Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters

Date

2014-01-01

Author

SHIN, Ilchung
RAY, Judhajeet
Gupta, Vinayak
İlgü, Müslüm
Beasley, Jonathan
BENDICKSON, Lee
MEHANOVIC, Samir
Kraus, George A.
Nilsen-Hamilton, Marit

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

72
views

0
downloads

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

Subject Keywords

Genetics

URI

https://hdl.handle.net/11511/47433

Journal

NUCLEIC ACIDS RESEARCH

DOI

https://doi.org/10.1093/nar/gku297

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Functional characterization of microrna-125b expression in MCF7 breast cancer cell line Tuna, Serkan; Erson Bensan, Ayşe Elif; Department of Biology (2010) microRNA dependent gene expression regulation has roles in diverse processes such as differentiation, proliferation and apoptosis. Therefore, deregulated miRNA expression has functional importance for various diseases, including cancer. miR-125b is among the commonly downregulated miRNAs in breast cancer cells . Therefore we aimed to characterize the effects of miR-125b expression in MCF7 breast cancer cell line (BCCL) to better understand its roles in tumorigenesis. Here, we investigated mir-125 family mem...
Cloning and expression of periplasmic (clp p-like) and membrane-bound serine protease genes of thermoplasma volcanium in escherichia coli Demirok, Burçak; Kocabıyık, Semra; Department of Biology (2006) Serine proteases are a family of proteases that utilize an activated serine residue in the substrate-binding pocket to catalytically hydrolyze peptide bonds. Enzymes which belong to this family, with a diverse array of metabolic and regulatory functions, play critical roles in cell physiology and pathology. ءClp̕s are a class of ATP dependent serine proteases which are composed of a protease (ClpP) and an ATPase (ClpA or ClpX) component. Their involvements in degrading proteins are especially implicated und...
Molecular recognition of poly(A) by small ligands: an alternative method of analysis reveals nanomolar, cooperative and shape-selective binding Persil Çetinkol, Özgül (Oxford University Press (OUP), 2009-02-01) A few drug-like molecules have recently been found to bind poly(A) and induce a stable secondary structure (T(m)approximate to 60 degrees C), even though this RNA homopolymer is single-stranded in the absence of a ligand. Here, we report results from experiments specifically designed to explore the association of small molecules with poly(A). We demonstrate that coralyne, the first small molecule discovered to bind poly(dA), binds with unexpectedly high affinity (K(a) >10(7) M(-1)), and that the crescent sh...
PCR cloning and heterologous expression of Scytalidium thermophilum laccase gene in Aspergillus sojae Koçlar, Gülden; Ögel, Zümrüt Begüm; Department of Biotechnology (2005) In this study, Scytalidium thermophilum laccase gene was first cloned into E. coli and then heterologously expressed in A. sojae. S. thermophilum is a thermophilic fungus with an important role in determining selectivity of compost produced for growing Agaricus bisporus. S. thermophilum laccase gene was first cloned by Novo Nordisk Bio Tech, Inc. in 1998. This laccase gene (lccS) has an open reading frame of 2092bp. It is composed of five exons punctuated by four small introns. The coding region, excluding ...
Molecular dynamics simulations and coupled nucleotide substitution experiments indicate the nature of A center dot A base pairing and a putative structure of the coralyne-induced homo-adenine duplex Joung, In Suk; Persil Çetinkol, Özgül; HUD, Nicholas V.; Cheatham, Thomas E. (Oxford University Press (OUP), 2009-12-01) Coralyne is an alkaloid drug that binds homo-adenine DNA (and RNA) oligonucleotides more tightly than it does Watson-Crick DNA. Hud's laboratory has shown that poly(dA) in the presence of coralyne forms an anti-parallel duplex, however attempts to determine the structure by NMR spectroscopy and X-ray crystallography have been unsuccessful. Assuming adenine-adenine hydrogen bonding between the two poly(dA) strands, we constructed 40 hypothetical homo-(dA) anti-parallel duplexes and docked coralyne into the s...

Citation Formats

I. SHIN et al., “Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters,” NUCLEIC ACIDS RESEARCH, pp. 0–0, 2014, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/47433.