Journal article
Analysis of Promoter Function in Aspergillus fumigatus
Eukaryotic cell, Vol.11(9), pp.1167-1177
09/2012
DOI: 10.1128/EC.00174-12
PMCID: PMC3445982
PMID: 22843562
Abstract
The filamentous fungus
Aspergillus fumigatus
is an important opportunistic pathogen that can cause high mortality levels in susceptible patient populations. The increasing dependence on antifungal drugs to control
A. fumigatus
has led to the inevitable acquisition of drug-resistant forms of this pathogen. In other fungal pathogens, drug resistance is often associated with an increase in transcription of genes such as ATP-binding cassette (ABC) transporters that directly lead to tolerance to commonly employed antifungal drugs. In
A. fumigatus
, tolerance to azole drugs (the major class of antifungal) is often associated with changes in the sequence of the azole target enzyme as well as changes in the transcription level of this gene. The target gene for azole drugs in
A. fumigatus
is referred to as
cyp51A
. In order to dissect transcription of
cyp51A
transcription and other genes of interest, we constructed a set of firefly luciferase reporter genes designed for use in
A. fumigatus
. These reporter genes can either replicate autonomously or be targeted to the
pyrG
locus, generating an easily assayable uracil auxotrophy. We fused eight different
A. fumigatus
promoters to luciferase. Faithful behaviors of these reporter gene fusions compared to their chromosomal equivalents were evaluated by 5′ rapid amplification of cDNA ends (RACE) and quantitative reverse transcription-PCR (qRT-PCR) analysis. We used this reporter gene system to study stress-regulated transcription of a Hsp70-encoding gene, map an important promoter element in the
cyp51A
gene, and correct an annotation error in the actin gene. We anticipate that this luciferase reporter gene system will be broadly applicable in analyses of gene expression in
A. fumigatus
.
Details
- Title: Subtitle
- Analysis of Promoter Function in Aspergillus fumigatus
- Creators
- Sanjoy Paul - Departments of Molecular Physiology and BiophysicsJ. Stacey Klutts - Pathology, Carver College of Medicine, University of IowaW. Scott Moye-Rowley - Departments of Molecular Physiology and Biophysics
- Resource Type
- Journal article
- Publication Details
- Eukaryotic cell, Vol.11(9), pp.1167-1177
- DOI
- 10.1128/EC.00174-12
- PMID
- 22843562
- PMCID
- PMC3445982
- NLM abbreviation
- Eukaryot Cell
- ISSN
- 1535-9778
- eISSN
- 1535-9786
- Publisher
- American Society for Microbiology; 1752 N St., N.W., Washington, DC
- Language
- English
- Date published
- 09/2012
- Academic Unit
- Molecular Physiology and Biophysics; Pathology; Internal Medicine
- Record Identifier
- 9984025348502771
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