Flanking Fragment Research Articles

Characterization of Protease Inhibitor Promoter from Black Gram (Vigna mungo L.) and gus Gene Expression in Transgenic Tobacco Plants

Like other legumes, black gram is also known to be source of plant protease inhibitors. Plant protease inhibitors (PIs) are small proteins which represent a class of well studied compounds of plant defense genes and are abundant in storage organs such as seeds and tubers. The protease inhibitor proteins serve various functions in the growth and developmental processes. Like other legumes black gram (Vigna mungo L.) is also known to be a source of plant protease inhibitors and considered to be involved in insect tolerance. In this study, promoter sequence consisting of 0.75 fragments of a 52 upstream flanking region preceding a protease inhibitor gene of black gram was cloned from genomic clones and its sequence is characterized. The spatial and temporal control of the 0.75 fragment promoter sequence was also further investigated for its regulation on expression of the â-glucuronidase (gus) reporter gene in transgenic tobacco. Analysis of gus activities showed that the 0.75 kb flanking fragment of the protease inhibitor gene actually drives the gus reporter gene expression in ovary, pistil and seeds of tobacco. In another case, promoter sequence consisting of 1.3 kb fragment of a 52 flanking region of black gram protease inhibitor gene was isolated by Genome Walker strategy and promoter sequence was analyzed in-silico by PLACE and PlantCARE database.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter.

ZmbZIP25 (Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5′ RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from −2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from −2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5′-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5′-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5′-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5′-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from −1117 to −957 that were responsible for the specificity of the ZmbZIP25 5′-flanking sequence.

Variation of genomic islands and flanking fragments in Vibrio parahaemolyticus isolates from environmental and clinical sources in Taiwan

Vibrio parahaemolyticus is a halophilic foodborne pathogenic bacterium that causes gastroenteritis; it has become an issue of global concern since the emergence and spread of pandemic O3:K6 strains. This study evaluated the role of Vibrio pathogenicity island (VPaI)-associated fragments in the genetic variation and grouping of this pathogen. Distribution of some VPaI fragments and flanking fragments (VPaI-1, VPaI-4, VPaI-5, VPaI-6 and VPaI-7) was determined in a total of 53 V. parahaemolyticus isolates from environmental and clinical sources in Taiwan, and supported by the sequences of seven fragments of VPaI-4 and its flanking fragment VP2145. As determined from the distribution of these VPaI-associated fragments, the clinical pandemic isolates were closely related in a single cluster; the clinical nonpandemic isolates were grouped into several clusters, while the environmental isolates were comparatively highly diversified. The profiles of virulence-associated genes of environmental pathogenic isolates varied, and were closer to those of clinical nonpandemic isolates than those of pandemic isolates. Isolates with atypical profiles of the VPaI-associated fragments and virulence-associated genes were identified. Sequences of VP2145 exhibited a close phylogenetic relationship among these local isolates, which were distinct from most V. parahaemolyticus strains from other geographic regions. This investigation demonstrated the application of VPaI-associated fragments in studying the genetic variation and clustering of V. parahaemolyticus isolates from different sources.

NaCl stress induces CsSAMs gene expression in Cucumis sativus by mediating the binding of CsGT-3b to the GT-1 element within the CsSAMs promoter

