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J H Doonan

Publications and source records attributed to J H Doonan.

32 records · Page 2Linked to original sources
PubMed1994

Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants.

Transcripts from five cell cycle related genes accumulate in isolated cells dispersed throughout the actively dividing regions of plant meristems. We propose that this pattern reflects gene expression during particular phases of the cell division cycle. The high proportion of isolated cells suggests that synchrony between daughter cells is rapidly lost following mitosis. This is the first time that such a cell specific expression pattern has been described in a higher organism. Counterstaining with a DNA specific dye revealed that transcripts from three genes (two mitotic cyclins and a cdc2-like gene) accumulate during part of interphase and early mitosis whereas transcripts from a histone H4 gene are preferentially detected only in interphase cells. Double labelling for cyclin and histone H4 transcripts confirms that these genes are expressed in different cells, and therefore at different phases of the cell cycle. The results suggest that transcriptional regulation of cell cycle related genes may be important in controlling cell division in plants, and that these genes are useful markers for identifying cells at specific phases of the cell cycle within plant meristems.

Amino Acid Sequence↗
PubMed1992

Cell division in Aspergillus.

Amenable to sophisticated genetic and molecular analysis, the simple filamentous fungus Aspergillus nidulans has provided some novel insights into the mechanisms and regulation of cell division. Mutational analysis has identified over fifty genes necessary for nuclear division, nuclear movement and cytokinesis. Molecular and cellular analysis of these mutants has led to the discovery of novel components of the cytoskeleton as well as to clarifying the role of established cytoskeletal proteins. Mutations leading to defects in the kinases (i.e. p34cdc2) and phosphatases (i.e. cdc25 and PP1), which are known to regulate mitosis in other eukaryotes, have been identified in Aspergillus. Additional, as yet novel, mitotic regulatory molecules, encoded by the nimA and bimE genes, have also been discovered in Aspergillus.

Aspergillus nidulans↗
PubMed1991

A cDNA encoding rabbit muscle protein phosphatase 1 alpha complements the Aspergillus cell cycle mutation, bimG11.

The bimG11 allele causes a conditional growth defect in the fungus Aspergillus nidulans preventing both progression through mitosis and normal polar growth. Previously, we have shown that the bimG11 mutation increases the phosphorylation of nuclear proteins and that the gene encodes a protein similar to mammalian type 1 protein phosphatase. Assay of protein phosphatase activity in protein extracts of Aspergillus demonstrates directly that type 1 phosphatase activity is greatly reduced in the mutant at restrictive temperature. Expression of a muscle type 1 protein phosphatase fully complements all aspects of the bimG11 phenotype, and restores the level of PP1 activity to nearly normal. Expression of the related phosphatase, PP2A, does not complement the bimG11 mutation, showing that complementation can only be achieved by the type 1 gene. This clearly demonstrates that the phenotype of bimG11 is due to reduced PP1 activity and that the PP1 catalytic subunit is functionally conserved over a wide span of evolution.

Alleles↗
PubMed1991

Two alpha-tubulin genes of Aspergillus nidulans encode divergent proteins.

We have isolated and analyzed the tubA and tubB alpha-tubulin genes of Aspergillus nidulans. The nucleotide sequences of these genes predict polypeptides of 447 amino acids for tubA and 450 for tubB. The predicted amino acids sequences exhibit 28% divergence between the two polypeptides. This is the second known case of such high divergence between alpha-tubulins within the same species. The tubB gene is unique in that it codes for an extra glycine residue between what are usually the second and third amino acids. RNA blot analysis demonstrates that the tubA and tubB transcripts are each 1.8 kb long. The level of tubA transcript remains the same throughout the cell cycle. The level of tubB transcript does not change at any particular stage in the cell cycle but increases continuously during spore germination. The tubA gene was previously mapped to linkage group eight, and we have now mapped the tubB gene to linkage group four. Gene disruption in heterokaryons suggests that the phenotypic consequences of disruption are different for the tubA and tubB genes. Molecular disruption of tubA results in a block in nuclear division whereas in tubB it gives rise to abnormal cell and nuclear morphology.

Amino Acid Sequence↗
PubMed1989

The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1.

