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This system consists of an SV40 early gene promoter, which drives the expression of wild-type and mutant CDC42HS and Rac 1 ; this first cistron is followed by the internal ribosomal entry site of encephalomyocarditis virus [S. K. Jang, M. V. Davies, R. J. Kaufman, E. Wimmer, J. Virol. 63, 1651 (1989)] and the gene for a mutant form of GFP with increased fluorescence properties (GFPS65T) [R. Heim, A. B. Cubitt, R. Y. Tsien, Nature 373, 663 (1995)]. The two genes are transcribed as a single dicistronic mRNA and therefore both proteins are synthesized independently but in a coupled manner. The backbone of the expression vector was from pSG5 (Stratagene). The sources of the different forms of CDC42Hs and Rac1 were as follows: CDC42HsWT, CDC42HsN17, CDC42HsS188, and CDC42HsL61 were obtained as Bam HI-PVu II fragments of pGEX-2TKCDC42, pGEX-2TKCDC42N17, pZipneoCDC42S188, and pGEX-2TKCDC42L61, respectively [S. Bagrodia, B. Dérijard, R. J. Davis, R. A. Cerione, J. Biol. Chem. 270, 27995 (1995)]; Rad WT and Rac1V12 were obtained as 600 bp Eco RI fragments from pEXVRac1 and pEXVRac1Val12, respectively; and Rac1N17 was obtained by PCR from pGEX2TRac1 [A. J. Ridley, H. F. Paterson, C. L. Johnston, D. Diekmann, A. Hall, Cell 70, 401 (1992)].
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This system consists of an SV40 early gene promoter, which drives the expression of wild-type and mutant CDC42HS and Rac 1 ; this first cistron is followed by the internal ribosomal entry site of encephalomyocarditis virus [S. K. Jang, M. V. Davies, R. J. Kaufman, E. Wimmer, J. Virol. 63, 1651 (1989)] and the gene for a mutant form of GFP with increased fluorescence properties (GFPS65T) [R. Heim, A. B. Cubitt, R. Y. Tsien, Nature 373, 663 (1995)]. The two genes are transcribed as a single dicistronic mRNA and therefore both proteins are synthesized independently but in a coupled manner. The backbone of the expression vector was from pSG5 (Stratagene). The sources of the different forms of CDC42Hs and Rac1 were as follows: CDC42HsWT, CDC42HsN17, CDC42HsS188, and CDC42HsL61 were obtained as Bam HI-PVu II fragments of pGEX-2TKCDC42, pGEX-2TKCDC42N17, pZipneoCDC42S188, and pGEX-2TKCDC42L61, respectively [S. Bagrodia, B. Dérijard, R. J. Davis, R. A. Cerione, J. Biol. Chem. 270, 27995 (1995)]; Rad WT and Rac1V12 were obtained as 600 bp Eco RI fragments from pEXVRac1 and pEXVRac1Val12, respectively; and Rac1N17 was obtained by PCR from pGEX2TRac1 [A. J. Ridley, H. F. Paterson, C. L. Johnston, D. Diekmann, A. Hall, Cell 70, 401 (1992)].
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We thank A, Abo, R. Cerione, R. Davis, and A. Hall for plasmids and cell lines, J. Lipsick for useful discussion, and D. Bar-Sagi for critical review of this manuscript. Supported by NIH grant GM52543 to J.E.G., who is an investigator of the American Heart Association.
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