临床分离嗜麦芽寡养单胞菌L1、L2酶基因克隆、测序及定位

目的了解临床分离嗜麦芽寡养单胞菌产LI、L2 β-内酰胺酶情况、酶基因定位及核苷酸序列进化和同源关系。方法PCR法扩增两种β-内酰胺酶基因，通过克隆、测序、序列比对确定其亚型、基因进化、同源关系和基因所在位置。结果细菌染色体DNA和质粒DNA扩增L1、L2酶基因阳性，其序列具有明显异质性。两种酶基因位于12kb大小的质粒上。结论嗜麦芽寡养单胞菌绝大多数产生两种β-内酰胺酶，酶基因变异和进化加速的驱动力部分可能与β-内酰胺类抗生素的长期使用有关。     

金银花DNA去甲基化酶基因的生物信息学分析

DNA去甲基化酶基因广泛存在于植物中,本文主要通过生物信息学的方法从金银花转录组中获得4个DNA去甲基化酶基因,分别为LJDME1、LJDME2、LJDME3和LJDME4,并预测其编码蛋白的理化性质和表达模式。系统进化树结果表明LJDME1、LJDME2聚为一类,LJDME3、LJDME4聚为一类,与拟南芥中的DME关系更为密切。基因表达分析表明,4个DNA去甲基化酶基因在变种间差异表达明显,LJDME1、LJDME2基因表达水平在红金银花中高于金银花,推测其可能参与调控金银花活性成分积累的过程,并且4个DNA去甲基化酶基因具有组织表达特异性。本研究为进一步解析金银花中DNA去甲基化酶的功能奠定了基础。     

治疗肿瘤的新方法——基因治疗

随着近代分子生物学的迅速发展和肿瘸发病分子机理的深入研究，一种治疗肿瘤的新方法——基因治疗，已开始由理论研究向临床试验过渡。基因治疗是指在DNA(RNA)水平上对疾病的控制与治疗，就是通过基因递呈(转移)包括转入外源正常DNA顺序以矫正缺失或突变的DNA序列，导入细胞因子或其他功能基因诱发免疫应答反应发挥抗细胞增殖作用，导入某些基因调控以控制基因的异常表达等方式来进行治疗。     

放射调控Bax基因表达的pEgr-Bax载体构建和鉴定

目的：探讨放射诱导肿瘤凋亡进而治疗肿瘤的新方法，构建含Egr-1启动子和Bax基因的表达载体。方法：以Egr-1启动子和Bax为靶基因，以pIRES—EGFP质粒为载体。根据Egr-1基因的DNA序列和Bax基因的c—DNA序列设计引物，分别从bab／c雌鼠的肝细胞提取含Egr-1启动子序列的DNA，从乳腺癌细胞（MCF-7）中提取含Bax—d基因序列目的基因，克隆到空载体pIRES—EGFP中，并转化至DH5α感受态细胞中，提取质粒，进行PCR鉴定和测序确认重组载体构建成功。结果：经PCR鉴定筛出的重组体的测序结果与目的基因序列相同．重组体构建成功。结论：利用克隆技术可成功构建放射调控Bax基因靶向表达的pEgr—Bax载体。     

miRNA与药物反应个体差异

目前关于药物反应个体差异研究集中在药物代谢和药物靶点基因的单核苷酸多态性位点(SNPs)和其他基因突变(例如拷贝数的变化)的表型效应,而仅分析DNA序列不能完全解释药物反应个体差异.miRNA的发现,为其提供了新的研究方向.miRNA是一类内源性的非编码小RNA,其与靶基因mRNA3'非翻译区(3'UTR)互补结合,引起mRNA切割或翻译抑制,从而在转录后水平下调基因表达.miRNA可负调控大量人类基因的表达,其中包括许多药物疗效相关基因.越来越多的研究结果表明,miRNA是导致药物反应个体差异的又一重要因素.本文从miRNA调控药物代谢、药物转运和药物靶点,miRNA及其结合位点多态性等方面对miRNA在药物反应个体差异中所起作用进行综述.     

