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N R Morris

Publications and source records attributed to N R Morris.

At least 55 records · Page 3Linked to original sources
PubMed1992

Mitotic regulation in Aspergillus nidulans.

The nimA and bimE genes of Aspergillus nidulans respectively encode a 79 kDa protein kinase that is a positive regulator of mitosis and a 229 kDa protein that is a negative regulator of mitosis. Either overproduction of nimA or inactivation of bimE can induce mitosis and override the checkpoint associated with incomplete DNA replication. Double mutants between temperature-sensitive nimA and bimE alleles undergo chromatin condensation and spindle polymerization at restrictive temperature, suggesting that the p79nimA kinase is not required for chromatin condensation and spindle polymerization when bimE function is defective. In contrast double mutants carrying ts bimE and nimEcyclinB mutations or bimE and nimTcdc25 mutations are blocked in interphase at restrictive temperature. These results indicate that the mitotic block caused by inactivation of bimE requires activation of the p34cdc2/MPF kinase for chromatin condensation and spindle polymerization to occur. Antibodies against bimE fusion proteins have been used to study p229bimE in wild-type cells and cells overexpressing the bimE gene.

Aspergillus nidulans↗
PubMed1992

An extra copy of nimEcyclinB elevates pre-MPF levels and partially suppresses mutation of nimTcdc25 in Aspergillus nidulans.

Previous work has shown that nimA encodes a cell cycle regulated protein kinase that is required along with the p34cdc2 histone H1 kinase (MPF) for mitosis in Aspergillus nidulans. We have now identified two other gene products required for mitosis in A.nidulans. nimT encodes a protein similar to the fission yeast cdc25 tyrosine phosphatase and is required for the conversion of pre-MPF to MPF and nimE encodes a B-type cyclin which is a subunit of MPF. A new genetic interaction between nimEcyclinB and nimTcdc25 type genes is reported. Increased copy number of nimEcyclinB can suppress mutation of nimTcdc25 and also lead to increased accumulation of tyrosine phosphorylated p34cdc2 (pre-MPF). This biochemical observation suggests an explanation for the genetic complementation. If nimEcyclinB recruits p34cdc2 for tyrosine phosphorylation to form pre-MPF it follows that increased expression of nimEcyclinB would increase the level of pre-MPF. The increased level of pre-MPF generated may then allow the mutant nimTcdc25 protein to convert enough pre-MPF to MPF and thus permit some mitotic progression. We also demonstrate that correct cell cycle regulation by the p34cdc2 protein kinase pathway is essential for correct developmental progression in A.nidulans.

Amino Acid Sequence↗
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↗
PubMed1991

The tubB alpha-tubulin gene is essential for sexual development in Aspergillus nidulans.

The filamentous fungus Aspergillus nidulans has two genes encoding alpha-tubulin, tubA and tubB. Mutational analysis of tubA has demonstrated that the tubA gene is essential for mitosis and nuclear migration. In this study we have deleted the tubB gene by replacing it with a selectable marker and have named this new allele tubB delta. The results demonstrate that the tubB gene is not required for vegetative growth or asexual reproduction, nor is it required for the initiation or early stages of sexual differentiation. Deletion of tubB, however, completely prevents ascosporogenesis, because tubB delta strains produce no sexual spores when self-crossed. These strains produce viable ascospores when outcrossed to tubB+ strains, indicating that the tubB delta mutation is recessive. We have studied the cytology of sexual development in wild-type strains and in the tubB mutant and have observed that tubB delta. strains develop normally to the stage of ascus formation. However, only a single nuclear mass is observed in the tubB delta ascus, indicating that either the two zygotic haploid nuclei are blocked in karyogamy or that karyogamy occurs but the resulting diploid nucleus is subsequently blocked in meiosis I.

Aspergillus nidulans↗
PubMed1991

bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans.

The recessive, temperature-sensitive bimA1 mutation of Aspergillus nidulans blocks nuclei in metaphase at restrictive temperature. To determine whether the bimA product is essential, integrative transformation was used to create a mutation in the bimA gene. The mutation was maintained in a heterokaryon and the phenotype of spores produced by the heterokaryon was analyzed. Molecular disruption of the wild-type bimA gene is recessive in the heterokaryon and causes a metaphase block, demonstrating that bimA is an essential gene for mitosis. bimA was cloned by DNA-mediated complementation of its mutant phenotype at restrictive temperature, and the nucleotide sequence of a full-length cDNA was determined. A single large open reading frame was identified in the cDNA sequence, which predicts a protein containing 806 amino acid residues that is related (30.4% identity) to the Schizosaccharomyces pombe nuc2+ gene product, which also is required for completion of mitosis. The sequence of the bimA gene indicates that it is a member of a family of mostly nuclear proteins that contain a degenerate 34 amino acid repeat, the TPR (tetratricopeptide repeat) gene family.

