U.S.-Federal Republic of Germany Cooperative Research: The Influence of Phosphorothioate on the Action of Restriction Endonucleases

该奖项支持冈萨加大学的Kay Nakamaye博士对位于德国哥廷根的马克斯普朗克实验医学研究所进行研究访问，以获得核苷酸合成方面的经验，并与核酸化学领域的领导者Fritz Eckstein博士合作，Fritz Eckstein博士率先使用了硫代酸盐。他们的合作研究将侧重于在改进重组DNA操作程序中使用硫代酸盐；具体来说，他们感兴趣的是结合到DNA中的硫代磷酸酯对限制性内切酶切割的影响。在之前的合作中，他们开发了一种简单而有效的方法，在分离的DNA片段或基因中产生位点特异性突变。然而，该技术的局限性之一是需要一个特定的DNA序列来使用限制性内切酶，最好是在预期突变位点附近。他们现在计划研究ii类限制性内切酶作为指导突变和内切酶切割位置的策略。这将使他们的诱变技术几乎适用于任何DNA和DNA序列。诱变技术的发展对蛋白质结构与功能关系、基因控制与调控、DNA/RNA结构等领域的研究至关重要。基因分离、表征和操作是许多生物技术的基本组成部分。这项工作的任何进一步技术将继续在基础和应用研究中具有重要价值。

This award supports a research visit by Dr. Kay Nakamaye of Gonzaga University to the Max Planck Institute for Experimental Medicine in Goettingen, FRG, to gain experience in the synthesis of nucleotides and to collaborate with Dr. Fritz Eckstein, a leader in the field of nucleic acid chemistry who has pioneered the use of phosphorothioates. Their cooperative research will focus on the use of phosphorothioates in improved procedures for recombinant DNA manipulation; specifically, they are interested in the effect of phosphorothioates incorporated into DNA on cleavage by restriction endonucleases. In previous collaboration, they developed a simple yet efficient method of producing site-specific mutations in isolated DNA fragments or genes. However, one of the limitations of the technique is the need for a specific DNA sequence for the restriction endonuclease to be used, preferably near the site of the intended mutation. They now plan to study class IIS restriction endonucleases as a strategy for directing both the mutation and the position of nicking by the endonuclease. This would make their mutagenesis technique nearly universal for any DNA and DNA sequence. Development of mutagenesis techniques has become essential to investigations in areas such as protein structure-function relationships, gene control and regulation, and DNA/RNA structure. Gene isolation, characterization and manipulation are fundamental components of many biotechnologies. Any further techniques for such work will continue to have great value in basic and applied research.