scholarly journals Isolation of mip (microtubule-interacting protein) mutations of Aspergillus nidulans.

1986 ◽  
Vol 6 (8) ◽  
pp. 2963-2968 ◽  
Author(s):  
C F Weil ◽  
C E Oakley ◽  
B R Oakley
Keyword(s):  
Aspergillus Nidulans ◽  
Chromosomal Rearrangements ◽  
Cold Sensitivity ◽  
Heat Sensitivity ◽  
Wild Type ◽  
Beta Tubulin ◽  
Interacting Protein ◽  
Allele Specific ◽  
Extragenic Suppressors ◽  
Microtubule Function

We identified four mutations in two previously undescribed loci involved in microtubule function in Aspergillus nidulans as extragenic suppressors of benA33, a heat-sensitive beta-tubulin mutation. Three of the four mutations map to a locus closely linked to riboB on linkage group VIII; we designated this locus mipA (for microtubule-interacting protein). We were not able to map the remaining suppressor because of chromosomal rearrangements. However, since it recombines with riboB at a significantly higher frequency than the mipA alleles, it is unlikely to be in mipA; thus, we designated it mipB1. The mip mutations are not allelic to the previously identified loci that encode alpha- and beta-tubulin, and it is likely that mipA and mipB encode previously unidentified nontubulin proteins involved in microtubule function. Each of the mip mutations suppresses the heat sensitivity conferred by benA33 and suppresses the blockage of nuclear division and movement conferred by this mutation at high temperatures. Interactions between mipA and benA are allele specific. All of the mipA mutations are cryptic in a wild-type benA background but cause cold sensitivity in combination with benA33. These mutations also confer cold sensitivity in combination with benA31 and benA32 and reduce the resistance conferred by these mutations to the antimicrotubule agent benomyl but do not suppress the heat sensitivity conferred by these alleles. Finally, the mipA alleles suppress the heat sensitivity conferred by benA11, benA17, and benA21 but do not confer cold sensitivity in combination with these alleles.

scholarly journals Isolation of mip (microtubule-interacting protein) mutations of Aspergillus nidulans

1986 ◽  
Vol 6 (8) ◽  
pp. 2963-2968
Author(s):  
C F Weil ◽  
C E Oakley ◽  
B R Oakley
Keyword(s):  
Aspergillus Nidulans ◽  
Chromosomal Rearrangements ◽  
Cold Sensitivity ◽  
Heat Sensitivity ◽  
Wild Type ◽  
Beta Tubulin ◽  
Interacting Protein ◽  
Allele Specific ◽  
Extragenic Suppressors ◽  
Microtubule Function

We identified four mutations in two previously undescribed loci involved in microtubule function in Aspergillus nidulans as extragenic suppressors of benA33, a heat-sensitive beta-tubulin mutation. Three of the four mutations map to a locus closely linked to riboB on linkage group VIII; we designated this locus mipA (for microtubule-interacting protein). We were not able to map the remaining suppressor because of chromosomal rearrangements. However, since it recombines with riboB at a significantly higher frequency than the mipA alleles, it is unlikely to be in mipA; thus, we designated it mipB1. The mip mutations are not allelic to the previously identified loci that encode alpha- and beta-tubulin, and it is likely that mipA and mipB encode previously unidentified nontubulin proteins involved in microtubule function. Each of the mip mutations suppresses the heat sensitivity conferred by benA33 and suppresses the blockage of nuclear division and movement conferred by this mutation at high temperatures. Interactions between mipA and benA are allele specific. All of the mipA mutations are cryptic in a wild-type benA background but cause cold sensitivity in combination with benA33. These mutations also confer cold sensitivity in combination with benA31 and benA32 and reduce the resistance conferred by these mutations to the antimicrotubule agent benomyl but do not suppress the heat sensitivity conferred by these alleles. Finally, the mipA alleles suppress the heat sensitivity conferred by benA11, benA17, and benA21 but do not confer cold sensitivity in combination with these alleles.

scholarly journals Systematic mutational analysis of the yeast beta-tubulin gene.