In Aspergillus nidulans, the temperature-sensitive, recessive cell cycle mutation bimG11 causes an elevated mitotic index at restrictive temperature and an inability to complete the anaphase separation of daughter nuclei. We have shown that this mutation has an abnormally high content of nuclear phosphoproteins and that the wild-type gene encodes a type 1 protein phosphatase. We conclude that dephosphorylation of a key protein(s) is required to complete mitosis.

Amino Acid Sequence↗
PubMed1989

The genetic analysis of mitosis in Aspergillus nidulans.

We describe here recent work on the molecular genetics of mitosis in the filamentous fungus Aspergillus nidulans. Aspergillus is one of three simple eukaryotes with powerful genetic systems that have been used to analyze mitosis. The modern molecular biological techniques available with this organism have made it possible to use mutations to identify genes and proteins that play an important role in mitosis. Three Aspergillus genes that affect mitosis are described. One gene, nimA, is specifically expressed late in the cell cycle and codes for a putative protein kinase that induces mitosis, even in cells blocked in S-phase. The second gene, bimG, codes for a putative phosphatase that interacts functionally with the nimA kinase. The third gene, bimE, codes for a protein that suppresses mitosis during interphase, apparently by keeping nimA turned off. None of these genes appear to be similar to any of the genes affecting mitosis that have been characterized in other eukaryotes, but rather appear to be elements of a system that prevents mitosis from occurring during interphase.

Amino Acid Sequence↗
PubMed1988

Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a gamma-actin.

The single actin gene from the filamentous fungus Aspergillus nidulans has been isolated and characterized. The only other organism reported to contain just one actin gene is another Ascomycete, the budding yeast Saccharomyces. The nucleotide sequence of the A. nidulans actin gene predicts a polypeptide containing the N-terminal sequence identifying the gamma-actin isotype. Until now this characteristic N terminus has only been reported to occur in vertebrate actin sequences. A monospecific anti-gamma-actin antiserum recognizes a single 42-kDa band in immunoblots of total Aspergillus protein. None of the six introns in the A. nidulans actin gene sequence aligns precisely with those found in other actin genes. One, unlike other known actin introns, is located in the 3'-untranslated region of the gene. The 5' and 3' ends of the gene have been characterized. The Aspergillus actin gene has a heterogeneous transcript size due to the presence of several different 3' termini. Of four characterized polyadenylated transcripts, only the longest contains a typical AATAAA polyadenylation signal near its 3' terminus. Using an integrative plasmid containing Aspergillus actin sequences and the pyr4 gene from Neurospora, the A. nidulans actin gene has been mapped to the first chromosome.

Actins↗
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↗
PubMed1987

An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus.

We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carota L.) using rhodaminyl lysine phallotoxin (RLP). In addition to conventional fixation with formaldehyde, we have used two different nonfixation methods before adding RLP: extracting cells in a stabilizing buffer; inducing transient pores in the plasma membrane with pulses of direct current (electroporation). These alternative methods for introducing RLP revealed additional features of the actin network not seen in aldehyde-fixed cells. The three-dimensional organization of this network in nonflattened cells was demonstrated by projecting stereopairs derived from through-focal series of computer-enhanced images. F-actin is present in interphase cells in four interconnected configurations: a meshwork surrounding the nucleus; thick cables in transvacuolar strands and deep in the cytoplasm; a finer network of bundles within the cortical cytoplasm; even finer filaments that run in ordered transverse array around the cell periphery. The actin network is organized differently during division but it does not disappear as do the cortical microtubules. RLP stains a central filamentous cortical band as the chromatin begins to condense (preprophase); it stains the mitotic spindle (as recently shown by Seagull et al. [Seagull, R. W., M. Falconer, and C. A. Weerdenburg, 1987, J. Cell Biol., 104:995-1004] for aldehyde fixed suspension cells) and the cytokinetic apparatus (as shown by Clayton, L., and C. W. Lloyd, 1985, Exp. Cell Res., 156:231-238). However, it is now shown that an additional network of F-actin persists in the cytoplasm throughout division associating in turn with the preprophase band, the mitotic spindle, and the cytokinetic phragmoplast.

Actin Cytoskeleton↗
PubMed1986

Anti-tubulin antibodies locate the blepharoplast during spermatogenesis in the fern Platyzoma microphyllum R.Br.: a correlated immunofluorescence and electron-microscopic study.

