Molecular Cloning And Differential Expression Patterns Of The Regulatory Subunit B′ Gene Of PP-2A In Gold Fish, Carassius Auratus

Protein dephosphorylation by serine/thronie phosphatases plays a critical role in regulating various intracellular processes. Protein Phosphatase 2A is one of the major serine/threonine phosphatases. Previous studies from our laboratory have established the expression patterns of the catalytic subunit for PP-2A (PP-2Acs) in goldfish and mouse eye. In the present thesis, I have focused on the study of the delta regulatory subunit for PP-2A. Using RACE cloning strategy, I first isolated the cDNA clone encoding the delta isoform of the B' family regulatory subunits of PP-2A from Goldfish. Then, I conducted RT-PCR and Western blot analysis and determined the differential expression patterns of the delta regulatory subunit in different tissues and in different developmental stages. Following results were obtained:(1) The full length cDNA for the delta isform of the B' regulatory subunits of PP2A in Goldfish contains 2415 bp with an ORF of 1668 bp coding for a deduced protein of 555 amino acids with a calculated molecular weight of 64.4 kDa. The amino acid sequence of the goldfish delta subunit bears a homology of 81.8%, 85.2% and 78.7% to that from human, mouse and xenopus, respectively. The presence of the high homology among different species suggests that the delta gene may be highly conserved during evolution in both structure and function. The differences in the amino and carboxyl termini imply that the B' subunits may have multiple functions, such as determining the substrate specificity and targeting cellular localization.(2) RT-PCR reveals that the highest level of mRNA is detected in brain, a less level detected in liver, testis, oocyte, kidney and gill, and the lowest detected in the fin. During different developmental stages of goldfish, the highest level of delta subunit mRNA expression is detected at the stages of two-cell, multiple-cell, blastula and gastrula, and a decreased level of B'δ. mRNA in other developmental stages. At the protein level, the highest expression of B'δprotein is found in testis, oocytes, brain and heart, a less amount found in liver and the lowest level detected in kidney, gill and fin. Developmentally, B' protein is strongly expressed at the stages of two-cell, multiple-cell, blastula, gastrula, neurula, and eye bud, and then decreased at the stages of brain differentiation and eye pigmentation. These results suggest that B'δappears to play a very important role during gold fish development and in adult tissue homeostasis.(3) In addition, a PP2A B'δantisense expression construct is established. With this construct, the role of B'δin regulating goldfish development is being explored.Together, these results have laid a foundation for the exploration of the detail functions of the B'δsubunit of PP-2A in lower vertebrates.