The Heterotrimeric G Protein Genes of Caenorhabditis e/egans
29
2.3.2 GFP Fusion Constructs
Routinely, a 3 kbp PCR fragment, containing approximately 2.5-3 kbp
of upstream sequences and the first 30--50 codons of the ex genes, was
fused in frame to the reporter gene GFP (vector pPD95.77; A. Fire, 1.
Ahnn, G. Seydoux, and S. Xu, personal communication). In most cases
the first intron was present. In this way fusion constructs were generated
for gpa-4, -6, -7, -8, -9, -10, -11, -13, -14, -15 and, -16. Promoter fusions
for gpa-1 and gpa-5 were generated by subcloning relevant regions from
cosmid clones. The gpa-1::GFP fusion construct contains 1.5 kbp of
upstream sequences, and the first eight exons of gpa-1. The gpa-5::GFP
construct contains 3.7 kbp upstream sequences and the first five exons
of gpa-5 (Jansen et al. 1999).
At least two independent transgenic lines were generated from at
least two independent clones of each of the gpa: :GFP fusion constructs,
to control for PCR induced sequence errors. Except for variation in
expression level, all transgenic lines generated for each of the Gex
subunits showed the same expression pattern. Subsequently, the transgenic array was integrated for one of the lines of each of the fusion
constructs. Particular cells were identified by using a combination of
their position and morphology (White et al. 1986). Expression of all
gpa::GFP fusions was observed in animals from the embryonic threefold stage onwards, or from the moment a postembryonic cell was born
(data not shown). Expression in males was examined for all GFP fusion
constructs except gpa-16: :GFP.
2.3.3 Isolation of Loss-of-Function Alleles
Deletion mutations of gpa-1, gpa-5, and gpa-8 were isolated as described: gpa-1(pk15), gpa-5(pk376), and gpa-8(pk345) (Zwaal et al.
1993). Deletion alleles of gpa-4, -6, -7, -9, -10, -11, -14, -15, and -16
were isolated using a recently developed method (Jansen et al. 1997)
that uses chemical mutagenesis: gpa-4(pk38l), gpa-6(pk480), gpa7(pk61 0), gpa-9(pk438), gpa-10(pk362), gpa-11(pk349), gpa12(pk322), gpa-13(pk482), gpa-14(pk347), gpa-15(pk477), and gpa16(pk481) (Jansen et al. 1999). Besides the deletion, the gpa-13 allele
seems to contain a complex rearrangement, which is currently under
29
2.3.2 GFP Fusion Constructs
Routinely, a 3 kbp PCR fragment, containing approximately 2.5-3 kbp
of upstream sequences and the first 30--50 codons of the ex genes, was
fused in frame to the reporter gene GFP (vector pPD95.77; A. Fire, 1.
Ahnn, G. Seydoux, and S. Xu, personal communication). In most cases
the first intron was present. In this way fusion constructs were generated
for gpa-4, -6, -7, -8, -9, -10, -11, -13, -14, -15 and, -16. Promoter fusions
for gpa-1 and gpa-5 were generated by subcloning relevant regions from
cosmid clones. The gpa-1::GFP fusion construct contains 1.5 kbp of
upstream sequences, and the first eight exons of gpa-1. The gpa-5::GFP
construct contains 3.7 kbp upstream sequences and the first five exons
of gpa-5 (Jansen et al. 1999).
At least two independent transgenic lines were generated from at
least two independent clones of each of the gpa: :GFP fusion constructs,
to control for PCR induced sequence errors. Except for variation in
expression level, all transgenic lines generated for each of the Gex
subunits showed the same expression pattern. Subsequently, the transgenic array was integrated for one of the lines of each of the fusion
constructs. Particular cells were identified by using a combination of
their position and morphology (White et al. 1986). Expression of all
gpa::GFP fusions was observed in animals from the embryonic threefold stage onwards, or from the moment a postembryonic cell was born
(data not shown). Expression in males was examined for all GFP fusion
constructs except gpa-16: :GFP.
2.3.3 Isolation of Loss-of-Function Alleles
Deletion mutations of gpa-1, gpa-5, and gpa-8 were isolated as described: gpa-1(pk15), gpa-5(pk376), and gpa-8(pk345) (Zwaal et al.
1993). Deletion alleles of gpa-4, -6, -7, -9, -10, -11, -14, -15, and -16
were isolated using a recently developed method (Jansen et al. 1997)
that uses chemical mutagenesis: gpa-4(pk38l), gpa-6(pk480), gpa7(pk61 0), gpa-9(pk438), gpa-10(pk362), gpa-11(pk349), gpa12(pk322), gpa-13(pk482), gpa-14(pk347), gpa-15(pk477), and gpa16(pk481) (Jansen et al. 1999). Besides the deletion, the gpa-13 allele
seems to contain a complex rearrangement, which is currently under