©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
elt-2, a Second GATA Factor from the Nematode Caenorhabditis elegans(*)

Mark G. Hawkins , James D. McGhee (§)

From the (1)Department of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously shown that a tandem pair of (A/T)GATA(A/G) sequences in the promoter region of the Caenorhabditis elegans gut esterase gene (ges-1) controls the tissue specificity of ges-1 expression in vivo. The ges-1 GATA region was used as a probe to screen a C. elegans cDNA expression library, and a gene for a new C. elegans GATA-factor (named elt-2) was isolated. The longest open reading frame in the elt-2 cDNA codes for a protein of M 47,000 with a single zinc finger domain, similar (approximately 75% amino acid identity) to the C-terminal fingers of all other two-fingered GATA factors isolated to date. A similar degree of relatedness is found with the single-finger DNA binding domains of GATA factors identified in invertebrates. An upstream region in the ELT-2 protein with the sequence C-X-C-X-C-X-C has some of the characteristics of a zinc finger domain but is highly diverged from the zinc finger domains of other GATA factors. The elt-2 gene is expressed as an SL1 trans-spliced message, which can be detected at all stages of development except oocytes; however, elt-2 message levels are 5-10-fold higher in embryos than in other stages. The genomic clone for elt-2 has been characterized and mapped near the center of the C. elegans X chromosome. ELT-2 protein, produced by in vitro transcription-translation, binds to ges-1 GATA-containing oligonucleotides similar to a factor previously identified in C. elegans embryo extracts, both as assayed by electrophoretic migration and by competition with wild type and mutant oligonucleotides. However, there is as yet no direct evidence that elt-2 does or does not control ges-1.


INTRODUCTION

The DNA sequence motif (A/T)GATA(A/G), hereafter referred to only as GATA, is known to be involved in lineage-specific gene expression in other organisms, most prominently in the control of globin gene transcription in vertebrates (reviewed in Refs. 1-3). ``GATA-binding proteins'' or ``GATA factors'' are a class of transcriptional activators that bind to GATA sequences in DNA. Six classes of GATA factors (termed GATA-1 through GATA-6) have now been defined in vertebrates(4) , and all have a pair of distinctive zinc finger domains. Several one-finger GATA factors have been identified in fungi (5-7), and two GATA factors have been identified in Drosophila, one with a single zinc finger (8) and one with a pair of zinc fingers(9, 10) . One GATA factor (named elt-1, standing for erythroid-like transcription factor 1) has been isolated from the nematode Caenorhabditis elegans on the basis of sequence homology with chicken GATA-1(11) .

We have been studying the C. elegans ges-1 (gut esterase 1) gene as an example of a terminally differentiated gene whose expression is restricted to the intestine. The ges-1 gene is expressed via transcription from the zygotic genome, beginning when the developing gut has only four to eight cells(12) . At this stage, the entire embryo has only 150-200 cells and ges-1 is one of the earliest markers of tissue-specific differentiation to appear in the C. elegans embryo. Our previous deletion-transformation analysis identified a region within the ges-1 promoter that appears to function both as a gut activator and as a pharynx/tail repressor(13) . A more detailed analysis of this region has revealed that the 30-base pair region controlling the spatial expression of ges-1 contains a tandem pair of (A/T)GATA(A/G) sequences(14) . Furthermore, nuclear extracts prepared from C. elegans embryos contain a factor that binds to these motifs(15) .

Judging from the sequence of the ges-1 control region (14-16), expression of the ges-1 gene is likely to involve a GATA factor. The obvious candidate for such a factor is the product of the elt-1 gene(11) . However, homozygous deficiency embryos lacking the elt-1 gene still express ges-1(15) , suggesting that elt-1 is not required zygotically for ges-1 expression. With the aim of identifying a factor that does control ges-1, we have probed a C. elegans cDNA expression library with multimerized ges-1 GATA sequences. The present report describes the result of this screen, the identification of a second C. elegans GATA factor elt-2.


