We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43°C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to Human tissue culture cells respond to heat shock, and certain other stimuli, by the induced synthesis of a small set of proteins (molecular weights 100,000, 70,000, and 37,000 [58]). The effect of heat shock on the pattern of protein synthesis in human cells is similar to that for other eucaryotic cells (reviewed in reference 55). This highly conserved response-the activation of a small number of genes and the repression of other normally active genes-has been most intensively studied in Drosophila (1). The altered protein synthetic pattern is, in general, a reflection of both the preferential transcription of heat shock genes and the selected translation of their mRNAs.The major heat shock protein synthesized by eucaryotic cells belongs to a family of 70,000-dalton proteins (HSP70). The conservation of HSP70 among species is revealed by the similar sizes, apparent isoelectric points, and tryptic peptide patterns (64). Indeed, polyclonal antibodies raised against chicken HSP70 cross-react with proteins of similar size from yeast, Drosophila, Xenopus, mice, and humans (32). In Drosophila, the HSP70 multigene family encodes two heat shock proteins, HSP68 and HSP70 (25), and three cognate proteins (27) whose synthesis occurs at normal temperature and which are not heat shock inducible. Drosophila melanogaster has five copies of the HSP70 gene, two at the 87A chromosomal locus and three at the 87C locus (25). Similarly, Saccharomyces cerevisiae contains two copies of the HSP70 gene (28).The sequence conservation of the HSP70 genes among species has been used to isolate the homologous genes from yeast (28) (31). In this study, we describe the structural features of the human HSP70 gene: the organization of the genomic clone and the location of the 5' and 3' termini of the heat shock-induced HSP70 transcript. We examine the expression of the human HSP70 gene in hamster cells and of a chimeric HSP70 bacterial chloramphenicol acetyltransferase (CAT) gene in human cells. MATERIALS AND METHODSGeneral methods. The human genomic lambda library (39) was generously provided by T. Maniatis. Approximately 5 x 105 recombinant phage were screened (22) for sequences homologous to a subclone (plasmid 232.1; 41) containing 1.1 kilobase (kb) of coding sequence adjacent to the 5' end of the Drosophila HSP70 gene. Hybridizing plaques were purified, and DNA was isolated from phage particles banded by equilibrium CsCl centrifugation (42).Genomic DNA was prepared from human placental tissue lysed with sodium dodecyl sulfate (SDS) and digested with proteinase K (8). Subclones of H3-1 were constructed with the v...

The expression of the human HSP70 gene is induced by a wide range of physiological stresses, including exposure to heat shock and heavy metals, or under nonstress conditions, such as in response to serum stimulation. We have previously demonstrated that in either case the regulated expression is at the primary level of transcription. To determine whether transcription is mediated through a single or multiple genetic elements, we have dissected the sequences upstream of the transcription start site of the human HSP70 gene by constructing chimeric genes retaining variable amounts of 5' flanking regions fused to the bacterial gene encoding chloramphenicol acetyltransferase. Transcription from the chimeric genes was determined by S1 nuclease analysis of separate stable transfectants. The sequences required for heat shock and cadmium induction lie between -107 and -68. Within this region is the sequence CTGGAATAT-TCCCG, which is identical in 12/14 positions with the heat shock element of Drosophila heat shock genes, and a separate sequence, CGNCCCGG, which is homologous to the core of the human metallothionein II metal-responsive element. The sequences required for serum-stimulated transcription are distinct from the heat shock element. The sequence CCAAT at -68 is required for high levels ofcorrectly initiated transcripts, and a purine-rich sequence, GAAGGGAAAAG, at -58 is required for serum stimulation. The human HSP70 promoter contains at least two regulatory domains-a distal domain responsive to heat shock or cadmium and a proximal domain responsive to stimulation by serum.Sequences defining eukaryotic promoters can be separated into three functional classes. The first class is enhancer elements: sequences that function independent of location or orientation and, in some cases, confer tissue specificity (1). The second class is elements that confer the basal activity of promoters and determine the start site of initiation; this class includes the "TATA box" (2-4) and the "CAAT homology" (5-9). These elements generally function in close proximity to the transcriptional start site. It is not clear whether these elements can play a role in regulation oftranscription. A third class of elements, regulatory elements, confers inducible regulation in response to a specific stimulus.Inducible promoters, such as those regulating metallothionein, mouse mammary tumor virus, interferon, and heat shock genes, have been particularly useful in studying rapid changes in transcription. Studies localizing regulatory elements responsive to specific inducers have identified the heat shock elements (HSE) for heat shock genes (10-13), metal-responsive elements (MRE) for metallothionein genes (14, 15), glucocorticoid-responsive elements (GRE) for mouse mammary tumor virus genes (16-18), and interferonregulatory elements (IRE) for the viral or poly(dI-dC) induction of a-and (3-interferon genes (19-21). One class of inducible genes with complex regulation includes the heat shock or stress-induced genes. The expression of heat shoc...

