人珠蛋白信使核糖核酸的制备及其体外翻译

本文以新生儿ABO血型不配溶血换得的血网织红细胞为材料,用酚-氯仿提取及Oligo(dT)纤维素亲和层析制备了人珠蛋白mRNA。并在变性条件下经琼脂糖凝胶电泳鉴定,证明它是9～10S的RNA。人珠蛋白mRNA在兔网织红细胞溶解物与麦胚体外翻译体系中的翻译活性与其浓度均有依赖关系。用SDS-聚丙烯酰胺凝胶不连续电泳及萤光显影鉴定了无细胞体系的翻译产物为人珠蛋白;并以醋酸纤维膜电泳分析了体外合成的人珠蛋白肽链组成,结果表明新生儿血中珠蛋白mRNA合成的α、β、γ珠蛋白肽链之比为3:1:2。     

人珠蛋白信使核糖核酸的制备及其体外翻译

本文以新生儿ABO血型不配溶血换得的血网织红细胞为材料,用酚-氯仿提取及Oligo(dT)纤维素亲和层析制备了人珠蛋白mRNA。并在变性条件下经琼脂糖凝胶电泳鉴定,证明它是9～10S的RNA。人珠蛋白mRNA在兔网织红细胞溶解物与麦胚体外翻译体系中的翻译活性与其浓度均有依赖关系。用SDS-聚丙烯酰胺凝胶不连续电泳及萤光显影鉴定了无细胞体系的翻译产物为人珠蛋白;并以醋酸纤维膜电泳分析了体外合成的人珠蛋白肽链组成,结果表明新生儿血中珠蛋白mRNA合成的α、β、γ珠蛋白肽链之比为3:1:2。     

北京鸭卵对蛋白mRNA的提纯及某些性质的鉴定

 本文报道了从产卵期北京鸭输卵管中提取总RNA,经Olilo(dT)-纤维素柱层析,再经sepharose 4B柱层析步骤,得到了纯化的鸭卵清蛋白mRNA。我们建立了麦胚无细胞体系并探索了鸭卵清蛋白mRNA在此体系中翻译的最适条件。在此条件下测定了各纯化步骤的总mRNA翻译活性,并用免疫沉淀法测定其中卵清蛋白mRNA的活性。测定结果表明我们从mRNA中分离得到了纯鸭卵清蛋白mRNA。用变性的琼脂糖凝胶电泳对各纯化步骤的核酸样品进行组成分析,确定出鸭卵清蛋白mRNA的大小约为21S。     

珠蛋白mRNA的分离和cDNA的合成

本文报道从人工贫血的兔与鸡的网织红细胞中提取珠蛋白mRNA,在麦胚无细胞体系中测定其蛋白翻译活力,与对照相比,兔、鸡珠蛋白mRNA促进~(14)C-亮氨酸参入新生蛋白质的活力分别达到32倍和10倍,所翻译的蛋白产物在聚丙烯酰胺凝胶上的行为与天然珠蛋白一致。上述mRNA在AMV反转录酶和DNA聚合酶的作用下,分别合成了单链和双链的cDNA及mRNA-cDNA杂交链,凝胶电泳分析其长度分别为500～700,550～800及500～800碱基对,550～800碱基对大小的双链cDNA约占总合成量的30%。     

珠蛋白mRNA的分离和cDNA的合成

本文报道从人工贫血的兔与鸡的网织红细胞中提取珠蛋白mRNA,在麦胚无细胞体系中测定其蛋白翻译活力,与对照相比,兔、鸡珠蛋白mRNA促进~(14)C-亮氨酸参入新生蛋白质的活力分别达到32倍和10倍,所翻译的蛋白产物在聚丙烯酰胺凝胶上的行为与天然珠蛋白一致。上述mRNA在AMV反转录酶和DNA聚合酶的作用下,分别合成了单链和双链的cDNA及mRNA-cDNA杂交链,凝胶电泳分析其长度分别为500～700,550～800及500～800碱基对,550～800碱基对大小的双链cDNA约占总合成量的30%。     

鲤鱼垂体mRNA反转录模板活性的研究

 本实验用不同方法研究鲤鱼垂体mRNA反转录为互补DNA的活性。用硫氰酸胍法,从垂体中提取总RNA,经过Oligo(dT)-纤维素柱层析,得到poly(A)~+RNA。 以mRNA为模板,30％反转录成单链cDNA。利用RNaseH-DNA聚合酶Ⅰ-E.coli DNA连接酶合成双链cDNA。单链cDNA拷贝成双链cDNA。经放射自显影分析,证明合成了全长cDNA。用水解法合成双链cDNA,大多数为不完整的双链cDNA。平末端的双链cDNA连接上Eco RI-linker经Sepharose-4B分离,收集大片段cDNA,与pUC 19载体相连接,经转化构建成10~5克隆/μg mRNA的cDNA文库。     