1994 ◽  
Vol 5 (1) ◽  
pp. 29-43 ◽  
Author(s):  
R A Reijo ◽  
E M Cooper ◽  
G J Beagle ◽  
T C Huffaker
Keyword(s):  
Mutational Analysis ◽  
Cold Sensitivity ◽  
Heat Sensitivity ◽  
Restrictive Temperature ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Systematic Strategy ◽  
Cytoplasmic Microtubules ◽  
Spindle Function ◽  
Beta Tubulin Gene

A systematic strategy was used to create a synoptic set of mutations that are distributed throughout the single beta-tubulin gene of Saccharomyces cerevisiae. Clusters of charged amino acids were targeted for mutagenesis and converted to alanine to maximize alterations on the protein's surface and minimize alterations that affect protein folding. Of the 55 mutations we constructed, three confer dominant-lethality, 11 confer recessive-lethality, 10 confer cold-sensitivity, one confers heat-sensitivity, and 27 confer altered resistance to benomyl. Only 11 alleles give no discernible phenotype. In spite of the fact that beta-tubulin is a highly conserved protein, three-fourths of the mutations do not destroy the ability of the protein to support the growth of yeast at 30 degrees C. The lethal substitutions are primarily located in three regions of the protein and presumably identify domains most critical for beta-tubulin function. Interestingly, most of the conditional-lethal alleles produce specific defects in spindle assembly at their restrictive temperature; cytoplasmic microtubules are relatively unaffected. The exceptions are two mutants that contain abnormally long cytoplasmic microtubules. Mutants with specific spindle defects were not observed in our previous collection of beta-tubulin mutants and should be valuable in dissecting spindle function.

scholarly journals Mitochondria and nuclei move by different mechanisms in Aspergillus nidulans.

1985 ◽  
Vol 101 (6) ◽  
pp. 2392-2397 ◽  
Author(s):  
B R Oakley ◽  
J E Rinehart
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungus ◽  
Colony Formation ◽  
Nuclear Division ◽  
Restrictive Temperature ◽  
Nuclear Movement ◽  
Beta Tubulin ◽  
Mitochondrial Movement ◽  
Tubulin Mutations ◽  
Microtubule Function

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.

scholarly journals Potential Link between the NIMA Mitotic Kinase and Nuclear Membrane Fission during Mitotic Exit in Aspergillus nidulans

2004 ◽  
Vol 3 (6) ◽  
pp. 1433-1444 ◽  
Author(s):  
Jonathan R. Davies ◽  
Aysha H. Osmani ◽  
Colin P. C. De Souza ◽  
Catherine Bachewich ◽  
Stephen A. Osmani
Keyword(s):  
Cell Cycle ◽  
Aspergillus Nidulans ◽  
Nuclear Membrane ◽  
Cell Cycle Progression ◽  
Colony Growth ◽  
Cold Sensitivity ◽  
Interacting Protein ◽  
Amino Terminal ◽  
Mitotic Kinase ◽  
Membrane Fission

ABSTRACT We have isolated TINC as a NIMA-interacting protein by using the yeast two-hybrid system and have confirmed that TINC interacts with NIMA in Aspergillus nidulans. The TINC-NIMA interaction is stabilized in the absence of phosphatase inhibitors and in the presence of kinase-inactive NIMA, suggesting that the interaction is enhanced when NIMA is not fully activated. TINC is a cytoplasmic protein. TINC homologues and a TINC-like protein (A. nidulans HETC) are conserved in other filamentous fungi. Neither deletion of tinC nor deletion of both tinC and A. nidulans hetC is lethal, but deletion of tinC does produce cold sensitivity as well as osmotic sensitivity. Expression of an amino-terminal-truncated form of TINC (ΔN-TINC) inhibits colony growth in Aspergillus and localizes to membrane-like structures within the cell. Examination of cell cycle progression in these cells reveals that they progress through multiple defective mitoses. Many cells contain large polyploid single nuclei, while some appear to have separated masses of DNA. Examination of the nuclear envelopes of cells containing more than one DNA mass reveals that both DNA masses are contained within a single nuclear envelope, indicating that nuclear membrane fission is defective. The ability of these cells to separate DNA segregation from nuclear membrane fission suggests that this coordination is normally a regulated process in A. nidulans. Additional experiments demonstrate that expression of ΔN-TINC results in premature NIMA disappearance in mitotic samples. We propose that TINC's interaction with NIMA and the cell cycle defects produced by ΔN-TINC expression suggest possible roles for TINC and NIMA during nuclear membrane fission.

scholarly journals The highly divergent beta-tubulins of Aspergillus nidulans are functionally interchangeable.

