Thermal Induction Research Articles

Cloning of a pair of genes encoding isoschizomeric restriction endonucleases from bacillus species: the BspEI and BspMII restriction and modification systems

The respective genes ( R− M) encoding restriction and modification systems from two Bacillus species which recognise the same nucleotide sequence, 5′-TCCGGA, have been cloned and expressed in Escherichia coli. The BspEI R-M genes were cloned on a 3.6-kb HindIII fragment, whereas the BspMII R-M genes were cloned on three contiguous HindIII fragments totalling 9.8 kb. Upon thermal induction, E. coli carrying the bspEIR clones under the control of the phage λ PL promoter, express high levels of R·BspEI (10 6 units/g wet cell paste). The bspMIIR gene, on the other hand, is only poorly expressed (about 4 × 10 3 units/g wet cell paste) following induction. Although the enzymes of both R-M systems recognize the same sequence and the restriction endonucleases (ENases) cleave DNA at the same position, the modification specified by the methyltransferases (MTases) differ. The internal cytosine is the site for M·BspMII modification (TC meCGGA), whereas the external cytosine is modified by M·BspEI (T meCCGGA). The two R-M systems probably evolved independently.

Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli

Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot. The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (lon protease- and heat shock protease-deficient). Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium. These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins.

Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli.

Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot. The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (Ion protease- and heat shock protease-deficient). Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium. These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins.

Functional analysis of the temperature-dependent expression of the barley Hvhsp17 gene promoter in monocot and dicot cell systems

The regulation of a barley heat shock gene ( Hvhsp17) has been investigated by means of transient expression assays with monocot and dicot protoplast systems. The complete 1700-bp 5′ region of Hvhsp17, and the deletions of 600 and 170 bp, respectively, were fused with the bacterial β-glucuronidase (β-GUS) marker gene, to give the following expression vectors: pHSGUS1 (−1700 pHS GUS fusion), pHSGD1 (−600 pHS GUS fusion) and pHSGD2 (−170 pHS GUS fusion). pHSGUS1 and pHSGD1 contain two heat shock elements (HSEs) located in position −238 and −168, respectively, while pHSGD2 contains only one HSE located near the TATA box. As control expression vector, plasmid pBI221 (Clontech) was used, in which the β-GUS gene is under the control of the CaMV 35S constitutive promoter. All expression vectors were transfected into protoplasts derived from leaf mesophyll cells of tobacco (dicot) and some cereal species (monocots). β-GUS activity was determined after a heat shock treatment of 1 h at 40°C. Transient expression assays showed that the promoter of Hvhsp17 was active and regulated by thermal induction only in monocot cell systems, while not expressed in tobacco cells. Analyses performed with the deletion constructs, indicated that thermal activation of β-GUS was obtained only when both the HSEs were present in the promoter region.

Mitomycin C stimulates thermally induced recombinant gene expression in Escherichia coli MC strains

The effects of mitomycin C on C1857-controlled recombinant gene expression have been explored in E. coli cultures when the drug was added simultaneously to the thermal induction. A significantly improved yield of homologous, heterologous and chimeric fusion proteins was observed in E. coli MC1061 and GE864 (a MC4100 derivative) thermoinduced cells. This feature was not detected in other E. coli strains and does not involve a gene dosage mechanism but a strain-dependent stimulation of gene expression unrelated to the RecA protease activity.

Influence of irradiation on the thermal decomposition induction period of some inorganic solids with polyvalent anions

Click to increase image sizeClick to decrease image sizeKey Words: Irradiation effectsgamma irradiationbarium bromatebarium titanyl oxalatedecompositionnucleation and growth

Overproduction, purification, and characterization of ferrochelatase from Escherichia coli.

To establish a system for overproduction of the ferrochelatase [EC 4.99.1.1] from Escherichia coli, a plasmid designated pFC3 was constructed. The 35-kDa protein was accumulated in E. coli DH5 alpha cells that harbored pFC3 to a level equal to approximately 9% of the total protein (roughly 50 mg/liter) upon thermal induction. This 35-kDa protein was identified as the ferrochelatase of E. coli by Western blotting and amino-terminal amino acid sequence analysis. The protein with ferrochelatase activity was purified from the cells by three simple steps with a yield of 17%. The optimum pH of the purified enzyme was around 8.0. The molecular weight of the enzyme was estimated to be 35-kDa from column chromatography on Sephacryl S-300, a value consistent with that estimated from SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a monomer. The isoelectric point of the enzyme was approximately 4.7. Determination of the far-ultraviolet circular dichroism spectrum allowed us to calculate the alpha-helix and beta-sheet contents of the enzyme as 10 +/- 0.2 and 39 +/- 0.2%, respectively. High-level production of the ferrochelatase from E. coli will greatly facilitate detailed structural analysis of this protein.

