Wild type C. elegans (N2 var Bristol), phaI (e2123) mutants and transgenic worms were cultured on Nematode Growth Medium (NGM) plates seeded with E. coli OP50 and maintained as described 24. Animals were cultured at 25°C (N2 var Bristol and transgenic worms) or at 15°C (phaI mutants).

An A. viteae genomic library which was constructed in λ-dash was screened by DNA-DNA hybridization using a PCR product of 405 bp amplified from Av17 cDNA (GenBank Accession # L43053) with primers (Av17fw, 5'-GTT TTG GTG CGC TGT GAA-3'; Av17rv, 5'-CAC TGA TGA GAG TAC TTC-3') spanning the region from bp 89 to bp 493 of the cDNA. PCR amplification was performed under the following cycling conditions: 94°C for 5 min; 35 cycles of 94°C for 45 sec, annealing for 1 min at 58°C, and extension at 72°C for 2 min, with a final extension at 72°C for 10 min. The resulting product was purified by separation through a 1% agarose gel and DIG-labelled with the DIG High Prime DNA labelling Kit (Roche). Positive clones were re-screened by PCR using combinations of the λ-dash universal primers T3 and T7 and the Av17 reverse primer. Cycling conditions were: 94°C for 1 min; 30 cycles of 94°C for 45 sec, annealing for 1 min at 53°C, and extension at 72°C for 2 min, with a final extension at 72°C for 20 min. A product of approximately 2800 bp, which was amplified using T3 and the Av17 reverse primers was subcloned into pGEM-T Easy (Clontech) and sequenced.

To generate cDNA of A. viteae, worms were disrupted by grinding in liquid nitrogen and then suspended in lysis buffer (RNAeasy RNA Extraction Kit, Qiagen). Total RNA was extracted according to the manufacturers instructions. After RNA isolation, any residual DNA contamination was removed by digestion with RNAse-free DNAse (Promega). The RNA was precipitated to remove the DNAse, dissolved in RNAse-free water, and used as a template for oligo-(dT) primed reverse transcriptions, with Moloney Murine Leukemia Virus H (-) Point Mutant RT (Promega). The cDNA synthesized was used for PCR reactions with different primers.

Sequence analysis was performed using the ABI Dye Terminator chemistry. The cloned fragment of the A. viteae gDNA spanning the putative 5' upstream genomic sequence of cystatin was analyzed for the presence of promoter elements and transcription factor binding sites by using the PromoterInspector and MatInspector public software at http://www.genomatix.de (Genomatix Software, Braunschweig, Germany).

For transfection of Cos7 cells, the pGEM-T plasmid containing the isolated genomic clone was used as a template in a PCR reaction to amplify the upstream genomic region using the plasmid-specific SP6 and a KpnI linked clone-specific reverse primer (Av17rvKpn, 5' TAT CCG GTA CCA TCG TCG TTA GCT TTG TTT 3'). The product was digested with SacI and KpnI and ligated into pEGFP-N1 (Clontech). The resulting plasmid was digested with SacI and AflII. The 1710 bp fragment encoding the potential Av17 promoter, EGFP and the SV40-poly A was purified by agarose electrophoresis and ligated into pSL1180 (Pharmacia). To construct a promoter-less negative control plasmid, pEGFP-N1 was digested with EcoRI and AflII. The 1010 bp fragment comprising the reporter gene and the SV40-polyA was then ligated into pSL1180. As positive control in transfection studies pEGFP-N1 was used.

To construct a Av17 promoter plasmid for the transformation of C. elegans, the pGEM-T plasmid containing the isolated genomic clone was used as a template in a PCR reaction to amplify the putative promoter region together with the first exon of Av17 using the specific primers (prAvfw, 5'-CCC AAG CTT TAA CCC TCA CTA AAG GGA-3' and prAvrv, 5'-AAC TGC AGA TTG CGT TCC TGC CAT CC-3'). The product was cloned into the vector pPD 95.77 (Fire lab kit, 1995), in-frame with the reporter gene GFP to obtain pPrAvGFP.

The Av17 cDNA and the pGEM-T clone containing the genomic sequence of Av17 were used as templates in PCR reactions with primers that incorporated a NheI restriction site at the 5' end of the sequence (Av17fwNhe, 5'-TAT TCA GGT AGC ATG ATG TTG TCA ATA AAG-3') in conjunction with a primer that incorporated a C-terminal 6X His tag and a KpnI restriction site at the 3' end (Av17rvHisKpn, 5'-TAT TCA CGG TAC CTC AAT GGT GAT GGT GAT GGT GAT GCA CTG ATG AGA GTA C-3'). The PCR products were cloned into the vector pPD49.83, which contains the C. elegans hsp16/41 promoter and a synthetic intron, to obtain p49Av17c (Av17 cDNA) and p49Av17g (Av17 gDNA). Protein expressed under the control of this promoter on heat shock is targeted to the gut cells of transgenic worms 25.

