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Cloning and analysis of an <i>Aspergillus nidulans</i> Sec7 domain coding geneYang, Yi 03 September 2003 (has links)
This study aimed to identify the genetic basis of the Aspergillus nidulans hypB5 mutant phenotype. A. nidulans is a filamentous fungus that is widely used as a cell biological and molecular genetic model system. Its hyphae grow by localized polar secretion, producing tubular cells. A. nidulans hypercellular strains define five unlinked genes, hypA1-hypE2, which cause hyphal morphogenesis defects at 42°C. hypA is orthologous to Saccharomyces TRS120, which mediates Golgi transit and is widely conserved. The hypB5 restrictive phenotype resembles hypA1: wide hyphae, short basal cells and small nuclei. Like hypA1, shifting hypB5 mutants from 28°C to 42°C causes cessation of tip growth but isotropic expansion of basal cells. A hypA1, hypB5 double mutant was impaired for growth at 28°C, suggesting these genes have related roles, but neither was epistatic at 37°C so they function in different pathways. The A. nidulans pRG3-AMA1 genomic library was used to clone hypB5 complementing DNA by phenotype rescue, and subcloned to a 5 kb KpnI fragment, pYY2. pYY2 was disrupted and sequenced by Tn1000 insertional mutagenesis. The pYY2 sequence is 4975 bp and encodes a putative Sec7 domain which has 81% identity to the Saccharomyces SEC7 domain. The Sec7 domain is highly conserved from yeasts to mammals. Saccharomyces SEC7 encodes a guanine nucleotide exchange factor involved in COPI vesicle formation and Golgi biogenesis. Insertions in the pYY2 non-Sec7 domain coding region complemented hypB5 efficiently, whereas those in the Sec7 domain did not, indicating that the Sec7 domain is sufficient for function. A point mutation was found in the hypB5 strain Sec7 domain, which could explain temperature sensitivity. However, the pYY2 sequence is found on chromosome I whereas hypB maps to chromosome VII. Although the origin and functional role of the point mutation in the hypB5 strain Sec7 protein remains unresolved, it appears that pYY2 contains an extragenic suppressor. Thus hypB likely encodes an element in the COPI vesicle assembly pathway.
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Cloning and analysis of an <i>Aspergillus nidulans</i> Sec7 domain coding geneYang, Yi 03 September 2003
This study aimed to identify the genetic basis of the Aspergillus nidulans hypB5 mutant phenotype. A. nidulans is a filamentous fungus that is widely used as a cell biological and molecular genetic model system. Its hyphae grow by localized polar secretion, producing tubular cells. A. nidulans hypercellular strains define five unlinked genes, hypA1-hypE2, which cause hyphal morphogenesis defects at 42°C. hypA is orthologous to Saccharomyces TRS120, which mediates Golgi transit and is widely conserved. The hypB5 restrictive phenotype resembles hypA1: wide hyphae, short basal cells and small nuclei. Like hypA1, shifting hypB5 mutants from 28°C to 42°C causes cessation of tip growth but isotropic expansion of basal cells. A hypA1, hypB5 double mutant was impaired for growth at 28°C, suggesting these genes have related roles, but neither was epistatic at 37°C so they function in different pathways. The A. nidulans pRG3-AMA1 genomic library was used to clone hypB5 complementing DNA by phenotype rescue, and subcloned to a 5 kb KpnI fragment, pYY2. pYY2 was disrupted and sequenced by Tn1000 insertional mutagenesis. The pYY2 sequence is 4975 bp and encodes a putative Sec7 domain which has 81% identity to the Saccharomyces SEC7 domain. The Sec7 domain is highly conserved from yeasts to mammals. Saccharomyces SEC7 encodes a guanine nucleotide exchange factor involved in COPI vesicle formation and Golgi biogenesis. Insertions in the pYY2 non-Sec7 domain coding region complemented hypB5 efficiently, whereas those in the Sec7 domain did not, indicating that the Sec7 domain is sufficient for function. A point mutation was found in the hypB5 strain Sec7 domain, which could explain temperature sensitivity. However, the pYY2 sequence is found on chromosome I whereas hypB maps to chromosome VII. Although the origin and functional role of the point mutation in the hypB5 strain Sec7 protein remains unresolved, it appears that pYY2 contains an extragenic suppressor. Thus hypB likely encodes an element in the COPI vesicle assembly pathway.
