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Chromosome-substituted haploid segregants were selected from among the benomyl-induced progeny of an interspecific hybrid produced by polyethylene-glycol-induced fusion of protoplasts of an Aspergillus nidulans master strain and an A. quadrilineatus auxotrophic mutant. These segregants were examined by RFLP, RAPD, and isoenzyme analysis. The A. nidulans ribosomal repeat unit was assigned to chromosome V, while the benA and the pyrG genes were assigned to linkage groups VIII and I, respectively, of A. nidulans. None of the other cloned genes tested (gdhA, amdS and 25s rRNA) showed polymorphism between the two parents. The method was also used to assign RAPD markers and isoenzyme bands of -arylesterase, phosphatases, NAD-dependent malate dehydrogenase, and cellulase, to A. nidulans chromosomes and/or to their A. quadrilineatus equivalents. The isoenzyme and DNA sequences assigned to chromosomes could be used to saturate the genetic map of A. nidulans, or could serve as starting points for the construction of a genetic map of A. quadrilineatus. No method affording the same possibilities has been described so far in Aspergilli. This chromosome-assay method may be a useful alternative to pulsed-field-gel electrophoretic procedures for the assignment of molecular markers to chromosomes. 相似文献
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Summary The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants. 相似文献
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Summary There are at least three alcohol dehydrogenases in Aspergillus nidulans. ADHIII has no obvious physiological function. We describe here the cloning of the ADHIII gene (alcC), its mapping on linkage group VII by reverse genetics, and the properties of multicopy transformants tested for their ability to grow on a range of alcohols (butan-1-ol being the best substrate tested for growth). We were unable to detect any obvious alteration in phenotype of a strain carrying a disrupted copy of the ADHIII gene. 相似文献
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Maria Amutan Eini Nyyssönen John Stubbs Maria R. Diaz-Torres Nigel Dunn-Coleman 《Current genetics》1996,29(5):468-473
Aspergillus niger var.awamori contains multiple copies of a transposable element, Vader. This element was detected as a 437-bp insertion in four independently isolated spontaneous mutants of theniaD (nitrate reductase) gene. The Vader element is present in approximately 15 copies in bothA. niger var.awamori andA. niger. A single copy of Vader was detected from only one of the two laboratory strains ofA. nidulans which were also examined. Insertion of the Vader element into theniaD gene ofA. niger var.awamori caused a 2-bp duplication (TA) of the target sequence. The Vader element is flanked by a 44-bp inverted repeat. The genetic stabilities of the inserted Vader elements atniaD were examined by studying reversion frequencies resulting in colonies able to grow on nitrate as a sole nitrogen source. MutantsniaD392 andniaD436 reverted at a frequency of 9x10-3 and 4x10-2, respectively. Two of the mutants,niaD587 andniaD410, reverted at a lower frequency of 6x10-4. 相似文献
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Generation of cytotoxic T cell responses directed to human leucocyte antigen Class I restricted epitopes from the Aspergillus f16 allergen 下载免费PDF全文
Ramadan G Davies B Kurup VP Keever-Taylor CA 《Clinical and experimental immunology》2005,140(1):81-91
Invasive aspergillosis (IA) is a major cause of infection-related mortality in patients with haematological malignancies, especially in recipients of haematopoietic stem cell transplants. We have prepared overlapping pentadecapeptides (11-aa overlap with previous peptide) spanning the entire 427-aa coding region of the Aspergillus allergen, Asp f16 shown previously in mice to induce Th1-type cell responses in vivo and in humans to induce proliferative and cytotoxic CD4(+) T cell responses. Mature dendritic cells (DC) pulsed with a complete pool of peptides were used to generate T cell lines. Two lines from HLA-B*3501(+) donors were found to be strongly cytotoxic to autologous Asp f16-peptide pool- and