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At least 19 recordsLinked to original sources
PubMed1985

Linear plasmids with terminal inverted repeats obtained from Streptomyces rochei and Kluyveromyces lactis.

Linear plasmids with inverted terminal repeats of 614 bp were obtained from Streptomyces rochei which produced lankacidin. The 5' ends were blocked by the association of a terminal protein. A DNA model of racket frame-like structure is presented implying the juxtaposition of 2 double-stranded DNAs of the same sequence through the binding of cohesive proteins which recognize and bind to the DNA. Two linear plasmids with the inverted terminal repeats of 202 and 184 bp were obtained from a yeast, Kluyveromyces lactis. A killer toxin was produced from the shorter plasmid. The protein toxin inhibited the adenylatecyclase activity of the yeast membrane.

Anti-Bacterial Agents↗
PubMed1991

Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast.

The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp). The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase. A 266-bp fragment (called Pk1) containing most of the terminal repeat sequence was isolated and examined for promoter activity. Pk1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn903. These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative (lac4), and a G418-sensitive K lactis host. Lac+ and G418-resistant transformants were obtained with either orientation of Pk1. The promoter activity of Pk1 fragment was independent of the presence or absence of killer plasmids. It is not known whether Pk1 can also function bidirectionally on the natural k1 plasmid. The possible functions of Pk1 for killer plasmid gene expression and plasmid replication are discussed.

Base Sequence↗
PubMed1987

The genome of lipid-containing bacteriophage PRD1, which infects gram-negative bacteria, contains long, inverted terminal repeats.

The bacteriophage PRD1 is a lipid-bearing phage that infects a wide variety of gram-negative bacteria, including Escherichia coli and Salmonella typhimurium when they contain the appropriate plasmid. It contains a linear duplex DNA molecule that is covalently bound by its 5' ends to a terminal protein. We report here that the PRD1 genome contains a 111-base-pair terminal inverted repeat which does not bear homology to that of any known linear duplex DNAs with terminal proteins. We further report that its 3' termini are susceptible to enzymatic digestion by exonuclease III.

Bacteriophages↗
PubMed1989

Characterization of the simian adenovirus type 30 inverted terminal repeat.

The presence of an inverted terminal repeat (ITR), which plays an important role in the initiation of DNA replication, is one of the characteristic properties of adenoviruses (Ads). We have established the nucleotide (nt) sequences for the ITR of simian adenovirus type 30 (SV30), a subgroup-III oncogenic virus. This repeat consists of 185 nt, representing the longest ITR found in an Ad so far. It contains multiple copies of internal repeats, as well as the consensus sequences of the putative binding sites for replication and transcription factors. The conserved features of the known ITRs are also found in SV30. Interestingly, the ITR of SV30 is more closely related to that of Ad5 (human), than to that of SA7 (simian).

Adenoviridae↗
PubMed1991

Eukaryotic transposable elements with short terminal inverted repeats.

Transposable elements with short terminal inverted repeats are believed to transpose directly from DNA to DNA via excision and integration. The cis/trans requirements for transposition have recently been characterized for some of these elements. Common features seem to emerge for the mechanisms of excision of these elements, with the mechanisms apparently similar for the different elements.

Animals↗
PubMed1987

Phylogenetic relationships between adenoviruses as inferred from nucleotide sequences of inverted terminal repeats.

