Identification of a gene for alpha-tubulin in Aspergillus nidulans.

This paper demonstrates that revertants of temperature-sensitive benA (β-tubulin) mutations in Aspergillus nidulans can be used to identify proteins which interact with β-tubulin. Three benomyl-resistant benA (β-tubulin) mutants of Aspergillus 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 temperature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had β-tubulins which differed from the β-tubulins of their parental strains by one (1^?) or two (2^?) negative charges on two-dimensional gel electrophoresis. Although the β-tubulins of the extragenic suppressor strains were all electrophoretically identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in α1-tubulin (1⁺). A heterozygous diploid between this strain and a strain with wild-type α1-tubulin was found to have both wild-type and mutant (1⁺) α1-tubulins. This experiment rules out post-translational modification as a possible cause of the α1-tubulin abnormality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for α1-tubulin. We propose that this gene be designated tubA to denote that it is a gene for α1-tubulin in A. nidulans.     

Identification of a gene for beta-tubulin in Aspergillus nidulans.

The tubulins of Aspergillus nidulans have been characterized in wild-type and ben A, B and C benomyl-resistant strains by two-dimensional gel electrophoresis, co-polymerization with porcine brain tubulin and peptide mapping. Four α-tubulins and at least four β-tubulins were resolved by two-dimensional gel electrophoresis of wild-type proteins. Eighteen of 26 benA mutants studied had electrophoretically abnormal β-tubulins. In these strains, one or more of the β-tubulins had either an altered isoelectric point or an altered electrophoretic mobility in the SDS gel dimension, or was diminished in amount. The a-tubulins were normal. Two-dimensional gels of protein extracts of a ben A/wild-type diploid strain demonstrated co-expression of the wild-type β-tubulins with the variant ben A tubulin. This experiment rules out post-translational modification as the source of the β-tubulin abnormalities in the benA mutants. We therefore conclude that benA must be a structural gene for β-tubulin. Due to the variety of abnormalities affecting β-tubulins in ben A mutants, and the absence of abnormalities affecting α-tubulins in any of the benomyl-resistant mutants, we also believe that the benomyl binding site must be located on the β-subunit of the tubulin dimer. The benA mutants of A. nidulans promise to be useful not only for characterizing the biochemical determinants of the benomyl binding site of tubulin but also for understanding the relationship between tubulin structure and function.     

Mitochondria and nuclei move by different mechanisms in Aspergillus nidulans

We have examined the effects of the antimicrotubule agent benomyl and several mutations on nuclear and mitochondrial movement in germlings of the filamentous fungus Aspergillus nidulans. While, as previously reported, benomyl inhibited nuclear division and movement, it did not inhibit mitochondrial movement. To test the effects of benomyl more rigorously, we germinated two benomyl super-sensitive, beta-tubulin mutants at a benomyl concentration 50-100 times greater than that required to inhibit colony formation completely. Again nuclear division and movement were inhibited, but mitochondrial movement was not. We also examined conditionally lethal beta-tubulin mutations that disrupt microtubule function under restrictive conditions. Nuclear division and movement were inhibited but, again, mitochondrial movement was not. Finally we examined the effects of five heat-sensitive mutations that inhibit nuclear movement but not nuclear division at restrictive temperatures. These mutations strongly inhibited nuclear movement at a restrictive temperature but did not inhibit mitochondrial movement. These data demonstrate that the mechanisms of nuclear and mitochondrial movement in Aspergillus nidulans are not identical and suggest that mitochondrial movement does not require functional microtubules.     

