用差异显示法从人胎脑基因文库分离一个编码序列

人１８周、２２周胎儿脑、肝肾组织总ｍ ＲＮＡ 用ＤＤＲＴ－ＰＣＲ显示出差异的条带,回收胎脑和肝肾特异性表达的４８７条电泳条带．其中某些条带用３种组织的ｃＤＮＡ 探针作点杂交,筛选只对胎儿脑总呈阳性的ＤＮＡ 片段．以其中某一条带ＤＮＡ 为探针,从胎儿脑ｃＤＮＡ 文库筛选阳性克隆,得到ＧＣ５８．经Ｎｏｒｔｈｅｒｎ 杂交和ＤＮＡ 测序,表明它是人脑表达的序列,与数据库中ＫＩＡＡ０５１５有同源性,并编码一个有２７４个氨基酸的蛋白质,该蛋白质序列尚未见报道．探讨了ＤＤＲＴ－ＰＣＲ的条件和假阳性问题．     

17个新的C2H2型锌指基因片段的分离与克隆

按照Ｃ２Ｈ２型锌指基因保守结构域的ＤＮＡ序列设计一对简并引物,以人基因组ＤＮＡ为模板进行ＰＣＲ同源扩增,将由此获得的锌指基因片段为探针,从人胎肾、骨骼肌、骨骼组织的ｃＤＮＡ分子库中筛选到２２个Ｃ２Ｈ２型锌指蛋白ｃＤＮＡ片段,经国际ＮＣＢＩ数据库查询检索,其中１７个为新的锌指基因片段。对从胎肾ｃＤＮＡ分子库中分离到的Ｋ３－４和Ｋ５－１２克隆进行了表达谱分析,发现Ｋ３－４在肾脏中的表达量明显高于其他几     

降低mRNA差异显示技术假阳性率的一种方法

为了探讨降低mRNA差异显示技术假阳性率的方法 ,进一步提高此技术的可靠性 ,提取了手术切除肝癌及非癌肝组织成对标本的总RNA ,逆转录获得cDNA片段 ,以mRNA差异显示方法筛选差异表达基因 ,选取较明显的一条差异表达条带 ,行进一步PCR扩增 .分别对PCR产物及其经TA克隆后随机挑选的 6个单克隆质粒DNA进行序列分析 ,并通过GenBank BLAST数据库进行序列的同源性比较 ,以Northern杂交予以来源确认 .自 72 0余条扩增条带中共选出 2 8条差异条带 .序列分析及同源性比较表明 ,所选择条带的PCR产物为一可能的新基因片段 ;而随机选择的 6个TA克隆质粒DNA中 ,有 4个为同一已知基因片段 ,一个为另一已知基因片段 ,一个为一可能的新基因片段 .同源性比较表明 ,PCR产物直接测序所得序列与TA克隆质粒DNA的 6个片段不具同源性 .结果表明 ,mRNA差异显示条带可能由 1条以上分子量相似的片段构成 ,直接对PCR产物行序列分析并以其为探针进行Northern杂交 ,是导致出现假阳性片段的原因之一 .将PCR产物进行TA克隆 ,对单克隆质粒DNA进行序列分析并以其为探针进行Northern杂交 ,可能是解决此问题的一种较好方法 .     

稻瘟病菌侵染诱导的水稻早期反应基因的cDNA片段克隆与序列分析

以稻瘟病菌感染水稻,利用ｍＲＮＡ 差异显示技术分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１（ｅａｒｌｙ ｒｅｓｐｏｎｓｉｖｅ ｇｅｎｅ） 的ｃＤＮＡ 片段。Ｎｏｒｔｈｅｒｎ ｂｌｏｔ 分析表明,ＥＲ１ 基因在稻瘟病菌侵染水稻叶片６ ｈ 后开始表达,８ ｈ 最强,１０ ～１２ ｈ 开始减弱,１６ ｈ 消失。Ｓｏｕｔｈｅｒｎ ｂｌｏｔ 分析表明,ＥＲ１ 基因属于水稻基因组。对ＥＲ１ 基因片段（２１９ ｂｐ） 进行了克隆和序列分析。经查询,在ＧｅｎＢａｎｋ 中没有与ＥＲ１ 同源的基因序列。     

