Yasutomo Nomura

Direct quantification of gene expression using fluorescence correlation spectroscopy.

Among the methods for single molecule detection in the field of medicinal chemistry, the importance of fluorescence correlation spectroscopy (FCS) is growing. FCS has the advantage of permitting us to determine the number of fluorescent molecules and the diffusion constant dependent on the molecular weight without any physical separation process such as gel electrophoresis. Thus this method is appropriate for studies on the hybridization of fluorescence-labeled oligonucleotides with RNA or DNA as well as gene expression through translation of a target protein linked with green fluorescent protein. Indeed, several groups have employed FCS for evaluation of gene expression in different ways. Many investigators are particularly interested in using FCS to quantitatively analyze mRNA just after transcription in the living cell. Technical advances in FCS have broadened the research spectrum in medicinal chemistry since it can also be used to study SNPs and molecular interactions between transcription factors and promoter sequences, as well as gene expression in living cells.

Genetic Oxygen Sensor

We report in this article a new method for in vivo oxygen measurement using green fluorescence protein (GFP). COS7 cells were transiently transfected with an expression vector, pCMX-GFP, using a polyethylenimine reagent and cultured for 48 hrs. After exposure of the cell to anoxic gas (O2 580 nm) that had been negligible before the photoactivation. This red shift of (green) GFP fluorescence was never observed in normoxia. We then examined the validity of this method in transgenic mice in which GFP is stably expressed (green mice). All the ventricular myocytes isolated from the green mice showed significant green fluorescence, although the intensity was ∼1/200 of the transiently GFP-expressing COS7 cells. The photoactivation in anoxia increased the red fluorescence in these cells, but the magnitude was much smaller than expected. In summary, GFP can be used as an in situ probe for hypoxia. In GFP-expressing transgenic animals, in vivo imaging of anoxic loci with a submicron spatial resolution may be possible.

Evaluation of Mitochondrial DNA Dynamics Using Fluorescence Correlation Analysis

Mitochondria are the sites of oxidative phosphorylation and generate ATP when electron is transferred from respiratory substrates to oxygen by a series of redox reaction in which respiratory enzymes pump protons across the mitochondrial inner membrane from the matrix space[1]. In isolated mitochondria as well as in intact cells, respiration frequently produces reactive oxygen species (ROS). Especially, ROS increased if respiration is perturbed, e.g., ischemia-reperfusion injury [2]. ROS can attack almost all biomolecules unspecifically. In case of DNA, ROS causes singleand double –strand breaks, and base damage[3]. Nevertheless mitochondria metabolize them only partially. Due to the defense system, mitochondrial DNA (mtDNA, ~17 kbp) that mitochondria contain independently of nucleus is particularly vulnerable because it is partially associated with the inner mitochondrial membrane as shown in Fig.(1A) [4]. Moreover, mtDNA repair system is weaker than that of nucleus[5]. Since mtDNA codes a part of respiratory enzymes[6], the damages would be harmful to mitochondrial function. On the other hand, because mtDNA forms a complex with proteins and is not naked, a concept that mtDNA have a resistance against ROS is also favored[7]. Therefore, one may need to reconsider mtDNA damage using a newly developed methodology which is able to detect symptoms failed to be found in the previous studies. Among symptoms, the changes in mtDNA dynamics are noticed.

Image Processing Methods for Quantitative Analysis of Mitochondrial DNA Dynamics

Mitochondria and mitochondrial DNA are known to be highly motile. Molecular analysis revealed that several proteins are bound between mitochondrial DNA and mitochondrial inner membrane. However, nucleoid formed by these proteins is involved in the replication and duplication of mitochondrial DNA and it remains unclear whether mitochondrial DNA keeps binding the mitochondrial inner membrane or not. When nucleoid is necessary during cell cycle, it would be formed probably. When nucleoid is formed in the manner dependent on mitochondrial DNA sequence, the changes in the sequence would result in the equilibrium shifted to dissociation. This implies that the important information on changes in mtDNA accompanied with various symptoms is obtained by quantitative analysis of mtDNA dynamics. In this review, theoretical background of two typical methods, i.e., particle trajectory and image correlation spectroscopy, and several results of the pilot experiment are introduced.