Undergraduate Experimental Biochemistry in Enzymology and Molecular Biology

该奖项为巴吞鲁日路易斯安那州立大学生物化学系提供资金，用于购买设备，以帮助升级核心生物化学实验室课程。这门本科实验生物化学课程将以酶学和分子生物学为重点，利用克隆的果蝇醇脱氢酶（ADH）全长cDNA提供实验材料。ADH将从携带ADHcDNA的大肠杆菌中纯化，该cdna克隆在表达载体中。将对纯化酶进行分析，以确定伯醇和仲醇的Km值以及竞争性和非竞争性抑制剂的Ki值。纯化的ADH将通过硫-二硫相互转化可逆地失活和再活化。通过比较携带突变adhcdna的大肠杆菌裂解物中的ADH活性和抗原反应，将检验无义、错义和缺失突变的影响。M13噬菌体中的ADH插入物将用于教授限制性内切图谱和DNA测序技术。在酸性、中性和碱性条件下，通过核糖核酸酶切、色谱和紫外光谱测定二和三核糖核苷酸的序列，说明了核酸化学的原理。将检查双链DNA的变性。在适当的实验中强调计算机分析和应用。受助人与非联邦来源的奖励相匹配。

This award provides funds to the Department of Biochemistry at Louisiana State University at Baton Rouge to purchase equipment that will help upgrade the core biochemistry lab course. This undergraduate Experimental Biochemistry course will focus on enzymology and molecular biology using a cloned full-length cDNA of Drosophila melanogaster alcohol dehydrogenase (ADH) to provide experimental materials. ADH will be purified from E. coli carrying the ADHcDNA cloned in an expression vector. The purified enzyme will be analyzed to determine the Km values for primary and secondary alcohols and the Ki values for competitive and non- competitive inhibitors. Purified ADH will be reversibly inactivated and reactivated through thio-disulfide interconversion. Effects of Nonsense, Missense and Deletion mutations will be examined by comparing ADH activity and antigenic response in the lysates of E. coli carrying the mutated ADHcDNAs. The ADH insert in M13 phage will be used to teach restriction mapping and DNA sequencing techniques. The principles of nucleic acid chemistry will be illustrated by determining the sequences of di- and tri- ribonucleotides with ribonuclease digestions, chromatography and UV spectra of the products under acidic, neutral and basic conditions. Denaturation of doubly-stranded DNA will be examined. Computer analysis and application are emphasized in appropriate experiments. The grantee is matching with award with non-Federal sources.