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At least 109 records · Page 6Linked to original sources
PubMed1988

DNA structural alterations in the SV40 enhancer region are retained in vivo.

Psoralen accessibility in the SV40 regulatory region has been analyzed in vivo on viral minichromosomes and extracellular virus particles using a psoralen mapping technique which we have recently described. This approach takes advantage of the fact that lambda exonuclease digestion of double-stranded DNA is inhibited by the presence of 5-methylisopsoralen monoadducts. Previous analysis of purified restriction fragments from this region irradiated in vitro revealed a region of psoralen hypersensitivity which encompassed the repeated 72-base-pair (bp) enhancers and sequences 150 bp upstream toward the late region. The studies reported here demonstrate that changes in the structure of the DNA helix due to supercoiling do not affect the pattern of psoralen accessibility in this region. Additionally, no significant difference in the precise pattern of the psoralen hypersensitive domain is observed between late nucleoprotein complexes and extracellular virus. Finally, virus particles that have been through a freeze-thaw cycle, a process which we have shown eliminates the preferential photoaddition of the regulatory region in chromatin, also exhibit the same pattern of psoralen hypersensitivity.

DNA, Viral↗
PubMed1991

HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles.

Recombinant human immunodeficiency virus type-1 (HIV-1)-like gag-env particles produced in mammalian cells were inoculated into two New Zealand white rabbits. In parallel, two control rabbits were inoculated with the homologous HIV-1 virions inactivated by ultra violet light (uv) and psoralen treatments. The humoral and cellular immune responses to HIV-1 were evaluated for both groups of animals. Recombinant particles elicited humoral immunity that was specific for all the viral structural proteins. The antibodies recognized both denatured and nondenatured proteins. Moreover, the sera neutralized the in vitro infectivity of the homologous virus in CEM cells. Importantly, the recombinant particles also generated a T helper response by priming with the HIV proteins. Similar results were observed with inactivated virus immunization. Therefore, our results suggest that the recombinant HIV-like particles elicit functional humoral immunity as well as cellular immunity and represent a novel vaccine candidate for AIDS.

Animals↗
PubMed1995

Identification of double Holliday junctions as intermediates in meiotic recombination.

During meiosis, branched DNA molecules containing information from both parental chromosomes occur in vivo at loci where meiosis-specific double-stranded breaks occur. We demonstrate here that these joint molecules are recombination intermediates: they contain single strands that have undergone exchange of information. Moreover, these joint molecules are resolved into both parental and recombinant duplexes when treated in vitro with Holliday junction-resolving endonucleases RuvC or T4 endo VII. Taken together with previous observations, these results strongly suggest that joint molecules are double Holliday junctions.

Alcohol Oxidoreductases↗
PubMed1995

Native DNA fragments photocrosslinked to psoralen binds to anti-B and anti-Z DNA antibodies.

Calf thymus DNA fragments of varying size (100-600 bp) were covalently linked to psoralen in the presence of 365 nm UV light. The crosslinked species were separated from the monoadducts by hydroxyapatite column chromatography. The DNA-psoralen crosslink showed binding with naturally occurring anti-DNA autoantibodies. This was substantiated by the recognition of affinity purified IgG by DNA-psoralen crosslinks of varying DNA size. The photo-crosslinks exhibited considerable recognition of both monoclonal and polyclonal anti-Z-DNA antibodies in inhibition ELISA and band shift assay.

Animals↗
PubMed1996

A non-radioactive method for identifying enzyme-amplified products of the reticuloendotheliosis proviral env and LTR genes using psoralen-biotin labelled probes.

A novel polymerase chain reaction (PCR) system based on the env gene of reticuloendotheliosis virus (REV) strain REV-A for the detection of proviral DNA is described. The designed PCR product of 807 bp was identified using an internal probe of 278 bp produced by nested PCR from REV-infected DNA CEF. The env-gene PCR was then compared with the previously described PCR for proviral REV-long terminal repeat and the PCR product served also as the probe. The probes were labelled with the psoralen-biotin system by photoactivation and the southern blot hybridization signal was detected colorimetricaly. The advantages of using a non-radioactive means of probe labelling were demonstrated clearly in that study, as well as the effective labeling of probes with psoralen-biotin and the simple colorimetric method of detection. The env-gene PCR detected all eleven REV strains used in the study. These included three REV prototype strains and eight Israeli REV isolates. Both PCR systems had similar levels of sensitivity.

