K253R和N184V点突变对葡萄糖异构酶热稳定性的影响

用人工合成的寡核苷酸突变引物,以双引物法于重组Ｍ１３ｍｐ１９载体上进行定点突变,分别得到了葡萄糖异构酶的突变体ＧＩＫ２５３Ｒ和ＧＩＮ１８４Ｖ。测定它们的热稳定性,并将之与野生型酶比较,结果表明：（１）ＧＩＫ２５３Ｒ于７０℃、８０℃下的热稳定性小于野生型,但在７０℃、１ｍｏｌ／ＬＬ－鼠李糖中,两者的失活速度相近。另外;ＧＩＫ２５３Ｒ的比活是野生型的１．５倍。（２）ＧＩＮ１８４Ｖ的比活和热稳定性都远低于野生型,Ａｓｎ１８４为酶活性中心构象的维持所必需．本文还根据动力学数据和分子结构模型对以上结果作了初步分析．     

用蛋白质工程方法改变葡萄糖异构酶最适pH和最适温度

用寡核苷酸诱导的定点突变方法构建了葡萄糖异构酶基因的突变体（Ｎ１８４Ｄ和Ａ１９８Ｃ）。含突变体的重组质粒ｐＴＫＤ－ＧＩ１（Ｎ１８４Ｄ）和ｐＴＫＤ－ＧＩ２（Ａ１９８ｃ）在Ｅ．ｃｏｌｉＫ３８菌株中表达,用ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＦＦ和Ｓｅｐｈａｃｒｙｌ Ｓ－３００ＨＲ柱层析分离纯化突变酶。与野生型葡萄糖异构酶比较实验表明：（１）突变酶Ｎ１８４Ｄ的最适ｐＨ值下降了１个单位;等电点下降了０．６个单位     

纤溶酶原激活物抑制因子—1突变体E350G,E351K的构建及性质研究

利用ＰＣＲ扩增和合成突变引物的方法,将ＰＡＩ－１的Ｇｌｕ３５０和Ｇｌｕ３５１分别突变为Ｇｌｙ和Ｌｙｓ在大肠杆菌中表达并分离纯化突变体ＰＡＩ－１（Ｅ３５０Ｇ,Ｅ３５１Ｋ）有 酸胍激活并以ＥＬＩＳＡ法确定它与野生型ｒＰＡＩ－１的相对含量,通过对ｕ－ＰＡ抑制的动力学研究表明,突变体与野生型ｒＰＡＩ－１相比,对ｕ－ＰＡ和ｔ－ＰＡ的抑制活性都有明显下降,由活性态向潜伏态转变的半寿期也由０．８３ｈ缩短为０．５     

PAI—2抑制细胞凋亡依赖其蛋白质结合功能域

将纤深酶原激活剂抑制物２型（ＰＡＩ－２）突变体ＰＡＩ－２ＣＤ和ＰＡＩ－２ＱｃＤＮＡ与真核表达载体ｐｃＤＮＡ３重组,构建真核表达质粒并转染ＨｅＬａ细胞,经Ｇ４１８筛选,Ｎｏｒｔｈｅｒｎ迷鉴定和ＥＬＩＳＡ检测,筛选出稳定表达ＰＡＩ－２ＣＤ和ＰＡＩ－２Ｑ的阳性细胞株。ＥＬＩＳＡ结果表明ＰＡＩ－２突变蛋白质在转染细胞中的表达量与相应野生型ＰＡＩ－２在阳性细胞克隆中的表达量基本相近,ＰＡＩ－２及其突变体在理     

纤溶酶原激活物抑制因子－1突变体E350G，E351K的构建及性质研究

利用ＰＣＲ扩增和合成突变引物的方法,将ＰＡＬ－１的Ｇｌｕ３５０和Ｇｌｕ３５１分别突变为Ｇｌｙ和Ｌｙｓ,在大肠杆菌中表达并分离纯化突变体ＰＡＬ－１（Ｅ３５０Ｇ,Ｅ３５１Ｋ）,用盐酸胍激活并以ＥＬＩＳＡ法确定它与野生型ｒＰＡＩ－１的相对含量。通过对ｕ－ＰＡ,ｔ－ＰＡ抑制的动力学研究表明,突变体与野生型ｒＰＡＩ－１相比,对ｕ－ＰＡ和ｔ－ＰＡ的抑制活性都有明显下降,由活性态向潜伏态转变的半寿期也由０．８３ｈ缩短为０．５７ｈ。     

用酵母双杂交系统研究胰岛素样生长因子—1突变体与其结合蛋白 …

为研究胰岛素样生长因子－１（ＩＧＦ１）及其突变体与ＩＧＦ结合蛋白－３（ＩＧＦＢＰ３）的相互作用,针对ＩＧＦ１的第３、４、１５、１６位氨基酸残基,采用定点突变的方法构建了「Ｙ１５Ｌ１６」ＩＧＦ１和「Ｑ３Ａ４Ｙ１５Ｌ１６」ＩＧＦ１。然后分别将ＩＧＦ１／ＩＧＦ１突变体和ＩＧＦＢＰ３ ｃＤＮＡ克隆至酵母表达载体ｐＧＢＴ９和ｐＡＣＴ２中,利用用酵母双杂交技术检测ＩＧＦ１／ＩＧＦ１突变体和ＩＧＦＢＰ３之间的相     

