Molecular characterization of a novel, developmentally regulated small embryonic chaperone from Caenorhabditis elegans.

Low molecular weight chaperones inhibit protein aggregation and facilitate refolding of partially denatured polypeptides in cells subjected to physical and chemical stresses. The nematode Caenorhabditis elegans provides a system amenable for investigations on roles for chaperone proteins in normal homeostasis and development. We characterized a C. elegans gene and cDNAs that encode a novel, small embryonic chaperone-like protein (SEC-1) that is composed of 159 amino acids. The central core of SEC-1 (residues 45-126) is ∼40% identical with a corresponding segment of mammalian Hsp27 and αB crystallin. Expression of SEC-1 in Escherichia coli confers thermotolerance on the bacterium. SEC-1 mRNA is evident only in C. elegans oocytes and developing embryos. Translation and accumulation of SEC-1 protein is temporally coupled with a prolonged burst of intense protein synthesis and rapid mitogenesis during early embryogenesis. As the rate of protein synthesis decreases during late embryogenesis, levels of SEC-1 and its cognate mRNA decline precipitously. Induction/deinduction of SEC-1 is precisely regulated by intrinsic developmental factors rather than extrinsic stresses. In vivo injection of C. elegans oocytes with antisense oligonucleotides that complement the 5′-end of SEC-1 mRNA arrests nematode development at an early stage after fertilization. Thus, SEC-1 appears to be adapted to perform essential functions in early embryogenesis.

Small heat shock proteins (sHsps) 1 are ubiquitously expressed in organisms ranging from Escherichia coli to humans (1)(2)(3)(4)(5). The principal sHsp prototypes in mammals are Hsp27 (ϳ200 -210 amino acid residues) and the ␣ crystallins (ϳ175 residues) (6,7). Human Hsp27 and ␣ crystallin are 35% identical in their central and COOH-terminal regions, and both proteins oligomerize to generate high molecular weight (Ͼ600,000) complexes (6 -8). ␣-crystallins and Hsp27 are often expressed at low levels in unstressed cells and tissues. However, the biosynthesis and accumulation of sHsps are induced by heat shock, interleukin-1, toxic chemicals (arsenite, oxidants), ATP depletion, endotoxins, hyperosmolarity, and other biological and physical stresses (5,9). Induction of sHsps often parallels the stress-induced accumulation of Hsp70 and Hsp90 molecular chaperones (1)(2)(3)(4). Recent investigations indicate that overexpression of Hsp27 confers resistance to heat stress in cultured cells and protects the actin cytoskeleton from disruption by ATP depletion, cytochalasin D, and other stresses (10 -12). Stabilization of the actin-based cytoskeleton may be related to the ability of Hsp27 to bind barbed ends of actin filaments (13,14). Phosphorylation of Hsp27 diminishes its affinity for F-actin, thereby suggesting that stabilization of cytoskeleton by sHsps is a reversible and regulated process (11)(12)(13). Ser 82 of Hsp27 is rapidly phosphorylated in cells exposed to heat stress, arsenite, or interleukin-1, and serves as a downstream target for a "stress-activated" protein kinase cascade that includes an extracellular regulated kinase homolog designated P38 and MAP kinase-activated protein kinase 2 (15)(16)(17). In vitro, Hsp27 and ␣ crystallin decelerate the aggregation of partially denatured proteins and promote correct ("native") folding of such proteins via cycles of reversible binding and release of the "client" proteins (8,18,19). The preceding observations suggest that sHsps are molecular chaperones that augment or complement functions of Hsp70 and Hsp90 proteins (1-5, 20 -22) at the levels of individual protein folding and the maintenance of multicomponent complexes (cytoskeleton). Interactions between partially folded proteins and members of the Hsp60/Hsp70 families are strictly dependent on the binding and hydrolysis of ATP by the chaperones; in contrast, sHsps promote folding in an ATP-independent manner (8,18,19).
Investigations on sHsps have focused principally on the ability of these proteins to protect cells against physical, chemical, and biological stresses. However, the properties of small molecular chaperones are also well suited for subserving physiological roles in unstressed cells. For example, the rate and level of protein synthesis are markedly elevated at various stages of embryogenesis in many organisms. As a consequence, cellular machinery that ensures efficient, high fidelity folding of polypeptides may be required for embryonic homeostasis. Hsp70 chaperones are absent during early developmental stages in mammals, and the forced expression of these proteins in Drosophila embryos results in lethality (1,4). Thus, embryonic expression of small molecular chaperone proteins, which inhibit nascent polypeptide aggregation and promote accurate folding of newly synthesized proteins, could potentially support and facilitate the rapid progression of early development. Small molecular chaperones that reversibly associate with actin filaments may also be intimately involved in the multiple cycles of cytoskeleton reorganization that accompany serial cell divisions and extensive cell differentiation in embryos.
