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U Kück

Publications and source records attributed to U Kück.

At least 37 records · Page 2Linked to original sources
PubMed1998

Transposons in filamentous fungi--facts and perspectives.

Transposons are ubiquitous genetic elements discovered so far in all investigated prokaryotes and eukaryotes. In remarkable contrast to all other genes, transposable elements are able to move to new locations within their host genomes. Transposition of transposons into coding sequences and their initiation of chromosome rearrangements have tremendous impact on gene expression and genome evolution. While transposons have long been known in bacteria, plants, and animals, only in recent years has there been a significant increase in the number of transposable elements discovered in filamentous fungi. Like those of other eukaryotes, each fungal transposable element is either of class or of class II. While class I elements transpose by a RNA intermediate and employ reverse transcriptases, class II elements transpose directly at the DNA level. We present structural and functional features for such transposons that have been identified so far in filamentous fungi. Emphasis is given to specific advantages or unique features when fungal systems are used to study transposable elements, e.g., the evolutionary impact of transposons in coenocytic organisms and possible experimental approaches toward horizontal gene transfer. Finally, we focus on the potential of transposons for tagging and identifying fungal genes.

Animals↗
PubMed1998

Distribution of the fungal transposon Restless: full-length and truncated copies in closely related strains.

The fungal transposon Restless of Tolypocladium inflatum ATCC 34921 is a member of the hAT family of mobile DNA elements. In order to study the distribution of this transposon we have looked at 13 fungal strains, most of which are taxonomically related to strain ATCC 34921. Three strains, which show identical banding patterns in a comparative RAPD analysis with strain ATCC 34921, similarly carry multiple copies of Restless. In addition, 1 T. inflatum strain and 2 Beauveria nivea strains contain only a few or even single copies of the transposon. Inverse PCR and DNA sequencing analysis revealed that 1 strain contains a nonmobile truncated version of the element, while the other one harbors a full-length transposon copy which was named Restless-2. The presence of a single transposon copy of Restless in a defined Beauveria strain indicates recent acquisition of this transposon, since class II transposons usually occur in several copies per haploid genome. Notably, the corresponding genomic location is not occupied by a transposon copy in strain ATCC 34921.

Base Sequence↗
PubMed1998

Splicing of the mitochondrial group-II intron rl1: conserved intron-exon interactions diminish splicing efficiency.

The mitochondrial intron rI1 is a self-splicing group-II intron of algal mitochondria that can be transferred into chloroplasts from the green alga Chlamydomonas reinhardtii for in vivo investigations (Herdenberger et al. 1994). Thus, rI1 is a suitable system to compare in vitro and in vivo RNA processing. Interestingly, rI1 shows correct RNA splicing, although typical cis-acting exon-sequences (IBS2, delta) of group-II introns are lacking. In order to examine the effect of these exon-intron interactions on splicing, we introduced the endogenous mitochondrial IBS2 sequence in order to produce optimal IBS2-EBS2 base pairing. In addition, the first nucleotide of the 3'exon (delta') was substituted to create an optimal delta-delta' interaction. Neither of the two mutations, nor a combination of both, had any effect on the precision of the splice-site selection. Unexpectedly, introduction of IBS2 led to a reduction in the efficiency of the second splicing step in vitro but not in vivo. These findings lead us to conclude that trans-acting factors are present in vivo to optimize splicing efficiency. The possibility is discussed that these factors may, for example, stabilize tertiary intron structures that are a prerequisite for correct RNA processing. Furthermore, our data indicate that similar trans-acting factors promote correct intron splicing in chloroplasts and mitochondria.

Alternative Splicing↗
PubMed1998

Evidence for circular transposition derivatives from the fungal hAT-transposon Restless.

The structure and function of eukaryotic hAT-transposons has already been elucidated; however, their transposition mechanism is barely understood. We recently have discovered Restless, a fungal member of the hAT-transposon family, which shows transposition in its host Tolypocladium inflatum. Investigations of two strains from T. inflatum, carrying either about 15 copies or only a single copy of the Restless element, indicate the presence of circular transposition intermediates. Using PCR technology, amplicons were identified which carry the joined end of the Restless transposon fused at its inverted repeats. All of eight sequenced PCR fragments contained the intact transposon ends with a short insertion of 1-93 bp of genomic DNA. Remarkably, one of the discovered genomic sequences matches a previously described integration site. Our data are discussed with respect to the transposition mechanism and the horizontal transfer of hAT-transposons.

