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Actin has a pivotal function in hyphal morphogenesis in filamentous fungi, but it is not certain whether its function is equivalent to that of a morphogen, or if it is simply part of a mechanism that executes orders given by another regulatory entity. To address this question we selected for cytochalasin A resistance and isolated act1, the first actin mutant in Neurospora crassa. This mutant branches apically and shows an altered distribution of actin at the tip. Based on the properties of this mutant, we propose a model of tip growth and branching in which actin effects tip growth by regulating the rate of vesicle flow from proximal to distal regions of a hypha, thereby controlling the tip-high gradient of cytoplasmic calcium. The actin-controlled calcium gradient at the tip is necessary for maintenance of tip growth as well as the dominance of one polarized site at the hyphal tip. The phenotype of act1 indicates that actin controls the balance between lateral and apical branching. [Copyright &y& Elsevier]

There are at least 100 loci encoding products that influence tip growth and branching in Neurospora crassa. The functional relationships between 38 of these loci were examined by an analysis of gene interaction in double mutants. A complex range of interactions was revealed. These have been grouped into full and partial epistasis, costasis, novel phenotypes, and synthetic lethality and sublethality. Epistasis was used to construct the simplest “pathway” that accommodated the results; this pathway was Y-shaped. If synthetic sublethality is interpreted to reflect mutations in the same pathway, the sublethal connections are compatible with the chart of epistasis. The gene interactions discovered represent candidates for future cell and molecular studies on the interaction of gene products in the control of tip growth and branching. [Copyright &y& Elsevier]

Many acyclovir-resistant herpes simplex virus isolates from patients contain insertions or deletions in homopolymeric sequences in the thymidine kinase (TK) gene (tk). Viruses that have one (G8) or two (G9) base insertions in a run of seven G's (G string) synthesize low levels of active TK (TK-low phenotype), evidently via ribosomal frameshifting. These levels of TK can suffice to permit reactivation from latently infected mouse ganglia, but in a majority of ganglia, especially with the G9 virus, reactivation of virus that has reverted to the TK-positive phenotype predominates. To help address the relative contributions of translational mechanisms and reversion in reactivation, we generated viruses with a base either inserted or deleted just downstream of the G string. Both of these viruses had a TK-low phenotype similar to that of the G8 and G9 viruses but with less reversion. Both of these viruses reactivated from latently infected trigeminal ganglia, albeit inefficiently, and most viruses that reactivated had a uniformly TK-low phenotype. We also generated viruses that have one insertion in a run of six C's or one deletion in a run of five C's. These viruses lack measurable TK activity. However, they reactivated from latently infected ganglia, albeit inefficiently, with the reactivating viruses having reverted to the wild-type TK phenotype. Therefore, for G-string mutants, levels of active TK as low as 0.25% generated by translational mechanisms can suffice for reactivation, but reversion can also contribute. For viruses that lack TK activity due to mutations on other homopolymeric sequences, reactivation can occur via reversion. [ABSTRACT FROM AUTHOR]

Abstract: In the fungus Neurospora crassa we have used RIP to obtain a presumptive null mutation of the phospholipase C-1 gene, thought to be important in intracellular calcium signaling, notably maintenance of the tip-high calcium gradient. The mutant is viable but has slow, aberrant growth and branching. Hence plc-1 is not required for polar growth at the tip, but is necessary to modulate growth to give normal form. The mutant has residual PLC activity suggesting that this enzyme function can be provided from other parts of the genome. Sequencing natural isolates has shown that the plc-1 gene is highly variable in nature. A large proportion of the variable sites are in a region that is unique to Neurospora. A phylogeny for this gene shows that New and Old World strains have diverged the most. Within the Americas, morphs are found throughout the continent suggesting extensive strain dispersal. [Copyright &y& Elsevier]

A single-cytosine-deletion in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. A laboratory strain engineered to carry this mutation did not generate sufficient TK activity for detection by plaque autoradiography, which detected 0.25% wild-type activity. However, a drug sensitivity assay suggested that extremely low levels of TK are generated by this virus. The virus was estimated to express 0.09% of wild-type TK activity via a ribosomal frameshift 24 nucleotides upstream of the mutation. Remarkably, this appeared to be sufficient active TK to support a low level of reactivation from latently infected mouse trigeminal ganglia. [ABSTRACT FROM AUTHOR]