低剂量重离子辐射对水稻种子和幼苗DNA甲基化的影响

为了研究低剂量重离子辐射对植物产生的表观遗传学效应,采用高传能线密度（62．2keV／μm）和低剂量（2Gy）的放射性束流^12C对水稻（Oryzasativa,japonica）的干种子和幼苗进行辐射。采用甲基化敏感限制性酶切多态性分析（MSAP）的方法对材料基因组CCGG位点的甲基化状态进行检测。共选用12对选择性引物扩增了共800个条带,其中有65个条带（8．13％）在种子辐射后发现呈多态性,而只有10个条带（1．3％）在幼苗辐射后发现呈多态性。统计学分析显示高能量低剂量的重离子对水稻种子和幼苗的基因组甲基化状态都产生了影响,而且种子受到辐射后产生的甲基化改变明显高于幼苗（P=0．011）。另外,甲基化和去甲基化变化类型的分析也表明种子和幼苗辐射后发生甲基化变化的趋势也不相同。     

组织培养导致的草莓DNA甲基化变异

以草莓品种‘丰香’和‘全明星’为材料,用甲基化敏感扩增多态性（MSAP）技术研究组织培养对草莓DNA甲基化的影响。结果表明,与普通苗相比,组织培养导致草莓试管苗的DNA甲基化水平下降,甲基化模式的变异以去甲基化为主。组织培养导致的DNA甲基化变异不稳定,在田间无性繁殖过程中,试管苗的无性繁殖后代DNA甲基化水平逐渐升高,仅部分变异的甲基化模式能够在试管苗的无性繁殖后代中稳定传递。两个品种之间,纽织培养对DNA甲基化变异程度的影响不同。     

Daily Variation in Global and Local DNA Methylation in Mouse Livers

DNA methylation is one of the best-characterized epigenetic modifications and has an important biological relevance. Here we showed that global DNA methylation level in mouse livers displayed a daily variation where the peak phases occurred during the end of the day and the lowest level at the beginning of the day in the light-dark or dark-dark cycles. Typical repeat sequence long interspersed nucleotide element-1 (LINE-1) had a similar methylation rhythm to global DNA. DNA methyltransferase 3A (DNMT3A) and ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) brought a relative forward daily variation to global DNA methylation, and the temporary change in ratio of SAM to SAH had no influence on the DNA methylation level. The rhythm of global DNA methylation was lost and DNA methylation level was increased in Per1^-/-Per2^-/- double knockout mice, which were in accordance with changes of Dnmt3a mRNA levels and its rhythm. Our results suggest that the daily variation in global DNA methylation was associated with the change of Dnmt3a expression rather than ratio of SAM to SAH.     

辐射诱导小鼠淋巴瘤细胞凋亡涉及NADPH氧化酶的活性

用60Coγ射线对小鼠淋巴瘤细胞3SB(p53 / )和1B1C4(p53-/-)进行照射处理。观察了辐射后细胞形态学变化,结果发现,3SB对γ射线诱导的凋亡非常敏感,而1B1C4细胞对辐射具有抗性。DNA片断化试验和藻红B染色试验的结果也证实这一点。进一步分析了凋亡信号途径上的caspase-3和p53。在3SB细胞中,检测到激活的caspase-3并发现p53的表达随辐射剂量的增加而增高。比较了两种细胞中caspase-3mRNA的表达,但未见差异。对两种细胞中的NADPH氧化酶活性的测定结果表明,凋亡的3SB细胞中的NADPH氧化酶活性增高。因此,在电离辐射诱导细胞凋亡的过程中,NADPH氧化酶参与了上游信号的转导。     

Synthesis of Immunoglobulins by Rat Thymus after Thymic Irradiation

THE thymus distinctively lacks plasma cells and pyroninophilic precursors, even after massive parenteral injection of protein^1–3. Direct injection of antigens into the thymus in guinea-pigs, however, can induce the formation of germinal centres, plasma cells and the production of antibodies⁴. An increase in the plasma cell population has been observed in the spleen, lymph nodes and bone marrow of chimpanzees⁵, rats^6–9 and germ-free mice¹⁰ after whole body irradiation.     