Animals↗
PubMed1989

Instability of the nuclear chromatin of procyclic Trypanosoma brucei brucei.

Digestion of nuclear chromatin of Trypanosoma brucei brucei procyclic culture forms with micrococcal nuclease yielded DNA fragments which formed DNA ladders in agarose gels, similar to those of rat liver. However, the chromatin of trypanosomes was digested more rapidly. The digestion of T. b. brucei chromatin yielded a large amount of DNA fragments of core-particle size. The numbers of base pairs per nucleosomal and linker DNA were identical in both species, if the digestion conditions were reduced in the case of T. b. brucei. Psoralen cross-linking of soluble chromatin of trypanosomes at 5 mM salt at pH 7 or pH 10 resulted in an irregular array of single-stranded (ss) bubbles separated by variable stretches of double-stranded (ds) DNA. The proportion of ss DNA was low compared with the ratio of ss/ds stretches in rat liver chromatin, which also showed regularly arranged nucleosomal DNA. Soluble chromatin of T. b. brucei, pre-treated with 500 mM NaCl to remove a potential H1 and psoralen cross-linked at 5 mM salt at pH 7 or pH 10 was to a great extent ds in both situations. The true nucleosome filament organization of T. b. brucei chromatin could only be shown by psoralen cross-linking the DNA in whole nuclei under physiological conditions. The results indicate that the chromatin of procyclic T. b. brucei differs significantly in its compaction pattern from rat liver chromatin; a typical histone H1 is not found, and the DNA-protein interactions are also less stable and can more easily be destabilized by experimental conditions.

Animals↗
PubMed1984

Psoralen-DNA crosslink repair in human lymphocytes. Comparison of alkaline elution with electron microscopy.

Much interest has surrounded the question of the removal of psoralen interstrand crosslinks in DNA of eukaryotic organisms. A commonly employed method for the study of psoralen repair is alkaline elution. In this study we have used alkaline elution to assess psoralen crosslink repair in human lymphocytes. The lymphocytes were treated with 8-methoxypsoralen or 4,5',8-trimethylpsoralen and allowed to repair for different periods of time. Analysis by alkaline elution showed elution patterns compatible with crosslink removal. When the crosslink removal under comparable conditions was studied by the use of electron microscopy under totally denaturing conditions, no repair of the crosslinks could be detected.

DNA↗
PubMed1986

Repair in E. coli of transforming plasmid DNA damaged by psoralen plus near-ultraviolet irradiation.

Treatment of DNA with psoralen plus near-ultraviolet irradiation gives rise to both monoadducts and cross-links. We have examined the repair of plasmid NTP16 DNA treated in this way in vitro and then used to transform E. coli. Monoadducts are found to be potentially lethal, and can be repaired by uvr-dependent and recA-dependent pathways. The presence of a related resident plasmid in the transformed cells can enhance the survival of the incoming damaged NTP16 DNA. This effect is not recA-dependent, and a similar effect (designated "resident enhanced repair") has been observed previously with UV-irradiated plasmids of this particular incompatibility group. Removal of unbound psoralen from the plasmid DNA and exposure to further NUV is known to increase the ratio of cross-links to monoadducts, and we demonstrate that such cross-linked plasmid DNA is not readily repaired following transformation. However in the presence of homologous DNA (related resident plasmid) there is evidence for the repair, and hence uptake by the cell, of cross-linked DNA.

Calcium↗
PubMed1990

Photosensitizing potential of benzanthrone.