Conditionally lethal tubA alpha-tubulin mutations in Aspergillus nidulans

Summary We have mapped 17 extragenic suppressors of benA 33, a heat-sensitive -tubulin mutation of Aspergillus nidulans, to the tubA tubulin locus. Fifteen of these tubA mutations cause cold sensitivity in a genetic background with benA 33 and appear to cause lethality in a background with the wild-type benA allele. We examined the microtubule-mediated processes, nuclear division and nuclear migration, in seven different cold-sensitive double mutants, each carrying benA 33 and a different cold-sensitive tubA allele. Nuclear division and migration were inhibited at a restrictive temperature in each case, suggesting that cold sensitivity is due to the inhibition of microtubule function at low temperatures. A single allele, tubA4, suppressed the heat sensitivity conferred by benA33 but did not confer cold sensitivity in a benA33 background, however in a wildtype benA background, tubA4 conferred supersensitivity to antimicrotubule agents and weak cold sensitivity. TubA4 did not suppress the heat sensitivity conferred by two other benA alleles. The cold sensitivity conferred by tubA4 was suppressed by the microtubule stabilizing agent deuterium oxide, and the suppression of heat sensitivity conferred by four other tubA mutations was reversed by deuterium oxide. These results suggest that these mutations may affect hydrophobic interactions between -and -tubulin.     

The Role of S. CEREVISIAE Cell Division Cycle Genes in Nuclear Fusion

Forty temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae were examined for their ability to complete nuclear fusion during conjugation in crosses to a CDC parent strain at the restrictive temperature. Most of the cdc mutant alleles behaved as the CDC parent strain from which they were derived, in that zygotes produced predominantly diploid progeny with only a small fraction of zygotes giving rise to haploid progeny (cytoductants) that signalled a failure in nuclear fusion. However, cdc4 mutants exhibited a strong nuclear fusion (karyogamy) defect in crosses to a CDC parent and cdc28, cdc34 and cdc37 mutants exhibited a weak karyogamy defect. For all four mutants, the karyogamy defect and the cell cycle defect cosegregated, suggesting that both defects resulted from a single lesion for each of these cdc mutants. Therefore, the cdc 4, 28, 34 and 37 gene products are required in both cell division and karyogamy.     

Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants

One hundred and forty-eight temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae have been isolated and characterized. Complementation studies ordered these recessive mutations into 32 groups and tetrad analysis revealed that each of these groups defines a single nuclear gene. Fourteen of these genes have been located on the yeast genetic map. Functionally related cistrons are not tightly clustered.

Mutations in different cistrons frequently produce different cellular and nuclear morphologies in the mutant cells following incubation at the restrictive temperature, but all the mutations in the same cistron produce essentially the same morphology. The products of these genes appear, therefore, each to function individually in a discrete step of the cell cycle and they define collectively a large number of different steps.

The mutants were examined by time-lapse photomicroscopy to determine the number of cell cycles completed at the restrictive temperature before arrest. For most mutants, cells early in the cell cycle at the time of the temperature shift (before the execution point) arrest in the first cell cycle while those later in the cycle (after the execution point) arrest in the second cell cycle. Execution points for allelic mutations that exhibit first or second cycle arrest are rather similar and appear to be cistron-specific. Other mutants traverse several cycles before arrest, and its suggested that the latter type of response may reveal gene products that are temperature-sensitive for synthesis, whereas the former may be temperature-sensitive for function.

The gene products that are defined by the cdc cistrons are essential for the completion of the cell cycle in haploids of a and α mating type and in a/α diploid cells. The same genes, therefore, control the cell cycle in each of these stages of the life cycle.

     

The Selection of S. CEREVISIAE Mutants Defective in the Start Event of Cell Division

Thirty-three temperature-sensitive mutations defective in the start event of the cell division cycle of Saccharomyces cerevisiae were isolated and subjected to preliminary characterization. Complementation studies assigned these mutations to four complementation groups, one of which, cdc28, has been described previously. Genetic analysis revealed that these complementation groups define single nuclear genes, unlinked to one another. One of the three newly identified genes, cdc37, has been located in the yeast linkage map on chromosome IV, two meiotic map units distal to hom2.—Each mutation produces stage-specific arrest of cell division at start, the same point where mating pheromone interrupts division. After synchronization at start by incubation at the restrictive temperature, the mutants retain the capacity to enlarge and to conjugate.     