Consistent and Heritable Alterations of DNA Methylation Are Induced by Tissue Culture in Maize

Plants regenerated from tissue culture and their progenies are expected to be identical clones, but often display heritable molecular and phenotypic variation. We characterized DNA methylation patterns in callus, primary regenerants, and regenerant-derived progenies of maize using immunoprecipitation of methylated DNA (meDIP) to assess the genome-wide frequency, pattern, and heritability of DNA methylation changes. Although genome-wide DNA methylation levels remained similar following tissue culture, numerous regions exhibited altered DNA methylation levels. Hypomethylation events were observed more frequently than hypermethylation following tissue culture. Many of the hypomethylation events occur at the same genomic sites across independent regenerants and cell lines. The DNA methylation changes were often heritable in progenies produced from self-pollination of primary regenerants. Methylation changes were enriched in regions upstream of genes and loss of DNA methylation at promoters was associated with altered expression at a subset of loci. Differentially methylated regions (DMRs) found in tissue culture regenerants overlap with the position of naturally occurring DMRs more often than expected by chance with 8% of tissue culture hypomethylated DMRs overlapping with DMRs identified by profiling natural variation, consistent with the hypotheses that genomic stresses similar to those causing somaclonal variation may also occur in nature, and that certain loci are particularly susceptible to epigenetic change in response to these stresses. The consistency of methylation changes across regenerants from independent cultures suggests a mechanistic response to the culture environment as opposed to an overall loss of fidelity in the maintenance of epigenetic states.     

Cytosine Methylation Changes in Rice Centromeric and Telomeric Sequences Induced by Foreign DNA Introgression

Cytosine methylation changes (hyper- or hypomethylation) in centromeric and telomeric sequences were observed in all three studied rice introgression lines containing DNA from wild rice, Zizania latifolia Griseb. The changed genomic Southern hybridization patterns were complex and non-concordant between a pair of isoschizomers (HpaII/MspI) digests, indicating methylation modifications at both the inner and outer cytosines of the CCGG sites. The changed patterns were inherited through generations. Possible mechanism for the methylation changes and their potential implications for the phenotypic variation and genome organization are discussed.     

DNA Methylation in Mouse Spermatozoa under Long-Term Modeling the Effects of Microgravity

Russian Journal of Developmental Biology - The mechanisms of the interaction between cells and the gravitational field are still unknown, and there are hardly any data on the effect of the gravity...     

辐射所致小鼠肾脏氧化损伤及川芎嗪的保护作用

目的:研究川芎嗪对辐射所致小鼠肾脏氧化损伤的预防和治疗作用。方法:采用60Co-γ射线5 Gy全身单次照射小鼠造模,在照射前和照射后分别于每天腹腔注射川芎嗪130 mg/kg,连续给药10 d,进行预防和治疗,并设对照组,观察肾组织中丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、还原型谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-Px)及总抗氧化力(T-AOC)的变化。结果:与阴性对照组比较,照射可显著增加肾组织中MDA的含量(P<0.05),降低SOD、CAT的活性(P<0.05),升高GSH-Px活性(P<0.05),降低GSH含量(P<0.05),使肾组织T-AOC下降(P<0.05),。与照射组比较,给予川芎嗪预防和治疗后,均可降低肾组织MDA含量(P<0.05),升高肾组织T-AOC(P<0.05),且治疗组优于预防组,与阴性对照组无显著性差异。同时,预防组可使SOD活性和GSH含量升高(P<0.05),治疗组可使SOD和CAT活性增高(P<0.05),但均对GSH-Px活性无显著影响(P>0.05)。结论:川芎嗪具有很好的抗氧化作用,无论预防和治疗均可降低辐射所致小鼠肾脏的氧化应激损伤,并且治疗效果优于预防效果。     

Changes in the Level of DNA Damage in Mouse Cells Induced by Atmospheric Factors

Biophysics - Abstract—Changes in the level of DNA damage induced by atmospheric factors were evaluated from the percentage of tail DNA (%TDNA) in the comet assay of mouse cells for blood...     

