Shaye and Greenwald
Supplementary Information 5
Genotypes of transgenic animals in figure 2:
Details of plasmid constructions are available upon request. All plasmids are
derived from pDS51, which contains a PCR product that corresponds to the egl-17
promoter, modified from ref. 1 according to M. Stern (personal communication). The egl17 promoter in pDS51 contains 3.9kb of sequence upstream of the initiating codon and
the coding region for the first 11 amino acids of EGL-17, with the initiating ATG and an
ATG coding for 9M of EGL-17 both mutated to ATC.
Generation of transgenic lines and plasmids co-injected with those listed below
are discussed in Methods.
Plasmid
injected
Protein Expressed
pDS58
sTM::GFP (sTM represents the SEL-1 signal sequence2, a
17 amino acid linker and a synthetic transmembrane
domain3)
pDS55.2
LIN-12(+)::GFP (GFP deletes 1322A to 1339S, which does not
affect LIN-12(+) function4)
pDS60
∆Extracellular::GFP (deletes amino acids 1M to 902T, which
are replaced by the SEL-1 signal sequence and an 11 amino
acid linker )
pDS61
LIN-12(intra)::GFP (amino acids 939M to 1429F)
pDS62
sTM::LIN-12(intra)::GFP (adding sTM deletes the first
amino acid of LIN-12(intra) so that this membrane targeted
form starts at 940I.)
pDS73.2
sTM::LIN-12(intra)∆r1::GFP (∆r1 deletes LIN-12 amino
acids 940I to 1029E.)
pDS83
sTM::LIN-12(intra)∆r2∆r3::GFP (∆r2 deletes LIN-12 amino
acids 1032G to 1266S and ∆r3 deletes from 1340R to 1429F)
pDS94
sTM::LIN-12(intra)∆DTS::GFP (deletes 957K to 971E)
pDS112
sTM::LIN-12(intra)LLAA::GFP (mutate 969L970L to AA)
*
All strains contain unc-4(e120).
Genotype*
arEx225
arEx226
arEx227
arEx228
arEx229
arEx230
arEx234
arEx235
arEx236
arEx237
arEx238
arEx239
arEx240
arEx241
arEx242
arEx260
arEx261
arEx262
arEx275
arEx276
arEx277
arEx281
arEx282
arEx283
arEx363
arEx364
arEx365
Shaye and Greenwald
Supplementary Information 5
Genotypes of transgenic animals in figure 3:
Details of plasmid constructions are available upon request. Generation of
transgenic lines and plasmids co-injected with those listed below are discussed in
Methods.
Plasmid injected Protein Expressed
Genotype*
arIs82
LIN-12(+)::GFP
pLin-12::GFP†
(lin-12g driven)
arIs84
arIs77
LIN-12(∆DTS)::GFP
pDS109.3‡
arIs79
(lin-12g driven)
arIs80
arEx229
LIN-12(+)::GFP
pDS55.2¶
(egl-17p driven)
arEx230
arEx389
LIN-12(∆DTS)::GFP
pDS118¶
(egl-17p driven)
arEx390
*
All strains contain unc-4(e120).
†
pLin-12::GFP is described in reference 4.
‡
pDS109.3 was made by deleting the DTS coding sequences from pLin-12::GFP.
¶
Derived from pDS51.
1.
2.
3.
4.
Burdine, R. D., Branda, C. S. & Stern, M. J. EGL-17(FGF) expression coordinates the attraction
of the migrating sex myoblasts with vulval induction in C. elegans. Development 125, 1083-93.
(1998).
Grant, B. & Greenwald, I. Structure, function, and expression of SEL-1, a negative regulator of
LIN-12 and GLP-1 in C. elegans. Development 124, 637-44. (1997).
Fire, A., Harrison, S. W. & Dixon, D. A modular set of lacZ fusion vectors for studying gene
expression in Caenorhabditis elegans. Gene 93, 189-98. (1990).
Levitan, D. & Greenwald, I. LIN-12 protein expression and localization during vulval
development in C. elegans. Development 125, 3101-9. (1998).