Sequence of inverted terminal repetitions from different adenoviruses: demonstration of conserved sequences and homology between SA7 termini and SV40 DNA.

We have established the nucleotide sequence for the inverted terminal repetition of human adenovirus type 3, a subgroup B adenovirus. The repetition, which is 136 bp long, shows a high degree of homology with the known sequence for the inverted repetition of adenovirus type 5 (Steenbergh et al., 1977) a subgroup C adenovirus. Partial sequence information convering 120 bp of the inverted terminal repetitions of human serotype 12, a subgroup A member, and of simian adenovirus type 7 has also been obtained. A comparison of the established sequences shows that the terminal repetitions, in particular the first 50 bp from the ends, contain sequences that have been well conserved in adenovirus evolution. For instance, only six mismatched base pairs were detected among the first 50 bp in the repetitions of simian adenovirus type 7 and human adenovirus type 5, although the homology between simian adenovirus 7 and human subgroup C adenoviruses was estimated to be only 30%. A 14 bp sequence located 9-22 nucleotides from the ends is present in DNAs from all the human serotypes examined as well as in simian adenovirus 7 DNA. Furthermore, the simian adenovirus 7 repetition contains a 21 bp sequence which is present in SV40 DNA, close to the origin of DNA replication.     

Polar encapsidation of adenovirus DNA: cloning and DNA sequence of the left end of adenovirus type 3

The left-end adenovirus type 3 DNA sequence is very similar to those of other subgroup B adenoviruses, especially in the area between the HinfI site (320 base pairs) and the early-region Ia gene. This segment of the genome has been implicated as necessary for the left-end polarity of adenovirus DNA encapsidation. This segment and the sequences flanking it are compared with the corresponding sequences of adenovirus type 5 and adenovirus type 12, and the extent and pattern of intersubgroup homologies are discussed.     

The nucleotide sequence at the termini of adenovirus type 5 DNA.

The sequences of the first 194 base pairs at both termini of adenovirus type 5 (Ad5) DNA have been determined, using the chemical degradation technique developed by Maxam and Gilbert (Proc. Nat. Acad. Sci. USA 74 (1977), pp. 560-564). The nucleotide sequences 1-75 were confirmed by analysis of labeled RNA transcribed from the terminal HhaI fragments in vitro. The sequence data show that Ad5 DNA has a perfect inverted terminal repetition of 103 base pairs long.     

MDBK nuclear factor-binding site of various serotypes of adenovirus DNA

A fraction with the ability to bind the terminal fragment of equine adenovirus (EAd) DNA was prepared from MDBK cell nuclei. The fraction (MDBK nuclear factor) bound to the terminal fragment of all human and animal adenovirus DNAs examined except avian adenovirus EDS-76. However, the terminal fragments of two animal adenoviruses, EAd and bovine adenovirus type 3 (BAd3), showed higher affinity for the nuclear factor than the others. The MDBK nuclear factor-binding site determined by footprinting analysis was the sequence located between nucleotides 22 and 46 in EAd, between 36 and 53 in canine adenovirus type 2, and between 20 and 46 in BAd3, counting from the terminus. The respective binding site contained a sequence resembling the consensus sequence. The binding site of Ad4 DNA was not within the inverted terminal repetition, but was located at least 550 base pairs apart from the terminus.     

Spacing requirements for simultaneous recognition of the adenovirus major late promoter TATAAAAG box and initiator element

The distance between the TATAAAAG box and initiator element of the strong adenovirus major late promoter was systematically altered to determine the optimal spacing for simultaneous recognition of both elements. We find that the TATAAAAG element is strongly dominant over the initiator for specification of the start site. The wild type spacing of 23 base pairs between TATAAAAG and +1A is optimal for promoter strength and selective recognition of the A-start. Initiation is constrained to a window spaced 19-26 base pairs downstream of (-31)-TATAAAAG-(-24), and A-starts are favored over alternate starts only when spaced between 21 and 25 base pairs downstream of TATAAAAG. We report an expanded TATAAAAG and initiator promoter consensus for vertebrates and plants. Plant promoters of this class are (A-T)-rich and have an A-rich (non-template strand) core promoter sequence element downstream of +1A.     

