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N R Morris

Publications and source records attributed to N R Morris.

At least 73 records · Page 4Linked to original sources
PubMed1988

Mitotic induction and maintenance by overexpression of a G2-specific gene that encodes a potential protein kinase.

There may be a causal relationship between expression of the G2-specific gene nimA and mitotic regulation in Aspergillus. To test this relationship we have introduced extra inducible copies of nimA into Aspergillus and determined the effect of nimA overproduction on mitotic regulation. The results show that nimA overexpression causes mitotic induction in less than a cell cycle and maintains chromatin in a condensed state. These effects occur even if cells are first blocked in S phase. Sequence analysis shows that the nimA gene encodes a potential protein kinase. These data indicate that there is indeed a causal relationship between expression of nimA and the regulation of mitosis and further implicate protein phosphorylation in mitotic control.

Amino Acid Sequence↗
PubMed1988

Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene.

In Aspergillus nidulans the temperature-sensitive cell cycle mutation bimE7 causes chromosome condensation and pre-anaphase spindle formation to occur at restrictive temperature. By constructing double mutants between bimE7 and S phase or G2 phase mutants and blocking DNA replication with hydroxyurea, we demonstrate that bimE7 can cause chromatin condensation and spindle formation in cells held in S or G2. Thus bimE7 overrides normal control systems that prevent mitosis from prematurely occurring during S or G2. We show that bimE7 is a loss of function mutation and propose that bimE normally functions to negatively control a positive mitotic inducing factor, possibly the cell cycle gene nimA.

Aspergillus nidulans↗
PubMed1988

Cell-cycle modulation of MPM-2-specific spindle pole body phosphorylation in Aspergillus nidulans.

MPM-2 is a monoclonal antibody that interacts with mitosis-specific phosphorylated proteins in many different organisms. Immunocytochemistry of tissue culture cells has shown that MPM-2 stains centrosomes, chromosomes, kinetochores, and spindles. In this paper, we demonstrate that MPM-2 staining colocalizes with the spindle pole body (SPB) of Aspergillus nidulans and that SPB staining varies during the mitotic cycle. In an unsynchronized population, about one-fourth to one-third of the cells stain with MPM-2 at the spindle plaques or SPBs. Nuclei in mitosis have two SPBs localized at the ends of the spindle, both of which stain with MPM-2. To determine when MPM-2 staining appears, we have examined the effects of temperature-sensitive cell-cycle mutations that block nuclear division in S or G2. Only a very small fraction of cells blocked in S-phase stain with MPM-2. In contrast, a large fraction of cells blocked in G2 stain brightly at the SPB. These data suggest that MPM-2 reactivity of SPBs appears in G2. Moreover, the fact that cells blocked in G2 showed MPM-2 staining but no spindles suggests that reactivity of SPBs occurs prior to mitosis but is not sufficient to trigger spindle formation. When G2-blocked cells were downshifted to permissive temperature, they generated a mitotic spindle with an SPB at each end. Both SPBs stained with MPM-2 in all of the mitotic cells.

Antibodies, Monoclonal↗
PubMed1988

Developmental regulation of a conidiation specific beta-tubulin in Aspergillus nidulans.

A beta-tubulin gene previously suggested to participate in conidial development in the filamentous fungus Aspergillus nidulans is shown to be developmentally regulated in its expression. A quantitative S1 assay was used to show that the abundance of the tubC messenger RNA increases during conidial development relative to the benA messenger RNA. Morphological analysis of cultures, used to prepare RNA for the S1 analysis, demonstrated that the increase in tubC messenger RNA was directly correlated with the appearance of conidiating cell types and structures and that as these cell types become more prevalent in the culture, the level of the messenger RNA increases. Two-dimensional gel electrophoresis of labeled proteins showed that the relative increase in tubC messenger RNA was reflected in an increase in the tubC protein beta 3. RNA blot hybridization analysis and in vitro translation of total RNA from mycelia and conidia were used to demonstrate that both benA and tubC beta-tubulin messenger RNAs are absent from conidia.

Aspergillus nidulans↗
PubMed1987

The unique histone H2A gene of Aspergillus nidulans contains three introns.

The histone H2A gene of the filamentous fungus Aspergillus nidulans has been cloned and sequenced. There is a single H2A gene in the genome of A. nidulans, and it contains three introns. The introns are 51 nucleotides (nt), 56 nt and 50 nt in length and split codons for amino acids (aa) 18, 48 and 116 of the predicted protein. The transcriptional start and termination points have been determined using an S1 nuclease protection assay. The predicted protein is 132 aa residues in length and surprisingly has a threonine after the initiator methionine instead of the usual serine. The sequence of the predicted histone H2A protein is compared to histone H2A proteins from Schizosaccharomyces pombe, Saccharomyces cerevisiae and calf thymus. Comparison of the amino acid sequence to these other H2A proteins shows that the divergence of amino acid sequences between H2A proteins is found in two clustered sites.

