Aspergillus nidulans FacB Transcriptional Activator and Neurospora crassa Acetate-induced Protein Compete for Their Respective Cognate Promoter DNAs
Martha Bibbins, Peter Sheffield, Lawrence Gainey, Ian Connerton. Food Macromolecular Sciences Department, Institute of Food Research, Earley Gate, Whiteknights Road, Reading RG6 6BZ, U.K.
The fac B gene product mediates acetate-induced gene expression in Aspergillus nidulans . Gel mobility shift scanning with the N-terminal DNA-binding domain of the FacB protein (FacB4-417) synthesised in Escherichia coli, demonstrates region-specific DNA-binding to the native acetate-inducible promoter acuD (gene coding for the isocitrate lyase) and a serie of analogous promoters from Neurospora crassa. Conversly a partially purified protein extract from N. crassa mycelium induced with acetate binds the corresponding A. nidulans promoter DNA fragments, Protein/DNA gel retardation assays with the respective isocitrate lyase gene promoter fragments identify the same 5' non -coding regions as those reported to be involved in acetate induction based on promoter deletion analyses. Competitive DNA-binding experiments between the FacB4-417 protein and the N. crassa protein extract demonstrate that each can effectively titre the other, implying they have DNA recognition motifs in common.
Cloning and Analysis of areA and creA -Like Genes from the Insect Pathogenic Fungus Metarhizium anisopliae
Screen,S.E., Cooper,R.M., Charnley,A.K. and Clarkson,J.M. School of Biology and Biochemistry, University of Bath,Bath BA2 7A Y, UK.
The insect pathogenic fungus Metarhizium anisopliae invades its host insect by direct penetration of the cuticle. a complex laminate structure comprising primarily chitin and protein. Several extracellular cuticle-degrading proteases have been characterised from Metarhizium and the role of one of these, PRI, in infection has been established. PRI production is regulated in response to C and N-derepression (and specific induction by insect cuticular protein) and consequently the pr1 promoter region was searched for the presence of putative binding sites for C and N response regulator proteins as defined in Aspergillus nidulans and Neurospora crassa. In A. nidulans, C-repression is mediated by CREA, a repressor protein which binds to the sequence 5' SYGGRG in C-repressed genes. N-derepression in N. crassa is effected by an activator protein NIT2 which binds to the sequence 5' GATA. 3 putative binding sites for a CREA-like protein were identified in the pr1 promoter. Two of these have been shown to bind CREA n vitro. Seven GATA sites were also identified, however, the DNA-binding requirement of NIT2 for 2 closely, spaced GATA sites would suggest that the pr1 promoter contains only 2- functional binding sites. PCR using oligos based on the zinc finger DNA-binding domain of CREA and MIGI (from S. cerevisiae) have been used to amplify fragments from Metarhizium genomic DNA. Partial sequencing of one of these fragments confirmed strong identity to CREA. A genomic clone was subsequently isolated and sequenced. This crea-like gene is present in a single copy in the Metarhizium genome. A similar approach using oligos based on homology between NIT2 and AREA (of Aspergillus) has allowed the isolation of a 800 bp PCR fragment which shows strong sequence similarity to AREA. The full length gene has been isolated from a Metarhizium genomic library and sequenced.
Increasing Expression of Neurospora crassa NADP+ -Dependent Glutamate Dehydrogenase
Helen Dunmore and Geoffrey Turner. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, SIO 2UH, UK
NADP+-dependent glutamate dehydrogenases (GDH) found in many bacteria, plants and fungi catalyse the reductive amination of 2-oxoglutarate to L-glutamate which is an important step in the amino acid assimilation pathways.
Work has been undertaken to increase the expression of the NADP+ dependent GDH protein, transcribed from the N. crassa am gene, as an aid to protein crystallisation studies of wt and mutant enzymes.
The am132 deletion strain of N. crassa was transformed with the am gene coupled to the strong promoters grg-1, and also crp-2. Integration of the am gene occurred and the corresponding protein assays showed high enzyme levels, but over successive subcultures the quantity of GDH enzyme declined, possibly because the gene expression was being affected by methylation or quelling. The same result was obtained with both N. crassa promoters. Stabilisation only occurred at levels of expression lower than those produced by the N crassa wt.
Expression of the am gene was also attempted in Aspergillus nidulans using the strong alcA promoter, but although levels of expression were obtained these were not higher than the N. crassa wt equivalent.
In vitro mutagenesis is now in progress to investigate subunit interaction in this hexameric protein.
