Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts.

An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O(2)) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

Caenorhabditis elegans as a model system for mtDNA replication defects.

Since the isolation and physical characterization of mammalian mitochondrial DNA (mtDNA) over 35 years ago, numerous studies have been conducted in order to understand its structure and properties, including mode of mtDNA replication and transcription. Even today, the mode of mtDNA replication is still a matter of intense debate. We believe that Caenorhabditis elegans holds the promise of identifying molecular mechanisms of mitochondrial replication. C. elegans is a simple and extremely powerful genetic and developmental model system. Their small size, rapid life cycle, the ability to self-fertilize and somatic tissues that consist of post-mitotic cells offer an efficient way to study mitochondrial metabolism. We have recently developed a number of methods in order to study mitochondrial DNA level and mtDNA maintenance during the development of C. elegans. We hope that the techniques described here can assist laboratories interested in understanding modes of mtDNA replication, distribution and mitochondrial morphology in C. elegans.

Mitochondrial energy metabolism and ageing.

Ageing can be defined as "a progressive, generalized impairment of function, resulting in an increased vulnerability to environmental challenge and a growing risk of disease and death". Ageing is likely a multifactorial process caused by accumulated damage to a variety of cellular components. During the last 20 years, gerontological studies have revealed different molecular pathways involved in the ageing process and pointed out mitochondria as one of the key regulators of longevity. Increasing age in mammals correlates with increased levels of mitochondrial DNA (mtDNA) mutations and a deteriorating respiratory chain function. Experimental evidence in the mouse has linked increased levels of somatic mtDNA mutations to a variety of ageing phenotypes, such as osteoporosis, hair loss, graying of the hair, weight reduction and decreased fertility. A mosaic respiratory chain deficiency in a subset of cells in various tissues, such as heart, skeletal muscle, colonic crypts and neurons, is typically found in aged humans. It has been known for a long time that respiratory chain-deficient cells are more prone to undergo apoptosis and an increased cell loss is therefore likely of importance in the age-associated mitochondrial dysfunction. In this review, we would like to point out the link between the mitochondrial energy balance and ageing, as well as a possible connection between the mitochondrial metabolism and molecular pathways important for the lifespan extension.

Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development.

A number of studies showed that the development and the lifespan of Caenorhabditis elegans is dependent on mitochondrial function. In this study, we addressed the role of mitochondrial DNA levels and mtDNA maintenance in development of C. elegans by analyzing deletion mutants for mitochondrial polymerase gamma (polg-1(ok1548)). Surprisingly, even though previous studies in other model organisms showed necessity of polymerase gamma for embryonic development, homozygous polg-1(ok1548) mutants had normal development and reached adulthood without any morphological defects. However, polg-1 deficient animals have a seriously compromised gonadal function as a result of severe mitochondrial depletion, leading to sterility and shortened lifespan. Our results indicate that the gonad is the primary site of mtDNA replication, whilst the mtDNA of adult somatic tissues mainly stems from the developing embryo. Furthermore, we show that the mtDNA copy number shows great plasticity as it can be almost tripled as a response to the environmental stimuli. Finally, we show that the mtDNA copy number is an essential limiting factor for the worm development and therefore, a number of mechanisms set to maintain mtDNA levels exist, ensuring a normal development of C. elegans even in the absence of the mitochondrial replicase.

Streptomyces durmitorensis sp. nov., a producer of an FK506-like immunosuppressant.

Screening of soil samples from the Durmitor National Park, Serbia and Montenegro, for strains producing immunosuppressants with a similar mechanism of action to FK506 resulted in the isolation of the actinomycete strain MS405(T). Isolate MS405(T) was found to have morphological and phenotypic properties that were consistent with its classification as a Streptomyces strain. The DNA G+C content of strain MS405(T) was 72 mol%. 16S rRNA gene sequence data confirmed the taxonomic position of the strain, following the generation of phylogenetic trees by using various treeing algorithms. On the basis of 16S rRNA gene sequence similarity, strain MS405(T) was shown to belong to the Streptomyces albidoflavus 'supercluster', being related to Streptomyces aureus DSM 41785(T) (99.59 % similarity) and Streptomyces kanamyceticus DSM 40500(T) (99.32 %). The 16S-23S rRNA internally transcribed spacer (ITS) region exhibited variations in length and sequence composition, showing limited usefulness in phylogenetic analyses. However, DNA relatedness values support the classification of this isolate within a novel species. A number of physiological and biochemical tests distinguished strain MS405(T) from its closest phylogenetic neighbours. Therefore, strain MS405(T) represents a novel species, for which the name Streptomyces durmitorensis sp. nov. is proposed, with the type strain MS405(T) (=DSM 41863(T) =CIP 108995(T)).