Table II. Plasmids used in this work.
| Plasmid | Description | Source/reference |
|---|---|---|
| pMVB12a | CDC12-His6 | This paper |
| pMVB13a | CDC12ΔC-His6 | This paper |
| pMVB14b | CDC12(S43V)-His6 | This paper |
| pMVB15b | CDC12(T48N)-His6 | This paper |
| pMVB16b | CDC12ΔC(S43V)-His6 | This paper |
| pMVB17b | CDC12ΔC(T48N)-His6 | This paper |
| pMVB120c | CDC11-His6 | This paper |
| pDT72d | pET24d-His6 vector | D. Truckses (this laboratory) |
| pBEG2e | His6-CDC3 | B. Gullbrand (this laboratory) |
| pBEG3e | His6-CDC10 | B. Gullbrand (this laboratory) |
| pMVB150 | His6-CDC10(S256A) | This paper |
| pMVB151 | His6-CDC10(S256A S312A) | This paper |
| pKM263 | His6-GST vector | Melcher, 2000 |
| pMVB7f | His6-GST-CDC3 | This paper |
| pMVB8f | His6-GST-CDC10 | This paper |
| pMVB10f | His6-GST-CDC11 | This paper |
| pGST-CDC11 | GST-CDC11 | D. Kellogg (University of California, Santa Cruz, Santa Cruz, CA) |
| pMVB24g | GST-CDC10 | This paper |
| pMVB25g | GST-CDC12 | This paper |
| pMVB27g | GST-CDC3 | This paper |
| pGEX-HSL1ΔN | GST-HSL1(833-1518) | Shulewitz, 2000 |
| pMVB80g | GST-CLA4 | This paper |
| pMVB82g | GST-CLA4(524–842) | This paper |
| pMVB81b | GST-CLA4(K594A) | This paper |
| pMVB83b | GST-CLA4(524–842)(K594A) | This paper |
| pLP17 | CEN, LEU2, CDC12-GFP | Lippincott and Li, 1998 |
| pLP29 | CEN, HIS3, CDC12-GFP | Lippincott and Li, 1998 |
| pMVB32b | CEN, LEU2, CDC12(T48N)-GFP | This paper |
| pMVB91b | CEN, HIS3, CDC12(T48N)-GFP | This paper |
| pMVB19h | pUC18-CDC12 | This paper |
| pMVB64h | pUC18-CDC12(S43V) | This paper |
| pMVB65h | pUC18-CDC12(T48N) | This paper |
| pMVB21i | pUC18-CDC12(S43V)-URA3 | This paper |
| pMVB22i | pUC18-CDC12(T48N)-URA3 | This paper |
| YCplac33 | CEN, URA3 vector | Gietz and Sugino, 1988 |
| pMVB39j | CEN, URA3, CDC12 | This paper |
| YEplac181 | 2 μm, LEU2 vector | Gietz and Sugino, 1988 |
| pMVB49k | 2 μm, LEU2, CDC12(T48N) | This paper |
| YIplac128 | LEU2 vector | Gietz and Sugino, 1988 |
| pMVB41l | LEU2, CDC10 | This paper |
| pMVB40b | LEU2, CDC10(S46N) | This paper |
| pMVB155b | LEU2, CDC10(S41V) | This paper |
| YIplac211 | URA3 vector | Gietz and Sugino, 1988 |
| pMVB69m | URA3, CDC10(S46N) | This paper |
| pMVB143m | LEU2, CDC10(S256A) | This paper |
| pMVB144m | LEU2, CDC10(S46N S256A) | This paper |
| pLA10 | CEN, URA3, CDC10-GFP | Cid et al., 1998 |
| pSB5 | CEN, URA3, CDC11-GFP | S. Bahmanyar (this laboratory) |
| CDC3-GFP | CEN, URA3, CDC3-GFP | B. Haarer |
| pCJ160 | CEN, URA3, GAL1-myc-STE20 | Shulewitz, 2000 |
| YCpUG-SKM1 | CEN, URA3, GAL1-myc-SKM1 | Shulewitz, 2000 |
| YEpUG-CLA4 | 2 μm, URA3, GAL1-myc-CLA4 | Shulewitz, 2000 |
| pMVB113n | CEN, URA3, GAL1-myc-CLA4 | This paper |
| pMVB112o | CEN, URA3, GAL1-myc-CLA4(K594A) | This paper |
| pMVB121p | pETDuet-His6 CDC12 | This paper |