Cloning of the GCN4/cpc-1 Homologue of Aspergillus niger
Sabine Eckert, Christoph Wanke, Wim v. Hartingsfeldt, Peter Punt, Cees van den Hondel and Gerhard Braus.
Friedrich-Alexander-University, Institute for Biochemistry, Microbiology and Genetics, Staudtstr. 5, 91058 Erlangen
TNO Nutrition and Food Research Institute, Molecular Genetics and Gene Technology, P.O.Box 5815, 2280 HV Rijswijk, The Netherlands
Imbalances in the pool of amino acids in certain fungi is known to result in a coordinated derepression of amino acid biosynthetic genes. This phenomenon, called General Control of amino acid biosynthesis in Saccharomyces cerevisiae (Hinnebusch, 1992) and Cross-Pathway control in Neurospora crassa (Carsiotis et al., 1974), was also known to exist in Aspergillus nidulans (Piotrowska 1980).
The genes coding for the main trans-acting factor mediating this control mechanism are identified in S. cerevisiae (aCN4) and N. crassa (cpc-1). These genes encode bZIP-type transcriptional activators which bind to palindromic general control recognition elements (GCRE's, consensus sequence 5' ATGA(C/G)TCAT 3'; Hope and Struhl, 1985, Arndt and Fink 1986; Ebbole et al., 1991) in the promoter sequence of many amino acid biosynthetic genes and stimulate their transcription.
Here we report the cloning and initial characterization of OcA, the GCN4/CPC-1 homologue of the filamentous fungus Aspergillus niger.
References:
Arndt, K. and Fink, G. R. (1 986): Proc. Natl. Acad. Sci. USA 83, 8516 - 8520.
Carsiotis, M., JonE,,s, R. F. and Wesseling, A. C. (1974): J. Bact. 119, 893 - 898.
Hinnebusch, A. G. (1 992): in: The molecular and cellular biology of the yeast Saccharomyces: Vol. 11, Gene expression (Jones, E. W., Pringle, J. R. and Broach, J. R., eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 319 - 414. Hope, 1. and Struhl, K. (1 985): Cell 43, 177 - 188.
Paluh, J. L. and Yanofsky, C. (1 991): Mol. Cell. Biol. 11, 935 - 944. Piotrowska, M. (1980): J. Gen. Microbiol. 116, 335 - 339.
The Phytoene Dehydrogenase Gene of Phycomyces: Regulation of the Expression by Blue Light and Vitamin A
M. J. Ruiz-Hidalgo 1, E. P. Benito 1, G. Sandmann 2 and A. P. Eslava. 1Area de Genetica. Departamento de Microbiologia y Genetica. Universidad de Salamanca, 37007 Salamanca, Spain. 2Botanisches Institut, J. W. Goethe Universitat, P. 0. Box 111932, D60054 Frankfurt/M., Germany
By using a PCR-based cloning strategy, we have isolated the first carotenogenic gene from the filamentous fungus Phycomyces blakesleeanus, encoding the enzyme phytoene dehydrogenase. The structural region of the gene includes 1935 nucleotides and consists of two exons separated by one intron, 183 bp in length, whose processing brings about a 1752-nucleotide open reading frame that encodes a 583-residue polypeptide with a predicted molecular mass of 65.9 kDa. The deduced protein showed a high homology to carotenoid dehydrogenases from other fungi and bacteria, specially in the amino-terminal region, and the main conserved regions found in other phytoene dehydrogenases, that are thought to be essential in the enzymatic activity, are present in the sequence from Phycomyces.
Heterologous expression of the Phycomyces gene in Escherichia coli showed that, like in other fungi and bacteria, a single phytoene dehydrogenase catalyzes the four dehydrogenations from phytoene to lycopene in the Phycomyces carotenoid biosynthetic pathway. RNA measurements showed that the level of expression of the phytoene dehydrogenase gene in wild-type mycelia increased in response to blue light. The kinetics of the mRNA levels of this gene after blue light induction (0.1 and 0.4 W/m2 ) exhibits a two-steps (biphasic) dependence on fluence rate, suggesting the possibility that there could be two separate components in the reception of the low and high blue light exposure. The presence of vitamin A in the medium stimulated the phytoene dehydrogenase transcript accumulation in wild-type and some carotenogenic mutants (genotype car). Diphenilamine, a phytoene dehydrogenase inhibitor, did not affect the level of transcription of this gene.
