scholarly journals Heterogeneity ofAspergillus nigerMicrocolonies in Liquid Shaken Cultures

2010 ◽  
Vol 77 (4) ◽  
pp. 1263-1267 ◽  
Author(s):  
Charissa de Bekker ◽  
G. Jerre van Veluw ◽  
Arman Vinck ◽  
L. Ad Wiebenga ◽  
Han A. B. Wösten
Keyword(s):  
Protein Production ◽  
Fluorescent Protein ◽  
Laser Microdissection ◽  
Pcr Analysis ◽  
Complex Object ◽  
Ferulic Acid Esterase ◽  
Mrna Accumulation ◽  
Esterase Gene ◽  
Green Fluorescent ◽  
Two Populations

ABSTRACTThe fungusAspergillus nigerforms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase geneglaAor the ferulic acid esterase genefaeAwere sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 μm in diameter. The population of small microcolonies of strains expressing GFP from theglaAorfaeApromoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies ofA. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production ofA. nigercan be improved in industrial fermentations by reducing the heterogeneity within the culture.

Rapid Optimization of Membrane Protein Production Using Green Fluorescent Protein-Fusions and Lemo21(DE3)

2011 ◽  
pp. 391-406
Author(s):  
Susan Schlegel ◽  
Mirjam Klepsch ◽  
Dimitra Gialama ◽  
David Wickström ◽  
David Drew ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Membrane Protein ◽  
Protein Production ◽  
Fluorescent Protein ◽  
Green Fluorescent

Elucidation of the distal convoluted tubule transcriptome identifies new candidate genes involved in renal Mg2+ handling

2013 ◽  
Vol 305 (11) ◽  
pp. F1563-F1573 ◽  
Author(s):  
Jeroen H. F. de Baaij ◽  
Marian J. Groot Koerkamp ◽  
Marla Lavrijsen ◽  
Femke van Zeeland ◽  
Hans Meijer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Candidate Genes ◽  
Transient Receptor Potential ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Distal Convoluted Tubule ◽  
Fine Tuning ◽  
Complex Object ◽  
New Genes ◽  
Green Fluorescent

The kidney plays a key role in the maintenance of Mg2+ homeostasis. Specifically, the distal convoluted tubule (DCT) is instrumental in the fine-tuning of renal Mg2+ handling. In recent years, hereditary Mg2+ transport disorders have helped to identify important players in DCT Mg2+ homeostasis. Nevertheless, several proteins involved in DCT-mediated Mg2+ reabsorption remain to be discovered, and a full expression profile of this complex nephron segment may facilitate the discovery of new Mg2+-related genes. Here, we report Mg2+-sensitive expression of the DCT transcriptome. To this end, transgenic mice expressing enhanced green fluorescent protein under a DCT-specific parvalbumin promoter were subjected to Mg2+-deficient or Mg2+-enriched diets. Subsequently, the Complex Object Parametric Analyzer and Sorter allowed, for the first time, isolation of enhanced green fluorescent protein-positive DCT cells. RNA extracts thereof were analyzed by DNA microarrays comparing high versus low Mg2+ to identify Mg2+ regulatory genes. Based on statistical significance and a fold change of at least 2, 46 genes showed differential expression. Several known magnesiotropic genes, such as transient receptor potential cation channel, subfamily M, member 6 ( Trpm6), and Parvalbumin, were upregulated under low dietary Mg2+. Moreover, new genes were identified that are potentially involved in renal Mg2+ handling. To confirm that the selected candidate genes were regulated by dietary Mg2+ availability, the expression levels of solute carrier family 41, member 3 ( Slc41a3), pterin-4 α-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1α ( Pcbd1), TBC1 domain family, member 4 ( Tbc1d4), and uromodulin ( Umod) were determined by RT-PCR analysis. Indeed, all four genes show significant upregulation in the DCT of mice fed a Mg2+-deficient diet. By elucidating the Mg2+-sensitive DCT transcriptome, new candidate genes in renal Mg2+ handling have been identified.

Rapid and massive green fluorescent protein production leads to formation of protein Y-bodies in plant cells

2012 ◽  
Vol 77 (6) ◽  
pp. 603-608 ◽  
Author(s):  
T. V. Komarova ◽  
E. V. Sheval ◽  
D. V. Pozdyshev ◽  
V. S. Kolesnikova ◽  
Yu. L. Dorokhov
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Production ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Green Fluorescent Protein Production ◽  
Green Fluorescent

Four novel splice variants of sulfonylurea receptor 1

2002 ◽  
Vol 283 (2) ◽  
pp. C587-C598 ◽  
Author(s):  
Annette Hambrock ◽  
Regina Preisig-Müller ◽  
Ulrich Russ ◽  
Anke Piehl ◽  
Peter J. Hanley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Splice Variants ◽  
Functional Characterization ◽  
Pcr Analysis ◽  
Sulfonylurea Receptor ◽  
Transmembrane Segments ◽  
Sulfonylurea Receptor 1 ◽  
Green Fluorescent ◽  
Splice Forms

ATP-sensitive K+ (KATP) channels are composed of pore-forming Kir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pig SUR1. Three splice forms originate from alternative splicing of the region coding for NBF1 and lack exons 17 (SUR1Δ17), 19 (SUR1Δ19), or both (SUR1Δ17Δ19). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31–39 containing the last two transmembrane segments and the COOH terminus of SUR1. RT-PCR analysis showed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy using enhanced green fluorescent protein-tagged SUR or Kir6.x did not provide any evidence for involvement of these splice forms in the mitochondrial KATP channel. Only SUR1 and SUR1Δ17 showed high-affinity binding of glibenclamide ( K d≈ 2 nM in the presence of 1 mM ATP) and formed functional KATPchannels upon coexpression with Kir6.2.

scholarly journals Application of RNA Interference Technology to Acroporid Juvenile Corals

2021 ◽  
Vol 8 ◽  
Author(s):  
Ikuko Yuyama ◽  
Tomihiko Higuchi ◽  
Michio Hidaka
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Stress Response ◽  
Large Scale ◽  
Elevated Temperatures ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Pcr Analysis ◽  
Genes Encoding ◽  
Green Fluorescent

Numerous genes involved in calcification, algal endosymbiosis, and the stress response have been identified in corals by large-scale gene expression analysis, but functional analysis of those genes is lacking. There are few experimental examples of gene expression manipulation in corals, such as gene knockdown by RNA interference (RNAi). The purpose of this study is to establish an RNAi method for coral juveniles. As a first trial, the genes encoding green fluorescent protein (GFP, an endogenous fluorophore expressed by corals) and thioredoxin (TRX, a stress response gene) were selected for knockdown. Synthesized double-stranded RNAs (dsRNAs) corresponding to GFP and TRX were transformed into planula larvae by lipofection method to attempt RNAi. Real-time PCR analysis to verify knockdown showed that GFP and TRX expression levels tended to decrease with each dsRNA treatment (not significant). In addition, stress exposure experiments following RNAi treatment revealed that planulae with TRX knockdown exhibited increased mortality at elevated temperatures. In GFP-knockdown corals, decreased GFP fluorescence was observed. However, the effect of GFP-knockdown was confirmed only in the coral at the initial stages of larval metamorphosis into polyps, but not in planulae and 1 month-old budding polyps. This study showed that lipofection RNAi can be applied to coral planulae and polyps after settlement, and that this method provides a useful tool to modify expression of genes involved in stress tolerance and fluorescence emission of the corals.

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