Temperature-dependent induction of an acid-inducible stimulon of Escherichia coli in broth.

The induction of the inducible lysyl-tRNA synthetase, LysU, and the inducible lysine and arginine decarboxylases of Escherichia coli K-12 grown in AC broth to a pH of 5.5 or less is temperature dependent, being distinctly lower at 24 than at 37 degrees C. This induction does not appear to be under HtpR control.     

Induction kinetics of RNA and proteins in exponentially growing organisms.

A mathematical model of the induction kinetics of RNAs and proteins in exponentially growing organisms is derived, and the cellular concentrations of the induced macromolecules at a given time after induction are related to three parameters: the fraction of the synthesis of these macromolecules in total synthesis, the half life of the inducible macromolecules, and the generation time of the organisms. The model predicts that the concentrations of the inducible macromolecules reach one half of the maximum induction level within one generation time after the onset of the induction. The model also predicts that induction curves of proteins are parabolic when their mRNAs are short-lived, but sigmoid when they are stable. Observed induction curves of beta-galactosidase in Escherichia coli cells fit in the theoretical induction curves.     

Activation of the human beta-interferon gene requires an interferon-inducible factor.

beta-Interferon (beta-IFN) gene expression can be induced by poly(I)-poly(C) or virus, but there is considerable variation in the extent of induction between different cell lines. We characterized two poorly inducible human cell lines, HeLa and 143 thymidine kinase negative (143 tk-), to define cellular factors involved in the activation of the beta-IFN gene. We show that the deficiency in beta-IFN induction in these cells can be complemented by fusion to highly inducible mouse cells. We conclude that the human cells are deficient in a trans-acting factor required for B-IFN gene activation. The level of induction of the beta-IFN gene in HeLa and 143 tk- cells can also be increased by priming with IFN before induction. If IFN priming is carried out in the presence of cycloheximide, a approximately 200-fold increase in induction is observed. We conclude that activation of the beta-IFN gene requires an IFN-inducible factor that is only expressed at low levels in unprimed HeLa and 143 tk- cells.     

Lack of adenosine-3',5'-monophosphate receptor protein and apparent lack of expression of adenosine-3',5'-monophosphate functions in Mycobacterium smegmatis CDC 46

The presence of adenosine-3',5'-monophosphate (cAMP), adenylate cyclase and the effect of glucose on cAMP levels in Mycobacterium spp have been reported earlier. To understand the role(s) of cAMP in these organisms, the induction of various enzyme systems was studied. Beta-galactosidase and L-tryptophanase were present at low levels of activity and could not be induced. Glycerokinase was inducible but the induction was not affected by glucose. The fructose uptake system was inducible, and the induction was lowered in the presence of glucose, but cAMP could not reverse the inhibition. cAMP binding protein was not detectable under a variety of conditions. On the basis of the lack of active cAMP binding protein, a model has been proposed to explain the apparent lack of expression of cAMP function in Mycobacterium smegmatis CDC 46.     

Expression and costs of induced defense traits in Alliaria petiolata,a widespread invasive plant

We used jasmonic acid to induce first-year plants of Alliaria petiolata, a European invader that largely escapes herbivory in North America, to examine continental, population, and environmental variation in the expression and costs of induced defense traits. While absolute levels varied among populations, the induction of trypsin inhibitor activity was strong and largely uniform across five native and seven invasive populations. Trichome densities varied across populations, were absent in two of them, and only tended to be inducible by jasmonic acid. Jasmonate induction was substantially costly to leaf growth and dry biomass production, the magnitude of which varied little among populations. Continental origin of the populations explained an insignificant amount of variation in any trait. Trypsin inhibitor activity was strongly inducible across a nutrient gradient, but induction was more costly to leaf growth at low soil nutrient levels. Our results show that A. petiolata displays defense traits that are strongly inducible by jasmonic acid across populations, that jasmonate induction is substantially costly to growth with little variation among populations, and that costs of induction increase with decreased soil nutrient availability. Escaping the need to express induced defense traits and their costs in the face of reduced herbivory in introduced habitats may benefit fitness of invasive plants even in the absence of any evolutionary change in resistance in these plants.     

Large scale production of cyclohexanone monooxygenase from Escherichia coli TOP10 pQR239

The cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus NCIMB 9871 has been cloned into Escherichia coli in an L-arabinose inducible vector. The recombinant E. coli containing the L-arabinose inducible CHMO was grown at 1.5 litres under controlled conditions to determine the parameters for growth and induction. It was found that induction with 0.1% (w/v) L-arabinose at late logarithmic phase of growth and growth for a further 2.5 to 3 h gave the optimal CHMO titre ( approximately 3500 U.l(-1,) 630 U. g dry cell weight(-1)). High dissolved oxygen concentrations were shown to be deleterious to the CHMO titre. This influenced the strategy for growth and induction, and was optimal when the oxygen uptake rate was maximized but the dissolved oxygen concentration was zero. Finally, a 300 litre scale fermentation was carried out giving a total CHMO titre of >8 x 10(5) U.     

