The C. elegans EMAP-like protein, ELP-1 is required for touch sensation and associates with microtubules and adhesion complexes

Background The founding member of the EMAP-like protein family is the Echinoderm Microtubule-Associated Protein (EMAP), so-named for its abundance in sea urchin, starfish, and sand dollar eggs. The EMAP-like protein family has five members in mammals (EML1 through EML5) and only one in both Drosophila (ELP-1) and C. elegans (ELP-1). Biochemical studies of sea urchin EMAP and vertebrate EMLs implicate these proteins in the regulation of microtubule stability. So far, however, the physiological function of this protein family remains unknown. Results We examined the expression pattern of C. elegans ELP-1 by means of transgenic gene expression in living embryos and adults, and by immunolocalization with an ELP-1-specific antibody in fixed tissues. In embryos, ELP-1 is expressed in the hypodermis. In larvae and adults, ELP-1 is expressed in the body wall, spermatheca and vulval muscles, intestine, and hypodermal seam cells. In muscle, ELP-1 is associated with adhesion complexes near the cell surface and is bound to a criss-crossing network of microtubules in the cytoplasm. ELP-1 is also expressed in a subset of mechanoreceptor neurons, including the ray neurons in the male tail, microtubule-rich touch receptor neurons, and the six ciliated IL1 neurons. This restricted localization in the nervous system implies that ELP-1 plays a role in mechanotransmission. Consistent with this idea, decreasing ELP-1 expression decreases sensitivity to gentle touch applied to the body wall. Conclusion These data imply that ELP-1 may play an important role during the transmission of forces and signals between the body surface and both muscle cells and touch receptor neurons.


Background
Microtubule networks are necessary for a variety of essential processes including cell polarity, migration, division and mechanotransduction [1][2][3]. Microtubule formation and function is regulated by a variety of proteins that mediate the structural and regulatory interactions between these microtubules and their cargo [4,5], and catalyze their assembly and disassembly [6]. In this report we examine the tissue-specific expression and the subcellular location of ELP-1, an EMAP-like protein from C. elegans and demonstrate that ELP-1 contributes to touch-sensitivity in a subset of mechanoreceptor neurons.
The founding member of the EMAP-like protein family is the 75 kDa Echinoderm Microtubule-Associated Protein (EMAP), so-named for its abundance in sea urchin, starfish, and sand dollar eggs [7]. Genes that code for EMAPlike proteins are conserved in the genomes of nematodes, chordates, insects, echinoderms, and platyhelminthes and several EMAP-like proteins have been shown to bind to microtubules in vitro and in vivo [8][9][10][11][12][13].
The in vivo function of EMAP and EMAP-like proteins is unknown. However there are indications that loss or alteration of EMAP function may lead to human disease. Human EML2 RNA is abundant in cancer cell lines including chronic myelogenous leukemia (K-562), lymphoblastic leukemia (MOLT-4), colorectal adenocarcinoma (SW480), and lung carcinoma (A549) [11]. Furthermore, in certain patients with T-cell acute lymphoblastic leukemia (TALL), the gene encoding the nonreceptor protein kinase (c-ABL1) is fused to the EML1 gene on chromosome 14, which causes expression of an EML1-ABL1 fusion protein, that functions as a dysregulated tyrosine kinase [14]. The EML1-ABL1 fusion protein constitutively activates the ERK, Stat5, and Src signalling pathways. The N-terminal coiled-coil domain of EML1 is required for kinase activation, which suggests that oligomerization of EML1 is required for the function of EML1-ABL1 fusion proteins.
Kinase oncogenes are not restricted to fusions with EML1. EML4 fusions with the anaplastic lymphoma kinase (ALK) occur in a subset of non-small cell lung cancers and adenocarcinomas of the lungs [15,16]. The amino portion of EML4 (residues 1-496) is fused to residues 1058-1620 of the intracellular domain of the ALK tyrosine kinase. The basic amino terminal domain of EML4 is critical to the catalytic activity of the ALK fusion [16]. These results imply that EML-kinase fusions and rearrangements may underlie other acquired solid tumours and blood related cancers.
