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Over the past decades, the heat-inducible (HI) promoter, derived from cI857 phage system has been widely utilized. However, the developments of alternative temperature-controlled promoters are limited. In this study, we explored a novel HI promoter through PCR amplification using the degenerate primer design. BLAST analysis identified this sequence as a partial pls B on Escherichia coli chromosome. Antisense RNAs targeting distinct regions of HI promoter were applied to examine the mechanism. The fluorescence of super-folder green fluorescent protein (sfGFP) driven by the HI promoter showed 5.5-fold increase from 30 to 42 °C, whereas the truncated HI yielded a trace amount of sfGFP. Among various E. coli strains, BL21 exhibited the highest fluorescence, reaching 3976 a.u. at 42 °C. Subsequently, the novel HI promoter was employed to drive T7RNA polymerase within a plasmid-driven T7 (PDT7) plasmid, serving its capability to express sfGFP and carbonic anhydrases (CA), respectively. The maximum intensity of sfGFP reached 38,702 a.u., and CA activity surged to 14286 WAU/mL in W3110 among other strains. Finally, the highest CA activity was 27,521 WAU/mL at pH 9. The promising results from the novel heat-inducible promoter-driven T7RNAP, incorporating the T7 terminator as HI-PDT7-TT, demonstrate potential for expressing more heterogeneous proteins across various chassis in the future. [Display omitted] • A new heat inducible (HI) promoter is explored for enhancing protein production. • Using HI plasmid-driven T7 system (HI-PDT7) improves 5-fold sfGFP in E. coli. • Terminator in HI-PDT7 is essential for plasmid stability and productivity. • HI-PDT7 expressing carbonic anhydrase (CA) has evaluated in different strains. • W3110 exhibits the highest CA at thermo- and alkaline conditions among all strains. [ABSTRACT FROM AUTHOR]

Background: Global warming is a major challenge for plant survival and growth. Understanding the molecular mechanisms by which higher plants sense and adapt to upsurges in the ambient temperature is essential for developing strategies to enhance plant tolerance to heat stress. Here, we designed a heat-responsive Arabidopsis thaliana reporter line that allows an in-depth investigation of the mechanisms underlying the accumulation of protective heat-shock proteins (HSPs) in response to high temperature. Methods: A transgenic Arabidopsis thaliana reporter line named "Heat-Inducible Bioluminescence And Toxicity" (HIBAT) was designed to express from a conditional heat-inducible promoter, a fusion gene encoding for nanoluciferase and d-amino acid oxidase, whose expression is toxic in the presence of d-valine. HIBAT seedlings were exposed to different heat treatments in presence or absence of d-valine and analyzed for survival rate, bioluminescence and HSP gene expression. Results: Whereas at 22 °C, HIBAT seedlings grew unaffected by d-valine, and all survived iterative heat treatments without d-valine, 98% died following heat treatments on d-valine. The HSP17.3B promoter was highly specific to heat as it remained unresponsive to various plant hormones, Flagellin, H2O2, osmotic stress and high salt. RNAseq analysis of heat-treated HIBAT seedlings showed a strong correlation with expression profiles of two wild type lines, confirming that HIBAT does not significantly differ from its Col-0 parent. Using HIBAT, a forward genetic screen revealed candidate loss-of-function mutants, apparently defective either at accumulating HSPs at high temperature or at repressing HSP accumulation at non-heat-shock temperatures. Conclusion: HIBAT is a valuable candidate tool to identify Arabidopsis mutants defective in the response to high temperature stress. It opens new avenues for future research on the regulation of HSP expression and for understanding the mechanisms of plant acquired thermotolerance. [ABSTRACT FROM AUTHOR]