At present, little is known about expression, structure, regulation, and possible functions of sHsp-related proteins during early embryogenesis in mammals and most other organisms. The nonparasitic nematode Caenorhabditis elegans is a good model system for investigations on these topics. Adult C. elegans are composed of 959 somatic cells, which are organized into highly specialized tissues that constitute reproductive, digestive, muscular, hypodermal, and nervous systems (23)(24)(25). The cellular and developmental biology of the nematode have been characterized in exceptional detail, and the lineage for each cell in the animal has been established (23)(24)(25). Moreover, the molecules and mechanisms that govern signal transduction and development in C. elegans are universal and control similar processes in higher organisms, including mammals (26,27). Embryogenesis in C. elegans is an invariant, stereotypical process that is completed over a period of 14 h (28). Approximately 60% of the adult's ultimate constellation of cells is generated from the zygote during the first 6 h of embryogenesis. Morphogenesis of the embryo into an L1 larva ensues in the subsequent 8 h. Compression of intensive embryonic protein synthesis and multiple cytoskeletal rearrangements into a relatively brief time frame creates an environment in which the demand for efficient and accurate protein folding is extremely high.
Previous investigations on C. elegans failed to identify small molecular chaperones that are expressed in normally developing, unstressed embryos. Candido and co-workers (29,30) characterized four homologous C. elegans genes (hsp16 -41, hsp16 -48, hsp16 -1, and hsp16 -2) that encode putative sHsps composed of 143 or 145 amino acid residues. Portions of the central and COOH-terminal regions of these predicted C. elegans polypeptides share substantial sequence identity/similarity with corresponding, highly conserved regions in mammalian Hsp27 and ␣ crystallin homologs. However, C. elegans hsp16 -1, hsp16 -2, hsp16 -41, and hsp16 -48 genes are fully repressed and uninducible during early embryogenesis (31). Moreover, accumulation of the various nematode HSP16 proteins at later stages of development is strictly dependent upon heat shock or other stresses. We now report the discovery of a C. elegans gene (sec-1) that encodes a novel, developmentally regulated chaperone protein (M r 18,000) that performs critical functions in early embryogenesis.

EXPERIMENTAL PROCEDURES
Growth of C. elegans-The Bristol N2 strain of C. elegans was grown at 20°C as described previously (32). To synchronize C. elegans for developmental studies, embryos were hatched in the absence of nutrients and then transferred to plates containing E. coli as a food source. Under these conditions, the worms develop synchronously into reproductive adults (33). L1 larvae were harvested 6 h after feeding, L2 larvae at 20 h, L3 at 29 h, L4 larvae at 40 h, young adult worms at 53 h, and egg-laying adult nematodes at 75 h. A purified population of embryos was obtained by alkaline hypochlorite treatment of gravid C. elegans, as described by Sulston and Hodgkin (34).
Isolation of cDNAs Encoding SEC-1-C. elegans were adapted to grow under conditions of heavy metal stress (2 mM CdCl 2 ) as described in a previous paper (35). A library that is enriched in stress-induced cDNAs was constructed in the bacteriophage ZAPII (Stratagene) using mRNA from CdCl 2 -treated nematodes as a template (35). The library was screened with single-stranded 32 P-labeled cDNAs that were generated from mRNAs that were isolated from control and cadmiumadapted C. elegans. Hybridization and washing conditions are given by Smith et al. (36). A group of recombinant phage clones that contain stress-induced cDNAs were obtained by this differential screening procedure. The cDNA inserts were subcloned into the plasmid pGEM7Z (Promega) and sequenced. One of the stress-induced cDNAs (Z500) encoded a novel partial protein that was related to sHsps from mammals and C. elegans. This protein was named SEC-1. A 325-bp fragment of SEC-1 cDNA was excised with SalI and used a template to generate a random-primed, 32 P-labeled probe. The probe was used to screen a more complete C. elegans cDNA library in the phage gt11 (Clontech), using the conditions described in Smith et al. (36). Four positive recombinant phage clones were plaque-purified, and the cDNAs were subcloned in pGEM7Z and sequenced. Further details are provided under "Results." Isolation of the sec-1 Gene-A 32 P-labeled, 325-bp SEC-1 cDNA fragment (see above) was used as a probe to screen a EMBL4 library that contains the C. elegans genome. The library and screening procedures are described in previous papers (32,37). A 2.4-kilobase pair fragment of genomic DNA, which includes the SEC-1 structural gene and 5Ј-and 3Ј-flanking sequences, was subcloned into the EcoRI site of pGEM7Z and sequenced. Further details are provided under "Results." Computer Analysis-Analysis of sequence data, sequence comparisons, and data base searches were performed using PCGENE-Intelli-Genetics software (IntelliGenetics, Mountain View, CA) and the BLAST and FASTA programs (38,39) provided by the NCBI (National Center For Biotechnology Information) server and the National Library of Medicine/National Institutes of Health.
Southern Gel Analysis-Fragments of C. elegans genomic DNA were generated by digestion with restriction endonucleases, fractionated in a 0.6% agarose gel, and transferred to a Nytran membrane as described previously (32). The Southern blot was probed with 32 P-labeled SEC-1 cDNA (2 ϫ 10 6 cpm/ml). Conditions for hybridization, as well as high and low stringency washing of the membrane, are given by Hu and Rubin (32).
Preparation of RNA and Northern Gel Analysis-Total C. elegans RNA was prepared as indicated in in a previous paper (32). Poly(A ϩ ) RNA was purified with a "Fast Track" Kit (Invitrogen) by following the manufacturer's instructions. Northern blot analysis was performed as described previously (40), using a 32 P-labeled, 262-bp 3Ј SalI fragment of SEC-1 cDNA as a probe. For some Northern analyses, nematodes were grown under environmental stresses prior to harvesting. Stressful conditions included heat shock (31°C for 1, 3, or 12 h), cold shock (4 or 10°C for 1, 2, or 12 h), starvation (22,48, or 63 h), or acute exposure to cadmium (0.2 mM CdCl 2 for 1, 2, or 24 h).