Mutational Analysis of the Proline Pathway Positive Regulator PRNA in Aspergillus nidulans
Pokorska A., Cazelle B., Gonzalez R., Scazzocchio C. Institut de Genetique et Microbiologie, Universite Paris XI, bat.409 91 405 Orsay, France
The Aspergillus nidulans proline catabolism is subject to three types of control:a general repression by glucose and ammonium and a specific induction by proline. At least three regulatory proteins are involved in this process: CreA, AreA and the specific regulator PrnA. The positive regulator PrnA is a 818 amino acid protein with a binuclear zinc complex and other domains common for regulatory proteins like a NLS or an activation domain. We study the PrnA domains for their interactions with other cellular factors. We are also interested in the role of the intramolecular interactions in the protein function.
This study is based on the analysis of cold- and thermosensitive mutations and their revertants. Six of these mutations are mapped and three are sequenced. Two cold-sensitive mutations appear to be localized in the bipartite nuclear entry signal and lead to an abolition of the consensus sequence. The prnA29 mutation changes an Arg into Leu and in the prnAI21 mutant the same Arg becomes a Pro. An aleatory mutagenesis was done on these mutant strains and gave several types of intragenic suppressors in the NLS spacer restoring the consensus sequence and the wild type phenotype of the mutant strains. We are now about to confirm the role of this sequence in the nuclear transport by using a tag construction and fusions with the Green Fluorescent Protein. We are also looking for some extragenic suppressors which could touch other proteins of the nuclear transport system.
We work also on the PrnA "central" region highly homologous to other specific regulators domains. The prnA27 mutation is localized in this region and changes a Thr into an Ala. We have found intragenic suppressors in other domains of the protein, upstream from this mutation. These results suggest the existence of some intramolecular interactions necessary for the protein function.
We are currently sequencing other prnA mutations. Some of them would be localized in the C-terminus and therefore could be in the glutamine-rich putative activation region of the protein.
Position Effects in the Transcriptional control of the Aspergillus nidulans prn Cluster
D. Gomez, R. Gonzalez, B. Cubero, V. Gavrias and C. Scazzocchio. Institut de Genetique et Microbiologie. Universite de Paris-Sud. 91405 Orsay, France
Proline catabolism in Aspergillus nidulans is subject to three levels of control: specific induction, mediated by the PrnA protein, carbon catabolite repression, mediated by the negatively acting protein CreA, and nitrogen metabolite repression, mediated by the positively-acting protein AreA. The 1.7 kb intergenic region between genes prnb and D has been shown to play a crucial role in the regulation of the expression of all prn genes with the exception of prnA. Deletion analysis in this region has confirmed the existence of a previously predicted enhancer element for prnC more than three kb upstream of its putative start of transcription. This enhancer is necessary for maximal transcription of prnC at 25 C but not at 37 C.
Using the same type of analysis we have confirmed our hypothesis that integration between carbon catabolite repression, and nitrogen metabolite repression is mediated by a factor, different from AreA, whose activity is counteracted by that of CreA. The binding site for this factor is located more than 500 pb appart from the CreA binding sites responsible "in vivo" for glucose repression.
We have used DNAsel footprint analysis with crude extracts of Aspergillus nidulans to localize some potential targets for both the enhancer and the other new factor in the regions defined by deletion analysis.
Other interesting observation from these deletions is that the binding of CreA shows a very stringent position requirement to be effective in repression. Chromatin structure analysis by DNAse1, micrococcal nuclease and several restriction enzymes also suggests the involvement of these regions in transcriptional control of proline catabolism. We propose a model integrating all these data and accounting for the positional requirement of the CreA binding sites.
A GATA-factor Encoding Gene (sreP) from Penicillium chrysogenum, Homologous to the Ustilago maydis urbs1 Gene
Hubertus Haas, Klaus Angermayr and Georg Stoffler. Department of Microbiology, University of Innsbruck, Medical School, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria,
GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus motif GATA. This group includes a range of major regulatory proteins from organisms as varied as fungi, mammals, birds, insects and plants. The different members of this family are related by a high degree of amino acid sequence identity within their DNA-binding domains, which consist of either one or two zinc chelating fingers. Intriguingly, in a number of different organisms multiple functionally distinct GATA factors with similarity of binding specificity have been reported. Recently, we have isolated and characterized the nre gene encoding the major nitrogen regulatory gene of Penicillium chrysogenum, containing a single Cys2/Cys2-type zinc finger motif which recognizes the consensus sequence GATA (1). To characterize additional Penicillium GATA factors we have used Polymerase Chain Reaction amplifications of cDNA using degenerate oligodeoxynucleotide primers derived from the conserved DNA binding domain of the GATA protein family. Using this approach the srep gene encoding a protein of 533 amino acids which contains two GATA type zinc finger motifs was isolated. The deduced amino acid sequence reveals 50% identity to the URBS I protein from Ustilago maydis over a stretch of 150 amino acids containing the two DNA binding motifs and the intervening sequence (2). The sequence homology to URBSI, which interacts directly or indirectly to repress biosynthesis of siderophores, suggests a similar role for sreP in Penicillium. In order to investigate the promoter region and structural organization of sreP gene, a genomic 10-kb fragment was isolated and partially sequenced. Comparison of the nucleotide sequence of the genomic fragment and the cDNA clones revealed the presence of two introns. In Northern blot analysis of total cellular RNA two transcripts, 2 kb and 2.5 kb in length respectively, were detected due to two different major transcriptional start sites.
