Showing papers on "Heat shock protein published in 1989"

Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells.

Abstract: Heat shock induces in cells the synthesis of specific proteins called heat shock proteins (HSPs) and a transient state of thermotolerance. The putative role of one of the HSPs, HSP27, as a protective molecule during thermal stress has been directly assessed by measuring the resistance to hyperthermia of Chinese hamster and mouse cells transfected with the human HSP27 gene contained in plasmid pHS2711. One- and two-dimensional gel electrophoresis of [3H]leucine- and [32P]orthophosphate-labeled proteins, coupled with immunological analysis using Ha27Ab and Hu27Ab, two rabbit antisera that specifically recognize the hamster and the human HSP27 protein respectively, were used to monitor expression and inducibility of the transfected and endogenous proteins. The human HSP27 gene cloned in pHS2711 is constitutively expressed in rodent cells, resulting in accumulation of the human HSP27 and all phosphorylated derivatives. No modification of the basal or heat-induced expression of endogenous HSPs is detected. The presence of additional HSP27 protein provides immediate protection against heat shock administered 48 h after transfection and confers a permanent thermoresistant phenotype to stable transfectant Chinese hamster and mouse cell lines. Mild heat treatment of the transfected cells results in an induction of the full complement of the endogenous heat shock proteins and a small increase in thermoresistance, but the level attained did not surpass that of heat-induced thermotolerant control cells. These results indicate that elevated levels of HSP27 is sufficient to give protection from thermal killing. It is concluded that HSP27 plays a major role in the increased thermal resistance acquired by cells after exposure to HSP inducers.

hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures.

Abstract: hsp82 is one of the most highly conserved and abundantly synthesized heat shock proteins of eucaryotic cells. The yeast Saccharomyces cerevisiae contains two closely related genes in the HSP82 gene family. HSC82 was expressed constitutively at a very high level and was moderately induced by high temperatures. HSP82 was expressed constitutively at a much lower level and was more strongly induced by heat. Site-directed disruption mutations were produced in both genes. Cells homozygous for both mutations did not grow at any temperature. Cells carrying other combinations of the HSP82 and HSC82 mutations grew well at 25 degrees C, but their ability to grow at higher temperatures varied with gene copy number. Thus, HSP82 and HSC82 constitute an essential gene family in yeast cells. Although the two proteins had different patterns of expression, they appeared to have equivalent functions; growth at higher temperatures required higher concentrations of either protein. Biochemical analysis of hsp82 from vertebrate cells suggests that the protein binds to a variety of other cellular proteins, keeping them inactive until they have reached their proper intracellular location or have received the proper activation signal. We speculate that the reason cells require higher concentrations of hsp82 or hsc82 for growth at higher temperatures is to maintain proper levels of complex formation with these other proteins.

Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins

Abstract: Cultured rat embryo cells were stimulated to rapidly release a small group of proteins that included several heat-shock proteins (hsp110, hsp71, hscp73) and nonmuscle actin. The extracellular proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. Heat-shocked cells released the same set of proteins as control cells with the addition of the stress-inducible hsp110 and hsp71. Release of these proteins was not blocked by either monensin or colchicine, inhibitors of the common secretory pathway. A small amount of the glucose-regulated protein grp78 was externalized by this pathway. The extracellular accumulation of these proteins was inhibited after they were synthesized in the presence of the lysine analogue aminoethyl cysteine. It is likely that the analogue-substituted proteins were misfolded and could not be released from cells, supporting our conclusion that a selective release mechanism is involved. Remarkably, actin and the squid heat-shock proteins homologous to rat hsp71 and hsp110 are also among a select group of proteins transferred from glial cells to the squid giant axon, where they have been implicated in neuronal stress responses (Tytell et al.: Brain Res., 363:161-164, 1986). Based in part on the similarities between these two sets of proteins, we hypothesized that these proteins were released from labile cortical regions of animal cells in response to perturbations of homeostasis in cells as evolutionarily distinct as cultured rat embryo cells and squid glial cells.

Cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mRNAs.

