scholarly journals Influence of Mouse-Strain-Specific Factors on Position-Dependent Transgene DNA Methylation Patterns

1996 ◽  
Vol 45 (1-2) ◽  
pp. 243-244
Author(s):  
P.A. Koetsier ◽  
W. Doerfler
Keyword(s):  
Dna Methylation ◽  
Genetic Background ◽  
Promoter Activity ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Adenovirus Type ◽  
Methylation Pattern ◽  
Parent Of Origin ◽  
Methylation Patterns

In previous work from this laboratory, an inverse dependence was established for the adenovirus type 2 E2A late promoter between sequence-specific DNA methylation and promoter activity [1-5; for reviews see ref. 6, 7]. The effect of DNA methylation on promoter activity was also assessed in the transgenic mice, which were obtained from microinjections of unmethylated or in vitro HpaII-premethylated pAd2E2AL-CAT DNA [1] into F2 zygotes from B6D2F, (C57BL/6 × DBA/2) hybrid mice. In CAT assays carried out on organ extracts from the pAd2E2AL-CAT mice, the inverse relationship was confirmed [2].We studied the stability of the pAd2E2AL-CAT DNA methylation patterns in up to eight mouse generations and assessed the influence of the strain-specific genetic background. Three pAd2E2AL-CAT mouse lines were crossed with inbred DBA/2, C57BL/6 or B6D2F, mice. Parent-of-origin effects were controlled by exclusive hemizygous transgene transmission either via females or males. The founder animal of line 7-1 carried two groups of transgenes (A and B) on separate chromosomes. The transgene methylation patterns of the 7-1B transgenes and those of the lines 5-8 and 8-1 were stably transmitted.Southern blot hybridization experiments [8, 9] revealed that the 7-1A transgene methylation pattern was a cellular mosaic. In mixed-genetic-background offspring from 7-1A animals, 10% carried transgenes with HpaII-DNA methylation levels that were reduced from 40 to 10-15%. This finding suggested that in this background the factors that supported high methylation levels were dominant. When inbred DBA/2 mice were the mates, 40% of the siblings carried demethylated transgenes, whereas this ratio amounted to only 10% in C57BL/6 offspring (comparable to B6D2F1 crossings). Transgene methylation patterns were not detectably influenced by the parent-of-origin.

scholarly journals Genome-wide DNA methylation patterns reveal clinically relevant predictive and prognostic subtypes in osteosarcoma

2020 ◽  
Author(s):  
Christopher E. Lietz ◽  
Erik T. Newman ◽  
Andrew D. Kelly ◽  
Santiago A. Lozano-Calderon ◽  
David H. Ebb ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Methylation Pattern ◽  
Transcriptional Analysis ◽  
Chemotherapy Response ◽  
Methylation Array ◽  
Genome Wide ◽  
Recurrent Mutations ◽  
Methylation Patterns ◽  
Genomic Methylation

ABSTRACTBackgroundOsteosarcoma (OSA) is an aggressive malignancy predominantly affecting children and young-adults. Genetic analysis has characterized very few recurrent mutations in OSA, and an improved understanding of interpatient tumor heterogeneity is needed for clinical management.MethodsWe analyzed genome-wide DNA methylation in primary OSA tumors from the NCI Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program (n = 83) profiled using the Illumina 450K methylation array. We tested if broad genomic methylation predicted outcomes and defined supervised methylomic signatures predictive of Recurrence Free Survival (RFS), Chemotherapy Response (CR), and Metastatic disease at Diagnosis (MetDx). We assessed methylation pattern reproducibility in two independent clinical datasets (n = 28 and 34) and in an in vitro dataset (n = 11). Correlations between genomic methylation and transcription were tested using TARGET RNA-seq data. An in silico pharmacogenomic screen was performed to identify agents for future stratified application.ResultsGenome-wide methylation defined two subgroups. Relatively hypomethylated tumors experienced better chemotherapy response (Odds Ratio = 6.429, Fisher’s p = 0.007), longer RFS (metastatic, median 2.3 vs 26.7 months, localized, median 63.5 vs 104.7 months, stratified log-rank p = 0.006), and Overall Survival (p = 5×10-4) than hypermethylated tumors. Robust genomic methylation signatures predictive of RFS and CR were defined, and the signatures’ methylation patterns were reproducible in the independent clinical and in vitro datasets. The RFS signature was enriched for intragenic sites, whereas the CR signature and clinically relevant genome-wide methylation patterns were enriched for intergenic sites. Normal-tissue-like methylation patterns were associated with poor prognosis and in vitro analysis suggested that the methylation signatures are associated with tumor aggressiveness. Downstream transcriptional analysis revealed that genes annotated to the RFS methylation signature were also predictive survival. The transcriptional program represented in the RFS signature included several critical cellular pathways, whereas the CR signature was associated with much fewer known pathways, possibly reflecting a much broader cellular “methylation state” related to chemoresponse. A pharmacogenomic screen identified potential therapies, including epigenomic modifiers, for future stratified clinical application.ConclusionGenomic methylation offers insight into patient prognosis and could be a useful tool for developing alternate adjuvant therapeutic strategies.

