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PubMed1991

Origin of adeno-associated virus DNA replication is a target of carcinogen-inducible DNA amplification.

DNA amplification of the helper-dependent parvovirus AAV (adeno-associated virus) can be induced by a variety of genotoxic agents in the absence of coinfecting helper virus. Here we investigated whether the origin of AAV type 2 DNA replication cloned into a plasmid is sufficient to promote replication activity in cells treated by the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A pUC19-based plasmid, designated pA2Y1, which contains the left terminal repeat sequences (TRs) representing the AAV origin of replication and the p5 and p19 promoter but lacks any functional parvoviral genes is shown to confer replication activity and to allow selective DNA amplification in carcinogen-treated cells. Following transfection of plasmid pA2Y1 or plasmid pUC19 as a control, density labeling by a bromodeoxyuridine and DpnI resistance assay suggested a semi-conservative mode of replication of the AAV origin-containing plasmid. Furthermore, the amount of DpnI-resistant full-length pA2Y1 DNA molecules was increased by MNNG treatment of cells in a dose-dependent manner. In addition, DNA synthesis of plasmid pA2Y1 was studied in vitro. Extracts derived from MNNG-treated CHO-9 and L1210 cells displayed greater synthesis of DpnI-resistant full-length pA2Y1 molecules than did nontreated controls. Experiments with specific enzyme inhibitors suggested that the reaction is largely dependent on DNA polymerase alpha, DNA primase, and DNA topoisomerase I. Furthermore, restriction endonuclease mapping analysis of the in vitro reaction products revealed the occurrence of specific initiation at the AAV origin of DNA replication. Though elongation was not very extensive, extracts from carcinogen-treated cells markedly amplified the AAV origin region. Our results, including electron microscopic examination, suggest that the AAV origin/terminal repeat structure is recognized by the cellular DNA replicative machinery induced or modulated by carcinogen treatment in the absence of parvoviral gene products.

Adenoviridae↗
PubMed1992

A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication.

We recently reported that human immunodeficiency virus type 1 (HIV-1) unintegrated linear DNA displays a discontinuity in its plus strand, precisely defined by a second copy of the polypurine tract (PPT) located near the middle of the genome (P. Charneau and F. Clavel, J. Virol. 65:2415-2421, 1991). This central PPT appears to determine a second initiation site for retrovirus DNA plus-strand synthesis. We show here that mutations replacing purines by pyrimidines in the HIV-1 central PPT, which do not modify the overlapping amino acid sequence, are able to significantly slow down viral growth as they reduce plus-strand origin at the center of the genome. One of these mutations, introducing four pyrimidines, results in a 2-week delay in viral growth in CEM cells and abolishes plus-strand origin at the central PPT. The introduction in this mutant of a wild-type copy of the PPT at a different site creates a new plus-strand origin at that site. This new origin also determines the end of the upstream plus-strand segment, probably as a consequence of limited strand displacement-synthesis. Our findings further demonstrate the role of PPTs as initiation sites for the synthesis of the retroviral DNA plus strand and demonstrate the importance of a second such origin for efficient HIV replication in vitro.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Two E2 binding sites (E2BS) alone or one E2BS plus an A/T-rich region are minimal requirements for the replication of the human papillomavirus type 11 origin.

Human papillomaviruses (HPVs) cannot be propagated in vitro, but the DNA can be replicated transiently in an assay in the presence of two trans-acting viral proteins, E1 and E2. Using this assay, we have defined the minimal cis-acting elements of the origin of replication of HPV type 11. Most HPV genomes are conserved at the origin of replication, and the core contains three E2 binding sites (E2BS) surrounding an A/T-rich spacer region. The present results show that the minimal requirement for replication is either two E2BS alone or the A/T-rich region plus one E2BS; in the latter case the relative position of the E2BS is important. In all the studies, the presence of both E1 and E2 proteins was essential for replication, yet only the E2BS was required at the origin. We have shown that E1, E2, and the origin of replication containing an E2BS from a complex in vitro, and our data are consistent with a model in which E2 acts to target E1 to the HPV type 11 replication origin.