Cdc20, a β-transducin homologue,links RAD9-mediated G2/M checkpoint control to mitosis in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the DNA damage-induced G2 arrest requires the checkpoint control genes RAD9, RAD17, RAD24, MEC1, MEC2 and MEC3. These genes also prevent entry into mitosis of a temperature-sensitive mutant, cdc13, that accumulates chromosome damage at 37^°?C. Here we show that a cdc13 mutant overexpressing Cdc20, a β-transducin homologue, no longer arrests in G2 at the restrictive temperature but instead undergoes nuclear division, exits mitosis and enters a subsequent division cycle, which suggests that the DNA damage-induced G2/M checkpoint control is not functional in these cells. This is consistent with our observation that overexpression of CDC20 in wild-type cells results in increased sensitivity to UV irradiation. Overproduction of Cdc20 does not influence the arrest phenotype of the cdc mutants whose cell cycle block is independent of RAD9-mediated checkpoint control. Therefore, we suggest that the DNA damage-induced checkpoint controls prevent mitosis by inhibiting the nuclear division pathway requiring CDC20 function.     

Induced transposition of Ds by a stable Ac in crosses of transgenic tobacco plants

Summary Rhizoxin and ansamitocin P-3 (a maytansinoid compound), potent inhibitors of mammalian brain tubulin assembly, inhibit growth of a variety of fungi including Aspergillus nidulans. Mutants of A. nidulans, benA10 which is a benomyl resistant -tubulin gene mutant and tubAl which is a benomyl supersensitive a-tubulin gene mutant, were both sensitive to rhizoxin and ansamitocin P-3 to the same extent as wild-type strains. We isolated 18 rhizoxin resistant mutants of A. nidulans. All of these mutants were cross-resistant to ansamitocin P-3, but not to benzimidazole antimitotic drugs. These mutants mapped to two loci, rhiA and rhiB, and all of those with high resistance mapped to rhiA. The fact that the protein extracts of rhiA mutants lost rhizoxin binding affinity and that rhiA was closely linked to benA, the major -tubulin gene in A. nidulans, indicated that rhiA must be a structural gene for -tubulin and that rhiA mutants are a new class of -tubulin gene mutants. All of this suggested that, in A. nidulans, these antimitotic drugs bind to -tubulin, and that rhizoxin and ansamitocin P-3 share the same binding site but the site does not overlap with the benzimidazole binding site. Protein extracts from a rhiB mutant retained rhizoxin binding affinity, therefore this rhizoxin resistance mechanism should not be a tubulin mediated process.     

Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis

By examining cytological phenotypes of 587 temperature-sensitive mutants of the fission yeast Schizosaccharomyces pombe, we obtained 18 mutants which cause cell division in the absence of nuclear division. By genetic analyses, these novel nuclear division arrest mutants can be classified into nine complementation groups (designated cut1 – cut9). The cytological phenotype of cut mutants is similar but not identical to that of DNA topoisomerase II mutants (top2). The cut1⁺ gene was cloned by transformation and shown to complement cut2 as well as cut1, indicating a functional relationship between the two genes. The cut genes are required for nuclear division, but their mutant phenotypes differ from most of the previously identified mutants which block nuclear division and also the subsequent cytokinesis. Fluorescence microscopy indicates that the mitotic chromosomes formed in cut mutant cells are abnormal and fail to separate properly. We suggest that cut mutations, like top2, block mitotic chromosome formation and concomitantly nuclear division, but that cytokinesis proceeds independently of the defects in nuclear division, demonstrating uncoordinated mitotic pathways. A novel mutant nuc1 is also described which shows a cytological phenotype similar to the double mutant of DNA topoisomerases I and II but contains normal levels of both DNA topoisomerase activities.     