The cloning and characterization of sreP and other GATA factor encoding genes from filamentous fungi will provide a suitable model system to investigate the regulation of iron metabolism as well as to enlighten how structurally related proteins maintain distinct roles within the same organism.
References
1)Haas, H. and Marzluf, G.A. (1995). Cuff. Genet. 28:177-183.
2)Voisard, C., Wang, J., McEvoy, J.L., Xu, P. and Leong, S.A. (1993). Mol. Cell. Biol. 13:7091-7100.
Promoter Analysis of a Heat Shock Gene (Hsp70) of Aspergillus nidulans.
Cas Kramer' , Jane A. Newbury', Theo Goosen 2 and John F. Peberdy'. 'Department of Life Science, University of Nottingham, Nottingham NG7 2RD, United Kingdom. 2Department of Genetics, Agricultural University Wageningen, 6703 HA Wageningen, The Netherlands.
A member of the hsp7O gene family of the filamentous fungus Aspergillus nidulans has been isolated and cloned (data being prepared for publication). The DNA sequence shows strong homology with hsp7O genes of other organisms, especially the ssb genes of Saccharomyces cerevisiae. Interestingly, this A. nidulans hsp7O gene contains several introns, which is an uncommon feature in highly expressed heat shock genes of higher eukaryotes.
Among other upstream sequence elements common to fungal promoters, six copies of the highly conserved Heat Shock Element (HSE) can be found in the 5' flanking sequences of this heat shock gene. A PCR amplification product of the promoter sequences has been fused in frame to the Escherichia coli lacZ gene, in order to study the promoter's functionality and efficiency. Site directed integration of a single copy of the vector pCK70 (carrying this promoter-lacZ fusion) into the Aspergillus genome has been accomplished. Enzyme assays to determine the -galactosidase activity of these fungal strains were performed under control conditions and after different heat shock treatments. Surprisingly, no significant increase in enzyme activity was apparent after heat stress. Although several HSEs can be found in the promoter sequences, this A. nidulans hsp7O gene seems to be constitutively expressed. Results of a promoter deletion study to elucidate this phenomenon are shown.
High levels of -galactosidase activity can be found under many different growth conditions. Little or no effect could be found after treatment with heat and other inducers, like heavy metals, ethanol and cold shock. However, growth phase related changes in enzyme activity were apparent and have been studied. Results of these studies will be presented and discussed.
SCONB, An Aspergillus nidulans Sulphur Metabolism Regulatory Gene Encodes a -Transducin-like Protein
Renata Natorff, Ma gorzata Piotrowska and Andrzej Paszewski. Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences 5A Pawinskiego Str, 02-106 Warsaw, Poland
Expression of genes coding for enzymes of sulphate assimilation and enzymes participating in the metabolism of sulphur amino acids are controlled by four regulatory scon (sulphur controller) genes. Mutations in these genes relieve sulphur metabolism from sulphur metabolite repression - a regulatory system which shuts off the synthesis of the enzymes of sulphate assimilation pathway when a favourable sulphur source such as methionine is available. Two of these genes (sconB and sconC) have been cloned by transformation of the A. nidulans with cosmid gene library.
Genomic and cDNA sequence of sconB+ gene indicated a 2034bp open reading frame interrupted by one intron of 47bp. It encodes a polypeptide of 678 amino acids which belongs to the expanding family of G-protein - subunit related eukaryotic regulatory proteins. Each -transducin-like protein is defined by the presence of four to eight highly conserved WD-40 repeats. The SCONB protein contains seven repeated G -homologous domains spanning the C-terminal half of the protein. It also possesses an amino-terminal domain found in several -transducin homologs including the mouse protein MD6, Xenopus protein BTrCP, Neurospora crassa protein SCON2 and the yeast proteins MET30 and CDC4. The SCONB protein shows considerable similarity to SCON2 protein of N. crassa (74%) and S. cerevisiae MET30 protein (63%); both are involved in the regulation of sulphur metabolism.
