The human genome must be packaged and organized in a functional manner for the regulation of DNA replication and transcription. The nuclear scaffold/matrix, consisting of structural and functional nuclear proteins, remains after extraction of nuclei and anchors loops of DNA. In the search for cis-elements functioning as chromatin domain boundaries, we identified 453 nuclear scaffold attachment sites purified by lithium-3,5-iodosalicylate extraction of HeLa nuclei across 30 Mb of the human genome studied by the ENCODE pilot project. The scaffold attachment sites mapped predominately near expressed genes and localized near transcription start sites and the ends of genes but not to boundary elements. In addition, these regions were enriched for RNA polymerase II and transcription factor binding sites and were located in early replicating regions of the genome. We believe these sites correspond to genome-interactions mediated by transcription factors and transcriptional machinery immobilized on a nuclear substructure. The eukaryotic nucleus has a complex architecture in which the genome must be packaged into higher ordered structures in an organized and accessible way that allows for the dynamic processes of DNA transcription, replication, and repair. While the mechanisms driving large-scale organization are unknown, it is well demonstrated that chromosomes occupy specific spatial territories within the nucleus that are positioned so that active and repressed regions of the genome often occupy different sub-nuclear compartments in vivo and to have functional consequences on genome organization and regulation. S/MARs, which are found in both genic and intergenic regions of the genome, correspond to boundaries between chromatin domains The idea of a nuclear structure to which chromatin loops are anchored has existed since the first demonstrations of a proteinaceous substructure that persists upon detergent or salt extraction of nuclei in vivo is still a controversial issue, the idea that genes and regulatory elements are tethered to immobilized active chromatin hubs and transcription factories is consistent with a nuclear substructure that is resistant to biochemical extractions. Regulation of gene expression involves long-distance interactions, often over tens to hundreds of kilobases, between locus control regions, enhancers, and promoters. The mechanisms driving such interactions have only recently begun to be uncovered as new techniques, such as chromosomal conformation and capture (3C), have evolved. A subset of these interactions corresponds to nuclear matrix attachment regions, as recently demonstrated by 3C experiments While the existence of a nuclear scaffold/matrix Great efforts have been made to map functional DNA elements and epigenetic modifications genome-wide 7 cells were isolated in CLB  and added to 1.25× SB  for 20 min on ice. To extract histones, stabilized nuclei were incubated in 100 mL LIS buffer  for precisely 10 min at room temperature. Scaffolds were pelleted by centrifugation at 2,615×g in a HB-6 swing-bucket rotor for 25 min and washed with MWB  followed by two washes with EcoRI Buffer . To fractionate scaffold-associated DNA from loop DNA, pellets were resuspended by gentle trituration in 5 mL of EcoRI buffer containing 0.025% Triton-X100 and digested with 5,000 units each of EcoR1 and HindIII restriction enzymes at 37°C for 1.5 h. Loop DNA released by digestion was separated from the nuclear scaffold by centrifugation at 2,615×g for 10 min. The pellet was again resuspended in 5 mL of EcoRI buffer containing 0.025% TritonX-100 and an additional 5,000 units of EcoR1 and HindIII enzyme was added. In addition, 2,500 units of HaeIII restriction enzyme were added to both the scaffold and loop fractions followed by incubation at 37°C for 1 h. For the last 15 min of incubation, RNAseA was added at final concentration of 20 ug/mL to both the scaffold and loop fractions. The nuclear scaffold fraction was again centrifuged to separate the second loop fraction, which was then combined with the first loop fraction. The proteinaceous pellet was resuspended in 1.5 mL of 300 mM NaCl, 2.5 mM EDTA, 10 mM Tris-HCL pH 8.0 and 4 ml of 1.5× PKB . Proteinase K was then added to a final concentration of 120 ug/mL and the reaction was incubated overnight at room temperature. The loop fraction was adjusted to contain 300 mM NaCl and 27 mM EDTA. DNA was recovered from the loop and scaffold fractions by extraction with phenol∶chloroform∶isoamyl alcohol (25∶24∶1) and ethanol precipitation. Precipitated DNA was dissolved in water and quantitated by OD and Pico Green quantification assay (Invitrogen). The quality of each nuclear scaffold preparation was monitored by performing qPCR to assess enrichment of the ApoB 3′MAR and a negative control from the ApoB locus.HeLa (ATCC CCL-2) cells were maintained in DMEM/high glucose  in the presence of 10% DCS and 1× penicillin/streptomycin (Gibco). Nuclear scaffolds was prepared according to the protocol of Nuclear scaffold DNA and total genomic DNA digested with EcoRI and HindIII were hybridized to ENCODE01-F  tiling microarrays as described previously −3 within a 1-kb window) of nuclear scaffold signal as compared to the genomic control were reported as scaffold attachment regions (SARs). These sites were then remapped to hg18 build of the genome using the UCSC Genome Browser liftover tool (http://genome.ucsc.edu). Many of the SAR sites called were in close proximity to each other (52% at 3 kb distance) and the intervening regions often corresponded to repetitive sequences that were not spotted on the microarray. To account for potential gaps in the data due to masked sequences, intervals from the MAT analysis that were less than 2501 bp apart were subsequently joined yielding 453 SARs (http://www.ncbi.nlm.nih.gov/geo/) and are accessible at accession number GSE26477. Inter-SAR distance was then calculated by determining the distance between endpoints of adjacent SARs within each ENCODE region. Since only distances between SARs can be considered, 26.89% of the 30 Mb covered by the ENCODE array representing the ends of each region were ignored during the analysis.Hybridization data were analyzed using Model-based analysis tool (MAT) for tiling arrays 453 SARs . Raw andEqual amounts of scaffold DNA and total genomic DNA digested with EcoRI and HindIII was used as template for quantitative PCR with an Applied Biosystems 7300 machine in triplicate reactions using PCR primers to amplify genomic regions of 100–300 bp. All primers used in this study are listed in www.genome.ucsc.edu). HeLa gene expression data, previously generated in our laboratory using the Human HG-U133 Plus 2.0 gene expression array (Affymetrix) All analysis was performed using in-house developed programs. Data sets corresponding to intergenic, genic, transcription start sites (TSS), and transcription end sites (TES) were generated from UCSC Known Gene track downloaded from the UCSC Genome Browser  was used to compare each SAR sequence against each other Table S1) with a median size of 3.4 kb covering 7.3% of the 30 Mb across the ENCODE pilot regions (To identify scaffold attachment regions (SARs), we purified nuclear scaffolds from HeLa nuclei using the LIS extraction method  regions Table 1.not associated with genes was 1876 bp.SARs are typically described to be 200–1000 bp long; however, matrix attachment regions of greater length have been reported As SARs are expected to represent the base of chromatin loops, we also assessed inter-SAR distances within the 44 different ENCODE regions interrogated as an estimate of DNA loop size Fig. 2D.Since S/MAR sequences are historically AT-rich, we determined the percent A+T content for each of the 453 nuclear scaffold attachment regions Fig. 2B.Upon comparison with genomic features, we found a significant association of SARs with regions of the genome that are actively transcribed. SARs were predominately found in genic areas of the ENCODE regions with 74% of sites residing within 5 kb of an annotated gene Fig. 3. We next evaluated the localization of SARs relative to transcription start sites (TSS). As shown in Given the strong association with actively transcribed genes, we investigated whether SARs were also preferentially distributed in regions with open chromatin structure by comparing SARs to DNA replication timing profiles that had been previously mapped in HeLa cells Evaluation of SARs and histone modifications also demonstrated an enrichment of SARs in euchromatic regions. As shown in Finally, we compared the localization of SARs within Lamin B-associated domains (LADs) that had been mapped in human lung fibroblasts Compartmentalization of the genome into functional domains may be mediated by recruitment of DNA-binding protein complexes to the nuclear scaffold/matrix in order to regulate gene expression, DNA replication, and chromatin structure. Thus, we investigated whether our SARs corresponded with known cis-elements that regulate such processes. Regulatory elements are often marked by DNAse I hypersensitivity and the absence of nucleosomes, which can be detected by formaldehyde-assisted isolation of regulatory elements (FAIRE) The vertebrate insulator protein CTCF is a multiple zinc finger DNA-binding protein that is believed to play a major role in the organization of the genome into functional domains by mediating chromatin loop formation Regulatory Factor Binding Regions (RFBR) as determined by the ENCODE consortium Given the association between SARs and transcription Fig. 3, In addition to the regulation of gene transcription, initiation of DNA replication has been proposed to be regulated by binding of replication machinery to the nuclear scaffold/matrix in situ hybridization studies of nuclear halos generated after high-salt extraction demonstrated the localization of several active genes on the nuclear matrix, while inactive gene loci were found in the extended DNA loops of the halo in vitro binding assays were within genic regions Here, we report the identification of 453 biochemically-defined nuclear scaffold attachment regions within 30 Mb of the human genome. A majority of these SARs localize to expressed genes, are enriched at the beginning and ends of transcripts, and are associated with RNA Pol II and transcription factor binding sites. Although we were hoping to isolate SARs that demarked chromatin domain boundaries, our data is consistent with numerous studies reporting the presence of active genes and transcriptional machinery in nuclear matrix and nucleoskeleton preparations. For example, fluorescence Transcription factories immobilize active RNA Pol II within the nucleus presumably to increase efficiency and to coordinately regulate the expression of co-localized genes. Genes whose entire length is retained on the scaffold may represent highly transcribed housekeeping genes localized to an immobilized transcription factory see Fig. 5A.Active chromatin hubs (ACH) refer to the clustering of interactions between promoters and regulatory elements involved in the regulation of many tissue-specific genes Attachment to the nuclear scaffold is not restricted to transcribed regions of the genome as nearly 40% of identified SARs resided in intergenic region or a non-transcribed gene. A number of these SARs may represent classical boundary elements and/or structural elements that contribute to overall genome organization. Of course, it is also possible that some of these sites do correspond to transcription as several studies suggest that the majority of the genome is transcribed at low levels and that transcription units exist that have not been annotated SARs are expected to mark the base of chromatin loops that range in size from 50–200 kb. Estimations of loop size in HeLa cells predict an average inter-S/MAR distance of 80–90 kb Although ultrastructural imaging demonstrates the presence of the peripheral lamina and nucleoli in LIS-prepared matrices ++et al. compared scaffold/matrix attachments mapped using two different isolation methods and found that only 52% of S/MARs between methods corresponded to each other The various isolation procedures utilized to purify a nuclear substructure and identify S/MAR sequences have received a fair amount of criticism due to the often contradictory results between different groups In conclusion, mapping nuclear scaffold attachment sites across 1% of the human genome reveals a strong association of transcription with a nuclear substructure. Many of these SARs have regulatory potential as they correspond to transcription factor binding sites and DNAse I hypersensitivity sites. In addition, nuclear scaffold attachment may act as an anchor point for active chromatin hubs and transcription factories, as suggested for the β-globin locus Table S1HeLa SARs identified across ENCODE pilot regions. See attached Excel file.(XLS)Click here for additional data file.Table S2Primers used for quantitative PCR in this study. See attached Excel file.(XLS)Click here for additional data file.


