creators_name: Kapoor, Divya
creators_name: Chandrayan, Sanjeev Kumar
creators_name: Ahmed, Shubbir
creators_name: Guptasarma, Purnananda
type: article
datestamp: 2011-11-30 07:06:14
lastmod: 2011-11-30 07:06:40
metadata_visibility: show
title: Using DNA sequencing electrophoresis compression artifacts as reporters of stable mRNA structures affecting gene expression.
ispublished: pub
subjects: QR
full_text_status: restricted
note: Copyright of this article belongs to Wiley.
abstract: The formation of secondary structure in oligonucleotide DNA is known to lead to "compression" artifacts in electropherograms produced through DNA sequencing. Separately, the formation of secondary structure in mRNA is known to suppress translation; in particular, when such structures form in a region covered by the ribosome either during, or shortly after, initiation of translation. Here, we demonstrate how a DNA sequencing compression artifact provides important clues to the location(s) of translation-suppressing secondary structural elements in mRNA. Our study involves an engineered version of a gene sourced from Rhodothermus marinus encoding an enzyme called Cel12A. We introduced this gene into Escherichia coli with the intention of overexpressing it, but found that it expressed extremely poorly. Intriguingly, the gene displayed a remarkable compression artifact during DNA sequencing electrophoresis. Selected "designer" silent mutations destroyed the artifact. They also simultaneously greatly enhanced the expression of the cel12A gene, presumably by destroying stable mRNA structures that otherwise suppress translation. We propose that this method of finding problem mRNA sequences is superior to software-based analyses, especially if combined with low-temperature CE.
date: 2007-11
date_type: published
publication: Electrophoresis
volume: 28
number: 21
publisher: Wiley
pagerange: 3862-7
refereed: TRUE
issn: 0173-0835
official_url: http://onlinelibrary.wiley.com/doi/10.1002/elps.200700359/pdf
related_url_url: http://onlinelibrary.wiley.com/doi/10.1002/elps.200700359/pdf
related_url_type: pub
citation:   Kapoor, Divya and Chandrayan, Sanjeev Kumar and Ahmed, Shubbir and Guptasarma, Purnananda  (2007) Using DNA sequencing electrophoresis compression artifacts as reporters of stable mRNA structures affecting gene expression.  Electrophoresis, 28 (21).  pp. 3862-7.  ISSN 0173-0835     
document_url: http://crdd.osdd.net/open/622/1/guptasarma07.1.pdf