Analisis ko-ekspresi gen-gen regulasi upstream dari gen Dehydrin di tanaman tebu (Saccharum officinarum L.) pada kondisi cekaman kekeringan

Sugarcane plantations in Indonesia have been expanded and shifted to the marginal land characterized by long drought period, therefore, an attempt has been initiated to generate drought tolerance varieties through genetic engineering. It could be conducted by inserting the gene that involve in plant adaptation response to drought stress such as dehydrin (DHN) into sugarcane genome. The promoter of sugarcane DHN gene was isolated and transformed into sugarcane in the previous research. This study aimed to demonstrate the functionality of sugarcane DHN promoter through expression analysis of DHN regulatory genes that play a role in response to drought stress. Expression analyses using RT-qPCR were also conducted on regulatory genes of sugarcane that inserted by Pr-1DHNSo construct treated with drought stress. The results showed that the expressions of SoMYB, SoWRKY, SoNAC, and SoDHN genes were escalated on sugarcane 16 days after stress treatment ranging from 353 to 4067 folds relatively to untreated samples in which SoNAC gene showed the highest expression. On the other hand, the analysis on transgenic sugarcane carrying DHNpromoter construct showed SoNAC and SoDREB expression increased after 72 hours under drought stress. The expression values of SoNAC in transgenic and non-transgenic plants under drought condition were 4.79 and 4.99, respectively. Meanwhile, the expression values of SoDREB in transgenic and non-transgenic plants under drought condition were 13.2 and 13.3, respectively. The results of these experiments showed that the promoter construct of Pr-1DHNSo was induced by drought stress treatments highlighting the regulation of several upstream genes of SoDHN.

Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

ABSTRACTPrecisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

Isolation and characterization of Dehydrin promoter region from sugarcane (Saccharum officinarum L.)

The development of molecular biology techniques nowadays has enabled to engineer drought tolerant sugarcane by genetic engineering to accelerate the breeding program. Dehydrin (DHN) is known to have an important role in plant response and adaptation to abiotic stresses (drought, high salinity, cold, heat, etc.). While plant tissues are subjected to drought stress (dehydration), DHN protein is accumulated to high content throughout all vegetative or generative tissues. The research aimed to isolate and characterize the DHN promoter from sugarcane that can be used as transformation material in generating drought tolerant sugarcane. Specific primers for DHN promoter amplification were designed and DHN promoter region was successfully isolated by PCR cloning method. Two putative promoter sequences were identified namely Pr-1DHNSo and Pr-2DHNSo. In silicoanalyses were carried out and cis-regulatory elements motifs that play a role in adaptation on abiotic stress as well as biotic stress including ABRE, MBS, CGTCA-motif, TGACG-motif, GARE-motif, P-box TCA-element and Box-W1 were identified. The promoter Pr-1DHNSo was then cloned into pBI121 expression vector by Overlap Extention PCR (OE-PCR) for further characterization. Functional test of the promoter construct pBI- Pr-1DHNSo was conducted through Agrobacterium transformation into sugarcane calli. GUS assay and PCR analysis showed that the DHN promoter was transformed and expressed in the sugarcane calli.

