A nearby inverted repeat of the terminal sequence of herpes simplex virus DNA.

When herpes simplex virus DNA is digested with λ-exonuclease, annealed, and then mounted for observation in the electron microscope, two types of molecules are seen. One type is circular DNA which forms because the enzyme has revealed the terminal repetition. The other type is full length linear duplex DNA with a short single-stranded loop on one end. This latter type, which apparently represents a fold-back reaction, forms because there is a nearby inverted repeat of the terminal sequence of herpes simplex virus DNA.     

Complementary strands of CELO virus DNA.

When alkali-denatured DNA from CELO virus (an avian adenovirus) was annealed for 15 min at 37 C in 0.1 M NaCl, 70% of the molecules formed single-stranded circles. This is probably due to base pairing of complementary sequences not more than 110 nucleotides long at the ends of the single strands and implies an inverted terminal repetition in the duplex DNA similar to that reported for the DNA from human adenoviruses. The circular molecules had a uniform length that was approximately the same as that of linear single-stranded molecules. The complementary strands of CELO virus DNA were separated on a preparative scale, and at least 40% of the heavy strands and 56% of the light strands were found to be intact as judged by the formation of single-stranded circles.     

Inverted terminal repetition in adeno-associated virus DNA: independence of the orientation at either end of the genome

Complementary strands of adeno-associated virus DNA labeled with 32P at the 5' ends were separated and then self-annealed to form single-stranded circles stabilized by hydrogen bonds between the complementary sequences in the inverted terminal repetitions. We have previously shown that there are two distinct sequences in the terminal repetition which represent an inversion of the first 125 nucleotides (E. Lusby et al., J. Virol. 34:402-409, 1980; I. S. Spear et al., Virology 24:627-634, 1977). Base pairing between terminal sequences of the same orientation leads to a normal double helical structure. If sequences of the opposite orientation pair, an aberrant secondary structure is formed. HpaII digestion of the self-annealed, single-stranded circles led to labeled terminal fragments that corresponded both to those generated from termini of a normal double helical structure and those generated from an aberrant terminal secondary structure. Thus, the orientation of the terminal repetition at one end of the genome is not influenced by the orientation at the other end.     

Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA.

The inverted terminal repetition in adeno-associated virus type 2 DNA has been sequenced. The terminal repetition contain 145 nucleotides of which the first 125 nucleotides can self-base pair to form a T-shaped hairpin structure. Both restriction endonuclease analysis with SmaI and BglI and direct sequence analysis of the SmaI fragments provide evidence for two sequences in the region of the terminal repetition between nucleotides 44 and 81. The two sequences represent an inversion of the first 125 nucleotides of the terminal repetition. Based on these data a model for adeno-associated virus DNA replication is presented which agrees in detail with a general model for eucaryotic DNA replication originally proposed by Cavalier-Smith (T. Cavalier-Smith, Nature [London] 18:672--684, 1976).     

Structure of the inverted terminal repetition of adenovirus type 2 DNA.

Several secondary structure features involving the ends of single strands of adenovirus type 2 DNA have been studied by electron microscopy by both the gene 32-ethidium bromide technique and a modification of the standard formamide-cytochrome c technique. A duplex stem of length 115 +/- 10 nucleotide pairs due to pairing between the two members of the inverted terminal repetition is observed in the single-stranded circles that form upon annealing single-stranded linear molecules. This duplex stem is shown to lie at the ends of the DNA by using several reference markers: (i) a newly discovered secondary structure feature (a loop of length ca. 500 nucleotides with a 20-nucleotide pair duplex stem) that maps 73% of the full length from the left end of the molecule and (ii) a duplex region due to a hybridized restriction fragment. There is also some secondary structure within each end of linear single strands. There is some variation in the morphology of the end strucures, and we propose that these involve base pairing, as in a tRNA clover leaf, rather than an exact single hairpin-type inverted repeat. These observations are consistent with the hypothesis that there is a foldback structure at the 3' ends of the DNA that functions as a primer for the initiation of replication.     

Physical map of bacteriophage BF23 DNA: terminal redundancy and localization of single-chain interruptions.

