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We have used genetic analysis to study the mode of action of two anti-microtubule herbicides, amiprophos-methyl (APM) and oryzalin (ORY). Over 200 resistant mutants were selected by growth on APM- or ORY-containing plates. The 21 independently isolated mutants examined in this study are 3- to 8-fold resistant to APM and are strongly cross-resistant to ORY and butamiphos, a close analog of APM. Two Mendelian genes, apm1 and apm2, are defined by linkage and complementation analysis. There are 20 alleles of apm1 and one temperature-sensitive lethal (33 degrees) allele of apm2. Mapping by two-factor crosses places apm1 6.5 cM centromere proximal to uni1 and within 4 cM of pf7 on the uni linkage group, a genetically circular linkage group comprising genes which affect flagellar assembly or function; apm2 maps near the centromere of linkage group VIII. Allele-specific synthetic lethality is observed in crosses between apm2 and alleles of apm1. Also, self crosses of apm2 are zygotic lethal, whereas crosses of nine apm1 alleles inter se result in normal germination and tetrad viability. The mutants are recessive to their wild-type alleles but doubly heterozygous diploids (apm1 +/+ apm2) made with apm2 and any of 15 apm1 alleles display partial intergenic noncomplementation, expressed as intermediate resistance. Diploids homozygous for mutant alleles of apm1 are 4-6-fold resistant to APM and ORY; diploids homozygous for apm2 are ts- and 2-fold resistant to the herbicides. Doubly heterozygous diploids complement the ts- phenotype of apM2, but they are typically 1.5-2-fold resistant to APM and ORY. From the results described we suggest that the gene products of apm1 and apm2 may interact directly or function in the same structure or process.

Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated by protein-protein interactions, protein phosphorylations, and nuclear localization of CDC2/CYCLINB. We previously reported a screen, in Aspergillus nidulans, for extragenic suppressors of nimX2 cdc2 that resulted in the identification of the cold-sensitive snxA1 mutation. We demonstrate here that snxA1 suppresses defects in regulators of the CDK1 mitotic induction pathway, including nimX2 cdc2 , nimE6 cyclinB , and nimT23 cdc25 , but does not suppress G2-arresting nimA1/nimA5 mutations, the S-arresting nimE10 cyclinB mutation, or three other G1/S phase mutations. snxA encodes the A. nidulans homolog of Saccharomyces cerevisiae Hrb1/Gbp2; nonessential shuttling messenger RNA (mRNA)-binding proteins belonging to the serine-arginine-rich (SR) and RNA recognition motif (RRM) protein family; and human heterogeneous ribonucleoprotein-M, a spliceosomal component involved in pre-mRNA processing and alternative splicing. snxA Hrb1 is nonessential, its deletion phenocopies the snxA1 mutation, and its overexpression rescues snxA1 and DsnxA mutant phenotypes. snxA1 and a second allele isolated in this study, snxA2, are hypomorphic mutations that result from decreased transcript and protein levels, suggesting that snxA acts normally to restrain cell cycle progression. SNXA HRB1 is predominantly nuclear, but is not retained in the nucleus during the partially closed mitosis of A. nidulans. We show that the snxA1 mutation does not suppress nimX2 by altering NIMX2 CDC2 /NIME CYCLINB kinase activity and that snxA1 or DsnxA alter localization patterns of NIME CYCLINB at the restrictive temperatures for snxA1 and nimX2. Together, these findings suggest a novel and previously unreported role of an SR/RRM family protein in cell cycle regulation, specifically in control of the CDK1 mitotic induction pathway. CONTROL of the eukaryotic G2/M transition by protein kinases has been widely studied and is highly conserved among all eukaryotes from the budding and fission yeasts and filamentous fungi to metazoans (for review, see Ma and Poon 2011). The CDK1/CYCLINB protein kinase complex is a major regulator of this transition in all eukaryotes and is responsible for the phosphorylations of numerous proteins, leading to massive nuclear and cytoplasmic reorganizations that regulate mitosis (for review, see Lindqvist et al. 2009). The complex itself is tightly regulated, both temporally and spatially, to allow mitotic entry.Although CDK1/CYCLINB activity is essential for mitotic entry in all eukaryotes, structural differences in the nucleus in various organisms result in "open" mitosis (more complex eukaryotes) or "closed" mitosis (budding yeasts); these differences likely affect the temporo-spatial functioning of CDK1/ CYCLINB. The partially closed mitosis of the filamentous fungus Aspergillus nidulans is an evolutionary intermediate between open and closed mitoses and provides a system for studying mitotic entry in organisms intermediate between budding ...