Generation of Amelx-iCre Mice Supports Ameloblast-Specific Role for Stim1

The identification and targeting of the molecular pathways regulating amelogenesis is an ongoing challenge in dental research, and progress has been restricted by the limited number of genetic tools available to study gene function in ameloblasts. Here, we generated 4 transgenic Cre-driver mouse lines that express improved Cre ( iCre)–recombinase from the locus of the mouse ameloblast-specific gene amelogenin X ( Amelx-iCre) with a large (250-kb) bacterial artificial chromosome DNA vector. All 4 Amelx-iCre transgenic lines were bred with ROSA26 reporter mice to characterize the iCre developmental pattern with the LacZ gene encoding β-galactosidase enzyme activity assay and Cre protein immunohistochemistry. From the 4 generated transgenic lines, 2 were selected for further analysis because they expressed a high amount of Cre recombinase exclusively in ameloblasts and showed developmental stage- and cell-specific β-galactosidase activity mimicking the endogenous amelogenin expression. To test the functionality of the selected transgenic models, we bred the 2 Amelx-iCre mice lines with stromal interaction molecule 1 ( Stim1) floxed mice to generate ameloblast-specific Stim1 conditional knockout mice ( Stim1 cKO). STIM1 protein serves as one of the main calcium sensors in ameloblasts and plays a major role in enamel mineralization and ameloblast differentiation. Amelx-iCre mice displayed exclusive CRE-mediated recombination in incisor and molar ameloblasts. Stim1 cKO mice showed a severely defected enamel phenotype, including reduced structural integrity concomitant with increased attrition and smaller teeth. The phenotype and genotype of the Amelx-iCre/Stim1 cKO showed significant differences with the previously reported Ker14- Cre/ Stim1 cKO, highlighting the need for cell- and stage-specific Cre lines for an accurate phenotype-genotype comparison. Furthermore, our model has the advantage of carrying the entire Amelx gene locus rather than being limited to an Amelx partial promoter construct, which greatly enhances the stability and the specificity of our Cre expression. As such, the Amelx-iCre transgenic lines that we developed may serve as a powerful tool for targeting ameloblast-specific gene expression in future investigations.

Impaired 11β-Hydroxysteroid Dehydrogenase Type 2 in Glucocorticoid-Resistant Patients

Context Six patients carrying heterozygous loss-of-function mutations of glucocorticoid (GC) receptor (GR) presented with hypercortisolism, associated with low kalemia, low plasma renin, and aldosterone levels, with or without hypertension, suggesting a pseudohypermineralocorticism whose mechanisms remain unclear. We hypothesize that an impaired activity of the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2; encoded by the HSD11B2 gene), catalyzing cortisol (F) inactivation, may account for an inappropriate activation of a renal mineralocorticoid signaling pathway in these GC-resistant patients. Objective We aim at studying the GR-mediated regulation of HSD11B2. Design The HSD11B2 promoter was subcloned and luciferase reporter assays evaluated GR-dependent HSD11B2 regulation, and 11β-HSD2 expression/activity was studied in human breast cancer MCF7 cells, endogenously expressing this enzyme. Results Transfection assays revealed that GR transactivated the long (2.1-kbp) HSD11B2 promoter construct, whereas a defective 501H GR mutant was unable to stimulate luciferase activity. GR-mediated transactivation of the HSD11B2 gene was inhibited by the GR antagonist RU486. A threefold increase in HSD11B2 mRNA levels was observed after dexamethasone (DXM) treatment of MCF7 cells, inhibited by RU486 or by actinomycin, supporting a GR-dependent transcription. Chromatin immunoprecipitation further demonstrated a DXM-dependent GR recruitment onto the HSD11B2 promoter. 11β-HSD2 activity, evaluated by the cortisone/F ratio, quantified by liquid chromatography/tandem mass spectrometry, was 10-fold higher in the supernatant of DXM-treated cells than controls, consistent with a GR-dependent stimulation of 11β-HSD2 catalytic activity. Conclusion Collectively, we demonstrate that 11β-HSD2 expression and activity are transcriptionally regulated by GR. In the context of GR haploinsufficiency, these findings provide evidence that defective GR signaling may account for apparent mineralocorticoid excess in GC-resistant patients.

Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1

Tumors can be therapeutically targeted with novel antifolates (e.g. AGF94) that are selectively transported by the human proton-coupled folate transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94. Progressive deletions of the hPCFT promoter construct (−2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions −50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by ∼5.1-, ∼44-, and ∼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antifolates.

Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct

The large-scale identification of putative alternative promoters study shows more than 52% of human genes are regulated by alternative promoters. The human myc-associated zinc finger protein (SAF/MAZ) gene have SAF-1 and SAF-3 variants transcripted from two transcription start sites (TSSs). By using SAF/MAZ promoter as a model, we set up an approach to probe how the alternative promoters are regulated in real time. We have constructed the bichromatic fluorescent reporter driven by SAF/MAZ 5′-proximal promoter plasmids from which transactivation status of SAF-1 and SAF-3 alternative promoter could be monitored by EGFP and DsRed expression respectively. The results showed that the SAF-3 expression is regulated by alternative promoters. When the bichromatic fluorescent reporter was driven by −1692/+277 or −1401/+277 SAF/MAZ promoter the dominant expression of SAF-3 would be observed in comparison with SAF-1 expression. We also identified that Elk-1 is an inhibitory transcription factor for SAF-3 expression. The temporal diversity of SAF-1 and SAF-3 expressions can be observed via bichromatic fluorescent reporters. These imply that the bichromatic fluorescent reporter driven by alternative promoter construct might be a useful tool for decoding the temporal regulatory repertoire of alternative promoter in human genes.

