用人DNA介导的基因转移修复中国仓鼠细胞的HPRT缺陷

 中国仓鼠卵巢细胞(CHO-K1)经N-甲基-N'-硝基-N-亚硝基胍(MNNG)诱变和6-巯基鸟嘌呤(6-TG)选择,得到稳定的次黄嘌呤磷酸核糖转移酶(HPRT)缺陷细胞株,酶活性仅为野生型的6.5％。用磷酸钙共沉淀法和电脉冲法向HPRT-细胞转移人宫颈癌细胞(HeLaS_3)基因组DNA,纠正了CHO细胞的HPRT缺陷。酶活性提高了6.9倍,达到野生型的45％。用Alu序列探针进行分子杂交,证实经过基因转移并连续传代15次以上的受体细胞中含人DNA序列。表明人的有关基因已稳定地整合到CHO细胞的染色体中。     

HeLa S3 DNA转染后XP细胞UV抗性的稳定增加和切除修复基因的定位探索

 通过带有选择标志的质粒pSV_2Neo的基因共转染和第一轮转化的XP细胞DNA的再转染,进一步证明了前文报道的HeLaS3 DNA的切除修复基因在XP细胞中的稳定表达,以及受体细胞UV抗性的稳定增加。通过五种限制性核酸内切酶酶切DNA片段的转染,初步寻找出切除修复基因位于Bg1 Ⅰ,xhoⅠ 酶切片段之中。     

紫外照射对DNA修复基因RAD24转录的影响

以酿酒酵母HY684为实验对象,用波长254nm紫外线分别在0,37,50和70J/m~2等强度下照射不同时间,提取总RNA,应用North-ern杂交方法检测RAD24基因转录水平的变化。结果显示37J/m~2、50J/m~2照射20分钟到120分钟明显提高RAD24基因转录水平,70J/m~2照射时,则恢复至正常水平,这说明低剂量紫外线照射可提高RAD24基因转录水平,该基因的表达具有损伤诱导性。     

肌肉介导的基因转移DNA药物及应用研究

介绍了肌肉介导的基因转移方法,探讨肌肉摄取DNA的机理,DNA药物的预防和治疗作用机制,DNA药物的具体应用以及影响DNA药物药效的因素,并讨论了DNA药物的窀生,可控性等问题。     

调控DNA损伤修复基因的微小RNA

随着对DNA损伤修复基因研究的深入,其信号转导路径及调控网络也进一步明了,调控DNA损伤修复基因的微小RNA(miRNA)也越来越多地被认识和发现。简要综述了DNA损伤途径中调控主要的损伤修复基因的miRNA,有助于深入阐明DNA损伤修复机制,为开发抗辐射药物和临床上DNA损伤修复异常相关肿瘤的基因治疗提供新的靶点。     

下调c—erbB—2对细胞DNA修复和凋亡的影响

将有义和反义c-erbB-2逆转录病毒体分别经脂质体包裹后转染入人胚肺二倍体成纤维细胞（2BS）。Southern印迹杂交表明,外源c-erbB-2 cDNA在转染细胞中已成功整合入基因组中。Northern印迹杂交显示,有义转染细胞的erbB-2表达上升57％,反义转染细胞erbB-2表达下降48％。与对照和空载体转染细胞相比,反义转染细胞的DNA损伤修理能力显著下降,凋亡可诱导性降低。这和我们观察到的反义转染细胞提早出现衰老表型相一致。     

SA脂质体介导DNA转染昆虫细胞的研究

ＳＡ脂质体介导ＤＮＡ转染昆虫细胞的研究张传溪（浙江农业大学应用昆虫学研究所杭州３１００２９）吴祥甫（中国科学院上海生物化学研究所上海２０００３１）杆状病毒昆虫表达系统是８０年代发展起来的高效真核表达系统,具有表达量高,表达产物后加工较完全等优点,因...     

