Worm Breeder's Gazette 12(5): 75 (February 1, 1993)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have chosen the nematode C. elegans as a model system for investigating the signal transduction pathway of the p21 ras-related rho subfamily.
Using the coding region of the human rac1 cDNA as probe, a C. elegans mixed stage cDNA library (from Stratgene) was screened under low stringency conditions. Sequence analysis of the positive clones indicated that we have cloned the nematode rac1 ,designated as CE rac1 (Chen et al., J. Biol. Chem. 268: 320-324,1993). The 1.6 kb cDNA clone contains a coding region of 191 amino acids with 82 and 79% identity to human rac1 and rac2 proteins, respectively. In collaboration with Dr. Coulson et al. at the MRC, UK, the cDNA was mapped to a position on chromosome IV close to cha-1 .The presence of rac1 isoforms is suggested by our genomic rac clone (derived from C. elegans genomic library screening using the same human probe and displaying 85% nucleotide sequence identity compared with CE rac1 ),which is mapped to a different location on chromosome IV. The CE rac1 cDNA hybridizes to two mRNAs (1.7 and 0.9 kb), their expression is developmentally regulated being the highest level at the embryonic stage and decreasing dramatically during development. The glutathione-S-transferase/CE rac1 fusion protein (46 kDa) expressed in E. coli binds GTP and exhibits intrinsic GTPase activity. The GTPase activity is stimulated by human n-chimaerin, a brain specific GTPase-activating protein for p21 rac.
We are currently analyzing other positive cDNA clones derived from the rac1 screening. Preliminary results indicate that we have probably cloned other members of the nematode rho subfamily.
In addition, the nematode rho GDI ( cm19a 7)and bcr-like (CELZC21) cDNAs have been expressed in E. coli, and their specific activity towards the CE rac1 proteins and the other nematode p21s is being determined. The conserved biochemical activity of CE rac1 protein suggests that further characterization of these p21 s and their regulatory proteins using genetic analysis, e.g. dominant negative mutant, would be helpful in elucidating not only their role in the signal transduction, but also the biological function of their mammalian homologues.