三种类型辐射对质粒超螺旋DNA损伤的研究

用ａｇａｒｏｓｅ电泳和图象处理技术比较了６０Ｃｏγ射线、ＵＶ及低能Ｎ＋离子处理ｐＵＣ１９ＤＮＡ超螺旋结构的损伤效应及若干自由基清除剂的保护效应。结果表明：（１）γ射线和ＵＶ照射干燥ＤＮＡ的损伤显著低于水溶液样品;（２）Ｎ＋离子注入后超螺旋ＤＮＡ的减少（ＳＣ％）与剂量呈良好线性关系,而γ射线和ＵＶ组的ＳＣ％随剂量升高呈指数下降;（３）干燥ＤＮＡγ辐照组的Ｄ３７值为８２０Ｇｙ,ＳＣ完全消失的剂量ＬＤ为３８１４Ｇｙ,ＬＤ／Ｄ３７＝４．６５;ＵＶ照射组的相应值分别为：１．６５Ｊ／ｃｍ２,７．６５Ｊ／ｃｍ２,４．６４;Ｎ＋离子组的相应值为：３．２×１０１５Ｎ＋／ｃｍ２,５．０×１０１５Ｎ＋／ｃｍ２和１．５６。虽然上述三种辐射的剂量单位不同,不能直接比较其相对生物学效应,但从ＬＤ与Ｄ３７的比值可反映ＤＮＡＳＣ破坏的程度和终点剂量（ＳＣ％＝０）的大小。从而看出,Ｎ＋离子（高ＬＥＴ辐射）比γ射线（低ＬＥＴ辐射）ＵＶ（非电离辐射）对ＤＮＡ损伤作用更强;（４）乙醇、甘露醇等自由基清除剂对电离辐射损伤有很强的保护作用,但对ＵＶ损伤未见明显的保护效应     

转译优化对EPO基因在COS7细胞中表达的影响

利用ＣＯＳ７细胞暂时表达系统,研究转译起始序列对ＥＰＯ－ｃＤＮＡ表达的影响。通过ＤＮＡ重组技术,构建了原ＥＰＯ－ｃＤＮＡ表达载体ｐＣＳＶ－ＥＰＯ（１）,其转译起始序列为５＇ＡＡＴＴＣＡＴＧＧ３＇。同时通过定点突变技术,将起始序列改变成５＇ＣＣＡＣＣＡＴＧＧ３＇,而构建了另一表达载体ＰＣＳＶ－ＥＰＯ（２）。后经序列分析证明无误后和前均通过ＤＥＡＥ－ｄｅｘｔｒａｎ法转染ＣＯＳ７细胞上清,测定结果为     

应用放射性自显影技术检测外源DNA与鸡精子的结合

应用放射性自显影技术检测外源ＤＮＡ与鸡精子的结合ＤＥＴＥＣＴＩＮＧＡＳＳＯＣＩＡＴＩＯＮＯＦＥＸＯＧＥＮＯＵＳＤＮＡＷＩＴＨＣＨＩＣＫＥＮＳＰＥＲＭＵＳＩＮＧＡＵＴＯＲＡＤＩＯＧＲＡＰＨＹ关键词鸡，精子，脂质体，ＤＮＡ与精子的结合ＫｅｙｗｏｒｄｓＣｈ...     

低氧对肺动脉内皮细胞PDGF—B链释放及平滑肌细胞生长的影响

应用蛋白ｄｏｔｂｌｏｔ技术检测了低氧内皮细胞条件培养液（ＨＥＣＣＭ）和常氧内皮细胞条件培养液（ＮＥＣＣＭ）内ＰＤＧＦ相对含量,并利用［３Ｈ］－ＴｄＲ掺入法和流式细胞术观察了ＨＥＣＣＭ和ＮＥＣＣＭ及加入特异ＰＤＧＦ抗体对肺动脉平滑肌细胞（ＰＡＳＭＣ）生长的影响。结果表明,ＨＥＣＣＭ中的ＰＤＧＦ含量明显高于ＮＥＣＣＭ;ＨＥＣＣＭ能明显增强ＰＡＳＭＣ内ＤＮＡ合成,促进ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期;当预先加入ＰＤＧＦ－Ｂ链抗体时,则会明显地抑制ＨＥＣＣＭ对ＰＡＳＭＣ的ＤＮＡ合成,阻止ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期。结果提示,低氧时ＰＡＳＭＣ增殖与肺动脉内皮细胞分泌释放ＰＤＧＦ增加有关     

