Application of the 32P-postlabelling technique to detect DNA-adducts by cisplatin and methylating agents.
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Alkylating Agents↗
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Results for “DNA Methylation Detection”
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Methylated purines in human liver DNA after probable dimethylnitrosamine poisoning.
DNA, isolated from two samples of human liver obtained from a suspected dimethylnitrosamine poisoning, contained 1363 to 1373 micromol of 7-methylguanine per mol of guanine and 273 to 317 micromol of O6-methylguanine per mol of guanine. Liver and kidney DNA obtained from unrelated cases contained no detectable methylated purines. From the DNA methylation levels, it is estimated that the dimethylnitrosamine-poisoning victim had been exposed to a dose of 20 mg or more of dimethylnitrosamine per kg of body weight. The results indicate for the first time that humans, like rodents, appear to activate dimethylnitrosamine metabolically to a strong methylating agent, resulting in methylation of liver DNA at both the 7- and O6 positions of guanine.
Adult↗
Molecular detection of p16 promoter methylation in the serum of colorectal cancer patients.
Assays based on the molecular detection of genetic changes in serum have been shown as potential diagnostic tools for colorectal cancer. We examined the methylation status of p16 in colorectal cancers using methylation-specific PCR (MSP). Forty-four of 94 (47%) cancer DNA exhibited abnormal promoter methylation of p16 gene while no corresponding normal DNA exhibited such methylation. Subsequently, we examined whether aberrant methylation could be detected in corresponding serum DNA, and found that 13 of 44 (30%) patients with p16 promoter methylation in tumor DNA demonstrated abnormal methylation in their serum DNA. Moreover, abnormal methylation was found in the serum of patients in all clinical stages, suggesting that early colorectal cancer could be detected using the MSP method.
Adenocarcinoma↗
Methylation profiling of urothelial carcinoma in bladder biopsy and urine.
OBJECTIVE: To test DNA methylation profiling in detection of urothelial carcinoma in urine. STUDY DESIGN: Thirty-three bladder specimens were analyzed for the DNA p16INK4a, RASSF1, APC, GSTP, E-Cad and CyclinD2 genes to determine if there is a difference in gene methylation between benign and malignant cases. Urine samples were analyzed in a feasibility study. Finally, methylation profiles of urine samples were obtained and compared with follow-up biopsy diagnoses. RESULTS: We found methylated genes in 18% benign, 37% urothelial carcinoma in situ and 93% infiltrating urothelial carcinoma cases (p = 0.001). Methylation profiles from the 18 urine samples revealed a significantly higher prevalence of methylated genes in carcinoma cases than benign cases (100% vs. 50%, p = 0.025). We analyzed methylation profiles in 37 cytologically atypical urine samples with malignant or benign diagnosis on surgical follow-up andfound that only APC (55% in malignant vs. 0% in benign, p=0.025) and CyclinD2 were differentially methylated (35% in malignant vs. 0% in benign, p=0.2) while p14ARF, p16INK4a, RASSF1, GSTP and E-Cad had similar methylation profiles. CONCLUSION: These results suggest that methylation of p14ARF, p16INK4a, RASSF1, GSTP and E-Cad genes may not accurately identify carcinoma, but methylated APC and CyclinD2 might be useful biomarkers for urothelial carcinoma in urine.
Aged↗
Foreign DNA integration. Genome-wide perturbations of methylation and transcription in the recipient genomes.
In hamster cells transgenic for the DNA of adenovirus type 12 (Ad12) or for the DNA of bacteriophage lambda, the patterns of DNA methylation in specific cellular genes or DNA segments remote from the site of transgene insertion were altered. In the present report, a wide scope of cellular DNA segments and genes was analyzed. The technique of methylation-sensitive representational difference analysis (MS-RDA) was based on a subtractive hybridization protocol after selecting against DNA segments that were heavily methylated and hence rarely cleaved by the methylation-sensitive endonuclease HpaII. The MS-RDA protocol led to the isolation of several cellular DNA segments that were indeed more heavily methylated in lambda DNA-transgenic hamster cell lines. By applying the suppressive subtractive hybridization technique to cDNA preparations from nontransgenic and Ad12-transformed or lambda DNA-transgenic hamster cells, several cellular genes with altered transcription patterns were cloned from Ad12-transformed or lambda DNA-transgenic hamster cells. Many of the DNA segments with altered methylation, which were isolated by a newly developed methylation-sensitive amplicon subtraction protocol, and cDNA fragments derived from genes with altered transcription patterns were identified by their nucleotide sequences. In control experiments, no differences in gene expression or DNA methylation patterns were detectable among individual nontransgenic BHK21 cell clones. In one mouse line transgenic for the DNA of bacteriophage lambda, hypermethylation was observed in the imprinted Igf2r gene in DNA from heart muscle. Two mouse lines transgenic for an adenovirus promoter-indicator gene construct showed hypomethylation in the interleukin 10 and Igf2r loci. We conclude that the insertion of foreign DNA into an established mammalian genome can lead to alterations in cellular DNA methylation and transcription patterns. It is conceivable that the genes and DNA segments affected by these alterations depend on the site(s) of foreign DNA insertion.