The conservation of the EMAP-like protein family amongst metazoans and the direct correlation between EML translocations and cancer indicates that this novel protein family may perform an important function in cells and tissues. To begin to understand the function of EMAP and EMAP-like proteins we undertook a molecular and cytological analysis of the elp-1 gene encoded by the ORF F38A6.2 in Caenorhabditis elegans. We determined whether ELP-1 bound to microtubules in vitro and in vivo. Furthermore, we took advantage of the transparency of the worm to examine the expression pattern of an ELP-1::GFP fusion protein in embryos and in adults. Our results indicate that ELP-1 is expressed in cells that make productive interactions with the extracellular matrix, including but not limited to the hypodermis, body wall muscles, male-specific sex muscles, and the microtubulerich touch receptor neurons. In addition, our behavioural studies show that wild-type levels of ELP-1 are needed for touch sensation in the worm.

elp-1 encodes the sole C. elegans member of the EMAPlike protein family
All of the EMAP-like proteins identified to date, including vertebrate EMLs and invertebrate ELPs, share a common domain organization with a short, 60-70 amino acid, hydrophobic EMAP-like protein (HELP) motif preceded by a series of WD repeat domains ( Figure 1). Although the function of the HELP motif is unknown, it is unique to this gene superfamily. There is evidence in the human genome for 6-8 EML genes, however at this time only EMLs 1-5 have a confirmed gene product. The apparent or predicted molecular mass of most EMAP-like proteins ranges from ~70 to ~120 kDa ( Figure 1). Human EML5 is the exception with a predicted Mr of 220 kDa and three repeats of the HELP and WD domains.
The C. elegans genome contains a single EMAP-like gene, elp-1, that maps to the right of unc-51 on the extreme right arm of linkage group (LG) V ( Figure 2). Genefinder™ analysis predicts that the elp-1 gene consists of 16 exons that encode an 891-amino acid polypeptide of 98 kDa. We used reverse transcription coupled to the polymerase chain reaction (RT-PCR) to show that there are at least two alternatively spliced variants: a full-length cDNA transcript composed of exons 1 through 16 (elp-1a); and an alternatively spliced transcript composed of 15 exons (elp-1b). The elp-1b transcript lacks exon 5, an 81 bp segment that encodes for a 27-amino acid sequence that is more than 46% serine and threonine residues ( Figure 2C). The elp-1b transcript is identical to the cDNA EST yk209e10 that is predicted to be translated into an 864-amino acid polypeptide of ~95 kDa [17].
Exon 5 is conserved in nematodes but not detected in other EMAP-like proteins. Twenty-four of the twentyseven predicted amino acids (88%) are identical in Caenorhabditis briggsae and C. elegans. All three substitutions are conservative (V15I, I16V, and S19N; numbers refer to the 27 amino acids coded by this exon). This domain shows a slightly higher level of sequence conservation when compared to the HELP domain protein sequence (81% identical) and the full-length protein sequence (80% identical) of C. elegans and C. briggsae. Although the function of this 27 amino acid region, rich in potential phosphorylation sites, is unknown, these observations indicate that exon 5 may be important for a nematode-specific function.
To learn more about the function of elp-1, we obtained a deletion allele, ok347, from the C. elegans Knockout Consortium. The ok347 allele deletes 1301 nucleotides, spanning intron 1 through intron 4 (Figure 2A Figure 2E). These transcripts are predicted to encode two proteins that include both the HELP and WD repeat domains, a finding which implies that these proteins could retain partial function.

Expression patterns of the elp-1 gene in embryos and adults
To learn how ELP-1 contributes to development, physiology and behaviour, we examined the expression pattern of the elp-1 gene. Three GFP reporter constructs were generated: an ELP-1 promoter construct [pKA99-3], a truncated ELP-1 with an NLS [pKA99-2]; and a full-length ELP-1 [pKA99-1] ( Figure 2B). All three constructs were expressed in the same cells and tissues. For clarity, the protein expressed from the pKA99-1 construct will be noted as ELP-1Δ::NLS::GFP and the full-length protein expressed from the pKA99-2 construct will be written as ELP-1::GFP.
In embryos, ELP-1::GFP expression first appeared during the comma stage. As the embryo matured into the 1-1/2 fold stage, the strongest expression was seen in the hypodermal cells, with only diffuse staining throughout the rest of the embryo ( Figure 3). During larval development, expression of the fusion protein was progressively refined to muscle, neurons and epithelial cells. In the adult, ELP-1::GFP and ELP-1Δ::NLS::GFP were expressed in body wall muscle, spermatheca, vulval muscle, seam cells, the intestine, touch receptor neurons (TRNs) and inner labial 1 (IL1) neurons of the head. Expression of ELP-1Δ::NLS::GFP is shown in Figure 4A-D. The truncated ELP-1 construct with the NLS was used in order to identify the neuronal cell bodies.

