Promoter activity, ARS function, dnaA binding sites and transposition of the activator (Ac) element --James H. Zhou and Alan G. Atherly Our previous observations showed that the end(s) of Ac exhibit: (i) prokaryotic promoter activity when fused to a promoter-less gene encoding chloramphenicol acetyltransferase (CAT) in E. coli, (ii) dnaA protein-binding repeats (TTATACACA), overlapped with the promoter-like repeats, and (iii) functional DNA autonomous replication sequences (ARS) when introduced into yeast cells. We now report construction of a set of defective Ac elements with internal replacements and subterminal deletions that we used to analyze the function of the terminal sequences of Ac. To test functionality each of the defective Ac elements was inserted into the XbaI site, in both orientations, between a CaMV 35S promoter and a GUS reporter gene in the Agrobacterium binary vector pZA3 (Zhou and Atherly, Plant Cell Reports, 1989, in press), and then co-transferred with a normal Ac-carrying plasmid pZAc20 into tobacco cells via T-DNA-mediated transformation. Expression of GUS activity was used to detect excision of the defective Ac elements. We found that a minimal 3'-terminal sequence of 95 bp, plus a short internal sequence between bp 3630 and 3644 (or 188 bp without any internal sequence) was required for transposition of the artificially defective Ac elements.

The deletion of the distal putative ARS on the 3'-end gave an approximate three-fold decrease in transposition frequency, and deletion of both the proximal and distal ARS's gave no transposition, suggesting the putative ARS's may be required for high efficiency of the transposition. Another very interesting finding was that the 3'-terminal sequence was found to be highly homologous to the region of DNA replication of maize strip virus (Dellaporta, 1989, personal communication), suggesting the origin of DNA replication at the 3'-end of Ac element may be needed for transposition.

Deletions of the dnaA protein-binding sequences in the 3'-end gave a read-through phenotype when in the same orientation with respect to the GUS gene, but not in the reverse orientation. This observation is analogous to that of prokaryotes, in which the DNA protein binding sites function as a terminator of transcription when present in one strand of the DNA double helix (Trends in Genetics 5:319-321, 1989). A surprising observation is that a short internal sequence between bps 3630-3644 was found to be a terminator that prevented read-through when present in the reverse orientation with respect to the GUS gene. But, both terminators were inactivated when an active Ac element was present in trans. However, one termination sequence (when in the reverse orientation) showed more Ac-dependence than the other. An analogous observation was made in the maize R locus where an Ac element was inserted into the regulatory sequence (Dellaporta, 1989, personal communication). We theorize that this may be due to the relaxation of the DNA double helix when the product of the Ac element interacts with the subterminal sequence of the defective Ac element.

We speculate from these data that the functional sequences observed within the terminal sequences of Ac (promoters; dnaA binding sequences, which also function as terminators in plants; and ARS's), that have evolved from three very divergent organisms (E. coli, yeast, and plants) play analogous functions in each of these organisms. Also, promoter activity identified at both ends of the element may correlate with transposition, and dnaA protein-binding sites, as well as ARS's may correlate with regulation of transposition frequency.

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