GFP转基因指示系统建立的研究

ＧＦＰ（绿色荧光蛋白）是存在于发光水母体内的一种吸收蓝光或紫外光（３９５ｎｍ）后能够发出绿色荧光的天然蛋白质。本文报道了将ＧＦＰ表达质粒导入ＢＨＫ、ＤＫ、ＲＫ等真核细胞,建立ＧＦＰ真核细胞转基因指示系统。研究表明,ＧＦＰ在ＢＨＫ、ＤＫ、ＲＫ等传代细胞中表达的最佳条件是３３℃,ｐＨ７．２、５％ＣＯ２和转染后４８ｈ观察等。并对野生型ＧＦＰ和突变型ＧＦＰ的发光强度进行了比较,结果表明,在相同条件下,突变型ＧＦＰ的荧光比野生型的强     

PAI—2及其突变体的生物化学性质

为研究和比较纤深酶原激活剂抑制物２型（ｐｌａｓｍｉｎｏｇｅｎ ａｃｔｉｖａｏｒ ｉｎｈｉｂｉｔｏｒ ｔｙｐｅ ２,ＰＡＩ－２）及其突变体的生物化学性质,用ＰＣＲ、体外定点突变技术构建ＰＡＩ－２突变体ＰＡＩ－２ＣＤ和ＰＡＩ－２ＱｃＤＮＡ,将突变体ｃＤＮＡ与原核表达载体重组并转化怕杆菌。经诱导表达,ＳＤＳ－ＰＡＧＥ证实表达出相应蛋白质,均占全菌总蛋白的１４％。Ｗｅｔｅｒｎ印迹、溶圈抑制及纤维蛋白翻转板     

组织型纤溶酶原激活剂A链与尿激酶原B链嵌合分子的构建与表达

利用 Ｄ Ｎ Ａ 重组技术和定位删除技术,将组织型纤溶酶原激活剂（ｔ Ｐ Ａ）的 Ａ 链（ Ｓｅｒｌ Ｔｈｒ２６３）基因与尿激酶原（ｐｒｏ Ｕ Ｋ）的 Ｂ链（ Ｓｅｒ１３８ Ｌｅｕ４１１）基因相连,得到嵌合分子基因ｔｕ ｐａ,并在昆虫杆状病毒系统中进行表达,表达量可达 ５００ Ｉ Ｕ／ｍ ｌ．经单克隆抗体免疫亲和层析纯化细胞表达上清液,得到ｔｕ Ｐ Ａ 嵌合分子,其比活为 ２００ ０００ Ｉ Ｕ／ｍ ｇ 蛋白． Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 及 Ｗ ｅｓｔｅｒｎ ｂｌｏｔ 鉴定证明此表达产物分子量约为 ６０ ｋ Ｄ,与预期值相符．纤维蛋白平板测活及纤维蛋白亲和性分析初步证明,此嵌合分子的溶纤活性与ｐｒｏ Ｕ Ｋ 相近,而纤维蛋白亲和性高于 ｐｒｏ Ｕ Ｋ．     

叶绿素缺乏的大麦突变体的光合作用和叶绿素荧光

和早熟３号大麦（野生型）（ＤＧ）相比,黄化大麦（突变体）（ＬＧ）叶片Ｃｈｌ含量低,而Ｃｈｌ ａ／ｂ较高;光合速率低,表观量子效率低,而饱和光强较高,气孔导度较高;荧光参数Ｆｏ低,而ＰＳⅡ的光化学效率（Ｆｖ／Ｆｍ）以及Ｔ１／２、Ｆｍ／Ｆｏ、Φｅ均高。以单位叶绿素计算,黄化大麦的ＰＳⅡ电子传递活性和全链电子传递活性较高,而ＰＳＩ电子传递活性较低。推测黄化大麦ＰＳⅡ的光化学效率增高可能是由于其ＰＳⅡ向Ｐ     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.     

History of the discovery of the mode of transmission of yellow fever virus

This essay documents and examines the historical circumstances and events surrounding the discovery of the mode of transmission of yellow fever virus in Cuba. Close scrutiny of the articles published by Walter Reed and his colleagues in 1900, 1901 and 1902 reveals their limitations as historic documents. Fortunately, other sources of information from that period survive in letters and documents written by individuals involved in the quest for the mode of transmission. Examination and comparison of those sources of information unveiled a fascinating story which reveals that misunderstandings engendered by published articles accorded merit where it was not fully due.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.     

Time of detection of recessive genes: effects of system of mating and number of examined individuals

Summary Anthers were cultured from two sets of seven lines of hexaploid wheat (Triticum aestivum L.) with different cytoplasms, the euplasmic nucleus donors, Siete Cerros 66 and Penjamo 62, as well as their six alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. Significant cytoplasmic and nuclear effects but no cytoplasmic-nuclear interaction were found for embryogenic anther response, with the best performance of Penjamo 62 in Ae. kotschyi cytoplasm. Plant regeneration was not affected significantly by the cytoplasmic background of the lines cultured. The possible genetic implications of the observed cytoplasmic and nuclear influences on the in vitro haploid induction of wheat are discussed.