In the plant genetic transformation process, single selection by a chemical-resistant marker gene occasionally allows the proliferation of non-transgenic cells, escaping selection pressure. The additional use of a visual marker gene is effective for accurate selection. For instance, R2R3-MYB genes are used for regulating anthocyanin biosynthesis; however, constitutive Myb expression in transgenic plants is not always desirable and may cause developmental abnormalities due to excess anthocyanin accumulation. To overcome the remaining problems in the use of Myb as a visible marker, we developed T-DNA. Ipomoea batatas Myb ( IbMyb ) and Cre expression cassettes were inserted between two loxP sequences, and the hygromycin phosphotransferase ( HPT ) and green fluorescent protein ( GFP ) expression cassettes were located outside of the loxP - IbMyb - Cre - loxP region. In the developed system, IbMyb and Cre were excised from the genomes of transgenic cells using heat-inducible Cre - loxP recombination. Upon heat treatment in a general incubator, green shoots emerged from purple tobacco transgenic calli that were pigmented with IbMyb expression. The excision of IbMyb from the genome of green shoots was confirmed using polymerase chain reaction (PCR) and sequencing. GFP expression was observed in the roots of the obtained green transgenic plants. We report that the system developed here operated successfully in tobacco, showing the potential to provide an easier and cheaper visual selection of transgenic cells in the genetic transformation process., Competing Interests: Conflict of interestThe authors have no conflicts of interest directly relevant to the content of this article., (© 2023 Japanese Society for Plant Biotechnology.)

A Bactrocera oleae genomic library was constructed and several genomic clones bearing hsp70 sequences were isolated. All clones were in situ hybridized on the major heat shock puff locus of the salivary gland polytene chromosomes. Restriction mapping of the isolated clones and genomic Southern hybridization indicated the presence of several putative hsp70 genes organized in a single cluster. Sequence analysis of an hsp70 transcription unit revealed a single 1905 nt long open reading frame that exhibits characteristic features of the inducible members of the HSP70 family. The presence and organization of many typical binding sites for the Heat Shock and GAGA factors suggest that the promoter of this gene is highly heat-inducible and could be used for conditional expression in transformation systems. Une banque génomique du Bactrocera oleae a été produite et plusieurs clones génomiques comprenant des séquences hsp70 ont été isolées. Tous les clones ont été employés en hybridations in situ et marquaient la principale boursouflure (« puff ») de choc thermique sur les chromosomes polytènes des glandes salivaires. La cartographie des sites de restriction sur ces clones ainsi que des hybridations Southern sur l’ADN génomique ont indiqué la présence de plusieurs gènes hsp70 putatifs organisés en un seul amas. L’analyse de la séquence nucléotidique d’une unité transcrite d’un gène hsp70 a révélé la présence d’un seul cadre de lecture de grande taille mesurant 1 905 nt et affichant les caractéristiques des membres inductibles de la famille HSP70. La présence et la disposition de plusieurs sites de liaison typiques des facteurs Heat Shock et GAGA suggère que le promoteur de ce gène est très inductible par la chaleur et pourrait s’avérer utile en vue d’une expression inductible dans des systèmes de transformation. [ABSTRACT FROM AUTHOR]

One of the key milestones that must be reached before gene therapy becomes feasible for clinical cancer treatment is to be able to regulate therapeutic gene expression. This is true for most current cancer gene therapy approaches, since the majority of therapeutic genes are toxic to both tumour and normal tissues. Among the wide array of strategies available for regulating gene expression, hyperthermia represents a unique approach. Hyperthermic regulation of gene therapy is feasible because of the widely conserved heat shock response, which allows therapeutic gene expression to be elevated to thousands of fold higher than background when temperature reaches 3–5°C over physiological temperature (37°C). In addition, because of the long history of experimental research on the use of hyperthermia as an approach for cancer therapy, it is now quite feasible to apply hyperthermia to a number of tumour sites and to achieve temperatures that are sufficient to induce a heat shock response. This review will attempt to discuss the current status of hyperthermia-regulated gene therapy, with special emphasis on hyperthermia-regulated immunogene therapy. [ABSTRACT FROM AUTHOR]