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 Identification and Characterization of a Novel Gene Involved in the trans-Specific Nematicidal Activity of Photorhabdus luminescens LN2

2009 ◽  
Vol 75 (12) ◽  
pp. 4221-4223 ◽  
Author(s):  
Xuehong Qiu ◽  
Richou Han ◽  
Xun Yan ◽  
Mingxing Liu ◽  
Li Cao ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transposon Mutagenesis ◽  
Nematicidal Activity ◽  
Photorhabdus Luminescens ◽  
Gene Encoding ◽  
Heterorhabditis Indica ◽  
Green Fluorescent ◽  
Identification And Characterization

ABSTRACT Photorhabdus luminescens subsp. akhurstii LN2 from Heterorhabditis indica LN2 showed nematicidal activity against axenic Heterorhabditis bacteriophora H06 infective juveniles (IJs). Transposon mutagenesis identified an LN2 mutant that supports the growth of H06 nematodes. Tn5 disrupted the namA gene, encoding a novel 364-residue protein and involving the nematicidal activity. The green fluorescent protein-labeled namA mutant was unable to colonize the intestines of H06 IJs.

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.

Transgenic expression of a GFP-rhodopsin COOH-terminal fusion protein in zebrafish rod photoreceptors

2002 ◽  
Vol 19 (3) ◽  
pp. 257-264 ◽  
Author(s):  
BRIAN D. PERKINS ◽  
PAMELA M. KAINZ ◽  
DONALD M. O'MALLEY ◽  
JOHN E. DOWLING
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Immunohistochemical Analysis ◽  
Rod Photoreceptor ◽  
Rod Photoreceptors ◽  
Transgenic Expression ◽  
Green Fluorescent ◽  
Identification And Characterization ◽  
Rod Cell

To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45–50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.

Identification and characterization of 2 types of erythroid progenitors that express GATA-1 at distinct levels

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3575-3583 ◽  
Author(s):  
Norio Suzuki ◽  
Naruyoshi Suwabe ◽  
Osamu Ohneda ◽  
Naoshi Obara ◽  
Shigehiko Imagawa ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Transferrin Receptor ◽  
Fluorescent Protein ◽  
Fetal Liver ◽  
Expression Level ◽  
Green Fluorescence ◽  
Erythroid Progenitors ◽  
Green Fluorescent ◽  
Identification And Characterization

AbstractTranscription factor GATA-1 is essential for the development of the erythroid lineage. To ascertain whether strict control of GATA-1 expression level is necessary for achieving proper erythropoiesis, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of the GATA-1 gene hematopoietic regulatory domain. We examined the GATA-1 expression level by exploiting the transgenic mice and found 2 GFP-positive hematopoietic progenitor fractions in the bone marrow. One is the GFPhigh fraction containing mainly CFU-E and proerythroblasts, which coexpress transferrin receptor, while the other is the GFPlow/transferrin receptor-negative fraction containing BFU-E. Since the intensity of green fluorescence correlates well with the expression level of GATA-1, these results indicate that GATA-1 is highly expressed in erythroid colony-forming unit (CFU-E) but low in erythroid burst-forming unit (BFU-E), suggesting that the incremental expression of GATA-1 is required for the formation of erythroid progenitors. We also examined GFP-positive fractions in the transgenic mouse spleen and fetal liver and identified fractions containing BFU-E and CFU-E, respectively. This study also presents an efficient method for enriching the CFU-E and BFU-E from mouse hematopoietic tissues. (Blood. 2003;102:3575-3583)

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