The nucleotide (nt) sequences of inverted terminal repeats (ITR) from human adenovirus (Ad) 19, bovine Ad1 (BAd1), bovine Ad3 (BAd3), canine Ad2 (CAd2) and an avian Ad, EDS-76, were determined. The length of the ITR sequence was 160 bp in Ad19, 159 bp in BAd1, 195 bp in BAd3, 196 bp in CAd2 and 52 bp in EDS-76. CAd2 had the longest ITR among the examined Ads, BAd3 the second longest, and EDS-76 had the shortest ITR. A TAAT sequence located between the 10th and 13th nt counted from the ends was conserved in all Ads examined so far. To determine phylogenetic relationships among human and animal Ads, sequences of their ITRs were compared, and a phylogenetic tree was constructed by using the maximum-likelihood method. It is the method involving statistical analysis of computing the probability of a particular set of sequences on a given tree and maximizing this probability over all evolutionary trees [Felsenstein, J. Mol. Evol. 17 (1981) 368-376]. From these analyses, it was found that members belonging to the same human Ad subgenus are related closely to each other, whereas representatives of different human subgenera are distributed rather divergently among animal Ads.

Adenoviridae↗
PubMed1989

Identification of nuclear proteins that specifically interact with adeno-associated virus type 2 inverted terminal repeat hairpin DNA.

A palindromic hairpin duplex containing the inverted terminal repeat sequence of adeno-associated virus type 2 (AAV) DNA was used as a substrate in gel retardation assays to detect putative proteins that specifically interact with the AAV hairpin DNA structures. Nuclear proteins were detected in extracts prepared from human KB cells coinfected with AAV and adenovirus type 2 that interacted with the hairpin duplex but not in nuclear extracts prepared from uninfected, AAV-infected, or adenovirus type 2-infected KB cells. The binding was specific for the hairpin duplex, since no binding occurred with a double-stranded DNA duplex with the identical nucleotide sequence. Furthermore, in competition experiments, the binding could be reduced with increasing concentrations of the hairpin duplex but not with the double-stranded duplex DNA with the identical nucleotide sequence. S1 nuclease assays revealed that the binding was sensitive to digestion with the enzyme, whereas the protein-bound hairpin duplex was resistant to digestion with S1 nuclease. The nucleotide sequence involved in the protein binding was localized within the inverted terminal repeat of the AAV genome by methylation interference assays. These nuclear proteins may be likely candidates for the pivotal enzyme nickase required for replication or resolution (or both) of single-stranded palindromic hairpin termini of the AAV genome.

Base Sequence↗
PubMed1985

Sequence of Dictyostelium DIRS-1: an apparent retrotransposon with inverted terminal repeats and an internal circle junction sequence.

The Dictyostelium discoideum transposon DIRS-1 contains long terminal repeats that are inverted (ITRs) and nonidentical. We show here that the internal sequence contains 4158 nucleotides and encodes three open reading frames (ORFs). Two of the ORFs (ORFs 2 and 3) are colinear and overlap for more than 2000 bases. Unusual sequence conservation between the two DIRS-1 elements in the overlap region is discussed. The conserved reading frame (ORF3) contains a 200 amino acid region that bears significant homology to retrovirus reverse transcriptase. Based on this homology, we classify DIRS-1 as a possible retrotransposon and propose a model by which the nearly genomic length 4.5 kb DIRS-1 RNA could be used to generate a genomic DNA copy of DIRS-1 with nonidentical inverted terminal repeats.

Base Sequence↗
PubMed1989

Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus.

To facilitate genetic analysis of the human pathogenic parvovirus B19, we constructed a hybrid B19 viral genome in which the defective B19 inverted terminal repeats were replaced with the full-length inverted terminal repeats from a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The hybrid AAV-B19 genome was rescued from a recombinant plasmid and then the DNA was replicated upon transfection into adenovirus 2-infected human KB cells in the presence of AAV genes coding for proteins required for AAV DNA replication (AAV-Rep proteins). In addition, in the presence of AAV genes coding for the viral capsid proteins (AAV-Cap proteins), the rescued/replicated hybrid AAV-B19 genomes were packed into mature AAV progeny virions, which were subsequently released into culture supernatants. The recombinant AAV-B19 progeny virions were infectious for normal human bone marrow cells and strongly suppressed erythropoiesis in vitro. The availability of an infectious recombinant B19 virus should facilitate the mutational analysis of the viral genome, which, in turn, may yield information on individual viral gene functions in B19-induced pathogenesis. The hybrid AAV-B19 genome may also prove to be a useful vector for gene transfer in human bone marrow cells.

