Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein.
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An expression cassette designed for high-level production of rabies virus glycoprotein (RG) could not be rescued into a replication-defective, adenovirus-based vector using standard procedures. To overcome this difficulty, a 293-based cell line, designated 293LAP13, was constructed that contained and expressed a derivative of the lac repressor protein. The lac operator sequence, to which the repressor binds, was incorporated into an expression cassette, containing a promoter and intron, designed for high-level production of RG. Insertion of a single operator sequence immediately downstream of the transcription start site and the use of the 293LAP13 cell line allowed recombinant viruses that could not be isolated with 293 cells to be rescued efficiently. The operator-containing virus reached higher titres in 293LAP13 than in parental 293 cells and also produced plaques more efficiently in 293LAP13 cells. Moreover, in non-complementing human and canine cell lines, adenovirus vectors with a promoter-intron expression cassette expressed RG at much higher levels than vectors lacking the intron. These observations, together with the demonstration that expression of RG by operator-containing vectors was repressed markedly in 293LAP13 cells and that this inhibition was relieved at least partly by IPTG, suggest that the 293LAP13 cell line may be useful for the rescue and propagation of many vectors in which high expression of the desired protein prevents vector rescue in 293 cells. (+info)
Hyperglycemia-induced embryonic dysmorphogenesis correlates with genomic DNA mutation frequency in vitro and in vivo.
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Congenital malformations affecting multiple organ systems are at least three times more common in infants of mothers with IDDM than in infants born to nondiabetic mothers. Numerous studies have confirmed the teratogenic effect of hyperglycemia on the developing embryo, although no direct mechanism has been determined. In this study, we aimed to correlate the frequency of lacI mutations with degree of hyperglycemic exposure and severity of malformations in mouse embryos from in vitro cultures. Day 8 transgenic mouse embryos cultured in 30 or 50 mmol/l glucose for 48 h exhibited a higher incidence of morphological abnormalities, as well as an increase in lacI mutation frequency, compared with embryos cultured in 10 mmol/l glucose with no abnormalities and a lower frequency of lacI mutations. We also used a transgenic lacI rat system to evaluate the relationship between abnormal embryonic development and DNA mutation frequency in day 11 embryos of severely diabetic rats (serum glucose >20 mmol/l). Compared with control embryos, the embryos from diabetic rats displayed significantly more malformations, shorter crown-rump lengths, fewer somites, and more than six times greater genomic DNA mutation frequency. Genetic analysis of the mutated lacI gene from both in vitro cultured mouse embryos and in vivo developed rat embryos revealed that the majority of mutations were due to base substitutions (transitions and transversions), but that the rate of large DNA mutations tended to increase in embryos exposed to a diabetic environment. Our results support the interrelationship between increased rates of congenital malformations and DNA mutations in the offspring of diabetic pregnancy. (+info)
Oxazepam is mutagenic in vivo in Big Blue transgenic mice.
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Although oxazepam (Serax), a widely used benzodiazepine anxiolytic, does not induce gene mutations in vitro or chromosomal aberrations in vivo, it was found to be a hepatocarcinogen in a 2 year bioassay in B6C3F1 mice. Thus, it was of interest to determine whether this carcinogen is mutagenic in vivo. Male B6C3F1 Big Blue transgenic mice were fed 2500 p.p.m. oxazepam or control diet alone for 180 days and killed on the next day. The mutant frequency (MF) of lacI in control mice was 5.02 +/- 2.4x10(5), whereas the MF in the oxazepam-treated mice was 9.17 +/- 4.82x10(-5), a significant increase (P < 0.05). Correction of the mutant frequency of lacI from the oxazepam-treated mice for clonality resulted in a decrease in the mean mutant frequency to 8.15 +/- 2. 54x10(-5). Although the mutant frequency difference was small, sequencing of a random collection of the mutants from each oxazepam-exposed mouse showed a significant difference (P < 0.015) in the mutation spectrum compared with that from control mice. In the oxazepam-exposed mice, an increase in G:C-->T:A and G:C-->C:G transversions and a concomitant decrease in G:C-->A:T transitions were observed. Clonal expansion of mutations at guanines in 5'-CpG-3' sequencing contexts at three sites was noted. It is postulated that some of the mutations found in the oxazepam-derived spectrum were due to oxidative damage elicited by induction of CYP2B isozymes as the result of chronic oxazepam administration. This study demonstrates that the in vivo Big Blue transgenic rodent mutation assay can detect mutations derived from a carcinogen that did not induce gene mutations in vitro or micronuclei in mouse bone marrow. Moreover, the sequencing of the recovered mutants can distinguish between the mutation spectrum from treated mice compared with that from control mice, thereby confirming the genotoxic consequences. (+info)
Tamoxifen induces G:C-->T:A mutations in the cII gene in the liver of lambda/lacI transgenic rats but not at 5'-CpG-3' dinucleotide sequences as found in the lacI transgene.
