电离辐射诱导的DNA双链断裂

利用γ射线和不同ＬＥＴ的碳离子幅照小鼠Ｂ１６黑色素瘤细胞、Ｈｅｌａ细胞、Ｖ７９中国仓鼠肺细胞和人肝癌ＳＭＭＣ－７７２１细胞的ＤＮＡ,采用脉冲场凝电泳结合荧光扫描技术研究了ＤＮＡ双链断裂（ＤＳＢ）片段的分布。结果发现ＤＳＢ片段是非随机分布的,而且这种分布与ＤＮＡ序列有关。原因可能在于沉积的能量直接或间接沿ＤＮＡ链迁移,链上相对较弱的化学键优先产生反应,并最终导致链的断裂,从而引起断裂的不均匀分布。     

H_2O_2－Fe~（3＋）所致人淋巴细胞DNA双链断裂损伤

采用脉冲电场凝胶电泳法检测Ｈ２Ｏ２－Ｆｅ（３＋）体系产生的ＯＨ对人淋巴细胞ＤＮＡ的双链断裂损伤．Ｈ２Ｏ２－Ｆｅ（３＋）浓度与ＤＮＡ双链断裂呈明显量效关系;随ＯＨ作用时间延长,细胞ＤＮＡ双链断裂加重;过氧化氢酶对ＯＨ损伤有明显抑制作用．脉冲电场凝胶电泳法可检测到的Ｈ２Ｏ２和ＦｅＣｌ３引起细胞ＤＮＡ双链断裂的最低浓度为０．３ｍｍｏｌ／Ｌ和６μｍｏｌ／Ｌ．     

碳离子诱导的DNA双链断裂

ＤＮＡ双链断裂（ＤＳＢｓ）是电离辐射诱导的最重要的原发损伤,研究ＤＳＢｓ有利于揭示细胞辐射敏感性的机理。用倒转脉冲场凝胶电泳结合荧光扫描进行ＤＮＡ定量研究７５ＭｅＶ／ｕ１２Ｃ６＋对小鼠Ｂ１６黑色素瘤细胞ＤＳＢｓ的诱导,结果表明：ＤＳＢｓ产额约为０．７４ＤＳＢｓ／１００Ｍｂｐ／Ｇｙ;ＤＮＡ片段分布在两个区域。大片段区分子量约为１．４Ｍｂｐ～３．２Ｍｂｐ,分子量小于１．２Ｍｂｐ的为小片段区;并且随着剂量的增加,大片段区ＤＮＡ含量逐渐下降,而小片段区的ＤＮＡ含量显著增加。表明Ｂ１６ＤＮＡ分子上可能存在对重离子较为敏感的位点。     

交变脉冲电场凝胶电泳与植物大分子DNA的制备

介绍了交变脉冲电场凝胶电泳的原理、方法及其在植物大分子ＤＮＡ制备方面的应用     

交变脉冲电场凝胶电泳与植物大分子DNA的制备

介绍了交变脉冲电场凝胶电泳的原理、方法及其在植物大分子DNA制备方面的应用。     

H2O2－Fe3+所致人淋巴细胞DNA双链断裂损伤

采用脉冲电场凝胶电泳法检测H₂O₂－Fe³⁺体系产生的OH^·对人淋巴细胞DNA的双链断裂损伤．H₂O₂－Fe³⁺浓度与DNA双链断裂呈明显量效关系;随OH^·作用时间延长,细胞DNA双链断裂加重;过氧化氢酶对OH^·损伤有明显抑制作用．脉冲电场凝胶电泳法可检测到的H₂O₂和FeCl₃引起细胞DNA双链断裂的最低浓度为0.3 mmol/L和6 μmol/L．     

用脉冲电场凝胶电泳分析棉病囊霉及其突变体的染色体DNA

采用交变脉冲电场凝胶电泳和碱变性交变脉冲电场凝胶电泳方法,分析了棉病囊霉酵母菌及其2个不同的突变菌株的核型,得知此菌株含有5条染色体 DNA,而2株突变体的染色体 DNA 都没有大片段的缺失或双链断裂,但其稳定性不如野生型菌株的 DNA,而且存在单链断裂等碱不稳定性位点.     

