Publications by authors named "Mark X Caddick"

22 Publications

  • Page 1 of 1

Histone mRNA is subject to 3' uridylation and re-adenylation in Aspergillus nidulans.

Mol Microbiol 2021 02 11;115(2):238-254. Epub 2020 Nov 11.

Institute of Systems, Molecular and Integrative Biology, The University of Liverpool, Liverpool, UK.

The role of post-transcriptional RNA modification is of growing interest. One example is the addition of non-templated uridine residues to the 3' end of transcripts. In mammalian systems, uridylation is integral to cell cycle control of histone mRNA levels. This regulatory mechanism is dependent on the nonsense-mediated decay (NMD) component, Upf1, which promotes histone mRNA uridylation and degradation in response to the arrest of DNA synthesis. We have identified a similar system in Aspergillus nidulans, where Upf1 is required for the regulation of histone mRNA levels. However, other NMD components are also implicated, distinguishing it from the mammalian system. As in human cells, 3' uridylation of histone mRNA is induced upon replication arrest. Disruption of this 3' tagging has a significant but limited effect on histone transcript regulation, consistent with multiple mechanisms acting to regulate mRNA levels. Interestingly, 3' end degraded transcripts are also subject to re-adenylation. Both mRNA pyrimidine tagging and re-adenylation are dependent on the same terminal-nucleotidyltransferases, CutA, and CutB, and we show this is consistent with the in vitro activities of both enzymes. Based on these data we argue that mRNA 3' tagging has diverse and distinct roles associated with transcript degradation, functionality and regulation.
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http://dx.doi.org/10.1111/mmi.14613DOI Listing
February 2021

Growing a circular economy with fungal biotechnology: a white paper.

Fungal Biol Biotechnol 2020 2;7. Epub 2020 Apr 2.

22Department of Biology, Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions. Fungal biotechnology has, thus, the potential to make a significant contribution to climate change mitigation and meeting the United Nation's sustainable development goals through the rational improvement of new and established fungal cell factories. The White Paper presented here is the result of the 2nd Think Tank meeting held by the EUROFUNG consortium in Berlin in October 2019. This paper highlights discussions on current opportunities and research challenges in fungal biotechnology and aims to inform scientists, educators, the general public, industrial stakeholders and policymakers about the current fungal biotech revolution.
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http://dx.doi.org/10.1186/s40694-020-00095-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140391PMC
April 2020

High-Quality Draft Genome Sequence and Annotation of the Basidiomycete Yeast CBS10092, a Producer of Mannosylerythritol Lipids.

Microbiol Resour Announc 2019 Oct 17;8(42). Epub 2019 Oct 17.

Institute of Integrative Biology, The University of Liverpool, Liverpool, United Kingdom

The basidiomycete (formally ) strain CBS10092 was originally isolated from an herbaceous plant in Russia. It is a known producer of mannosylerythritol lipids (MELs), the main component being MEL-C. Here, we present the 19.9-Mb draft genome sequence, which comprises 6,602 genes, including those encoding the MEL biosynthetic pathway.
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http://dx.doi.org/10.1128/MRA.00479-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797523PMC
October 2019

Similarity regression predicts evolution of transcription factor sequence specificity.

Nat Genet 2019 06 27;51(6):981-989. Epub 2019 May 27.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

Transcription factor (TF) binding specificities (motifs) are essential for the analysis of gene regulation. Accurate prediction of TF motifs is critical, because it is infeasible to assay all TFs in all sequenced eukaryotic genomes. There is ongoing controversy regarding the degree of motif diversification among related species that is, in part, because of uncertainty in motif prediction methods. Here we describe similarity regression, a significantly improved method for predicting motifs, which we use to update and expand the Cis-BP database. Similarity regression inherently quantifies TF motif evolution, and shows that previous claims of near-complete conservation of motifs between human and Drosophila are inflated, with nearly half of the motifs in each species absent from the other, largely due to extensive divergence in C2H2 zinc finger proteins. We conclude that diversification in DNA-binding motifs is pervasive, and present a new tool and updated resource to study TF diversity and gene regulation across eukaryotes.
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http://dx.doi.org/10.1038/s41588-019-0411-1DOI Listing
June 2019

The role of the GATA transcription factor AreB in regulation of nitrogen and carbon metabolism in Aspergillus nidulans.

