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An ortholog of farA of Aspergillus nidulans is implicated in the…
An ortholog of farA of Aspergillus nidulans is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast Yarrowia lipolytica
Introduction
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In fungi
fatty acid utilization in transcriptionally regulated by transcription factors belonging to 2 distinct families
af1p-Pip2p system, most extensively characterized in the budding yeast Saccharomyces cerevisiae, in which the Zn2Cys6 transcription factor, Oaf1p and Pip2p together with C2H2 zinc portein Adr1p, activate the transcription of target genes in response fatty acids
alternative system has been reported in filamentous fungus Aspergillus nidulans, in FarA and its paralog FarB, belongign to another Zn2Cys6 transcription factor family, control the transcription of genes involved in the metabolism of fatty acids
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Ctf1p, an ortholog of FarA in
Candida albicans, is involved in fatty acid response
orthologs in Fusarium species, CTF1 alpha and CTF1 beta of F. solani and Ctf1 of F. oxysporum are engaged in the transcriptional regulation of cutinase genes
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In this study, characterized of the ortholog of FarA in Y.lipolytica, Por1p (primary oleate regulator)
results indicate that Por1p- required for growth on fatty acids and for the transcriptional activation of genes involved in B-oxidation and peroxisome proliferation
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Discussion
POR1- a gene encoding the ortholog of farA that encodes the transcription factor involved in fatty acid metabolism and peroxisomal function in A. nidulans
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profound detects in the transcriptional induction of genes involved in fatty acid utilization by oleic acid were observed in the deletion mutant of POR!
results imply that mutants YALI0F13321g, YALI0D17988g, and/or YALI0D10681g that exhibited low similarity to Oaf1p and Pip2p in the zinc cluster domains showed no significant defects on oleic acid -less probable that orthologs of Oaf1p and Pip2p function in fatty acid response in Y. lipolytica
In F. solani - expression of cutinase genes cut! and cut2 is differentially regulated by CTF1 alpha and CTF beta
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A. nidulans, deletion of farA - caused defects in the induction by both short- and long-chain fatty acids
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unidentified functional homolog may involved in the transcriptional activation in response in fatty acids in Y. lipolytica
Transcription
Transcription of POT1, PAT1POX2 and PEX5- induced by both oleic acid and n-decane
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transcription of genes could in part be activated by fatty acids produced in the process of oxidation of n-alkanes by action of Por1p
considerable growth of the deletion mutant of POR1 on n-alkanes suggests that the expression og genes involved in beta-oxidation and peroxisome
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slight growth impairment of *por1 strain on n-decane could be due to the accumulation of harmful metabolites of n-decane caused by decrease in the expression of genes
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northen blot analysis
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significant increase in the beta-galactosidase activity was observed on n-decane in the reporter assay using PAT1 promoter
post -transcriptional regulation by carbon sources might be involves iin fatty acid response in Y. lipolytica
ALK1
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Yas2p and Yas3p are orthologs of Ino4p,Ino2p and Opi1p nvolved in transcriptional regulation of phospholipid biosynthetic genes in S.cerevisae
transcriptional repressor Opi1p localizes to the ER membrane by binding to phosphtidic acid (PA) and membrane protein scs2p in the absence of inositol and Ino2p and Ino4p activate the transcription of phospholipid synthetic gene
presence of inositol, PA is consumed for
phosphatidylinositol synthesis,and Opi1p is released from ER membrane and repressed the transcription of target genes by binsing to Ino2p in the nucleus
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deletion of POR1 could cause an indirect effect on the amount of the potential ligand of Yas3p in ER membrane
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fatty acids
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variety of yeasts and filamentous fungi appear to have acquired FarA systems for transcriptional regulation of genes involved in fatty acids utilization
Material and methods
Yeast strains and media
Y. lipolytica strain CXAU/A1 (ura3, ade1::ADE1) used as a
wild-type strain
for liquid media: appropriate carbon source was added to 0.67% YNB (Yeast Nitrogen Base without amino acids)
For solid media: 0.2% fatty acids were dispersed by adding 1% Triton X100. n-Alkanes were supplied by vapor to YNB solid media
-deletion mutant of POR1 was constructed.
-amplified fragments were digested with BamHI and XbaI or EcoRI, and cloned into the XbaI-EcoRI sites to obtain pULP1
The deletion cassette was obtained by digestion of pDP1 with XbaI and EcoRV and then introduced into the CXAU1
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Plasmid construction
plasmid, pPOR1, to express POR1 under its native promoter
in Y. lipolytica was constructed
amplified fragment was digested with XbaI and BglII,
and cloned into XbaI-BamHI sites of pSUT5
The plasmid, pPpro-LacZ, to analyze promoter activity of PAT1
using lacZ as a reporter gene
The amplified fragment was digested with
XbaI and StuI, and cloned into XbaI-StuI sites of pSUT5-LacZ
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Northern blot analysis
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Hybridization and detection were performed by using digoxigenin-labeled DNA probes with PCR DIG probe synthesis kit
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