scholarly journals Genetically encoded betaxanthin-based small-molecular fluorescent reporter for mammalian cells

2020 ◽  
Vol 48 (12) ◽  
pp. e67-e67
Author(s):  
Pascal Stücheli ◽  
Simon Sieber ◽  
David W Fuchs ◽  
Leo Scheller ◽  
Tobias Strittmatter ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Human Model ◽  
Fluorescence Labeling ◽  
Application Software ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Amanita Muscaria ◽  
Standard Application ◽  
Fungal Pigments ◽  
Heterologous Pathway

Abstract We designed and engineered a dye production cassette encoding a heterologous pathway, including human tyrosine hydroxylase and Amanita muscaria 4,5-DOPA dioxygenase, for the biosynthesis of the betaxanthin family of plant and fungal pigments in mammalian cells. The system does not impair cell viability, and can be used as a non-protein reporter system to directly visualize the dynamics of gene expression by profiling absorbance or fluorescence in the supernatant of cell cultures, as well as for fluorescence labeling of individual cells. Pigment profiling can also be multiplexed with reporter proteins such as mCherry or the human model glycoprotein SEAP (secreted alkaline phosphatase). Furthermore, absorbance measurement with a smartphone camera using standard application software enables inexpensive, low-tech reporter quantification.

scholarly journals High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effect

2021 ◽  
Author(s):  
Huawei Tong ◽  
Jia Huang ◽  
Qingquan Xiao ◽  
Bingbing He ◽  
Xue Dong ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Basic Research ◽  
Rna Degradation ◽  
High Fidelity ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Major Barrier ◽  
Collateral Effects ◽  
Collateral Effect

CRISPR-Cas13 systems have recently been employed for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has imposed a major barrier for their in vivo applications. We designed a dual-fluorescent reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants (including Cas13d and Cas13X) exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. Importantly, high-fidelity Cas13 variants show comparable RNA knockdown activity with wild-type Cas13 but no detectable collateral damage in transgenic mice and adeno-associated virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effect are now available for targeted degradation of RNAs in basic research and therapeutic applications.

scholarly journals DnaK response to expression of protein mutants is dependent on translation rate and stability

2021 ◽  
Author(s):  
Signe Christensen ◽  
Sebastian Rämisch ◽  
Ingemar André
Keyword(s):  
Large Degree ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Protein Abundance ◽  
Reporter System ◽  
Thermal Stabilities ◽  
Translation Rate ◽  
Fluorescent Reporter ◽  
Unfolded Protein ◽  
Protein Mutants

AbstractChaperones play a central part in the quality control system in cells by clearing misfolded and aggregated proteins. The chaperone DnaK acts as a sensor for molecular stress by recognising short hydrophobic stretches of misfolded proteins. As the level of unfolded protein is a function of protein stability, we hypothesised that the level of DnaK response upon overexpression of recombinant proteins would be correlated to stability. Using a set of mutants of the λ-repressor with varying thermal stabilities and a fluorescent reporter system, the effect of stability on DnaK response and protein abundance was investigated. Our results demonstrate that the initial DnaK response is largely dependent on protein synthesis rate but as the recombinantly expressed protein accumulates and homeostasis is approached the response correlates strongly with stability. Furthermore, we observe a large degree of cell-cell variation in protein abundance and DnaK response in more stable proteins.

scholarly journals Staphylococcal DNA repair is required for infection

2020 ◽  
Author(s):  
Kam Pou Ha ◽  
Rebecca S. Clarke ◽  
Gyu-Lee Kim ◽  
Jane L. Brittan ◽  
Jessica E. Rowley ◽  
...  
Keyword(s):  
Human Blood ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Gram Positive ◽  
Fluorescent Reporter ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Transposon Mutants

AbstractThe repair of DNA damage is essential for bacterial viability and contributes to adaptation via increased rates of mutation and recombination. However, the mechanisms by which DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double strand breaks via the oxidative burst, which are repaired by RexAB, leading to induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted survival of these pathogens in human blood, suggesting that DNA double strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.

A novel reporter system for bacterial and mammalian cells based on the non-ribosomal peptide indigoidine

2012 ◽  
Vol 14 (4) ◽  
pp. 325-335 ◽  
Author(s):  
Marius Müller ◽  
Simon Ausländer ◽  
David Ausländer ◽  
Christian Kemmer ◽  
Martin Fussenegger
Keyword(s):  
Mammalian Cells ◽  
Reporter System

scholarly journals Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Nicolas Lemus-Diaz ◽  
Kai O. Böker ◽  
Ignacio Rodriguez-Polo ◽  
Michael Mitter ◽  
Jasmin Preis ◽  
...  
Keyword(s):  
Single Cell ◽  
Mirna Gene ◽  
Gene Repression ◽  
Single Cell Level ◽  
Reporter System ◽  
Cell Level ◽  
Fluorescent Reporter

scholarly journals Stress-Inducible Alternative Translation Initiation of Human Cytomegalovirus Latency Protein pUL138

