Self-renaturing fractions in the separated strands of mouse satellite deoxyribonucleic acid.
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ABSTRACT:
SUBMITTER: Sutton WD
PROVIDER: S-EPMC1173754 | biostudies-other | 1972 Jun
REPOSITORIES: biostudies-other
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Project description:1. DNA polymerase activity is present in both nuclear and supernatant fractions prepared from rapidly dividing L929 mouse cells. 2. Nuclear preparations are 2-5 times more active with added native DNA as template and the supernatant fractions show an equivalent preference for heat-denatured DNA. 3. Isolated nuclei can carry on limited DNA synthesis in the absence of added template but are stimulated five- to ten-fold by addition of 50mug of native DNA per assay. 4. DNA polymerase activity can be released from intact nuclei by ultrasonic treatment or by extraction with 1.5m-potassium chloride. 5. The activities in nuclear and supernatant fractions, with their preferred templates, respond similarly to changes in pH and Mg(2+) and K(+) concentrations. 6. Maximal enzyme activity is approached with 40mug of DNA per assay and activation of the DNA template by treatment with deoxyribonuclease does not decrease the amount of DNA required to reach saturation. 7. The nuclear enzyme, incubated with native DNA, is markedly inhibited by the addition of heat-denatured DNA to the assay. In contrast, the supernatant DNA polymerase activity on denatured templates is not affected by the presence of native DNA. 8. The nuclear enzyme exhibits high activity in the absence of one or more deoxyribonucleoside triphosphates but this is much diminished after partial purification of the enzyme by precipitation at pH5 and fractionation on Sephadex G-200 columns. 9. The (3)H-labelled DNA products formed by Sephadex-purified nuclear and supernatant fractions, with their preferred templates, were found to be resistant to treatment with exonuclease I. Alkali-denaturation of the (3)H-labelled DNA products rendered them susceptible to attack by exonuclease I. 10. Analysis of the products on alkaline sucrose density gradients suggests that the newly synthesized material may not be covalently bound to the original DNA template. 11. By using their preferred templates the specific activity of supernatant fractions varies markedly with the position of the cells in the cell-cycle, but the specific activity of nuclear fractions varies only slightly.
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Project description:Purified satellite DNA from melon (Cucumis melo) was shown to contain at least two components from thermal-denaturation and renaturation studies. Two components were separated after partial renaturation, a fast-renaturing fraction similar in complexity to mouse satellite DNA, and one with 6000 times greater complexity. Both components renatured very accurately, indicating a minimum of sequence divergence. Centrifugation of the purified satellite DNA in Ag+/Cs2SO4 gradients resolved two major and several minor fractions. The two major fractions were only slightly enriched for fast- or slow-renaturing sequences.
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Project description:Two different methods were used to isolate high-molecular-weight heavy satellite DNA from the spermatozoa of the sea-urchin Lytechinus variegratus. Purification of satellite rDNA (ribosomal DNA) by selective denaturation of the spermatozoal DNA followed by a separation of the native and denatured DNA in an aqueous mixture of poly(ethylene glycol)/dextran is affected by the molecular weight of the spermatozoal DNA. Enrichment of satellite rDNA by selective precipitation of main-band DNA by poly-L-lysine is cumbersome but more suitable.
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Project description:Aberrant methylation has been regarded as a hallmark of cancer. 5-hydroxymethylcytosine (5hmC) is recently identified as the ten-eleven translocase (ten-eleven translocase)-mediated oxidized form of 5-methylcytosine, which plays a substantial role in DNA demethylation. Cell-free DNA has been introduced as a promising tool in the liquid biopsy of cancer. There are increasing evidence indicating that 5hmC in cell-free DNA play an active role during carcinogenesis. However, it remains unclear whether 5hmC could surpass classical markers in cancer detection, treatment, and prognosis. Here, we systematically reviewed the recent advances in the clinic and basic research of DNA 5-hydroxymethylation in cancer, especially in cell-free DNA. We further discuss the mechanisms underlying aberrant 5hmC patterns and carcinogenesis. Synergistically, 5-hydroxymethylation may act as a promising biomarker, unleashing great potential in early cancer detection, prognosis, and therapeutic strategies in precision oncology.