Production of recombinant proteins in <Emphasis Type="Italic">E. coli</Emphasis> by the heat inducible expression system based on the phage lambda pL and/or pR promoters

The temperature inducible expression system, based on the pL and/or pR phage lambda promoters regulated by the thermolabile cI857 repressor has been widely use to produce recombinant proteins in prokariotic cells. In this expression system, induction of heterologous protein is achieved by increasing the culture temperature, generally above 37°C. Concomitant to the overexpression of heterologous protein, the increase in temperature also causes a variety of complex stress responses. Many studies have reported the use of such temperature inducible expression system, however only few discuss the simultaneous stress effects caused by recombinant protein production and the up-shift in temperature. Understanding the integral effect of such responses should be useful to develop improved strategies for high yield protein production and recovery. Here, we describe the current status of the heat inducible expression system based on the pL and/or pR λ phage promoters, focusing on recent developments on expression vehicles, the stress responses at the molecular and physiological level that occur after heat induction, and bioprocessing factors that affect protein overexpression, including culture operation variables and induction strategies.     

CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock

Escherichia coli contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible. The cspI gene is located at 35.2 min on the E. coli chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively. Analyses of cspI-lacZ fusion constructs and the cspI mRNA revealed that cspI is cold shock inducible. The 5'-untranslated region of the cspI mRNA consists of 145 bases and causes a negative effect on cspI expression at 37 degrees C. The cspI mRNA was very unstable at 37 degrees C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15 degrees C. Taking these results together, E. coli possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10 degrees C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10 degrees C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10 degrees C).     

Role of inducible heat shock protein 70 in radiation-induced cell death

We previously demonstrated the protective effect of inducible heat shock protein 70 (Hsp70) against gamma radiation. Herein, we extend our studies on the possible role of Hsp70 to ionizing radiation-induced cell cycle regulation. The growth rate of inducible hsp70-transfected cells was 2-3 hours slower than that of control cells. Flow cytometric analysis of cells at G1 phase synchronized by serum starvation also showed the growth delay in the Hsp70-overexpressing cells. In addition, reduced cyclin D1 and Cdc2 levels and increased dephosphorylated phosphoretinoblastoma (pRb) were observed in inducible hsp70-transfected cells, which were probably responsible for the reduction of cell growth. To find out if inducible Hsp70-mediated growth delay affected radiation-induced cell cycle regulation, flow cytometric and molecular analyses of cell cycle regulatory proteins and their kinase were performed. The radiation-induced G2/M arrest was found to be inhibited by Hsp70 overexpression and reduced p21Waf induction and its kinase activity by radiation in the Hsp70-transfected cells. In addition, radiation-induced cyclin A or B1 expressions together with their kinase activities were also inhibited by inducible Hsp70, which represented reduced mitotic cell death. Indeed, hsp70 transfectants showed less induction of radiation-induced apoptosis. When treated with nocodazole, radiation-induced mitotic arrest was inhibited by inducible Hsp70. These results strongly suggested that inducible Hsp70 modified growth delay (increased G1 phase) and reduced G2/M phase arrest, subsequently resulting in inhibition of radiation-induced cell death.     

The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA

In many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis. Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild-type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis.     

Copper Contamination Impairs Herbivore Initiation of Seaweed Inducible Defenses and Decreases Their Effectiveness

Seaweed-herbivore interactions are often mediated by environmental conditions, yet the roles of emerging anthropogenic stressors on these interactions are poorly understood. For example, chemical contaminants have unknown consequences on seaweed inducible resistance and herbivore response to these defenses despite known deleterious effects of contaminants on animal inducible defenses. Here, we investigated the effect of copper contamination on the interactions between a snail herbivore and a brown seaweed that displays inducible resistance to grazing. We examined seaweed inducible resistance and its effectiveness for organisms exposed to copper at two time points, either during induction or after herbivores had already induced seaweed defenses. Under ambient conditions, non-grazed tissues were more palatable than grazed tissues. However, copper additions negated the preference for non-grazed tissues regardless of the timing of copper exposure, suggesting that copper decreased both how herbivores initiated these inducible defenses and their subsequent effectiveness. Copper decreased stimulation of defenses, at least in part, by suppressing snail grazing pressure—the cue that turns inducible defenses on. Copper decreased effectiveness of defenses by preventing snails from preferentially consuming non-grazed seaweed. Thus, contaminants can potentially stress communities by changing seaweed-herbivore interactions mediated via inducible defenses. Given the ubiquity of seaweed inducible resistance and their potential influence on herbivores, we hypothesize that copper contamination may change the impact of these resistant traits on herbivores.     