Amino Acid Sequence↗
PubMed1987

Aspergillus nidulans beta-tubulin genes are unusually divergent.

Aspergillus nidulans has two beta-tubulin genes: benA, which is involved in both vegetative growth and asexual sporulation, and tubC, which is involved mainly in asexual sporulation. Both genes have now been cloned and sequenced. benA encodes a polypeptide of 447 amino acids (aa) and tubC encodes one of 449 aa. The two polypeptides differ by 78 aa residues but the net charge for the two proteins remains the same. The divergence between the amino acid sequences of the Aspergillus beta-tubulins is greater than that for any other two beta-tubulins yet described in the same organism. The benA gene has eight introns and the tubC gene has five, all of which correspond in amino acid position to introns in benA. The positions of some of these introns are conserved in other beta-tubulin genes. The 5'-splice site, internal, and 3'-splice site consensus sequences are similar to those found in other fungal introns. The transcriptional start points for each gene have been determined using primer extension and/or S1 nuclease mapping. Neither the benA gene nor the tubC gene contains a TATA sequence in its 5'-flanking region. The tubC gene has two repeated upstream sequences which are not found in benA. The sites of polyadenylation have been determined for each gene using S1 nuclease mapping. Neither gene contains a polyadenylation signal, AATAAA, typical of other eukaryotic genes.

Amino Acid Sequence↗
PubMed1987

Regulation of the mRNA levels of nimA, a gene required for the G2-M transition in Aspergillus nidulans.

The temperature-sensitive cell cycle mutation nimA5 causes nuclei of Aspergillus nidulans to be blocked in late G2 at restrictive temperature. Under these conditions the spindle pole body divides but does not separate and the mitotic index drops to zero. If nimA5 is blocked for more than one doubling time and then shifted from restrictive to permissive temperature, nuclei immediately enter mitosis, the mitotic spindle forms, and the chromosomes condense (Oakley, B. R., and N. R. Morris, 1983, J. Cell Biol., 96:1155-8). We have cloned the wild-type nimA gene by DNA-mediated complementation of the nimA5 mutant phenotype and have characterized nimA mRNA expression by Northern blot analysis. The transcript is 3.6 kb in length and is under tight nuclear cycle regulation. In synchronously dividing cells, the levels of nimA mRNA become elevated as cells enter mitosis and drop sharply as cells progress through mitosis. Cells blocked in S-phase with hydroxyurea have very low levels of nimA mRNA. Cells blocked in mitosis, either by the antimitotic agent benomyl or by the cell cycle mutation bimE7, maintain elevated levels of the nimA transcript. These data demonstrate not only that nimA is required for entry into mitosis, but because the transcript is normally expressed cyclically and is under tight cell cycle control, they suggest that nimA may play a regulatory role in the initiation of mitosis.

Aspergillus nidulans↗
PubMed1985

Involvement of a particular species of beta-tubulin (beta 3) in conidial development in Aspergillus nidulans.

Strains of Aspergillus containing the benA22 mutation are resistant to benomyl for vegetative growth but do not produce conidia. To test whether conidiation involved an additional benomyl-sensitive tubulin (i.e., was mediated by a tubulin other than the tubulins coded for by the benA locus), a collection of mutants was produced that formed conidia in the presence of benomyl, i.e., were conidiation-resistant (CR-) mutants. We analyzed the tubulins of these CR- mutants using two-dimensional gel electrophoresis and found that the mutants lacked one species of beta-tubulin (designated beta 3). We have examined two of these mutants in detail. In crosses with strains containing wild-type tubulins, we found that the absence of the beta 3-tubulin co-segregated perfectly with the CR- phenotype. In diploids containing both the benA22 and CR- mutations, we found that the CR- phenotype was recessive and that beta 3-tubulin was present on two-dimensional gels of tubulins prepared from these diploids. In another set of crosses, these two CR- strains and seven others were first made auxotrophic for uridine and then crossed against strains that had homologously integrated a plasmid containing an incomplete internal fragment of the beta 3-tubulin gene and the pyr4 gene of Neurospora crassa (which confers uridine prototrophy on transformants). If the CR- phenotype were produced by a mutation in a gene distinct from the structural gene for beta 3-tubulin (designated the tubC gene), then crossing over should have produced some CR+ segregants among the uridine auxotrophic progeny of the second cross. All of the uridine auxotrophs from this type of cross, however, showed the CR- phenotype, suggesting that the mutation in these strains is at or closely linked to the tubC locus. The most obvious explanation of these results is that beta 3-tubulin is ordinarily used during conidiation and the presence of this species of beta-tubulin renders conidiation sensitive to benomyl. In the CR- mutants, beta 3-tubulin is absent, and in the presence of the benA22 mutation the benomyl-resistant beta 1-and/or beta 2-tubulin substitutes for beta 3 to make conidiation benomyl resistant. We discuss these results and give two models to explain the interactions between these beta-tubulin species.