The DNA of bacteriophage BF23 possesses two structural features, localized single-chain interruptions and a large terminal repetition, previously described for T5, a closely related virus. As is the case for T5, single-chain interruptions occur with variable frequencies at a small number of fixed sites within one strand of the double-stranded BF23 genome. The sites where interruptions occur with the highest frequencies were napped by an electrophoretic analysis of the single-stranded fragments produced by denaturation of BF23 DNA. The positions of these fragments were determined by degrading BF23 DNA to various extents with lambda exonuclease and observing the relative order with which they were (i) degraded or (ii) released intact from the undenatured duplex. The exact locations of the interruptions were determined from analysis of analogous duplex fragments produced by degrading exonuclease III-treated BF23 DNA with a single-strand-specific endonuclease. BF23 has five principal sites (located at 7.9, 18.7, 32.4, 65.8, and 99.6% from the left end of the DNA) where interruptions occur in most molecules. The principal interruptions in T5 DNA occur at similar positions. The locations of eight secondary interruptions in BF23 DNA were also determined. In general, BF23 DNA has fewer secondary interruptions than t5 dna, although there is at least one location where an interruption occurs with a greater frequency in BF23. The presence of a terminal repetition in BF23 DNA was demonstrated by annealing ligase-repaired molecules that had been partially digested with lambda exonuclease. If the complementary sequences at both ends of the DNA were exposed by exonuclease treatment, the duplex segment that resulted from annealing could be released by digestion with a single-strand-specific endonuclease. This segment was analyzed by agarose gel electrophoresis and found to represent 8.4% of BF23 DNA.     

Tandem repeats within the inverted terminal repetition of vaccinia virus DNA

A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approximately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restriction analysis of cloned DNA fragments and by hybridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abundance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites occurred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of approximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence containing single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replication.     

Conformation takes precedence over sequence in adeno-associated virus DNA replication.

Deletion of an 11-base symmetrical sequence in the inverted terminal repetition of the adeno-associated virus 2 genome inhibits DNA replication. Substitution of either an 8- or a 12-base symmetrical sequence unrelated to the original 11-base sequence restores DNA replication.     

Adenovirus DNA replication in vitro: duplication of single-stranded DNA containing a panhandle structure

Adenovirus DNA replicates by displacement of one of the parental strands followed by duplication of the displaced parental single strand (complementary strand synthesis). Displacement synthesis has been performed in a reconstituted system composed of viral and cellular proteins, employing either the viral DNA-terminal protein complex as template or linearized plasmids containing the origin. Previously, evidence was obtained that in vivo complementary strand synthesis requires formation of a panhandle structure originating from hybridization of the inverted terminal repeats. To study the conditions for complementary strand synthesis in vitro, we have constructed an artificial panhandle molecule that contains a double-stranded inverted terminal repetition (ITR) region and a single-stranded loop derived from the left and right terminal XmaI fragments of Ad2. Such a molecule appeared to be an efficient template and could initiate by the same protein-priming mechanism as double-stranded DNA, employing the precursor terminal protein. The efficiency of both types of template was comparable. Like for replication of the duplex molecule initiation of panhandle replication was stimulated by nuclear factors I and III, proteins that bind to specific double-stranded regions of the ITR. The Ad DNA-binding protein is essential and the 39 kDa C-terminal domain of this protein that harbors the DNA-binding properties is sufficient for its function. These results support the hypothesis that panhandle formation is required for duplication of the displaced strand.     

In vitro synthesis of a 9 kbp terminally redundant DNA carrying the infectivity of Moloney murine leukemia virus.

Detergent-disrupted virions of Moloney murine leukemia virus synthesize a 9 kbp double-stranded infectious DNA. It contains mainly full-length, single-stranded DNA, and its infectivity and size are insensitive to digestion by the single-strand-specific S1 nuclease. Analysis of fragmentation of the DNA using restriction endonucleases has shown that it is indistinguishable from the linear double-stranded DNA synthesized in infected cells. On the basis of the positions of the cleavage sites for a number of enzymes, the 9 kbp DNA has a 575 base direct terminal repetition. It is longer than the viral RNA at both ends, evidently due to repetitive copying of segments of the RNA. Virions also synthesize an 8.4 kbp double-stranded circular DNA that lacks one copy of the terminal repetition, as well as viral DNA longer than 9 kbp. The enzymatic machinery in the virions of retroviruses therefore appears to be responsible for all the steps involved in making fully double-stranded linear and one form of circular DNA.     