cDNA RDA法克隆小鼠鼻咽部组织特异性表达基因

采用ｃＤＮＡ代表性差异分析法（ｃＤＮＡ ＲＤＡ）克隆小鼠鼻咽部组织特异性表达基因。通过三轮消减杂交后,获得了七个差异片段。随机选择两个差异片段ＴＤＰ１和ＴＤＰ４,经ＤＮＡ印迹和ＲＮＡ印迹证实了ＴＤＰ１在鼻咽部特异性表达,而ＴＤＰ４在鼻咽部及食道均有表达,并没有特异性。进一步对这两片段测序分析,并与Ｇｅｎ－Ｂａｎｋ等数据库同源比较,推测ＴＤＰ１和ＴＤＰ４是两个新的基因片段,但是仅ＴＤＰ１在小鼠鼻咽部     

稻瘟病菌侵染诱导的水稻早期反应基因的cDNA片段克隆与序列分析

以稻瘟病菌感染水稻,利用ｍＲＮＡ差汞显示技术分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１的ｃＤＮＡ片段。Ｎｏｒｔｈｅｒｎ ｂｌｏｔ分析表明,ＥＲ１基因在稻瘟病菌侵染水稻叶片６ｈ后开始表达,８ｈ最强,１０￣１２ｈ开始减弱,１６ｈ消失。Ｓｏｕｔｈｅｍ ｂｌｏｔ分析表明,ＥＲ１基因属于水稻基因组。对ＥＲ１基因片段进行了克隆和序列分析。经查询,在ＧｅｎＢａｎｋ中没有与ＥＲ１同源的基因序列。     

用基因表达谱芯片研究人正常肝和肝细胞癌中差异表达的基因

以基因表达谱芯片对人正常肝及肝癌组织基因表达的差异性进行了研究比较。奖４０９６条人ｃＤＮＡ用点样仪点在特制玻片上制备成表达谱芯片;利用肝和肝癌组织的ｍＲＮＡ通过逆转录方法,将Ｃｙ３和Ｃｙ５２种荧光分别标记到两种组织的ｃＤＮＡ上,制备成ｃＤＮＡ探针,并与表达谱芯片进行杂交及扫描,重复４次实验,通过计算机数据处理判定基因是否在上述２种组织中有表达差异,筛选出差异表达的基因共９０３条。基因芯片技术可同时     

LDL受体基因cDNA的RT—PCR分离,克隆及其在RFLP中的应用

利用ＲＴ－ＰＣＲ技术分离低密度脂蛋白受体基因ｃＤＮＡ,将长为２６０５ｂｐ的ｃＤＮＡ插入质粒ｐＪＮ６,并经全序列测定证实,该序列与已报道序列相比,存在两个变异碱基,即第７５４位Ｃ→Ｔ,和１６５４位的Ｇ→Ａ,这两个变异碱基并不改变编码的氨基酸,利用ＬＤＬ受体基因ｃＤＮＡ中的ＣｌａＩ片段的作为探针,检测到ＬＤＬ受体基因上外显子８的ＳｔｕＩ位点是个限制性片段长度多态性位点,所克隆的ｃＤＮＡ含有可译框架的全     

