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Cloning of theRhoB Gene from the Mouse Genome and Characterization of Its Promoter Region
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
226, 688–694 (1996)
1415
Cloning of the RhoB Gene from the Mouse Genome and Characterization of Its Promoter Region Takao Nakamura,* Midori Asano,* Nobuko Shindo-Okada,† Susumu Nishimura,* and Yoshiaki Monden*,1 *Banyu Tsukuba Research Institute in Collaboration with Merck Research Laboratories, Okubo 3, Tsukuba 300-26, Japan; and †Biology Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan Received August 16, 1996 Rho proteins have been implicated in a variety of cytoskeletal functions, but it is unclear how Rho proteins regulate these cellular functions and how Rho proteins are regulated. In this study, we cloned the rhoB gene from the mouse genome and characterized its promoter region. The predicted amino acid sequence was identical to that encoded by the human rhoB gene. A site for initiation of transcription was found at position 0376 relative to the site for initiation of translation. Deletion analysis of the 5*-flanking region of the rhoB gene revealed that the minimum region of the promoter was located between positions 0507 and 0376. Northern blotting analysis showed that the expression of the mouse rhoB gene was induced by serum, suggesting that expression of the rhoB gene might be controlled by some signal-responsive element(s). q 1996 Academic Press, Inc.
The Rho family is a member of the Ras-related superfamily of small GTP-binding proteins. The rho family itself consists of three subfamilies, rho, rac and cdc42. The rho subfamily has three closely related members, rhoA, rhoB and rhoC (1,2). Rho proteins have been implicated in the regulation of various actin-dependent cytoskeletal functions, such as control of cell morphology (3,4), formation of stress fibers and focal adhesions (4-6) and cell motility (7,8). Function of Rho has been shown to be associated with cellular transformation by Ras and overexpression of Rho induces apoptosis (9,10). We reported previously that azatyrosine, which can convert transformed cells to a normal phenotype, induces the expression of rhoB in c-Ha-ras-transformed cells (11). We also showed that stimulation of the expression of rhoB is involved in the biological action of azatyrosine (12). Thus, Rho proteins appear likely to function in both cellular transformation and apoptosis. Several enzymes appear to act downstream of Rho protein, namely, a genistein-sensitive kinase (13), phosphatidylinositol 4-phosphate 5-kinase (14) and phosphatidylinositol 3-kinase (15,16), and protein kinase N (PKN) and a PKN-related protein, rhophilin, have been identified as direct downstream targets of Rho protein (17,18). However, the way in which Rho proteins regulate cellular functions and the way in which Rho protein is regulated remain unclear. We have performed a detailed analysis of the promoter region of rhoB in an effort to increase our understanding of the mechanism of action of the rhoB gene. MATERIALS AND METHODS Isolation of the mouse rhoB gene. A genomic library of mouse fibroblast NIH 3T3 cells in the phage l FixII (Stratagene, La Jolla, CA, USA) was screened with complementary DNA (cDNA) for the human rhoB gene as the probe. The cDNA was kindly provided by Dr. P. Madaule (Dept. of Pharmacology, Kyoto University). Oligonucleotide
1
To whom correspondence should be addressed. Fax.: 81-298-77-2027. 688
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primers for sequencing were synthesized at approximately 300-bp intervals with a DNA synthesizer (Applied Biosystems, Inc., Foster City, CA, USA). DNA sequencing was performed with the Sequenase version 2 kit (Amersham, Little Chalfont, Buckinghamshire, U.K.) and the Taq cycle sequencing kit (Takara, Kyoto, Japan). The DNA sequence was scanned for putative binding sites for transcription factors using a list of consensus sequences for transcriptionregulatory elements (19). Construction of promoter-luciferase plasmids. A 4-kbp fragment (positions 03952 to /85) was subcloned into the BglII site of a plasmid vector that included a reporter gene for luciferase, pGL2-E (Promega, Madison, WI, USA). Deletions in the rhoB promotor region were generated with a Kilo-Sequence Deletion kit (Takara) or by taking advantage of SmaI, PstI, HincII, HindIII, StuI and ScaI sites. Transient transfection and assay of luciferase activity. Cells were transfected with plasmids using the lipofectoamine reagent (Gibco BRL, Gaithersburg, MD, USA). NIH 3T3 cells were seeded in 35-mm dishes at