Worm Breeder's Gazette 12(1): 24 (September 1, 1991)

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.

Tc1 insertions in the gpa-genes

Richard Zwaal[1], Alice Rushforth[2], Phil Anderson[2], Ronald Plasterk[1]

[1]Netherlands Cancer Institute, Amsterdam, Holland
[2]University of Wisconsin, Madison, WI

Guanine nucleotide binding proteins (G-proteins) play a role in a variety of transmembrane signal transduction pathways. They consist of 3 subunits, the alpha-, ß-, and gamma-subunit, of which the alpha-subunit contains the guanine nucleotide binding site, determining the activity or inactivity of the protein. The variability in structure of the alpha-subunits seems to cause the variability in G-protein function. For much of the Galpha-subunits however the specific signal transduction pathway they participate in is not known, nor is the receptor- or effector molecule they interact with.

In C. elegans a number of alpha-subunit genes has been discovered. The genomic sequence of novel, possibly C. elegans specific genes, gpa-1 , gpa-2 and gpa-3 ,is known, as well as cDNA sequences of goa-1 ,gq-1 and gsa-1 ,genes homologous to resp. mammalian Goalpha, Gqalpha and GSalpha (da Silva and Plasterk, JMB 215, 483-487 (1990); Lochrie et al., Cell Regulations 2, 135-154 (1991); Mendel and Sternberg pers. comm.; Oshima, pers. comm.).

To investigate the functions of the different alpha-subunits we are trying to obtain Tc1 knock-out mutants in the C. elegans specific gpa-genes, using the PCR technique to screen populations of worms for Tc1 insertions, followed by sib-selection to isolate the mutant animal (as described by Rushforth et al., WBG 11/5, 65).

Via this method, starting with approximately 25,000 animals of the MT3126 strain, a worm was obtained in which a Tc1 element is inserted in the gpa-2 gene between the translation stop codon and the first putative poly-adenylation site. The animal seems to have a wild-type phenotype although RNA levels are not checked yet to see if it really is a knock-out.

In the same screen a second Tc1 insertion was found in the gpa-2 gene in an intron and one was found in the gpa-1 gene. Both of these are inheritable and are now being purified.

A general conclusion can be that the method of PCR detection and sib-selection of Tc1 insertions seems widely applicable. We found inheritable insertions in both genes we looked at, at a frequency that makes purification of the mutant relatively easy. Elaborating this approach we hope to be able to systematically study the role of different G-proteins in C. elegans development.