Salmonella biofilm formation, virulence gene expression and the production of the major surface antigen present on the cell surface: flagellin. At the top of a flagellar regulatory hierarchy is the master operon, flhDC, which encodes the FlhD4C2 transcriptional complex required for the expression of flagellar, chemotaxis and Salmonella pathogenicity island 1 (Spi1) genes. Of six potential transcriptional start-sites within the flhDC promoter region, only two, P1flhDC and P5flhDC, were functional in a wild-type background, while P6flhDC was functional in the absence of CRP. These promoters are transcribed differentially to control either flagellar or Spi1 virulent gene expression at different stages of cell growth. Transcription from P1flhDC initiates flagellar assembly and a negative autoregulatory loop through FlhD4C2-dependent transcription of the rflM gene, which encodes a repressor of flhDC transcription. Transcription from P1flhDC also initiates transcription of the Spi1 regulatory gene, hilD, whose product, in addition to activating Spi1 genes, also activates transcription of the flhDC P5 promoter later in the cell growth phase. The regulators of flhDC transcription  also exert their control at different stages of the cell growth phase and are also subjected to cell growth phase control. This dynamic of flhDC transcription separates the roles of FlhD4C2 transcriptional activation into an early cell growth phase role for flagellar production from a late cell growth phase role in virulence gene expression.The flagellar regulon controls  Salmonella pathogenesis, which takes the lives of hundreds of thousands of people each year. The genes of the Salmonella pathogenicity island 1 and those of the flagellar regulon are part of the same transcriptional hierarchy. We report the novel finding where the key control of this network takes place at the flhDC promoter region. We followed the transcription from the two “live” flhDC promoters as a function of the cell growth phase. P1 comes on early in the cell cycle, while P5 comes on late. Transcription of P5 is HilD dependent, which represents a totally new finding and Salmonella specific: there is no HilD in E. coli flhDC control, no P5 transcription. P1 & P5 can express flhDC to equivalent levels, yet only P1- dependent expression produces motility UNLESS we artificially induce P5 EARLY in the cell cycle. This work is the foundation for the cell cycle stages a Salmonella bacterium experiences during host infection. This is a significant conceptual advance in Salmonella pathogenesis: one can no longer consider gene regulation at 37°C and OD 0.6 as a reflection of the Salmonella infection cycle; the whole cell growth cycle must be considered in understanding this complex biological processes.Flagellar-mediated motility is fundamental to  Salmonella enterica, occur worldwide every year killing more than a hundred thousand people annually . Typhoid fever caused by Salmonella Typhi kills an equivalent number of people each year. A prominent player in Salmonella pathogenesis is the bacterial flagellum. The bacterial flagellum is an ion-powered, complex motor organelle that endows bacterial cells, such as Escherichia coli and Salmonella enterica, with the ability to propel themselves through liquid medium and across hydrated surfaces Tens of millions of human cases of Salmonellosis, a foodborne gastroenteritis caused by Salmonella virulence genes identified a transposon insertion in the flagellar filament cap gene, fliD, as defective for survival of cells in macrophages fliD is in an operon with the fliT gene whose product is a regulator of the flagellar and Spi1 virulence genes master regulatory complex FlhD4C2fliD was polar on fliT gene expression and thus identified regulation of FlhD4C2 activity as critical for Salmonella virulence. The two proteins that make up the FlhD4C2 transcriptional regulatory complex are co-expressed from the flhDC operon, class 1 promoter, which is at the top of a complex transcriptional hierarchy for both flagellar and Spi1 virulence genes expression. The decision whether or not to produce flagella is regulated at the levels of flhDC transcription, translation, FlhD4C2 assembly and stability flhDC operon transcription include cAMP-CRP, Fis, Fur, H-NS and QseB flhDC transcription. These factors include, LrhA, RcsB, RtsB, SlyA, DskA, PefI-SrgD, FimZ, HdfR, OmpR and RflM 4C2 activity generates an auto-regulatory loop by activating transcription of the rflM gene encoding a LysR-type DNA binding protein RflM, which in turn inhibits the transcription of flhDCflhDC include, CsrA 4C2 repressed gene, ydiV4C2, in its free or DNA-bound form, remove FlhD4C2 from DNA and serves as an adapter that targets FlhD4C2 for ClpXP-dependent degradation Early work on the discovery of Salmonella, an initial characterization of the flhDC promoter region identified six transcriptional start sites (TSSs) Salmonella flhDC regulatory region combined with the presence of DNA binding sites of CRP, LrhA, RtsB, HilD, RcsB, HNS and others indicated a complex level of the flhDC transcriptional regulation.In Salmonella enterica is an intracellular facultative pathogen causing a range of diseases in a variety of hosts Salmonella invasion of epithelial cells and development of Salmonellosis are encoded within the Salmonella pathogenicity island 1 (Spi1) genes. Spi1 encodes a virulence-associated type III secretion system (T3SS) as part of an injectisome structure required for the secretion and injection of multiple effector proteins into the cytoplasm of host cells hilC and hilD genes whose products are members of the Ara/XylS family of transcriptional regulators that control hilA gene transcription. HilD is at the top of the regulatory network controlling Spi1 expression because most regulators controlling hilA transcription appears to be HilD-dependent flhDC also target the main transcriptional regulators of Spi1, such as HilA and HilD 4C2 and HilD Salmonella, expression of Spi1 genes is activated by FliZ fliAZY operon. FilZ activates the hilD gene expression at the posttranslational level and HilD in turn promotes transcription of the rtsAB operon, which encodes a pathogenesis-related DNA-binding regulatory proteins. RtsA and RtsB reciprocally regulate both the Spi1 and flagellar genes flhDC promoter region inhibits flhDC transcription and motility It is noteworthy to mention that many protein components of the Spi1 and flagella T3SS exhibit a significant degree of amino acid identity, leading to the production of remarkably similar T3SS structures flhDC expression in Salmonella enterica. We explored how and when positive and negative regulators affect flhDC expression throughout the cell growth cycle. We measured the effect of RcsB, LrhA, RflM, SlyA, RtsB and HilD regulatory factors on flhDC operon transcription at different cell growth phases. We characterized the specific TSSs within the flhDC promoter region and their involvement in the positive and negative control of flhDC cell-cycle dependent transcription. Finally, we examined how the individual TSSs and protein regulatory factors controlled the interconnection between the flagellar and Spi1 regulons.We decided to investigate how input from different regulatory factors might integrate multiple environmental or cell cycle signals into the control of flhDC operon transcription at different phases of the cell growth, we constructed a transcriptional fusion of the flhDC promoter region to the luciferase operon of Photorhabdus luminescence (luxCDBAE operon). Because the flhDC operon is autoregulated negatively by RflM and positively by HilD, we designed strains harboring an intact copy of the flhDC operon under the control of its native promoter (PflhDC) and an in-frame fusion of a second copy of the promoter region of flhDC (through the first 272 nucleotides of flhD coding sequence) to the luciferase operon: DUP[(PwtflhDC-luxCDBAE)*Km*(PwtflhDC-+CflhD+)] *Km*(PwtflhDC-+CflhD+)] construct as PwtflhDC.To investigate hD+C+)] . Thus, iflhDC strain into fresh media with shaking at 30°C, transcription of the flhDC genes declined 4-fold during the initial lag phase transition to log phase growth to a minimal value . The diversity of transcription factors controlling expression of flhDC reflects the complexity of flhDC transcriptional regulation and suggests that flhDC transcription is controlled when Salmonella cells are experiencing different metabolic or environmental states, or different growth conditions under which these transcriptional factors are active. We examined both the timing and magnitude of individual regulatory proteins on flhDC transcriptional control throughout the cell's growth phase. We tested flhDC transcriptional levels as a function of the cell's growth phase in strains missing the individual negative regulators RcsB, LrhA, RflM, RtsB, SlyA and the positive regulator HilD , this result suggests that flhDC auto-inhibition through RflM occurs during early exponential phase to control when full FlhD4C2-dependent gene expression occurs at log phase. The negative effect of RtsB and SlyA on flhDC transcription was detected as cells enter early stationary phase. We also observed that the maximum flhDC transcription level peaked earlier for both the hilD and rflM mutants at OD 1, while the wild type and mutants in rcsB, lrhA, slyA and rtsB peaked around OD 1.2.In tor HilD . As was ild type . This suflhDC transcription is kept low by a combination of repressors including at least RcsB and LrhA. Initial FlhD4C2 expression during the stationary to log phase transition produces enough RflM to maintain a low level of flhDC transcription until an OD of ∼0.3 is reached. After OD 0.3, flhDC transcription increased significantly, but RflM, RcsB and LrhA reduce the overall level. Interestingly, the wild-type level is balanced by the presence of the HilD activator of flhDC transcription, the hilD-activated inhibitor of flhDC transcription RtsB and by the virulence associated factor SlyA  with continuous shaking at 150 rpm. We observed the same trend of regulation of the flhDC operon as seen in batch cultures for lrhA, rcsB  where bacterial cells are grown in much lower volumes and likely to be subjected to different oxygen levels in the medium compared to batch cultures. It has been shown that activation of flgA, a gene under the control of flhDC, under static conditions (no shaking of 96 well