a density of 1.51105 cells per dish. The cells were washed with serum-free Dulbecco’s modified Eagle’s medium (DMEM) and incubated with 2 mg of DNA-liposome complexes for 3 h at 37 7C. Then they were cultured in DMEM that contained 5% calf serum (CS) for 24 h. Luciferase activity was determined by incubating 25 ml of a cell lysate with 175 ml of luciferase reaction buffer (25 mM glycylglycine, pH 7.8, 15 mM MgSO4 , 4 mM EGTA, 15 mM KH2PO4 , 1 mM dithiothreitol, 2 mM ATP) and it was measured after automated injection of 100 ml of 0.5 mM luciferin with a luminometer (Autolumat LB953; Berthold, Wildbad, Germany). For normalization of the efficiency of transfection, 1 mg of a lacZexpression plasmid was cotransfected and b-galactosidase activity was measured with the Luminescent b-Galactosidase Genetic Reporter System (Clontech, Palo Alto, CA, USA) S1 nuclease mapping. Total cellular RNA was extracted from NIH 3T3 cells as described by Chomczynski and Sacchi (20). A 459-base SacI fragment (positions 0464 to 06) or a 125-base chemically synthesized polynucleotide (positions 0406 to 0282) was used as the probe. The 5*-end 32P-labeled probe and 20 mg of RNA were incubated at 80 7C for 10 min and then allowed to hybridize in 20 ml of hybridization buffer (80 % formamide, 0.4 M NaCl, 1 mM EDTA, 50 mM Pipes, pH 6.4). Digestion with S1 nuclease was performed as described previously (21). Samples were separated by electrophoresis on a 6% polyacrylamide gel. The dried gel was subjected to autoradiography. Northern blot analysis. Total RNA prepared from NIH 3T3 cells was fractionated on a 1% agarose-formaldehyde gels and transferred to a nylon membrane (Hybond-N/; Amersham). Hybridization was performed with randomly primed 32P-labeled probes in Rapid hybridization buffer (Amersham) at 65 7C. The membrane was then washed sequentially with 21 standard saline citrate (SSC) plus 0.1% sodium dodecyl sulfate (SDS) at room temperature, 11 SSC plus 0.1% SDS at 65 7C and 0.11 SSC plus 0.1% SDS at 65 7C, with subsequent autoradiography. A 2.8-kb HindIII fragment containing the coding region of the mouse rhoB gene (Fig. 1) and a 1.0-kb fragment of cDNA for human glyceraldehyde-3-phosphate dehydrogenase (G3PDH; Clontech) were used as probes.
RESULTS AND DISCUSSION
Isolation of mouse rhoB genomic clones. To isolate the rhoB gene from the mouse genome, we screened a mouse fibroblast genomic library using cDNA for the human rhoB gene as the probe. Ten positive phages were obtained from 8.4 1 105 phages. Restriction enzyme mapping of these phages showed that nine of the phages could be classified as representatives of four clones (Fig. 1) with extensive overlap. For DNA sequence analysis, we subcloned EcoRI fragments from partially digested clone I and clone IV into pUC119 and determined the DNA sequences of a 4.6-kbp region (Fig. 1). Part of the DNA sequence (3 kbp) is shown in Figure 2. Comparison of the sequence with that of the cDNA for human rhoB revealed an open reading frame of 588 bp, uninterrupted by any introns, which was 94.9 % homologous to the sequence of the human cDNA. The predicted amino acid sequence of the protein encoded by mouse rhoB was identical to that encoded by the human rhoB gene. The complete conservation of amino acid sequences between the human and mouse proteins suggests that the RhoB protein has an important function in mammalian cells and that its function is strictly regulated. Identification of the site for initiation of transcription. The site for initiation of transcription in the mouse rhoB gene was mapped by S1 nuclease analysis. The 5*-end-labeled SacI fragment (positions 0406 to 06) was allowed to hybridize with total RNA from NIH 3T3 cells and then digested with S1 nuclease. For size markers, products of dideoxy-sequencing reaction were subjected to polyacrylamide gel electrophoresis with products of the digestion by S1 nuclease. One protected fragment was observed (Fig. 3A), starting at approximately 0375 relative to the site of initiation of translation. To identify the exact initiation site, we prepared 689
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FIG. 1. Restriction map of mouse genomic DNA that includes the rhoB gene. A white arrow represents the predicted coding region of the mouse rhoB gene and the direction of its translation. Restriction sites are abbreviated as follows: Hind III, H; EcoR I, E; and BamH I, B. The four bars under the restriction map indicate regions of the rhoB gene in the genomic clones. The DNA sequence of the region indicated by the double-headed arrow was determined in this study. Scale bar, 1 kbp.