The cDNA sequence of Av17 was also amplified using the specific primers, 5'-TAT TCA GCT AGC ATG CAC CAT CAC CAT CAC CAT ATG ATG TTG TCA ATA AAG-3' (Av17fwHisNhe) incorporating NheI sequence and an N-terminal 6XHIS tag and 5'-TCC CCC CGG GTC ACA ATG TAC TTT A-3' (Av17rvUTRSma) incorporating the 3'UTR of the Av17 gene and SmaI restriction site. The product was cloned into the vector pPD103.05 to obtain p103Av173'. This plasmid was chosen since the let-858 promoter ubiquitously expresses in all C. elegans tissues 26.

Cos7 cells were maintained in RPMI 1640 (Biochrom) supplemented with 10% FCS (Biochrom) under standard tissue culture conditions and were transfected using lipofectamin reagent (Invitrogen). Cells were plated out (10⁵ per well) in 12-well plates 24 h prior to transfection. Two μg of the desired plasmid and 8 μl lipofectamin were resuspended in 100 μl serum-free medium separately. After 30 min incubation at room temperature (RT) the solutions were mixed and incubated for another 30 min at RT. To this, 800 μl serum-free medium was added and applied to the cells. After 6 h of incubation under standard tissue culture conditions the transfection reagent was removed and the cells were maintained in complete RPMI 1640 before microscopic examination after 48 h.

For promoter studies, young adult hermaphrodite wild-type (WT) worms were immobilized on agarose pads overlayed with mineral oil and the construct pPRAvGFP along with the marker plasmid pRF4 (a kind gift from James M. Kramer, Northwestern University Medical School) was microinjected into the gonads at a concentration of 100 ng/μl. The injection was done at a pressure of 460 psi using femtotips (Eppendorf, Germany). The transformation buffer contained 2% w/v PEG 6000, 20 mM KH₂PO₄ and 3 mM potassium citrate. The marker plasmid confers transgenic worms with a roller phenotype. Only worms of the second and subsequent generations, which showed both the roller phenotype and GFP expression were used for further analysis of the Av17 promoter. As a negative control, WT worms were microinjected with pRF4 and promoter-less pPD95.77.

For the expression of Av17, C. elegans, pha-1mutants (e-2123) were transformed by microinjecting the constructs p49Av17c, p103Av173' and p49Av17g into the gonads together with the marker plasmid pBX at a concentration of 100 ng/μl each. The transformed worms were selected by their ability to survive and reproduce at 25°C. The selected worms 103cAv173', 49cAv17 and 49gAv17 were maintained as discrete lines at 25°C. Integration of the extrachromosomal arrays was achieved by gamma irradiation of the transgenic worms with 35 Gy. The progeny of these worms was then screened for 100% transmittance to obtain lines with chromosomally integrated transgens. To verify if the transformed C. elegans contained the Av17 construct, single worm PCR was done on the F2 generation using the same primers as for the RT-PCR. Single worms were picked from NGM plates and suspended in 2.5 μl of lysis buffer (50 mM KCl, 10 mM Tris-HCl pH 8, 2.5 mM MgCl₂, 0.45% w/v Nonidet P 40. 0.45% v/v Tween 20, 0.1% gelatin and 200 μg/ml Proteinase K) and frozen on dry ice for 20 min. The samples were then incubated at 60°C for 1 h for protein digestion and at 95°C for 15 min to inactivate Proteinase K. The resulting DNA was mixed with PCR master mix, cycling conditions were 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min and a final extension of 72°C for 10 min. The products were resolved on 1% agarose gel.

Light and fluorescence microscopy observations for EGFP detection in Cos7 cells were made using a Zeiss Axioplan fluorescent microscope (Oberkochen, Germany) with a blue (number 487909) excitation filter set. The transgenic C. elegans worms with promoter-reporter constructs were immobilized on agarose pads and observed using a Leica Confocal Microscope (TCS SP2) (Heidelberg, Germany).

Transcription of the injected construct was analyzed by RT-PCR using total RNA prepared from 0.5 mg wet weight of transformed worms (103Av173', 49cAv17 and 49gAv17) as described above. Transgenic lines 49cAv17 and 49gAv17 were given a heat shock at 33°C for 3 h before total RNA isolation. The synthesized cDNA was used as template in a PCR reaction using Av17 specific primers (Av17fwNhe and Av17rv, see above). Amplification from non-transgenic worms was included as control. PCR was performed with the following thermal cycling conditions: 94°C, 1 min, 30 cycles of 94°C for 1 min, annealing 1 min 57°C, and extension at 72°C for 2 min, final extension 72°C for 10 min. The products were separated on 1% agarose gel.

Transgenic worms were grown in large numbers in NGM liquid cultures at 25°C with E. coli OP50 as a source of food. Worm line with the Av17 gene downstream of the inducible hsp promoter (49cAv17) was given a heat shock at 33°C for 3 h. The main contaminants from the nematode culture were cleaned by centrifugation through 60% sucrose in 0.1M NaCl 27. The worms were frozen in liquid nitrogen and homogenized with a mortar and pestle in lysis buffer (20 mM NaH₂PO₄, 300 mM NaCl, pH 7.4). The lysate was separated on a 14% SDS-polyacrylamide gel. Western Blot analysis was performed as described 28. The blot was incubated with a 1:5000 dilution of monoclonal anti-His mouse antibody (Qiagen, Germany) and alkaline phosphatase coupled goat anti-mouse antibody diluted 1:5000 was used as conjugate.