Aspergillus nidulans↗
PubMed1985

Identification and functional analysis of beta-tubulin genes by site specific integrative transformation in Aspergillus nidulans.

We have cloned two different beta-tubulin sequences from the filamentous fungus Aspergillus nidulans. Each was used in the construction of transforming plasmids that carry the pyr4 gene of Neurospora crassa. We used these plasmids to transform a pyrG-strain of Aspergillus to uridine prototrophy. Both plasmids were shown to integrate site specifically into the homologous chromosomal sequences. We then used transformant strains in genetic crosses to demonstrate that one of the cloned beta-tubulin sequences was the benA beta-tubulin gene, which codes for the beta 1-and beta 2-tubulins. The other cloned beta-tubulin sequence was shown to be the structural gene for beta 3-tubulin by gene disruption and to participate in conidial development. This is the first report of a gene disruption by site specific, integrative recombination in Aspergillus nidulans.

Aspergillus nidulans↗
PubMed1984

Aspergillus contains multiple tubulin genes.

Previous work with benomyl-resistant mutants of Aspergillus nidulans has demonstrated that the benA locus is a structural gene for beta-tubulin. Two of the benA mutants, benA22 and benA85, show altered electrophoretic mobilities on two-dimensional gels for two beta-tubulins (designated beta 1 and beta 2). These shifts of the beta 1- and beta 2-tubulins uncover a spot in the region where wild-type beta-tubulins migrate that is occluded on gels of wild-type extracts by the beta 1- and beta 2-tubulins. Evidence has now been obtained indicating that this spot represents an additional beta-tubulin (designated beta 3). Tubulin was partially purified from Aspergillus and run on one- and two-dimensional gels and the band or spot uncovered by the shift of the beta 1- and beta 2-tubulins was identified as a beta-tubulin by immunoblotting with monoclonal and affinity-purified polyclonal anti-tubulin antibodies and by one-dimensional peptide mapping. These observations show that Aspergillus contains at least two structural genes for beta-tubulins. Similar techniques have also been applied to a mutant showing altered alpha-tubulins to confirm and modify earlier observations suggesting that at least two structural genes for alpha-tubulins are also present.

Antibodies, Monoclonal↗
PubMed1984

Effects of mitotic and tubulin mutations on microtubule architecture in actively growing protoplasts of Aspergillus nidulans.

We used immunofluorescent microscopy to characterize microtubule (MT) architecture in wild-type and mutant protoplasts of Aspergillus nidulans at interphase and at mitosis. Because the visualization of MTs by immunofluorescence is technically difficult in intact hyphae of A. nidulans, we developed a method for removing the cell wall under conditions that do not perturb cell physiology, as evidenced by the fact that the resulting protoplasts undergo nuclear division at a normal rate and that cell cycle mutant phenotypes are expressed at restrictive temperature. Interphase cells exhibited an extensive network of cytoplasmic MTs. During mitosis the cytoplasmic MTs mostly disappeared and an intranuclear mitotic spindle appeared. We have previously shown that the benA 33 beta-tubulin mutation causes hyperstabilization of the mitotic spindle, and we have presented additional indirect evidence that suggested that the tubA1 and tubA4 alpha-tubulin mutations destabilize spindle MTs. In this paper, we show that the benA33 mutation increases the stability of cytoplasmic MTs as well as spindle MTs and that the tubA1 and tubA4 mutations destabilize both spindle and cytoplasmic MTs.

Aspergillus nidulans↗
PubMed1984

S-phase, G2, and nuclear division mutants of Aspergillus nidulans.