The analysis of the sconC gene is in progress.
Identification of a Regulatory Gene in Ustilago maydis That Affects the Expression of Genes Regulated by the B Locus
C. Quadbeck-Seeger, F. Schauwecker, J. Kamper und R. Kahmann. Institut fur Genetik und Mikrobiologie der Universitat Munchen, Maria-Ward-Str. la, 80638 Munchen, Germany
In the maize pathogen Ustilago maydis mating, filamentous growth and pathogenic development are controlled by the two mating type loci a and b. The a locus exists in two allles and controls fusion of haploid cells while the multiallelic b locus regulates pathogenicity and sexual development. Filamentous growth requires the concerted action of the a and b locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW. These proteins dimerize only when they are derived from different alleles and are then able to switch on pathogenic development.
One of the genes regulated by the b locus is egl1 a gene encoding an endoglucanase. egl1 is expressed only in the filamentous phase when an active bE/bW heterodimer is present in the cell. Expression of egl1 can easily be monitored in a plate-assay. egl1 was used as a reporter to screen for mutants which allow egl1 expression in haplid cells where egl1 is normally not expressed. With this screen one mutant was found which expresses egl1 constitutively. Northern analysis showed that the mutation leads to constitutive expression of several b-dependent genes. This indicates that the mutation has affected a gene with a more general regulatory function. Assays in planta showed that the mutation does not attenuate pathogenicity. Interestingly, however, formation of spores was abolished. The mutation was complemented with a cosmid library. After recovery of the cosmid a complementing subclone containing a 8.9 kb Bglll-Xbal fragment was isolated. We will provide the sequence of the respective gene and discuss how this negative regulator fits into our current scheme on gene regulation exerted by the bE/bW heterodimer.
Cpc-3 of Neurospora cassa Is Homologous to Yeast GCN: an eIF-2 Kinase Involved in General Amino Acid Control
Evelyn Sattlegger and Ilse Babette Barthelmess. Institut fur Angewandte Genetik, Universitat Hannover, Herrenhauser Str.2, 30419 Hannover, Germany
In lower eukaryotes, like Neurospora crassa and Saccharomyces cerevisiae, starvation for any one of a number of amino acids leads to simultaneous induced transcription and derepression of enzymes in many amino acid biosynthetic pathways. This mechanism is called general amino acid control. The transcriptional activator of the regulated genes is encoded by the homologous genes cpc-1 (N. crassa ,l) and GCN4 (S. cerevisiae,2), respectively. In S. cerevisiae expression of GCN4 itself is under translational control mediated by the GCN2 encoded eIF-2 kinase (3).
To find out whether substantial differences exist in the details of general control between the two ascomycetes we cloned the GCN2 homolog of N. crassa, called cpc-3.
cpc-3 was identified using PCR and degenerate primers representing highly conserved sequences of eIF-2 kinases. The open reading frame encodes a putative protein with a two domain structure, an eIF-2 kinase and juxtaposed histidyl-tRNA synthetase-like domain, and shows 29% amino acid identity over the entire length with yeast GCN2p.
The N. crassa genomic sequence was used to engeneer a disruption mutation of the cpc-3 gene. Strains carrying the recessive cpc-3 allele were unable to induce increased transcription and derepression of amino acid biosynthetic enzymes under conditions of amino acid starvation indicating that CPC3p like yeast GCN2p is involved in the mechanism of general amino acid control. The cpc-3 mutation does not interfere with cpc-1 transcriptional regulaton.
(1)Barthelmess 1982 Genet. Res 39:169-185 Paluh et al. 1988 Proc. Natl. Acad. Sci. USA 85:3728-3732
(2)review: Hinnebusch 1994 TIBS 19:409-414
(3)review: Wek 1994 TIBS 19:491-496
Control of SC3 Expression in Schizophyllum commune
Theo A. Schuurs, Eveline A.M. Schaeffer en Joseph G.H. Wessels. Department of Plant Biology, University of Groningen, Haren, The Netherlands
The monokaryon of the basidiomycetous fungus Schizophyllum commune abundantly expresses the SC3 hydrophobin gene which is accompanied by intensive formation of aerial hyphae. In the thn mutant SC3 expression is suppressed. Also in a common-MATA heterokaryon with two different MA7B mating-type genes present, SC3 is down-regulated. In both the thn mutant and the common-MATA heterokaryon, few aerial hyphae are formed.