plates) occurred immediately after dilution of an overnight culture into LB-1% Salt flhDC operon in standing batch culture in LB, we observed that flhDC transcription increased at OD∼0.12 . We monitored the activities of these constructs in 96 well plates over time. We observed that the transcription of the autoregulated gene lrhA is immediately activated following dilution from an overnight culture, and before the activation of flhDC (rcsD (which is the first gene of the rcsDB operon transcribed from the rcsD promoter) also initiated before flhDC  appeared to be activated at the same time as hilD  at different optical densities , and increased at OD∼0.4 followed by a quick decay during the rest of the cell's growth phase. HilD protein, the positive activator of flhDC transcription, was detected at OD∼0.4 and increased at stationary phase (flhDC at OD∼1). In contrast, the negative regulator SlyA was produced during all the phases of cell growth, with a sharp increase at OD∼1. These results demonstrate a hierarchy at the level of expression of flhDC regulators that specifically mimics the differential dynamics of flhDC operon transcription.We next asked if the protein levels of the regulatory factors controlling ensities . We estary phase . RtsB, win vivo binding dynamics within the flhDC promoter region by these regulatory factors. At different optical densities (0.4 to 1.4), chromatin immunoprecipitations (ChIP) were conducted using strains with individually tagged transcriptional factors, RcsB, RflM, HilD, RtsB, LrhA and SlyA  followed by decreased binding at latter stages of growth. The transcriptional regulator RflM binding to DNA was detected at OD∼0.4 with maximal binding at OD∼0.6, but was no longer bound the flhDC promoter region beyond OD∼0.6. HilD, a transcriptional activator of flhDC, was bound to the flhDC promoter region at OD∼0.4 increasing to a maximum bound level at OD∼0.8 and followed by absence of bound HilD at OD∼1. SlyA was not physically associated with the flhDC promoter at OD∼0.4 and ∼0.6, but was bound to the flhDC promoter region at OD∼0.8. There was no binding of RtsB to the flhDC promoter at an early time point of cell growth OD∼0.4 to 0.6. Binding by RtsB had initiated by OD 0.8 and increased through OD 1.4. We were unable to immunoprecipitate LrhA tagged protein because C-terminal or N-terminal tagged LrhA behaved like lrhA null mutant . Mutations of wild-type sequence P1flhDC (TATAGT) to GTTGGT (P1−.1flhDC); TCTAGC (P1−.2flhDC) or TCTAGT (P1−.3flhDC) but not TACAGT (P1−.4flhDC) were associated with a significant reduction of flhDC transcription (flhDC (TATGCT) to TCTGCC (P5−.2flhDC) or TCTGCT (P5−.3flhDC) but not to TACGCT (P5−.4flhDC) reduced significantly the transcription of flhDC to the same extent as the mutation of −10 to GTTGGT (P5−.1flhDC)  . These rflhDC and P6flhDC (overlapping with the CRP binding site which is required for the transcription of flhDC from P1flhDC promoter) and P3flhDC (overlapping with the LrhA binding site) and P4flhDC were not conclusive . We established that P1+flhDC and P5+flhDC were able to promote flhDC operon transcription but to a lesser extent to what is observed in the wild-type strain   is inhibited by CRP, because in a crp null mutant there was an increase of transcription of P6+flhDC compared to wild-type (We further investigated the authenticity of the six putative TSSs of the e strain . The tra strain) . These rild-type .flhDC and P5flhDC promoters significantly affected the expression of flhDC, we would expect the level of transcription of flhDC operon in the absence of both P1 and P5 promoters to be similar to the level of transcription of flhDC operon in the absence of all flhDC promoters (P1 through P6). To investigate this hypothesis, we measured the luciferase activity in a strain with combined mutations in P1flhDC and P5flhDC promoters (P1−P5−flhDC) and compared it to the luciferase activity of a wild-type strain and to a strain with all six promoters mutated (strain AP's). We observed that transcription of flhDC operon in strain P1−P5−flhDC was totally abolished to the same levels observed in a strain with all flhDC promoters mutated (flhDC and P5flhDC are the major promoters driving transcription of flhDC operon. We concluded that transcription of the flhDC operon in strain P1−flhDC (harboring mutations of the −10 box of P1flhDC) is driven from the P5flhDC promoter and that transcription of the flhDC operon in strain P5−flhDC (harboring mutations of −10 box of P5flhDC) is driven from P1flhDC.Since only mutations in P1 mutated . These rflhDC and P5flhDC are the main promoters driving transcription of the flhDC operon, we monitored the expression of the P1flhDC and P5flhDC promoters at different optical densities using PwtflhDC, P1−flhDC and P5−flhDC constructs (flhDC operon in strains P1−flhDC (P5-expressed) and P5−flhDC (P1-expressed) demonstrated that both promoters are required for transcription of flhDC because the expression of flhDC operon in constructs P1−flhDC (P5-expressed) and P5−flhDC (P1-expressed) did not reach the expression levels of the wild-type strain, PwtflhDC (both P1 and P5 are expressed) (flhDC operon from P1flhDC is activated earlier than P5flhDC because (i) the transcription profile of the flhDC operon in construct P5−flhDC (P1-expressed) overlapped with that of the wild-type strain from OD 0.1 to OD 0.4, (flhDC operon in construct P1−flhDC (P5-expressed) where the transcription started taking place at OD∼0.35 (flhDC and P5−flhDC (P1-expressed) strains (OD∼0.2). The same hierarchy of expression of P1 and P5 was observed in batch culture (flhDC operon in P5−flhDC (P1-expressed) started declining at OD∼0.4–0.5, meanwhile, transcription of flhDC operon in strain P1−flhDC (P5-expressed) was more pronounced at a later growth stage accounting for ∼60% relative to the wild-type at OD∼0.6  . Moreove OD 0.4,  and (ii) OD∼0.35  compared culture . The trat OD∼0.6 . It is a P1flhDC . These rflhDC transcription  reduced PwtflhDC promoter transcription; (ii) abolished the transcription of flhDC operon in construct P1−flhDC (P5-expressed) (flhDC operon in construct P5−flhDC (P1-expressed)   and (iiipressed) . These rflhDC operon is subjected to negative feedback by RflM, which is activated at the transcriptional level by FlhD4C2flhDC operon transcription kinetics, we monitored the transcription profile, over time, in the three strains PwtflhDC, P1−flhDC and P5−flhDC in the absence and presence of RflM. We established that there was an increase in the transcription from PwtflhDC in the absence of RflM (flhDC promoter is under negative autoregulation by RflM because the expression of flhDC operon in strain P1−flhDC (P5-expressed) was similar between the wild-type and its isogenic rflM null mutant (flhDC because flhDC transcription in strain P5−flhDC (P1-expressed) increased in the absence of RflM  ParaBAD::FCF (ii) ParaBAD::rflM+. We observed that under conditions that overproduce RflM, presence of arabinose, there was an inhibition of transcription of the autoregulated gene hilD  ParaBAD::FCF PflhDC::T-POP and (ii) ParaBAD::rflM+ PflhDC::T-POP. For the PflhDC::T-POP backgrounds the flhDC operon is transcribed from the tetracycline(Tc)-inducible tetA promoter, and as such are flhDC− in the absence of tetracycline and flhDC+ in the presence of tetracycline. First, we observed that flhDC controlled transcription of the hilD gene, because in the absence of Tc, there was a 2-fold decrease in the PhilD transcription level in the PflhDC::TPOP strain background  in strains defective in either the rcsB, lrhA, slyA or rtsB genes. We observed increased transcription in the P5−flhDC background, in either rcsB, rtsB or lrhA null mutants compared to the wild-type strain −) − present in the P1+flhDC construct −) . HoweverflhDC operon in construct P1−flhDC (P5-expressed) in strains lacking either the rcsB, lrhA, slyA or rtsB genes. We demonstrate that the transcription from P5flhDC promoter is regulated by RcsB, LrhA and SlyA proteins, because transcription of flhDC in construct P1−flhDC (P5-expressed) increased in rcsB (2-fold), lrhA (2-fold) and slyA (1.8-fold) mutant strains (flhDC promoter is not regulated by RtsB protein because transcription of flhDC in construct P1-flhDC (P5-expressed) was independent of RtsB −), we observed the same regulation as with P1−flhDC  in the ΔrcsB mutant was different than that of the PwtflhDC or the P5−flhDC (P1-expressed) in the absence of RcsB. While, there was a relief in the inhibition of transcription for the PwtflhDC or P5−flhDC (P1-expressed) constructs at earlier time points of cell growth, the rcsB mutation resulted in increased transcription in the construct P1−flhDC (P5-expressed) only at later stage of growth -dependent transcription of the rcsC gene hilD transcription. We used a fliZ null mutant to detect if the effect of RcsB on hilD is through the flhDC regulated gene fliZ, which is the post-translational activator of the autoregulated hilD gene. We observed that FliZ regulated transcription of hilD, because there was a 2-fold reduction of PhilD-dependent transcription in the strain lacking fliZ compared to the fliZ+ background , the transcription of hilD was abolished  were non-motile while P5flhDC defective strains (only P5 is mutated) exhibited no apparent reduction of motility . The motility of P3− and P4− were not significantly different from the wild-type strain. These results confirmed that in the wild-type background transcription from P1flhDC is a prerequisite for motility while P5flhDC is not required for motility. These results also suggested that the right timing of expression of flhDC is essential for motility. If this hypothesis is correct, we could expect that if flhDC is expressed from P5flhDC promoter at an early time point it should confer a motility phenotype. To test this hypothesis we used the non-motile strain P5+  (flhDC that would otherwise suppress motility + strain (hilD gene (addition of a thymine residue at position −51 from the start codon of HilD and resulting in higher expression of hilD (labeled hilDup)). The isolation of this mutation confirmed that HilD regulates the P5flhDC promoter. If the hypothesis that the timing of expression of flhDC as a prerequisite for motility is correct, then a hilD-up mutation should promote transcription of flhDC operon from P5 promoter at early growth phase. To test this hypothesis we used a