02578naa a2200145 a 450000100080000000500110000800800410001910000160006024500830007626000090015952019120016870000150208070000160209577303210211113150802008-10-07       2008    bl uuuu     u00u1 u    #d1 aNIVA, C. C.  aPCNA as a cell proliferation marker in the oligochaete Enchytraeus japonensis.  c2008  aPCNA (proliferating cell nuclear antigen) is a protein involved in DNA replication expressed in dividing cells which has been used as a cell proliferation marker for clinical studies of neoplastic tissues in vertebrates, and also for developmental studies in several vertebrates and invertebrates. Among invertebrates, PCNA has been detected by immunohistochemical methods in several organisms, including the fragmenting oligochaete annelid E. japonensis. Despite the apparent positive cross-reaction with commercially available antibodies, it was unknown whether PCNA protein was really synthesized by E. japonensis. The objective of this study was to clarify this question and verify whether PCNA could be used as a reliable molecular marker for proliferative cells during E. japonensis regeneration, growth and embryo development at the RNA level. For that purpose we cloned a cDNA encoding PCNA from E. japonensis by carrying out degenerate PCR with primers designed from the conserved regions of known PCNAs using a cDNA library of regenerating E. japonensis as the template. Two full-length cDNA sequences with identical deduced amino acid sequence were obtained and the amino acid sequence showed high similarity to known PCNAs of other organisms. We analyzed the gene expression by RT-PCR and in situ hybridization in whole mounts (WISH) and paraffin sections in intact and regenerating worms. The results showed a strong expression of pcna gene in the developing growth zone of intact worms. In regenerating fragments, pcna expression was first detected in a few cells in the vicinity of the amputated surface, and then clearly seen in cell agglomerates in the blastema as regeneration progressed. Embryos also showed strong expression. We conclude that pcna is a good cell proliferation marker for E. japonensis and may be a good biomarker to study effects of stressors on developmental processes.1 aLEE, J. M.1 aMYOHARA, M.  tIn: INTERNATIONAL COLLOQUIUM ON SOIL ZOOLOGY, 15; INTERNATIONAL COLLOQUIUM ON APTERYGOTA, 12., 2008, Curitiba. Biodiversity, conservation and sustainabele management of soil animal: abstracts. Colombo: Embrapa Florestas. Editors: George Gardner Brown; Klaus Dieter Sautter; Renato Marques; Amarildo Pasini. 1 CD-ROM.