ELP-1 is expressed in a subset of mechanoreceptor neurons
As indicated above ELP-1::GFP and ELP-1Δ::NLS::GFP were expressed in cells that were identified as mechanoreceptor neurons ( Figures 4E &4F). Although the NLS did not restrict expression to the nucleus, it was generally easier to identify neurons with this construct than with the full-length ELP-1::GFP construct. Specifically, ELP-1Δ::NLS::GFP was found in the six touch receptor neurons (ALML/R, AVM, PVM, and PLML/R) responsible for detecting light touch applied to the body surface ( Figure  4E, F) [18]. These cells were identified by the position of their cell bodies and the long nerve cell processes that extend anteriorly or posteriorly over half of the body length. Touch receptor neurons (TRNs) are not ciliated or organized into sensilla, but extend neurites along the midline and lateral line of the worm with their dendritic receptors lying within 150 nm of the inner border of the cuticle [19].
ELP-1::GFP was also expressed in the dendrites and the ciliated endings of six neurons in the head ( Figure 4G, J-L).
Candidates for these six neurons included the six inner labial 1 neurons (IL1s), six inner labial 2 neurons (IL2s), and six outer labial neurons (2OLLs and 4 OLQs) [20]. To determine the identity of these neurons, the worms were labeled with the lipophilic tracer molecule DiD which gains access to the sensory cilia that project through an opening in the cuticle. In the presence of calcium acetate, DiD is exclusively taken up into the six IL2 neurons, although occasional dye-filling of the amphid neurons can be observed (personal communication, Elizabeth Ryder, Worcester Polytechnic Institute). The ciliated nerve endings of IL2 and IL1 both extend through the inner labial sensillum. However, only the IL2 nerve endings extend through a hole in the cuticle and are able to take up the dye. Dye-filling in the presence of calcium acetate allowed visualization of the IL2 neurons and thus allowed for a comparison to be made between the position of the IL2 neurons relative to the ELP-1::GFP expressing neurons ( Figure 4J-L). DiD staining in the presence of calcium acetate showed that the IL2 neuronal cell bodies were located anterior to the ELP-1::GFP expressing neurons. This finding suggests that these head neurons are the mechanosensory IL1 neurons. We confirmed this identification by crossing ELP-1::GFP worms with deg-1(u38) mutations. The toxic DEG-1 protein caused the degeneration of the neurons which normally express the deg-1 gene: IL1, PVC, AVG and AVD [21]. In the deg-1(u38) background, we found fewer than three GFP-labelled neurons in the head (Table 1). These results confirm that the IL1 mechanosensory neurons express ELP-1::GFP.
In addition to the mechanoreceptor neurons described above, ELP-1::GFP was associated with the nine pairs of rays of the male tale ( Figure 4H). The male tale is a sensory apparatus used to find the hermaphrodite vulva during mating. The relatively diffuse and broad band of fluorescence appears to originate from the hypodermal cells (hyp7) and one or more of the neuronal cells of the ray (RnA, RnB).

ELP-1 is associated with adhesion sites
At the subcellular level, ELP-1::GFP was associated with adhesion sites in both males and hermaphrodites. In the hermaphrodite body wall muscle, ELP-1::GFP was associated with the muscle arms that terminate in neuromuscular junctions ( Figure 5). The muscle arms are long cellular processes that extend to make contact with motor axons. The muscle cell shown in Figure 5A shows four muscle arms, which is consistent with the three to five muscle arms classically observed per body wall muscle cell [22,23]. The ELP-1::GFP fluorescence is found throughout the muscle arm. Figure 5B shows a male with ELP-1::GFP associated with repeating focal attachment points in the sex-specific muscles of the worm. These muscles are located in the posterior end of the worm and function in various phases of male mating behaviour [24]. ELP-1 expression was prominent at the adhesion sites located at the sarcolemma. Unlike the dense body adhesion sites described below, these sites occur at the muscle ends in a plane perpendicular to the long axis of the sarcomere.
Hermaphrodites were examined by fluorescent microscopy with the optical axis perpendicular to the longitudinal axis of a body wall muscle cell. Affinity-purified antibodies against ELP-1 and the full-length ELP-1::GFP fusion protein localized to thin linear rows near the cell surface that were not quite parallel to the long axis of the muscle cell ( Figure 6A, B). At higher resolution there was a periodicity to these lines that approximated the distance between the dense body adhesion sites ( Figure 6C, D).
