Comparative study of the expression of the native,modified, and hybrid <Emphasis Type="Italic">cry</Emphasis>3a genes of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in prokaryotic and eukaryotic cells

The cry3a gene of Bacillus thuringiensis was cloned. Based on sequence analysis of this gene, a modified gene, cry3aM, was constructed, which has the optimal codon composition for effective expression in eukaryotic cells. Hybrid genes cry3a-licBM2 and cry3aM-licBM2 were constructed, in which the sequences of the native and modified genes are fused with the reporter gene for thermostable lichenase in the reading frame. We have shown that the expression levels of hybrid genes cry3a-licBM2 and cry3aM-licBM2 in Escherichia coli are comparable, being 5% of those for reporter gene licBM2. In cells of a lower eukaryote Saccharomyces cerevisiae, the expression of hybrid gene cry3aM-licBM2, which contains the modified gene, considerably exceeded the level of expression of cry3a-licBM2 containing the native gene. The presence of lichenase in the composition of hybrid proteins was shown to facilitate selection and analysis of the expression level of hybrid proteins in transgenic organisms.Translated from Genetika, Vol. 41, No. 2, 2005, pp. 171–177.Original Russian Text Copyright © 2005 by Salehi Jozani, Komakhin, Piruzian.     

Expression of hybrid <Emphasis Type="Italic">cry</Emphasis>3aM–<Emphasis Type="Italic">lic</Emphasis>BM2 genes in transgenic potatoes (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>)

The full-modified Bacillus thuringiensis cry3a (cry3aM) gene was designed and synthesized for effective expression in plants. A plant expression vector pC29RBCS-leader-cry3aM–licBM2 was constructed for potato transformation. In this vector, the cry3aM sequence was fused in reading frame with a new reporter gene (licBM2) and a leader sequence for the rbcs gene. The reporter gene encoded thermostable lichenase and the leader sequence encoded a signal peptide for transporting protein product to chloroplasts. The vector contained the light-inducible promoter for rbcs gene isolated from Arabidopsis thaliana. Transgenic plants were obtained by Agrobacterium mediated transformation using microtuber explants. Transgenic plantlets were selected by kanamycin resistance and confirmed as transgenic by PCR with specific primers, evaluation of lichenase activity, and bioassay of Colorado potato beetle neonate larvae. Promoter activity assays under light induction (kinetic analysis) using lichenase activity and bioassay both showed high and stable expression of hybrid genes in transgenic plantlets. Furthermore, the presence of lichenase as a reporter protein in the composition of hybrid protein was shown to facilitate selection and analysis of the expression level of hybrid genes in transgenic plants.     

Expression of Acyl-lipid △12-desaturase Gene in Prokaryotic and Eukaryotic Cells and Its Effect on Cold Stress Tolerance of Potato

We report the expression profile of acyl-lipid Δ12-desaturase (desA) gene from Synechocystis sp. PCC6803 and its effect on cell membrane lipid composition and cold tolerance in prokaryotic (Escherichia coli) and eukaryotic (Solanum tuberosum) cells. For this purpose, a hybrid of desA and reporter gene encoding thermostable lichenase (licBM3) was constructed and used to transform these cells. The expression of this hybrid gene was measured using qualitative (Petri dish test, electrophoregram and zymogram) and quantitative methods (spectrometry and gas liquid chromatography assays). The maximum level of linoleic acid in the bacterial cells containing hybrid gene was 1.9% of total fatty acids. Cold stress tolerance assays using plant damage index and growth parameters showed that cold tolerance was enhanced in primary transgenic lines because of increased unsaturated fatty acid concentration in their lipids. The greatest content of 18:2 and 18:3 fatty acids in primary transgenic plants was observed for lines 2 (73%) and 3 (41%). Finally, our results showed that desaturase could enhance tolerance to cold stress in potato, and desaturase and lichenase retain their functionality in the structure of the hybrid protein where the enzymatic activity of target gene product was higher than in the case of reporter lichenase gene absence in the construction.     

