Browsing by Author "Chanchao, Chanpen"
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- Generation of cDNA libraries of amoeba, 8 hour, and 12 hour stages of Dictyostelium discoideumChanchao, Chanpen (Virginia Tech, 1996-08-09)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.
- The Role of 5' Nucleotidase in the Regulation of Morphogenesis in Dictyostelium DiscoideumChanchao, Chanpen (Virginia Tech, 1999-06-28)5' Nucleotidase (5NU) in Dictyostelium discoideum is an enzyme that shows high substrate specificity to 5'AMP. The enzyme has received considerable attention in the past because of the critical role played by cyclic AMP in cell differentiation in this organism. Degradation of cAMP by cAMP phosphodiesterase (PDE) produces 5'AMP, the substrate of 5NU. Dictyostelium switches its genetic program from growth to cellular differentiation when nutrients become limited. During the time course of development, the activity of 5NU is high and becomes restricted to a narrow band of cells that form the interface between the prestalk/prespore zones. Understanding how this gene is regulated will provide knowledge underlying the process of cell differentiation. In order to understand the functional significance of the 5NU, I first purified of the 5NU protein using an artificial substrate p-nitrophenol phosphate (pNPP). An activity stain on non-denaturing gels with Nitro Blue Tetrazolium (NBT) and 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) as the substrate was also used. A polypeptide of approximately 90 kDa was associated with 5NU enzyme activity after gel filtration chromatography and denaturing gel electrophoreses. Protein sequence of this peptide was obtained from Mass Spectrometry and Edmund Degradation. Various databanks were searched for similar sequences, but no matches with high identity were obtained. However, a search of the sequences of an ongoing cDNA project at the University of Tsukuba in Japan revealed a clone that corresponded to the peptide sequence of 5NU. In addition, a clone was found that corresponded to the classical "alkaline phosphatase" found in several organisms. Analysis of the expression of the 5NU and AP during Dictyostelium development by Northern blotting determined that the 5NU is developmentally regulated while the AP is expressed at all stages of the life cycle. Southern blot analysis showed a single form of the gene for both 5NU and AP. Targeted gene disruption and knockout mutagenesis using the 5NU sequences flanking a blasticidin-resistant cassette was attempted. Analysis of the transformants showed the 5NU gene was not disrupted, and that the blasticidin-resistant cassette was randomly inserted into the genome.