EXPERIMENTAL PROCEDURES

General Materials and Methods

Unless otherwise stated, all DNA manipulations were carried out as described in Ausubel et al.(17) and Sambrook et al.(18) . Sequencing reactions were performed using Taq DyeDeoxy Terminator Cycle sequencing kits (Applied Biosystems) in a GeneAmp 9600 PCR()system (Perkin Elmer) and analyzed by automated sequencer (Applied Biosystems model 373A). Routine handling of C. elegans was performed as described by Brenner(19) .

Isolation of cDNA and Genomic Clones

A mixed stage C. elegans gt11 cDNA library (obtained from Dr. P. Okkema, Carnegie Institute of Washington, Baltimore, MD) was screened with a P-labeled multimerized GATA-containing double-stranded oligonucleotide using the method of Vinson et al.(20) ; the exact oligonucleotide sequences are given under ``Results.'' A single positive clone was isolated and subsequently used as a probe to isolate further clones from an independent C. elegans mixed stage cDNA library prepared in ZAP(21) . The probe was radioactively labeled with [-P]dCTP using a Prime-it II kit (Stratagene), and hybridizations were performed at 65 °C for 18 h (6 SSC, 1% SDS, 5 Denhardt's solution, 100 µg/ml sonicated salmon sperm DNA). The filters were washed at high stringency (0.1 SSC, 1% SDS, 65 °C) and exposed to x-ray film overnight (X-Omat; Eastman Kodak Co.). The longest such cDNA clone was sequenced on both strands using a combination of unidirectional nested deletions (22) and custom synthesized primers (Regional Oligonucleotide Synthesis Unit, University of Calgary). The 5` and 3` ends of all the other purified ZAP clones were also sequenced.

Genomic clones were isolated from a C. elegans EMBL-4 library (obtained from Dr. C. Link, University of Colorado, Boulder, CO). The library was screened with a full-length elt-2 cDNA fragment, radioactively labeled using random oligonucleotide primers; hybridization and washing conditions were as described above. Once the gene sequence had been determined (see below), Southern blotting confirmed that restriction fragments of the lengths predicted from the sequence could indeed be identified in the appropriate digests of C. elegans genomic DNA.

Amplification of 5` End of mRNA Transcript

Poly(A) mRNA was prepared from a mixed population of N2 worms using a Micro-Fastrack mRNA isolation kit (Invitrogen), and cDNA was synthesized as described by Frohman et al.(23) . PCR amplifications were performed using the C. elegans trans-spliced primers SL1 (24) or SL2 (25) and several elt-2-specific antisense internal primers. PCR products generated were cloned and sequenced.

In Vitro Transcription-Translation of cDNA Clones and Gel Mobility Shifts

ELT-2 protein was produced from elt-2 cDNA contained in the vector pBluescript SK (Stratagene) using the TNT-coupled transcription-translation system (Promega). Deletion constructs of ELT-2, in which either amino acids 1-174 or amino acids 243-433 had been removed, were also produced by in vitro transcription and translation. DNA binding activity was analyzed by gel mobility shift assays, essentially as described by Stroeher et al.(15) . Each reaction typically contained 2 ng of double-stranded oligonucleotide probe, 100 ng of poly[d(I-C)] competitor DNA (unless specified otherwise), and either 1 µl of in vitro transcription/translation reaction (full-length ELT-2 or deletion construct as appropriate) or 10 µg of nuclear extract, prepared from fluorodeoxyuridine-blocked embryos as described by Stroeher et al.(15) .

Northern Blotting

RNA was isolated from staged worms using a total RNA purification kit (Clontech) and analyzed for levels of elt-2 message by Northern hybridization using a Hybond N+ nylon membrane (Amersham International plc.) according to the manufacturer's instructions. The full-length elt-2 cDNA, random oligo-labeled as described above, was used as a probe. After hybridization and washing (using the same conditions described above), filters were exposed to X-Omat x-ray film (Kodak) with intensifying screens at -70 °C for 1-30 days.


