Worm Breeder's Gazette 14(4): 38 (October 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Molecular Parasitology Group, New England Biolabs, Beverly, MA 01915
Filariasis afflicts over 300 million people worldwide causing severe and debilitating disease with symptoms such as lymphoedema, elephantiasis and blindness. Filariasis is caused by several species of parasitic nematodes including Brugia malayi, Brugia timori, Wuchereria bancrofti, and Onchocerca volvulus, transmitted to man by mosquitoes or black flies. Knowledge of the molecular mechanisms involved in parasite development may suggest strategies for parasite control. A useful model system for the study of these parasites is the closely related parasite, Dirofilaria immitis, the causative agent of dog heartworm disease. D. immitis undergoes four molts from the microfilarial to the adult stage. Many insects undergo a superficially similar series of molts during their development. The steroid hormone 20-hydroxyecdysone (hereafter referred to as ecdysone) has a well-characterized developmental role in insects, where it is necessary for molting and metamorphosis. The molecular basis of this response during metamorphosis has been extensively studied and many of the relevant genes have been cloned (reviewed in Andres and Thummel. 1992, TIG 8:132-138). Briefly, ecdysone exerts its effect by binding to the functional ecdysone receptor, which is composed of a heterodimer between the Ecdysone Receptor and Ultraspiracle gene products. This complex activates a small set of early genes, many of which also encode transcription factors. These transcription factors negatively autoregulate their own expression as well as activating a large set of late genes which are thought to encode the structural proteins responsible for metamorphosis. Ecdysone has also been shown to have an effect on the development of D. immitis. Specifically, molting from the L3 larva, the infective form, to the L4 larva can be prematurely induced in culture by ecdysone (Warbrick et al. 1993, Parasitology 107: 459-463). Homologs of both the ecdysone receptor and ultraspiracle genes have been identified in D. immitis (Richer et al. 1993, WBG 13: 86; Hough et al. 1993, WBG 13:87). In order to determine how much of the gene hierarchy is conserved between Drosophila and D. immitis, we initiated a search for homologs of known Drosophila early genes. A number of these genes are members of the nuclear hormone receptor (NHR) superfamily. NHRs are thought to play a role in the development of a wide variety of multicellular organisms. Over 40 putative NHRs have been identified in C. elegans so far. We used degenerate primers in the highly conserved DNA binding domain to perform PCR on D. immitis genomic DNA. Primers derived from the E75 and E78 ecdysone primary response genes were successful in identifying putative homologs in D. immitis. The E75 homolog, dinhr6, exhibits 83% amino acid identity to Drosophila E75 in the DNA binding domain (Figure 1A). The E78 homolog, dinhr7, exhibits 83% amino acid identity to Drosophila E78 in the DNA binding domain (Figure 1B). In addition, a C. elegans E78 homolog, CNR14, has been identified (Kostrouch et al. 1995, PNAS 92: 156-159) which exhibits 79% identity to E78 and 71% identity to dinhr7 in the DNA binding domain. It is interesting to note that CNR14 and dinhr7 are more closely related to Drosophila E78 than to each other. The presence of these genes in D. immitis and in C. elegans supports the ever expanding role of both nuclear hormone receptors and steroid-regulated gene cascades in development. We are currently cloning full-length cDNAs for dinhr6 and dinhr7, determining their expression patterns and asking whether they are responsive to ecdysone in D. immitis. Figure 1: Alignment of dinhr6 and dinhr7 DNA binding domains to Drosophila E75 and E78. A. E75 CGDKASGFHYGVHSCEGCKGFFRRSIQQKIQYRPCTKNQQCSILRINRNRCQYCRLKKCIAVGM ||| |||||||| ||||||||||||||||||||||| ||| | |||||| ||| ||| ||| dinhr6 CGDRASGFHYGVFACEGCKGFFRRSIQQKIQYRPCTKSQQCIVARNNRNRCQHCRLQKCIRVGM B. E78 CKVCGDKASGYHYGVTSCEGCKGFFRRSIQKQIEYRCLRDGKCLVIRLNRNRCQYCRFKKCLSAGM ||||||| || ||||| ||||||||||||||| |||||||||| |||||||| ||| ||| || dinhr7 CKVCGDKSSGFHYGVTACEGCKGFFRRSIQKQMEYRCLRDGKCHIHRLNRNRCQFCRFRKCLAVGM