scholarly journals Proteome-Level Responses ofEscherichia colito Long-Chain Fatty Acids and Use of Fatty Acid Inducible Promoter in Protein Production

2008 ◽  
Vol 2008 ◽  
pp. 1-12 ◽  
Author(s):  
Mee-Jung Han ◽  
Jeong Wook Lee ◽  
Sang Yup Lee ◽  
Jong Shin Yoo
Keyword(s):  
Oleic Acid ◽  
Fluorescent Protein ◽  
Inducible Promoter ◽  
Biological Research ◽  
Cell Physiology ◽  
Altered Expression ◽  
Chain Acyl ◽  
Proteome Level ◽  
Green Fluorescent ◽  
Long Chain

InEscherichia coli, a long-chain acyl-CoA is a regulatory signal that modulates gene expression through its binding to a transcription factor FadR. In this study, comparative proteomic analysis ofE. coliin the presence of glucose and oleic acid was performed to understand cell physiology in response to oleic acid. Among total of 52 proteins showing altered expression levels with oleic acid presence, 9 proteins including AldA, Cdd, FadA, FadB, FadL, MalE, RbsB, Udp, and YccU were newly synthesized. Among the genes that were induced by oleic acid, the promoter of thealdAgene was used for the production of a green fluorescent protein (GFP). Analysis of fluorescence intensities and confocal microscopic images revealed that soluble GFP was highly expressed under the control of thealdApromoter. These results suggest that proteomics is playing an important role not only in biological research but also in various biotechnological applications.

scholarly journals Murine bubblegum orthologue is a microsomal very long-chain acyl-CoA synthetase

2004 ◽  
Vol 377 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Peter FRAISL ◽  
Sonja FORSS-PETTER ◽  
Mihaela ZIGMAN ◽  
Johannes BERGER
Keyword(s):  
Fluorescent Protein ◽  
Neurodegenerative Disorder ◽  
Direct Interaction ◽  
Confocal Laser ◽  
Synthetase Activity ◽  
Wild Type ◽  
Protein Fusion ◽  
Chain Acyl ◽  
Green Fluorescent ◽  
Long Chain

It has been suggested that a gene termed bubblegum (Bgm), encoding an acyl-CoA synthetase, could be involved in the pathogenesis of the inherited neurodegenerative disorder X-ALD (X-linked adrenoleukodystrophy). The precise function of the ALDP (ALD protein) still remains unclear. Aldp deficiency in mammals and Bgm deficiency in Drosophila led to accumulation of VLCFAs (very long-chain fatty acids). As a first step towards studying this interaction in wild-type versus Aldp-deficient mice, we analysed the expression pattern of the murine orthologue of the Bgm gene. In contrast with the ubiquitously expressed Ald gene, Bgm expression is restricted to the tissues that are affected by X-ALD such as brain, testis and adrenals. During mouse brain development, Bgm mRNA was first detected by Northern-blot analysis on embryonic day 18 and increased steadily towards adulthood, whereas the highest level of Ald mRNA was found on embryonic day 12 and decreased gradually during differentiation. Protein fractionation and confocal laser imaging of Bgm–green fluorescent protein fusion proteins revealed a microsomal localization that was different from peroxisomes (where Aldp is detected), endoplasmic reticulum and Golgi. Mouse Bgm showed acyl-CoA synthetase activity towards a VLCFA substrate in addition to LCFAs, and this activity was enriched in the microsomal compartment. Speculating that Bgm expression could be regulated by Ald deficiency, we compared the abundance of Bgm mRNA in wild-type and Ald knockout mice but observed no difference. Although mouse Bgm is capable of activating VLCFA, we conclude that a direct interaction between the mouse Bgm and the Aldp seems unlikely.

scholarly journals Tb3+-doped fluorescent glass for biology

2021 ◽  
Vol 7 (2) ◽  
pp. eabd2529
Author(s):  
Kazuki Okamoto ◽  
Teppei Ebina ◽  
Naoki Fujii ◽  
Kuniaki Konishi ◽  
Yu Sato ◽  
...  
Keyword(s):  
Single Cell ◽  
Fluorescent Protein ◽  
Biological Research ◽  
Optical Investigation ◽  
Patch Clamp Recording ◽  
Rare Earth Ion ◽  
Cell Transcriptome ◽  
Green Fluorescent ◽  
Doped Glass