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The sumoylation and neddylation networks in Aspergillus nidulans developmentHarting, Rebekka 19 June 2013 (has links)
Proteine können post-translational durch Ubiquitin und Ubiquitin-ähnliche Proteine modifiziert werden. Dies erfordert die Aktivität dreier Enzyme. Das Protein wird durch ein E1 Enzym aktiviert, an ein E2 Enzym übertragen und im letzten Schritt mit der Hilfe von E3 Ligasen kovalent an das Substrat gebunden. Dieser Prozess der posttranslationalen Modifikation ist reversibel durch Isopeptidasen. Zu der Proteinfamilie gehören unter anderem Sumo und Nedd8. Beide Proteine sind im Modellorganismus Aspergillus nidulans konserviert. Während die Deletion des nedd8 Homologs neddH zum Zelltod führt, können Pilze ohne SumO überleben. Diese Stämme weisen jedoch Defekte in der sexuellen und asexuellen Entwicklung auf. In dieser Arbeit wurde die NeddH E3 Ligase DcnA/RbxA untersucht. DcnA interagierte mit der Neddylierungs-Maschinerie und die Deletion des Gens führte zu einer leichten Reduktion der Neddylierung von Cullinen. Diese verminderte Neddylierung hatte jedoch keine Auswirkungen auf die pilzliche Entwicklung unter Laborbedingungen. Das RING-finger Protein RbxA zeigt eine E3 Ligaseaktivität sowohl in der Ubiquitinierung als auch in der Neddylierung. Eine Deletion des betreffenden Gens führte zum Zelltod. In einer vorangegangenen Studie mit einem Stamm mit Defekt in der Isopeptidase CSN wurden Substratadaptoren des SCF Ubiquitin-E3-Ligase Komplexes (Fbox-Proteine) identifiziert. In dieser Arbeit wurde festgestellt, dass die biochemische Anreicherung von Fbox15 nicht auf eine generelle Stabilisierung des Proteins zurückzuführen ist, sondern wahrscheinlich auf eine Stabilisierung des SCF Komplexes mit Fbox15. Zusätzlich wurde der Prozess der Sumoylierung in A. nidulans untersucht. Unter normalen Wachstumsbedingungen ist nur ein kleiner Anteil der zellulären Proteine sumoyliert. Um diesen zu erhöhen, wurden die zwei SumO Isopeptidasen UlpA und UlpB untersucht. Durch biochemische Experimente im Wildtyp und einem Stamm, welchem die Isopeptidase UlpA fehlt, konnte ein komplexes SumO Netzwerk identifiziert werden. Zu diesem gehören neben den sumoylierenden Enzymen (E1, E2 und E3), Histon modifizierende Enzyme/Enzymkomplexe, andere Transkriptionsregulatoren, Proteine, die eine Rolle in der RNA-Reifung oder Stressantwort spielen, sowie Wechselwirkungen mit den Prozessen der Ubiquitinierung und Neddylierung. Eine wichtige Schnittstelle zwischen Sumoylierung und Histonmodifikation könnte hierbei der COMPASS Komplex sein. Dieser Komplex ist involviert in Histonmethylierung und damit in die Regulation der Transkription. Um ein besseres Verständnis für die Rolle des Komplexes in der Regulation der pilzlichen Entwicklung zu bekommen, wurde die Kerneinheit SetA deletiert. Der resultierende Stamm zeigte Defekte in der frühen sexuellen Entwicklung, im Koloniewachstum und Sekundärmetabolismus. SetA wurde als wichtiger Faktor für die richtige Positionierung der asexuellen Sporenträger identifiziert.