The discovery that the monoclonal anti-tubulin antibody YOL 1/34 recognizes a microtubule organizing centre, the blepharoplast (which arises de novo during the latter stages of spermatogenesis in the fern, Platyzoma microphyllum), has enabled us to follow it and associated microtubules throughout most of its ontogeny. By correlating electron-microscopic and immunofluorescence observations, YOL 1/34 is seen to stain the blepharoplast uniformly at a time when no microtubules are present within the organelle. Later, staining becomes intense at the surface, concomitant with the re-location of cylindrical channels to the periphery of the blepharoplast. During anaphase of the ultimate division of the spermatid mother cell the blepharoplast moves to the spindle poles and sharpens the otherwise barrel-shaped mitotic apparatus. Prior to this stage the blepharoplast is, however, off-centre and at variable positions around the poles. Later still, in the differentiating spermatids, the blepharoplast is the focus for radiating cytoplasmic microtubules that abut directly onto the electron-dense organelle, penetrating the ribosome-free halo. The three main conclusions are: that tubulin in a pre-microtubular form is associated with the cylindrical channels that arise de novo within the previously amorphous blepharoplast and act as a template in basal body formation; that the late appearance of the blepharoplast as a focus for the spindle poles during the final mitosis provides strong argument against its functioning during spindle pole initiation (despite its ability to sharpen the poles at anaphase); that the blepharoplast does seem to act as a microtubule organizing centre in the mitotically quiescent spermatid.

Antibodies, Monoclonal↗
PubMed1985

The cytoskeleton underlying side walls and cross walls in plants: molecules and macromolecular assemblies.

Plant cells organize their growth by reinforcing side walls during interphase (causing them to elongate) and by positioning and orienting the cross wall at cytokinesis. In the first part of this presentation we review progress made in identifying different cytoskeletal components that underlie side walls and that are involved in the deposition of the cross wall. During interphase, the cortical microtubule arrays co-distribute with an antigen recognized by a 'universal' monoclonal antibody to intermediate filaments. Using rhodaminyl-lysine-phalloidin no F-actin could be detected at the cortex but endoplasmic, axial cables were found. The cytokinetic apparatus--the phragmoplast--contains microtubules and we find that F-actin and the intermediate filament antigen also co-distribute with this array. We describe the three-dimensional arrangement of microtubules forming the interphase array in cells enlarging by both tip-growth and intercalary growth. In root hairs of higher plants and in apical cells of the filamentous stage of moss Physcomitrella patens, microtubules (MT) are detected at the apices and it is suggested from this that fragmentation of microtubules and absence of MTs from the tip are preparation artefacts. Using human serum from a scleroderma patient, possible microtubule nucleating sites are detected in meristematic cells; these segregate with the broad spindle poles and they surround the nucleus during early interphase--implying a peri-nuclear origin for the cortical MT array. The interphase microtubule array is described in terms of a dynamic helical model, which proposes: that the MT array is an integral complex; that microtubules form helices; that helices can change their pitch--the array converting to the various conformations.

Actins↗
PubMed1985

Immunofluorescence microscopy of microtubules in intact cell lineages of the moss, Physcomitrella patens. I. Normal and CIPC-treated tip cells.

Monoclonal antibodies to yeast tubulin have been used to visualize the distribution of microtubules in the intact filamentous protonemata of the moss Physcomitrella patens. Protonemata were prepared for immunofluorescence by fixation in formaldehyde and cells were made permeable with Driselase. Extensive cell files were preserved by 'blotting' the moss onto glutaraldehyde-derivatized coverslips. Problems due to fluorescence from chloroplasts were obviated by extraction with dimethyl sulphoxide and the non-ionic detergent, Nonidet NP40. These improvements allowed us to determine that microtubules were present throughout the cell cycle in the apical dome of caulonemal tip cells, that was a pronounced association of microtubules with the nucleus, that 'astral' microtubules were associated with the mitotic spindle and during anaphase may be involved in reorientation of the spindle before an oblique cytokinesis in caulonemata and that the cytokinetic phragmoplast appeared identical to the structure described for higher plants. Microtubules appeared to converge at the very tip of apical caulonemal cells and this was studied further by treating cells with CIPC--a drug that is known to produce multiple microtubule-organizing centres--and which here produces multiple foci for microtubules at the tip. These observations emphasize the involvement of microtubules in tip growth, alignment of the cell plate and nuclear migration--processes that are fundamental to the morphogenesis of filamentous organisms.

Antibodies, Monoclonal↗