Cold-Sensitive Cell-Division-Cycle Mutants of Yeast: Isolation, Properties, and Pseudoreversion Studies

We isolated 18 independent recessive cold-sensitive cell-division-cycle (cdc) mutants of Saccharomyces cerevisiae, in nine complementation groups. Terminal phenotypes exhibited include medial nuclear division, cytokinesis, and a previously undescribed terminal phenotype consisting of cells with a single small bud and an undivided nucleus. Four of the cold-sensitive mutants proved to be alleles of CDC11, while the remaining mutants defined at least six new cell-division-cycle genes: CDC44, CDC45, CDC48, CDC49, CDC50 and CDC51.—Spontaneous revertants from cold-sensitivity of four of the medial nuclear division cs cdc mutants were screened for simultaneous acquisition of a temperature-sensitive phenotype. The temperature-sensitive revertants of four different cs cdc mutants carried single new mutations, called Sup/Ts to denote their dual phenotype: suppression of the cold-sensitivity and concomitant conditional lethality at 37°. Many of the Sup/Ts mutations exhibited a cell-division-cycle terminal phenotype at the high temperature, and they defined two new cdc genes (CDC46 and CDC47). Two cold-sensitive medial nuclear division cdc mutants representing two different cdc genes were suppressed by different Sup/Ts alleles of another gene which also bears a medial nuclear division function (CDC46). In addition, the cold-sensitive medial nuclear division cdc mutant csH80 was suppressed by a Sup/Ts mutation yielding an unbudded terminal phenotype with an undivided nucleus at the high temperature. This mutation was an allele of CDC32. These results suggest a pattern of interaction among cdc gene products and indicate that cdc gene proteins might act in the cell cycle as complex specific functional assemblies.     

Regulation of mating in the cell cycle of Saccharomyces cerevisiae

The capacity of haploid a yeast cells to mate (fuse with a haploid strain of alpha mating type followed by nuclear fusion to produce a diploid cell) was assessed for a variety of temperature-sensitive cell division cycle (cdc) mutants at the permissive and restrictive temperatures. Asynchronous populations of some mutants do not mate at the restrictive temperature, and these mutants define genes (cdc 1, 4, 24, and 33) that are essential both for the cell cycle and for mating. For most cdc mutants, asynchronous populations mate well at the restrictive temperature while populations synchronized at the cdc block do not. Populations of a mutant carrying the cdc 28 mutation mate well at the restrictive temperature after synchronization at the cdc 28 step. These results suggest that mating can occur from the cdc 28 step, the same step at which mating factors arrest cell cycle progress. The cell cycle interval in which mating can occur may or may not extend to the immediately succeeding and diverging steps (cdc 4 and cdc 24). High frequency mating does not occur in the interval of the cell cycle extending from the step before the initiation of DNA synthesis (cdc 7) through DNA synthesis (cdc 2, 8, and 21), medial nuclear division (cdc 13), and late nuclear division (cdc 14 and 15).     

Staphylococcus aureus chromosomal mutation specifically affecting the copy number of Inc3 plasmids

A chromosomal mutation leading to an important increase in the copy number of plasmid pT181 and its derivatives has been isolated from Staphylococcus aureus strain 8325. The amplification effect in the mutant strain SA1350 was found to be specific for plasmids of the Inc3 group, to which belongs pT181. There are some other differences in the behavior of Inc3 plasmids between SA1350 and 8325, including stable maintenance in SA1350 at high copy number of temperature-sensitive replication mutants at restrictive temperatures, and altered incompatibility properties. Derivatives of SA1350 carrying only Inc3 plasmid mutants with high copy numbers (Cop mutants) could not be obtained, suggesting a lethal runaway plasmid replication in this situation. SA1350 expressed also a temperature-sensitive phenotype. The relationship of this character to the plaC1 mutation determining the amplification of Inc3 plasmids has not yet been elucidated.     