用DNA扩增法检测镰状细胞基因

本文报道应用DNA扩增技术对国内首例镰状细胞特征患者(Hb s杂合子)进行基因诊断。方法是从患者干血标本中微量抽提基因组DNA,通过聚合酶链反应(PCR)扩增其β珠蛋白基因,经限制性内切酶MstⅡ消化后作电泳分析直接检测Hb S基因。本文介绍的DNA诊断技术快速、灵敏、简便,它不需要放射性同位素标记的探针,可以采用干血抽提的DNA,因此,对遗传病基因诊断和携带者的筛查具有重要价值。     

谷子花粉介导法转几丁质酶基因的研究

以晋谷21号A不育系材料为受体,以西粒pGLⅡ-RC-1为供体,在10%浓度的蔗糖溶液中,加入花粉与含有质粒DNA,应用YKH-1型液体快速混合器进行震荡处理得到花粉处理液,然后辅以人工授粉的方法将花粉处理液滴入晋谷21号不育系的柱头上获得种子,将F1的种子经除草剂筛选和PCR检测,有目的基因特异带出现,实践证明,利用花粉介导法进行谷子几丁质酶基因转移方法可行,为谷子转基因提供了一个新方法。     

Loss of the Xeroderma Pigmentosum Group A Gene (XPA) Enhances Apoptosis of Cultured Cerebellar Neurons Induced by UV but Not by Low-K+ Medium

Abstract: To study the involvement of the xeroderma pigmentosum group A gene ( XPA ) in neuronal apoptosis, we cultured cerebellar neurons from mice lacking XPA gene ( XPA ^−/−) and induced apoptosis by exposure to UV irradiation or medium containing a low concentration of potassium (low-K⁺ medium). When cerebellar neurons from postnatal days 15–16 wild-type mice were treated with UV irradiation, apoptotic neuronal death was observed after 24–48 h. About 60% of neurons survived 48 h after UV irradiation at a dose of 5 J/m². On the other hand, neurons from XPA ^−/− mice showed a significantly increased vulnerability to UV irradiation, and >90% of neurons died 48 h after UV irradiation at a dose of 5 J/m². In contrast, low-K⁺ medium induced apoptosis of neurons from mice of each genotype with the same kinetics. These results suggest that the XPA gene is involved in neuronal DNA repair and that it thereby influences apoptosis induced by DNA damage in cultured cerebellar neurons.     

以精子为载体的体外转基因猪胚胎研究

去精清精子与外源DNA共孵育,使其携带外源DNA,再与成熟卵母细胞进行体外受精,生产转基因猪胚胎,为通过胚胎移植生产转基因猪奠定基础。结果:通过PCR从不同时期胚胎中检测出携带外源DNA的阳性胚,说明精子与外源DNA共孵育能使精子携带外源DNA,并通过体外受精技术使外源DNA进入早期胚胎。     

腺相关病毒介导基因组来源的基因转移与整合

构建一个带β-珠蛋白基因组序列的腺相关病毒载体AV53HS2Δβ2Neo.经包装成重组腺相关病毒后,转导红系细胞.DNA印迹证实包含红系增强子、β-珠蛋白基因和筛选标志基因的前病毒基因组完整整合于红系细胞基因组中.结果说明腺相关病毒载体能介导基因组序列来源的目的基因稳定整合于受体细胞基因组中.     

An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells: NUCLEOTIDE EXCISION REPAIR,DNA DAMAGE CHECKPOINT,AND APOPTOSIS*

DNA damage by UV and UV-mimetic agents elicits a set of inter-related responses in mammalian cells, including DNA repair, DNA damage checkpoints, and apoptosis. Conventionally, these responses are analyzed separately using different methodologies. Here we describe a unified approach that is capable of quantifying all three responses in parallel using lysates from the same population of cells. We show that a highly sensitive in vivo excision repair assay is capable of detecting nucleotide excision repair of a wide spectrum of DNA lesions (UV damage, chemical carcinogens, and chemotherapeutic drugs) within minutes of damage induction. This method therefore allows for a real-time measure of nucleotide excision repair activity that can be monitored in conjunction with other components of the DNA damage response, including DNA damage checkpoint and apoptotic signaling. This approach therefore provides a convenient and reliable platform for simultaneously examining multiple aspects of the DNA damage response in a single population of cells that can be applied for a diverse array of carcinogenic and chemotherapeutic agents.     

