RESUMO
PURPOSE: To investigate the molecular characteristics of mutations induced by repeated low doses of X-rays in spleen, liver, brain and testis of mice. MATERIALS AND METHODS: Muta mice, which harbour the lacZ gene contained in the lambda genome, were irradiated with 0.15 Gy every Monday, Wednesday and Friday for 6 months starting at 10 weeks of age for a total of 78 times. Four months after the last irradiation, DNAs were isolated from the four different tissues and the mutant frequencies of lacZ were determined. Next, the nucleotide sequences of the mutant lacZ genes were determined and compared with that of the wild-type to identify the molecular changes in the mutants. The frequencies of different types of mutations were compared with those found in age-matched non-irradiated mice. They were also compared with those found in mice irradiated with a single high dose. RESULTS: The repeated low-dose irradiation resulted in slight increases in the mutant frequency in the four kinds of tissues. The spleen, liver and brain in repeatedly irradiated mice showed higher frequencies of deletion type mutations than those of non-irradiated mice. In testis, however, the level of the increase was modest and not statistically significant. Complex type mutations were observed only in irradiated tissues. The characteristics observed in somatic tissues were similar to those induced by a single high dose of irradiation. CONCLUSIONS: The results suggest that the mechanism of mutation induction in vivo is similar between low- and high-dose irradiation in spleen, liver and brain. The low induction of deletion mutations in testis with low-dose irradiation suggests that spermatogonial cells have a unique DNA repair system against low-dose radiation-induced damage.
Assuntos
Encéfalo/efeitos da radiação , Fígado/efeitos da radiação , Mutação/genética , Baço/efeitos da radiação , Testículo/efeitos da radiação , Animais , Reparo do DNA , Relação Dose-Resposta à Radiação , Óperon Lac , Masculino , Camundongos , Camundongos Transgênicos , Raios XRESUMO
Recently, the use of restriction enzymes has been extended to studies in which rare events such as mutation and mistakes in DNA repair are examined. In these studies, the specificity of restriction enzymes becomes critical. To clarify the nature of the rare unexpected events occurring in the process of cutting of DNA with restriction enzymes then ligating it, we studied the molecular characteristics of unexpected plasmid DNAs that were retrieved as mutants of the plasmid after transfection to E. coli. The plasmid used was pUR288, containing lacZ as a marker of mutation. It was digested with restriction enzymes under the conditions recommended by the supplier of the enzymes and under the presence of DMSO, which is known to induce star activity of the enzymes. Comparisons of mutant frequencies and of nucleotide sequences of the mutants found in the different conditions indicated that nonspecific endonucleolytic activity similar to that found under star activity was present under the recommended conditions and, further, was responsible for the creation of deletion-type mutations. The frequency of these events ranged from 10(-5) to 10(-3), depending on the kind of restriction enzymes analyzed. Although the levels of the nonspecificity were not high, they should be considered in assays such as mutation and mistakes in DNA repair, where rare events are examined.
Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA/metabolismo , Mutação , Plasmídeos , Sequência de Bases , Primers do DNA , TransfecçãoRESUMO
Depletion of the stratospheric ozone layer leads to an increase in ambient UV loads, which are expected to raise skin cancer incidences. Tumor development in the skin could be a multistep process in which various genetic alterations, such as point mutations and deletions, occur successively. Here, we demonstrate that UVB irradiation efficiently induces deletions in the epidermis using a novel transgenic mouse, gpt delta. In this mouse model, deletions in lambda DNA integrated in the chromosome are preferentially selected as Spi(-) (sensitive to P2 interference) phages, which can then be subjected to molecular analysis. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m(2). After 4 weeks, lambda phage was rescued from the genomic DNA of the epidermis by in vitro packaging reactions. The mutant frequencies of Spi(-) with large deletions in the epidermis increased >15-fold at a UVB dose of 0.5 kJ/m(2) over the control. Molecular sizes of most of the large deletions were >1000 bp. More than one-half of the large deletions occurred between short direct-repeat sequences from 1 to 6 bp, and the remainder had flush ends. In the unirradiated mouse, almost all of the Spi(-) mutants were 1-bp frameshifts in runs of identical bases. These results suggest that UVB irradiation induces deletions in the murine epidermis, and most of the deletions are generated through end-joining of double strand breaks in DNA.