Mitochondrial DNA synthesis in cell cycle mutants of saccharomyces cerevisiae

Mitochondrial DNA replication was examined in mutants for seven different Saccharomyces cerevisiae genes which are essential for nuclear DNA replication. In cdc8 and cdc21, mutants defective in continued replication during the S phase of the cell cycle, mitochondrial DNA replication ceases at the nonpermissive temperature. Replication is temperature sensitive even when these mutants are arrested in the G1 phase of the cell cycle with α factor, a condition where mitochondrial DNA replication continues for the equivalent of several generations at the permissive temperature. Therefore the cessation of replication results from a defect in mitochondrial replication per se, rather than from an indirect consequence of cells being blocked in a phase of the cell cycle where mitochondrial DNA is not normally synthesized. Since the temperature-sensitive mutations are recessive, the products of genes cdc8 and cdc21 must be required for both nuclear and mitochondrial DNA replication. In contrast to cdc8 and cdc21, mitochondrial DNA replication continues for a long time at the nonpermissive temperature in five other cell division cycle mutants in which nuclear DNA synthesis ceases within one cell cycle: cdc4, cdc7, and cdc28, which are defective in the initiation of nuclear DNA synthesis, and cdc14 and cdc23, which are defective in nuclear division. The products of these genes, therefore, are apparently not required for the initiation of mitochondrial DNA replication.     

Genetic and enzymatic characterization of conditional lethal mutants of the yeast Schizosaccharomyces pombe with a temperature-sensitive DNA ligase.

The DNA ligase activities of wild type and temperature-sensitive lethal cdc 17 mutants of Schizosaccharomyces pombe have been studied by measuring effects on the conversion of relaxed DNA circles containing a single nick to a closed circular form. Such assays have revealed that all cdc 17 mutants have a thermosensitive DNA ligase deficiency, that this deficiency cosegregates 2:2 with their temperature-sensitive cdc-lethality in three tetrads derived from a cross against wild type, and that genetic reversion of the temperature-sensitive cdc^? phenotype is accompanied by a restoration of DNA ligase activity; all of which implies that the temperature-sensitive cdc^? phenotype of cdc 17 mutants is due to a single nuclear mutation causing a DNA ligase deficiency. Both wild type and mutant enzymes have been partially purified by chromatography in heparin/agarose columns. The wild-type enzyme is completely stable in vitro at both permissive (25 °C) and restrictive (35 °C) temperatures, whereas that of two different mutants, though completely stable at 25 °C, is rapidly inactivated at 35 °C, implying that their mutations are located in the structural gene for DNA ligase.     

Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization.

We have isolated mutants defective in DNA topoisomerases and an endonuclease from the fission yeast Schizosaccharomyces pombe by screening individual extracts of mutagenized cells. Two type I topoisomerase mutants (top1) and three endonuclease mutants (end1) were all viable. The double mutant top1 end1 was also viable and, in its extract, Mg2+- and ATP- dependent type II activity could be detected. Three temperature-sensitive (ts-) mutants having heat-sensitive (hs-) type II enzymes were isolated, and the ts- marker cosegregated with the hs- type II activity. All the ts- mutations fell in one gene (top2) tightly linked to leul in chromosome II. The nuclear division of single top2 mutants was blocked at the restrictive temperature, but the formation of a septum was not inhibited so that the nucleus was cut across with the cell plate. In contrast, the double top1 top2 mutants were rapidly arrested at various stages of the cell cycle, showing a strikingly altered nuclear chromatin region. The type II topoisomerase may have an essential role in the compaction and/or segregation of chromosomes during the nuclear division but also complement the defect of the type I enzyme whose major function is the maintenance of chromatin organization throughout the cell cycle.     

Division septation in the absence of chromosome termination in Bacillus subtilis.

