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1.
绿色荧光蛋白的发光机制   总被引:1,自引:0,他引:1  
从多管水母(Aequoreavictoria)中分离纯化的绿色荧光蛋白(GFP)是由238个氨基酸残基组成的单链多肽,分子量约27kD,1992年其cDNA被克隆[1]。1994年重组野生型GFP(WtGFP)在异源细胞中表达[2]。野生型GFP被紫外光和蓝光激发后能发出绿色荧光,最大荧光吸收/激发峰在395nm,在475nm有一个肩峰,荧光发射峰为508nm。GFP的结构和光致荧光非常稳定,而且因GFP生色团的形成是自催化的,检测GFP的光致荧光不需要外加底物和辅因子,便于活体观察[2]。如今…  相似文献   

2.
利用GFP示踪细胞内源性P53活性检测DNA损伤   总被引:2,自引:1,他引:1  
DNA损伤的检测对预防癌症和遗传病等非常重要。采用分子克隆技术,将报告基因—绿色荧光蛋白(GFP)置于SV40基本启动子调控下,构建成对照载体pSV-GFP。在SV40基本启动子上游插入寡核苷酸P53RE,构建成示踪载体p53RE-GFP。转染NIH3T3细胞,以GFP示踪细胞内源性P53的转录激活活性。紫外线照射或H2O2处理转化细胞使DNA损伤,诱导细胞内源性P53的表达。用激光扫描共聚焦成像系统(LSCIS)对细胞进行红、绿、蓝三色光融合成像,并测定GFP经488nm激发后发出的绿色荧光光密度,验证GFP示踪P53的特异性。p53RE-GFP转化细胞3T3-REG经紫外线照射或H2O2处理后,GFP的表达增高,处理后1hr光密度即达到最高水平,随后逐渐降低。血清“饥饿”—非DNA损伤处理的3T3-REG细胞,以及经紫外和H2O2处理的对照载体pSV-GFP转化细胞3T3-SVG,GFP的表达无明显增强。实验表明:GFP示踪内源性P53转录激活活性用于检测DNA损伤有很高的灵敏度和特异性,适宜推广应用。  相似文献   

3.
绿色荧光蛋白的特性及其在信号转导中的应用   总被引:3,自引:0,他引:3  
细胞和分子生物学的最终目标是 ,明确细胞内的各种事件是如何发生的 ,明了细胞内复杂的动态变化的生化机制。绿色荧光蛋白 (greenfluorescentprotein ,GFP)自从克隆、表达之后 ,以其良好的物理特性及荧光特性而成为良好的报告基因和荧光标记分子 ,并在探索生命现象过程中得到了非常广泛的应用。GFP作为报告基因 ,可用在活细胞中直接观察蛋白质向细胞器 ,如细胞核、内质网中运动 ;作为荧光标记分子 ,GFP既具有敏感的标记检测率 ,又没有放射性的危害 ;最近又发现GFP是一个良好的细胞间信号传递的动态标记…  相似文献   

4.
活细胞的分子探针——绿色荧光蛋白   总被引:14,自引:0,他引:14  
来自水母的绿色荧光蛋白(GFP),在不加外源物质的情况下,紫外光激发后,能在原核和玩具核活细胞中发绿色荧光,荧光性质稳定,突变蛋白发光效率提高,激发和发射光谱明显改变。GFP是一种十分有用的活细胞分子探针,在基因表达调控,转基因动物研究,蛋白在细胞中功能定位,迁移变化,病原菌侵入活细胞的分子过程等诸多方面均有广泛的用途。  相似文献   

5.
绿色荧光蛋白及其在植物研究中的应用   总被引:11,自引:1,他引:10  
绿色荧光蛋白(GFP)是海洋生物水母(Aequoreavictoria)体内的一种发光蛋白,分子量27kD,由238个氨基酸组成。该蛋白65~67位SerTyrGly三种氨基酸环化加氧形成特殊的生色团结构。野生型GFP发光较弱,而且gfpcDNA含有隐蔽型剪切位点,而加工改造的GFP在植物中能够正常表达并且加强了荧光信号。GFP作为新的报告基因和遗传标记被广泛应用于植物研究之中。  相似文献   

