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A new mouse gene, SRG3, related to the SWI3 of Saccharomyces cerevisiae, is required for apoptosis induced by glucocorticoids in a thymoma cell line.

Jeon SH, Kang MG, Kim YH, Jin YH, Lee C, Chung HY, Kwon H, Park SD, Seong RH - J. Exp. Med. (1997)

Bottom Line: The expression of anti-sense RNA to SRG3 mRNA in a thymoma cell line, S49.1, reduced the expression level of the SRG3 protein, and decreased the apoptotic cell death induced by glucocorticoids.These results suggest that the SRG3 protein is involved in the glucocorticoid-induced apoptosis in the thymoma cell line.This implicates that the SRG3 may play an important regulatory role during T cell development in thymus.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Genetics and Department of Molecular Biology, Seoul National University, Seoul 151-742, Korea.

ABSTRACT
We isolated a new mouse gene that is highly expressed in thymocytes, testis, and brain. This gene, SRG3, showed a significant sequence homology to SWI3, a yeast transcriptional activator, and its human homolog BAF155. SRG3 encodes 1,100 amino acids and has 33-47% identity with SWI3 protein over three regions. The SRG3 protein contains an acidic NH2 terminus, a myb-like DNA binding domain, a leucine-zipper motif, and a proline- and glutamine-rich region at its COOH terminus. Rabbit antiserum raised against a COOH-terminal polypeptide of the SRG3 recognized a protein with an apparent molecular mass of 155 kD. The serum also detected a 170-kD protein that seems to be a mouse homologue of human BAF170. Immunoprecipitation of cell extract with the antiserum against the mouse SRG3 also brought down a 195-kD protein that could be recognized by an antiserum raised against human SWI2 protein. The results suggest that the SRG3 protein associates with a mouse SWI2. The SRG3 protein is expressed about three times higher in thymocytes than in peripheral lymphocytes. The expression of anti-sense RNA to SRG3 mRNA in a thymoma cell line, S49.1, reduced the expression level of the SRG3 protein, and decreased the apoptotic cell death induced by glucocorticoids. These results suggest that the SRG3 protein is involved in the glucocorticoid-induced apoptosis in the thymoma cell line. This implicates that the SRG3 may play an important regulatory role during T cell development in thymus.

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Immunoblotting and immunoprecipitation of the SRG3  protein. The overexpressed COOH-terminal part of SRG3 gene in Escherichia coli system was used to immunize rabbits to produce the polyclonal  antiserum. When thymus and lymph node extract were blotted with the  SRG3 antiserum, bands at 155 and 170 kD were detected (A). When the  extract was blotted with the hSWI2 antiserum, a band at 195 kD (B, top)  was detected. After immunoprecipitating the extract with the SRG3 antiserum, the precipitates were blotted with the SRG3 antiserum (B, bottom)  or the hSWI2 antiserum (B, top), displaying the 155- and 195-kD bands, respectively. Immunoprecipitation with preimmune serum and blotting with  the SRG3 and hSWI2 antiserum dose not show any band (B). TCL, total  cell lysate; IP:Pre, immunoprecipitation with the pre immune serum; IP: anti-SRG3, immunoprecipitation with the SRG3 antiserum.
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Figure 3: Immunoblotting and immunoprecipitation of the SRG3 protein. The overexpressed COOH-terminal part of SRG3 gene in Escherichia coli system was used to immunize rabbits to produce the polyclonal antiserum. When thymus and lymph node extract were blotted with the SRG3 antiserum, bands at 155 and 170 kD were detected (A). When the extract was blotted with the hSWI2 antiserum, a band at 195 kD (B, top) was detected. After immunoprecipitating the extract with the SRG3 antiserum, the precipitates were blotted with the SRG3 antiserum (B, bottom) or the hSWI2 antiserum (B, top), displaying the 155- and 195-kD bands, respectively. Immunoprecipitation with preimmune serum and blotting with the SRG3 and hSWI2 antiserum dose not show any band (B). TCL, total cell lysate; IP:Pre, immunoprecipitation with the pre immune serum; IP: anti-SRG3, immunoprecipitation with the SRG3 antiserum.

