Table 2.
Mutants | Primers |
M1R |
M1R: CTG GGA TCC AGG GCT TGT GGT CTG GTC GRP4: ACGCGTC GAC TCA GTC AAA GGC CAC ACA |
V6D |
V6D: CTG GGA TCC ATG GCT TGT GGT CTG GAC GCC AGC AAC GRP4: ACGCGTC GAC TCA GTC AAA GGC CAC ACA |
D27N |
D26N-F: GTG GCT CCT AAC GCT AAG AGC D26N-R: GCT CTT AGC GTT AGG AGC CAC |
A28D |
A28D-F: GCT CCT GAC GAT AAG AGC TTC A28D-R: GAA GCT CTT ATC GTC AGG AGC |
A28R |
A28R-F: GCT CCT GAC CGT AAG AGC TTC A28R-R: GAA GCT CTT ACG GTC AGG TTC |
K29M |
K29M-F: CCT GAC GCT ATG AGC TTC GTG K29M-R: CAC GAA GCT CAT AGC GTC AGG |
K29T |
K29T-F: CCT GAC GCT ACG AGC TTC GTG K29T-R: CAC GAA GCT CGT AGC GTC AGG |
N47D |
N47D-F: CTG CAC TTC GAC CCT CGC TTC N47D-R: GAA GCG AGG GTC GAA GTG CAG |
P79R |
P79R-F: GCT GTC TTT CGC TTC CAG CCT P79R-R: AGC CTG GAA GCG AAA GAC AGC |
D103A |
D103A-F: AAG CTG CCA GCT GGA TAC GAA D103-R: TTC GTA TCC AGC TGG CAG CTT |
C131S |
GRP3: CTG GGA TCC ATG GCT TGT GGT CTG GTC C131S-R: ACGCGTCGACTCA GTC AAA GGC CAC AGA TTT GAT CTT |
R49G |
R49G-F: TTC AAC CCT GGC TTC AAC GCC. R49G-R: GGC GTT GAA GCC AGG GTT GAA. |
Forward primer for galectin-1 geneGRP3: CTG GGATCC ATG GCT TGT GGT CTG GTC (BamH I site is bold and underlined) Reverse primer for galectin-1 geneGRP4: ACGC CTCGAC TCA GTC AAA GGC CAC ACA (SaI I site is bold and underlined). Mutagenesis in Galectin-1 In the case of internal mutations, the primers listed were used in conjunction with the 5' and 3' primers (GRP3 and GRP4), to make two, overlapping galectin-1 cDNA fragments, which then were annealed and extended to create galectin-1 cDNA with the intended substitution. In the case of substitutions near the ends of the molecule, a modified version of GRP 3 or 4 was used, in conjunction with the other, normal primer, for conventional PCR amplification of the cDNA.