scholarly journals Combination of Two Regulatory Elements in the Tetrahymena thermophila HSP70-1 Gene Controls Heat Shock Activation

2007 ◽  
Vol 7 (2) ◽  
pp. 379-386 ◽  
Author(s):  
Sabrina Barchetta ◽  
Antonietta La Terza ◽  
Patrizia Ballarini ◽  
Sandra Pucciarelli ◽  
Cristina Miceli
Keyword(s):  
Heat Shock ◽  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Regulatory Elements ◽  
Mobility Shift ◽  
Functional Link ◽  
In Vivo Analysis ◽  
Mobility Shift Assay ◽  
Green Fluorescent

ABSTRACT The induction of heat shock genes (HSPs) is thought to be primarily regulated by heat shock transcription factors (HSFs), which bind target sequences on HSP promoters, called heat shock elements (HSEs). In this study, we investigated the 5′ untranslated regions of the Tetrahymena thermophila HSP70-1 gene, and we found, in addition to the canonical and divergent HSEs, multiple sets of GATA elements that have not been reported previously in protozoa. By means of in vivo analysis of a green fluorescent protein reporter transgene driven by the HSP70-1 promoter, we demonstrate that HSEs do not represent the minimal regulatory elements for heat shock induction, since the HSP70-1 is tightly regulated by both HSE and GATA elements. Electrophoretic mobility shift assay also showed that HSFs are constitutively bound to the HSEs, whereas GATA elements are engaged only after heat shock. This is the first demonstration by in vivo analysis of functional HSE and GATA elements in protozoa. Furthermore, we provide evidence of a functional link between HSE and GATA elements in the activation of the heat shock response.

scholarly journals Longitudinal evaluation of expression of virally delivered transgenes in gerbil cone photoreceptors

2008 ◽  
Vol 25 (3) ◽  
pp. 273-282 ◽  
Author(s):  
MATTHEW C. MAUCK ◽  
KATHERINE MANCUSO ◽  
JAMES A. KUCHENBECKER ◽  
THOMAS B. CONNOR ◽  
WILLIAM W. HAUSWIRTH ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Imaging System ◽  
Regulatory Elements ◽  
Meriones Unguiculatus ◽  
Opsin Gene ◽  
Post Injection ◽  
Cone Photoreceptors ◽  
Long Wavelength ◽  
Green Fluorescent

Delivery of foreign opsin genes to cone photoreceptors using recombinant adeno-associated virus (rAAV) is a potential tool for studying the basic mechanisms underlying cone based vision and for treating vision disorders. We used an in vivo retinal imaging system to monitor, over time, expression of virally-delivered genes targeted to cone photoreceptors in the Mongolian gerbil (Meriones unguiculatus). Gerbils have a well-developed photopic visual system, with 11–14% of their photoreceptors being cones. We used replication deficient serotype 5 rAAV to deliver a gene for green fluorescent protein (GFP). In an effort to direct expression of the gene specifically to either S or M cones, the transgene was under the control of either the human X-chromosome opsin gene regulatory elements, i.e., an enhancer termed the locus control region (LCR) and L promoter, or the human S-opsin promoter. Longitudinal fluorescence images reveal that gene expression is first detectable about 14 days post-injection, reaches a peak after about 3 months, and is observed more than a year post-injection if the initial viral concentration is sufficiently high. The regulatory elements are able to direct expression to a subpopulation of cones while excluding expression in rods and non-photoreceptor retinal cells. When the same viral constructs are used to deliver a human long-wavelength opsin gene to gerbil cones, stimulation of the introduced human photopigment with long-wavelength light produces robust cone responses.

scholarly journals In vivo analysis of human nucleoporin repeat domain interactions

2013 ◽  
Vol 24 (8) ◽  
pp. 1222-1231 ◽  
Author(s):  
Songli Xu ◽  
Maureen A. Powers
Keyword(s):  
Fluorescent Protein ◽  
Live Cells ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Nucleocytoplasmic Trafficking ◽  
Repeat Domain ◽  
Domain Interactions ◽  
In Vivo Analysis ◽  
Green Fluorescent

The nuclear pore complex (NPC), assembled from ∼30 proteins termed nucleoporins (Nups), mediates selective nucleocytoplasmic trafficking. A subset of nucleoporins bear a domain with multiple phenylalanine–glycine (FG) motifs. As binding sites for transport receptors, FG Nups are critical in translocation through the NPC. Certain FG Nups are believed to associate via low-affinity, cohesive interactions to form the permeability barrier of the pore, although the form and composition of this functional barrier are debated. We used green fluorescent protein–Nup98/HoxA9 constructs with various numbers of repeats and also substituted FG domains from other nucleoporins for the Nup98 domain to directly compare cohesive interactions in live cells by fluorescence recovery after photobleaching (FRAP). We find that cohesion is a function of both number and type of FG repeats. Glycine–leucine–FG (GLFG) repeat domains are the most cohesive. FG domains from several human nucleoporins showed no interactions in this assay; however, Nup214, with numerous VFG motifs, displayed measurable cohesion by FRAP. The cohesive nature of a human nucleoporin did not necessarily correlate with that of its yeast orthologue. The Nup98 GLFG domain also functions in pore targeting through binding to Nup93, positioning the GLFG domain in the center of the NPC and supporting a role for this nucleoporin in the permeability barrier.

