用于蛋白质体外分子进化研究的DNA随机突变技术

蛋白质体外分子进化是模拟自然的进化过程,利用基因随机突变和定向筛选（选择）技术,以获得具有预期新功能的突变体分子。虽然体外进化近几年才产生,但已成为医药和工业领域中筛选具有特殊催化性质的酶的最重要的方法之一。ＤＮＡ随机突变技术是蛋白质体外分子进化研究的基础,本文将对几种最重要的突变方法：倾向错误的ＰＣＲ、ＤＮＡ重排、模板交错延伸反应和随机延伸突变的原理和应用等加以介绍。     

山茶属CHS基因家族的组成和分子进化初探

用PCR方法从4种山茶属(Camellia)(山茶科)(Theaceae)植物的总DNA中分别扩增到CHS基因外显子2的部分序列,经克隆、测序得到16个该基因的序列,这些序列与来自GenBank的该属另一种植物的3个序列及作为外类群的大豆(Glycine max (L.) Merr.)的2个序列一起进行分析.研究表明,山茶属CHS基因家族在进化过程中已分化为A、B、C三个亚家族,包括A1、A2、A3、B1、B2、C 等6类不同的基因成员;其中只有A2类成员为全部被研究的5种植物所共有,而其他5类成员只在部分被研究的植物中发现;所有这些CHS成员具有很高的同源性:在核苷酸水平上同一亚家族内基本上高于90%,不同亚家族间也在78%以上.从推测的氨基酸组成看,山茶属内CHS基因的功能已发生了分化,各类成员的碱基替代率有较大差异; 从分子系统发育树和可能的氨基酸组成分析,山茶属具有新功能的基因成员是在经过基因重复后,或是由少数几个位点的突变而成,或是由逐渐积累的突变而形成的.进一步分析认为,该属CHS基因的分化直到近期还在活跃地进行,并且不同种的进化式样有一定的差别,这种不同的进化式样可能是物种形成后受不同环境因素影响而形成的.     

基于细菌人工染色体的大基因质粒制备及应用研究

细菌人工染色体(BAC)最多可克隆300 kb的DNA片段且遗传特性稳定,是目前常用的克隆载体.但如何高效提取BAC装载的大基因及其大基因操作等方面还需不断的摸索完善.本研究采用超大基因质粒提取法从BAC中提取出165 kb的大基因,经Nanodrop测定浓度、酶切、脉冲场电泳,表明获得目标基因;将获得的大基因质粒转染...     

季节性温度变化对CANON型潮汐流人工湿地脱氮的影响

探究了温度的季节性变化对基于亚硝化的全程自养脱氮(CANON)型潮汐流人工湿地(TFCW)脱氮性能及其微生物特性的影响。CANON型TFCW中的脱氮微生物群落在温度的季节性变化下会发生不同程度的改变,其脱氮途径及性能随之会出现周期性的波动。填料层温度在20.0 ℃以上时对TFCW脱氮性能及其中的优势脱氮菌群无显著影响,CANON作用是系统脱氮的主要途径。当填料层温度低于20.0 ℃时,厌氧氨氧化菌丰度与活性显著降低,在9.3~20.0 ℃时,亚硝酸盐氧化菌(NOB)的增殖及其活性的提高使TFCW中脱氮的主要途径由CANON作用演替为硝化/反硝化作用,系统对总氮(TN)的去除率仅为(34.8±13.0)%;在2.2~9.0 ℃时,TFCW中的厌氧氨氧化菌在受到抑制的同时仍保持着对NOB和反硝化菌群的相对竞争优势,系统脱氮重新依赖于CANON作用,其对TN的去除率为(54.8±4.8)%。该研究可为CANON型TFCW工艺的优化及工程化应用提供参考。     

基于CRISPR/Cas9技术的基因敲入/敲除策略

成簇的规律间隔性短回文序列(CRISPR)基因编辑系统,因其设计简单操作方便和无种属限制,已成为一种广泛应用的基因组定点编辑工具,在复杂的基因组编辑,例如基因的人源化改造以及条件等位基因的构建中有所应用。在自然界中,CRISPR系统拥有多种类别。其中,CRISPR/Cas9系统是研究最深入、应用最成熟的一种。本文针对CRISPR/Cas9系统,分别从基因敲入/敲除片段的大小、同源臂长短、构型即递送方式等技术环节进行综述,阐述不同设计及操作条件下由CRISPR/Cas9系统介导的基因敲入/敲除的效率差异。     

耐辐射球菌基因DRB0099缺失突变株的构建及逆境分析

耐辐射球菌(Deinococcus radiodurans R1)有着极强的辐射抗性.研究其抗辐射的机理对于处理放射性废料有着潜在的应用价值.在耐辐射球菌的基因组中,许多序列的功能未知.其中DRB0099尤为引人注意.将DRB0099缺失突变构建该基因的突变株.对野生型和突变体进行比较后发现,在正常生长条件下的前期阶段(0～16 h),突变体生长速度比野生型慢.16 h以后,野生型逐渐进入稳定生长期.这时,突变株的生长速度高于野生型.但是,野生型的浓度一直高于突变株.表明在DRB0099被删除后,耐辐射球菌的生长可能受到了阻滞.在紫外线照射的条件下,尽管野生型随着照射剂量的增加,存活率越来越低,但是要比突变体高许多.野生型具有比突变体更强的修复DNA双链断裂的能力.DRB0099可能直接参与了对DNA的修复.突变体对H2O2的敏感程度高于野生型,表明野生型耐辐射球菌在对抗活性氧保护其蛋白质、DNA或者DNA修复方面具有比突变体更强的功能.在低浓度H2O2处理条件下,尽管野生型和突变体的存活率都出现下降趋势,但二者的差值并不大.随着H2O2剂量的增加,二者的差值越来越大.表明随着活性氧浓度的增加,蛋白质和DNA损伤的数量增加,失去DRB0099基因功能的突变体比野生型更容易受到损伤.在紫外线照射处理或者H2O2处理条件下,DRB0099能够保护蛋白质和DNA.     