FEMS Microbiol Lett 2019 03;366(6)

Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, ul. Pawińskiego 5A, 02-106 Warsaw, Poland.

In Aspergillus nidulans, nitrogen and carbon metabolism are under the control of wide-domain regulatory systems, including nitrogen metabolite repression, carbon catabolite repression and the nutrient starvation response. Transcriptomic analysis of the wild type strain grown under different combinations of carbon and nitrogen regimes was performed, to identify differentially regulated genes. Carbon metabolism predominates as the most important regulatory signal but for many genes, both carbon and nitrogen metabolisms coordinate regulation. To identify mechanisms coordinating nitrogen and carbon metabolism, we tested the role of AreB, previously identified as a regulator of genes involved in nitrogen metabolism. Deletion of areB has significant phenotypic effects on the utilization of specific carbon sources, confirming its role in the regulation of carbon metabolism. AreB was shown to regulate the expression of areA, tamA, creA, xprG and cpcA regulatory genes suggesting areB has a range of indirect, regulatory effects. Different isoforms of AreB are produced as a result of differential splicing and use of two promoters which are differentially regulated by carbon and nitrogen conditions. These isoforms are likely to be functionally distinct and thus contributing to the modulation of AreB activity.
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http://dx.doi.org/10.1093/femsle/fnz066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494665PMC
March 2019

Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper.

Fungal Biol Biotechnol 2016 31;3. Epub 2016 Aug 31.

Ceratium Limited, The Haven, West Kirby, CH48 8AP UK.

The EUROFUNG network is a virtual centre of multidisciplinary expertise in the field of fungal biotechnology. The first academic-industry Think Tank was hosted by EUROFUNG to summarise the state of the art and future challenges in fungal biology and biotechnology in the coming decade. Currently, fungal cell factories are important for bulk manufacturing of organic acids, proteins, enzymes, secondary metabolites and active pharmaceutical ingredients in white and red biotechnology. In contrast, fungal pathogens of humans kill more people than malaria or tuberculosis. Fungi are significantly impacting on global food security, damaging global crop production, causing disease in domesticated animals, and spoiling an estimated 10 % of harvested crops. A number of challenges now need to be addressed to improve our strategies to control fungal pathogenicity and to optimise the use of fungi as sources for novel compounds and as cell factories for large scale manufacture of bio-based products. This white paper reports on the discussions of the Think Tank meeting and the suggestions made for moving fungal bio(techno)logy forward.
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http://dx.doi.org/10.1186/s40694-016-0024-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611618PMC
August 2016

Transcriptome analysis of the filamentous fungus Aspergillus nidulans directed to the global identification of promoters.

BMC Genomics 2013 Dec 3;14:847. Epub 2013 Dec 3.

Institute of Integrative Biology, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, UK.

Background: The filamentous fungus Aspergillus nidulans has been a tractable model organism for cell biology and genetics for over 60 years. It is among a large number of Aspergilli whose genomes have been sequenced since 2005, including medically and industrially important species. In order to advance our knowledge of its biology and increase its utility as a genetic model by improving gene annotation we sequenced the transcriptome of A. nidulans with a focus on 5' end analysis.

Results: Strand-specific whole transcriptome sequencing showed that 80-95% of annotated genes appear to be expressed across the conditions tested. We estimate that the total gene number should be increased by approximately 1000, to 11,800. With respect to splicing 8.3% of genes had multiple alternative transcripts, but alternative splicing by exon-skipping was very rare. 75% of annotated genes showed some level of antisense transcription and for one gene, meaB, we demonstrated the antisense transcript has a regulatory role. Specific sequencing of the 5' ends of transcripts was used for genome wide mapping of transcription start sites, allowing us to interrogate over 7000 promoters and 5' untranslated regions.

Conclusions: Our data has revealed the complexity of the A. nidulans transcriptome and contributed to improved genome annotation. The data can be viewed on the AspGD genome browser.
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http://dx.doi.org/10.1186/1471-2164-14-847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046813PMC
December 2013

RrmA regulates the stability of specific transcripts in response to both nitrogen source and oxidative stress.

Mol Microbiol 2013 Sep 29;89(5):975-88. Epub 2013 Jul 29.

Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, ul. Pawińskiego 5A, 02-106, Warsaw, Poland.

