Plasmids and transgenes
| Plasmid (concentration injected) . | Protein expressed . | Lines analyzed . | Figure . | lin-12(0)rescue¶(number of lines/total) . |
|---|---|---|---|---|
| pLIN12GFP* | LIN-12(+)::GFP (wild type) | arEx541 | 2 | 6/7 |
| (5 μg/ml) | 952EEKNRKNHQSITSSQHSLLEASYDGYIKRQ981 | arEx542 | ||
| arEx543 | ||||
| p410† | LIN-12(LLtoA)::GFP | arEx495 | 2 | 4/5 |
| (5 μg/ml) | 952EEKNRKNHQSITSSQHSAAEASYDGYIKRQ981 | arEx496 | ||
| arEx497 | ||||
| p411† | LIN-12(S/TtoA)::GFP | arEx498 | 2 | 5/6 |
| (5 μg/ml) | 952EEKNRKNHQAIAAAQHALLEASYDGYIKRQ981 | arEx499 | ||
| arEx500 | ||||
| p413† | LIN-12(KtoA)::GFP | arEx514 | 2 | 7/8** |
| (5 μg/ml) | 952EEANRANHQSITSSQHSLLEASYDGYIARQ981 | arEx515 | ||
| arEx516 | ||||
| p415* | LIN-12(S/TtoD)::GFP | arEx524 | 3 | 7/11 |
| (5 μg/ml) | 952EEKNRKNHQDIDDDQHDLLEASYDGYIKRQ981 | arEx525 | ||
| arEx526 | ||||
| p405‡ | LIN-12(ΔEΔDTS)::GFP†† | arEx409 | 6 | |
| (20 μg/ml) | arEx410 | |||
| p406‡ | LIN-12(extra)::TM::GFP | arEx411 | 6 | |
| (20 μg/ml) | arEx412 | |||
| p408§ | LIN-12(extra)::TM::DTS::GFP | arEx482 | 6 | |
| (20 μg/ml) | arEx483 | |||
| arEx485 | ||||
| p409§ | LIN-12(extra)::TM::region1::GFP | arEx492 | 6 | |
| (20 μg/ml) | arEx493 |
| Plasmid (concentration injected) . | Protein expressed . | Lines analyzed . | Figure . | lin-12(0)rescue¶(number of lines/total) . |
|---|---|---|---|---|
| pLIN12GFP* | LIN-12(+)::GFP (wild type) | arEx541 | 2 | 6/7 |
| (5 μg/ml) | 952EEKNRKNHQSITSSQHSLLEASYDGYIKRQ981 | arEx542 | ||
| arEx543 | ||||
| p410† | LIN-12(LLtoA)::GFP | arEx495 | 2 | 4/5 |
| (5 μg/ml) | 952EEKNRKNHQSITSSQHSAAEASYDGYIKRQ981 | arEx496 | ||
| arEx497 | ||||
| p411† | LIN-12(S/TtoA)::GFP | arEx498 | 2 | 5/6 |
| (5 μg/ml) | 952EEKNRKNHQAIAAAQHALLEASYDGYIKRQ981 | arEx499 | ||
| arEx500 | ||||
| p413† | LIN-12(KtoA)::GFP | arEx514 | 2 | 7/8** |
| (5 μg/ml) | 952EEANRANHQSITSSQHSLLEASYDGYIARQ981 | arEx515 | ||
| arEx516 | ||||
| p415* | LIN-12(S/TtoD)::GFP | arEx524 | 3 | 7/11 |
| (5 μg/ml) | 952EEKNRKNHQDIDDDQHDLLEASYDGYIKRQ981 | arEx525 | ||
| arEx526 | ||||
| p405‡ | LIN-12(ΔEΔDTS)::GFP†† | arEx409 | 6 | |
| (20 μg/ml) | arEx410 | |||
| p406‡ | LIN-12(extra)::TM::GFP | arEx411 | 6 | |
| (20 μg/ml) | arEx412 | |||
| p408§ | LIN-12(extra)::TM::DTS::GFP | arEx482 | 6 | |
| (20 μg/ml) | arEx483 | |||
| arEx485 | ||||
| p409§ | LIN-12(extra)::TM::region1::GFP | arEx492 | 6 | |
| (20 μg/ml) | arEx493 |
Transgenes containing unc-4(+) were analyzed in an unc-4(e120) background. Transgenes containing pha-1(+) were analyzed in a pha-1(e2123ts)background. Constructs driven by lin-12 genomic sequences (Figs 2, 3) were analyzed at 20°C. Constructs driven by the egl-17 promoter(Fig. 6) were analyzed at 25°C.
DTS is underlined.
Mixed with unc-4(+) (30 μg/ml pNC4.21) (see Miller and Niemeyer, 1995), ttx-3p::gfp (60 μg/ml pTTX-3GFP) (see Hobert et al., 1997) and egl-17p::lacZ (20 μg/ml pNH291) (see Burdine et al., 1998).
Mixed with unc-4(+) (30 μg/ml), ttx-3p::gfp (60 μg/ml) and pBluescript (Stratagene, USA)carrier (20 μg/ml).
Mixed with unc-4(+) (30 μg/ml), egl-17p::lacZ (20 μg/ml) and pBluescript carrier (30μg/ml).
Mixed with pha-1(+) (50 μg/ml pBX) (see Granato et al., 1994), ttx-3p::gfp (60 μg/ml) and egl-17p::lacZ (20 μg/ml).
Rescue was assessed in a lin-12(n941) background, and the effects on downregulation and lateral signaling were assessed in a lin-12(+) background (see Shaye and Greenwald,2002).
Essentially all hermaphrodites carrying this transgene display a Multivulva(Muv) phenotype, which suggests elevated lin-12 activity (see text).
The DTS, 957K through 971E, was deleted and replaced by four amino acids, MAAG. LIN-12(ΔEΔDTS)::GFP is highly toxic but causes a Muv phenotype when expressed using regulatory sequences from the sel-12 gene, which drives expression in all VPCs and many other cells(data not shown).