The Heterotrimeric G Protein Genes of Caenorhabditis e/egans
15
markably, C. elegans has 20 ex subunits, of which 16 cannot be classified
in one of the mammalian classes. In this study we focus on the complete
family of ex subunit genes of the nematode.
Functional genomics should ideally follow closely in the tracks of
structural genomics, in that the same advantages of scale are sought for
gene function analysis as were found for DNA sequencing. In the case
of phenotypic analysis this is probably impossible. Even the description
of expression patterns is not so easily scaled up. Ideally one would need
to detect expressed proteins, but the generation of large series of wellidentified specific antibodies is a major undertaking, and in gene families there is ample chance of overlapping specificities. Therefore we
chose to determine a first approximation of the expression patterns by
gene fusion studies, realizing that these may not always identify the
definitive pattern. This approach, however, can be scaled up: PCR-amplified putative promoter regions of the G protein genes were fused in
frame to the GFP reporter gene (Chalfie et al. 1994; A Fire, J Ahnn, G
Seydoux, and S Xu, personal communication). These fusion constructs
were used to generate transgenic animals. This procedure allowed us to
determine in a short period of time the expression patterns for all Gex
genes.
Similarly, reverse genetic analysis by gene inactivation needed to be
scaled up. For this purpose we developed a method for target-selected
gene deletion after chemical mutagenesis (Jansen et al. 1997). The
principle of this method is as follows: animals are mutagenized by a
chemical that is known to generate deletions, and a collection of frozen
mutant lines is established. A corresponding set of (pooled) DNA samples is available for inspection by PCR. This inspection uses a selective
PCR with primer pairs that flank the gene of interest; deletions are
observed as PCR products that are more easily amplified than the larger
wild-type DNA. In some cases we also used a related method that
employs the Tel transposon as mutagen, which was developed by us
previously (Zwaal et al. 1993).
Finally, we analyzed the effect of gain-of-function mutations for
most genes by overproduction of the wild-type proteins in transgenic
animals (Mello et al. 1991). To minimize aspecific effects we always
used the endogenous promoter sequences to drive the expression of the
overexpression or dominant active constructs. Since G proteins are
molecular switches, one might expect that loss- and gain-of-function
15
markably, C. elegans has 20 ex subunits, of which 16 cannot be classified
in one of the mammalian classes. In this study we focus on the complete
family of ex subunit genes of the nematode.
Functional genomics should ideally follow closely in the tracks of
structural genomics, in that the same advantages of scale are sought for
gene function analysis as were found for DNA sequencing. In the case
of phenotypic analysis this is probably impossible. Even the description
of expression patterns is not so easily scaled up. Ideally one would need
to detect expressed proteins, but the generation of large series of wellidentified specific antibodies is a major undertaking, and in gene families there is ample chance of overlapping specificities. Therefore we
chose to determine a first approximation of the expression patterns by
gene fusion studies, realizing that these may not always identify the
definitive pattern. This approach, however, can be scaled up: PCR-amplified putative promoter regions of the G protein genes were fused in
frame to the GFP reporter gene (Chalfie et al. 1994; A Fire, J Ahnn, G
Seydoux, and S Xu, personal communication). These fusion constructs
were used to generate transgenic animals. This procedure allowed us to
determine in a short period of time the expression patterns for all Gex
genes.
Similarly, reverse genetic analysis by gene inactivation needed to be
scaled up. For this purpose we developed a method for target-selected
gene deletion after chemical mutagenesis (Jansen et al. 1997). The
principle of this method is as follows: animals are mutagenized by a
chemical that is known to generate deletions, and a collection of frozen
mutant lines is established. A corresponding set of (pooled) DNA samples is available for inspection by PCR. This inspection uses a selective
PCR with primer pairs that flank the gene of interest; deletions are
observed as PCR products that are more easily amplified than the larger
wild-type DNA. In some cases we also used a related method that
employs the Tel transposon as mutagen, which was developed by us
previously (Zwaal et al. 1993).
Finally, we analyzed the effect of gain-of-function mutations for
most genes by overproduction of the wild-type proteins in transgenic
animals (Mello et al. 1991). To minimize aspecific effects we always
used the endogenous promoter sequences to drive the expression of the
overexpression or dominant active constructs. Since G proteins are
molecular switches, one might expect that loss- and gain-of-function