2010 ◽  
Vol 84 (18) ◽  
pp. 9472-9486 ◽  
Author(s):  
Lora Grainger ◽  
Louis Cicchini ◽  
Michael Rak ◽  
Alex Petrucelli ◽  
Kerry D. Fitzgerald ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Translation Initiation ◽  
Rna Splicing ◽  
Mammalian Cells ◽  
Open Reading Frames ◽  
Reporter System ◽  
Entry Site ◽  
Sequence Element ◽  
Low Passage

ABSTRACT We have previously characterized a 21-kDa protein encoded by UL138 (pUL138) as a viral factor inherent to low-passage strains of human cytomegalovirus (HCMV) that is required for latent infection in vitro. pUL138 is encoded on 3.6-, 2.7-, and 1.4-kb 3′ coterminal transcripts that are produced during productive and latent infections. pUL138 is encoded at the 3′ end of each transcript and is preceded by an extensive 5′ sequence (∼0.5 to 2.5 kb) containing several putative open reading frames (ORFs). We determined that three putative ORFs upstream of UL138 (UL133, UL135, and UL136) encode proteins. The UL138 transcripts are polycistronic, such that each transcript expresses pUL138 in addition to the most-5′ ORF. The upstream coding sequences (CDS) present a significant challenge for the translation of pUL138 in mammalian cells. We hypothesized that sequences 5′ of UL138 mediate translation initiation of pUL138 by alternative strategies. Accordingly, a 663-nucloetide (nt) sequence overlapping the UL136 CDS supported expression of a downstream cistron in a bicistronic reporter system. We did not detect cryptic promoter activity or RNA splicing events that could account for downstream cistron expression. These data are consistent with the sequence element functioning as an internal ribosome entry site (IRES). Interestingly, pUL138 expression from the 3.6- and 2.7-kb transcripts was induced by serum stress, which concomitantly inhibited normal cap-dependent translation. Our work suggests that an alternative and stress-inducible strategy of translation initiation ensures expression of pUL138 under a variety of cellular contexts. The UL138 polycistronic transcripts serve to coordinate the expression of multiple proteins, including a viral determinant of HCMV latency.

MVP interacts with YPEL4 and inhibits YPEL4-mediated activities of the ERK signal pathway

2010 ◽  
Vol 88 (3) ◽  
pp. 445-450 ◽  
Author(s):  
Pei Liang ◽  
Yongqi Wan ◽  
Yan Yan ◽  
Yuequn Wang ◽  
Na Luo ◽  
...  
Keyword(s):  
Metal Binding ◽  
Mammalian Cells ◽  
Mapk Signaling ◽  
Reporter System ◽  
Biochemical Assays ◽  
Metal Binding Domain ◽  
Erk Signal Pathway ◽  
Gene Maps ◽  
Putative Zinc Finger ◽  
Two Hybrid

Human YPEL4 is a member of YPEL family. It contains a Yippee domain, which is a putative zinc-finger-like, metal-binding domain. The human YPEL4 gene maps to chromosome 11q12.1, is ubiquitously expressed in adult tissues, and encodes a nuclear protein of 127 amino acids, the function of which remains unknown. To gain insights into the cellular function of this protein, we searched for YPEL4-interacting proteins using a yeast two-hybrid screen. The major vault protein (MVP), a lung resistance associated protein, was identified as a binding partner of YPEL4. The interaction between YPEL4 and MVP in mammalian cells was further demonstrated by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, co-immunoprecipitation assay, and immunocytochemistry. Using a reporter system, we found that MVP can inhibit YPEL4’s ability to activate Elk-1 in the MAPK signaling pathway. This study provides new clues for understanding the molecular mechanism of YPEL4 in cell division and signal transduction pathways and should be helpful for understanding molecular functions of the YPEL family.

scholarly journals Analysis of Proteasome-Dependent Proteolysis in Haloferax volcanii Cells, Using Short-Lived Green Fluorescent Proteins

2004 ◽  
Vol 70 (12) ◽  
pp. 7530-7538 ◽  
Author(s):  
Christopher J. Reuter ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Specific Inhibitor ◽  
Haloferax Volcanii ◽  
Reporter System ◽  
Reporter Protein ◽  
Protein Variant ◽  
Fluorescent Reporter ◽  
New Genes ◽  
Green Fluorescent

ABSTRACT Proteasomes are energy-dependent proteases that are central to the quality control and regulated turnover of proteins in eukaryotic cells. Dissection of this proteolytic pathway in archaea, however, has been hampered by the lack of substrates that are easily detected in whole cells. In the present study, we developed a convenient reporter system by functional expression of a green fluorescent protein variant with C-terminal fusions in the haloarchaeon Haloferax volcanii. The levels of this reporter protein correlated with whole-cell fluorescence that was readily detected in culture. Accumulation of the reporter protein was dependent on the sequence of the C-terminal amino acid fusion, as well as the presence of an irreversible, proteasome-specific inhibitor (clasto-lactacystin β-lactone). This inhibitor was highly specific for H. volcanii 20S proteasomes, with a Ki of ∼40 nM. In contrast, phenylmethanesulfonyl fluoride did not influence the levels of fluorescent reporter protein or inhibit 20S proteasomes. Together, these findings provide a powerful tool for the elucidation of protein substrate recognition motifs and the identification of new genes which may be involved in the proteasome pathway of archaea.

Sign in / Sign up

Export Citation Format

Share Document