Biomedical subjects

H D Osiewacz

Publications and source records attributed to H D Osiewacz.

At least 37 records · Page 2Linked to original sources

PubMed1997

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea.

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5' upstream region of the grisea ORF. One mutation is a G --> A transition at the 5' splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

Ascomycota↗

PubMed1997

Genetic regulation of aging.

Aging of biological systems is a complex process that is controlled by both environmental factors and the genetic constitution of the individual. Although the molecular mechanisms have not been elucidated for any system in detail, it is clear that various genetic traits are involved in the modulation of life span. In particular, the genetic information located in the mitochondria has been identified as a major genetic component. Instabilities of the mitochondrial DNA (mtDNA) lead to mitochondrial dysfunction and increased oxidative stress. In some cases mtDNA instabilities are related to the activity of mobile genetic elements. In addition, nuclear genes appear to be crucially involved in mtDNA maintenance. Furthermore, the initial analysis of a few cloned nuclear genes affecting life span suggests a cellular machinery dealing with various stress situations as a major component involved in the genetic control of aging. This conclusion may hold true for all biological systems and be related to a unified mechanism of aging. However, in the various lineages this mechanism may be superimposed by other species or lineage-specific mechanisms.

Aging↗

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↗

PubMed1997

DNA reorganization and biological aging. A review.

Biological aging is a fundamental process observed in almost all living beings. It is characterized by a progressive impairment of biological systems leading to an increase in age-related mortality. The understanding of the mechanisms of aging is of particular interest not only from a general point of view, but it has wide implications for both the human individual and society. Specifically, understanding the basic mechanisms leading to human aging will certainly provide novel strategies to deal with the many different and severe age-related diseases (e.g., different types of cancer, Alzheimer's disease, cardiovascular diseases) and can be expected to increase the quality of life in old age significantly. Despite this obvious significance, it is amazing how little is known about the basis of aging. Currently, it is not clear whether there exists a single conserved mechanism by which all biological systems age, nor is it clear whether or not such a mechanism accounts for aging in a single species. However, beside these and many other open issues, it is clear today that there is a genetic component of aging. It is also clear that age-dependent changes of genetic information play an important role. The processes leading to the observed age-related accumulation of DNA alterations is dependent on two major factors: the rate at which the alterations occur and the ability of the biological system to repair them. In addition, there are a number of different types of DNA alterations known. One group of changes are unspecific and occur randomly. Other alterations result from the differential activity of specific genetic traits (e.g., age-related genes, mobile genetic elements). Moreover, DNA changes may be subtle, occurring at the nucleotide level, or may include larger regions of the genetic information. In this review we focus on the latter type of age-related DNA reorganizations. We will refer to reorganizations which are of special relevance in different biological systems.

Aging↗

PubMed1996

GRISEA, a putative copper-activated transcription factor from Podospora anserina involved in differentiation and senescence.

Podospora anserina is a filamentous fungus with a limited lifespan. Lifespan is controlled by both environmental and genetic factors. Using a combination of genetic and molecular approaches we have cloned one of these factors, gerontogene grisea. The cloned wild-type copy of grisea complements the altered morphological characteristics (e.g., colony and ascospore color), the defect in gametangia development, and the increased lifespan of the pleiotropic mutant grisea. A molecular analysis revealed that grisea is a discontinuous gene with a single intron. The deduced amino acid sequence shows significant homology to MAC1, ACE1 and AMT1, indicating that GRISEA, like the proteins from Saccharomyces cerevisiae (MAC1 and ACE1) and Candida glabrata (AMT1), codes for a copper-activated transcription factor. This conclusion is consistent with the pleiotropic nature of the grisea phenotype. We suggest that the gerontoprotein GRISEA is one component of a transcription apparatus involved in the genetic control of morphogenesis and aging.

Amino Acid Sequence↗

PubMed1996

Induction of longevity by cytoplasmic transfer of a linear plasmid in Podospora anserina.

In Podospora anserina the longevity inducing linear plasmid pAL2-1 was transferred from the extrachromosomal long-lived mutant AL2 to the short-lived wild-type strain A. The resulting strain, AL2-IV, exhibited the long-lived phenotype. In the short-lived progeny of crosses between this strain and wild-type strain A, the plasmid was absent. In contrast, all long-lived progeny contained both the autonomous plasmid as well as copies of it integrated in the mitochondrial DNA (mtDNA). Molecular analysis revealed that the integrated plasmid copies most likely resulted from a de novo integration of the autonomous element and the generation of AT-linker sequences at the integration site. We conclude that once the plasmid is present in mitochondria of a particular genetic background, it is able to integrate into the mtDNA and to induce longevity.

Ascomycota↗

PubMed1996

Telomere length does not change during senescence of the ascomycete Podospora anserina.

