MultipleCis-acting DNA elements that regulate transcription of the adenovirus 12E1A gene

Abstract

To delineate cis-acting elements for adenovirus (Ad) 12 E1A gene transcription, we transfected HeLa and NIH3T3 cells with DNAs having various deletions in the 5'-upstream region linked to the chloramphenicol acetyltransferase gene. Deletions in the regions between nucleotide (nt) positions 54 and 166, and 167 and 200, with respect to the left end of the viral genome at nt position 1, caused a two- to three-fold reduction in transcription. Transcription decreased to an almost undetectable level with loss of the region between nt positions 201 and 282. The effect of these mutations was almost consistent between both cell lines. The region between nt positions 77 and 94 stimulated transcription when situated upstream of the simian virus 40 early promoter in either orientation. Transcription was stimulated about ninefold in the presence of the DNA that encodes the product of the 13S, but not the 12S mRNA of the Ad12 E1A gene. These results indicate that transcription of the Ad12 E1A gene is regulated by multiple cis-acting elements and is stimulated by its own gene product.

A cis-acting element 7 bp upstream of the ESF-1-binding motif is involved in E1A 13S autoregulation of the adenovirus 12 TS2 promoter.

“…To investigate whether the E1A TS2 promoter is also regulated by the 13S mRNA product, and to determine the elements required for promoter activity stimulated by this E1A protein, we carried out transient CAT expression assays. In this context it should be noted that the 12S mRNA product of Ad12 was previously shown not to be involved in positive regulation of E1A gene expression (Yamazaki et al, 1992). A pRc\RSV-13S cDNA expression vector, encoding the 13S mRNA of the Ad12 E1A gene, was co-transfected into human A549, Raji and HeLa cells together with the TS2 promoter constructs (shown in Fig.…”

“…Evidence from transient expression assays suggests that Ad12 E1A transcription is positively autoregulated by its largest gene product, the 13S protein (Kirch et al, 1993 ;Yamazaki et al, 1992), but is not affected through the protein translated from the 12S mRNA (Yamazaki et al, 1992).…”

Transcription stimulation of the adenovirus type 12 E1a gene in vitro by the 266-amino-acid E1A protein

“…A recombinant DNA was prepared and expressed by the procedure described by Koikeda et al (22). Briefly, a DNA fragment encompassing the entire coding sequence of the Adl2 266-amino-acid ElA protein (266-R E1A) was amplified by PCR with the p12H13S DNA (47,60) and ligated with the pGa DNA (22) so that the Ela insert was headed by the prepro-sequence of the Saccharomyces cerevisiae ot-factor and tailed by the transcription terminator sequence of the S. cerevisiae Pgk gene. The resulting DNA, pGoaElA13, was inserted into the yeast expression vector pAM82 (34), in which the inducible PHOS promoter directs transcription of the insert when the phosphate concentration in the medium is lowered.…”

“…The intensity of the signals derived from the accurate transcripts was determined by using an image analyzer (BA 100; Fuji Photo Film). The structure of the template DNAs was as described previously (60).…”

“…However, the regulation of Ela gene expression is not fully understood. We previously showed by DNA transfection that the 13S but not the 12S mRNA product of the Ela gene of the highly oncogenic Adl2 stimulates the expression of its own gene (60). Positive autoregulation of the Ela gene of nononcogenic Ad serotypes has also been demonstrated (19).…”