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.

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.

Aspergillus galactose metabolism is more complex than that of Saccharomyces: the story of GalDGAL7 and GalEGAL1

Botany ◽  
2013 ◽  
Vol 91 (7) ◽  
pp. 467-477 ◽  
Author(s):  
Md. Kausar Alam ◽  
Susan G.W. Kaminskyj
Keyword(s):  
Aspergillus Nidulans ◽  
Minimal Medium ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Galactose Metabolism ◽  
Sequence Identity ◽  
Functional Homology ◽  
Wild Type Phenotype ◽  
Polymerase Chain ◽  
Green Fluorescent

Saccharomyces cerevisiae Hansen GAL1 (galactokinase) generates galactose-1-phosphate; GAL7 (galactose-1-phosphate uridylyltransferase) transfers UDP between galactose or glucose and their respective sugar-1-phosphate conjugates, and both are essential on galactose. Aspergillus nidulans ANID_04957 has 41% amino acid sequence identity with GAL1; ANID_06182 has 50% sequence identity with GAL7. The names Aspergillus nidulans GalE (galactokinase) and GalD (galactose-1-phosphate uridylyltransferase) are consistent with prior studies. Complemented galDΔ:ScGAL7 and galEΔ:ScGAL1 strains had wild-type phenotype, demonstrating functional homology. The galD5 and galE9 alleles were truncated. Strains galDΔ and galD5 were impaired on minimal medium containing 1% galactose (MM-Gal) at pH 7.5 and did not grow on MM-Gal pH 4.5. Strains galEΔ and galE9 grew on MM-Gal at both pH levels. Strains galDΔ and galEΔ produced wild-type conidiophores on minimal medium containing 1% glucose (MM-Glu) but few spores; for both, sporulation was lower on MM-Gal pH 7.5. GalD-GFP (green fluorescent protein) and GalE-GFP were cytosolic and upregulated on MM-Gal, consistent with quantitative real-time polymerase chain reaction. Galactofuranose immunolocalization in galDΔ resembled wild type on MM-Glu but was reduced on MM-Gal. The galEΔ strains had immunolocalizable Galf on all these media. Strains galDΔ and galEΔ were more sensitive to calcofluor, caspofungin, and itraconazole on MM-Gal. Neither galD nor galE is essential on galactose at high pH, implying additional routes for galactose metabolism in Aspergillus. Aspergillus galactose metabolism is more complex than that of S. cerevisiae.

scholarly journals Identification and characterization of sequence signatures in the Bacillus subtilis promoter Pylb for tuning promoter strength

2019 ◽  
Vol 42 (1) ◽  
pp. 115-124
Author(s):  
Jiangtao Xu ◽  
Xiaoqing Liu ◽  
Xiaoxia Yu ◽  
Xiaoyu Chu ◽  
Jian Tian ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Fluorescent Protein ◽  
Gc Content ◽  
Organophosphorus Hydrolase ◽  
Promoter Strength ◽  
Green Fluorescent ◽  
Mutant Promoter ◽  
Identification And Characterization ◽  
Sequence Signatures

Abstract Objective To thoroughly characterize the Pylb promoter and identify the elements that affect the promoter activity. Result The sequences flanking the − 35 and − 10 box of the Pylb promoter were divided into six segments, and six random-scanning mutant promoter libraries fused to an enhanced green fluorescent protein EGFP were made and analyzed by flow cytometry. Our results showed that the four nucleotides flanking the − 35 box could mostly influence the promoter activity, and this influence was related to the GC content. The promoters mutated in these regions were successfully used for expressing the gene ophc2 encoding organophosphorus hydrolase (OPHC2) and the gene katA encoding catalase (KatA). Conclusion Our work identified and characterized the sequence signatures of the Pylb promoter that could tune the promoter strength, providing further information for the potential application of this promoter. Meanwhile, the sequence signatures have the potential to be used for tuning gene expression in enzyme production, metabolic engineering, and synthetic biology.

scholarly journals Tortoise, a Novel Mitochondrial Protein, Is Required for Directional Responses of Dictyostelium in Chemotactic Gradients

2001 ◽  
Vol 152 (3) ◽  
pp. 621-632 ◽  
Author(s):  
Saskia van Es ◽  
Deborah Wessels ◽  
David R. Soll ◽  
Jane Borleis ◽  
Peter N. Devreotes
Keyword(s):  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
Wild Type ◽  
Cytosolic Ph ◽  
Spatial Gradients ◽  
Similar Phenotype ◽  
Green Fluorescent ◽  
Triton X ◽  
Shape Changes

We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA− partially restores chemotaxis, whereas overexpression of TorA in mek1− does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA–green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

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