Base Sequence↗
PubMed1993

cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen.

Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.

Amino Acid Sequence↗
PubMed1994

Expression of the varicella-zoster virus origin-binding protein and analysis of its site-specific DNA-binding properties.

The varicella-zoster virus (VZV) genome contains homologs to each of the seven herpes simplex virus (HSV) genes that are required for viral DNA synthesis. VZV gene 51 is homologous to HSV UL9, which encodes an origin of DNA replication binding protein (OBP). It was previously shown, by using a protein A fusion protein, that the product of gene 51 is a site-specific DNA-binding protein which binds to sequences within the VZV origin (Stow et al., Virology 177:570-577, 1990). In this report, gene 51 was expressed in an in vitro translation system. Rabbit antiserum raised against the carboxyl-terminal 20 amino acids was used to confirm expression of the full-length gene 51 protein, and site-specific DNA-binding activity was demonstrated in a gel retardation assay. The origin-binding domain was located within a 263-amino-acid region of the carboxyl terminus by using a series of deletion mutants. The affinity of binding of the VZV OBP to the three binding sites in the VZV origin was found to be similar. In addition, as with UL9, a CGC triplet within a 10-bp consensus sequence is critical to the interaction between the OBP and the origin. The HSV and VZV OBPs, therefore, appear to have virtually identical recognition sequences despite only 33% identity and 44% similarity in the primary structure of their site-specific DNA-binding domains.

Amino Acid Sequence↗
PubMed1995

Two E2 binding sites alone are sufficient to function as the minimal origin of replication of human papillomavirus type 18 DNA.

Replication of papillomaviruses requires an origin of replication and two virus-encoded proteins, E1 and E2. Using a transient replication assay for human papillomavirus type 18 (HPV-18) DNA, we have found that two adjacent sequences present within the origin of replication can independently support replication. The first, a 77-bp region, contains one E2 binding site (E2BS) and a 16-bp inverted repeat element that probably corresponds to the E1 binding site (E1BS). The other, an 81-bp region, includes two E2BS but lacks the putative E1BS. A synthetic 33-bp oligonucleotide containing two high-affinity E2BS was also found to function as an origin of replication. Replication of all these plasmids was absolutely dependent on the presence of the HPV-18 E1 and E2 proteins. The HPV-1a E1 and E2 proteins were also found to support replication of a plasmid containing the complete HPV-18 origin but failed to replicate a plasmid containing two E2BS alone. Our results suggest that the E2 protein can target E1 to the origin through the formation of an E1-E2 complex which is likely to be involved the initiation of replication.

Base Sequence↗
PubMed1997

Purification of the simian virus 40 (SV40) T antigen DNA-binding domain and characterization of its interactions with the SV40 origin.

To better define protein-DNA interactions at a eukaryotic origin, the domain of simian virus 40 (SV40) large T antigen that specifically interacts with the SV40 origin has been purified and its binding to DNA has been characterized. Evidence is presented that the affinity of the purified T antigen DNA-binding domain for the SV40 origin is comparable to that of the full-length T antigen. Furthermore, stable binding of the T antigen DNA-binding domain to the SV40 origin requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix and positioned in a head-to-head orientation. Although two pairs of pentanucleotides are present in the SV40 origin, footprinting and band shift experiments indicate that binding is limited to dimer formation on a single pair of pentanucleotides. Finally, it is demonstrated that the T antigen DNA-binding domain interacts poorly with single-stranded DNA.

Antigens, Polyomavirus Transforming↗
PubMed1998

High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin.

Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5' end of the DNA at the nick and providing a 3' hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays to study ATP-dependent initiation within the right-hand (5') MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3') viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.

Animals↗
PubMed1990

Mapping an origin of DNA replication at a single-copy locus in exponentially proliferating mammalian cells.