Optical investigation and manipulation constitute the core of biological experiments. Here, we introduce a new borosilicate glass material that contains the rare-earth ion terbium(III) (Tb3+), which emits green fluorescence upon blue light excitation, similar to green fluorescent protein (GFP), and thus is widely compatible with conventional biological research environments. Micropipettes made of Tb3+-doped glass allowed us to target GFP-labeled cells for single-cell electroporation, single-cell transcriptome analysis (Patch-seq), and patch-clamp recording under real-time fluorescence microscopic control. The glass also exhibited potent third harmonic generation upon infrared laser excitation and was usable for online optical targeting of fluorescently labeled neurons in the in vivo neocortex. Thus, Tb3+-doped glass simplifies many procedures in biological experiments.

scholarly journals The Reporter System for GPCR Assay with the Fission YeastSchizosaccharomyces pombe

Scientifica ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Shintaro Sasuga ◽  
Toshiya Osada
Keyword(s):  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Biological Activities ◽  
Inducible Promoter ◽  
Upstream Region ◽  
Reporter Plasmid ◽  
Reporter System ◽  
High Background ◽  
Downstream Region ◽  
Green Fluorescent

G protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR pathway. As a control region of GFP expression on the reporter plasmid, we focused on seven endogenous genes specifically activated through the pathway. When upstream regions of these genes were used as an inducible promoter in combination with LPI terminator, themam2upstream region produced GFP most rapidly and intensely despite the high background. Subsequently, LPI terminator was replaced with the corresponding downstream regions. The SPBC4.01 downstream region enhanced the response with the low background. Furthermore, combining SPBC4.01 downstream region with the sxa2 upstream region, the signal to noise ratio was obviously better than those of other regions. We also evaluated the time- and dose-dependent GFP productions of the strains transformed with the reporter plasmids. Finally, we exhibited a model of simplified GPCR assay with the reporter plasmid by expressing endogenous GPCR under the control of the foreign promoter.

scholarly journals Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases

2000 ◽  
Vol 347 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Brian S. FINLIN ◽  
Haipeng SHAO ◽  
Keiko KADONO-OKUDA ◽  
Nan GUO ◽  
Douglas A. ANDRES
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Dissociation Constants ◽  
Intrinsic Rate ◽  
Biochemical Properties ◽  
Cell Physiology ◽  
Gtp Binding ◽  
C Terminus ◽  
Green Fluorescent

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, ad and G-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

Human Genetics

2005 ◽  
Author(s):  
Antonio Regalado
Keyword(s):  
Heart Attack ◽  
Fluorescent Protein ◽  
Human Genetics ◽  
New Technology ◽  
Genetic Research ◽  
Biological Research ◽  
Scientific Journals ◽  
Big Picture ◽  
New Gene ◽  
Green Fluorescent

Genetic research is moving faster than a nematode poked by a platinum needle. Every week, the scientific journals report a score of new gene discoveries made in mice, worms, and men. How can a science journalist cover it all? It's hopeless, of course. So one thing I always keep in mind is it's often the methods or scientific tools behind these molecular discoveries, not the discoveries themselves, that present the best story possibilities. Examples of topics for such “tool stories” include DNA chips, proteomics, and new imaging technologies like the green-fluorescent protein used to make zebrafish and other laboratory critters glow. In writing about the technologies that drive biological research, I've found a formula that has worked well for me, time and again. Of course, not every story fits the same mold, and the best ones break it. But it's important to be familiar with how a tool story typically comes to be, and how to write one. I like to think about biology as a big onion that's rapidly being peeled. There are tens of thousands of biologists peeling away every day, figuring out all of life's working parts. But I never saw much sense in inspecting every peel for its news potential. (And some editors I know refer dismissively to the latest uncovering of a gene for heart attack or schizophrenia as “gene-of-the-week” stories.) It's better, sometimes, to focus on the new techniques and ideas for peeling the onion. Tool stories are big-picture stories that can be newsy, but the trends tend to have a long shelf life. They endure through numerous news cycles, and ultimately nearly every outlet in the journalistic food chain will cover the big ones. Your decision is when to catch the wave. Some reporters put a big emphasis on being first, but others will be content to watch the story unfold and cover their piece of it when it's right for whatever market they happen to be writing for. Either way, a tale of how a new technology is changing biological research is a great way to teach your readers—and yourself—about how science really works.

scholarly journals Tissue-specific expression of ferritin H regulates cellular iron homoeostasis in vivo

2006 ◽  
Vol 395 (3) ◽  
pp. 501-507 ◽  
Author(s):  
John Wilkinson ◽  
Xiumin Di ◽  
Kai Schönig ◽  
Joan L. Buss ◽  
Nancy D. Kock ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Protein Activity ◽  
Specific Expression ◽  
Cellular Iron ◽  
Tissue Iron ◽  
Green Fluorescent ◽  
Ferritin H

