1. Volume 4, Issue 3, Pages 353-363 (September 1999)
Directed Evolution to Bypass Cyclin Requirements for the Cdc28p Cyclin-Dependent Kinase 
Kristi Levine, Lee Kiang, Matthew D Jacobson, Robert P Fisher, Frederick R Cross 
Molecular Cell 
Volume 4, Issue 3, Pages (September 1999)
DOI: /S (00)

2. Figure 1 Comparison of CDC28-SKE Alleles and CDC28-BYC Alleles
(A) CDC28-SKE (suppressor of cln2-KAEA) alleles suppress the growth defect of a cln1,2,3, cdc28-csr1 pcln2-KAEA strain and are dependent upon pcln2KAEA for viability. Strain B pKL033-1 (cln1,2,3, cdc28-csr1 GAL1::CLN3 pcln2KAEA-URA3) was transformed with the TRP1 plasmid RS414 (vector), SF19 (CDC28-w.t.-HA), T411 (CLN2-3P), and the indicated CDC28-SKE alleles. The cdc28-csr1 allele was included in the strain to tighten the plasmid CDC28 requirement (Levine et al. 1998). Sequences were as follows: SKE1, K41E I42T; SKE2, K63N I124T; SKE3, K63N I124T N145S R226I; SKE13, R43K K63R E64Q S79T; SKE15, L44Q K83E G123S; SKE19, P52L K187R; SKE22, L93M K96E N145S N207K; SKE27, L61I K83E; SKE28, K83R Q188L; SKE35, S46I R97G; SKE37, D75G K83D K104E; SKE38, S53I G101D; and SKE42 P52L G101C.
(B) CDC28-BYC (bypass of CLN s) alleles suppress the growth defect of a cln1,2,3, pcln2KAEA strain and are independent of pcln2-KAEA for viability. Strain B pKL033-1 (see A) was transformed with the TRP1 plasmid RS414 (vector), SF19 (CDC28-w.t.-HA), T411 (CLN2-3P), and the indicated SKE and BYC alleles of CDC28. Sequences were as follows: BYC5, L44Q K83E K96E N145S; BYC6, L61I K83E K96E; BYC8, L61I D75G K83E N145S; BYC10, L44P L61I K83E N145S; BYC15, T18A K41E I42T L61I K96E; BYC16, L44P L61I K83E K96E; and BYC19, G3A V14I L44Q L61I K83E K96E N145S.
Transformants were selected on ScGal-Trp-Ura, pooled into patches and grown on SCGal-Trp-Ura, and then replica plated onto YEPGal, YEPD, and FOADex. The YEPGal and YEPDex plates, and FOADex plate were incubated at 30°C for 2 and 3 days, respectively.
Molecular Cell 1999 4, DOI: ( /S (00) )

3. Figure 2
Comparison of CDC28-SKE Mutant Alleles and Selected CDC28 Point Mutant Alleles
Strain B pKL033-1 (cln1,2,3, cdc28-csr1 GAL1::CLN3 pcln2-KAEA/URA3) was transformed with TRP1/CDC28 plasmids containing the indicated mutations (see Figure 1 legend for SKE mutations) and controls. Transformants were selected on ScGal-Trp-Ura and pooled. For each transformed strain, 10-fold serial dilutions were plated onto a YEPGal plate (GAL1::CLN3 ON) and a YEPD plate (GAL1::CLN3 OFF) and incubated for 2 days at 30°C.
Molecular Cell 1999 4, DOI: ( /S (00) )

4. Figure 3
Generation of Recombinant Pools of CDC28 Mutant Alleles Allows the Identification of Highly Active CLN-Independent CDC28 Alleles
(A) Recombinant Pool I, input and most highly active alleles identified. The possible mutations in each segment are indicated (wild type was included in all segments). The recombinant pool was constructed by amplification of each segment separately from pooled templates including wild type, followed by splice overlap extension sequentially recombining the segments to reconstruct full-length CDC28. Sequences of the two most highly active CLN-independent alleles identified from this pool are shown (CDC28-BYC19-3 and CDC28-BYC29). (BYC19-3 is BYC19 [Figure 1B legend] with the irrelevant G3A and V14I mutations removed).
(B) Recombinant Pool II, input and most highly active alleles identified. DNA fragments of Recombinant Pool II were fused to either a wild-type or an L157S, A160V–containing 3′ end. Sequences of highly active CLN-independent alleles identified from this pool are shown.
(C) Characterization of the BYC phenotype by plating efficiency assays of cln1,2,3, GAL1::CLN3 transformants: comparison of BYC19-3, BYC29, P3A3, P3A4, P3C4, and P3C16. Strain D (cln1,2,3 GAL1::CLN3) was transformed with the TRP1 plasmid T411(CLN2-3P-HA), SF19 (CDC28-HA), or the indicated CDC28-BYC plasmids. Pooled transformants were grown on SCGal-Trp medium, and 10-fold serial dilutions were plated onto YEPGal (GAL1::CLN3 ON) and YEPD (GAL1::CLN3 OFF) plates, and allowed to incubate for 3 and 2 days, respectively, at 30°C.
Molecular Cell 1999 4, DOI: ( /S (00) )