Examination of the dynamics of global DNA methylation pattern establishment during spermatogenesiss

2007 ◽  
Vol 30 (4) ◽  
pp. 90
Author(s):  
Kirsten Niles ◽  
Sophie La Salle ◽  
Christopher Oakes ◽  
Jacquetta Trasler
Keyword(s):  
Dna Methylation ◽  
Germ Cell ◽  
Germ Cells ◽  
Epigenetic Modification ◽  
Methylation Pattern ◽  
Chromosome 9 ◽  
Specific Gene ◽  
Global Methylation ◽  
Dna Methylation Pattern ◽  
Methylation Patterns

Background: DNA methylation is an epigenetic modification involved in gene expression, genome stability, and genomic imprinting. In the male, methylation patterns are initially erased in primordial germ cells (PGCs) as they enter the gonadal ridge; methylation patterns are then acquired on CpG dinucleotides during gametogenesis. Correct pattern establishment is essential for normal spermatogenesis. To date, the characterization and timing of methylation pattern acquisition in PGCs has been described using a limited number of specific gene loci. This study aimed to describe DNA methylation pattern establishment dynamics during male gametogenesis through global methylation profiling techniques in a mouse model. Methods: Using a chromosome based approach, primers were designed for 24 regions spanning chromosome 9; intergenic, non-repeat, non-CpG island sequences were chosen for study based on previous evidence that these types of sequences are targets for testis-specific methylation events. The percent methylation was determined in each region by quantitative analysis of DNA methylation using real-time PCR (qAMP). The germ cell-specific pattern was determined by comparing methylation between spermatozoa and liver. To examine methylation in developing germ cells, spermatogonia from 2 day- and 6 day-old Oct4-GFP (green fluorescent protein) mice were isolated using fluorescence activated cell sorting. Results: As compared to liver, four loci were hypomethylated and five loci were hypermethylated in spermatozoa, supporting previous results indicating a unique methylation pattern in male germ cells. Only one region was hypomethylated and no regions were hypermethylated in day 6 spermatogonia as compared to mature spermatozoa, signifying that the bulk of DNA methylation is established prior to type A spermatogonia. The methylation in day 2 spermatogonia, germ cells that are just commencing mitosis, revealed differences of 15-20% compared to day 6 spermatogonia at five regions indicating that the most crucial phase of DNA methylation acquisition occurs prenatally. Conclusion: Together, these studies provide further evidence that germ cell methylation patterns differ from those in somatic tissues and suggest that much of methylation at intergenic sites is acquired during prenatal germ cell development. (Supported by CIHR)

scholarly journals Methylation pattern polymorphism of cyp19a in Nile tilapia and hybrids

2018 ◽  
Vol 13 (1) ◽  
pp. 327-334 ◽  
Author(s):  
Xiaowu Chen ◽  
Yonghua Zhao ◽  
Yudong He ◽  
Jinliang Zhao
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Methylation Level ◽  
Molecular Mechanisms ◽  
Methylation Pattern ◽  
Male Ratio ◽  
Sex Development ◽  
Pattern Polymorphism ◽  
Fish Hybrids

AbstractSkewed sex development is prevalent in fish hybrids. However, the histological observation and molecular mechanisms remain elusive. In this study, we showed that the interspecific hybrids of the two fish species, Oreochromis niloticus and Oreochromis aureus, had a male ratio of 98.02%. Microscopic examination revealed that the gonads of both male and female hybrids were developmentally retarded. Compared with the ovaries, the testes of both O. niloticus and hybrids showed higher DNA methylation level in two selected regions in the promoter of cyp19a, the gonadal aromatase gene that converts androgens into estrogens, cyp19a showed higher level gene expression in the ovary than in the testis in both O. niloticus and hybrid tilapia. Methylation and gene expression level of cyp19a were negative correlation between the testis and ovary. Gene transcription was suppressed by the methylation of the cyp19a promoter in vitro. While there is no obvious difference of the methylation level in testis or ovary between O. niloticus and hybrids. Thus, the DNA methylation of the promoter of cyp19a may be an essential component of the sex maintenance, but not a determinant of high male ratio and developmental retardation of gonads in tilapia hybrids.

Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination

1983 ◽  
Vol 3 (11) ◽  
pp. 1943-1948
Author(s):  
L J Kelly ◽  
R Kelly ◽  
H L Ennis
Keyword(s):  
Dictyostelium Discoideum ◽  
Spore Germination ◽  
Developmental Regulation ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Molecular Weights ◽  
Multiple Copies

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.

scholarly journals DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sebastian Canovas ◽  
Elena Ivanova ◽  
Raquel Romar ◽  
Soledad García-Martínez ◽  
Cristina Soriano-Úbeda ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Rna Seq ◽  
Methylation Analysis ◽  
Number Of Children ◽  
Methylation Patterns

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

scholarly journals Application of the MSAP Technique to Evaluate Epigenetic Changes in Plant Conservation

2020 ◽  
Vol 21 (20) ◽  
pp. 7459
Author(s):  
María Elena González-Benito ◽  
Miguel Ángel Ibáñez ◽  
Michela Pirredda ◽  
Sara Mira ◽  
Carmen Martín
Keyword(s):  
Dna Methylation ◽  
In Situ Conservation ◽  
Plant Conservation ◽  
Ex Situ ◽  
Conservation Strategies ◽  
Regenerated Plants ◽  
Before And After ◽  
Methylation Patterns

Epigenetic variation, and particularly DNA methylation, is involved in plasticity and responses to changes in the environment. Conservation biology studies have focused on the measurement of this variation to establish demographic parameters, diversity levels and population structure to design the appropriate conservation strategies. However, in ex situ conservation approaches, the main objective is to guarantee the characteristics of the conserved material (phenotype and epi-genetic). We review the use of the Methylation Sensitive Amplified Polymorphism (MSAP) technique to detect changes in the DNA methylation patterns of plant material conserved by the main ex situ plant conservation methods: seed banks, in vitro slow growth and cryopreservation. Comparison of DNA methylation patterns before and after conservation is a useful tool to check the fidelity of the regenerated plants, and, at the same time, may be related with other genetic variations that might appear during the conservation process (i.e., somaclonal variation). Analyses of MSAP profiles can be useful in the management of ex situ plant conservation but differs in the approach used in the in situ conservation. Likewise, an easy-to-use methodology is necessary for a rapid interpretation of data, in order to be readily implemented by conservation managers.

145 THE METHYLATION PATTERNS OF POTENTIAL DIFFERENTIALLY METHYLATED REGIONS IN BOVINE Xist, Impact, NDN, AND H19 GENES

2007 ◽  
Vol 19 (1) ◽  
pp. 190
Author(s):  
N. T. D'Cruz ◽  
K. J. Wilson ◽  
M. K. Holland
Keyword(s):  
Genomic Dna ◽  
Bisulfite Sequencing ◽  
Cpg Island ◽  
Methylation Pattern ◽  
Imprinted Genes ◽  
Aberrant Methylation ◽  
Differentially Methylated Regions ◽  
Methylation Patterns ◽  
The Impact

Clinical and laboratory-assisted reproductive techniques such as ICSI have recently been associated with an increased incidence of several syndromes associated with defects in genomic imprinting. Genomically imprinted genes are expressed from only one parental allele and act to regulate growth of the fetus and placenta and brain development/ function. Imprinted genes are controlled by differentially methylated regions (DMRs), whereby one parental allelle (i.e. either maternal or paternal) is epigenetically silenced via methylation. Studies conducted in vitro suggest that culture of embryos and embryo manipulations may perturb the imprinting process. In the current study, the genomic DNA methylation patterns of CpG islands within bovine H19 (27 CpGs analyzed), Impact (36 CpGs), NDN (22 CpGs), and Xist (21 CpGs) were analyzed by bisulfite sequencing. Genomic DNA from a female fibroblast cell line and sperm were chosen for analysis. Potential DMRs for the 4 genes were identified, and semi-nested PCR primers were designed surrounding those regions. Second-round PCR products (2 separate reactions) were mixed, subcloned, and sequenced (n ≥ 10). The fibroblast methylation pattern of the Xist DMR showed consistent methylation in 50% of sequenced clones, with no methylation observed in sperm. The H19 DMR in fibroblast DNA also showed consistent methylation in 25% of sequenced clones, with sperm DNA fully methylated. These results confirm previous studies showing that Xist and H19 are imprinted in cattle. Sequencing of the putative Impact DMR clones indicated no methylation in either cell type, suggesting no imprinting in cattle, tissue-specific imprinting, or that this CpG island (15 bp post ATG) is not the DMR that controls imprinted expression of the Impact gene. The NDN DMR (500 bp post ATG) in sperm was not methylated, whereas the fibroblast cells had a variable methylation pattern. This may be for the same reasons suggested for Impact, but the variability within the CpG island may also be due to in vitro culture conditions resulting in aberrant methylation. This possible culture effect is currently being confirmed through bisulfite sequencing of the gene in an adult tissue. The investigation of methylation patterns in oocytes is also underway. Together, the information gathered will be used to determine the imprinting status of several bovine genes and, in the future, whether any of these imprinted genes are responsible for the increased pregnancy loss and calf abnormalities associated with advanced reproductive technologies.