a 125-base fragment (positions 0406 to 0282). The S1 mapping analysis with this fragment yielded a major product that started at 0376 (Fig. 3B). The results suggest that a major site of initiation of transcription of the mouse rhoB gene is located at position 0376. There were two less intense bands that represented fragments starting at positions 0377 and 0375, which might represent alternative initiation sites or artifactual products of digestion by S1 nuclease. The sequence upstream of the site of initiation of transcription includes two TATA-like boxes (approximately 30 bp and 50 bp upstream, respectively). Identification of the promoter region of the mouse rhoB gene. To identify the promoter region of the mouse rhoB gene, we subcloned a 4-kbp fragment (03952 to /85) that contained the site for initiation of transcription and that for the initiation of translation into the luciferase reporter plasmid (pGL2-E). We designated the product pGE-I. This construct or the control plasmid pGL2-C, which expressed the reporter gene for luciferase under the control of an SV40 promoter, was used to transiently transfect NIH 3T3 cells. The lysate of cells transfected with pGE-I had obvious luciferase activity, as compared with lysates of cells transfected with pGL2-C (Fig. 4), indicating that the promoter region of the mouse rhoB gene was located within the 4-kbp fragment. The minimum promoter region was analyzed with deletion constructs derived from pGE-I (Fig. 4). The pGE-X construct yielded little luciferase activity, resembling the promoterless plasmid (pGL2-E). By contrast, the pGE-IX construct, which contained 128 bp of the region upstream of the site of initiation of transcription yielded obvious activity. Thus, the promoter region of the mouse rhoB gene appeared to be included in this 128-bp fragment. Cells transfected with pGE-VII had the greatest activity of all of the transfected cells. The activity of cells transfected with pGE-VI was 60% of that of cells transfected with pGE-VII. The addition of 506 bp in pGE-V resulted in a further increase in activity. Moreover, the promoter activities of the longer constructs decreased as the 5*-flanking region was extended. The transcription of the mouse rhoB gene might thus be controlled by some regulatory elements located upstream of the minimum promoter. Induction of expression of the rhoB gene by serum. To examine the expression of the rhoB gene in mouse cells, we caused confluent NIH3T3 cells to become quiescent by serum starvation and then treated them with 5% calf serum. Northern blotting revealed that the level of expression of the rhoB gene in quiescent NIH 3T3 cells was quite low but it increased dramatically 690
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FIG. 2. Part of sequence of the 5*-flanking region and the coding region of the mouse rhoB gene. Nucleotides are numbered relative to the site of initiation of translation, as indicated by numbers on the left. The amino acid (a. a.) sequence deduced from the coding region is given in the single-letter code with positions on the right. The major predicted site for initiation of transcription is indicated by an arrow. TATA-like boxes and predicted consensus sequences for binding of transcription factors are underlined and labelled. Accession number (EMBL): X99963.
30 min after addition of serum (Fig. 5). This result indicates that the mouse rhoB gene is an ‘‘early-response’’ gene. The DNA sequence of the 5*-flanking region that we determined in this study does not include a serum-responsive element or an AP-1-binding element but it does include AP-2-binding elements. The expression of rhoB that was induced by serum might be controlled by AP-2 and/or some unidentified transcription-regulatory factor(s). The difference in functional properties between members of the rho subfamily is unclear. However, the actin-dependent cytoskeletal functions that are regulated by Rho proteins are closely associated with the presence of serum or certain growth factors in the environment of the cells (22). The expression of the mouse rhoB gene was induced by serum (Fig. 5). It has been reported that expression of the rat rhoB gene is induced by serum, by epidermal growth 691
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FIG. 3. S1 mapping analysis of the mouse rhoB gene using the 459-base Sac I fragment (panel A) and the 125base fragment (panel B) as probes. The mRNA from NIH 3T3 cells was hybridized with each probe and treated with (lane 2) or without (lane 1) S1 nuclease. DNA sequencing ladders (G, A, T and C ) for the 5*-flanking region of the mouse rhoB gene obtained with 026 to 06 (A) and 0298 to 0282 (B) oligonucleotides as sequencing primers are shown. The shaded arrows indicate the protected products. The solid arrows indicate the no-digested probes.
