Yukinao Sakai

Isolation and characterization of a mouse SRY-related cDNA, mSox7

SOX is a family of SRY-related genes, which encode transcriptional factors involved in development. In this study, we newly isolated and sequenced mouse cDNA clones for mSox7. The mSox7 gene encodes 380 amino acids containing an SRY-type HMG box. Genomic Southern analysis suggested that the mSox7 gene was a single-copy gene. Tissue specific expression of mSox7 was investigated by Northern analysis. The expression was restricted to the ovary and heart, and the size of the transcripts was estimated to be 3.6 knt. Electrophoretic mobility shift assay indicated that recombinant mSox7 polypeptide was capable of binding to a nucleotide sequence, AACAAT. Immunohistochemical study revealed that mSox7 protein was localized in oocytes in the mouse ovary.

Cloning and characterization of Xenopus laevis xSox7 cDNA.

A family of SRY-related genes has been termed SOX. We have isolated and sequenced a cDNA encoding a novel Sox protein from Xenopus laevis ovary. This cDNA contains an open reading frame (ORF) coding for 362 amino acids, which encompasses an HMG box and exhibits a strong (90%) identity to that of mouse Sox7; the cDNA was named xSox7 in this study. Northern analysis revealed that the xSox7 mRNA was 2.0 kb in length. Various adult frog tissues were tested by reverse transcription/polymerase chain reaction for xSox7 mRNA, and the results showed that xSox7 is expressed in a wide range of tissues. Furthermore, electrophoretic mobility shif assay indicated that recombinant xSox7 is capable of binding to AACAAT sequence.

XLS13A and XLS13B: SRY-related genes of Xenopus laevis

SRY-related cDNAs, XLS13A and XLS13B, have been isolated from Xenopus laevis ovary. The cDNAs encode polypeptides of 382 and 375 amino acids, respectively. Nucleotide sequences of the two cDNAs are highly homologous to each other. The type-A and type-B XLS13 proteins, and xSox13 reported previously share an identical high mobility group (HMG) box at the amino acid level, although they contain silent nucleotide alterations. The HMG box exhibits strong similarity (> 93% amino acid identity) to those of mouse Sox4/human SOX4 and chicken Sox11/human SOX11. The size of XLS13A/XLS13B mRNA was estimated to be 2.8 knt in Xenopus ovary by Northern analysis. Reverse transcription/polymerase chain reaction (RT/PCR) assay indicated that XLS13A and XLS13B mRNAs are present in various tissues of adult frog. The mRNAs of XLS13A and XLS13B of maternal origin found in unfertilized eggs disappear in the early stages of the Xenopus embryo. DNA-binding properties of the XLS13 HMG domain were examined by electrophoretic mobility shift assay (EMSA). The HMG domain preferentially binds to the canonical target sequence of SOX proteins, AACAAT, in vitro.

The mouse Sox5 gene encodes a protein containing the leucine zipper and the Q box.

The nucleotide sequence of mouse Sox5 (mSox5) cDNA derived from the testis has been reported by Denny et al. (EMBO J. 11 (1992) 3705-3712). We newly isolated an mSox5 cDNA derived from 8.5-day mouse embryo. Our cDNA encodes a protein of 763 amino acids, which is considerably larger in size than the previous one (392 amino acids) derived from the adult mouse testis. The most significant difference between the embryo-derived and testis-derived mSox5 cDNAs is that the embryonic one encodes a leucine zipper motif and a neighboring glutamine-rich sequence stretch (named Q box), but the testis-derived one does not. The leucine zipper and the Q box are highly conserved among type-D SOX proteins including mSox5. mSox5 was suggested to be a single-copy gene by Southern analysis. With reverse transcription/polymerase chain reaction, we found that not only mouse embryo, but also the adult mouse testis, express mSox5 mRNA species encoding the leucine zipper and the Q box.

PLP-I: a novel prolactin-like gene in rodents.

In this report, we describe molecular cloning and characterization of cDNAs encoding a novel rat prolactin-like protein. The rat cDNAs were isolated from the decidua and the gene was named PLP-I. cDNAs for the mouse equivalent were also cloned by the cross-hybridization technique. Pregnancy-specific expression of the rat PLP-I gene was observed in the rat placenta by Northern analysis. Location of signal peptide cleavage sites in rat and mouse pre-PLP-I proteins was predicted using a theoretical method. A molecular phylogenetic tree for the growth hormone-prolactin superfamily including the novel member, PLP-I, constructed using the neighbor-joining method, places rat/mouse PLP-I closest to rat/mouse placental lactogen I and II.

Isolation and expression of a human SRY-related cDNA, hSOX20

SOX is a family of genes related to the testis-determining gene, SRY. We have isolated and sequenced an hSOX20 cDNA from a cell line of human embryonic carcinoma. This cDNA contains an open reading frame (ORF) encoding 233 amino acids. The protein encompasses an SRY-type HMG box exhibiting strong homologies to those of mouse Sox15 and Sox16. Various adult and fetal tissues were tested for hSOX20 mRNA by Northern analysis. Its expression is restricted to the fetal testis and the size of the transcript is 1.5 knt. Electrophoretic mobility shift assay indicated that recombinant hSOX20 polypeptide is capable of binding to AACAAT sequence.

Cloning and characterization of mouse mSox13 cDNA

A novel SRY-related cDNA, mSox13, was isolated from a lambda phage library derived from mouse embryo. The cDNA encodes a protein of 595 amino acids containing the SRY-type high mobility group (HMG) box and a putative leucine zipper motif. A sequence comparison of mSox13 and other type-D SOX proteins shows that the leucine zipper and a neighboring glutamine-rich sequence stretch, which was named Q box, are well conserved among known type-D SOX proteins. The expression of mSox13 is restricted to the kidney and ovary. The electrophoretic mobility shift assay indicates that the recombinant mSox13 protein is capable of binding to the AACAAT sequence.

mRNA sequence of the Xenopus laevis paxillin gene and its expression.

Paxillin is a cytoskeletal protein found in structures of focal adhesions where cells adhere to the extracellular matrix. We isolated paxillin cDNA from the Xenopus laevis ovary. The cDNA sequence encodes a protein of 539 amino acids with four LIM and five LD motifs. 80% of the amino acids of frog paxillin are shared by human and chicken paxillins. Northern analysis showed that the frog gene is expressed in the spleen, kidney, testis and ovary. Immunocytochemistry showed that paxillin protein is accumulated in the nucleus as well as in the periphery of the cytoplasm of the A6 cell. This intriguing result shows that paxillin, which has been characterized as a cytoskeletal protein, is capable of translocating to the nucleus.

Molecular cloning and expression of Xenopus laevis Requiem cDNA

Abstract Requiem is an apoptosis-associated gene, which was originally identified in mouse (T.G. Gabig et al., J. Biol. Chem. 269 (1994) 29515–29519). In this study, we isolated five independent cDNA clones for frog Requiem ( xReq ) from Xenopus laevis ovary. Sequence analysis of the multiple cDNAs has suggested that Xenopus genome contains at least two non-allelic copies of the xReq gene. The amino acid sequence deduced from the cDNAs contained a single Kruppel -type zinc-finger motif and two PHD-finger motifs. Northern analysis revealed that the ovary expressed xReq transcripts with different sizes.