DNA Transposable Elements↗
PubMed1998

The Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase is encoded by a light-regulated gene in Chlamydomonas reinhardtii.

We have studied the light-dependent expression of the Chlamydomonas reinhardtii csbp gene encoding sedoheptulose-1,7-bisphosphatase (SBPase), an enzyme of the pentose-phosphate pathway. Expression studies using light/dark-synchronized cultures revealed that csbp mRNA abundance increases significantly during illumination. We have used a 1.4 kb region upstream of the csbp gene in transcriptional fusions to the homologous arylsulfatase-encoding reporter gene (ars). In transformants carrying the chimeric csbp/ars reporter gene, arylsulfatase activity is detected in the absence of sulfate, a condition under which the endogenous ars gene is repressed. Moreover, ars mRNA accumulation is dramatically stimulated by light, indicating that 1.4 kb of the csbp 5'-untranslated region are sufficient to confer light-dependent expression on the ars reporter gene.

Animals↗
PubMed1997

Efficient synthesis of the blood-coagulation inhibitor hirudin in the filamentous fungus Acremonium chrysogenum.

The isopenicillin-N-synthetase-encoding pcbC gene from the filamentous fungus Acremonium chrysogenum is differentially expressed in strains showing either a high or low cephalosporin C production. For a case study to demonstrate heterologous protein synthesis in A. chrysogenum, we have chosen a synthetic 195-bp gene encoding the thrombin inhibitor hirudin from the leech Hirudo medicinalis. The hirudin gene was fused with the 5' and 3' regions of the pcbC gene, resulting in four different expression vectors, which we named pHIR1 to pHIR4. In order to achieve secretion of the heterologous polypeptide, two out of four vectors carry, in addition, secretion signal sequences of an alkaline protease gene originating either from Fusarium sp. or from A. chrysogenum. After DNA-mediated transformation of the two A. chrysogenum strains, transformants were further analysed on the transcriptional and translational level. Irrespective of the vector used for transformation, all transformants show a hirudin-gene-specific transcript in Northern hybridizations. In further analysis, hirudin synthesis was determined with a thrombin-inhibition assay, but was detectable only in those strains carrying expression plasmids with the secretion signals. In this case, hirudin was secreted into the culture medium. Transformants from strains with a high cephalosporin C production showed a three- to eightfold higher expression of the hirudin gene compared to low cephalosporin-C-producing strains. The amount of recombinant hirudin was quantified further by ELISA and Western blotting, using a monoclonal antibody directed against recombinant hirudin. Finally, the time course of hirudin gene expression was investigated in a selected transformant that has hirudin activities of 8.0 ATU/ml culture medium. Northern hybridization experiments revealed the highest hirudin transcript level after 2-5 days of cultivation, showing the strongest signal after 3 days. After 4-5 days, we detected the highest hirudin activity, as was confirmed by Western blotting. The level of heterologous hirudin synthesis in A. chrysogenum is discussed in relation to other eukaryotic expression systems.

Acremonium↗
PubMed1997

Parental and novel copies of the mitochondrial orf25 gene in the hybrid crop-plant triticale: predominant transcriptional expression of the maternal gene copy.

Triticale, an intergeneric hybrid crop-plant, is generated when female wheat lines are fertilised with pollen from rye. We have investigated the mitochondrial DNA organisation and the expression of a total of 11 different triticale genotypes, varying in their nuclear and cytoplasmic backgrounds. In Southern hybridisations using probes homologous to the upstream flanking sequences, mtDNA fragments characteristic of both wheat and rye mtDNA can be detected in all triticale lines analysed. In addition, clones isolated fom a triticale lambda library exhibit either a maternal-like or paternal-like organisation of the orf25 gene region. By PCR cloning, four different orf25 gene copies were identified in triticale, three of which correspond to maternal (85%) or paternal (12%) orf25 sequences. Three percent of all clones represent a novel type, that might have arisen by homologous recombination. Although these data suggest biparental inheritance of mtDNA in wheat/rye crosses, paternal-like gene copies can also be detected in maternal wheat mitochondria. Their stoichiometry as assayed by competitive PCR is about 0.1% of total orf25 gene copies. The high abundance of paternal-like sequences in the F1 hybrid might therefore be due to either the transmission of rye mtDNA in the intergeneric cross and/or the amplification of sequences in triticale that persist in sub-stoichiometric amounts in wheat. These data suggest that amplification and recombination of sub-genomic mitochondrial molecules are affected by different nuclear genotypes. Interestingly, sequence analysis of triticale RT-PCR clones indicates a selective transcription of maternal-like orf25 gene copies in triticale. Mitochondrial gene expression may therefore possess mechanisms to compensate for the variation of mtDNA organisation.