Propagation of phage Mu in IHF-dencient Escherichia coli in the absence of the H-NS histone-like protein

Integration host factor (IHF) is known to be required for the expression of early genes and formation of the transpososome of mutator phage Mu. Prophage Mucts62 was stably maintained at 30°C and proliferated effectively after thermal induction at 42°C in an Escherichia coli mutant defective in the histone-like H-NS and IHF proteins. No IHF activity was detected in cells lacking H-NS and IHF; cells could not be transformed with plasmid pCL1920, which is based on the pSC101 replicon whose replication requires IHF. No difference in the superhelical densities of the reporter plasmid was detected in the H-NS, IHF null mutant and parental cells. From these results it is concluded that IHF is not essential for Mu development. These results also suggest that H-NS may function as a silencer for P e operon expression and that IHF overcomes the inhibitory effect of H-NS.

The Growth Transformation of Human B Cells Involves Superinduction of hsp70 and hsp90

Epstein-Barr virus (EBV) is a latent human herpes virus associated with a range of malignant and non-malignant disorders. EBV binds to CD21 virus receptors on B lymphocytes and growth transforms these cells; in susceptible (e.g., immunodeficient) hosts such cells rapidly expand into fatal lymphomas. Virus binding and infection trigger a cascade of cellular events which are transformation prerequisite and analogous to non-oncogenic cell activation events but which differ in several quantitative or qualitative respects. Unique trans -membrane Ca 2+ currents, Na +/H + exchange, as well as tyrosine phosphorylation and p56 lck -gene induction suggest that even early on the transformation process has oncogenic specificity. In this report we describe that two additional cellular gene families, the stress proteins hsp 70 and hsp 90 , are coordinately induced at mRNA and protein levels and, quite different from hsp induction by thermal stress, this induction is dependent on EBV-induced trans-membrane Ca 2+ currents. Blockade of hsp induction prevents transformation. The kinetics and induction prerequisites set this response well apart from reported responses to thermal or viral stress protein induction. Like p56 lck , hsp induction is purely a post-receptor binding event and not dependent on expression of any viral gene. The induction kinetics, with a peak at ∼ 12-16 hr and subsequent decline to control levels, considerably extend the chronological map of elements in the CD21-dependent branch of the transformation pathway and suggest a specific role of induced hsp different from the cell cycle-related functions observed in other cell systems.

Lambda Kil-Mediated Lysis Requires the Phage Context

The λ kil gene has been shown to be responsible for premature lysis effected by addition of chloramphenicol between 15 and 20 min after thermal induction of a λ prophage. Here, we localized the kil reading frame. The kil gene, represented by λ orf47, overlaps genes cIII and gam. Expression of the plasmid-borne kil gene resulted in growth arrest, a reduction of colony-forming units and filament formation. However, kil-mediated cell lysis could not be triggered by chloramphenicol when the plasmid borne kil gene was expressed, suggesting that kil-induced cell lysis requires the phage context.

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.

Overproduction of recombinant proteins by escherichia coli — employing the combinations of two and three different types of multicopy plasmids — in a 60-litre working volume airlift tower loop reactor

Bioprocess aspects of the overproduction of recombinant proteins were investigated in a 60-litre working volume airlift tower loop reactor by monitoring the cell mass concentration ( X), total cell count (TCC), number of colony-forming units (CFU), plasmid copy numbers, and the production of EcoRI and the fusion protein (EcoRI::SpA) activity produced by Escherichia coli JM 103 carrying the combination of the expression plasmids pRTF309 + or pMTC48, the repression plasmids pcI857 or pRK248cI, and the protection plasmid pEcoR4. The investigations were carried out in shake flasks, a 1·5-litre stirred-tank reactor and in a 60-litre airlift tower loop reactor (ATLR). In the absence of the protection plasmid, the cell mass concentration X, total cell count TCC, the number of colony-forming units CFU, and the product concentrations were very low due to the toxicity of EcoRI. The combination of expression, repression and protection plasmids yielded high enzyme activities in the shake flasks and in the small stirred-tank reactor with temperature shift induction of gene expression. By applying the expression plasmid pMTC48, it was possible to compare the enzyme activities with the indiction of the gene expression by the P R promotor (by thermal induction) as well as by the P LAcUV5 promotor (by chemical induction). The optimum thermal and chemical induction conditions were evaluated. In the 1·5-litre stirred tank, the optimal inducer (isopropylthiogalactoside, IPTG) concentration was much lower (0·5 m M) than that in the 60-litre airlift tower loop reactor (4 m M). In small reactors, the enzyme activity with thermal induction was five times higher than that with chemical induction and in agreement with the ratio of the corresponding promotor strengths. In the 60-litre reactor, however, the enzyme activities with chemical induction were higher than those with thermal induction. After chemical induction, the cell mass, total cell count, and number of CFU increased. After thermal induction, the cell concentration and total cell count stagnated and the number of CFU decreased to very low values, which could explain the lower performance in this reactor.