Mentions: To identify the protein product of the SRG3 gene, polyclonal antibody was produced against the SRG3–GST fusion protein. The COOH-terminal part of the SRG3 gene was inserted inframe into the pGEX4T-2 plasmid containing the glutathione-S-transferase (GST) gene. The overexpressed GST– 3C fusion protein with ∼85 kD of molecular mass was used to immunize rabbits through subcutaneous injection. After primary and booster injections, polyclonal antiserum against GST–3C fusion protein was obtained. The antiserum was confirmed to recognize specifically the fusion protein (data not shown). To identify the SRG3 gene product, immunoblot analysis was performed with crude extracts prepared from thymus and lymph node. As shown in Fig. 3 A, two bands of ∼155 and 170 kD were observed; the 155-kD protein is likely to be SRG3. This is supported by the observations that the protein matches well to the size of the BAF155 in human (42), that the antiserum immunoprecipitates the 155-kD protein (Fig. 3 B), and that the intensity of the 155-kD protein band was specifically reduced when anti-sense RNA to the SRG3 is expressed in a cell line (see Fig. 6 A). The antiserum also recognized a 170-kD protein that seemed to be similar to the SRG3 protein in its structure. Considering the results of human SWI3 homologues (42), it is likely that the 170-kD protein may be the mouse counterpart of the human BAF170 protein. The SRG3 and BAF170 are quite similar to each other over regions I (86%), II (93%), and III (89%) (see Fig. 2 B), and anti-SRG3 antiserum seems to recognize a murine BAF170like protein as well as SRG3. Interestingly, SRG3 is expressed at a three times higher level in thymus than in lymph nodes; however, the 170-kD protein is expressed at similar levels in both tissues.


A new mouse gene, SRG3, related to the SWI3 of Saccharomyces cerevisiae, is required for apoptosis induced by glucocorticoids in a thymoma cell line.

Jeon SH, Kang MG, Kim YH, Jin YH, Lee C, Chung HY, Kwon H, Park SD, Seong RH - J. Exp. Med. (1997)