日本血吸虫26kD抗原基因在BCG中的表达

研究了外源基因日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）在卡介苗（ｂａｃｉｌｕｓＣａｌｍｅｔｅ－Ｇｕｅｒｉｎ,ＢＣＧ）、耻垢分枝杆菌（Ｍ．ｓｍｅｇｍａｔｉｓ）和大肠杆菌（Ｅ．ｃｏｌｉ）中的表达．运用重组ＤＮＡ和聚合酶链反应（ＰＣＲ）等分子生物学技术,以表达Ｓｊ２６ＧＳＴ的Ｅ．ｃｏｌｉｐＧＥＸ衍生质粒为模板,经ＰＣＲ得到编码Ｓｊ２６ＧＳＴ的全长ｃＤＮＡ片段．将其按正确的阅读框顺序,克隆到人结核杆菌热休克蛋白（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ,ＨＳＰ）７０的启动子下游,再将ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因一起亚克隆到Ｅ．ｃｏｌｉ－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中,得到Ｅ．ｃｏｌｉ－分枝杆菌穿梭表达质粒ｐＢＣＧ－Ｓｊ２６．ｐＢＣＧ－Ｓｊ２６电转化入ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓｍｃ２１５５中表达Ｓｊ２６ＧＳＴ抗原,所表达的天然重组Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白,在ＳＤＳ－ＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带．其表达量分别占ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓ菌体总蛋白的１５％和１０％．可见,Ｓｊ２６ＧＳＴ基因能在ＢＣＧ中高效表达．     

酵母金属硫蛋白对g12细胞抗γ射线损伤的作用

用胞质阻断法微核试验和单细胞凝胶电泳法检测酵母金属硫蛋白（ＢＤ１０１Ｃｕ－ＭＴ）对(6０)Ｃｏ－γ射线诱发的ｇ１２细胞微核形成和ＤＮＡ链断裂的影响。结果表明１０和５０μｇ／ｍｌＢＤ１０１Ｃｕ－ＭＴ处理,对１或３Ｇｙγ射线诱发的双核微核细胞率和１Ｇｙγ射线诱发的彗星细胞频率及核ＤＮＡ迁移距离的增加有明显的抑制作用．提示酵母金属硫蛋白对γ射线诱发ｇ１２细胞的遗传损伤有拮抗作用．     

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。     

高效毛细管电泳在DNA序列分析中的应用

高效毛细管电泳在ＤＮＡ序列分析中的应用种康（兰州大学化学系,兰州７３００００）ＡＰＰＬＩＣＡＴＩＯＮＯＦＨＩＧＨ－ＰＥＲＦＯＲＭＡＮＣＥＣＡＰＩＬＬＡＲＹＥＬＥＣＴＣＴＯＲＨＯＲＥＳＩＳＯＮＤＮＡＳＥＱＵＥＮＣＩＮＧ￥ＣｈｏｎｇＫａｎｇ（Ｄｅｐａｒｔ...     

DNA分析技术及其在植物系统学研究中的应用

ＤＮＡ分析技术及其在植物系统学研究中的应用贺新强,李法曾（山东师范大学生物系,济南２５００１４）ＤＮＡＡＮＡＬＹＺＩＮＧＴＥＣＨＮＩＱＵＥＳＡＮＤＩＴＳＡＰＰＬＩＣＡＴＩＯＮＩＮＰＬＡＮＴＳＹＳＴＥＭＡＴＩＣＳＲＥＳＥＡＲＣＨ￥ＨｅＸｉｎ－ｑｉａｎｇ...     

