胸苷酸合成酶mRNA亲和多肽的鉴定及其对翻译过程的抑制作用

胸苷酸合成酶(thymidylate synthase, TS)是催化生物体内胸苷酸合成所必需的酶, 多年来一直作为肿瘤化疗的靶点. 研究表明, TS是一种RNA结合蛋白, 可以与其自身mRNA的2个位点结合, 使mRNA翻译受阻. 本文以mRNA体外展示技术进行了由大容量多肽库(>10¹³)中筛选胸苷酸合成酶mRNA亲和多肽的研究, 对随机库进行了12轮循环的选择及扩增. 结果表明, 与初始库相比, 经选择循环之后, 碱性氨基酸及芳香族的苯丙氨酸含量明显增加, 它们在TS RNA与蛋白质的相互作用中扮演着重要角色. 按其理化特性对每一随机位点的氨基酸进行分类, 并与初始库比较, 发现位点1, 12, 17和18具有明显的带正电荷的特性, 表明碱性侧链参与了与RNA的结合. 二级结构预测表明, 随着筛选的进行, 与TS mRNA 亲和的多肽显示出明显的螺旋倾向, 而且形成螺旋的区域富含碱性氨基酸. 凝胶迁移及体外翻译实验证实, 选择循环之后的多肽能够与TS mRNA高度亲和, 并能抑制TS mRNA的翻译. 本研究表明mRNA体外展示方法得到的亲和多肽可以用作新的TS RNA的翻译抑制剂, 并有可能成为一类新型的抗肿瘤药物.     

低温诱导唐古特红景天细胞分泌抗冻蛋白

选择青海高原海拔4000m高山上生长的唐古特红景天为实验材料,以叶片为外植体,在MS+BA₂+NAA_0.2固体培养基上诱导出黄绿色、松脆愈伤组织.愈伤组织细胞在同样成分的液体培养基中培养获得成功.在悬浮培养液中可检测到分泌蛋白的存在.经SDS-聚丙烯酰胺凝胶电泳分析表明,低温锻炼或脱落酸（ABA）诱导后,细胞分泌蛋白的多肽谱带数增加.与此相对应的是,细胞的抗冻能力也明显提高.PAS染色揭示多肽中均含有糖基.通过测定热滞值,确信细胞分泌蛋白是具有抗冻活性的糖蛋白.     

植物抗冻机理研究新进展：抗冻基因表达及其功能

综述了迄今业已分离和鉴定的低温诱导表达的抗冻基因,以及通过染色体基因制图方法揭示和证实的抗冻性基因。并着重地介绍了这些抗冻基因在提高植物抗冻能力上的功能。这些抗冻基因表达合成的新多肽具有高度的亲水性,起着保护和稳定细胞膜结构的作用,从而防止冰冻伤害、提高植物的抗冻能力。同时还介绍了抗冻基因表达中的某些调节因子,其中尤其值得注意的是,CBF基因似乎有可能作为抗冻基因表达的“主开关”,以及Ca2+作为植物细胞的第二信使在传递低温信号、启动和调节抗冻基因表达中可能的重要作用。这些研究结果,不仅为阐明植物的抗冻机理提供了新的证据,而且为改良农作物的抗寒性提供了新的启示。     

北京鸭珠蛋白mRNA的分离纯化及其cDNA的合成

 从人工贫血的北京鸭网织红细胞中直接提取总RNA,经Oligo(dT)-纤维素柱层析分离获得珠蛋白mRNA,并经蔗糖密度梯度离心首次得到了电泳单一条带的北京鸭球蛋白mRNA。从凝胶电泳以及蔗糖密度梯度离心鉴定其沉降系数为9S。在麦胚无细胞体外翻译体系中测定了它们的蛋白翻译活力。鸭珠蛋白mRNA促进了~3H-亮氨酸参入新生蛋白的活力,达到对照组的10倍。所翻译的蛋白产物在SDS-聚丙烯酰胺凝胶上的电泳行为与天然鸭珠蛋白一致。 经Oligo(dT)-纤维素及蔗糖密度梯度离心提纯的珠蛋白mRNA,在AMV反转录酶及DNA聚合酶的作用下,分别合成了单链及双链cDNA。其双链链长,经凝胶电泳分析,约为500碱基对。     

植物抗冻机理研究新进展：抗冻基因表达及其功能

综述了迄今业已分离和鉴定的低温诱导表达的抗冻基因,以及通过染色体基因制图方法褐示和证实的抗冻性基因,并着重地介绍了这些抗冻基因在提高植物抗冻能力上的功能。这些抗冻基因表达合成的新多肽具3有高度的亲水性,起着保护和稳定细胞膜结构的作用,从而防止冰冻伤害,提高植物的抗冻能力,同时还介绍了抗冻基因表达中的某些调节因子,其中尤其值得注意的是,CBF基因似乎有可能作为抗冻基因表达的“主开关”,以及Ca^2 作为植物细胞的第二信使在传递低温信号,启动和调节抗冻基因表达中可能的重要作用,这些研究结果,不仅为阐明植物的抗冻机理提供了新的证据,而且为改良农作物的抗寒性提供了新的启示。     

