International Symposium on “ Germline Mutagenesis and Biodiversification ”

Arikuni Uchimura*, Yuichiro Hara†, Yoichi Gondo and Yusaku Nakabeppu Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minami, Chuo-ku, Kobe, Hyogo 650-0047, Japan Mutagenesis and Genomics Team, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan Research Center for Nucleotide Pool and Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan


I. Mechanisms of Germline Mutagenesis
When considering mechanisms of germline mutagenesis, there are two types of causes: exogenous and endogenous causes.This session consisted of two talks related to exogenous causes (irradiation and mutagenic chemi-cals), two related to endogenous causes (endogenous damage and DNA replication errors) and one about a new technology to detect germline mutagenesis.
The first talk was delivered by Ohtsura Niwa (Fukushima Medical University) and entitled "The repetitious sequence mutation and the radioresponse of the germline to early embryonic stages."He showed the difference between the effects of irradiation of two types of male germ cells, spermatozoa and spermatogonia, on genomic instability, especially in non-irradiated maternal alleles during early embryogenesis.This implied that there were complex mechanisms in germline mutagenesis.He suggested that the genomic instability involved the p53 gene and sister chromatid exchange.He also pointed to the existence of an unknown repair mechanism in the germline that responds to radiation and future research on germline mutagenesis.
Takehiko Nohmi (National Institute of Health Sciences; Germ cell and somatic cell mutagenesis in gpt delta rodents) talked about mutagenic environmental chemicals.He reviewed the latest knowledge about the mutagenic effects of various chemicals, including the differences in sensitivity between germ cells and somatic cells.He also described new international guidelines regulating commercially available novel chemicals.His established transgenic gpt delta rodents were adopted in an official protocol in the new guidelines.In his presentation, differences in the mutagenic effects of chemicals among species were of great interest to many people in the audience.
Yusaku Nakabeppu (Kyushu University; Oxidation of nucleic acids and control mechanisms of genetic diversity in mammals) talked about the relationship between oxidative stress and germline mutagenesis.He focused on 8-oxoguanine (8-oxoG, one of the major oxidation products in DNA and nucleotides) and its involvement in genomic diversity in mammals.He first showed that regions with a high frequency of recombination and single nucleotide A. UCHIMURA et al. polymorphisms (SNPs) are preferentially located within chromosomal regions with a high density of 8-oxoG in human cells, and then assessed the mutagenic potential of 8-oxoG through experiments using triple-knockout mice that lack three enzymes (MTH1, OGG1 and MUTYH) involved in the removal of 8-oxoG.Notably, an increase of meiotic chromosome recombination observed in the mutant mouse germ cells, and an estimation of the exceptionally high mutation rate derived from analyses of the pedigree of the mutant mice, stimulated interest in many of the participants.
Arikuni Uchimura (Osaka University) presented a talk entitled "DNA replication errors that fuel genetic and phenotypic diversities in mammals."He began by introducing his "Mouse Evolution Project," a new experimental model to investigate evolutionary processes directly using mutator mouse (DNA polymerase δ-modified mice, which display lower fidelity during DNA replication) breeding lines.He showed the first direct estimation of the spontaneous germline mutation rate in wild type mice (in addition to the increased rate in the mutator mice) by high-throughput sequencer.This mouse mutation rate is actually much lower than the rate in the mutant mice presented by Yusaku Nakabeppu in the preceding talk.He also showed differences between phenotypic changes observed in the mutator mice and wild type mice after long-term breeding.
As the last talk in this session, Yuichiro Hara (RIKEN CDB) gave a presentation entitled "Search for germline mutations from next-generation sequencing analysis."First, he reviewed recent advances in high-throughput sequencers.He then showed his new approach to detect mutagenesis, especially "ultramicro inversion," which can occur preferentially in male germ cells, using next-generation sequencers.The development of new detection technology is essential for a better understanding of the mechanism of germline mutagenesis.His talk emphasized that various approaches using high-throughput sequencers should provide a major impetus for germline mutagenesis research.