RESULTS

Isolation of a cDNA Clone by Probing C. elegans Expression Libraries with GATA Sequences from the Gut Activator Region of the ges-1 Gene

We have identified a tandem pair of GATA sequences required for the gut-specific expression of the ges-1 gene in the C. elegans embryo(14) . Both single GATA motifs and the double GATA motif (see Fig. 1) appear to bind the same protein present in embryo extracts(15) . The downstream GATA sequence (with terminal extensions added to ensure directional cloning; see Fig. 1) was ligated into 11 tandem copies, kinase-labeled with [-P]ATP, and used as probe to screen an expression library prepared in gt11 from mixed stage C. elegans RNA (26). One positive clone was isolated from approximately 300,000 plaques screened; partial sequence analysis revealed that the clone did indeed code for a ``GATA factor'' protein, as will be discussed in detail below. The gene has been named elt-2 in accordance with C. elegans nomenclature conventions(27) .


Figure 1: DNA sequence of the C. elegans ges-1 gut activator region. Nucleotides 2170-2208 from GenBank entry no. M96145 (16) are shown, along with the oligonucleotides used for probing cDNA libraries and gel mobility shift assays. Terminal extensions (atgc) were added to ensure directional cloning (see ``Results''). Complementary oligonucleotides were synthesized as appropriate, and double-stranded oligonucleotides were used in all experiments.



The insert from this original elt-2 cDNA clone was amplified by PCR and used as probe to screen a mixed-stage cDNA library prepared in ZAP(21) . Eleven positive clones were isolated from approximately 350,000 plaques screened under high stringency conditions; partial sequence analysis and restriction mapping indicated that all of these clones were derived from the same transcript.

The longest elt-2 cDNA clone is 1560 bp (excluding the poly(A) tail) and is essentially full-length. Reverse transcriptase-PCR (23) showed that elt-2 mRNA is trans-spliced to the SL1 leader sequence(24) , and the last three nucleotides of the SL1 leader sequence can indeed be identified at the 5` end of the longest cDNA clone. Reverse transcriptase-PCR using the SL2 trans-splice primer (25) instead of SL1 did not produce a product (data not shown). As will be shown below, the length of the longest cDNA agrees well with the estimated size of the elt-2 message detected by Northern blotting.

Position of the elt-2 Gene on the Physical Map of the C. elegans Genome

The isolated insert from the longest cDNA clone was used as a probe to screen a C. elegans genomic library under high stringency conditions. Six positive phage were isolated and restriction mapping showed them all to be identical or overlapping. A total of 5433 bp of elt-2 genomic sequence, including the entire gene, 3.5 kilobase pairs of upstream flanking region and 0.3 kilobase pair of downstream flanking region, has been obtained and deposited in GenBank (accession no. U25175). The genomic clones were used to locate the elt-2 gene on the physical map of the C. elegans genome (Dr. A. Coulson, MRC, Cambridge, UK). elt-2 lies just right of center on the X chromosome (cosmid C12H7 on the C. elegans physical map), close to the gene mab-18. No obvious genetic candidate for elt-2 has been identified in this vicinity.

Twelve examples of the sequence (A/T)GATA(A/G) can be identified in the elt-2 5`-flanking region (not shown), approximately twice as many as would be expected by chance. During erythropoiesis in vertebrates, expression of GATA-1 is autoregulated through flanking GATA motifs(28, 29) , and elt-2 may be similarly autoregulated

Alignment of elt-2 Genomic, cDNA, and Protein Sequences

Comparison of the genomic and cDNA sequences allows unambiguous assignment of the intron-exon structure of the elt-2 gene, as shown schematically in Fig. 2A. The elt-2 cDNA and protein sequences are aligned on Fig. 2B. The longest open reading frame (433 amino acid residues, producing a predicted protein of M 47,000) begins with a putative initiator methionine residue 1 base pair downstream of the SL1 trans-splice acceptor site; this reading frame includes the distinctive GATA-factor DNA binding domain, as will be discussed in detail in the next section. A potential poly(A) addition site (AATAAA) is present 237 nucleotides downstream of the proposed translation termination codon and 11 nucleotides upstream of the poly(A) sequence found in the cDNA clones.