Base Sequence↗
PubMed1997

Mating-type genes from the homothallic fungus Sordaria macrospora are functionally expressed in a heterothallic ascomycete.

Homokaryons from the homothallic ascomycte Sordaria macrospora are able to enter the sexual pathway and to form fertile fruiting bodies. To analyze the molecular basis of homothallism and to elucidate the role of mating-products during fruiting body development, we cloned and sequenced the entire S. macrospora mating-type locus. Comparison of the Sordaria mating-type locus with mating-type idiomorphs from the heterothallic ascomycetes Neurospora crassa and Podospora anserina revealed that sequences from both idiomorphs (A/a and mat-/mat+, respectively) are contiguous in S. macrospora. DNA sequencing of the S. macrospora mating-type region allowed the identification of four open reading frames (ORFs), which were termed Smt-a1, SmtA-1, SmtA-2 and SmtA-3. While Smt-a1, SmtA-1, and SmtA-2 show strong sequence similarities with the corresponding N. crassa mating-type ORFs, SmtA-3 has a chimeric character. It comprises sequences that are similar to the A and a mating-type idiomorph from N. crassa. To determine functionality of the S. macrospora mating-type genes, we show that all ORFs are transcriptionally expressed. Furthermore, we transformed the S. macrospora mating-type genes into mat- and mat+ strains of the closely related heterothallic fungus P. anserina. The transformation experiments show that mating-type genes from S. macrospora induce fruiting body formation in P. anserina.

Amino Acid Sequence↗
PubMed1996

Altered expression of nuclear genes encoding chloroplast polypeptides in non-photosynthetic mutants of Chlamydomonas reinhardtii: evidence for post-transcriptional regulation.

In photoautotrophic organisms it is well documented that the expression of nuclear genes encoding plastid proteins can be regulated at various levels. We present here the analysis of a non-photosynthetic strain (CC1051) of the green unicellular alga Chlamydomonas reinhardtii; this strain carries a mutation in the newly identified Cen gene involved in the co-regulated expression of several different nuclear genes encoding plastid proteins. We performed a differential screening strategy to isolate cDNAs corresponding to genes that are differentially expressed in mutant and wild-type strains. Extensive hybridization experiments revealed that the 15 cDNA clones isolated represent five different mRNAs that fail to accumulate in the non-photosynthetic mutant. Comparative analysis of DNA sequencing data showed that they all code for plastid proteins. In particular, we identified genes for the chlorophyll a/b binding protein of the light-harvesting complex II (LHCII), for subunits II and III of photosystem I (PsaD, PsaF), for pentose-5-phosphate 3-epimerase (PPE), an enzyme of the Calvin cycle, and for an unidentified 7 kDa protein with a suggested lumenal location. With the exception of the gene for LHCII, all proteins are encoded by single-copy genes. Evidence from run-on transcription experiments is presented showing that expression of the above mentioned plastid proteins is affected at the post-transcriptional level in the mutant strain CC1051 with a defect in the Cen gene. Our results suggest that the product of the Cen gene is involved in stabilization and/or processing of transcripts from nuclear genes encoding chloroplast proteins.

Amino Acid Sequence↗
PubMed1996

Efficient synthesis of a 72-kDa mitochondrial polypeptide using the yeast Ty expression system.

Using the Ty system from yeast we report the efficient expression of a heterologous eukaryotic gene encoding a 72 kDa mitochondrial polypeptide. The pFM2IIBgIII expression vector was initially modified for this purpose by inserting the factor X(a) protease cleavage site. The TyA gene, which encodes the structural component of the yeast virus-like particles (VLPs), and the eukaryotic yst1 gene, encoding a 72 kDa mitochondrial tyrosyl-tRNA synthetase from the filamentous fungus Podospora anserina, were subsequently fused to the factor X(a) cleavage site. The resulting chimeric gene, in which the two polypeptide coding sequences are separated by the factor X(a) cleavage site, was expressed in yeast. High yield expression of this foreign protein, which was isolated from yeast transformants as hybrid TyVLPs, was verified after factor X(a) treatment by SDS polyacrylamide gel electrophoresis and antibody detection. The strategy presented here should be useful for expressing a wide variety of eukaryotic genes.