scholarly journals Pituitary Corticotroph Ontogeny and Regulation in Transgenic Zebrafish

2003 ◽  
Vol 17 (5) ◽  
pp. 959-966 ◽  
Author(s):  
Ning-Ai Liu ◽  
Haigen Huang ◽  
Zhongan Yang ◽  
Wiebke Herzog ◽  
Matthias Hammerschmidt ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Cell Mass ◽  
Gene Promoter ◽  
Genetic System ◽  
Regulatory Elements ◽  
Transgenic Zebrafish ◽  
Posterior Pituitary ◽  
Gfp Expression ◽  
Green Fluorescent

Abstract We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18–20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and αMSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10−5m) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.

scholarly journals Control of Microtubule Dynamics by Stu2p Is Essential for Spindle Orientation and Metaphase Chromosome Alignment in Yeast

2001 ◽  
Vol 12 (9) ◽  
pp. 2870-2880 ◽  
Author(s):  
Karena A. Kosco ◽  
Chad G. Pearson ◽  
Paul S. Maddox ◽  
Peijing Jeremy Wang ◽  
Ian R. Adams ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Microtubule Dynamics ◽  
Spindle Orientation ◽  
Cytoplasmic Microtubule ◽  
Chromosome Alignment ◽  
In Vivo Analysis ◽  
Cytoplasmic Microtubules ◽  
Green Fluorescent

Stu2p is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast. Here, we report the first in vivo analysis of microtubule dynamics in cells lacking a member of this protein family. For these studies, we have used a conditional Stu2p depletion strain expressing α-tubulin fused to green fluorescent protein. Depletion of Stu2p leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells. The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing. These changes have significant consequences for the cell because they impede the ability of cytoplasmic microtubules to orient the spindle. In addition, recovery of fluorescence after photobleaching indicates that kinetochore microtubules are no longer dynamic in the absence of Stu2p. This deficiency is correlated with a failure to properly align chromosomes at metaphase. Overall, we provide evidence that Stu2p promotes the dynamics of microtubule plus-ends in vivo and that these dynamics are critical for microtubule interactions with kinetochores and cortical sites in the cytoplasm.

The Mouse GATA-2 Gene is Expressed in the Para-Aortic Splanchnopleura and Aorta-Gonads and Mesonephros Region

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4196-4207 ◽  
Author(s):  
Naoko Minegishi ◽  
Jun Ohta ◽  
Hironori Yamagiwa ◽  
Norio Suzuki ◽  
Shimako Kawauchi ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fetal Liver ◽  
Alternative Promoters ◽  
Endogenous Expression ◽  
Hypersensitive Sites ◽  
Promoter Construct ◽  
In Vivo Analysis ◽  
Green Fluorescent ◽  
Almost All

We previously reported that the mouse GATA-2 gene is regulated by two alternative promoters (Minegishi et al, J Biol Chem, 273:3625, 1998). Although the more proximal IG (general) promoter is active in almost all GATA-2–expressing cells, the distal IS (specific) promoter activity was selectively detected in hematopoietic tissues but not in other mesodermal tissues. We report here in vivo analysis of the GATA-2 locus and its regulatory characteristics in hematopoietic tissues of transgenic mice. Transgenes containing 6 or 7 kbp of sequence flanking the 5′ end of the IS first exon direct expression of β-galactosidase or green fluorescent protein (GFP) reporter genes specifically to the para-aortic splanchnopleura, aorta-gonads, and mesonephros (AGM) region, and in the neural tissues. In situ hybridization analysis showed that reporter gene expression specifically recapitulates the endogenous expression profile of GATA-2 in these tissues. The flk-1, CD34, c-kit, and CD45 antigens were identified in the GFP-positive cells from the AGM region and fetal liver, indicating that GATA-2 is expressed in immature hematopoietic cells. Deletion of 3.5 kbp from the 5′ end of the 6.0 kbp IS promoter construct, including one of the DNase I hypersensitive sites, completely abolished hematopoietic expression. These experiments describe an early developmental GATA-2 hematopoietic enhancer located between 6.0 and 2.5 kbp 5′ to the IS exon.

scholarly journals Characterization of the groESL Operon inListeria monocytogenes: Utilization of Two Reporter Systems (gfp and hly) for Evaluating In Vivo Expression

2001 ◽  
Vol 69 (6) ◽  
pp. 3924-3932 ◽  
Author(s):  
Cormac G. M. Gahan ◽  
James O'Mahony ◽  
Colin Hill
Keyword(s):  
Heat Shock ◽  
Fluorescent Protein ◽  
Ethanol Stress ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Intracellular Expression ◽  
Green Fluorescent ◽  
Shock Proteins ◽  
Transcriptional Fusions