To study the regulation of SC3, the availability of a reporter-gene system would be convenient. However in S. commune foreign genes are mostly not expressed. To overcome this problem a genomic SC3 clone was introduced into a SC3 disruptant. Restoration of SC3 expression could be, detected on RNA as well on protein level. In these cotransformation experiments using the UR,41 gene as an auxotrophic marker, reconstitution of SC3 expression was very poor. DNA analysis showed no correlation between copy number and SC3 expression. Using the phleomycin resistance gene as a selective marker on the same vector as the SC3 genomic fragment, gave much better SC3 expression after transformation to the SC3 disruptant. Not only did more transfortnants show SC3 expression, also the average level of expression was higher. Southern Blot analysis again showed no clear correlation between copy number and the level of SC3 expression. These results taken together strongly suggest position-dependent expression of integrated DNA.
However, regulation of the introduced SC3 gene was as the original endogenous gene both with respect to temporal and mating-type gene regulation. Apparantly all cis-regulatory sequences were still present on the transformed genomic fragment. Experiments using this reporter system with promoter deletion constructs of the SC3 gene, are currently underway.
Cloning and Heterologous Expression of the creA Gene of Penicillium chrysogenum
T. Stanzer, E. Schreiner and H. Schwab. Institut fur Biotechnologie, TU Graz, Petersgasse 12, 8010 Graz, Austria
As an enzyme saving response, in the presence of less favoured carbon sources microorganisms usually turn off the synthesis of a range of enzymes when more readily utilized carbon sources such as glucose are present by carbon catabolite repression. The mig1 gene of Saccharomyces cerevisiae and the creA genes of Aspergillus nidulans and A. niger are the best studied genes mediating carbon catabolite repression in fungi.
As our intentions are directed towards Penicillium chrysogenum, it was our aim to identify, clone and characterize creA gene of P. chrysogenum.
Performing a high stringency plaque hybridization, a DIG labeled NcoI-NcoI fragment containig the zinc finger domain of the creA gene of A. nidulans was found to hybridize to a genomic Lambda EMBL3 DNA library of P. chrysogenum. From one of five selected positive plaques DNA was isolated and sequenced directly, starting with primers designed from sequences within the highly conserved regions known from the creA genes of A. nidulans and A. niger respectively from the crel genes of T. reesei and T. harzianum. The complete coding region of the P. chrysogenum crea gene was amplified using a PCR reaction and the product was cloned into the prokaryotic protein expression vectors pSE420 and pMS470.
At the moment overexpression using E. coli BL21 and TOP1OF' hosts is under investigation.
Glucose Repression of xyn1 (Xylanase I Encoding Gene) Expression by Crel in Trichoderma reesei Requires Binding of a Protein Complex to an Inverted Repeat of the Crel Consensus Sequence.
Robert L. MACH, Joseph STRAUSS, Susanne ZEILINGER, Martin SCHINDLER, and Christian P. KUBICEK
Abteilung fur Mikrobielle Biochemie, Institut fur Biochemische Technologie and Mikrobiologie, TU Wien, A-1060 Wien, Austria
To degrade xylan in plant hemicelluloses, the filamentous fungus Trichoderma reesei forms a xylanase enzyme system comprising two specific, inducible xyianases encoded bv xyn1 and xyn2. Their expression is affected by glucose in different wavs: xyn1 transcript is absent in the presence of glucose as sole carbon source, whereas xyn2 is expressed at a low constitutive level under these conditions. xyn1 induction by xylan is not affected by the simultaneous presence of glucose, whereas induction of xyn2 is prevented. Repression of xyn1 is mediated bv the carbon catabolite repressor protein Crel. Binding of Crel, either overexpressed as a truncated fusion to GST in E coli, or present in cell-free extracts, to the xynl promoter requires the consensus binding site 5'-SYGGRG-3'. In the xynl promoter, in vivo functionality of this consensus is dependent upon arrangement as inverted repeat: T. reesei strains, bearing a xynl:hph construct in which four nucleotides from the center of the repeat has been removed, expresses hph on glucose at a level comparable to that observed under carbon catabolite derepressing conditions (e.g. growth on lactose). Conversely, the insertion of a double consensus into the xyn2 promoter does not result in glucose repressability of constitutive xyn2 transcript formation. We conclude that that the mere presence of an inverted repeat Crel binding site is not sufficient to mediate glucose repression but the functionality of such site is dependent on the nucleotide context and/or on the position in the promoter. Native Crel protein, present in cell free extracts of T. reesei participates in formation of a Crel specific multi-protein complex, regardless the presence of a functional or non-functional (four base-pair delition) double consensus site. From these results we conclude that Crel strongly interacts with other components of the transcriptional machinery but functionality as a repressor is dependent on binding of Crel to its target.