80 DNA METHYLATION OF INSULIN-LIKE GROWTH FACTOR 2 (IGF2) GENE IN DAY 14 IN VITRO-PRODUCED BOVINE EMBRYOS OF DIFFERENT SIZES

2015 ◽  
Vol 27 (1) ◽  
pp. 133
Author(s):  
J. O. Carvalho ◽  
M. M. Franco ◽  
G. M. Machado ◽  
M. A. N. Dode
Keyword(s):  
Dna Methylation ◽  
Growth Factor ◽  
Embryo Development ◽  
Methylation Pattern ◽  
Lower Percentage ◽  
Insulin Like Growth Factor ◽  
Bovine Embryos ◽  
Igf2 Gene ◽  
Exon 10

In mammals, a correct DNA methylation reprogramming and the maintenance of genomic imprinting after fertilization are essential for embryo development and pregnancy. One important imprinted gene, related to embryo development and placentation, is the insulin-like growth factor 2 (IGF2) gene. Therefore, embryos with different sizes could show differences in the methylation pattern of IGF2 gene. The aim of this study was to evaluate the methylation pattern of the differentially methylated region (DMR) located within exon 10 of the IGF2 gene, of in vitro-produced Nellore bovine embryos that were different in size on day D14 of development. The embryos were produced from oocytes obtained by follicular aspiration of slaughter house ovaries. On D7 after in vitro fertilization only grade I blastocysts were selected and, in groups of 10 embryos, were transferred non-surgically to the uteri of previously synchronized recipients with similar conditions. Seven days after being transferred, embryos were collected (Day 14 of development) and measured using Motic Images Plus 2.0 program (Motic, Richmond, BC, Canada). Embryos >45 mm were considered large (L) and those <25 mm were considered small (S). After being measured, a portion of each trophoblast layer was biopsied and used to determine the methylation status of the IGF2 gene by bisulfite sequencing. The methylation pattern was evaluated on individual embryos considered as separate replicates. At least 5 to 8 clones were evaluated per embryo and the sequences were analysed with the BiQAnalyser software (Max-Planck-Institut für Informatik, Saarbrücken, Germany), using the GenBank sequence NM_174087.3 as reference. The methylation pattern of the different groups was compared using Kruskal-Wallis test (P < 0.05). No differences in DNA methylation were found between S (26.7 ± 8.3%, n = 37 clones, 5 embryos) and L (34.8 ± 2.9%, n = 20 clones, 4 embryos) embryos. It is already known that the region studied is hypermethylated in sperm and hypomethylated in oocytes and, in some somatic cell types, it is expected to be around 50% methylated, being an imprinted region. Although we found a lower percentage of methylation than that expected for an imprinted region, this pattern may be the physiological pattern for trophoblast cells. This is the first report describing the methylation pattern of this region of the IGF2 gene in Day 14 bovine embryos of different sizes. It can be concluded that the methylation pattern of the intragenic DMR on exon 10 of IGF2 gene of in vitro-produced embryos on Day 14 of development is not affected by embryo size.This work was supported by CNPq, FAP-DF.

53 IMPACTS OF USING PROCAINE AS A DNA-DEMETHYLATING AGENT IN IN VITRO CULTURE OF BOVINE CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 132
Author(s):  
V. A. Michalczechen-Lacerda ◽  
F. C. Rodrigues ◽  
R. V. de Sousa ◽  
R. Rumpf ◽  
M. M. Franco
Keyword(s):  
Dna Methylation ◽  
Cell Viability ◽  
In Vitro Culture ◽  
Cytogenetic Analysis ◽  
Imprinted Genes ◽  
Demethylating Agent ◽  
Chromosome Integrity ◽  
Methylation Patterns ◽  
Number Of Cells