Differential regulation of transcript stability is an effective means by which an organism can modulate gene expression. A well-characterized example is glutamine signalled degradation of specific transcripts in Aspergillus nidulans. In the case of areA, which encodes a wide-domain transcription factor mediating nitrogen metabolite repression, the signal is mediated through a highly conserved region of the 3' UTR. Utilizing this RNA sequence we isolated RrmA, an RNA recognition motif protein. Disruption of the respective gene led to loss of both glutamine signalled transcript degradation as well as nitrate signalled stabilization of niaD mRNA. However, nitrogen starvation was shown to act independently of RrmA in stabilizing certain transcripts. RrmA was also implicated in the regulation of arginine catabolism gene expression and the oxidative stress responses at the level of mRNA stability. ΔrrmA mutants are hypersensitive to oxidative stress. This phenotype correlates with destabilization of eifE and dhsA mRNA. eifE encodes eIF5A, a translation factor within which a conserved lysine is post-translationally modified to hypusine, a process requiring DhsA. Intriguingly, for specific transcripts RrmA mediates both stabilization and destabilization and the specificity of the signals transduced is transcript dependent, suggesting it acts in consort with other factors which differ between transcripts.
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http://dx.doi.org/10.1111/mmi.12324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282371PMC
September 2013

Cytoplasmic mRNA 3' tagging in eukaryotes: does it spell the end?

Biochem Soc Trans 2012 Aug;40(4):810-4

Institute of Integrative Biology, The University of Liverpool, Liverpool, UK.

Although functional RNA is generally protected against degradation, defects or irregularity during RNA biogenesis lead to rapid degradation. Cellular surveillance mechanisms therefore need to distinguish aberrant, erroneous, damaged or aging transcripts from normal RNAs in order to maintain fidelity and control of gene expression. The detection of defects seems to be primarily based on functionality or aberrant rates of a given step in RNA biogenesis, allowing efficient detection of many different errors without recognition of their specific nature. We propose that the addition of non-templated nucleotides to the 3' end of mRNAs and small non-coding RNAs, 3' tagging, is the primary means by which malfunctioning RNAs are labelled, promoting their functional repression and degradation. However, the addition of non-templated nucleotides to transcripts can have diverse effects which vary with location, length, substrate and sequence.
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http://dx.doi.org/10.1042/BST20120068DOI Listing
August 2012

mRNA 3' tagging is induced by nonsense-mediated decay and promotes ribosome dissociation.

Mol Cell Biol 2012 Jul 30;32(13):2585-95. Epub 2012 Apr 30.

The University of Liverpool, Institute of Integrative Biology, Liverpool, United Kingdom.

For a range of eukaryote transcripts, the initiation of degradation is coincident with the addition of a short pyrimidine tag at the 3' end. Previously, cytoplasmic mRNA tagging has been observed for human and fungal transcripts. We now report that Arabidopsis thaliana mRNA is subject to 3' tagging with U and C nucleotides, as in Aspergillus nidulans. Mutations that disrupt tagging, including A. nidulans cutA and a newly characterized gene, cutB, retard transcript degradation. Importantly, nonsense-mediated decay (NMD), a major checkpoint for transcript fidelity, elicits 3' tagging of transcripts containing a premature termination codon (PTC). Although PTC-induced transcript degradation does not require 3' tagging, subsequent dissociation of mRNA from ribosomes is retarded in tagging mutants. Additionally, tagging of wild-type and NMD-inducing transcripts is greatly reduced in strains lacking Upf1, a conserved NMD factor also required for human histone mRNA tagging. We argue that PTC-induced translational termination differs fundamentally from normal termination in polyadenylated transcripts, as it leads to transcript degradation and prevents rather than facilitates further translation. Furthermore, transcript deadenylation and the consequent dissociation of poly(A) binding protein will result in PTC-like termination events which recruit Upf1, resulting in mRNA 3' tagging, ribosome clearance, and transcript degradation.
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http://dx.doi.org/10.1128/MCB.00316-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434495PMC
July 2012

The GATA factors AREA and AREB together with the co-repressor NMRA, negatively regulate arginine catabolism in Aspergillus nidulans in response to nitrogen and carbon source.

Fungal Genet Biol 2012 Mar 28;49(3):189-98. Epub 2012 Jan 28.

Institute of Genetics and Biotechnology, University of Warsaw, ul. Pawińskiego 5A, 02-106 Warsaw, Poland.

