The PHOA and PHOB Cyclin-Dependent Kinases Perform an Essential Function in Aspergillus nidulans

Genetics ◽  
2003 ◽  
Vol 165 (3) ◽  
pp. 1105-1115
Author(s):  
Xiaowei Dou ◽  
Dongliang Wu ◽  
Weiling An ◽  
Jonathan Davies ◽  
Shahr B Hashmi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Aspergillus Nidulans ◽  
Nuclear Division ◽  
Cell Cycle Control ◽  
Dominant Negative ◽  
Null Alleles ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Cyclin Dependent Kinase ◽  
Essential Function

Abstract Unlike Pho85 of Saccharomyces cerevisiae, the highly related PHOA cyclin-dependent kinase (CDK) of Aspergillus nidulans plays no role in regulation of enzymes involved in phosphorous acquisition but instead modulates differentiation in response to environmental conditions, including limited phosphorous. Like PHO85, Aspergillus phoA is a nonessential gene. However, we find that expression of dominant-negative PHOA inhibits growth, suggesting it may have an essential but redundant function. Supporting this we have identified another cyclin-dependent kinase, PHOB, which is 77% identical to PHOA. Deletion of phoB causes no phenotype, even under phosphorous-limited growth conditions. To investigate the function of phoA/phoB, double mutants were selected from a cross of strains containing null alleles and by generating a temperature-sensitive allele of phoA in a ΔphoB background. Double-deleted ascospores were able to germinate but had a limited capacity for nuclear division, suggesting a cell cycle defect. Longer germination revealed morphological defects. The temperature-sensitive phoA allele caused both nuclear division and polarity defects at restrictive temperature, which could be complemented by expression of mammalian CDK5. Therefore, an essential function exists in A. nidulans for the Pho85-like kinase pair PHOA and PHOB, which may involve cell cycle control and morphogenesis.

scholarly journals MEIOTIC RECOMBINATION AND DNA SYNTHESIS IN A NEW CELL CYCLE MUTANT OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1978 ◽  
Vol 90 (1) ◽  
pp. 49-68
Author(s):  
Yona Kassir ◽  
Giora Simchen
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Division ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Essential Function ◽  
Normal Meiosis

ABSTRACT Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.

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

1987 ◽  
Vol 104 (6) ◽  
pp. 1495-1504 ◽  
Author(s):  
S A Osmani ◽  
G S May ◽  
N R Morris
Keyword(s):  
Cell Cycle ◽  
Aspergillus Nidulans ◽  
Cell Cycle Control ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Mrna Levels ◽  
Antimitotic Agent

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.

scholarly journals Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

Genetics ◽  
1993 ◽  
Vol 134 (1) ◽  
pp. 63-80 ◽  
Author(s):  
T A Weinert ◽  
L H Hartwell
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Control ◽  
Dna Lesions ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
A Cell ◽  
G2 Phase ◽  
Rad Mutants

Abstract In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

scholarly journals Mitotic mutants ofAspergillus nidulans

1975 ◽  
Vol 26 (3) ◽  
pp. 237-254 ◽  
Author(s):  
N. Ronald Morris
Keyword(s):  
Aspergillus Nidulans ◽  
Nuclear Division ◽  
Complementation Group ◽  
Gene Symbol ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Temperature Sensitive Mutants ◽  
Ofaspergillus Nidulans ◽  
Complementation Groups ◽  
Genetic Clustering

SUMMARYForty-five temperature-sensitive mutants ofAspergillus nidulanswhich are defective in nuclear division, septation or distribution of nuclei along the mycelium have been isolated, and most have been subjected to complementation analysis and mapped to chromosome. Thirty-five of the mutants were unable to complete nuclear division at the restrictive temperature. Twenty-six of these mutants exhibited a co-ordinate drop in both spindle and chromosome mitotic indices at 42 °C, indicating that they fail to enter mitosis. These mutants have been assigned to the gene symbolnim. Nine mutants exhibited a co-ordinate rise in spindle and chromosome mitotic indices at 42 °C, indicating that they are arrested in mitosis. These mutants were assigned the gene symbolbim. Five mutants failed to form septa and were given the gene symbolsep; and five mutants had an abnormal nuclear distribution and were given the gene symbolnud. All of the mutations were recessive. Most of the mutants were in different complementation groups. Mutants in the same complementation groups were phenotypically similar, but phenotypically similar mutants were not necessarily or usually in the same complementation group. There was no evidence for genetic clustering of phenotypically similar mutants. The mutants were located on all eight chromosomes.