transcriptional lac fusion to fliL, a class 2 promoter that is positively regulated by FlhD4C2, as readout to determine the expression of the P5 promoter transcription. The transcription of fliL indicates the presence of FlhD4C2-dependent transcription. Transcription of fliL in the P5+ strain was very low during early growth phases and increased when cells reached an OD of 1.4   to isola+ strain  and mappD of 1.4 . These re strain . These rflhDC operon Salmonella flhDC operon is primarily transcribed from two promoters, P1flhDC and P5flhDC. The activities of these two promoters are coupled to five stages controlling flhDC transcription and each stage is differentially controlled by a set of transcriptional factors: (1) repression of transcription of flhDC during the initial growth phase by LrhA and RcsB (2) repression by RflM at early exponential phase (3) activation through the action of HilD at mid exponential phase (4) repression by SlyA and RtsB at the onset of stationary phase, and finally (5) shut down at late stationary phase.The complex networks and the number of factors necessary for the production of functional flagella and the resulting motility, though beneficial for the bacteria, represent a significant requirement on the cell's resources flhDC regulation is controlled by two transcription factors, RcsB and LrhA. Null mutation in any of these transcriptional regulators, promoted flhDC transcription early in the growth phase and this inhibition was maintained throughout the rest of the growth phase . We found that the effect of LrhA and RcsB was coincident with activation of transcription of their respective genes. As cell densities reached an OD of 0.2–0.3, transcription of flhDC increased. The increased flhDC transcription resulted in transcription of rflM, which in turn resulted in the feedback inhibition of flhDC transcription. This effect was consistent with the concurrent transcriptional activation of flhDC and rflM, where a surge of transcription of rflM mimicked that of flhDC and decayed quickly compared to the rest of the regulators controlling flhDC transcription. At the protein level, RflM appeared to follow the same early production and a quick decay as observed at the transcriptional level. We conclude that RflM limits flhDC transcription perhaps to efficiently control the kinetic expression of the middle and late flagellar class genes to facilitate flagellum assembly. Class 2 promoters respond differently to FlhD4C2 levels allowing the cell to control the timing of an individual class 2 operon transcription with respect to the other class 2 operons. Auto-repression at the transcriptional level has been shown to reduce relative variance and duration of fluctuations, and consequently limits noise in downstream expression fliC, encoding the filament component of the flagellum, has been demonstrated to be bistable flhDC operon transcription. In support of this hypothesis, a null mutation of rflM gene has been shown to increase heterogeneity of fliC expression in a cell population when compared to wild-type The pre-log steady state transcription of flhDC operon transcription. This control is positive, and is brought on by the effect of a virulence-associated transcription factor, HilD. There was a delay in the positive effect of HilD compared to the negative control exerted by RcsB, LrhA and RflM. This delayed HilD effect on flhDC operon transcription was due to the time required to activate HilD expression through FlhD4C2-dependent FliZ production. FlhD4C2 activates fliZ gene transcription from a flagellar class 2 promoter and FliZ, in turn, activates hilD expression at the post-translational level flhDC transcription takes place and, unexpectedly, is also controlled by HilD. HilD activates the transcription of two regulatory factor genes, rtsBslyA (data not shown). RtsB and SlyA are two DNA binding regulators, which then act to inhibit flhDC transcription.Once bacteria reach mid-exponential phase growth, there is a second layer of control on Salmonella virulence flhDC operon and keeping a check on the flagellar synthesis as well as FlhD4C2 production.There is no doubt that flagellar motility provides a significant survival advantage over non-motile bacteria in many environmental situations. Furthermore, the link between production of flagella and other regulatory networks flhDC promoter region flhDC and P5flhDC promoters are functional in a wild-type strain under laboratory growth conditions. First, we demonstrated that there was a reduction in flhDC operon transcription in the absence of P1flhDC or P5flhDC compared to the wild-type strain  does not impede swimming. FlhD4C2 activity is also required in other cell processes such as Spi1 gene expression and other genes less characterized such as the srfABC operon modABC operon flhDC is required for flagella production and P5flhDC is required for growth in various environmental conditions such as in biofilms or in host cells. One possibility is that the activation of flhDC transcription from P5flhDC might represent a mechanism of protein amplification by a surge of transcription, when it is necessary to turn on the Spi1 regulatory network, even under conditions where flagella synthesis is inhibited at the level of fliA and fliC. This scenario can be very useful after infection when the bacteria requires expression of virulence factors to survive the physical and immune clearance of the eukaryotic host.The three promoter classes of the flagellar regulon, class 1, class 2 and class 3; are expressed in a temporal cascade that coincides with flagellum assembly flhDC operon from the P1 promoter provides the transcription factor, FlhD4C2 for expression of functional flagellar machinery to reassemble filaments and allow bacterial cells to swim to selected points of entry into epithelia cells. At the time of invasion, expression of both T3SS1 and flagella has been shown to be required. Thus, in the second step, the already expressed flhDC from P1flhDC promoter activates transcription of fliZ, the posttranslational regulator of HilD. In turn, HilD promotes transcription of Spi1 genes, leading to invasion. Thus P1-expressed flhDC fulfills two functions: driving the cells near the point of entry and also boosting the expression of Spi1, necessary for invasion, through its effect on HilD. It is noteworthy to mention that invasion of epithelial cells is a rapid process occurring within 10 to 15 minutes after introduction of S. typhimurium into the intestinal lumen Salmonella can replicate within two distinct intracellular environments: intravacuolar and cytosolic flhDC and down-regulates the transcription of P1flhDC in an RtsB-dependent manner. The transcription from P5flhDC is bistable leading to two populations of cells, one is flagellated and the other is not (∼10% of cells being flagellated). This bistable expression of P5flhDC is reminiscent with the bistable expression of Spi1. We suggest that the presence of two populations inside epithelial cells could be explained by the bistability from P5flhDC promoter and the consequent downregulation of P1flhDC might represent a mechanism to limit the number of flagellated cells. The cytosolic growth of Salmonella leads to the extrusion of epithelial cells as a host defense mechanism Salmonella with a fitness advantage to use the energy-taxis mechanism to benefit from inflammation flhDC represent a mechanism by which bacteria can disseminate and increase transmission by fecal shedding. These hypotheses are under investigation.Flagella appear to be required for reaching and selecting point of entry of bacteria into host cells flhDC has no effect on the synthesis of flagella but rather leads to the production of single subunits of the active transcriptional complex FlhD4C2. It has been shown that the inhibition of FlhD4C2-dependent transcription inside host cells is due to the effect of YdiV-mediated ClpXP degradation of the FlhD4C2 complex. The expression from P5flhDC late during cell growth will not allow for motility because the activation of the ClpXP leads to the degradation of the complex. However, ClpXP in addition to degrading the FlhD4C2complex also degrades the FlhC single subunit but not FlhD. This leads to the hypothesis that single FlhD or FlhC subunits might activate transcription of other genes required for virulence An additional scenario is that the transcription from P5flhDC by inhibiting transcription from P1flhDC and P5flhDC. The effect of RcsB is more dominant on P1flhDC then on P5flhDC, whereas LrhA represses more strongly P5flhDC than P1flhDC. Transcription activation of P1flhDC by CRP leads to a rapid transcription of rflM, which in turn reduces transcription of P1flhDC, and limits a rapid class 2 and class 3 genes expression. The FlhD4C2 complex, already produced, allows motility to proceed and also promotes activation of HilD at the posttranslational level through FliZ, ultimately leading to activation of transcription from the P5flhDC promoter. This positive autoregulation also generates a subsequent inhibition of flhDC operon transcription, of both P1flhDC and P5flhDC promoters, by two HilD-induced regulatory factors SlyA and RtsB, themselves regulated by different environmental cues. The activation of transcription from P5flhDC would lead to higher expression of FlhD4C2. Though not necessary for motility, it could affect expression of HilD. Because, HilD is required for Salmonella survival inside host cells, this positive circle of activation might be well suited for virulence.Our finding can be rationalized in terms of a model . Two regS. typhimurium P22HT105/1 int-201 was used in all transductional crosses Bacterial strains and primers used in this study are listed in flhDC8093-luxCDBAE)*Km*(PwtflhDC-flhD+C+)] primers 5104 and 5103  demonstrated the correct integration of the plasmid at the flhDC promoter region. A second PCR reaction using [Primers 267 (Km) and 1403 (luxE)] demonstrated the correct placement of Km cassette after the luciferase operon. Amplification with primers, 1403 and 1401, indicated a single copy integration of the plasmid without its origin of replication. Five candidates were obtained having a single integration of PwtflhDC-luciferase into the chromosome. One of the five candidates was sequenced and used in this study (TH18684). The Duplication of PflhDC was maintained in the presence of 5–10 µg/ml Km.For the construction of strain