6.
绿色荧光蛋白基因在青蒿转基因芽中的表达   总被引:5,自引:1,他引:4  
将改良的绿色荧光蛋白(GFP)基因,插入到植物表达载体中,构建双CaMV35S启动子驱动下的植物表达载体pBIGFP,在Kam浓度为20mg/L的筛选培养基上,用含有pBIFP质粒的根癌农杆菌LBA4404感染青蒿叶片,获得5个抗Kan阳性丛生芽系。Southern blotting分析表明,外源GFP基因已整合到青蒿转基因芽G-1系的基因组中。在OLYMPUS-BH2型荧光显微镜下,观察到转基因  相似文献   

7.
EGFP基因在粉纹夜蛾细胞中的高效表达   总被引:7,自引:0,他引:7  
绿色荧光蛋白(greenfluorescentprotein,GFP)基因是从一种水母体内分离的新型报告基因(reportgene)(Chalfie等,1994),其编码的、由238个氨基酸组成的GFP是一种对光稳定的可溶性蛋白,在长波紫外光或蓝光激发下就能发出绿色荧光,而不需添加任何酶或底物,因而检测十分方便快速,无背景干扰。同时GFP也是目前唯一能在异源细胞内表达后自发产生荧光的蛋白质。EGFP基因是由野生型GFP基因经人工改造后更适合于在真核细胞中表达的突变体,基因编码区长717bp,编码…  相似文献   

8.
能发荧光的转基因家蚕   总被引:3,自引:0,他引:3  
利用同源重组的方法研究了家蚕丝心蛋白链基因的定点替代。将IE启动子带动的GFP基因插入蚕丝心慢白质链基因的上、下游序列之间,构民 重组质粒。将该质粒线性化后导入早期受精卵。当家蚕饲养至五龄期时用紫外灯检测,在约5000条蚕中发现3条有绿色荧光斑块。PCR检测证明GFP已整合到家蚕基因组中,对其中一条蚕的Southern杂交分析表明丝心蛋白重链基因已被成功敲除并被GFP报告基因所替代。这条转基因蚕能  相似文献   

9.
绿色荧光蛋白在植物细胞生物学中的应用   总被引:3,自引:0,他引:3  
克隆于海洋动物水母 (Aequoreavictori a)的绿色荧光蛋白 (greenfluorescentprotein ,GFP)作为一种新型的非酶性报告基因具有检测简便 ,结果真实可靠 ,不需要任何外源底物或辅助因子的特点 ,自出现以来它已引起人们的广泛兴趣 ,目前已经应用于烟草、柑橘、拟南芥、玉米、水稻、大豆、苜蓿等多种植物材料的研究中。GFP含有特殊的六肽生色团结构 ,用蓝紫光激发即能发出肉眼清晰可见的绿色荧光 ,而无需任何底物或辅助因子。GFP能与多种不同蛋白质的N端或C端融合而保持与天然蛋白质相似的荧…  相似文献   

10.
通过基因工程操作,使乙型肝炎病毒e抗原(HBeAg)基因与绿色荧光蛋白基因(GFP)融合,用新型Bac-to-Bac杆状病毒表达系统在昆虫细胞中高效表达了HBeAg-GFP双功能融合蛋白。经ELISA法和荧光显微镜观察证实,表达产物既能发射易于检测的绿色荧光,又具有HBV的e抗原活性,为免疫诊断新方法的建立进行了有益的探索  相似文献   

11.
The possible relationship between cell surface receptor numbers, receptor gene expression for low density lipoprotein (LDL), insulin and epidermal growth factor (EGF), and differentiation capacity has been studied in normal and SV40 transformed (SVK14) keratinocytes, various squamous carcinoma cell (SCC) lines and A431 cells. Our recent studies demonstrated that an inverse relationship exists between LDL- and EGF-receptor binding and the ability to differentiate of both normal and transformed keratinocytes. In the present study cloned LDL- and EGF-receptor complementary DNAs were used as probes to identify both LDL and EGF receptor gene fragments on genomic DNA blots. The extent of hybridisation was found to be increased to the highest extent in A431 cells and decreased in other cells in the following order SCC-4 greater than SCC-15. In SCC-12F2, SVK14 and normal keratinocytes no increase has been observed. The increased hybridisation of LDL- and EGF-receptors in A431, SCC-4 and SCC-15 cells was found to be due to gene amplification and not to aneuploidy. In contrast to the LDL- and EGF-receptor binding, no correlation has been found between insulin receptor binding and ability of cells to differentiate. Furthermore, no amplification of insulin receptor gene has been observed in any of the cells under study.  相似文献   

