Expression, activity, and subcellular localization of the Yin Yang 1 transcription factor in Xenopus oocytes and embryos.

Yin Yang 1 (YY1) is a multifunctional transcription factor that acts as an activator, repressor, or initiator of transcription of numerous cellular and viral genes. Previous studies in tissue culture model systems suggest YY1 plays a role in development and differentiation in multiple cell types, but the biological role of YY1 in vertebrate oocytes and embryos is not well understood. Here we analyzed expression, activity, and subcellular localization profiles of YY1 during Xenopus laevis development. Abundant levels of YY1 mRNA and protein were detected in early stage oocytes and in all subsequent stages of oocyte and embryonic development through to swimming larval stages. The DNA binding activity of YY1 was detected only in early oocytes (stages I and II) and in embryos after the midblastula transition (MBT), which suggested that its potential to modulate gene expression may be specifically repressed in the intervening period of development. Experiments to determine transcriptional activity showed that addition of YY1 recognition sites upstream of the thymidine kinase promoter had no stimulatory or repressive effect on basal transcription in oocytes and post-MBT embryos. Although the apparent transcriptional inactivity of YY1 in oocytes could be explained by the absence of DNA binding activity at this stage of development, the lack of transcriptional activity in post-MBT embryos was not expected given the ability of YY1 to bind its recognition elements. Subsequent Western blot and immunocytochemical analyses showed that YY1 is localized in the cytoplasm in oocytes and in cells of developing embryos well past the MBT. These findings suggest a novel mode of YY1 regulation during early development in which the potential transcriptional function of the maternally expressed factor is repressed by cytoplasmic localization.

Glycogen synthase kinase 3beta negatively regulates both DNA-binding and transcriptional activities of heat shock factor 1.

Stress activation of heat shock factor (HSF1) involves the conversion of repressed monomers to DNA-binding homotrimers with increased transcriptional capacity and results in transcriptional up-regulation of the heat shock protein (hsp) gene family. Cells tightly control the activity of HSF1 through interactions with hsp90 chaperone complexes and through integration into a number of different signaling cascades. A number of studies have shown that HSF1 transcriptional activity is negatively regulated by constitutive phosphorylation in the regulatory domain by glycogen synthase kinase (GSK3) isoforms alpha/beta. However, previous studies have not examined the ability of GSK3 to regulate the DNA-binding activity of native HSF1 in vivo under heat shock conditions. Here we show that GSK3beta inhibits both DNA-binding and transcriptional activities of HSF1 in heat-shocked cells. Specific inhibition of GSK3 increased the levels of DNA binding and transcription after heat shock and delayed the attenuation of HSF1 during recovery. In contrast, the overexpression of GSK3beta resulted in significant reduction in heat-induced HSF1 activities. These results confirm the role of GSK3beta as a negative regulator of HSF1 transcription in cells during heat shock and demonstrate for the first time that GSK3beta functions to repress DNA binding.

Constitutive and heat-inducible heat shock element binding activities of heat shock factor in a group of filamentous fungi.

This study represents the initial characterization of the heat shock factor (HSF) in filamentous fungi. We demonstrate that HSFs from Beauveria bassiana, Metarhizium anisopliae, Tolypocladium nivea, Paecilomyces farinosus, and Verticillium lecanii bind to the heat shock element (HSE) constitutively (non-shocked), and that heat shock resulted in increased quantities and decreased mobility of HSF-HSE complexes. The monomeric molecular mass of both heat-induced and constitutive HSFs was determined to be 85.8 kDa by UV-crosslinking and the apparent molecular masses of the native HSF-HSE complexes as determined by pore exclusion gradient gel electrophoresis was 260 and 300 kDa, respectively. Proteolytic band clipping assays using trypsin and chymotrypsin revealed an identical partial cleavage profile for constitutive and heat-induced HSF-HSE complexes. Thus, it appears that both constitutive and heat-inducible complexes are formed by trimers composed of the same HSF molecule which undergoes conformational changes during heat shock. The mobility difference between the complexes was not abolished by enzymatic dephosphorylation and deglycosylation, indicating that the reduced mobility of the heat-induced HSF is probably due to a post-translational modification other than phosphorylation or glycosylation.

Multiple components of the HSP90 chaperone complex function in regulation of heat shock factor 1 In vivo.