DNA Sequence Analysis-SEC-1 cDNAs and genomic DNA fragments containing the sec-1 gene were subcloned into the plasmid pGEM7Z. DNA inserts were sequenced by the dideoxynucleotide chain termination procedure of Sanger et al. (41) using T7, SP6, and custom oligonucleotide primers as described previously (32,37). Sequencing reagents were from Amersham Corp.
Expression and Purification of Recombinant SEC-1 Fusion Protein-A 507-bp XbaI-EcoRI restriction fragment of SEC-1 cDNA was subcloned into the pRSET-C expression plasmid (Invitrogen). This places the entire coding region of SEC-1 cDNA downstream from the T7 RNA polymerase promoter and 45 codons that constitute an NH 2terminal fusion peptide. The peptide contains a stretch of six consecutive His residues, which form a nickel binding domain. E. coli BL21(DE3) was transformed with the expression plasmid and induced with 1 mM isopropyl-1-thio-␤-D-galactopyranoside (IPTG) for 1 h at 37°C. The host bacterium contains a chromosomal copy of the phage T7 RNA polymerase gene under the control of the lac promoter. Bacteria were harvested, disrupted, and separated into soluble and particulate fractions as described for previous studies (43). The SEC-1 fusion protein was recovered in the pellet fraction, because it is insoluble in the standard 20 mM Tris-HCl buffer system (pH 8) used for bacterial lysis. Recombinant SEC-1 was dissolved in 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl supplemented with 8 M urea and purified to near homogeneity by nickel-chelate chromatography (in the presence of 8 M urea) as described previously (43). When urea was eliminated by extensive dialysis against 50 mM sodium acetate, pH 4.9, the SEC-1 fusion protein remained soluble (see "Results" for details). Approximately 5 mg of highly purified SEC-1 fusion protein was obtained from a 500-ml culture of E. coli.
Production of Antibodies Directed against SEC-1-Samples of the SEC-1 fusion protein were injected into rabbits (0.2-mg initial injection; 0.2 mg for each of three booster injections) at Hazelton Corning Laboratories (Vienna, VA) for the generation of antisera. Serum was collected at 3-week intervals.
Electrophoresis of Proteins and Western Immunoblot Assays-Samples of proteins were denatured in gel loading buffer and subjected to electrophoresis in a 12% polyacrylamide gel containing 0.1% SDS as described previously (44). Albumin (M r 67,000), ovalbumin (M r 43,000), carbonic anhydrase (M r 29,000), myoglobin (M r 17,000), and cytochrome C (M r 12,000) were used as standards for the estimation of M r values. The cytosolic and particulate fractions of C. elegans homogenates were prepared as described previously (45). Western blots of C. elegans proteins and SEC-1 fusion protein were blocked, incubated with antiserum (1:2000), and washed as described previously (46). SEC-1 was visualized by an indirect chemiluminescence procedure as previously reported (46).
Thermotolerance Studies-E. coli BL21(DE3) was transformed with pRSET-C expression plasmids that contained no insert, a cDNA encoding full-length SEC-1, or an unrelated cDNA (clone YPP, a cDNA encoding a portion of the cytoplasmic domain of a C. elegans proteintyrosine phosphatase). In the latter two instances, the recombinant proteins were constitutively expressed in the absence of inducer (see "Results"). Bacteria were grown to A 600 ϭ 0.3. Next, the cultures were diluted 1:4000 into fresh medium and incubated for 1 h at 28°C. Subsequently, the bacteria were subjected to heat stress by shifting the incubation temperature to 58°C for 30 min. After rapid cooling, the cells were maintained at 28°C for 48 h. Growth was monitored by determination of A 600 values at various times.
Ablation of SEC-1 Function by Antisense Oligonucleotides-Phosphorothioate oligonucleotides were obtained from Gene Link Inc. (Thornwood, NY). Two antisense oligonucleotides were employed. ␣S-1 (5ЈGACGGCCAGTGTATGGGCAGA-3Ј) corresponds to the inverse complement of nucleotides 17-37 in SEC-1 cDNA (Fig. 1); ␣S-2 (5Ј-TGAGCCCAGTATGGCATCAT-3Ј) corresponds to the inverse complement of nucleotides 67-86 in SEC-1 cDNA (Fig. 1). S1 (5Ј-TCTGC-CCATACACTGGCCGTC-3Ј), which corresponds to nucleotides 17-37 in SEC-1 cDNA (Fig. 1), served as a "sense" control. A mixture of antisense oligonucleotides (5 M ␣S1 plus 5 M ␣S2) was injected into the gonadal syncytium of 25 young adult C. elegans as described previously (40). The control, sense oligonucleotide (10 M S1) was injected into a second group of 25 animals. Injected oligonucleotides diffuse throughout a common cytoplasm that is associated with many oocyte nuclei. As oocyte differentiation proceeds, plasma membranes are generated, and the oligonucleotides are sequestered in individual oocytes. These oocytes are destined for fertilization and externalization (as developing embryos) within a 24-h period. Individual injected C. elegans were placed on plates containing E. coli as a food source. After 24 h, embryonic nematodes derived from injected parental C. elegans were examined for abnormalities via microscopy. Results were recorded on film as described previously (40).

