Worm Breeder's Gazette 13(1): 80 (October 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.
Heterotrimeric G proteins (subunits alpha, ß, and gamma) transduce and amplify signals between receptors and intracellular effectors. The alpha subunit is a GTPase and regulates the 'on' or 'off' state of its signal transduction pathway. We have been studying the expression and function of four cloned G protein a subunit genes: goa-1 , gpa-1 , gpa-2 and gpa-3 .Cloning of these genes has been reported previously (Fino Silva and Plasterk, 1990, J. Mol. Biol. 215, 483-487; Lochrie et al., 1991, Cell Reg. 2,135-154). Here we report the apparent expression pattern of gpa-2 and gpa-3 and the phenotype of dominant mutations in these two genes.
Apparent expression patterns were determined using Andy Fire's lacZ fusion vectors, pPD21 .28and pPD22 .11.The upstream region and a portion of the coding region of each gene was inserted into the vector as a translational fusion. The fusion for gpa-3 includes 6 kb of 5' flanking DNA and terminates in the first exon; that for gpa-2 includes 2.4 kb of 5' flanking DNA and terminates in the third exon. Neither fusion contains its full complement of intron and flanking DNA, so the expression of the lacZ fusion may not be identical to that of the native protein. The usual caveats concerning levels and perdurance of expression should also be noted.
The lacZ fusion for gpa-3 is expressed in the neurons of the amphid and phasmid. Nine anterior and two posterior nuclei per side stain for ß galactosidase activity. Eight of the anterior nuclei show the characteristic positioning of those of the amphid; we will be confirming this assignment with cell ablation. In the absence of the nuclear localization signal, staining is seen anteriorly running through the amphid channel up to the amphid opening, and posteriorly through the phasmid channel. The apparent expression pattern for gpa-2 is more complex. A number of nuclei (about 20) in the nerve ring show staining. Additional staining is seen in PVT, the anal sphincter and the phasmid neurons. In the absence of the nuclear localization signal, staining is seen in three anteriorly directed neural processes that run near the amphid channel. These processes terminate prior to the tip of the nose, and do not appear to exit through the amphid pore as do those of gpa-3 .
We have constructed dominant mutations in gpa-3 and gpa-2 by substituting codons for leucine for those of glutamine in the conserved sequence DVGGQR (position 205 in gpa-3 ,207 in gpa-2 ).This alteration is equivalent to activating mutations in p21r asand in mammalian Gs-alpha and Gi-alpha (e. g. Landis, et al. 1989, Nature 340, 692-696; Wong et al., 1991, Nature 361, 63-65). The intrinsic GTPase activity of the alpha subunit is reduced by this mutation, presumably resulting in constitutive activation of its signal transduction pathway. The mutant genes were injected individually as genomic fragments containing the same extent of 5' flanking DNA as the lacZ fusions described above. The mutant constructs have their full complement of intron sequences as well as approximately 2 kb of 3' flanking DNA, so their expression patterns may not be identical to those of the lacZ fusions.
Transgenic animals carrying either the gpa-3 or gpa-2 dominant mutation become dauer larvae under non-inducing conditions. For both genes the penetrance of the dauer constitutive phenotype is low: 7% for our strongest gpa-3 (Q205L)line and 40% for our strongest gpa-2 (Q207L)line (at 25°). Dauer formation is enhanced at higher temperatures, and the dauers look like typical dauers and are SDS-resistant. gpa-3 Q205L-and gpa-2 Q207L-induceddauers are not identical: gpa-3 Q205L-induceddauers fail to recover at 16°, but recover slowly (24-48 hours) at 25°, while gpa-2 Q207L-induceddauers recover slowly (24-48 hours) at 16° and fail to recover at 25°. We are currently determining whether there is any synergy or epistasis between these two mutations, and will be determining their relationship to other genes in the dauer pathway.