Methylation analysis by DNA immunoprecipitation

DNA methylation regulates gene expression primarily through modification of chromatin structure. Global methylation studies have revealed biologically relevant patterns of DNA methylation in the human genome affecting sequences such as gene promoters, gene bodies, and repetitive elements. Disruption of normal methylation patterns and subsequent gene expression changes have been observed in several diseases especially in human cancers. Immunoprecipitation (IP)‐based methods to evaluate methylation status of DNA have been instrumental in such genome‐wide methylation studies. This review describes techniques commonly used to identify and quantify methylated DNA with emphasis on IP based platforms. In an effort to consolidate the wealth of information and highlight critical aspects of methylated DNA analysis, sample considerations, experimental and bioinformatic approaches for analyzing genome‐wide methylation profiles, and the benefit of integrating DNA methylation data with complementary dimensions of genomic data are discussed. J. Cell. Physiol. 222: 522–531, 2010. © 2009 Wiley‐Liss, Inc.     

cis-Acting Signal for Inheritance of Imprinted DNA Methylation Patterns in the Preimplantation Mouse Embryo

The inheritance of gametic methylation patterns is a critical event in the imprinting of genes. In the case of the imprinted RSVIgmyc transgene, the methylation pattern in the unfertilized egg is maintained by the early mouse embryo, whereas the sperm’s methylation pattern is lost in the early embryo. To investigate the cis-acting requirements for this preimplantation stage of genomic imprinting, we examined the fate of different RSVIgmyc methylation patterns, preimposed on RSVIgmyc and introduced into the mouse zygote by pronuclear injection. RSVIgmyc methylation patterns with a low percentage of methylated CpG dinucleotides, generated by using bacterial cytosine methylases with four-base recognition sequences, were lost in the early embryo. In contrast, methylation was maintained when all CpG dinucleotides were methylated with the bacterial SssI (CpG) methylase. This singular maintenance of RSVIgmyc methylation preimposed with SssI methylase appears to be specific to the early, undifferentiated embryo; differentiated NIH 3T3 fibroblasts transfected with methylated versions of RSVIgmyc maintained all methylation patterns, independent of the level of preimposed methylation. The methylation pattern of the RSVIgmyc allele in adult founder transgenic mice that was produced by pronuclear injection of an SssI-methylated construct could not be distinguished from the maternal RSVIgmyc methylation pattern. Thus, a highly methylated allele in adult mice, normally generated by transmission of RSVIgmyc through the female germ line, was also produced in founder transgenic mice by bypassing gametogenesis and introducing a highly methylated RSVIgmyc into the mouse zygote. These results suggest that RSVIgmyc methylation itself is a cis-acting signal for the preimplantation maintenance of the oocyte’s methylation pattern and, therefore, a cis-acting signal for RSVIgmyc imprinting. Furthermore, our inability to identify a sequence element within RSVIgmyc that was absolutely required for its imprinting suggests that the extent of RSVIgmyc methylation, rather than a particular pattern of methylation, is the principal feature of this imprinting signal.     

重离子辐照酿酒酵母DNA损伤修复途径研究进展

重离子辐照通过直接和间接作用导致生物体DNA产生损伤,包括DNA的链断裂、碱基的插入或丢失以及氧化损伤等.DNA损伤直接影响复制、转录和蛋白质合成,同时还是突变的重要原因,因此,DNA损伤修复系统尤为重要.在酿酒酵母中,这些损伤主要是通过同源重组修复(homologous recombination repair,HRR)、碱基错配修复(mismatch repair,MMR)和碱基切除修复(base excision repair,BER)等途径来修复的.作为真核生物研究的模式生物,对于酿酒酵母DNA损伤修复的HRR、MMR和BER途径研究颇多,也不断有一些新的成果出现,特别是对于相关途径的完善和相关蛋白的深化更是研究热点,在此对近年来有关重离子辐照酿酒酵母DNA损伤修复途径方面的研究做一综述.     

