Generation of cDNA libraries of amoeba, 8 hour, and 12 hour stages of Dictyostelium discoideum

Abstract

A critical event during the life cycle of Dictyostelium discoideum is glycogen turnover. This process is catalyzed by glycogen phosphorylase-2 (gp-2). Since gp-2 expression is first induced during the transition from growth to differentiation, understanding how this gene is controlled may provide some insight into the process of differentiation. In order to identify the trans-acting factors responsible for activating gp-2 expression, cDNA plasmid libraries of amoebae, and cells at 8 h and 12 h of development were generated. The long-term goals of this project involve screening expression libraries with identified cis-acting elements from the gp-2 promoter to yield the DNA binding proteins responsible for gene regulation. For this approach to succeed, a high-quality cDNA library is essential. The library must contain full-length cDNA that represents the complexity of mRNA present during the developmental stage of interest. Hence, all three libraries were subjected to extensive testing prior to and following cloning. RNA quality and the fidelity of the time points were determined by Northern blot analysis and by RTPCR for several marker genes. Following cDNA synthesis, the cDNA was assessed for complexity and full-length synthesis by PCR and radioactive primer extension, respectively. Ligation of the cDNA into a vector was performed using several ratios of vector:insert in order to ensure that long cDNA species were included in the plasmid library. Finally, the presence of the marker genes was confirmed by PCR amplification of plasmid extracted from bacteria transformed with the plasmid library.