Rapid and transient activation of heat shock genes in response to stress is mediated in eukaryotes by the heat shock transcription factor HSF1. It is well established that cells maintain a dynamic equilibrium between inactive HSF1 monomers and transcriptionally active trimers, but little is known about the mechanism linking HSF1 to reception of various stress stimuli or the factors controlling oligomerization. Recent reports have revealed that HSP90 regulates key steps in the HSF1 activation-deactivation process. Here, we tested the hypothesis that components of the HSP90 chaperone machine, known to function in the folding and maturation of steroid receptors, might also participate in HSF1 regulation. Mobility supershift assays using antibodies against chaperone components demonstrate that active HSF1 trimers exist in a heterocomplex with HSP90, p23, and FKBP52. Functional in vivo experiments in Xenopus oocytes indicate that components of the HSF1 heterocomplex, as well as other components of the HSP90 cochaperone machine, are involved in regulating oligomeric transitions. Elevation of the cellular levels of cochaperones affected the time of HSF1 deactivation during recovery: attenuation was delayed by immunophilins, and accelerated by HSP90, Hsp/c70, Hip, or Hop. In immunotargeting experiments with microinjected antibodies, disruption of HSP90, Hip, Hop, p23, FKBP51, and FKBP52 delayed attenuation. In addition, HSF1 was activated under nonstress conditions after immunotargeting of HSP90 and p23, evidence that these proteins remain associated with HSF1 monomers and function in their repression in vivo. The remarkable similarity of HSF1 complex chaperones identified here (HSP90, p23, and FKBP52) and components in mature steroid receptor complexes suggests that HSF1 oligomerization is regulated by a foldosome-type mechanism similar to steroid receptor pathways. The current evidence leads us to propose a model in which HSF1, HSP90 and p23 comprise a core heterocomplex required for rapid conformational switching through interaction with a dynamic series of HSP90 subcomplexes.

HSP90 interacts with and regulates the activity of heat shock factor 1 in Xenopus oocytes.

Transcriptional activation of heat shock genes is a reversible and multistep process involving conversion of inactive heat shock factor 1 (HSF1) monomers into heat shock element (HSE)-binding homotrimers, hyperphosphorylation, and further modifications that induce full transcriptional competence. HSF1 is controlled by multiple regulatory mechanisms, including suppression by additional cellular factors, physical interactions with HSP70, and integration into different cellular signaling cascades. However, the signaling mechanisms by which cells respond to stress and control the HSF1 activation-deactivation pathway are not known. Here we demonstrate that HSP90, a cellular chaperone known to regulate several signal transduction molecules and transcription factors, functions in the regulation of HSF1. The existence of HSF1-HSP90 heterocomplexes was shown by coimmunoprecipitation of HSP90 with HSF1 from unshocked and heat-shocked nuclear extracts, recognition of HSF1-HSE complexes in vitro by using HSP90 antibodies (Abs), and recognition of HSF1 in vivo by HSP90 Abs microinjected directly into oocyte nuclei. The functional impact of HSP90-HSF1 interactions was analyzed by using two strategies: direct nuclear injection of HSP90 Abs and treatment of cells with geldanamycin (GA), an agent that specifically blocks the chaperoning activity of HSP90. Both HSP90 Abs and GA delayed the disassembly of HSF1 trimers during recovery from heat shock and specifically inhibited heat-induced transcription from a chloramphenicol acetyltransferase reporter construct under control of the hsp70 promoter. HSP90 Abs activated HSE binding in the absence of heat shock, an effect that could be reversed by subsequent injection of purified HSP90. GA did not activate HSE binding under nonshock conditions but increased the quantity of HSE binding induced by heat shock. On the basis of these findings and the known properties of HSP90, we propose a new regulatory model in which HSP90 participates in modulating HSF1 at different points along the activation-deactivation pathway, influencing the interconversion between monomeric and trimeric conformations as well as transcriptional activation. We also put forth the hypothesis that HSP90 links HSF1 to cellular signaling molecules coordinating the stress response.

Induction of the DNA-binding and transcriptional activities of heat shock factor 1 is uncoupled in Xenopus oocytes.

