CELⅠ酶的粗提取及其活性检测

CEL I酶是第一个从真核生物中提取的用于高效特异切割DNA双链碱基错配和DNA扭曲的内切酶, 因而也是TILLING技术中用到的一种关键酶。文章对CELI酶的粗提取及其活性检测进行了研究。错配切割实验表明, CELI酶在包含有G→A点突变的杂合双链中, 能有效地在错配位点进行切割, 并可以通过ABI377测序仪获得直观的检测结果, 从而可以用于TILLING分析。     

小麦TILLING分析中CELⅠ酶切及PCR反应体系的优化

在小麦中建立稳定的基于CELⅠ酶切的目的基因突变位点检测技术,有助于高通量鉴定目的基因片段的点突变及提高突变检测效率。本研究以冬小麦品种新麦18空间诱变SP2群体为材料,以小麦糯质基因Waxy为目标片段,通过优化基因组DNA提取方法、调整PCR反应体系中dNTP、Mg2+及引物浓度、改变目标片段CELⅠ酶切缓冲液成分,以及调整纯化过程中的空气相对湿度等方式,优化了小麦TILLING技术体系。在利用PVP-40法提取DNA过程中,研磨器振动频率提高到30/s,KAc溶液的反应时间延伸为20min时,基因组DNA质量和纯度最佳;在设定的浓度范围内dNTP和Mg2+浓度对产物影响差异不明显,均能高效扩增出目的条带。引物浓度对产物影响差异显著,最佳引物浓度为0.4μmol/L。20μl酶切体系中,最佳CEL I酶浓度为0.1U且利用超纯水代替CELⅠ缓冲液。最终在小麦中建立起了基于CELⅠ酶切的高通量TILLING筛选技术体系。     

采用CELⅠ酶粗提物高效鉴定CRISPR/Cas9介导的基因突变

CRISPR/Cas9是新兴的基因组编辑技术,该技术操作简单、效率高,已成为目前最主流的基因组编辑技术。利用该技术进行突变体创制,对基因功能研究和育种应用具有重要的意义,而快速、高效、低成本的基因编辑个体鉴定是其中的重要环节。本研究对影响CELⅠ粗提物鉴定CRISPR/Cas9介导的水稻基因编辑个体的条件,包括蛋白用量及作用时间、PCR反应缓冲液等条件进行了探索,并将整个检测体系集成于一管操作。同时,采用CELⅠ粗提物检测了CRISPR/Cas9介导水稻stn1突变的T_0代植株及后代,对杂合突变、纯合突变及双等位突变的鉴定策略进行了探讨;该方法检测正确率经测序验证达100%。上述结果表明,采用CELⅠ粗提物检测突变体与已有方法比较具有廉价、快速和高效的特点。     

An autoantibody against N(ε)-(carboxyethyl)lysine (CEL): possible involvement in the removal of CEL-modified proteins by macrophages

Advanced glycation end products (AGEs) are believed to play a significant role in the development of diabetic complications. In this study, we measured the levels of autoantibodies against several AGE structures in healthy human plasma and investigated the physiological role of the autoantibodies. A high titer of the autoantibody against N(ε)-(carboxyethyl)lysine (CEL) was detected in human plasma compared with other AGE structures such as CML and pentosidine. The purified human anti-CEL autoantibody reacted with CEL-modified human serum albumin (CEL-HSA), but not CML-HSA. A rabbit polyclonal anti-CEL antibody, used as a model autoantibody against CEL, accelerated the uptake of CEL-HSA by macrophages, but did not enhance the uptake of native HSA. Furthermore, when (125)I-labeled CEL-HSA was injected into the tail vein of mice, accumulation of (125)I-CEL-HSA in the liver was accelerated by co-injection of the rabbit anti-CEL antibody. These results demonstrate that the autoantibody against CEL in plasma may play a role in the macrophage uptake of CEL-modified proteins.     

