Molecular Characterization of Mouse CREB3 Regulatory Factor in Neuro2a Cells

2021 ◽  
Author(s):  
Kentaro Oh-hashi ◽  
Tomoyuki Hasegawa ◽  
Yoshihisa Naruse ◽  
Yoko Hirata
Keyword(s):  
Luciferase Activity ◽  
Intracellular Localization ◽  
Full Length ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Regulatory Factor ◽  
Neuro2a Cells ◽  
Positive Effect ◽  
Stress Pathway

Abstract We performed expression and functional analysis of mouse CREB3 regulatory factor (CREBRF) in Neuro2a cells by constructing several expression vectors. Overexpressed full-length CREBRF protein was stabilized by MG132; however, the intrinsic CREBRF expression in Neuro2a cells was negligible under all conditions. On the other hand, N- or C-terminal deletion of CREBRF influenced its stability. Cotransfection of CREBRF together with GAL4-tagged full-length CREB3 increased luciferase reporter activity, and only the N-terminal region of CREBRF was sufficient to potentiate luciferase activity. Furthermore, this positive effect of CREBRF was also observed in cells expressing GAL4-tagged cleaved CREB3, although CREBRF hardly influenced the protein stability of NanoLuc-tagged cleaved CREB3 or intracellular localization of EGFP-tagged one. In conclusion, this study suggests that CREBRF, a quite unstable proteasome substrate, positively regulates the CREB3 pathway, which is distinct from the canonical ER stress pathway in Neuro2a cells.

Cloning and characterization of the light-inducible Gacab promoter from Gossypium arboreum

2005 ◽  
Vol 2 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Li Wei-Min ◽  
Wang Zhi-Xing ◽  
Pei Xin-Wu ◽  
Jia Shi-Rong
Keyword(s):  
Promoter Sequence ◽  
Inducible Promoter ◽  
Regulatory Elements ◽  
Gus Expression ◽  
Full Length ◽  
Expression Vectors ◽  
35S Promoter ◽  
Gossypium Arboreum ◽  
Gus Histochemical Assay

AbstractA 1009 bp promoter sequence of cab gene, which encodes chlorophyll a/b binding protein belonging to a class of light-inducible proteins, was cloned from Gossypium arboreum. Sequence analysis showed that it had no obvious homology with previously published cab promoters. The full-length Gacab promoter and 5′ deletions with length of 197, 504 and 779 bp were fused with gus (uid A) gene, respectively, and plant expression vectors were used for transformation of Nicotiana tabacum cv. NC89. β-Glucuronidase (GUS) histochemical assay of transgenic tobacco plants showed that GUS was expressed specifically in leaves and young green tissues. GUS was not detected in the leaves of transgenic plants grown in the dark for 6 days. However, it was highly expressed in the leaves of these plants after induction with light for another 6 days, demonstrating that the full-length Gacab promoter is a light-inducible promoter. Transient GUS expression in rice calli indicated that the expression level of Gacab504::gus was the highest and stronger than that of the CaMV 35S promoter, while expression was reduced for Gacab197::gus, Gacab779::gus and Gacab1009::gus constructs. This suggests that −197 bp to −1 bp is a basic promoter of Gacab, some positive regulatory elements may exist in −504 bp to −197 bp, and the fragment −1009 bp to −504 bp may contain negative elements.

Regulation of the Cyclin D1 Proto-Oncogene by Cell Cycle Regulatory MicroRNAs.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1801-1801
Author(s):  
Anagha Borwankar ◽  
Alessandro Pastore ◽  
Aniruddha Deshpande ◽  
Yvonne Zimmermann ◽  
Christian Buske ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Binding Sites ◽  
Cell Cycle Progression ◽  
Luciferase Activity ◽  
Full Length ◽  
Luciferase Reporter ◽  
Microrna Target ◽  
The Impact

