Primers listed below are for making mutants with altered CACC repeat motif in MT-1 3′UTR Δ28,295′-gtgctgctgccctcagccgtaaataatttccggac-3′5′-gtccggaaattatttacggctgagggcagcagcac-3′ Δ68,695′-cagagtcttgccgtacggctccttccagtttac-3′5′-gtaaactggaaggagccgtacggcaagactctg-3′ Δ74,755′-gtcttgccgtacacctccttccagtttactaaaccccg-3′5′-cggggtttagtaaactggaaggaggtgtacggcaagac-3′ Each site-directed mutagenesis reaction effected a single nucleotide
IGF-1 DES when producing shortened stem MT-1 3′UTR mutants or replacement of two nucleotides when making MT-1 3′UTR mutants containing altered CACC repeat motif. The designated removal or substitution of several bases was achieved by sequential PCR with the forward and reverse primers given above. Full-size table Table optionsView in workspaceDownload as CSV In vitro transcription of radiolabelled and unlabelled RNAs. Templates for transcription of rat MT-1 3′UTR
nucleotides 1–111 and the various mutants were generated by PCR from pcMTfull, or the appropriate mutant construct, with the following primers;- T7 Forward 5′-TAATACGACTCACTATAGGAGTGACGAACAGTGCTGCTG-3′ containing the promoter sequence (underlined) and Reverse 5′-CACATGCTCGGTAGAAAACGG-3′. PCR products were purified using QIAquick columns (Qiagen). For electrophoretic mobility-shift assays RNA corresponding to nucleotides 1–111 of the MT-1 3′UTR (MT-111) was synthesized incorporating radiolabelled [α-32P]CTP (GE Healthcare) using the MAXIscript? kit (Ambion, Applied Biosystems). The synthesized MT-111 mRNA was purified by phenol–chloroform extraction, and ethanol precipitated. Unlabelled RNA or transcripts incorporating biotin-16UTP (Roche) were produced using the MEGAshortscript kit (Ambion) and quantified spectrophotometrically.