TH18684 DUP/pKD46) as the electroporation recipient. Individual fusion constructs with specific promoter regions were designed as follows: the rcsB promoter region included 400 bp upstream of the start codon through 230 bp of coding region, the rcsD promoter region included 466 bp upstream of the start codon through 260 bp of coding region, the slyA promoter region included 258 bp upstream of the start codon and 290 bp of the coding region, the hilD promoter region included 300 bp upstream of the start codon through 240 bp of coding region, the rtsA promoter region included 264 bp upstream of the start codon through 290 bp of coding region, the lrhA promoter region included 880 bp upstream of the start codon through 200 bp of coding region and the rflM promoter region included 460 bp upstream of the start codon through 284 bp of coding region. The promoter regions defined above were amplified by PCR using the respective primers listed in tetRA element with a PCR-amplified DNA fragment Mutations in the promoter region of PChromosomal FLAG-tagged HilD, RcsB and chromosomal HA-tagged RtsB, SlyA, RflM, HilD and LrhA were generated by the Lambda-Red recombinase system, as described previously flhDC. After background correction, relative light units (Arbitrary Units) were calculated by dividing the lights reading with its corresponding OD 595 nm. The OD 595 nm in our setting of the PolarStar Optima reader corresponds to ∼1.69 factor of the OD 595 nm read with 1 ml spectrophotometer.LB+Km medium containing 1% tryptone, 0.5% yeast extract, and 0.5% NaCl was used for growth of all bacterial cultures to determine the transcription activities of luciferase. Overnight cultures in LB+Km cultures were adjusted to the same OD 595 nm, then, 8-ml glass tubes containing 2 ml of LB+Km were inoculated with a 500-fold dilution of the bacterial suspensions and incubated at 30°C in a water bath with shaking at 250 rpm. For determination of luciferase activity in batch cultures, samples (200 µl) were taken at different time point and the light production along with the OD595 were measured in 96 well plates in a microplate reader (PolarStar Optima). For the determination of luciferase activity in 96 well plates, adjusted OD595 of overnight bacterial cultures at 37°C were diluted 500-fold in LB+Km and 200 µl of diluted bacteria were added to 96 well dark plates (Greiner). The plates were sealed with breathe easy membrane  and incubated in a chamber/shaker of a PolarStar Optima microplate reader (BMG labtech) set at 30°C. The conditions of the plate reader to determine the light production and OD 595 nm were as follow: orbital Shaking for 300 s at 150 rpm, 5 s stop and 95 s for luciferase light reading of the wells. For normalization of results a 0.1 s integration time was used. The OD 595 nm and light production (luciferase) was measured over time using a PolarStar Optima microplate reader (BMG labtech). For the background, we took the average measurements of the strain (TH18402) harboring mutations in all the promoters of Whole-cell extracts were prepared from samples of cultures grown in LB. 500-ml flasks containing 100 ml of LB were inoculated with a 500-fold dilution of the bacterial suspensions and incubated at 30°C in an orbital shaker at 150 rpm. Cells were collected at different optical densities  and washed twice with ice cold PBS. Pellets were lysed, at room temperature for 15 minutes, using B-PER reagent  with freshly added lysozyme (1 mg/ml) and protease inhibitors (Roche). The lysates were clarified by centrifugation at 4°C for 10 minutes. Supernatants were transferred to new eppendorfs and the extracted proteins were quantified using the BSA assay (BioRad). Samples, containing 50 µg of total protein per lane, were electrophoresed onto 12% to 14% Tris/SDS gels. To detect RtsA-HA a 15% Tricine-SDS gel was used as described flhDC were used  were normalized  to the Input DNA fraction Ct values by substracting the Ct-values of the sample from the corresponding no antibody control. The percentage input of each ChIP fraction was calculated using 2 and adjusted to the normalized background (No antibody) using the following formula: ΔΔCt(Chip) = ΔCt−ΔCt. The IP fold enrichment was then calculated using 2(−ΔΔCt(ChIP/NAC)) to evaluate the fold amount of starting material of the sample applied in the real-time PCR.CHIP was performed as in ere used . PCR conFigure S1Effect of static culture growth on the transcription of the flhDC operon in Salmonella enterica serovar Typhimurium. This plot represents the luminescence divided by the corresponding OD595 (A.U.) of a static culture. An overnight culture of strain PwtflhDC (TH18684) at 37°C was diluted 1 to 500 into fresh LB media. Cells were then incubated statically at 30°C and luminescence was recorded along with the OD595. OD values are shown at the bottom of the chart. Values are the average of two experiments done in duplicate.(TIF)Click here for additional data file.Figure S2Transcription dynamics of factors that regulate flhDC transcription mimic the time in the cell growth phase where their effect on flhDC operon transcription is exerted. Luciferase activity was investigated in wild-type strain harboring PwtflhDC-luxCDBAE-PwtflhDCflhD+C+ (TH18684), lrhA-luxCDBAEP (TH20540), rcsD-luxCDBAEP (TH20087), slyA-luxCDBAEP (TH19426), hilD-luxCDBAEP (TH19425) rtsA-luxCDBAEand P (TH19664). Luciferase activity was recorded and plotted as described in A) Transcription of the auto-regulated lrhA gene promoter was activated immediately after dilution of an overnight culture into LB media and earlier than the transcription of the flhDC operon. (B) The transcriptional profiles of rflM and rcsD promoters are shown in the second axis along with the PwtflhDC. The activation of the rflM promoter transcription, expressed from an FlhD4C2-dependent promoter, was concomitant with that of flhDC operon transcription (PwtflhDC), happening at earlier time point of the cell growth phase. The transcription from the rflM promoter (PrflM) diminished before cells enter stationary phase compared to promoter transcription for other regulator factors shown in this figure. Transcription from the rcsD promoter (PrcsD) (shown in the second axis) was activated before that of PwtflhDC. (C & D) Activation of promoters of the virulence related genes, implicated in the regulation of flhDC transcription, took place after initiation of flhDC transcription. A representative growth curve is shown in . For (B), the OD595 is shown at the bottom of the chart.(TIF)Click here for additional data file.Figure S3Analysis of mutations of the putative promoters P2, P3, P4 and P6. (A) The wild-type sequence of −10 box of the putative TSSs and their mutant alleles are shown.  Charts represent the luciferase activities of the PwtflhDC-luxCDBAE-PwtflhDCflhD+C+ reporter construct in wild-type and isogenic strains carrying mutations in individual start-site −10 boxes. Cells were grown overnight in LB and diluted 1 to 500 in fresh media, and grown at 30°C with shaking and luciferase activities were recorded at two optical densities . Charts of luciferase activity in strains with mutations in the P2 (B), P3 (C), P4 (D), P6 (E) promoters of flhDC operon compared to the wild-type flhDC promoter activity that was set at 100%. Each specific mutation is indicated under their corresponding bars. (F) Luciferase activity of strains P5+P6+P2+  and P6+P2+P1+ . Results are the average of three independent experiments done in duplicate. Error bars represent standard deviation. (G & H) Mutations in the flhDC P2flhDC and P6flhDC promoter start-sites inhibit CRP-mediated transcriptional activation of P1flhDC start-site. (G) CRP does no longer affect transcription of flhDC in strains deficient in P1, P2 and P6 promoters. Luciferase activity of PwtflhDC, P1−flhDC, P2−flhDC and P6−flhDC was measured in two genetic backgrounds: wild-type (wt) and its isogenic null mutant crp (crp::Tn10). Plots represent the ratio of the luciferase activity measured in wild-type strain relative to crp null mutant. (H) CRP represses transcription of P6flhDC promoter. Luciferase activity of P1+flhDC (only P1 is active the rest of the promoters are mutated) and P6+flhDC (only P6 is active the rest of the promoters are mutated) was measured in two genetic backgrounds: wild-type and its isogenic null mutant crp. Plots represent the ratio of the luciferase activity measured in crp null mutant relative to the wild-type.(TIF)Click here for additional data file.Figure S4Effects of RcsB, LrhA, RtsB and SlyA regulators on flhDC P1flhDC and P5flhDC transcription. For these assays, we compared the transcription of flhDC promoter region constructs (A) The P5+flhDC  promoter constructs transcribed flhDC primarily from the P5 start-site. (B) The P1+flhDC  promoter constructs transcribed flhDC primarily from the P1 start-site. (A) RcsB and LrhA but not RtsB or SlyA repressed transcription of flhDC in P1+flhDC construct. Luciferase activity of P1+flhDC-luxCDBAE- PwtflhDCflhD+C+ transcriptional fusion was investigated in five genetic backgrounds: wild-type (TH18901), ΔrcsB::tetRA (TH19217), rtsB::T-POP (TH19176), lrhA::T-POP (TH19603), slyA::T-POP (TH19618). (B) RcsB, LrhA and SlyA but not RtsB repressed transcription of flhDC in P5+flhDC construct. Luciferase activity of P5+flhDC -luxCDBAE-PwtflhD+C+ transcriptional fusion was measured in wild-type (TH18905), ΔrcsB::tetRA (TH19221), rtsB::T-POP (TH19180), lrhA::T-POP (TH19607) and slyA::T-POP (TH19619). Plots represent luciferase activity divided by the OD595 and plotted against the OD595 values shown at the bottom of the chart.(TIF)Click here for additional data file.Figure S5Effects of HA or Flag-tagged regulators, LrhA, RcsB, SlyA, HilD, RtsB and RflM on flhDC operon transcription. Luciferase activity of PwtflhDC -luxCDBAE-PwtflhD+C+ transcriptional fusion was measured in wild-type, lrhA-HA, rcsB-Flag, slyA-HA, hilD-Flag, rtsB-HA and rflM-HA. Cells were diluted 1 to 500 from an overnight culture into LB and grown at 30°C. Plots represent luciferase activity measured at OD 1 compared to the wild-type expression set at 100%.(TIF)Click here for additional data file.Table S1List of strains used in this study.(DOCX)Click here for additional data file.Table S2List of primers used in this study.(DOCX)Click here for additional data file.Text S1Analysis of the promoters of the flhDC operon.(DOCX)Click here for additional data file.