Assuntos
Epiderme/efeitos da radiação , Proteínas , Deleção de Sequência , Raios Ultravioleta/efeitos adversos , Animais , Proteínas de Bactérias/genética , Bacteriófago lambda/genética , Sequência de Bases , DNA Viral/genética , DNA Viral/efeitos da radiação , Relação Dose-Resposta à Radiação , Epiderme/fisiologia , Escherichia coli/genética , Escherichia coli/virologia , Proteínas de Escherichia coli , Feminino , Mutação da Fase de Leitura , Masculino , Camundongos , Camundongos Transgênicos , Pentosiltransferases , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genéticaRESUMO
We have developed a new genomic sequencing method for detecting, with resolution at the nucleotide level, the interstrand DNA cross-links induced by 4,5',8-trimethylpsoralen along single-copy genes in mammalian cells. The cross-links (diadducts) initially formed are converted into monoadducts by alkali reversal prior to the use of terminal transferase-dependent PCR (TD-PCR). After alkali reversal, but not before, the DNA strands can be separated and used as templates for gene-specific primer extension, which is the first step in the TD-PCR procedure. The converted psoralen adducts block primer extension, and the prematurely terminated single-stranded products are then amplified by TD-PCR and visualized on a sequencing gel. Adducts formed by angelicin, a psoralen derivative that forms only monoadducts, were also investigated by use of TD-PCR. Comparison of the adduct distribution patterns of in vivo-treated DNA with those of in vitro-treated DNA revealed that the binding of transcription factors inhibited both psoralen cross-linking and angelicin monoadduct formation in the c-JUN and c-FOS promoters in living human cells. Adduct formation was also inhibited in the region of a putative positioned nucleosome in the c-FOS promoter. These methods should be of general use for study of in vivo protein-DNA interactions and DNA repair.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Adutos de DNA/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Trioxsaleno/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Reagentes de Ligações Cruzadas/química , Adutos de DNA/antagonistas & inibidores , Pegada de DNA , DNA Nucleotidilexotransferase/metabolismo , Furocumarinas/metabolismo , Humanos , Substâncias Intercalantes/metabolismo , Dados de Sequência Molecular , Nucleossomos/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trioxsaleno/análogos & derivados , Trioxsaleno/químicaRESUMO
PURPOSE: To study the dose-response and molecular nature of radiation-induced mutations in the spleen and brain of lacZ transgenic mice. MATERIALS AND METHODS: Line 60 transgenic mice containing the bacterial lacZ gene in a plasmid background were used. Mutants were selected using phenyl-beta-D-galactoside. The nature of mutants was determined by sequencing DNAs of mutant lacZ genes found in control and irradiated tissues. RESULTS: X-ray irradiation at 50 and 100 Gy showed linear dose-responses for mutation induction in both tissues. The slope, however, was about twice as steep in spleen than in brain. DNA sequence analyses showed that the predominant type of mutation induced by radiation in both tissues were large deletions. CONCLUSIONS: Radiation induces mutations in spleen and brain at different efficiencies but the molecular nature of the induced mutations are similar in the two issues.
