Automated agarose gel electrophoresis of dsDNA fragments on a commercial DNA sequencer.

vectors may also be affected depending on whether these regions contribute to this activity (14). Interestingly , although pGL3-basic (Promega, Madison, WI, USA) is derived from pUC vectors, it lacks many of the AP1 motifs found in pBLCAT3; however, it is still transactivated in experiments where other transcription factors such as NF-Y, c-Ets-2, PEA3, Sp1, or GATA-4 are overexpressed (D.A. Young, unpublished observations). This research suggests that caution is necessary when overexpressing transcription factors with reporter plasmids during promoter analysis. Furthermore, it underlines the necessity of including a vector-only control in such studies. Several groups have designed new CAT vectors by deleting prokaryotic sequences immediately upstream of the CAT gene and replacing them with fragments containing a tandem dimer of the SV40 early polyadenylation signal (1,3,11,14). This prevents the processing of mature CAT mRNA from the vector and subsequent translation. Indeed, this can reduce pBLCAT3 vector read-through by up to 8-fold (3). CAT vectors such as pBLCAT5 (ATCC reference no. 77412) and pBLCAT6 (ATCC reference no. 77413) (1), which are derived from pBLCAT2 and pBLCAT3 or other modified vectors such as pJFCAT1 (3), have these modifications. However, these vectors may also need to be checked for transcription factor inducibility before being used in promoter analysis. 1992. Reporter constructs with low background activity utilizing the cat gene. Gene 110:129-130.ciferase reporter gene vectors that lack potential AP-1 sites. BioTechniques 27:220-222. and glucocorticoids:novel effects of fos family proteins. 1988. c-Jun dimer-izes with itself and with c-Fos, forming complexes of different DNA binding affinities. Cell 55:917-924.latory elements lurking in plasmid DNA: the activator protein-1 site in pUC. Mol. En-docrinol. 8:405-407.bell, and Z. Werb. 1996. Synergistic transcrip-tional activation of the tissue inhibitor of metal-loproteinases-1 promoter via functional interaction of AP-1 and Ets-1 transcription factors. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 15:5490. A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plas-mid-initiated transcription. BioTechniques 7: 276-280. 1999. A sequence-selective single-strand DNA binding protein regulates basal transcription of the murine tissue in-hibitor of metalloproteinases-1 (Timp-1) gene. Cryptic promoter activity within the backbone of a plasmid commonly used to prepare pro-moter/reporter gene constructs. In Vitro Cell. Electrophoretic separation of dsD-NA molecules, such as PCR products and restriction digest fragments, is usually accomplished in agarose and sometimes in composite agarose-polyacry-lamide gels (3). Under nondenaturing conditions, the electrophoretic mobility of dsDNA fragments …