Amino Acid Sequence↗
PubMed1990

A negative regulator of mitosis in Aspergillus is a putative membrane-spanning protein.

The temperature-sensitive cell cycle mutation bimE7 of Aspergillus nidulans causes cells to become blocked in mitosis at a restrictive temperature. Previous work has shown that this mitotic block is induced even when cells are arrested in the S or G2 phase. The mitotic block is also observed in cells carrying a null mutation in bimE, obtained by molecular disruption of the gene (Osmani, S.A., Engle, D.B., Doonan, J.H., and Morris, N.R. (1988) Cell 52, 241-251), indicating that a lack of bimE function is responsible for the phenotype. We have cloned the bimE gene by complementation of the mutant phenotype and have isolated and sequenced its corresponding cDNA. The gene product is encoded by a 6.5-7-kilobase mRNA. The deduced amino acid sequence suggests a protein with three transmembrane domains. The sequence contains numerous potential N-glycosylation sites and several putative cAMP-dependent phosphorylation sites. No homologous protein sequences were found in the common data bases. The bimE gene product is a novel component in the regulation of mitosis.

Amino Acid Sequence↗
PubMed1990

Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans.

In A. nidulans, the temperature-sensitive cell cycle mutation bimC4 causes an elevated mitotic index at restrictive temperature. Under restrictive conditions the mutation interferes with separation of the spindle pole bodies, causes abnormal spindle morphology, and prevents nuclear division. We have cloned and sequenced the wild-type bimC gene. The predicted protein product has homology to Drosophila kinesin heavy chain. We conclude that this kinesin-like protein has an important role in nuclear division in Aspergillus.

Adenosine Triphosphatases↗
PubMed1990

Increasing tubC beta-tubulin synthesis by placing it under the control of a benA beta-tubulin upstream sequence causes a reduction in benA beta-tubulin level but has no effect on microtubule function.

We have constructed a chimeric beta-tubulin gene that places the structural gene for the tubC beta-tubulin of Aspergillus nidulans under the control of the benA beta-tubulin promoter. Introduction of either this chimeric gene or a second wild-type benA gene into a benomyl-resistant benA22 strain causes it to become benomyl sensitive, indicating that the introduced genes are functional. Analysis of the tubulin proteins synthesized in benA22 strains into which a second wild-type benA beta-tubulin gene was transformed showed that the total amount of beta-tubulin protein was the same as in the parental strain with a single benA gene. Thus the level of beta-tubulin must be regulated. This was also true of transformants carrying an extra copy of the chimeric beta-tubulin gene. The total amount of beta-tubulin was the same as in the parental strain. Two-dimensional gel analysis showed that the endogenous benA22 and the introduced chimeric tubC gene contributed equally to the total beta-tubulin pool. The fact that one-half of the benA beta-tubulin could be replaced by tubC beta-tubulin with no effect on the growth of the cells suggests that the benA and tubC beta-tubulins are functionally interchangeable.

Amino Acid Sequence↗
PubMed1990

The molecular cloning and identification of a gene product specifically required for nuclear movement in Aspergillus nidulans.

A temperature-sensitive mutation in the nudC gene (nudC3) of Aspergillus nidulans specifically prevents the microtubule-based movement of nuclei in this organism at the restrictive temperature. The mutation does not affect short term growth, nuclear division, or the movement of other subcellular organelles. Immunofluorescence analysis of cells blocked at the restrictive temperature, using antitubulin antibodies, shows that the inability of nuclei to move under these conditions is not related to an inability of a particular class of microtubule to form. The inability to move nuclei in this mutant is also shown to be independent of both mitosis and the number of nuclei in the cell as a double mutant carrying both nudC3 and a cell cycle-specific mutation blocks with a single immotile nucleus at the restrictive temperature. The molecular cloning of the nudC gene and sequence analysis reveal that it encodes a previously unidentified protein of 22 kd. Affinity-purified antisera reactive to the nudC protein cross reacts to a single protein of 22 kD in Aspergillus protein extracts. This purified sera failed to reveal a subcellular location for the nudC protein at the level of indirect immunofluorescence. The data presented suggest that the 22-kD nudC gene product functions by interacting between microtubules and nuclei and/or is involved in the generation of force used to move nuclei during interphase.

Amino Acid Sequence↗
PubMed1989

Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes.

Plasmids have been constructed in which expression of a gene can be placed under the control of the inducible promoter of the alcA gene encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain alpha-peptide expression. The beta-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of beta-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.

Alcohol Dehydrogenase↗
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↗