Body wall muscle cells are anchored along their length to the basement membrane by the dense bodies, finger-like projections analogous to the vertebrate Z-lines. Anchorage of actin in the myofilament lattice to the dense bodies is necessary for force transduction in body wall muscle. Dense body puncta are distinctive because they are aligned in a row that runs at a 6-degree pitch from the longitudinal axis of the muscle cell [25].
In Figure 6E, individual dense bodies are shown as phasedark puncta. The fluorescent image of ELP-1::GFP was examined at the same focal plane ( Figure 6F) and these micrographs were overlaid and shown in Figure 6G. The ELP-1::GFP fluorescence slightly overlaps the edges of the dense bodies but is not superimposable with the dense bodies at this angle. These results indicate that ELP-1 is unlikely to be a component of the dense bodies.
To further examine the association of ELP-1 with dense bodies, we double-stained muscle cells with anti-ELP-1 antibodies and a monoclonal antibody MH25 against β integrin (PAT-3), an integral membrane component that anchors the dense bodies to the sarcolemma. Figure 6 H-J shows that integrin and ELP-1 have overlapping staining patterns at this level of resolution. Because of the relatively impenetrable cuticle that surrounds the nematode, these worms were frozen and cracked open prior to antibody staining. Occasionally a portion of the muscle cell membrane was removed carrying along its complement of dense bodies (see white outline in Figure 6J). In areas lacking dense bodies, ELP-1::GFP was sometimes lost. However most of the ELP-1::GFP was retained in a linear pattern. These results indicate that the membrane and dense bodies can be separated from the ELP-1::GFP.

ELP-1 is associated with microtubules
Although the truncated construct and the full-length construct were expressed in the same cells and tissues, only the full length ELP-1::GFP construct localized to an elaborate array of fluorescent filaments. These filaments, which resembled microtubules, were obvious in the larger cells of the worm including the body wall muscle (Figure 7) ELP-1 is expressed in diverse muscle cellular junctions and intestine (Figure 8). An obliquely striated fluorescent pattern emerges approximately 0.8 μm apically from the base of the dense body. Deeper within the muscle body the filaments appear to separate from the linear track and branch out into the cytoplasm.
To test the idea that the filaments decorated by ELP-1::GFP were microtubules, we treated worms with the microtubule inhibitor, nocodazole. Because the worm cuticle is an effective barrier to nocodazole, we gently pressed worms between a slide and coverslip to extrude their intestines. This process was done in the presence of levamisole, an acetylcholine agonist, which was used to paralyze the worms for microscopy. In Figure 8, the worm was flattened and the intestines were gently extruded. The filaments in the extruded layer were stable for 30 minutes or more in the absence of nocodazole. The decorated filaments began to degrade within seconds and were entirely de-polymerized after two minutes in nocodazole. When the fluorescence disappeared the intestinal fragments noticeably flattened and the remnants of the worm con-tracted. This result indicates that ELP-1::GFP associates with microtubules in situ.
Finally, we show that ELP-1 was enriched in preparations of paclitaxel-stabilized microtubules in vitro. Paclitaxelstabilized microtubules were prepared from a mixed-stage worm preparation and examined by means of Western blotting with an affinity-purified antisera against a bacterially expressed ELP-1 fusion protein (Figure 9). An ELP-1reacting band co-purified with microtubules and migrated at ~100 kDa, a mass that corresponded to the Gene-finder™ predictions for the larger ELP-1a polypeptide. Two smaller and less abundant proteins that cross-react with the ELP-1 antibodies also co-purified with microtubules. These may be isoforms generated by alternative splicing (i.e. ELP-1b) or proteolytic fragments of ELP-1a. This experiment and the one described above showed that ELP-1 is microtubule-associated both in situ and in vivo.
Whether ELP-1 binds directly to tubulin remains to be determined.

ELP-1 is needed for normal touch-sensitivity
Based on its expression in the TRNs and its intimate association with microtubules, we hypothesized that ELP-1 plays a role in gentle-touch sensation. We tested this idea by measuring touch-sensitivity in mutants carrying defects in the elp-1 gene and in animals treated with RNAi directed against ELP-1. We found that the ok347 allele significantly increases the proportion of touch-insensitive animals ( Figure 10). This is unlikely to be the null phenotype, however, since ok347 mutants retain two transcripts predicted to encode nearly full-length ELP-1 proteins containing both the HELP and WD repeat domains ( Figure  2E). Thus, ok347 is predicted to be a partial loss-of-function allele. Consistent with this idea, the proportion of touch-insensitive animals was dramatically increased when the elp-1(ok347) allele was placed in trans to a deficiency that covers the elp-1 gene (ozDf1) and when ELP-1 expression was decreased by feeding RNAi-expressing bacteria ( Figure 10). These results demonstrate that wild-type ELP-1 is needed for normal touch sensitivity in C. elegans and indicate that EMAP-like proteins could play a critical role in sensory mechanotransmission.