The filamentous fungus Aspergillus nidulans can utilize arginine both as a nitrogen and carbon source. Analysis of areA and areB single and double mutants has shown that the two GATA transcription factors AREA and AREB negatively regulate the expression of arginine catabolism genes agaA and otaA under nitrogen repressing conditions. AREA is necessary for the ammonium repression of agaA and otaA under carbon repressing conditions, while AREB is involved under carbon-limiting conditions. The ability of both AREA and AREB to sense the status of carbon metabolism is most probably dependent on NMRA, and not on the transcription factor CREA, which mediates general carbon catabolite repression in A. nidulans. NMRA is a co-repressor which has previously been shown to bind the C-terminus of AREA and inhibits its activity under conditions of nitrogen sufficiency, in response to high intracellular glutamine levels. We therefore propose a novel function for NMRA, the modulation of AREA and AREB activity in response to the carbon status of the cell.
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http://dx.doi.org/10.1016/j.fgb.2012.01.004DOI Listing
March 2012

The bZIP transcription factor MeaB mediates nitrogen metabolite repression at specific loci.

Eukaryot Cell 2010 Oct 20;9(10):1588-601. Epub 2010 Aug 20.

Institut für Botanik der Westfälischen Wilhelms-Universität Münster, Germany.

In Fusarium fujikuroi, bikaverin (BIK) biosynthesis is subject to repression by nitrogen. Unlike most genes subject to nitrogen metabolite repression, it has been shown that transcription of bik biosynthetic genes is not AreA dependent. Searching for additional transcription factors that may be involved in nitrogen regulation, we cloned and characterized the orthologue of Aspergillus nidulans meaB, which encodes a bZIP transcription factor. Two transcripts are derived from F. fujikuroi meaB: the large transcript (meaB(L)) predominates under nitrogen-sufficient conditions and the smaller transcript (meaB(S)) under nitrogen limitation, in an AreA-dependent manner. MeaB is specifically translocated to the nucleus under nitrogen-sufficient conditions in both F. fujikuroi and A. nidulans. Deletion of meaB resulted in partial upregulation of several nitrogen-regulated genes, but only in the ΔmeaB ΔareA double mutant were the bikaverin genes significantly upregulated in the presence of glutamine. These data demonstrate that MeaB and AreA coordinately mediate nitrogen metabolite repression and, importantly, that independently of AreA, MeaB can mediate nitrogen metabolite repression at specific loci in F. fujikuroi.
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http://dx.doi.org/10.1128/EC.00146-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2950422PMC
October 2010

Distinct roles for Caf1, Ccr4, Edc3 and CutA in the co-ordination of transcript deadenylation, decapping and P-body formation in Aspergillus nidulans.

Mol Microbiol 2010 Apr 10;76(2):503-16. Epub 2010 Mar 10.

School of Biological Sciences, The University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.

Transcript degradation is a key step in gene regulation. In eukaryotes, mRNA decay is generally initiated by removal of the poly(A) tail mediated by the Ccr4-Caf1-Not complex. Deadenylated transcripts are then rapidly degraded, primarily via the decapping-dependent pathway. Components of this pathway can be localized into highly dynamic cytoplasmic foci, the mRNA processing (P)-bodies. We have undertaken confocal fluorescence microscopy to monitor P-bodies in Aspergillus nidulans. As in other organisms a dynamic shift in P-body formation occurs in response to diverse physiological signals. Significantly, both this cellular response and the signalled degradation of specific transcripts are dependent on the nuclease activity of Caf1 but not Ccr4. P-body formation is disrupted in A. nidulans strains deleted for Edc3, an enhancer of decapping, or CutA, which encodes a nucleotidyltransferase that triggers mRNA decapping by the addition of a CUCU tag to the poly(A) tail. As with DeltacutA, Deltaedc3 led to reduced rates of transcript degradation. These data link P-bodies to both the optimization and regulation of transcript degradation.
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http://dx.doi.org/10.1111/j.1365-2958.2010.07118.xDOI Listing
April 2010

CUCU modification of mRNA promotes decapping and transcript degradation in Aspergillus nidulans.

Mol Cell Biol 2010 Jan 9;30(2):460-9. Epub 2009 Nov 9.

University of Liverpool, School of Biological Sciences, Crown Street, Liverpool L69 7ZB, United Kingdom.

