Jörg Servos

Gene SNQ2 of Saccharomyces cerevislae, which confers resistance to 4-nitroquinoline-N-oxide and other chemicals, encodes a 169 kDa protein homologous to ATP-dependent permeases

SummaryThe yeast gene SNQ2 confers hyper-resistance to the mutagens 4-nitroquinoline-N-oxide (4-NQO) and Triaziquone, as well as to the chemicals sulphomethuron methyl and phenanthroline when present in multiple copies in transformants of Saccharomyces cerevisiae. Subcloning and sequencing of a 5.5 kb yeast DNA fragment revealed that SNQ2 has an open reading frame of 4.5 kb. The putative encoded polypeptide of 1501 amino acids has a predicted molecular weight of 169 kDa and has several hydrophobic regions. Northern analysis showed a transcript of 5.5 kb. Haploid cells with a disrupted SNQ2 reading frame are viable. The SNQ2-encoded protein has domains believed to be involved in ATP binding and is likely to be membrane associated. It most probably serves as an ATP-dependent permease.

Isolation and characterization of additional genes influencing resistance to various mutagens in the yeast Saccharomyces cerevisiae

SummaryScreening of a multi-copy vector-based yeast genomic library in haploid cells of wild-type Saccharomyces cerevisiae yielded transformants hyper-resistant to various chemical mutagens. Genetical analysis of the yeast insert DNAs revealed three genes SNG1, SNQ2, and SNQ3 that confer the phenotype hyper-resistance to MNNG, to 4-NQO and triaziquone, and to mutagens 4-NQO, MNNG, and triaziquone, respectively. Integration of the gene disruption-constructs into the haploid yeast genome yielded viable null-mutants with a mutagen-sensitive phenotype. Thus, copy number of these non-essential yeast genes determines the relative resistance to certain chemical mutagens, with zero copies yielding a phenotype of mutagen sensitivity and multiple copies one of mutagen hyper-resistance, respectively.

A Genome-Wide Longitudinal Transcriptome Analysis of the Aging Model Podospora anserine

Aging of biological systems is controlled by various processes which have a potential impact on gene expression. Here we report a genome-wide transcriptome analysis of the fungal aging model Podospora anserina. Total RNA of three individuals of defined age were pooled and analyzed by SuperSAGE (serial analysis of gene expression). A bioinformatics analysis identified different molecular pathways to be affected during aging. While the abundance of transcripts linked to ribosomes and to the proteasome quality control system were found to decrease during aging, those associated with autophagy increase, suggesting that autophagy may act as a compensatory quality control pathway. Transcript profiles associated with the energy metabolism including mitochondrial functions were identified to fluctuate during aging. Comparison of wild-type transcripts, which are continuously down-regulated during aging, with those down-regulated in the long-lived, copper-uptake mutant grisea, validated the relevance of age-related changes in cellular copper metabolism. Overall, we (i) present a unique age-related data set of a longitudinal study of the experimental aging model P. anserina which represents a reference resource for future investigations in a variety of organisms, (ii) suggest autophagy to be a key quality control pathway that becomes active once other pathways fail, and (iii) present testable predictions for subsequent experimental investigations.

Mitochondrial pathways governing stress resistance, life, and death in the fungal aging model Podospora anserina

Work from more than 50 years of research has unraveled a number of molecular pathways that are involved in controlling aging of the fungal model system Podospora anserina. Early research revealed that wild‐type strain aging is linked to gross reorganization of the mitochondrial DNA. Later it was shown that aging of P. anserina does also take place, although at a slower pace, when the wild‐type specific mitochondrial DNA rearrangements do not occur. Now it is clear that a network of different pathways is involved in the control of aging. Branches of these pathways appear to be connected and constitute a hierarchical system of responses. Although cross talk between the individual pathways seems to be fundamental in the coordination of the overall system, the precise underlying interactions remain to be unraveled. Such a systematic approach aims at a holistic understanding of the process of biological aging, the ultimate goal of modern systems biology.

Catalytically active soluble ecto-5'-nucleotidase purified after heterologous expression as a tool for drug screening

To establish a procedure for reproducible production of a soluble enzymatically active form of rat ecto‐5′‐nucleotidase, we heterologously expressed the polypeptide of the mature protein in insect cells. An expression construct was created consisting of this polypeptide and glutathione‐S‐transferase from Schistosoma japonicum as a fusion partner. Although infected insect cells did not secrete the enzyme into the medium, considerable amounts of recombinant protein were detected in the whole cell extract. A fraction of soluble protein yielding 5′‐nucleotidase activity could be purified from cells infected with recombinant baculovirus bearing the glutathione‐S‐transferase fusion construct. The catalytic properties of this recombinant protein correspond to those of the native protein isolated from animal tissues. Potential agonists and inhibitors of P2 receptors can function as inhibitors of ecto‐5′‐nucleotidase. The glutathione‐S‐transferase fusion protein may be used in experiments for drug screening, for the study of the interaction of immobilized ecto‐5′‐nucleotidase with other proteins, or for application in tissue culture experiments. Drug Dev. Res. 45:269–276, 1998.

A differential genome-wide transcriptome analysis: impact of cellular copper on complex biological processes like aging and development.

The regulation of cellular copper homeostasis is crucial in biology. Impairments lead to severe dysfunctions and are known to affect aging and development. Previously, a loss-of-function mutation in the gene encoding the copper-sensing and copper-regulated transcription factor GRISEA of the filamentous fungus Podospora anserina was reported to lead to cellular copper depletion and a pleiotropic phenotype with hypopigmentation of the mycelium and the ascospores, affected fertility and increased lifespan by approximately 60% when compared to the wild type. This phenotype is linked to a switch from a copper-dependent standard to an alternative respiration leading to both a reduced generation of reactive oxygen species (ROS) and of adenosine triphosphate (ATP). We performed a genome-wide comparative transcriptome analysis of a wild-type strain and the copper-depleted grisea mutant. We unambiguously assigned 9,700 sequences of the transcriptome in both strains to the more than 10,600 predicted and annotated open reading frames of the P. anserina genome indicating 90% coverage of the transcriptome. 4,752 of the transcripts differed significantly in abundance with 1,156 transcripts differing at least 3-fold. Selected genes were investigated by qRT-PCR analyses. Apart from this general characterization we analyzed the data with special emphasis on molecular pathways related to the grisea mutation taking advantage of the available complete genomic sequence of P. anserina. This analysis verified but also corrected conclusions from earlier data obtained by single gene analysis, identified new candidates of factors as part of the cellular copper homeostasis system including target genes of transcription factor GRISEA, and provides a rich reference source of quantitative data for further in detail investigations. Overall, the present study demonstrates the importance of systems biology approaches also in cases were mutations in single genes are analyzed to explain the underlying mechanisms controlling complex biological processes like aging and development.