• Proceedings of the KSAR Conference
• /
• 2001.10a
• /
• pp.14-17
• /
• 2001

Positional cloning (map-based cloning) of mutations or genetic variations has been served as an invaluable tool to understand in-vivo functions of genes and to identify molecular components underlying phenotypes of interest. Mice homozygous for the cerebellar deficient folia (cdf) mutation are ataxic, with cerebellar hypoplasia and abnormal lobulation of the cerebellum. In the cdf mutant cerebellum approximately 40% of Purkinje cells are ectopically located within the white matter and the inner granule cell layer (IGL). To identify the cdf gene, a high-resolution genetic map for the cdf-gene-encompassing region was constructed using 1997 F2 mice generated from C3H/HeSnJ-cdf/cdf and CAST/Ei intercross. The cdf gene showed complete linkage disequilibrium with three tightly linked markers D6Mit208, D6Mit359, and D6Mit225. A contig using YAC, BAC, and P1 clones was constructed for the cdf critical region to identify the gene. A deletion in the cdf critical region on chromosome 6 that removes approximately 150kb of DNA was identified. A gene associated with this deletion was identified using cDNA selection. cdf mutant mice with the transgenic copy of the identified gene restored the brain abnormalities of the mutant mice. The positional cloning of cdf gene provides a good example showing the identification of a gene could lead to finding a new component of important molecular pathways.

• Proceedings of the Korean Society of Crop Science Conference
• /
• 2017.06a
• /
• pp.113-113
• /
• 2017

Downy mildew (DM), caused by several species in the Peronosclerospora and Scleropthora genera, is a major maize (Zea mays L.) disease in tropical or subtropical regions. DM is an obligate parasite species in the higher plants and spreads by oospores, wind, and mycelium in seed surface, soil, and living hosts. Owing to its geographical distribution and destructive yield reduction, DM is one of the most severe maize diseases among the maize pathogens. Positional cloning in combination with phenotyping is a general approach to identify disease resistant gene candidates in plants; however, it requires several time-consuming steps including population or fine mapping. Therefore, in the present study, we suggest a new combination strategy to improve the identification of disease resistant gene candidates. Downy mildew (DM) resistant maize was selected from five cultivars using the spreader row technique. Positional cloning and bioinformatics tools identified the DM resistant QTL marker (bnlg1702) and 47 protein coding genes annotations. Eventually, 5 DM resistant gene candidates, including bZIP34, Bak1, and Ppr, were identified by quantitative RT-PCR without fine mapping of the bnlg1702 locus. Specifically, we provided DM resistant gene candidates with our new strategy, including field selection by the spreader row technique without population preparation, the DM resistance region identification by positional cloning using bioinformatics tools, and expression level profiling by quantitative RT-PCR without fine mapping. As whole genome information is available for other crops, we propose applying our novel protocol to other crops or for other diseases with suitable adjustment.

• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2001.10a
• /
• pp.14-17
• /
• 2001

Positional clonging (map-based cloning) of mutations or genetic variations has been served as an invaluable tool to understand in-vivo functions of genes and to identify molecular components underlying phenotypes of interest. Mice homozygous for the cerebellar deficient folia (cdf) mutation are ataxic, with cerebellar hypoplasia and abnormal lobulation of the cerebellum. In the cdf mutant cerebellum approximately 40% of Purkinje cells are ectopically located within the white matter and the inner granule cell layer (IGL). To identify the cdf gene, a high-resolution genetic map for the cdf-gene-encompassing region was constructed using 1997 F2 mice generated from C3H/HeSnJ-cdf/cdf and CAST/Ei intercross. The cdf gene showed complete linkage disequilibrium with three tightly linked markers D6Mit208, D6Mit359, and D6Mit225. A contig using YAC, BAC, and P1 clones was constructed for the cdf critical region to identify the gene. A deletion in the cdf critical region on chromosome 6 that removes approximately 150 kb of DNA selection. cdf mutant mice with the transgenic copy of the identified gene restored the brain abnormalities of the mutant mice. The positional cloning of cdf gene provides a good example showing the identification of a gene could lead to finding a new component of important molecular pathways.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2003.10a
• /
• pp.24-25
• /
• 2003