Abstract: In heat-shocked tomato cell cultures, cytoplasmic heat shock granules (HSGs) are tightly associated with a specific subset of mRNAs coding mainly for the untranslated control proteins. This messenger ribonucleoprotein complex was banded in a CsCl gradient after fixation with formaldehyde (approximately 1.30 g/cm3). It contains all the heat shock proteins and most of the RNA applied to the gradient. During heat shock, a reversible aggregation of HSGs from 15S precursor particles can be shown. These pre-HSGs are not identical to the 19S plant prosomes. Ultrastructural analysis supports the ribonucleoprotein nature of HSGs and their composition of approximately 10-nm precursor particles. A model summarizes our results. It gives a reasonable explanation for the striking conservation of untranslated mRNAs during heat shock and may apply also to animal cells.

Human major histocompatibility complex contains genes for the major heat shock protein HSP70

Abstract: Little is known as to why a large number of human diseases are influenced by the major histocompatibility complex. In some cases, a direct involvement of the products of the polymorphic class I and class II, aas well as the less variable products of the class III, genes has been proposed. During characterization of the class III region for the presence of additional loci, we have located a duplicated locus encoding the major heat shock protein HSP70 between the complement and tumor necrosis factor genes. The HSP70 loci are 12 kilobases apart and lie 92 kilobases telomeric of the C2 gene. As HSP70 proteins have been linked with a protective role during and after cellular stress, and HSP70 analogues are often presented as antigens in bacterial and protozoal infections, this finding may have major implications with regard to the major histo-compatibility complex and associated diseases.

T cells against a bacterial heat shock protein recognize stressed macrophages.

Abstract: Heat shock proteins are evolutionarily highly conserved polypeptides that are produced under a variety of stress conditions to preserve cellular functions. A major antigen of tubercle bacilli of 65 kilodaltons is a heat shock protein that has significant sequence similarity and cross-reactivity with antigens of various other microbes. Monoclonal antibodies against this common bacterial heat shock protein were used to identify a molecule of similar size in murine macrophages. Macrophages subjected to various stress stimuli including interferon-gamma activation and viral infection were recognized by class I-restricted CD8 T cells raised against the bacterial heat shock protein. These data suggest that heat shock proteins are processed in stressed host cells and that epitopes shared by heat shock proteins of bacterial and host origin are presented in the context of class I molecules.

The molecular chaperone concept.

Abstract: Molecular chaperones are a ubiquitous family of proteins whose proposed role is to mediate the folding and assembly of other proteins into oligomeric structures. The essential function of molecular chaperones is to prevent the formation of incorrect structures which may result from the transient exposure of charged or hydrophobic surfaces normally involved in interactions between or within polypeptide chains. Such transient exposure may occur during the synthesis of polypeptides, the unfolding and refolding that occurs during their transport across membranes, the association of polypeptides made in one subcellular compartment with those made in another, changes in protein-protein interactions during the normal functioning of a complex, and recovery from stresses such as heat shock. Three classes of molecular chaperone are discussed: the nucleoplasmins, the BiP group, and the chaperonins.

A peptide binding protein having a role in antigen presentation is a member of the HSP70 heat shock family.

Abstract: The T cell recognition of globular protein antigens requires the processing and presentation of the antigen by Ia-expressing APCs. Processing is believed to involve the uptake of antigen into an acidic compartment where proteolysis occurs. The resulting peptides containing the T cell antigenic determinant are associated with Ia and presented at the cell surface to the specific T cells. The mechanisms by which antigenic peptides become associated with Ia is not known. We previously described a peptide binding protein of 72/74 x 10(3) Mr (PBP72/74) that plays a role in antigen presentation as shown by the ability of an antiserum raised in rabbits to affinity-purified PBP72/74 to block presentation of cytochrome c to a cytochrome c-specific T cell hybrid. Here we show that PBP72/74 is recognized by mAbs specific for members of the HSP70 family of proteins. In Western blots PBP72/74 is bound by mAb 7.10, specific for an evolutionarily conserved epitope of HSP proteins and by mAb N27, specific for both the constitutively expressed and inducible 72/73 x 10(3) Mr HSP70 proteins. In addition, PBP72/74 shares a second common feature of the HSP proteins, that of binding to ATP. Indeed, ATP causes the release of PBP72/74 from binding to a peptide fragment of cytochrome c (Pc 81-104) and PBP72/74 can be eluted from ATP columns by Pc 81-104. Finally, a portion of PBP72/74 is shown to be present on B cell surfaces by immunofluorescence staining. Thus, it appears that characteristics of the heat shock proteins are shared by a protein playing a role in antigen presentation, suggesting some commonality in function.

Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein.

Abstract: Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

Stress proteins may provide a link between the immune response to infection and autoimmunity

Abstract: Stress proteins are frequently the target of humoral and cell-mediated immune responses to infection. These proteins belong to highly conserved gene families and there is substantial sequence homology between antigens produced by pathogenic organisms and the corresponding proteins from mammalian cells. Human T cells from sites of infectious and autoimmune lesions proliferate in response to stress proteins, and mapping of antigenic determinants on a mycobacterial stress protein shows that both species specific and highly conserved, 'self-like', regions of the molecule can take part in immune recognition. It is proposed that the lymphocyte population induced in response to stress proteins of pathogens during infection includes cells capable of autoimmune recognition of the corresponding self protein. Local accumulation of self stress proteins--in response to viral infection, for example--may subsequently provide a stimulus for proliferation of such autoreactive lymphocytes, thereby triggering a cycle of events which may contribute to the pathological damage associated with autoimmune disease.

T lymphocytes from healthy individuals with specificity to self-epitopes shared by the mycobacterial and human 65-kilodalton heat shock protein.

Abstract: The immune response to mycobacterial pathogens comprises a significant percentage of T cells with specificity for a 65-kDa heat shock protein (hsp) which is highly conserved in bacteria and man. PBMC were activated in vitro with killed Mycobacterium tuberculosis and afterward tested for CTL activity on autologous target cells primed with 1) killed M. tuberculosis, 2) intact recombinant 65-kDa hsp of Mycobacterium bovis/M. tuberculosis; or 3) tryptic fragments of the recombinant 65-kDa hsp. Strong CTL activity was observed on targets primed with killed M. tuberculosis or with tryptic fragments of the 65-kDa hsp, but not on those primed with the intact 65-kDa hsp. M. tuberculosis activated T cells from 2/13 donors tested exerted killer activity against unprimed targets. To assess whether T cell responses were directed against self-epitopes shared by the mycobacterial and human 65-kDa hsp, four peptides of at least 10 amino acids length were synthesized corresponding to fully or almost identical regions of these molecules. Peripheral blood T cells from 8/9 individuals tested, after activation with killed M. tuberculosis, expressed strong CTL activity toward autologous targets primed with one or more of these synthetic peptides. By using HLA-DR transfected murine L cells we found that the epitopes were recognized in the context of histocompatible HLA-DR (class II) molecules. We conclude that the demonstration of T cells with specificity to self-epitopes in vitro is not indicative for autoimmune disease. However, if at certain stages of infection such T cells are activated by crossreactive microbial epitopes they could cause autoimmune responses.

A global response induced in Escherichia coli by redox-cycling agents overlaps with that induced by peroxide stress

Abstract: Escherichia coli treated with nontoxic levels of the superoxide-generating redox-cycling agents menadione and paraquat showed dramatic changes in protein composition as monitored by two-dimensional gel analysis. The distribution of proteins synthesized after treatment with these agents overlapped significantly with that seen after hydrogen peroxide treatment, and it included all the proteins in the oxyR regulon. The redox-cycling agents also elicited the synthesis of at least 33 other proteins that were not seen with hydrogen peroxide, including three heat shock proteins, the Mn-containing superoxide dismutase, the DNA repair protein endonuclease IV, and glucose-6-phosphate dehydrogenase. At least some of these redox-inducible proteins appear to be part of a specific response to intracellular superoxide. E. coli is thus equipped with a network of inducible responses against oxidative damage, controlled in multiple regulatory pathways.

Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism.

