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  • 1
    ISSN: 1617-4623
    Keywords: Key words Neurospora ; Mating type ; Mutations ; Heterokaryon incompatibility
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The 293-amino acid mt A-1 ORF of the A mating-type idiomorph of Neurospora crassa is multifunctional. It confers A mating identity and is responsible for heterokaryon incompatibility. The goal of this study was to dissect the functional regions of mt A-1. New mutants of mt A-1 selected for loss of the incompatibility function were obtained. One new mutant, A m99 , was partially fertile as a maternal parent. This is the first time that fertility and incompatibility functions have been separated for the A idiomorph. In this mutant, the mt A-1 ORF is truncated after the first 85 amino acids, indicating that this N-terminal region is minimally sufficient for female fertility. A series of deletion constructs and frameshift alleles of mt A-1 was obtained and tested for male-mating activity and vegetative incompatibility in transformation experiments. These experiments showed that a region from position 1 to 111 is sufficient to confer incompatibility, while amino acids from position 1 to 227 are required for mating activity. A transcriptional analysis of mt A-1 showed that the mRNA is expressed both before and after fertilization. This, together with the phenotype of the A m99 mutant, suggests a post-fertilization function for mt A-1.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1617-4623
    Keywords: Key wordsN. crassa ; Osmotic-remedial temperature-sensitive mutation ; Ribonucleotide reductase ; Repeat-induced point mutation (RIP) ; Hydroxyurea
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An osmotic-remedial, temperature-sensitive conditional mutant (un-24) was generated by Repeat Induced Point mutation (RIP) from a cross between a wild-type N. crassa strain and a strain carrying a ≈250-kb duplication of the left arm of linkage group II (LGII). The mutation was mapped to the duplicated segment, within 2.6 map units of the heterokaryon incompatibility locus het-6. DNA transformation identified a 3.75-kb fragment that complemented the temperature-sensitive phenotype. A large ORF within this fragment was found to have a high degree of sequence identity to the large subunit of ribonucleotide reductase (RNR) from diverse organisms. Conserved amino acids at the active site and the allosteric activity sites are also evident. An unusual feature of the Neurospora sequence is a large insertion near the C-terminus relative to otherwise homologous sequences from other organisms. Three transition mutations, indicative of RIP, were identified in the N-terminal region of the temperature-sensitive mutant allele. One of these mutations results in a non-conservative amino acid substitution within the four-helix bundle that is important in the allosteric control of ribonucleotide reductase activity. This substitution appears to disrupt proper folding of the allosteric activity site during synthesis of the protein.
    Type of Medium: Electronic Resource
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