Plant tissue-specific promoters can drive gene expression in Escherichia coli

Plant transgenesis often requires the use of tissue-specific promoters to drive the transgene expression exclusively in targeted tissues. Although the eukaryotic promoters are expected to stay silent in Escherichia coli, when the promoter-transgene units within the plant transformation vectors are constructed and propagated, some eukaryotic promoters have been reported to be active in prokaryotes. The potential activity of plant promoter in E. coli cells should be considered in cases of expression of proteins that are toxic for host cells, environmental risk assessment or the stability in E. coli of plant vectors for specific Cre/loxP applications. In this study, DNA fragments harbouring four embryo- and/or pollen-specific Arabidopsis thaliana promoters were investigated for their ability to drive heterologous gene expression in E. coli cells. For this, they were fused to gfp:gus reporter genes in the pCAMBIA1304 vector. Although BPROM, bacterial sigma70 promoter recognition program identified several sequences with characteristics similar to bacterial promoters including -10 and -35 sequences in each of tested fragments, the experimental approach showed that only one promoter fragment was able to drive relatively strong- and one promoter fragment relatively weak-GUS expression in E. coli cells. Remaining two tested promoters did not drive any transgene expression in bacteria. Our results also showed that cloning of a shorter plant promoter sequence into vectors containing lacZ α-complementation system can increase the probability of gene expression driven by upstream located lac promoter. This should be considered when cloning of plant expression units, the expression of which is unwanted in E. coli.     

Pollen- and anther-specific chi promoters from petunia: tandem promoter regulation of the chiA gene.

We have analyzed the spatial and temporal activities of chalcone flavanone isomerase (chi) A and B gene promoters from petunia. To study the tandem promoter regulation of chiA, various chiA promoter fragments were fused with the beta-glucuronidase (GUS) reporter gene. Analysis of transgenic plants containing these chimeric genes provided definitive proof that the chiA coding region is regulated by two distinct promoters (designated PA1 and PA2). We also showed that both promoters can function independently and that the chiA PA1 promoter is expressed in limb (epidermal and parenchyma cells), tube (inner epidermal and parenchyma cells), seed (seed coat, endosperm, and embryo), sepal, leaf, and stem. The use of chiA and chiB promoters in the regulation of anther- and pollen-specific gene expression has been studied. By analyzing transgenic plants containing chimeric genes consisting of chiA and B promoter fragments and the GUS reporter gene, we were able to identify a 0.44-kilobase chiA PA2 promoter fragment that drives pollen-specific gene expression and a 1.75-kilobase chiB PB promoter fragment that confers anther-specific (pollen and tapetum cells) expression to the GUS gene.     

A rice promoter containing both novel positive and negative cis-elements for regulation of green tissue-specific gene expression in transgenic plants

The tissue-specific expression of transgenes is essential in plant breeding programmes to avoid the fitness costs caused by constitutive expression of a target gene. However, knowledge on the molecular mechanisms of tissue-specific gene expression and practicable tissue-specific promoters is limited. In this study, we identified the cis -acting elements of a tissue-specific promoter from rice, P_D54O , and tested the application of original and modified P_D54O and its cis -elements in the regulation of gene expression. P_D54O is a green tissue-specific promoter. Five novel tissue-specific cis -elements (LPSE1, LPSE2, LPSRE1, LPSRE2, PSE1) were characterized from P_D54O . LPSE1 activated gene expression in leaf and young panicle. LPSRE2 suppressed gene expression in leaf, root, young panicle and stem, and PSE1 suppressed gene expression in young panicle and stem. LPSRE1 and LPSE2 had dual roles in the regulation of tissue-specific gene expression; both functioned as activators in leaf, but LPSRE1 acted as a repressor in stem and LPSE2 as a repressor in young panicle and root. Transgenic rice plants carrying cry1Ac encoding Bacillus thuringiensis endotoxin, regulated by P_D54O , were resistant to leaf-folders, with no Cry1Ac protein found in endosperm or embryo. A reporter gene regulated by a series of truncated P_D54O showed various tissue-specific expression patterns. Different fragments of P_D54O fused with the constitutive cauliflower mosaic virus 35S promoter suppressed 35S -regulated gene expression in various tissues. P_D54O , truncated P_D54O and the tissue-specific cis -elements provide useful tools for the regulation of tissue-specific gene expression in rice breeding programmes.     

SARS冠状病毒N蛋白激活hfgl2凝血酶原酶基因的研究

研究鉴定激活hfgl2凝血酶原酶基因的SARS冠状病毒结构蛋白。从SARS尸检肺组织中抽提RNA后制备cDNA,分别扩增SARS-CoV的N、S2和M全长基因序列,再分别克隆到真核表达载体pcDNA3.1( )上。应用免疫组织化学分析鉴定pcDNA3.1-N、pcDNA3.1-M和pcDNA3.1-S2的表达。构建人纤维介素(hfgl2)启动子荧光素酶报告基因质粒,并将SARS冠状病毒结构蛋白表达质粒分别与其共转染以明确激活hfgl2基因转录的SARS冠状病毒结构蛋白。将目的片段克隆至pcDNA3.1( ),经酶切鉴定和测序鉴定无误;免疫组织化学染色可见明显的CHO细胞胞浆棕染。与hfgl2启动子共转染实验阐明SARS冠状病毒膜(M)蛋白和刺突糖(S2)蛋白对hfgl2基因的激活与对照组无显著差异,而SARS冠状病毒核心(N)蛋白可激活hfgl2启动子,使其转染活性提高4.6倍。SARS冠状病毒N蛋白可增强hfgl2基因的转录活性。     

Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120

Abstract The strengths of several promoters were assessed in the cyanobacterium Anabaena PCC 7120 by fusing them to luxAB , encoding bacterial luciferase. Two promoters, P _tac and P _psbA , with sequences nearly identical to consensus Escherichia coli σ ⁷⁰ promoters, gave as high or higher expression than the strong Anabaena promoter, P _rbc . P _npt , the natural promoter driving expression of the kanamycin-resistance determinant from Tn5, was poorly expressed in Anabaena . The Lac repressor partially repressed expression from P _tac , permitting regulated expression in Anabaena after induction with isopropyl thiogalactoside to a level 4–5-fold higher than without inducer.     

Improved baculovirus system for transferring genes into insect and mammalian cells

A new set of eukaryotic expression vectors was constructed on the basis of baculoviruses. EcoRI fragments S, J, and P with the genes for late viral proteins p35 (polyhedrin), p39, and p10 were cloned from genomic DNA of the nuclear polyhedrosis virus. The promoter regions of these genes were used to construct double-and triple-promoter expression vectors. Baculovirus vectors containing an expression cassette with the cytomegalovirus promoter and the green fluorescent protein reporter gene were designed to express the cloned genes in cultured mammalian cells.     

Growth phase-dependent gene regulation in vivo in Sulfolobus solfataricus

Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1-based lacS reporter gene constructs containing the Sulfolobus 16S/23S rRNA gene core promoter, the TF55α core promoter, or the native lacS promoter were tested in Sulfolobus solfataricus cells lacking the lacS gene. The 42-bp 16S/23S rRNA gene and 39-bp TF55α core promoters are sufficient for gene expression in S. solfataricus. However, only gene expression driven by the 16S/23S rRNA gene core promoter is dependent on the culture growth phase. This is the smallest known regulated promoter in Sulfolobus. To our knowledge, this is the first study to show growth phase-dependent rRNA gene regulation in Archaea.