硼替佐米增强P16蛋白表达诱导骨髓瘤细胞衰老

目的：研究蛋白酶体抑制剂硼替佐米诱导骨髓瘤RPMI8226、MMH929细胞衰老作用,并进一步探讨其作用机制。方法：硼替佐米0.1-100nmol/L处理骨髓瘤RPMI8226、MMH929细胞48、72h,MTT法检测细胞存活率、药物IC50值。选择药物IC50值1/10剂量处理骨髓瘤RPMI8226、MMH929细胞0、24、48H后检测衰老相关β-半乳糖苷酶染色率。流式细胞术检测细胞周期情况及凋亡率。Western-blot检测相关蛋白表达。结果：硼替佐米处理骨髓瘤细胞RPMI8226、MMH929后48小时IC50值：RPMI8226：19.05 nmol/L,MMH929：18.45nmol/L。以硼替佐米2 nmol/L处理骨髓瘤RPMI8226、MMH929细胞0、24、48H后发现β-半乳糖苷酶染色率、细胞G0/G1期比例明显上升与药物作用时间呈正相关,Western-blot检测细胞周期调控蛋白发现P53、PTEN蛋白无变化,P16蛋白与药物作用时间正相关。结论：硼替佐米通过增强P16蛋白表达诱导骨髓瘤细胞RPMI8226、H929衰老。     

Role of DNA Methylation in Cell Cycle Arrest Induced by Cr (VI) in Two Cell Lines

Hexavalent chromium [Cr(IV)], a well-known industrial waste product and an environmental pollutant, is recognized as a human carcinogen. But its mechanisms of carcinogenicity remain unclear, and recent studies suggest that DNA methylation may play an important role in the carcinogenesis of Cr(IV). The aim of our study was to investigate the effects of Cr(IV) on cell cycle progress, global DNA methylation, and DNA methylation of p16 gene. A human B lymphoblastoid cell line and a human lung cell line A549 were exposed to 5–15 µM potassium dichromate or 1.25–5 µg/cm² lead chromate for 2–24 hours. Cell cycle was arrested at G₁ phase by both compounds in 24 hours exposure group, but global hypomethylation occurred earlier than cell cycle arrest, and the hypomethylation status maintained for more than 20 hours. The mRNA expression of p16 was significantly up-regulated by Cr(IV), especially by potassium dichromate, and the mRNA expression of cyclin-dependent kinases (CDK4 and CDK6) was significantly down-regulated. But protein expression analysis showed very little change of p16 gene. Both qualitative and quantitative results showed that DNA methylation status of p16 remained unchanged. Collectively, our data suggested that global hypomethylation was possibly responsible for Cr(IV) - induced G₁ phase arrest,but DNA methylation might not be related to up-regulation of p16 gene by Cr(IV).     

DNA Methylation in Mammals

DNA methylation is one of the best characterized epigenetic modifications. In mammals it is involved in various biological processes including the silencing of transposable elements, regulation of gene expression, genomic imprinting, and X-chromosome inactivation. This article describes how DNA methylation serves as a cellular memory system and how it is dynamically regulated through the action of the DNA methyltransferase (DNMT) and ten eleven translocation (TET) enzymes. Its role in the regulation of gene expression, through its interplay with histone modifications, is also described, and its implication in human diseases discussed. The exciting areas of investigation that will likely become the focus of research in the coming years are outlined in the summary.     

植物DNA甲基化

DNA甲基化是造成植物转录水平基因沉默的主要原因。从DNA甲基化的发生机理,DNA甲基化抑制基因转录以及调控基因转录的方式简要地介绍了真核生物中DNA甲基化的功能和调控机制方面的一些研究进展。