The Xenopus Sox3 gene expressed in oocytes of early stages
Introduction
Sex determination of mammals depends on the sex chromosome. Recently, SRY, the sex-determining region of the Y chromosome, has been identified as the best candidate for the testis-determining gene in mammals and encodes a protein with an HMG box, a DNA-binding domain (Sinclair et al., 1990). In addition to SRY, several SRY-type HMG box (Sox) genes have been isolated from widespread organisms including insects (Denny et al., 1992a), fish (Ito et al., 1995; Takamatsu et al., 1995), amphibians (Denny et al., 1992a), birds (Uwanogho et al., 1995) and mammals (Denny et al., 1992a; Goze et al., 1993; Wright et al., 1993). Some of these Sox genes are expressed early in development and may play roles in development and differentiation as a transcription factor. It has been shown that the expression of the chicken Sox-2, Sox-3 and Sox-11 genes is restricted to neuronal tissues in embryogenesis (Uwanogho et al., 1995), and mouse SOX-LZ and Sox-5 may be involved in spermatogenesis (Denny et al., 1992b; Takamatsu et al., 1995).
In amphibians, regulation of genes involved in sex determination is still unclear. The knowledge on subsequent gametogenesis following sex determination has been accumulated rapidly only in the last few years (Cooley, 1995). However, most advances in understanding amphibian gametogenesis are limited to oocyte maturation and maternally expressed genes, and there is little evidence for early gametogenesis. To understand sexual development or gametogenesis in Xenopus, we have sought Sox genes expressed in immature ovary. In this paper, we describe the isolation and characterization of a Xenopus Sox gene, XSox3, which is homologous with mammalian SOX3 genes (Stevanovic et al., 1993). Northern and Western blot analyses show that the XSox3 mRNA and protein are restricted to the early stages during oocyte maturation. The sequence-specific DNA-binding activity of the recombinant XSox3 protein implies the role of XSox3 as a transcription factor in early oogenesis of Xenopus.
Section snippets
Construction of a Xenopus ovary cDNA library
Poly(A)+RNA was purified using Oligotex-dT30 (Takara Shuzo) from total RNA extracted from an immature ovary by the acid guanidium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). The immature ovary of the young adult Xenopus laevis frogs (head to anus 4 cm) were obtained from Hamamatsu Seibutsu Kyozai (Maisaka, Japan). The poly(A)+RNA was converted into double-stranded cDNA using a cDNA synthesis kit (Amersham). Then, cDNA was ligated with EcoRI-digested λZAPII DNA
Isolation of XSox3 cDNA
To investigate the roles of Sox genes during oogenesis in X. laevis, we performed PCR to amplify cDNA fragments encoding SRY-type HMG boxes from a Xenopus immature ovary cDNA using primers based on the mammalian SRY-type HMG boxes (see Section 2). After being subcloned, PCR products were sequenced, and two kinds of SRY-type HMG box sequences were revealed. One was identical to the HMG box sequence of XSox11 reported by Denny et al. (1992a). The other was a novel type of HMG box sequence in
Discussion
From a Xenopus ovary cDNA library, we have isolated an SRY-type HMG box cDNA, XSox3. In the HMG box region, the XSox3 protein has 98.2% amino-acid sequence homology with XSox11 (data not shown), which was cloned only in the HMG box region by PCR from Xenopus oocyte cDNA (Denny et al., 1992a), suggesting the possibility of their diversification by gene duplication. In addition, the XSox3 protein shows overall high homology (62–77%) with the human and chicken Sox3 proteins (Fig. 2), and so the
Acknowledgements
We thank T. Noyama for technical assistance. We also thank Drs. E. Terada and Y. Sakurai for generation of antiserum. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture, Japan, by a grant pioneering research project in biotechnology from the Ministry of Agriculture, Forestry and Fisheries, Japan, and by a grant from the Kitasato Research Foundation, All Kitasato Project Study and research grants of the Kanagawa Academy of Science and Technology
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