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This article investigates three teachers' conceptions and use of inquiry-based instructional strategies throughout a professional development program. The professional development program consisted of a 2-week summer inquiry institute and research experience in university scientists' laboratories, as well as three academic year workshops. Insights gained from an in-depth study of these three secondary teachers resulted in a model of teacher conceptions that can be used to direct future inquiry professional development. Teachers' conceptions of inquiry teaching were established through intensive case-study research that incorporated extensive classroom observations and interviews. Through their participation in the professional development experience, the teachers gained a deeper understanding of how to implement inquiry practices in their classrooms. The teachers gained confidence and practice with inquiry methods through developing and presenting their institute-developed inquiry lessons, through observing other teachers' lessons, and participating as students in the workshop inquiry activities. Data analysis revealed that a set of four core conceptions guided the teachers' use of inquiry-based practices in their classrooms. The teachers' conceptions of science, their students, effective teaching practices, and the purpose of education influenced the type and amount of inquiry instruction performed in the high school classrooms. The research findings suggest that to be successful inquiry professional development must not only teach inquiry knowledge, but it must also assess and address teachers' core teaching conceptions.

The heat shock transcription factor (HSF) is a trimer that binds to DNA containing inverted repeats of the sequence nGAAn. HSF can bind DNA with the sequence nGAAnnTTCn or with the sequence nTTCnnGAAn, with little preference for either sequence over the other. However, (nGAAnnTTCn)2 is considerably less active as a heat shock response element (HSE) than is (nTTCnnGAAn)2. The electrophoretic mobilities of DNA-protein complexes and chemical cross-linking between protein monomers indicate that the sequence (nGAAnnTTCn)2 is capable of binding a single HSF trimer. In contrast, the sequence with higher biological activity, (nTTCnnGAAn)2, is capable of binding two trimers. Thus, the ability of four-nGAAn-element HSEs to bind one or two trimers depends on the permutation with which the elements are presented. A survey of naturally occurring HSEs shows the sequence (nTTCnnGAAn)2 to be the more prevalent. We suggest that the greater ability of one permutation over the other to bind two HSF trimers accounts for the initial identification of the naturall occurring heat shock consensus sequence as a region of dyad symmetry.The eukaryotic heat shock transcription factor (HSF) is the main transcription factor responsible for expression of heat shock protein (hsp) genes during periods of stress. The activity of HSF is exquisitely sensitive to the presence of unfolded, denatured, or aberrant proteins, which appear to act as an intracellular signal to induce hsp gene transcription. It is likely that this signal acts on the posttranslational regulation of HSF activity, at least in part, through one of the heat shock proteins, hsp7O (reviewed in reference 16). Association of hsp70 with HSF appears to be necessary to sequester HSF in a form that is unable to activate transcription effectively. Any stress-induced increase in the concentration of aberrant proteins is thought to result in the titration of hsp70 and the accumulation of HSF in an active form.In vertebrate and Drosophila cells, the inactive form of HSF is a non-DNA-binding monomer (5,20,29). Heat shock causes the assembly of HSF monomers into trimers, which are then competent to bind DNA (5,20,29). In the yeast Saccharomyces cerevisiae, HSF is constitutively competent to bind DNA (11) and is trimeric even before heat shock (25).The observation that HSF is trimeric raises some interesting questions with regard to its DNA interaction. The initial analyses of natural heat shock elements (HSEs) resulted in the description of a consensus sequence, CTnGAAnnTTC nAG (17,18,27). This consensus HSE is palindromic, reminiscent of binding sites for dimeric proteins. It has more recently been suggested that functional HSEs are more properly described as inverted repeats of the sequence module nGAAn (2, 31). Within an array of nGAAn repeats, it is likely that each nGAAn element is contacted by a single HSF monomer; Perisic et al. (19) observed that repeats of two to three nGAAn elements are bound by a single trimer of Drosophila HSF, repeats of four to six elements are bound by two tri...

