CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA.

The major regulatory protein in carbon repression in Aspergillus nidulans is CreA. Strains constitutively over-expressing creA show normal responses to carbon repression, indicating that auto-regulation of creA is not essential for CreA-mediated regulation. In these strains, high levels of CreA are present whether cells are grown in repressing or derepressing conditions, indicating large-scale degradation of CreA does not play a key role. CreA is located in the nucleus and cytoplasm in cells when grown in either repressing or derepressing conditions, and absence of CreB, CreD or AcrB does not affect either the localisation or amount of CreA. Therefore, CreA must require some modification or interaction to act as a repressor. Deletion analysis indicates that a region of CreA thought to be important for repression in Trichoderma reesei and Sclerotina sclerotiorum CreA homologues is not critical for function in Aspergillus nidulans.     

CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA

The major regulatory protein in carbon repression in Aspergillus nidulans is CreA. Strains constitutively over-expressing creA show normal responses to carbon repression, indicating that auto-regulation of creA is not essential for CreA-mediated regulation. In these strains, high levels of CreA are present whether cells are grown in repressing or derepressing conditions, indicating large-scale degradation of CreA does not play a key role. CreA is located in the nucleus and cytoplasm in cells when grown in either repressing or derepressing conditions, and absence of CreB, CreD or AcrB does not affect either the localisation or amount of CreA. Therefore, CreA must require some modification or interaction to act as a repressor. Deletion analysis indicates that a region of CreA thought to be important for repression in Trichoderma reesei and Sclerotina sclerotiorum CreA homologues is not critical for function in Aspergillus nidulans.     

Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans.

The complete nucleotide sequence derived from a genomic clone and two cDNA clones of the creA gene of Aspergillus nidulans is presented. The gene contains no introns. The derived polypeptide of 415 amino acids contains two zinc fingers of the C2H2 class, frequent S(T)PXX motifs, and an alanine-rich region indicative of a DNA-binding repressor protein. The amino acid sequence of the zinc finger region has 84% similarity to the zinc finger region of Mig1, a protein involved in carbon catabolite repression in yeast cells, and it is related both to the mammalian Egr1 and Egr2 proteins and to the Wilms' tumor protein. A deletion removing the creA gene was obtained, by using in vitro techniques, in both a heterokaryon and a diploid strain but was unobtainable in a pure haploid condition. Evidence is presented suggesting that the phenotype of such a deletion, when not complemented by another creA allele, is leaky lethality allowing limited germination of the spore but not colony formation. This phenotype is far more extreme than that of any of the in vivo-generated mutations, and thus either the gene product may have an activator activity as well as a repressor function or some residual repressor function may be required for full viability.     

ADHII in Aspergillus nidulans is induced by carbon starvation stress

In Aspergillus nidulans there are three NAD(+)-dependent alcohol dehydrogenases (ADHs) that are capable of utilizing ethanol as a substrate. ADHI is the physiological enzyme of ethanol catabolism and ADHIII is induced under conditions of anaerobiosis. The physiological role of ADHII (structural gene alcB) is unknown. We have measured beta-galactosidase in a transformant with an alcB::lacZ fusion and have shown that alcB is maximally expressed under conditions of carbon starvation. The behavior of the alcB::lacZ transformant suggests a hierarchy of repressing carbon sources characteristic of repression by the general carbon catabolite repressor protein, CreA, but in a creA(d)30 background the transformant shows only partial derepression of beta-galactosidase on 1% glucose compared to the creA+ strain. Our results suggest that, in addition to carbon catabolite repression acting via CreA, a CreA-independent mechanism is involved in induction of alcB on carbon starvation.     

Transcription analysis using high-density micro-arrays of Aspergillus nidulans wild-type and creA mutant during growth on glucose or ethanol

Here, we describe how the recently published Aspergillus nidulans genome sequence [Galagan, J.E., Calvo, S.E., Cuomo, C., Li-Jun, M., Wortman, J.R., et al., 2005. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438 (7071), 1105-1115] was used to design a high-density oligo array with probes for 3,278 selected genes using the Febit Geniom One array system. For this purpose, the program OligoWiz II was used to design 24,125 probes to cover the 3,278 selected genes. Subsequently, the Febit system was used to investigate carbon catabolite repression by comparing the gene expression of a creA deleted mutant strain with a reference strain grown either with glucose or ethanol as the sole carbon source. In order to identify co-regulated genes and genes influenced by either the carbon source or CreA, the most significantly regulated genes (p相似文献   