factor and by platelet-derived growth factor, while that of rhoA and rhoC is not (23). It seems likely that rhoB might be the most important member of the rho subfamily with respect to many kinds of cellular function. Further analysis of the regulation of expression of rhoB and
FIG. 4. Deletion analysis of the promoter of the mouse rhoB gene. Fragments of the 5*-flanking region of the rhoB gene included in deletion constructs are indicated on the left. Relative promoter activities in cells transfected with deletion constructs are shown on the right as percentages of that in cells transfected with the pGE-I plasmid. A plasmid with a gene for b-galactosidase was used as an internal control in all experiments to allow normalization of the variations in transfection efficiency. The results are averages { SD of results from three experiments. 692
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FIG. 5. Northern blot analysis of mouse rhoB mRNA from quiescent NIH 3T3 cells that had been incubated with or without serum. (A) Confluent NIH 3T3 cells were rendered quiescent by incubation in medium that contained 0.5% calf serum (CS) for 48 h and then they were supplied with medium that contained 0.5% CS (lanes 1 and 2) or 5% CS (lanes 3 and 4). The expression of the mouse rhoB gene was analyzed with the mouse rhoB probe. (B) mRNA for G3PDH was probed as a control to monitor loading of RNA. Positions of markers, with sizes in kb, are indicated on the left.
its function in the actin-dependent formation of the cytoskeleton, as well as in transformation and apoptosis, seems to be worthwhile. ACKNOWLEDGMENTS The authors thank Dr. Pascal Madaule for providing the cDNA for rhoB and Dr. Yoshimi Takai of the Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, and Drs. Eisaku Yoshida and Hideaki Higashi of the Banyu Tsukuba Research Institute for helpful discussions.
REFERENCES 1. Hall, A. (1990) Science 249, 653–640. 2. Takai, Y., Kaibuchi, K., Kikuchi, A., and Kawata, M. (1992) Int. Rev. Cytol. 133, 187–230. 3. Chardin, P., Boquet, P., Madaule, P., Popoff, M. R., Rubin, E. J., and Gill, D. M. (1989) EMBO J. 8, 1087– 1092. 4. Ridley, A. J., and Hall, A. (1992) Cell 70, 387–399. 5. Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K., and Hall, A. (1990) J. Cell. Biol. 111, 1001– 1007. 6. Miura, Y., Kikuchi, A., Musha, T., Kuroda, S., Yaku, H., Sasaki, T., and Takai, Y. (1993) J. Biol. Chem. 268, 510–515. 7. Takaishi, K., Kikuchi, A., Kuroda, S., Kotani, K., Sasaki, T., and Takai, Y. (1993) Mol. Cell. Biol. 13, 72–79. 8. Aepfelbacher, M., Vauti, F., Weber, P. C., and Glomset, J. A. (1994) Proc. Natl. Acad. Sci. USA 91, 4263–4267. 9. Qiu, R-G., Chen, J., McCormick, F., and Symons, M. (1995) Proc. Natl. Acad. Sci. USA 92, 11781–11785. 10. Jime´nez, B., Arends, M., Esteve, P., Perona, R., Sa´nchez, R., Cajal, S. R., Wyllie, A., and Lacal, J. C. (1995) Oncogene 10, 811–816. 11. Shindo-Okada, N., Makabe, O., Nagahara, H., and Nishimura, S. (1989) Mol. Carcinogenesis. 2, 159–167. 12. Monden, Y., Nakamura, T., Kojiri, K., Shindo-Okada, N., and Nishimura, S. (1996) Oncol. Rep. 3, 49–55. 693
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13. Ridley, A. J., and Hall, A. (1994) EMBO J. 13, 2600–2610. 14. Chong, L. D., Traymor-Kaplan, A., Bokoch, G. M., and Schwartz, M. A. (1994) Cell 79, 507–513. 15. Zhang, J., King, W. G., Dillon, S., Hall, A., Feig, L., and Rittenhouse, S. E. (1993) J. Biol. Chem. 268, 22251– 22254. 16. Kumagai, N., Morii, N., Fujisawa, K., Nemoto, Y., and Narumiya, S. (1993) J. Biol. Chem. 268, 24535–24538. 17. Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648–650. 18. Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645–648. 19. Locker, J. (1993) in Gene Transcription (Hames, B. D., and Higgins, S. J., Eds.), pp. 321–339, Oxford Univ. Press, New York. 20. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159. 21. Mason, P. J., Enver, T., Wilkinson, D., and Williams, J. G. (1993) in Gene Transcription (Hames, B. D., and Higgins, S. J., Eds.), pp. 22–38, Oxford Univ. Press, New York. 22. Nobes, C. D., and Hall, A. (1995) Cell 81, 53–62. 23. Ja¨hner, D., and Hunter, T. (1991) Mol. Cell. Biol. 11, 3682–3690.