Sensitivity of hyperthermia trial outcomes to temperature and time: Implications for thermal goals of treatment

Purpose : In previous work we have found that the cumulative minutes of treatment for which 90% of measured intratumoral temperatures (T 90) exceeded 39.5°C was highly associated with complete response of superficial tumors. Similarly, the cumulative time for which 50% of intratumoral temperatures (T 50) exceeded 41.5°C was highly associated with the presence of > 80% necrosis in soft tissue sarcomas resected after radiotherapy and hyperthermia. In the present work we have calculated the time for isoeffective treatments with T 90 = 43°C and T 50 = 43°C, respectively, using published thermal isoeffective dose formulae. The purpose of these calculations was to determine the sensitivity of treatment outcome to variations in thermal isoeffective dose. Methods and Materials : The basis for the calculations were the thermal parameters and treatment outcomes in three patient populations: 44 patients with moderate or high grade soft tissue sarcoma treated preoperatively with hyperthermia and radiation; 105 patients with superficial tumors treated with hyperthermia and radiation, and 59 patients with deep tumors treated with hyperthermia and radiation. Results : The thermal dose values calculated are strongly associated with outcome in multivariate logistic regression analysis. Simple dose-response equations result from the analysis, and we use these equations to assess the sensitivity of outcome upon variations in thermal dose. This information, in turn, allows us to estimate the number of patients required in Phase II and III trials of hyperthermia and radiation therapy. Conclusions : For regimens of 5 to 10 hyperthermia treatments, improvements in median T 90 (superficial tumors) and T 50 (deep tumors) parameters by 1.2 – 1.5°C could result in response rates high enough (compared to radiotherapy alone) to justify Phase III trials. A similar improvement in response rates would require an increase in overall duration of treatment by a factor of 3 to 5. This would be difficult to achieve while also avoiding thermal tolerance induction. Achieving these temperature goals may be possible with improvements in hyperthermia technology. Alternatively, there may be ways to increase the sensitivity of cells to temperatures that can be achieved currently, such as pH reduction or chemosensitization.

Gene expression following transfection of fish cells

Various genes containing different transcriptional regulatory elements (TRE) and the bacterial marker gene coding for chloramphenicol acetyl transferase were transfected into several fish cell lines to evaluate the efficiency of expression in comparison with mammalian cells. The CMV and RSV TRE were the most efficient non-inducible promoters in directing reporter gene expression. RSV and CMV appeared of similar potency in a stable fish cell line. The human HSP-70 promoter showed high potency in a carp and in a trout cell line after thermal induction. This promoter also induced the synthesis of human growth hormone directed by the corresponding cDNA, but not by the gene. RSV TRE was also able to drive the synthesis of bovine growth hormone when attached directly to the cDNA but not to the gene. These data suggest that non-fish gene TRE can be used to express foreign genes in fish cells or transgenic fish; however, in most cases they are relatively inefficient. The data also suggest that the translation and secretion machinery of fish cells can express efficiently foreign genes but that mammalian introns might be not processed properly in some cases.

A large decrease in heat-shock-induced proteolysis after tryptophan starvation leads to increased expression of phage lambda lysozyme cloned in Escherichia coli.

The R gene coding for phage lambda lysozyme (lambda L), cloned under the control of the PL promoter on a multicopy vector, is expressed in an Escherichia coli strain auxotrophic for tryptophan. Induction by a thermal shift after tryptophan supplementation in a culture initially brought into stationary phase by tryptophan starvation leads to highly increased expression. A thermally unstable mutant protein, difficult to obtain under standard conditions, can be easily produced by post-stationary-phase expression. It is shown that this is due to a drastic decrease in the heat-shock-induced proteolysis normally observed on thermal induction. These data are discussed in relation to our present knowledge of stringent and heat-shock responses.

Enriched sources of Escherichia coli replication proteins : The dnaG primase is a zinc metalloprotein

Primase, the product of the Escherichia coli dnaG gene, is the enzyme responsible for RNA primer synthesis on both template strands at replication forks during chromosomal DNA synthesis. The dnaG gene was modified by replacement of the natural ribosome-binding site with one complementary to the 3′ end of 16S rRNA, and then inserted downstream of tandem bacteriophage λ P R and P L promoters in the pUC9-derived vector pCE30. Following thermal induction of transcription, the resulting plasmid pPL195 directed synthesis of primase activity to levels corresponding to approx. 120000 molecules per cell. The overproduced protein was soluble and was readily purified in high yield (31 mg per 1 of culture). Purified primase was monomeric, was fully active in priming replication at the bacteriophage G4 complementary strand origin, and was shown to contain 0.92 ± 0.08 g atom of tightly-bound zinc per mol of protein. Potential zinc-binding amino-acid residues near the N-terminus of the protein were identified. Although a mutant primase lacking 27 amino acid residues from the N-terminus was partly soluble, it was completely inactive.