Abstract: DnaK is a major heat shock protein of Escherichia coli and has been previously reported to be essential for growth at high temperatures. We systematically investigated the role of DnaK in cellular metabolism at a wide range of growth temperatures by analyzing cellular defects caused by deletion of the dnaK gene (delta dnaK52). At intermediate temperatures (30 degrees C), introduction of the delta dnaK52 allele into wild-type cells caused severe defects in cell division, slow growth, and poor viability of the cells. delta dnaK52 mutants were genetically unstable at 30 degrees C and frequently acquired secondary mutations. At high (42 degrees C) and low (11 and 16 degrees C) temperatures the delta dnaK52 allele could only be introduced into the subpopulation of wild-type cells that had duplicated the dnaK region of their chromosome. delta dnaK52 mutants isolated at 30 degrees C were cold sensitive as well as temperature sensitive for growth. Cell division defects of delta dnaK52 mutants at 30 degrees C were largely suppressed by overproduction of the FtsZ protein, which is normally required for septation during cell division; however, slow growth and poor viability at 30 degrees C and cold sensitivity and temperature sensitivity of growth were not suppressed, indicating that delta dnaK52 mutants had additional defective cellular functions besides cell division. Images

Stress-resistance conferred by high level of bcl-2 alpha protein in human B lymphoblastoid cell.

Abstract: High levels of human bcl-2 protein(s) result in (i) the tumorigenic conversion of mouse NIH3T3 cells, (ii) the better survival of mouse myeloid cells in the absence of the required growth factor and (iii) give a growth advantage to human EBV-lymphoblastoid B cells both in low serum medium and limiting dilutions. The effect of the high levels of bcl-2 protein in EBV-B cells was further investigated. This revealed that high levels of bcl-2 alpha protein made EBV-B cells more resistant to a variety of stresses including the application of heat shock, ethanol, methotrexate and the absence of serum. Stress resistance was not observed in EBV-B cells with elevated level of c-myc protein. The mechanism of stress resistance conferred by the bcl-2 alpha protein is yet to be determined although the resistance does not seem to be the result of an increase in major heat shock proteins, hsp70 and hsp90, nor the arrest of cells in G1/G0 phase. The increased viability was observed in control transfectants but not in bcl-2 transfectants when cells are seeded at higher density in the absence of serum. Thus the improved survival of cells as a result of high levels of the bcl-2 alpha protein is not specific to the absence of growth factor but is found to occur with a variety of stresses.

Protection against streptococcal cell wall-induced arthritis by pretreatment with the 65-kD mycobacterial heat shock protein.

Abstract: We report that streptococcal cell wall (SCW)-induced arthritis in rats, a T cell-dependent chronic, erosive polyarthritis, can be prevented by pretreatment of the rats with the mycobacterial 65-kD heat shock protein. This 65-kD protein shows extensive amino acid homology with prokaryotic and eukaryotic 65-kD heat shock proteins and is a ubiquitous bacterial common antigen. Both the clinical and histopathologic manifestations of the arthritis were prevented completely when rats were pretreated with 50 micrograms of 65-kD protein intraperitoneally at 35, 25, 15, or 5 d before administration of SCW. In such protected rats, SCW-specific T cell responses were suppressed, as compared with responses in arthritic rats. Pretreatment with 65-kD protein had no effect on the production of antibodies against SCW, on a nonspecific inflammatory reaction (zymosan-induced arthritis), or on general cellular immunity in vivo (delayed type hypersensitivity reaction to a nonrelated protein antigen). Furthermore, the protection against SCW arthritis was transferable by splenic T cells to naive recipients. Our data show that pretreatment with the 65-kD mycobacterial heat shock protein protects rats against a subsequent bacterium-induced arthritis. This protection is immunologically specific and resides in the lymphoid cell population.

Protein synthesis and protein phosphorylation during heat stress, recovery, and adaptation.