Characterization of cDNAs That Encode a Novel Small
Chaperone from C. elegans-A cDNA library was prepared in the bacteriophage ZAPII, using template mRNA isolated from nematodes that were stressed with 2 mM CdCl 2 (35). Recombinant phage that contain cDNAs encoding stress-induced proteins were identified by differential screening. A 521-bp cDNA insert from a recombinant phage designated Z500 encodes a partial polypeptide (amino acid residues 4 -159, Fig. 1) with a novel sequence. A translation termination codon and 3Ј-untranslated nucleotides followed the open reading frame. Four additional cDNAs were retrieved from another C. elegans library in bacteriophage gt11 by screening with 32 P-labeled probes corresponding to Z500 cDNA. The largest cDNA (Fig.  1) encodes a polypeptide that is composed of 159 amino acids and has a calculated M r of 17,839. The remaining cDNA sequences were included within the sequence presented in Fig. 1. As the result of overlaps, the cDNA sequence was determined three times, and no discrepancies were detected.
An initiator ATG codon (nucleotides 7-9) lies within the context of a consensus C. elegans translation start site (AN-NATGT). Determination of the transcription start site and genomic DNA sequencing (see Fig. 3; see below) disclosed that the proposed translation start codon is preceded by a short 5Ј-untranslated region (47 bp) that lacks an ATG trinucleotide. A 3Ј-untranslated region (57 bp) follows the translation stop codon (nucleotides 484 -486) and terminates with a poly(A) tail. A classical poly(A) addition signal (AATAAA, nucleotides 527-532) precedes the poly(A) tail by 11 nucleotides.
Nomenclature-The novel C. elegans polypeptide (Fig. 1) exhibits structural and functional features associated with chaperones and is expressed only during embryogenesis. Therefore, it is named SEC-1 2 for small embryonic chaperone-1. Data documenting the developmentally controlled expression of SEC-1 and various structural and functional properties of the protein are presented in Figs. 2-7, Table I, and text below.
Structural Features of the SEC-1 Polypeptide-Amino acid sequences of SEC-1 (Fig. 1) and proteins in standard data bases were compared by using BLASTX, BLASTP, and FASTA homology search computer programs (38,39). The searches indicated that SEC-1 is a unique, previously uncharacterized protein. However, the SEC-1 sequence has motifs that are conserved in two families of proteins: C. elegans HSP16 isoforms (32-38% overall identity with SEC-1) and vertebrate low molecular weight chaperones (23-28% overall identity with SEC-1).
The predicted pI for SEC-1 is 8.2, whereas pI values for HSP16 isoforms lie within the range of 5.0 -5.9. Since the internal pH in C. elegans is ϳ6.2 (49), SEC-1 will exhibit a net positive charge in the physiological milieu. HSP16 proteins will be almost neutral or negatively charged. SEC-1 is enriched 2-5-fold in His residues relative to HSP16 proteins. Imidazole side chains will contribute a substantial level of positive charge to SEC-1 at the physiological pH, which may promote interactions with unfolded acidic proteins.
Characterization of the C. elegans sec-1 Gene-When Southern blots of restriction enzyme digests of C. elegans DNA were probed with 32 P-labeled SEC-1 cDNA under conditions of high stringency, a simple pattern of hybridizing fragments was observed (Fig. 3A). This pattern was not altered when low stringency conditions were employed (data not shown). Thus, it is likely that the sec-1 gene occurs as a single copy in the C. elegans genome.
A segment of DNA that contains the SEC-1 coding region was isolated from a C. elegans genomic library in the bacteriophage EMBL4. The sequence for 729 bp of 5Ј-flanking DNA, the sec-1 gene, and 251 bp of 3Ј-flanking DNA is presented in Fig. 4. Comparison of the cDNA and gene structures revealed that the primary transcript has one short intron (56 bp) that interrupts codon 93. The transcription start site was mapped to an A residue 47 bp upstream from a potential ATG codon by primer extension analysis (Fig. 3B). A classical TATAA box sequence (Fig. 4, nucleotides Ϫ31 to Ϫ27) precedes the transcription initiation site by 27 bp. The promoter/enhancer region for the sec-1 gene lacks cis sequences that mediate heat-induced (29 -31, 50, 51) and cadmium-activated gene transcription (40). 4 Excision of the intron from the primary transcript via splicing yields a mature SEC-1 mRNA that contains a . Fragments resolved on a 0.6% agarose gel were transferred to a Nytran membrane and were probed with 32 P-labeled cDNA corresponding to nucleotides 1-380 in Fig. 1. An autoradiogram for a filter washed at high stringency is shown. The gel was calibrated with a standard series of HindIII fragments of DNA as described previously (32). The sizes determined for the hybridizing DNA fragments in lanes 1-5 were 1.5, 4.0, 5.3, 3.4, and 5.0 kb, respectively. B, primer extension analysis was performed as described under "Experimental Procedures." An autoradiogram of the relevant portion of a 6% polyacrylamide, 7 M urea gel is shown. Lanes 1-4 received standard dideoxynucleotide sequencing reactions that were used to calibrate the gel for measuring the size of the extended product (lane P). A single extended product (see "Experimental Procedures") was detected (lane P). Its length indicates that the transcription initiation site lies 47 nucleotides upstream from the initiator ATG codon. No larger extension products were observed in the upper portion of the gel (data not shown). No signals were obtained when template was omitted, an unrelated primer was used, or an excess of nonradioactive primer was employed (data not shown). 480-nucleotide coding region flanked by 5Ј-and 3Ј-untranslated sequences that are 47 and 57 nucleotides long, respectively. If the transcript receives a typical poly(A) tail (ϳ70 A residues) for C. elegans, the size of SEC-1 mRNA will be ϳ0.65 kb.
