A distinct intron-DNA structure in simian virus 40 T-antigen and adenovirus 2 E1A genes

Abstract

Distinct structures delineating the introns of simian virus 40 T-antigen and adenovirus 2 El A genes have been discovered. The structures, which are centered around the branch points of the genes inserted in supercoiled double-stranded plasmids, are specifically targeted through photoactivated strand cleavage by the metal complex tris(4,7-diphenyl-l,10-phenanthroline)rhodium(III). The DNA sites that are recognized lack sequence homology but are similar in demarcating functionally important sites on the RNA level. The single-stranded DNA fragment corresponding to the coding strand of the El A gene was also found to fold into a structure apparently identical to that in the supercoiled ElA gene based on the recognition by the metal complex. Further investigation of different single-stranded DNA fragments showed that the structure requires the sequences at both ends of the intron plus the flanking sequences but not the middle of the intron. These results provide direct evidence that the positions of these introns are structurally encoded on the DNA level.Since the discovery of introns, much attention has been focused on the mechanism of the processing of the nascent transcript to the messenger RNA, a process which involves a myriad of factors and precise splicing of the folded RNA molecule (Padgett et al., 1986;Green, 1986;Maniatis & Reed, 1987; Cech, 1986). Since most eukaryotic genes have been found to contain introns, their persistence in a higher order structure of genes suggests that intrans confer an evolutionary advantage to the organism, an advantage great enough to justify maintaining the elaborate splicing machinery. But what function(s) does (do) intrans serve? Here we report finding an unusual structure in the intron DNA of two mammalian viral genes. The results we describe establish that in these genes the position of intrans are encoded structurally in the DNA.Structural probing of the intron DNA was carried out with tris( 4, 7-diphenyl-l, 10-phenanthroline) rhodium(III) [Rh(DIP)J 3 +] (Figure 1), a metal complex designed in our laboratory which lacks hydrogen-bonding functionalities and t We are grateful to the NIH for their support of this work.

Chemical probing of tDNAPhe with transition metal complexes: A structural comparison of RNA and DNA

“…In several RNAs, Rh(DIP) 33+ has been shown to target the base to the 3'-side of a GU mismatch within double-helical regions . In the absence of a strong recognition site, on DNAs a faint reaction at guanines has generally been found (Lee & Barton, 1993).…”

“…0006-2960/93/0432-11029S04.00/0 © 1993 American Chemical Society has been shown to intercalate in the major groove of doublehelical DNA at 5'-pyr-pyr-pur-3'steps (Pyle etal., 1989(Pyle etal., ,1990Sitlani et al, 1992; David & Barton, 1993) and, consistent with this recognition, to target sites of tertiary interaction in folded RNAs (Chow & Barton, 1990;Chow et al, 1992a,b). Rh(DIP)33+ targets unusual DNA tertiary structures, such as cruciforms, Z-DNA, and Holliday junctions (Kirshenbaum et al, 1988;Lee & Barton, 1993), while it targets primarily GU mismatches in RNAs . Ru(TMP)32+ binds against the shallow minor groove of RNA and DNA double helices which are A-like in character (Mei & Barton, 1986Chow & Barton, 1990).…”

Guanine Specific DNA Cleavage by Photoirradiation of Dibenzoyldiazomethane−Oligonucleotide Conjugates

“…Various strategies for photoinduced DNA cleavage have been intensively investigated. These included a generation of active oxygen species − or photoreactive metal center, an electron transfer from DNA nucleobases, − hydrogen abstraction from DNA sugar backbone by photogenerated radicals, − and a photoinduced DNA alkylation. − Among these approaches DNA alkylation by highly electrophilic species generated by light-triggered reactions is particularly attractive, since electrophilic species have an intrinsic reaction selectivity to nucleophilic nucleobases such as guanine and adenine. Photochemical methods for the generation of such electrophiles so far reported are (i) the photodecomposition of arylazide eventually producing azaheptatetraene via aryl nitrene,20a (ii) photoheterolysis of quinolylmethylisothioronium salt generating quinolylmethyl carbocation,20b (iii) photoenolization of 5-methylnaphthoquinone giving a quinone methide, and (iv) the generation of oxonium cation from hemithioacetal by photoinduced electron transfer …”

Transient foamy virus vector production by adenovirus vectors

“…Although these cells do not contain the adenoviral E1 gene, they do express the SV40 large T antigen that has been shown to share sequence homology and some functionality with the adenovirus E1A gene. [39][40][41][42][43] However, the characteristic adenovirus cytopathic effect (CPE) was not observed during the experiment. Cells (Cos-7), seeded at 8 Â 10 5 cells/well of a six-well plate 1 day prior to transfection, were transfected with 4 mg pAd-MD9, 6 mg pAd-GagPolDPacI and 3 mg pAd-Env using 10 mM polyethylenimine (PEI) (Sigma, UK).…”