Adenoviridae↗
Can we improve the cytologic examination of malignant pleural effusions using molecular analysis?
BACKGROUND: Currently, 40% of patients remain undiagnosed after routine cytologic examination for malignant pleural effusions. Deoxyribonucleic acid (DNA) methylation is a robust strategy for detecting cancer early in tissue. We hypothesized that DNA methylation would be more sensitive in diagnosing patients with malignant pleural effusions than cytology. METHODS: We conducted a prospective cohort study of 31 inpatients with pleural effusions (24 malignant pleural effusions metastatic from 10 different organs and 7 benign) over 18 months. Aspirated pleural fluid underwent cytologic examination and DNA extraction for nested methylation-specific polymerase chain reaction (PCR). We assayed for promoter hypermethylation in 8 genes known to be methylated in many cancers. Pleural fluid was considered positive if 2 or more genes were methylated by methylation-specific PCR. RESULTS: Cytology alone confirmed malignant pleural effusions in 15 of 24 patients (sensitivity 63%), whereas methylation alone positively identified 16 of 24 patients (sensitivity 67%). Both tests had 100% specificity in predicting benign effusions. If cytology and methylation were considered together, they exhibited 88% sensitivity and 100% specificity in discriminating benign and malignant effusions. Combined, the two assays were more sensitive than either test alone. Although the positive predictive value of each test was 100%, the negative predictive value of cytology and methylation combined was 78%, better than 47% and 44% for methylation and cytology alone, respectively. CONCLUSIONS: Epigenetic analysis of pleural fluid can detect malignant DNA from a variety of neoplasms, provide complementarity with cytology, and improve the diagnostic yield of the current standard examination of pleural fluid.
Breast Neoplasms↗
Disordered beta-catenin expression and E-cadherin/CDH1 promoter methylation in gastric carcinoma.
AIM: To investigate the distribution of beta-catenin in nuclei or membrane/cytoplasm of gastric carcinoma cells, the relationship between E-cadherin gene methylation and its expression, and the role of beta-catenin and E-cadherin as potential molecular markers in predicting tumor infiltration. METHODS: Twenty-nine cases of gastric carcinoma, classified as diffuse and intestinal variants, were selected for study. Nuclear and cytoplasmic proteins were purified and beta-catenin content was detected by ELISA. DNA methylation of E-cadherin/CDH1 gene promoter was studied by methylation-specific PCR and compaired with E-cadherin expression detected by immunohistochemistry. RESULTS: In 27 cases of gastric carcinoma, the ratio of beta-catenin content between nuclei and membrane/cytoplasm was correlated with the T-classification (r = 0.392, P = 0.043). The significance was present between T2 and T3 groups. No correlation was detected between diffuse and intestinal variants in terms of their beta-catenin distribution. In 21 cases of diffuse variants of gastric carcinoma, there was a difference in E-cadherin expression between CDH1 gene-methylated group and non-methylated group (29 % vs 71 %, P = 0.027). No correlation between CDH1 gene methylation and T-classification was found, neither was the significance between E-cadherin expression and tumor infiltration grade. CONCLUSION: Comparative analysis of nuclear and membrane/cytoplasmic beta-catenin can predict local tumor infiltration. E-cadherin/CDH1 gene methylation is an important cause for its gene silence in diffuse variant gastric carcinoma. Methylation of CDH1 gene in the absence of E-cadherin is an early event in gastric carcinogenesis.
Antigens, CD↗
Engineered zinc finger proteins that respond to DNA modification by HaeIII and HhaI methyltransferase enzymes.