The DNA-binding and transcriptional activities of the heat shock transcription factor 1 (HSF1) are repressed under normal conditions and rapidly upregulated by heat stress. Here, we tested for the ability of various stress agents to activate HSF1 in the Xenopus oocyte model system. The HSE-binding activity of HSF1 was induced by a number of chemical stresses including cadmium, aluminum, iron, mercury, arsenite, ethanol, methanol, and salicylate. HSE-binding was not induced by several stresses known to induce the synthesis of hsps in other cell types in different organisms including zinc, copper, cobalt, manganese, recovery from anoxia, UV-irradiation, and increased pH. The inability of several known inducers of the stress response to activate the HSE-binding ability of HSF1 suggests that certain stress activation pathways may be absent or inactive in oocytes. The transcriptional activity of oocyte HSF1 was induced by heat, cadmium, and arsenite, but many of the agents that induced HSE-binding failed to stimulate HSF1-mediated transcription. The apparent uncoupling of inducible HSE-binding and transcriptional activities of HSF1 under a variety of stress regimes indicates that these events are regulated by independent mechanisms in the oocyte.

Xenopus heat shock factor 1 is a nuclear protein before heat stress.

Stress-induced expression of the heat shock (hs) genes in eukaryotes is mediated by a transcription factor known as heat shock factor 1 (HSF1). HSF1 is present in a latent, monomeric form in unstressed metazoan cells and upon exposure to heat or other forms of stress is converted to an "active" trimeric form, which binds the promoters of hs genes and induces their transcription. The conversion of HSF1 to its active form is hypothesized to be a multistep process involving (i) oligomerization of HSF1, plus (ii) additional changes in its physical conformation, (iii) changes in its phosphorylation state, and for some species (iv) translocation from the cytoplasm to the nucleus. Oligomerization of HSF appears to be essential for high affinity DNA binding, but it remains unclear whether the other steps occur in all organisms or what their mechanistic roles are. In this study we have examined if heat-induced cytoplasmic-nuclear translocation of HSF1 occurs in Xenopus oocytes. We observed that germinal vesicles (nuclei) that were physically dissected from unshocked Xenopus laevis oocytes contain no HSF1 binding activity. Interestingly, in vitro heat shock treatments of isolated nuclei from unshocked oocytes activated HSF1 binding, indicating that HSF1 must have been present in the unshocked nuclei prior to isolation. Induction of HSF1 binding was not observed in enucleated oocytes. Western blot analysis using an affinity-purified polyclonal antibody made against X. laevis HSF1 showed that HSF1 is present in equal amounts in unshocked and shocked oocytes and isolated nuclei. HSF1 was not detected in enucleated oocytes. These results clearly demonstrate that HSF1 is a nuclear protein in oocytes prior to exposure to stress. In Xenopus oocytes, therefore, HSF1 translocation from the cytoplasm to the nucleus is not part of the multistep process of HSF1 activation. These results also imply that the signals and/or factors involved in HSF1 activation must have their effect in the nuclear compartment.

A conserved element in the protein-coding sequence is required for normal expression of replication-dependent histone genes in developing Xenopus embryos.

Replication-dependent histone genes in the mouse and Xenopus share a common regulatory element within the protein-encoding sequence called the CRAS alpha element (coding region activating sequence alpha) which has been shown to mediate normal expression in vivo and to interact with nuclear factors in vitro in a cell cycle-dependent manner. Thus far, the alpha element has only been studied in rodent cells in culture, and its effect on histone gene expression during development has not been determined. Here we examine the role of the alpha element in histone gene expression during Xenopus development which features a switch in histone gene expression from a replication-independent mode in oocytes to a replication-dependent mode in embryos after midblastula stage. In vivo expression experiments involving wild-type or alpha-mutant mouse H3.2 genes show that mutation of the CRAS alpha element results in a fourfold decline of expression in embryos, but does not affect expression in oocytes. Two distinct alpha sequence-specific binding activities were detected in both oocyte and embryonic extracts. A slowly migrating DNA-binding complex was present at relatively constant levels throughout development from the earliest stages of oogenesis through larval stages. In contrast, levels of a rapidly migrating complex were high in stage I and II oocytes, declined in stage II-VI oocytes, remained low in unfertilized eggs and cleavage stage embryos, and rose dramatically after the midblastula transition. The molecular masses of the factors forming the slow and rapidly migrating complexes were estimated to be approximately 110 and 85 kDa, respectively. DNA-binding activity of the 85 kDa alpha-binding factor was affected by phosphorylation, binding with higher affinity in the dephosphorylated state. The abrupt increase in DNA-binding activity of the 85-kDa alpha-binding factor at late blastula coincides with the switch to the replication-dependent mode of histone gene expression. We propose that the conserved alpha element present in the coding sequence of mouse and Xenopus core histone genes is required for normal replication-dependent histone expression in the developing Xenopus embryo.