A Ring-finger Protein Involved in the Regulation of the Biosynthesis of Carotenoids in the Fungus Mucor circinelloides
Eusebio Navarro and Santiago Torres-Martinez. Departamento de Genetica y Microbiologia Facultad de Biologia, Universidad de Murcia, 30071 Murcia (Spain)
Most of the mucorales species that produces carotenoids accumulate -carotene as the end product. Mucor circinelloides is one of the few mucorales that is suitable for genetic transformation and can therefore be used for gene cloning. M. circinellioides possesses a light-regulated carotenoid biosynthetic pathway like other related species, including Phycomyces blakesleeanus. We have obtained a collection of M. circinelloides mutants blocked in each of the enzymatic steps from famesyl pyrophosphate to -carotene and three different regulatory mutants affected in the total production of carotenes or/and in their regulation by light.
By complementation of this collection of mutants with a gene bank, we have already isolated a truncated sequence that produces a high increasing in the production of -carotene, both in the dark and light conditions, when introduced in the wild type strain of M. circinelloides. The protein deduced from this truncated sequence includes three main features: (i) a RING-finger motif at the amino end, (ii) an acidic domain and, (iii) a poly-glutamine stretch. Experiments carried out to elucidate the function of the gene which codes for this sequence will be presented.
Control of Taka-amylase A gene: CCAAT binding protein in A. nidulans
Norihiro Tsukagoshi. Department of Applied Biological Sciences, School of Agriculture, Nagoya University, Nagoya 464-01, Japan
Taka-amylase A, TAA, is a typical inducible enzyme induced by starch and repressed by glucose. The deletion analysis of Taa gene revealed that a 55bp DNA fragment containing a CCAAT sequence (312/-308) conferred starch inducibility. This prompted us to identify regulatory proteins which bind in a sequence-specific manner to the promoter region.
A.nidulans carrying Taa gene isolated from A. oryzae was grown under either inducible or repressive conditions, from which nuclear protein was prepared. Protein was further fractionated on a heparin Sepharose column and used for both gel shift and DNase I footprinting assays. A nuclear protein (AnCPI) extracted ftom starch-grown mycelia was found to bind to a DNA fragment containing CCAAT element. Its precise binding sequence was determined by DNase I footprinting analysis and found to be a 14 -9 bp sequence containing CCAAT element. A nuclear protein (AnCP2) isolated from glucose-grown mycelia protected almost the same region protected by AnCPl against DNase I digestion, suggesting that both factors might be the same protein. This further suggests that CCAAT binding protein could be constitutively synthesized. Purification of AnCPI is in progress. The other CCAAT binding protein (AnCF) has been also detected in A. nidulans using amdS gene. Although the relationship between AnCP and AnCF remains to be determined, AnCP protected a sequence containing CCAAT element on amdS gene as well as that on gatA gene. Therefore, AnCP would function as a global regulatory protein.
As to carbon catabolite repression of Taa gene, CREA was found to bind to three sites with high affinity and one site with low affinity, indicating creA gene product participates in the repression of Taa gene. All these sites contain sequences related to the CREA consensus binding sequence, SYGGRG.
Regulation of the Gibberellin Biosynthesis in Gibberella fujikuroi
Bettina Tudzynski, Katrin Mende and Solveig Woitek. Westfalische Wilhelms-Universitat, Institut fur Botanik, Schlossgarten 3, 48149 Munster, Germany
The rice pathogen Gibberella fujikuroi ( imperfect stage: Fusarium moniliforme ), produces high amounts of gibberelins,a group of diterpenic plant hormones.The economic importance of these plant growth regulators has led to an extensive study of the regulation of gibberellin biosynthesis by physiological methods in the last years. It is well known that ammonium and glucose in high concentrations drastically reduce the yield of gibberellin formation. (Bruckner, 1992; Candau et al., 1992) However, the target genes of nitrogen and carbon catabolite repression are still unknown.
