Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene.

Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor.     

Wound-responsive cis-element in the 5'-upstream region of cucumber ascorbate oxidase gene

The cucumber (Cucumis sativas) AAO1 gene (former name, Aso1) encodes an ascorbate oxidase that catalyzes the oxidation by molecular oxygen of ascorbic acid to dehydroascorbate. CsAAO1 mRNA concentrations rose rapidly after mechanical wounding of cucumbers. To study the wound-responsive expression of CsAAO1 in detail, we examined transgenic tobacco plants harboring a CsAAO1 promoter-beta-glucuronidase fusion gene. CsAAO1 promoter activity in leaves of the tobacco was induced by wounding. Analysis of the regulatory properties of 5'-deleted promoter fragments showed that a putative wound-responsive cis-element (WRE) was located -736 to -707 bp from the translation initiation site. DNA binding factors that bound specifically to the putative WRE sequence were identified in tobacco nuclear extracts by gel retardation assays.     

N6-methyldeoxyadenosine residues at specific sites decrease the activity of the E1A promoter of adenovirus type 12 DNA

The activity of eukaryotic promoters is highly sensitive to site-specific modifications by DNA methylations. We have used the E1A promoter of adenovirus type 12 (Ad12) DNA to investigate the effects of methylations at different promoter sites on its activity. The chloramphenicol acetyltransferase gene has served as an activity indicator. Activity of the E1A promoter is lost or markedly decreased by deoxycytidine methylation of two HpaII (5'-C-C-G-G-3') or seven HhaI (5'-G-C-G-C-3') sites upstream from the 3' located T-A-T-A signal. There are two T-A-T-A signals in the E1A promoter of adenovirus type 12 DNA, one T-A-T-T-A-T sequence starting at nucleotide 276 (5' located), a second T-A-T-T-T-A-A sequence starting at nucleotide 414 (3' located). Deoxycytidine methylations at two AluI (5'-A-G-C-T-3') sites downstream from the 5' located T-A-T-A signal have no effect on promoter activity. When one EcoRI (5'-G-A-A-T-T-C-3') or one TaqI (5'-T-C-G-A-3') sequence at 281 base-pairs upstream or 61 base-pairs downstream from the 5' located E1A T-A-T-A signal, respectively, is deoxyadenosine methylated, the promoter becomes inactive. Deoxyadenosine methylation at one MboI (5'-G-A-T-C-3') site, which is located 127 nucleotides downstream from the 5' located T-A-T-A signal, fails to decrease E1A promoter activity. There is no conspicuous anatomical relation of any of these sites to the two presumptive enhancer sequences in the E1A promoter. We conclude that 5-deoxymethylcytidine or N6-methyldeoxyadenosine residues have to be introduced at highly specific promoter sites to inactivate the promoter. These sites are probably different for different promoters.     

Production of a monospecific antiserum against the early region 1A proteins of adenovirus 12 and adenovirus 5 by an adenovirus 12 early region 1A-beta-galactosidase fusion protein antigen expressed in bacteria.

Antisera were prepared against the amino acid sequences encoded within the N-terminal half of the adenovirus 12 (Ad12) early region 1A (E1A) gene. This was accomplished by construction of a plasmid vector which encoded the N-terminal 131 amino acids of Ad12 E1A joined in frame to the coding sequence of beta-galactosidase. After induced synthesis in Escherichia coli, the Ad12 E1A-beta-galactosidase fusion protein (12-1A-FP) was extracted with urea and used to raise antibodies in rabbits. The 12-1A-FP antisera immunoprecipitated major phosphoproteins of 39,000 and 37,000 apparent molecular weights from Ad12-transformed and infected cells. The 12-1A-FP antisera also immunoprecipitated E1A phosphoproteins from Ad5-transformed and infected cells. Immunospecificity of the 12-1A-FP antisera was demonstrated by the ability of 12-1A-FP antigen to block immunoprecipitation of E1A proteins. Furthermore, E1A proteins immunoprecipitated from in vivo-labeled cells comigrated with those translated in vitro by RNA that had been hybridization selected to E1A DNA.     

Xenobiotic responsive element in the 5'-upstream region of the human P-450c gene.

The nucleotide sequence of the 5'-upstream region up to about -4.1 kb of the human P-450c gene was determined. Two kinds of repetitive sequences were located; one was the Alu sequence which was inserted at three positions (-3127 to -3038, -3017 to -2770, and -2167 to -1851), and the other was the SINE-R element located just upstream of the most distal Alu sequences. The region other than the two repeated sequences showed an overall similarity of 70% to that of the rat P-450c gene. Survey of XRE or its homologues, responsible for the inducible expression of the rat P-450c gene, revealed eight XRE core sequences in this region of the human P-450c gene. Three of them were carried in the Alu sequences. A fusion gene which was constructed by ligating the upstream region of the human P-450c gene to the chloramphenicol acetyltransferase (CAT) gene expressed the CAT activity in response to the inducer, methylcholanthrene, when transfected into Hepa-1 cells. Stepwise decrease in CAT activity in three regions was observed as the 5'-upstream sequence containing XRE motifs was removed. However, the XRE core sequence in the Alu sequences seemed inactive, because elimination of the three elements in the Alu sequences did not affect the expressed CAT activity. In accordance with this observation, competition experiments using gel mobility shift assay showed that XRE core sequences in the Alu sequences could not compete with the XRE sequence for the inducer-bound receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a nondefective recombinant between adenovirus type 5 and early region 1A of adenovirus type 12.

A nondefective recombinant between adenovirus type 5 (Ad5) and type 12 (Ad12), rc-1 (Ad5 dl312, carrying the Ad12 E1A gene), was isolated from hamster cell foci transformed by a defective recombinant, rcB-1 (dl312, carrying the Ad12 E1 gene). The recombinant rc-1 grew in human embryo kidney and KB cells in the absence of helper and synthesized Ad12 T antigen g, the product of the E1A gene. The genome of rc-1 has a deletion between 79.9 and 82.5 map units of Ad5 dl312 DNA with an insertion of 0.1 to 5.5 map units of Ad12 DNA at the deletion site. The mRNAs of Ad12 E1A were transcribed from the Ad12 E1A promoter, and unusual RNAs were abundantly transcribed from the Ad5 E3 promoter on the opposite strand. The frequency of cell transformation with rc-1 was lower than those with Ad5 and Ad12 wild types.