In eukaryotes, mRNA decay is generally initiated by the shortening of the poly(A) tail mediated by the major deadenylase complex Ccr4-Caf1-Not. The deadenylated transcript is then rapidly degraded, primarily via the decapping-dependent pathway. Here we report that in Aspergillus nidulans both the Caf1 and Ccr4 orthologues are functionally distinct deadenylases in vivo: Caf1 is required for the regulated degradation of specific transcripts, and Ccr4 is responsible for basal degradation. Intriguingly disruption of the Ccr4-Caf1-Not complex leads to deadenylation-independent decapping. Additionally, decapping is correlated with a novel transcript modification, addition of a CUCU sequence. A member of the nucleotidyltransferase superfamily, CutA, is required for this modification, and its disruption leads to a reduced rate of decapping and subsequent transcript degradation. We propose that 3' modification of adenylated mRNA, which is likely to represent a common eukaryotic process, primes the transcript for decapping and efficient degradation.
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http://dx.doi.org/10.1128/MCB.00997-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798463PMC
January 2010

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88.

Nat Biotechnol 2007 Feb 28;25(2):221-31. Epub 2007 Jan 28.

DSM Food Specialties, PO Box 1, 2600 MA Delft, The Netherlands.

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.
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http://dx.doi.org/10.1038/nbt1282DOI Listing
February 2007

Opposing signals differentially regulate transcript stability in Aspergillus nidulans.

Mol Microbiol 2006 Oct;62(2):509-19

The University of Liverpool, School of Biological Sciences, Biosciences Building, Crown Street, Liverpool, L69 7ZB, UK.

A good model for gene regulation, requiring the organism to monitor a complex and changing environment and respond in a precise and rapid way, is nitrogen metabolism in Aspergillus nidulans. This involves co-ordinated expression of hundreds of genes, many dependent on the transcription factor AreA, which monitors the nitrogen state of the cell. AreA activity is in part modulated by differential degradation of its transcript in response to intracellular glutamine. Here we report that glutamine triggers synchronized degradation of a large subset of transcripts involved in nitrogen metabolism. Among these are all four genes involved in the assimilation of nitrate. Significantly, we show that two of these transcripts, niaD and niiA, are stabilized by intracellular nitrate, directly reinforcing transcriptional regulation. Glutamine-signalled degradation and the nitrate-dependent stabilization of the niaD transcript are effected at the level of deadenylation and are dependent on its 3' UTR. When glutamine and nitrate are both present, nitrate stabilization is predominant, ensuring that nitrate and the toxic intermediate nitrite are removed from the cell. Regulated transcript stability is therefore an integral part of the adaptive response. This represents the first example of distinct physiological signals competing to differentially regulate transcripts at the level of deadenylation.
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http://dx.doi.org/10.1111/j.1365-2958.2006.05383.xDOI Listing
October 2006

Nonsense-mediated mRNA decay mutation in Aspergillus nidulans.

Eukaryot Cell 2006 Nov 8;5(11):1838-46. Epub 2006 Sep 8.

Department of Molecular Microbiology and Infection, Flowers Building, Imperial College London, Armstrong Road, London SW7 2AZ, United Kingdom.

An Aspergillus nidulans mutation, designated nmdA1, has been selected as a partial suppressor of a frameshift mutation and shown to truncate the homologue of the Saccharomyces cerevisiae nonsense-mediated mRNA decay (NMD) surveillance component Nmd2p/Upf2p. nmdA1 elevates steady-state levels of premature termination codon-containing transcripts, as demonstrated using mutations in genes encoding xanthine dehydrogenase (hxA), urate oxidase (uaZ), the transcription factor mediating regulation of gene expression by ambient pH (pacC), and a protease involved in pH signal transduction (palB). nmdA1 can also stabilize pre-mRNA (unspliced) and wild-type transcripts of certain genes. Certain premature termination codon-containing transcripts which escape NMD are relatively stable, a feature more in common with certain nonsense codon-containing mammalian transcripts than with those in S. cerevisiae. As in S. cerevisiae, 5' nonsense codons are more effective at triggering NMD than 3' nonsense codons. Unlike the mammalian situation but in common with S. cerevisiae and other lower eukaryotes, A. nidulans is apparently impervious to the position of premature termination codons with respect to the 3' exon-exon junction.
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http://dx.doi.org/10.1128/EC.00220-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1694799PMC
November 2006

Gene regulation in Aspergillus: From genetics to genomics.

Med Mycol 2006 Sep;44(Supplement_1):S13-S16

School of Biological Sciences, University of Liverpool, Liverpool, UK.