Abstract Mutations, activation or overexpression of cyclin D1 are common features of several human cancers including mantle cell lymphoma (MCL) which bears the characteristic t(11;14) translocation juxtaposing the cyclin D1 gene downstream of the immunoglobulin heavy chain enhancer. The loss of the 3’UTR of this gene has been reported in a majority of MCL patients as well as in cell lines. In order to assess the impact of the 3’UTR on cyclin D1 expression levels, we used YFP tagged cyclin D1 reporter plasmids to quantify cyclin D1 expression in cell lines with different mutations of the 3’UTR. Interestingly, protein expression was significantly higher upon deletion of the cyclin D1 3’UTR compared to the full-length cyclin D1 gene as assessed by flow cytometry (2.1 fold; n=3, P < 0.05). Applying a more sensitive dual-luciferase reporter assay where a constitutively expressed luciferase gene was fused to the cyclin D1 3’UTR, the normalized firefly luciferase activity was reduced significantly to 23% as compared to luciferase only (the empty vactor). We then introduced 3’UTR mutations observed in MCL patients (insertion of adenosine between nucleotides 2308 and 2309 and a deletion of the tri-nucleotide sequence TCA from 2309–2311 of the full length cyclin D1-YFP reporter cDNA), which resulted in a significant increase of cyclin D1 expression (1.3 fold both in Ins308 and Δ309-311) compared to full length cyclin D1, (P< 0.05) showing that these mutations contribute to cyclinD1 overexpression in these patients. Subsequently, the 3’UTR was scanned for elements potentially regulating cyclin D1 expression, and putative microRNA binding sites were identified using the TargetScan and PicTar microRNA target prediction software. The most interesting candidate microRNAs include the miR-15/16 family and the miR-17–92 cluster, both of which have been shown to be involved in lymphoid malignancies and regulate cell cycle progression. In order to confirm whether the cyclin D1 3’UTR is a direct target of these microRNAs, we cloned the cyclin D1 3’UTR target region containing putative miR-15/16 or miR-17/20a binding sites and transfected these reporter constructs into HeLa cells. Upon introduction of oligonucleotide mimics of the miR15/16 microRNAs or a plasmid expressing microRNAs of the miR-17 cluster, the normalized luciferase activity of the respective luciferase reporters was reduced significantly to 41% (miR-15), 33% (miR-16) and 79% (miR-17/20a), respectively. Moreover, introduction of mutations in the seed sequences of the putative microRNA recognition sites rendered these constructs insensitive to inhibition by these microRNAs, confirming the specificity of the microRNA::target interaction. These data confirm that the binding of these microRNAs play an important role in the repression of cyclin D1 mediated by the 3’UTR and mutation or deletion result in cyclin D1 overexpression in MCL as well as other human tumors.

4 CHARACTERIZATION OF LENTIVIRAL SHORT-HAIRPIN RNA EXPRESSION VECTORS CONTAINING SINGLE OR MULTIPLE BOVINE POLYMERASE III PROMOTERS

2010 ◽  
Vol 22 (1) ◽  
pp. 160
Author(s):  
M. Peoples ◽  
M. Westhusin ◽  
M. Golding ◽  
C. Long
Keyword(s):  
Luciferase Activity ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Second Phase ◽  
Shrna Expression ◽  
Significant Difference ◽  
Short Hairpin ◽  
Pol Iii