Base Sequence↗
PubMed1996

Transcript mapping and processing of mitochondrial RNA in the chlorophyte alga Prototheca wickerhamii.

The detailed transcript map of the circular 55328 bp mitochondrial (mt) genome from the colourless chlorophycean alga Prototheca wickerhamii has been determined. On each half of this genome the genes are encoded only on one DNA strand, forming transcriptional units comprising variable numbers of genes. With the exception of four genes coding for ribosomal proteins, transcripts of the three rRNA genes and all protein-coding genes have been detected by both northern analysis and primer extension experiments. Polycistronic transcripts of protein coding and tRNA genes were verified by northern analyses, primer extension and RNAse mapping experiments. The 5' and 3' ends of different RNA species are often located in close proximity to putative stem-loop structures and some 5' termini of mRNAs coincide with the 3' end of tRNAs located immediately upstream. Transcript mapping in a putative promoter region revealed two different possible transcription initiation sites; no significant sequence homology to putative mt promoters from higher plants could be found. In addition, two out of three group I introns residing in the cox1 gene were found to be self-splicing in vitro under reaction conditions developed for related mt introns from a filamentous fungus. Mitochondrial gene expression of P. wickerhamii and of filamentous fungi has several features in common, such as intron splicing and the processing of longer polycistronic transcripts. The similarities in RNA maturation between higher-plant and P. wickerhamii mitochondria are less pronounced, since plants rarely use tRNAs as processing signals for their relatively short mitochondrial co-transcripts.

Base Sequence↗
PubMed1996

restless, an active Ac-like transposon from the fungus Tolypocladium inflatum: structure, expression, and alternative RNA splicing.

Elements of the hAT transposon family, such as the maize activator (Ac), have been discovered in a large number of eukaryotic species. This type of class II transposon, present in both plants and animals, has not been previously detected in any fungal organism. However, using a differential screening method to detect repetitive DNA, we have identified a hAT transposon in the hyphomycete Tolypocladium inflatum. The transposon, which we named restless, is 4,097 bp long, carries 20-bp inverted repeats and an 8-bp target site duplication, and encodes a long open reading frame which is interrupted by a single intronic sequence. The derived mRNA exhibits alternative splicing, resulting in the formation of two transcripts that may be translated into polypeptides of 157 or 803 amino acids. The predicted amino acid sequence of the larger polypeptide demonstrates significant homology with transposases from the hAT transposon family. A chromosomal analysis using pulsed-field gel electrophoresis showed that all seven chromosomal bands carry copies of the 4.1-kb transposon. This was confirmed in hybridization experiments with rare-cutting restriction endonucleases which indicate that about 15 copies are present in T. inflatum. The genomic distribution of restless and its transcriptional expression, alternative mRNA splicing, and genomic mobility all imply a potential role for this element in developing a transposon tagging system for use in filamentous fungi.

Alternative Splicing↗
PubMed1995

A unique repeated DNA sequence in the cyclosporin-producing strain of Tolypocladium inflatum (ATCC 34921).

Recombinant lambda clones containing repeated DNA sequences were isolated from the cyclosporin A-producing fungus Tolypocladium inflatum (ATCC 34921) by differential hybridization with total fungal DNA and rDNA probes. From this survey 1% of the lambda clones appeared to contain repeated sequences. Subsequent analysis led to the identification of a dispersed repetitive DNA element. It was named CPA element (cyclosporin production associated) and appears to be strain specific, since it is absent from other related strains or fungi. Hybridization with chromosomal restriction fragments indicates an equal distribution of the CPA element in the genome. The copy number was estimated to be between 20 and 30 per haploid genome. Sequence analysis of a 0.9-kb XhoI fragment from three copies of the CPA element revealed strong conservation of this sequence among all copies. A 200-bp region exhibits similarities to a repeated sequence from Zea diploperennis. The use of this DNA sequence as a molecular marker for identification of this cyclosporin-producing strain ATCC 34921 is discussed as is the relevance of repeated DNA sequences for rearrangements of fungal karyotypes.