5. Figure 4
Characterization of the Efficiency of CLN Bypass by CDC28-BYC Alleles
Strain D (cln1,2,3 GAL1::CLN3) was transformed with the TRP1 plasmid T411 (CLN2-3P-HA), or the indicated CDC28-BYC alleles. Pooled transformants were grown to log phase in YEPD medium (GAL1::CLN3 OFF). Log phase cultures were analyzed (see Experimental Procedures) for cell volume (CV), doubling time (DT), percentage of unbudded cells (%UB), and for DNA content by flow cytometry.
Molecular Cell 1999 4, DOI: ( /S (00) )

6. Figure 5
A Test of CDC28-BYC Activity in Cyclin-Specific Genetic Assays
(A–F) cln1 cln2 cln3 GAL1::CLN1 strains containing null mutations in additional genes that increase the stringency and specificity of the requirement for CLN function (Levine et al. 1996) were transformed with the indicated plasmids. (A) clb5 clb6, (B) swi4, (C) swi6, (D) pcl1 pcl2, (E) bud2, (F) bck2. Plating efficiency assays were performed as in Figure 3C. The CLN3 plasmid was KL001 (CLN3-3P-HA) (Levine et al. 1996). (D) and (E) were incubated at 38°C; others at 30°C. CDC28-P3C4 and -P3C16 produced variably sized, and generally much smaller, transformant colonies in the cln1,2,3 clb5,6 strain even with GAL1::CLN1 on, suggesting a toxic effect of these alleles in this background (data not shown).
(G–J) Strains containing integrated GAL1::SIC1 or GAL1::SIC1-Δ3P as indicated. (G and H) cln1 cln2 CLN3 strains were transformed with the indicated plasmids and tested directly on YEPD (GAL1::SIC1 off) versus YEPGal (GAL1::SIC1 on). (I) A cln1 cln2 cln3 GAL1::SIC1 strain with a URA3/CLN3 plasmid was transformed with the indicated TRP1 plasmids, and loss of the URA3/CLN3 plasmid selected on FOAD. The transformants were then tested on YEPD versus YEPGal. (J) Similar to (I) except that a cln1 cln2 cln3 clb5 clb6 GAL1::SIC1 strain containing a URA3/CLN2 plasmid was used.
Molecular Cell 1999 4, DOI: ( /S (00) )

7. Figure 6
Clb-Bound Mutant Cdc28p Has Wild-Type Levels of Kinase Activity that Is Sic1p Sensitive
(A) CLN1,2,3+ cdc28::HIS3 transformants with the indicated CDC28 plasmids were transformed with the URA3 plasmid RS416 (vector), CE119-4 (GAL1::CLB5-HA), or 143 (GAL1::CLB2-HA). Ura+ transformants were pooled, and grown overnight in SCGal-Ura medium. HA-tagged cyclin proteins were immunoprecipitated and kinase assays and immunoblotting performed.
(B) The GAL1::CLB5-HA transformants in (A) were extracted. A cln1 cln2 CLN3 GAL1::SIC1 culture grown in YEP-raffinose was induced for 3 hr with galactose and extracted. (+) Equal amounts of protein from the Clb5p-HA-containing extracts and the Sic1p-containing extracts were mixed prior to immunoprecipitation; (−) buffer alone was added to the Clb5p-HA-containing extracts. Extracts from sic1 strains have no effect on Clb5p-associated kinase activity in this assay (Schwob et al. 1994; S. Gray and F. R. C., unpublished data).
Molecular Cell 1999 4, DOI: ( /S (00) )

8. Figure 7 Gel Filtration Analysis of Kinase Activity of Cdc28-Byc19-3
(A) CLN1,2,3+ cdc28::HIS3 transformants with the wild-type (SF19) or BYC19-3 CDC28-HA plasmids were grown to log phase in YEPD. Extracts were prepared and fractionated by Superdex 75 gel filtration chromatography. Cdc28p-HA was immunoprecipitated from the indicated fractions, and kinase assays and immunoblotting were performed.
(B) The experiment in (A) was repeated using the same strains (Run 1). Eighty percent of fraction #22 was refractionated by gel filtration chromatography (Run 2). Cdc28p-HA was immunoprecipitated from the remaining 20% of fraction #22 and from 20% of each of the other indicated fractions from Run 1, and from all of the corresponding fractions from Run 2. Samples were analyzed as in (A).
(C) CLN1,2,3+ cdc28::HIS3 transformants with wild-type CDC28 or the indicated CDC28-BYC alleles (lacking the HA epitope tag) were transformed with a URA3/GAL1::cln2-K129A,E183A-HA plasmid (KL044). Trp+Ura+ transformants were pooled, and grown overnight in SCGal-Ura medium. HA-tagged Cln2-KAEA protein was immunoprecipitated, and kinase assays and immunoblotting were performed.
Molecular Cell 1999 4, DOI: ( /S (00) )