黑曲霉3.795glaA基因及其5'调控区的克隆和序列分析

黑曲霉Ｔ２１是由黑曲霉３．７９５经诱变育种获得的糖化酶高产菌株,为阐明其高产的分子机制,由黑曲霉３．７９５克隆了糖化酶结构基因及其５′旁侧序列,并与黑曲霉Ｔ２１的相应序列进行了比较．由黑曲霉３．７９５菌丝体分离染色体ＤＮＡ,Ｓｏｕｔｈｅｒｎ杂交分析表明,糖化酶结构基因位于～２．５ｋｂ的ＥｃｏＲⅠ－ＥｃｏＲⅤ染色体ＤＮＡ片段上,在此ＥｃｏＲⅠ位点上游约１．０ｋｂ处有一ＳａｌⅠ位点．为构建糖化酶结构基因及其５′旁侧序列的基因组文库,该染色体ＤＮＡ分别用ＥｃｏＲⅠ＋ＥｃｏＲⅤ和ＥｃｏＲ＋ＳａｌⅠ消化,琼脂糖凝胶电泳分离并回收长度在１．０ｋｂ左右和２．５ｋｂ左右的ＤＮＡ片段,分别与ｐＵＣ１９载体连接后转化入Ｅ．ｃｏｌｉＤＨ５．用原位杂交方法筛选到了携带糖化酶基因编码区及其１５０５ｂｐ５′旁侧序列的阳性克隆．对克隆片段的ＤＮＡ序列进行了测定并与黑曲霉Ｔ２１的相应序列进行了比较,结果表明,在糖化酶基因编码区及其１５０ｂｐ３′非编码区内,未发现碱基差异,但在－３４０～－１５０５的５′上游区内发生了９个位置的碱基变化,包括缺失、插入和替换．这些结果表明,黑曲霉Ｔ２１与３．７９５的糖化酶产量的差异与其结构基因无关,但可能与其     

LDL受体基因cDNA的RT－PCR分离、克隆及其在RFLP中的应用

利用ＲＴ－ＰＣＲ技术分离低密度脂蛋白受体基因ｃＤＮＡ,将长为２６０５ｂｐ的ｃＤＮＡ插入质粒ｐＪＮ６,并经全序列测定证实,该序列与已报道序列相比,存在两个变异碱基,即第７５４位Ｃ→Ｔ,和１６５４位的Ｇ→Ａ,这两个变异碱基并不改变编码的氨基酸,利用ＬＤＬ受体基因ｃＤＮＡ中的ＣｌａⅠ片段作为探针,检测到ＬＤＬ受体基因上外显子８的ＳｔｕⅠ位点是个限制性片段长度多态性位点。所克隆的ｃＤＮＡ含有可译框架的全部密码,因此可作为基因表达材料。     

差异显示技术及其研究进展

差异显示技术是分离差异表达基因的重要方法与手段,同其它研究基因差异表达的方法如RDA,SSH,SAGE相比,差异显示技术是其中使用频率较高的方法。该技术自1992年创立以来,经过各国研究者不断完善与改进,现已大大克服以往诸多不足,扩大了应用领域。本文就差异显示技术的原理及主要优缺点作以简要概述,并着重就引物设计、降低假阳性、鉴定差异表达基因及差异显示技术的衍生技术这4个方面的研究进展作以详尽介绍。     

Multiomics analysis on DNA methylation and the expression of both messenger RNA and microRNA in lung adenocarcinoma

Lung adenocarcinoma (LUAD) poses a significant threat to public health worldwide, while the genetic and epigenetic abnormalities involved in the oncogenesis of LUAD remains unknown. This study aimed to identify and validate key genes during the development and progression of LUAD by multiomics analysis. First, Empirical Analysis of Digital Gene Expression Data in R (EdgeR) was used to identify differentially regulated genes between normal samples and LUAD samples. Then significance analysis of microarrays (SAM) was used to identify differentially methylated genes and regulated microRNAs (miRNAs) between normal samples and LUAD samples. Following that, Kyoto Encyclopedia of Genes and Genomes (KEGG)-enrichment analysis was used to analyze the function that these genes enriched in. A total of 4,816 genes, 419 miRNAs, and 4,476 methylated genes that were significantly differentially expressed corresponding to the normal tissues in LUAD were obtained, and some of the pathways these genes enriched in were the same. Moreover, 255 genes differentially methylated and expressed at the same time were also found, and these 255 genes were the target genes of the miRNAs differentially expressed in LUAD. Finally, nine genes (BRCA1, COL1A1, ESR1, FGFR2, HNF4A, IGFBP3, MET, MMP3, and PAK1) network analysis, and two of which were found to be related to the survival of LUAD patients. In summary, a total of nine genes that may play important roles in the development of LUAD were identified, and two (PAK1 and FGFR2) of them can be served as prognostic biomarkers for LUAD patients. The genes found in this study played different roles in the tumor progression of LUAD, indicating these genes may be considered as potential target genes for LUAD treatment.     