Diamond film synthesis on Mo in thermal RF plasma

Deposition of diamond films was performed in a sub-atmospheric thermal RF induction plasma reactor. The pressure varied from 600 to 200 mbar (60 to 20 k Pa) and the axial distance of the substrate from the plasma centre varied from 6 to 15 cm. The deposits varied from individual diamond crystallites to very smooth and homogeneous diamond films. The grain size varied from about 10 μm to between 0.01 and 0.1 μm in the finest case depending on deposition conditions. The deposition rate of the films varied between 4 and 11 μm/h.

A survey of the heat shock response in four Streptomyces species reveals two groEL-like genes and three groEL-like proteins in Streptomyces albus

A survey of the heat shock response was carried out in a series of streptomycetes. Four major heat shock proteins (HSPs) were observed in each of four species (Streptomyces albus, S. lividans, S. parvulus, S. viridochromogenes) after pulse labeling with [35S]methionine and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three corresponded to the major procaryotic HSPs Lon, DnaK, and GroEL on the basis of their apparent molecular masses (94 to 100, 70, and 56 to 58 kDa, respectively). In addition, a smaller protein (16 to 18 kDa) was detected in all species but was most dramatically induced in S. albus. Consequently, studies focused on this species. As in other procaryotic systems, thermal induction (elicited by a shift from 30 degrees C to 41 degrees C) of the 70- and 94-kDa proteins was transient and expression returned to uninduced levels after 60 min. In contrast, the 56- to 58-kDa (GroEL) and 18-kDa proteins (HSP18) remained induced for more than 2 h. Two-dimensional gel electrophoresis allowed resolution of at least eight S. albus HSPs. HSP56-58 was composed of multiple acidic protein species, whereas HSP18 appeared to be basic. In spite of these differences in their physical characteristics, the N-terminal peptide sequence of HSP18 was similar to those of GroEL-like proteins found in other organisms and identical to one of the HSP56-58 species. In fact, N-terminal amino acid analysis of the S. albus 56- to 58-kDa species showed that it was composed of two proteins that differed in 3 of 10 positions, an observation that was supported by the detection of two groEL-like genes by Southern hybridization. The amino acid sequence of one of these proteins was identical to that of HSP18. Pulse-chase experiments did not reveal evidence of posttranslational processing of either HSP56-58 or HSP18.

Mutation in the Escherichia coli htpR locus results in stabilization of recombinant expression products that are susceptible to proteolytic degradation

We compared the expression and degradation of three cloned malarial proteins in a pair of isogeneic strains of Escherichia coli that differed at the htpR locus. The htpR locus encodes an alternate σ factor necessary for the transcription of heat shock promoters. Plasmodium sequences were cloned from polymerase chain reaction-amplified DNA initiated by oligonucleotide primers that were specific for the gene coding regions to be expressed. The amplified DNA was cloned and expressed in a vector that encodes a strong T7 promoter and translation-initiation signal. The total cell yield of two of the expressed proteins was found to be increased when synthesis occurred in a E. coli htpR mutant. Pulse-chase experiments showed that the increased protein yield correlated with a decrease in the degradation of the protein in the htpR strain. A two- to seven-fold increase in the half-life of the malaria proteins was observed in the E. coli htpR − background as compared to htpR +. We found no difference in survival of the E. coli K165 htpR mutant and isogeneic parent during thermal induction. Since the synthesis of the heat shock σ factor did not significantly influence survival of E. coli and htpR expression results in increased degradation of foreign proteins, the E. coli htpR mutant was a valuable host strain for production of foreign proteins.

Pleiotropic changes resulting from depletion of Era, an essential GTP‐binding protein in Escherichia coli

Phenotypic analysis of a temperature-sensitive era mutant strain indicates that Escherichia coli cells depleted of Era undergo many physiological changes. At 43 degrees C, a completely non-permissive temperature, growth is arrested because of loss of the gene and depletion of the Era protein. Depletion of Era at 43 degrees C results in depressed synthesis of heat-shock proteins DnaK, GroEL/ES, D33.4 and C62.5, lack of thermal induction of ppGpp pool levels, and increased capacity for carbon source metabolism through the citric acid cycle. Thus, in addition to inhibition of cell growth and viability, loss of Era function results in pleiotropic changes including abnormal adaptation to thermal stress.