The sec-1 gene was mapped to the right central region of chromosome III by the fingerprinting procedure of Coulson et al. (52). The sec-1 locus is in close proximity with the mel-23 and unc-49 genes.
Effects of Cadmium, Heat Stress, and Stage of Development on the Accumulation of SEC-1 mRNA-The 0.65-kb SEC-1 mRNA is evident in a mixed population (embryos plus larvae and adults) of unstressed C. elegans (Fig. 5A, lane 2). Incubation of the nematodes with 0.2 mM CdCl 2 for 24 h (Fig. 5A, lane 1) or 72 h (data not shown) had little effect on the level of SEC-1 mRNA. Moreover, when C. elegans were exposed to an elevated temperature for 1 or 3 h the relative content of SEC-1 mRNA declined ϳ40 -50% (Fig. 5C, compare lane 3 with lanes 1 and 2).
Total RNA was also prepared from purified populations of L1-L4 larvae, gravid adults, and embryos. Northern gel analysis revealed that SEC-1 mRNA content is subject to stringent developmental/temporal control (Fig. 5B). C. elegans embryos contain SEC-1 mRNA, but the transcript is not detected as postembryonic development (larval stages L1-L4) proceeds. As expected, SEC-1 mRNA accumulates in adult C. elegans that contain developing embryos (data not shown, but see Fig. 9).
Production and Characterization of Antibodies Directed against SEC-1-A cDNA fragment that encodes the full-length SEC-1 polypeptide was cloned into the expression plasmid pR-SET-C (see "Experimental Procedures"). A "His-tagged" SEC-1 fusion protein (apparent M r ϳ25,000) was synthesized constitutively in the bacterium (Fig. 6A, lanes 1 and 2). This is probably due to the binding of endogenous lac repressor protein by lac promoter/operator sequences in the high copy number pRSET plasmid. Partial depletion of the repressor results in inducer-independent synthesis of T7 RNA polymerase, which efficiently utilizes the plasmid T7 promoter to initiate transcription of SEC-1 mRNA. Nevertheless, incubation of transformed E. coli with inducer for 1 h doubled the level of SEC-1 fusion protein (Fig. 6A, lanes 2 and 3). Recombinant SEC-1 protein was dissolved in buffer containing 8 M urea and purified to homogeneity via nickel chelate chromatography, under denaturing conditions. Dialysis of purified SEC-1 fusion polypeptide against 10 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl resulted in quantitative precipitation of the protein (Fig. 6B, lanes 1 and 2). In contrast, dialysis against 50 mM sodium acetate, pH 4.9, eliminated urea and yielded a highly purified soluble preparation of SEC-1 (Fig. 6B, lanes 3  and 4).
Purified SEC-1 was used to immunize rabbits. The resulting antibodies avidly bound recombinant SEC-1, readily detecting 1 ng of antigen at high dilution (Fig. 7A). Moreover, the antibodies complexed a single 18-kDa polypeptide in cytosol derived from embryos and a mixed population of C. elegans (Fig.  7B, lanes 3 and 4). No signals were observed when the antiserum was preincubated with a large excess of recombinant SEC-1 (Fig. 7B, lanes 1 and 2).
