TAGGING GENES AND EXPRESSION OF 5' NUCLEOTIDASE IN DICTYOSTELIUM DISCOIDEUM

点击翻译按钮获取中文摘要

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