应用荧光原位杂交和RFLP标记检测多小穗小麦新种质10—A中的黑麦染 …

利用ＡＰＡＧＥ,荧光原位杂交技术和ＲＦＬＰ标记,对导入黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）多小穗等性状创制的小麦新种质１０－Ａ进行了分子标记检测,ＡＰＡＧＥ分析发现,１０－Ａ与其他１ＲＳ／１ＢＬ易位系一样,含有１ＲＳ的醇溶蛋白标记位点Ｇｌｄ１Ｂ３。以黑麦基因组总ＤＮＡ作探针,用中国春（Ｔｒｉｔｉｃｕｍａｅｓｔｉｕｍｃｖ．ＣｈｉｎｅｓｅＳｐｒｉｎｇ）基因组ＤＮＡ作封阻,与１０－Ａ根尖细胞有丝分裂染     

不同浓度红景天苷对γ射线和质子辐照质粒DNA的影响

利用14．2MeV的质子和60↑Co产生的γ射线在200Gy的剂量下分别对添加了不同浓度红景天苷的pUC19质粒DNA样品进行了辐照,凝胶电泳分析结果表明,红景天苷在两种辐照过程中均对DNA具有一定的保护作用,并且随着浓度的增加保护作用增强。同时还发现质子辐照后DNA样品中出现了线性形态的,而γ射线辐照后则没有出现,再次证实质子对DNA的损伤作用强于γ射线,这应该和质子与物质相互作用复杂以及质子对物质的直接电离作用相关。     

鸡发育过程肌组织核提抽物结合活性与AChRγ亚基基因持续表达的关系

烟碱样乙酰胆碱受体 ( n ACh R)是由 4种亚基组成的五聚体 .哺乳类动物出生后γ亚基由ε亚基取代 ,迄今为止鸡 n ACh Rγ基因是否存在上述置换规律尚无定论 .为探索发育过程鸡骨骼肌n ACh R基因表达是否存在γ/ε亚基的置换及其机制 ,采用 RT- PCR技术和凝胶阻滞试验检测了鸡胚发育 9d至出生后 6周小鸡 γ基因表达的动力学及骨骼肌核抽提物的 DNA结合活性 .RT-PCR检测结果显示 ,在鸡胚发育 9d至出生后 6周的雏鸡骨骼肌组织均检出有 γ亚基 m RNA转录 .提示与哺乳类不同 ,出生前后鸡骨骼肌组织 ACh Rγ亚基基因持续表达 ,不存在 γ/ε亚基的置换表达规律 .以 γ基因 - 2 60 /- 2 4 0 (含 E盒与 M- CAT盒重叠序列 )和 - 2 39/- 50 (含 M- CAT盒及 GC富含区 )片段为探针 ,分别与鸡胚发育 9d至出生后 2周小鸡骨骼肌的核抽提物进行凝胶阻滞试验 .在发育各阶段的骨骼肌核抽提物中均有识别 - 2 39/- 50片段的结合活性存在 ,但在出生后 2周小鸡骨骼肌的核抽提物中未检出 - 2 60 /- 2 4 0结合活性 .结果提示 ,在出生后第 1 4d的肌核抽提物中存在的、识别并结合 - 2 39/- 50片段的活性物质与鸡 ACh Rγ基因在出生后持续表达有关 .     

DNA损伤生物学反应中ATM对p21~(WAF1/CIP1)蛋白的直接磷酸化

毛细血管扩张性共济失调症突变蛋白 (mutatedinataxiatelangiectasia ,ATM)是直接感受DNA双链断裂损伤并起始诸多DNA损伤信号反应通路的主开关分子 .已有研究发现 ,DNA损伤生物学反应中 ,ATM可通过磷酸化活化p5 3,继而转录活化细胞周期检查点蛋白p2 1WAF1 CIP1的表达 ,而对于ATM是否直接参与p2 1WAF1 CIP1的早期活化迄今尚无实验证明 .通过免疫共沉淀反应 ,检测到细胞电离辐射 (ionizingradiation ,IR)反应早期ATM与p2 1WAF1 CIP1蛋白存在相互作用 .将p2 1WAF1 CIP1蛋白编码基因全长克隆入原核表达载体pGEX4T 2 ,经诱导表达及亲和层析纯化获取GST p2 1融合蛋白作为磷酸化底物 .体外磷酸化实验检测证明 ,IR活化的ATM具磷酸化p2 1WAF1 CIP1蛋白的功能 ,并且此磷酸化功能可被PI3K家族特异性抑制剂Wortmannin所抑制 .结果揭示了IR后ATM可通过直接磷酸化p2 1WAF1 CIP1蛋白 ,在IR致DNA损伤生物学反应早期调控p2 1WAF1 CIP1蛋白的快速活化过程     

Measurement of the initial levels of DNA damage in human lymphocytes induced by 29 kV X rays (mammography X rays) relative to 220 kV X rays and gamma rays

