The expression of the hsp16 gene family in Caenorhabditis elegans has been examined by introducing hsp16-lacZ fusions into the nematode by transformation. Transcription of the hsp16-lacZ transgenes was totally heat-shock dependent and resulted in the rapid synthesis of detectable levels of beta-galactosidase. Although the two hsp16 gene pairs of C. elegans are highly similar within both their coding and noncoding sequences, quantitative and qualitative differences in the spatial pattern of expression between gene pairs were observed. The hsp16-48 promoter was shown to direct greater expression of beta-galactosidase in muscle and hypodermis, whereas the hsp16-41 promoter was more efficient in intestine and pharyngeal tissue. Transgenes that eliminated one promoter from a gene pair were expressed at reduced levels, particularly in postembryonic stages, suggesting that the heat shock elements in the intergenic region of an hsp16 gene pair may act cooperatively to achieve high levels of expression of both genes. Although the hsp16 gene pairs are never constitutively expressed, their heat inducibility is developmentally restricted; they are not heat inducible during gametogenesis or early embryogenesis. The hsp16 genes represent the first fully inducible system in C. elegans to be characterized in detail at the molecular level, and the promoters of these genes should find wide applicability in studies of tissue- and developmentally regulated genes in this experimental organism.

The small heat shock protein (smHSP) and ␣-crystallin genes encode a family of 12-43-kDa proteins which assemble into large multimeric structures, function as chaperones by preventing protein aggregation, and contain a conserved region termed the ␣-crystallin domain. Here we report on the structural and functional characterization of Caenorhabditis elegans HSP16-2, a 16-kDa smHSP produced only under stress conditions. A combination of sedimentation velocity, size exclusion chromatography, and cross-linking analyses on wild-type HSP16-2 and five derivatives demonstrate that the Nterminal domain but not most of the the C-terminal extension which follows the ␣-crystallin domain is essential for the oligomerization of the smHSP into high molecular weight complexes. The N terminus of HSP16-2 is found to be buried within complexes which can accommodate at least an additional 4-kDa of heterologous sequence per subunit. Studies on the interaction of HSP16-2 with fluorescently-labeled and radiolabeled actin and tubulin reveal that this smHSP possesses a high affinity for unfolded intermediates which form early on the aggregation pathway, but has no apparent substrate specificity. Furthermore, both wild-type and C-terminally-truncated HSP16-2 can function as molecular chaperones by suppressing the thermally-induced aggregation of citrate synthase. Taken together, our data on HSP16-2 and a unique 12.6-kDa smHSP we have recently characterized demonstrate that multimerization is a prerequisite for the interaction of smHSPs with unfolded protein as well as for chaperone activity.Molecular chaperones belong to a class of proteins whose function is to interact with and stabilize proteins that are partially or totally unfolded, as is the case when proteins are in the process of being synthesized, translocated across a membrane, or damaged by conditions of cellular stress. Many chaperones are expressed at higher levels during biological stresses, and are members of heat shock protein (HSP) 1 families (1-4). Whereas some chaperones (HSP70, HSP40, and HSP60) are involved in protein folding under normal conditions in vivo (5-7), others such as HSP104 (8 -10), inducible HSP70s (11), and small HSPs (12-15) are known to play important roles in protecting organisms from stress.The small HSPs (smHSPs) form a structurally divergent protein family with members present in Archaea, Bacteria, and Eukarya (16, 17). The presence of an evolutionarily conserved ␣-crystallin domain distinguishes all smHSPs and ␣-crystallins (18 -20). This domain is preceded by an N-terminal domain, which is highly variable in size and sequence, and is followed by a short, poorly conserved C-terminal extension. Some smHSP genes contain an intron which delineates the N-terminal and ␣-crystallin domains (21, 22), and structural studies support a two-domain structure (20,23,24) consisting mostly of ␤-sheets (25, 26) for smHSPs. The C-terminal extensions of smHSPs appear relatively unstructured (27,28) and are known to undergo numerous modifications, including truncati...

Exposure to microwave radiation enhances the aggregation of bovine serum albumin in vitro in a time-and temperature-dependent manner. Microwave radiation also promotes amyloid ¢bril formation by bovine insulin at 60 ‡C. These alterations in protein conformation are not accompanied by measurable temperature changes, consistent with estimates from ¢eld modelling of the speci¢c absorbed radiation (15^20 mW kg 31 ). Limited denaturation of cellular proteins could explain our previous observation that modest heat-shock responses are induced by microwave exposure in Caenorhabditis elegans. We also show that heat-shock responses both to heat and microwaves are suppressed after RNA interference ablating heat-shock factor function. ß

Caenorhabditis elegans is a small, free‐living hermaphroditic nematode that is widely used for the investigation of basic biological phenomena at the genetic and molecular levels. The hsp16 genes in this system encode a family of stress‐inducible 16‐kDa proteins. Stable transgenic nematode lines were derived that carry fusions of the hsp16 genes to the Escherichia coli lacZ reporter gene. These transgenic strains express high levels of β‐galactosidase in the nucleus, in response to a heat shock or to a variety of chemical stressors. Agents tested to date that induce the stress response in these animals include Cd2+, Cu2+, Hg2+, Pb2+, Zn2+, AsO2−, and the herbicide paraquat. Some of these agents yield distinct tissue patterns of stress induction (e.g., Pb2+ in the posterior pharynx, Cd2+ throughout the pharynx, Hg2+ in intestine), suggesting that classification of stress agents in complex mixtures may be a useful feature of this biomonitoring system. Using a soluble β‐galactosidase substrate, an assay was developed that allows the magnitude of the stress response to be measured. Stress reporter gene induction always occurred below the LC50 of the test substance, suggesting that this assay is a more sensitive and rapid indicator of stress than current LC50 assays using Caenorhabditis elegans.

Small heat shock proteins (smHSPs) and ␣-crystallins constitute a family of related molecular chaperones that exhibit striking variability in size, ranging from 16 to 43 kDa. Structural studies on these proteins have been hampered by their tendency to form large, often dynamic and heterogeneous oligomeric complexes. Here we describe the structure and expression of HSP12.6, a member of a novel class of smHSPs from the nematode Caenorhabditis elegans. Like other members of its class, HSP12.6 possesses a conserved ␣-crystallin domain but has the shortest N-and C-terminal regions of any known smHSP. Expression of HSP12.6 is limited to the first larval stage of C. elegans and is not significantly upregulated by a wide range of stressors. Unlike other smHSPs, HSP12.6 does not form large oligomeric complexes in vivo. HSP12.6 was produced in Escherichia coli as a soluble protein and purified. Cross-linking and sedimentation velocity analyses indicate that the recombinant HSP12.6 is monomeric, making it an ideal candidate for structure determination. Interestingly, HSP12.6 does not function as a molecular chaperone in vitro, since it is unable to prevent the thermally induced aggregation of a test substrate. The structural and functional implications of these findings are discussed.