Distinct stress-inducible and developmentally regulated heat shock transcription factors in Xenopus oocytes.

The presence of a maternal pool of heat shock factor (HSF) in Xenopus oocytes has been suggested by two lines of evidence from previous studies. First, heat shock response element (HSE)-binding activity is induced in heat-shocked eggs and embryos prior to expression of zygotic HSF. Second, expression from microinjected heat shock protein promoters in oocytes is induced upon heat shock. To date, however, endogenous oocyte HSF molecules have not been detected, nor has induction of HSE-binding activity been directly demonstrated. Here we report the detection of distinct stress-inducible and developmentally regulated HSE-binding activities of endogenous oocyte factors. Exposure of defolliculated oocytes to heat, cadmium, and arsenite resulted in the formation of an HSE-specific complex detectable by gel mobility shift assay. Induction of HSE-binding activity by each of these stressors corresponded to increased expression from a microinjected hsp70 promoter. The stress-inducible HSE-binding complex was recognized by antiserum against mammalian HSF1, but not by HSF2 antiserum, suggesting that a Xenopus homologue of HSF1 is the major component of this activity. The HSE-binding activity of HSF1 was induced by stress treatments of stage I through VI oocytes, an indication that it is responsive to stress throughout oogenesis. During recovery from heat shock, the HSF1-HSE complex rapidly declined to control levels, but was induced for prolonged periods in oocytes exposed to continuous stress, a pattern unlike the transient activation previously observed in fertilized eggs or embryos. The kinetics of HSF1 activation in oocytes suggests that a key protein(s) regulating attenuation of the stress response is present at exceedingly low levels or is somehow modified during preembryonic development. We also detected an unusual constitutive HSE-binding complex in unstressed stage I and II oocytes, but not in later stage oocytes, eggs, developing embryos, or A6 cells. This constitutive complex was unaffected by heat or chemical treatments and was not recognized by either HSF1 or HSF2 antiserum. Appearance of the constitutive HSE-binding activity during oogenesis corresponded closely with peak levels of hsp70 mRNA detected by Northern blot analysis of RNA from staged oocytes. We suggest that the constitutive HSE-binding activity in early oocytes is formed by a unique developmentally regulated heat shock factor that may play a role in the expression of heat shock proteins during early stages of oogenesis.

Preferential activation of HSF-binding activity and hsp70 gene expression in Xenopus heart after mild hyperthermia.

We have examined the effect of mild hyperthermia on the pattern of heat shock transcription factor (HSF) binding activity, heat shock protein 70 (hsp70) and hsp30 gene expression and protein denaturation in selected tissues of adult Xenopus namely, heart, hind limb muscle, eye, liver and spleen. In these studies it was found that heart tissue was the most thermally sensitive of all of the tissues examined since maintenance of adult frogs at 26 degrees C resulted in a preferential activation of HSF binding. Thus, heart has a lowered set point temperature for HSF activation compared to the other tissues examined. At 30 degrees C HSF activation was observed in all of the tissues examined. Heart HSF activation at 26 degrees C was correlated with an increase in hsp70 mRNA and Hsp70 protein accumulation. At 28 degrees C the largest amount of hsp70 and hsp30 mRNA accumulation was detected in heart and skeletal muscle compared to other tissues while hsp70 mRNA accumulation was relatively low in spleen and hsp30 mRNA accumulation was not detectable in eyes, liver and spleen. Incubation of adult frogs at 30 degrees C resulted in enhanced hsp70 and hsp30 mRNA accumulation in all of the tissues. Finally, we have used differential scanning calorimetry (DSC) to compare the temperatures at which protein denaturation occurs in heart and liver tissue. The onset of protein denaturation (T0) occurred approximately 8.5 degrees C lower in heart compared to liver. Also the midpoint of the DSC profile (T1/2) was approximately 10.4 degrees C lower in heart than in liver. Thus, heart proteins are generally more thermolabile than proteins in liver tissue. Taken together these data suggest that heart is more sensitive than the other tissues examined with respect to moderate increases in environmental temperature.

Gene activation in response to neural induction.