All strains of the filamentous fungus Podospora anserina are characterized by a well defined life span. Senescence is controlled by nuclear and extranuclear genetic traits. In order to test whether or not the ends of the chromosomes of this ascomycete shorten during senescence and thus telomere shortening may be linked to the well analyzed, age-related reorganizations of the mitochondrial DNA (mtDNA), we analyzed the genomic DNA of P. anserina wild-type strain s. We found that, although the mtDNA becomes reorganized when cultures age, the telomeres remain constant suggesting that telomere shortening does not play a major role in normal aging of this particular biological system.

Ascomycota↗

PubMed1995

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina.

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wat, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5' ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extra-chromosomal genetic traits.

Ascomycota↗

PubMed1995

Evidence for giant linear plasmids in the ascomycete Podospora anserina.

In the extrachromosomal mutant AL2 of the ascomycete Podospora anserina longevity is correlated with the presence of the linear mitochondrial plasmid pAL2-1. In addition to this autonomous genetic element, two types of closely related pAL2-1-homologous molecules were detected in the high-molecular-weight mitochondrial DNA (mtDNA). One of these molecules is of linear and the other of circular structure. Both molecules contain pAL2-1 sequences which appear to be integrated at the same site in the mtDNA. Sequence analysis of a DNA fragment cloned from one of these molecules revealed that it contains an almost full-length copy of pAL2-1. At the site of plasmid integration a 15-nucleotide AT-spacer and long inverted mtDNA sequences were identified. Finally, two giant linear plasmid-like DNAs of about 50 kbp and 70 kbp were detected in pulsed-field gels of mutant AL2. These molecules are composed of mtDNA and pAL2-1-specific sequences and may result from the integration of mtDNA sequences into linear plasmid pAL2-1.

Base Sequence↗

PubMed1994

Nucleotide sequence of the petB gene encoding cytochrome b6 from the mesophilic cyanobacterium Synechocystis PCC 6803: implications for evolution and function.

The gene encoding the cytochrome b6 subunit (petB) of the cytochrome b6f complex has been isolated, cloned and sequenced by nonradioactive methods from genomic DNA of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 666 nucleotides, coding for a polypeptide with a molecular mass of 25.02 kDa. In contrast to higher plant petB sequences an aminoterminal extension of seven amino acids occurs. Aminoterminal sequencing of the isolated protein excludes--different from higher plants--the existence of an intron after the first amino acids but indicate the posttranslational removal of three amino acids from the amino terminus. The aminoterminal extension--found only in non-nitrogen-fixing, unicellular cyanobacteria--shows a high degree of homology between different species.

Amino Acid Sequence↗

PubMed1994

Evidence for a life span-prolonging effect of a linear plasmid in a longevity mutant of Podospora anserina.

The linear mitochondrial plasmid pAL2-1 of the long-lived mutant AL2 of Podospora anserina was demonstrated to be able to integrate into the high molecular weight mitochondrial DNA (mtDNA). Hybridization analysis and densitometric evaluation of the mitochondrial genome isolated from cultures of different ages revealed that the mtDNA is highly stable during the whole life span of the mutant. In addition, and in sharp contrast to the situation in certain senescence-prone Neurospora strains, the mutated P. anserina mtDNA molecules containing integrated plasmid copies are not suppressive to wild-type genomes. As demonstrated by hybridization and polymerase chain reaction (PCR) analysis, the proportion of mtDNA molecules affected by the integration of pAL2-1 fluctuates between 10% and 50%. Comparative sequence analysis of free and integrated plasmid copies revealed four differences within the terminal inverted repeats (TIRs). These point mutations are not caused by the integration event since they occur subsequent to integration and at various ages. Interestingly, both repeats contain identical sequences indicating that the mechanism involved in the maintenance of perfect TIRs is active on both free and integrated plasmid copies. Finally, in reciprocal crosses between AL2 and the wild-type strain A, some abnormal progeny were obtained. One group of strains did not contain detectable amounts of plasmid pAL2-1, although the mtDNA was clearly of the type found in the long-lived mutant AL2. These strains exhibited a short-lived phenotype. In contrast, one strain was selected that was found to contain wild-type A-specific mitochondrial genomes and traces of pAL2-1. This strain was characterized by an increased life span. Altogether these data suggest that the linear plasmid pAL2-1 is involved in the expression of longevity in mutant AL2.

Base Sequence↗

PubMed1994

Three mitochondrial unassigned open reading frames of Podospora anserina represent remnants of a viral-type RNA polymerase gene.

The mitochondrial DNA of Podospora anserina is complex, consisting of a characteristic set of genes with a large number of introns and a substantial amount of sequence of unknown function and origin. In addition, as indicated by various types of reorganization, this genome is highly flexible. Here we report the identification of three unassigned mitochondrial open reading frames (ORF P', ORF Q', ORF 11) as remnants of a rearranged viral-type RNA polymerase gene. These ORFs are not transcribed and may be derived from the integration of a linear plasmid of the type recently identified in a mutant of P. anserina.