The CsSAMs promoter is a salt-stress-inducible promoter containing three GT-1 elements that are sufficient for the salt-stress response. The transcription factor CsGT-3b was found to bind to the GT-1 element. The S-adenosyl-L-methionine synthase (SAMs) gene is among the functional genes induced during environmental stress. However, little is known about the regulatory mechanism and upstream regulators of this salt-inducible gene in cucumber plants. Thus, it is necessary to understand the characteristics of the SAMs gene by analyzing its promoter and transcription factors. In this study, we isolated and functionally analyzed a 1743-bp flanking fragment of the CsSAMs gene from Cucumis sativus. To examine promoter activity, the full-length promoter, as well as different promoter fragments, were fused to the β-glucuronidase (GUS) reporter gene and introduced into the tobacco genome. The full-length promoter displayed maximal promoter activity, whereas the P4 promoter, containing 321bp of upstream sequence, showed no basal promoter activity. In addition, the CsSAMs promoter exhibited stress-inducible regulation rather than tissue-specific activity in transgenic tobacco. Histochemical analysis revealed strong GUS staining in leaves, stems, and roots, especially in the veins of leaves, the vascular bundle of stems, and root tip zones following NaCl stress. A transient expression assay confirmed that the 242-bp region (-1743 to -1500) was sufficient for the NaCl-stress response. Yeast one-hybrid assays further revealed interaction between the NaCl-response protein CsGT-3b and the GT-1 (GAAAAA) element within the 242-bp region. Taken together, we revealed the presence of four salt-stress-responsive elements (GT-1 cis-elements) in the CsSAMs promoter and identified a transcription factor, CsGT-3b, that specifically binds to this sequence. These results might help us better understand the intricate regulatory network of the cucumber SAMs gene.

Identification and functional characterization of the NAC gene promoter from Populus euphratica.

The PeNAC1 promoter is a non-tissue-specific and stress-inducible promoter containing a GA-responsive element and a MYB recognition sequence that are responsible for induced expression patterns. NAC transcription factors play vital roles in complex signaling networks during plant stress responses. Promoters as crucial molecular switches are involved in the transcriptional regulation of gene activities dynamic network controlling a variety of biological processes, such as developmental processes, responses to hormone and abiotic stress. In this study, a 1217-bp flanking fragment of the stress-responsive NAC gene PeNAC1 was isolated from Populus euphratica. In transgenic Arabidopsis, this promoter fragment was found to have a higher activity than the cauliflower mosaic virus 35S promoter and remained active throughout the plant life cycle, particularly in the spiral vessels and cortical cells of vascular tissues of various organs. We identified a gibberellic acid-responsive element, required for response to gibberellic acid and involved in the salt-stress signaling pathway, and a MYB recognition sequence, which has an important role in promoter response to drought stress, in the PeNAC1 promoter. These results suggest that the PeNAC1 promoter is more effective, non-tissue-specific, and inducible. In addition, the presence of a putative NAC protein-binding motif in the PeNAC1 promoter indicates that PeNAC1 is either regulated by other NAC transcription factors or is self-regulated. Our research will help reveal the regulatory mechanism of the upstream region of the PeNAC1 gene and provide a foundation for the use of the PeNAC1 promoter in molecular breeding.

BdBRD1, a brassinosteroid C-6 oxidase homolog in Brachypodium distachyon L., is required for multiple organ development

Brassinosteroids (BRs), known as a kind of phytohormones, play essential roles in plant growth and development. Although the studies on the BR biosynthesis and signaling are extensive in Arabidopsis, little is known in temperate cereals. In this study, bdbrd1-1, a T-DNA insertion mutant from Brachypodium distachyon, was isolated and characterized in details. The bdbrd1-1 mutant showed lots of cellular and morphogenetic defects, including shortened cell shapes, severe dwarfing, twisted leaves and sterile spikes. Sequencing the flanking fragment of the T-DNA and complementation by genomic DNA in the mutant, confirmed that the developmental defects are caused by the T-DNA insertion in BdBRD1, a possible brassinosteroid C-6 oxidase gene. Application of 24-epicastasterone could partly rescue the bdbrd1-1 dwarfing phenotype. Expression analysis of BdBRD1 suggested that bdbrd1-1 is probably a null mutant and its wild type transcript is expressed in various tissues and highest in the leaf sheaths. Meanwhile, measurements on leaf numbers of the main stems or days to the emergence of the inflorescences suggested that bdbrd1-1 is late-flowering. The late-flowering phenotype could be converted by vernalization treatment, although there lacks a typical FLC gene in B. distachyon. The current data provide an insight into the relationship between BRs biosynthesis and individual development in B. distachyon, an emerging model plant for the temperate cereals.

Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains

In Yarrowia lipolytica, targeted gene replacement occurs only with long length (1kb) homologous flanking fragments, as this yeast preferentially uses the non-homologous end-joining mechanism (NHEJ) for DNA repair over homologous recombination (HR). To improve the frequency of HR, we identified and disrupted the KU70 and KU80 genes responsible for double strand break repair in the NHEJ pathway in Y. lipolytica. In ku70∆ HR of URA3 marker at the ADE2 locus occurred with 43% frequency with as little as 50bp long flanking regions. The number of Ura(+) transformants was reduced to 1% of the Po1d (ura3-302) wild type-like strain level, regardless of the flanking fragment length. On the contrary, even though HR was not improved in ku80∆, Ura(+) transformants was 60% lower compared to the wild type.

Identification and Functional Analysis of Variant Haplotypes in the 5′-Flanking Region of Protein Phosphatase 2A-Bδ Gene

Serine-threonine protein phosphatase 2A (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of cell growth, differentiation, and apoptosis. The substrate specificity and (sub)cellular localization of the PP2A holoenzymes are highly regulated by interaction with a family of regulatory B subunits (PP2A-Bs). The regulatory subunit PP2A-B/PR55δ (PP2A-Bδ) is involving in the dephosphorylation of PP2A substrates and is crucial for controlling entry into and exit from mitosis. The molecular mechanisms involved in the regulation of expression of PP2A-Bδ gene (PPP2R2D) remain largely unknown. To explore genetic variations in the 5′-flanking region of PPP2R2D gene as well as their frequent haplotypes in the Han Chinese population and determine whether such variations have an impact on transcriptional activity, DNA samples were collected from 70 healthy Chinese donors and sequenced for identifying genetic variants in the 5′-flanking region of PPP2R2D. Four genetic variants were identified in the 1836 bp 5′-flanking region of PPP2R2D. Linkage disequilibrium (LD) patterns and haplotype profiles were constructed for the genetic variants. Using serially truncated human PPP2R2D promoter luciferase constructs, we found that a 601 bp (−540 nt to +61 nt) fragment constitutes the core promoter region. The subcloning of individual 5′-flanking fragment revealed the existence of three haplotypes in the distal promoter of PPP2R2D. The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. The EMSA revealed that the −462 G>A variant influences DNA-protein interactions involving the nuclear factor 1 (NF1). In vitro reporter gene assay indicated that cotransfection of NF1/B expression plasmid could positively regulate the activity of PPP2R2D proximal promoter. Introduction of exogenous NF1/B expression plasmid further confirmed that the NF1 involves in the regulation of PPP2R2D gene expression. Our findings suggest that functional genetic variants and their haplotypes in the 5′-flanking region of PPP2R2D are critical for transcriptional regulation of PP2A-Bδ.

Activity of dlx5a/ dlx6a regulatory elements during zebrafish GABAergic neuron development

