Journal of Biochemistry and Molecular Biology, Vol. 38, No. 3, May 2005, pp. 328-333 Expression of Sara2 Human Gene in Erythroid Progenitors Denis Leonardo Fontes Jardim, Anderson Ferreira da Cunha, Adriana da Silva Santos Duarte, Camila Oresco dos Santos, Sara Terezinha Olalla Saad and Fernando Ferreira Costa* State University of Campinas - Center of Hemotherapy and Hematology. Campinas, SP, 13083-970, Brazil Received 14 October 2004, Accepted 23 December 2004 A human homologue of Sar1, named Sara2, was shown to be preferentially expressed during erythropoiesis in a culture stimulated by EPO. Previous studies, in yeast, have shown that secretion-associated and Ras-related protein (Sar1p) plays an essential role in protein transport from the endoplasmic reticulum to the Golgi apparatus. Here, we report the molecular analysis of Sara2 in erythroid cell culture. A 1250 bp long cDNA, encoding a 198 amino-acid protein very similar to Sar1 proteins from other organisms, was obtained. Furthermore, we also report a functional study of Sara2 with Real-time quantitative PCR analysis, demonstrating that expression of Sara2 mRNA increases during the initial stages of erythroid differentiation with EPO and that a two-fold increase in expression occurs following the addition of hydroxyurea (HU). In K562 cells, Sara2 mRNA was observed to have a constant expression and the addition of HU also up-regulated the expression in these cells. Our results suggest that Sara2 is an important gene in processes involving proliferation and differentiation and could be valuable for understanding the vesicular transport system during erythropoiesis. Keywords: Erythropoiesis, Hydroxyurea, K562 cells, Sar1, Vesicular traffic Introduction Erythroid differentiation, one of the few human processes of continuous non-malignant cellular proliferation and differentiation, is stimulated by erythropoetin (EPO), which possibly regulates the expression of many genes interacting with specific high affinity receptors on erythroid progenitors cells (Spivak et al., 1991). The identification of genes preferentially expressed during human erythroid proliferation and differentiation, particularly in the presence of EPO stimulus, could provide further insight into the molecular mechanisms that underlie hematopoietic differentiation and the appearance of hematopoietic malignancies. A transcriptional profile of genes from peripheral blood mononuclear cells, arising only in response to EPO, has been published (Gubin et al., 1999). Among the 719 expressed sequence tags (ESTs) found in this study, available at the Hembase database, we focused our attention on three related ESTs named Ax35f04, Ax35g04 and Ax40e08. These ESTs are related to the human gene, Sara2, homologous to the S. cerevisiae Secretion-associate and Ras-related gene (Sar1). The Sar1 gene, originally isolated as a multiple copy suppressor of a temperature sensitive mutant of the Sec12 gene (Nakano and Muramatsu, 1989), is required for transport from the endoplasmatic reticulum to the Golgi apparatus in the yeast, S.cerevisiae (Nakano et al., 1988; Takai et al., 2001). Sar1 protein is a member of the small GTPases family, which are very important in mediating multiple signaling transduction pathways involved in the regulation of cellular proliferation and differentiation (Barbacid 1987). The function of Sar1 in vesicle budding has been extensively characterized in the yeast, S. cerevisiae, but is less studied in mammals (Schekman and Orci 1996). Two Sar1 related genes (Sara1 and Sara2) have been found in mammals and in humans (Shen et al., 1993; He et al., 2002; Jones et al., 2003). Sara2 was first sequenced in humans in pituitary tumor (GenBank accession no. AF092130). This gene was further described as highly expressed in human liver cancer (He et al., 2002) and, recently, mutations in Sara2 gene have been associated with some lipid absorption disorders (Jones et al., 2003). To the best of our knowledge, there is no description available of Sara2 gene expression in hematopoietic tissue. Thus, we carried out cDNA cloning and the first functional study of the Sar1 human homologue Sara2 during erythropoiesis “in vitro”. The expression pattern of this gene was analyzed during erythroid differentiation with EPO stimulus and with the addition of hydroxyurea (HU), a drug used to treat sickle cell disease. The precise mechanism by *To whom correspondence should be addressed. Tel: 55-19-37888734; Fax: 55-19-32891089 E-mail: ferreira@unicamp.br