Dissertation/Thesis Abstract

Isolation and Characterization of pco-1, which Encodes a Regulatory Protein that Controls Purine Degradation in Neurospora crassa
by Liu, Ta-Wei D., Ph.D., The Ohio State University, 2003, 149; 10834915
Abstract (Summary)

A feature of the nitrogen regulatory circuit in filamentous fungi is that pathway-specific control genes mediate induction of enzymes by substrates in specific pathways. The gene encoding a new pathway-specific factor involved in purine degradation pathway, pco-1, was isolated from Neurospora using a PCR-mediated method. The open reading frame of the pco-1 gene is interrupted by two introns which were identified by comparing the genomic DNA sequence and the cDNA sequence obtained by RT-PCR. The predicted PCO1 protein contains 1101 amino acids and appears to possess a single Zn(II)2/Cys6 binuclear-type zinc cluster. A coiled-coil domain was predicted by computer-aided sequence analysis, suggesting that PCO1 might function as a dimer. A chemical crosslinking assay indicated PCO1 does dimerize in vitro. Deletion of the coiled-coil domain completely abolished the activity of PCO1. A loss of function pco-1 mutant was created by the rip procedure. Analysis of pco1- strains revealed that PCO1 acts as a positive regulator of the purine degradation pathway. Results of mobility shift assays indicate that PCO1 specifically binds to TCGG-N6-CCGA DNA sequences which exist in promoter regions of the structural genes it regulates. The C-terminus of PCO1 features two glutamine-rich regions which are commonly found in activation domains of transcription factors and a polyglycine stretch. The PCO1 protein with one of the glutamine-rich regions deleted was still partially functional. Removing both of them completely abolished the activity of PCO1. This domain shows higher homology to NIT4, the Neurospora pathway-specific factor in the nitrate assimilation pathway, than to UAY, its counterpart in Aspergillus nidulans, suggesting transcription factors in N. crassa may share similar activation regions. A feature of the nitrogen regulatory circuit in filamentous fungi is that pathway-specific control genes mediate induction of enzymes by substrates in specific pathways. The gene encoding a new pathway-specific factor involved in purine degradation pathway, pco-1, was isolated from Neurospora using a PCR-mediated method. The open reading frame of the pco-1 gene is interrupted by two introns which were identified by comparing the genomic DNA sequence and the cDNA sequence obtained by RT-PCR. The predicted PCO1 protein contains 1101 amino acids and appears to possess a single Zn(II)2/Cys6 binuclear-type zinc cluster. A coiled-coil domain was predicted by computer-aided sequence analysis, suggesting that PCO1 might function as a dimer. A chemical crosslinking assay indicated PCO1 does dimerize in vitro. Deletion of the coiled-coil domain completely abolished the activity of PCO1. A loss of function pco-1 mutant was created by the rip procedure. Analysis of pco1- strains revealed that PCO1 acts as a positive regulator of the purine degradation pathway. Results of mobility shift assays indicate that PCO1 specifically binds to TCGG-N6-CCGA DNA sequences which exist in promoter regions of the structural genes it regulates. The C-terminus of PCO1 features two glutamine-rich regions which are commonly found in activation domains of transcription factors and a polyglycine stretch. The PCO1 protein with one of the glutamine-rich regions deleted was still partially functional. Removing both of them completely abolished the activity of PCO1. This domain shows higher homology to NIT4, the Neurospora pathway-specific factor in the nitrate assimilation pathway, than to UAY, its counterpart in Aspergillus nidulans, suggesting transcription factors in N. crassa may share similar activation regions.

Indexing (document details)
Advisor: Marzluf, George
Commitee:
School: The Ohio State University
Department: Biochemistry
School Location: United States -- Ohio
Source: DAI-B 79/09(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Biochemistry
Keywords: Binuclear zinc cluster, Dna-binding protein, Fungi, Neurospora, Transcription factor
Publication Number: 10834915
ISBN: 9780355946956
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