人线粒体tRNALeu(UUR)基因A3243G点突变对其亮氨酰化活性的影响

化学法合成人线粒体野生型与A3243G点突变型tRNALeu(UUR)基因,体外转录生成相应的tRNALeu(UUR),表达并纯化人线粒体亮氨酰tRNA合成酶(mtLeuRS),用mtLeuRS催化野生型与突变型tRNALeu(UUR)与亮氨酸结合,分别检测两种类型tRNALeu(UUR)的氨酰化动力学常数。结果表明,野生型tRNALeu(UUR)的Km/Kcat仅为突变型tRNALeu(UUR)的63.9%,A3243G点突变使tRNALeu(UUR)接受亮氨酸的能力明显下降,提示此为A3243G点突变致病机制之一。Abstract:The wild-type and mutant-type human mitochondrial tRNALeu(UUR) genes were synthesized and transcribed in vitro with T7 RNA polymerase.The kinetic parameters of human mitochondrial leucyl-tRNA synthetase(mtLeuRS) were determined with wild-type and mutant-type human mitochondrial tRNALeu(UUR) respectively.The results show that the value of Km/Kcat of mtLeuRS for the mutant-type tRNALeu(UUR) is 63.9% as compared with the wild-type.Human mitochondrial tRNALeu(UUR) gene A3243G point mutant can remarkably reduce it′s aminoacylation activity,suggesting it would be one of the mechanisms that the mutation could produce such clinical phenotypes.     

shRNA抑制活化诱导的胞苷脱氨酶（AID）对前列腺癌细胞C4-2表型的影响

目的:探讨AID在前列腺癌中的表达情况,AID对前列腺癌细胞C4-2的侵袭、迁移、增殖以及凋亡方面的影响。方法:应用靶向敲减AID的慢病毒对前列腺癌细胞C4-2进行干扰,运用Western-blot、免疫组化、平板克隆形成、流式、Transwell实验对前列腺癌组织和前列腺癌细胞C4-2表型的变化情况进行研究。结果:临床前列腺癌样本中AID高表达,良性前列腺增生组织中AID低表达,正常前列腺组织不表达(*P0.05);shRNA干扰以后的shAICDA-C4-2单克隆细胞株中AID的表达量显著降低,其增殖、迁移和侵袭能力阳性对照组(Monoclonal6)与阴性对照组(NC)相比分别降低49%、80%、63%,凋亡率阳性对照组(Monoclonal6)为阴性对照组(NC)的3.2倍。结论:前列腺癌组织中AID高表达,AID在促进前列腺癌细胞的增殖、迁移、侵袭,抑制前列腺自细胞的凋亡中具有极其重要的作用。AID表达极可能与前列腺癌的进展、预后明显相关。     

Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line

A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies (Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on.     

AID expression in B-cell lymphomas causes accumulation of genomic uracil and a distinct AID mutational signature

The most common mutations in cancer are C to T transitions, but their origin has remained elusive. Recently, mutational signatures of APOBEC-family cytosine deaminases were identified in many common cancers, suggesting off-target deamination of cytosine to uracil as a common mutagenic mechanism. Here we present evidence from mass spectrometric quantitation of deoxyuridine in DNA that shows significantly higher genomic uracil content in B-cell lymphoma cell lines compared to non-lymphoma cancer cell lines and normal circulating lymphocytes. The genomic uracil levels were highly correlated with AID mRNA and protein expression, but not with expression of other APOBECs. Accordingly, AID knockdown significantly reduced genomic uracil content. B-cells stimulated to express endogenous AID and undergo class switch recombination displayed a several-fold increase in total genomic uracil, indicating that B cells may undergo widespread cytosine deamination after stimulation. In line with this, we found that clustered mutations (kataegis) in lymphoma and chronic lymphocytic leukemia predominantly carry AID-hotspot mutational signatures. Moreover, we observed an inverse correlation of genomic uracil with uracil excision activity and expression of the uracil-DNA glycosylases UNG and SMUG1. In conclusion, AID-induced mutagenic U:G mismatches in DNA may be a fundamental and common cause of mutations in B-cell malignancies.     

Aberrant AID expression and human cancer development

Cancer develops via a multistep process that occurs through the accumulation of somatic mutations of tumor-related genes that govern cell proliferation, regeneration, and apoptosis. The question how normal cells acquire the genetic changes that lead to malignant transformation is, however, unknown at present. Activation-induced cytidine deaminase (AID) produces immune-diversity by inducing somatic hypermutations and class-switch recombinations in human immunoglobulin genes. Unfortunately, this function of AID as a genome mutator could aim at the generation of somatic mutations in various host genes of non-lymphoid tissues and contribute to tumorgenesis. Notably, aberrant AID expression can be triggered by several pathogenic factors, including Helicobacter pylori infection and proinflammatory cytokine stimulation, in human epithelial cells, whereas AID expression is absent in those cells under physiologic conditions. Thus, aberrant AID activity in epithelial tissues may provide the critical link between inflammation, somatic mutations, and cancer development.