In Neurospora crassa, crosses between normal sequence strains and strains bearing some translocations can yield progeny bearing a duplication (Dp) of the translocated chromosome segment. Here, 30 breakpoint junction sequences of 12 Dp-generating translocations were determined. The breakpoints disrupted 13 genes (including predicted genes), and created 10 novel open reading frames. Insertion of sequences from LG III into LG I as translocation T(UK8-18) disrupts the eat-3 gene, which is the ortholog of the Podospora anserine gene ami1. Since ami1-homozygous Podospora crosses were reported to increase the frequency of repeat-induced point mutation (RIP), we performed crosses homozygous for a deficiency in eat-3 to test for a corresponding increase in RIP frequency. However, our results suggested that, unlike in Podospora, the eat-3 gene might be essential for ascus development in Neurospora. Duplication-heterozygous crosses are generally barren in Neurospora; however, by using molecular probes developed in this study, we could identify Dp segregants from two different translocation-heterozygous crosses, and using these we found that the barren phenotype of at least some duplication-heterozygous crosses was incompletely penetrant.
Repeat-induced point mutation (RIP) is a sexual stage-specific mutational process of Neurospora crassa and other fungi that alters duplicated DNA sequences. Previous studies from our laboratory showed that chromosome segment duplications (Dps) longer than (approx.) 300 kbp can dominantly suppress RIP, presumably by titration of the RIP machinery, and that although Dps less than 200 kbp did not individually suppress RIP, they could do so in homozygous and multiply heterozygous crosses, provided the sum of the duplicated DNA exceeds (approx.) 300 kbp. Here we demonstrate suppression of RIP in a subset of progeny carrying the normally sub-threshold 154 kbp Dp(R2394) from a cross of T(R2394) to the wild isolated Carrefour Mme. Gras strain (CMG). Thus, the CMG strain contains a factor that together with Dp(R2394) produces a synthetic RIP suppressor phenotype. It is possible that the factor is a cryptic Dp that together with Dp(R2394) can exceed the size threshold for titration of the RIP machinery and thereby causes RIP suppression.
Isolated cell wall preparations of N. crassa bind significant levels of Ca, Mg and other divalent cations. Enzymatic treatment of the cell wall with beta-(1,3)-glucanase, but not with chitinase, resulted in solubilization of only the calcium-binding protein fraction. A calcium-binding protein (CaBP) was purified by metal-chelate affinity chromatography and reversed phase HPLC. CaBP has an Mr of around 6 kDa on SDS-PAGE and mass spectrometry showed that it has a molecular mass of 5744 Da. One mole of CaBP binds 2 moles of calcium and is partially inhibited (15-50%) by other divalent cations (Mg, Ni and Cu). Quenching of tryptophan fluorescence was observed upon copper binding but not calcium binding. This is a first report of a calcium binding protein from the cell wall of fungi.
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