cDNA末端快速扩增技术及其应用*

摘要：cDNA末端快速扩增（RACE）技术是一种快速获得cDNA的3′和5＇端的方法。本文从RACE的原理出发,指出其技术本身存在的优缺点,阐述了RACE操作中须注意和不容忽视的技术要点,并对前人对RACE技术所做的改进加以总结,最后对RACE技术的应用前景给予了展望。Abstract: Rapid amplification of cDNA end (RACE) technique is a method of which the 3’ and 5’ fragments of cDNA can be rapidly obtained. In this review, the advantages and shortcomings RACE manipulation were pointed out and some important technical points in RACE protocols in the previous literatures were summarized.     

海南岛早志留世晚期腕足类Xinanospirifer的发现--兼论南好组

海南岛保亭县毛感乡南兵至南好公路边南好组以往被确认为下石炭统岩关阶 ,并认为与其下的上志留统足赛岭组呈角度不整合接触。著者最近在该剖面南好组中发现兰多维列世特里奇期晚期 (LateTelychian)Xi nanospirifer腕足动物群和三叶虫Latiproetuscf.latilimbatus,证明久归于下石炭统岩关阶南好组的地质时代应改归于早志留世 (Llandoverian) ;海南岛地区在早志留世明显属于扬子地台区的范畴 ;从地质时间上还暗示南好组与其下伏的足赛岭组不可能存在角度不整合接触 ;     

抗癌胚抗原免疫毒素生物学功能的研究

为了研究免疫毒素发挥生物学作用的全过程,构建了两种不同形式的基于假单胞菌外毒素(Pseudomonas exotoxin A,PE)的抗癌胚抗原（carcinoembryonic antigen, CEA）免疫毒素CEA(Fv)/PE38/KDEL和PE35/CEA(Fv)/KDEL.用流式细胞术经间接免疫荧光法测定免疫毒素的抗原结合活性.免疫毒素的内化(internalization)通过间接免疫荧光标记的方法在激光共聚焦显微镜下进行检测,采用流式细胞术进行免疫毒素内化的定量分析.细胞凋亡采用FITC-annexin V/PI双荧光染色方法进行分析.用噻唑蓝(MTT)法测定免疫毒素的靶细胞杀伤功能.对两种形式的免疫毒素在各个功能阶段的性质进行研究和比较,全面展现了免疫毒素发挥生物学功能的过程及各个生物作用过程之间的相互影响,为详尽的机制研究和免疫毒素的进一步优化改造提供了重要的依据.     

蛋白磷酸酯酶2A调节微管相关蛋白1b的磷酸化

微管相关蛋白MAP1b的生物学活性受其磷酸化修饰的调节,后者则受相应的蛋白激酶和蛋白磷酸酯酶(PP)调控.为研究蛋白磷酸酯酶在脑内对MAP1b磷酸化的调控作用,采用有代谢活性的大鼠脑片作为模型,分别应用冈田酸(okadaic acid)和cyclosporin A选择性地抑制PP2A 和PP2B活性,来研究其对脑内蛋白磷酸酯酶MAP1b磷酸化的调控.采用特异性的MAP1bⅠ型磷酸化依赖性抗体522和免疫印迹技术检测MAP1bⅠ型磷酸化.结果表明,当PP2A被okadaic acid选择性抑制后,MAP1bⅠ型磷酸化明显增加.而PP2B被选择性地抑制后,MAP1b磷酸化的变化不大.免疫组化染色显示,MAP1b广泛分布于鼠大脑神经元和突起中,与对照组相比,在PP2A抑制的脑片中抗体522的免疫活性在神经元中明显升高.上述结果表明,PP2A是脑中调控MAP1bⅠ型磷酸化的主要蛋白磷酸酯酶.     

Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility.     

Effects of AF64A on Gene Expression of Choline Acetyltransferase (ChAT) in the Septo-Hippocampal Pathway and Striatum In Vivo

AF64A (ethylcholine mustard aziridinium ion) was stereotaxically administered bilaterally (1 nmol/side) into rat lateral cerebral ventricles. Choline acetyltransferase (ChAT) activity and ChAT mRNA levels were measured at predetermined time points in the septo-hippocampal pathway and striatum, both well identified as rich in cholinergic neurons. AF64A caused a rapid but transient increase in ChAT mRNA (167%, P < 0.05) and ChAT activity (164%, P < 0.01) in the septum. By day 7 post treatment, there was a significant decrease in ChAT mRNA (42.5% of control, P < 0.05) in the septum although the ChAT activity still stayed high. This decreased ChAT mRNA level in the septum lasted for at least four weeks, and was paralleled by a long-lasting decrease in ChAT activity in the hippocampus. In the striatum, on the other hand, there were no observed changes in either ChAT activity or ChAT mRNA. These data suggest that the long term effect of AF64A on the septo-hippocampal cholinergic pathway may, at least in part, be due to an action of AF64A on gene expression in the cholinergic neuron. The difference in the response to AF64A between the septo-hippocampal and striatal cholinergic systems might be due to their difference in neuron types.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

哺乳动物DGAT基因及其生物学功能研究进展

二酰基甘油酰基转移酶(DGAT, EC2.3.1.20)是一种微粒体酶, 与脂肪代谢、脂类在组织中的沉积有很大关系, 它的主要作用机制是使二酰甘油加上脂肪酸酰基形成三酰甘油。DGAT在细胞甘油代谢中起根本性的作用, 并在高等真核生物甘油三酯代谢途径如肠脂肪吸收、脂蛋白集合、脂肪形成和泌乳中发挥着重要的功能, 提示DGAT不仅是调控甘油三酯与脂肪酸之间的关键因子, 而且可能在动物脂肪沉积中起着关键的调控作用。     

铀的生物成矿作用及成矿过程中矿质元素循环

概述了微生物成矿作用,并介绍了硫杆菌、硫酸盐还原菌、铁细菌、硝化细菌、反硝化细菌等各种细菌在与铀的生物成矿过程相关的地质元素循环中所起到的作用。并展望了今后矿床中微生物种群的研究方向。