Drosophila, dissected tissues or cultured cells. Transgenic integration of the reporters into the Drosophila germline was performed in a site-directed manner, using ΦC31 integrase. This strategy avoids variable position effects and assures low base level activity and high signal responsiveness. Defined integration sites furthermore enable the experimenter to compare the activity of different reporters in one organism. The reporter constructs have a modular design to facilitate the combination of promoter elements , reporter genes , and genomic integration sites. The system was used to analyze and compare the activity and signal response profiles of two stress inducible transcription factors, AP-1 and Nrf2. To complement the transgenic reporter fly lines, tissue culture assays were developed in which the same synthetic ARE and TRE elements control the expression of firefly luciferase.This paper describes the construction and characterization of a system of transcriptional reporter genes for monitoring the activity of signaling pathways and gene regulation mechanisms in intact  Such research has yielded detailed molecular descriptions of many signaling and gene regulation processes. Questions that are, however, much less well understood include: How are spatial and temporal patterns of gene expression regulated in the two and three dimensions of epithelia or tissues? How do multiple signaling systems and transcription factors interact and form complex gene control networks in the intact organism? Addressing such questions requires experimental tools with which the activity of signal-dependent transcription factors can be monitored in live tissues with good temporal and spatial resolution. To this end, reporter genes that can register the activity of transcription factors or other signaling proteins have been designed. A widespread type of reporter relies on fusion genes in which synthetic DNA elements, that represent defined binding sites for a transcription factor of interest, are inserted upstream of sequences encoding protein products such as fluorescent proteins and enzymes, which are easy to visualize or quantify, are not normally present in the test organism, and for which sensitive assays are available such as chloramphenicol acetyl transferase or luciferase. Such reporter genes have been used in tissue culture or in transgenic animals Drosophila GFP or RFP reporters have been employed in the analysis of specific signaling pathways and regulatory processes in the context of an intact organism Signal transduction mechanisms that modulate transcription profiles in response to extracellular inputs control all major biological processes, including development, growth and homeostasis. Our knowledge of signal-dependent transcription control has been derived from studies Drosophila reporter lines published so far is that they have been generated by random P-element integration into the genome of a recipient strain. Consequently, the insertion sites and their genomic environment are undefined and these reporters can be subject to variable and unpredictable position effects. The influence of nearby gene regulatory elements or the overall epigenetic state at the integration site can substantially affect the expression characteristics and response patterns of transgenes One technical limitation of most transgenic Drosophila germline can alleviate problems arising from random position effects. The feasibility of such strategies has recently been documented in vivo assays by providing platforms for rapid quantitative analyses and high throughput screens. This standardized system permits comparative reporter analysis across different signaling pathways using in vivo and in vitro platforms.The development of targeted methods for site specific transgene integration into the The continued integrity of cells and organisms depends on their ability to protect themselves against oxidative stress and fluctuations in redox state. Multiple signaling systems have evolved that can sense oxidative and genotoxic stresses and orchestrate rapid and effective responses in order to deflect or repair potentially harmful damage to cells, tissues and organisms. Conserved stress-inducible signaling systems include: the JNK, p38, p53, NF-kB and Nrf2 pathways  is the eponymous enzyme of a MAP kinase pathway that has been implicated in stress responses, embryonic development, wound healing, apoptosis and cancer cis-linked genes. The JNK signaling system in the fruitfly is structurally and functionally similar to that of mammals. The Drosophila homolog of JNK is encoded by the gene basket (bsk) Drosophila Jun and Fos homologs are regulated by JNK phosphorylation JNK , a kinase that selectively phosphorylates JNK and causes its robust activation Drosophila Nrf2 pathway was genetically activated, by overexpression of CncC, enhanced ARE but not TRE activity was observed  that serves as a background control.Compared to existing P-element based systems, the reporters described here offer several advantages: The The two integration sites chosen are on the two arms of the second chromosome. Pairs of red and green reporters can thus be easily combined, generating stable dual reporter stocks. In this manner, Nrf2 and JNK activity can be tracked independently in the same specimen. It is also possible to combine ARE or TRE reporters with tissue or cell type specific red or green markers or with the control mRE vector. The similar maturation time of eGFP and DsRed.T4 further assists this comparative analysis.Drosophila transgene technology, the system can be adapted to track other signaling pathways or transcription factors.The plasmid constructions are designed in a way that the system can easily accommodate other synthetic or natural promoter or enhancer sequences. Using straightforward recombinant DNA and in vivo reporters. For example, both in vivo and in vitro assays document that Nrf2 and JNK responses are regulated by independent upstream pathways. The S2 cell system can be used for medium or high throughput RNAi or drug screens. The integrated reporter system allows rapid in vivo validation of genes or mechanisms pinpointed by cell based screens.Luciferase versions of the same reporter genes show the regulatory characteristics and signal responsiveness in S2 cell transfections that was predicted from the behavior of the Drosophila. For example, Tsai et al While JNK, as well as Nrf2, reporters have been constructed for mammals, there are no reports of a comprehensive, standardized and directly comparable system like the one we have generated for Drosophila Hsp70 minimal promoter driving the expression of firefly luciferase gene in vivo reporter plasmids were generated in multiple steps. 4×ARE was transferred into the pGreen H-Pelican vector to generate pGreen-ARE. The attB sequence from piB-GFP Synthetic double stranded oligonucleotides, carrying four high affinity AP-1 or Nrf2 binding sites or a mutant control . Individual reporter fly lines were then recombined with other reporter fly lines to facilitate the simultaneous study of two different promoter elements in the same organism. For overexpression or RNAi experiments in which genetic effects on reporter activity were analyzed, the reporter lines were also recombined with different driver lines .Plasmids maps and sequence files are included as Drosophila S2 cells were transiently transfected with firefly luciferase reporter constructs using the calcium phosphate method. A co-transfected actin-driven renilla luciferase reporter plasmid (pAct-RL) 6 cells/ml concentration 6 hours before transfection. The transfection mix was prepared by combining Solution A  and Solution B . Solution A was slowly added drop wise to an equal volume of Solution B with continuous vortexing and the mixture was incubated at room temperature for 30 minutes to allow formation of a fine precipitate. The mixture was briefly vortexed before adding it drop wise to the seeded cells with constant swirling. 1 ml of seeded S2 cells was incubated with 0.5 ml of transfection mix  at 25°C for 16 hrs before the calcium phosphate solution was aspirated, cells were washed with 1×PBS and fresh culture medium was added. The Solution A for CncC and Hepact over expression experiments contained 0.25 M CaCl2, 0.5 µg/ml of the firefly luciferase reporter construct, 0.2 µg/ml pAct-RL, 0.5 µg/ml pAct-Gal4 and 1 µg/ml pUAST-CncC or pUAST-Hepact. pUAST-CncC and pUAST-Hepact were replaced with 1 µg/ml salmon sperm DNA in the control group of these experiments.To study the effect of different oxidative stressors on reporter gene expression in transgenic flies, 1 week old flies that were mated for one day and then separated into males and females, were exposed to different oxidative stressors. The animals were starved for 2 hours and then fed a solution of 5% sucrose ±20 mM Paraquat . To examine the effect of oltipraz on reporter flies, similarly collected reporter flies were fed food supplemented with 1 mM oltipraz  for 48 hours. 15–20 flies were used in each group for these experiments and 3–5 representative flies were chosen randomly for imaging. Similar results were seen in multiple experiments. Western blot for reporter proteins on pools of flies also confirmed the induction of reporter transgenes with different treatments (data not shown).2 ) and were incubated at 25°C for 24 hrs.To assess the effect of different oxidative stressors and chemicals on the cell-based reporters S2 cells were transiently transfected with the reporter plasmids by the calcium phosphate method. 8 hours after the PBS wash and medium change, the transfected cells were transferred to 96-well plates and treated with 25 µM oltipraz, 100 µM DEM, 100 µM NaAsO6 cells/ml concentration) were bathed with dsRNAs in 12-well plates (8 µg dsRNA/well) as described dsRNAs were synthesized and purified following the protocol provided by Sandra Steinbrink and Michael Boutros The activities of cell-based reporters were measured by ‘Dual Glo Luciferase Assay System’ kit (Promega). 75 µl of transfected S2 cells  were transferred to wells of 96-well luciferase assay plates and luciferase assay was carried out in triplicate following the protocol provided by the supplier.To study JNK activity during dorsal closure TRE-red-2R/SGMCA stage 13 embryos were collected on apple plates and were dechorionated manually on double sided tape. Live embryos were mounted in PCTFE oil . Confocal images were collected using a Leica TCS SP5 system and were processed using Adobe Photoshop.RNAi; UAS-dKeap1RNAi/+ stage 2 larvae were treated with inducer RU486  by addition of 2 mg/ml RU486 in 50% ethanol to the food. The control group of the same genotype was treated with 50% ethanol. Both the control and treated larvae were dissected at stage 3 in PBS and salivary glands and guts were fixed at room temperature for 30 minutes in 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO4, 4 mM sodium phosphate, 1 mM MgCl2, and 4% formaldehyde. DNA was stained by Hoechst. Confocal images were collected and processed as above.To study CncC activity tubGS-Gal4, ARE-green-2L/UAS-dKeap1Figure S1Insertion site or type of reporter gene do not affect in vivo ARE activity. ARE-red-2L, ARE-green-2R double Nrf2 reporter flies were exposed to DEM stress. Both basal level and DEM-induced activities of ARE reporters at two different sites, attP40 (2L) and attP16 (2R), containing different reporter genes (eGFP and DsRedT4) were similar.(TIF)Click here for additional data file.Text S1Plasmid maps and sequences of in vivo reporters. The maps and sequences for plasmids pB-ARE-Green, pB-ARE-Red, pB-TRE-Green, pB-TRE-Red and pB-mRE-Red are shown. These plasmids were inserted in attP40 and attP16 sites to generate transgenic reporter fly lines.(PDF)Click here for additional data file.Table S1Primers used to generate amplicons for dsRNA synthesis.(DOC)Click here for additional data file.


Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.In order to better understand the influence of sesquiterpene synthases on artemisinin yield in  Secondary metabolites released from aerial organs into air and roots into soil play important roles in plant responsiveness to various external signals, such as direct and indirect defenses, plant reproduction and plant-plant interactions. Many of these secondary products are terpenoids. The most common emitted terpenoids are mono- and sesquiterpenes. The constituents of such emissions differ for different varieties and even in different tissues of the same plant Artemisia annuaE)-β-CPS is suggested to be involved in indirect defenses of maize and rice, even though it is not expressed in all varieties of maize E)-β-Farnesene synthase (FS), which can be induced by herbivore in some maize genotypes, was cloned and characterized from maize A. annuaCitrus junosMany terpene synthases involved in the biosynthesis of the emitted terpenoids have been cloned and characterized. For instance, β-caryophyllene synthase (CPS) and (3R)-linalool synthase (LIS) were found to be involved in responsiveness to wounding and fungal elicitors in E)-farnesene, (E)-nerolidol, and -farnesol after herbivore attack ArabidopsisMany terpene synthases catalyze the formation of a single product while some terpene synthases are involved in the formation of product mixtures. In maize, such an enzyme (terpene synthase I) catalyzes the formation of (A. annua. Artemisinin Combination Therapies (ACTs) are and continue to be effective against multi-resistant malaria parasites and are at present the preferred drug to fight this disease Artemisinin is an endoperoxide sesquiterpene lactone of high value isolated from the Chinese herbal medicinal plant A. annua have been characterized. One is high in artemisinic acid and arteannuin B  and the other is high in dihydroartemisinic acid and artemisinin  A. annuaThe biosynthesis of artemisinin is fairly well understood as far as the enzymology is concerned. Cyclization of farnesyl diphosphate (FDP) to amorpha-4-11-diene by amorph-4,11-diene synthase (ADS) is the initial step of the artemisinin biosynthetic pathway A. annua and may reduce the yield of artemisinin due to a competition for the substrate FDP (epi-cedrol synthase (ECS) A. annua plants leading to improved yields of artemisinin. Furthermore, over-expression of the endogenous farnesyl diphosphate synthase (FDS) The first and rate-limiting enzyme of artemisinin biosynthesis is ADS converting FDP to amorpha-4,11-diene. A number of other enzymes using FDP as substrate are present in rate FDP . These iA. annua, which varies between different cultivars. In most cultivars of A. annua, germacrene D, β-caryophyllene and β-farnesene are main constituents A. annua.The activity of various sesquiterpene synthases is reflected in the composition of extracts and essential oils from A. annua have been reported. The relative expression of ADS, β-CPS, and ECS transcripts in different tissues were studied by qPCR to determine the relative expression of these enzymes Some studies on the expression of sesquiterpene synthases in A. annuaCPS, ECS and FS genes. These three genes were randomly selected from the sesquiterpene synthase that have been cloned from A. annua. An additional sesquiterpene synthase (germacrene A synthase) has been cloned from A. annua but this enzyme was not included in the promoter studies. The cloned promoters have been fused to the GUS reporter gene and transgenic A. annua plants expressing these fusions have been established. Here we report on studies on the activity of these promoters in different tissues as reported by GUS staining. The response of the wild-type promoters to wounding and methyl jasmonate treatment have been studied by qPCR.We have previously reported on studies on the ADS promoter using a promoter-GUS fusion in transgenic A. annua var. Chongqing were obtained from the Southwest University in Chongqing China. The Chongqing cultivar is a high artemisinin producer.Seeds of A. annua were grown under 16 h light and 8 h dark at 22 °C to a height of approximately 1 m followed by flower buds induction at 8 h light and 16 h dark at 22 °C. Flower buds, young leaves, old leaves, stems and roots were collected for GUS staining. Samples of these tissues were also frozen in liquid nitrogen and ground to a fine powder for RNA extraction.Plants of A. annua using the CTAB method CPS, ECS and FS genes by PCR using primers listed in EcoRI and NcoI restriction site for cloning of the fragments into the modified plant transformation vector pCAMBIA1381Z.Genomic DNA was extracted from fresh young leaves of A. annua. This vector had previously been modified in that the plant selection gene was changed from hygromycin to kanamycin The pCAMBIA1381Z vector (CambiaLabs) carrying the GUS-gene was used for the transformation of NcoI/EcoRI and separately inserted into the modified pCAMBIA1381Z vector digested with the same restriction enzymes. The plant transformation vectors obtained  were introduced into E. coli BL21, which was grown on LB medium containing kanamycin (50 mg/l). pCAMBIA1381Z::GUS lacking a promoter was used as control. The vectors were purified using the GeneJet Plasmid Miniprep Kit (Fermentas) and the plant transformation vectors were introduced into A. tumefaciens EHA105 by the freeze and thaw method. A. tumefaciens EHA105 carrying the plant transformation vectors were grown on YEP medium containing kanamycin (100 mg/L) and rifampicin (40 mg/L) at 28°C to an OD600  =  0.8–1. The cells were collected by centrifugation and resuspended in MSMO liquid medium to an OD600  =  0.3–0.5.The cloned promoter regions were double-digested with Seed sterilization and germination as well as plant transformation was carried out as previously described A. annua plants to perform GUS analysis. GUS histochemical staining of the various plant tissues was carried out as previously described Leaf primordia at the apex and expanded leaves at different nodes, stems, roots from shoots and flowering plants, and flower buds were sampled from transgenic A. annua plants were sampled to analyze expression pattern by qPCR using β-actin as reference gene. RNA was extracted using Purelink™ Plant RNA Reagent (Invitrogen) according to the manufacture’s instruction. Genomic DNA was removed by treatment with DNase I (Fermentas). RNA (1 µg) was reverse transcribed using RevertAid™ H Minus-MuLV reverse transcriptase (Fermentas) primed with 0.5 µg oligo(dT)18 primer. RNA was removed from the cDNA obtained by treatment with RNase H (Fermentas).Different tissues of transgenic The qPCR was performed on a 7500 qPCR thermocycler (Applied Biosystems) using primers for β-actin, ADS, CPS, ECS, GUS Propagation of transgenic lines was carried out by shoot cuttings. Plants with similar morphology were selected 2 months after roots had appeared and were used for wounding and hormone experiments.A. annua plants were wounded at vegetative or reproductive stage by cutting (3–5 mm) along the midrib with a razor blade. Wound responsiveness was checked by GUS staining. Three transgenic lines were tested and three plants of the same variety transformed with the control vector were used as control. The response of the wild-type promoters to wounding was determined by qPCR using non-transgenic plants.Leaves of transgenic of Transgenic plants were sprayed with 300 µM methyl jasmonate (MeJA) + 0.01% Tween 20. Each plant was sprayed with 80 mL MeJA solution from 4 directions to ensure treatment of the entire plant. Three control plants of the same transgenic line were sprayed with 0.01% Tween 20. Treated plants were kept in open air for 3 h until there was no liquid on leaf surfaces. Subsequently, the plants were returned to standard growth conditions for 69 h. Middle leaves were sampled for expression analysis by GUS-staining. For each variety, three lines were checked. For each line, three technical replicates were carried out. The response of the wild-type promoters to MeJA was determined by qPCR using non-transgenic plants.EcoRI or HindIII, and eletrophoretically separated on an agarose gel , then transferred to a positively charged nylon membrane (Boheringer Mannheim GmbH). The 686 bp NPTII probe was labeled with digoxigenin by PCR using primers 15 and 16  phenyl phosphate (CSPD)  was used as substrate for chemiluminescent detection, after high stringency washes. The membrane was exposed to X-ray film for 30-60 min to obtain the desired image strength.Genomic DNA was digested completely by 5 and 16 . Hybridimean | /s) was used to test for outliers. The outliers were rejected if Gcalculated > Gcritical at P  =  0.05. The critical value of G is 1.155 when the sample size is three Grubbs test  as summarized in http://www.dna.affrc.go.jp/PLACE/) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) databases, we localized putative TATA- and CAAT-boxes of the recombinant promoters as summarized in The nucleotide sequences of the promoter regions upstream to the start codon were cloned by the genome walking method. The transcription start site (TSS) (labeled +1) of the cloned promoters was predicted using the TSSP software  for A. annua. Some of these TFs are involved in the regulation of the promoters studied.In plants, sesquiterpene synthase genes are involved in the biosynthesis of communication and defense related terpenoids. Defense terpenoids may be induced by insects, pathogens, wounding or MeJA. We will restrict our discussion of putative  factors . There aArabidopsis thaliana, there are six signal-responsive genes (AtSR1 to 6), which are related to the tobacco ethylene-responsive gene NtER1 encoding a calmodulin-binding protein AtSR genes are rapidly and differentially induced by environmental signals such as extreme temperatures, UV-B, salt, and wounding, hormones such as ethylene and abscisic acid (ABA), and signal molecules such as MeJA, H2O2, and salicylic acid (SA) cis-acting elements are found in promoters of genes involved in ethylene, ABA and SA signaling, and light signal perception. One putative CGCG-box was found in the ADS and ECS promoters (In romoters .APETALA2/ ethylene response factor TFs (AP2/ERF), i.e. AaERF1 and AaERF2, have recently been cloned from A. annuaTwo MeJA-responsive AaERF2 and a pADS::GUS construct in tobacco resulted in a three-fold increased activity of the ADS promoter. Over-expression of AaERF1 or AaERF2 in transgenic A. annua plants resulted in elevated transcript levels of ADS (2- to 8-fold) and in an increased accumulation of artemisinin The effects of the AaERF1 and AaERF2 have been studied on the ADS promoter A. annua TF database. Most bHLH TFs bind to the E-box sequence (CANNTG), which also is recognized by MYB and bZIP TFs. The number of E-boxes found in the studied promoters varied; ten, seven, three and one for the ADS, CPS, ECS and FS promoters, respectively (A. annua to JA.Basic/helix-loop-helix (bHLH) TFs may be involved in the JA responsiveness of plants ectively . These Ecis-acting regulatory elements in the sesquiterpene synthase promoters. The CGTCA motif, involved in