Blotting, Southern↗
PubMed1978

Isolation and characterization of a highly repetitious inverted terminal repeat sequence from Oxytricha macronuclear DNA.

The low-complexity "gene-sized" linear DNA duplexes of the Oxytricha macronucleus sport short inverted terminal repeats; thus, each single strand is capable of forming a circle held together by a duplex "neck" [Wesley, R. D. (1975) Porc. Natl. Acad. Sci. USA 72, 678--682]. We have isolated necks from total, circularized, single-stranded macronuclear DNA by treatment with nuclease S1. Necks represent at least 2.2% of the total DNA, are homogeneous in size (23 base pairs), melt at 55 degrees in 0.18 M Na+, and reassociate extremely rapidly at 22 degrees (Cot1/2 = 1.1 X 10(-5) mol-liter-1.sec) to form hybrid necks of the same thermal stability. From these and other results, we conclude that all necks on all the many thousands of different single-stranded circles are the same. The neck sequence is therefore highly repetitious--found in multiple copies (as inverted terminal repeats at flush duplex ends and probably also internally) on each natural "gene-sized" macronuclear DNA molecule--implying the possible participation of this sequence both in the general vegetative metabolism of macronuclear DNA and in the pre-vegetative process whereby macronuclear DNA is excised from the total Oxytricha genome.

Animals↗
PubMed1989

The adenovirus inverted terminal repeat functions as an enhancer in a cell-free system.

Two binding sites for EivF, a factor involved in transcription from the adenovirus early promoter iv (Eiv), were mapped within the adenovirus inverted terminal repeats (ITR). Consistent with the observation that EivF was required to initiate transcription from the Eiv promoter and with the demonstration that two EivF binding sites were present in the ITR, we show that the inverted terminal repeat region was able to promote transcription from the CAP site of the Eiv promoter in vitro and in an EIa-dependent fashion in vivo. The minimum sequence within the ITR capable of directing EIa-dependent transcription consists of forty nine nucleotides comprising two EivF binding sites and at least one Sp1 binding site. This 49-base pair fragment possesses the characteristics of an enhancer which is induced by EIa. The enhancer is active in HeLa cell nuclear extracts. Transcription directed by the ITR required EivF and the general transcription factors. The addition of purified Sp1 factor specifically stimulated transcription which correlates with the presence of Sp1 binding sites between the two EivF recognition sites.

Adenoviridae↗
PubMed1991

Redundant elements in the adenovirus type 5 inverted terminal repeat promote bidirectional transcription in vitro and are important for virus growth in vivo.

The adenovirus inverted terminal repeat (ITR) contains a number of cis-acting elements that are involved in the initiation of viral DNA replication, as well as multiple binding motifs for the cellular transcription factors SP1 and ATF. In this study, we utilized a Hela cell transcription extract to demonstrate that the adenovirus type 5 ITR promotes bidirectional transcription in vitro. Primer extension analyses demonstrated that the ITR directed transcription at initiation sites both within the terminal repeat and at fixed distances outside of the ITR. The ITR also strongly stimulated transcription at the early region 1A (E1A) initiation site when it was situated immediately upstream of the E1A TATA box region. Deletion and point mutational analyses demonstrated that two distinct cis-acting elements were involved in these ITR-dependent transcriptional activities in vitro. Cellular transcription factors SP1 and ATF were previously shown to bind to these two regions. Analysis of viral mutants in vivo demonstrated that the NFIII/OCT-1 binding site and a conserved ATF motif were important for efficient viral growth. Regulatory elements in the ITR flanking region were found to functionally substitute for these sites.

Adenoviruses, Human↗
PubMed1991

Terminal region recognition factor 1, a DNA-binding protein recognizing the inverted terminal repeats of the pGKl linear DNA plasmids.