DNA断裂检测方法──单细胞凝胶电泳法

单细胞凝胶电泳(single cell gel electrophoresis assay,SCGE)也叫彗星试验(comet assay),是一种快速、敏感、简便、廉价的检测单个哺乳动物细胞DNA断裂的技术,目前已用于检测氧化、紫外线和电离辐射引起的损伤,以及三氯乙烷、丙烯酰胺等化学物及老化、吸烟所致损害的研究.文章介绍SCGE的发展、检测分析方法、原理及其在DNA损伤与修复、生物监测、遗传毒理研究、肿瘤治疗方案优化和疗效研究方面的应用前景．     

电离辐射诱导DNA链断裂的动力学研究

通过测试γ射线辐照下超螺旋ｐＢＲ３２２ ＤＮＡ分子单链断裂（ＳＳＢ）,双链剂量效应,得到ＳＳＢ、αＤＳＢ产额与ＤＮＡ现含一定浓度甘露醇的ＤＮＡ溶液体系中,Ｇ（ＳＳＢ）、Ｇ（αＤＳＢ）的例数与ｃ（ＤＮＡ）的倒数成线性关系,并以二级动力学描述了ＤＮＡ和甘露醇分子对．ＯＨ的竞争反应,得到．ＯＨ引起ＳＳαＤＳＢ的速率常数及其效率。     

Interaction of Microwaves and a Temporally Incoherent Magnetic Field on Single and Double DNA Strand Breaks in Rat Brain Cells

The effect of a temporally incoherent magnetic field noise on microwave-induced DNA single and double strand breaks in rat brain cells was investigated. Four treatment groups of rats were studied: microwave-exposure (continuous-wave 2450-MHz microwaves, power density 1 mW/cm², average whole-body specific absorption rate of 0.6 W/kg), noise-exposure (45 mG), microwave + noise-exposure, and sham-exposure. Animals were exposed to these conditions for 2h. DNA single- and double-strand breaks in brain cells of these animals were assayed 4h later using a microgel electrophoresis assay. Results show that brain cells of microwave-exposed rats had significantly higher levels of DNA single- and double-strand breaks when compared with sham-exposed animals. Exposure to noise alone did not significantly affect the levels (i.e., they were similar to those of the sham-exposed rats). However, simultaneous noise exposure blocked microwave-induced increases in DNA strand breaks. These data indicate that simultaneous exposure to a temporally incoherent magnetic field could block microwave-induced DNA damage in brain cells of the rat.     

脉冲场凝胶电泳技术及其在真菌学研究中的应用

脉冲电泳是用于分离大分子量DNA的一种电泳技术,已广泛用于真菌的核型分析,种群特异性鉴定,基因定位及遗传分析的研究。介绍了脉冲电泳的原理,发展和基本操作程序,并阐述了脉冲电泳技术在真菌分子生物学研究领域的应用。     

Histochemical Demonstration of DNA Double Strand Breaks by in Situ 3'-tailing Reaction in Apoptotic Endometrium

A new histochemical technique, called in situ 3'-tailing reaction (ISTR), to detect DNA double strand breaks (DSB) was developed and applied to tissue sections of apoptotic endometrium. To demonstrate DSB, biotin-labeled and unlabeled dATPs with terminal deoxynucleotidyl transferase (TdT) were added to the many 3-hydroxyl termini of DNA fragments generated in the apoptotic cells. For an efficient 3'-end labeling, it was necessary to treat the sections with λ-exonuclease (λEx) prior to the TdT reaction to generate 3'-protruding ends. The λEx-TdT reaction specifically labeled nuclear fragments in the apoptotic cells in paraformaldehyde fixed frozen sections. In paraffin sections, pretreatment with proteinase K was effective for 3'-tailing reaction. ISTR should be a useful tool for detecting dying cells in both physiological and pathological states.     