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Tamoxifen, a rat liver carcinogen, can induce mutations in the lacI gene in the livers of lambda/lacI transgenic rats. However, the presence of persistent tamoxifen adducts on the liver DNA raises the possibility that some contribution to the mutagenesis from ex vivo mutations during the in vitro lacI assay cannot be ruled out. To address this issue, mutagenesis at the cII gene of the transgenic shuttle vector was determined using a selection based assay which is unaffected by the presence of tamoxifen-DNA adducts. Female lambda/lacI transgenic rats were dosed orally with tamoxifen (20 mg/kg body wt) daily for 6 weeks, causing a 3.2-fold increase in the mutant frequency (MF) in the cII gene compared with that obtained with solvent treated animals. This was similar to the MF found previously at the lacI gene and confirms that tamoxifen is mutagenic in vivo. The major class of mutation induced by tamoxifen in the cII gene was G:C-->T:A transversions as was found previously in the lacI gene. However, in the one unreplicated study of mutations in the p53 gene of liver tumours induced by tamoxifen, no G:C-->T:A transversions were found; possible differences between mutagenesis in normal and tumour tissues are explored. The major proportion of the G:C-->T:A transversions occurred at 5'-CpG-3' dinucleotide (CpG) sites in the lacI gene, but not at such sites in the cII gene. The methylation of CpG sites greatly enhances the targeting of deoxyguanosine by carcinogens, thus this finding might be explained by differences in the methylation patterns at their respective CpG sites; however, nothing is known about the methylation status of either the lacI nor the cII gene in this transgenic rat. This study raises the important issue of which target genes (mammalian or transgenic) should be used as endpoints in mammalian mutagenesis assays. (+info)
Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain.
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Analysis of the relationship between transcriptional activators and chromatin organization has focused largely on lower levels of chromatin structure. Here we describe striking remodeling of large-scale chromatin structure induced by a strong transcriptional activator. A VP16-lac repressor fusion protein targeted the VP16 acidic activation domain to chromosome regions containing lac operator repeats. Targeting was accompanied by increased transcription, localized histone hyperacetylation, and recruitment of at least three different histone acetyltransferases. Observed effects on large-scale chromatin structure included unfolding of a 90-Mbp heterochromatic chromosome arm into an extended 25-40-micrometers chromonema fiber, remodeling of this fiber into a novel subnuclear domain, and propagation of large-scale chromatin unfolding over hundreds of kilobase pairs. These changes in large-scale chromatin structure occurred even with inhibition of ongoing transcription by alpha-amanitin. Our results suggest a functional link between recruitment of the transcriptional machinery and changes in large-scale chromatin structure. Based on the observed long-range propagation of changes in large-scale chromatin structure, we suggest a possible rationale for the observed clustering of housekeeping genes within Mbp-sized chromosome bands. (+info)
Elevated mutant frequencies in lymphoid tissues persist throughout plasmacytoma development in BALB/c.lambdaLIZ mice.
