Tests of a cellular model for constant branch distribution in the filamentous fungus Neurospora crassa.

The growth of mycelial fungi is characterized by the highly polarized extension of hyphal tips and the formation of subapical branches, which themselves extend as new tips. In Neurospora crassa, tip growth and branching are crucial elements for this saprophyte in the colonization and utilization of organic substrates. Much research has focused on the mechanism of tip extension, but a cellular model that fully explains the known phenomenology of branching by N. crassa has not been proposed. We described and tested a model in which the formation of a lateral branch in N. crassa was determined by the accumulation of tip-growth vesicles caused by the excess of the rate of supply over the rate of deposition at the apex. If both rates are proportional to metabolic rate, then the model explains the known lack of dependence of branch interval on growth rate. We tested the model by manipulating the tip extension rate, first by shifting temperature in both the wild type and hyperbranching (colonial) mutants and also by observing the behavior of both tipless colonies and colonyless tips. We found that temperature shifts in either direction result in temporary changes in branching. We found that colonyless tips also pass through a temporary transition phase of branching. The tipless colonies produced a cluster of new tips near the point of damage. We also found that branching in colonial mutants is dependent on growth rate. The results of these tests are consistent with a model of branching in which branch initiation is controlled by the dynamics of tip growth while being independent of the actual rate of this growth.

Action of repeat-induced point mutation on both strands of a duplex and on tandem duplications of various sizes in Neurospora.

In Neurospora crassa, DNA sequence duplications are detected and altered efficiently during the sexual cycle by a process known as RIP (repeat-induced point mutation). Affected sequences are subjected to multiple GC-to-AT mutations. To explore the pattern in which base changes are laid down by RIP we examined two sets of strains. First, we examined the products of a presumptive spontaneous RIP event at the mtr locus. Results of sequencing suggested that a single RIP event produces two distinct patterns of change, descended from the two strands of an affected DNA duplex. Equivalent results were obtained using an exceptional tetrad from a cross with a known duplication flanking the zeta-eta (zeta-eta) locus. The mtr sequence data were also used to further examine the basis for the differential severity of C-to-T mutations on the coding and noncoding strands in genes. The known bias of RIP toward CpA/TpG sites in conjunction with the sequence bias of Neurospora accounts for the differential effect. Finally, we used a collection of tandem repeats (from 16 to 935 bp in length) within the mtr gene to examine the length requirement for RIP. No evidence of RIP was found with duplications shorter than 400 bp while all longer tandem duplications were frequently affected. A comparison of these results with vegetative reversion data for the same duplications is consistent with the idea that reversion of long tandem duplications and RIP share a common step.

Spontaneous mutation during the sexual cycle of Neurospora crassa.

The DNA sequences of 42 spontaneous mutations of the mtr gene in Neurospora crassa have been determined. The mutants were selected among sexual spores to represent mutations arising in the sexual cycle. Three sexual-cycle-specific mutational classes are described: hotspot mutants, spontaneous repeat-induced point mutation (RIPs) and mutations occurring during a mutagenic phase of the sexual cycle. Together, these three sexual-cycle-specific mutational classes account for 50% of the mutations in the sexual-cycle mutational spectrum. One third of all mutations occurred at one of two mutational hotspots that predominantly produced tandem duplications of varying lengths with short repeats at their end-points. Neither of the two hotspots are present in the vegetative spectrum, suggesting that sexual-cycle-specific mutational pathways are responsible for their presence in the spectrum. One mutant was observed that appeared to have been RIPed precociously. The usual prerequisite for RIP, a duplication of the affected region, was not present in the parent stocks and was not detected in this mutant. Finally, there is a phase early in the premeiotic sexual cycle that is overrepresented in the generation of mutations. This "peak" appears to represent a phase during which the mutation rate rises significantly. This phase produces a disproportionally high fraction of frame shift mutations (3/6). In divisions subsequent to this, the mutation rate appears to be constant.