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Genome biology and technology innovation have accelerated development and expansion of biology dramatically for last 20 years. In physics, many laws had been established during 16th to 17th century based one the analysis of previously accumulated observations and now is the period for the biology changing from accumulation of data to discovery of laws. Before the genome analysis, small number of targets had been focused in biology to be analyzed but now, thanks to the achievements of genome and post-genomic projects, entire set of genes in a certain organism can be investigated on the global aspect point of view. In other word, until now we focused on the research of gpartsh, such as genes and proteins but current biology can provide us the possibilities to do research for the systems consist of parts. Last half century, many efforts have been performed for elucidation of the function and structure of genes in molecular biology and now the functional relationships between genes, how systems, assembled by gene or gene products, are working, etc. can be analyzed in current biology. This is systems biology.
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- Analysis of genetic interaction network
Our target organism is Escherichia coli and the reasons why we use this small bug are, 1) one of the best-studied organisms, 2) uni-cellular system. For complete understanding of cellular system, accumulation of knowledge about parts, such as genes and proteins, of the system is essential. And uni-cellular system has a complete set of a life to survive a certain environment in a single cell level although multi-cellular system requires network between cells to be alive as an independent organism.
Microorganisms are thought to be simple, however even in E. coli, more than 4000 protein coding genes are on the genome. Since we had finished to determine the genome sequence of this bacterium, we had started to construct comprehensive experimental resources, such as plasmid clone libraries and single deletion mutation collections of protein coding genes, to make global and systematic analysis available on the same plat form. Now we have had a couple of valuable resources, called ASKA libraries and Keio collection. Microarray analysis was one the applications using ASKA library launched 10 years ago in Japan. Construction of deletion strains revealed about 300 essential genes that cannot be removed from the chromosome. In other word, the rest of genes can be eliminated and most of those deletion strains did show little phenotype changes. Normally single gene deletion can be compensated by other gene(s) and this feature is called grobustnessh. And the relationship of these two genes is called ggenetic interactionh. To clear the molecular mechanisms of grobustnessh, one of the simple but powerful method is identification of two genes combination that compensate deletion effect each other (see Fig. 1). The concept is simple but practically very difficult to analyze entire combination even in E. coli, because 16 million combinations of two genes should be analyzed. To make it possible, we have had long efforts to develop the efficient system using conjugation. Now the method has been established and the partial genetic network structure is shown in Fig. 2.
- Quantitative metabolic network analysis
Not only the qualitative analysis of network structure, but quantitative features of metabolic network is so important to understand cellular metabolic activities. Metabolic network is built up by reactions of enzymes and each of the enzymatic reaction defined by concentrations of substrates and enzymes and enzymatic parameters. We are now trying to obtain these quantitative data and to make a model of cellular activities based on these numerical data. This approach may be the fundamental platform to analyze and predict the cell behavior in variety of environmental changes.
- Development of transfer system for huge DNA between species
Industrial microbes, such as Streptomyces sp., are so valuable for production of chemical materials. Such microbes, however, are sometimes so inefficient for genetic modification. Streptomyces sp. is also in such case. On the other hand, Escherichia coli is widely used as a tool for genetic manipulation due to the accumulation of genetic methods. To combine the advantages of these organisms, the systems having a big capacity of DNA size of cloning and transferring from E. coli to production microbe by conjugation are now developing (see Fig. 3).
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- Tohsato Y, et al., J Bioinform Comput Biol, 8 Suppl 1: 83-99, 2010.
- Rajagopala SV, et al., BMC Genomics, 11: 470, 2010.
- Yamamoto N, et al., Mol Syst Biol, 5: 335, 2009.
- Typas A, et al., Nat Methods, 5: 781-787, 2008.
- Butland G, et al., Nat Methods, 5: 789-795, 2008.
- Baba T, et al., Mol Syst Biol, 2: 2006 0008, 2006.
- Arifuzzaman M, et al., Genome Res, 16: 686-691, 2006.
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Fig. 1 Concept of genetic network.
Normally cell has multiple pathways to produce an essential substrate for cellular life. Single gene knockout destroys one path but another route is still active and cell can survive. The opposite case may show the same situation. But both of pathways are shutdown together, cell cannot alive any more. These two genes are defined to have a genetic interaction.
Fig. 2 The genetic network structure of central metabolic genes.
We performed the genetic interaction analysis using genes related to glycolysis, TCA cycle and pentose phosphate pathways as query genes. Green nodes represent genes and arch shows genetic interaction. Shorted arch means similar genetic interaction profiles to have.
Fig. 3 Transfer system between species.
RPs is a plasmid carrying wider range of host organism than F plasmid conjugation system. In this case, BAC plasmid carrying transfer origin of RP4 might be transferred into the production strain, such as Streptomyces sp. by conjugation supplied from the tra genes operon on the host cell chromosome.
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