Euchromatin and heterochromatin organisation define the specificity of each cell type. This structure is controlled by epigenetic modifications and the DNA methylation is one of the best known for inducing transcriptional repression. Recently, procaine was uncovered as a DNA-demethylating agent, but there are few reports about its dynamic epigenetic action on somatic cells. Mono-allelic expression of imprinted genes is controlled by DNA methylation and inherited to somatic tissues of a sex-specific manner. The aim was to investigate the effects of using procaine, a DNA-demethylating agent, in in vitro culture of bovine (Bos taurus indicus) fibroblast for 72 h (passage 4). We have evaluated cell viability, chromosome integrity, and DNA methylation patterns. To evaluate cell viability, we have used trypan blue 0.4%. To evaluate chromosome integrity, we have used conventional cytogenetic analysis. To investigate DNA methylation patterns, we have analysed 2 differentially methylated regions (DMR) located into the exon 10 of IGF2 and exon 1 of XIST imprinted genes, using the bisulfite sequencing method (EZ DNA methylation kit, Zymo Research, Orange, CA, USA). After bisulfite treatment and nested-PCR, the amplicons were separated in agarose gel electrophoresis, purified with GenClean III kit (MP Biomedicals, Irvine, CA, USA), cloned in a pGEM-T easy vector system (Promega, Madison, WI), and sequenced. The DNA sequences were analysed using the BiQ Analyzer v. 2.0 (2008) software. The cell viability data were analysed using ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney tests, and the methylation status were analysed using Student’s t-test or Mann-Whitney tests in the Prophet software (BBN Systems and Technologies). Cell culture using 0.1 mM or 0.5 mM of procaine were viable and the number of cells with intact membrane was higher than the control and 2.0 mM of procaine groups (P ≤ 0.05). The total number of cells was lower in the group with 2.0 mM of procaine (P ≤ 0.01). Cytogenetic analysis showed no differences among the groups, with no chromosome abnormalities detected. The methylation pattern was not different for both DMR evaluated among the groups. We have observed that there was a beneficial effect to the cells that have received supplementation with 0.1 mM or 0.5 mM of procaine, because there was an increase in the number of viable cells without chromosomal abnormalities. We cannot ignore that a global DNA demethylation may have occurred, which was not detected in the specific analysed regions. The results obtained here may contribute to improving the efficiency of animal cloning, transgenic animal production, and the knowledge about stem cells. Supported by Embrapa Genetic Resources and Biotechnology and CAPES.

DNA methylation pattern during the encystment of Physarum flavicomum

1990 ◽  
Vol 68 (6) ◽  
pp. 944-948 ◽  
Author(s):  
Chengming Zhu ◽  
Henry R. Henney Jr.
Keyword(s):  
Dna Methylation ◽  
High Performance ◽  
Restriction Enzymes ◽  
Methylation Pattern ◽  
Dna Base Composition ◽  
Metabolic Role ◽  
Growing Cell ◽  
Dna Base ◽  
Intracellular Content ◽  
Methylation Patterns

In Physarum flavicomum Berk., haploid myxamoebae convert to dormant microcysts under conditions of nutrient imbalance. Exogenous adenine increases the intracellular content of S-adenosylmethionine (SAM) and inhibits this process. However, treatments that reduce the intracellular SAM levels relieve the inhibition of encystment induced by adenine. SAM plays a major metabolic role in cellular transmethylation reactions. In this study, we compared the DNA methylation patterns of growing cells, encysting cells, adenine-inhibited cells, and cysts using three different approaches: incubation of the cells with [14C]methylmethionine and detection of the labeled methyl group in purified DNA samples; analyses of DNA base composition by high performance liquid chromatography; and restriction endonuclease analyses of DNA. We found that DNA from the adenine-treated cells was labelled 1.3 times more with [14C]methylmethionine than was the DNA of untreated encysting cells. The DNA G + C content of this species was about 41%. The DNA of growing cells had the highest 5-methylcytosine (5MC) content, while DNA from the cysts had the lowest (about 27% that of growing cells). Adenine-inhibited cells had about 1.2 times more DNA-5MC than did encysting cells. Using the restriction enzymes SmaI, PvuI, and XhoI (which are inhibited by C residue methylation), we found that cyst DNA had more cutting sites than did amoebal DNA. By using the restriction enzyme DpnI which cuts DNA at GmATC sites, we found that cyst DNA, but not growing cell DNA, contained N6-methyladenine.Key words: amoebae, cysts, methylation, 5-methylcytosine, N6-methyladenine, DNA, encystment, Physarum flavicomum, development, inhibition.

Sign in / Sign up

Export Citation Format

Share Document