Undergraduate Experimental Biochemistry in Enzymology and Molecular Biology

该奖项为路易斯安那州立大学巴吞鲁日分校的生物化学系提供资金，用于购买有助于升级核心生物化学实验室课程的设备。这门本科实验生物化学课程将以克隆的果蝇黑腹果蝇乙醇脱氢酶(ADH)全长cDNA为实验材料，重点讲授酶学和分子生物学。ADH将从携带克隆在表达载体中的ADH基因的大肠杆菌中纯化。将对纯化的酶进行分析，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将被可逆灭活，并通过硫代二硫化物相互转化重新激活。无义突变、错义突变和缺失突变的影响将通过比较携带突变的ADHcDNA的大肠杆菌裂解产物中的ADH活性和抗原反应来检验。在M13噬菌体中插入ADH将被用来教授限制性内切酶图谱和DNA测序技术。在酸性、中性和碱性条件下，用核糖核酸酶消化测定二和三核糖核苷酸的序列，以及产物的层析和紫外光谱，从而阐明核酸化学的原理。将检测双链DNA的变性。在适当的实验中强调计算机的分析和应用。受赠人正在与非联邦来源的奖项进行匹配。

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.