Characterization of the adenosine deaminase-related growth factor (ADGF) gene family in Drosophila Stephanie A. Maier, Lynn Podemski, Sean W. Graham, Heather E. McDermid, John Locke * Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada Received 25 June 2001; received in revised form 1 October 2001; accepted 19 October 2001 Received by D. Finnegan Abstract A novel family of growth factors, with sequence similarity to adenosine deaminase, has been identified in various organisms including flesh fly, tsetse fly, sand fly, mollusk and human. The human homologue, CECR1, is a candidate gene for the genetic disorder cat eye syndrome. Here, we describe six members of this growth factor family in Drosophila and two in vertebrates. The six Drosophila genes, named adenosine deaminase-related growth factors (ADGF), are found at three different chromosomal locations, with one singleton, two in an inverted orientation, and three in a tandem arrangement. These genes show distinct patterns of expression as measured by RT-PCR and Northern blots, indicating gene-specific function. The presence of six ADGF genes in the Drosophila genome suggests that gene duplication and divergence has been important for these growth factors in insect development. Phylogenetic analysis of the 14 extant ADGF-like gene products shows there are at least three major groups, two of which are found in Drosophila. The third appears specific to the vertebrate line. Seven gene duplications are inferred among the ADGF-like genes, most of which occurred long before the origin of Drosophila. Our analysis predicts the existence of several other unsampled ADGF-like genes, both within the species examined here, and in other related invertebrates. q 2001 Published by Elsevier Science B.V. Keywords: Adenosine deaminase; CECR1; Insect-derived growth factor; Mollusk-derived growth factor; Male-specific insect-derived growth factor; LuloADA; Tsetse salivary growth factor 1. Introduction Growth factors are proteins that bind to receptors on the cell surface and act to cause cell proliferation and/or differ- entiation. Recently, a family of secreted growth factors that share sequence similarity to adenosine deaminase (ADA) have been described. The founding member of this novel growth factor family is insect-derived growth factor (IDGF) (Homma et al., 1996). IDGF was purified from an embryo- nic cell line of Sarcophaga peregrina (flesh fly), and was shown to have growth factor properties, as indicated by an increased growth rate of this cell line when purified IDGF was added (Homma et al., 1996). Amino acid sequence comparisons have identified other members that are similar to IDGF in a wide variety of invertebrates, including mollusk-derived growth factor (MDGF; Akalal and Nagle, 2001; formerly known as AGSA, or atrial gland granual- specific antigen; Sossin et al., 1989) from Aplysia califor- nica (sea hare, mollusk), tsetse salivary growth factor (TSGF-1 and -2) from Glossina morsitans morsitans (tsetse fly) (Li and Aksoy, 2000), and LuloADA from Lutzomyia longipalpis (sand fly) (Charlab et al., 2000, 2001). This protein family is not limited to invertebrates, however, as these members also show significant amino acid similarity to human protein CECR1 (Riazi et al., 2000). CECR1 is a candidate gene for cat eye syndrome, which is a rare dupli- cation disorder characterized by defects of the eyes, heart, anus, kidneys, face and mental development (Schinzel et al., 1981). Also, there are expressed sequence tags (ESTs) with similarity to this family in various other organisms (pig, Sus scrofa, Accession number: BF192876; cow, Bos taurus, Accession number: BF076055; zebrafish, Danio rerio, Accession number: AW077621; frog, Xenopus laevis, Accession number: BG233900; and Dictyostelium discoi- deum, Accession number: C89929), indicating a widely Gene 280 (2001) 27–36 0378-1119/01/$ - see front matter q 2001 Published by Elsevier Science B.V. PII: S0378-1119(01)00762-4 www.elsevier.com/locate/gene Abbreviations: aa, amino acid(s); ADGF, adenosine deaminase-related growth factor; bp, base pairs; cDNA, DNA complementary to RNA; DNase, deoxyribonuclease; dNTP, deoxyribonucleoside triphosphate; EST(s), expressed sequence tag(s); kb, kilo bases; kbp, kilo base pairs or 1000 bp; mRNA, messenger RNA; ORF, open reading frame; PCR, poly- merase chain reaction; Poly(A) 1 , polyadenylated RNA; RT-PCR, reverse transcriptase-polymerase chain reaction; ssDNA, single-stranded DNA; UTR, untranslated region * Corresponding author. Tel.: 11-780-492-2193; fax: 11-780-492-9234. E-mail address: john.locke@ualberta.ca (J. Locke).