Recently we have isolated the Gibberella fujikuroi HMG-CoA-reductase- FPPS-, and GGPPS-genes. Northern analyses showed that those genes are expressed constitutively as expected for genes which play a role in primary and secondary metabolism as well.Therefore, the target genes for both of the different regulation types should be in the more specific part of the gibberellin pathway,e.g. in the kaurene synthetase gene coding the formation of the first intermediate with the specific gibberellin sceleton. In order to study the nitrogen and glucose repression of gibberellin formation in more detail in next future, we isolated and sequenced the global nitrogen regulatory gene areA. Furthermore, cloning of the carbon-catabolite repression gene ( creA ) is on the way.
Bruckner,B., 1992.Regulation of gibberellin formation by the fungus Gibberella fujikuroi, in: Secondary metabolites:their function and evolution, John Wiley and Sons
Candau,R.,Avalos,J. and Cerda-Olmeda,E., 1992. Regulation of gibberellin biosynthesis in Gibberella fujikuroi, Plant Physiol., 100, 1184-1188.
Regulation of Expression of a Cytochrome P450 Enzyme System in Aspergillus niger
Hans (J.).M. van den Brnk, Cees.A.M.J.J van den Hondel and Robert.F.M. van Gorcom
TNO Nutrition and Food Research, Department of Molecular Genetics and Gene technology, P.O. box 5815, 2280 HV Rijswijk, the Netherlands
Cytochrome P450 enzyme systems comprise two elements; cytochrome P450 reductase (CPR), a generally acting electron donor and the reaction specific cytochrome P450 enzyme. In previous work we have identified the A.niger cytochrome P450 gene encoding benzoate para-hydroxylase (bpha) and the gene encoding cytochrome P450 reductase (cprA). Expression of both genes was shown to be regulated at the transcriptional level by benzoate. However, some indications were obtained that regulation also might occur at post-transcriptional level.
To study the exact mechanism underlying the regulation of gene expression of both genes, the gene control region of both genes were fused to a reporter gene followed by generation of progressive deletions. Using this strategy we were able to identify regions in both gene control regions involved in benzoate dependent induction of gene expression. In gel mobility shift assays, using specific DNA fragments obtained from both promoters, we were able to further localize these Benzoate Responsive Elements (BRE's).
Another mechanism involved in regulation of the BPH enzyme system is the use of different promoters'). Clear differences in mRNA size was observed between cprA and bphA mRNA obtained from induced and from non-induced mycelium. Using 5'-RACE we were able to determine the different transcription start points.
1)Promoter is defined as the part of the gene expression control region where the general transcription factors and the RNA polymerase assemble.
Cloning of cpcB Encoding a WD - Protein from Aspergillus nidulans
Christoph Wanke, Kirsten Kirchner, Claus Frick & Gerhard Braus. Friedrich Alexander University, Institute for Biochemistry, Microbiology, and Genetics Staudstr. 5, 91058 Erlangen, Germany
Imbalances in the pool of amino acids in fungi result in a coordinated derepression of amino acid biosynthetic genes (general control) and has been described in Neurospora crassa, Asperaillus nidulans, and Saccharomyces cerevisiae. Recently, a gene (cpc-2) from Neurospora crassa has been cloned (MGG, 248, 162-173) which influences general control as well as formation of female sexual organs (protoperithecia).
Here, we report the cloning of cpcB, the Neurospora crassa cpc-2 homologue from Aspergillus nidulans. cpcB encodes a protein entirely composed of WD-repeats and is highly homologous to similar proteins from eukaryotes including fungi, plants, and animals, indicating a conserved function during evolution.
The Elongation Factor 1 (EF-1 ) of Schizophyllum commune
Wendland, J. and Kothe, E. Dept. of Genetics, Philipps-University, Karl von Flisch Str., 35032 Marburg, Germany
The translation elongation factor lot (EF-l ) plays an essential role during protein synthesis by transferring aminoacyl-tRNA into the acceptor site of the ribosome in a process requiring GTP. In evolutionary terms the amino acid sequence of EF-l has been highly conserved. PCR using degenerate oligonucleotide primers allowed the amplification of a 750 bp fragment from genomic DNA. This PCR fragment was then used to obtain genomic clones containing the entire tef-locus. By sequence analysis seven introns interrupting the tefl open reading frame were identified. Southern blot analysis revealed only one copy of the tefl-gene in genomic DNA. The level of tefl mRNA is very high in growing cells allowing for the use of the tefl promoter for gene expression in S. commune. Amino acid sequence alignments of Tefl of S. commune showed a high degree of similarity to other fungal EF1- sequences.
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