1989 ◽  
Vol 109 (5) ◽  
pp. 2267-2274 ◽  
Author(s):  
G S May
Keyword(s):  
Aspergillus Nidulans ◽  
Nuclear Division ◽  
Selectable Marker ◽  
Wild Type ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Tubulin Genes ◽  
Internal Gene ◽  
Type Strains ◽  
Beta Tubulin Gene

An internal 1.4-kb Bst EII fragment was used to disrupt the benA gene and establish heterokaryons. The heterokaryons demonstrated that the molecular disruption of benA results in a recessive benA null mutation. Conidia from a heterokaryon swell and germinate but cannot undergo nuclear division and are thus inviable. A chimeric beta-tubulin gene was constructed with the benA promoter driving the tubC structural gene. This chimeric gene construction was placed on a plasmid containing a selectable marker for Aspergillus transformation and the gene disrupting fragment of benA. Integration of this plasmid at benA by the internal gene disrupting fragment of benA simultaneously disrupts the benA gene and replaces it with the chimeric beta-tubulin gene, rescuing the benA null generated by the integration. Strains generated by this procedure contain only tubC beta-tubulin for all beta-tubulin functions. Strains having only tubC beta-tubulin are viable and exhibit no detectable microtubule dysfunction though they are more sensitive than wild-type strains to the antimicrotubule drug benomyl. It is concluded that the two beta-tubulin genes of Aspergillus nidulans, though highly divergent, are interchangeable.

scholarly journals MITOCHONDRIAL FOUR-POINT CROSSES IN ASPERGILLUS NIDULANS: MAPPING OF A SUPPRESSOR OF A MITOCHONDRIALLY INHERITED COLD-SENSITIVE MUTATION

Genetics ◽  
1983 ◽  
Vol 103 (3) ◽  
pp. 409-428
Author(s):  
Richard B Waring ◽  
Claudio Scazzocchio
Keyword(s):  
Aspergillus Nidulans ◽  
Frequency Distribution ◽  
Cold Sensitivity ◽  
Wild Type ◽  
Selection Technique ◽  
Marker Selection ◽  
Cold Sensitive ◽  
Original Point

ABSTRACT Four-point mitochondrial crosses were conducted in heterokaryons of Aspergillus nidulans. The mutations used were (oliA1), conferring resistance to oligomycin, (camA112), conferring resistance to chloramphenicol; (cs-67), conferring cold-sensitivity, and (sumD16), a suppressor of (cs-67). Initially, the crosses were conducted by observing the segregation of extranuclear markers in heterokaryotic sectors emerging from the original point of heterokaryosis. This showed that (camA112), (cs-67) and (sumD16) were linked but were probably all unlinked to (oliA1). Second, four-point crosses were conducted using a double marker selection technique, in which (camA112) and (oliA1) were always set in repulsion and the frequency of the phenotypes produced by the segregation of the mutant and wild-type alleles of (cs-67) and (sumD) were observed in (camA112 oliA1) recombinants. From these results we concluded that (camA112), (cs-67) and (sumD16) were linked and probably mapped in the order given. It was observed that the two nuclear types of conidia from a heterokaryon often had a dissimilar frequency distribution of the segregants of a mitochondrial cross.

scholarly journals The SONBNUP98 Nucleoporin Interacts With the NIMA Kinase in Aspergillus nidulans

Genetics ◽  
2003 ◽  
Vol 165 (3) ◽  
pp. 1071-1081
Author(s):  
Colin P C De Souza ◽  
Kevin P Horn ◽  
Kathryn Masker ◽  
Stephen A Osmani
Keyword(s):  
Aspergillus Nidulans ◽  
Nuclear Accumulation ◽  
Cold Sensitivity ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Physical Interaction ◽  
Restrictive Temperature ◽  
Histone H2a ◽  
Cold Sensitive ◽  
Extragenic Suppressors

Abstract The Aspergillus nidulans NIMA kinase is essential for mitotic entry. At restrictive temperature, temperature-sensitive nimA alleles arrest in G2, before accumulation of NIMA in the nucleus. We performed a screen for extragenic suppressors of the nimA1 allele and isolated two cold-sensitive son (suppressor of nimA1) mutants. The sonA1 mutant encoded a nucleoporin that is a homolog of yeast Gle2/Rae1. We have now cloned SONB, a second nucleoporin genetically interacting with NIMA. sonB is essential and encodes a homolog of the human NUP98/NUP96 precursor. Similar to NUP98/NUP96, SONBNUP98/NUP96 is autoproteolytically cleaved to generate SONBNUP98 and SONBNUP96. SONBNUP98 localizes to the nuclear pore complex and contains a GLEBS domain (Gle2 binding sequence) that binds SONAGLE2. A point mutation within the GLEBS domain of SONB1NUP98 suppresses the temperature sensitivity of the nimA1 allele and compromises the physical interaction between SONAGLE2 and SONB1NUP98. The sonB1 mutation also causes sensitivity to hydroxyurea. We isolated the histone H2A-H2B gene pair as a copy-number suppressor of sonB1 cold sensitivity and hydroxyurea sensitivity. The data suggest that the nucleoporins SONAGLE2 and SONBNUP98 and the NIMA kinase interact and regulate nuclear accumulation of mitotic regulators to help promote mitosis.