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226, 688–694 (1996)
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Cloning of the RhoB Gene from the Mouse Genome and Characterization of Its Promoter Region Takao Nakamura,* Midori Asano,* Nobuko Shindo-Okada,† Susumu Nishimura,* and Yoshiaki Monden*,1 *Banyu Tsukuba Research Institute in Collaboration with Merck Research Laboratories, Okubo 3, Tsukuba 300-26, Japan; and †Biology Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan Received August 16, 1996 Rho proteins have been implicated in a variety of cytoskeletal functions, but it is unclear how Rho proteins regulate these cellular functions and how Rho proteins are regulated. In this study, we cloned the rhoB gene from the mouse genome and characterized its promoter region. The predicted amino acid sequence was identical to that encoded by the human rhoB gene. A site for initiation of transcription was found at position 0376 relative to the site for initiation of translation. Deletion analysis of the 5*-flanking region of the rhoB gene revealed that the minimum region of the promoter was located between positions 0507 and 0376. Northern blotting analysis showed that the expression of the mouse rhoB gene was induced by serum, suggesting that expression of the rhoB gene might be controlled by some signal-responsive element(s). q 1996 Academic Press, Inc.
The Rho family is a member of the Ras-related superfamily of small GTP-binding proteins. The rho family itself consists of three subfamilies, rho, rac and cdc42. The rho subfamily has three closely related members, rhoA, rhoB and rhoC (1,2). Rho proteins have been implicated in the regulation of various actin-dependent cytoskeletal functions, such as control of cell morphology (3,4), formation of stress fibers and focal adhesions (4-6) and cell motility (7,8). Function of Rho has been shown to be associated with cellular transformation by Ras and overexpression of Rho induces apoptosis (9,10). We reported previously that azatyrosine, which can convert transformed cells to a normal phenotype, induces the expression of rhoB in c-Ha-ras-transformed cells (11). We also showed that stimulation of the expression of rhoB is involved in the biological action of azatyrosine (12). Thus, Rho proteins appear likely to function in both cellular transformation and apoptosis. Several enzymes appear to act downstream of Rho protein, namely, a genistein-sensitive kinase (13), phosphatidylinositol 4-phosphate 5-kinase (14) and phosphatidylinositol 3-kinase (15,16), and protein kinase N (PKN) and a PKN-related protein, rhophilin, have been identified as direct downstream targets of Rho protein (17,18). However, the way in which Rho proteins regulate cellular functions and the way in which Rho protein is regulated remain unclear. We have performed a detailed analysis of the promoter region of rhoB in an effort to increase our understanding of the mechanism of action of the rhoB gene. MATERIALS AND METHODS Isolation of the mouse rhoB gene. A genomic library of mouse fibroblast NIH 3T3 cells in the phage l FixII (Stratagene, La Jolla, CA, USA) was screened with complementary DNA (cDNA) for the human rhoB gene as the probe. The cDNA was kindly provided by Dr. P. Madaule (Dept. of Pharmacology, Kyoto University). Oligonucleotide
1
To whom correspondence should be addressed. Fax.: 81-298-77-2027. 688