Most members of the superfamily associate with microtubules [8,9,[11][12][13] and several are implicated in regulating microtubule stability during mitosis [8,9,11,27]. Their expression is not limited to dividing cells, however. The function of EMAP-like proteins in post-mitotic cells such as neurons is poorly understood. To learn more, we combined molecular and behavioural genetics with cell biological approaches to analyze ELP-1, the sole EMAP-like protein in the C. elegans genome. Because all 959 somatic cells in each adult hermaphrodite are post-mitotic, this analysis offers new insight into the cell biology and physiology of ELP-1 in post-mitotic cells and tissues.
We show here that ELP-1 is prominently expressed in cells that make productive interactions with the cuticle. In males and hermaphrodites, these include mechanoreceptor neurons such as the TRNs and diverse muscle cell types. In males, ELP-1 is found in the hypodermal cells and neurons of the male sensory rays. Additionally, ELP-1 is found in the cell bodies, throughout the neuronal processes, and in the ciliated endings of all six IL1 neurons. The IL1 neurons are putative mechanoreceptor neurons needed for wild-type foraging movements and for sensing touch applied to the nose [32].
ELP-1 is a promising candidate for regulating microtubule function in mechanoreceptor cells. For example, the TRNs are unique in that their sensory processes contain 15-protofilament microtubules rather than 11-protofilament microtubules observed in the majority of C. elegans cells [33]. Approximately 450 of these large-diameter microtubules are bundled together and constrained to move as a single structure by distinct 10 nm filaments [19,32,34]. The bundles are also linked to the plasma membrane through a series of 14 nm filaments [19]. ELP-1 is an attractive candidate to bundle microtubules. In addition, the HELP motif, rich in hydrophobic amino acids, could link the bundles to the plasma membrane. In either scenario, ELP-1 would contribute to TRN function by amplifying the forces generated at the cuticle. Consistent with this idea, reducing ELP-1 levels decreased touch-sensitivity.
In addition to mechanoreceptor neurons, ELP-1 is expressed in cells that utilize cadherin-based apical junctions and or fibrous organelles for cell adhesion and attachment to the basal lamina. These include the cells of the intestine, body wall, vulval, and male-specific sex Analysis of subcellular filaments in a single body wall muscle cell of a hermaphrodite Figure 7 Analysis of subcellular filaments in a single body wall muscle cell of a hermaphrodite. These three panels (A-C) were part of a Z-series of images that were taken every 0.2 μm from the muscle cell membrane towards the interior of the muscle cell. The first image in Panel A was taken approximately 0.8 μm from the cell surface (4 sections down from the surface). At this focal plane, ELP-1::GFP is associated with oblique linear striations overlap with the anti-β integrin (MH25) staining pattern shown in Figure 6. In successive focal planes (B and C) the filaments are no longer in linear arrays and have branched off into a criss-crossing array. Bar represents 10 μm.
muscles, hypodermis and seam cells [35]. The hemidesomosome-like fibrous organelles, formed by the hypodermis, transmit cuticle deformation to the touch receptor neurons and muscle tension to the cuticle [36,37]. Microtubules are interspersed with actin filaments near these fibrous organelles, however it is not known how they might be anchored to the plasma membrane [35]. We speculate that ELP-1 is involved in the anchoring or bundling of microtubules to the plasma membrane and perhaps the transmission of forces therein.
ELP-1 may also be involved in force transmission and synapse formation in body wall muscle. C. elegans has four longitudinal muscle quadrants that are anchored to the cuticle through the hypodermis in order to generate loco-motion [38]. Specifically, the myofibrils are anchored to the extracellular matrix by dense bodies, structures similar to integrin-based, vertebrate focal adhesions [39]. In addition, muscle arms project towards motor axons in the nerve cord to form the postsynaptic element of the neuromuscular junction [32,40]. ELP-1 is found in the muscle arms and at the dense bodies of body wall muscle cells. In muscle cells ELP-1 may function to link the microtubules to the adhesion sites, regulate the assembly and disassembly of microtubules, or even deliver or remove a regulatory molecule to adhesion sites ( Figure 11).