Zinc finger modules are capable of specifically interacting with DNA that contains 5-methylcytosine (5-mC) in place of cytosine, suggesting that zinc finger-DNA binding could be regulated by extrinsic methylation of DNA. Here, we have used phage display to engineer zinc finger proteins that detect and discriminate DNA methylation by the prokaryotic enzymes HaeIII and HhaI. In these systems, zinc finger-DNA complexes are induced by DNA modification using the appropriate enzyme, which can therefore act as a switch. To further develop the specificity of the switch, zinc finger discrimination between 5-mC and thymine in DNA sequences is demonstrated despite the presence of the characteristic major groove methyl group that is common to both bases. Specificity was achieved using a DNA-binding strategy involving synergy between adjacent zinc fingers. We propose that engineered zinc fingers that recognise particular DNA modifications, such as sequence-specific DNA methylation, could be integrated into artificial regulatory circuits for the control of gene expression and other biological processes.
Bacteriophages↗
Direct assay for O6-methylguanine-DNA methyltransferase and comparison of detection methods for the methylated enzyme in polyacrylamide gels and electroblots.
We describe in detail a direct assay for the substrate-inactivated DNA-repair enzyme, O6-methylguanine-DNA methyltransferase (O6-MT), which measures the transfer of radiolabelled methyl groups from a prepared O6-methylguanine-DNA substrate to the protein fraction of an enzyme-containing cell/tissue extract. This assay, a modification of a previously suggested method for monitoring O6-ethylguanine-DNA repair [Renard, Verly, Mehta & Ludlum (1983) Eur. J. Biochem. 136, 461-467], is sensitive, highly reproducible, accurate and is, as described here and relative to previously published methods, well suited for use with a large number of test samples. We identified two problems with the O6-[Me-3H]methylguanine-DNA substrate used in the present work and in other reported assay: firstly, that of progressively higher assay backgrounds with increasing age of substrate, which was nullified by once-only purification of the double-stranded substrate by hydroxyapatite chromatography; secondly, a substrate of high specific radioactivity (30 Ci/mmol), made with freshly prepared tritiated methylnitrosourea, behaved as a substrate of 5 Ci/mmol when referenced against radiolabelled O6-methylguanine-DNA made with either [3H]- or [14C]-methylnitrosourea at the lower specific radioactivities of 1 Ci/mmol and 61 mCi/mmol respectively. This apparently stemmed from the known instability of high-specific-radioactivity [3H]methylnitrosourea and indicated that an expected increase in sensitivity of the assay does not necessarily result from increasing the specific radioactivity of substrates above approx. 1 Ci/mmol. Although O6-MT was stable to preincubation at 25 degrees C, marked losses of activity were observed at 37 degrees C, and more so at 45 degrees C. Enzyme lability at the higher temperatures was not, however, seen during preincubation in the presence of its substrate. O6-[Me-3H]methylguanine-DNA, which apparently protected O6-MT against thermal inactivation. As previously seen with other human cells and tissues, extracts of human spleen in the present study showed wide interindividual differences in O6-MT specific activity (18-fold), which spanned the range 50-900 fmol/mg of protein. Cultured human lymphoblastoid Jurkat cells contained approx. 57,000 enzyme molecules/cell. Substrate-inactivated [Me-3H]methylated O6-MT was analysed by SDS/PAGE and electroblotting. The different but similarly sized forms of this enzyme that we previously detected in human spleen [Major, Gardner, Carne & Lawley (1990) Nucleic Acids Res. 18, 1351-1359] were clearly resolved by fluorography of electroblots, but only at considerable expense of time. As expected, scintillation counting of the protein extracted from gel slices and linear-wire scanning of enzyme-associated radioactivity on electroblots were quicker methods for detecting the [Me-3H]methylated inactivated O6-MT.
Bacterial Proteins↗
Detection of aberrant p16 methylation in the serum of colorectal cancer patients.