A general method for determining the physical location of an origin of bidirectional DNA replication has been developed recently and shown to be capable of correctly identifying the simian virus 40 origin of replication (L. Vassilev and E. M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989). The advantage of this method over others previously reported is that it avoids the use of metabolic inhibitors, the requirement for cell synchronization, and the need for multiple copies of the origin sequence. Application of this method to exponentially growing Chinese hamster ovary cells containing the nonamplified, single-copy dihydrofolate reductase gene locus revealed that DNA replication begins bidirectionally in an initiation zone approximately 2.5 kilobases long centered about 17 kilobases downstream of the DHFR gene, coinciding with previously described early replicating sequences. These results demonstrate the utility of this mapping protocol for identifying cellular origins of replication and suggest that the same cellular origin is used in both the normal and the amplified DHFR locus.

Animals↗
PubMed1994

A mammalian origin of bidirectional DNA replication within the Chinese hamster RPS14 locus.

Two complementary experimental approaches have been used to identify a chromosomal origin of bidirectional DNA replication within or immediately downstream of the Chinese hamster ribosomal protein S14 gene (RPS14). The replication origin, designated oriS14, maps within a 1.6- to 2.0-kbp region of RPS14 that includes the gene's third and fourth introns, exons IV plus V, and approximately 500 bp of proximal downstream flanking DNA. The nucleic acid sequence encoding oriS14 closely resembles the other mammalian chromosomal replication origins whose primary structures are known. It contains DNA binding sites for a large number of transcription factors, replication proteins, and mammalian oncogenes as well as several dinucleotide repeat motifs, an AT-rich region, and a sequence that is likely to bend the DNA. In contrast to the other well-characterized mammalian replication origins, which are autosomal and therefore carried as two copies per somatic cell, oriS14 is encoded by single-copy DNA within a hemizygous segment of chromosome 2q in CHO-K1 cells. Also, other known mammalian replication origins are situated in nontranscribed, intergenic DNA, whereas the DNA sequence encoding oriS14 substantially overlaps the transcribed portion of a constitutively expressed housekeeping gene.

Animals↗
PubMed1987

Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.

Adenoviruses, Human↗
PubMed1988

Sequence and structural requirements of a herpes simplex viral DNA replication origin.

Herpes simplex virus (HSV) types 1 and 2 contain two classes of origins of DNA replication, oriS and oriL, which are closely related. A series of plasmids was constructed which contained specifically altered versions of the HSV type 2 oriS replication origin. Their ability to replicate in an in vivo replicon assay allowed a core origin of 75 base pairs (bp) to be defined. It included both arms of a 56-bp palindrome and from 13 to 20 bp of sequence leftward of the palindrome. The AT-rich sequence at the center of the palindrome was essential. Sequences on either side of the core origin enhanced replication. When additional copies of the -AT-dinucleotide were introduced progressively into the center of the palindrome, an oscillating effect on origin function was observed. These and other data implicate a linear rather than a cruciform conformation of the oriS palindrome in the initiation of HSV replication.

Base Sequence↗
PubMed1993

Effects of time, sex, ethnic origin, and area of residence on prevalence of asthma in Israeli adolescents.

OBJECTIVES: To study effects of time, sex, ethnic origin, and area of residence on prevalence of asthma in Israeli adolescents. DESIGN: Retrospective survey of asthma from computerised medical draft records of conscripts examined up to the end of 1989. SETTING: Five regional centres in Israel. SUBJECTS: 443 186 conscripts (262 836 males and 180 350 females) aged 17-18 who were born over a nine year period. MAIN OUTCOME MEASURES: Asthma determined by medical history, physical examination, and lung function tests at rest and after exercise. RESULTS: Asthma was more prevalent in males than females (26.5/1000 v 21.4/1000, relative risk 1.25 (95% confidence interval 1.19 to 1.32)). Subjects were split into three groups according to year of birth, and prevalence of asthma increased over time from 18/1000 to 24/1000 to 36/1000 (risk of asthma 0.56 (0.54 to 0.59) for first birth group relative to last birth group and 0.69 (0.66 to 0.72) for second group relative to last group). Risk of asthma was also affected by ethnic origin (highest for Western origin and lowest for north African origin, relative risk 1.63 (1.56 to 1.71)) and area of residence (highest in the central coastal region--risk of 1.24 (1.19 to 1.30) relative to the north coastal region--and lowest in inland areas--risk of 0.67 (0.64 to 0.70) relative to north coastal region). CONCLUSIONS: Prevalence of asthma in Israel is increasing and is higher in males, in people of Western origin, and in those living in the most industrialised coastal region.