In order to study the molecular processes involved in the final steps of neural induction, we are investigating the transcriptional regulation of the Xenopus laevis neural cell adhesion molecule (NCAM) gene. NCAM is expressed very early during neural development and its expression is dependent on neural induction signals. Using microinjection of reporter gene constructions into fertilized frog eggs, we have shown that cloned copies of the NCAM promoter respond appropriately to neural induction signals. Examination of the Xenopus NCAM promoter reveals a number of potential regulatory elements. We have directed our attention to a small region of the promoter that is highly conserved over large evolutionary distances. This region contains an enhancer, the OZ element, and also a silencer element, the N-box. We have purified DNA DNA proteins that interact specifically with both the OZ enhancer and the N-box silencer sequences. A model suggesting a role for these regulatory factors during neural induction is presented.

Examination of the DNA sequence-specific binding properties of heat shock transcription factor in Xenopus laevis embryos.

The binding of heat shock transcription factor (HSF) to the heat shock element (HSE) is necessary for transcriptional activation of eukaryotic heat shock protein (HSP) genes. The properties of Xenopus embryo HSF were examined by DNA mobility shift analysis employing a synthetic oligonucleotide corresponding to the proximal HSE in the promoter of the Xenopus HSP70B gene. Heat shock induced activation of HSF binding in Xenopus neurulae was not affected by an inhibition of protein synthesis, indicating that the mode of activation may be posttranslational. Also, while HSF binding was activated in control Drosophila cell extracts by in vitro heat shock or other chemical treatments, HSF binding in Xenopus embryo or somatic cell extract was not. Thus, the activation of Xenopus HSE-HSF binding may occur via a different mechanism compared with Drosophila. Furthermore, we determined that the native size of heat-induced HSF in pre- and post-midblastula stage Xenopus embryos is approximately 530 kilodaltons (kDa), which corresponds to a hexamer made up of 88 kDa monomers. Finally, the slower accumulation of HSP70 mRNA to peak levels found at lower heat shock temperatures was not correlated with HSE-HSF binding activity.

A maternal factor, OZ-1, activates embryonic transcription of the Xenopus laevis GS17 gene.

We describe the identification of an enhancer sequence and a sequence-specific DNA-binding protein required for developmental expression of the Xenopus laevis GS17 gene. Using microinjection of recombinant plasmids into fertilized frog eggs, we have shown that a 14 base pair CT-rich sequence element, normally located about 700 bases upstream of the GS17 promoter, is sufficient to activate transcription of a heterologous reporter gene in gastrula stage embryos. This regulatory element has been called the OZ sequence. Sequences closely related to OZ are located in the promoter regions of several other genes expressed during Xenopus development. Extracts prepared from Xenopus embryos show the presence of a DNA-binding factor, OZ-1, that specifically recognizes the OZ sequence. Mutations within the OZ element that abolish OZ-1 binding also abolish enhancer activity. The OZ-1 factor contains at least two proteins of approximate M(r) 76 x 10(3) and 100 x 10(3). The sequence-specific binding activity accumulates during oogenesis and remains present at approximately constant levels throughout early development.

Compensatory effect of distal promoter sequences on the basal expression of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis.

The promoter sequences involved in the basal expression of a human 70-kilodalton heat shock protein (HSP70) gene during Xenopus embryogenesis were analyzed by microinjection of mutant promoters of a HSP70--chloramphenicol acetyltransferase fusion gene into fertilized eggs and following their expression during early development. While deletion of the HSP70 gene promoter to--100 base pairs (bp) did not affect basal transcription in postmidblastula stage embryos, linker-scanner mutations in the CCAAT and purine box elements blocked expression. However, extension of the 5' boundary to--188 bp restored full wild-type expression to these mutants. These results suggest that multiple redundant cis-acting regulatory elements present in the human HSP70 gene promoter can function during Xenopus embryogenesis.

Analysis of CCAAT box transcription factor binding activity during early Xenopus laevis embryogenesis.

Unfertilized eggs and pre-midblastula (MBT) stage Xenopus embryos were found to contain a large pool of maternally derived CCAAT box-binding transcription factor (CBTF). DNA mobility shift experiments using embryonic extracts prepared with either low or high salt buffers suggest that Xenopus CBTF may not interact with embryonic DNA until the late blastula stage, a time point coincident with the increase in zygotic transcription. Additionally, photoaffinity-labeling experiments revealed that both pre- and post-MBT CBTF-binding activities were composed of at least three proteins having relative molecular masses of 68, 52, and 42 kDa.

DNA sequence-specific binding activity of the heat-shock transcription factor is heat-inducible before the midblastula transition of early Xenopus development.