During vertebrate forebrain formation, Dlx homeobox genes play essential roles in the differentiation, migration and survival of subpallial precursor cells that will later give rise to diverse subtypes of γ-aminobutyric acid (GABA)-expressing neurons, including inhibitory cortical interneurons in mammals. They also participate in the regulation of the Gad genes encoding the enzymes necessary for GABA synthesis. In mice, at least four cis-regulatory elements (CREs) control Dlx expression in the telencephalon and diencephalon: URE2 and I12b in the Dlx1/ Dlx2 bigene cluster, and I56i and I56ii in the Dlx5/ Dlx6 bigene cluster. However, little is known so far with respect to the function of orthologous dlx genes and their regulatory elements during zebrafish GABAergic neuron development. To investigate whether similar dlx-mediated pathways exist in the early developing zebrafish forebrain, we generated independent lines of transgenic zebrafish carrying two distinct GFP reporter constructs driven by a β-globin minimal promoter: one containing a ∼1.4 kb dlx5a/ dlx6a intergenic sequence (encompassing I56i and I56ii) and one with a ∼1.1 kb fragment containing only the I56i CRE, respectively. The expression patterns of these two transgenes were compared with that obtained with another construct containing the ∼1.4 kb dlx5a/ dlx6a intergenic sequence and driven by a ∼3.5 kb dlx6a 5′-flanking fragment. Our comparative analysis showed that GFP expression of the three transgene is largely overlapping throughout the ventral forebrain. Intriguingly, the dlx6a 5′-flanking fragment has a major impact on transgene expression in the mesencephalic tectum. Furthermore, comparison of transgene expression between the ∼1.4 kb and ∼1.1 kb intergenic fragments did not show any specific spatial expression conferred by I56ii. Almost all GFP-expressing cells in the transgenic zebrafish are GABA-positive and also express various GABAergic interneuron markers. Together, our data suggest that zebrafish dlx5a/ dlx6a intergenic CREs may be involved in a conserved genetic pathway necessary for proper dlx expression during zebrafish GABAergic neuron development.

Regulation of aldo–keto reductase AKR1B10 gene expression: Involvement of transcription factor Nrf2

Aldo–keto reductase 1B10 (AKR1B10) is an aldose reductase-like oxidoreductase of human origin. The expression of AKR1 B10 is highly induced in the cells of various cancers such as lung non-small-cell carcinoma and hepatocellular carcinoma. Since the enzyme exhibits broad substrate specificities toward various xenobiotics such as anti-tumor drugs or various endogenous compounds such as retinaldehyde, AKR1B10 may play an important role in tumor progression or drug resistance. However, very little is known about its gene regulation. In this study, we investigated the regulation of AKR1 B10 expression. A −3282 bp of the 5′-flanking fragment of AKR1 B10 gene was isolated from A549 lung carcinoma cells. This region contains several putative regulatory motifs such as AP-1, NF-κB and antioxidant response element. In addition, a complex polymorphic microsatellite with repetitive sequences enriched with C and T was found. However, luciferase reporter assay revealed that the microsatellite polymorphism did not influence the basal promoter activity. We found that an antioxidant ethoxyquin induced the AKR1 B10 expression based on RT-PCR analysis and luciferase reporter assay. Since ethoxyquin is known to activate the gene expression mediated through transcription factor Nrf2, the involvement of Nrf2 was examined. Forced expression of dominant-negative Nrf2 mutant suppressed the ethoxyquin-induced AKR1 B10 expression, and co-introduction of Nrf2 expression plasmid into the cells significantly augmented the luciferase reporter activity. Deletion analysis revealed that Nrf2-regulating cis-element(s) lay within −539 bp of the 5′-flanking region. These results suggest that Nrf2 is one of the major factors involved in the AKR1 B10 gene regulation.

Induction and identification of disulfide-intact and disulfide-reduced β-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS and top-down proteomics

The disulfide-intact and disulfide-reduced β-subunit of Shiga toxin 2 (β-Stx2) from Escherichia coli O157:H7 (strain EDL933) has been identified by matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomic analysis using software developed in-house. E. coli O157:H7 was induced to express Stx2 by culturing on solid agar media supplemented with 10-50 ng mL(-1) of ciprofloxacin (CP). Bacterial cell lysates at each CP concentration were analyzed by MALDI-TOF-MS. A prominent ion at mass-to-charge (m/z) ~7820 was observed for the CP concentration range: 10-50 ng mL(-1), reaching a maximum signal intensity at 20 ng mL(-1). Complex MS/MS data were obtained of the ion at m/z ~7820 by post-source decay resulting in top-down proteomic identification as the mature, signal peptide-removed, disulfide-intact β-Stx2. Eight fragment ion triplets (each spaced Δm/z ~33 apart) were also observed resulting from backbone cleavage between the two cysteine residues (that form the intra-molecular disulfide bond) and symmetric and asymmetric cleavage of the disulfide bond. The middle fragment ion of each triplet, from symmetric disulfide bond cleavage, was matched to an in silico fragment ion formed from cleavage of the backbone at a site adjacent to an aspartic acid or glutamic acid residue. The flanking fragment ions of each triplet, from asymmetric disulfide bond cleavage, were not matched because their corresponding in silico fragment ions are not represented in the database. Easier to interpret MS/MS data were obtained for the disulfide-reduced β-Stx2 which resulted in an improved top-down identification.

