Biological activity of a putative maize Adh-1S promoter determined by transient expression in corn protoplasts

Maize alcohol dehydrogenase (Adh) genes constitute one of the most well characterized gene systems in plants. The Adh-1S allele has been cloned, sequenced and a 1.4 kb (BamHI-HindIII) fragment was identified as the promoter region (Dennis et al., Nucl. Acid. Res. 12:3983, 1984). We demonstrate biological activity of this fragment by a transient transformation assay described below.

A recombinant plasmid pSH 17 was constructed. This was a derivative of pSV cat plasmids used in transient expression studies in mammalian cells (Gorman et al., Molec. Cell. Biol. 2:1044, 1982) and Drosophila cell lines (DiNocera and Dawid, PN.A.S. 80:7095, 1983). The promoter fragment of the Adh1-S allele (described above) was placed at the 5' end of the CAT (Chloramphenicol Acetyl Transferase) coding region. This construct has the globin gene splice signals and polyadenylation sequences from the original pSV cat plasmid at the 3' end of the CAT coding region. The control plasmid pCM 7 has CAT coding region at the 3' end of the Tetracycline (Tc) resistance gene promoter. Purified plasmid DNA was delivered as a calcium phosphate co-precipitate, as described by Graham and VanderEb (Virology 52:456, 1973), to corn protoplasts and three days later the protoplasts were homogenized and assayed for CAT activity using 14C-chloramphenicol, as described by Shaw and Brodsky (J. Bact. 95:28, 1968). The reaction products of CAT activity (chloramphenicol-1-acetate, 3-acetate and 1,3-diacetate) were resolved by thin layer chromatography and visualised by autoradiography (Figure 1).

The pSH 17 treated corn protoplasts show CAT activity, while protoplasts treated with pCM 7 did not. Interestingly, pSH 17 produced very little CAT in E. coli (basal level), while pCM 7 produced a significant amount of CAT (data not shown). These results indicate that the 1.4 kb fragment contains a biologically active promoter sequence, however it is not known if the total sequence is required for expression.


Narayana R. Isola, Prem S. Chourey, Diana Z. Sharpe and Elizabeth S. Dennis

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