277 zyxwvutsrqp Biochtmica et Biophysics Acfa, 1088 (1991) 277-284 0 1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 AD0Nl.S 016747819100086X BBAEXP 92219 Cloning of genomic and complementary DNA encoding insect pheromone binding proteins: evidence for microdiversity J. Krieger, K. Raming and H. Breer Uniuersq Srut:gort-Hohenheim Institute of Zoophysrologv, Stuttgart (F: R.G.) (Received 18 September 1990) KQ words: Binding protein; Molecular cloning; Gene structure: Sequence similarity; (Silk moth) zyxwvutsrqponmlkjihgfedcb Cenomic DNA from the silk moth Anttrerueu pen?yi bearing the gene of a pheromone binding protein has been isolated from a pariid yuomic libraq using sr~ .- tfic cDN4 probes. The DNA spans 3.5 kilobases, contains three exons and two intervening sequences that interrupt the protein coding region of the gene. A DNA fragment of a second gene was isolated and the complete primary structure of a corresponding cDNA clone was unravelled. Tbe expression of two different genes, giving rise to different pheromone binding proteins, implies a more specific function of these proteins than was hitherto assumed. Introduction The ability of insects to detect and iden:ify minu!e amounts of compounds of behavioural importance is particularly well developed for moth sex pheromones (1). Pheromones are perceived by male moth through specialized sensilla arranged in dense arrays on the antennae of moth. The sensory sensilla of the silk moth A~&YWXI are hollow, cuticular structures, each about 300 pm long and 6 pm in diameter [2]; their lumen contains the sensory dendrites of 2-3 receptor neurons bathing in a proteinaceous fluid, the sensillum lymph. This receptor lymph between cuticle and sensory den- drites provides a hydrophilic barrier between airborne pheromone molecules and their hypothetical receptors in the dendritic membranes; this fluid thus represents an equivalent of the nasal mucus of vertebrate olfactory epithelia. It has been proposed that a soluble phero- mone-binding protein (PBP), which is abundantly found in the sensillum fluid, may shuttle the pheromone mole- cules across the lymph barrier. The PBPs of several moths have been identified as 15 kDa polypeptides with a pl of 4.7 [3,4]. Recently, the Abbreviation: PBP, pheromone-binding protein Correspondence: H. Breer, UniversitBt Stuttgart-Hoheaheim. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG lnstitut Wr Zc a p hysio lo g ie , G a rb e nsrr. 30, 7000 Stuttg a rt 70. F.R.G. complete amino acid sequence of PBPs from the moth species Munducu sexlo [S], An&eruea potjphemw [6]. and Antheraeo pernyi [7] have been elucidated using recombinant DNA techniques. The primary structures of these proteins show a high degree of similarity and display hydrophilic and hydrophobic domains which enable the protein to meet its specific requirements, i.e., forming binding pockets for hydrophobic pheromone molecules and to be in solution at rather high con- centrations. However. neither the chemical analysis of PBPs nor the molecular studies have unequivocally clarified if only one polypeptide exists in the sensillum lymph binding the two or three different sex pheromones found in each moth species [8]. Alternatively. specific PBPs may exist which are tuned to different ligands; the resulting microdiversity of PBPs would probably not have been detectable by the analytical approaches previ- ously employed. In this case, homologous PBPs may be encoded by multigene families, the members of which are differentially expressed to give rise to different isoforms, or one gene may express multiple isoforms of the same protein by differential splicing of the primary transcript from a single gene. To better understand if and how isoforms of PBPs are implemented, we are investigating the structure of PBP genes. Here, we report the complete exon organiza- tion of the gene encoding the pheromone binding pro- tein APR-I from A. pernyi [7]; in addition, evidence is presented for a second gene which expresses a different PBP.