Structure and promoter characterization of aldo–keto reductase family 1 B10 gene

Aldo–keto reductase family 1 member B10 ( AKR1B10) is overexpressed in human hepatocellular carcinoma, lung squamous carcinoma, and lung adenocarcinoma in smokers. Our recent studies have showed that AKR1B10 plays a critical role in the growth and proliferation of cancer cells by detoxifying reactive carbonyls and regulating fatty acid biosynthesis. However, little is known about the regulatory mechanisms of AKR1B10 expression. In this study, we determined the structure of AKR1B10 gene and characterized its promoter. The results demonstrated that AKR1B10 consists of 10 exons and 9 introns, stretching approximately 13.8 kb. A 5′-RACE study determined the transcriptional start site of AKR1B10 at 320 bp upstream of the ATG translational start codon. A TATA-like (TAATAA) and a CAAT box are present from − 145 to − 140 bp and − 193 to − 190 bp upstream of the transcriptional start site, respectively. Motif analysis recognized multiple putative oncogenic and tumor suppressor protein binding sites in the AKR1B10 promoter, including c-Ets-1, C/EBP, AP-1, and p53, but osmolytic response elements were not found. A − 4091 bp of the 5′-flanking fragment of the AKR1B10 gene was capable of driving GFP and luciferase reporter gene expression in HepG2 cells derived from human hepatocellular carcinoma; progressive 5′-deletions revealed that a − 255 bp fragment possesses full promoter activity.

Isolation and Characterization of a Curcin Promoter from Jatropha curcas L. and Its Regulation of Gene Expression in Transgenic Tobacco Plants

Ribosome-inactivating proteins (RIPs) represent those proteins that universally depurinate conserved α-sarcin loops of large rRNAs. In this study, a 0.6-kb fragment of a 5′ flanking region preceding a curcin gene, encoding a type I RIP curcin, of Jatropha curcas L. endosperm was cloned, and its regulation of expression of the β-glucuronidase (GUS) reporter gene was investigated in transgenic tobacco. Analysis of GUS activities showed that the 0.6-kb flanking fragment of the curcin gene was sufficient to drive the GUS reporter gene expression in tobacco seed. The activity of this flanking fragment was analyzed at different stages of seed development. Histochemical localization of GUS activity indicated that the promoter was specifically active in the endosperm tissue of the dicotyledonous tobacco embryo. Moreover, this activity was first initiated at the heart-shaped embryonic stage during seed development.

Identification and isolation of Mu-flanking fragments from maize

Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics. Isolation of genomic segments flanking the insertion Mutator ( Mu) is a key step in insertion mutagenesis studies. Herein, we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize. The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl II/ Msp I and ligation of digested fragments to the Bgl II- and Msp I-adaptors; 2) enrichment of a subset of Bgl II/ Msp I fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking regions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions. Using this approach, we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-induced mutant, and one such fragment, M196-FF, is found to contain a partial sequence of the DNA topoisomerase I gene Top1. Moreover, the modified AFLP method including all restriction enzymes, adaptors and primers has been optimized in this study. The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.

