大鼠胚胎和成年组织中EGFR基因转录分析

本文报告用2.4Kb EGFR cDNA PE7为探针,分析大鼠胚胎发育过程中全胚组织、成年大鼠七种组织和再生肝组织中EGFR基因转录产物的水平。实验结果说明:(1)用人EGFRcDNA探针可以检测到大鼠肝脏细胞转录约为5.6Kb的EGFRmRNA。(2)大鼠胚胎发育过程中,EGFR基因转录活性显示骤然变化。10—12天胚胎中EGFR mRNA出现高峰。(3)成年大鼠肝、肾、肺、脑、脾、心脏和睾丸组织中均有EGFR基因的转录,转录水平各有差异,以肾为最高。(4)大鼠肝脏再生过程中,EGFR基因转录呈现时相调节的特征,部分肝切除刺激EGFR基因转录活性的增高,8小时达到高峰后又回落到接近正常的水平。这些EGFR基因转录的变化可能与大鼠组织和细胞的生长及分化的调控有关。     

大鼠C3基因TATA区结合核蛋白与其转录活性关系的研究

大鼠Ｃ３基因的转录受雄激素调控且只在大鼠腹侧前列腺中表达。本文报道用凝胶电泳带漂移分析和离体ＤＮａｇｅＩ足迹法分析研究了大鼠Ｃ３基因ＴＡＴＡ区结合核蛋白与其组织专一的和雄激素调控的转录活性间的关系。结果表示：相同的（－５０到－１０）４ｂｐ的ＴＡＴＡ区ＤＮＡ片段在不同组织的细胞核中结合的核蛋白是不同的。通过与一个仅在ＴＡＴＡ区－２９．位核苷酸Ａ变为Ｇ的天然存在的不表达的Ｃ３（２）等位基因上此片段的核蛋白结合能力作比较后提示在Ｃ３基因唯一表达的组织—腹侧前列腺中，结合在此区的核蛋白是－２９位Ａ依赖的，而在所研究的不表达组织—肝和睾丸中则否。而且此类蛋白质还可能具有与雄激素调控此基因表达有关的其他反式作用因子相互作用的结构域．     

绵羊EGFR基因的克隆、表达及序列分析

表皮生长因子受体(epidermal growth factor receptor,EGFR)是酪氨酸激酶受体家族成员之一,不仅参与细胞增殖、生长和凋亡等多种生命活动,也可调节哺乳动物的乳腺发育及泌乳维持,但对绵羊EGFR基因的序列特征及组织表达情况鲜有报道.本试验以高泌乳量的小尾寒羊(泌乳高峰期和空怀期)及低泌乳量的甘肃高山细毛羊(泌乳高峰期)母羊为研究对象,利用RT-PCR、克隆及测序技术获得绵羊EGFR基因完整的CDS区,分析了 EGFR蛋白的结构特征及理化性质,利用RT-qPCR技术研究了基因的组织表达情况.结果表明,绵羊EGFR基因CDS区全长为3 627 bp,编码1 208个氨基酸.绵羊EGFR的氨基酸序列在各物种间较保守,与黄牛EGFR的氨基酸序列同源性最高.EGFR为跨膜蛋白,包含111个磷酸化位点,二级结构以α螺旋和无规则卷曲为主.网络互作分析表明EGFR蛋白与肝素结合表皮生长因子(HB-EGF)、表皮调节素(EREG)、双调蛋白(AREG)及生长因子受体结合蛋白2(GRB2)结合发挥作用.EGFR主要参与MAPK,PI3K/AKT,JAK/STAT及Wnt信号通路,从而参与了动物的乳腺发育及泌乳功能的调节.RT-qPCR结果表明,绵羊EGFR基因的表达具有组织特异性、时空特异性和品种特异性.该基因在所研究的8个组织中均表达,但在肾脏、卵巢、肝脏、乳腺和肺脏组织中的表达量较高;在小尾寒羊的乳腺组织中,该基因在空怀期的表达量显著高于泌乳高峰期的(P＜0.05);在泌乳高峰期的乳腺组织中,该基因在小尾寒羊中的表达量高于甘肃高山细毛羊的.本试验为深入研究绵羊EGFR基因的泌乳生物学功能提供了基础数据.     