Construction and Transient Expression of Chimeric Cassettes Containing CaMV 35S or OsAER1 Promoter and GUS Gene Fusion in Tobacco

<p>Reporter gene assays are commonly used to study the expression pattern of a gene and the promoter activity. The purpose of this study was to assemble the chimeric gene constructs consisting of CaMV 35S promoter orOsAER1 gene promoter connected to the β-glucuronidase (GUS) reporter gene encoding the β-glucuronidase enzyme and to obtain an efficient method for Agrobacterium tumefaciens-mediated transient transformation of tobacco sprouts. The CaMV 35S promoter fragment reamplified from pCAMBIA1301 binary vector and the OsAER1 gene promoter fragment amplified from rice cv. Awan Kuning were ligated into pCAMBIA1300int::gus::tNOS to produce binary vectors pCAMBIA1300int::p35S::gus::tNOS and pCAMBIA1300int::prOsAER1::gus::tNOS. The vectors were used for transient transformation of 5–day old tobacco seedlings. The transformation was carried out using two bacterial cultures with densities of OD600 = 0.5 or OD600 = 1.0 combined with a vacuum for 15 or 30 minutes. Tobacco seedlings transformed with pCAMBIA1300int::p35S::gus::tNOS showed higher transformation efficiency as compared tothe ones transformed with pCAMBIA1300int::prOsAER1::gus::tNOS. A higher efficiency was obtained from transformation using bacterial culture with density of OD600 = 0.5 in combination with a vacuum for 30 minutes. Expression of GUS gene in the tobacco sprouts transformed with CaMV 35S promoter construct was observed through out the sprouts area (root, hypocotyl, cotyledon, and leaf), where as expression of GUS gene was observed in root, hypocotyl, and cotyledon, but not in leaf on tobacco sprouts transformed with OsAER1 promoter construct. These results indicate that the transient transformation is a quick and simple method for testing a chimeric gene construct.</p>

Increased KCNJ18 promoter activity as a mechanism in atypical normokalemic periodic paralysis

ObjectiveTo identify the genetic basis of a patient with symptoms of normokalemic sporadic periodic paralysis (PP) and to study the effect of KCNJ18 mutations.MethodsA candidate gene approach was used to identify causative gene mutations, using Sanger sequencing. KCNJ18 promoter activity was analyzed in transfected HEK293 cells with a luciferase assay, and functional analysis of Kir2.6 channels was performed with the two-electrode voltage-clamp technique.ResultsAlthough we did not identify harmful mutations in SCN4A, CACNA1S, KCNJ2 and KCNE3, we detected a monoallelic four-fold variant in KCNJ18 (R39Q/R40H/A56E/I249V), together with a variant in the respective promoter of this channel (c.-542T/A). The exonic variants in Kir2.6 did not alter the channel function; however, luciferase assays revealed a 10-fold higher promoter activity of the c.-542A promoter construct, which is likely to cause a gain-of-function by increased expression of Kir2.6. We found that reducing extracellular K+ levels causes a paradoxical reduction in outward currents, similar to that described for other inward rectifying K+ channels. Thus, reducing the extracellular K+ levels might be a therapeutic strategy to antagonize the transcriptionally increased KCNJ18 currents. Consistently, treatment of the patient with K+ reducing drugs dramatically improved the health situation and prevented PP attacks.ConclusionsWe show that a promoter defect in the KCNJ18 gene is likely to cause periodic paralysis, as the observed transcriptional upregulation will be linked to increased Kir2.6 function. This concept is further supported by our observation that most of the PP attacks in our patient disappeared on medical treatment with K+ reducing drugs.