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The spraygene was cloned, and wildtype and mutant alleles were sequenced. Spray+has a 3452-bp open reading frame plus seven introns. The spraymutant had a T → G transversion close to the carboxyl end, creating a stop codon (TGA). The sequence shows no match to genes of known function, but the carboxyl end shows seven transmembrane domains and matches putative membrane proteins of yeast. The most abundant transcript detected was 4.4 kb in size. Repeat-induced point mutagenesis produced the mutant sprayphenotype. Electrophysiological analysis showed that ion fluxes in the sprayplasma membrane are normal; furthermore, whereas the spraymutant was known to have no organelle-based calcium fluorescence, the cytosol shows a tip-high calcium gradient. The spraymutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is located in an organellar membrane, regulating the distribution of Ca2+via calcineurin.

The Neurospora crassamutant frosthas a hyperbranching phenotype that can be corrected by adding Ca2+, suggesting that characterization of this gene might clarify the mechanism of Ca2+-dependent tip growth. The wild-type allele was cloned by sib selection using protoplasts from arthroconidia. RFLP analysis revealed that the cloned DNA fragment mapped to the frlocus. The nucleotide sequence of genomic and cDNA was determined. The deduced amino acid sequence showed homology to the SaccharomycescerevisiaeCDC1 protein, implicated in manganese homeostasis. The frmutant was sensitive to Mn2+, and a revertant allele whose product differs by one amino acid was tolerant to Mn2+. Mn2+depletion induced the wild-type strain to hyperbranch, resulting in a morphology similar to that of fr. The frmutant was also sensitive to calcineurin inhibitors. These results suggest that fris involved in Mn2+homeostasis and point to a role for Mn2+in Neurosporabranching.

The kalilovariants of Neurospora contain a cytoplasmic genetic factor that causes senescence. This factor is a 9.0 kb transposable element (kaIDNA) that lacks nucleotide sequence homology with mtDNA and is inserted into the mitochondrial chromosome, often at sites located within the open reading frame in the intron-DNA of the mitochondrial 25S-rRNA gene. Genomes containing the “foreign” DNA insert accumulate during growth, and death occurs as the cells become deficient in functional large and small subunits of mitochondrial ribosomes. The kaIDNA transposon may be an “activator” element that causes breaks in mtDNA. Nonsenescing [+] strains of Neurospora do not contain kaIDNA.