Modification of the Sunflower Defensin SD2 Gene Sequence and Its Expression in Bacterial and Yeast Cells

To achieve broader range of the defensin antimicrobial activity, based on the sd2 gene sequence, the modified gene, sd2mod, was constructed. Hybrid genes, sd2-licBM2, licBM2-sd2, licBM2-sd2mod, and sd2mod-licBM2, in which the wild-type and modified gene sequences were fused in frame with the reporter gene encoding thermostable lichenase, were constructed. Expression of the wild-type, modified, and hybrid genes was examined in the cells of pro- and eukaryotes. It was demonstrated that these genes were efficiently expressed in the cells of lower eukaryotes, the yeast. Inhibiting effect of the SD2 and SDmod proteins as the components of the hybrid proteins, SD2-LicBM2 and SD2mod-LicBM2, on the growth of the Fusarium culmorum hyphae was similar to that of the wild-type and modified proteins. It was shown that the presence of lichenase in the hybrid proteins facilitated selection and analysis of the hybrid proteins expression in transgenic organisms.     

Comparative expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> of the native and hybrid genes for acyl-lipid Δ<Superscript>9</Superscript> desaturase

Expression of the desC gene coding for acyl-lipid Δ⁹ desaturase of thermophilic cyanobacterium Synechocystis sp. PCC6803 was studied in Escherichia coli cells. In a hybrid gene constructed (desC-licBM3), a sequence of the native acyl-lipid Δ⁹ desaturase was fused in frame with the reporter gene coding for thermostable lichenase. Lichenase contained in the hybrid protein simplified selection and analysis of the expression of membrane desaturase in the heterologous host. Comparisons of the expression for the native and hybrid genes in bacterial cells showed that lichenase remained active and thermostable in the hybrid protein, while desaturase retains the capability of introducing a double bound in the corresponding position of fatty acid residues.     

Bifunctional Reporter Genes: Construction and Expression in Prokaryotic and Eukaryotic Cells

Bifunctional reporter proteins were constructed to combine Clostridium thermocellum lichenase (LicBM2) with Aequorea victoria green fluorescent protein (GFP) or with Escherichia coli -glucuronidase (GUS). The major properties of the initial proteins were preserved in the hybrid ones: LicBM2 was active at 65°C, GFP fluoresced, and GUS hydrolyzed its substrates. LicBM2 remained active after extension of its C or N end. Bifunctional reporter systems were shown to provide a convenient tool for studying the gene expression regulation in prokaryotic (E. coli) and eukaryotic (Saccharomyces cerevisiae, mammalian) cells, advantages of one reporter compensating for drawbacks of the other.     

Thermostable lichenase as a translational reporter

Hybrid genes containing the reporter gene for thermostable lichenase and model genes recA, recA1, cry3a, cry3aM, and ssp1 were constructed. The expression of these genes was studied in prokaryotic and eukaryotic cells. The presence of lichenase in the hybrid proteins was shown to facilitate analysis of the hybrid protein expression in transgenic organisms. Owing to high relative activity and thermostability of lichenase, the activity of this enzyme can be measured by simple, rapid and sensitive qualitative and quantitative methods that do not require costly equipment and reagents. Using the zymograms method, molecular masses of the lichenase-containing hybrid proteins can be precisely estimated. This method is proposed instead of Western blotting using lichenase as a translational reporter. Our results showed that the use of thermostable lichenase as a translational reporter yields the data that are problematic to obtain using traditional methods of gene expression analysis, which is of importance for fundamental and applied research.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 30–39.Original Russian Text Copyright © 2005 by Komakhin, Abdeeva, Salehi Dzhuzani, Goldenkova, Zhuchenko.     

A thermostable Clostridium thermocellum lichenase-based reporter system for studying the gene expression regulation in prokaryotic and eukaryotic cells

A new reporter system was developed to study the gene expression regulation in prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells. The system was based on the modified bacterial lichenase gene (licBM2), which was shown to meet the requirements for a reporter. The gene product was active and did not undergo modification in heterologous hosts. Simple and sensitive methods were used to detect and to quantitate the lichenase activity. Inducible licBM2 expression was demonstrated with E. coli and yeast cells, allowing the system to be employed in dynamic studies.     