In skin-photosensitization studies on guinea-pigs benzanthrone induced significant erythema and oedema, depending upon the doses both of benzanthrone and of sunlight or artificially simulated UVA radiation. Maximum sensitization and delayed tanning response on the guinea-pig skin were observed 24-36 hr after irradiation. Photosensitized benzanthrone was found to produce significant amounts of singlet oxygen in vitro, as assessed by the bleaching of N,N-dimethyl-p-nitrosoaniline. As with haematoporphyrin and rose bengal, both of which are potent generators of singlet oxygen, the production of singlet oxygen by benzanthrone was dependent on both the concentration of the test chemical and the dose of solar radiation. Benzanthrone also produced a significant yield of superoxide anion radicals on exposure to simulated solar radiation or sunlight. Photosensitized benzanthrone induced photohaemolysis of rat erythrocytes and lipid peroxidation of erythrocyte ghosts, in vitro, probably largely through involvement of singlet oxygen (1O2). The rate of lipid peroxidation by photosensitized benzanthrone was inhibited by 64-80% with 1,4-diazabicyclo(2,2,2)octane and sodium azide, 15% with superoxide dismutase but was not affected by mannitol and sodium benzoate. Equimolar concentrations of haematoporphyrin and rose bengal also produced considerable photohaemolysis of erythrocytes and lipid peroxidation in erythrocyte ghosts; in both cases rose bengal was the most active and benzanthrone the least active of the three compounds.

Animals↗
PubMed1989

Effects of active compounds isolated from Angelica shikokiana on lipid metabolism in fat cells.

It was found that the EtO Ac fraction of the roots of Angelica shikokiana inhibited adrenaline-induced lipolysis in rat fat cells, while having no effect on ACTH-induced lipolysis. On the other hand, the MeOH fraction was found to inhibit ACTH-induced lipolysis but not to inhibit adrenaline-induced lipolysis. The active substances isolated from this root were elucidated to be psoralen (I), bergapten (II), isopteryxin(3'-angeloyloxy-4'-acetoxy-3',4'-dihydroseseli n) (III) and 3'-epoxyangeloyloxy-4'-acetoxy-3',4'-dihydroseselin (IV), respectively. The two furocoumarins, psoralen and bergapten, were found to enhance ACTH- and adrenaline-induced lipolysis in fat cells respectively. In contrast, a coumarin derivative of khellactone type, isopteryxin (III), was found to inhibit the adrenaline-induced lipolysis but with no effect on ACTH-induced lipolysis and insulin-induced lipogenesis from glucose. The other coumarin of khellactone type, 3'(R), 4'(R)-3'-epoxyangeloyloxy-4'-acetoxy-3',4'-dihydroseseline (IV), was found to inhibit the insulin-induced lipogenesis from glucose but it had no effect on adrenaline- and ACTH-induced lipolysis.

5-Methoxypsoralen↗
PubMed1989

Identification of a HeLa mRNA fraction which can correct the DNA-repair defect in Fanconi anaemia fibroblasts.

Injection of Fanconi anaemia (complementation group A) fibroblasts with HeLa mRNA is shown to correct their abnormal response to a psoralen cross-linking challenge, namely permanent repression of DNA synthesis. Injection of gradient-fractionated mRNA led to identification of a single fraction, containing mRNA of approximately 650 bases, which is responsible for this effect. This finding suggests that Fanconi anaemia (group A) cells are deficient in a small protein, up to 20 kDa in size, which is involved in the cellular response to DNA interstrand cross-links.

Adenine↗
PubMed1992

Psoralen damage-induced plasmid recombination in Saccharomyces cerevisiae: dependence on RAD1 and RAD52.

Photoreaction with psoralen, a DNA-crosslinking reagent, induces mitotic recombination in the yeast Saccharomyces cerevisiae. Psoralen damage-induced recombination was studied with non-replicating plasmids, which transform yeast cells by undergoing recombination events with chromosomal DNA. When plasmid DNA was photoreacted with psoralen in vitro and transformed into yeast cells, transformation was stimulated by psoralen modification in a dose-dependent manner. The stimulation by psoralen damage requires RAD52 gene function and is partially dependent on RAD1. Analysis of transformants indicates that plasmid integration occurs at the homologous chromosomal loci. Multiple tandem integrations are common in repair-proficient cells, with more than 20 copies of integrated plasmid seen in some transformants. Multiple integration depends on RAD1 function; only 9% of rad1 transformants, compared to 80% of RAD transformants, contained multiple plasmid copies, while 52% of the rad1 transformants were produced by gene conversion.

DNA Damage↗
PubMed1992

Defective DNA endonuclease activities in Fanconi's anemia cells, complementation groups A and B.