The yeast linear DNA plasmids pGKl1 and pGKl2 contain inverted terminal repeats (ITRs) and terminal proteins covalently bound to the 5' termini of each plasmid. The presence of these features suggests a protein-primed mechanism of DNA replication, similar to that exemplified by mammalian adenovirus and phi 29 phage of Bacillus subtilis. In this paper, we report the identification of an activity in cytoplasmic extracts of yeast harboring the pGKl plasmids that recognizes the termini of both pGKl1 and pGKl2. We call this activity TRF1, for terminal region recognition factor 1. Deletion analyses and DNase I protection experiments demonstrate that the activity recognizes base pairs 107-183 within the ITR of pGKl1, and base pairs 126-179 within the ITR of pGKl2. The presence of T-tracts within these two regions, but otherwise dissimilar nucleotide sequences, suggests that TRF1 recognizes a common structural feature within the ITRs of the two plasmids. TRF1 has been partially purified from yeast cytoplasmic extracts and Southwestern analysis indicates that the apparent molecular mass of the protein is 16 kDa. By expressing three open reading frames from pGKl2 in Escherichia coli, we found that open reading frame 10 (ORF10) of pGKl2 encodes TRF1. The sequence of the ORF10 gene product indicates that TRF1 is a highly basic protein of small molecular mass. Comparison of TRF1 with other DNA-binding proteins known to recognize the terminal regions of linear DNAs, such as NFI and NFIII involved in adenovirus DNA replication, and phi 29 p6, involved in phi 29 DNA replication, indicates that TRF1 has a different mode of binding.

Amino Acid Sequence↗
PubMed1986

Viable viruses with deletions in the left inverted terminal repeat define the adenovirus origin of DNA replication.

A series of human adenovirus type 2 genomes with deletions in the left inverted terminal repeat (ITR) have been constructed. Viral genomes that contained a minimum of 45 base pairs (bp) from the terminus of the genome were fully infectious and gave rise to progeny virus which maintained the deletion. In contrast, genomes containing 36 bp or less from the termini of the genome were not infectious. The boundary of a cis-acting element required for viral replication is therefore between 36 and 45 bp from the adenovirus termini and corresponds to the previously identified viral origin of replication, defined using a transfection assay to measure ori activity in vivo. The growth parameters of viruses with deletions in the left ITR were examined. These deletions had no measurable effect on plaque formation or morphology, viral DNA synthesis or early viral mRNA synthesis. Thus, it appears that DNA sequences in the left ITR, outside the replication origin, are completely dispensable for lytic viral growth in tissue culture cells.

Adenoviruses, Human↗
PubMed1989

Tissue-specific DNA binding of nuclear proteins that bind to the adenovirus inverted terminal repeat.

Adenovirus (Ad) DNA replication requires nuclear factor I (NFI), a cellular sequence-specific DNA binding protein which binds to the Ad DNA inverted terminal repeat (ITR). The NFI binding consensus sequence, TGGN6-7GCCAA, possibly includes the CCAAT-box which is often observed in regulatory elements of transcription. Adjacent to the NFI binding site on the Ad ITR is the binding site for nuclear factor III (NFIII), another cellular factor that stimulates Ad DNA replication in a cell-free system. Using gel retardation assay, we have examined the tissue specificity of these DNA binding activities in mouse. High NFI activity was detected in nuclear extracts from mouse liver, kidney, and spleen. Competition gel retardation assay with double-stranded oligonucleotides containing herpes simplex virus thymidine kinase (HSV-TK) gene or hsp70 gene CCAAT-box showed no effect on mouse NFI binding to its binding site. In the course of competitive DNA-binding assay, we detected a novel DNA binding protein in mouse kidney nuclear extracts, designated nuclear factor K (NFK). The NFK binding site is included in the NFIII binding site on the Ad ITR. NFK seems to be different from NFIII, in mode of binding to its binding site.