A newly isolated densonucleosis virus from Pseudoplusia includens (Lepidoptera: Noctuidae)

An icosahedral DNA virus isolated from the soybean looper, Pseudoplusia includens, was characterized. Purified virus had a diameter of 20 ± 1 nm and negatively stained preparations showed a trend to form linear to three-dimensional crystals. The virus had a sedimentation coefficient of 120 ± 3 S and a buoyant density of 1.40 ± 0.01 g/cm³. The DNA content of the virus was 37.8 ± 0.1% and the absorption spectrum showed it to be a typical nucleoprotein. Viral DNA in situ was shown to be single-stranded by staining the virus with acridine orange as well as by reaction to formaldehyde. Evidence of inverted terminal repetition of the DNA was observed by electron microscopy. The terminal repetition comprises ca. 6–7% of the genome. The molecular weight of the ssDNA was 2.0 ± 0.1 × 10⁶ as determined by agarose gel electrophoresis or 2.1 ± 0.1 × 10⁶ as determined by electron microscopy. Four virion proteins with molecular weights of 46.5 ± 0.1, 54.0 ± 0.1, 64.0 ± 0.2, and 87.0 ± 0.1 × 10³ were detected by 9% SDS-polyacrylamide gel electrophoresis. Double-diffusion tests showed the virus to be serologically related but not identical to DNV-1. Ultrathin sections showed that the nucleus of the hemocyte, muscle, hypodermal, and fat body cells contained virus-like particles. The chromatin of an infected nucleus always underwent a margination and the nucleoplasm was often replaced largely by virions.Data indicate that the virus belongs to the Densovirus of the family Parvoviridae.     

A common sequence in the inverted terminal repetitions of human and avian adenoviruses

The termini of the avian chick embryo lethal orphan (CELO) virus DNA have been sequenced. The results revealed a 63-bp-long inverted terminal repetition (ITR) which shared the sequence ATAATA with all adenovirus termini, thus far analyzed. The CELO virus ITR differed from those of the mammalian adenoviruses in two major aspects: (i) it is not a perfect duplication; (ii) it begins with a 5'-guanylic acid residue instead of the cytidylic acid normally observed in adenoviruses.     

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.     

The nucleotide sequences at the termini of adenovirus-2 DNA.

The nucleotide sequence of the first 156 residues from the left end and the first 134 residues from the right end of adenovirus-2 DNA have been determined by direct DNA sequencing techniques. The inverted terminal repetition is 102 nucleotide pairs long. The 5′-ends of the intact DNA are resistant to the action of T4 polynucleotide kinase and the 5′ → 3′ exonucleases from phages lambda and T7. This resistance is most likely due to the covalent attachment of the 5′-terminal C residue to the terminal protein. No significant self-complementarity exists within the inverted terminal repetition, making terminal initiation of DNA replication via a self-priming mechanism unlikely. However, the terminal A + T-rich region followed immediately by a very G + C-rich region is consistent with other schemes for adenovirus-2 DNA replication. The left end of adenovirus-2 DNA contains extensive sequence repetition.     

Linear mitochondrial DNAs of yeasts: closed-loop structure of the termini and possible linear-circular conversion mechanisms.

The terminal structure of the linear mitochondrial DNA (mtDNA) from three yeast species has been examined. By enzymatic digestion, alkali denaturation, and sequencing of cloned termini, it was shown that in Pichia pijperi and P. jadinii, both termini of the linear mtDNA were made of a single-stranded loop covalently joining the two strands, as in the case of vaccinia virus DNA. The left and right loop sequences were in either of two orientations, suggesting the existence of a flip-flop inversion mechanism. Contiguous to the terminal loops, inverted terminal repeats were present. The mtDNA from Williopsis mrakii seems to have an analogous structure, although terminal loops could not be directly demonstrated. Electron microscopy revealed the presence, among linear molecules, of a small number of circular DNAs, mostly of monomer length. Linear and circular models of replication are considered, and possible conversion mechanisms between linear and circular forms are discussed. A flip-flop inversion mechanism between the inverted repeat sequences within a circular intermediate may be involved in the generation of the linear form of mtDNA.     