肿瘤表观遗传与代谢研究进展

表观遗传学是研究在基因核苷酸序列不发生改变的情况下基因表达可遗传的变化的一门学科,是生命科学领域的热点学科。其中,基于表观遗传学的肿瘤治疗更是近年来研究的热点前沿。本文从DNA与RNA的甲基化（包括甲基化介导的基因表达调控）、组蛋白修饰（与调控基因的相互作用交流）、染色质重塑相关基因的表达调控、代谢重编程及基于表观遗传调控的肿瘤治疗等几个方面对近年来肿瘤表观遗传热点问题与相关重大进展进行总结和讨论,以期为肿瘤的基础研究和临床治疗提供一定的借鉴。     

4′，5，7-三羟基异黄酮抑制肿瘤细胞核因子-KB活化的作用机制

核因子KB（NF-KB）是一类能特异性地识别结合DNA的Rel类蛋白质二聚体转录因子。在静息细胞中，NF-KB与抑制性蛋白IKBs结合形成复合物，并被滞留于细胞质中而处于非活化状态；当细胞受到各种胞内外刺激时，IKBs被迅速地降解，NF-KB得以释放并进入细胞核，从而发挥其转录调节功能。NF-KB通过调控众多靶基因的转录表达而在免疫、炎症反应、细胞增殖与凋亡及肿瘤发生等许多生理学过程中发挥重要作用。4′，5，7-三羟基异黄酮（genistein）是大豆异黄酮（soybeanisoflavones）的成分之一，是大豆中一类重要非营养素成分，近年来研究显示其具有显著的防治癌症效果。     

产毒性大肠杆菌不耐热和耐热肠毒素基因克隆及序列分析

目的:用基因工程方法克隆产毒性大肠杆菌(ETEC)不耐热肠毒素(LT)和耐热肠毒素(ST)基因,以制备检测ETEC的单克隆抗体.方法:利用PCR技术扩增LT和ST基因并将它们插入T-easy载体中,采用全自动测序仪进行核苷酸序列分析.结果:DNA序列分析表明,克隆的ETEC-LT-B DNA序列与GenBank公布序列比较在17、69、101、102位点有碱基变异,同源性98.9%;ETEC-STDNA序列与GenBank公布序列一致.结论:成功获得正确的ETEC的LT和ST基因,为其重组表达及后续研究奠定了实验基础.     

中国恒河猴指名亚种基因中BamHⅠ重复序列的克隆与结构分析

探索灵长类细胞基因中重复序列结构与基因启动调控序列的关系,获得具有启动子功能的基因结构。方法：使用酶切－ＰＣＲ技术,从中国恒河猴指名亚种基因中,克隆ＢａｍＨⅠ基因重复序列,并对其结构进行序列测定、分析和ＤＮＡ结合实验。结果：克隆到两个ＢａｍＨⅠ基因重复序列片段。结论：提示这两个重复序列片段 中具有启动调控的典型结构,因而具备用于构建真核表达载体的可能性。     

21世纪的药物疗法

21世纪的药物疗法主要是以查明病态机制为基础的治疗，用药物释放系统（DDS）特异性受体、反义寡核耷酸和载体的治疗。这些疗法特异性高，副作用小，效果好。20世纪后，细胞生物学知识剧增，遗传工程技术突飞猛进，使临床上有用的重组产品得以生产和实用化，且在基因水平上查明病态机制和以此为基础的基因水平的治疗成为可能。例如，DNA或RNA中存在着与特定核着酸相对的反义寡核音酸。如果能启动反义寡核管酸，就会抑制特定的DNA或由其而来的mRNA。到21世纪时，会有各种反义寡核青酸作为新的药物。应用DDS的治疗，现已用脂质体包裹…     

MicroRNAs and cancer epigenetics

The term epigenetics refers to all heritable changes in gene expression that are not associated with concomitant alterations in the DNA sequence. Reversible DNA methylation and histone modifications are the hallmarks of epigenetic gene regulation. MicroRNAs (miRNAs) are a recently discovered category of noncoding RNAs with important regulatory functions. Aberrancies in both the epigenetic and in the miRNA regulation of genes have been documented in cancer. An increasing number of studies are showing that the abnormalities of the epigenome and of the miRNome are not independent and could be explained both by an epigenetic regulation of miRNA expression and by miRNA control on components of the epigenetic machinery. A deeper understanding of this correlation could lead to new therapeutic avenues.     

