Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The specificity of N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (G-8-AAF) adducts in double-stranded DNAs from M13mp8 and M13mp9 bacteriophage was determined following transfection of modified DNA with multiple adducts into competent JM103 cells. Mutant phages were selected by phenotypic screening for colorless or light blue plaques indicating a defective beta-galactosidase marker enzyme. Mutation frequencies of phage DNA with G-8-AAF adducts were increased up to 8-fold in SOS-induced host cells as compared to the uninduced JM103 host cells. DNA sequencing of mutants from SOS-induced host cells indicated approximately 52% frameshifts and 39% base substitutions in M13mp8 DNA and 65% frameshifts and 25% base substitutions in M13mp9 DNA. Mutation spectra exhibited mutations at many sites within the bp 6200-6400 region; one mutational hotspot at position 6343-6347 (5' GGGGG 3') for frameshifts was also observed. The G-8-AAF adduct induced mostly single base deletions at this site. In contrast, a deacetylated adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene (G-8-AF) in our previous experiments induced mostly single base additions at the same position indicating the ability of adduct structure to modulate the specificity of frameshift mutations. A number of other frameshift mutations (11 out of 29) were observed within non-repetitive and non-palindromic sequences. Molecular mechanisms for the induction of these mutations by DNA perturbations produced by the G-8-AAF adducts are discussed.
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PMID:Induction of mutations by N-acetoxy-N-acetyl-2-aminofluorene modified M13 viral DNA. 202 46

An SV40-based shuttle vector, pZ189, carrying a bacterial suppressor tRNA target gene (supF) was treated with radiolabeled polycyclic aromatic carcinogens and the number of covalently bound residues (adducts) per plasmid was determined. The plasmids were transfected into the human embryonic kidney cell line 293 and allowed to replicate. The progeny plasmids were rescued and assayed for the frequency of supF mutants by being used to transform indicator bacteria carrying an amber mutation in the beta-galactosidase gene. The agents tested were the 7,8-diol-9,10-epoxide of benzo[a]pyrene (BPDE); 1-nitrosopyrene (1-NOP); N-acetoxy-2-acetylaminofluorene (N-AcO-AAF); and its trifluoro-derivative (N-AcO-F3-AAF) which yields deacetylated adducts. With each agent there was a linear increase in the frequency of supF mutants as a function of the number of DNA adducts formed, reaching frequencies as high as 20 x 10(-4) to 40 x 10(-4), with a background frequency of 1.4 x 10(-4). When compared on the basis of adducts formed per plasmid, BPDE, which forms its principal DNA adduct at the N2 position of guanine, was approximately 4 times more mutagenic than 1-NOP, N-AcO-AAF and N-AcO-F3-AAF, which bind principally or exclusively to the C8 position of guanine. This difference in mutagenic effectiveness may reflect intrinsic differences in the nature of the adducts and their location in the DNA molecule. It could also reflect a difference in the rate of removal of particular adducts by nucleotide excision repair since the 293 host cell line excised BPDE-induced adducts from genomic DNA at least 3 times slower than 1-NOP-induced adducts. Agarose gel electrophoresis and DNA sequencing analysis of 35 mutants derived from untreated plasmids showed that the majority (70%) involved deletions, insertions, or altered gel mobility (gross rearrangements). In contrast, the majority of those derived from carcinogen-treated plasmids were base-substitutions. DNA-sequencing of 86 unequivocally independent mutants derived from BPDE-treated plasmids and 60 from 1-NOP-treated plasmids indicated that 60% and 80%, respectively, contained a single base-substitution, 5-10% had two base-substitutions, and 4-10% had small insertions or deletions (one or two base pairs).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Comparing the frequency and spectra of mutations induced when an SV-40 based shuttle vector containing covalently bound residues of structurally-related carcinogens replicates in human cells. 253 43

Escherichia coli that are lysogenic for a lambda-lacZ fusion phage produce beta-galactosidase, product of the lacZ gene, upon induction of the prophage by DNA-damaging agents. The miniaturization of a quantitative, colorimetric beta-galactosidase (prophage) induction assay (BIA) is presented. Induction assays are performed in microtiter wells with the aid of multichannel pipetting devices. Results are shown with screening strain BR513 (uvrB delta envA) and a strain, BR339 (uvrB delta lexA3ind-) which exhibits enhanced induction. A method developed for strain BR339 utilizes bacteria stored frozen in log phase, permeabilized in vitro, and used immediately; with this method, 2 consecutive assays may be completed in 1 working day. Mutagens utilized for the model studies included 4NQO, ENNG, daunorubicin, bleomycin, acetoxy-AAF, B[a]P, DMBA, and DEN (the last three in the presence of liver S9). Induced levels of beta-galactosidase were monitored using a vertical light path photometer that measured color absorbance in each microtiter well. Alternatively, color intensity could be determined by using a color chart prepared for this assay. These values were then plotted to generate dose-response curves. Considerable savings in labor and materials are achieved with the method described, one which may be used as a screen for DNA-damaging chemicals. Automated equipment and computers may be used to advantage with this assay, but they are not required.
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PMID:Micro-BIA, a colorimetric microtiter assay of lambda prophage induction. 293 52

Genetic labeling using recombinant retroviruses is a powerful strategy for the study of cell lineage in the liver. However, this type of vector is only able to infect dividing cells. The synthetic steroid cyproterone acetate (CPA) is mitogenic and carcinogenic in the adult rat liver. In this study, we used retroviral vectors carrying the nuclear targeted beta-galactosidase gene to selectively label and follow the fate of hepatocytes dividing on administration of CPA. Labeled cells as well as those in mitosis were preferentially located around the portal tract, whereas apoptotic bodies were predominant in the pericentral area. Labeled hepatocytes did not disappear after apoptosis, suggesting a preferential elimination of nontransduced cells. The presence of labeled binucleated hepatocytes showed the persistence of a binucleation process. Finally, we performed long-term analysis of labeled cells in transgenic animals tolerant for beta-galactosidase and treated with 2-acetylaminofluorene (2-AAF) to promote the growth of CPA-initiated hepatocytes. The presence of beta-galactosidase-positive hepatocyte clones showed that hepatocytes divided during treatment with 2-AAF. Only 3% of beta-galactosidase clones were positive for the placental form of glutathione S-transferase (GSTp), indicating the absence of a preferential appearance of preneoplastic foci in the population of beta-galactosidase-labeled hepatocytes. In conclusion, our results show that the mitogenic and tumor-initiating activities of CPA are directed toward different hepatocyte populations.
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PMID:In vivo cell lineage analysis in cyproterone acetate-treated rat liver using genetic labeling of hepatocytes. 1182

Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the beta-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we observed clusters of beta-galactosidase-positive hepatocytes, some of them also expressing preneoplastic markers such as gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature hepatocytes may occur during the course of carcinogenic regimen.
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PMID:In vivo cell lineage analysis during chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes. 1206 89