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Three independent pleiotropic drug-resistance (pdr) mutants were isolated by selecting for resistance to the anti-microtubule herbicides amiprophos-methyl (APM) and oryzalin (ORY). These three mutants and a previously isolated mutant, ani1 (anisomycin resistance), were semi-dominant in heterozygous diploids, and they displayed varying degrees of resistance to structurally and functionally unrelated inhibitors such as cycloheximide, cryptopleurine, emetine, atrazine, and nonidet P-40. Linkage analysis and genetic mapping suggested that three of the four mutants, including ani1, define a single locus, here named pdr1. The fourth mutant defined a new locus, pdr2, which is located on the left arm of linkage group VI. One pdr1 mutant exhibited unusual genetic interactions, including enhanced ts-lethality and synergistic increases in drug resistance, when combined with pdr2-1 and with herbicide-resistant alleles of three other genes.

Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimE APC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQ MCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G 1 /S by lack of nimQ MCM2 contain p34 cdc2 /cyclin B, but p34 cdc2 remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQ MCM2 and bimE APC1does not abrogate the S phase arrest checkpoint over mitosis. nimQ MCM2 , likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34 cdc2 during the G 1 to S transition, which may occur by inactivation of nimT cdc25 . Cells lacking both nimQ MCM2 and bimE APC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34 cdc2 and the function of bimE APC1 . Initiation of DNA replication, which requires nimQ MCM2 , is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34 cdc2 at G 1 /S. We also show that cells arrested at G 1 /S due to lack of nimQ MCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function. Promotion of mitosis is controlled by coordinate activation of the p34cdc2 /cyclin B (1, 2) and NIMA protein kinases (3, 4). In many systems, the key step for mitotic initiation in G 2 is Tyr-15 dephosphorylation of p34 cdc2 /cyclin B (5-11) to cause activation of this kinase complex (known as mitosis-promoting factor or MPF 1 ). Rapid tyrosine dephosphorylation of p34 cdc2 occurs by tipping the balance between the Wee1 kinase and the Cdc25 phosphatase to favor dephosphorylation during G 2 /M (9, 11).During S phase and G 2 , tyrosine phosphorylation of p34 cdc2 acts to prevent premature entry into mitosis, particularly after DNA damage or when DNA replication is perturbed (12-17), although this level of regulation is lacking in Saccharomyces cerevisiae (18, 19).Exit from mitosis is controlled by proteolysis (20 -23) of key mitotic regulatory proteins mediated by the anaphase-promoting complex (APC) or cyclosome (24 -29). The APC is thought to promote degradation of proteins such as Pds1 (30 -32) and Cut2 (33) to assist anaphase, and then cyclin B to allow exit from mitosis into G 1 (21-23, 34). Components of the APC have also been implicated in interphase cell cycle control (13,35,36).During progression into G 1 and S phase, cell cycle-specific proteolysis and tyrosine phosphorylation of p34 cdc2 need to be coordinated in some way to ensure that mitosis does not occur during G 1 or before DNA replication has been completed. For instance, if the APC failed to function during late mitosis or G 1 , then accumulation of cyclin B could potentially form a complex with p34 cdc2 and, if the balance between Wee1 and Cdc25 st...

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii was isolated by using the amiprophos-methyl-resistant mutation apm1-18 as a background to select new mutants that showed increased resistance to the drug. The upA12 mutation caused twofold resistance to amiprophos-methyl and oryzalin, and twofold hypersensitivity to the microtubule-stabilizing drug taxol, suggesting that the mutation enhanced microtubule stability. The resistance mutation was semi-dominant and mapped to the same interval on linkage group III as the alpha 1-tubulin gene. Two-dimensional gel immunoblots of proteins in the mutant cells revealed two electrophoretically altered alpha-tubulin isoforms, one of which was acetylated and incorporated into microtubules in the axoneme. The mutant isoforms co-segregated with the drug-resistance phenotypes when mutant upA12 was backcrossed to wild-type cells. Two-dimensional gel analysis of in vitro translation products showed that the non-acetylated variant alpha-tubulin was a primary gene product. DNA sequence analysis of the alpha 1-tubulin genes from mutant and wild-type cells revealed a single missense mutation, which predicted a change in codon 24 from tyrosine in wild type to histidine in mutant upA12. This alteration in the predicted amino acid sequence corroborated the approximately +1 basic charge shift observed for the variant alpha-tubulins. The mutant allele of the alpha 1-tubulin gene was designated tua1-1.