0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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primers for sequencing were synthesized at approximately 300-bp intervals with a DNA synthesizer (Applied Biosystems, Inc., Foster City, CA, USA). DNA sequencing was performed with the Sequenase version 2 kit (Amersham, Little Chalfont, Buckinghamshire, U.K.) and the Taq cycle sequencing kit (Takara, Kyoto, Japan). The DNA sequence was scanned for putative binding sites for transcription factors using a list of consensus sequences for transcriptionregulatory elements (19). Construction of promoter-luciferase plasmids. A 4-kbp fragment (positions 03952 to /85) was subcloned into the BglII site of a plasmid vector that included a reporter gene for luciferase, pGL2-E (Promega, Madison, WI, USA). Deletions in the rhoB promotor region were generated with a Kilo-Sequence Deletion kit (Takara) or by taking advantage of SmaI, PstI, HincII, HindIII, StuI and ScaI sites. Transient transfection and assay of luciferase activity. Cells were transfected with plasmids using the lipofectoamine reagent (Gibco BRL, Gaithersburg, MD, USA). NIH 3T3 cells were seeded in 35-mm dishes at a density of 1.51105 cells per dish. The cells were washed with serum-free Dulbecco’s modified Eagle’s medium (DMEM) and incubated with 2 mg of DNA-liposome complexes for 3 h at 37 7C. Then they were cultured in DMEM that contained 5% calf serum (CS) for 24 h. Luciferase activity was determined by incubating 25 ml of a cell lysate with 175 ml of luciferase reaction buffer (25 mM glycylglycine, pH 7.8, 15 mM MgSO4 , 4 mM EGTA, 15 mM KH2PO4 , 1 mM dithiothreitol, 2 mM ATP) and it was measured after automated injection of 100 ml of 0.5 mM luciferin with a luminometer (Autolumat LB953; Berthold, Wildbad, Germany). For normalization of the efficiency of transfection, 1 mg of a lacZexpression plasmid was cotransfected and b-galactosidase activity was measured with the Luminescent b-Galactosidase Genetic Reporter System (Clontech, Palo Alto, CA, USA) S1 nuclease mapping. Total cellular RNA was extracted from NIH 3T3 cells as described by Chomczynski and Sacchi (20). A 459-base SacI fragment (positions 0464 to 06) or a 125-base chemically synthesized polynucleotide (positions 0406 to 0282) was used as the probe. The 5*-end 32P-labeled probe and 20 mg of RNA were incubated at 80 7C for 10 min and then allowed to hybridize in 20 ml of hybridization buffer (80 % formamide, 0.4 M NaCl, 1 mM EDTA, 50 mM Pipes, pH 6.4). Digestion with S1 nuclease was performed as described previously (21). Samples were separated by electrophoresis on a 6% polyacrylamide gel. The dried gel was subjected to autoradiography. Northern blot analysis. Total RNA prepared from NIH 3T3 cells was fractionated on a 1% agarose-formaldehyde gels and transferred to a nylon membrane (Hybond-N/; Amersham). Hybridization was performed with randomly primed 32P-labeled probes in Rapid hybridization buffer (Amersham) at 65 7C. The membrane was then washed sequentially with 21 standard saline citrate (SSC) plus 0.1% sodium dodecyl sulfate (SDS) at room temperature, 11 SSC plus 0.1% SDS at 65 7C and 0.11 SSC plus 0.1% SDS at 65 7C, with subsequent autoradiography. A 2.8-kb HindIII fragment containing the coding region of the mouse rhoB gene (Fig. 1) and a 1.0-kb fragment of cDNA for human glyceraldehyde-3-phosphate dehydrogenase (G3PDH; Clontech) were used as probes.