Identification of single nucleotide polymorphisms of the human metabotropic glutamate receptor 1 gene and pharmacological characterization of a P993S variant

mGluR1 receptors are believed to play major roles in the pathophysiology of diseases such as anxiety and chronic pain and are being actively investigated as targets for drug development. Sequence polymorphisms can potentially influence the efficacy of drugs in patient populations and are therefore an important consideration in the drug development process. To identify DNA sequence variants of the mGluR1 receptor, comparative DNA sequencing was performed on DNA samples (n = 186) from apparently healthy subjects representing two ethnic groups. In total, eight non-synonymous single nucleotide polymorphisms (SNPs) were identified and one SNP (c2977 > T) was found to be particularly common, this SNP results in a proline to serine substitution at residue 993 (P993S). The WT (P993) and S993 variants were expressed in an inducible system which allowed us to titrate gene expression to equivalent levels and were pharmacologically characterized. We determined the potency and affinity of standard antagonist compounds as well as the potency and efficacy of the endogenous ligand glutamate and other agonist compounds at both receptor variants. Agonist evoked increases in intracellular Ca²⁺ were measured by fluorometric imaging plate reader (FLIPR). The potency of mGluR1 antagonists was evaluated by their ability to inhibit quisqualate induced increases in intracellular Ca²⁺, while their affinities were determined by radio-ligand binding studies. This study demonstrates that the Pro993Ser amino acid exchange is highly frequent in the human mGluR1 gene. This polymorphism however, does not appear to affect the potency of agonist compounds or the potencies or affinities of small molecule antagonist compounds.     

Interaction of human estrogen receptors alpha and beta with the same naturally occurring estrogen response elements

Estrogen receptors are derived from two different gene products referred to as estrogen receptor-alpha (ER-alpha) and ER-beta. Both receptors bind to the consensus estrogen response element (ERE) present in the vitellogenin gene, but their binding to hormone response elements present in other estrogen responsive genes has not been reported yet. Using in vitro expressed human receptors, we now show that ER-beta binds to a panel of six endogenous hormone response elements (vitellogenin, c-fos, c-jun, pS2, cathepsin D, and choline acetyltransferase) already known to bind ER-alpha and confer estrogen inducibility to reporter constructs. Binding of ER-alpha and ER-beta occurred at similar DNA concentrations for some EREs, but different DNA concentrations were required to form complexes of the two receptors with other elements. These results illustrate for the first time by direct receptor-DNA binding studies that both ER-alpha and ER-beta bind to a number of EREs present in endogenous hormone regulated genes, and further suggest that the two forms of the receptor display different patterns of affinities for naturally occurring hormone response elements.     

Discovery of two novel, small-molecule inhibitors of DNA methylation

DNA methyltransferases are promising targets for cancer therapy. In many cancer cells promoters of tumor suppressor genes are hypermethylated, which results in gene inactivation. It has been shown that DNA methyltransferase inhibitors can suppress tumor growth and have significant therapeutic value. However, the established inhibitors are limited in their application due to their substantial cytotoxicity. To discover novel compounds for the inhibition of human DNA methyltransferases, we have screened a set of small molecules available from the NCI database. Using a 3-dimensional model of the human DNA methyltransferase 1 and a modified docking and scoring procedure, we have identified a small list of molecules with high affinities for the active site of the enzyme. The two highest scoring structures were found to inhibit DNA methyltransferase activity in vitro and in vivo. The newly discovered inhibitors validate our screening procedure and also provide a useful basis for further rational drug development.