Twenty-two temperature-sensitive cell cycle mutants of the fungus Aspergillus nidulans, which block in interphase at restrictive temperature, were analyzed by the reciprocal shift method of Jarvik and Botstein (Proc. Nath Acad. Sci. U.S.A. 70:2046-2050, 1973) and Hereford and Hartwell (J. Mol. Biol. 84:445-461, 1974) to determine whether these mutations were blocked at the G1, S, or G2 phase of the cell cycle. We found five mutants to be blocked in S and nine to be blocked in G2. Two of the G2 mutants were atypical in that they were not able to accomplish the G2 to M transition at restrictive temperature but nevertheless could initiate subsequent cycles of DNA replication. None was blocked in G1. There were nine strains that could not be classified. The block imposed by restrictive temperature was irreversible in three of these strains, and the six other strains were unclassifiable due to their aberrant terminal nuclear phenotypes.

Aspergillus nidulans↗
PubMed1983

A mutation in Aspergillus nidulans that blocks the transition from interphase to prophase.

In order to develop a method for obtaining mitotic synchrony in aspergillus nidulans, we have characterized previously isolated heat-sensitive nim mutations that block the nuclear division cycle in interphase at restrictive temperature. After 3.5 h at restrictive temperature the mitotic index of a strain carrying one of these mutations, nimA5, was 0, but when this strain was subsequently shifted from restrictive to permissive temperature the mitotic index increased rapidly, reaching a maximum of 78 percent after 7.5 min. When this strain was examined electron-microscopically, mitotic spindles were absent at restrictive temperature. From these data we conclude that at restrictive temperature nimA5 blocks the nuclear division cycle at a point immediately preceding the initiation of chromosomal condensation and mitotic microtubule assembly, and upon shifting to permissive control over the initiation of microtubule assembly and chromosomal condensation in vivo through a simple temperature shift and, consequently, nimA5 should be a powerful tool for studying these processes. Electron-microscopic examination of spindles of material synchronized in this manner reveals that spindle formation, although very rapid, is gradual in the sense that spindle microtubule numbers increase as spindle formation proceeds.

Aspergillus nidulans↗
PubMed1983

Kinetics of the nuclear division cycle of Aspergillus nidulans.

We have analyzed the cell cycle kinetics of Aspergillus nidulans by using the DNA synthesis inhibitor hydroxyurea (HU) and a temperature-sensitive cell cycle mutant nimT that blocks in G2. HU rapidly inhibits DNA synthesis (S), and as a consequence progression beyond S to mitosis (M) is blocked. Upon removal of HU the inhibition is rapidly reversible. Conidia (asexual spores) of nimT were germinated at restrictive temperature to synchronize germlings in G2 and then downshifted to permissive temperature in the presence of HU. This procedure synchronizes the germlings at the beginning of S in the second cell cycle after spore germination. We have measured the total duration of S, G2, and M as the time required for these cells to recover from the HU block and undergo the next nuclear division. The duration of S was defined by the time course of sensitivity to reintroduction of HU during recovery from the initial HU block. The cell cycle time was measured as the nuclear doubling time, and the duration of mitosis was determined from the mitotic index. The duration of G1 was calculated by subtracting the combined durations of S, G2, and M from the nuclear doubling time, and the length of G2 was calculated by subtracting S and M from the aggregate length of S, G2, and M. We have also determined the duration of the phases of the cell cycle during the first cycle after spore germination. In these experiments spores were germinated directly in HU without first being blocked in G2. Because the durations of G1, S, G2, and M for the first cell cycle after spore germination were identical with those previously determined for spores presynchronized at the beginning of S in the second cell cycle, we conclude that dormant conidia of A. nidulans are arrested at, or before, the start of S.

Aspergillus nidulans↗
PubMed1981

A beta-tubulin mutation in Aspergillus nidulans that blocks microtubule function without blocking assembly.

We have isolated a heat-sensitive beta-tubulin mutation, benA33, that blocks nuclear division and nuclear movement at restrictive temperature. This blockage demonstrates that the beta tubulin encoded by the benA gene is essential to both processes. The blockage of both processes is suppressed by the alpha-tubulin mutation, tubA1; thus the alpha tubulin encoded by the tubA gene must also be involved in both processes. When benA33 is shifted from a permissive to restrictive temperature, nuclei are blocked in mitosis. Light microscopy of blocked nuclei reveals that benA33 inhibits movement of chromosomes to the poles, and electron microscopy of blocked nuclei shows that they contain apparently normal spindles. Thus benA33 does not block microtubule assembly but, directly or indirectly, blocks microtubule disassembly. BenA33 also confers resistance to several antimicrotubule agents and the heat sensitivity conferred by benA33 is suppressed by each of these agents. We suggest that the simplest explanation for these effects is that benA33 causes a temperature-dependent hyperstabilization of microtubules that blocks chromosomal movement by blocking microtubule disassembly.

Aspergillus nidulans↗