Experiments using the alkaline comet assay, which measures all single-strand breaks regardless of their origin, were performed to evaluate the biological effectiveness of photons with different energies in causing these breaks. The aim was to measure human lymphocytes directly for DNA damage and subsequent repair kinetics induced by mammography 29 kV X rays relative to 220 kV X rays, 137Cs gamma rays and 60Co gamma rays. The level of DNA damage, predominantly due to single-strand breaks, was computed as the Olive tail moment or percentage DNA in the tail for different air kerma doses (0.5, 0.75, 1, 1.5, 2 and 3 Gy). Fifty cells were analyzed per slide with a semiautomatic imaging system. Data from five independent experiments were transformed to natural logarithms and fitted using a multiple linear regression analysis. Irradiations with the different photon energies were performed simultaneously for each experiment to minimize interexperimental variation. Blood from only one male and one female was used. The interexperimental variation and the influence of donor gender were negligible. In addition, repair kinetics and residual DNA damage after exposure to a dose of 3 Gy were evaluated in three independent experiments for different repair times (10, 20, 30 and 60 min). Data for the fraction of remaining damage were fitted to the simple function F(d) = A/(t + A), where F(d) is the fraction of remaining damage, t is the time allowed for repair, and A (the only fit parameter) is the repair half-time. It was found that the comet assay data did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiation types, 220 kV X rays and the gamma rays of 137Cs and 60Co, either for the total dose range or in the low-dose range. These results are, with some restrictions, consistent with physical examinations and predictions concerning, for example, the assessment of the possible difference in effectiveness in causing strand breaks between mammography X rays and conventional (150-250 kV) X rays, indicating that differences in biological effects must arise through downstream processing of the damage.     

DNA repair after gamma irradiation in lymphocytes exposed to low-frequency pulsed electromagnetic fields

The effect of exposure to extremely low-frequency pulsed electromagnetic fields (EMFs) on DNA repair capability and on cell survival in human lymphocytes damaged in vitro with gamma rays was studied by two different micromethods. In the first assay, which measures DNA repair synthesis (unscheduled DNA synthesis, UDS), lymphocyte cultures were stimulated with phytohemagglutinin (PHA) for 66 h and then treated with hydroxyurea (which blocks DNA replication), irradiated with 100 Gy of 60Co, pulsed with [3H]thymidine ([3H]TdR), and then exposed to pulsed EMFs for 6 h (the period in which cells repaired DNA damage). In the second assay, which measures cell survival after radiation or chemical damage, lymphocytes were first irradiated with graded doses of gamma rays or treated with diverse antiproliferative agents, and then stimulated with PHA, cultured for 72 h, and pulsed with [3H]TdR for the last 6 h of culture. In this case, immediately after the damage induced by either the radiation or chemicals, cultures were exposed to pulsed EMFs for 72 h, during which cell proliferation took place. Exposure to pulsed EMFs did not affect either UDS or cell survival, suggesting that this type of nonionizing radiation--to which humans may be exposed in the environment, and which is used for both diagnostic and therapeutic purposes--does not affect DNA repair mechanisms.     

Measurement of DNA damage in mammalian cells using flow cytometry

A technique for the detection of DNA damage induced by radiation insult has been developed. Cells were lysed with a buffer containing 2 M sodium chloride to release the DNA in a supercoiled form, the nucleoid. These were stained with the DNA intercalating dye, ethidium bromide, and exposed to laser light within a flow cytometer. Scattered and fluorescent light was analyzed from the laser/nucleoid interaction following irradiation of viable cells with gamma rays. The addition of ethidium bromide to prepared nucleoids caused a reduction in scattered light due to condensation of the nucleoid. Irradiation of cells prior to nucleoid production and ethidium bromide treatment restricted this condensation and produced a dose-dependent increase in laser scatter. Nucleoids derived from human lymphocytes showed enhanced light scatter from 5 Gy, compared to Chinese hamster ovary (CHO) fibroblasts where doses above 10 Gy were required. Up to 30 Gy CHO nucleoids showed a dose-dependent reduction in the ethidium bromide fluorescence. This technique allows detection of altered light scattering and fluorescent behavior of nucleoids after cellular irradiation; these may be related to structural changes within the nucleus induced by the radiation. The use of flow cytometry compared to other methods allows a rapid analysis of nuclear damage within individual cells.     

