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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 56 (2005), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Fungal morphogenesis and secondary metabolism are frequently associated; however, the molecular determinants connecting both processes remain largely undefined. Here we demonstrate that CPCR1 (cephalosporin C regulator 1 from Acremonium chrysogenum), a member of the winged helix/regulator factor X (RFX) transcription factor family that regulates cephalosporin C biosynthesis, also controls morphological development in the β-lactam producer A. chrysogenum. The use of a disruption strain, multicopy strains as well as several recombinant control strains revealed that CPCR1 is required for hyphal fragmentation, and thus the formation of arthrospores. In a ΔcpcR1 disruption strain that exhibits only hyphal growth, the wild-type cpcR1 gene was able to restore arthrospore formation; a phenomenon not observed for ΔcpcR1 derivatives or non-related genes. The intracellular expression of cpcR1, and control genes (pcbC, egfp) was determined by in vivo monitoring of fluorescent protein fusions. Further, the role of the forkhead transcription factor AcFKH1, which directly interacts with CPCR1, was studied by generating an Acfkh1 knockout strain. In contrast to CPCR1, AcFKH1 is not directly involved in the fragmentation of hyphae. Instead, the presence of AcFKH1 seems to be necessary for CPCR1 function in A. chrysogenum morphogenesis, as overexpression of a functional cpcR1 gene in a ΔAcfkh1 background has no effect on arthrospore formation. Moreover, strains lacking Acfkh1 exhibit defects in cell separation, indicating an involvement of the forkhead transcription factor in mycelial growth of A. chrysogenum. Our data offer the potential to control fungal growth in biotechnical processes that require defined morphological stages for optimal production yields.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0983
    Keywords: Evolution of mitochondria ; Heterotrophic alga Prototheca wickerhamii ; Mitochondrial SSUrRNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The gene for the mitochondrial small subunit rRNA (SSUrNA) from the heterotrophic alga Prototheca wickerhamii has been isolated from a gene library of extranuclear DNA. Sequence and structural analyses allow the determination of a secondary structure model for this rRNA. In addition, several sequence motifs are present which are typically found in SSUrRNAs of various mitochondrial origins. Unexpectedly, the Prototheca RNA sequence has more features in common with mitochondrial SSUrRNAs from plants than with that from the green alga Chlamydomonas reinhardtii. The phylogenetic relationship between mitochondria from plants and algae is discussed.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 19 (1991), S. 73-76 
    ISSN: 1432-0983
    Keywords: Acremonium (Cephalosporium) species ; Cephalosporin C producer ; Electrophoretic karyotype
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A restriction fragment length polymorphism (RFLP) analysis was performed on six related Acremonium strains. With respect to the restriction fragment pattern, all strains of A. chrysogenum were indistinguishable from each other but showed distinctive differences from those of A. strictum, A. flavum and Cephalosporium polyvaleurum. Using pulsed-field gel electrophoresis, we obtained different chromosome patterns from most of the Acremonium strains. Remarkably, the pattern varies in three related A. chrysogenum strains which also differ in their rate of cephalosporin C biosynthesis. The electrophoretic karyotyping was confirmed by the location of rDNA genes on separate chromosomes. Our data indicate that chromosome translocations in industrial strains may be responsible for increased β-lactam synthesis.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Applied microbiology and biotechnology 23 (1986), S. 462-469 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary With the ultimate intent to establish a transformation system for eukaryotic organelles, the structure and organization of mitochondrial genes from the unicellular algaChlamydomonas reinhardii has been investigated. Using DNA hybridisation and DNA sequencing techniques, 3.9 kb of DNA, comprising about 25% of the mitochondrial genome, have been analysed in detail. By comparing the primary structure of homologous genes from other eukaryotic systems, we were able to identify the continuous genes coding for cytochrome oxidase subunit I (COI) and a NADH dehydrogenase subunit (ND5). The two genes are coded by opposite DNA strands and are not overlapping. The COI and the ND5 genes code for 505 and 567 amino acids, respectively. Interestingly, the comparative analysis with homologous genes from other eukaryotes shows that the universal genetic code is used in mitochondria ofC. reinhardii. This situation is different from all other mitochondrial systems studied so far. The results provide evidence thatC. reinhardii would be the appropriate organism for development of a transformation and expression system, where foreign genes, translated via the universal genetic code, are introduced into mitochondria.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The electropheretic karyotype for the imperfect filamentous fungus Tolypocladium inflatum and for six related strains is presented. Pulsed-field gel electrophoresis was used with improved separation conditions to separate DNA from 6.6 Mb to 1.05 Mb in size. Using probes encoding rRNA or the β-tubulin gene from T.iinflatum the corresponding genes were detected on designated chromosomes. In addition, two recombinant lambda clones, carrying T. inflatum chromosomal DNA, were used as chromosome-specific probes. Although all strains investigated are very similar in their morphology, significant chromosome-lengt polymorphisms were detected, allowing easy strain differentiation. The polymorphism was confirmed using an rRNA probe for genomic mapping. All strains contain a homologous “minichromosome” of 1.05 Mb. Finally, the resolution of very large DNA enabled us to separate a 6.6-Mb DNA band that specifically hybridizes with mitochondrial gene probes. The electrophoretic karyotyping presented here may be regarded as a reliable molecular tool to differentiate morphologically very similar filamentous fungi.