Elsevier

Gene

Volume 211, Issue 2, 12 May 1998, Pages 251-257
Gene

Rainbow trout Sox24, a novel member of the Sox family, is a transcriptional regulator during oogenesis

https://doi.org/10.1016/S0378-1119(98)00100-0Get rights and content

Abstract

We isolated a cDNA clone encoding a novel SRY-type HMG box (Sox) protein, designated Sox24, from a rainbow trout ovary cDNA library. On the basis of the HMG box amino acid sequence, Sox24 can be categorized into the same subgroup of Sox proteins as SOX4, SOX11, and SOX22. The proteins in this group also share a highly conserved sequence at the C-terminus. The Sox24 mRNA is expressed at high levels in the ovary, and in-situ hybridization localized its expression to oocytes. The recombinant protein containing the Sox24 HMG box region bound to an AACAAT sequence strongly in a gel retardation assay. Upon co-transfection into CHO cells, the full-length Sox24 transactivated transcription from a reporter plasmid through the AACAAT binding motif. We used GAL4/Sox24 chimeras with the DNA binding domain of yeast GAL4 at the N-terminus to map the transactivation function to the C-terminal region, which included the conserved sequence. These results suggest that Sox24 plays a role as a transcriptional regulator during oogenesis.

Introduction

The SRY (sex-determining region of the Y chromosome) gene has been identified as a testis determining factor in mammals (Gubbay et al., 1990; Sinclair et al., 1990). This gene encodes a DNA binding domain known as the HMG (high mobility group) box. The discovery of SRY/Sry in mouse and human rapidly led to the isolation of Sox genes, encoding a protein with an SRY-type HMG box in a wide variety of organisms (Denny et al., 1992a). The SRY-type HMG boxes bind to the minor groove of the (A/T)(A/T)CAAG motif (Denny et al., 1992b; Harley et al., 1992, Harley et al., 1994; van de Wetering et al., 1992), and induce a sharp bend in the DNA helix (Ferrari et al., 1992; Giese et al., 1992; Connor et al., 1994). The Sox genes are expressed during the embryonic development and also in some adult tissues, and are considered to play diverse roles in development as a transcription factor. In chicken, the Sox2 gene is expressed in the early developing lens and is implicated in the regulation of crystallin genes (Kamachi et al., 1995). The mouse Sox4 gene is involved in cardiac development as well as lymphocyte differentiation (Schilham et al., 1996), and human SOX4 can transactivate the CD2 gene (Wotton et al., 1995). The human SOX9 gene was identified as a causal gene for autosomal sex reversal and campomelic dysplasia (Foster et al., 1994; Wagner et al., 1994), and human SOX9 is likely to be a regulator of the type II collagen gene, COL2A1 (Lefebvre et al., 1997; Ng et al., 1997).

In salmonid ovaries, the follicles develop with a degree of synchrony, and large numbers of follicles at approximately the same stage of development can be obtained from a single animal, which is an enormous advantage for biochemical studies. Since in fish, as in other vertebrates, oogenesis can be divided into two parts, the growth phase and the maturation phase, the studies in fish could contribute to an understanding of oogenesis in vertebrates. However, in contrast to the understanding of endocrine regulation of oocyte growth and maturation in fish (Nagahama et al., 1995), the underlying gene regulation remains to be elucidated. The observation that between fish and mammals, the SOX-LZ (SOX6) and SOX9 genes show high degrees of conservation of gene structure and expression in spite of the evolutionary distance (Takamatsu et al., 1995Takamatsu et al., 1997) led us to seek for Sox genes expressed in rainbow trout ovary to understand their roles in oogenesis in fish. Here, we describe the isolation of a novel Sox cDNA from a rainbow trout ovary cDNA library and the characterization of its expression and transactivation activity.

Section snippets

Isolation of rainbow trout Sox24 cDNA clones

SRY-type HMG box sequences were amplified by PCR from rainbow trout ovary cDNA as described by Takamatsu et al. (1995), and one of the obtained sequences, Sox24, was used to screen a rainbow trout ovary cDNA library constructed with λZAPII vector (Stratagene). Positive clones were isolated, and rescued as pBluescript plasmids by in-vivo excision. The nt sequence was determined by using ssDNA by the dideoxy method (Sanger et al., 1997) with T7 DNA polymerase.

Northern (RNA) blot analysis

Total RNA was prepared by the

Isolation of rainbow trout Sox24 cDNA

By a PCR-based strategy with degenerate oligos corresponding to highly conserved aa sequences within the SRY-type HMG boxes, we obtained three kinds of Sox sequences from rainbow trout ovary cDNA as described by Takamatsu et al. (1995). Using one of the Sox sequences, designated Sox24, as a probe, we isolated a full-length cDNA clone (pRTSox24) from a rainbow trout ovary cDNA library. Sequence analysis revealed that the rainbow trout Sox24 is 367 aa and has an acidic region (66%) (aa 188–211) in

Acknowledgements

This work was supported in part 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.

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