Identification of amdX, a new Cys-2–His-2 (C2H2) zinc-finger gene involved in the regulation of the amdS gene of Aspergillus nidulans

The acetamidase-encoding amdS gene of Aspergillus nidulans has been shown to be controlled by multiple regulatory genes. A new gene, amdX , involved in amdS regulation was identified during the characterization of a translocation affecting amdS control. The amdX gene is predicted to encode a 1150-amino-acid polypeptide which contains two Cys-2–His-2 (C2H2) zinc finger DNA-binding motifs. Insertional inactivation of amdX and the phenotypes of transformants containing multiple copies of the amdX gene show that it is an activator of amdS expression. A fusion protein containing the AmdX zinc fingers was found to bind to sequences in the 5' region of amdS which overlap binding sites for the CreA and AmdA regulatory proteins. Evidence is presented for AmdX acting at these sites in vivo .     

The wide-domain carbon catabolite repressor CreA indirectly controls expression of the Aspergillus nidulans xlnB gene, encoding the acidic endo-beta-(1,4)-xylanase X(24)

The Aspergillus nidulans xlnB gene, which encodes the acidic endo-beta-(1,4)-xylanase X(24), is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation creA(d)30 results in considerably elevated levels of xlnB mRNA indicates a role for the wide-domain repressor CreA in the repression of xlnB promoter (xlnBp) activity. Functional analyses of xlnBp::goxC reporter constructs show that none of the four CreA consensus target sites identified in xlnBp are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence xlnB expression by acting directly on xlnB.     

Gibberellin Biosynthesis in Plants and Fungi: A Case of Convergent Evolution?

As well as being phytohormones, gibberellins (GAs) are present in some fungi and bacteria. Indeed, GAs were first discovered in the fungus Gibberella fujikuroi, from which gibberellic acid (GA3) and other GAs are produced commercially. Although higher plants and the fungus produce structurally identical GAs, there are important differences in the pathways and enzymes involved. This has become particularly apparent with the identification of almost all of the genes for GA-biosynthesis in Arabidopsis thaliana and G. fujikuroi, following the sequencing of the Arabidopsis genome and the detection of a GA-biosynthesis gene cluster in the fungus. For example, 3b-hydroxylation occurs early in the pathway in G. fujikuroi and is catalyzed by a cytochrome P450 monooxygenase, whereas it is usually the final step in plants and is catalyzed by 2-oxoglutarate-dependent dioxygenases. Similarly, 20-oxidation is catalyzed by dioxygenases in plants and a cytochrome P450 in the fungus. Even where cytochrome P450s have equivalent functions in plants and Gibberella, they are unrelated in terms of amino acid sequence. These profound differences indicate that higher plants and fungi have evolved their complex biosynthetic pathways to GAs independently and not by horizontal gene transfer.     

菊芋叶片提取物抑菌活性与化学成分的研究

为了开发新型植物源杀菌剂和充分利用菊芋(Helianthus tuberosus L.)资源,本文尝试用石油醚、乙醚、乙酸乙酯和水等4种溶剂对菊芋叶片进行平行提取,采用生长速率法测定菊芋叶片提取物对水稻纹枯菌(Rhizoc-tonia solaniKühn)、小麦赤霉菌[Gibberella zeae(Schw.)Petch]、番茄早疫菌[Alternaria solani(Ellis et Martin)Jones et Grour]和番茄灰霉菌(Botrytis cinerea Pers.)生长量的抑制活性;并通过试管法和滤纸法对菊芋叶片内化学成分进行初步预试。抑菌实验结果表明:(1)菊芋叶片各溶剂提取物处理与对照处理相比差异显著;(2)菊芋叶片水提取物处理与各有机溶剂提取物处理差异也基本显著;(3)菊芋叶片各溶剂提取物同浓度处理对水稻纹枯菌、番茄早疫菌和番茄灰霉菌抑制效果较好;(4)菊芋叶片乙酸乙酯提取物抑菌效果最为显著,浓度为20mg/mL时对番茄早疫菌和番茄灰霉菌已达到完全抑制,对水稻纹枯菌抑制率也达到77.91%。初步化学预试结果说明,菊芋叶片中含有蛋白质、氨基酸、还原糖类、有机酸、酚类和鞣质、黄酮类、内酯类、强心甙以及油脂等化学成分。     

Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi

The gene areA-GF, a homologue of the major nitrogen regulatory genes nit-2, areA, nre and NUT1 of Neurospora crassa, Aspergillus nidulans, Penicillium chrysogenum and Magnaporthe grisea, respectively, was cloned from the gibberellin (GA)-producing rice pathogen Gibberella fujikuroi.areA-GF encodes a protein of 972 amino acid residues which contains a single putative zinc finger DNA-binding domain that is at least 98% identical to the zinc finger domains of the homologous fungal proteins. The areA-GF gene has been shown to be functional in N. crassa by heterologous complementation of a RIP induced nit-2 mutant. The transformation rate was nearly as high as in a homologous complementation control. Transformants were able to utilize nitrate and expressed a normally regulated nitrate reductase activity. To generate areA-GF  ⁻ mutants, gene replacement experiments were performed using a linearized replacement vector carrying the hygromycin B phosphotransferase (hph) gene. The replacement of the zinc finger by the hygromycin cassette resulted in transformants which were unable to utilize nitrogen sources other than ammonium and glutamine, and gave significantly reduced gibberellin production yields. Complementation of such a mutant with the wild-type gene led to the full recovery of gibberellin production. Received: 23 April 1998 / Accepted: 16 October 1998     

The DNA-binding domain of the gene regulatory protein AreA extends beyond the minimal zinc-finger region conserved between GATA proteins

The AreA protein of Aspergillus nidulans regulates the activity of over 100 genes involved in the utilisation of nitrogen, and has a limited region of homology with the vertebrate family of GATA proteins around a zinc finger (Zf) motif. A 66 amino acid (a.a.) residue fragment (Zf(66)) corresponding to the zinc finger, a 91 a.a fragment (Zf(91)) containing an additional 25 a.a. at the C-terminus, and a much larger 728 a.a. sequence (3'EX) corresponding to the 3'exon have been over-expressed as fusion proteins in E. coli and purified. The DNA-protein complexes formed by these proteins have been examined by gel retardation analysis. The 91 a.a. protein forms a discrete shifted species with a GATA-containing DNA fragment with high affinity (K(d)=0.15 nM), whereas the 66 a.a. protein has very low ( approximately microM) affinity for the same sequence. The results show that the region of AreA required for high affinity DNA binding extends beyond the zinc finger motif that is homologous to GATA-1, requiring in addition a region within the 25 a.a. sequence C-terminal to the zinc finger. Using hydroxyl radical and ethylation interference footprinting, the minimal Zinc finger protein (Zf(66)) shows no appreciable interference effects whereas Zf(91) shows much stronger interference effects, identical to those of the larger protein. These effects extend over sequences up to two nucleotides either side of the GATA site, and indicate contacts additional to those observed in the three-dimensional structure of the complex of the minimal zinc-finger protein with DNA. We suggest that these additional contacts are responsible for the enhanced DNA binding affinity of the extended zinc-finger protein Zf(91).     

Effect of silicone oil on the gibberellic acid production by Gibberella fujikuroi and Aspergillus niger

The effect of silicone oil on gibberellic acid production was investigated using Gibberella fujikuroi and Aspergillus niger fungi. Silicone oil was used as an oxygen vector in the gibberellic acid production process. When silicone oil was used, gibberellic acid concentration increased with respect to the control run having A. niger and G. fujikuroi microorganisms 4.6 and 6.7 times, respectively.     

Evidence for similarities and differences in the biosynthesis of fungal sterols

The sterol composition of two ascomycetous fungi, Saccharomyces cerevisiae and Gibberella fujikuroi, was examined by chromatographic (TLC, GLC, and HPLC) and spectral (MS and 1H-NMR) methods. Of notable importance was that both fungi produced cholesterol and a homologous series of long chain fatty alcohols (C22 to C30). In addition to ergosterol two novel sterols, ergosta-5,7, 9(11), 22-tetraenol and ergosterol endoperoxide, were isolated as minor compounds in growth-arrested cultures of yeast and in mycelia of G. fujikuroi. 24-Ethylidenelanosterol was also detected in mycelia of G. fujikuroi. A shift in sterol biosynthesis was observed by treatment with 24 (RS), 25-epiminolanosterol (an inhibitor of the S-adenosylmethionine C-24 transferase) and by monitoring the sterol composition at various stages of development. The results are interpreted to imply that the genes for 24-desalkyl, e.g., cholesterol, and 24-alkyl sterols, e.g., 24 beta- methyl cholesterol and 24-ethyl cholesterol, are distributed (but not always expressed) generally throughout the fungi but the occurrence of one or another compounds is influenced by the fitness (structure and amount) for specific sterols to act functionally during fungal ontogeny; sterol fitness is coordinated with Darwinian selection pressures.