Figure 2: Alignment of the elt-2 genomic, cDNA, and protein sequences. A, schematic alignment of the elt-2 genomic and cDNA structures. The elt-2 gene contains nine exons; the length of each exon in base pairs is noted above the genomic clone. The lengths of the eight introns (in base pairs) are listed in italics below the 1.6-kilobase pair schematic diagram of the elt-2 cDNA. Intron 6 interrupts the conserved zinc finger domain (shaded). B, nucleotide sequence of the elt-2 cDNA and deduced amino acid sequence. Numbers on the right refer to the amino acid and nucleotide sequences, respectively. The longest open reading frame is 433 amino acids, beginning at the first ATG codon after the SL1 trans-splice leader sequence (underlined). A putative polyadenylation site (aataaa) is double-underlined and is 237 nucleotides downstream of the proposed translation termination codon. The conserved zinc finger DNA binding domain (amino acids 237-261) is shaded. Intron positions are indicated by the arrowheads.



The elt-2 Gene Encodes a Distinctive Zinc Finger Domain of the Type Found in GATA Factors

The ELT-2 protein contains a region whose sequence is clearly related to the zinc finger DNA binding domains of all GATA factors yet reported. The majority of GATA factors have two such domains, but ELT-2 appears to only have one (as do several factors isolated from fungi and from Drosophila, as noted earlier). Fig. 3A aligns the sequence of the single zinc finger domain of ELT-2 (amino acids 237-261) with the sequence of the C-terminal finger of the previously isolated GATA factors elt-1 from C. elegans(11) and pannier from Drosophila(9, 10) and with the sequence of the C-terminal fingers found in each of the six classes of chicken GATA factors(4, 30, 31) ; four examples of single zinc finger factors from invertebrates (5, 6, 7, 8) are also included in the alignment. Levels of sequence matches are in the range of 72-84% amino acid identity (76-92% similarity) when compared to vertebrate GATA factors and in the range of 56-72% identity (68-85% similarity) when compared to invertebrate factors. A similar alignment of the zinc finger domain of ELT-2 with the N-terminal zinc fingers of two-finger GATA factors reveals 48-56% identity (68-72% similarity). Unlike other known GATA factors, the zinc finger domain of ELT-2 is contained on two separate introns. As shown in Fig. 3B, all of the proteins listed above, including ELT-2, share a highly conserved region (36-75% identity, 54-88% similarity) extending 25 amino acids to the C-terminal side of the zinc finger; this region has been shown to be necessary for DNA binding(32) .


Figure 3: The elt-2 gene encodes a distinctive zinc finger domain of the type found in GATA factors. A, amino acid sequence alignment of the zinc finger domain of ELT-2 (residues 237-261) aligned with the C-terminal zinc finger domains from a number of two-finger GATA factors (elt-1, C. elegans, residues 272-296) (11); pannier, Drosophila, residues 226-250 (9, 10); GATA-1 to GATA-6 from chicken, residues 164-188 (cGATA-1) (30), 335-359 (cGATA-2) (31), 317-342 (cGATA-3) (31), 211-235 (cGATA-4) (4), 239-263 (cGATA-5) (4), 235-259 (cGATA-6) (4), and zinc finger domains from several single-finger GATA factors (ABF, Drosophila, residues 318-342 (8); nit-2, Neurospora crassa, residues 743-767 (6); areA, Aspergillus nidulans, residues 516-540 (5); GLN3, Saccharomyces cerevisiae, residues 306-330 (7)). Residues identical to aligned residues in ELT-2 are shaded, and the percentage amino acid identity to ELT-2 is listed to the right of each alignment. B, amino acid sequence alignment of the 25 amino acids immediately C-terminal to the zinc finger domain of ELT-2 (residues 262-286) and equivalent residues from other GATA-factors; references to individual sequences are listed in A. Residues identical to aligned residues in ELT-2 are shaded, and the percentage of amino acid identity to ELT-2 is listed to the right of each alignment.



ELT-2 Protein Produced in Vitro Binds to the GATA Sequences of the C. elegans ges-1 Gene

