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Given a large number of genes with unknown functions in model organisms, collections of mutants are valuable resources for studying gene function. For the mouse, embryonic stem cell technology offers the possibility to manipulate the genome and select for mutations in vitro. Mutant mice can then be generated from clones of interest to study the phenotype of these animals. We manipulate the genome of mouse embryonic stem (ES) cells chemically using the mutagen trimethylpsoralen (TMP). TMP predominantly causes deletions in the genome of Caenorhabditis elegans and Escherichia coli, but has not been established as a mutagen in mammalian systems yet. We have characterized TMP as a mutagen for mouse ES cells regarding death rates, mutation frequencies, and mutation spectrum. Allowing for 12.5% of cell survival, the mutation frequency at the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus was 3.5 x 10(-5) on average. The characterization of a non-redundant set of 17 Hprt-deficient ES clones revealed that only 12% of clones contained genomic deletions and almost 50% were point mutations. Base substitutions were mostly transversions and all affected AT base pairs. We conclude that the mutation spectrum of TMP in mouse ES cells is different from that observed in C. elegans and E. coli.

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The genome project of the nematode Caenorhabditis elegans is completed. It is important and useful to disrupt nematode genes to know their function. We treated wild-type animals with potential candidates for mutagens for reverse genetics, EMS (ethyl methanesulfonate), short-wavelength UV, and long-wavelength UV in the presence of TMP (trimethylpsoralen). We estimated forward mutation rates by counting the occurrence of a marker unc-22 mutation. We found that the forward mutation rate by TMP/UV could be comparable with EMS by improving the frequency one order higher than before. We next isolated mutants of another marker gene ben-1 and examined the probability for the deletion mutations by PCR and sequencing. Deletion mutations were found only by TMP/UV method, which suggested TMP/UV is the choice for deletion mutagenesis among these methods. As a pilot experiment, we could isolate actual deletion mutations at a much higher frequency than previously.

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In a recent paper, the hypothesis of 'conservative pairing' between complementary DNA strands belonging to both sister chromatids has been proposed as a phenomenon that could account for, at least in part, sister chromatid pairing in late G2/mitosis. The hypothesis was verified through a cytogenetic approach, studying the so-called 'sister chromatid chromatin bridges' (SCCBs), induced in the previous G2/mitosis by a crosslinking (TMP + UVA 365 nm) treatment in CHO cells (Rizzoni, M., E. Cundari, P. Perticone and B. Gustavino (1993) Chromatin bridges between sister chromatids induced in late G2 mitosis in CHO cells by trimethylpsoralen + UVA. Experimental Cell Res., 209, 149-155; [1]). The purpose of the present paper is the study of the relationship between chromatin bridges without fragments in ana-telophase, which were demonstrated to be SCCBs, and chromosomal aberrations, in order to investigate their mechanism of induction. The evolution along the time of the two classes of mitotic anomalies was studied and a comparison was carried out to verify whether the bridges rise as a direct and immediate effect of the treatment or represent the misrepair-mediated effect of it. The present data show that single bridges without fragments come from a direct effect of photoinduced crosslinks in late G2/mitosis. Moreover TMP + 365 nm UVA treatment shows an S-dependent clastogenic effect. The proposed hypothesis of 'conservative pairing' between DNA strands of sister chromatids is further supported.

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Psoralens react photochemically with DNA to form interstrand crosslinks as well as two types of monoadduct (furan-side and pyrone-side adducts). To investigate the relative roles of these adducts in toxicity, we have studied the interaction of 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP) with bacteriophage T7. These two derivatives differ in the fraction of pyrone-side monoadducts formed, TMP producing very small amounts of this type of adduct. The results show similar phage survival for the two psoralen analogs at equivalent numbers of crosslinks per DNA molecule. However, the survival fraction of treated phage is significantly lower than the fraction of noncrosslinked DNA molecules. Phage survival decreases after secondary irradiation which is used to transform monoadducts into crosslinks, but this decrease is not due solely to crosslinks; at doses beyond that required to transform all crosslinkable monoadducts into crosslinks, phage survival continues to decrease, pointing to the production of other genotoxic lesions during secondary irradiation. These results indicate that, although crosslinks can kill phage T7, as shown by the secondary irradiation results, they are not sufficient in number to explain the psoralen toxicity after primary irradiation. Therefore monoadducts, both furan-side and pyrone-side types, must in large part be responsible for phage inactivation.

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Inactivation of the blood-borne parasite Trypanosoma cruzi by UVA and 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 micrograms/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type photo-reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi, the trypomastigote, was achieved at a UVA (320-400 nm radiation) fluence of 4.2 J/cm2. We note that while the infectivity of the parasite is eliminated at 4.2 J/cm2 the trypomastigote motility continues for at least 16 h-post-treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of 3H-AMT indicated that at the lethal UVA influence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi, the causative agent of Chagas disease.

