Specific activation of the hb4 gene in the Xenopus oocyte through a Nobox-binding element located at the proximal promoter sequence

Zygote ◽  
2019 ◽  
Vol 27 (4) ◽  
pp. 195-202
Author(s):  
Masanori Nakamigawa ◽  
Takumi Kondo ◽  
Mitsugu Maéno
Keyword(s):  
Transcriptional Activation ◽  
Fluorescent Protein ◽  
Developmental Process ◽  
Promoter Sequence ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Flanking Sequence ◽  
Conserved Sequence ◽  
Series Of Experiments ◽  
Green Fluorescent

SummaryWe isolated and characterized Xenopus tropicalis hb4 flanking DNA and showed that the −3076/+29 sequence was able to drive stage-specific transcription in the developmental process. Transgenic reporter analysis indicated that green fluorescent protein was expressed in the ovaries of female frogs at 3 months of age and in both the ovaries and testis of frogs at 6 months of age. A series of experiments with deletion of the flanking sequence and a subsequent luciferase reporter assay revealed that there were two positive regulatory regions and that the most proximal sequence of the promoter region had a certain level of transcriptional activity in oocytes. Subsequently, we showed that a conserved sequence containing Nobox-binding element (NBE) was essential for transcriptional activation and that Nobox expressed in the ovary had a crucial role in hb4 transcription through the NBE sequence.

Characterization of the promoter activity of a poxvirus conserved element

2008 ◽  
Vol 54 (6) ◽  
pp. 483-488
Author(s):  
Heather E. Eaton ◽  
Julie Metcalf ◽  
Craig R. Brunetti
Keyword(s):  
Fluorescent Protein ◽  
Promoter Sequence ◽  
Promoter Strength ◽  
Wild Type ◽  
Sequence Element ◽  
Promoter Function ◽  
Conserved Sequence ◽  
Polymerase Chain ◽  
Green Fluorescent

The conserved sequence element (CSE) is a highly conserved 42-bp poxvirus sequence that can function as a poxvirus promoter element. The CSE is composed of 2 repeats, each containing the highly conserved late poxvirus promoter sequence TAAAT. To define the location of the nucleotides critical for promoter function, polymerase chain reaction was carried out using primers that inserted modified versions of the CSE upstream of the green fluorescent protein (GFP), and the constructs were transiently transfected into cells by using GFP levels as a measure of promoter function. The results of this analysis revealed that the second TAAAT sequence, but not the first TAAAT sequence, is critical for promoter function of the CSE. Furthermore, deletion of half of the intervening sequence, i.e., from 10 to 5 nt, increases the promoter strength of the CSE as compared with the wild-type CSE. These results indicate the potential of this novel poxvirus promoter for driving high levels of gene expression.

Regulation of apo A-IV transcription by lipid in newborn swine is associated with a promoter DNA-binding protein

2003 ◽  
Vol 284 (2) ◽  
pp. G248-G254 ◽  
Author(s):  
Song Lu ◽  
Ying Yao ◽  
Heng Wang ◽  
Songmei Meng ◽  
Xiangying Cheng ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Promoter Sequence ◽  
Dietary Lipid ◽  
Lipid Absorption ◽  
Proximal Promoter ◽  
Significant Shift ◽  
Human Sequence ◽  
Nuclear Extracts ◽  
Promoter Dna ◽  
Isocaloric Diets

Dietary lipid acutely upregulates apolipoprotein (apo) A-IV expression by sevenfold at the pretranslational level in neonatal swine jejunum. To determine the mechanism of this regulation, two-day-old female swine received intraduodenal infusions of low- and high-triacylglycerol (TG) isocaloric diets for 24 h. Nuclear runoff assay confirmed apo A-IV gene transcriptional regulation by the high-TG diet. Footprinting analysis using the swine apo A-IV proximal promoter sequence (+14 to −246 bp) demonstrated three regions protected by the low-TG extracts. Of these three motifs, only ACCTTC showed 100% homology to the human sequence and was further studied. EMSA was performed using probes containing wild-type (WT) and mutant (M) motifs. A shift was noted with the low-TG nuclear extracts with the WT probe but not with the M probe. Excess unlabeled free WT probe competed out the shift, whereas the M probe did not. No significant shift occurred with either probe using high-TG extracts. These results suggest that a repressor protein binds to the ACCTTC motif and becomes unbound during lipid absorption, allowing transcriptional activation of the apo A-IV gene in newborn swine small intestine.

scholarly journals Regulation of RamA by RamR in Salmonella enterica Serovar Typhimurium: Isolation of a RamR Superrepressor

