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.

Search for homologs of Drosophila ecdysone response genes in the parasitic nematode Dirofilaria immitis

Kirsten Crossgrove, Claude V. Maina

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