Separation of Different Conformations of Plant Mitochondrial DNA Molecules by Field Inversion Gel Electrophoresis

Mitochondrial (mt) DNA structure in higher plants is still unclear as to the circularity or linearity of the genome. We have developed a system to electrophoretically separate distinct populations of mtDNA, with some populations enriched for networked linear and circular DNA molecules. Using field inversion gel electrophoresis (FIGE) and electron microscopy (EM), we have identified four distinct populations of mtDNA from two Brassica species. Using FIGE, two slow migrating mtDNA populations ran faster than a 66 kbp Escherichia coli circular plasmid marker, while these same populations comigrated in the compression zone in contour-clamped homogeneous electrophoretic field (CHEF) gels. A fast-migrating mtDNA population was also resolved by FIGE as a diffuse band between 20 to 70 kbp when compared with linear lambda () markers. FIGE resolved the 66 kbp circular marker into several multimers, while CHEF resolved only open-circular monomers and linears. In agreement with FIGE results, EM analysis indicated the two slow migrating mtDNA populations contained circular (both supercoiled and relaxed circles) and free linear molecules of 10-60 kbp, and networked linear molecules of 45–140 kbp total size that may represent recombination intermediates. The fast migrating population consisted of 10–50 kbp linear molecules. Well-bound mtDNA showed only long linear molecules of 40–150 kbp with no detection of circles or complex/rosette molecules. This report shows that FIGE has clear advantages over CHEF for separating large DNA molecules with different conformations, and may be very useful for studies to characterize genome structure in complex systems such as plant mitochondria.     

一种提高质粒DNA凝胶电泳检测灵敏度的方法

比较了凝胶电泳示检测质粒ＤＮＡ时不同激发波长对ＤＮＡ－ＥＢ荧光强度的影响,发现短波长激发光可增加ＤＮＡ的探测灵敏度。采用２６０ｎｍ作为激光光时可探测到少至０．７ｎｇ的线性ＤＮＡ。且在很广的ＤＮＡ的质量范围内,ＤＮＡ－ＥＢ荧光强度 与ＤＮＡ量或正比。以此改进方法检测电离辐射诱导的ＤＮＡ单、双链断裂岢得到与其它研究结果相一致的Ｇ（ＳＳＢ）和Ｇ（ＤＳＢ）值。     

衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

Ionizing radiation-induced growth in soft agar is associated with miR-21 upregulation in wild-type and DNA double strand break repair deficient cells

DNA double strand breaks (DSBs) are a severe threat to genome integrity and a potential cause of tumorigenesis, which is a multi-stage process and involves many factors including the mutation of oncogenes and tumor suppressors, some of which are transcribed microRNAs (miRNAs). Among more than 2000 known miRNAs, miR-21 is a unique onco-miRNA that is highly expressed in almost all types of human tumors and is associated with tumorigenesis through its multiple targets. However, it remains unclear whether there is any functional link between DSBs and miR-21 expression and, if so, does the link contribute to DSB-induced genomic instability/tumorigenesis. To address this question, we used DNA-PKcs-/- (deficient in non-homologous end-joining (NHEJ)) and Rad54-/- (deficient in homologous recombination repair (HRR)) mouse embryonic fibroblasts (MEFs) since NHEJ and HRR are the major pathways for DSB repair in mammalian cells. Our results indicate that levels of miR-21 are elevated in these DSB repair (DSBR) deficient cells, and ionizing radiation (IR) further increases these levels in both wild-type (WT) and DSBR-deficient cells. Interestingly, IR stimulated growth in soft agar and this effect was greatly reduced by blocking miR-21 expression in both WT and DSBR-deficient cells. Taken together, our results suggest that either IR or DSBR-deficient can lead to an upregulation of miR-21 levels and that miR-21 is associated with IR-induced cell growth in soft agar. These results may help our understanding of DSB-induced tumorigenesis and provide information that could facilitate the development of new strategies to prevent DSB-induced carcinogenesis.     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.