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Folylpolyglutamate synthesis in Neurospora crassa: transformation of polyglutamate-deficient mutants
I J Atkinson1, F E Nargang, E A Cossins
1Department of Biological Sciences, University of Alberta, Edmonton, Canada.
Abstract:
The methionine auxotrophy of Neurospora crassa met-6 and mac mutants is related to an inability to synthesize long-chained folylpolyglutamates. Both of these lesions affect folylpolyglutamate synthetase activity, but it is not clear whether these mutations occur in different genes or in functional domains of the same gene. To address this question, copies of the met-6+ gene have been introduced into both mutants using plasmid and cosmid vectors. Transformation to prototrophy was achieved in both mutants. The ability of these mutant and transformant strains to synthesize folylpolyglutamates was assessed by HPLC analysis of folate cleavage products. Mycelial extracts of the wild type revealed a folate pool dominated by folylhexaglutamates. These folates were also detected in the transformants but were lacking in both mutants. In the latter strains, the conjugated folates were mainly di- and triglutamates. When incubated for 24 hr with [14C]p-aminobenzoate, transformant and wild type cultures synthesized long chain folates, ca60-80% of these being hexaglutamyl derivatives. In contrast, the labelled folates of mac and met-6 were mainly mono- and diglutamyl derivatives, respectively. Polyglutamate synthesis was also studied in vitro by partial purification and characterization of mycelial folylpolyglutamate synthetase protein. Mycelial extracts of the wild type and transformant cultures utilized 5,10-methylenetetrahydrofolate monoglutamate and its diglutamate as substrates in this synthetase reaction. In contrast, extracts of met-6 and mac mycelia utilized only one of these folate substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Neurospora crassa mutants met-6 and mac cannot synthesize long-chained folylpolyglutamates due to impaired folylpolyglutamate synthetase activity. Introducing the met-6+ gene restored this ability, indicating mutations may affect the same gene.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Methionine auxotrophy in Neurospora crassa mutants met-6 and mac is linked to defective long-chained folylpolyglutamate synthesis.
- Folylpolyglutamate synthetase activity is impaired in these mutants, but it's unclear if mutations are in different genes or functional domains of the same gene.
Purpose of the Study:
- To investigate whether the met-6 and mac mutations in Neurospora crassa affect the same gene or different genes.
- To elucidate the role of the met-6+ gene in folylpolyglutamate synthesis.
Main Methods:
- Genetic complementation by introducing met-6+ gene into met-6 and mac mutants using plasmid and cosmid vectors.
- High-performance liquid chromatography (HPLC) analysis of folate cleavage products to assess folylpolyglutamate synthesis in wild-type, mutant, and transformant strains.
- In vitro characterization of mycelial folylpolyglutamate synthetase activity using radiolabeled folate substrates.
Main Results:
- Transformation with met-6+ restored prototrophy and folylpolyglutamate synthesis in both met-6 and mac mutants.
- Wild-type and transformant strains synthesized long-chained folylhexaglutamates, while mutants predominantly produced di- and triglutamates.
- Mutant extracts showed altered substrate utilization for folylpolyglutamate synthetase compared to wild-type and transformant extracts.
Conclusions:
- The met-6+ gene is essential for the synthesis of long-chained folylpolyglutamates in Neurospora crassa.
- The results suggest that the met-6 and mac mutations may occur within the same gene or functionally related domains affecting folylpolyglutamate synthetase.
- Restoration of folylpolyglutamate synthesis via gene transformation confirms the role of met-6+ in folate metabolism.