N. Ronald Morris, Margaret H. Lai and C. Elizabeth Oakley Department of Pharmacology CMDNJ-Rutgers Medical School Piscataway, New Jersey 08854 Summary This paper demonstrates that revertants of tem- perature-sensitive benA (p-tubulin) mutations in Aspergillus nidulans can be used to identify pro- teins which interact with /3-tubulin. Three beno- myl-resistant benA (P-tubulin) mutants of Asper- gillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts-) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts+ were isolated by selection at the restrictive tem- perature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had p-tubulins which differed from the p-tubulins of their parental strains by one (l-) or two (2-) negative charges on two-dimensional gel electro- phoresis. Although the P-tubulins of the extra- genie suppressor strains were all electrophoreti- tally identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in al-tubulin (1). A heterozygous diploid between this strain and a strain with wild-type al-tubulin was found to have both wild-type and mutant (l+) rul-tubulins. This experiment rules out post-translational modifica- tion as a possible cause of the al-tubulin abnor- mality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for al-tubulin. We propose that this gene be designated tubA to denote that it is a gene for cul-tubulin in A. nidulans. Introduction The assembly of tubulin into microtubules involves it in a large number of a cellular processes. Al- though a great deal is known about microtubules both morphologically and biochemically, there are large gaps in our knowledge. For example, micro- tubule assembly and dynein-dependent microtu- bule-associated movement have been extensively studied in vitro, but almost nothing is known about the regulation of microtubule assembly, disassem- bly or microtubule-dependent transport in the liv- ing cell (Snyder and McIntosh, 1976); and although the molecular architecture of microtubules has been the subject of elegant ultrastructural investi- gation (Snyder and McIntosh, 1976), the molecular details of the interactions between LY- and P-tubu- lins and microtubule-associated proteins are un- known. The genetics of tubulin and microtubules have also not been explored. This paper demon- strates that genetic studies can be used to provide information about the interactions between micro- tubule proteins. Mutants of the fungus Aspergillus nidulans se- lected for resistance to the antimitotic benzimida- zole compounds benomyl or thiabendazole may carry a mutation at any one of three loci, benA, B or C (Van Tuyl, 1977). In a previous paper, we showed that most benA mutants have electropho- retically abnormal p-tubulins and that benA is a structural gene for P-tubulin in A. nidulans (Sheir- Neiss, Lai and Morris, 1978). The present paper extends the potential usefulness of the benA sys- tem for analyzing tubulin structure and function by isolating and characterizing a number of revertants and suppressors of benA mutations. Since the original benA mutants were drug-resistant, it was not possible to isolate revertants directly, but be- cause most of the mutants studied electrophoreti- tally appeared to have missense mutations in the benA gene and because missense mutations may result in destabilization of protein structure, we tested a series of benA mutants for their ability to grow at elevated temperatures with the expectation of finding one or more temperature-sensitive (ts-) strains. Three such ts- mutants were found and characterized, from these a series of revertants to ts+ have been isolated. Among 18 revertants from ts- to ts+, four have intragenic back-muta- tions in p-tubulin and 14 have extragenic suppres- sor mutations. One of the extragenic suppressor strains has electrophoretically abnormal al-tubu- lit-r. The suppressor mutation this strain has been identified as a structural gene for cul-tubulin in A. nidulans. Results BEN 9,155 and 19 Are Temperature-Sensitive Twenty six benA mutants of A. nidulans originally isolated by Van Tuyl (1977) were tested for inability to grow at high temperature. The three strains BEN 9, 15 and 19, carrying the mutations benAll, benA and benA21, were found to be tempera- ture-sensitive (ts-) for growth at 44°C. At this tem- perature, the wild-type parental strain B3 and other benA mutants grew about half as rapidly at 32”C, whereas the three ts- strains were inhibited by -90% (Figure 1). To determine whether the tem-
Abstract The effect of 5-bromodeoxyuridine (BrdUrd) on the ability of the glial tumor cell line, C-6, to elevate its adenosine 3',5'-monophosphate (cAMP) content in response to norepinephrine has been examined. Increasing concentrations of BrdUrd were found to inhibit the norepinephrine response progressively, with a maximal inhibition of approximately 80%. No effect of BrdUrd was seen on basal cAMP levels. Analysis of individual enzymes demonstrated that BrdUrd increased the cAMP phosphodiesterase activities but had no effect on the adenylate cyclase or its sensitivity to norepinephrine. In addition, BrdUrd caused a change in the shape of the cells. The effect on shape also occurred in confluent cells, whereas the effect on the norepinephrine response required dividing cells, suggesting that incorporation of the BrdUrd into DNA was required. N6, O2'-Dibutyryl adenosine 3',5'-monophosphate also increased the cAMP phosphodiesterase activities of the cells, and caused a similar change in their morphology. Although BrdUrd had no effect on the adenylate cyclase, variations in the culture conditions were found to have a large effect on the enzyme's norepinephrine sensitivity. Following subculturing, there was little elevation of cAMP in response to norepinephrine until after the cells began to enter the stationary growth phase. Phosphodiesterase activity was found to remain constant throughout this period. The results suggest that cell-to-cell contact may regulate certain cellular functions.
