Worm Breeder's Gazette 13(1): 87 (October 1, 1993)

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 RXR Homologs In Nematodes.

David M. Hough, Jennifer Richer, Claude V. Maina

Figure 1

Parasitology Group, New England Biolabs, Beverly MA 01915.

We are studying the genetic regulation of molting in the nematodes C. elegans and Dirofilaria Immitis through the use of ecdysone and its receptor, EcR (see abstract by Richer et al., this issue). EcR is a member of the nuclear hormone receptor (NHR) superfamily. It requires the presence of ecdysone and another Drosophila protein ultraspiracle, usp, in order to activate transcription. usp, also a member of the NHR superfamily (Yao et al. 1992, Cell 71:63-72), is a homolog of the vertebrate retinoid X receptor (RXR, Oro et al. 1990, Nature 347:298-301). To determine if a nematode EcR behaves as does the Drosophila EcR, we have initiated a search for nematode usp homologs.

We have used the Drosophila usp to search for D. immitis and C. elegans homologs using several approaches, the most fruitful being PCR with degenerate primers to the DNA binding domain. Two degenerate primers (C1 and C3 )encoding the amino acids shown in the figure were used in PCR experiments with genomic DNA from both D. immitis and C. elegans. All PCR products were cloned into pUC19 and sequenced.

The C1 /C3primer combination amplified 2 DNA fragments from C. elegans ( crf-6 and crf-7 ,in keeping with the nomenclature for nematode NHR s established by Sluder et al. WBG 11, #3) and 1 from D. immitis (dirf-4), ranging in size from 137 to 190 bps. The predicted amino acid sequence for each of the 3 shows significant similarity to the NHR superfamily. 1 of the 3 contains a putative intron of 50 bps at the site indicated by the arrow. The sequence of dirf-4 shows 93% similarity at the amino acid level to the Drosophila usp. While the crf-4 NHR shows less similarity to usp (68%), we are pursuing it further because it shows cross hybridization to the D. immitis dirf-4 done and most of the matches in a TFASTA search of GenBankre to RXR sequences.

Neither of the C. elegans NHR s have been described previously and they have been mapped to the genome (see figure). crf-6 maps to chromosome III and cosmid DF12 . crf-7 shows hybridization to 2 noncontiguous regions of the genome, strong hybridization to chromosome II and weak hybridization to chromosome IV. We have obtained a cDNA for 1, crf-6 ,from a screen of a mixed stage cDNA library (Stratagene). The D. immitis RXR homolog is the first to be described for a nematode and we are presently screening an adult female D. immitis cDNA library (Grandea et al 1989, Mol Biochem Parasitol 35:31-42) for a cDNA copy of this gene.

Figure 1