Sp1 is involved in 8-chloro-adenosine-upregulated death receptor 5 expression in human hepatoma cells

8-Chloro-adenosine (8-Cl-Ado) is an adenosine derivative, which inhibits proliferation and induces apoptosis in various tumor cells. Subtoxic concentration of 8-Cl-Ado sensitizes human hepatoma cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-triggered apoptosis. However, the molecular mechanism by which TRAIL cytotoxicity is amplified by 8-Cl-Ado is unknown. In the present study, we demonstrated by Western blot and real-time PCR that 8-Cl-Ado selectively up-regulated death receptor 5 (DR5), but not death receptor 4 (DR4), at both protein and RNA levels in human hepatoma cell line BEL-7402. Analysis of the transcriptional regulation of DR5 expression by using Dual-Luciferase reporter assay system demonstrated that the 5'-flanking fragment -207 to -145 upstream to the ATG site within the DR5 promoter region was responsible for the 8-Cl-Ado-upregulated DR5 expression. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) confirmed that 8-Cl-Ado treatment facilitated transcription factor Sp1 binding to its cis-element -198/-189 in the DR5 promoter, suggesting that Sp1 is at least one of the 8-Cl-Ado-responsive transcription factors. However, we observed that nuclear factor kappaB (NF-kappaB) activity remained invariable in the cells treated with 8-Cl-Ado. These data allowed us to draw a conclusion that 8-Cl-Ado-enhanced DR5 expression is regulated by Sp1 binding to the -198/-189 cis-element in DR5 promoter without affecting NF-kappaB activity in the hepatoma cells. This study may shed light on further screening the regulators of DR5 expression and developing novel therapeutic drugs for liver cancer.

Isolation of the maize Zpu1 gene promoter and its functional analysis in transgenic tobacco plants

By screening a genomic library of maize, a 2.2 kb 5' flanking fragment of Zpu1 gene, encoding the pullulanase-type starch debranching enzyme, was isolated. Promoter fragments of various lengths, including the full 5' flanking sequence (-2267 to -1) (Z1), a 3' deletion (-2267 to -513) (Z5) and three 5' deletions extending to -1943 (Z2), -1143 (Z3) and -516 (Z4) upstream of the translational initiation codon (ATG), were fused to the GUS reporter gene and introduced into tobacco. When these constructs were tested in transgenic tobacco plants, seed-preferred GUS activity was observed in pZ1-transgenic lines. In pZ2-transgenic lines, the GUS activity was not only restricted to seeds, but was also detected in calyxes, petals, stamens and mature leaves. At the same time, negligible GUS activity was detected in roots, stems, young leaves, stigmas and ovaries from the transgenic tobacco plants, which had integrated the full isolated sequence of Zpu1 promoter or its deletions. Deletion analysis indicated that the promoter contained a putative positive cis-regulatory element and the proximal region (-516 to -1) was essential for directing the expression of gus reporter gene. Analysis of GUS activity during the fruit development and seed germination suggested that Zpu1 promoter is active both in starch anabolism and in starch catabolism, which is consistent with the function of the endogenous gene in maize. GUS activity in leaves under light and darkness confirmed that Zpu1 promoter functions in the starch degradation of photosynthetic tissues in the dark phase of the diurnal cycle.

Isolation and characterization of Brittle2 promoter from Zea Mays and its comparison with Ze19 promoter in transgenic tobacco plants

ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression in embryos, which is different from similar promoters tested in maize.

Characterization of the human intestinal CD98 promoter and its regulation by interferon-γ