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Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, the mean mutant frequencies in the target gene, lacI, were found to be significantly increased in lymphoid tissues of congenic BALB/c.lambdaLIZ N5 mice in the terminal stage of a plasmacytoma induction experiment, 213-280 days after the first i.p. injection of the plasmacytomagenic agent pristane (2,6,10,14-tetramethylpentadecane). In plasmacytoma-bearing mice (n = 7), mutant frequencies in the spleens and mesenteric lymph nodes were elevated 2.46-fold and 5.35-fold, respectively, when compared with age-matched controls. In plasmacytoma-negative mice (n = 11), mutant frequencies were increased 2.30-fold (spleens) and 3.48-fold (mesenteric nodes). These results, interpreted in conjunction with our previous findings (K. Felix et al., Cancer Res., 58: 1616-1619, 1998) of approximately 3-fold elevations in pristane-induced splenic mutagenesis on day 42 postpristane, indicate that increased mutant levels in lymphoid tissues persist throughout plasmacytomagenesis in genetically susceptible BALB/c mice. (+info)
Hydrogen peroxide and coffee induce G:C-->T:A transversions in the lacI gene of catalase-defective Escherichia coli.
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The mutagenicity of hydrogen peroxide (H2O2) was compared with that of coffee, a complex mixture which generates H2O2. An Escherichia coli strain defective in catalase activity (katG katE double mutant) and carrying a single copy mucAB (pRW144) plasmid was constructed to enhance the mutagenic response to oxidants. The ability of the mucAB genes to influence the type, frequency and distribution of H2O2-induced mutations was also investigated in isogenic bacteria lacking pRW144. Induced mutational spectra were characterized and compared with that of spontaneous mutagenesis. A total of 444 independent forward mutations affecting the first 210 bp of the lacI gene were identified by DNA sequence analysis. The spontaneous mutation spectrum showed no bias (P = 0.52) for substitutions at G:C base pairs. In contrast, in the H2O2-induced spectrum substitutions occurred preferentially at G:C base pairs (P < 0.0001) with a preponderance of G:C-->T:A transversions (43.4% of H2O2-induced mutants versus 17.3% of spontaneous mutants). These data support the view that 7,8-dihydro-8-oxoguanine is the main premutagenic lesion induced by H2O2 and that catalase-defective bacteria have elevated levels of 8-oxoguanine in chromosome DNA after H2O2 exposure. Coffee produced a similar distribution of mutational events as H2O2 (P > 0.05), suggesting that this compound may be the main cause of the coffee-induced mutagenesis. The presence of plasmid pRW144 did not affect the frequency of H2O2-induced G:C-->T:A transversions, but caused an increase in A:T-->T:A transversions and a decrease in -1 base frameshifts. Although the frequencies of G:C-->T:A transversions were similar in all three induced spectra (H2O2 and coffee +/- pRW144), differences were observed in location of mutations throughout the target gene. (+info)
Positive regulation of Bacillus subtilis sigD by C-terminal truncated LacR at translational level.
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DegR is a positive regulator for degradative enzyme synthesis in Bacillus subtilis. The degR gene is transcribed by RNA polymerase containing delta D, and the level of its expression is low in a mecA-deficient mutant. In a search for suppressors of the mecA effect through mini-Tn10 transposon mutagenesis, a lacR mutation designated lacR288 was discovered. The B. subtilis lacR gene encodes the repressor for lacA which specifies beta-galactosidase, and therefore, inactivation of the lacR gene results in overproduction of the enzyme. In the lacR288 mutant, however, the expression of lacA was at a negligible level, indicating that the repressor activity was not destroyed by the mutation. The putative gene product of the lacR288-containing gene is a 288-amino acid protein lacking the C-terminal 42 amino acids of intact LacR and carries no extra amino acids derived from the transposon sequence. The suppression by lacR288 of the decreased degR expression in the mecA background was found to be caused by an increase in the delta D level as shown by Western blot analysis. Furthermore, the increase was due to post-transcriptional regulation of sigD, the gene encoding delta D, as revealed by using both transcriptional and translational sigD-lacZ fusions. The lacR288 mutation had no effect on the stability of the delta D protein. Based on these results we conclude that the lacR288 mutation stimulates sigD expression at the translational level. (+info)