Regulation of expression of cytochromes P450 family 1 in cell cultures at different stages of tumor transformation

Xenobiotic lipophilic compounds, including carcinogenic substances and antitumor agents, are metabolized by isoforms of cytochrome P450 (CYP). The constitutive and induced expression of genes for CYP in tumors is known to decrease as compared to that in homologous normal tissue; this determines to a significant degree the higher resistance of tumors to the effects of some cytostatics. To reveal the tumor transformation stage at which changes in the level of CYP of family 1 take place, we compared the levels of mRNA expression of the genes for CYP1A1, CYP1B1, and that of intracellular proteins regulating the CYP synthesis: Ah-receptor, ARNT, and AHRR. We studied embryonic fibroblast-like cells, the same cells immortalized either by Rauscher virus or spontaneously after passage of a crisis, as well as the cells of three transformed clones (K1, K2, and K8) obtained by action of benzo(a)pyrene on the cells immortalized by Rauscher virus. The constitutive level of expression of the studied genes was revealed in all cell cultures. Benz(a)anthracene induction increased the mRNA expression level for all inducible genes (CYP1A1, 1B1, and AHRR) in the initial culture immortalized by Rauscher virus and in the transformed clone K2. In the culture of the spontaneously immortalized cells and in the transformed clone K1 there was induction only of the CYP1B1 gene. In the cell culture of transformed clone K8, induction of all the inducible genes did not occur. This absence of induction was not caused by hyper expression of the AHRR-protein which blocks induction. The results indicate that the ability of immortalized cells to induce CYP isoforms is determined not only by the property of immortality, but also by how this property was produced. It was also shown that transformed clones, in spite of their common origin, differ from each other in their induction of CYP1 isoforms. The same level of mRNA expression of the genes Ah-receptor and ARNT occurred in cells in which induction of all inducible genes took place, and in those in which induction of all inducible genes did not take place, indicating that, apart from the known participants in the transduction of the induction signal, some other, thus far unknown, factors also take part.     

Induction of adaptive response by low-dose radiation in RIF cells transfected with Hspb1 (Hsp25) or inducible Hspa (Hsp70)

An adaptive response results in a reduced effect of a high challenging dose of a stressor after a smaller, inducing dose has been applied a few hours earlier. Radiation-induced fibrosarcoma (RIF) cells did not show an adaptive response, i.e. a reduced effect from a high challenging dose (2 Gy) of a radiation after a priming dose (1 cGy) had been applied 4 or 7 h earlier, but cells of a thermoresistant clone (TR) derived from RIF cells did. Since the expression of inducible Hspa (also known as Hsp70) and Hspb1 (also known as Hsp25) was different in these two cell lines, the role of inducible Hspa and Hspb1 in the adaptive response was examined. When RIF cells were transfected with inducible Hspa or Hspb1, both radioresistance measured by clonogenic assays and a reduction of apoptosis were detected. The adaptive response was also acquired by these two cell lines. The inducible Hspa transfectant showed a more pronounced adaptive response than the Hspb1 transfectant. Based on these results, it appears that inducible Hspa and Hspb1 are at least partly responsible for the induction of the adaptive response in these cells. Moreover, when inducible Hspa or Hspb1 was transfected into RIF cells, co-regulation of the two genes was detected. Heat-shock factor (Hsf) was found to be at least partially responsible for the induction of the adaptive response in these cells.     

Involvement of poly-A in selective gene expression: suppression of enzyme induction in neural retina by inhibitors of poly-A synthesis

The hormonal induction og glutamine synthetase (GS) in embryonic neural retina involves selective accumulation of stable and active RNA templates for GS synthesis. Cordycepin, (3′-deoxyadenosine) suppresses this induction in close correlation with its inhibition of poly-adenylate (poly-A) synthesis. Similarly ethidium bromide, which also reduces poly-A synthesis, suppresses the induction of GS. Both agents inhibit GS induction by acting at a pre-translational level. The overall results suggest that formation of poly-A is required for the induction of GS; they provide the first indication of a relationship between poly-A synthesis and a specific, inducible, gene-controlled aspect of cell differentiation.     

Mix.1, a homeobox mRNA inducible by mesoderm inducers, is expressed mostly in the presumptive endodermal cells of Xenopus embryos

In frogs, mesoderm presumably derives from presumptive ectoderm by induction under the control of diffusible substances produced by the endoderm. To analyze the early phase of mesoderm induction, I have isolated cDNA copies of mRNAs induced in presumptive ectoderm by mesoderm inducing factor secreted by the XTC cell line. One of the inducible mRNAs encodes a homeodomain-containing protein that is likely to play a regulatory role in development. Mix.1 behaves as an immediate early response to induction, and its kinetics of expression suggest a major role for MBT in the control of inducible gene expression. Unexpectedly, Mix.1 is expressed mostly in the future endoderm, suggesting that endoderm may be formed by induction in a similar way as mesoderm.