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DNA repair and mutagenesis in the UV-sensitive mutant UVSI of Aspergillus nidulansChae, Suhn-Kee January 1993 (has links)
The effects of a newly mapped DNA repair-defective mutant, uvsI, on mutagen sensitivities and mutation were investigated. Results showed that uvsI differs for most of the investigated properties from other uvs mutants of A. nidulans which are known to belong to three different epistatic groups, "UvsF", "UvsC", and "UvsB". Most of these mutants are sterile and many of them alter mitotic recombination frequencies, while uvsI exhibits normal levels of meiotic and mitotic recombination. In addition, uvsI strains are not more sensitive than wild type to MMS (methyl methanesulfonate) to which all other uvs strains are sensitive. However, the uvsI mutant was found to be very sensitive to the killing effects of UV light and the chemical mutagen, 4-NQO (4-nitro-quinoline-N-oxide). In line with the distinct phenotype of uvsI, no epistatic interactions were found for this mutant with any members of the established three epistatic groups. The effects of uvsI on mutagenesis are highly specific and dependent on the mutational test systems. In the uvsI mutant, two types of forward mutation were not affected, but spontaneous and UV-induced reversion frequencies of choA1 and pabaA1 were significantly reduced. Specific effects were further demonstrated in reversion tests of various sC alleles originally isolated as selenate resistant mutants by treatment with EMS (ethyl methanesulfonate), which leads mainly to G:C to A:T transitions. After EMS treatment uvsI mutants showed highly reduced reversion frequencies for all these sC alleles (except one) compared to $uvs sp+$ strains. These results suggest that the uvsI mutation may be defective in AT to GC transition mutagenesis, while increasing transversion(s) from A:T base pairs. In contrast, uvsI affected the frequencies of spontaneous and UV-induced reversions for these sC alleles in a variety of ways. Thus, uvsI may well represent a fourth functional and epistatic group of DNA repair and possibly be involved in a minor mutagenic DN
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Influences of the translocation T2 (1; VIII) on mitotic and meiotic recombination in Aspergillus nidulans.Ma, Gloria Ching Lai January 1972 (has links)
No description available.
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Induction and genetic analysis of UV-sensitive muitants in Aspergillus nidulans.De la Torre, Rosa Ana. January 1971 (has links)
No description available.
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Modification of iron binding ligands in isopenicillin N synthaseSami, Malkit January 1998 (has links)
Isopenicillin N synthase (IPNS) is a non-haem iron dependent dioxygenase which catalyses the oxidative conversion of anddelta;-(L-andalpha;-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN). Sequence comparisons between IPNS isozymes reveal the complete conservation of two histidine (His214, His270), one aspartate (Asp216) [also known as the '2-His-l-carboxylate' motif] and one glutamine (Gln330) residue. The crystal structure of IPNS (Aspergillus nidulans) active site (in the absence of ACV) revealed an octahedrally coordinated manganese atom surrounded by these four protein ligands and two water molecules. The role of the four conserved metal binding ligands was investigated using site directed mutagenesis. The results demonstrated that ligation of the iron with Gln330 was not essential for the catalytic activity of IPNS. In contrast, ligation of the iron with the three remaining metal ligands was indispensable for catalytic activity. Additionally, it was demonstrated that the conserved Asp216 residue may be substituted by a glutamate residue (D216E) with significant retention of catalytic activity. Crystallographic and spectroscopic evidence suggested that the D216E mutant bound both iron and ACV in a similar way to wild-type IPNS. The inactivation of wild-type IPNS was examined under in vitro assay conditions. This study showed that inactivation of IPNS results (minimally) from a slow non-oxidative pathway (in buffer alone) and a fast oxidative pathway via Udenfriend's chemistry (ferrous iron, ascorbate, and oxygen). The oxidative inactivation pathway was substantially reduced by the inclusion of catalase in the assay mixture, thus indicating that oxidative IPNS inactivation results (in part) from the generation of hydrogen peroxide in solution. Inactivation was also accompanied by a slow fragmentation of intact IPNS into (at least) five oligopeptides (observed by sodium dodecyl sulphate polyacrylamide gel electrophoresis). N-Terminal sequencing analyses confirmed that the fragmentation resulted from at least two cleavage sites within the active site (between Asp216-Val217 and Val272-Lys273).
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Mutational analysis of creA, the mediator of carbon catabolite repression in Aspergillus nidulans / Robert Shnoff.Shnoff, Robert January 1997 (has links)
Addendum pasted on front end paper. / Copies of author's previously published articles inserted. / Bibliography: leaves 180-196. / xi, 198, [23] leaves, [16] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to define functionally important regions within the CreA protein. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1998
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The cloning and preliminary characterization of the creA gene from Aspergillus nidulans / by Celia E.A. Dowzer.Dowzer, Celia E. A. (Celia Elizabeth Anne) January 1991 (has links)
Bibliography: leaves 164-199. / ix, 199, [19] leaves, [17] leaves of plates : ill. (some. col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Studies the cloning and characterisation of the Aspergillus nidulans creA gene whose product is suggested as a negatively acting regulatory protein in carbon catabolite repression. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1991
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The DNA damage response of Aspergillus nidulans links to programmed cell death and hyphal morphogenesis /Semighini, Camile P. January 1900 (has links)
Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed Feb. 19, 2008). PDF text: vi, 222 p. : ill. (some col.) ; 58 Mb. UMI publication number: AAT 3271919. Includes bibliographical references. Also available in microfilm and microfiche formats.
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