• 대한생식의학회:학술대회논문집
• /
• 2001.06a
• /
• pp.27-35
• /
• 2001

• Proceedings of the Zoological Society Korea Conference
• /
• 2003.08a
• /
• pp.52-52
• /
• 2003

No Abstract, See Full Text

• Proceedings of the Zoological Society Korea Conference
• /
• 1999.10b
• /
• pp.31-31
• /
• 1999

Characterization of mutant mice has been utilized as an animal model for the study of human inherited diseases. In addition to the pathogenesis stduy using the mutant mice, the mice have been used for the identification of the genes causing the phenotypes. Functional cloning and positional cloning are two approaches, depending on the phenotypes of the mutant mice. Though it takes a long time positional cloning has been well used to identify the gene of which function can not be presumed from the mouse phenotype. Recently by the advance of the molecular tools and the human genome project close to 10,000 genetic markers are developed to make the procedure faster. We obtained a new mutant mouse, sims, spontaneously arose and the affected mouse has a mild tremor and seizure was observed. Homozygote in either sex is sterile since uterus growth in female and seminal vesicle in male are not induced for the growth in puberty, implying the abnormal hormonal regulation during puberty. Supporting this, there is no detectable testosterone in the serum of the mutant male and the brain of the mutant is 30% heavier than littermate. To identify the location of the mutated gene, intraspecies cross to CAST/Ei was carried out and the 37 affected mice was analyzed for the linkage. The gene was mapped on chromosome 18, 20 cM from the centromere. More than 500 F2 progenies have been analyzed for the linkage and the locus becomes narrow within 3cM between Egrl and Fgf gene.f gene.

• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2003.10a
• /
• pp.24-25
• /
• 2003

The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. I will talk about positional cloning of two developmental mutants in zebrafish. The first mutant is headless: The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo by virtue of its distinct head and trunk inducing properties. Wingless/Wntantagonists secreted by the organizer have been identified as head inducers. Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation. These observations suggest that the ability of head inducers to inhibit Wntsignalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals. I describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in T-cell factor-3 (Tcf3), a member of the Tcf/Lef family. Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. I provide genetic evidence that a component of the Wntsignalling pathway is essential in vertebrate head formation and patterning. Second mutant is mind bomb: Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneuralgene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. (중략)

• Development and Reproduction
• /
• v.7 no.2
• /
• pp.69-80
• /
• 2003

The zebrafish(Danio rerio) is a pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. We show examples of positional cloning in two developmental mutants in zebrafish. headless: The severe head defects in headless(hdl) mutants are due to a mutation in T-cell factor-3(Tcf3). Loss of Tcf3 function in the hdl mutant reveals that Hdl represses Wnt target genes. The results provide genetic evidence that a component of the Wnt signaling pathway is essential in vertebrate head formation and patterning. mind bomb: Reduced lateral inhibition in mind bomb(mib) mutants permits too many neural precursors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a ubiquitin E3 ligase. Mib interacts with the intracellular domain of Delta to promote its internalization. The results suggest a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell.

• Genomics & Informatics
• /
• v.4 no.2
• /
• pp.80-86
• /
• 2006

It is clear that the construction of large insert DNA libraries is important for map-based gene cloning, the assembly of physical maps, and simple screening for specific genomic sequences. The bacterial artificial chromosome (BAC) system is likely to be an important tool for map-based cloning of genes since BAC libraries can be constructed simply and analyzed more efficiently than yeast artificial chromosome (YAC) libraries. BACs have significantly expanded the size of fragments from eukaryotic genomes that can be cloned in Escherichia coli as plasmid molecules. To facilitate the isolation of molecular-biologically important genes in Ashbya gossypii, we constructed Ashbya chromosome-specific BAC libraries using pBeloBAC11 and pBACwich vectors with an average insert size of 100 kb, which is equivalent to 19.8X genomic coverage. pBACwich was developed to streamline map-based cloning by providing a tool to integrate large DNA fragments into specific sites in chromosomes. These chromosome-specific libraries have provided a useful tool for the further characterization of the Ashbya genome including positional cloning and genome sequencing.