The tumor suppressor p53 is a nuclear phosphoprotein with characteristics of a transcription factor. It displays sequence-specific DNA binding, contains a potent transactivation domain, and has been implicated as both a transcriptional activator and a repressor. Transcription of the human hsp70 gene is stimulated by adenovirus E1a protein. This E1a transactivation of the hsp70 promoter is mediated by CCAAT binding factor (CBF). It is demonstrated here that p53 both represses transcription from the human hsp70 promoter and also interacts with CBF. Thus, the repression of the hsp70 promoter by p53 may be mediated by direct protein-protein interaction with CBF. These results suggest that protein-protein interaction between p53 and specific transcription factors may be an additional mechanism by which p53 regulates gene expression.

We have examined the expression of the heat shock protein (hsp7O) gene in human cells. The transcription of the hsp7O gene and accumulation of cytoplasmic hsp70 mRNA is induced by serum stimulation. Populations of HeLa cells and human embryonic kidney cells (cell line 293) were serum starved. Upon serum stimulation, the level of hsp70 mRNA transiently increases between 12 and 18 hr to a 10-fold higher level. The increased levels of hsp70 mRNA can be accounted for by a 10-to 15-fold increase in the rate of transcription of the hsp7O gene. When cells were serum-stimulated in the presence of an inhibitor of DNA synthesis, 1-p-D-arabinofuranosylcytosine (araC), the levels of hsp70 mRNA were induced to only 20% of the maximal level detected in the absence of the inhibitor. This suggests that the expression of the hsp7O gene is coupled to DNA synthesis. The cloned human hsp7O gene contains regulatory sequences that confer serum-stimulated transcriptional control. The endogenous hsp7O gene and the transfected chimeric gene containing sequences upstream of the hsp7O gene fused to bacterial chloramphenicol acetyltransferase are both temporally expressed in stable transfectants of cell line 293 cells. The endogenous hsp70 mRNA and the chimeric mRNA reach maximum levels 12-18 hr after serum stimulation.

Expression of the human gene encoding the major heat shock protein, HSP70, was induced during cell growth by serum stimulation and after infection with adenovirus 5. In this study we showed that HSP70 gene expression could be induced by adenovirus 5 infection, even in the absence of exogenous serum factors. Whereas serum stimulation induced the expression of the endogenous HSP70 gene, it had no effect on early adenovirus promoters. However, expression of both the cellular HSP70 gene and the adenovirus E3 promoter were activated during adenovirus infection. By using a collection of reconstructed mutant viruses, we identified the 13S product of the ElA region as the specific transcriptional trans-activator of the HSP70 gene.Although the sequence of events during productive adenovirus infection of human cells has been well documented (36), little is known about the events leading to adenovirusmediated cellular transformation. Genetic studies have revealed that the viral ElA gene is necessary for cellular transformation (6,14,18,22,30,32). ElA has complex effects on transcription: it stimulates transcription of early adenoviral (1,17,23,(26)(27)(28) and cellular (9,12,19,25,34,35) promoters but represses the effects of viral enhancers (4, 37). A particularly attractive hypothesis for the mechanism by which viral transforming gene products mediate cellular transformation predicts that this trans-regulatory activity may alter cellular gene expression. Specifically, those genes promoting cell growth would be stimulated, and genes restricting cell growth would be repressed. To date, four endogenous cellular genes have been shown to be responsive to ElA: a rat class I MHC gene is repressed (31), whereas a human P-tubulin gene (34), a human HSP70 gene (19, 25), and a mouse MHC H-2K gene (29) are stimulated. The induced synthesis of human HSP70 during adenovirus 5 (AdS) infection is dependent on ElA gene expression and is due to an increase in both the rate of transcription and the accumulation of HSP70 mRNA (19,25).The human HSP70 gene is a good candidate for a cellular gene that is growth regulated and induced after virus infection. Expression of human HSP70 has been shown to be stimulated during AdS infection (19,25), and HSP70 is expressed at high levels in AdS-transformed human embryonic kidney cells (cell line 293) (11,25,40 three culture conditions for Ad5 infection of HeLa cells: (i) conditioned medium, (ii) fresh medium, and (iii) serum-free medium. HeLa cells were grown to 80% confluence, and the medium was saved (conditioned medium). The cells were either mock treated or infected with AdS at a multiplicity of infection of 10 PFU per cell for 30 min in serum-feee medium. After mock or viral infection, the cells were fed either conditioned medium, fresh medium containing 10% serum, or serum-free medium. To facilitate our analysis, we used a HeLa cell line, 27-T, derived by cotransfection with the pSV2neo selectable marker and pKCAT23, in which expression of chloramphenicol acetyltransferase (CAT) is dependent on t...