组蛋白在胚胎早期发育转录调控中的作用

组蛋白是一组等电点大于１０．０的碱性蛋白质,在进化上十分保守。真核生物的染色体上主要含有５种组蛋白,即核心组蛋白Ｈ２ａ、Ｈ２ｂ、Ｈ３、Ｈ４及连接组蛋白Ｈ１。核心组蛋白八聚体、蛋白Ｈ１和２００ｂｐ的ＤＮＡ共同组成了染色体的基本单位——核小体。组蛋白不仅...     

昆明小鼠体内发育早期胚胎葡萄糖代谢关键酶转录产物的分析

首次报道了昆明小鼠体内发育的早期胚胎1-细胞至桑椹期阶段葡萄糖代谢的3种关键酶-6-磷酸葡萄糖脱氢酶（G6PDH）、6-磷酸果糖激酶（PFK）和磷酸葡萄糖变位酶（PGM）的基因转录情况,其分别体现了磷酸戊糖、糖酵解、糖原的合成和分解等途径,根据G6PDH、PFK、PGM的cDNA序列分别设计和合成3套共6对内、外引物,采用巢式RT-PCR方法对其进行检测。结果表明：早期胚胎1-8细胞阶段均有G6PDH基因的转录,叠椹期胚胎不存在该基因的转录,说明早期胚胎1-8细胞阶段可能存在磷酸戊糖,而桑椹期则不存在;1-细胞至桑椹期均存在PFK基因的转录,说明该阶段的胚胎可能存在糖酵解代谢途径;1-细胞至桑椹期均不存在PGM基因的转录,说明该阶段的胚胎可能不存在糖原的合成与分解代谢途径。     

通过胚胎干细胞途径获得的嵌合体小鼠中neo基因在各组织中的分布

为探讨小鼠胚胎干细胞参与早期胚胎发育的潜能,以neo基因作为目的基因进行了胚胎干细胞的转染,经G418的筛选,获得表达neo基因的单克隆细胞,挑取部分克隆扩大,进行小鼠囊胚腔注射,再重新植入小鼠子宫,共注射了60个囊胚,分别回输到5只假孕小鼠,在子代中得到了携带有neo基因的嵌合体小鼠。通过对嵌合体小鼠各组织PCR分析发现,neo基因可以在皮肤、肝脏、血液等多种组织中存在。     

真核细胞中基因的转录调控

叙述了真核细胞三种RNA聚合酶合成的基因的转录调控.由于真核细胞DNA含量非常大,其基因的转录调控具有以下特点：参与的转录因子多;与顺式DNA序列元件结合呈一定顺序.这反映了真核细胞中基因的转录调控是由多个转录因子间的相互作用来实现的．     

大鼠iNOS基因上游调控区在转录激活中的作用

为了探讨大鼠ｉＮＯＳ基因上游调控区不同部位在对细胞因子诱导应答中所起的作用,将调控区不同部位插入ｐＳＶ０－ＣＡＴ报告基因载体,转染体外培养的血管平滑肌细胞（ＶＳＭＣ）,经ＩＬ－１β诱导后,采用氯霉素乙酰转移酶（ＣＡＴ）活性测定和Ｎｏｒｔｈｅｒｎ印迹杂交,检查了调控区各部位在ＩＬ－１β诱导ｃａｔ表达中所起的作用．结果表明,被转染的细胞在未经ＩＬ－１β刺激时,各种调控区序列启动ｃａｔ表达的活性均很低．在ＩＬ－１β作用下,调控区远端序列（－１０３７～－４３８）、近端序列（－４３７～＋４６）和全长序列（－１０３７～＋４６）均能独立激活ｃａｔ表达,其中以全长序列的作用最强,表明ｉＮＯＳ基因表达调控区远端和近端序列均具有启动子和增强子样功能．同时证实,远端序列和近端序列单独启动ｃａｔ表达的活性分别为全长序列的９１．３％和６７．１％,揭示大鼠ｉＮＯＳ基因调控区远端序列在介导ＩＬ－１β的应答反应中发挥更重要作用．     

大鼠肝tRNA^Ile基因的克隆与体外转录

采用ＰＣＲ扩增出大鼠肝ｔＲＮＡ＾Ｉｌｅ基因,构建了重组质粒ｐＧＷＩＷ,并用Ｔ７ＲＮＡ聚合酶／启动子系统对其进行了体外表达。经过对转录产物片段大小及运用Ｎｏｒｔｈｅｒｍｂｌｏｔ鉴定,证明了获得了不含修饰核苷酸的大鼠肝ｔＲＮＡ＾Ｉｌｅ。生物学活性检测显示：合成基因体外转录产物氨基酰经活性是天然ｔＲＮＡ的４０％,提示修饰核苷酸在哺乳动物Ｉｌｅ－ｔＲＮＡ合成酶的识别过程中起重要作用。     