Adolescent↗
PubMed1998

Studies of non-disjunction in trisomies 2, 7, 15, and 22: does the parental origin of trisomy influence placental morphology?

Recently, there have been several molecular studies of trisomic fetuses and liveborns which have examined the parent and meiotic stage of origin of nondisjunction. However, little is known about the possible phenotypic effects of the origin of trisomy. For trisomic spontaneous abortions, no distinct phenotype has been described, although some have been reported to have features, such as trophoblastic hyperplasia, similar to hydatidiform moles. In the present report, we describe molecular and histological studies of spontaneous abortions with trisomies 2, 7, 15, or 22, conditions occasionally linked to trophoblastic hyperplasia. Our results provide strong evidence for chromosome specific mechanisms of nondisjunction, with trisomy 2 having a high frequency of paternally derived cases and trisomy 7 typically originating postzygotically. In studies correlating parental origin of trisomy with phenotype, we found no difference in the proportion of cases with trophoblastic hyperplasia, fetal tissue, nucleated red blood cells, or hydropic villi among paternally or maternally derived trisomies 2, 7, 15, or 22. However, paternally derived trisomies tended to abort earlier than maternally derived trisomies. This suggests that parental origin might affect the developmental stage at which abortion occurs but not other features of placental phenotype.

Abortion, Spontaneous↗
PubMed2004

Neurogenic pain relief by repetitive transcranial magnetic cortical stimulation depends on the origin and the site of pain.

OBJECTIVE: Drug resistant neurogenic pain can be relieved by repetitive transcranial magnetic stimulation (rTMS) of the motor cortex. This study was designed to assess the influence of pain origin, pain site, and sensory loss on rTMS efficacy. PATIENTS AND METHODS: Sixty right handed patients were included, suffering from intractable pain secondary to one of the following types of lesion: thalamic stroke, brainstem stroke, spinal cord lesion, brachial plexus lesion, or trigeminal nerve lesion. The pain predominated unilaterally in the face, the upper limb, or the lower limb. The thermal sensory thresholds were measured within the painful zone and were found to be highly or moderately elevated. Finally, the pain level was scored on a visual analogue scale before and after a 20 minute session of "real" or "sham" 10 Hz rTMS over the side of the motor cortex corresponding to the hand on the painful side, even if the pain was not experienced in the hand itself. RESULTS: and discussion: The percentage pain reduction was significantly greater following real than sham rTMS (-22.9% v -7.8%, p = 0.0002), confirming that motor cortex rTMS was able to induce antalgic effects. These effects were significantly influenced by the origin and the site of pain. For pain origin, results were worse in patients with brainstem stroke, whatever the site of pain. This was consistent with a descending modulation within the brainstem, triggered by the motor corticothalamic output. For pain site, better results were obtained for facial pain, although stimulation was targeted on the hand cortical area. Thus, in contrast to implanted stimulation, the target for rTMS procedure in pain control may not be the area corresponding to the painful zone but an adjacent one. Across representation plasticity of cortical areas resulting from deafferentation could explain this discrepancy. Finally, the degree of sensory loss did not interfere with pain origin or pain site regarding rTMS effects. CONCLUSION: Motor cortex rTMS was found to result in a significant but transient relief of chronic pain, influenced by pain origin and pain site. These parameters should be taken into account in any further study of rTMS application in chronic pain control.

Adult↗
PubMed1997

Renal artery origins: location and distribution in the transverse plane at CT.