光/暗对高粱叶片磷酸烯醇式丙酮酸羧化酶基因表达的调节

高粱幼苗黄化叶片经照光转绿后,其PEP-Case活性提高4～15倍,mRNA含量提高了1.03倍,并测定出PEPCase mRNA的分子量为3.4kb。以等量的总RNA及mRNA进行体外翻译,发现转绿后PEPCase专一性翻译活性提高了51％～53％。这表明光照可以在转录水平上调节PEP-Case的基因表达。     

大鼠前列腺前叶细胞两种受雄激素调节的mRNA核前体

在大鼠前列腺前叶细胞,雄激素在转录水平调节六种已知多肽的合成:前列腺结合蛋白(PBP)的三个亚肽、一种22KDa糖蛋白、亚精胺结合蛋白(SBP)和29KDa蛋白质。此外,尚有9.3KDa和13.5KDa的多肽,其相应的mRNA已由克隆的cDNA(~*119和~*92)杂交以及体外翻译系统证实。     

mRNA展示技术

mRNA展示技术是一种新兴的体外筛选多肽和蛋白质的有力工具.在筛选过程中,mRNA与其编码的多肽或蛋白质共价结合,形成mRNA-蛋白质融合体,能在大容量的多肽文库(1013～1015)中筛选具有特定生物学功能的多肽和蛋白质.目前,mRNA展示技术主要应用于各种靶分子的多肽和蛋白质适体的发现以及蛋白质相互作用机制的阐明和分析.由于其自身的巨大发展潜力,mRNA展示技术具有更为广阔的应用前景.     

草鱼出血病病毒多肽的基因定位

用聚丙烯酰胺凝胶电泳分离纯化的草鱼出血病病毒ＧＣＨＶ８７３的基因组ｄｓ－ＲＮＡ的１１个片段,分别在麦胚无细胞翻译体系中进行翻译。其翻译产物经ＳＤＳ－ＰＡＧＥ系统分析。结果表明,基因组片段１、２、３、４、５、和１０分别编码病毒核心衣壳的结构多肽ＶＰ１、ＶＰ２、ＶＰ３、ＶＰ４、ＶＰ５和ＶＰ１０,片段６和７分别编码病毒外层衣壳的结构多肽ＶＰ６和ＶＰ７。片段８和９分别编码５２ｋＤ和４１ｋＤ的多肽,片段１１编码两种多肽,分子量分别为２９ｋＤ和１９．５ｋＤ,它们与病毒结构多肽无明显对应关系。病毒基因组与多肽大体是一一对应的关系。     

同义密码子使用模式对蛋白产物表达及构象形成的影响*

鉴于遗传密码子的简并性能够将基因遗传信息的容量提升,同义密码子使用偏嗜性得以在生物体的基因组中广泛存在。虽然同义密码子之间碱基的变化并不能导致氨基酸种类的改变,在研究mRNA半衰期、编码多肽翻译效率及肽链空间构象正确折叠的准确性和翻译等这一系列过程中发现,同义密码子使用的偏嗜性在某种程度上通过精微调控翻译机制体现其遗传学功能。同义密码子指导tRNA在翻译过程中识别核糖体的速率变化是由氨基酸的特定顺序决定,并且在新生多肽链合成时,蛋白质共翻译转运机制同时调节其空间构象的正确折叠从而保证蛋白的正常生物学功能。某些同义密码子使用偏嗜性与特定蛋白结构的形成具有显著相关性,密码子使用偏嗜性一旦改变将可能导致新生多肽空间构象出现错误折叠。结合近些年来国内外在此领域的研究成果,阐述同义密码子使用偏嗜性如何发挥精微调控翻译的生物学功能与作用。     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

A comparison of rapid electrophoretic and chromatographic methods for the investigation of common histological dyes

Summary The compositions of fifty-nine common histological dyes, as well as duplicate samples of several dyes from different suppliers, have been studied by agar gel electrophoresis, agarose gel electrophoresis, paper electrophoresis, paper chromatography and thin layer chromatography. Tables are presented to show the number of components present in each dye as disclosed by the different methods; the cases where duplicate samples were available are summarised in a separate table.On the basis of effectiveness and convenience agar gel electrophoresis and thin layer chromatography were by far the best methods. The Chromatographic method was of slightly wider applicability but as electrophoretic methods gave information on dye charge, agar gel electrophoresis was the best single method.     

Proteins and glycoproteins of the erythrocyte membrane

Erythrocyte membranes from several species were prepared by three different methods of hypotonic hemolysis and examined for variations in protein and glycoprotein content by acrylamide gel electrophoresis in sodium dodecyl sulfate. Significant variations were noted in morphology of the membranes prepared by the different methods without attendant variations in protein patterns of the major membrane proteins for most cases observed, which show a similar pattern of nine common bands for all of the species observed. The significant difference in protein pattern which was noted was attributed to proteolytic digestion of membranes which were fragmented during preparation. Failure to remove white blood cells from membrane preparations was shown to be a significant source of the problem with proteolytic digestion. Glycoproteins were analyzed by acrylamide gel electrophoresis or by column chromatography. Each species appears to have a different major glycoprotein (or group of closely related glycoproteins). Molecular weights of glycoproteins calculated from acrylamide gel electrophoresis were found to vary with the percentage of acrylamide in the gel, indicating that these proteins do not behave in a normal fashion in this electrophoresis system. The molecular weight calculated from gel filtration data for the human membrane glycoproteins (26,000) was quite disparate from those calculated from gel electrophoresis (88,000 to 62,000 in 5 to 10% gels).     