OBJECTIVES: To detect aberrant p16 promoter methylation in serum of patients with colorectal cancer (CRC), and to explore the possibility of using this assay in early detection or as a prognostic marker. METHODS: Methylation-specific PCR was used to detect p16 methylation in DNA extracted from 52 CRCs and corresponding serum samples. Serum samples from 34 patients with adenomatous polyps and 10 healthy individuals were used as controls. The association of p16 hypermethylation in serum DNA of CRC patients with clinicopathological characteristics was analyzed. RESULTS: p16 methylation was found in 38% (20 of 52) of CRC tissues. Among the 20 patients with aberrant methylation in the tumor tissues, similar changes were also detected in the serum of 14 (70%) patients. No methylated p16 sequences were detected in the peripheral serum of the 32 CRC patients without these changes in the tumor, in 34 paitents with adenomatous polyps, or in 10 healthy controls. Clinicopathological analysis revealed that p16 methylation in serum was significantly associated with later Dukes' stage (chi(2) = 5.7, P = 0.03). CONCLUSION: This assay offers a potential means for the serum-based detection and/or monitoring of CRC patients.
Colorectal Neoplasms↗
Lack of evidence for methylation of parental and newly synthesized adenovirus type 2 DNA in productive infections.
Methylations of highly specific sites in the promoter and 5' regions of eucaryotic genes have been shown to shut off gene activity and thus play a role in the long-term regulation of gene expression. It was therefore of interest to investigate whether site-specific DNA methylations could also play a role in adenovirus DNA in productive infections. It has been reported earlier that adenovirion DNA is not detectably methylated (U. Günthert, M. Schweiger, M. Stupp, and W. Doerfler, Proc. Natl. Acad. Sci. USA 73:3923-3927, 1976). In the present study, evidence for the methylation of cytidine residues in 5'-CCGG-3' and 5'-GCGC-3' sequences or the methylation of adenine residues in 5'-GATC-3' and 5'-TCGA-3' sequences in intranuclear adenovirus type 2(Ad2) DNA isolated and analyzed early (5 h) or late (24 h) after infection could not be obtained. In Ad2 DNA, 22.5% of all 5'-CG-3' dinucleotides reside in 5'-CCGG-3' and 5'-GCGC-3' sequences. Intranuclear viral DNA was examined by restriction endonuclease cleavage by using HpaII, MspI, HhaI, DpnI, or TaqI and Southern blot hybridizations. The HindIII fragments of Ad2 DNA served as hybridization probes. The data rendered it very unlikely that free intracellular adenovirus DNA in productively infected cells was extensively methylated. Thus, DNA methylation was not a likely element in the regulation of free adenovirus DNA expression in productively infected cells.
Adenoviruses, Human↗
Methylational urinalysis: a prospective study of bladder cancer patients and age stratified benign controls.
Tumour suppressor gene (TSG) methylation has been proposed as a diagnostic marker for urothelial cancer (UC). Here, we compare the frequency of urinary TSG methylation in young and elderly patients, with and without UC. Urine samples were obtained prospectively from 35 UC patients, 35 benign controls over the age of 70 years and 34 healthy volunteers under the age of 40 years. Methylation analysis was performed for eight gene promoters using quantitative methylation-specific PCR. Methylation was detected in urine DNA from all three patient groups. The highest frequencies were seen in UC patients. Significantly less methylation was present in control samples than UC cases for RASSF1a and APC (P < 0.034). The 'methylation index' and level of methylation was highest in the UC group and lowest in the young control group. A marker panel of RASSF1a, E-cad and APC generated a sensitivity of 69%, a specificity of 60% and a diagnostic accuracy of 86%. TSG methylation is detectable in urine DNA from patients with and without bladder cancer. The frequency and extent of methylation appears to increase with age and malignancy. The lack of tumour specificity suggests that further investigation is required before this test is introduced into clinical practice.
Adult↗
Endogenous and background DNA adducts by methylating and 2-hydroxyethylating agents.
