TABLE 1.
Yeast E2 expression plasmidsa
| Group | rad6ΔC
|
ubc1ΔC/UBC4
|
||
|---|---|---|---|---|
| Substitution type | Substitutions | Substitution type | Substitutions | |
| A | 1 | C-terminal truncation (residues 154–172), S(TCC)2S(AGC), T(ACA)3S(TCA), G(GGA)51G(GGC), T(ACT)52A(GCC), P(CCA)/98P(CCC), M(ATG)153I(ATC) | 2 | ubc1ΔC; C-terminal truncation (residues 151–215), S(TCT)2S(AGC), R(AGG)3S(TCA)∗ |
| 3 | UBC4; S(TCT)2S(AGT)∗ | |||
| B | 4 | E(GAA)61T(ACA) | ||
| 5 | N(AAT)65F(TTT)∗ | |||
| 6 | Y(TAT)82N(AAC)∗ | |||
| 7 | Q(CAG)93K(AAG) | |||
| 8 | S(TCG)120D(GAT) | |||
| 9 | N(AAC)123V(GTC) | |||
| 10 | D(AAC)132T(ACT) | |||
| 11 | N(AAC)32D(GAC), N(AAC)37Q(CAG) | |||
| 12 | N65F, Y82N∗ | |||
| 13 | S120D, N123V | |||
| 14 | F(TTT)13L(TTG),R(CGT)15D(GAT), V(GTA)24C(TGT) | |||
| 15 | Y(TAT)100L(TTG), D(GAT)101T(ACT); T(ACA)107L(TTA), Q(CAA)110C(TGT) | |||
| 16 | R(AGG)54F(TTC), L(TTG)73T(ACT), E(GAA)75K(AAA), T(ACG)144W(TGG), V(GTA)145T(ACA), S(TCT)148Y(TAT), W(TGG)149A(GCA) | 18 | ubc4; F(TTC)63N(AAC), N(AAT)80Y(TAT), D(GAT)118S(TCT), V(GTA)121N(AAT)∗ | |
| 17 | R(AGG)8Q(CAG) | |||
| C | 19 | rad6ΔC1–10 (rad6 residues 1–10 are replaced by residues 1–8 of Ubc4) | 22 | ubc41–8 (Ubc4 residues 1–8 replaced by residues 1–10 of RAD6) |
| 20 | rad6ΔCCdc34a (Cdc34 residues 101–112 between rad6Δ Q93 and N94) | 23 | ubc41–50 (Ubc4 residues 1–50 replaced by residues 1–52 of Rad6) | |
| 21 | rad6ΔCCdc34b (Cdc34 tail residues 171–295 after rad6Δ), N65F | |||
| D | 24 | ubc1ΔC; R(AGG)6A(GCA) | ||
| 25 | ubc1ΔC; S(TCG)97R(AGG) | |||
| 26 | ubc1ΔC; A(GCG)111R(AGG) | |||
| 27 | ubc1ΔC; E(GAA)125A(GCC) | |||
| 28 | ubc1ΔC; E(GAA)125A(GCT), H(CAC)129A(GCC), L(TTA)131A(GCA), R(CGG)132A(GCG), E(GAA)135A(GCA) | |||
| 29 | ubc1ΔC; R(AGA)134A(ACG) | |||
a
Shown are the codon and residue changes for each of the yeast-high copy E2 expression plasmids used in the present study. Plasmids are grouped (A to D) and numbered (1 to 29) according to substitution type. A, E2 catalytic domains used as positive controls; B, reciprocal amino acid substitutions made between rad6ΔC and UBC4. C, transposition of amino acid stretches; D, selected nonreciprocal substitutions. Asterisks indicate plasmids created and tested in both high-copy and low-copy forms.