2012 ◽  
Vol 56 (11) ◽  
pp. 6037-6040 ◽  
Author(s):  
Vito Ricci ◽  
Stephen J. W. Busby ◽  
Laura J. V. Piddock
Keyword(s):  
Transcription Factor ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Promoter Sequence ◽  
Palindromic Sequence ◽  
Content Type ◽  
Gfp Reporter ◽  
Serovar Typhimurium ◽  
Green Fluorescent

ABSTRACTRamA is a transcription factor involved in regulating multidrug resistance inSalmonella entericaserovar Typhimurium SL1344. Green fluorescent protein (GFP) reporter fusions were exploited to investigate the regulation of RamA expression by RamR. We show that RamR represses theramApromoter by binding to a palindromic sequence and describe a superrepressor RamR mutant that binds to theramApromoter sequence more efficiently, thus exhibiting aramAinactivated phenotype.

scholarly journals The miR-26b-5p/KPNA2 Axis Is an Important Regulator of Burkitt Lymphoma Cell Growth

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1464
Author(s):  
Fubiao Niu ◽  
Marta Kazimierska ◽  
Ilja M. Nolte ◽  
Miente Martijn Terpstra ◽  
Debora de Jong ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Burkitt Lymphoma ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Luciferase Reporter ◽  
Argonaute 2 ◽  
A Genome ◽  
Green Fluorescent ◽  
Competition Assays

The expression of several microRNAs (miRNAs) is known to be changed in Burkitt lymphoma (BL), compared to its normal counterparts. Although for some miRNAs, a role in BL was demonstrated, for most of them, their function is unclear. In this study, we aimed to identify miRNAs that control BL cell growth. Two BL cell lines were infected with lentiviral pools containing either 58 miRNA inhibitors or 44 miRNA overexpression constructs. Eighteen constructs showed significant changes in abundance over time, indicating that they affected BL growth. The screening results were validated by individual green fluorescent protein (GFP) growth competition assays for fifteen of the eighteen constructs. For functional follow-up studies, we focused on miR-26b-5p, whose overexpression inhibited BL cell growth. Argonaute 2 RNA immunoprecipitation (Ago2-IP) in two BL cell lines revealed 47 potential target genes of miR-26b-5p. Overlapping the list of putative targets with genes showing a growth repression phenotype in a genome-wide CRISPR/Cas9 knockout screen, revealed eight genes. The top-5 candidates included EZH2, COPS2, KPNA2, MRPL15, and NOL12. EZH2 is a known target of miR-26b-5p, with oncogenic properties in BL. The relevance of the latter four targets was confirmed using sgRNAs targeting these genes in individual GFP growth competition assays. Luciferase reporter assay confirmed binding of miR-26b-5p to the predicted target site for KPNA2, but not to the other genes. In summary, we identified 18 miRNAs that affected BL cell growth in a loss- or gain-of-function screening. A tumor suppressor role was confirmed for miR-26b-5p, and this effect could at least in part be attributed to KPNA2, a known regulator of OCT4, c-jun, and MYC.

scholarly journals Knockdown of tyrosine hydroxylase in the nucleus of the solitary tract reduces elevated blood pressure during chronic intermittent hypoxia

2013 ◽  
Vol 305 (9) ◽  
pp. R1031-R1039 ◽  
Author(s):  
Chandra Sekhar Bathina ◽  
Anuradha Rajulapati ◽  
Michelle Franzke ◽  
Kenta Yamamoto ◽  
J. Thomas Cunningham ◽  
...  
Keyword(s):  
Tyrosine Hydroxylase ◽  
Transcriptional Activation ◽  
Intermittent Hypoxia ◽  
Fluorescent Protein ◽  
Nucleus Tractus Solitarius ◽  
Cardiovascular Responses ◽  
Ventrolateral Medulla ◽  
Chronic Intermittent Hypoxia ◽  
Sprague Dawley ◽  
Green Fluorescent

Noradrenergic A2 neurons in nucleus tractus solitarius (NTS) respond to stressors such as hypoxia. We hypothesize that tyrosine hydroxylase (TH) knockdown in NTS reduces cardiovascular responses to chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia observed during sleep apnea in humans. Adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters and adeno-associated viral constructs with green fluorescent protein (GFP) reporter having either short hairpin RNA (shRNA) for TH or scrambled virus (scRNA) were injected into caudal NTS. Virus-injected rats were exposed to 7 days of CIH (alternating periods of 10% O2 and of 21% O2 from 8 AM to 4 PM; from 4 PM to 8 AM rats were exposed to 21% O2). CIH increased mean arterial pressure (MAP) and heart rate (HR) during the day in both the scRNA ( n = 14, P < 0.001 MAP and HR) and shRNA ( n = 13, P < 0.001 MAP and HR) groups. During the night, MAP and HR remained elevated in the scRNA rats ( P < 0.001 MAP and HR) but not in the shRNA group. TH immunoreactivity and protein were reduced in the shRNA group. FosB/ΔFosB immunoreactivity was decreased in paraventricular nucleus (PVN) of shRNA group ( P < 0.001). However, the shRNA group did not show any change in the FosB/ΔFosB immunoreactivity in the rostral ventrolateral medulla. Exposure to CIH increased MAP which persisted beyond the period of exposure to CIH. Knockdown of TH in the NTS reduced this CIH-induced persistent increase in MAP and reduced the transcriptional activation of PVN. This indicates that NTS A2 neurons play a role in the cardiovascular responses to CIH.