Assuntos
Encéfalo/efeitos da radiação , Óperon Lac/genética , Óperon Lac/efeitos da radiação , Mutação , Baço/efeitos da radiação , Sequência de Aminoácidos , Animais , Sequência de Bases , Análise Mutacional de DNA , Relação Dose-Resposta à Radiação , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de Órgãos/efeitos da radiação , Deleção de Sequência , Irradiação Corporal Total , Raios XRESUMO
Accumulation of mutation has long been hypothesized to be a cause of aging and contribute to many of the degenerative diseases, which appear in the senescent phase of life. To test this hypothesis, age-associated changes in spontaneous mutation in different tissues of the body as well as the molecular nature of such changes should be examined. This kind of approach has become feasible only lately with a development of new transgenic mice suitable for mutation assay. Here, using one of these transgenic mice harboring lacZ gene, we have shown that the age-associated increase in spontaneous mutant frequency is common to all tissues examined; spleen, liver, heart, brain, skin and testis, while the rates of increase in mutant frequency differed among the tissues. DNA sequencing of the 496 lacZ mutants recovered from the tissues of newborn and old mice has revealed that spectra of mutations are similar at the two age points with G:C to A:T transition at CpG site being a predominant type of mutation. Furthermore, some mutations in old tissues are complex type and not found in tissues of newborn mice. These results suggest that similar mechanisms may be operating for mutation induction in fetal and postnatal aging process. In addition, the appearance of complex types of mutations in the old tissues suggests a unique cause for these mutations in aging tissues.
Assuntos
Envelhecimento , Óperon Lac/genética , Mutação/genética , Animais , Animais Recém-Nascidos , Sequência de Bases , Encéfalo/metabolismo , DNA/química , DNA/genética , Análise Mutacional de DNA , Feminino , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Pele/metabolismo , Baço/metabolismo , Testículo/metabolismoRESUMO
In our previous study using transgenic Muta mice, G:C --> A:T transitions at 5'-CG-3' (CpG) sites, which are the most common mammalian spontaneous mutation, were detected in 197 of 330 spontaneous lacZ mutants. These transitions were recovered at only 27 of the 357 mutable G:C pairs within CpG sites where the transition could produce a missense or termination codon in the lacZ gene. To address the underlying mechanism for the uneven distribution of mutated CpG sites, the CpG methylation status of the Muta lacZ gene was analyzed by a bisulfite method. All the CpG sites examined in the coding region were evenly methylated at a high level, and no site-specific methylation was evident. Analysis of the sequence context around the mutated CpG sites, however, revealed that 21 of these 27 sites contained a CpG flanked by a pyrimidine on the 5' side, and that 187 of the 197 mutants resulted from substitutions at these sites. Moreover, we found five hotspots among those sites, the location of which was intimately related to the enzymatic activity of the gene product: one site produced a nonsense codon; three sites, one of which corresponded to the nucleophile at the active site, resided in the substrate-binding pocket; and the other site was located in a region conserved in the beta-galactosidase family. These results strongly suggest that recovery of lacZ mutations at each site largely depend on the adjacent sequence context and the extent to which the mutation damages the enzymatic activity of the gene product.
Assuntos
Ilhas de CpG , Metilação de DNA , Óperon Lac , Mutação , Proteínas Repressoras/genética , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos MutantesRESUMO
Ultraviolet light B (UVB)-induced mutagenesis was studied in gpt delta transgenic mice, which contain the lambdaEG10 shuttle vector as a transgene. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m(2). At 4 weeks after irradiation, the mutant frequencies (MF) of the gpt gene were determined in the epidermis and the dermis, and the gpt mutations in the epidermis were identified by DNA sequencing. The epidermis exhibited a higher sensitivity to UVB than the dermis at doses of 0.3 and 0.5 kJ/m(2) UVB: the MF of the epidermis were more than nine times higher than those of the nonirradiated mice, whereas the MF of the dermis were only two to three times higher than the nonirradiated level at the doses used. The UVB-induced mutation spectrum in the epidermis was dominated by G:C to A:T transitions at dipyrimidine sites, such as 5'-TC-3', 5'-CC-3', and 5'-T/C-CG-3'. Tandem transitions such as CC to TT were also observed. Interestingly, a remarkable bias towards the template strand of the gpt gene was observed in the single transitions at 5'-TC-3' and 5'-CC-3' sites, but not at 5'-T/C-CG-3' site. In contrast, G:C to A:T transitions at CpG sites and deletions were observed in nonirradiated mice. Hot spots of transitions were observed at different sites in UVB-irradiated and nonirradiated mice. These results indicate that gpt delta transgenic mouse is a suitable model for studying in vivo UVB-induced mutations at the molecular level.
