Table 2.
Constructs, primers and transgenes used in this study.
| Construct | Vector | Parent plasmid |
Primers (5′-3′)1 | Transgene psEx# |
Background/ coinjection marker(ng.μl−1)2 |
ng. μl−1 |
|---|---|---|---|---|---|---|
| pnc-1 genomic | pMC1 | pPD95.67 | Restriction linker F GCATGCGCTAGCCTGCAGGCGGCCGCTCTAGA Restriction linker R TCTAGAGCGGCCGCCTGCAGGCTAGCGCATGC pnc-1 F (Pst I) AACTGCAGAATCGCCAACTGGATCTTC pnc-1 R (Not I) AAGCGGCCGCCTTCTTCACGATCCTTTGAACCCATTC pnc-1 Fa (Sph I) GCATGCCAGGCTTTTCTTGACTTTCTGTTACTTTTT pnc-1 Ra (Nhe I) GCTAGCATTTTACCTCTCTAAACTCCGAAAAC pnc-1 F9 (Nhe I) AAGCTAGCGTAAAATGAATGTTTCCGTGTTGTTGTTGTG pnc-1 R18 (Pst I) AACTGCAGCCCAAGCCAAGATTCCATAATCCTCT |
216-9 | unc-119; pnc-1/unc-119(+)(60) | 20 |
| 251-2, 265-6, 268 | pnc-1/sur-5::gfp(60) and pBS KS-(39) | 1 | ||||
|
| ||||||
| pnc-1 genomic ΔSS | pMC11 | pPD95.67 |
pnc-1 F17 (Kas I) GAGGCGCCACATCTCTTCCC pnc-1 R25 (EcoR V) CTGGATATCGTTTTAAACGCTCTAAACGAG pnc-1 F18 (mutagenesis linker) GCATTTATTGaGCaCAagTGGGTGA3 pnc-1 R26 (mutagenesis linker) TCACCCActTGtGCtCAATAAATGC |
246-7 | unc-119/unc-119(+)(60) | 20 |
| 249, 253, 271, 276-8 |
pnc-1/sur-5::gfp(60) and pBS KS-(39) |
1 | ||||
|
| ||||||
| pnc-1a | pMC4 | pPD95.69 | Common to pMC4/5/6 pnc-1 F16 (EcoR I) AAGAATTCAAGTAATAATAAAATATTGTGCATAATTTTGTAAT pnc-1 R24 (Spe I) AAACTAGTGAGGAGTACACGGCGCGT pPD118.20 R1 CCAAGCGAGGACAATTCTCATCG GFP F7 ATGGGTTCAAAGGATCGTGAAGAAGATGAGTAAAGGAGAAGAACTTTTCACTGG pnc-1a/b CM R4 CCAGTGAAAAGTTCTTCTCCTTTACTCATCTTCTTCACGATCCTTTGAACCCAT Specific to pMC4 pnc-1a/b CM F8 ATGTTTCCCTGCCCAAAGCTTATATTCC pnc-1 F3 (Pst I) AACTGCAGTTTTCCAGGCTTTTCTTGACTTTCTGTTAC pnc-1 R4 (Xba I) AATCTAGACAAGAGAATTTGATCTAAGAATGTATTCTCCAG |
213-5, 220-1, 228 | unc-119; pnc-1/unc-119(+)(60) | 1 |
| 238 | “ | 60 | ||||
|
| ||||||
| pnc-1b.1 | pMC5 | pPD95.69 | Common to pMC5 and 6 pnc-1a/b CM F9 CAGCTAATTCCATTTTTGCTCCTAGC Specific to pMC5 pnc-1 F4 (Pst I) AACTGCAGGTGAGTTTTGGTGGATAGGACGGT pnc-1 R5 (Nhe I) AAGCTAGCCTTCGTTTCCGCGGAAACGAGTG |
222-5 | pnc-1/- | 10 |
|
| ||||||
| pnc-1b.2 | pMC3 | pPD95.69 |
pnc-1 F2 (Pst I) CTGCAGGTAAAATGAATGTTTCCGTGTTGTTGTTGTG pnc-1 R3 (Sal I) GTCGACCATCTACAACAATACAAACTATTTGCGAACAAC |
207-8 | unc-119/unc-119(+)(60) | 20 |
| pMC6 | 217-9 | pnc-1/- | 10 | |||
|
| ||||||
| daf-7 pnc-1a/ b | pMC13/14 | TU#61/2 |
daf-7 CF12 (Sph I) GCATGCGTAAACACTTTTCAGATTTTTCAAATTTCAG daf-7 CR14 (Xma I) CCCGGGGAACATGGCTGAACTTCAAGC |
pnc-1a: 283-6 | unc-119; pnc-1/unc-119(+)(60) | 10 |
|
pnc-1b: 287–290 |
||||||
|
| ||||||
| gcy-8 pnc-1a/ b | pMC15/16 | TU#61/2 |
gcy-8 F1 (Sph I) GCATGCCTTCTCCTTGACGAGTCTTCC gcy-8 R1 (Xma I) CCCGGGTTTGATGTGGAAAAGGTAGAATCG |
pnc-1a: 291 | unc-119; pnc-1/unc-119(+)(60) | 10 |
| pnc-1b: 292 | ||||||
1
Restriction enzymes in parentheses refer to the enzymes used in the initial cloning step with fragments generated using these primers. Subsequent sub-cloning steps are detailed in the methods section.
2
Coinjection markers used were either pDP#MM016B (unc-119(+)) DNA (Maduro and Pilgrim, 1995) or sur-5::gfp DNA (pTG96_1 (Gu et al., 1998)) and pBluescript KS- to a total of 100ng.ul−1.
3
Nucleotides in lowercase are those changed to inactivate the pnc-1a signal sequence.