Analysis of the function and regulation of the 94kDa glycoprotein of the glucose-regulated protein family. - Page 31 |
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primer 5’ CCtgtcgactCGCCGCACCACGCAC-3? (lower case denotes mutated
sequence) and an upstream CAT primer (Wooden et al., 1991) were used with
pGRP94 (-357) CAT as template to amplify a 200-bp region o f the promoter by
polymerase chain reaction (PCR). By using LS primer 5r-CGagtcgacaGGGTT
CATGTTCCC 3’ and a CAT 22-nucleotide oligonucleotide primer (Wooden et
al., 1991), a 300-bp region was similarly amplified. The two amplified
fragments were then mixed together and reannealed, generating a template
where the CCAAT site contained within -18 to -71 was mutated and was
flanked by the wild-type promoter sequence. Using this as template and the
CAT and reverse CAT primers, we amplified a 500-bp region in which the
grp94 C 1 CCAAT motif was mutated. This fragment was cloned into the Hind
III site o f the pSVOCAT vector as previously described (Resendez et al., 1985).
The 94 C 1 mutation and orientation were confirmed by restriction mapping with
the Sal 1 site introduced in the mutated sequence.
1.2.3 Transient cotransfections
In these transfection experiments, 10 pg o f the grp-promoter/CAT fusion
plasmids were co-transfected with 5 pg o f either pMT2, pSB479 or pRB9221
per dish using the calcium phosphate precipitation method. In each transfection
mixture, 3 pg o f HeLa carrier DNA was added. Cells were shocked with 15%
glycerol for 4 h following transfection, to optimize for the transfection
15
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| Title | Analysis of the function and regulation of the 94kDa glycoprotein of the glucose-regulated protein family. - Page 31 |
| Repository email | cisadmin@lib.usc.edu |
| Full text | primer 5’ CCtgtcgactCGCCGCACCACGCAC-3? (lower case denotes mutated sequence) and an upstream CAT primer (Wooden et al., 1991) were used with pGRP94 (-357) CAT as template to amplify a 200-bp region o f the promoter by polymerase chain reaction (PCR). By using LS primer 5r-CGagtcgacaGGGTT CATGTTCCC 3’ and a CAT 22-nucleotide oligonucleotide primer (Wooden et al., 1991), a 300-bp region was similarly amplified. The two amplified fragments were then mixed together and reannealed, generating a template where the CCAAT site contained within -18 to -71 was mutated and was flanked by the wild-type promoter sequence. Using this as template and the CAT and reverse CAT primers, we amplified a 500-bp region in which the grp94 C 1 CCAAT motif was mutated. This fragment was cloned into the Hind III site o f the pSVOCAT vector as previously described (Resendez et al., 1985). The 94 C 1 mutation and orientation were confirmed by restriction mapping with the Sal 1 site introduced in the mutated sequence. 1.2.3 Transient cotransfections In these transfection experiments, 10 pg o f the grp-promoter/CAT fusion plasmids were co-transfected with 5 pg o f either pMT2, pSB479 or pRB9221 per dish using the calcium phosphate precipitation method. In each transfection mixture, 3 pg o f HeLa carrier DNA was added. Cells were shocked with 15% glycerol for 4 h following transfection, to optimize for the transfection 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. |