Aspergillus culture extract-pulsed targets after 4-5 weekly primings. Cytotoxic T lymphocyte (CTL) culture supernatant killed Aspergillus conidia, and cells directly killed Aspergillus hyphae. Cytotoxic activity and interferon (IFN)-gamma production were mediated exclusively by CD8(+) T cells in response to pool-pulsed targets. Interleukin (IL)-4 production was not detected. CTL activity was restricted by HLA-B*3501 and based on peptide prediction programmes was most probably directed to YFKYTAAAL (YFK), LPLCSAQTW (LPL) and GTRFPQTPM (GTR) in one donor, while only LPL was recognized by CTL from the second donor. Pool-pulsed B*3503(+) BLCL but not B*3502(+) or B*3508(+) BLCL presented peptide to donor no. 1. B*3503(+) BLCL presented YFK and to a lesser extent GTR, but not peptide LPL. Our data show that in addition to our previously identified Class II restricted peptide response, DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing polyclonal, HLA-Class I-restricted, Aspergillus-specific T cells that may be capable of conferring immunity to IA. 相似文献
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BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) is characterized by a heightened Th2 CD4+ T-cell response to Aspergillus fumigatus (Af) allergens and a hyper-immunoglobulin E (IgE) state compared with cystic fibrosis patients without ABPA. The IgE serologic differentiation of ABPA from atopic CF patients can be difficult. We propose as the reactivity with purified antigens varies qualitatively and quantitatively and that the antibody response is more specific than with crude Af antigen extract, the IgE responses to purified recombinant Af allergens may differentiate ABPA from atopic CF patients. METHODS: Serum IgE reactivity to seven recombinant purified allergens and to a crude extract of Af was measured in 15 ABPA, in 23 Af skin test positive (ST+), and in 19 Af skin test negative (ST-) CF patients. Four of the ABPA CF patients were studied before and after developing ABPA. Nine ABPA patients were studied during flares and remissions of ABPA. RESULTS: Allergic bronchopulmonary aspergillosis patients had significantly increased IgE reactivity to Asp f2, f3, f4, f6, and f16 compared with the Af ST+ and ST- non-ABPA CF patients. In the ABPA patients studied before and after developing ABPA, IgE reactivity also increased to Asp f2, f3, f4, and f6, and to the crude extract. In ABPA CF patients, IgE reactivity to Asp f1, f2, f3, and f6 significantly increased during periods of ABPA flares compared with periods of remission. Analysis of the receiver operating curve demonstrated that IgE reactivity to Asp f3 and f4 gave the best sensitivity and specificity and were better than IgE reactivity to a crude extract of Aspergillus. Furthermore, in ABPA patients studied during periods of remission the IgE reactivity to Asp f3 and f4 remained significantly elevated compared with Af ST+ non-ABPA patients. The IgE responses when considered either to be positive or negative to Asp f3 and f4 significantly differentiated ABPA from Af ST+ and ST- non-ABPA CF patients. In contrast, IgE reactivity was considered positive to the crude extract in 89% of ABPA, 61% of Af ST+, and 0% of Af ST- non-ABPA CF patients. CONCLUSIONS: Immunoglobulin E reactivity to a panel of purified Af allergens, especially to Asp f3 and f4, differentiates ABPA from atopic Af ST+ non-ABPA CF patients. Serial determinations of IgE reactivity to individual purified Aspergillus antigens, especially Asp f3, demonstrates that increases in IgE reactivity may provide improved distinction between stages of flares and remission compared with changes in IgE reactivity to a crude Aspergillus extract. 相似文献
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目的研究低聚果糖产生菌黑曲霉 (Aspergillusniger)SIPI 6 0 2来源的 β 果糖基转移酶 (β FT)的性质。 方法经典的酶学研究方法。结果 β FT催化蔗糖转化成蔗果三糖的Km和Vmax分别为 2 6 8.37g L和 2 .37g (min·L) ;将蔗果三糖转化成蔗果四糖的Km和Vmax分别为 173.0 8g L和 0 .10g (min·L)。葡萄糖是 β FT的竞争性抑制剂 ,其Ki=2 4 5 .2 3g L。β FT反应的最适pH为 4 .5~ 6 .0 ,最适温度为 5 5℃。β FT在中性条件、5 0℃以下稳定性良好。β FT转化蔗糖生成低聚果糖的反应进程实验表明 ,高浓度的底物有利于提高反应速度和转化率 ,并且产物中果糖浓度较低浓度底物中低。结论黑曲霉SIPI 6 0 2来源的 β FT适合用于工业化生产低聚果糖 相似文献
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