| pMVB122p | pETDuet-His6 CDC12-CDC3 | This paper |
| pMVB123p | pACYDuet-CDC10 | This paper |
| pMVB124p | pACYDuet-CDC10-CDC11 | This paper |
| pMVB125p | pETDuet-His6 CDC12T48N-CDC3 | This paper |
| pMVB126p | pACYDuet-CDC10S46N-CDC11 | This paper |
CDC12 was amplified using a forward primer, 5′-GGAATTCCAT ATGAGTGCTGCCACTGC-3′ (NdeI site in italics, start codon in bold) and as reverse primer, 5′-TATCCGCTCGAGAGATCCACGTGGAACCAGTTTTAAATGGGATTTTTTTACTT-3′ (XhoI site in italics). CDC12(Δ339–407), abbreviated Cdc12ΔC, was amplified using the same forward primer and a different reverse primer, 5′-TATCCGCTCGAGAGAGCCACGCGGAACTAAGTGTGACAATTTCCTTGCT-3′. Both reverse primers encode a thrombin cleavage site. The resulting CDC12 and CDC12ΔC derivatives were inserted as NdeI–XhoI fragments into the corresponding sites of pET-24b (Novagen) upstream and in-frame with a His6 tag.
These constructs were created via site-directed mutagenesis of the indicated codons using mismatch primers.
Derived from pET-23d (Novagen).
The NcoI–BamHI fragment of pET-24d was replaced with an NcoI–BamHI fragment of pBH4 (a gift of K. Prehoda and W. Lim, UCSF) that contains six tandem His codons and a sequence encoding the cleavage site for TEV.
Derived from pDT72.
Derived from pKM263.
Derived from pGEX3 (Amersham Biosciences).
CDC12, including 500 bp of its promoter region, was amplified and inserted in pUC18. Mutations S43V and T48N were introduced separately into Cdc12, as described above. To permit subsequent integration at the CDC12 locus on chromosome VIII, 446 bp of the 3′-untranslated region of CDC12 was inserted in the corresponding constructs.
To provide a selectable marker for integration, the URA3 gene, excised from pJJ244 (Jones and Prakash, 1990) was inserted into the naturally occurring SnaBI site present in the 3′-flanking region of CDC12.
A fragment of pMVB19 carrying CDC12 and its promoter was inserted into the corresponding sites in YCplac33.
A fragment of pMVB22 carrying CDC12(T48N) and its promoter was inserted into the corresponding sites in YEplac181.
CDC10, including 500 bps of its promotor region and 250 bp 3′ to its stop codon, was amplified and cloned into YIplac128.
A fragment containing cdc10S46N and its promotor was subcloned from pMVB40 into YIplac211. The cdc10S256A mutation was introduced via site-directed mutagenesis.
The GAL1-Myc-CLA4 fragment from YEpUG-Myc-CLA4 was subcloned into YCplac33.
GAL1-Myc-CLA4 was subcloned from pMVB113 to pUC18, which was used as a template in a site directed mutagenesis reaction, changing Lys 594 to Ala; GAL1-Myc-CLA4(K594A) was then inserted into YCplac33.
These vectors are based on pETDuet-1 and pACYDuet-1 (Novagen). The His6 tag in pACYDuet was entirely deleted in pACYDuet1 using PCR mutagenesis. The GTP binding mutants were introduced by site-directed mutagenesis.