Yoshiki Hiraoka

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.

CLONING AND EXPRESSION OF XENOPUS LAEVIS XSOX12 CDNA

A family of SRY-related genes has been termed SOX. We have isolated and sequenced a cDNA encoding xSox12 from Xenopus laevis ovary. The cDNA contained an open reading frame (ORF) coding for 470 amino acids encompassing an HMG box characteristic of the SOX family, a leucine zipper motif and glutamine-rich segments. The size of the xSox12 mRNA was determined to be 3.0 knt by Northern analysis. The ovary was the most prominent in the expression of the Sox mRNA among the various tissues of adult frog as far as examined.

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.

Expression and characterization of Xenopus laevis SRY-related cDNAs, xSox17α1, xSox17α2, xSox18α and xSox18β

Abstract Sox is a large family of genes related to the sex-determining region Y gene (designated as the SRY gene). Sox genes encoding DNA-binding transcriptional factors are found in many animals and are involved in developmental events. In this study, we newly isolated and sequenced novel Sox cDNAs from African clawed frog (Xenopus laevis). Five clones isolated here were classified into four distinct Sox genes designated as xSox17α1, xSox17α2, xSox18α and xSox18β. All four belong to a subtype of SOX family, type F. The cDNA xSox17α1 contains essentially the same nucleotide sequence as that identified as Sox17α in a previous work (Cell 91 (1997) 397), whereas xSox17α2 is a distinct gene with high homology to xSox17α1. The clones, xSox18α and xSox18β, are highly homologous to each other over the entire nucleotide sequences. The xSox18α and xSox18β genes encode 363 and 361 amino acids, respectively. Genomic Southern hybridization analysis showed the existence of two copies of the xSox18. Northern analysis indicated that the xSox18 gene was expressed in the spleen and kidney and the size of the transcript was estimated to be 2.4 knt. Electrophoretic mobility shift assays indicated that recombinant xSox18 polypeptide was capable of binding to the HMG consensus nucleotide sequence, AACAAT.

Synthesis of human gamma-glutamyl transpeptidase (GGT) during the fetal development of liver

We have determined expression of human GGT gene encoding gamma-glutamyl transpeptidase (GGT) during fetal development of liver using the Northern-blot analysis with a cloned human GGT cDNA and immunohistochemistry with a monoclonal antibody. GGT mRNA could be detected as early as the 12th week of gestation. It then increased gradually to a peak of approx. threefold the amount at week 12, at week 40, just before birth. The size of the mRNA in the fetal liver was 2.7 kb and mRNA of the same size was detected both in the human fetal kidney and human hepatocellular carcinoma as well as normal adult liver. Immunohistochemical analyses show that GGT increased as the fetal liver developed in parallel with the increase in mRNA. Histochemically, GGT was shown to be located in the wall of bile canaliculi when synthesis was low in early development, but to be distributed, in addition, all over the cell membrane of the fetal hepatocytes when synthesis was high at the later stage of development.

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.

Cloning and expression of a human/mouse polycomb group gene, ENX-2/Enx- 2

The Drosophila Polycomb group (Pc-G) genes encode transcriptional factors involved in development. Little is known about members of the vertebrate Pc-G genes. In this study, we have isolated a cDNA encoding a human Pc-G protein and the mouse equivalent. The human and mouse genes, which were named ENX-2 and Enx-2, encode 702 and 750 amino acids, respectively. ENX-2/Enx-2 protein exhibits a high homology (53-55% identity) to Drosophila Enhancer of zeste [E(z)] protein belonging to the Pc-G. The expression of Enx-2 was observed in mouse kidney, adrenal gland, testis and brain at high levels by Northern blot analysis. A cell line of mouse neuroblastoma, Neuro-2a, also expresses Enx-2 mRNA and its level is elevated by induction of neuronal differentiation of the cell.

Immunohistochemical study of localization of γ-glutamyl transpeptidase in the rat brain

Abstract γ-glutamyl transpeptidase (γ-GTP) is a membrane-bound enzyme which is known to play a crucial role in active transport of amino acids across membrane barriers. We prepared a monoclonal antibody recognizing specifically rat γ-GTP and investigated localization of the enzyme in the rat brain by immunohistochemistry with this antibody. The antigen was localized on the ependyma, epithelia of the choroid plexus and microvessels. More precise localization of γ-GTP was examined with immuno-electron microscopy. The antigen was recognized on the microvilli and cilia of the ependymal cells, microvilli of the choroid epithelial cells and luminal membranes of the vascular endothelial cells.