ABSTRACT The bimE (blocked-in-mitosis) gene appears to function as a negative mitotic regulator because the recessive bimE7 mutation can override certain interphase-arresting treatments and mutations, causing abnormal induction of mitosis. We have further investigated the role of bimE in cell cycle checkpoint control by: (1) coordinately measuring mitotic induction and DNA content of bimE7 mutant cells; and (2) analyzing epistasis relationships between bimE7 and 16 different nim mutations. A combination of cytological and flow cytometric techniques was used to show that bimE7 cells at restrictive temperature (44°C) undergo a normal, although somewhat slower cell cycle prior to mitotic arrest. Most bimE7 cells were fully reversible from restrictive temperature arrest, indicating that they are able to enter mitosis normally, and therefore require bimE function in order to finish mitosis. Furthermore, epistasis studies between bimE7 and mutations in cdc2 pathway components revealed that the induction of mitosis caused by inactivation of bimE requires functional p34cdc2 kinase, and that mitotic induction by bimE7 depends upon several other nim genes whose functions are not yet known. The involvement of bimE in S phase function and mitotic checkpoint control was suggested by three lines of evidence. First, at restrictive temperature the bimE7 mutation slowed the cell cycle by delaying the onset or execution of S phase. Second, at permissive temperature (30°C) the bimE7 mutation conferred enhanced sensitivity to the DNA synthesis inhibitor hydroxyurea. Finally, the checkpoint linking M phase to the completion of S phase was abolished when bimE7 was combined with two nim mutations that cause arrest in G1 or S phase. A model for bimE function based on these findings is presented.
The filamentous fungus Aspergillus nidulans has two genes encoding alpha-tubulin, tubA and tubB, which are differentially required at distinct stages during the life cycle. The tubA gene is required during vegetative growth for mitosis and nuclear migration (B. R. Oakley, C. E. Oakley, and J. E. Rinehart, Mol. Gen. Genet. 208:135-144, 1987; P. Doshi, C. A. Bossie, J. H. Doonan, G. S. May, and N. R. Morris, Mol. Gen. Genet. 225:129-141, 1991). The tubB gene is not required for any detectable aspect of vegetative growth or asexual reproduction but is essential during sexual development prior to the first meiotic division (K. E. Kirk and N. R. Morris, Genes Dev. 5:2014-2023, 1991). In this study, we determined whether the role of each alpha-tubulin gene is to provide a specific isotype necessary for a particular microtubule function or whether either alpha-tubulin isotype, if present in sufficient quantities, can participate effectively in all types of microtubule. Strains carrying a deletion allele of tubB (tubB delta) produce no ascospores from a cross. When one copy of a plasmid containing the region upstream of the tubB gene fused to the tubA coding region was integrated into a tubB delta strain, ascosporogenesis proceeded beyond the tubB delta block and resulted in the formation of sexual spores. However, irregular numbers of spores formed in some asci during development, and the ascospores had greatly diminished viability and aberrant morphologies. These defects were nearly corrected when two additional copies of the tubA coding region were integrated into the tubB delta strain. These results indicate that the tubA alpha-tubulin isotype can form functional microtubules during sexual development in the absence of tubB protein. In a reciprocal set of experiments, we examined whether upregulation of tubB can complement the tubA4 mutation, which causes supersensitivity to benomyl during vegetative growth. When tubA4 strains integrated a plasmid containing an alcohol-inducible promoter joined to the tubB coding region and subsequently overexpressed the tubB isotype, the benomyl supersensitivity normally caused by the tubA4 allele was relieved. These results indicate that when enough tubB alpha-tubulin is supplied, strains lacking functional tubA isotype can still form microtubules which effectively carry out mitosis and nuclear migration.
