International Mammalian Genome Society

The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

I19 Identification of New Genes Located Near the Highly Methylated Region Screened by RLGS-M Method from the Liver Tumor of the SV40T Antigen-Transformed C57BL/6J Mouse

Minako Tateno1,2, Yoshifumi Fukunishi1,2, Sei Komatsu2, Kazuhiro Shibata1,2, Yasushi Okazaki2 and Yoshihide Hayashizaki1,2. 1CREST, Japan Science and Technology Corporation (JST); 2Laboratory for Genome Exploration Research Project, Genomic Sciences Center (GSC) and Genome Science Laboratory, Tsukuba Life Science Center, the Institute of Physical and Chemical Research (RIKEN)

DNA methylation has an essential regulatory function in gene expression and the alterations in methylation pattern have been reported in a variety of tumors. In the previous study using restriction landmark genomic scanning-methylation (RLGS-M) method, we identified highly methylated spots in common with 30 liver tumors from the SV40T antigen-transformed C57BL/6J mice (Cancer Res.57.3294-3299, 1997). To identify the genes located in the methylated regions, we screened the BAC library (RPCI23) to identify the BAC clones covering the RLGS-M spots, and the shotgun sequence of the BAC clones was performed by 6x redundancies using 384 capillary sequencer (RISA) developed by us. BLAST analysis was performed against non-redundant EST databases with the contigs. We identified tulip1 (tuberin-like protein 1) gene from the BAC clone covering the spot B236, which is located 3.6 kb upstream of the spot B236. RT-PCR analysis showed that tulip 1 transcripts were significantly repressed in the liver tumor compared to the normal one. A new ORF is predicted downstream to the B236 region. This ORF involves a nuclear transport signal, Snail/Gfi-1 type repressor domain, and Cys2-His2 class zinc finger domains, suggesting to encode a repressor-like protein. We are further analyzing the expression of the new gene. We will also discuss the efficiency of the shot gun sequence without finishing for the identification of the new genes in a target region.

[We thank Noriko Kikuchi and Norihito Hayatsu for their technical assistance.]


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