Experimental models for creation of transgenic plants resistant to stressors

Experimental models of primary potato transgenic plants that express the cry3aM-licBM2 hybrid gene were created. The molecular analysis and biotests of the experimental models allow a new system of cry genes expression in plants to be proposed. This system is based on the expression of hybrid genes containing the reporter lichenase gene sequence and the use of a light-induced promoter ensuring preferential expression of the regulated genes only in green plant tissues (leaves), the target tissues for pests, as a regulatory element. In is shown that the presence of lichenase in hybrid proteins facilitates selection and analysis of the level of expression of hybrid proteins in transgenic plants. Judging by the properties of the reporter protein lichenase in hybrid proteins, it seems possible to use this reporter system for transgene monitoring in agrocenosis, because this system is fairly simple and precise and does not need considerable material and time expenses.     

Control of gene expression in plant cells using a 434:VP16 chimeric protein

The herpes simplex virus type 1 VP16 polypeptide is a potent trans-activator of viral gene expression. We have tested the ability of the VP16 activation domain to activate gene expression in plant cells. A plasmid encoding a translational fusion between the full-length 434 repressor and the C-terminal 80 amino acids of VP16, was constructed. When expressed in Escherichia coli, the chimeric protein binds efficiently to 434-binding motifs (operators). For expression in plant cells, the chimeric activator gene was placed between the cauliflower mosaic virus (CaMV) 35S promoter and nos terminator sequences in a pUC-based plasmid. The 434 operators were placed upstream of a minimal CaMV 35S promoter linked to the E. coli gus reporter gene. This reporter-expression cassette was then incorporated into the same plasmid as the 434 cI/VP16 activator-expression cassette. Two control plasmids were also constructed, one encoding the 434 protein with no activator domain and the second a chimeric activator with no DNA-binding domain. The chimeric activator was tested for its ability to activate gene expression in a tobacco protoplast transient assay system. Results are presented to show that we can obtain in plant cells significant activation of gene expression that is dependent on both DNA-binding and the presence of the activator domain.     

Expression of Soluble Active Interferon αA in <Emphasis Type="Italic">Escherichia coli</Emphasis> Periplasm by Fusion with Thermostable Lichenase Using the Domain Insertion Approach

A recombinant DNA in which the interferon αA (IFN–αA) gene sequence is integrated into a loop region of the gene coding thermostable lichenase was constructed. This approach of insertion fusion with thermostable lichenase is advantageous in terms of increasing the solubility, stability, and production of the fusion partner in soluble form in general and in the periplasm of bacterial cells in particular. Thus, the insertion of IFN–αA into the loop (53 a.a.) of thermostable lichenase from Clostridium thermocellum resulted in effective expression of the soluble form of the recombinant protein in the periplasm of Escherichia coli without any compromise in biological activity of IFN–αA, while the thermostable lichenase retained its ability for functional folding without dramatic loss of its basic activity and thermostability.     

A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains

A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu₉₁ and Glu₂₂₂ may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A.     

Thermostable lichenase as a translational reporter

Hybrid genes containing the reporter gene for thermostable lichenase and model genes recA, recA1, cry3a, cry3aM, and ssp1 were constructed. The expression of these genes was studied in prokaryotic and eukaryotic cells. The presence of lichenase in the hybrid proteins was shown to facilitate analysis of the hybrid protein expression in transgenic organisms. Owing to high relative activity and thermostability of lichenase, the activity of this enzyme can be measured by simple, rapid and sensitive qualitative and quantitative methods that do not require costly equipment and reagents. Using the zymograms method, molecular masses of the lichenase-containing hybrid proteins can be precisely estimated. This method is proposed instead of Western blotting using lichenase as a translational reporter. Our results showed that the use of thermostable lichenase as a translational reporter yields the data that are problematic to obtain using traditional methods of gene expression analysis, which is of importance for fundamental and applied research.     