RESULTS AND DISCUSSION
Isolation of mouse rhoB genomic clones. To isolate the rhoB gene from the mouse genome, we screened a mouse fibroblast genomic library using cDNA for the human rhoB gene as the probe. Ten positive phages were obtained from 8.4 1 105 phages. Restriction enzyme mapping of these phages showed that nine of the phages could be classified as representatives of four clones (Fig. 1) with extensive overlap. For DNA sequence analysis, we subcloned EcoRI fragments from partially digested clone I and clone IV into pUC119 and determined the DNA sequences of a 4.6-kbp region (Fig. 1). Part of the DNA sequence (3 kbp) is shown in Figure 2. Comparison of the sequence with that of the cDNA for human rhoB revealed an open reading frame of 588 bp, uninterrupted by any introns, which was 94.9 % homologous to the sequence of the human cDNA. The predicted amino acid sequence of the protein encoded by mouse rhoB was identical to that encoded by the human rhoB gene. The complete conservation of amino acid sequences between the human and mouse proteins suggests that the RhoB protein has an important function in mammalian cells and that its function is strictly regulated. Identification of the site for initiation of transcription. The site for initiation of transcription in the mouse rhoB gene was mapped by S1 nuclease analysis. The 5*-end-labeled SacI fragment (positions 0406 to 06) was allowed to hybridize with total RNA from NIH 3T3 cells and then digested with S1 nuclease. For size markers, products of dideoxy-sequencing reaction were subjected to polyacrylamide gel electrophoresis with products of the digestion by S1 nuclease. One protected fragment was observed (Fig. 3A), starting at approximately 0375 relative to the site of initiation of translation. To identify the exact initiation site, we prepared 689
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FIG. 1. Restriction map of mouse genomic DNA that includes the rhoB gene. A white arrow represents the predicted coding region of the mouse rhoB gene and the direction of its translation. Restriction sites are abbreviated as follows: Hind III, H; EcoR I, E; and BamH I, B. The four bars under the restriction map indicate regions of the rhoB gene in the genomic clones. The DNA sequence of the region indicated by the double-headed arrow was determined in this study. Scale bar, 1 kbp.
a 125-base fragment (positions 0406 to 0282). The S1 mapping analysis with this fragment yielded a major product that started at 0376 (Fig. 3B). The results suggest that a major site of initiation of transcription of the mouse rhoB gene is located at position 0376. There were two less intense bands that represented fragments starting at positions 0377 and 0375, which might represent alternative initiation sites or artifactual products of digestion by S1 nuclease. The sequence upstream of the site of initiation of transcription includes two TATA-like boxes (approximately 30 bp and 50 bp upstream, respectively). Identification of the promoter region of the mouse rhoB gene. To identify the promoter region of the mouse rhoB gene, we subcloned a 4-kbp fragment (03952 to /85) that contained the site for initiation of transcription and that for the initiation of translation into the luciferase reporter plasmid (pGL2-E). We designated the product pGE-I. This construct or the control plasmid pGL2-C, which expressed the reporter gene for luciferase under the control of an SV40 promoter, was used to transiently transfect NIH 3T3 cells. The lysate of cells transfected with pGE-I had obvious luciferase activity, as compared with lysates of cells transfected with pGL2-C (Fig. 4), indicating that the promoter region of the mouse rhoB gene was located within the 4-kbp fragment. The minimum promoter region was analyzed with deletion constructs derived from pGE-I (Fig. 4). The pGE-X construct yielded little luciferase activity, resembling the promoterless plasmid (pGL2-E). By contrast, the pGE-IX construct, which contained 128 bp of the region upstream of the site of initiation of transcription yielded obvious activity. Thus, the promoter region of the mouse rhoB gene appeared to be included in this 128-bp fragment. Cells transfected with pGE-VII had the greatest activity of all of the transfected cells. The activity of cells transfected with pGE-VI was 60% of that of cells transfected with pGE-VII. The addition of 506 bp in pGE-V resulted in a further increase in activity. Moreover, the promoter activities of the longer constructs decreased as the 5*-flanking region was extended. The transcription of the mouse rhoB gene might thus be controlled by some regulatory elements located upstream of the minimum promoter. Induction of expression of the rhoB gene by serum. To examine the expression of the rhoB gene in mouse cells, we caused confluent NIH3T3 cells to become quiescent by serum starvation and then treated them with 5% calf serum. Northern blotting revealed that the level of expression of the rhoB gene in quiescent NIH 3T3 cells was quite low but it increased dramatically 690
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FIG. 2. Part of sequence of the 5*-flanking region and the coding region of the mouse rhoB gene. Nucleotides are numbered relative to the site of initiation of translation, as indicated by numbers on the left. The amino acid (a. a.) sequence deduced from the coding region is given in the single-letter code with positions on the right. The major predicted site for initiation of transcription is indicated by an arrow. TATA-like boxes and predicted consensus sequences for binding of transcription factors are underlined and labelled. Accession number (EMBL): X99963.