12.
The REC104 gene was initially defined by mutations that rescued the inviability of a rad52 spo 13 haploid strain in meiosis. We have observed that rec104 mutant strains undergo essentially no induction of meiotic gene conversion, and we have not been able to detect any meiotic crossing over in such strains. The REC104 gene has no apparent role in mitosis, since mutations have no observable effect on growth, mitotic recombination, or DNA repair. The DNA sequence of REC104 reveals that it is a previously unknown gene with a coding region of 549-bp, and genetic mapping has localized the gene to chromosome VIII near FUR1. Expression of the REC104 gene is induced in meiosis, and it appears that the gene is not transcribed in mitotic cells. Possible roles for the REC104 gene product in meiosis are discussed.  相似文献   

13.
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15.
Whether drug-selectable genes can influence expression of the beta-globin gene linked to its LCR was assessed here. With the tkNeo gene placed in cis and used to select transfected cells, the beta-globin gene was expressed fourfold lower when it was positioned upstream of the LCR rather than downstream. This difference did not occur when the pgkPuro gene replaced tkNeo. Moreover, the beta-globin gene situated upstream of the LCR was transcribed without position effects when it was cotransfected with a pgkPuro-containing plasmid, whereas cotransfection with a tkNeo plasmid gave measurable position effects. Previous results from transfected cells selected via a linked tkNeo gene suggested that the 3' end of the beta-globin gene has no impact on LCR-enhanced expression. Here, removal of the 3' end of the beta-globin gene resulted in lower and much more variable expression in both transgenic mice and cells cotransfected with pgkPuro. Together, the results suggest that tkNeo, but not pgkPuro, can strongly influence expression of the beta-globin gene linked to its LCR. The findings could partly explain why data on beta-globin gene regulation obtained from transfected cells have often not agreed with those obtained using transgenic mice. Hence, one must be careful in choosing a drug-selectable gene for cell transfection studies.  相似文献   

16.
M E Rogel  L I Wu    M Emerman 《Journal of virology》1995,69(2):882-888
Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that can cause extensive cytopathicity in T cells. However, long-term productive infection of T-cell lines has been described. Here we show that although Vpr has no effect on the initial cytopathic effect of HIV-1, viruses that contain an intact vpr gene are unable to establish a chronic infection of T cells. However, virus with a mutated vpr gene can readily establish such long-term cultures. The effect of Vpr is independent of the env gene and the nef gene. Furthermore, expression of Vpr alone affects the progression of cells in the cell cycle. These results suggest that HIV-1 has evolved a viral gene to prevent chronic infection of T cells.  相似文献   

17.
18.
Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.  相似文献   

19.
Thymidine kinase (TK)-deficient human cells were infected with ultraviolet light-inactivated Herpes simplex virus type 1, and "transformed" cells that expressed Herpes TK activity were isolated. Purified metaphase chromosomes were isolated from the transformed human line and incubated with TK-deficient mouse cells. TK+ cells were selected, and it was shown that these cells were gene transferents which expressed Herpes TK activity, identical to that found in the transformed human cells. The gene transferents contained no intact human chromosomes. When removed from selective pressure, the gene transferents rapidly lost the TK+ phenotype. However, upon continued growth in nonselective medium, a subpopulation in which the TK+ phenotype had become more stabilized appeared. These results suggest that the Herpes gene for thymidine kinase has integrated into the genome of the HSV-transformed human cells and that it can be transferred to other cells by means of purified metaphase chromosomes.  相似文献   

20.

Background  

Horizontal Gene Transfer (HGT) is beneficial to a cell if the acquired gene confers a useful function, but is detrimental if the gene has no function, if it is incompatible with existing genes, or if it is a selfishly replicating mobile element. If the balance of these effects is beneficial on average, we would expect cells to evolve high rates of acceptance of horizontally transferred genes, whereas if it is detrimental, cells should reduce the rate of HGT as far as possible. It has been proposed that the rate of HGT was very high in the early stages of prokaryotic evolution, and hence there were no separate lineages of organisms. Only when the HGT rate began to fall, would lineages begin to emerge with their own distinct sets of genes. Evolution would then become more tree-like. This phenomenon has been called the Darwinian Threshold.  相似文献   

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