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The heat shock transcription factor (HSF) of the yeast Saccharomyces cerevisiae is posttranslationally modified. At low growth temperatures, it activates transcription of heat shock genes only poorly; after shift to high temperatures, it activates transcription readily. In an effort to elucidate the mechanism of this regulation, we constructed a series of HSF-VP16 fusions that join the HSF DNA-binding domain to the strong transcriptional activation domain from the VP16 gene of herpes simplex virus. Replacement of the endogenous C-terminal transcriptional activation domain with that of VP16 generates an HSF derivative that exhibits behavior reminiscent of HSF itself: low transcriptional activation activity at normal growth temperature and high activity after heat shock. HSF can thus restrain the activity of the heterologous VP16 transcriptional activation domain. To determine what is required for repression of activity at low temperature, we deleted portions of HSF from this HSF-VP16 fusion to map the regulatory domain. We also isolated point mutations that convert the HSF-VP16 fusion into a constitutive transcriptional activator. We conclude that the central, evolutionarily conserved domain of HSF, encompassing the DNA-binding and multimerization domains, contains a major determinant of temperature-dependent regulation.Stress proteins, or heat shock proteins, are highly regulated, exhibiting low-level expression at low growth temperatures, intermediate levels at intermediate temperatures, and high levels at high temperatures. For Escherichia coli and the yeast Saccharomyces cerevisiae, the response to a temperature upshift is similar: a rapid increase in expression well above the original basal level, followed by adaptation to a new basal level that is only fewfold above the original.In eukaryotes, the major regulatory protein involved in heat shock protein expression is the transcriptional activator heat shock transcription factor (HSF). Indeed, the addition of a synthetic oligonucleotide bearing the HSF-binding site (a heat shock element) suffices to bring a heterologous gene under the control of the heat shock system (19, 29; this report). Therefore, to understand the regulation of heat shock protein expression, it is necessary to know how the activity of HSF is modulated such that it stimulates different levels of transcription at different temperatures.In Drosophila (30,39) example, reference 6) or of transcription factor activity (18,26,33,36) by phosphorylation. In most of these cases, it is either known or speculated that the effect of phosphorylation is to stabilize a conformation of the protein that exhibits higher (or lower) activity. It is relatively easy to imagine that HSF, whether from humans, flies, or yeasts, undergoes a conformational change upon phosphorylation and that the new conformation exhibits higher activity in DNA binding, transcriptional activation, or both (17,38).Existing data suggest that yeast HSF almost certainly undergoes a conformational change upon temperature upshift. First,...

Fractionation of heat-shocked Drosophila melanogaster Kc cells reveals that both the small heat shock proteins (hsp28, -26, -23, and -22) and vnmentin-like intermediate filament proteins (EFPs) are abundantly represented in the nuclear fraction. Cofractionation of the IFPs with nuclei is due to the collapse of the IFP network against the nucleus upon heat shock, raising the possibility that cofractionation of the small hsps is by a similar mehanis. Indirect Immunofluorescence supports this possibility. In salivary glands, both the hsps and the IFPs are cytoplasmic after mild-to-moderate heat shocks and only enter the nucleus upon severe-indeed, lethal-shocks. Double-label experiments with Schneider line 2 cells show that the IFPs and small hsps colocalize to the some perinuclear aggregates in 70% of the cells examined. Thus, the small hsps are associated with the cytoskeleton rather than with nuclear structures.The heat shock response is a ubiquitous cellular response to physiological stress characterized by the rapid induction of a small number of heat shock proteins (hsps; reviewed in refs. 1-3). The extreme conservation of the response and of the hsps themselves suggests that these proteins carry out essential functions. However, aside from their role in the development of thermotolerance-the ability to withstand a second heat shock at an otherwise lethal temperature (4-10)-little is known about their function(s). Thus far, the most progress has been made for hsp70. In yeast, replacement of several members of the hsp7O gene family with mutant genes indicates a role for hsp70 in growth at high temperatures (11). Likewise, mutants in dnaK (the Escherichia coli counterpart ofhsp70) are temperature-sensitive for growth (12) and are unable to recover normal protein synthesis following heat shock (13). hsp70 appears to play a similar role in Drosophila (14, 15) and apparently carries out this function within the nucleus (16).As a means of addressing the function of the low molecular weight family of hsps, several investigators have examined the intracellular distribution of these proteins. Several reports have argued for an association with chromatin and nucleoli (8,17). Yet, others appear to rule out association with chromatin and suggest that these proteins are part of the nuclear matrix (18,19 (20)(21)(22)(23). This cofractionation appears to be due to the collapse of the IFP network against the nucleus within 5 min of heat shock (20-23). In order to assess the contribution of the collapse of the IF cytoskeleton on the localization of the small hsps, we have reinvestigated the intracellular distribution of the small hsps ofDrosophila by biochemical fractionation and indirect immunofluorescence. The data best support association of these proteins with the IF cytoskeleton. For heat shock, cells were incubated at 370C for 15 min prior to harvest. Cells were rinsed with Echalier's medium, concentrated to 1 ml, and labeled with 50 ,uCi (1 Ci = 37 GBq) of [35S]methionine (Amersham) at 250C or 37TC for 1 hr. T...