Cells from patients with the inherited disorder, Fanconi's anemia (FA), were analyzed for endonucleases which recognize DNA interstrand cross-links and monoadducts produced by psoralen plus UVA irradiation. Two chromatin-associated DNA endonuclease activities, defective in their ability to incise DNA-containing adducts produced by psoralen plus UVA light, have been identified and isolated in nuclei of FA cells. In FA complementation group A (FA-A) cells, one endonuclease activity, pI 4.6, which recognizes psoralen intercalation and interstrand cross-links, has 25% of the activity of the normal human endonuclease, pI 4.6, on 8-methoxypsoralen (8-MOP) plus UVA-damaged DNA. In FA complementation group B (FA-B) cells, a second endonuclease activity, pI 7.6, which recognizes psoralen monoadducts, has 50% and 55% of the activity, respectively, of the corresponding normal endonuclease on 8-MOP or angelicin plus UVA-damaged DNA. Kinetic analysis reveals that both the FA-A endonuclease activity, pI 4.6, and the FA-B endonuclease activity, pI 7.6, have decreased affinity for psoralen plus UVA-damaged DNA. Both the normal and FA endonucleases showed approximately a 2.5-fold increase in activity on psoralen plus UVA-damaged reconstituted nucleosomal DNA compared to damaged non-nucleosomal DNA, indicating that interaction of these FA endonucleases with nucleosomal DNA is not impaired. These deficiencies in two nuclear DNA endonuclease activities from FA-A and FA-B cells correlate with decreased levels of unscheduled DNA synthesis (UDS), in response to 8-MOP or angelicin plus UVA irradiation, in these cells in culture.

Cell Line↗
PubMed1992

Xeroderma pigmentosum endonuclease complexes show reduced activity on and affinity for psoralen cross-linked nucleosomal DNA.

Two DNA endonuclease complexes have been isolated from the chromatin of normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells which are active on DNA damaged with psoralen plus long wavelength ultraviolet radiation (UVA). In both normal and XPA cells, one endonuclease complex, pI 4.6, recognizes the psoralen cross-link and the other endonuclease complex, pI 7.6, recognizes the psoralen monoadduct. The levels of activity of these complexes from both normal and XPA cells are similar on damaged naked DNA. Kinetic analysis of assays using graduated concentrations of substrate revealed that selective activity of these endonuclease complexes on 8-MOP plus UVA treated DNA correlates with a reduction in Km of these complexes, indicating an increased affinity for, or rate of association with, damaged naked DNA. When the damaged substrates were reconstituted into core nucleosomes (without histone H1), both normal endonuclease complexes showed a 2.5-fold enhancement of activity, which correlated kinetically with a further increase in affinity, or rate of association (decreased Km), for this damaged nucleosomal substrate. This increase in activity and in affinity was reduced but not eliminated when histone H1 was present. By contrast, neither XPA endonuclease complex showed this enhanced activity on, or affinity for, damaged core nucleosomal DNA, and actually showed decreased activity, and affinity, when histone H1 was present. Introduction, via electroporation, of either of the normal complexes into 8-MOP plus UVA treated XPA cells in culture corrected their DNA-repair defect, further confirming the role of these complexes in the repair process.

Cell Line, Transformed↗
PubMed1995

Pulsed-field gel electrophoresis analysis of the repair of psoralen plus UVA induced DNA photoadducts in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, double-strand breaks (DSB) have been observed during the DNA repair of psoralen plus UVA induced lesions. In the present paper, we analyzed this repair step in some detail using pulsed-field gel electrophoresis (CHEF) to get a better understanding of this phenomenon with regard to the type of lesions induced and the repair pathways involved. The results confirm that, during post-treatment incubation of Saccharomyces cerevisiae cells, DSB are formed. Their appearance is dose-dependent and the rate of induction is comparable in large (chromosome IV) and small (chromosome III) chromosomes. The formation of DSB is evidenced by the breakage of linear chromosomes III and IV, but also, after high doses, by the linearization of a circular form of chromosome III. The induction of DSB appears to be highly dependent on the induction of interstrand cross-links since they are clearly present after treatments with 8-MOP plus 365 nm radiation (inducing monoadducts and cross-linking in DNA), but practically absent after treatment with 8-MOP plus 405 nm radiation (inducing predominantly monoadducts) at comparable levels of photoadducts. The occurrence of DSB is dependent on the RAD2 and RAD52, but not on the RAD6 gene. It is likely that the specific processing of DNA lesions involving DSB is related to the genotoxic consequences observed.

DNA Repair↗