Ferritin is a ubiquitously distributed iron-binding protein. Cell culture studies have demonstrated that ferritin plays a role in maintenance of iron homoeostasis and in the protection against cytokine- and oxidant-induced stress. To test whether FerH (ferritin H) can regulate tissue iron homoeostasis in vivo, we prepared transgenic mice that conditionally express FerH and EGFP (enhanced green fluorescent protein) from a bicistronic tetracycline-inducible promoter. Two transgenic models were explored. In the first, the FerH and EGFP transgenes were controlled by the tTACMV (Tet-OFF) (where tTA and CMV are tet transactivator protein and cytomegalovirus respectively). In skeletal muscle of mice bearing the FerH/EGFP and tTACMV transgenes, FerH expression was increased 6.0±1.1-fold (mean±S.D.) compared with controls. In the second model, the FerH/EGFP transgenes were controlled by an optimized Tet-ON transactivator, rtTA2S-S2LAP (where rtTA is reverse tTA and LAP is liver activator protein), resulting in expression predominantly in the kidney and liver. In mice expressing these transgenes, doxycycline induced FerH in the kidney by 14.2±4.8-fold (mean±S.D.). Notably, increases in ferritin in overexpressers versus control littermates were accompanied by an elevation of IRP (iron regulatory protein) activity of 2.3±0.9-fold (mean±S.D.), concurrent with a 4.5±2.1-fold (mean±S.D.) increase in transferrin receptor, indicating that overexpression of FerH is sufficient to elicit a phenotype of iron depletion. These results demonstrate that FerH not only responds to changes in tissue iron (its classic role), but can actively regulate overall tissue iron balance.

scholarly journals Subcellular Localization of TatAd of Bacillus subtilis Depends on the Presence of TatCd or TatCy

2009 ◽  
Vol 191 (13) ◽  
pp. 4410-4418 ◽  
Author(s):  
Anja N. J. A. Ridder ◽  
Esther J. de Jong ◽  
Jan D. H. Jongbloed ◽  
Oscar P. Kuipers
Keyword(s):  
Bacillus Subtilis ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Inducible Promoter ◽  
Specific Protein ◽  
Positive Bacterium ◽  
Protein Substrates ◽  
Gfp Fusion Protein ◽  
Dual Localization ◽  
Green Fluorescent

ABSTRACT The gram-positive bacterium Bacillus subtilis contains two minimal Tat translocases, TatAdCd and TatAyCy, which are each involved in the secretion of one or more specific protein substrates. We have investigated the subcellular localization of the TatA components by employing C-terminal green fluorescent protein (GFP) fusions and fluorescence microscopy. When expressed from a xylose-inducible promoter, the TatA-GFP fusion proteins displayed a dual localization pattern, being localized peripherally and showing bright foci which are predominantly located at the division sites and/or poles of the cells. Importantly, the localization of TatAd-GFP was similar when the protein was expressed from its own promoter under phosphate starvation conditions, indicating that these foci are not the result of artificial overexpression. Moreover, the TatAd-GFP fusion protein was shown to be functional in the translocation of its substrate PhoD, provided that TatCd is also present. Furthermore, we demonstrate that the localization of TatAd-GFP in foci depends on the presence of the TatCd component. Remarkably, however, the TatAd-GFP foci can also be observed in the presence of TatCy, indicating that TatAd can interact not only with TatCd but also with TatCy. These results suggest that the formation of TatAd complexes in B. subtilis is controlled by TatC.

scholarly journals Genetics of single-cell protein abundance variation in large yeast populations

2013 ◽  
Author(s):  
Frank Albert ◽  
Sebastian Treusch ◽  
Arthur H Shockley ◽  
Joshua S Bloom ◽  
Leonid Kruglyak
Keyword(s):  
Single Cell ◽  
Fluorescent Protein ◽  
Single Cell Protein ◽  
Cell Protein ◽  
Cell Physiology ◽  
Protein Abundance ◽  
Large Populations ◽  
Abundance Variation ◽  
Green Fluorescent ◽  
Fluorescent Protein Tags

Many DNA sequence variants influence phenotypes by altering gene expression. Our understanding of these variants is limited by sample sizes of current studies and by measurements of mRNA rather than protein abundance. We developed a powerful method for identifying genetic loci that influence protein expression in very large populations of the yeast Saccharomyes cerevisiae. The method measures single-cell protein abundance through the use of green-fluorescent-protein tags. We applied this method to 160 genes and detected many more loci per gene than previous studies. We also observed closer correspondence between loci that influence protein abundance and loci that influence mRNA abundance of a given gene. Most loci cluster at hotspot locations that influence multiple proteins—in some cases, more than half of those examined. The variants that underlie these hotspots have profound effects on the gene regulatory network and provide insights into genetic variation in cell physiology between yeast strains.

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