterns of mammals can vary among individuals and are subject to dynamic changes during development. In order to gain a better understanding of this variation, we have analyzed patterns of cytosine methylation within a 200 bp region at the CpG island of the human FMR1 gene from leukocyte DNA. FMR1 is normally methylated during inactivation of the X chromosome in females and it is also methylated and inactivated upon expansion of CGG repeats in fragile-X syndrome. Patterns of methylation (epigenotypes) were determined by the sequencing of bisulfite-treated alleles from normal males and females and alleles from a family of five brothers who are methylation mosaics and are affected to various degrees by the fragile-X syndrome. Our data indicate that: (i) methylation of individual CpG cytosines is strikingly variable in hypermethylated epigenotypes obtained from a single individual, suggesting that maintenance of cytosine methylation is a dynamic process; (ii) methylation of non-CpG cytosines in the region studied may occur but is rare; (iii) mosaicism of methylation in the analyzed fragile-X males is remarkably similar to that found for the active X and inactive X alleles in normal females, suggesting that the methylation mosaicism of some fragile-X males reflects similar on and off states of FMR1 expression that exist in normal females; (iv) hypermethylation is slightly more pronounced on fragile-X alleles than on normal inactive X alleles of females; (v) the general dichotomy of hypo- and hypermethylated alleles persisted over the 5 year period that separated samplings of the fragile-X males; (vi) methylation variability was most pronounced at a consensus binding sequence for the alpha-PAL transcription factor, a sequence that may play a role in regulating expression of FMR1.

Alleles↗

PubMed2024

An epigenome-wide study of selenium status and DNA methylation in the Strong Heart Study.

BACKGROUND: Selenium (Se) is an essential nutrient linked to adverse health endpoints at low and high levels. The mechanisms behind these relationships remain unclear and there is a need to further understand the epigenetic impacts of Se and their relationship to disease. We investigated the association between urinary Se levels and DNA methylation (DNAm) in the Strong Heart Study (SHS), a prospective study of cardiovascular disease (CVD) among American Indians adults. METHODS: Selenium concentrations were measured in urine (collected in 1989-1991) using inductively coupled plasma mass spectrometry among 1,357 participants free of CVD and diabetes. DNAm in whole blood was measured cross-sectionally using the Illumina MethylationEPIC BeadChip (850 K) Array. We used epigenome-wide robust linear regressions and elastic net to identify differentially methylated cytosine-guanine dinucleotide (CpG) sites associated with urinary Se levels. RESULTS: The mean (standard deviation) urinary Se concentration was 51.8 (25.1) μg/g creatinine. Across 788,368 CpG sites, five differentially methylated positions (DMP) (hypermethylated: cg00163554, cg18212762, cg11270656, and hypomethylated: cg25194720, cg00886293) were significantly associated with Se in linear regressions after accounting for multiple comparisons (false discovery rate p-value: 0.10). The top hypermethylated DMP (cg00163554) was annotated to the Disco Interacting Protein 2 Homolog C (DIP2C) gene, which relates to transcription factor binding. Elastic net models selected 425 hypo- and hyper-methylated DMPs associated with urinary Se, including three sites (cg00163554 [DIP2C], cg18212762 [MAP4K2], cg11270656 [GPIHBP1]) identified in linear regressions. CONCLUSIONS: Urinary Se was associated with minimal changes in DNAm in adults from American Indian communities across the Southwest and the Great Plains in the United States, suggesting that other mechanisms may be driving health impacts. Future analyses should explore other mechanistic biomarkers in human populations, determine these relationships prospectively, and investigate the potential role of differentially methylated sites with disease endpoints.

Humans↗

PubMed1992

Effects of mutagen-sensitive mus mutations on spontaneous mitotic recombination in Aspergillus.

Methyl methane-sulfonate (MMS)-sensitive, radiation-induced mutants of Aspergillus were shown to define nine new DNA repair genes, musK to musS. To test mus mutations for effects on mitotic recombination, intergenic crossing over was assayed between color markers and their centromeres, and intragenic recombination between two distinguishable adE alleles. Of eight mutants analyzed, four showed significant deviations from mus+ controls in both tests. Two mutations, musK and musL, reduced recombination, while musN and musQ caused increases. In contrast, musO diploids produced significantly higher levels only for intragenic recombination. Effects were relatively small, but averages between hypo- and hyperrec mus differed 15-20-fold. In musL diploids, most of the rare color segregants resulted from mitotic malsegregation rather than intergenic crossing over. This indicates that the musL gene product is required for recombination and that DNA lesions lead to chromosome loss when it is deficient. In addition, analysis of the genotypes of intragenic (ad+) recombinants showed that the musL mutation specifically reduced single allele conversion but increased complex conversion types (especially recombinants homozygous for ad+). Similar analysis revealed differences between the effects of two hyperrec mutations; musN apparently caused high levels solely of mitotic crossing over, while musQ increased various conversion types but not reciprocal crossovers. These results suggest that mitotic gene conversion and crossing over, while generally associated, are affected differentially in some of the mus strains of Aspergillus nidulans.

Aspergillus nidulans↗

PubMed1972

Synthesis of ribonucleic acid by isolated rat liver mitochondria.

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.

Acridines↗

PubMed1997

Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors.

Methylation of CpG sites in the genome, which is generally conserved during cell replication, is considered to play important roles in cell differentiation and carcinogenesis. However, investigations on changes in methylation status have been limited to known genes. To make a genome-wide search for differentially methylated genes, we developed a methylation-sensitive-representational difference analysis (MS-RDA) method. The representation of the genome was prepared using the methylation-sensitive restriction enzyme HpaII, and the mixture ratio of tester and driver DNAs was optimized to detect differences in methylation status of a single copy per diploid mammalian genome. By performing comparative MS-RDA of one hepatocellular carcinoma and of background liver tissue of one mouse treated with a food carcinogen (2-amino-3,4-dimethylimidazo[4,5-f] quinoline), we were able to identify (i) extensive hypomethylation of long interspersed nuclear element repetitive sequences in a number of hepatocellular carcinomas, (ii) reduction of the gene dosage of their mitochondrial DNA, and (iii) a hypermethylated DNA fragment of unknown origin. Furthermore, by adding the clones obtained in the first MS-RDA to the driver DNA [MS-RDA with elimination of excessive clones (MS-RDA-WEEC)], nine DNA fragments that could not be detected at the first MS-RDA were isolated as differentially methylated DNA fragments. MS-RDA, combined with MS-RDA-WEEC, is thus a promising approach to identify DNA fragments differentially methylated in two DNA sources.

Animals↗

PubMed1992

Carrier detection in agammaglobulinemia by X chromosome inactivation analysis.

Using a recently developed strategy to analyze patterns of X chromosome inactivation in cell populations, we found that two mothers and a sister were carriers in three atypical or sporadic cases of patients with agammaglobulinemia, two of whom were brothers. In this study, a phosphoglycerate kinase 1 (PGK1) gene probe was used to detect patterns of methylation of X-chromosome genes. A random pattern of X inactivation was observed in isolated peripheral blood granulocytes. In contrast, one of the two X chromosomes was preferentially active in the Epstein-Barr virus (EBV)-transformed peripheral B cells of the family members of these patients. The volume of the blood specimen could be significantly reduced using EBV-transformed B cell lines which contained multiple clones. The analysis described here can be used to distinguish between X-linked agammaglobulinemia (XLA) and other forms of a- or hypo-gammaglobulinemia as well as to detect the carrier state.

Adolescent↗