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Identification and Characterization of Evolutionarily Conserved Pufferfish, Zebrafish, and Frog Orthologs of GASZ¹

Departments of Pathology,³ Molecular and Human Genetics,⁴ and Molecular and Cellular Biology,⁵ Baylor Collegeof Medicine, Houston, Texas 77030

We previously identified Gasz (a erm cell-specific gene encoding a protein containing four nkyrin repeats, a terile- motif, and a basic leucine ipper) in six mammalian species. Here, we report GASZ orthologs in pufferfish (Fugu rubripes), zebrafish (Danio verio), and frog (Xenopus laevis). Sequences of the three Gasz cDNAs were determined by database mining and 5'- and 3'-rapid amplification of cDNA ends (RACE) followed by sequencing. The three orthologous vertebrate genes encode proteins structurally similar to mammalian GASZ and contain the characteristic four ankyrin repeats (ANKs) and sterile- motif (SAM). Their ANK and SAM domains share 55– 74% and 38–55% amino acid identity with those in human GASZ, respectively. Similar to human and mouse Gasz genes, pufferfish Gasz is composed of 13 exons, spanning approximately 12 kilobases, and flanked by Cftr at its 5'-end and Wnt2 at its 3'-end. Northern and Western blot analyses detect frog Gasz expression only in testis and ovary. In situ hybridization and immunohistochemical analyses show that frog Gasz mRNA and protein expression is confined to pachytene spermatocytes in the testis and to oocytes in the ovary. In frog oocytes, GASZ protein appears to localize to a cytoplasmic structure resembling the Balbiani body, a postulated mRNA transport organizer in the cytoplasm. The high evolutionary conservation and germ cell specificity suggest that GASZ plays an essential role in gametogenesis. The data presented here are important for future studies of the physiological roles of GASZ using fish and amphibians as animal models.

¹ Supported in part by the NIH Specialized Cooperative Centers Program in Reproduction Research (HD-07495). W.Y. is supported by a postdoctoral fellowship from the Ernst Schering Research Foundation. C.A.Z. has been supported by NEI grants R01 EY12505 and T32 EY67102. The cDNA sequences for pufferfish, zebrafish, and frog Gasz have been deposited into GenBank with the accession numbers AY273804, AY273805, and AY273806, respectively.

² Correspondence: Martin M. Matzuk, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. FAX: 713 798 5833; mmatzuk{at}bcm.tmc.edu