History of Modern Chromosomal Analysis. Differential Staining of Plant Chromosomes

Differential staining methods found extensive use in karyotype studies of many plant and animal species and provide for reliable identification of all chromosomes of the organism. Below we describe the most widespread methods and history of their advent. In addition, we discuss specific structure of the chromosomes and possible mechanisms responsible for differential segmentation.     

不同抗逆性菠萝品种的差异基因和差异代谢物分析

该研究对2个不同抗逆性的菠萝品种PZ2(抗逆性较差)和PZ3(抗逆性强)进行转录组和代谢组比较分析,以探索品种间抗逆性差异的分子生理机制,为菠萝抗性育种提供理论依据。(1)转录组结果显示,以PZ2为对照,在2个品种间共筛选到1 667个差异表达基因,上调和下调的差异表达基因分别为770个和897个;其中筛选到20个与抗逆性相关的差异表达基因,包括WRKY和MYB转录因子基因以及ASR3、SOD1、POD48、HSP20、GSTF1、GSTU17等基因。(2)代谢组分析表明,以PZ2为对照,在2个品种间共筛选到208个差异代谢物,含量上调和下调的差异代谢物分别为98个和110个,其中22种差异代谢物与抗逆性相关,包括氨基酸及其衍生物、脂类、黄酮类、糖类和糖苷等。(3)qRT PCR分析验证表明,所选的8个差异表达基因在菠萝品种PZ2与PZ3中的差异表达趋势与转录组测序中差异基因表达水平变化趋势基本一致。(4)关联分析表明,差异表达基因和差异代谢物共同富集的通路有类黄酮生物合成,苯丙素生物合成,莨宕烷、哌啶和吡啶丙酸盐生物合成,半胱氨酸与蛋氨酸代谢等4条。研究发现,抗逆性强的菠萝品种的非生物逆境反应相关基因、抗氧化酶基因、逆境响应结构基因和调控基因的表达水平高于抗逆性较差的品种,且黄酮类、氨基酸衍生物类及木脂素、脂质等代谢物的含量也高于抗逆性较差的品种。     

The dynamic systems approach to control and regulation of intracellular networks

Systems theory and cell biology have enjoyed a long relationship that has received renewed interest in recent years in the context of systems biology. The term 'systems' in systems biology comes from systems theory or dynamic systems theory: systems biology is defined through the application of systems- and signal-oriented approaches for an understanding of inter- and intra-cellular dynamic processes. The aim of the present text is to review the systems and control perspective of dynamic systems. The biologist's conceptual framework for representing the variables of a biochemical reaction network, and for describing their relationships, are pathway maps. A principal goal of systems biology is to turn these static maps into dynamic models, which can provide insight into the temporal evolution of biochemical reaction networks. Towards this end, we review the case for differential equation models as a 'natural' representation of causal entailment in pathways. Block-diagrams, commonly used in the engineering sciences, are introduced and compared to pathway maps. The stimulus-response representation of a molecular system is a necessary condition for an understanding of dynamic interactions among the components that make up a pathway. Using simple examples, we show how biochemical reactions are modelled in the dynamic systems framework and visualized using block-diagrams.     

