Worm Breeder's Gazette 15(5): 46 (February 1, 1999)
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.
The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Post-sequence genetics offers us the possibility to analyze complete gene families at once. We studied the family of heterotrimeric G proteins. The C. elegans genomic sequence was screened using previously identified G alpha, beta and gamma sequences. This identified 20 G protein alpha subunit genes: one clear homologue of each of the four mammalian classes Go/Gi, Gs, Gq, G12: goa-1, gsa-1, egl-30, and gpa-12, and sixteen new genes that cannot clearly be classified in one of the mammalian classes (gpa-1 to 16 and odr-3). There are 2 G beta genes, gpb-1 and 2. Inactivation of gpb-1 leads to embryonic lethality as a result of inappropriate orientation of the planes of cell division (Zwaal et al. 1996, Cell 86, 619-629). The second G beta subunit is not required for viability (abstract by F. Simmer et al.). Furthermore, we could identify 2 G gamma subunits, gpc-1 and 2. As these latter genes are short, and poorly conserved, additional G gamma subunit genes may remain to be found.
Six of the G alpha genes were previously studied: goa-1 (Ségalat et al. 1995, Science 267, 1648-1651; Mendel et al. 1995, Science 267, 1652-1655), gsa-1 (Park, J-H. et al. 1997, Gene 194, 183-190; Korswagen et al. 1997, Genes Dev. 11, 1493-1503), egl-30 (Brundage et al. 1996, Neuron 16, 999-1009), gpa-2 and 3 (Zwaal et al. 1997, Genetics 145, 715-727) and odr-3 (Roayaie et al. 1998, Neuron 20, 55-67). To functionally characterize the family of G alpha genes we determined the expression patterns of the remaining genes. While gpa-7 and the conserved G alpha subunits are expressed in many neurons and muscle cells, GFP fusions indicate that 14 of the new G alpha genes are expressed almost exclusively in a small subset of the amphid cells, sensory neurons in the head of the nematode, or other putative sensory neurons.
Next, we isolated null alleles, using target selected gene inactivation. Gain-of-function alleles were generated by introducing the wild type genes as transgenes (XS). None of the mutations generated in this study resulted in a lethal phenotype. gpa-7 animals were egg-laying defective and gpa-7XS animals were hyperactive regarding egg-laying, as measured by the number of eggs in utero. No significant difference could be observed in body wall muscle activity. These results indicate that gpa-7 possesses a stimulatory function in muscle cells and/or neurons, like gsa-1 and egl-30.
The specific expression of 14 of the 16 new G alpha genes in amphid neurons, suggests they might be involved in perception, or in the development of sensory neurons. Therefore all mutants were tested for chemotaxis to water soluble and volatile attractants and repellents, and dye-filling. Surprisingly we found only few phenotypes for loss-of-function alleles: gpa-3, 6 and odr-3 affect water soluble chemoattraction; gpa-3, 7 and odr-3 (Roayaie et al. 1998) affect avoidance; gpa-2, 5 and odr-3 (Roayaie et al. 1998) affect olfaction. The absence of phenotypes for most of the knock-out alleles suggests that there probably is functional redundancy. Given the expression patterns of multiple subunit genes per neuron this seems certainly possible. In contrast, 8 of the 10 gain-of-function alleles tested show phenotypes in these assays. Although we cannot extrapolate gene function from dominant mutants with any certainty, these phenotypes can provide hints about the involvement of G proteins in specific cellular functions.
Taken together, the expression patterns and phenotypes give a
consistent pattern of at least 4 G alpha genes regulating muscle
and neuron activity and at least 14 G alpha genes acting in perception.