30 min after addition of serum (Fig. 5). This result indicates that the mouse rhoB gene is an ‘‘early-response’’ gene. The DNA sequence of the 5*-flanking region that we determined in this study does not include a serum-responsive element or an AP-1-binding element but it does include AP-2-binding elements. The expression of rhoB that was induced by serum might be controlled by AP-2 and/or some unidentified transcription-regulatory factor(s). The difference in functional properties between members of the rho subfamily is unclear. However, the actin-dependent cytoskeletal functions that are regulated by Rho proteins are closely associated with the presence of serum or certain growth factors in the environment of the cells (22). The expression of the mouse rhoB gene was induced by serum (Fig. 5). It has been reported that expression of the rat rhoB gene is induced by serum, by epidermal growth 691
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FIG. 3. S1 mapping analysis of the mouse rhoB gene using the 459-base Sac I fragment (panel A) and the 125base fragment (panel B) as probes. The mRNA from NIH 3T3 cells was hybridized with each probe and treated with (lane 2) or without (lane 1) S1 nuclease. DNA sequencing ladders (G, A, T and C ) for the 5*-flanking region of the mouse rhoB gene obtained with 026 to 06 (A) and 0298 to 0282 (B) oligonucleotides as sequencing primers are shown. The shaded arrows indicate the protected products. The solid arrows indicate the no-digested probes.
factor and by platelet-derived growth factor, while that of rhoA and rhoC is not (23). It seems likely that rhoB might be the most important member of the rho subfamily with respect to many kinds of cellular function. Further analysis of the regulation of expression of rhoB and
FIG. 4. Deletion analysis of the promoter of the mouse rhoB gene. Fragments of the 5*-flanking region of the rhoB gene included in deletion constructs are indicated on the left. Relative promoter activities in cells transfected with deletion constructs are shown on the right as percentages of that in cells transfected with the pGE-I plasmid. A plasmid with a gene for b-galactosidase was used as an internal control in all experiments to allow normalization of the variations in transfection efficiency. The results are averages { SD of results from three experiments. 692
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FIG. 5. Northern blot analysis of mouse rhoB mRNA from quiescent NIH 3T3 cells that had been incubated with or without serum. (A) Confluent NIH 3T3 cells were rendered quiescent by incubation in medium that contained 0.5% calf serum (CS) for 48 h and then they were supplied with medium that contained 0.5% CS (lanes 1 and 2) or 5% CS (lanes 3 and 4). The expression of the mouse rhoB gene was analyzed with the mouse rhoB probe. (B) mRNA for G3PDH was probed as a control to monitor loading of RNA. Positions of markers, with sizes in kb, are indicated on the left.
its function in the actin-dependent formation of the cytoskeleton, as well as in transformation and apoptosis, seems to be worthwhile. ACKNOWLEDGMENTS The authors thank Dr. Pascal Madaule for providing the cDNA for rhoB and Dr. Yoshimi Takai of the Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, and Drs. Eisaku Yoshida and Hideaki Higashi of the Banyu Tsukuba Research Institute for helpful discussions.
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13. Ridley, A. J., and Hall, A. (1994) EMBO J. 13, 2600–2610. 14. Chong, L. D., Traymor-Kaplan, A., Bokoch, G. M., and Schwartz, M. A. (1994) Cell 79, 507–513. 15. Zhang, J., King, W. G., Dillon, S., Hall, A., Feig, L., and Rittenhouse, S. E. (1993) J. Biol. Chem. 268, 22251– 22254. 16. Kumagai, N., Morii, N., Fujisawa, K., Nemoto, Y., and Narumiya, S. (1993) J. Biol. Chem. 268, 24535–24538. 17. Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648–650. 18. Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645–648. 19. Locker, J. (1993) in Gene Transcription (Hames, B. D., and Higgins, S. J., Eds.), pp. 321–339, Oxford Univ. Press, New York. 20. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159. 21. Mason, P. J., Enver, T., Wilkinson, D., and Williams, J. G. (1993) in Gene Transcription (Hames, B. D., and Higgins, S. J., Eds.), pp. 22–38, Oxford Univ. Press, New York. 22. Nobes, C. D., and Hall, A. (1995) Cell 81, 53–62. 23. Ja¨hner, D., and Hunter, T. (1991) Mol. Cell. Biol. 11, 3682–3690.
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