Identification and characterisation of imprinted genes in the mouse.

Imprinted genes are expressed specifically from one or other parental allele. Over 70 are now known, and about one-half of these are expressed from the paternal allele and one-half from the maternal allele. Most imprinted genes are clustered within imprinting regions of the mouse genome, regions which are associated with abnormal phenotypes when inherited uniparentally. Imprinted genes have been identified from surveys based on differential expression or differential methylation according to parental origin, as well as analyses of candidate genes, mutants and imprinted gene clusters. Many imprinted genes affect growth and development, and more than 25 per cent determine non-coding RNAs that may have a function in controlling imprinted gene expression.     

纯种与杂种鸡之间肝脏组织基因差异表达及其与肉用性状杂种优势的关系

以白洛克肉鸡 (EE)、中国丝羽乌骨鸡 (CC)、农大褐 (DD)和白来航 (AA) 4个纯种鸡为材料 ,进行 4× 4完全双列杂交 ,共得到 16种杂交组合。应用mRNA差异显示技术 (DDRT PCR)检测了 8周龄纯种和杂种鸡之间肝脏组织基因的差异表达。结果表明 ,在纯种和杂种间共有 8种 15类基因差异表达模式 ,杂种和纯种之间基因表达存在明显的差异。对各种基因差异表达模式与 10个肉用性状的杂种优势率进行相关分析发现 ,表达一致型P8(t1111)与肉用性状的杂种优势率相关不显著 (P >0 0 5) ,这说明杂种优势的形成与某些基因的差异表达有关 ;正交或反交特异表达型P4(t0 10 0、t0 0 10 )与 8周龄个体重、腿肌重、半净膛重、全净膛重相关显著 (P <0 0 5) ,与胸肌重相关极显著 (P <0 0 1) ;单亲特异表达型P1(t10 0 0、t0 0 0 1)与腹脂重相关显著 (P <0 0 5) ,与体斜长相关极显著 (P <0 0 1) ;双亲特异表达型P7(t10 0 1)与腿肌重、翅重、半净膛重、肌间脂宽的杂种优势率相关显著 (P <0 0 5) ;正交或反交单亲表达一致型P2 (t110 0、t0 0 11、t10 10、t0 10 1)与肌间脂宽的杂种优势率相关显著 (P <0 0 5) ;单亲表达一致型P5(t1110、t0 111)胫骨长的杂种优势率相关显著 (P <0 0 5)。     

置换层析——重新崛起的生物物质分离技术

置换层析是一种非线性层析技术,它产生于４０年代末,只是在近十几年里,置换层析才显示了其在生物物质特别是蛋白质的制备性分离中的重要潜力。由于这一技术与传统的洗脱层析技术相比,有着明显的优点,即高上样量、高产率、高分辨率、易于操作,以及被分离样品在分离过程中的自行浓缩效应,因此这一技术已越来越引起人们的关注。     

Intermediate variables in the explanation of differential fertility: Results of a village study in rural Java

A tendency for fertility to be higher among middle-class women than among lower-class women has been found in all the recent sources of data on fertility levels and patterns in Indonesia. After briefly reviewing these findings, this article presents the results of a community study in rural Java which systematically investigates the positive relation between fertility and socioeconomic status, focusing on the so- called intermediate variables (first inventoried by Davis and Blake in 1956) which directly determine fertility. Differential patterns of marital stability, secondary sterility, and the practice of postpartum abstinence in particular were found to contribute to the contrasts in fertility levels observed. The use of modern contraceptives, a recent innovation at the time of the study, was found to a greater extent among middle-class women, and may prove to be an important determinant of future patterns of fertility and fertility differentials.This article is based on a study of fertility patterns undertaken in Yogyakarta during 1972–1973. The research was funded by the Australian National University. A complete account of the research findings is contained in Hull (1975), and a detailed analysis of the topics discussed here can be found in Hull (1976).