微生物发酵生产十一烷1，1 1二羧酸的研究

从热带假丝酵母(Candiada tropicalis)T25—14经过紫外线和亚硝酸的多次诱变,获得4株产十一烷l,11二羧酸(DC₁₃)较多的突变株,其中最优的NP-159株以20％(V／V)正十三烷(nC₁₃)为碳源摇瓶发酵4天,DC₁₃达80g／L左右。在16L罐上,以30％(V／V)nC₁₃发酵6天,DC₁₃高达139g／L,回收残烃后,对nC₁₃的转化率为80％以上。后处理收率为78．9％,DC₁₃的纯度为95．3％。     

Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A)

Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO₃⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO₃⁻ exchange at the basolateral membrane and failure of proton (H⁺) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.     

Niemann–Pick C1 Like 1 (NPC1L1) an intestinal sterol transporter

Niemann–Pick C1 Like 1 (NPC1L1) has been identified and characterized as an essential protein in the intestinal cholesterol absorption process. NPC1L1 localizes to the brush border membrane of absorptive enterocytes in the small intestine. Intestinal expression of NPC1L1 is down regulated by diets containing high levels of cholesterol. While otherwise phenotypically normal, Npc1l1 null mice exhibit a significant reduction in the intestinal uptake and absorption of cholesterol and phytosterols. Characterization of the NPC1L1 pathway revealed that cholesterol absorption inhibitor ezetimibe specifically binds to an extracellular loop of NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice are resistant to diet-induced hypercholesterolemia, and when crossed with apo E null mice, are completely resistant to the development of atherosclerosis. Intestinal gene expression studies in Npc1l1 null mice indicated that no exogenous cholesterol was entering enterocytes lacking NPC1L1, which resulted in an upregulation of intestinal and hepatic LDL receptor and cholesterol biosynthetic gene expression. Polymorphisms in the human NPC1L1 gene have been found to influence cholesterol absorption and plasma low density lipoprotein levels. Therefore, NPC1L1 is a critical intestinal sterol uptake transporter which influences whole body cholesterol homeostasis.     

细胞色素P450 1A1和1B1靶向抗肿瘤前药研究进展

细胞色素P450（CYP）能催化各种内源性及外源性化合物的代谢,与多种肿瘤发生有关。其中CYP1A1参与多种前致癌物和致突变物的代谢活化,CYP1B1被认为在许多人癌细胞中特异性表达,参与药物的氧化代谢和前药的活化。CYP1A1和181已成为靶向抗肿瘤前药研究的新靶点。相继有大量相关研究报道,本文就近年来文献报道的CYP1A1和1B1靶向抗肿瘤前药研究进展。     

Tbf1 or not Tbf1?

In this issue of Molecular Cell, Hogues et al. (2008) demonstrate a wholesale shift in the key regulatory protein involved in ribosomal protein (RP) synthesis during the evolution of S. cerevisiae and, en passant, raise interesting questions about the relationship between RP genes and telomeres.     

CYP1B1基因与青光眼

遗传性青光眼包括两种主要的类型,原发性开角型青光眼(primary open angle glaucoma,POAG)和原发性先天型青光眼(primary congenital glaucoma,PCG).眼前节发育不良(anterior segment dysgenesis,ASD)是眼发育异常的遗传异质性病,与增长的眼内压和青光眼有关,包括Peter's异常、Rieger's异常、无虹膜和虹膜发育不全.CYPIB1基因是PCG的致病基因,也有少数报道是POAG的修饰基因,或是POAG和ASD的致病基因.本文就CYP1B1基因突变与遗传性青光眼和ASD发育不全的关系及其遗传特点作一综述.     

问题解答1

问 :蛋白质、核酸都是通过缩聚反应形成的吗 ?答 :由于人们对教材内容机械引入或对教材语意曲解 ,因而众多的复习资料无论在介绍蛋白质、核酸的结构还是生命起源时都人为地强调氨基酸“缩合”形成蛋白质、核苷酸“聚合”形成核酸。“缩合”与“聚合”似乎成了风马牛不相及的两个概念 ,更谈不上去探索其有机联系了。缩合源于生物化学中蛋白质 (或肽 )的形成 ,它可以指两个或多个氨基酸形成肽时脱去一个或多个水分子的反应。正由于它脱去小分子物质 ,而有别于加成反应 ,同时它发生在有机的小分子之间 ,且反应结果并没有形成不饱合键而不同于消…     