Abstract: Incubating cells at elevated temperatures causes an inhibition of protein synthesis. Mild heat stress at 41-42 degrees C inhibits the fraction of active, polysomal ribosomes from greater than 60% (preheating) to less than 30%. A return to 37 degrees C leads to an increase in protein synthesis, termed "recovery." Continuous incubation at 41-42 degrees C also leads to a gradual restoration of protein synthesis (greater than 70% of ribosomes reactivated by 2-4 h), termed "adaptation". Protein synthesis inhibition and reactivation is prestressed, recovered cells that contain elevated levels of the heat stress proteins occur to the same extent and at the same rate as in "naive" cells. The adaptation response requires transcription of new RNA whereas recovery does not. A large number of phosphorylation changes are induced by severe heat stress and occur with kinetics similar to the inhibition of protein synthesis. These include phosphorylation of eukaryotic protein synthesis initiation factor (eIF)-2 alpha and dephosphorylation of eIF-4B and eIF-4Fp25 (eIF-4E). However, the extent to which the modification occurs is proportional to the severity of the stress, and, under mild (41-42 degrees C) heat stress conditions, these initiation factor phosphorylation changes do not occur. Similarly, under conditions of severe heat stress eIF-2 alpha and eIF-4B frequently recover to their prestress phosphorylation state before the recovery of protein synthesis. eIF-4E dephosphorylation likewise does not occur under mild heat stress conditions. Therefore, these changes in phosphorylation states, which are thought to be sufficient cause, are not necessary for the inhibition of protein synthesis observed.

Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation

Abstract: To test the idea that unfolded protein might act as an intracellular signal for induction of the heat shock response in Escherichia coli, we examined the synthesis of several heat shock proteins after expression of an unfolded variant of the amino-terminal domain of lambda repressor. These experiments show that expression of a single mutant protein, and not its wild-type counterpart, is sufficient to induce a heat shock-like response. In addition, by measuring the abilities of unfolded variants of differing proteolytic susceptibilities to induce heat shock protein synthesis and by monitoring heat shock protein synthesis as a function of the amount of a single unfolded protein, we show that it is the concentration of unfolded protein in the cell, and not its degradation, that is important for inducing the heat shock-like response.

Heat shock and the sorting of luminal ER proteins.

Abstract: When I arrived at the MRC Laboratory of Molecular Biology in 1981 1 was keen to study the transcriptional regulation of a protein-coding gene; I had just spent two years working with Donald Brown on 5S rRNA synthesis and wanted to try something different. I began looking for a gene that might be regulated when transiently expressed in the newly developed monkey COS cell system. There were rather few to choose from, but a good candidate was the hsp7O heat shock gene from Drosophila, which had just been shown to respond to heat shock when introduced into animal cells (Corces et al., 1981). Heat shock genes have a venerable history, having been discovered in fruit flies as early as 1962 (Ritossa, 1962), but only in 1978 did it become apparent that they existed in organisms other than flies (reviewed by Ashburner, 1982); it is now known that they are present in essentially all living cells. They encode a small family of evolutionarily conserved proteins that are expressed when cells are heated or exposed to various other stresses. Thanks to the pioneering work of Alfred Tissieres, the Drosophila heat shock genes were amongst the first protein-coding genes to be cloned (Schedl et al., 1978), and they were well characterized by 1981. More importantly, they were available: Mariann Bienz, who had started working in the Cell Biology Division with John Gurdon, had tried to express the Drosophila genes in Xenopus oocytes, but after some unpromising results had turned her attention instead to the heat shock response of the oocytes themselves. So I tried expressing the genes in COS cells, and after a few attempts found that they were indeed heat-inducible. Deletion analysis of the hsp7O promoter was an obvious approach. To my amazement, I found that I had picked an extremely simple eukaryotic promoter: when assayed in animal cells, heat-activation of the Drosophila gene required only a TATA box and a short regulatory sequence about 20 base pairs further upstream (Pelham, 1982). After gazing at the sequences of other heat shock promoters, I guessed a symmetric consensus for this regulatory sequence and begged a synthetic decameric oligonucleotide from the chemists in the Structural Studies Division of the laboratory. Synthesis was a laborious manual process at that time, and I was worried that the guess might be wrong, so I picked a sequence that consisted of two overlapping restriction sites, so that if it didn't work as a promoter element it could still be used as a cloning adaptor. In fact, when placed upstream of the TATA box of the herpes thymidine kinase gene, the synthetic sequence did confer heat inducibility, much to my relief and excitement (Pelham and Bienz, 1982). Such an experiment now seems commonplace, but it was the first time a promoter element had been identified in this way.

In vitro responses to a 65-kilodalton mycobacterial protein by synovial T cells from inflammatory arthritis patients.

