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Whole genome approaches to development, evolution, and physiology: Progresses and Prospects
Organizer: Takano-Shimizu, Toshiyuki
(National Institute of Genetics)

This workshop is aimed at genome-wide scanning studies. With the availability of a complete DNA sequence for Drosophila genome (following genomic and cDNA libraries, this may be the third library that we have gotten, and the forth is coming as protein library) and soon for human genome, many researchers are interested to develop genome-wide approaches for a better understanding of developmental and evolutionary processes and of molecular bases of genetic diseases. This is not just to pick up your own genes, but new methods are expected to give insight into whole pictures of the processes. On the other hand, functional characterization of genes and assessment of allele effects cannot fully be done in isolation, but it should be done in networks of genes, molecules, cells, individuals, and even populations. I put here great emphasis on interactions and there are indeed so many different interactions: allele-by-allele interaction (which includes dominant forms of alleles and transvection), gene-by-gene interaction, protein-by-protein interaction, gene-by-environment (which includes internal and external environments), cell-by-cell interaction, tissue-by-tissue interaction (e.g., interactions at compartment boundaries), and so on. Fruit flies (and mouse) have been among the tops in model organisms for genetic study, but some (or many) people doubt the future of this champion of model organisms. However, I have no doubt that Drosophila has still been among the champions of model organisms to study interactions and to explore the roles of genes (alleles) and environment in determining characteristics of organisms. Five speakers invited for this workshop will give us their findings and perspectives of what we get from different genome-wide screening studies.

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S1-1 Probing the genome for pattern elements: a lesson from a large scale enhancer trap screen.
Hayashi, Shigeo
(Invertebrate Genetics Lab, National Institute of Genetics and RIKEN CDB)
S1-2 Drosophila pharmacogenomics: quantitative expression profiling and association studies.
Gibson, Greg, Naruo Nikoh, Rebecca Riley, Gisele Passador-Gurgel, and Roland Carrillo.
(Dept. of Genetics, North Carolina State University)
S1-3 Genomic incompatibility between D. melanogaster and D. simulans
Sawamura, Kyoichi and Masa-Toshi Yamamoto
(Drosophila Genetic Resource Center, Kyoto Institute of Technology)
S1-4 The gene search system as a tool for functional genomics in Drosophila.
Aigaki, Toshiro
(Dept. of Biological Sciences, Tokyo Metropolitan University)
S1-5 Genome-wide mouse mutagenesis: gene-driven vs. phenotype-driven.
Gondo, Yoichi
(Population and Quantitative Genomics Team, RIKEN Genomic Sciences Center)
S1-6 A proposal of genome-wide linkage disequilibrium scan.
Takano-Shimizu, Toshiyuki
(Dept. of Population Genetics, National Institute of Genetics)
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S1-1
Probing the genome for pattern elements: a lesson from a large scale enhancer trap screen.
Hayashi, Shigeo
(Invertebrate Genetics Lab, National Institute of Genetics and RIKEN CDB)

One of surprises that immediately came from the sequencing of the Drosophila genome was that the estimated gene number (about 13600) is much fewer than that of C. elegans (about 19000). The result suggests that gene number is not a good measure of organismal complexity. It is likely that the functional diversification of limited number of genes contributes to increased complexities in cell type, body plan, neural diversity and others. Use of the same gene in multiple contexts is one solution for diversification of gene functions. To understand the gene regulatory capacity of the genome, a catalogue of transcriptional enhancers will be necessary. Unlike coding regions that are often conserved enough to allow identification from a sequence comparison, transcriptional enhancers are highly divergent in sequence and are difficult to identify. P element has a tendency to insert into transcriptional regulatory regions, and therefore might be useful as a biological probe to search enhancers in the genome. To obtain a resource for identifying enhancers and for other purposes, we analyzed 4528 lines of Gal4 enhancer trap generated by NP consortium. Genomic sequences flanking P element insertion sites of more than 4000 lines were determined and mapped onto specific locations of the genome map. These insertions were clustered into 2051 separate sites, most of which were found in the proximity of transcription units. We also studied expression patterns of enhancers detected by those insertions. We will summarize the results of the screen and discuss the power and limitation of P element in detecting enhancer elements.
S1-2
Drosophila pharmacogenomics: quantitative expression profiling and association studies.
Gibson, Greg, Naruo Nikoh, Rebecca Riley, Gisele Passador-Gurgel, and Roland Carrillo.
(Dept. of Genetics, North Carolina State University)