ELT-2 protein was produced in a coupled transcription-translation reaction and used in electrophoretic mobility shift experiments with double-stranded oligonucleotide probes. As shown in Fig. 4A, ELT-2 protein binds to the downstream GATA sequence of the ges-1 gene (the sequence used as probe for the library screening), and this binding is resistant to a large excess of the nonspecific competitor poly[d(I-C)]. Formation of the complex is competed effectively with the unlabeled oligonucleotide, but is competed ineffectively by an oligonucleotide in which the TGATAA sequence has been mutated to GTCGCC (Fig. 4B). This specific binding behavior mimics the binding properties of the GATA-binding factor identified in nuclear extracts prepared from C. elegans embryos (see Fig. 5B of Ref. 15). Fig. 4C shows that elt-2 also binds to the upstream GATA motif from ges-1. Fig. 4D shows that an elt-2-GATA oligonucleotide complex has an electrophoretic mobility very close to that of the complex containing the binding protein present in embryonic nuclear extracts(15) . Numerous repetitions of this experiment have never reliably distinguished between the two complexes. Any migration differences seen in individual experiments are slight, and we judge them to lie within the uncertainty associated with comparing crude extracts and in vitro translation products.


Figure 4: Gel mobility shift analysis of in vitro expressed ELT-2 protein binding to the putative gut activator region of the C. elegans ges-1 gene. A, double-stranded downstream GATA probe (see Fig. 1 for oligonucleotide sequences) incubated with in vitro expressed full-length ELT-2 protein in reactions containing X-fold excess (by weight) of nonspecific competitor DNA. B, double-stranded downstream GATA probe incubated with in vitro expressed full-length ELT-2 protein in reactions containing X-fold molar excess of unlabeled double stranded wild-type (downstream GATA oligo) or mutant competitor oligonucleotide. C, in vitro expressed full-length ELT-2 protein, incubated in reactions with either double-stranded upstream or downstream GATA oligonucleotides. D, double-stranded downstream GATA oligonucleotide incubated in reactions with either nuclear extracts from fluorodeoxyuridine-blocked embryos (15) or in vitro expressed full-length ELT-2 protein.




Figure 5: A possible second zinc finger domain in the elt-2 gene. A, amino acid sequence of the putative upstream zinc finger domain of ELT-2 (residues 153-176) aligned with the N-terminal zinc finger domains from several two-finger GATA factors; for references to individual sequences, see legend to Fig. 3A. Residues that are identical to the aligned residues in ELT-2 are shaded; overall percentage identitity is listed on the right of each example. B, gel mobility shift assay of reactions containing double-stranded gut activator oligonucleotides binding to in vitro expressed full-length ELT-2 protein, putative zinc finger construct (amino acids 243-433 deleted) and true zinc finger construct (amino acids 1-174 deleted). Left-hand three lanes use the upstream GATA oligonucleotide as the probe; the right-hand three lanes use the tandem GATA oligonucleotide as the probe (see Fig. 1 for sequences).



A Possible Second Zinc Finger Domain in the ELT-2 Protein

Standard alignment programs identify only a single zinc finger domain in the ELT-2 protein. However, visual inspection reveals the sequence C-X-C-XC-X-C (amino acids 153-176), which looks sufficiently like a second zinc finger domain to warrant further investigation. Fig. 5A shows the best alignments that could be obtained between this region of ELT-2 and the N-terminal fingers of other GATA factors (25-30% identity; 42-48% similarity). This upstream region lacks the distinctive residues usually found between the two cysteine pairs in GATA factors, but it could still be involved in DNA-binding nonetheless. To test this possibility, ELT-2 mutant proteins were produced by in vitro transcription and translation and then assayed for DNA binding ability by gel mobility shift. Three constructs were assayed: (i) full-length ELT-2 protein; (ii) a truncated version containing the upstream putative zinc finger domain, but lacking the downstream ``true'' zinc finger; and (iii) a truncated version containing the downstream true zinc finger but lacking the upstream putative finger. The results of this experiment are shown on Fig. 5B. The protein containing only the upstream putative finger does not bind to the GATA-containing oligonucleotides; in contrast, the protein containing only the downstream ``true'' finger shows easily detectable binding.

In summary, the upstream putative finger does not appear to bind GATA sequences by itself nor does it appear necessary for GATA-sequence binding by the downstream true finger.

Expression of elt-2 during C. elegans Development

Total RNA was isolated from the different stages of the C. elegans life cycle and analyzed by Northern blotting, using the full-length cDNA as a probe. As shown on Fig. 6, elt-2 mRNA sequences can be detected at all stages of development, except oocytes (even after 1-month exposure of the autoradiograph). However, the highest level of elt-2 mRNA is present in embryos, 5-10-fold higher than in other stages. Approximately equal amounts of RNA were loaded for each developmental stage, at least as judged by the hybridization intensity produced by probing with the rp21 ribosomal protein gene(11) . Finally, the size of the elt-2 message, as shown in Fig. 6, is within experimental error of the size predicted from the full-length elt-2 cDNA.


