Partial identification by site-directed mutagenesis of a cell growth inhibitory site on the human galectin-1 molecule

Table 2 Oligonucleotide Primers used for Overlap Extension

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 gene GRP3: CTG GGATCC ATG GCT TGT GGT CTG GTC (BamH I site is bold and underlined) Reverse primer for galectin-1 gene GRP4: 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.

ISSN: 2661-8850