Expression of SEC-1 Protein during C. elegans Development-Western immunoblot analysis was used to monitor SEC-1 expression at six distinct stages of C. elegans development (Fig. 8A). A substantial level of 18-kDa SEC-1 protein is evident in embryos. However, SEC-1 is absent (Ͻ0.0025% of total protein) when embryogenesis terminates and larvae hatch (L1). L2, L3, and L4 larvae also fail to express SEC-1 protein. Subsequently, SEC-1 reappears de novo and becomes  6. Expression and purification of recombinant SEC-1 fusion protein. A, E. coli BL21 was transformed with a recombinant expression plasmid that encodes the His-tagged SEC-1 fusion protein described under "Experimental Procedures" and "Results." A control transformation was performed with pRSET-C plasmid that lacks a cDNA insert. Transformed E. coli were grown to A 600 ϭ 0.6. Subsequently, aliquots of the cultures were incubated in the presence or absence of 1 mM IPTG. After 1 h at 37°C, the bacteria were harvested, and E. coli proteins were fractionated by electrophoresis on a 0.1% SDS-12% polyacrylamide gel (see "Experimental Procedures"). Polypeptides were visualized by staining with Coomassie Blue. Lane 1 received 50 g of protein from E. coli harboring the control (no cDNA insert) plasmid; lanes 2-4 contained 50, 50, and 25 g of protein, respectively, from E. coli transformed with recombinant pRSET-C that encodes the SEC-1 fusion protein (apparent M r 25,000). Samples applied to lanes 1 and 2 were obtained from uninduced E. coli; samples in lanes 3 and 4 were from E. coli that were induced with 1 mM IPTG. B, The SEC-1 fusion protein from IPTG-induced E. coli was dissolved and purified, via nickel chelate affinity chromatography, in buffer containing 8 M urea (see "Experimental Procedures") and "Results"). Urea was removed from replicate samples of purified fusion protein by dialysis against either 10 mM sodium phosphate, pH 7.4, 0.15 M NaCl or 50 mM sodium acetate, pH 4.9. Dialyzed samples were centrifuged at 30,000 ϫ g for 10 min, and supernatant and pellet fractions were collected. After boiling in SDS loading buffer, aliquots (25 l) of these fractions were analyzed by denaturing electrophoresis as described above. Lanes 1 and 2 contained the pellet and supernatant fractions, respectively, obtained by dialysis at pH 7.4; lanes 3 and 4 received the pellet and supernatant fractions, respectively, isolated after dialysis at pH 4.9. abundant as nematodes acquire the ability to reproduce (Fig.  8A, lane EL). Coomassie Blue staining of polyacrylamide gels disclosed typical patterns and sizes of intact polypeptides at each stage of C. elegans development (data not shown). The quality and integrity of the protein samples for the larval (L1-L4) stages were also assessed in an independent immunoblot assay (Fig. 8B). Antibodies directed against the catalytic subunit of C. elegans protein kinase A (54), a nonabundant protein unrelated to SEC-1, were used. Fig. 8B shows that the 40-kDa kinase accumulates in L2-L4 larvae, whereas embryos lack the enzyme. These results replicate published data (54) and confirm that the absence of signals for SEC-1 in larvae is not due to artifactual degradation of proteins.
Distribution of SEC-1 mRNA and Protein in Situ-In situ hybridization analysis was used to monitor SEC-1 mRNA accumulation in embryos and adult nematodes. C. elegans engaged in early and middle phases of embryogenesis stain in-tensely for SEC-1 mRNA (Fig. 9A, arrows). SEC-1 mRNA content declines precipitously to background levels as the embryos undergo morphogenesis (elongation and folding) to generate L1 larvae (Fig. 9A, arrowheads). Immunofluorescence microscopy demonstrated that SEC-1 protein is abundant and widely distributed in ellipsoid embryos that have not yet entered the postproliferative, morphogenesis stage (Fig. 9C, arrows). Animals at later stages of embryogenesis (Fig. 9C, arrowheads) have markedly lower or undetectable amounts of SEC-1 protein. In adult nematodes SEC-1 mRNA is highly expressed in the gonadal arms (developing oocytes) (Fig. 9B). The transcript is not detected in any other cells of adult C. elegans.
SEC-1 Confers Thermotolerance on E. coli-The recombinant from embryos (E) and L2-L4 larvae were assayed for the expression of the 40-kDa catalytic subunit of protein kinase A by Western immunoblot analysis as described previously (46). The lane marked Std received 60 ng of purified catalytic subunit.

FIG. 9. Detection of SEC-1 mRNA and protein in situ.
For A and B, C. elegans were fixed, permeabilized, and then hybridized with digoxigenin-labeled, SEC-1 antisense DNA as described previously (55). RNA-DNA complexes were visualized by incubating the specimens serially with antidigoxigenin IgGs coupled to alkaline phosphatase and a chromogenic substrate. Alkaline phosphatase catalyzes the synthesis of an insoluble reaction product (shown as a black precipitate) in cells expressing SEC-1 mRNA. A, immature embryos (arrows) stain intensely for SEC-1 mRNA. Embryos that have undergone morphological transitions that result in the development of the classical C. elegans body shape (arrowheads) have low or undetectable levels of SEC-1 mRNA. B, L4 larvae and adult (not shown) C. elegans selectively express SEC-1 mRNA in developing oocytes in the gonad. Specimens in A and B were photographed using Nomarski interference optics with a Zeiss Axioscop microscope as described previously (40). C, C. elegans embryos were fixed, permeabilized, and incubated with antibodies directed against SEC-1 as described previously (45). Subsequently, samples were incubated with fluorescein isothiocyanate-coupled goat IgGs directed against rabbit immunoglobulins (45). Fluorescence signals corresponding to SEC-1-IgG complexes were obtained with a Bio-Rad MRC 600 laser scanning confocal microscope as described previously (45). Immature embryos (arrows) contain substantial levels of SEC-1 protein, whereas nematodes undergoing morphogenesis stain weakly (arrowheads) or remain unstained (10 additional mature embryos, which are observed in this field by phase microscopy, are not visualized by immunofluorescence) .   FIG. 7. Characterization of antibodies directed against SEC-1. A, samples of highly purified recombinant SEC-1 were subjected to electrophoresis in a 0.1% SDS, 12% polyacrylamide gel and transferred to an Immobilon P membrane (Millipore Corp.) as described under "Experimental Procedures." The membrane was sequentially incubated with rabbit antiserum directed against SEC-1 (diluted 1:2,000) and peroxidase-conjugated antibodies directed against rabbit IgGs (see "Experimental Procedures"). Antigen-antibody complexes were visualized by an enhanced chemiluminescence procedure as described previously (46). The amounts of recombinant SEC-1 protein used were 0.  1 and 3) or a mixed population containing C. elegans at all developmental stages (lanes 2 and 4) were subjected to Western immunoblot analysis as described in A. Antiserum directed against SEC-1 was used at a dilution of 1:2,000; excess SEC-1 antigen (3 g) was added to the antiserum that was used to probe lanes 1 and 2. Endogenous SEC-1 exhibits a M r of 18,000 (lanes 3 and 4), in agreement with its amino acid composition (Fig. 1).