Furthermore, the progeny of the transgenic worms expressing Av17 constitutively (103cAv173') was quantified and compared to the uninduced transgenic worms with Av17 downstream of hsp promoter (49cAv17). Young adult hermaphrodite (six of each) worms were transferred to NGM plates and the number of eggs and of larvae at every molt were quantified.

After screening of ~10,000 plaques of an A. viteae λ-dash genomic library, a positive clone (gAv17) was obtained. The insert of gAv17 was ~2.8 kb in length, and when sequenced it was found to include the entire Av17 gene. Comparison to the published cDNA sequence of the A. viteae Av17 revealed that the genomic sequence is comprised of 702 bp of the putative promoter region, 4 exons of 166 bp, 158 bp, 51 bp and 97 bp, interspersed by 3 introns of sizes 222 bp, 158 bp and 1020 bp, followed by 191 bp of 3' untranslated sequence (Fig. 1).

Several putative regulatory sequences could be identified by computer analysis in the 702 bp upstream genomic sequence (Fig. 1). A putative TATA-box was located at position -296. Consensus recognition sites for the transcription factors NF-Y and AP-1 were identified at positions -37, -353 and -583, respectively. An inverted CCAAT box was located at position -603.

The putative promoter region of 702 bp was capable of promoting transient expression of the reporter EGFP in mammalian cells. EGFP fluorescence was detected in 5 % of Cos7 cells transfected with the promoter-EGFP construct (data not shown) with the same intensity as the positive control. Cos7 cells transfected with a promoter-less control plasmid did not show any EGFP fluorescence.

C. elegans was used as an heterologous system to analyze the ability of the upstream genomic sequence of Av17 to promote the expression of the reporter gene, GFP. The promoter construct was microinjected into the gonads of young wild type C. elegans together with the marker plasmid. Transgenic animals from three transformed lines were examined. All the lines showed GFP expression in all stages of the worms, L1, L2, L3 and adults, but not in the eggs. Strongest expression was observed in the gland cells of the pharynx, which consist of 2 cells (g1 and g2; Fig. 2A,B). Expression of GFP was also observed in the rectal glands (Fig. 2C,D), which consists of three large cells (recD, recVL, rectVR) and are connected to the intestinal lumen just posterior to the rectal valve.

The temperature sensitive pha I mutant of C. elegans was used for microinjection experiments to facilitate selection of the transgenic worms expressing Av17. The transcription factor pha-1 is required for the morphogenesis of the pharynx 2930. In the pha-1 mutant worms the pharynx fails to undergo differentiation and the mutation interferes with the embryonic development at 25°C making it easier for the selection of transgenic worms, that are co-transfected with the rescue plasmid. Transgenic worms lines, 103cAv173' (injected with p103Av17), 49cAv17 (injected with p49Av17c) and 49gAv17 (injected with p49Av17g) were established and maintained as discrete lines. In order to integrate the plasmid constructs into the genome, worms were exposed to γ-irradiation and subjected to another round of screening. The F2 and subsequent generations of transgenic lines were used for further analysis. Single worm PCRs to test for the presence of injected constructs showed that the worms (49cAv17, 103cAv173' and 49gAv17) were transgenic. No product was amplified from the pha-1 control worms (Fig. 3A).

The transcription of Av17 in C. elegans was shown by RT-PCR. A 475 bp product consistent with the predicted size of the coding region was obtained from the clones containing the cDNA sequence of Av17 (Fig. 3B, lane 2). In worms with the genomic sequence (49gAv17), transcripts of a larger size were amplified, which on sequencing were found to include a part of the first intron (Fig. 3B, lane 3). Further analysis revealed that the conserved intron splice donor site AG/gt was apparently read through and the second AG/gt downstream in the first intron was recognised as the splice donor site. The remaining introns were spliced out correctly to obtain a transcript of 626 bp (Fig. 4, ii) including 152 bp of the first intron, instead of a transcript size of 475 bp, if splicing had occurred correctly. No amplification product was obtained from the control pha-1 worms.

In a western blot, anti-His antibodies specifically recognized a band of 17 kDa in the transgenic C. elegans lines 103cAv173' and 49cAv17 expressing Av17 constitutively and under heat shock (Fig. 3C). No expression was seen in pha-1 control worms and 49gAv17.

In worms expressing Av17 constitutively (103cAv173') of 20 ± 15.9 eggs only 5.2 ± 3.8 developed to adults. The reduction in the ability of the eggs to develop to adults is significant (p < = 0.0129) when compared to worms transformed with Av17 downstream of hsp promoter but uninduced, where of 24 ± 16.4 eggs, 19 ± 10.2 developed to adults (data not shown).