Assuntos
Proteínas de Bactérias/genética , Mutação , Proteínas , Pele/efeitos da radiação , Animais , Sequência de Bases , DNA , Relação Dose-Resposta à Radiação , Proteínas de Escherichia coli , Camundongos , Camundongos Transgênicos , Pentosiltransferases , Raios UltravioletaRESUMO
DNA sequences of 103 spontaneous mutants and 102 X-ray-induced mutants of the lacZ transgene from spleen, liver, and brain of the MutaMouse were examined and compared to elucidate characteristics of radiation-induced mutations in vivo. The radiation-induced mutants were isolated from genomic DNA of each tissue collected at 3.5 days after 200 Gy of whole body irradiation. Base substitution was predominant (80% or more) in nonirradiated tissues, while deletion was prevalent (about 55%) in irradiated tissues. The other types of mutation appeared at similar frequencies in both control and irradiated tissues. The size of the deletions was smaller than 438 nucleotides, with a predominance of one basepair deletions in both control and irradiated tissues. A close look at the nucleotides at the deletion endpoints revealed that many of the radiation-induced deletions did not have repeated sequences at the break point termini, whereas all deletions found in unirradiated tissues showed one or more bases of repeated sequences at the termini. Further, eight complex-type deletion mutations were found only in irradiated tissues. Comparison among the three types irradiated tissue did not reveal any tissue-specificity. The data indicate that the molecular nature of mutations induced in tissues with ionizing radiation is different from that of spontaneous mutations.
Assuntos
Encéfalo/efeitos da radiação , Fígado/efeitos da radiação , Mutação , Baço/efeitos da radiação , Animais , Sequência de Bases , Primers do DNA , Relação Dose-Resposta à Radiação , Óperon Lac , Camundongos , Camundongos Transgênicos , Deleção de Sequência , Raios XRESUMO
ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have been shown to have sequences homologous to the catalytic domains of mammalian phosphatidylinositol 3-kinase (PI3-kinase). In order to determine the contribution of ATM and DNA-PKcs to the increased sensitivity of cells to DNA-damaging agents observed in the presence of PI3-kinase inhibitors, we examined the effects of a PI3-kinase inhibitor, wortmannin, on cellular sensitivity to bleomycin (BLM), mitomycin C (MMC), X-irradiation and ultraviolet (UV)-irradiation using 2 human tumor cell lines (T98G and A172), a human fibroblast cell line (LM217), an ataxia telangiectasia (AT) cell line (AT3BISV), a scid murine cell line (SCF) and a control murine cell line (CBF). Wortmannin sensitized all of the cells, including AT3BISV and SCF, to BLM and X-irradiation, but not to MMC or UV-irradiation. Hypersensitivity to BLM and X-irradiation and normal sensitivity to MMC and UV-irradiation are characteristic phenotypes of both AT and scid mice. DNA-dependent protein kinase (DNA-PK) activity was suppressed by wortmannin to 45-65% of the control values in all of the cells except SCF, in which DNA-PK activity was not detected. Wortmannin also induced radioresistant DNA synthesis, which is a cellular phenotype of AT, in T98G and SCF cells, but did not change the DNA synthesis rates after X-irradiation in AT3BISV. Our data suggest that wortmannin decreases the activities of both the ATM protein and DNA-PK, indicating that it might be of use as a sensitizing agent for radiotherapy and chemotherapy.
