Molecular cloning and differential expression patterns of the regulatory subunit B′ gene of PP2A in goldfish, Carassius auratus

Abstract

It is well established that the protein serine/threonine phosphatase 2A (PP2A) plays very important roles in many different cellular processes, including cell proliferation and differentiation, gene expression, neurotransmission, apoptosis, and aging. PP2A consists of three heterogenic subunits: the scaffold subunit A, the catalytic subunit C, and the regulatory subunit B. While both the scaffold and the catalytic subunits contain only two forms, at least four families of the regulatory subunits, B, B', B'', and B''' have been identified. These regulatory subunits from different families are encoded by different genes and bear other functions besides directing the specificity of PP2A. To study the functions of the regulatory subunits of PP2A in lower vertebrates, we have cloned the full-length cDNA sequence of the gene encoding the regulatory subunit B'delta of PP2A from gold fish, Carassius auratus using 3'-RACE and 5'-RACE cloning strategies. Our results revealed that the full-length B'delta cDNA contains 2415 bp and encodes a protein of 555 amino acids. The B'delta protein displays a very high level of sequence identity with the B'delta regulatory subunit from other species of vertebrates. Regarding its expression pattern, RT-PCR revealed that the highest level of mRNA was detected in brain, a less level detected in liver, spermary, ovary, kidney and gill, and the lowest level detected in the fin. During different developmental stages of gold fish, the highest level of mRNA expression was detected at the stages of two-cell, multiple-cell, blastula and gastrula, and a decreased level of B'gamma mRNA was detected in other developmental stages. At the protein level, the highest expression level of B'delta protein was found in spermary, ovary, brain and heart, a less amount found in liver and the lowest level detected in kidney, gill and fin. Developmentally, B'delta protein was strongly expressed at the stages of two-cell, multiple-cell, blastula, gastrula, neurula, and optic vesicle, and then decreased at the stages of brain differentiation and eye pigmentation. These results suggest that B'delta appears to play a very important role during gold fish development and also in adult tissue homeostasis.