scholarly journals In vivo analysis of key elements within the renin regulatory region

2008 ◽  
Vol 35 (3) ◽  
pp. 243-253 ◽  
Author(s):  
Sean T. Glenn ◽  
Craig A. Jones ◽  
Li Pan ◽  
Kenneth W. Gross
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Region ◽  
Renin Angiotensin System ◽  
Artificial Chromosome ◽  
Proximal Promoter ◽  
Gfp Expression ◽  
Renin Gene ◽  
Green Fluorescent

Renin is responsible for initiating the enzymatic cascade that results in the production of angiotensin II, the major effector molecule of the renin-angiotensin system (RAS). Extensive information on the regulatory region of the renin gene has been derived by transient transfection studies in vitro, particularly using the As4.1 cell line. To verify key factors within the regulatory region of renin in vivo, homologous recombination was used to introduce a green fluorescent protein (GFP) cassette into exon one of the renin gene contained within a 240 kb bacterial artificial chromosome (BAC) to create a construct that has GFP expression controlled by the renin regulatory region (RenGFP BAC). Within the regulatory region of the RenGFP BAC construct we independently deleted the enhancer, as well as mutated the HOX-PBX site within the proximal promoter element. Transgenic lines were generated for each of these BAC constructs and GFP expression was analyzed throughout a spectrum of tissues positive for renin expression including the kidney, adrenal gland, gonadal artery, and submandibular gland. The results described within this manuscript support the interpretation that the renin enhancer is critical for regulating baseline expression where as the Hox/Pbx site is important for the tissue specificity of renin expression.

scholarly journals Molecular Mechanism of the Inhibition of Estradiol-Induced Endometrial Epithelial Cell Proliferation by Clomiphene Citrate

Endocrinology ◽  
2010 ◽  
Vol 151 (1) ◽  
pp. 394-405 ◽  
Author(s):  
Mitsuyoshi Amita ◽  
Toshifumi Takahashi ◽  
Seiji Tsutsumi ◽  
Tsuyoshi Ohta ◽  
Keiko Takata ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cell ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Clomiphene Citrate ◽  
Immunofluorescence Assay ◽  
Epithelial Cell Proliferation ◽  
Endometrial Epithelial Cell ◽  
Green Fluorescent

Abstract We examined the molecular mechanisms of the antiestrogenic effects of clomiphene citrate (CC) in the endometrium using two types of cell lines, Ishikawa and EM-E6/E7/hTERT cells. CC or ICI182780 inhibited 17β-estradiol (E2)-induced endometrial cell proliferation and transcriptional activation of the estrogen response element (ERE) gene. We directly visualized the ligand-estrogen receptor (ER)α interaction using green fluorescent protein (GFP)-tagged ERα in a single living cell. Whereas E2 changed the nuclear localization of GFP-ERα to a punctate distribution within 5 min, CC or ICI182780 changed the slower and less mobilization of GFP-ERα compared with E2. Pretreatment with CC or ICI182780 partly prevented the E2-induced nuclear redistribution of GFP-ERα. Fluorescence recovery after photobleaching revealed that GFP-ERα mobility treated with E2 was more rapid than that treated by CC or ICI182780. As coactivator recruitment to the ER is essential for ER-dependent transcription, we examined the interaction between ERα and steroid receptor coactivator-1 (SRC-1). The complex formation between ERα and SRC-1 was significantly increased by E2 but was prevented in the presence of CC or ICI182780 by coimmunoprecipitation. Moreover, the E2-induced colocalization of GFP-ERα and SRC-1 was prevented in the presence of CC or ICI182780 according to an immunofluorescence assay. We also observed that the reduction of SRC-1 using small interfering RNA for SRC-1 resulted in the inhibition of E2-induced cell proliferation and transcriptional activation of the ERE gene. Collectively, these results suggest that CC may inhibit E2-induced endometrial epithelial cell proliferation and ERE transactivation by inhibiting the recruitment of SRC-1 to ERα.

scholarly journals Detection on Surfaces and in Caco-2 Cells of Campylobacter jejuni Cells Transformed with New gfp, yfp, andcfp Marker Plasmids