DNA damage response by single-strand breaks in terminally differentiated muscle cells and the control of muscle integrity

DNA single-strand breaks (SSB) formation coordinates the myogenic program, and defects in SSB repair in post-mitotic cells have been associated with human diseases. However, the DNA damage response by SSB in terminally differentiated cells has not been explored yet. Here we show that mouse post-mitotic muscle cells accumulate SSB after alkylation damage, but they are extraordinarily resistant to the killing effects of a variety of SSB-inducers. We demonstrate that, upon SSB induction, phosphorylation of H2AX occurs in myotubes and is largely ataxia telangiectasia mutated (ATM)-dependent. However, the DNA damage signaling cascade downstream of ATM is defective as shown by lack of p53 increase and phosphorylation at serine 18 (human serine 15). The stabilization of p53 by nutlin-3 was ineffective in activating the cell death pathway, indicating that the resistance to SSB inducers is due to defective p53 downstream signaling. The induction of specific types of damage is required to activate the cell death program in myotubes. Besides the topoisomerase inhibitor doxorubicin known for its cardiotoxicity, we show that the mitochondria-specific inhibitor menadione is able to activate p53 and to kill effectively myotubes. Cell killing is p53-dependent as demonstrated by full protection of myotubes lacking p53, but there is a restriction of p53-activated genes. This new information may have important therapeutic implications in the prevention of muscle cell toxicity.     

聚腺苷二磷酸核糖基聚合酶──在DNA损伤修复及程序性死亡中的作用

聚腺苷二磷酸核糖基聚合酶(poly (ADP-ribose) polyerase, PARP)是存在于多数真核细胞中的一个蛋白质翻译后修饰酶,它可催化组蛋白H₁等重要核蛋白及它自身的聚腺苷二磷酸核糖基化作用.细胞受到外界损伤因子作用时, DNA发生链断裂,PARP结合到DNA断裂口,其催化活性被激活,修饰受体蛋白,进而引发一系列级联反应.这种性质使PARP有可能作为细胞内的分子感受器和传感器,启动细胞内对损伤作出反应的信号传导机制,从而根据细胞受损程度决定细胞的命运：修复或是死亡．     

乳腺癌易感蛋白1在DNA损伤修复中的作用

人类乳腺癌易感基因1(breast cancer susceptibility gene 1,BRCA1)首先是在乳腺癌家族中发现的,是具有遗传倾向的乳腺癌和卵巢癌易感基因,其基因的突变与家族性乳腺癌及卵巢癌的发生有密切联系。BRCA1是一种抑癌基因,其基因产物可以参与维持基因组稳定性的多条细胞信号通路,例如DNA损伤诱导的细胞周期调控、DNA损伤修复、基因转录调节、细胞凋亡、泛素化等重要的细胞活动。本文就近几年来BRCA1在DNA损伤修复中的作用的研究进展作一综述,包括DNA损伤诱导的细胞周期检查点的激活和DNA损伤修复两方面。     

Functional aspects of PARylation in induced and programmed DNA repair processes: Preserving genome integrity and modulating physiological events

To cope with the devastating insults constantly inflicted to their genome by intrinsic and extrinsic DNA damaging sources, cells have evolved a sophisticated network of interconnected DNA caretaking mechanisms that will detect, signal and repair the lesions. Among the underlying molecular mechanisms that regulate these events, PARylation catalyzed by Poly(ADP-ribose) polymerases (PARPs), appears as one of the earliest post-translational modification at the site of the lesion that is known to elicit recruitment and regulation of many DNA damage response proteins.