ABSTRACT The ability of intracellular pathogens to sense and adapt to the hostile environment of the host is an important factor governing virulence. We have sequenced the operon encoding the major heat shock proteins GroES and GroEL in the gram-positive food-borne pathogenListeria monocytogenes. The operon has a conserved orientation in the order groES groEL. Upstream ofgroES and in the opposite orientation is a gene encoding a homologue of the Bacillus subtilis protein YdiL, while downstream of groEL is a gene encoding a putative bile hydrolase. We used both reverse transcriptase-PCR (RT-PCR) and transcriptional fusions to the UV-optimized Aequorea victoria green fluorescent protein (GFPUV) to analyze expression of groESL under various environmental stress conditions, including heat shock, ethanol stress, and acid shock, and during infection of J774 mouse macrophage cells. Strains harboring GFPUV transcriptional fusions to the promoter region ofgroESL demonstrated a significant increase in fluorescence following heat shock that was detected by both fluorimetry and fluorescence microscopy. Using both RT-PCR and GFP technology we detected expression of groESL following internalization by J774 cells. Increased intracellular expression of dnaK was also determined using RT-PCR. We have recently described a system which utilizes L. monocytogenes hemolysin as an in vivo reporter of gene expression within the host cell phagosome (C. G. M. Gahan and C. Hill, Mol. Microbiol. 36:498–507, 2000). In this study a strain was constructed in which hemolysin expression was placed under the control of the groESL promoter. In this strain hemolysin expression during infection also confirms transcription from the groESL promoter during J774 and murine infection, albeit at lower levels than the known virulence factorplcA.

scholarly journals Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras.

1995 ◽  
Vol 130 (3) ◽  
pp. 639-650 ◽  
Author(s):  
K R Olson ◽  
J R McIntosh ◽  
J B Olmsted
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Microtubule Organization ◽  
Microtubule Binding ◽  
Binding Characteristics ◽  
Basic Domain ◽  
In Vivo Analysis ◽  
Projection Domain ◽  
Green Fluorescent

MAP 4 is a ubiquitous microtubule-associated protein thought to play a role in the polymerization and stability of microtubules in interphase and mitotic cells. We have analyzed the behavior of protein domains of MAP 4 in vivo using chimeras constructed from these polypeptides and the green fluorescent protein (GFP). GFP-MAP 4 localizes to microtubules; this is confirmed by colocalization of GFP-MAP 4 with microtubules that have incorporated microinjected rhodamine-tubulin, and by loss of localized fluorescence after treatment of cells with anti-microtubule agents. Different subdomains of MAP 4 have distinct effects on microtubule organization and dynamics. The entire basic domain of MAP 4 reorganizes microtubules into bundles and stabilizes these arrays against depolymerization with nocodazole. Within the basic domain, the PGGG repeats, which are conserved with MAP 2 and tau, have a weak affinity for microtubules and are dispensable for microtubule binding, whereas the MAP 4-unique PSP region can function independently in binding. The projection domain shows no microtubule localization, but does modulate the association of various binding subdomains with microtubules. The acidic carboxy terminus of MAP 4 strongly affects the microtubule binding characteristics of the other domains, despite constituting less than 6% of the protein. These data show that MAP 4 association with microtubules is modulated by sequences both within and outside the basic domain. Further, our work demonstrates that GFP chimeras will allow an in vivo analysis of the effects of MAPs and their variants on microtubule dynamics in real time.

scholarly journals A Simple Method to Detect the Inhibition of Transcription Factor-DNA Binding Due to Protein–Protein Interactions In Vivo

Genes ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 684 ◽  
Author(s):  
Guangzhe Yang ◽  
Dong Chao ◽  
Zhenhua Ming ◽  
Jixing Xia
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Regulatory Elements ◽  
Inhibitory Effect ◽  
Protein Protein Interactions ◽  
Mobility Shift ◽  
Simple Method ◽  
Mobility Shift Assay ◽  
Aureobasidin A

Binding of transcription factors (TFs) to cis-regulatory elements (DNA) could modulate the expression of downstream genes, while interactions between TFs and other proteins might inhibit them binding to DNA. Nowadays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) approaches are usually employed to detect the inhibitory effect. However, EMSA might not reflect the inhibitory effect in vivo. ChIP requires preparation of specific antibody or stable genetic transformation and complicated experimental steps, making it laborious and time-consuming. Here, based on the yeast one-hybrid (Y1H) system, we present a simple method to detect the inhibition of TF–DNA binding due to protein–protein interactions in vivo. When interactions between TFs and other proteins inhibit TFs binding to DNA, the reporter (Aureobasidin A resistance) gene is not activated, thereby inhibiting yeast growth on media containing the AbA antibiotic. Two examples were tested with the newly developed method to demonstrate its feasibility. In conclusion, this method provides an alternative strategy for detecting the inhibition of DNA-binding of TFs due to their interactions with other proteins in vivo.

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