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Project description:XAD-8 adsorption technique coupled with stepwise elution using pyrophosphate buffers with initial pH values of 3, 5, 7, 9, and 13 was developed to isolate Chinese standard fulvic acid (FA) and then separated the FA into five sub-fractions: FApH3, FApH5, FApH7, FApH9 and FApH13, respectively. Mass percentages of FApH3-FApH13 decreased from 42% to 2.5%, and the recovery ratios ranged from 99.0% to 99.5%. Earlier eluting sub-fractions contained greater proportions of carboxylic groups with greater polarity and molecular mass, and later eluting sub-fractions had greater phenolic and aliphatic content. Protein-like components, as well as amorphous and crystalline poly(methylene)-containing components were enriched using neutral and basic buffers. Three main mechanisms likely affect stepwise elution of humic components from XAD-8 resin with pyrophosphate buffers including: 1) the carboxylic-rich sub-fractions are deprotonated at lower pH values and eluted earlier, while phenolic-rich sub-fractions are deprotonated at greater pH values and eluted later. 2) protein or protein-like components can be desorbed and eluted by use of stepwise elution as progressively greater pH values exceed their isoelectric points. 3) size exclusion affects elution of FA sub-fractions. Successful isolation of FA sub-fractions will benefit exploration of the origin, structure, evolution and the investigation of interactions with environmental contaminants.
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Project description:Organization of gold nanoobjects by oligonucleotides has resulted in many three-dimensional colloidal assemblies with diverse size, shape, and complexity; nonetheless, autonomous and temporal control during formation remains challenging. In contrast, living systems temporally and spatially self-regulate formation of functional structures by internally orchestrating assembly and disassembly kinetics of dissipative biomacromolecular networks. We present a novel approach for fabricating four-dimensional gold nanostructures by adding an additional dimension: time. The dissipative character of our system is achieved using exonuclease III digestion of deoxyribonucleic acid (DNA) fuel as an energy-dissipating pathway. Temporal control over amorphous clusters composed of spherical gold nanoparticles (AuNPs) and well-defined core-satellite structures from gold nanorods (AuNRs) and AuNPs is demonstrated. Furthermore, the high specificity of DNA hybridization allowed us to demonstrate selective activation of the evolution of multiple architectures of higher complexity in a single mixture containing small and larger spherical AuNPs and AuNRs.
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Project description:Diethylstillboestrol, a synthetic and carcinogenic hormone, binds to DNA as a result of incubation with a liver microsomal preparation in vitro and on incubation with primary mouse foetal cells in culture. Enzymic digestion of DNA samples thus prepared gives several covalent deoxyribonucleoside-diethylstilboestrol products from the microsomal system. One of these is produced in small but significant yield in the tissue-culture system.
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Project description:Enhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells and during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates such as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes.
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Project description:ObjectiveTo investigate whether deoxyribonucleic acid (DNA) methylation at birth and in childhood differ by conception using assisted reproductive technologies (ART) or ovulation induction compared with those in children conceived without fertility treatment.DesignUpstate KIDS is a matched exposure cohort which oversampled on newborns conceived by treatment.SettingNew York State (excluding New York City).Patient(s)This analysis included 855 newborns and 152 children at approximately 9 years of age.Intervention(s)None.Main outcome measure(s)DNA methylation levels were measured using the Illumina EPIC platform. Single CpG and regional analyses at imprinting genes were conducted.Result(s)Compared to no fertility treatment, ART was associated with lower mean DNA methylation levels at birth in 11 CpGs (located in/near SYCE1, SPRN, KIAA2013, MYO1D, GET1/WRB-SH4BGR, IGF1R, SORD, NECAB3/ACTL10, and GET1) and higher mean methylation level in 1 CpG (KLK4; all false discovery rate P<.05). The strongest association (cg17676129) was located at SYCE1, which codes for a synaptonemal complex that plays a role in meiosis and therefore infertility. This CpG remained associated with newborn hypomethylation when the analysis was limited to those conceived with ICSI, but this may be because of underlying male infertility. In addition, nine regions in maternally imprinted genes (IGF1R, PPIEL, SVOPL GNAS, L3MBTL, BLCAP, HYMAI/PLAGL1, SNU13, and MEST) were observed to have decreased mean DNA methylation levels among newborns conceived by ART. In childhood, hypomethylation of the maternally imprinted gene, GNAS, persisted. No CpGs or regions were associated with ovulation induction.Conclusion(s)ART but not ovulation induction was associated with hypomethylation at birth, but only one difference at an imprinting region appeared to persist in childhood.
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