Lentiviral vectors have become an important and efficient molecular biology tool to integrate foreign DNA into target genomes. These vectors have been previously used in our laboratory to make cloned transgenic fetuses expressing short-hairpin RNAs (shRNAs) targeting the caprine prion mRNA (Golding et al. 2006 Proc. Natl. Acad. Sci. USA 103, 5285-5290) and bovine myostatin mRNA. Specially designed shRNAs have a robust ability to decrease protein expression by initiating a mRNA destruction pathway or by translational inhibition. However, initial experiments targeting foot and mouth (FMDV) viral RNA have indicated that polymerase (Pol) II promoters may be unable to produce enough mature shRNA particles to significantly knock down viral replication in vitro. The goal of this research project was to identify and utilize bovine Pol III promoters to express shRNAs in lentiviral vectors and to express multiple unique shRNAs from a single lentiviral vector using different Pol III promoters. This goal is particularly important to the successful reduction of FMDV replication in a cell, as it limits random mutations from escaping the shRNA-mediated viral genome destruction. The 3 bovine Pol III promoters we selected were 7sk, U6-2, and H1. They were individually amplified from the same genomic DNA preparation. The promoters were inserted immediately upstream of our shRNA expression sequence, resulting in lentiviral vectors designated GT-b7sk, GT-bU6-2, and GT-bH1.To confirm that the promoters were functional, a luciferase reporter assay was performed in HEK 293T cells, where each vector expressed either a shRNA targeting luciferase (luc) or a non-specific shRNA.All promoters expressing luc shRNA resulted in significant reduction of luciferase activity between 68 and 80% compared with non-targeting controls. In addition, there was no significant difference between Pol III promoters when analyzing reduced luciferase activity. In the second phase of the study, we developed 7 unique combinations of 2 or 3 Pol III shRNA expression cassettes to test individual shRNA function with one shRNA designed to target luciferase and the others non-targeting. In multiple Pol III expression constructs, the U6-2 and 7sk promoters resulted in the greatest reduction of luciferase activity at 89 and 95%, respectively. In addition, luciferase activity was reduced to the greatest extent when the luc shRNA was expressed from the second (82%) or third (87%) Pol III cassette. Overall, bovine Pol III-based promoters are effective at expressing shRNAs from a lentiviral vector. In addition, multiple Pol III shRNA expression cassettes can be inserted into a single lentiviral vector and still achieve significant reduction of target protein. These vectors will be used to create transgenic cattle and pigs that express multiple shRNAs targeting the FMDV genome with hopes of creating animals that are resistant to FMDV.

Abstract 234: Defining Domains In The Transcription Factor Grainyhead-like 3 Required For Protective Functions In The Endothelium

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Sabrina Farrokh ◽  
Niloofar Ale-Agha ◽  
Judith Haendeler ◽  
Joachim Altschmied
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Large Scale ◽  
Target Genes ◽  
Intracellular Localization ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Deletion Mutants ◽  
Nuclear Localization Signals ◽  
Apoptosis Level

Important aspects during aging of human endothelial cells (EC) are an increased apoptosis level and a reduced migratory capacity. In a screening for anti-apoptotic genes we have identified the transcription factor Grainyhead-like 3 (GRHL3) and demonstrated its anti-apoptotic and pro-migratory effects after overexpression and knockdown in EC. To define the domains in GRHL3 responsible for these - potentially also extranuclear - functions we have cloned large scale deletion mutants and mutants with deletions of putative nuclear localization signals (NLS) and analyzed them for their intracellular localization and functional properties. Immunostaining of transfected EC were used to examine the subcellular distribution. Two mutants with deletions in a bioinformatically predicted bipartite NLS were localized predominantly in the cytoplasm. To corroborate these data, lysates of the cells were fractionated biochemically. Western blotting confirmed the immunostaining data, indicating that we have identified the major NLS in GRHL3. To analyze the transcriptional properties of these mutants, we constructed a GRHL3-specific luciferase reporter containing a tandem GRHL3 binding sites in front of a minimal promoter and cotransfected it with expression vectors for the GRHL3 deletion mutants. Besides the previously described activation domain we identified another region required for transcriptional upregulation of target genes in the N-terminal half of the protein. We will now use these mutants to further dissect the regions in GRHL3 necessary for its pro-migratory and anti-apoptotic activities in endothelial cells. This will also allow us to investigate if all of these functions are mediated solely by the activation of target genes or by other mechanisms coupled to a non-nuclear function of GRHL3.