猪垂体总mRNA的提取、鉴定及其cDNA的合成

 用改进的LiCl沉淀法和寡聚(dT)-纤维素亲和层析法由猪垂体制得总mRNA。在兔网织红细胞无细胞翻译体系中进行体外翻译的结果表明,制得的总mRNA具有一定的翻译活力。翻译产物与兔抗猪生长激素抗血清发生免疫沉淀,沉淀物占总翻译产物的10％左右。SDS聚丙烯酰胺凝胶电泳的结果表明翻译产物有一条很深的带,分子量约为24,000道尔顿,与猪前生长激素的分子量相近。以制备的mRNA为模板反转录合成了双链cDNA。第一链的合成产率为10—35％,第二链的合成产率为84—115％。cDNA的平均分子长度为825bp。     

野生大豆未成熟种子总mRNA的分离及其cDNA的分子克隆

用氯化锂沉淀法从野生大豆(G.soja)未成熟种子中制备总RNA,经oligo(dT)-纤维素柱亲和层析,获得总mRNA,在兔网织红细胞体系中表现出一定翻译活性。以总mRNA为模板,oligo(dT)_(12)(?)为引物,反转录酶催化合成第一链cDNA,RNase H-DNA聚合酶Ⅰ协同合成第二链cDNA。双链cDNA的长度大约为200—5000 bp,且不存在发夹结构。将双链cDNA修补后钝端连接到pUC 19质粒的Sma 1位点,转化E.coli JM107,获得800多个白色重组子克隆。快速电泳检测及酶切分析表明,多数重组子带有插入片段,其中3个重组子的插入片段长度大致为1700 bp、2600 bp和1400 bp。     

杜氏盐藻完整叶绿体的分离及其蛋白提取

应用高压破碎及蔗糖密度梯度离心的方法,分离出杜氏盐藻的完整叶绿体,随后用冻融法和研磨法分别提取叶绿体蛋白,并通过蛋白定量和SDS-PAGE凝胶电泳对两种蛋白提取方法进行比较,确立了一套适用于杜氏盐藻叶绿体分离和蛋白提取、定量以及电泳的方法.结果表明:用高压破碎结合蔗糖密度梯度离心的方法能够获得完整且较纯的盐藻细胞叶绿体;SDS-PAGE凝胶电泳结果显示,冻融法提取叶绿体蛋白效率高,电泳条带清晰,蛋白数量较多,蛋白齐全.为下一步在亚细胞水平进行杜氏盐藻耐盐机制的蛋白组学研究奠定了基础.     

从大肠杆菌B／r中同时制备ATP：RNA腺苷酰转移酶和多核苷酸磷酸化酶

本文报道了从大肠杆菌B／r中同时制备ATP：RNA腺苷酰转移酶和PNPase的简便方法。菌体经压力破碎,聚乙二醇、葡聚糖分相抽提及磷酸纤维素柱层析得到ATP：RNA腺苷酰转移酸。其滤液经DEAE纤维素柱层析和Scphadex G-200凝胶过滤得到PNFasc。用Oligo dT纤维素和聚丙烯酰胺凝胶电泳对酶反应产物作了初步鉴定。     

Nuclear steady-state RNA from chicken immature red blood cells. Distribution of globin-coding and poly (A) sequences

Nuclear steady-state RNA and polysomal RNA of chicken immature red blood cells were isolated and separated on formamide sucrose gradients. For comparison the distribution of 9 S globin mRNA was investigated by gradient centrifugation of 125I-labelled mRNA. The material was either pooled into two fractions (less than 20 S; greater than 20 S) and translated in an Ehrlich ascites cell-free system or each gradient fraction was analyzed by hybridization with [3H]-poly (U) or [3H]-labelled DNA complementary to purified 9 S globin mRNA (globin cDNA). In neither case could evidence be obtained for the existence of a high molecular weight RNA as a probable globin mRNA precursor. Further analysis was performed by electrophoresis of RNA on exponential polyacrylamide gels in formamide and subsequent hybridization with cDNA. The results are consistent with those of gradient centrifugation and demonstrate that the distribution of globin-coding sequences in nuclear steady state RNA corresponds to that of cytoplasmic 9 S globin mRNA.     

Specific hydrolysis of rabbit globin messenger RNA by S1 nuclease.

S1 nuclease isolated from Aspergillus oryzae has been used to investigate the secondary structure of rabbit globin messenger RNA (mRNA). The enzyme, which is specific for single stranded nucleotides, digests globin mRNA to a limited extent, with 65-75% of the mRNA nucleotides resistant to digestion under mild conditions. This limited digestion is not due to enzyme inactivation, but rather to the normal activity of the single-strand nuclease. The reaction was studied as a function of temperature, salt and enzyme concentration. Analysis of the products of digestion on 20% acrylamide- 7M urea slab gels reveals a stable pattern of unique fragments ranging in size from 9 to 71 nucleotides. Separated alpha and beta globin mRNAs show similar, but not identical gel patterns, indicating strong structural similarities between the two species. The high degree of nuclease resistance, along with the fragment patterns seen on polyacrylamide gels, gives evidence to support a model of rabbit globin mRNA which contain specific, rather than random, helical structure.     

