Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


Genomic comparative analysis of the Fugu rubripes homologue of ETV6, a gene frequently rearranged in human leukemias

Alexandre Montpetit and Daniel Sinnett

Division of Hemato-Oncology, Charles-Bruneau Cancer Center, Sainte-Justine Hospital, Montreal, Canada and
Department of Biochemistry, University of Montreal, Montreal, Canada

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. Little is known on the molecular pathogenesis of this disease. Recently, loss of heterozygosity (LOH) studies have reported deletions of the chromosome 12p12.3 in 15-47% of pre-B ALL patients. This region was also found deleted in several other hematological malignancies as well as a variety of solid tumors suggesting the presence of a putative tumor suppressor gene. The chromosomal region containing this tumor suppressor locus was restricted to a ~750kb interval that includes the gene ETV6, encoding an ets-like transcription factor required for hematopoiesis in bone marrow. Accumulating evidences suggest that ETV6 is not the tumor suppressor gene targeted by the deletions. However this gene is frequently found translocated with different partners leading to the creation of chimeric products in hematological disorders suggesting that this region might be intrinsically unstable. The search for genes in large mammalian genomic region is the limiting step in positional cloning. We propose that this task could be facilitated by genomic comparative studies of the region of interest in the compact genome of Fugu rubripes that is 7.5 times smaller than that of human but contains a similar repertoire of genes. Here we report the characterization of the Fugu homologue of ETV6 (frETV6) that constitutes the initial step toward the comparative mapping of the human chromosome 12p12.3 tumor suppressor locus. Two Fugu genomic libraries were screened with a human ETV6 cDNA leading to the identification of 6 genomic clones that are part of a contig covering more than 175 kb. The 8 exons of frETV6 were identified and located within a 15kb region. Compared to the human homologue, which is spread over 350 kb, this represents a substantial 23-fold compaction. At the protein level, we observed an overall 57% sequence identity between both species. In particular, the conserved ETS and PNT domains showed 95% and 69% amino acid identity, respectively. A phylogenetic analysis conducted on ETV6 sequences from human, mouse, chicken, Fugu and zebrafish revealed a close relationship between the two teleost fishes. The comparative mapping of the suppressor locus will be extended to the region flanking frETV6 in order to identify candidate genes that could be involved in ALL and/or other cancers as well as to get clues about the cause of the chromosomal instability affecting this region.

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