Assuntos
Androstadienos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Proteínas de Ligação a DNA , DNA/biossíntese , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Radiossensibilizantes/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Proteína Quinase Ativada por DNA , Humanos , Camundongos , Camundongos SCID , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas/análise , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , WortmaninaRESUMO
Ionizing radiation and hydrogen peroxide (H2O2) produce many types of oxidative DNA damage such as strand breaks, apurinic/apyrimidinic (AP) sites, base modifications and 3'-blocking damage such as 3'-phosphoglycolated and 3'-phosphorylated termini. AP sites and 3'-blocking damage are repairable by exonuclease III and endonuclease IV in Escherichia coli. XthA-nfo double mutants of E. coli, which are deficient in exonuclease III and endonuclease IV, were highly sensitive to lethal and mutagenic effects of H2O2, compared with the wild-type strains. The pNT180 and pNT186 plasmids containing wild-type nfo and mutant nfo-186 gene, respectively, were introduced into the xthA-nfo mutant. The nfo-186 gene product, Nfo186, retained normal AP endonuclease activity but could not remove 3'-blocking damage from DNA. The pNT180 corrected the sensitivity of the xthA-nfo mutant to lethal and mutagenic effects of H2O2. On the other hand, the pNT186 did not have any complementation effects. From these results it was concluded that 3'-blocking damage rather than an AP site is the primary lesion responsible for both lethal and mutagenic effects of H2O2.
Assuntos
Dano ao DNA , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Mutação , Escherichia coli/genéticaRESUMO
To evaluate the usefulness of the transgenic Muta mouse for investigating radiation-induced mutations in vivo, we have examined the effects of whole-body X irradiation and compared them to the effects of ultraviolet light. The spontaneous mutation frequencies in young adults were about 7 x 10(-5) in the spleen, liver and skin. The mutation frequencies 1 week after a lethal dose of X radiation (8 Gy) were 3.2, 2.6 and 2.7 times the spontaneous levels in the spleen, liver and skin, respectively. When the skin was irradiated with 10 kJ m-2 of UVB, the mutation frequency increased about 6 times. The mutation frequencies induced by an acute dose of 4 Gy or by a fractionated dose of 11.7 Gy (0.15 Gy x 78 times, 3 times/week) in the spleen and liver were less than 2-fold the spontaneous levels at 16 weeks after irradiation. A comparison of X and UV radiation was also conducted with cultured cells derived from the mouse embryo. UVC of 5 J m-2 raised the mutation frequency to 15 times that of unirradiated cells, while 10 Gy X rays raised it 2.6 times. The findings indicate that the Muta mouse is less sensitive to X-ray-induced mutation than UV-induced mutation.
Assuntos
Mutação/efeitos da radiação , Animais , Células Cultivadas , Camundongos , Raios Ultravioleta , Raios XRESUMO
In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature. To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39 degrees C, for only a limited period after inducing DNA damage. We used ts mutants defective in genes for ubiquitin-activating enzyme (E1), DNA polymerase alpha, and p34(cdc2) kinase. Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39 degrees C for limited periods after UV exposure. Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39 degrees C, and more than 36 h at 39 degrees C did not produce any further sensitization. Moreover, while the 39 degrees C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype. These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating enzyme (E2), to which E1 is known to transfer ubiquitin. These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.
Assuntos
Reparo do DNA/fisiologia , Ligases/genética , Mutagênese , Temperatura , Raios Ultravioleta , Animais , Proteína Quinase CDC2/genética , Carcinoma , Dano ao DNA , DNA Polimerase II/genética , Neoplasias Mamárias Experimentais , Camundongos , Mutagênicos/farmacologia , Mutação , Tioguanina/farmacologia , Células Tumorais Cultivadas , Enzimas Ativadoras de Ubiquitina , Ubiquitina-Proteína Ligases , Raios XRESUMO
Spontaneous mutant frequency of lacZ gene in spleen of transgenic MutaTM mouse was examined at different ages. It was (3.2 +/- 1.3 (SD)) x 10(-5) at newborn and increased almost linearly with age up to (8.3 +/- 1.8) x 10(-5) at one year. Since the mutation of the gene is not likely to be subject to selection in vivo, the data support the idea that spontaneous mutation takes place throughout aging process and accumulates with age if not selected out by cell death.