Role of Putrescine in the Regulation of the Expression of the Oxidative Stress Defense Genes of Escherichia coli

The role of putrescine in the adaptive response of Escherichia coligrown aerobically in synthetic M9 medium with glucose to the H₂O₂-induced oxidative stress was studied. Under oxidative stress, the expression of the single-copy reporter gene fusions oxyR"::lacZand katG"::lacZwas found to undergo biphasic changes, which were most pronounced in glucose-starved E. colicells. The concentration-dependent activating effect of putrescine on the expression of the OxyR regulon genes was maximum when theoxyRgene was inhibited by high concentrations of hydrogen peroxide.     

Chromosomal integration and expression of green fluorescent protein in Zymomonas mobilis

An integrative vector was constructed to allow expression of heterologous proteins into the adhB locus of Zymomonas mobilis. As a reporter gene, the ORF of a bright variant of green fluorescent protein from Aequorea victoria (GFPuv) was fused to the adhB strong promoter from Z. mobilis by using a two-step PCR strategy. Z. mobilis recombinant strains that were stably marked by precise gene replacement at adhB locus with a single chromosomal copy of gfpuv. Protein expression was confirmed by fluorescence microscopy and measured by fluorescence spectroscopy, showing high expression levels (12 to 30 times higher than those obtained in E. coli) without affecting the host growth.     

Development ofgusA reporter gene constructs for cereal transformation: Availability of plant transformation vectors from the CAMBIA Molecular Genetic Resource Service

The use of reporter genes to characterise sequence elements that act to regulate gene expression in transgenic plants has been vital to the development of foreign gene expression strategies for use in cereal transformation. ThegusA locus ofEscherichia coli, which encodes the enzyme-glucuronidase (GUS), is by far the most popular reporter gene used in plant transformation. In this paper we extend the utility of the GUS reporter gene system in cereal transformation by describing and evaluating a number of novel constructs suitable for use in direct gene transfer experiments. These plasmids are all available from the Molecular Genetic Resource Service of the Center for the Application of Molecular Biology to International Agriculture.     

Co-expression of P450 BM3 and glucose dehydrogenase by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in an NADPH-dependent indigo production system

P450 BM3 mutant can catalyze indole to indoxyl, and indoxyl can dimerize to form indigo. But the reaction catalyzed by P450 BM3 requires NADPH, as coenzyme regeneration is very important in this system. As we know, when glucose dehydrogenase oxidizes glucose to glucolactone, NADH or NADPH can be formed, which can contribute to NADPH regeneration in the reaction catalyzed by P450 BM3. In this paper, a recombinant Escherichia coli BL21 (pET28a (+)-P450 BM3-gdh0310) was constructed to co-express both P450 BM3 gene and glucose dehydrogenase (GDH) gene. To improve the expression level of P450 BM3 and GDH in E. coli and to avoid the complex and low-efficiency refolding operation in the purification procedure, the expression conditions were optimized. Under the optimized conditions, the maximum P450 BM3 and GDH activities amounted to 8173.13 and 0.045 U/mg protein, respectively. Then bioconversion of indole to indigo was carried out by adding indole and glucose to the culture after improved expression level was obtained under optimized conditions, and 2.9 mM (760.6 mg/L) indigo was formed with an initial indole concentration of 5 mM.     

Overproduction and secretion of Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium

A gene coding for endo--1,3-1,4-glucanase (lichenase) containing a recombinant plasmid, pLL200K, was transferred from Bacillus circulans into a new shuttle plasmid, pLLS920, by ligating linearized DNAs of pLL200K and pUB110. B. subtilis RM125 and B. megaterium ATCC14945 transformed with pLLS920 produced the endo--1,3-1,4-glucanase. The enzyme was produced during active growth with maximum activity. The B. subtilis (pLLS920) enzyme was 83 times (8522 mU ml^–1) more active than that of the gene donor cells (103 mU ml^–1). The B. megaterium (pLLS920) enzyme was 7 times (735 mU ml^–1) more active than that of the gene donor cells. While E. coli secreted only about 10% of the produced enzyme, B. subtilis excreted the enzyme completely into the medium and B. megaterium by about 98%. The plasmid pLLS920 was stable in B. megaterium (98%), and in B. subtilis (51%) but not in E. coli (29%).