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dc.contributor.advisorRangarajan, P N
dc.contributor.authorNallani, Vijay Kumar
dc.date.accessioned2016-05-04T10:34:07Z
dc.date.accessioned2018-07-30T14:13:44Z
dc.date.available2016-05-04T10:34:07Z
dc.date.available2018-07-30T14:13:44Z
dc.date.issued2016-05-04
dc.date.submitted2012-11
dc.identifier.urihttp://etd.iisc.ac.in/handle/2005/2535
dc.identifier.abstracthttp://etd.iisc.ac.in/static/etd/abstracts/3286/G25598-Abs.pdfen_US
dc.description.abstractPichia pastoris, a methylotrophic yeast is widely used for recombinant protein production. It has a well characterized methanol utilization (MUT) pathway, the enzymes of which are induced when cells are cultured in the presence of methanol. In this study, we have identified an unannotated zinc finger protein, which was subsequently named ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) and characterized its function. ROP expression is induced in P. pastoris cells cultured in biotin depleted glucose ammonium medium as well as a medium containing methanol as the sole source of carbon. In glucose-abundant, biotin depleted cultures, ROP induces the expression of a number of genes including that encoding PEPCK. Interestingly, a strain in which the gene encoding ROP is deleted (ΔROP) exhibits biotin-independent growth. Based on a number of studies, it was proposed that the ability of ΔROP to grow in the absence of biotin is due to the activation of a pyruvate carboxylase-independent pathway of oxaloacetate biosynthesis. It was also proposed that PEPCK, which normally functions as a gluconeogenic enzyme, may act as an anaplerotic enzyme involved in the synthesis of oxaloacetate. ROP was shown to be a key regulator of methanol metabolism when P. pastoris cells are cultured in YPM medium containing yeast extract, peptone and methanol but not YNBM medium containing yeast nitrogen base and methanol. In P. pastoris cells cultured in YPM, ROP functions as a transcriptional repressor of genes encoding key enzymes of the methanol metabolism such as the alcohol oxidase I. (AOXI). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion while overexpression of ROP results in repression of AOXI and retardation of growth of P. pastoris cultured in YPM medium. Subcellular localization studies indicate that ROP translocates from cytosol to nucleus in cells cultured in YPM but not YNBM. To understand the mechanism of action of ROP, we examined its DNA-binding specificity. The DNA-binding domain of ROP shares 57% amino acid identity with that of Mxr1p, a master regulator of genes of methanol metabolism. We demonstrate that the DNA-binding specificity of ROP is similar to that of Mxr1p and both proteins compete with each other for binding to AOXI promoter sequences. Thus, transcriptional interference due to competition between Mxr1p and ROP for binding to the same promoter sequences is likely to be the mechanism by which ROP represses AOXI expression in vivo. Mxr1p and ROP are examples of transcription factors which exhibit the same DNA-binding specificity but regulate gene expression in an antagonistic fashion.en_US
dc.language.isoen_USen_US
dc.relation.ispartofseriesG25598en_US
dc.subjectMethylotrophic Yeastsen_US
dc.subjectMethanol-Inducible Zinc Finger Proteinen_US
dc.subjectMethanol Metabolism - Gene Repressionen_US
dc.subjectMethylotrophic Yeasts - Methanol Metabolismen_US
dc.subjectMethylotrophic Yeasts - Methanol-Inducible Genes - Regulationen_US
dc.subjectPichia pastorisen_US
dc.subjectYeasts - Biotinen_US
dc.subjectRepressor of Phosphoenolpyruvate Carboxykinase (ROP)en_US
dc.subjectYeasts - Methanol Metabolism - Transcriptional Repressoren_US
dc.subjectBiotin Biosynthesisen_US
dc.subjectZinc Finger Proteinen_US
dc.subject.classificationMycologyen_US
dc.titleTranscriptional Regulation By A Biotin Starvation- And Methanol-Inducible Zinc Finger Protein In The Methylotrophic Yeast, Pichia Pastorisen_US
dc.typeThesisen_US
dc.degree.namePhDen_US
dc.degree.levelDoctoralen_US
dc.degree.disciplineFaculty of Scienceen_US


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