In vitro DNA-binding assays demonstrate that the heat shock transcription factor (HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation when stressed. This conformation, reflected in a change in electrophoretic mobility, requires that two HSF trimers be bound to DNA. Single trimers do not show this change, which appears to represent an alteration in the cooperative interactions between trimers. HSF isolated from stressed cells displays a higher propensity to adopt this altered conformation. Purified HSF can be stimulated in vitro to undergo the conformational change by elevating the temperature or by exposing HSF to superoxide anion. Mutational analysis maps a region critical for this conformational change to the flexible loop between the minimal DNA-binding domain and the flexible linker that joins the DNA-binding domain to the trimerization domain. The significance of these findings is discussed in the context of the induction of the heat shock response by ischemic stroke, hypoxia, and recovery from anoxia, all known to stimulate the production of superoxide. INTRODUCTIONSince its discovery in 1962 (Ritossa, 1962), the heat shock response has been the focus of intensive investigation, leading to significant insights into protein folding and global gene regulation. In virtually all organisms, the heat shock response is manifested as the stress-induced, rapid, and dramatic increase in synthesis rates of a small number of protein chaperones. The chaperones bind to partially unfolded proteins and act to prevent their aggregation and to facilitate their refolding. Thus, this highly conserved system serves as an intricate means to protect cells against damage resulting from environmental stress.The most common stresses that induce the heat shock response are elevated temperature and oxidative stress. The latter is particularly important medically, because it typically results from the production of superoxide anion that occurs during partial oxygen deprivation or during the recovery from anoxia that occurs upon reperfusion after ischemia. Induction of the heat shock system upon recovery from anoxia may be universal; it has been described not only in mammalian systems but in Drosophila (Ritossa, 1964) and yeast (Brazzell and Ingolia, 1984). It has been unclear, however, how reperfusion triggers the heat shock response. Superoxide is rapidly converted to hydrogen peroxide, which can stimulate the production of the highly reactive hydroxyl radical, which, in turn, causes considerable "reperfusion damage." Thus, the heat shock response could be induced directly via one or more of these reactive oxygen species, or it could be induced by the protein damage caused by the hydroxyl radical.In eukaryotes, the heat shock response depends on modulating the activity (rather than the concentration) of a transcription factor. The heat shock transcription factor (HSF) is synthesized constitutively; its activity is regulated posttranslationally. Despite this commonality, different species show remarkably dis...

Two lines of evidence derived from fusion gene constructs indicate that sequences residing in the 5'-nontranslated region of a cell cycle-dependent human H3 histone mRNA are involved in the selective destabilization that occurs when DNA synthesis is terminated. The experimental approach was to construct chimeric genes in which fragments of the mRNA coding regions of the H3 histone gene were fused with fragments of genes not expressed in a cell cycle-dependent manner. After transfection in HeLa S3 cells with the recombinant plasmids, levels of fusion mRNAs were determined by S1 nuclease analysis prior to and following DNA synthesis inhibition. When the first 20 nucleotides of an H3 histone mRNA leader were replaced with 89 nucleotides of the leader from a Drosophila heat-shock (hsp70) mRNA, the fusion transcript remained stable during inhibition of DNA synthesis, in contrast to the rapid destabilization of the endogenous histone mRNA in these cells. In a reciprocal experiment, a histone-globin fusion gene was constructed that produced a transcript with the initial 20 nucleotides of the H3 histone mRNA substituted for the human f3-globin mRNA leader. In HeLa cells treated with inhibitors of DNA synthesis and/or protein synthesis, cellular levels of this histone-globin fusion mRNA appeared to be regulated in a manner similar to endogenous histone mRNA levels. These results suggest that the first 20 nucleotides of the leader are sufficient to couple histone mRNA stability with DNA replication.The human histone genes are a moderately repeated gene family that encode the major structural proteins ofchromatin. It has been well established that histone gene expression and DNA replication are temporally and functionally coupled. The synthesis of most histone proteins (1-6) and the steadystate levels of histone mRNAs (7-12) are closely correlated with DNA synthesis in the S phase of the cell cycle. At the natural end of S phase or following inhibitor-induced termination of DNA synthesis, there is a coordinate and stoichiometric decrease in histone mRNA levels and histone protein synthesis (8)(9)(10)(11)(12)(13). The rapid loss of histone mRNA under these conditions is in contrast to minimal changes in nonhistone mRNA levels. The selective destabilization of histone mRNA during DNA synthesis inhibition is posttranscriptionally mediated; destabilization is not dependent on transcription (11) but requires protein synthesis (11-15). The cellular and molecular basis for histone mRNA turnover, however, remains unresolved.To address molecular mechanisms operative in the selective destabilization of histone mRNAs, we are attempting to identify regions of a cloned, cell cycle-dependent human H3 histone gene (16,17) that are involved in the destabilization of its transcripts. Our approach is to construct fusion genes, in which fragments ofthe mRNA coding regions ofthe cloned human H3 histone gene are fused with fragments of other genes not expressed in a cell cycle-dependent manner. After transfection into HeLa S3 cells, le...