Amino Acid Sequence↗

PubMed1994

A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes.

A shuttle cosmid vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi. This vector contains two cos sequences separated by a single XbaI restriction site. pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15-20 micrograms of genomic DNA. Due to the presence of a functional hygromycin B phosphotransferase gene (hph) transformation of fungal protoplasts with pAN-sCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants. The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts.

Cloning, Molecular↗

PubMed1993

Differential expression of the psbA genes in the cyanobacterium Synechocystis 6803.

The 5' region and transcription initiation sites of the psbA-2 and psbA-3 genes of Synechocystis 6803 were determined. The otherwise highly homologous genes were shown to diverge significantly in the 5' noncoding regions. The transcription start site for the psbA-2 gene was mapped to position -49 upstream of the coding region and for the psbA-3 gene to position -88, i.e. 38 bp upstream of the psbA-2 transcription start point. Both genes exhibit promoter elements, which conform in sequence and position to Escherichia coli consensus motifs. The two genes share identical -35 sequences but differ in their -10 sequences. Primer extension analysis demonstrated that the psbA-2 and psbA-3 genes are differentially expressed, with > 90% of the total psbA transcripts being produced by the psbA-2 gene and the rest by the psbA-3 gene. Inactivation of the psbA-2 gene resulted in an eightfold up-regulation of the psbA-3 gene. The strikingly higher stability of the psbA transcripts in darkness compared to light, and the accumulation of a specific decay intermediate under dark conditions was reported previously. We show here that this dark-stability applies to both the psbA-2 and psbA-3 transcripts. The psbA-3 transcript did not appear to produce the processed intermediate, arguing for the involvement of the 5' non-coding region as a determinant in psbA transcript degradation.

Bacterial Proteins↗

PubMed1992

Evolution of linear plasmids.

Linear plasmids are genetic elements commonly found in yeast, filamentous fungi, and higher plants. In contrast to all other plasmids they possess terminal inverted repeats and terminal bound proteins and encode their own DNA and RNA polymerases. Here we present alignments of conserved amino acid sequences of both the DNA and RNA polymerases encoded by those linear plasmids for which DNA sequence data are available. Additionally these sequences are compared to a number of polymerases encoded by related viral and cellular entities. Phylogenetic trees have been established for both types of polymerases. These trees appear to exhibit very similar subgroupings, proving the accuracy of the method employed.

Amino Acid Sequence↗

PubMed1992

Construction of insertion mutants of Synechocystis sp. PCC 6803: evidence for an essential function of subunit IV of the cytochrome b6/f complex.

The gene encoding subunit IV of the cytochrome b6/f complex (petD) has been isolated from a genomic library of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 480 nucleotides and can code for a polypeptide with a molecular weight of 17.5 kDa. The deduced amino acid sequence shows high identity with the corresponding sequences of both the photoautotrophic prokaryote Nostoc sp. PCC 7906 as well as of lower and higher photoautotrophic eukaryotes (e.g. Chlorella prototecoides, Nicotiana tabacum). Transformation of Synechocystis sp. PCC 6803 with a plasmid containing the cloned petD gene in which the coding sequence is interrupted by the aminoglycoside 3'-phosphotransferase gene (aph) from Tn903 resulted in the formation of km resistant transformants. The molecular analysis of independent transformants revealed that all clones were merodiploid containing both uninterrupted wild-type as well as interrupted mutant petD copies. Approaches to segregate these two genomes were unsuccessful implying an essential function of the petD gene product in Synechocystis sp. PCC 6803.

Amino Acid Sequence↗

PubMed1992

The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase.

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5' termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3'-5' exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.

Amino Acid Sequence↗

PubMed1992

Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency.

The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of Podospora anserina has been isolated from a genomic library by heterologous hybridization with the corresponding gene of Curvularia lunata. The coding region consists of 1014 nucleotides and is interrupted by a single intron. The amino-acid sequence encoded by the gpd gene shows a high degree of sequence identity with the corresponding gene products of various fungi. Multiple alignments of all fungal GPD sequences so far available resulted in the construction of a phylogenetic tree. The evolutionary relationships of the various fungi belonging to different taxa will be discussed on the basis of these data. Sequence analysis of 1.9 kbp of the 5' non-coding region revealed the presence of typical fungal promoter elements. Utilizing different parts of the 5' regulatory sequence of the Podospora gpd gene, expression vectors containing a dominant selectable marker gene (hygromycin B phosphotransferase) have been constructed for the transformation of P. anserina protoplasts. The use of these homologous gpd regulatory sequences resulted in a significant increase in transformation efficiencies compared to those obtained with vectors in which the selectable marker gene is under the control of the corresponding heterologous promoter of Aspergillus nidulans.

Amino Acid Sequence↗