the MeJA-responsive gene expression in barley (Hordeum vulgare) Arabidopsis involved in JA signaling In addition to the JA-responsive motifs discussed above, we have also found some other JA-responsive cis-acting elements, which may be involved in the response of A annua plants to MeJA. In fact, treatment of plants of A. annua with MeJA results in the induction of ADS and other enzymes of artemisinin biosynthesis such as FDS, CYP71AV1, DBR2 and ALDH1 leading to increased production of artemisinin Thus, the four sesquiterpene synthase promoters carry a number of JA responsive cis-acting element (PyACGTGGC) in ABA-responsive gene expression. One and three such ABA-responsive elements (TACGTG) were found in the CPS and ECS promoters, respectively (cis-element such as coupling element 1 (CE1) (TGCCACCGG) CGCGTGTCCTCA) ACCGACAT) cis-acting elements may both be involved in the response of the promoters studied to ABA.The ABA-responsive element (ABRE) is a major ectively . A singlA. annua plants with ABA (10 µM) resulted in the induction of a number of genes encoding enzymes involved in artemisinin biosynthesis Treatment of GAGAAGAATA) is involved in responsiveness of genes to SA cis-acting element involved in salicylic acid responsiveness and systemic acquired resistance in wild cabbage (Brassica oleracea) GAGAAGAAAA. Treatment of A. annua plants with 1 mM SA resulted in a temporary peak in the expression of the ADS gene The TCA-element  is found in the ADS (one copy) and CPS (one copy) promoters. The ACTTTA motif is essential for tissue-specific and auxin-regulated expression of the A. annua with gibberellic acid 3 (GA3) results in an increased formation of artemisinin, indicating an increased expression of the key regulatory enzyme ADS 3 by soil drenching Furthermore, the putative gibberellin (GA)-responsive element (the GARE-motif AAACAGA) is present in the ADS and FS promoters (one copy each). In addition, the FS promoter carries one copy of the (GA)-responsive pyrimidine box ; a conserved element in the upstream sequence of GA inducible genes in cereal seeds ATTTTCTCCA) was previously described in tobacco (Nicotiana tabacum) as a cis-acting element involved in defense and stress responsiveness Only the ADS promoter contains the putative ethylene responsiveness ERE-element (ATTTCAAA) (one copy). The TC-rich repeat  has been cloned The WRKY TFs constitute a large family of TFs in plants Arabidopsis with members of the WRKY transcription factor family to mediate responses to wounding or pathogen responses A. annua transcription factor AaWRKY in agroinfiltrated leaves of A. annua resulted in increased levels of ADS (7x) and other biosynthetic enzymes, such as HMGR (5x), CYP71AV1 (3x) and DBR2 (4x) indicating that several of the genes encoding enzymes involved in artemisinin biosynthesis are co-induced by binding of AaWRKY to W-boxes et al., unpublished] promoters also carry two box-W1 motifs each. A recent study showed that foliar application of an elicitor (chitosan) resulted in increase of dihydroartemisinic acid and artemisinin by 72% and 53%, respectively, in A. annuaThe fungal elicitor responsive box-W1 element, which interacts in Verticillium dahliae induces CPS in leaves of A. annuacis-acting regulatory AG-motif (AGATCCAA) that confers responsiveness to wounding and elicitors MYB gene NtMYB2 is up-regulated by an AG-motif binding protein (AGP1), which is a GATA-type zinc finger. The other three promoters do not carry any EL1-box3 sequence or AG-motif.An elicitor preparation from the phytopathogenic fungus i.e. 9, 5, 9 and 6 copies (A. annua leaves CPS gene is induced by wounding ECS or FS gene by wounding have been published.The WUN motif (consensus AATTTCC) is present in the four promoters in a relative large number, 6 copies  in the AArabidopsis, MBS sequences are the binding site for the proteins AtMYB1 and AtMYB2 involved in regulation of genes that are responsive to water stress Petunia hybrida for MYB.Ph3 involved in regulation of flavonoid biosynthesis cis-acting element (AAAAAATTTC) involved in heat stress responsiveness.In addition, the E-boxes discussed above are also involved in responses to abiotic elicitors. They are MYC recognition site conferring dehydration, cold and freezing induction. The MYB binding site  is involved in drought-inducibility. In et al. A annua cultures. Using qPCR, they observed after 24 h 11- and 7-fold increases in ADS and CYP, respectively, in cultures chilled to 4°C for 30 min compared to non-chilled cultures. Higher temperatures (42°C) did not seem to affect the transcript levels of either ADS or CYP71AV1. Yang et al. Lulu EcoRI or HindIII, and the fragments obtained were analyzed by Southern blotting using a 686 bp hybridization probe binding to the NPTII gene (pCPS::GUS and pFS::GUS constructs) or two copies (the pECS::GUS and pADS::GUS constructs)  .CPS gene, transgenic A. annua plants carrying the pCPS::GUS construct were produced using A. tumefaciens. Twenty-three positive T0 transgenic lines were selected by PCR (primers listed in ::GUS (lacking a promoter) were obtained and five randomly selected lines were used as controls.In order to study the temporal and spatial expression of the Tissues of primary transgenic lines were tested for CPS promoter activity by histochemical GUS activity staining. The same expression pattern was observed for all transgenic lines but the intensity of the staining varied. For further studies, we selected the five lines showing the strongest staining. GUS expression controlled by the CPS promoter was exclusively located to T-shaped trichomes (TSTs) of leaf primordia and expanded leaves . No GUS et al.CPS gene is expressed in all tissues of A. annua except in roots. In this study, the CPS promoter is specifically active in TSTs of leaves, stem, basal bract of flower buds and some florets cells but not in GSTs.Except for the expression in roots, the results obtained are in good agreement with those of Cai A. annua plants carrying the pECS::GUS construct were produced using A. tumefaciens as described above for the CPS promoter. Twenty-six positive T0 transgenic lines were selected by GUS staining and PCR (primers listed in ::GUS (lacking a promoter) construct were obtained and five randomly selected lines were used as controls.Transgenic A. annua plants at vegetative stage (two stages) and at the reproductive stage were sampled for histochemical GUS staining. At an early vegetative stage, GUS expression was exclusively located to TSTs of leaf primordia and expanded leaves  construct were used as controls.Transgenic All transgenic lines showed similar expression patterns, but different expression levels in individual plants. GUS expression was observed in leaf cells of shoots including GSTs and TSTs . FS is tA. annua was determined by measuring the transcript levels of the gene by qPCR. β-Actin was used as reference gene. The relative activity of the wild-type promoters in different tissues was compared with the activity of the β-actin promoter, which was set to 1.0. Relative activities were calculated using the 2-tΔΔC method http://www.REST.de.com) was used to analyze the data.The activity of the wild-type sesquiterpene synthase promoters in different tissues of pCPS) was active in all plant tissues except in roots  was highly active in flower buds and old leaves while it was moderately activity in flowers and leaf primordia (pECS in old leaves was ∼ 30 times higher than that in leaf primordia (The wild-type ECS promoter (rimordia . The relrimordia . Very lopFS) was highly active in flowers  (The four promoters studied carry a number of putative  motifs) . In orde motifs)  and 9.pCPS::GUS fusion showed rapid GUS expression at the site of wounding. Strong GUS staining was observed immediately after wounding, i.e. around 10 minutes . However, recently it was shown that ADS is induced by wounding and that the amount of artemisinin is increased around 50% 4 h after wounding Wounded leaves carrying the A. annua was determined by qPCR. β-Actin was used as reference gene. The relative activity was compared with that of the β-actin promoter, which was set to 1.0   ; Table 3i.e. after MeJA treatment GUS expression was observed in the same tissues as for non-treated plants.GUS-staining of treated transgenic plants showed that no difference in the expression patterns of GUS could be observed in any of the plants, Since it is impossible to quantitatively determine the relative levels of GUS expression in transgenic plants by staining, the activity of the wild-type sesquiterpene synthase promoters were determined by qPCR after MeJA-treatment, using β-actin as reference gene. The relative activity was compared with that of β-actin promoter, which was set to 1.0 . The fouADS gene, some studies on the effect of MeJA have been carried out, which are in agreement with the results presented here CPS gene was given as the amount of caryophyllene increased 3-fold after the MeJA treatment A. annua) is induced by the MeJA treatment.In the case of the A. annua plants of both chemotypes were treated with 100 µM MeJA every second day. After two weeks a 6- and 3-fold increase of ADS transcripts was observed in the HAP and LAP variety, respectively In another study, A. annua by qPCR. The relative amount of the β-actin transcript was set to 1.0  using primers as reported previously t to 1.0 . Flower In flower buds, the relative amounts of GAS, FS, ECS, CPS and ADS transcript were 0.14-, 0.74-, 0.01-, 0.20- and 0.46-fold the β-actin transcript level, respectively . The relA. annua plants leading to improved yields of artemisinin The four cloned sesquiterpene synthase promoters show different expression patterns as reported by the histochemical GUS staining. It seems that CPS and ECS do not influence the biosynthesis of artemisinin to any great extent due to their low expression in glandular trichomes  and 5. HGAS, ECS or CPS genes may not improve artemisinin production in A. annua to any great extent. However, down-regulation of the FS gene may have an effect on the yield of artemisinin.In conclusion, the five sesquiterpene synthases exhibited different expression pattern  and 12. Figure S1Colour-code for cis-acting elements present in the four cloned promoters.(PDF)Click here for additional data file.Figure S2Nucleotide sequence of the cloned ADS promoter with putative cis-acting elements shown. Putative TSS is shown in bold. Putative TATA- and CAAT-boxes are underlined.(PDF)Click here for additional data file.Figure S3Nucleotide sequence of the cloned CPS promoter with putative cis-acting elements shown. Putative TSS is shown in bold. Putative TATA- and CAAT-boxes are underlined.(PDF)Click here for additional data file.Figure S4Nucleotide sequence of the cloned ECS promoter with putative cis-acting elements shown. Putative TSS is shown in bold. Putative TATA- and CAAT-boxes are underlined.(PDF)Click here for additional data file.Figure S5Nucleotide sequence of the cloned FS promoter with putative cis-acting elements shown. Putative TSS is shown in bold. Putative TATA- and CAAT-boxes are underlined.(PDF)Click here for additional data file.