Insect Biochemistry and Molecular Biology 34 (2004) 531–542 www.elsevier.com/locate/ibmb Immunocharacterization of the DNA puff BhC4-1 protein of Bradysia hygida (Diptera: Sciaridae) 5 N. Monesi a , J.A. Silva Jr. b , P.C.M. Martins b , A.B. Teixeira a , E.C. Dornelas b , J.E. Moreira b , M.L. Pac ¸o ´ Larson b, a Departamento de Ana ´lises Clı ´nicas, Toxicolo ´gicas e Bromatolo ´gicas, Faculdade de Cie ˆncias Farmace ˆuticas de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo, Ribeira ˜o Preto, SP, Brazil b Departamento de Biologia Celular e Molecular e de Bioagentes Patoge ˆnicos, Faculdade de Medicina de Ribeira ˜o Preto, Universidade de Sa ˜oPaulo, Ribeira ˜o Preto, SP, CEP 14049-900, Brazil. Received 5 September 2003; accepted 13 February 2004 Abstract The DNA puff BhC4-1 gene is amplified and highly expressed in the salivary gland of Bradysia hygida late larvae. Using affin- ity-purified polyclonal antibodies we have identified the product of the BhC4-1 gene as a 43 kDa polypeptide which is present in extracts of salivary glands from late fourth instar larvae and in the corresponding gland secretion, but not in glands from earlier stages. We also demonstrate that this protein is produced mainly in the S1 and S3 regions of the salivary gland, where BhC4-1 amplification levels are more pronounced and larger amounts of mRNA are produced. By immunoelectron microscopy the BhC4-1 protein was detected in secretory granules of the S1 and S3 regions, and localized in fibrous structures present in the saliva. # 2004 Elsevier Ltd. All rights reserved. Keywords: DNA puff; Salivary gland; Secreted protein 1. Introduction DNA puffs are formed in the polytene chromosomes of sciarid salivary glands at the end of the larval stage, and are sites of developmentally regulated gene amplifi- cation and transcription (Glover et al., 1982; DiBarto- lomeis and Gerbi, 1989; Pac ¸o ´-Larson et al., 1992; Fontes et al., 1992; Wu et al., 1993). The extra syn- thesis of DNA and RNA in DNA puffs has been inter- preted as gene amplification exerting a regulatory role on the synthesis of high levels of specific proteins that are needed in a short period of time (Breuer and Pavan, 1955). Several polypeptides have been identified as DNA puff gene product candidates based on tem- poral correlations established between the patterns of DNA puff formation and changes in the profiles of protein synthesis in late larvae salivary gland extracts (Winter et al., 1977a; Laicine et al., 1984). Some of these polypeptides are also present in the salivary gland secretion (Winter et al., 1977b; de Almeida, 1997), including the product of DNA puff BhB10-1 gene, pre- viously identified in immunoblots as a 23 kDa polypep- tide (Fontes et al., 1999). Polypeptide sequences deduced from full-length cDNAs of DNA puffs present no significant matches with proteins of known function (DiBartolomeis and Gerbi, 1989; Frydman et al., 1993; Monesi et al., 1995; Penalva et al., 1997; Fontes et al., 1999). A compara- tive sequence analysis has identified three families of DNA puff proteins based on similarity of the deduced amino acid sequences, families I, II and III. The protein coded by DNA puff C4 of Bradysia hygida, 5 We dedicate this paper to the memory of Dr Roberto Vicente Santelli, who was one of the leading researchers in Brazil in this field, an inspiration to the authors and a dedicated Biochemistry Associate Professor in the University of Sa ˜o Paulo.  Corresponding author. Departamento de Biologia Celular e Mol- ecular e de Bioagents Patoge ˆnicos, Faculdade de Medicina de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo, Ribeira ˜o Preto, SP, CEP 14049-900, Brazil. Tel.: +55-16-602-3032/602-3347; fax: +55-16-633- 1786. E-mail address: mlplarso@fmrp.usp.br (M.L. Pac ¸o ´ Larson). 0965-1748/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.02.010