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary We have performed a comparative transcript analysis with RNA from two different strains of Cephalosporium acremonium, using synthetic oligonucleotides as specific probes for the isopenicillin N-synthetase gene (pcbC). Strain DSM 2353 shows a considerably higher amount of the pcbC transcript than strain ATCC 14553. Subsequently, a genomic library from C. acremonium strain DSM 2353 DNA was constructed using lambda vector EMBL4. We have isolated five recombinant clones containing identical copies of the pcbC gene as was confirmed by partial DNA sequencing. The 5′ region of the pcbC gene was fused with the prokaryotic gene for hygromycin B phosphotransferase (hph) using a synthetic oligonucleotide linker. The resulting plasmid pMW1 can be used for high frequency transformations of the filamentous fungus Aspergillus niger (about 10000 transformants/μg plasmid DNA). From Southern hybridization analysis it can be concluded that all transformants tested contain vector DNA integrated into the genomic DNA. The expression of the prokaryotic hph gene in A. niger was conclusively demonstrated with an assay specific for hygromycin B phosphotransferase.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 25 (1994), S. 34-40 
    ISSN: 1432-0983
    Keywords: Acremonium chrysogenum ; β-tubulin gene ; Homologous transformation system ; Benomyl resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Theβ−tubulin gene was isolated from the filamentous fungusAcremonium chrysogenum using a heterologous gene probe to screen anA. chrysogenum lambda library. Sequencing of theA. chrysogenum gene revealed a mosaic gene which contains five exons and four intervening sequences. The exons encode for a polypeptide of 447 amino-acid residues which showed a high degree of similarity when compared with amino-acid sequences from β-tubulins of other eukaryotes. The introns are characterized by typical consensus sequences found in intervening sequences from other filamentous fungi. In-vitro mutagenesis of codon 167 of the β-tubulin gene resulted in the substitution of a phenylalanine by a tyrosine in the corresponding polypeptide sequence. The mutated gene was used successfully in the transformation and co-transformation ofA. chrysogenum to benomyl resistance. The molecular analysis of transformants provided evidence that they contain the mutated β-tubulin gene in addition to the wild-type gene, as was proved by Southern-hybridization analysis and direct sequencing of PCR amplification products.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1432-0983
    Keywords: Key wordsChlamydomonas reinhardtii ; Chlorophyll-a/b-binding protein ; Light regulation ; Plastid signal ; Gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In Chlamydomonas reinhardtii, expression of the lhcb1 gene encoding a chlorophyll-a/b-binding protein of photosystem II is highly regulated by light, inhibitors of chlorophyll synthesis, as well as by circadian rhythms. In light/dark synchronized cultures, the rapid increase of lhcb1 mRNA levels during the light phase is regulated primarily at the transcriptional level. We have used the arylsulphatase (ars) reporter gene to analyze the lhcb1 5′ upstream sequences for the presence of light-responsive elements. In transformants carrying chimeric reporter genes, accumulation of lhcb1/ars mRNA is markedly stimulated by light, with a time course similar to that of transcripts from the endogenous lhcb1 gene. Promoter deletion studies revealed that a 255-bp fragment of the lhcb1 5′ upstream region is sufficient to confer proper light regulation on the promoterless ars gene. Moreover, the region between positions –255 and –122 with respect to the start site of translation were found to contain one or more light-responsive elements. Strikingly, these sequences also seem to be involved in chloroplast-dependent lhcb1 gene expression as indicated by Northern analyses of transformants with photo-oxidatively damaged chloroplasts. This suggests that both light- and chloroplast-dependent expression of the lhcb1 gene are mediated by the same cis-acting elements.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 5 (1982), S. 143-147 
    ISSN: 1432-0983
    Keywords: Podospora anserina ; Mitochondrial DNA cloning ; Restriction and gene mapping
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In order to develop an eukaryotic vector with the Podospora plasmid, further characterization is required of the mitochondrial DNA into which this plasmid is integrated, a physical map (restriction sites) of the Podospora chondriome (size 95 kb) has been completed. As prerequisite for the establishment of a genetic (functional) map, 70% of the chondriome was cloned in E. coli vectors. Using mitochondrial genes from Saccharomyces cerevisiae, six structural genes were located on the Podospora chondriome by cross hybridization experiments. There is strong evidence that the plasmid is inserted into the cytochrome b gene. A comparison of the genetic map of the Podospora chondriome with those of Neurospora crassa and Aspergillus nidulans exhibits a rather good accordance with respect to the sequence of genes.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1432-0983
    Keywords: Sordaria macrospora ; DNA transformation ; pcbC Promoter ; Heterologous integration ; Pulsed-field gel electrophoresis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. marcospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5 Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora
    Type of Medium: Electronic Resource
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