The nucleotide sequence of the transforming BglII-H fragment of adenovirus type 7 DNA

The nucleotide sequence of the leftmost BglII-H fragment (0--4.5%) of weakly oncogenic human adenovirus serotype 7 (Ad7) has been determined (1568 base pairs). This is the shortest Ad7 DNA fragment reported to transform primary rat cells into an immortal cell line (Dijkema et al., 1979). The l-strand of BhlII-H was found to contain the complete information for a polypeptide of at most 28 051 daltons, followed by the putative promoter site of the next gene. Comparison of the determined Ad7 sequence with that of the corresponding region of non-oncogenic Ad5 (Van Ormondt et al., 1978; Maat and Van Ormondt, 1979) showed that the over-all organization of the two DNAs is quite similar, but that the sequences, except in regions of suspected strategic importance, diverge considerably.     

Initiation of adenovirus DNA replication.

In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared with those obtained in a soluble nuclear extract competent for viral DNA replication. It was observed that in vitro DNA replication, which is dependent on the exogenously added viral DNA-protein complex as its optimal template, occurs in a manner apparently indistinguishable from the situation in virus-infected cells. This includes the presence of proteinaceous material on the molecular termini of newly initiated viral DNA.     

Adenovirus type 5 virions can be assembled in vivo in the absence of detectable polypeptide IX.

Mutant dl313 is an adenovirus type 5 deletion mutant which lacks 2,307 base pairs, including the 5' portion of the polypeptide IX gene. Mutant virions did not contain detectable levels of this polypeptide. They were substantially more thermolabile than wild-type particles and did not produce hexon nonomers upon pyridine disruption.     

Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein.

Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When XbaI digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9-22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4-8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication.     

Mutant carrying deletions in the two simian virus 40 early genes.

We isolated a simian virus 40 mutant, dl2194, which carried deletions in both early genes. One deletion removed 234 base pairs in the 54/59 region within the small-t-antigen coding sequence and the large-T-antigen gene intron. The second deletion removed 57 base pairs at the C terminus of the large-T-antigen coding sequence (0.20 map unit). dl2194 was a viable mutant, it carried a normal helper function for adenovirus growth on monkey cells, and it displayed the transformation properties of a small-t-antigen-negative single mutant. Therefore, none of the known large-T-antigen functions seemed to be altered by the C-terminal deletion.     

Adenovirus chromatin structure at different stages of infection.

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.     

Structure and function of the adenovirus origin of replication

Efficient initiation of adenovirus DNA replication requires the presence of specific terminal nucleotide sequences that collectively constitute the viral origin of replication. Using plasmids with deletions or base substitutions in a cloned segment of DNA derived from the terminus of the adenovirus 2 genome, we have demonstrated that the origin contains two functionally distinct regions. The first 18 bp of the viral genome are sufficient to support a limited degree of initiation. However, the presence of a sequence in the region between nucleotides 19 and 67 greatly enhances the efficiency of the initiation reaction. This region contains a specific binding site for a protein present in uninfected cells (KD = 2 X 10(-11) M). The bound protein protects the DNA segment between base pairs 19 and 43 from attack by DNAase I. Studies with deletion mutants indicate that binding of the cellular protein is responsible for the enhancement of initiation.     

Adenoassociated virus has a unique chromatin structure

The organization of intranuclear adenoassociated virus DNA (AAV) was examined following micrococcal nuclease digestion of nuclei prepared from cells coinfected with AAV type 2 (AAV-2) and adenovirus type 2 (Ad2). Blot-hybridization analysis of the DNA with AAV-2, Ad2, and cellular DNA probes revealed that AAV-2 chromatin has a unique structure, which upon nuclease digestion gives rise to a smear of oligomeric DNA fragments from 600-2200 base pairs in length with only a very faint band about 160 base pairs and no discrete multimers. This structure was similar to, but distinguishable from, Ad2 chromatin and completely unrelated to eukaryotic chromatin.     

An adenovirus agnogene.

The nucleotide sequence of a 550 base pairs long segment, located between map positions 21 and 22.5 in the adenovirus type 2 genome has been determined. S1 nuclease mapping and sequence analysis of cDNA copies of adenovirus mRNA demonstrated that the established sequence includes the i-leader which is spliced to the 5'-end of certain adenovirus mRNAs (1). The i-leader which is 440 nucleotides long contains an open translational reading frame which is preceded by an AUG triplet and which terminates in the third segment of the tripartite leader. A polypeptide with the same molecular weight as predicted from the DNA sequence was identified by in vitro translation of mRNA which had been selected by hybridization to DNA fragments, containing sequences from the i-leader. The results thus suggest that the i-leader, unlike other adenovirus leader segments, is used for translation.     