scholarly journals An Extragenic Suppressor of the Mitosis-Defective bimD6 Mutation of Aspergillus nidulans Codes for a Chromosome Scaffold Protein

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 777-787 ◽  
Author(s):  
Cydne L Holt ◽  
Gregory S May
Keyword(s):  
Aspergillus Nidulans ◽  
Chromosome Segregation ◽  
Chromosome Structure ◽  
Heat Sensitivity ◽  
Restrictive Temperature ◽  
Mitotic Spindles ◽  
Normal Number ◽  
Cold Sensitive ◽  
Extragenic Suppressors ◽  
Chromosome Scaffold

Abstract We previously identified a gene, bimD, that functions in chromosome segregation and contains sequences suggesting that it may be a DNA-binding protein. Two conditionally lethal mutations in bimD arrest with aberrant mitotic spindles at restrictive temperature. These spindles have one-third the normal number of microtubules, and the chromosomes never attach to the remaining microtubules. For this reason, we hypothesized that BIMD functioned in chromosome segregation, possibly as a component of the kinetochore. To identify other components that function with bimD, we conducted a screen for extragenic suppressors of the bimD5 and bimD6 mutations. We have isolated seven cold-sensitive extragenic suppressors of bimD6 heat sensitivity that represent three or possibly four separate sud genes. We have cloned one of the suppressor genes by complementation of the cold-sensitive phenotype of the sudA3 mutation. SUDA belongs to the DA-box protein family. DA-box proteins have been shown to function in chromosome structure and segregation. Thus bimD and the sud genes cooperatively function in chromosome segregation in Aspergillus nidulans.

scholarly journals Involvement of Very Short DNA Tandem Repeats and the Influence of the RAD52 Gene on the Occurrence of Deletions in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 549-557 ◽  
Author(s):  
Anne J Welcker ◽  
Jacky de Montigny ◽  
Serge Potier ◽  
Jean-Luc Souciet
Keyword(s):  
Fusion Proteins ◽  
Tandem Repeats ◽  
Chromosomal Rearrangements ◽  
Reversion Rate ◽  
Wild Type ◽  
Recombination Mechanism ◽  
Nonhomologous Recombination ◽  
Deletion Rate ◽  
Dna Tandem Repeats

Abstract Chromosomal rearrangements, such as deletions, duplications, or Ty transposition, are rare events. We devised a method to select for such events as Ura+ revertants of a particular ura2 mutant. Among 133 Ura+ revertants, 14 were identified as the result of a deletion in URA2. Of seven classes of deletions, six had very short regions of identity at their junctions (from 7 to 13 bp long). This strongly suggests a nonhomologous recombination mechanism for the formation of these deletions. The total Ura+ reversion rate was increased 4.2-fold in a rad52Δ strain compared to the wild type, and the deletion rate was significantly increased. All the deletions selected in the rad52Δ context had microhomologies at their junctions. We propose two mechanisms to explain the occurrence of these deletions and discuss the role of microhomology stretches in the formation of fusion proteins.

scholarly journals Isolation of a gene required for programmed initiation of development by Aspergillus nidulans.

1992 ◽  
Vol 12 (9) ◽  
pp. 3827-3833 ◽  
Author(s):  
T H Adams ◽  
W A Hide ◽  
L N Yager ◽  
B N Lee
Keyword(s):  
Life Cycle ◽  
Aspergillus Nidulans ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Nutrient Deprivation ◽  
Deletion Mutants ◽  
Wild Type ◽  
Microbial Development ◽  
Type Strains ◽  
Posttranscriptional Level

In contrast to many other cases in microbial development, Aspergillus nidulans conidiophore production initiates primarily as a programmed part of the life cycle rather than as a response to nutrient deprivation. Mutations in the acoD locus result in "fluffy" colonies that appear to grow faster than the wild type and proliferate as undifferentiated masses of vegetative cells. We show that unlike wild-type strains, acoD deletion mutants are unable to make conidiophores under optimal growth conditions but can be induced to conidiate when growth is nutritionally limited. The requirement for acoD in conidiophore development occurs prior to activation of brlA, a primary regulator of development. The acoD transcript is present both in vegetative hyphae prior to developmental induction and in developing cultures. However, the effects of acoD mutations are detectable only after developmental induction. We propose that acoD activity is primarily controlled at the posttranscriptional level and that it is required to direct developmentally specific changes that bring about growth inhibition and activation of brlA expression to result in conidiophore development.

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