Adenoviridae↗
PubMed2026

Modified Plasmids and Inverted Terminal Repeats Enhance Adeno-Associated Virus Production and Performance.

Recombinant adeno-associated virus (rAAV) is a preferred vector in gene therapy, although high production costs inhibit widespread adoption. The most common approach for rAAV production involves transfection of HEK293 cells with three plasmids: pTransgene, pRep/Cap and pHelper. Producing sufficient amounts of these plasmids accounts for up to 40% of total batch costs. Initially, this work aimed to increase plasmid yields by replacing the backbones. While this approach increased pHelper yields, pRep/Cap and pTransgene yields were unaffected. A possible reason was identified: pTransgene contains inverted terminal repeat (ITR) sequences that are essential for rAAV production. ITRs have strong secondary structures (including hairpin loops termed B and C arms) that likely interfere with plasmid production. Therefore, targeted deletions were performed within the ITRs. Partial deletions in both the B and C arms of the ITR were most beneficial, as both plasmid yield and transgene expression increased. Importantly, partial deletions did not reduce rAAV yield, as had been previously observed when the B and C arms were fully deleted. In summary, we report a 140% increase in pHelper plasmid production, while the most successful ITR variant increased pTransgene plasmid yields by 57% and transgene expression by 28%, without reducing rAAV yields or transduction efficiency.

Humans↗
PubMed1991

Vaccinia virus homologues of the Shope fibroma virus inverted terminal repeat proteins and a discontinuous ORF related to the tumor necrosis factor receptor family.

Nucleotide sequencing data from a region extending 35 kb inward from the right inverted terminal repeat (ITR) of the vaccinia virus (VV) genome established the presence of VV homologues of the Shope fibroma virus (SFV) ITR proteins. The nucleotide sequences, comprising a total of 8.6 kb, and the amino acid translations for nine predicted open reading frames (ORFs) (designated SalF4L, SalF 19R, SalF21R, B4R, B8R, B9R, B10R, and B14R) are presented. Eight of the nine VV genes and all the SFV ORFs are transcribed towards their genomic termini. However, the relative positions of the VV genes (genus Orthopoxvirus) are different than those of the corresponding ORFs in SFV (genus Leporipoxvirus), indicating complex rearrangements of DNA in the genome of one or both of these viruses subsequent to their divergence from a common ancestor. Several other features of the VV ORFs were noted. SalF4L, B7R, B8R, and B9R have hydrophobic amino-terminal signal sequences but lack discernible membrane anchor domains suggesting that the proteins may be secreted. VV ORF SalF19R has a single cysteine-rich region homologous to the multiple domains of nerve growth factor receptor (NGFR), CD40, OX40 (a glycoprotein from the surface of activated murine T lymphocytes), and the recently described tumor necrosis factor receptors. Just downstream of the ORF SalF19R and in a different reading frame, there are another two related cysteine-rich domains, indicating that SalF19R was once a larger gene. B4R has homology to the host range gene of cowpox virus and to related genes near the opposite end of the vaccinia virus genome, and contains regions homologous to the repeat domains of erythrocyte ankyrin. In addition, several of the VV ORFs have homology to ORFs from near the opposite end of the VV genome, thus increasing the number of known VV gene families.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence of the adenovirus type 40 inverted terminal repeat: close relation to that of adenovirus type 5.

Human adenovirus type 40 (Ad40) is a pathogen that causes acute infantile gastroenteritis. Ad40 has the distinct characteristic of being difficult to propagate in conventional cultured human cells. The nucleotide sequence of the inverted terminal repeat (ITR) of Ad40, which includes the origin of adenoviral DNA replication, was determined using recombinant plasmid DNA. By using our newly developed program to express the ITR homologies simply, we found that the ITR of Ad40, which is 163 nucleotides long, was related most closely to that of adenovirus type 5, which replicates efficiently.

Adenoviruses, Human↗