Reciprocal adaptive response of human peripheral lymphocytes induced by bleomycine or gamma rays.

The adaptive response and reciprocal adaptive response induced in vitro by exposure to low doses of gamma rays (0.05 Gy) or bleomycin (0.05 microg/ml) in human peripheral blood lymphocytes were assessed by the frequency of chromosome aberrations. Gamma rays (1.5 Gy) or bleomycin (1.5 microg/ml) were used as the challenge doses. In the experiments, blood samples from 5 healthy donors were investigated. It has been found that low doses of bleomycin and gamma rays induced a reciprocal adaptive response to high doses of gamma rays or bleomycin. Moreover, the results confirmed that the adaptive response did not correlate with the radiosensitivity of the peripheral blood lymphocytes.     

Dose-dependent misrejoining of radiation-induced DNA double-strand breaks in human fibroblasts: experimental and theoretical study for high- and low-LET radiation

Misrejoining of DNA double-strand breaks (DSBs) was measured in human primary fibroblasts after exposure to X rays and high-LET particles (helium, nitrogen and iron) in the dose range 10-80 Gy. To measure joining of wrong DNA ends, the integrity of a 3.2-Mbp restriction fragment was analyzed directly after exposure and after 16 h of repair incubation. It was found that the misrejoining frequency for X rays was nonlinearly related to dose, with less probability of misrejoining at low doses than at high doses. The dose dependence for the high-LET particles, on the other hand, was closer to being linear, with misrejoining frequencies higher than for X rays, particularly at the lower doses. These experimental results were simulated with a Monte Carlo approach that includes a cell nucleus model with all 46 chromosomes present, combined with realistic track structure simulations to calculate the geometrical positions of all DSBs induced for each dose. The model assumes that the main determinant for misrejoining probability is the distance between two simultaneously present DSBs. With a Gaussian interaction probability function with distance, it was found that the data for both low- and high-LET radiation could be fitted with an interaction distance (sigma of the Gaussian curve) of 0.25 microm. This is half the distance previously found to best fit chromosomal aberration data in human lymphocytes using the same methods (Holley et al., Radiat. Res. 158, 568-580, 2002). The discrepancy may indicate inadequacies in the chromosome model, for example insufficient chromosomal overlap, but may also be partly due to differences between fibroblasts and lymphocytes.     

Bleomycin, in contrast to gamma irradiation, induces extreme variation of DNA strand breakage from cell to cell

Chinese hamster ovary cells grown in vitro were treated with bleomycin or irradiated with high doses of 60Co gamma rays (200 and 400 Gy). DNA strand breaks in single cells were analysed by using our newly introduced microelectrophoretic technique. Bleomycin seems to act in a selective manner so that in some cells the DNA is heavily degraded while in others there is only moderate or no measurable damage. In contrast, a uniform response was found after gamma irradiation. To achieve the same magnitude of DNA fragmentation as in the most severely bleomycin-damaged cells, irradiation with more than 200 Gy is required. Some 8000 double-strand breaks per cell are produced by 200 Gy which will convert the molecular weight of the DNA to the range of 10(8)-10(9) dalton, and free migration of DNA fragments occurs during electrophoresis. We include also a detailed study of the DNA migration pattern following doses of 0-100 Gy gamma rays.     

The effects of X rays on BCNU-induced DNA crosslinking

We have used the technique of alkaline elution to study DNA interstrand crosslinking in 9L rat brain tumor cells treated with combinations of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and X rays. Irradiation with doses as low as 50 rad of X rays immediately or 6 hr after a 1-hr treatment with 60, 80, or 100 microM BCNU produced a significant increase in BCNU-induced DNA interstrand crosslinking. If cells were irradiated before BCNU treatment, the amount of crosslinking was not affected compared with BCNU alone. Cell survival experiments using 600 rad of X rays and 1-hr treatments with 0-30 microM BCNU were also performed. As found in the crosslinking studies, irradiation immediately or 6 hr after the BCNU treatment produced enhanced cell kill, but irradiation 6 hr before BCNU treatment did not produce enhanced cell kill. Therefore, the X-ray-mediated increase in BCNU-induced DNA interstrand crosslinking may be the mechanism through which cell kill is increased by combination treatment with the agents.