Mechanisms of Leukemogenesis by MLL-CALM: Characterization of a CALM-Derived Transcriptional Regulatory Domain.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3387-3387
Author(s):  
Mwe Mwe Chao ◽  
Emily J. Fox ◽  
Daniel S. Wechsler
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Transcriptional Activity ◽  
Hox Genes ◽  
Luciferase Activity ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Fusion Partner ◽  
Cytoplasmic Localization

Abstract Background: MLL translocations are common in infant leukemias, and >50 distinct translocation partners have been described. We recently identified the CALM gene as a novel MLL partner in an infant with aggressive AML. Interestingly, CALM was first discovered as a translocation partner for AF10, which had previously been identified as an MLL fusion partner in aggressive leukemias and lymphomas. The native CALM protein exhibits predominantly cytoplasmic localization, and participates in clathrin-dependent endocytosis and intracellular vesicle transport. We have previously shown that expression of MLL-CALM immortalizes murine hematopoietic progenitors, and that fusion of the carboxy terminus of CALM to MLL alters MLL transcriptional activity. We hypothesize that CALM possesses a specific transcriptional activation domain (TAD) which modulates MLL transcriptional activity of HOX genes, thereby contributing to leukemogenesis. Objectives: 1) To determine whether native CALM localizes to the nucleus, 2) To delineate specific CALM domains which constitute the CALM TAD, and 3) To determine whether MLL-CALM activates transcription through the murine HOXA7 promoter. Methods: Human fibroblast cells were treated with Leptomycin B (an antifungal antibiotic which specifically inhibits nuclear export) and stained with an anti-CALM antibody. We prepared a set of expression vectors in which various portions of CALM are fused to a GAL4 DNA-binding domain. These vectors were co-transfected with a GAL4-luciferase reporter plasmid into COS7 cells, and luciferase activity was measured 48 hours after transient transfection. Luciferase assays were also performed using MSCV-MLL-CALM or MSCV-CALM plasmids co-transfected with a HOXA7 promoter-luciferase reporter construct. Results: After inhibition of nuclear export, native CALM localized to both the nucleus and cytoplasm. Significant luciferase activity was only observed with constructs containing distal CALM carboxy amino acids (aa 436–660). Mutation of an NR (Nuclear Receptor) Box motif (aa 510–514) did not affect CALM-dependent transcription. We found that two endocytosis-related NPF domains play opposite roles: deletion of NPF#1 (aa 437–439) dramatically reduced, while mutation of NPF#2 (aa 639–641) increased transcriptional activity. Expression constructs lacking GAL4 DNA binding domains had no effect on transcription, and GAL4 binding sites were required for luciferase activity in this system. Finally, MLL-CALM activated transcription of the murine HOXA7 promoter in comparison with native CALM or empty vector. Conclusions: We have confirmed that native CALM is able to localize to the nucleus, and we have begun to identify specific critical residues in the CALM TAD. The presence of a CALM TAD in MLL-CALM suggests that altered transcriptional regulation of MLL-dependent HOX genes may play an important role in MLL-CALM dependent transformation. Our observations raise the possibility that other MLL partners with native cytoplasmic localization may possess unrecognized transcriptional activity, and provide new insight into both MLL-CALM and CALM-AF10 mediated leukemogenesis.

Molecular characterization of mouse CREB3 regulatory factor in Neuro2a cells

2021 ◽  
Author(s):  
Kentaro Oh-hashi ◽  
Tomoyuki Hasegawa ◽  
Yoshihisa Naruse ◽  
Yoko Hirata
Keyword(s):  
Molecular Characterization ◽  
Regulatory Factor ◽  
Neuro2a Cells

scholarly journals Immunological Characterization of Escherichia coli O157:H7 Intimin γ1

2002 ◽  
Vol 9 (1) ◽  
pp. 46-53 ◽  
Author(s):  
W.-G. Son ◽  
T. A. Graham ◽  
V. P. J. Gannon
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Full Length ◽  
Expression Vectors ◽  
Protein Fusion ◽  
Western Blots ◽  
E Coli ◽  
Specific Pcr ◽  
His Tag

ABSTRACT Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a(+) expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a(+). Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin typesα (O serogroups 86, 127, and 142), β1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), γ1 (O serogroups 55, 145, and 157), γ2 (O serogroups 111 and 103), and ε (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intγ1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin γ1-producing E. coli strains such as E. coli O157:H7.

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

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