Figure 6: Northern blot analysis of elt-2 expression during C. elegans development. Five µg of total RNA isolated from staged populations of worms were electrophoresed on each gel lane, blotted, and probed with the full-length elt-2 cDNA clone as described under ``Experimental Procedures'' (7-day exposure). The blot was also probed with a ribosomal protein gene (rp21, obtained from Dr. J. Spieth, Indiana University, Bloomington, IN) to confirm RNA loading quantities (overnight exposure). The size of the elt-2 mRNA was estimated from RNA standards run on the same gel (not shown). Stages are represented as follows: Oo = oocytes, prepared as in Stroeher et al. (15); Em = embryos, isolated by alkaline hypochlorite digestion of gravid adults; L1, L2, L3, and L4 = larval stages; Gr = gravid adults; and Mx = mixed stage population.




DISCUSSION

Sequence analysis and DNA binding assays have shown that ELT-2 clearly belongs to the GATA factor family of DNA-binding proteins. ELT-2 appears slightly more closely related to GATA-5 than to any of the other GATA factors (84% identity, 92% similarity in the DNA binding domain). Considering that elt-2 was isolated by binding to an endoderm specific gene in nematodes (i.e. ges-1), it is interesting that GATA-5 also appears to be expressed in the endoderm of vertebrates(4) .

The majority of GATA factors contain two highly conserved zinc finger domains, but ELT-2 contains only one such domain. Although the diverged sequence C-X-C-X-C-X-C was detected in ELT-2 just upstream of the true finger, there was no evidence that this region bound GATA-containing oligonucleotides. On the other hand, the upstream fingers of two-finger GATA factors also do not appear to bind DNA in the absence of the downstream finger(33, 34) . Thus it remains possible that this upstream domain in ELT-2 somehow contributes to binding strength or binding specificity. A different view is that this motif is an evolutionary relic of a former finger.

Our central concern is whether elt-2 does indeed control expression of the ges-1 gene in vivo. Although both in vitro expressed ELT-2 and a factor present in C. elegans embryonic extracts (15) bind GATA sequences in a similar manner, this is only indirect evidence that two proteins could be identical. Thus a suitably cautious statement is that elt-2 has not yet been eliminated as a candidate for ges-1 control. In any case, the necessary proof must come from a genetic experiment that assays ges-1 expression in an elt-2 mutant. We have identified a Tc1 transposon insertion into the fifth intron of the elt-2 gene, using the system of Zwaal et al.(35) . It should now be possible to produce an elt-2 null mutation by imprecise transposon excision events. Until such a mutant has been identified, we leave open the question how (or even if) elt-2 is involved in ges-1 control.

elt-2 is the second GATA factor that has been cloned from C. elegans. Our low stringency screens of genomic DNA, using elt-2 cDNA as a probe, have not yet revealed obviously cross- hybridizing species (data not shown), nor did previous attempts using the elt-1 gene as a probe(11) . However, both elt-1 and elt-2 are expressed predominantly in embryos, and we would be surprised if C. elegans did not have additional GATA factors involved in gene expression during other stages of the life cycle.


FOOTNOTES

*
This work was supported by the Medical Research Council of Canada, the Alberta Heritage Foundation for Medical Research, the Science and Engineering Research Council of Great Britain (SERC), the North Atlantic Treaty Organization (NATO), and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U25175.

§
To whom correspondence should be addressed: Dept. of Medical Biochemistry, University of Calgary, Health Sciences Centre, Rm. 2265, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1. Tel.: 403-220-4476; Fax: 403-270-0737.

The abbreviation used is: PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Drs. Peter Okkema and Robert Barstead for providing cDNA libraries, Dr. Chris Link for providing the C. elegans genomic library, Barbara Goszczynski for providing total RNA from staged populations of worms, and Fran Allen for her help in sequencing the elt-2 genomic clone.


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