scholarly journals hyp Loci Control Cell Pattern Formation in the Vegetative Mycelium of Aspergillus nidulans

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 669-680
Author(s):  
Susan G W Kaminskyj ◽  
John E Hamer
Keyword(s):  
Aspergillus Nidulans ◽  
Tip Growth ◽  
Nuclear Division ◽  
Apical Cell ◽  
Control Cell ◽  
Temperature Shift ◽  
Growth Cycle ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Apical Cells

Abstract Aspergillus nidulans grows by apical extension of multinucleate cells called hyphae that are subdivided by the insertion of crosswalls called septa. Apical cells vary in length and number of nuclei, whereas subapical cells are typically 40 μm long with three to four nuclei. Apical cells have active mitotic cycles, whereas subapical cells are arrested for growth and mitosis until branch formation reinitiates tip growth and nuclear divisions. This multicellular growth pattern requires coordination between localized growth, nuclear division, and septation. We searched a temperature-sensitive mutant collection for strains with conditional defects in growth patterning and identified six mutants (designated hyp for hypercellular). The identified hyp mutations are nonlethal, recessive defects in five unlinked genes (hypA-hypE). Phenotypic analyses showed that these hyp mutants have aberrant patterns of septation and show defects in polarity establishment and tip growth, but they have normal nuclear division cycles and can complete the asexual growth cycle at restrictive temperature. Temperature shift analysis revealed that hypD and hypE play general roles in hyphal morphogenesis, since inactivation of these genes resulted in a general widening of apical and subapical cells. Interestingly, loss of hypA or hypB function lead to a cessation of apical cell growth but activated isotropic growth and mitosis in subapical cells. The inferred functions of hypA and hypB suggest a mechanism for coordinating apical growth, subapical cell arrest, and mitosis in A. nidulans.

Cytokinesis in Aspergillus nidulans is controlled by cell size, nuclear positioning and mitosis

1996 ◽  
Vol 109 (8) ◽  
pp. 2179-2188 ◽  
Author(s):  
T.D. Wolkow ◽  
S.D. Harris ◽  
J.E. Hamer
Keyword(s):  
Aspergillus Nidulans ◽  
Cell Size ◽  
Nuclear Division ◽  
Size Control ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Nuclear Positioning ◽  
Septum Formation ◽  
Nuclear Distribution ◽  
Cellular Compartments

The mycelium of Aspergillus nidulans is composed of multinucleate cellular compartments delimited by crosswalls called septa. Septum formation is dependent on mitosis and requires the recruitment of actin to the site of septum formation. Employing a collection of temperature sensitive nuclear distribution (nudA2, nudC3 and nudF7), nuclear division (nimA5, hfaB3), and septation (sepD5, sepG1) mutants, we have investigated the interdependency among nuclear positioning, mitosis, and cell growth in structuring the cellular compartments of A. nidulans. The cellular compartments of nud+ strains were highly uniform with regard to nuclear distribution and averaged 38 microns in length. Incubation of nud mutants at semi-restrictive temperature resulted in aberrant nuclear distribution that appeared to direct the formation of variable-sized cellular compartments, ranging from 5 microns to greater than 81 microns. In germinating spores, the first septum forms at the basal end of the germ tube following the third round of nuclear division. Germlings must undergo mitosis in order to form a septum. Temperature-sensitive mitotic mutants were used to show that a single nuclear division is sufficient to activate septum formation, provided a critical cell size has been attained. In mitotic mutants and wild-type cells, delays in nuclear division resulted in the misplacement of the first septum. These results strongly support the role of mitotic nuclei in determining septal placement, and suggest that cell size control is post-mitotic in A. nidulans.

scholarly journals Regulation of mating in the cell cycle of Saccharomyces cerevisiae.