Localization of pan-cadherin immunoreactivity in adult rat tissues

Cadherins, being responsible for selective cell recognition and normal tissue integrity in adults, regulate morphogenesis in a variety of organs during development. In this study, anti-rat pan-cadherin antibody, specific to all subgroups of the cadherin family, was used to map the distribution of the pan-cadherin immunoreactivity in adult rat organs. Pan-cadherin immunoreactivity positive tissues were: secretory cells of the adenohypophysis, autonomic nerve, corneal epithelium, oesophageal nerve plexus, stomach and pyloric glandular cells, epithelium of the ileum and its nerve plexus, alveolar cells of the lung, proximal convoluted tubules of the kidney, islet cells of Langerhans, and the acinar cells of the exocrine pancreas. For the first time, positive pan-cadherin immunoreactivity was demonstrated in the epithelial cells of the corpus ciliaris and in the nerve plexus of corpus cavernosum of the penis. In conclusion, our results suggest that cells in many tissues and organs of the adult rat synthesize cadherins.     

Identification of microRNAs from different tissues of chicken embryo and adult chicken

We report for the first time the identification of 25 microRNAs from tissues originating from chicken embryo and adult chicken. Most of the cloned microRNAs are expressed in both adult chickens and chicken embryos. Fourteen were identified without any prior prediction. One microRNA, miR-757, is thought to be chicken-specific. Three of the microRNAs appear to be extremely tissue specific.     

Expression of the zebrafish Staufen gene in the embryo and adult

Staufen, a double stranded RNA binding protein, has been shown to be involved in creating and maintaining cellular asymmetry in the Drosophila oocyte, neuroblast, and mammalian neuron. Staufen binds to the 3' UTR of specific mRNAs and acts in their localization and anchoring to various subcellular domains. Staufen's molecular interactions during development have been limited to investigations in Drosophila melanogaster. Since a vertebrate Staufen has not been studied in a developmental system, the aim of this study was to clone and characterize a staufen orthologue gene in the vertebrate developmental model, zebrafish. The zebrafish staufen-like sequence shows a 64% homology to the human staufen with a 81.2% homology in the highly conserved double stranded RNA binding domain (dsRBDs). Staufen maps on the LN54 radiation hybrid panel to linkage group 6, 16.25 cR from Z265 between fb22h06 and fi16e01. Northern blot and in situ hybridization showed that staufen is expressed both maternally and zygotically. Zygotically expressed staufen is localized to the developing nervous system and at 24 h is highly concentrated in the subventricular zone of the developing brain. Maternally expressed staufen is dispersed in the mature oocyte and early embryo. In the adult, staufen is expressed in specific brain nuclei, the testis, neurons and Leydig cells.     

Specificity of transcription of single-copy DNA in different rat tissues

• 1.1. The amount of single-copy DNA sequences transcribed in normal tissues of adult rats (brain and liver) and in the Guerin ascites tumor (GAT) was determined by hybridization of in vitro labelled ¹²⁵I-single-copy rat DNA with a vast excess of total nuclear RNA to very high Rot values (up to 350,000).
• 2.2. The tissue specificity of total nuclear RNA (nRNA) was estimated by annealing of single-copy DNA to a mixture of nuclear RNAs of two different organs (brain + GAT; liver + GAT).
• 3.3. Liver and GAT RNAs annealed to about 12% of the single-copy DNA.
• 4.4. Hybridization with a mixture of the two RNAs increased slightly the amount of hybridization.
• 5.5. In contrast to other tissues, brain nuclear RNA hybridized to a much higher level (20% of the single copy DNA). Addition of GAT RNA did not increase this value.

     

Culture of cells from zebrafish (Brachydanio rerio) embryo and adult tissues

The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1 Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.[/ p]Abbreviations TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin - EROD, 7-ethyoxyresorufin - HDPDS, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - EDTA, ethylanediaminetetraacetic acid - FBS, fetal bovine serum - LDF, limit dilution factor - DMSO, dimethyl sulfoxide - ES, embryonal stem - PAH, polycylic aromatic hydrocarbons - ZG, zebrafish gill - ZBF, zebrafish pelvic fin - ZV, Zebrafish viscera - ZCF, zebrafish caudal fin - ZEM, diploid blastula-derived