Detection of 7-alkylguanine DNA adducts is useful to assess human exposure to and the resulting DNA damage caused by simple alkylating agents. The background 7-methylguanine (7-MG) and 7-hydroxyethylguanine (7-HEG) adduct levels were determined in human and rat tissues, using thin-layer chromatography (TLC) combined with high pressure liquid chromatography (HPLC). In addition, these two adduct levels were also compared in various tissues between smokers and non-smokers. The results demonstrated that the background level of 7-alkylguanine adducts in WBC and lung tissues of non-smokers was 2.9 and 4.0 adducts/107 nucleotides, respectively. In smokers with lung cancers 7-MG adduct level in lung samples (6.3+/-1.9 adducts/107 nucleotides) and in bronchus samples (6.1+/-1.5 adducts/107 nucleotides) was significantly higher than that in WBC samples (3.3+/-0.9 adducts/107 nucleotides). 7-HEG adduct levels obtained from the same individuals were 0.8+/-0.3 in lung, 1.0+/-0.8 in bronchus and 0.6+/-0.2 adducts/107 nucleotides in WBC, respectively. Animal studies showed that background levels of 7-MG (2.1-2.5 adducts/107 nucleotides) in control rats were approximately 2-4-fold higher than 7-HEG levels (0.6-0.9 adducts/107 nucleotides). After a 3-day exposure to 300 ppm ethene, 7-HEG adducts accumulated to a similar extent in different tissues of rats, with the mean adduct level of 5.6-7.0 in liver, 7.4 in lymphocytes and 5.5 adducts/107 nucleotides in kidney.
Animals↗
Epigenetic profiling of circulating cell-free DNA for early detection and minimal residual disease assessment in lung cancer: a focus on DNA methylation.
Lung Cancer (LC) continues to be the biggest cause of cancer-related deaths around the world, mostly because of delayed diagnosis. Even if tissue biopsies and circulating tumor DNA (ctDNA) tests have revolutionized clinical management of LC patients, their effectiveness is restricted in settings with lower tumor burden, molecular heterogeneity, and bias in sampling approaches. In this scenario, the epigenetic profiling of cell-free DNA (cfDNA) stands out as a promising, less invasive approach, accurately detect cancer traces. Evidence from stage I-II disease and CT-detected pulmonary nodules supports the diagnostic potential of cfDNA methylation, although further validation in prospective screening cohorts remains necessary. Beyond genomic alterations, cfDNA epigenetic changes, including DNA methylation, chromatin organization, nucleosome positioning, and fragmentation patterns, reflect multi-dimensional complexity of tumor biology. These properties convey both the functional status and the origin of the circulating DNA fragments, accelerating for tumor integrating genomic analysis. Within this group, DNA methylation is the biologically robust and clinically well-established epigenetic marker, as alterations in methylation linked to cancer often occur in the early stages of tumorigenesis and are commonly found across different cancer cell types. Here, we explored the biological and clinical relevance of the epigenetic landscape of cfDNA in LC patients, particularly focusing on DNA methylation-based biomarkers and their evolving applications towards early diagnosis and post-surgical monitoring of minimal residual disease (MRD). We aimed to comprehensively overview analytical approaches for cfDNA methylation analysis, including targeted and genome-wide profiling strategies, and discuss their integration with machine learning (ML) and multi-omics frameworks in order to improve diagnostic performance and clinical applicability in LC management.
DNA methylation↗
Epigenetic profiling of synchronous colorectal neoplasias by quantitative DNA methylation analysis.
Transcriptional inactivation of tumor-suppressor genes by promoter CpG island methylation is thought to be an important mechanism in human carcinogenesis. The CpG island methylator phenotype (CIMP) with extensive promoter methylation appears to be a distinct epigenetic subtype of colorectal carcinoma. Most previous studies on CpG island methylation in colorectal carcinoma used methylation-specific PCR, which may detect low levels of DNA methylation with little or no biological significance. In contrast, quantitative DNA methylation assays have been shown to provide useful information beyond that which can be achieved with methylation-specific PCR. Synchronous neoplasias provide a unique model for investigators to examine molecular alterations in multistep tumorigenesis within one individual. However, no study to date has quantified DNA methylation of CIMP-specific promoters in synchronous colorectal neoplasias. Utilizing real-time PCR (MethyLight), we quantified DNA methylation in five CIMP-specific gene promoters [CACNA1G (calcium channel, voltage-dependent, T type alpha-1G subunit), CDKN2A (p16/INK4A), CRABP1 (cellular retinoic acid binding protein-1), MLH1 and NEUROG1 (neurogenin 1)] and MGMT in six synchronous carcinoma pairs (12 carcinomas) and eight synchronous carcinoma and adenoma pairs (16 tumors). We found that while some synchronous tumor pairs showed discordant promoter methylation patterns, other tumor pairs showed similar, but not exactly identical, patterns of promoter methylation. All but two pairs showed concordant patterns of CIMP status (CIMP positive vs CIMP negative) (P = 0.05 in cancer pairs). BRAF mutations were present in only CIMP-positive tumors. A high degree of microsatellite instability (MSI-H) was observed in both CIMP-positive and CIMP-negative tumors. KRAS mutations were not concordant in any synchronous neoplasia pair. In conclusion, epigenetic alterations at CIMP-specific promoter CpG islands in synchronous colorectal neoplasias likely have both random and nonrandom components.