1977 ◽  
Vol 75 (2) ◽  
pp. 355-365 ◽  
Author(s):  
B J Reid ◽  
L H Hartwell
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
High Frequency ◽  
Nuclear Division ◽  
Diploid Cell ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Haploid Strain ◽  
Cdc 2

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).

scholarly journals Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis.

Genetics ◽  
1994 ◽  
Vol 136 (2) ◽  
pp. 517-532 ◽  
Author(s):  
S D Harris ◽  
J L Morrell ◽  
J E Hamer
Keyword(s):  
Aspergillus Nidulans ◽  
Nuclear Division ◽  
Temperature Shift ◽  
Mitotic Division ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
The Third ◽  
Septum Formation ◽  
New Genes

Abstract Filamentous fungi undergo cytokinesis by forming crosswalls termed septa. Here, we describe the genetic and physiological controls governing septation in Aspergillus nidulans. Germinating conidia do not form septa until the completion of their third nuclear division. The first septum is invariantly positioned at the basal end of the germ tube. Block-and-release experiments of nuclear division with benomyl or hydroxyurea, and analysis of various nuclear division mutants demonstrated that septum formation is dependent upon the third mitotic division. Block-and-release experiments with cytochalasin A and the localization of actin in germlings by indirect immunofluorescence showed that actin participated in septum formation. In addition to being concentrated at the growing hyphal tips, a band of actin was also apparent at the site of septum formation. Previous genetic analysis in A. nidulans identified four genes involved in septation (sepA-D). We have screened a new collection of temperature sensitive (ts) mutants of A. nidulans for strains that failed to form septa at the restrictive temperature but were able to complete early nuclear divisions. We identified five new genes designated sepE, G, H, I and J, along with one additional allele of a previously identified septation gene. On the basis of temperature shift experiments, nuclear counts and cell morphology, we sorted these cytokines mutants into three phenotypic classes. Interestingly, one class of mutants fails to form septa and fails to progress past the third nuclear division. This class of mutants suggests the existence of a regulatory mechanism in A. nidulans that ensures the continuation of nuclear division following the initiation of cytokinesis.

scholarly journals The Prpl  + Gene Required for Pre-mRNA Splicing in Schizosaccharomyces pombe Encodes a Protein That Contains TPR Motifs and Is Similar to Prp6p of Budding Yeast

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 101-115 ◽  
Author(s):  
Seiichi Urushiyama ◽  
Tokio Tani ◽  
Yasumi Ohshima
Keyword(s):  
Cell Cycle ◽  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Protein Interactions ◽  
Nuclear Export ◽  
Cell Cycle Progression ◽  
Synthetic Lethality ◽  
Cell Cycle Control ◽  
Mrna Splicing ◽  
Restrictive Temperature

Abstract The prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature. One of the prp mutants, prp1-4, also has a defect in poly(A)+ RNA transport. The prp1  + gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions. The amino acid sequence of Prplp shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly. No functional complementation was observed between S. pombe prp1  + and S. cerevisiae PRP6. We examined synthetic lethality of prp1-4 with the other known prp mutations in S. pombe. The results suggest that Prp1p interacts either physically or functionally with Prp4p, Prp6p and Prp13p. Interestingly, the prp1  + gene was found to be identical with the zer1  + gene that functions in cell cycle control. These results suggest that Prp1p/Zer1p is either directly or indirectly involved in cell cycle progression and/or poly(A)+ RNA nuclear export, in addition to pre-mRNA splicing.

The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis

1995 ◽  
Vol 108 (2) ◽  
pp. 475-486 ◽  
Author(s):  
F. al-Khodairy ◽  
T. Enoch ◽  
I.M. Hagan ◽  
A.M. Carr
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Cell Cycle Control ◽  
S Phase ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Ubiquitin Conjugating Enzyme ◽  
Efficient Recovery ◽  
Mediate Cell

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called ‘checkpoints’, mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

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