pRSET plasmid that mediates constitutive expression of recombinant SEC-1 protein in E. coli (see Fig. 6; see above) was exploited to test a possible functional role for the C. elegans protein. A highly diluted sample of transformed E. coli was heated at 58°C for 30 min and then incubated in standard growth medium at 28°C for 40 h. E. coli transformed with pRSET containing a cDNA encoding an unrelated protein or pRSET lacking an insert were used as controls. Bacteria that accumulated the SEC-1 protein (Fig. 6) grew to a high density after the intense thermal stress (Table I). E. coli transformed with the control plasmids did not survive. The ability of SEC-1 to protect E. coli from heat shock demonstrates that the C. elegans protein can perform a chaperone-associated function in intact cells.
SEC-1 Is Essential for the Progression of Embryogenesis-Expression of the SEC-1 polypeptide during embryogenesis was inhibited by introducing into oocytes antisense DNA oligonucleotides, which complement codons near the 5Ј-end of SEC-1 mRNA. Oligonucleotides were microinjected into the gonadal syncytium at a site where multiple oocyte nuclei share a common cytoplasm. The subsequent generation of oocyte plasma membranes during a later step in oogenesis permanently sequesters the antisense oligonucleotides in numerous individual oocytes. Inspection of embryos produced (as externally deposited eggs) by injected animals revealed that inhibition of SEC-1 expression yielded aborted embryos that are composed of a disorganized mass of cells (Fig. 10, A and B). Injection of sense oligonucleotides had no effect on embryogenesis (Fig. 10C). Thus, SEC-1 appears to mediate an essential function(s) that is crucial for the normal progression of embryogenesis.

DISCUSSION
The C. elegans sec-1 gene encodes a novel 18-kDa polypeptide that is selectively expressed during embryogenesis. SEC-1 mRNA is produced in the maternal gonad and packaged into oocytes. SEC-1 protein is abundant during the time period when a fertilized oocyte rapidly develops into an embryo containing a precisely arranged constellation of ϳ550 cells. Since most C. elegans genes are transcriptionally silent during early embryogenesis (28), it is probable that all or most of the SEC-1 protein is derived from translation of maternally synthesized mRNA molecules. However, the possibility that early sec-1 gene transcription augments SEC-1 mRNA and protein content in embryos has not been excluded.
The size and structural properties of SEC-1 suggest that the 18-kDa protein is a member of the small chaperone/sHsp superfamily. The central core region (residues 45-126) of SEC-1 shares high levels of sequence identity with analogous segments of both mammalian chaperones and four C. elegans sHSP proteins. Similarities among SEC-1, chaperones, and nematode heat shock proteins include motifs that promote correct refolding of denatured proteins in stressed cells (5). The evolutionary conservation of structural features is consistent with the idea that C. elegans SEC-1 mediates protein folding and/or inhibits polypeptide aggregation in vivo (8,18,19). Furthermore, SEC-1 confers thermotolerance, a chaperone-associated function (1)(2)(3)(4)(5), on E. coli (Table I). This parallels the ability of mammalian Hsp27 and crystallin transgenes to protect various cells and organisms against heat and chemical stresses (9 -12).
Results in this paper indicate that physiological roles of SEC-1 are not redundant with functions performed by C. elegans HSPs 16 -1, 16 -2, 16 -41 and 16 -48. Several structural features suggest a functional divergence between SEC-1 and other chaperones. Sequences at the NH 2 and COOH termini of SEC-1 are not highly related to sequences in any other proteins. The four C. elegans HSP16 isoforms are acidic proteins with pI values in the range of 5-6. In contrast, SEC-1 is a basic polypeptide that is highly enriched in His residues. Thus, SEC-1 contains distinct subsets of amino acid side chains that may generate novel higher order structures and/or surfaces. Such domains could bind proteins that are not complexed by HSP16 isoforms or large chaperones.
Aspects of the organization and regulation of the C. elegans sec-1 gene are unique. Like genes encoding the C. elegans HSP16 isoforms, the sec-1 gene contains a single intron. However, the sec-1 intron is inserted within codon 93. This generates two exons that encode similarly sized segments of the conserved central core region of the SEC-1 protein (Figs. 1-3). The nematode hsp16 genes encode the entire conserved core domain in a single exon (29,30). This leads to speculation that the C. elegans sec-1 and hsp16 genes evolved from an intronless ancestor gene and acquired intervening sequences after they diverged.
During the course of our studies, the DNA sequence corresponding to the sec-1 gene was deposited in the EMBL data base (Accession number Z35640) by the C. elegans Genome Sequencing Project (56). The sequence determined for the sec-1 structural gene and its associated 5Ј-and 3Ј-flanking DNA in this laboratory and the data provided by the sequencing project are in complete agreement. No other information on the structure, regulation, and function of the sec-1 gene or SEC-1 mRNA and protein has been previously published or deposited in data bases.
