Before 2001, our research was focused on the structure, evolution and regulation of expression of the chorion genes superfamily of the silkmoth.
Since 1989, an interest began to grow in our lab regarding the animal mitochondrial DNA (mtDNA). MtDNA is an alternative parsimony model compared to the nuclear DNA, both from its gene content and gene organization point of view.
In the last 10 years, our lab has been focusing on four biological subjects:
1. The investigation of the molecular base of the unorthodox phenomenon of Doubly Uniparental Inheritance of animal mtDNA
2. Structure, gene organization and evolution of animal mtDNA.
3. Phylogeographic analysis of organisms based on mitochondrial and nuclear gene sequences.
4. Molecular diagnosis of deletions in mitochondrial DNA of sperm of infertile and normal men as an attempt to associate deletions with male infertility in connection with the results of assisted reproduction methods.
1. Doubly Uniparental Inheritance (DUI)
There is a growing realization that, apart from its role in energy production, mitochondrial DNA (mtDNA) plays a role in a number of important cellular and organismal functions, such as apoptosis, hormone regulation and senescence. The list of abnormalities in which this genome is involved (myopathies, neurodegenerative diseases, certain types of cancer, male infertility) is growing. Its role in some important applications, such as cloning, is also important. A general rule in eukaryotic organisms is that biparental (i.e. sexual) inheritance of nuclear DNA is accompanied with uniparental inheritance of cytoplasmic DNA. Thus, animals have strict maternal inheritance (SMI) of mtDNA. The mechanism and the implications of SMI are not fully understood. A major exception to SMI is known in bivalvian mollusks. These organisms possess two rather distinct mitochondrial genomes, one that is transmitted through the female line (egg—as in any other animal species) and the other through the male line (sperm). These genomes are known as F and M, respectively. The phenomenon is known as doubly uniparental inheritance (DUI).
Upon fertilization all embryos receive mtDNA from both parents. In females the M genome disappears or becomes an insignificant minority. In male embryos the F genome becomes dominant or exclusive in somatic tissues and the M genome becomes the exclusive genome of the germ line and, eventually, the sperm. The phenomenon has been named Doubly Uniparental Inheritance (DUI) (Zouros et al. 1994a) to emphasize the fact that, as in the common case of strict maternal inheritance (SMI), uniparentality is preserved, however in DUI two rather than one mitochondrial genomes are transmitted uniparentally.
We adopt the strategy of understanding the rule through its exceptions. By studying DUI we will help understand some of the most fundamental aspects of mtDNA biology, such as: Why is SMI of mtDNA the rule in animals? What are the molecular underpinning of SMI and how this mechanism has been modified in species with DUI? Are there hitherto unrecognized roles of mtDNA “hidden” in its coding or non-coding sequences? What are the implications of SMI vis-à-vis DUI in evolution and phylogeny?
We focus on the molecular and developmental mechanism of DUI, which remains basically unexplored.
We currently study the full transcriptome of the maternally and paternally inherited mitochondrial genomes of the mussel Mytilus, search for sequence motifs that are responsible for these genome’s separate transmission routes and explore nuclear/mtDNA interactions that are characteristic for the biparental mtDNA inheritance in this species.
2. & 3. MtDNA analysis & Phylogeographic genetic population analysis
The main experimental model of this research subject was the mtDNA of the land snail Albinaria (GAstropoda: Clausiliidae).
First of all, the full sequencing and gene annotation of mtDNA of the species Albinaria coerulea was performed, as well as the partial sequencing and annotation of the mtDNA of A. turrita. The extensive analysis of these sequences resulted in the inference of interesting conclusions, such as the fact that the mtDNA of Albinaria is the shortest between animals (~14 kb) and also that some tRNA genes are incomplete as far as length is concerned, but they complete their sequence through polyadenylation of the 3’ end after cleavage of the primary transcript.
The molecular phylogenesis of the land snail Albinaria as a paleogeographic indicator in the Aegean region was a new phylogeographic dimension of the mtDNA research of the lab. The result of this research was the indication of the usefulness of molecular phylogenesis as an indicator of “historical” paleogeographic changes, through the “traces” that these changes leave on the evolutionary course of organisms. From this point of view, the Aegean (a region with severe geological changes and inadequately determined phylogeography) is a model area for phylogeographic analyses.
In addition, new molecular data emerged from this research, regarding studied genes, as well as the land snails themselves—organisms least studied from a molecular approach. Moreover, the population genetics analysis allowed the investigation of the demographic parameters that govern the evolution potential of land snails, with the application of a modern approach including simulations in a computer, based on the coalescence theory.
The phylogenetic—phylogeographic study of other species (e.g. the genus Podacris), is also a research subject for our lab.
4. Molecular diagnosis of deletions in human mtDNA
In human, a large variety of shorter in length mtDNA molecules, has been detected, mainly from pathological tissues. However, it is unclear whether the occurrence of such deletions is caused by spontaneous recombinations or whether nuclear factors also play a role.
We navigated our research towards the “applied”, as it is called research. In particular, we focused on the detection of deletions in mtDNA of spermatozoa of infertile men, in order to investigate the possible relationship between infertility and the increase of the appearance frequency of deletions. This choice was based on the fact that for the particular system, there is no clear data internationally, contrary to cases of deletions in the muscle or neural tissue for example.
Due to the fact that the existing data is unclear regarding the frequency and the percentage of deletion appearance, we stated the hypothesis that at least some of the published results have ensued as artefacts of the widely used experimental approach of diagnostic PCR. Therefore, the first question that we attempted to answer was whether the PCR method can be a safe, routine diagnostic tool.
The up-to-date research, using approximately 150 samples, allowed us to challenge the correctness of the up-to-date published results. At the same time, we developed a new and safe methodology (i.e. without artefacts) the application of which showed a strong connection between the presence and the percentage of the deletion of 4977 bp and the pathological phenotype (according to WHO) of the sperm of infertile men. In addition, we presented for the first time strong evidence for the involvement of this deletion in the result of in vitro fertilization (IVF). This connection does not exist when the method of Intra Cytoplasmic Sperm Injection (ICSI) is applied.
Based on the up-to-date results and conclusions, we think that we have set the basis for the reduction of such a method to a safe diagnostic tool for inspection of the potency of the sperm that is destined for fertilization.
Created by: Eleni Kyriakou
NATIONAL AND KAPODISTRIAN UNIVERSITY OF ATHENS
School of science
Faculty of biology
Department of biochemistry and molecular biology
Molecular Biology and Evolution Laboratory
Professor George C. Rodakis