Abstract: Bacterial Ag, especially those of mycobacteria, have been implicated in the pathogenesis of experimental inflammatory arthritis in rodents, while in man, reactive arthritis has a clear temporal relationship to infection with particular bacteria. To investigate the role of immune responses to bacterial Ag in inflammatory arthritis, we have examined the proliferative responses of paired synovial fluid and PBMC when stimulated with 1) suspensions of irradiated or heat-killed bacteria associated with reactive arthritis (ReA), 2) purified protein derivative, 3) a recombinant 65-kDa heat shock protein of Mycobacterium leprae. The 65-kDa Ag was stimulatory to synovial fluid mononuclear cells, but not PBMC, from patients with different arthropathies, including most of those with ReA, but also some with rheumatoid arthritis. Furthermore, the magnitude of these responses correlated more closely with responses to ReA-associated bacteria (such as Salmonella), than with responses to the mycobacterial Ag represented in purified protein derivative. These results suggest that the 65-kDa molecule, which is common to a wide range of bacteria, may be an important immunogen for the T cell-mediated immune responses within the joint in different clinically defined inflammatory arthropathies.

Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells

Abstract: Prostaglandins (PGs) A1 and J2 were found to potently suppress the proliferation of human K562 erythroleukemia cells and to induce the synthesis of a 74-kDa protein (p74) that was identified as a heat shock protein related to the major 70-kDa heat shock protein group. p74 synthesis was stimulated at doses of PGA1 and PGJ2 that inhibited cell replication, and its accumulation ceased upon removal of the PG-induced proliferation block. PGs that did not affect K562 cell replication did not induce p74 synthesis. p74 was found to be localized mainly in the cytoplasm of PG-treated cells, but moderate amounts were found also in dense areas of the nucleus after PGJ2 treatment. p74 synthesis was not necessarily associated with cytotoxicity or with inhibition of cell protein synthesis. The results described support the hypothesis that synthesis of the 70-kDa heat shock proteins is associated with changes in cell proliferation. The observation that PGs can induce the synthesis of heat shock proteins expands our understanding of the mechanism of action of these compounds whose regulatory role is well known in many physiological phenomena, including the control of fever production.

Periodic segmental anomalies induced by heat shock in the chick embryo are associated with the cell cycle

Abstract: This study provides evidence that cells destined to segment together into somites have a degree of cell division synchrony. We have measured the duration of the cell division cycle in somite and segmental plate cells of the chick embryo as 9.5 h using [3H]thymidine pulse- and-chase. Treatment of embryos with any of a variety of inhibitors known to affect the cell division cycle causes discrete periodic segmental anomalies: these anomalies appear about 6-7 somites after treatment and, in some cases, a second anomaly is observed 6 to 7 somites after the first. Since somites take 1.5 h to form, the 6- to 7- somite interval corresponds to about 9-10 h, which is the duration of the cell cycle as determined in these experiments. The anomalies are similar to those seen after heat shock of 2-day chick embryos. Heat shock and some of the other treatments induce the expression of heat-shock proteins (hsp); however, since neither the expression nor the distribution of these proteins relate to the presence or distribution of anomalies seen, we conclude that hsps are not responsible for the pattern of segmental anomalies observed. The production of periodic segmental anomalies appears to be linked to the cell cycle. A simple model is proposed, in which we suggest that the cell division cycle is involved directly in gating cells that will segment together.

Modulation of stability of the Escherichia coli heat shock regulatory factor sigma.

Abstract: The heat shock response of Escherichia coli is under the positive control of the sigma 32 protein (the product of the rpoH gene). We found that overproduction of the sigma 32 protein led to concomitant overproduction of the heat shock proteins, suggesting that the intracellular sigma 32 levels limit heat shock gene expression. In support of this idea, the intracellular half-life of the sigma 32 protein synthesized from a multicopy plasmid was found to be extremely short, e.g., less than 1 min at 37 and 42 degrees C. The half-life increased progressively with a decrease in temperature, reaching 15 min at 22 degrees C. Finally, conditions known previously to increase the rate of synthesis of the heat shock proteins, i.e., a mutation in the dnaK gene or expression of phage lambda early proteins, were shown to simultaneously result in a three- to fivefold increase in the half-life of sigma 32. Images