TAGGING GENES AND EXPRESSION OF 5' NUCLEOTIDASE IN DICTYOSTELIUM DISCOIDEUM

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。要了解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。我们的目标是确定这样的突变体，这些突变体可以用来鉴定5-核苷酸酶正常运行所需的功能基因，以及细胞正确分化所必需的功能基因。将限制性内切酶与线性化的质粒一起引入，可在基因组限制位点整合高比例的转化子中的DNA。限制性内切酶介导的整合(REMI)以一种明显随机的方式产生插入到基因组限制酶位点的片段，其中一些会导致突变。将分析表现出停滞或异常发育的网柄网柄菌转化子。整合后的质粒连同侧翼基因组DNA将从其中一些突变体中分离出来，克隆到大肠杆菌中，然后用于转化其他Dictyostelius细胞。侧翼序列中的同源重组导致与原始突变体显示的相同表型，直接表明受影响的基因对特定的形态表型负责。使用REMI将提供更多关于5-NT在网柄苔藓中的特定功能和作用的信息，并可能提供更多信息来帮助理解其他生物体的功能，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