We have examined the activity of the Xenopus heat-shock transcription factor (HSF) in extracts from stressed and unstressed embryos at various stages of development using DNA mobility shift analysis. A specific interaction between HSF and a synthetic oligonucleotide corresponding to the proximal heat-shock element (HSE) of the Xenopus HSP70B gene was greatly enhanced in heat-shocked embryos compared to controls. HSF binding was inducible at all developmental stages examined including pre-midblastula transition (MBT) stages which are incapable of expressing HSP genes. In time-course experiments with both cleavage and neurula stage embryos, the activation of HSF binding was rapid and transient. Removal of cleavage and neurula stage embryos from heat stress resulted in a rapid loss of binding activity. The molecular mass of HSF, as determined by comparative gel electrophoresis of photoaffinity-labeled factor was 88 x 10(3) in both heat-shocked cleavage and neurula stage embryos. These experiments suggest that maternally derived HSF is stored in pre-MBT embryos in a heat-activatable form and may function in the regulation of heat-shock genes immediately after the MBT.

cis-acting sequences and trans-acting factors required for constitutive expression of a microinjected HSP70 gene after the midblastula transition of Xenopus laevis embryogenesis.

Microinjected human HSP70 promoter-chloramphenical acetyl transferase (CAT) chimeric genes are constitutively expressed immediately after the midblastula transition of Xenopus embryogenesis. Analysis of a series of 5'-deletion mutants in the HSP70 promoter revealed that sequences within 74 bases of the transcriptional start site were sufficient for strong basal activity. We investigated the role of specific sequences in the basal promoter by injecting HSP70-CAT vectors containing linker-scanner mutations in the basal elements (CCAAT, purine-rich element, GC-element, ATF/AP1, and TATA). Our data reveal that deletion of any of these cis-acting elements in the basal promoter prevents expression after the midblastula stage of development. Furthermore, we have identified specific binding activities in embryonic nuclear extracts that complex with basal promoter elements (CCAAT, ATF, and GC) of the heterologous HSP70 promoter. These trans-acting factors are detectable in nuclear extracts of early blastula embryos, and their respective binding activity increases dramatically after the midblastula transition. The expression of the human HSP70 gene after the midblastula transition of Xenopus embryogenesis requires an array of cis-acting elements, which interact with specific Xenopus transcription factors.

Heat shock-induced accumulation of ubiquitin mRNA in Xenopus laevis embryos is developmentally regulated.

Exposure of Xenopus laevis embryos to heat shock induced the accumulation of ubiquitin mRNA (size range, 1.7-3.5 kb) in a developmental stage-dependent fashion. While constitutive ubiquitin transcripts were detectable throughout development, heat shock-induced accumulation did not occur until the fine-cell blastula stage. Continuous exposure of neurulae to heat shock (33 degrees C) induced a transient accumulation of ubiquitin mRNA with peak levels occurring after 2 hr. Finally, placement of Xenopus neurulae at 22 degrees C after a 1-hr heat shock at 33 degrees C produced a decrease in ubiquitin mRNA levels to near control levels by 24 hr.

Decay of the oocyte-type heat shock response of Xenopus laevis.

Xenopus oocytes have a complex heat shock response. During transition of the oocyte into fertilized egg, the heat shock response undergoes several qualitative and quantitative changes culminating in its complete extinction. Heat shock induces oocytes to synthesize four heat shock proteins (hsps): 83, 76, 70, and 57. After ovulation, two additional proteins (hsps 22 and 16) are inducible. The heat shock response of spawned eggs can be modified by changing the ionic configuration of the external medium and by adding pyruvate and oxaloacetate to the media. Since Xenopus eggs do not synthesize mRNA, these modifications to the external medium apparently alter the utilization of preexisting messenger RNAs in protein synthesis. Artificial activation terminates inducibility of hsps 76, 57, and 16 and diminishes the hsp 70 response. Two new heat shock proteins-66 and 48-are also inducible in artificially activated eggs. Fertilization, on the other hand, terminates the heat shock response; no hsps can be induced. However, hsp 70 appears to be made constitutively in fertilized eggs. RNA blot analyses reveal that oogenic hsp 70 messenger RNA is retained in eggs and early embryos. This messenger is apparently used for heat-induced synthesis of hsp 70 before fertilization and for constitutive synthesis of hsp 70 in zygotes.