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAATbinding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.Expression of the human hsp70 gene is cell cycle regulated (24) and is induced by both serum and the adenovirus Ela protein (37,39). This regulated expression is conferred by the basal promoter, whose activity is predominantly controlled by the CCAAT element at position -70 relative to the transcription initiation site (14,35,38). The CCAAT homology was originally identified as a potential cis-acting promoter element for a number of eucaryotic genes (2, 9), and since then multiple CCAAT-binding activities have been reported (1,6,7,29). Although it is difficult to assess the relationship among these activities, the data suggest that different CCAAT elements are recognized in vivo by different factors.The best-characterized CCAAT-binding proteins are CCAAT transcription factor/nuclear factor 1 (CTF/NF1) and CCAAT/enhancer-binding protein (C/EBP). CTF/NF1 is a set of HeLa cell nuclear polypeptides ranging from 52 to 66 kDa that function both as transcription factors for in vitro transcription of a-globin promoter templates and as in vitro initiation factors for adenovirus DNA replication (18). C/EBP is a heat-stable, 42-kDA (42K) rat liver nuclear protein that shows selective binding to the CCAAT homology of several viral promoters as well as to the core homology common to many viral enhancers (13,17 MATERIALS AND METHODS Bacterial expression. pGEX-N1/534 and pGEX-CBF were constructed in plasmid pGEX2T (33). pGEX-N1/534 encodes the fusion protein glutathion-S-transferase (GST)-N1/ 534, containing residues 1 to 534 of CBF, and was used to generate a rabbit polyclonal antiserum. pGEX-CBF encoded fusion protein GST-CBF, containing residues 1 to 999 of CBF, and was used for in vitro binding assays.Induction and purification of the fusion proteins were accomplished by a procedure modified from that of Smith and Johnson (33). Cells were induced with 3 mM isopropyl-3-D-thiogalactopyranoside (IPTG) for 3 h at 37°C before being harvested by centrifugation. The pellet from a 300-ml culture was resuspended in 8 ml of 8.5% sucrose-2 mM MgCl2-0.25 M Tris (pH 8.0) and lysed with 32 mg of lysozyme. Incubation on ice with 3.2 ml of 0.25 M EDTA for 30 min was done prior to the addition of 9 ml of 0.5% Triton X-100-62.5 mM EDTA-50 mM Tris (pH 8.0). Phenylmethylsulfonyl fluoride, dithiothreitol, and ethyleneglycoltetraacetic acid were added to final concentrations of 1, 10, and 20 mM, respectively. The cell lysate was cleared by centrifugation at 26,800 x g...

Bayesian inference provides a powerful approach to system identification and damage assessment for structures. The application of Bayesian method is motivated by the fact that inverse problems in structural engineering, including structural health monitoring, are typically ill-conditioned and ill-posed when using noisy incomplete data because of various sources of modeling uncertainties. One should not just search for a single ''optimal'' value for the vector of model parameters but rather attempt to describe the whole family of plausible model parameters based on measured data using a Bayesian probabilistic framework. In this article, the fundamental principles of Bayesian analysis and computation are summarized; then a review is given of recent state-of-the-art practices of Bayesian inference in system identification and damage assessment for civil infrastructure. Discussions of the benefits and deficiencies of these approaches, as well as potentially useful avenues for future studies, are also provided. Our focus is on meeting challenges that arise from system identification and damage assessment for the civil infrastructure but our presented theories also have a considerably broader applicability for inverse problems in science and technology.

The HSP70 family of heat-shock proteins constitutes the major proteins synthesized in response to elevated temperatures and other forms of stress. In eukaryotes members of the HSP70 family also include a protein similar if not identical to bovine brain uncoating ATPase and glucose-regulated proteins. An intriguing relation has been established between expression of heat-shock proteins and transformation in mammalian cells. Elevated levels of HSP70 are found in some transformed cell lines, and viral and cellular gene products that are capable of transforming cells in vitro can also stimulate transcription of HSP70 genes. To determine the organization of this complex multigene family in the human genome, we used complementary approaches: Southern analysis and protein gels of Chinese hamster-human somatic cell hybrids, and in situ hybridization to human chromosomes. We demonstrate that functional genes encoding HSP70 proteins map to human chromosomes 6, 14, 21, and at least one other chromosome.