Biochemical Identification of the Two Races of Radopholus similis by Polyacrylamide Gel Electrophoresis

Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as diagnostic. Resolution of protein bands was greatly improved by SDS and gradient slab gel electrophoresis, but no differences could be detected among the proteins resolved between the two rares with these techniques. Two-dimensional gels revealed a large number of proteins, but background staining obscured them hindering interpretation. When nematode races were reared on three different host plants, no differences in protein patterns were detected between them, indicating host preferences does not play a role in determining the types proteins occurring in these nematodes.     

A simple electrophoretic procedure for the determination of the polypeptide composition of the subunits of Fraction 1 protein

A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 m urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques.     

Acid-base and optical properties of heparin.

Sodium dodecylsulfate (SDS) can be removed from protein by gel electrophoresis. This principle is useful for separating protein bands which are close to each other in SDS gel electrophoresis. We accomplished this by “two-stage” gel electrophoresis. In this system, SDS gel electrophoresis was carried out as the first step. Gel electrophoresis was then continued (after replacing the buffer) without SDS. SDS was then eluted from the gel into the lower buffer during the second stage. Separation of the subunits was significantly improved relative to simple SDS gel electrophoresis.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

Isolation of the smallest component of silk protein.

Silk proteins were solubilized from cocoons with ethylenediamine/cupric hydroxide solution. A series of polymers of the smallest component, detected by polyacrylamide-gel electrophoresis, could be converted into the smallest component by reduction and aminoethylation. Fibroin and sericin fractions were separated by precipitation of sericin at pH 5.5. On gel electrophoresis, sericin showed distinct bands but fibroin did not. The components of fibroin and sericin were fractionated by gel filtration on Sepharose 6B. The smallest component in the sericin fraction was purified by rechromatography and showed a single band on gel electrophoresis. Its mol. wt. was 24 000, and its amino acid composition was determined.     

New allelic product of the PRH1 locus coding for salivary acidic proline-rich proteins

A new polymorphic acidic proline-rich protein (As) was found in human parotid saliva by SDS and basic polyacrylamide gel electrophoresis. The phenotypic relationships and family studies support the hypothesis that the As protein is another allelic product of the PRH1 locus. The As protein could not be discriminated from the parotid isoelectric focusing (PIf) protein by isoelectric focusing gel electrophoresis due to similar migration of the two proteins. In order to determine salivary PRH1 phenotypes it is necessary to use SDS or basic gel electrophoresis in addition to the isoelectric focusing gel electrophoresis. The As protein was not found in Caucasians. The allele frequencies of the PRH1 locus in Japanese were PRH1 (double-band protein) = 0.035, PRH1(2) (acidic protein) = 0.193, PRH1(4) (PIf) = 0.751, and PRH1(5) (As) = 0.021.     

Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium

In human unbilical artery and vein endothelial cell conditioned medium fibrinolytic inhibitors have been detected by two different techniques. A fast-acting inhibitor of tissue-type plasminogen activator (t-PA)_and urokinase has been detected and quantified by its capacity to neutralize the above-mentioned plasminogen activators in a kinetic assay. By reverse fibrin autography after SDS-polyacrylamide gel electrophoresis a fibrinolytic inhibitor can be detected with a molecular mass of 52 kDa. The mutual relationship between these two inhibitors was studied. Neutralization of the fast-acting inhibitor by t-PA results in the formation of a complex with a molecular mass of 100 kDa. The t-PA added to endothelial cell conditioned medium in excess of the fast-acting inhibitor is fully stable. However, the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography is not affected by complete neutralization of the fast-acting inhibitor, and removal of the formed complexes by immune adsorption with immobilized anti-t-PA IgG. This suggests that the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography does not react with t-PA. Moreover, endothelial cell conditioned medium that is depleted of the fast-acting inhibitor does not show lysis resistance when directly applied to the reverse fibrin autography indicator gel (without previous electrophoresis), although the inhibitor is still present in the zymogram after SDS-polyacrylamide gel electrophoresis. This suggests that the inhibitor is induced by the SDS treatment. Heating the endothelial cell conditioned medium for 15 min at 70°C fully destroys the fast-acting inhibitory activity, but leaves the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography unaffected. Moreover, at least one additional fibrinolytic inhibitor is detected in the zymogram after SDS-polyacrylamide gel electrophoresis. We conclude that the fast-acting inhibitor is not the same as the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography; the latter inhibitor is not operational in endothelial cell conditioned medium, but is induced by SDS-polyacrylamide gel electrophoresis.