We are studying pharmacogenomics in Drosophila along two broad lines: (i) linkage disequilibrium mapping to test for association between SNP variation in neurotransmitter receptors, potassium channels, and other candidate genes, and two phenotypes, heart rate and drug sensitivity; and (ii) cDNA microarray analysis of transcriptional differences among lines associated with exposure to the drugs dopamine, nicotine, and caffeine. A new mixed model ANOVA statistical procedure has been developed that allows us to partition the variance contributions of sex, genotype and drug on the expression of individual transcripts, and to assign significance to the effects. With moderate levels of replication, changes in transcription as small as 1.2 fold can be shown to be highly significant, while sex and genotype are both seen to contribute to transcriptional variance of between one quarter and two thirds of all genes. Since we have shown that there is high heritability for survival times on food supplemented with a variety of drugs, and low genetic correlation in the responses to different drugs, the possibility arises that we will be able to identify genes whose expression correlates with the phenotypic response across genotypes. Results describing the distribution of transcriptional responses to three different monoamines, caffeine and nicotine will be presented. Candidate genes identified with this approach will be sequenced to identify SNP variation, which will also be tested for association with physiological responses. Sequence analysis to date indicates that there is considerably more protein variation (indel and amino acid replacement polymorphisms) encoded within the serotonin receptors in Drosophila than in the dopamine receptors, while variation in potassium channels is restricted in distribution. Molecular evolutionary and population genetic strategies are being combined with quantitative genetics to gain insight into the mechanisms that maintain variation in genetic modifiers of pharmacology.
S1-3
Genomic incompatibility between D. melanogaster and D. simulans
Sawamura, Kyoichi and Masa-Toshi Yamamoto
(Drosophila Genetic Resource Center, Kyoto Institute of Technology)

Once biological populations acquire species status by completing reproductive isolating mechanisms, each genome evolves independently. Thus it cannot be guaranteed that interspecific substitution of a part of the genome works appropriately as a developmental system. Such systems as interspecific hybrids or introgressions will provide us a good opportunity to analyze genetic mechanisms difficult to dissect by single gene mutations. The "model organism" D. melanogaster together with its sibling species D. simulans has contributed much to our knowledge of such genomic incompatibility. In the cross of D. melanogaster females and D. simulans males, male hybrids die at the larval/pupal stage while female hybrids are viable though poorly viable at high temperature (late inviability). In the reciprocal cross, male hybrids are viable but the majority of female hybrids die as embryos (early inviability). The genetic bases of the hybrid inviability have been investigated by analyzing genes whose mutations rescue the hybrids from inviability. The late inviability is the result of hyperactivity of the wild type allele of a D. melanogaster gene, Hybrid male rescue (Hmr), which is compensated by a second site mutation of D. simulans, Lethal hybrid rescue (Lhr). On the other hand, the early inviability is caused by the incompatibility between maternal products encoded by the wild type allele of a D. simulans gene, maternal hybrid rescue (mhr), and the zygotic hybrid rescue (zhr) locus of D. melanogaster embodied in the X chromosome heterochromatin consisting of 359 bp repetitive sequences (1.688 satellite DNA).
The barrier that had prevented further genetic analyses of hybrid inviability and sterility was removed by the recent discovery of a D. simulans strain which restores the fertility of female hybrids. We succeeded to introgress a part of the left arm of D. simulans chromosome 2 into the D. melanogaster genetic background. Flies heterozygous for the introgression are fertile while homozygotes are sterile both in females and males. We are currently identifying the genes responsible for the sterility by RFLP-assisted recombination and complementation with a series of deficiency chromosomes and sterility mutations of D. melanogaster. The high resolution mapping of 2.5 Mb region around the Alcohol dehydrogenase (Adh) gene has indicated that no single introgressed genes alone cause male sterility. Alternatively, hybrid male sterility is the consequence of synergistic effect of multiple genes. On the contrary, the responsible gene(s) of the female sterility have been localized in the 170 kb region containing only 20 ORFs, which suggests a single gene with a major effect. The genetic analysis of hybrid inviability is also feasible in this introgression system. The Lhr mutation rescues F1 male hybrids from the inviability but not the introgressioin-bearing male hybrids because of recessive gene(s) of hybrid inviability on the introgression. The responsible gene(s) have not been separated by recombination from the hybrid female sterility gene(s) at the present level of resolution, which suggests a close linkage of these two or a pleiotropy of genes of reproductive isolation.
S1-4
The gene search system as a tool for functional genomics in Drosophila.
Aigaki, Toshiro
(Dept. of Biological Sciences, Tokyo Metropolitan University)