2000 ◽  
Vol 66 (12) ◽  
pp. 5426-5436 ◽  
Author(s):  
William G. Miller ◽  
Anne H. Bates ◽  
Sharon T. Horn ◽  
Maria T. Brandl ◽  
Marian R. Wachtel ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Promoter Sequence ◽  
Broth Culture ◽  
Single Strain ◽  
Shuttle Vectors ◽  
Chicken Skin ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

ABSTRACT We have developed two sets of Campylobacter shuttle vectors containing either the gfp (green fluorescent protein), yfp (yellow fluorescent protein), orcfp (cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus Campylobacterpromoter sequence (Pc). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments. C. jejuni cells transformed with the Pc fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each C. jejuni strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many C. jejuni strains, >91% of the cells transformed with the Pc fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent C. jejuni transformants suggest that aggregates containing two or more strains of C. jejuni may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of C. jejuni, by selecting single colonies, from an environmental sample may not always yield a single strain.

scholarly journals Identification of a novel distal enhancer in human adiponectin gene

2008 ◽  
Vol 200 (1) ◽  
pp. 107-116 ◽  
Author(s):  
Katsumori Segawa ◽  
Morihiro Matsuda ◽  
Atsunori Fukuhara ◽  
Kentaro Morita ◽  
Yosuke Okuno ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Promoter Region ◽  
Luciferase Activity ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Adiponectin Gene ◽  
Distal Enhancer

Adiponectin is exclusively expressed in adipose tissue and secreted from adipocytes, and shows anti-diabetic and anti-atherogenic properties. However, the precise transcriptional mechanism of adiponectin remains elusive. In this study, the 5′ flanking promoter region of human adiponectin gene was analyzed using UCSC genome browser, and a 10 390-bp fragment, containing an evolutionally conserved region among species, was investigated. The luciferase reporter assay using this fragment identified a novel distal enhancer of human adiponectin gene. Promoter constructs with the distal enhancer exhibited high promoter activities in 3T3-L1 mature adipocytes. However, no such activity was observed in other types of cell lines. The distal enhancer is highly conserved, and contains two completely conserved CCAAT boxes. In 3T3-L1 mature adipocytes, deletion or each point mutation of these CCAAT boxes markedly reduced luciferase activity driven by adiponectin promoter. Knockdown of CCAAT/enhancer-binding protein α (CEBPA; also known as C/EBPα) using small interfering RNA diminished adiponectin mRNA expression and luciferase activity driven by adiponectin promoter with the distal enhancer. However, adiponectin promoter with each mutation of two CCAAT boxes in the distal enhancer did not respond to knockdown of CEBPA expression. Furthermore, CEBPA bound to the distal enhancer both in vitro and in vivo. We also identified a proximal promoter region responsible for transcriptional activation by the distal enhancer in human adiponectin gene. Our results indicate that CEBPA plays a pivotal role in the transcription of human adiponectin gene via the distal enhancer and proximal region in its promoter.

Transcriptional activation by the human Hsp70-associating protein Hap50

2001 ◽  
Vol 114 (10) ◽  
pp. 1839-1845
Author(s):  
Y. Niyaz ◽  
M. Zeiner ◽  
U. Gehring
Keyword(s):  
Transcriptional Activation ◽  
Fluorescent Protein ◽  
Biological Effects ◽  
Cell Types ◽  
Mrna Stabilization ◽  
Activation Of Transcription ◽  
Enhanced Expression ◽  
Endogenous Genes ◽  
Green Fluorescent ◽  
Different Cell Types

We investigated human Hap50, the large isoform of the previously characterized Hsp70/Hsc70-associating protein Hap46, also called BAG-1, for effects on transcriptional activities. Overproduction by transient transfection led to enhanced expression of reporter gene constructs in various cell types using different promoters, suggesting independence of promoter type. Similarly, overexpression of Hap50 resulted in increased levels of poly(A)(+)mRNAs in HeLa, COS-7, 3T3 and HTC cells. Concomitantly, the expression of some selected endogenous genes, such as those coding for c-Jun and the glucocorticoid receptor, was enhanced significantly relative to actin. Nuclear runoff transcription assays using HeLa cells showed that the effect is caused by increased transcription rates rather than mRNA stabilization. Activation of transcription by Hap50 occurred at 37 degrees C and did not require prior thermal stress, as is the case for Hap46. In accordance with these biological effects, Hap50 is localized exclusively in the nuclear compartment of different cell types, whereas Hap46 is mostly cytoplasmic in unstressed cells, as revealed by use of fusion constructs with green fluorescent protein. High cellular levels of Hap50 were found to make cells less susceptible to adverse environmental effects such as heat stress. Our data suggest that Hap50 is a nuclear protein that acts in cells to increase the transcription of various genes.

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