Nucleotide sequence required for resolution of the concatemer junction of vaccinia virus DNA.

The mature form of the vaccinia virus genome consists of a linear, 185,000-base-pair (bp) DNA molecule with a 10,000-bp inverted terminal repetition and incompletely base-paired 104-nucleotide hairpin loops connecting the two strands at each end. In concatemeric forms of intracellular vaccinia virus DNA, the inverted terminal repetitions of adjacent genomes form an imperfect palindrome. The apex of this palindrome corresponds in sequence to the double-stranded form of the hairpin loop. Circular plasmids containing palindromic concatemer junction fragments of 250 bp or longer are converted into linear minichromosomes with hairpin ends when they are transfected into vaccinia virus-infected cells, providing a model system with which to study the resolution process. To distinguish between sequence-specific and structural requirements for resolution, plasmids with symmetrical insertions, deletions, and oligonucleotide-directed mutations within the concatemer junction were constructed. A sequence (ATTTAGTGTCTAGAAAAAAA) located on both sides of the apex segment was found to be critical for resolution. Resolution was more efficient when additional nucleotides, TGTG, followed the run of A residues. Both the location and sequence of the proposed resolution signal are highly conserved among poxviruses.     

Deletion of a 9,000-base-pair segment of the vaccinia virus genome that encodes nonessential polypeptides.

Deletions contained within the genomes of unstable and stable variants of vaccinia virus (strain WR) were analyzed. Restriction endonuclease mapping and hybridization to specific 32P-labeled DNA probes indicated that more than 6 X 10(6) daltons of DNA were deleted from the variants. In each case, the deletion occurred on the left side of the genome and started very close to the junction of the inverted terminal repetition and unique sequence. Both variants also contained a new SstI side on the right side of the genome. Hybridization selection and cell-free translation experiments indicated that these variants lost the ability to synthesize at least eight early mRNA's mapping within the deleted region. Although the deleted DNA was not essential for replication of the WR strain of vaccinia virus under laboratory conditions of infection, it presumably has a defined role under other circumstances. This conclusion was based on the conservation within the Elstree strain of vaccinia, the Utrecht strain of rabbitpox, and the Brighton strain of cowpox virus of sequences homologous to the deleted DNA. Moreover, mRNA's that hybridized to the deleted vaccinia virus DNA segment and encoded similar size polypeptides were made in cells infected with rabbitpox and cowpox viruses.     

自身互补型腺相关病毒载体发展趋势

重组腺相关病毒(Recombinant adeno-associated virus,rAAV)可以作为基因运载工具将目的基因运送入靶器官并对多种疾病发挥治疗作用。以rAAV为载体进行基因治疗的关键是病毒基因组由单链变为双链,否则不能适时、有效表达目的基因。自身互补型rAAV(scrAAV)载体基因组本身以双链形式存在,与常规的单链rAAV(ssrAAV)载体相比,无论在表达时间还是表达强度上都有十分明显改善,可显著降低在疾病治疗过程中由于载体本身所诱发的免疫反应。目前,scrAAV已经在肝脏疾病、中枢神经系统疾病、眼部疾病、干细胞修饰以及RNA干扰、核酶技术等领域得到应用。以下在介绍scrAAV载体构建、表达、定位的基础上,以血友病B为主要对象,阐述scrAAV的应用潜力及发展趋势。     

A linear DNA plasmid from Streptomyces rochei with an inverted terminal repetition of 614 base pairs.

The terminal structure of a linear plasmid pSLA2 , which was isolated from Streptomyces rochei , was analysed. The 5' ends of pSLA2 DNA were blocked by the association of a protein probably covalently bonded with the DNA. This block is removed by alkali treatment and blunt ends with 5'-phosphate and 3'-hydroxy termini were released. The two terminal fragments of pSLA2 were cloned and the nucleotide sequence was determined. An inverted terminal repetition of 614 bp was found along with the presence of further interrupted homologous sequences beyond this area up to 800 bp. These are the first inverted terminal repeat sequences found in microbial linear plasmids.