Immunoblotting and immunoprecipitation of the SRG3  protein. The overexpressed COOH-terminal part of SRG3 gene in Escherichia coli system was used to immunize rabbits to produce the polyclonal  antiserum. When thymus and lymph node extract were blotted with the  SRG3 antiserum, bands at 155 and 170 kD were detected (A). When the  extract was blotted with the hSWI2 antiserum, a band at 195 kD (B, top)  was detected. After immunoprecipitating the extract with the SRG3 antiserum, the precipitates were blotted with the SRG3 antiserum (B, bottom)  or the hSWI2 antiserum (B, top), displaying the 155- and 195-kD bands, respectively. Immunoprecipitation with preimmune serum and blotting with  the SRG3 and hSWI2 antiserum dose not show any band (B). TCL, total  cell lysate; IP:Pre, immunoprecipitation with the pre immune serum; IP: anti-SRG3, immunoprecipitation with the SRG3 antiserum.
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Figure 3: Immunoblotting and immunoprecipitation of the SRG3 protein. The overexpressed COOH-terminal part of SRG3 gene in Escherichia coli system was used to immunize rabbits to produce the polyclonal antiserum. When thymus and lymph node extract were blotted with the SRG3 antiserum, bands at 155 and 170 kD were detected (A). When the extract was blotted with the hSWI2 antiserum, a band at 195 kD (B, top) was detected. After immunoprecipitating the extract with the SRG3 antiserum, the precipitates were blotted with the SRG3 antiserum (B, bottom) or the hSWI2 antiserum (B, top), displaying the 155- and 195-kD bands, respectively. Immunoprecipitation with preimmune serum and blotting with the SRG3 and hSWI2 antiserum dose not show any band (B). TCL, total cell lysate; IP:Pre, immunoprecipitation with the pre immune serum; IP: anti-SRG3, immunoprecipitation with the SRG3 antiserum.
Mentions: To identify the protein product of the SRG3 gene, polyclonal antibody was produced against the SRG3–GST fusion protein. The COOH-terminal part of the SRG3 gene was inserted inframe into the pGEX4T-2 plasmid containing the glutathione-S-transferase (GST) gene. The overexpressed GST– 3C fusion protein with ∼85 kD of molecular mass was used to immunize rabbits through subcutaneous injection. After primary and booster injections, polyclonal antiserum against GST–3C fusion protein was obtained. The antiserum was confirmed to recognize specifically the fusion protein (data not shown). To identify the SRG3 gene product, immunoblot analysis was performed with crude extracts prepared from thymus and lymph node. As shown in Fig. 3 A, two bands of ∼155 and 170 kD were observed; the 155-kD protein is likely to be SRG3. This is supported by the observations that the protein matches well to the size of the BAF155 in human (42), that the antiserum immunoprecipitates the 155-kD protein (Fig. 3 B), and that the intensity of the 155-kD protein band was specifically reduced when anti-sense RNA to the SRG3 is expressed in a cell line (see Fig. 6 A). The antiserum also recognized a 170-kD protein that seemed to be similar to the SRG3 protein in its structure. Considering the results of human SWI3 homologues (42), it is likely that the 170-kD protein may be the mouse counterpart of the human BAF170 protein. The SRG3 and BAF170 are quite similar to each other over regions I (86%), II (93%), and III (89%) (see Fig. 2 B), and anti-SRG3 antiserum seems to recognize a murine BAF170like protein as well as SRG3. Interestingly, SRG3 is expressed at a three times higher level in thymus than in lymph nodes; however, the 170-kD protein is expressed at similar levels in both tissues.

Bottom Line: The expression of anti-sense RNA to SRG3 mRNA in a thymoma cell line, S49.1, reduced the expression level of the SRG3 protein, and decreased the apoptotic cell death induced by glucocorticoids.These results suggest that the SRG3 protein is involved in the glucocorticoid-induced apoptosis in the thymoma cell line.This implicates that the SRG3 may play an important regulatory role during T cell development in thymus.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Genetics and Department of Molecular Biology, Seoul National University, Seoul 151-742, Korea.

ABSTRACT
We isolated a new mouse gene that is highly expressed in thymocytes, testis, and brain. This gene, SRG3, showed a significant sequence homology to SWI3, a yeast transcriptional activator, and its human homolog BAF155. SRG3 encodes 1,100 amino acids and has 33-47% identity with SWI3 protein over three regions. The SRG3 protein contains an acidic NH2 terminus, a myb-like DNA binding domain, a leucine-zipper motif, and a proline- and glutamine-rich region at its COOH terminus. Rabbit antiserum raised against a COOH-terminal polypeptide of the SRG3 recognized a protein with an apparent molecular mass of 155 kD. The serum also detected a 170-kD protein that seems to be a mouse homologue of human BAF170. Immunoprecipitation of cell extract with the antiserum against the mouse SRG3 also brought down a 195-kD protein that could be recognized by an antiserum raised against human SWI2 protein. The results suggest that the SRG3 protein associates with a mouse SWI2. The SRG3 protein is expressed about three times higher in thymocytes than in peripheral lymphocytes. The expression of anti-sense RNA to SRG3 mRNA in a thymoma cell line, S49.1, reduced the expression level of the SRG3 protein, and decreased the apoptotic cell death induced by glucocorticoids. These results suggest that the SRG3 protein is involved in the glucocorticoid-induced apoptosis in the thymoma cell line. This implicates that the SRG3 may play an important regulatory role during T cell development in thymus.

Show MeSH
Related in: MedlinePlus