It is intriguing that ELP-1 is associated with adhesion sites similar to mammalian focal adhesions. It has been known for some time that microtubules can locate or target focal ELP-1::GFP is associated with microtubules adhesions [41] and that depolymerization of microtubules enhance actomyosin-based cell contractility [42]. The increase in cell contractility is mediated by GEF-H1 [43,44], a microtubule-associated guanine nucleotide exchange factor that activates the small G-protein, RhoA [45]. Upon microtubule depolymerization, GEF H1 is released and activates the Rho-associated kinase (ROCK) that phosphorylates the myosin regulatory light chain (MLC) resulting in increased contractility [46,47]. Whether a similar pathway is functional in C. elegans muscle remains to be determined.

Conclusion
In C. elegans, ELP-1 is the sole member of a highly conserved family of metazoan microtubule-binding proteins.
In adults, ELP-1 is expressed in post-mitotic cells that make productive interactions with the cuticle such as body wall muscle and mechanoreceptor neurons, suggesting a role in force generation and sensing. In support of this idea, disrupting ELP-1 expression by mutation and RNAi renders animals insensitive to gentle body touch.
Three-dimensional sketch illustrating the relationship of ELP-1-associated microtubules and the dense bodies (green) Figure 11 Three-dimensional sketch illustrating the relationship of ELP-1-associated microtubules and the dense bodies (green). A transverse section through a single body wall muscle cell is shown. The thin filaments, which are attached to the dense bodies, and the thick filaments attached to the M-lines, have been eliminated from the sketch for clarity. ELP-1 associated microtubules (magenta) are associated with the apical portion of the dense body. Neighbouring dense bodies are linked by microtubules giving rise to the linear staining patterns in Figure 6. The microtubules extend into the cytosol that contains the nucleus, mitochondria, and other membranes (not shown).
This study is a critical first step toward elucidating the function of EMAP-like proteins in post-mitotic cells, including many mechanoreceptor neurons.  The elp-1(ok347) strain, RB639, was obtained from the C. elegans Gene Knockout Consortium at the Oklahoma Medical Research Foundation (Oklahoma City, OK), outcrossed four times and the exact location of the deletion was confirmed by sequencing a PCR product that spanned the deletion ( Figure 2D).

Tissue-specific expression
Vectors that express ELP-1 fused to GFP were created from the Fire Lab Vectors http://www.addgene.org by PCR amplification or restriction digestion of the genomic cosmid, F38A6.2. Standard molecular techniques were used and all PCR products were cloned and sequenced to be certain that no errors were introduced during amplification. The P elp-1 ::elp-1(Δ11-16)::nls::gfp construct (pKA99-1) was generated by ligating a SphI-BamHI genomic fragment that contains 4 kb of the elp-1 5'UTR region (P elp-1 ) and 3 kb of the elp-1 gene (exons 1-10) into the pPD95.67 vector upstream of a nuclear localization sequence (NLS) and the gfp gene. The construct expresses a truncated ELP-1 protein fused to GFP with an NLS under the endogenous elp-1 promoter. The P elp-1 ::elp-1::gfp construct (pKA99-2) contains 9 kb of the elp-1 gene with the endogenous elp-1 promoter (P elp-1 ) inserted into the SphI-XbaI site in the pPD95.75 vector and expresses the fulllength ELP-1 protein (exons 1-16) fused to GFP. elp-1 was amplified from the F38A6 cosmid with the Expand Long Template PCR system (Roche-Boehringer Mannheim; Alameda, CA) with the primers, EMAPUSGFP1-5', corresponding to sequence upstream of the SphI genomic restriction site (5'-AACACCGAACTTGATGAAATATTCG-GTGCAAC-3') and EMAPSTP2GFP2-3' (5'CCTCTCTAGATCCGCTTCCAGGCACCATTCAAAAAC-CGAATTATCAG-3'), corresponding to the sequence at the 3' end of the coding region of the gene, and substituting an XbaI restriction site for the stop codon. The P elp-1 ::gfp construct (pKA99-3) expresses GFP under the control of the 4 kb elp-1 promoter region. For this construct the promoter region was amplified with primer, EMAPUSGFP1-5' and primer, EMAPSTRTGFP1-3' (5'-CGGGGATCCTCCATTTTTTTGAAGAATTTTTGCAAATTT-TCTCCTGCAAC-3'), corresponding to sequence at the start site of the gene, with a BamHI tag (GGGATCC) used to ligate into the pPD95.75 vector.