沉默DEPDC1抑制鼻咽癌细胞HNE-1和CNE-1侵袭迁移

DEPDC1(DEP domain containing 1)是一个新的肿瘤相关基因,在多种恶性肿瘤的发生发展进程中起着重要作用。我们前期工作中在鼻咽癌细胞内沉默了DEPDC1的表达,发现抑制细胞增殖并诱发细胞凋亡。本研究旨在探讨沉默DEPDC1表达后,对鼻咽癌细胞HNE-1和CNE-1侵袭迁移能力的影响及其分子机制。结果显示,siRNA介导DEPDC1表达沉默后,细胞侧向运动能力、侵袭及迁移能力显著降低。qRT-PCR及Western印迹检测发现DEPDC1沉默导致EMT上游关键转录因子Twist1及间质细胞标志分子Vimentin表达显著下调。这些研究表明,鼻咽癌细胞中DEPDC1通过调节Twist1等EMT关键分子的表达在细胞侵袭转移过程中起关键作用。推测DEPDC1在鼻咽癌中高表达可能对于促进其侵袭转移具有重要作用,进而促进肿瘤发生发展,但具体分子机制仍有待更深入研究。     

Mutators in SACCHAROMYCES CEREVISIAE: MUT1–1, MUT1–2 and MUT2–1

The purpose of this study was to characterize two mutator stocks of yeast which were induced and selected on the basis of high spontaneous reversion rates of the suppressible "ochre" nonsense allele lys1-1. In the mutator stock VA-3, a single mutation, designated mut1-1, is responsible for the increase in the reversion rate of the ochre alleles lys1-1 and arg4-17. In stock VA-105, there are two separate mutator mutations. Tetrad analysis data showed these two loci are loosely linked. Based on complementation data, one of these mutations is at the same locus as mut1-1 and designated mut1-2. The second mutator of stock VA-105 was designated mut2-1. All three mutators are recessive. Both mut1-1 and mut1-2 give a high mutation rate for ochre nonsense suppressor (SUP) loci, but not for the ochre nonsense alleles. On the contrary, the mutation rates of the ochre alleles are greatly reduced. With the mutant mut2-1 there were mutations at both the lys1-1 site and its suppressors; mut2-1 is as effective as mut1-2 but not as effective as mut1-1 in inducing reversions of a missense mutant, his1-7. Neither mut1-1, mut1-2 nor mut2-1 were effective in inducing reversions of a putative frameshift mutation, hom3-10, or in inducing forward mutations to canavanine resistance.     

IBVS1基因表达载体PG-S1的构建

以含有鸡传染性支气管炎病毒S1基因的载体P^IBV-Z和含有CMV启动子的栽体质粒P^EGFR-C1为材料,构建了可表达IBV S1基因的表达载体P^G-S1,经酶切、电泳和PCR检测结果证实,构建的载体符合目的要求,可以作为表达载体用于鸡的抗病育种。     

2009甲型H1N1流感病毒研究进展

2009年3月在美国和墨西哥爆发的新型甲型H1N1流感在很短的时间内便扩散到世界多个国家,形成了流感的大流行,引起世界卫生组织和各国的高度重视。综述新型甲型H1N1流感病毒的基因组来源、目前主要的检测手段,并对预防和治疗的方法进行简单介绍。     

应用寡核苷酸芯片并行检测CYP1A1和 GSTM1基因多态性

利用寡核苷酸芯片检测方法分析CYP1A1单核苷酸多态性 (SNP)和GSTM1缺失与否 ,实验结果证明了寡核苷酸芯片技术可并行、准确、高效地检测基因的单核苷酸多态性和其他类型的基因多态型 ,可为疾病遗传易感性及单体型的研究提供强有力的研究工具。采用该寡核苷酸芯片 ,检测了 84份正常人的血液DNA样本 ,其中GSTM1基因缺失率达到 4 7 6 % ,接近报道数值。统计分析发现 ,CYP1A1m1 m2的 3种基因型组合TT AG、TT GG和TC GG的发生频率都为 0 ,而根据实验得到的m1和m2各自基因型数据计算 ,它们的发生频率应是11 4 %、2 6 %和 3 1% ,所以推测在所检测的样本中没有T(m1位点 )和G(m2位点 )的连锁组合 ,即m1和m2位点的组合只有 3种单体型 :T A、C A和C G ,其发生频率分别是 6 9 6 %、7 7%和 2 2 6 %。