Table 2.
Plasmids used in this study
| Plasmid | Description | Source/reference |
|---|---|---|
| pMVB12 | CDC12-His6 | Versele and Thorner, 2004 |
| pMVB13 | CDC12ΔC-His6 | Versele and Thorner, 2004 |
| pMVB120 | CDC11-His6 | Versele and Thorner, 2004 |
| pBEG2 | His6-CDC3 | Versele and Thorner, 2004 |
| pBEG2 | His6-CDC10 | Versele and Thorner, 2004 |
| pGST-CDC11 | GST-CDC11 | D. Kellogg (University of California, Santa Cruz, CA) |
| pMVB29a | GST-CDC11ΔC | This study |
| pMVB24 | GST-CDC10 | Versele and Thorner, 2004 |
| pMVB25 | GST-CDC12 | Versele and Thorner, 2004 |
| pMVB26a | GST-CDC12ΔC | This study |
| pMVB27 | GST-CDC3 | Versele and Thorner, 2004 |
| pMVB30a | GST-CDC3ΔC | This study |
| pBEG4b | GST-SHS1 | This study |
| pBEG24b | GST-SHS1ΔC | This study |
| pMVB172c | His6-GST-CTECdc12 | This study |
| pMVB121 | pETDuet-His6CDC12 | Versele and Thorner, 2004 |
| pMVB128d | pETDuet-His6CDC12-CDC10 | This study |
| pMVB134d | pETDuet-His6CDC10 | This study |
| pMVB132d | pACYDuet-CDC3 | This study |
| pMVB133d | pACYDuet-CDC3-CDC11 | This study |
| pMVB127d | pACYDuet-CDC11 | This study |
| pMVB122 | pETDuet-His6CDC12-CDC3 | Versele and Thorner, 2004 |
| pMVB129d | pETDuet-His6CDC12ΔC | This study |
| pMVB130d | pETDuet-His6CDC12ΔC-CDC10 | This study |
| pMVB135d | pETDuet-His6CDC12ΔC-CDC3 | This study |
| pMVB45c | CEN, LEU2, CDC12 | This study |
| pMVB48e | CEN, LEU2, CDC12ΔC | This study |
| pMVB52e | CEN, LEU2, CDC12ΔC(S43V) | This study |
| pMVB53e | CEN, LEU2, CDC12ΔC(T48N) | This study |
| pLP17 | CEN, LEU2, CDC12-GFP | Lippincott and Li, 1998 |
| pMVB33f | CEN, LEU2, CDC12ΔC-GFP | This study |
| pMVB62g | 2μ, LEU2, CDC12-GFP | This study |
| pMVB63g | 2μ, LEU2, CDC12ΔC-GFP | This study |
| pMVB23h | URA3, CDC12ΔC | This study |
| pMVB54i | 2μm, LEU2, CDC12ΔC | This study |
| pMVB2j | CEN, URA3, GAL1-CDC12-GFP | This study |
| pMVB160j | CEN, URA3, GAL1-CDC12ΔC-GFP | This study |
| pMVB162k | CEN, LEU2, GAL1-CDC12 | This study |
| pMVB164k | CEN, LEU2, GAL1-CDC12ΔC | This study |
| pSB5 | CEN, URA3, CDC11-GFP | Versele and Thorner, 2004 |
| CDC3-GFP | CEN, URA3, CDC3-GFP | B. Haarer (SUNY Upstate Med. Ctr., Syracuse, NY) |
| pLA10 | CEN, URA3, CDC10-GFP | Cid et al., 1998 |
| pMVB38l | 2μm, LEU2, P(CDC12)-GFP-CTECdc12 | This study |
| pMVB100m | CEN, URA3, CDC3 | This study |
| pMVB102m | CEN, URA3, CDC3ΔC | This study |
| pSB1n | CEN, URA3, CDC11 | This study |
| pSB2n | CEN, URA3, CDC11ΔC | This study |
| YCpUG-CDC11o | CEN, URA3, GAL1-CDC11 | This study |
| YCpUG-CDC11ΔCo | CEN, URA3, GAL1-CDC11ΔC | This study |
CDC11(Δ357-415), CDC12(Δ339-407), or CDC3(Δ427-521) were cloned into pGEX3 (Amersham Biosciences)
SHS1 or SHS1(Δ418-551) were cloned into pGEX4T-1 (Amersham Biosciences)
The C-terminus of Cdc12 (aa 339-407) was cloned in pKM263 (Melcher, 2000)
These vectors are based on pETDuet-1 and pACYDuet-1 (Novagen). The His6-tag in pACYDuet-1 was entirely deleted by PCR mutagenesis
CDC12, CDC12(Δ339-407), CDC12(Δ339-407)S43V, and CDC12(Δ339-407)T48N, including 500 bp of its promoter region, were cloned into YCplac111 (Gietz and Sugino, 1988)
CDC12(Δ339-407) was cloned into pLP17, digested with PstI/BamHI to remove full-length CDC12, but leaving GFP intact and in frame
CDC12-GFP and CDC12(Δ339-407)-GFP were subcloned from pLP17 and pMVB33, respectively, to pRS425 (Sikorski and Hieter, 1989)
CDC12(Δ339-407), including 500-bp promoter region, was cloned in pUC18, and 446 bp of the terminator sequence of CDC12 was inserted. Then, the URA3 gene, excised from pJJ244 (Jones and Prakash, 1990) was inserted into the SnaBI site in the terminator region of CDC12
CDC12(Δ339-407) was cloned into YEplac181 (Gietz and Sugino, 1988)
CDC12 and CDC12(Δ339-407) were cloned in frame with GFP in TS395 (a gift from Tim Stearns, Stanford University)
CDC12 and CDC12(Δ339-407) were cloned in TS395 without the GFP fusion; in addition, a LEU2 marker was inserted at the URA3 locus of these plasmids
GFP, under the control of the CDC12 promoter, was fused to residues 337-407 of CDC12, and cloned into YEplac181 (Gietz and Sugino, 1988)
CDC3 and CDC3(Δ440-520) were cloned into YCplac33 (Gietz and Sugino, 1988)
CDC11 and CDC11(Δ372-415) were cloned into pRS316 (Sikorski and Hieter, 1989)
CDC11 and CDC11(Δ372-415) were cloned into YCpUG (Bardwell et al., 1998)