II. The Way from Mutagenesis to Evolution
This session included two presentations dealing with mutagenesis related to evolution.The two talks were from different standpoints, providing the audience with a broad understanding of the relationship between mutagenesis and evolution.
The first speaker was Yoko Satta (The Graduate University for Advanced Studies; Germline mutations and evolutionary genetics).She started by talking about the divergence between human and chimpanzee, and provided some estimates of evolutionary rate, the proportions of sites that are susceptible to natural selection, and the proportion of mutations co-segregating in the human population among newly arising mutations.She then dis-cussed the evolutionary process quantitatively, using her original simulation model with simple prerequisites.Understanding the relationship between recombination rate and/or selective coefficient and variance of observed mutation number seemed to stimulate the audience into new ways of thinking about mutagenesis and the evolutionary process.
The second speaker was Mitsuru Furusawa (Neo-Morgan Laboratory Inc.; A novel theory for accelerating evolution and its industrial application).He introduced his evolutionary theory, the "Disparity Theory of Evolution," which focused on the unbalanced DNA synthesis mechanism in the leading strand and lagging strand during DNA replication, and asserted the importance of the differential mutation rate in the two strands for the production of genetic variation among populations.He showed theoretical and experimental results of the theory.Notably, the fact that its industrial application has provided fruitfully developed and optimized strains in various fields (agriculture, green energy, production of useful compounds, etc.) persuaded the audience that investigations into germline mutagenesis are important for creating successful future industrial applications.
After this session, Arikuni Uchimura chaired the first day's general discussion.He introduced the topic of the "too-high mutation rate" (per generation) in the human population and raised the question of whether or not mutation accumulation (mutational load) is a risk for the human population.Numerous views with different standpoints were expressed by the participants.For example, the view that the efficiency of purifying selection against deleterious mutations is an important consideration in evaluating such a problem was proposed by an evolutionary scientist.Another scientist stressed the importance of flexible adaptation in the human social system to overcome such a risk.Overall, most views were optimistic in this session, but continuing the discussion seemed to be important.

III. Physiological Functions of Mutations
The session on physiological functions of mutations, comprising three talks, was held in the morning of the second day.Each speaker gave a presentation about their research involved in clarifying the genetic causes of human diseases using mutant mice as a model.Since the three speakers work in bioresource facilities of mutant mice in China, Korea and Japan, they also introduced their activities in these large-scale projects.This session was opened by Yoichi Gondo (RIKEN BRC) with his remarks about the history of mutagenesis studies, molecular genetics, and genomics.
Xiang Gao (Nanjing University) gave a talk entitled "Mouse mutagenesis and functional genomics in China."He presented his current research about elucidation of the mechanisms of metabolic syndromes involving genetic Symposium on Germline Mutagenesis variation.Under rapidly increasing demands of mouse mutagenesis studies and the archiving and distribution of mutant mice in the early 2000s in China, he founded the National Resource Center for Mutant Mice (NRCMM) and the Model Animal Research Center (MARC) in Nanjing.He introduced these facilities and his activities in establishing mutagenesis technologies and bioresource storage in China.
Je Kyung Seong (Seoul National University) gave a talk entitled "Systemic mouse mutagenesis and phenotyping in Korea."Through his mouse mutagenesis research, he has extended the traditional view of genetics of "genotypes-to-phenotypes" to the more modern "genotypes-plus-environments-to-phenotypes."He investigated phenotypic changes caused by spatiotemporal dosageeffects of gene expression derived from the complex interaction between germline mutations and environments.He integrated his advanced achievements into analytic pipelines for clarifying the genetic causes of human diseases based on mouse mutagenesis research in the Korea Mouse Phenotyping Consortium (KMPC), which he has been directing.
The final talk of this symposium was given by Yoichi Gondo (RIKEN BRC) and titled "Mouse germline mutagenesis and future perspectives."He established a mutant mice library in which mutations were induced by ENU (N-ethyl-N-nitrosourea).This library is publicly accessible and, from 10,000 archived mutant mice, several mice have so far been established as models of human genetic diseases.He also introduced his current project of whole-exome sequencing of G1 ENU-mutant mice without any backcrosses.From such a wholegenome approach, multifactorial phenotypic changes arising from multiple ENU-induced de novo mutations will be identified.
A general discussion chaired by Yuichiro Hara was held at the end of the symposium.He proposed a fourth aspect of mutagenesis in addition to the three themes described above: (iv) how robust our genomes are.This is because some mutants can survive and even be fertile under high mutation rates, implying the existence of mechanisms to stabilize the genome even in the presence of numerous mutations.In addition, he offered the suggestion of searching for new model organisms that have suitable phenotypes for germline mutagenesis studies.

IV. Concluding Remarks
Thanks to dramatically rapid progress in genomic sciences from the 1990s onward, research and analytical methods at the molecular level in genetics have also advanced.Technical innovations in reverse genetics such as transgenic and knockout organisms, for example, have now become conventional tools in various studies.At the same time, such expansions of genetic tools have, inversely, caused segmentation in the research field of genetics.In this Symposium, these segmented researchers gathered again to contemplate de novo germline mutations and genetics.The forthcoming experimental outcomes of de novo germline mutation studies encouraged this gathering of researchers from various fields, from basic to applied sciences: molecular biology, development, evolution and physiology, as well as environmental sciences and medicine.We hope that a "germ" of mutagenesis research will soon flourish into a new interdisciplinary science of genetics in the 21 century.