Genome-scale generation and mapping of mutations onto the genome would greatly facilitate the investigation of individual genes' function. P-element insertional mutagenesis appears to be the most suitable method for this purpose, since it can be easily mobilized to generate new insertion lines, and the insertion sites can be rapidly mapped on the genome based on the vector-flanking sequences. However, there is a limitation of loss-of-function mutagenesis: only one-thirds of all genes are thought to be mutable to visible phenotype (Ashburner et al., 1999). Another potential problem is that a significant fraction of P-element-induced mutations is not associated with the insertions. These drawbacks are complemented by gain-of-function mutagenesis involving misexpression of a gene nearby P-elements insertion site. Commonly used misexpression vectors contain UAS (Upstream Activating Sequence), which would induce GAL4-dependent expression of vector-flanking gene. Due to a conditional feature of misexpression phenotypes, it is possible to study the function of genes in later developmental stage and aging process. Although a gain-of-function phenotype alone is not sufficient to define the normal function of gene, it is useful to find candidate genes that might be involved in the biological processes of interest.
We have developed the gene search system involving several versions of P-element based Gene Search (GS) vectors containing UAS. We use the system as a high throughput method to obtain functional information of genes in the genome (Fly Gene Search Project). The GS stocks serve as a library to screen for misexpression phenotypes as well as insertion homozygous phenotypes. We plan to use systematically collected data to analyze phenotypic similarity among the GS lines. We are currently developing an on-line application that allows clustering of genes based on the phenotypic information in addition to the protein motif data. Up-to-date information about the Fly GS project will be presented in the workshop.
S1-5
Genome-wide mouse mutagenesis: gene-driven vs. phenotype-driven.
Gondo, Yoichi
(Population and Quantitative Genomics Team, RIKEN Genomic Sciences Center)

A large-scale mouse mutagenesis is one of the focuses in the post genome project. A number of mutant mice should provide a useful tool to understand the gene function of the mammalian genome. It also becomes an animal model system for the studies of human diseases. One of the most potent chemical mutagen, N-ethyl-N-nitrosourea (ENU), which mainly induces point mutations, is administered to male mice. Their first generation (G1) mice are the candidates for dominant mutants. Extensive screenings of G1 mice for early-onset phenotypes which may appear within three months old are the major driving force of the world-wide projects; thus, it is a phenotype-driven approach to elucidate the gene function. In addition to this kind of general mutagenesis approaches, we have several unique features including: 1) The screening of late-onset phenotypes like tumorigenesis and senescence, that requires the longer observation of G1 mice up to 18 months, and 2) a development of a high throughput screening system for recessive mutations based upon a gene-driven approach. In order to make late-onset screenings feasible, all G1 male sperms are stored in a liquid nitrogen tank at the age of 12 weeks. Therefore, we could recover candidate strains at any time even after original G1 males may exhibit severe sterility or lethality accompanied to the late-onset phenotype(s) due to either mutations or just by aging. At the same time, genomic DNA is isolated from individual G1 males. Target genes will be amplified by PCR and newly-arisen point mutations would be identified by a molecular genotyping. When a critical base substitution is identified in a G1 genome, its offspring will be restored from the corresponding frozen sperms.
S1-6
A proposal of genome-wide linkage disequilibrium scan.
Takano-Shimizu, Toshiyuki
(Dept. of Population Genetics, National Institute of Genetics)

Much of current Drosophila research focuses on functionally characterizing all Drosophila genes by mutational analysis, but it is not our final goal. From the standpoint of evolutionary and disease genetics, assessment of natural variants is an important task in the coming generation of research and it cannot be fully done by in-lab-made disruptants. Quantitative genetics has uncovered, for instance, the predominance of sex-specific effects of many variations in nature which are easily missed in many loss-of-function mutations with large effects, although little is known yet about how much epistatic interactions exist between minor-effect mutations at different loci. This does not, of course, dampen our activity to characterize Drosophila genes by loss-of- and gain-of-function mutations, which certainly help subsequent allele assessment. To date, P-element-based mutagenesis has been used for many large scale screens for mutations, but unfortunately no one knows for sure how many genes are tagged by this element alone. An upper limit might be as small as 5000 genes. Thus, development of alternative approaches for genetic screens is highly desirable. The completion of Drosophila genome sequences greatly facilitates the construction of molecular markers exemplified by SNPs, which will, in turn, make it feasible to map any mutagen-mediated mutations. The availability of high dense makers also stimulates population and quantitative geneticists to so-called association studies and interaction-detection studies. Indeed, besides genetic and biochemical methods, population genetic approaches for gene-by-gene interactions are theoretically available, but their success has been so limited because of sparsity of markers along chromosomes. In this workshop, I will discuss the need of dense SNP markers and propose a linkage disequilibrium scan study of Drosophila genome for detecting allele specific interactions in nature.

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