A fundamental aspect of any organism's success is the ability to monitor and respond effectively to its environment, a process which is largely achieved through the appropriate regulation of gene expression. There are few better examples than fungi, which inhabit diverse and often hostile environments, ranging from leaf litter to the human body. Regulation can occur at many levels, and as we investigate specific genes in detail, the paradigm is one of increasing complexity. We will briefly review the different levels at which regulation is known to occur in Aspergillus and the insights gained from the available genome sequences.
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http://dx.doi.org/10.1080/13693780600835781DOI Listing
September 2006

Genetic analysis of the TOR pathway in Aspergillus nidulans.

Eukaryot Cell 2005 Sep;4(9):1595-8

The University of Liverpool, School of Biological Sciences, The Biosciences Building, Crown Street, Liverpool L69 7ZB, United Kingdom.

We identified five genes encoding components of the TOR signaling pathway within Aspergillus nidulans. Unlike the situation in Saccharomyces cerevisiae, there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.
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http://dx.doi.org/10.1128/EC.4.9.1595-1598.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1214207PMC
September 2005

The alc-GR system: a modified alc gene switch designed for use in plant tissue culture.

Plant Physiol 2005 Jul;138(3):1259-67

Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom.

The ALCR/alcA (alc) two-component, ethanol-inducible gene expression system provides stringent control of transgene expression in genetically modified plants. ALCR is an ethanol-activated transcription factor that can drive expression from the ALCR-responsive promoter (alcA). However, the alc system has been shown to have constitutive expression when used in plant callus or cell suspension cultures, possibly resulting from endogenous inducer produced in response to lowered oxygen availability. To widen the use of the alc system in plant cell culture conditions, the receptor domain of the rat glucocorticoid receptor (GR) was translationally fused to the C terminus of ALCR to produce ALCR-GR, which forms the basis of a glucocorticoid-inducible system (alc-GR). The alc-GR switch system was tested in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells using a constitutively expressed ALCR-GR with four alternative alcA promoter-driven reporter genes: beta-glucuronidase, endoplasmic reticulum-targeted green fluorescent protein, haemagglutinin, and green fluorescent protein-tagged Arabidopsis (Arabidopsis thaliana) Arath;CDKA;1 cyclin-dependent kinase. Gene expression was shown to be stringently dependent on the synthetic glucocorticoid dexamethasone and, in cell suspensions, no longer required ethanol for induction. Thus, the alc-GR system allows tight control of alcA-driven genes in cell culture and complements the conventional ethanol switch used in whole plants.
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http://dx.doi.org/10.1104/pp.105.059659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1176399PMC
July 2005

Characterization of the ethanol-inducible alc gene expression system in tomato.

J Exp Bot 2005 Jun 25;56(416):1635-42. Epub 2005 Apr 25.

School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK.

The efficacy of the ethanol-inducible alc transgene expression system, derived from the filamentous fungus Aspergillus nidulans, has been demonstrated in transgenic tomato. Two direct comparisons have been made. First, this study has utilized two transgenic lines carrying distinct reporter genes (chloramphenicol acetyltransferase and beta-glucuronidase) to distinguish aspects of induction determined by the nature of the gene/gene product rather than that of the plant. Second, comparisons have been made to data generated in other species in order to identify any species-specific effects. The induction profiles for different genes in different species have shown remarkable similarity indicating the broad applicability of this gene switch. While there are minor differences observed between species, these probably arise from diversity in their metabolism. A series of potential alternative inducers have also been tested, revealing that ethanol (through metabolism to acetaldehyde) is better than other alcohols and ketones included in this study. Expression driven by alc was demonstrated to vary spatially, the upper younger leaves having higher activity than the lower older leaves; this will be important for some applications, and for experimental design. The highest levels of activity from ethanol-inducible transgene expression were determined to be the equivalent of those from the constitutive Cauliflower Mosaic Virus 35S promoter. This suggests that the alc system could be an important tool for plant functional genomics.
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http://dx.doi.org/10.1093/jxb/eri159DOI Listing
June 2005

Re: Watts et al. Proteins 2002;48:161-168.

Proteins 2003 Aug;52(2):125-8

Department of Infectious Diseases and Microbiology, Faculty of Medicine, Imperial College of Science, Technology, and Medicine, London, United Kingdom.

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http://dx.doi.org/10.1002/prot.10447DOI Listing
August 2003