PURPOSE: To determine the in situ location and distribution of the renal artery origins in the transverse plane with computed tomography (CT). MATERIALS AND METHODS: CT scans of the paired main renal arteries in 200 patients (89 men, 111 women) were retrospectively reviewed. The locations of the renal artery origins, defined on the basis of their optimal profile angles, and the angle between them were measured. The degree of aortic atherosclerosis was graded in 119 of the 200 patients. RESULTS: The origins of 400 paired main renal arteries were identified. A statistically significant difference was found between the average best profile angle on the right (24 degrees [range, 26 degrees-70 degrees]) and that on the left (5 degrees [range, -75 degrees to 38 degrees]) (P < .001). Truly laterally located renal arteries were seen on the right in 11 (5%) of 200 right renal arteries and on the left in 56 (28%) of 200 left renal arteries. One hundred eighty-six (93%) of 200 right ostia and only 40 (20%) of 200 left ostia were in an anterolateral location. One hundred four (52%) of 200 left ostia and three (2%) of 200 right ostia were in a posterolateral location. The prevalence of truly opposite renal arteries was 17%. The average profile angle between the renal artery origins was 161 degrees (range, 72 degrees-225 degrees) and was significantly larger in women (P = .001). No relationship was found between ostial location and patient age or atherosclerotic grade. CONCLUSION: In the transverse plane, the location of the origin of the right renal artery tended to be anterolateral and of the left renal artery tended to be posterolateral or lateral. The variation in location and distribution width was great.

Aortic Diseases↗
PubMed1983

Quantitative relation between sites of atrial impulse origin and cycle length.

Having previously described the multicentric origin of the atrial impulse from sites widely distributed over the right atrium as well as an intrinsic link between these sites of origin and cycle length (CL), we undertook a quantitative study of this relationship. In 132 dogs anesthetized with pentobarbital sodium or fentanyl citrate, we recorded atrial activation sequence maps from 360 closely positioned electrodes and determined locations of impulse origin at heart rates between 80 and 240 (CL between 750 and 250). We used cardiac nerve stimulation and agonist-antagonist infusion to produce changes in CL and impulse origin. Results demonstrate a significant correlation between site of impulse origin and CL. These sites, associated with both the sinus node (SN) and extranodal sites, function predictably and consistently controlling impulse initiation at heart rates above and below rates at which the SN predominates. This relationship can be used to specify an anatomic-functional model of atrial pacemaker hierarchy and to quantitate the response of different atrial regions to specific pharmacological and physiological interventions.

Animals↗
PubMed2006

Acinar origin of CFTR-dependent airway submucosal gland fluid secretion.

Cystic fibrosis (CF) airway disease arises from defective innate defenses, especially defective mucus clearance of microorganisms. Airway submucosal glands secrete most airway mucus, and CF airway glands do not secrete in response to VIP or forskolin. CFTR, the protein that is defective in CF, is expressed in glands, but immunocytochemistry finds the highest expression of CFTR in either the ciliated ducts or in the acini, depending on the antibodies used. CFTR is absolutely required for forskolin-mediated gland secretion; we used this finding to localize the origin of forskolin-stimulated, CFTR-dependent gland fluid secretion. We tested the hypothesis that secretion to forskolin might originate from the gland duct rather than or in addition to the acini. We ligated gland ducts at various points, stimulated the glands with forskolin, and monitored the regions of the glands that swelled. The results supported an acinar rather than ductal origin of secretion. We tracked particles in the mucus using Nomarski time-lapse imaging; particles originated in the acini and traveled toward the duct orifice. Estimated bulk flow accelerated in the acini and mucus tubules, consistent with fluid secretion in those regions, but was constant in the unbranched duct, consistent with a lack of fluid secretion or absorption by the ductal epithelium. We conclude that CFTR-dependent gland fluid secretion originates in the serous acini. The failure to observe either secretion or absorption from the CFTR and epithelial Na(+) channel (ENaC)-rich ciliated ducts is unexplained, but may indicate that this epithelium alters the composition rather than the volume of gland mucus.

Animals↗