The four C. elegans hsp16 genes are organized as tandem head to head pairs (hsp16 -1 linked to hsp16 -48; hsp16 -2  10. Effects of SEC-1 antisense oligonucleotides on embryonic development. Sense and antisense SEC-1 oligonucleotides were injected into the gonadal syncytium (oocytes) of C. elegans; progeny derived from the injected nematodes were examined by Nomarski interference microcopy, using a magnification factor of 1000 (see "Experimental Procedures"). C. elegans that were injected with SEC-1 antisense oligonucleotides produced non-viable embryos that contained either small (A) or large (B) masses of disorganized cells when development ceased. These embryos failed to undergo morphogenesis and did not hatch. C. elegans injected with SEC-1 sense oligonucleotides generated embryos indistinguishable from wild type embryos (28). These embryos (C) subsequently progressed through L1 larval morphogenesis and then hatched and developed into wild type adult C. elegans. paired with hsp16 -41) that share overlapping promoter regions (29,30). Transcription of each hsp16 gene is controlled by heat-inducible promoter elements that precede TATAA boxes by ϳ20 bp (29,30). Heat stress induces hsp16 gene transcription and HSP16 isoform accumulation in virtually every somatic cell in larval and adult nematodes (31). No expression is observed in the absence of stress. In contrast, sec-1 is a single copy gene that is expressed in unstressed oocytes and embryos. The sec-1 structural gene is preceded by a TATAA box, but the 5Ј-flanking DNA lacks both cis heat-inducible promoter elements and sequences that govern cadmium-activated transcription. 4 The absence of stress-activated enhancers is confirmed by the observations that SEC-1 mRNA is not induced by elevated temperature or CdCl 2 (Fig. 5). Expression of the sec-1 gene is regulated by developmental factors (presumably transcription factors). SEC-1 mRNA is abundant in adult gonad (especially in oocytes) and in early to midstage embryos. Neither SEC-1 mRNA nor SEC-1 protein was detected in nongonadal tissues of adult C. elegans. Both macromolecules remain undetectable throughout larval development (L1-L4 larvae). Thus, the sec-1 gene and its protein product appear to be specifically adapted for functions associated with embryogenesis. SEC-1 cDNAs were discovered in a screen for cadmium (stress)-induced gene products. However, CdCl 2 does not elicit an increase in SEC-1 mRNA content. Together the physiological adaptation of C. elegans to CdCl 2 and the pattern of SEC-1 expression during development described herein provide an explanation for the paradoxical enrichment of SEC-1 transcripts in the "stress-induced" cDNA library. Nematodes adapt to CdCl 2 by ceasing to expel developing embryos (as eggs) to the external milieu. Instead, the eggs hatch internally, and the adults are destroyed by larvae shortly thereafter. In addition, larvae may have a reduced survival rate as they feed in the presence of CdCl 2 . Thus, the relative proportion of embryos in the population rises as the numbers of adult and larval C. elegans decline. Since SEC-1 mRNA is abundant in developing embryos, its concentration will increase substantially relative to its level in a control mixed population of C. elegans. Thus, SEC-1 cDNA is enriched in the library and detected by differential hybridization because it is elevated by indirect consequences of heavy metal stress.
C. elegans HSP16 isoforms are not expressed during early embryogenesis (31). Furthermore, when the hsp16 genes subsequently become competent for transcription, accumulation of HSP16 mRNAs and protein isoforms is strictly dependent upon the presence of an external heat or chemical stress (31). Thus, it is likely that the principal role of HSP16 isoforms is to protect and rescue certain nematode proteins after the organism receives an external insult. HSP16 isoforms provide a protective system that is switched on in response to external signals. The properties of the sec-1 gene are fundamentally different. Expression of the gene appears to be driven by a regulated developmental program (presumably governed by the availability of transcription factors). Moreover, introduction of SEC-1 antisense oligonucleotides into oocytes has catastrophic consequences for developing embryos (Fig. 10). Embryos arrest as a disorganized mass of cells and are unable to proceed to the larval stages. Thus, SEC-1 appears to be essential for the normal progression of embryogenesis. We propose the working hypothesis that SEC-1 is needed to facilitate high fidelity folding of large quantities of nascent polypeptides that are produced during the intense biosynthetic phase of early embryonic development. By analogy with mammalian small chaperones (10 -14) SEC-1 may also play a central role in regulating the assembly and disassembly of cytoskeleton as embryonic cells rapidly proliferate and differentiate. Thus, SEC-1 may be ex-pressed to prevent, rather than respond to, biological stress. SEC-1 may function independently or in concert with C. elegans Hsp60 and Hsp70 chaperones. C. elegans genes encoding the larger chaperones have been cloned and characterized (50,51), but the expression and physiological roles of the Hsp60 and Hsp70 proteins during embryogenesis have not been systematically explored. Hsp70 is not normally expressed in Drosophila embryos. Moreover, forced constitutive expression of this chaperone during early embryogenesis resulted in lethality (4). Whether SEC-1 functions in concert with large chaperones or independently, it is the only candidate small chaperone in C. elegans for a key role in early development. Finally, in view of the potential importance of SEC-1 and a lack of knowledge regarding roles for small chaperones in mammalian development (1)(2)(3)(4)(5), it might be fruitful to search for SEC-1 homologs or analogs in higher organisms.