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Using the 4,5',8-trimethylpsoralen in combination with the reirradiation protocol, we show that, in normal human lymphoblasts, the cytotoxic potential of photoinduced cross-links (CL) is higher than that of monoadducts (MA). In contrast to cytotoxicity, the significant increase in the proportion of CL, at a constant level of total adducts, had no effect on the induction of mutations at the HPRT locus. Comparison with the data obtained in yeast and rodent cells using the same double irradiation protocol shows that the mutagenic potential of CL versus MA varies between species. This suggests that the equilibrium between the excision, the recombinational and the mutagenic components of the repair pathways which probably determine the mutagenic efficiency of CL versus MA is likely to be species-dependent.

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We have examined the rate and extent of removal of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) cross-linkable monoadducts and interstrand cross-links from restriction fragments within the amplicon containing the dihydrofolate reductase (DHFR) gene in the Chinese hamster ovary (CHO) cell line B11. The rate and extent of removal of HMT cross-links was significantly greater in an actively transcribed fragment than in a nontranscribed extragenic fragment of similar size. For the 5' half of the DHFR gene, approximately 80% of the HMT cross-links were removed in 8 h, in agreement with results reported by Vos and Wauthier [Vos, J. M., & Wauthier, E. L. (1991) Mol. Cell Biol. 11, 2245-2252, 1991]. However, few cross-links were removed in that period from the nontranscribed fragments, whose 5' end is approximately 7 kb downstream from the DHFR transcription unit and which includes a putative replication initiation site. Even after 24 h, only about 50% of the cross-links had been removed from this fragment. In contrast, both the rate and the extent of removal of cross-linkable HMT monoadducts were similar in the two fragments with 50% of the cross-linkable monoadducts removed in 24 h. Moreover, monoadducts formed in the bulk of the genome were removed in 24 h. Moreover, monoadducts formed in the bulk of the genome were removed at a slightly slower rate and to a lesser extent (30% in 24 hours) than those from either of these specific sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

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Psoralens and UVA light have been used in the laboratory to study the inactivation of viruses that may be infrequently present in platelet concentrates that are prepared for transfusion. In order to evaluate safety aspects of the treatment of platelet suspensions with 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), we have investigated the residual levels and mutagenic potential of AMT after UVA phototreatment. 4'-aminomethyl-4,5',8-trimethylpsoralen, at a final concentration of 40 micrograms/mL, was added to platelet suspensions which contained 16% plasma and a synthetic medium. Platelet suspensions containing AMT were irradiated with up to 7.2 J/cm2 UVA light under normal oxygen levels. Residual levels of AMT were determined by HPLC and a bioassay based on bacteriophage phi 6 inactivation. The photodestruction of AMT or its activity by UVA was characterized by a D37 value of 0.6 and 0.3 J/cm2 with HPLC or bioassay, respectively. At 2.4 J/cm2 UVA, which results in approximately 5 log10 inactivation of vesicular stomatitis virus (VSV) and retention of platelet in vitro properties, 12% (HPLC) to 9% (bioassay) AMT remained. Like other psoralens, AMT was found to bind to serum proteins as shown by ultrafiltration. Results are consistent with approximately 36% of the initial drug load binding primarily to serum albumin. It was determined using 3H-AMT that 9 to 18% of radioactivity was bound to platelets in the absence of irradiation. Similar fractions (13 to 18%) of AMT were bound to platelets after 3.6 J/cm2 UVA irradiation, and 8 to 10% of total AMT was associated with saline-washed irradiated platelets and is presumably tightly bound.(ABSTRACT TRUNCATED AT 250 WORDS)

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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.

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The minimal phototoxic dose values for UVA radiation of psoralen-treated skin and of oral mucous membrane were studied in 16 healthy volunteers. A commercial 0.01% trioxalen ointment was used as the topical photosensitizer. In all 16 persons the radiation dose needed to induce erythema was greater for the buccal mucosa than for the skin, and the average buccal minimal phototoxic dose was 2.3-fold that of the cutaneous minimal phototoxic dose.

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Fanconi's anaemia (FA) cells are hypersensitive to the lethal effect of DNA cross-linking compounds. Herpes simplex virus (HSV) has been used here as a probe to monitor in FA cells repair of psoralen damage of which cross-links are a part. The replication of HSV is impaired when its DNA contains covalently photobound psoralen molecules. In comparison to other psoralens, 4,5',8-trimethylpsoralen (4,5',8-TMP) is one of the most photoreactive psoralens and it forms a relatively high proportion of DNA interstrand cross-links. TMP-damaged HSV is efficiently reactivated by multiple infection in human fibroblasts. The extent of multiplicity reactivation is greater in cells from FA donors (five strains tested) than in normal cells (three strains). Mutagenesis studied in the viral thymidine kinase locus revealed that: (i) spontaneous viral mutation rate is lower in FA than in normal cells; and (ii) under conditions of multiple infection, the mutation rate is either greater (normal cells) or unchanged (FA cells) in the progeny from psoralen-damaged HSV compared to that from untreated virus. Taken together, these observations suggest that the pathway underlying multiplicity reactivation of psoralen-damaged HSV is error-free in FA cells relative to normal cells.

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