Adenocarcinoma↗
Prognostic DNA methylation marker in serum of cancer patients.
Changes in the status of DNA methylation are among the most common molecular alterations in human neoplasia. Recent demonstrations of tumor-derived methylated DNA in the blood stream of cancer patients allow the use of these epigenetic markers for risk assessment in cancer patients. We were interested in evaluating the prognostic value of several methylated genes in the serum of cancer patients. Using MethyLight, a high-throughput DNA methylation assay, we analyzed 215 serum samples from patients with cervical (n = 93) or breast cancer (n = 122) for DNA methylation changes. In cervical cancer, hypermethylation of three genes (MYOD1, CDH1, and CDH13) in pretreatment sera was statistically significantly associated with a poorer disease outcome. Additionally, for the first time we used a so-called gene evaluation set to identify the most important DNA methylation changes in the serum of breast cancer patients from a long list of candidate genes. In the gene evaluation set, we detected five genes (ESR1, APC, HSD17B4, HIC1, and RASSF1A) using our criteria for further analysis. Finally, two of the evaluated genes (APC and RASSF1A) proved to be independent prognostic parameters in breast cancer patients. In summary, we detected several prognostic DNA methylation markers in the serum of cervical and breast cancer patients. This finding indicates great potential for the use of these epigenetic markers in clinical, routine risk assessment in patients with various malignancies.
Biomarkers, Tumor↗
Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression.
Inorganic arsenic, a human carcinogen, is enzymatically methylated for detoxication, consuming S-adenosyl-methionine (SAM) in the process. The fact that DNA methyltransferases (MeTases) require this same methyl donor suggests a role for methylation in arsenic carcinogenesis. Here we test the hypothesis that arsenic-induced initiation results from DNA hypomethylation caused by continuous methyl depletion. The hypothesis was tested by first inducing transformation in a rat liver epithelial cell line by chronic exposure to low levels of arsenic, as confirmed by the development of highly aggressive, malignant tumors after inoculation of cells into Nude mice. Global DNA hypomethylation occurred concurrently with malignant transformation and in the presence of depressed levels of S-adenosyl-methionine. Arsenic-induced DNA hypomethylation was a function of dose and exposure duration, and remained constant even after withdrawal of arsenic. Hyperexpressibility of the MT gene, a gene for which expression is clearly controlled by DNA methylation, was also detected in transformed cells. Acute arsenic or arsenic at nontransforming levels did not induce global hypomethylation of DNA. Whereas transcription of DNA MeTase was elevated, the MeTase enzymatic activity was reduced with arsenic transformation. Taken together, these results indicate arsenic can act as a carcinogen by inducing DNA hypomethylation, which in turn facilitates aberrant gene expression, and they constitute a tenable theory of mechanism in arsenic carcinogenesis.
Animals↗
Maternal inheritance of the mouse mitochondrial genome is not mediated by a loss or gross alteration of the paternal mitochondrial DNA or by methylation of the oocyte mitochondrial DNA.
To evaluate whether the absence or modification of paternal mitochondrial DNA or methylation of the oocyte mitochondrial DNA could be the molecular basis for maternal inheritance of mitochondria in mammals, the mitochondrial genome has been analyzed in four meiotic and postmeiotic testicular cell types, and in oocytes from the mouse. All four testicular cell types including spermatozoa contain mitochondrial DNA. Between meiosis and the end of spermatogenesis the number of mitochondrial genomes per haploid genome decreases 8- to 10-fold with spermatozoa containing approximately one copy of the mitochondrial genome per mitochondrion. Restriction enzyme digestions with six different enzymes indicate no gross differences in DNA sequence in the testicular mitochondrial DNA from meiotic cells, early haploid cells, late haploid cells, and spermatozoa. By the criterion of differential digestion with the isoschizomers, MspI and HpaII, the mitochondrial DNA is not differentially methylated during spermatogenesis. No methylation differences were detected in mitochondrial DNA from sperm and oocytes following digestion with seven methylation-sensitive restriction enzymes.
Animals↗