Germinating spores of the temperature-sensitive DNA initiation mutants of Bacillus subtilis, TsB134 and dna-1(Ts), were allowed to undergo a single round of replication by shifting to the restrictive temperature shortly after its initiation. To monitor the progress of the round 5-bromouracil was added at various times and DNA extracted after a further time, sufficient to allow completion of the chromosome. Average replication was measured from the relative amounts of LL and LH material in Cs₂SO₄ gradients. The replication state of origin (purA), intermediate (leuA) and terminus (metB) markers at the times of 5-bromouracil addition were obtained from genetic analysis of the density species fractionated in gradients of CsCl.The DNA replication inhibitor, 6-(p-hydroxyphenylazo)-uracil (HPUra), was added at various stages of the single round and the outgrown cells examined at later times for the frequency and type of septation. Under the conditions of the experiment, central division septation was blocked if HPUra (20 μm) was added before 70% (approximately) of the chromosome was replicated. Using higher concentrations of HPUra, 40 and 100 μm, it was shown that central division septation would occur at about its normal time if replication was blocked after this 70% stage but before termination. In these circumstances there was a distinct tendency for the DNA to remain close to the septum on both sides of it. The B. subtilis spore contains a single chromosome, which means that the central septum that forms in the absence of termination must pass through a partially completed chromosome. Electron microscopic evidence for such a situation has already been described (Van Iterson &; Aten, 1976). It is concluded that, at least under the restrictive conditions of the present experiments, termination of chromosome replication is not obligatory for the formation of the division septum with which it is normally coupled.     

Novel mutations in β-tubulin gene in Trichoderma harzianum mutants resistant to methyl benzimidazol-2-YL carbamate

Twelve low resistant (LR) mutants of Trichoderma harzianum with the capability of grow fast at 0.8 μg/mL methyl benzimidazol-2-yl carbamate (MBC) were obtained using UV mutagenesis. MR and HR mutants which could grow fast at 10 and 100 μg/mL MBC, respectively, were isolated by step-up selection protocols in which UV-treated mutants were induced and mycelial sector screening was made in plates with growth medium. Subsequently, β-tubulin genes of 14 mutants were cloned to describe the molecular lesion likely to be responsible for MBC resistance. Comparison of the β-tubulin sequences of the mutant and sensitive strains of T. harzianum revealed 2 new MBC-binding sites differed from those in other plant pathogens. A single mutation at amino acid 168, having Phe (TTC) instead of Ser (TCC), was demonstrated for the HR mutant; a double mutation in amino acid 13 resulting in the substitution of Gly (GGC) by Val (GTG) was observed in β-tubulin gene of MR mutant. On the other hand, no substitutions were identified in the β-tubulin gene and its 5’-flanking regions in 12 LR mutants of T. harzianum.     

Mutations affecting cell division in Tetrahymena pyriformis, syngen 1. II. Phenotypes of single and double homozygotes.

The surface patterns associated with arrest of cell division by temperature-sensitive mutations at five different loci are described. Mutations at the mo1 locus prevent both the subdivision of ciliary meridians that mark the fission zone and the subsequent furrowing. Mutations at mo8 and mo12 cause abnormal configurations in the fission zone and aborted furrowing. Mutations in mo3 and mo6 bring about fission arrest with associated elongation (mo3) or twisting (mo6), even though complete fission zones do develop. The defects in mo1, mo3, and mo12 are expressed in the first division after shift to restrictive temperature, whereas expression of mo6 and of one allele of mo8 are delayed. Following preincubation in an amino acid-free medium at the restrictive temperature, mo8 causes arrest at the first division after readdition of nutrients, while mo6 blocks fission only after one or more divisions at the restrictive temperature. Double homozygotes were constructed containing the mo3^a mutation and mutations at each of the other loci. In addition, mo1^a was combined with mo8^a. In each of the double homozygotes, the characteristic phenotypes of both mutations were simultaneously expressed, and the penetrance of division blockage was very greatly enhanced. The results suggest that the functions of these five loci are not ordered in a single dependent sequence of steps, but rather that these loci probably mediate independent processes required for cytokinesis.