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Supporting Methods
The rat ASBT (rASBT) full-length 3’ untranslated region (3’UTR) was cloned into pCRII-TOPO (pCRIIrASBT3’/3.1)(Invitrogen, Carlsbad, CA) by RT-PCR and TA cloning, using IEC-6 cell mRNA template
(primers:
5’-ACACTAAGGAGGCAAAAGAAGAAAGC-3’
and
5’-
CTTTCCTTTAAAATATAAATATTC-3’ (DNA Sequencing Core Facility, Mount Sinai School of
Medicine, New York, NY). The 2.1 kb human ASBT (hASBT) full-length 3’ untranslated region
(3’UTR) was subcloned into XbaI/KpnI sites of the in vitro transcription vector pSPT18 (Roche, Nutley,
NJ), by RT-PCR and subcloning techniques, using Caco-2 cell mRNA template and primers: 5’GCTCTAGAACATCAAGTGGACAAAACAGACG-3’
and
5’-
GCGGTACCTAAGCCTTTAAAATAAAATATTC-3’ (Integrated DNA Technologies, Coralville, IA).
The correct sequence of the resultant construct pSPT18-hASBT3’/2.1 was confirmed by DNA sequencing
(Genomics and Proteomics Core Laboratories, University of Pittsburgh, Pittsburgh, PA). Three
mammalian cell expression vectors as described below were used to make hybrid constructs, with the
insertion of cDNA fragments corresponding to the rASBT 3’UTR sequences 1155-4270 (3.1 kb, the full
3’UTR region), 2434-4128 (1.7 kb) and 2114-2434 (0.3 kb) (GenBank NM_017222) (Figure 1A). To
generate the series of pGL3 constructs that include rat ASBT sequences in the 3’ UTR of luciferase
mRNA (rASBT3’-luciferase), pCRII-rASBT3’/3.1 was digested with XbaI, that produced DNA
fragments containing the 1.7 kb and 0.3 kb 3’UTR.
The constructs rASBT3’-luciferase/1.7 and
rASBT3’-luciferase/0.3 were generated by subcloning of the 1.7 kb and 0.3 kb XbaI fragments,
respectively, into an XbaI-digested pGL3-Promoter vector (Promega, Madison, WI), in an orientation so
that the 3’UTR sense strand cDNA downstream of a firefly luciferase gene was transcribed by an SV40
promoter. The construct rASBT3’-luciferase /3.1 was generated by blunt-end ligation of the 3.1 kb
3’UTR fragment with the XbaI-nicked vector, following fill-in of the vector’s two 5’-protruding cohesive
ends by Klenow Fragment (Invitrogen, Carlsbad, CA). The intact vector pGL3-Promoter was used as a
control construct in luciferase assays. In order to prepare a similar series of constructs where the 3’UTR
was inserted downstream of a ß-globin reporter, pCRII-rASBT3’/3.1 was used as a template and the
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following three pairs of DNA primers were in vitro synthesized (DNA Sequencing Core Facility, Mount
Sinai School of Medicine, New York, NY. Production of the 3.1, 1.7 and 0.3 kb 3’UTRs utilized the
following primers respectively: (3.1 kb) 5’-CGACACTAAGGAGGCAAAAGAAG-3’ and 5’CGGGTACCCTTTCCTTTAAAATATAAATATTC-3’;
(1.7
kb)
and
CGTCTAGAAAATGACAATGGAGAG-3’
5’-
(0.3
CGGGTACCTCTAGAGTATTAGGGGAAAAATAAAC-3’;
5’-
kb)
5’-
CGTCTAGAAGTAGAAATGAGAGA-3’ and 5’-CGGGTACCTCTAGACTGATACCTTTTTTCC-3’.
The three PCR-produced 3’UTR fragments were digested with KpnI to generate a 3’-protruding terminus
at their 3’ ends. The vector pBBB was digested with BglII/KpnI, and the linearized vector was bluntended by fill-in with Klenow fragment at the BglII-end with a protruding 5' terminus, generating the
vector pBBB-BglII(b)/KpnI. The BglII/KpnI digestion of pBBB resulted in a nearly complete removal of
the β-globin 3’UTR (88 bp out of 94 bp), starting from the 7th bp downstream of the translation
termination codon. The constructs rASBT3’-ßglobin/3.1, rASBT3’-ßglobin/1.7 and rASBT3’-ßglobin/0.3
were prepared by insertion of the three rASBT 3’UTR fragments, respectively, into the nicked vector
pBBB-BglII(b)/KpnI, in a sequence in that the rASBT 3’UTR was located immediately downstream of
the β-globin coding region. The construct pBBB-G3’d88 was made by self blunt-end ligation of pBBBBglII(b)/KpnI, following 3’-5’ digestion by DNA polymerase I of the KpnI 3’-protruding end prior to
addition of deoxynucleoside triphosphates (Invitrogen, Carlsbad, CA).
control construct in β-globin half-life assays.
pBBB-G3’d88 was used as a
In the preparation of the series of pSPT18 in vitro
transcription constructs, pSPT18-rASBT3’/1.7 and pSPT18-rASBT3’/0.3 were produced by subcloning
of the 1.7 kb and 0.3 kb XbaI fragments, respectively, into an XbaI site in the polylinker of the vector
pSPT-18 (Roche, Nutley, NJ), in a direction in that the sense strand 3’UTR cDNA was in-vitro
transcribed by either T7 or SP6 promoter flanking the polylinker.
The full-length 3’UTR construct
pSPT18-rASBT3’/3.1 and pSPT18-hASBT3’/2.1 were obtained by insertion of the 3.1 or 2.1 kb PCR
fragment into a SmaI-digested pSPT-18 vector. All the constructs described above were examined by
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restriction enzyme digestions and confirmed by DNA sequencing, in order to identify the correct
construction and insert orientation.
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