The subunit interface of the Escherichia coli ribosome. Crosslinking of 30 S protein S9 to proteins of the 50 S subunit.

Purified 50 S ribosomal subunits were found to contain significant amounts of protein coincident with the 30 S proteins S9 and/or S11 on two-dimensional polyacrylamide/urea electropherographs. Peptide mapping established that the protein was largely S9 with smaller amounts of S11. Proteins S5 and L6 were nearly coincident on the two-dimensional polyacrylamide/urea electropherographs. Peptide maps of material from the L6 spot obtained from purified 50 S subunits showed the presence of significant amounts of the peptides corresponding to S5. Experiments in which ³⁵S-labelled 30 S subunits and non-radioactive 50 S subunits were reassociated to form 70 S ribosomes showed that some radioactive 30 S protein was transferred to the 50 S subunit. Most of the transferred radioactivity was associated with two proteins, S9 and S5. Sulfhydryl groups were added to the 50 S subunit by amidination with 2-iminothiolane (methyl 4-mercaptobutyrimidate). These were oxidized to form disulfide linkages, some of which crosslinked different proteins of the intact 50 S ribosomal subunit. Protein dimers were partially fractionated by sequential salt extraction and then by electrophoresis of each fraction in polyacrylamide gels containing urea. Slices of the gel were analysed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the constituent proteins in each dimer by two-dimensional polyacrylamide/urea gel electrophoresis showed that 50 S proteins L5 and L27 were crosslinked to S9. The evidence suggests that proteins S5, S9, S11, L5 and L27 are located at the interface region of the 70 S ribosome.     

猕猴(Macaca mulatta)肝5S核糖核蛋白体RNA的分离提纯

应用酚-去污剂和1摩尔/升NaCl抽提低分子量RNA,通过DEAE-葡聚糖A-50离子交换层析提纯后,经含有7摩尔/升尿素的10%聚丙烯酰胺凝胶垂直板电泳制备纯化猕猴肝5S核糖核蛋白体RNA是简易而行之有效的方法。制纯的5SrRNA经聚丙烯酰胺凝胶电泳鉴定呈现单一的一条区带。与大肠杆菌5SrRNA标准品具有相同的电泳迁移率。     

Initial synthesis of globin peptide chains in differentiating embryonic red blood cells

The initial time of synthesis of globin polypeptide chains in differentiating red cells has been delineated. Three analytical techniques were used, namely, SDS-polyacrylamide gel electrophoresis, acid urea polyacrylamide gel electrophoresis, and two-dimensional gel electrophoresis. The results indicate that globin peptide chains are synthesized in proerythroblasts at the initial time when globin mRNA first enters into the cytoplasm, and there is no apparent time gap between these two events.     

Direct microinjection of rabbit globin mRNA into mouse 3T3 cells. Analysis of the polypeptides synthesized in vivo

3T3 cells grown attached to 9 mm² coverslips have been microinjected in the cytoplasm with total rabbit globin mRNA and the polypeptides synthesized after injection have been labelled with [³⁵S]-methionine under conditions in which the product of as few as 100 cells could be analysed by high resolution two-dimensional gel electrophoresis followed by 10 days' fluorography. Microinjection of rabbit globin mRNA results in the synthesis of a basic polypeptide of mol. wt 15 K that is not present in control cells, and that co-migrates with purified [³H]leucine-labelled globin as determined by high resolution two-dimensional gel electrophoresis (NEPHGE). Visual inspection of the fluorograms revealed that the injection of globin mRNA (up to 14000 molecules/cell) does not alter significantly the relative intensity of the major acidic (IEF) and basic (NEPHGE) polypeptides synthesized by the cells.     

Organization of sequences of avian globin mRNA.

Formamide polyacrylamide gel electrophoresis shows that chicken globin mRNA contains about 6.50 nucleotides, and since only 435 of these code for globin, a further 215 are not translated, and their function and position are not known. This work has produced the following conclusions. 1. 45-50 of these untranslated nucleotides are present as poly (A) at the 3' terminus. 2. The 3' untranslated region of chicken globin mRNA is at least 90 nucleotides in length. This minimal estimate is based on data derived from hybridization of defined lenghts of chicken globin cDNA to rabbit globin mRNA. The percentage of avian globin cDNA sequences which hybridize to rabbit globin mRNA is directly proportional to the length of the cDNA in each case. This relationship holds for lengths of cDNA from 115 up to 620 nucleotides. The low percentage homology for short cDNA molecules is not due to their being short per se. In homologous mRNA excess hybridizations (chicken cDNA/chicken mRNA), all cDNA preparations were completely protected from S1 nuclease digestion. 3. It is probable that there is greater evolutionary divergence in the 3' untranslated region of chicken and rabbit globin mRNA when compared with the coding regions of these molecules; The combined data is sued to formulate a regional map of chicken globin mRNA,