Growing evidence that epithelial CD98 plays an important role in intestinal inflammation focused our interest to investigate the transcriptional regulation of CD98. Our mouse-based in vivo and in vitro experiments revealed that epithelial colonic CD98 mRNA expression was transcriptionally increased in intestinal inflammation. We then isolated and characterized a 5'-flanking fragment containing the promoter region required for CD98 gene transcription. Primer extension and rapid amplification of 5'-cDNA ends were used to map a transcriptional initiation site 129 bp upstream from the translational start codon (ATG). Direct sequencing of the 5'-flanking region revealed the presence of four GC-rich stimulating protein (Sp)1 binding domains, one NF-kappaB binding domain, and no TATA box. Binding of Sp1 [Sp1(-874), SP1(-386), Sp1(-187), and Sp1(-177)] and NF-kappaB [NF-kappaB(-213)] to the promoter was confirmed by EMSA and supershift assays. Furthermore, chromatin immunoprecipitation experiments showed the in vivo DNA-Sp1 and DNA-NF-kappaB interactions under basal and IFN-gamma-stimulated conditions. Reporter genes driven by serially truncated and site-mutated CD98 promoters were used to examine basal and IFN-gamma-responsive transcription in transiently transfected Caco2-BBE cells. Our results revealed that Sp1(-187), Sp1(-177), and the NF-kappaB binding site were essential for basal and IFN-gamma-stimulated CD98 promoter activities, whereas Sp1(-874) and Sp1(-386) were not. The results from additional site-mutated CD98 promoters suggested that Sp1(-187), Sp1(-177), and the NF-kappaB site may cooperate in mediating basal and IFN-gamma-stimulated CD98 promoter activities. Finally, we demonstrated that a reduction of Sp1 or NF-kappaB expression reduced CD98 protein expression in unstimulated and IFN-gamma-stimulated Caco2-BBE cells. Collectively, these findings indicate that the Sp1 and NF-kappaB transcription factors are likely to play a significant role in IFN-gamma-mediated transcriptional regulation of CD98 in the intestinal epithelium, providing new insights into the regulation of CD98 expression in intestinal inflammation.

Characterization of the mitochondrial aconitase promoter and the identification of transcription factor binding

Mitochondrial (m) aconitase plays an important role in the unique pathway of citrate accumulation in prostate epithelial cells through its limited activity. In the current study, we characterized the human m-aconitase gene promoter. A 1,411-bp 5'-flanking fragment of the human m-aconitase gene was cloned, followed by 5' serial deletion analysis of promoter activity. Transcriptional start sties and transcription factors bound to the promoter were identified by 5' RACE, DNA pull-down assay and transcription factor array analysis. Two transcriptional start sites were identified. The promoter fragment pulled down 15 transcription factors, some without consensus sequences in the promoter. Deletion of one Sp1 site eliminated all promoter activity. The m-aconitase promoter is contained in a 153-bp 5' fragment lacking a TATA or CAAT sequence. Sp1 binding to a specific Sp1 site is required for promoter activity while other transcription factors are recruited through protein-protein interactions.

Regulatory sequences driving expression of the sea urchin Otp homeobox gene in oral ectoderm cells

PlOtp (Orthopedia), a homeodomain-containing transcription factor, has been recently characterized as a key regulator of the morphogenesis of the skeletal system in the embryo of the sea urchin Paracentrotus lividus. Otp acts as a positive regulator in a subset of oral ectodermal cells which transmit short-range signals to the underlying primary mesenchyme cells where skeletal synthesis is initiated. To shed some light on the molecular mechanisms involved in such a process, we begun a functional analysis of the cis-regulatory sequences of the Otp gene. Congruent with the spatial expression profile of the endogenous Otp gene, we found that while a DNA region from −494 to +358 is shown to drive in vivo GFP reporter expression in the oral ectoderm, but also in the foregut, a larger region spanning from −2044 to +358 is needed to give firmly established tissue specificity. Microinjection of PCR-amplified DNA constructs, truncated in the 5′ regulatory region, and determination of GFP mRNA level in injected embryos allowed the identification of a 5′-flanking fragment of 184bp in length, essential for expression of the transgene in the oral ectoderm of pluteus stage embryos. Finally, we conducted DNAse I-footprinting assays in nuclear extracts for the 184bp region and detected two protected sequences. Data bank search indicates that these sites contain consensus binding sites for transcription factors.