Studies on the Mechanism of Replication of Adenovirus DNA II. The Nature of Single-Stranded DNA in Replicative Intermediates

Replicative intermediates of adenovirus type 5 DNA contain large stretches of single-stranded DNA. We have shown that this single-stranded DNA is mainly of parental origin, whereas all new DNA synthesized during one round of replication has a double-stranded structure. Hybridization experiments of the single-stranded DNA with isolated complementary strands of adenovirus type 5 DNA showed that this DNA hybridized only with the viral L-strand (the strand with the lower equilibrium density in alkaline CsCl) indicating that it represents the viral H-strand. This observation implies that replication always starts from one and the same molecular end. Electron microscopy of partially denatured Y-shaped intermediates confirmed this and showed that replication started from the molecular right end (the end richest in A-T base pairs). In conclusion, we have shown that replication of adenovirus type 5 DNA starts at the molecular right end, displacing the parental H-strand.     

A two-base-pair substitution in T7 promoter by SP6 promoter-specific base pairs alone abolishes T7 promoter activity but reveals SP6 promoter activity.

The phage T7 and SP6 RNA polymerase-promoter systems are very similar in many characteristics, but maintains stringent specificity for each. In order to identify the base pair element that distinguishes between T7 and SP6 promoters, the base pairs at -12, -10, -9, and -8 of the T7 promoter consensus sequence were changed singly and multiply to the SP6 promoter-specific base pairs, and assayed for T7 and SP6 promoter activities. The results indicate that the primary discrimination element is the base pairs at -8 and -9. The two-base-pair substitution alone in T7 promoter by SP6-specific base pairs is sufficient to make the T7 variant be a SP6 promoter, abolishing T7 promoter activity.     

Genetic and quantum chemical basis of the mutagenicity of nitroarenes for adenine-thymine base pairs

The mutagenicity of nitroarenes for Salmonella typhimurium strains with adenine-thymine base pairs at the mutational site is dependent upon enzymic reduction of the nitro function. Although the electrophilic metabolites of nitroarenes are capable of mutating adenine-thymine base pairs, they show a marked preference for guanine-cytosine pairs when given a choice. Quantum chemical calculations indicate the reactivity order for nucleophilic sites in an AT run of base pairs to be the N-7 of adenine (N7(A)) first, followed by an approximately equal reactivity for C-8 of adenine (C8(A)) and O4 of thymine (O4(T)). Given the low probability of reaction of electrophilic metabolites of nitroarenes with adenine-thymine base pairs, the mutagenic potency of nitroarenes for strains with adenine-thymine base pairs at the mutational site is remarkable.     

NMR investigation of Hoogsteen base pairing in quinoxaline antibiotic--DNA complexes: comparison of 2:1 echinomycin, triostin A and [N-MeCys3,N-MeCys7] TANDEM complexes with DNA oligonucleotides.

Hoogsteen base pairs have been demonstrated to occur in base pairs adjacent to the CpG binding sites in complexes of triostin A and echinomycin with a variety of DNA oligonucleotides. To understand the relationship of these unusual base pairs to the sequence specificity of these quinoxaline antibiotics, the conformation of the base pairs flanking the YpR binding sites of the 2:1 drug-DNA complexes of triostin A with [d(ACGTACGT)]2 and of the TpA specific [N-MeCys3, N-MeCys7] TANDEM with [d(ATACGTAT)]2 have been studied by 1H NMR spectroscopy. In both the 2:1 triostin A-DNA complex and the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the terminal A.T base pairs are Hoogsteen base paired with the 5' adenine in the syn conformation. This indicates that both TpA specific and CpG specific quinoxaline antibiotics are capable of inducing Hoogsteen base pairs in DNA. However, in both 2:1 complexes, Hoogsteen base pairing is limited to the terminal base pairs. In the 2:1 triostin A complex, the internal adenines are anti and in the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the internal guanines are anti regardless of pH, which indicates that the central base pairs of both complexes form Watson-Crick base pairs. This indicates that the sequence dependent nature of Hoogsteen base pairing is the same in TpA specific and CpG specific quinoxaline antibiotic-DNA complexes. We have calculated a low resolution three-dimensional structure of the 2triostin A-[d(ACGTACGT)]2 complex and compared it with other CpG specific quinoxaline antibiotic-DNA complexes. The role of stacking in the formation of Hoogsteen base pairs in these complexes is discussed.