Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth.

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.     

Optimization of affinity and ion-exchange chromatographic processes for the purification of proteins

This study documents several alternative approaches for the optimization of the ion-exchange and affinity chromatographic purification of proteins. In these approaches, the chromatographic process has been treated as a four-stage (adsorption, washing, elution, and regeneration) operation. Central to these investigations has been the elaboration of practical iterative procedures based on the use of theoretical models describing each of these stages. Predictions derived from these models have then been evaluated in terms of experimental data obtained using batch adsorption measurements in finite bath configurations and frontal breakthrough measurements with packed beds of different dimensions, containing nonporous and porous adsorbents of different selectivities and capacities for proteins. Commencing with the kinetic and distribution parameters derived from batch equilibrium measurements, the effect of the initial concentration of the target protein, the solid-liquid volume ratio, the superficial velocity and the column dimensions on the pressure drop, production rate, concentration profile, column utilization, and yield have been determined with packed beds. The potential of these iterative approaches to simplify the determination of key mass transfer and interaction parameters required for scale-up and economic optimization of chromatographic purifications of proteins has been examined using ion exchange, immobilized metal ion affinity, and triazine dye pseudo-affinity adsorbents of different selectivity and adsorption capacities. (c) 1996 John Wiley & Sons, Inc.     

地膜条件下土壤生态因子与玉米产量的关系

研究干旱地区地膜条件下,土壤生态因子对玉米产量的影响．结果表明,最佳种植模式为作物距地膜中心线２１．３—３３．２Ｃｍ,施纯Ｎ＋Ｐ为５９８—７４３ｋｇ·ｈａ－１,玉米产量为１４４９２≤Ｙ佳≤１６５９ｋｇ·ｈ－１．该种植模式有利于提高作物产量及残膜回收．     

激光照射茶树花药培养及其过氧化物酶同工酶的分析

Ｈｅ－Ｎｅ和ＣＯ２激光对茶树花药培养的药裂率和出愈率的影响,既有促进作用,也有抑制作用。按出愈率和愈伤组织生长状况的序列：Ｈｅ－Ｎｅ：２０Ｊ／ｃｍ＾２〉ＣＫ〉Ｈｅ－Ｎｅ：３０Ｊ／ｃｍ＾２〉ＣＯ２：３５．４Ｊ／ｃｍ＾２。对Ｈｅ－Ｎｅ激光照射花药及其愈伤组织的过氧化物酶同工酶分析,主要表现在弱酶带的变化上,无规律差异。     

川中丘陵人工幼草本层动态研究初报

 川中丘陵为长江中上游防护林体系建设重点区域。本文对荒山荒坡种植柏木、桤木及补播牧草后的植被动态进行了研究。结果表明：在幼林期,随树木郁闭度的增加,林下草本植物的盖度也随之提高,生物量增大,林草之间具良好的共生效益;补播草种可大大加快幼林地植被覆盖。     

抗鞘翅目δ－内毒素 及毒素基因文库的构建

研究了对鞘翅目昆虫有毒效的5个苏云金芽孢杆菌新菌株YM-03、Sph04-04、YK14-01、SH11-05、Sph16-01伴孢晶体的蛋白质组成,以柳蓝叶甲为供试虫测定了它们的LC_(50)值,其中YM-03毒力最高。测定了YM-03晶体蛋白N-末端部分氨基酸序列。用琼脂糖凝胶电泳快速检测了5株菌的质粒组成,证明其质粒图型各不相同。以广谱的cosmid质粒pLAFRI为载体,通过限制酶EcoRI部分酶解获得“目的”DNA片段,构建了菌株YM-03的总DNA文库,对17个抗性克隆的质粒进行酶切分析表明,其中含有外源片段的克隆占总数的76％,超过要求的理论值。以人工合成的杀鞘翅目基因的18bp保守序列片段为探针,筛选了近1200个抗性克隆,获得了3个阳性克隆,LE392(PBYM2)、LE392(pBYM3)和LE392(pBYM4),毒力测定试验表明LE392(pBYM3)和LE392(pBYM4)有一定表达,表明其携带有δ-内毒素基因。     

人重组IL6／IL2融合蛋白的变性、复性及纯化

经超声破碎,分离已表达CH925包涵体,较系统地研究变性剂浓度、融合蛋白浓度对蛋白折叠的影响.在还原型及氧化型谷胱甘肽复性条件下,成功地将融合蛋白CH925折叠成具有IL6及IL2双活性蛋白,IL6的比活为2.3×10⁸U/mg, IL2比活为2.2×10⁶U/mg.经阴离子交换、凝胶过滤层析,获得一定纯度的CH925,配合反相HPLC.洗脱收集蛋白峰,CH925纯度为98%.     

内毒素致大鼠肺损伤时肺组织β受体的变化与膜磷脂代谢的关系

采用放射性配基结合分析法,现察内毒大致大鼠急性肺损伤时肺β－肾上腺素能受体（β－ＡＲ）的变化。分别用荧光偏振法和高效液相色谱测定肺组织细胞膜脂流动性和磷脂含量。结果显示：（１）静脉注射内毒素后４ｈ,大鼠肺β－ＡＲ的最大结合容量明显降低,较对照组减少４７％;（２）内毒素组肺膜脂流动性和磷脂含量均明显降低,同时伴有肺组织磷脂酶Ａ２（ＰＬＡ２）活性升高。提示：（１）β－ＡＲ下调导致其介导的功能减弱,在内毒素诱导的大鼠急性肺损伤发病机理中起一定作用;（２）ＰＬＡ２激活是膜磷脂减少的重要原因,后者可导致膜脂流动性降低,结果引起β－ＡＲ的侧向扩散和旋转运劝减弱,从而减少β－ＡＲ与配基结合的机率,出现β－ＡＫ下调。     

重组人IL6／2融合蛋白对6.5Gy照射小鼠造血功能恢复的影响

观察了ｈＦＰＩＬ６／２对６．５Ｇｙγ线照射ＮＩＨ小鼠第１０天造血功能恢复的影响。结果表明：照射小鼠连续４ｄ给予ｈＦＰＩＬ６／２２５０μｇ·ｋｇ－１·ｄ－１,其脾重、ＣＦＵ－８、骨髓有核细胞数及ＣＭ－ＣＦＵ分别比对照组增加５９．０％、２７８．５％、５７．９％和１３８．２％,统计学处理均有显著差异;对此四项指标的改善也明显优于２５μｇ组。另外,２５０μｇ剂量组小鼠外周血象３０ｄ的动态观察结果表明,ｈＦＰＩＬ６／２不但能明显提高红细胞和血红蛋白的最低值,而且能使血小板的恢复提前。提示ｈＦＰＩＬ６／２在促进血小板生成和促进红系造血方面可能具有良好的应用前景。     

茶碱诱导野生毛尖蘑子实体形成的研究

５ｍｇ／Ｌ—１０ｍｇ／Ｌ的茶碱在０℃─１０℃变温条件下诱导野生毛尖蘑形成了实体原基。分析证明,此条件下毛尖蘑菌丝体呼吸强度提高,体内可溶性蛋白质增加,而ＤＮＡ与ＲＮＡ总量却稍有下降之势。本研究为珍品毛尖蘑人工驯化做了先导性工作。