TABLE 2.
Primers used in this study
| Primer | Sequence (5′-3′)i |
|---|---|
| spn1Dfwa | CAGATCTCATGGCGTCAATGGTACTCG (BglII) |
| spn1Drva | CTCGAGTAAGGAGACTATTTAAAAAACTTCC (XhoI) |
| spn2Dfwa,g | CAGATCTAATGGAAGTTCCTTCTGCAGTTACC (BglII) |
| spn2Drva,g | CTCGAGGAAGAAGACTATTGAGCGGTG (XhoI) |
| spn3Dfwa | CAGATCTAATGTGGTATACATATAACGAAGAC (BglII) |
| spn3Drva | CTCGAGCACCATTAAATTGGTTATGACTGG (XhoI) |
| spn4Dfwa | AAACCCGGGATGAATGAAGAAGAGACAAACTTCG (SmaI) |
| spn4Drva | AAATCTAGACTAACGTTTGCGAGAGCTGGTAGC (XbaI) |
| spn5Dfwa,g | CAGATCTGATGGATAGCTCAAATTTGTCTTCATC (BglII) |
| spn5Drva,g | CTCGAGTTTATGCTAAGATGCCTGC (XhoI) |
| spn6Dfwa,g | TTAGATCTCACGATGTCTTTGACTGAAAACCTTCAATTAT (BglII) |
| spn6Drva,g | TTCCCGGGTCATTTTTTATGACCGCGGCCCTTGT (SmaI) |
| spn7Dfwa,g | CAGATCTGATGAATAAAGGCCCAAGACATCG (BglII) |
| spn7Drva,g | CTCGAGCACTCTCAAATGTTTAATTTAGC (XhoI) |
| pik1Dfwa | AAGGATCCGATGCCATCTTCGAATTCGGG (BamHI) |
| pik1Drva | TTGCCAAATCGCAGATCTTCTGCGC |
| spn2KDfwb | TACAATGTTGACTTGGCCAAAACCAACAAATTCCAAATTCATTCAATTCGCAACTTGCAACGGATCCCCGGGTTAATTAA |
| spn2KCfwc | ATGGGAAGTCCCGCACCTGTGTACCCTTCTGAACCACATCTCCATACAGCCACCGCTCAACGGATCCCCGGGTTAATTAA |
| spn2KDrvb,c | GACAGTCATACAAATGGTTTAGTCTTTGTTCTAAACATACTATATATTACCTTAGGAAGAGAATTCGAGCTCGTTTAAAC |
| spn7KCfwc | ACAATACGACAAAGGAATTGGAGATGAAGAAAATGGATGATTTGTCTCATGAGCGTTACGAAAACCTCCCTTTCTATCGCCGGATCCCCGGGTTAATTAA |
| spn7KDrvc | GATTAAGCAATTAAAAAGAAATCAAAAGACTTTTCATGAACTTATACTAAATTATTCTTTTGATTGTTTATAATTTGCACGAATTCGAGCTCGTTTAAAC |
| ura4checkrvd | TCTTTGGCTACTGGTTCCTACAC |
| kancheckrvd | CGGATAAAATGCTTGATGGTCGGAAGAGG |
| GFPfwe | CCCCTCGAGTATGAGTAAAGGAGAA (XhoI) |
| GFPrve | CCCGGATCCGTCGACTTGTATAGTTCATCCATGCCATGTGTAATCCC (BamHI) |
| psy1fwe | CCCGTCGACAATGAATAAAGCAAACGAT (SalI) |
| psy1rve | CCCGAGCTCATCTAACCGGCCATATCACT (SacI) |
| propsy1fwe | CCCCTCGAGTGGTGCATCCAATCTCTG (XhoI) |
| propsy1rve | CCCCTCGAGTTTGTTATATTTATCTGTTTAAA (XhoI) |
| spn2Gfwf | AAGGATCCAGCGCTAATTATTTGTGAATCTGG (BamHI) |
| spn2Grvf | TTGCGGCCGCTGAGCGGTGGCTGTATGGAG (NotI) |
| spn5Gfwf | AAGGATCCTCCCGCCACTTCACTAAG (BamHI) |
| spn5Grvf | TTGCGGCCGCGCTAAGATGCCTGCTTTTTTC (NotI) |
| spn6Gfwf | AAAGATCTATTCGATCCTTGACACTGTC (BglII) |
| spn6Grvf | TTGCGGCCGCTTTTTATGACCGCGGCCCTTG (NotI) |
| spn7Gfwf | AAAGATCTTAAACCTGCAGCTCAACTC (BglII) |
| spn7Grvf | TTGCGGCCGCCGATAGAAAGGGAGGTTTTCG (NotI) |
| spn2mQQh | AACCAACAGTTAATTCAACAAGGT |
Used to construct the plasmids for the gene disruptions described in Fig. 1.
Used to generate strain MO684 (spn2Δ::kanMX6).
Used to generate strains MO701 (spn2+-GFP::kanMX6), MO815 (spn2+-mRFP::kanMX6), MO832 (spn2+-mRFP::kanMX6), and JW193 (spn7+-GFP:kanMX6).
Used to confirm successful disruption/tagging by the ura4+ or kanMX6 marker cassette. ura4checkrv was used with the appropriate Dfw series primer; kancheckrv was used with Spn2Gfw to check the spn2 deletion and with Spn2Dfw to check the C-terminal tagging.
Used to construct plasmid pBR(GFP-Psy1).
Used to construct the pAL(spn-GFP) plasmids.
Used to amplify cDNAs for construction of pMAL(spnN) and pREP41(GFP-spn) plasmids.
Used for mutagenesis of spn2+ to generate spn24Q.
Underlining indicates restriction enzyme site (corresponding enzyme is indicated in parentheses).