Assuntos
Envelhecimento/genética , Óperon Lac , Mutação/genética , Baço/metabolismo , Transgenes , Animais , Feminino , Hipoxantina Fosforribosiltransferase/genética , Masculino , Camundongos , Camundongos Transgênicos/genéticaRESUMO
DNA polymerases (pol) alpha, delta and epsilon of a mouse cell line FM3A and its temperature-sensitive derivative tsFT20, which is defective in DNA replication at a non-permissive temperature, were purified by chromatographic procedures monitored by a set of relatively specific assays for the respective DNA polymerase activities. The pol epsilon activity was separated into two fractions with similar enzymatic properties except for their optimal KCl concentrations and processivities. The fractions of pol delta and epsilon were not homogeneous, but their identities were confirmed by their sensitivities to DNA polymerase inhibitors, their associated 3'-->5' exonuclease activities, optimal concentrations of salts, dependencies on the proliferating cell nuclear antigen and processivities in polymerization, which also excluded significant contamination with other DNA polymerases. Of the DNA polymerases prepared from tsFT20 cells, only pol alpha showed greatly decreased activity and remarkable sensitivity to the non-permissive temperature, demonstrating that pol delta and epsilon, the other polymerases supposed to be involved in nuclear DNA replication, are unequivocally different entities from pol alpha. The level of pol epsilon activity tsFT20 was also significantly lower than in the parental cells, suggesting cooperation and/or interaction between pol alpha and epsilon, and some relevance of pol epsilon to DNA replication.
Assuntos
DNA Polimerase II/análise , DNA Polimerase Dirigida por DNA/análise , Animais , DNA Polimerase II/isolamento & purificação , DNA Polimerase II/metabolismo , DNA Polimerase III , Replicação do DNA , DNA Polimerase Dirigida por DNA/isolamento & purificação , DNA Polimerase Dirigida por DNA/metabolismo , Estabilidade Enzimática , Camundongos , Camundongos Endogâmicos C3H , Mutação , Temperatura , Células Tumorais CultivadasRESUMO
Altered sequences were determined of 52 independent spontaneous mutations occurring in a cDNA of the human hypoxanthine phosphoribosyltransferase (hprt) gene, which was integrated into chromosomal DNA of the mouse cell as a part of the retroviral shuttle vector. Spontaneous mutations comprised a variety of events: base substitutions, frameshifts, deletions, duplications, and complex mutational events, and were distributed randomly over the coding region of the gene. Frameshifts were the most frequent mutational event (38%), and base substitutions were the next most frequent (25%), followed by deletions (19%). Frameshift and deletion mutations commonly occurred preferentially at sites flanked by short direct repeats. Short inverted repeats were frequently found to be associated with duplication and complex mutational events. Analysis of the sequence alterations in the mutant genes suggests that misalignment mutagenesis represents an important molecular mechanism for the generation of spontaneous mutations in eukaryotic cells.
Assuntos
DNA/genética , Hipoxantina Fosforribosiltransferase/genética , Mutação , Animais , Sequência de Bases , Linhagem Celular , Deleção Cromossômica , Vetores Genéticos , Humanos , Camundongos , Dados de Sequência MolecularRESUMO
cDNA of the human hprt gene was introduced into the BamHI cloning site of the retroviral shuttle vector pZipNeoSV(X)1. The mouse cell line 2TGOR, a hypoxanthine phosphoribosyltransferase-deficient derivative of Balb/c 3T3, was transformed with the vector and some stably transformed HATrNEOr clones were established. One of the clones, VH-12, contained a single copy of the vector integrated stably into a chromosome in a proviral form. From this clone, we were able to recover efficiently the vector sequence preserving its intact structure by use of COS cell fusion. The relatively small size of the hprt cDNA (657 base pairs for the coding region) allowed quick determination of the entire DNA sequence. It was also notable that use of 6TG NEO double selection for mutant isolation could eliminate the 6TGr derivatives of VH-12 cells which arose from loss of the total vector sequence or from some epigenetic event, because such alterations would lead to inactivation of the neo gene as well as the hprt cDNA. The properties of our shuttle vector system were particularly useful for analysis of the molecular mechanisms of mutational events in chromosomal DNA of mammalian cells.