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Hagiuda J
,
Hiraoka Y
,
Hasegawa M
,
Ogawa M
,
Aiso S
.
Abstract
The sex-determining region Y (SRY) gene and its related Sox genes encode transcriptional regulatory factors. In this study, we isolated and sequenced a novel Sox cDNA from African clawed frog (Xenopus laevis). The Sox gene was named xSox33. xSox33 was revealed to encode 244 amino acids. Reverse transcription-polymerase chain reaction (RT-PCR) showed that xSox33 was expressed at very low levels in some frog tissues including lung, ovary, skeletal muscle, testis, brain and heart. Its embryonic expression was also studied by RT-PCR. After the mid-blastula transition, the zygotic expression was initiated during gastrulation and the level was elevated as the embryogenesis proceeded. Electrophoretic mobility shift assay (EMSA) indicated that a recombinant xSox33 polypeptide was capable of binding to the nucleotide sequence AACAAT.
Fig. 1.
Nucleotide sequence of the xSox33 gene and the deduced amino acid sequence. The nucleotide sequence was assembled from the cDNA clone XS and genomic clone XG for xSox33. The cDNA clone XS contains the first 681 nt of the sequence shown. The remaining 3â² half of the transcribable region was determined by RT-PCR using forward primers (F1 and F2) and reverse primers (B1âB4). Primer combinations used in this experiment were: F1/B1, F2/B2, F2/B3 and F2/B4. The transcribable region is indicated with capital letters; the sequence at the 3â² end, which is indicated with lower-case letters, is derived from the genomic clone. Nucleotides are numbered in parentheses at right. The deduced aa sequence is shown above the nt sequence. Amino acid numbers are indicated in brackets at right. Upstream and downstream in-frame stop codons are indicated by asterisk. The SRY-related HMG box is underlined. The putative poly(A) signal is boxed.
Fig. 2.
Southern blot analysis for the xSox33 gene. Genomic DNA (10 μg) from the muscle of an adult female frog (X. laevis) was digested with BamHI, BglII, EcoRI or HindIII. The digest was electrophoresed on a 0.7% agarose gel. A short portion (421 bp) of the xSox33 cDNA was amplified by PCR using a pair of primers, 5â²-AAG AAT TCG AGA GCA AAA CTC GGG CCA-3â² and 5â²-AAA AGC TTC TAT GGC TCA GTA GGA AAA CA-3â². The PCR products were labeled with 32P and used as a probe.
Fig. 3.
Comparison of amino acid sequences of Sox proteins. (a) The HMG box amino acid sequences from the xSox33 found in this study and known Sox proteins identified previously were aligned (all the known Sox sequences have been cited in [4] ; [9]). Homology (%) means percentage amino acid identity between the xSox33 HMG box and that from each of the known Sox proteins. The previously defined Sox subtype names AâJ are shown at right. (b) The entire amino acid sequence of xSox33 is compared with those of major subtype-C SOX proteins previously identified. Amino acid residues shared by xSox33 and the other subtype-C members are highlighted. The HMG box region is boxed.
Fig. 4.
Tissue-specific and embryonic expression of xSox33. (a) Total RNAs from various adult frog tissues were studied by RT-PCR. Total RNA (50 μg) was pre-treated with RNase-free DNase I for removing contaminated genomic DNA and then reverse-transcribed into single-stranded cDNA according to a standard method. The cDNA was used as a template in the subsequent PCR assay. PCR was carried out by using a pair of primers: 5′-GAG AGC AAA ACT CGG GCC A-3′ and 5′-CTA TGG CTC AGT AGG AAA ACA-3′ specific to xSox33. The thermal cycling profile was: preheat at 95 °C for 9 min; 37 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 2 min and extension at 68 °C for 3 min. The band corresponding to xSox33 is 415 bp in size. (b) Total RNAs from Xenopus embryos at various stages were studied by RT-PCR assay. PCR was carried out using a pair of primers: 5′-GCA CAA TGC TGA CAT CTC TC-3′ and 5′-CTA TGG CTC AGT AGG AAA ACA-3′ specific to xSox33. The thermal cycling profile employed for xSox33 was: preheat at 95 °C for 9 min; 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 2 min and extension at 68 °C for 2 min. The band corresponding to xSox33 is 605 bp in size. Control RT-PCR experiments were also carried out using primers specific to the ornithine decarboxylase (ODC) gene, which is expressed in frog eggs [21]. The thermal cycling profile for ODC was: preheat at 95 °C for 9 min; 25 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min and extension at 72 °C for 2 min.
Fig. 5.
EMSAs of oligonucleotide binding by xSox33. Nucleotides 366â647 from the xSox33 cDNA, encoding aa 30â123, were amplified by PCR using primers, 5â²-AAG GAT CCA GCT GGT GCA AGA CCT CCA-3â² and 5â²-AAG AAT TCG GCC CGA GTT TTG CTC TCT-3â². The PCR products were digested with EcoRI, and the resulting fragments were subcloned into the pGEX-2T expression vector (Pharmacia). GSTâxSox33 and non-fused GST proteins expressed in E. coli were purified by affinity chromatography on glutathioneâSepharose 4B. (a) DNA sequences of EMSA probes are aligned. Nucleotides flanking the mutated position in each probe are identical to those of WT probe and are indicated by hyphen. Names of probes are shown at right. Procedures for EMSA were described in detail previously [5]; [6]; [7]; [8]; [9] ; [10]. (b) Purified GST (lane 2) or GST fusion protein (lanes 3 and 4) was mixed with 32P-labeled oligonucleotide probe WT. The reaction for lane 1 was performed in the absence of protein. Unlabeled competitor oligonucleotide WT was included in the control reaction (lane 4). (c) Specificity of sequence recognized by xSox proteins was assessed by EMSA using the series of probes shown in (a). The probes used are indicated above the respective lanes.
