TAGGING GENES AND EXPRESSION OF 5' NUCLEOTIDASE IN DICTYOSTELIUM DISCOIDEUM

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。了解5-核苷酸酶基因及其蛋白产物的功能，需要对影响该基因表达的各种突变体进行体内和体外分析。我们的目标是鉴定出这样的突变体，这些突变体可以用来鉴定5-核苷酸酶正常运作所需的功能基因和细胞正常分化所必需的功能基因。在线性化质粒的基础上引入限制性内切酶，使质粒DNA在基因组限制性内切位点的整合率很高。限制性内切酶介导的整合（REMI）以一种明显随机的方式在基因组限制性内切位点上产生插入，其中一些插入会导致突变。对表现出发育停滞或异常的盘基柱变形体进行分析。整合的质粒以及两侧的基因组DNA将从其中一些突变体中切除，克隆到大肠杆菌中，然后用于转化其他盘基骨柱细胞。在侧翼序列内的同源重组导致与原始突变体显示相同的表型，直接证明受影响的基因负责特定的形态表型。使用REMI将提供更多关于5-nt在盘齿柱菌中的具体功能和作用的信息，并可能有助于了解其他生物的功能，如

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Understanding the function of 5-nucleotidase gene and its protein product require both in vivo and in vitro analysis of various mutants that affect the expression of the gene. Our goal is to identify such mutants that could be used to identify functional genes that are required for proper operation of 5-nucleotidase and necessary for the proper differentiation of the cells. Introduction of a restriction enzyme along with linearized plasmid results in integration of the plasmid DNA at the genomic restriction site in a high proportion of the resulting transformants. Restriction enzyme mediated integration (REMI) generates insertions into the genomic restriction sites in an apparently random manner, some of which cause mutations. The Dictyostelium transformants that show arrest or aberrant development will be analyzed. The integrated plasmid, along with flanking genomic DNA will be then excised from some of these mutants, cloned in E. coli, and then used to transform other Dictyostelium cells. Homologous recombination within the flanking sequence that result in the same phenotypes displayed by the original mutants, directly demonstrating that the affected genes were responsible for the specific morphological phenotypes. Using REMI will give more information about the specific function and role of 5-nt in Dictyostelium and probably more information to help understand the functions in other organisms like