Base Sequence↗
PubMed1995

DNA-mediated transformation of a fungus employing a vector devoid of bacterial DNA sequences.

Acremonium chrysogenum, a producer of cephalosporin C, was subjected to DNA-mediated transformations using a vector without bacterial DNA sequences. Recombinant fungal strains were generated with a gel-purified DNA fragment, carrying only the mutated beta-tubulin gene from A. chrysogenum. The lack of any bacterial DNA was verified by Southern hybridization analysis and polymerase chain reaction amplifications to detect even residual DNA sequences. This procedure can be referred to as a self-cloning experiment for which less restricted working regulations are needed. Finally, the transfer of a synthetic hirudin gene by cotransformation demonstrated that any DNA molecule can be introduced into the A. chrysogenum genome without bacterial marker genes. This seems to be highly relevant for biotechnical processes in which safe recombinant producer strains are required to satisfy governmental restrictions.

Acremonium↗
PubMed1995

The mitochondrial atpA/atp9 co-transcript in wheat and triticale: RNA processing depends on the nuclear genotype.

The gene region coding for subunits alpha and 9 of the mitochondrial ATP synthase exhibit an identical DNA sequence in wheat, rye, and the intergeneric hybrid triticale (xTriticosecale Wittmack). However, co-transcripts containing both genes show different sizes depending on the nuclear genotype. To investigate nuclear-mitochondrial interactions leading to this variation, we performed a comparative transcript analysis with various lines carrying defined nuclear and cytoplasmic genotypes. Northern analyses showed that all wheat lines investigated possess a single atpA/atp9 mRNA of 2.6kb, whereas in rye and five independent triticale lines an additional transcript of 2.35kb appeared. Primer-extension and RNase-protection analyses indicate that the co-transcripts of this gene have staggered 5' termini in some lines, whereas the 3' termini seem to be similar in wheat, rye, and triticale. Transcription is initiated at position -338/-339 upstream of the atpA gene in all lines investigated, giving rise to a 2.6-kb mRNA. In rye and triticale, staggered 5' termini were observed closer to the translational start. The DNA sequences upstream of these termini exhibit homology to plant mitochondrial-processing sites, therefore the proximal 5' ends are most probably generated by RNA processing. As the processing event occurs more frequently in triticale carrying the Triticum timopheevi cytoplasm, trans-acting factors from rye are likely to interact with other cytoplasmic factors resulting in the observed RNA modification. Most interestingly, the T. timopheevi cytoplasm inducing male sterility in alloplasmic wheat, fails to generate the CMS phenotype in triticale. The data support our hypothesis that nuclear factors affect mitochondrial gene expression and thus control sexual fertility in wheat and triticale.

Arabidopsis Proteins↗
PubMed1995

Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis.

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. macrospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5Mb. 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.

Ascomycota↗
PubMed1994

Correct in vivo RNA splicing of a mitochondrial intron in algal chloroplasts.

The self-splicing group II intron (rl1) from Scenedesmus obliquus mitochondria together with its 6 bp intron binding site (IBS1) were inserted in the correct and inverse orientation into the chloroplast tscA gene from C.reinhardtii. Precursor RNA derived from the chimeric tscA-rl1 gene can be used to demonstrate in vitro self-splicing of the rl1 intron RNA. Using the particle bombardment technique, the tscA-rl1 construct was transferred into the chloroplast of the unicellular alga Chlamydomonas reinhardtii. We recovered transformants which contain the chimeric tscA-rl1 gene as shown by Southern analysis. Hybridization and PCR analysis of transcripts confirmed that the heterologous intron is correctly spliced in vivo. From sequencing of cDNA clones we conclude that the IBS1 sequence is sufficient for correct splicing of the mitochondrial intron in C. reinhardtii chloroplasts. Using specific probes, we demonstrate by Northern hybridization that the mature RNA, as well as an intron-3' exon intermediate, accumulate in transformants containing the rl1 intron, correctly inserted into the tscA gene. As expected, no RNA splicing at all was observed when the intron had an inverted orientation within the tscA gene. In addition, a mutated intron RNA with an altered 3' terminal nucleotide was tested in vivo. In contrast to similar mutants examined in vitro, this mutated RNA shows accumulated intron and intron-3' exon intermediates, but no ligated exons at all. Our approach should prove useful for elucidating nucleotide residues involved in splicing of organelle introns in vivo.

Animals↗