Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into ... more Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into Bacillus subtilis. Four of these plasmids (pUBllO, pCM194, pSA2100, and pSA0501) have been selected for further study. These plasmids replicate as multicopy autonomous replicons in both Rec+ and Rec-B. subtilis strains. They may be transduced between B. subtilis strains or transformed at a frequency of 104 to 105 transformants per,g of DNA. The molecular weights of these plasmids were estimated, and restriction endonuclease cleavage site maps are presented. Evidence is given that pSA2100, an in vivo recombinant of pSA0501 and pCM194 (S. Iordanescu, J. Bacteriol. 124:597-601, 1975), arose by a fusion of the latter plasmids, possibly by insertion of one element into another as a translocatable element. Genetic information from three other S. aureus plasmids (pK545, pSH2, and pUB101) has also been introduced into B. subtilis, although no covalently closed circular plasmid DNA was recovered.
ABSTRACT The transformation of Bacillus subtilis competent cultures by plasmid DNA is a recE inde... more ABSTRACT The transformation of Bacillus subtilis competent cultures by plasmid DNA is a recE independent first order process which requires multimeric CCC DNA. A model of plasmid transformation is presented which accounts for these properties. Transformation of recipients carrying a homologous resident plasmid by linear plasmid DNA, on the other hand, car. occur by a process which is recE dependent and which requires that homologous sequences flank the selectea donor marker. Evidence is presented that transformation by linear plasmid occurs by recombination and is a valid model for the study of bacterial transformation. The knowledge, obtained in this study is used to develop a system for the cloning of foreign DNA in B. subtilis.
Lysyl oxidase is an extracellular enzyme involved in connective tissue maturation that also acts ... more Lysyl oxidase is an extracellular enzyme involved in connective tissue maturation that also acts as a phenotypic suppressor of the ras oncogene. To understand how this suppressor is controlled, gene transcription was studied and the promoter was characterized. Nuclear runoff transcription assays indicated that the markedly reduced amounts of lysyl oxidase message detected after ras transformation resulted from inhibition of lysyl oxidase transcription. Interferon-mediated phenotypic reversion of ras transformed cells, in which the ras oncogene continued to be expressed, was accompanied by the restoration of lysyl oxidase transcription. Reporter gene assay of a transfected mouse lysyl oxidase promoter indicated that it was active in the transformed background, despite the silencing of the endogenous lysyl oxidase promoter. The detection of comparable amounts of mRNA for transcription factors IRF-1 and IRF-2 in normal and ras-transformed cell lines suggests that the differential trans...
Interferons were first described in 1957, but it was not until 34 years after their discovery tha... more Interferons were first described in 1957, but it was not until 34 years after their discovery that sufficient quantities of it were available for treatment of hepatitis C virus (HCV) infections, Clinicians now have an excellent understanding of the basis for the effectiveness of interferon alpha (IFN-) in the therapy of this disease. Treatment with IFN- is more efficient when it complemented by the antiviral ribavirin and the IFN- is conjugated with polyethylene glycol to form peginterferon. In the near future treatment of HCV with IFN- may involve new anti-HCV agents that are currently under development.
H-Ras functions as a signal switch molecule in numerous signaling pathways in the cytoplasm, requ... more H-Ras functions as a signal switch molecule in numerous signaling pathways in the cytoplasm, requiring H-Ras localization to the inner surface of the cytoplasmic membrane, and H-Ras is considered to be a cytoplasmic protein. Immunoblot studies of cells transformed by overexpression of c-H-ras indicated that H-Ras protein was present in both cytoplasmic and nuclear extracts, suggesting a possible correlation of nuclear H-Ras and cellular transformation. Unexpectedly, additional studies revealed that H-Ras protein was also present in the nuclei of nontransformed and primary mouse cells, which do not overexpress H-Ras. Mouse fibroblast NIH 3T3 cells, L cells, and a primary fibroblast line all had H-Ras present in both cytoplasmic and nuclear extracts. Nuclear extracts of cells synchronized by growth without serum displayed an increasing amount of H-Ras and cyclin D1 as cells grew after serum addition. Treatment with farnesyltransferase inhibitor caused loss of H-Ras from the nucleus. Immunofluorescence in situ studies of nuclei from synchronized cultures showed that H-Ras protein appeared in and disappeared from the nuclei as the cells moved through the growth cycle. This cycling occurred in both nontransformed and ras-transformed cells. Flow cytometry measurements on parallel cultures revealed that the time point at which the greatest percentage of cells were in S phase, for each line, corresponded to appearance of a noticeably stronger in situ signal for H-Ras. H-Ras may participate in nuclear signaling pathways associated with replication in addition to its cytoplasmic signaling functions.
A partial complementary DNA was isolated for a gene (rrg) that is normally expressed in mouse NIH... more A partial complementary DNA was isolated for a gene (rrg) that is normally expressed in mouse NIH 3T3 cells, but is down-regulated after cellular transformation by long terminal repeat (LTR)-activated c-H-ras (LTR-c-H-ras). This gene was reexpressed in a nontumorigenic persistent revertant cell line created by prolonged treatment of the transformed cells with mouse interferon alpha/beta. Persistent revertants stably transfected with rrg complementary DNA antisense expression vectors appeared transformed, had decreased amounts of rrg messenger RNA, and were tumorigenic in nude mice. Stable transfection with sense constructs did not alter the normal morphology, message level, or nontumorigenicity of the persistent revertant cell line.
For more than 20 years after the excitement engendered by their discovery in 1957 as antiviral ag... more For more than 20 years after the excitement engendered by their discovery in 1957 as antiviral agents, there were no significant clinical uses of interferons; however, following their cloning they have been employed as effective treatment for several viral, autoimmune, and neoplastic diseases.
The difficulty experienced in the shotgun cloning of chromosomal DNA on plasmid vectors in Bacill... more The difficulty experienced in the shotgun cloning of chromosomal DNA on plasmid vectors in Bacillus subtilis is analyzed and an explanation for this difficulty is offered based on an inherent property of competent cells which imposes a requirement of plasmid multimers in transformation of plasmidfree recipients (Canosi et al,, 1978). A stratagem which uses cloning by recombination between the vector and a resident homologous plasmid is tested and shown to be successful. Several recombinant plasmids are obtained containing Bacillus lichen(formis DNA fragments which complement aromatic amino acid mutants of Bacillus subtilis. The yield of recombinant clones ranges from 6.7 to 210 per gg of chromosomal DNA, depending on the selection and the restriction endonuclease. The various trp clones obtained after cutting chromosomal DNA with BgllI and BclI do not complement trpE and exhibit both orientations with respect to the vector. The location of several restriction endonuclease cleavage sites in the cloned trp fragments is presented, and their relationship to the genetic map of Bacillus licheniformis is described.
Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has... more Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (e) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 103 to 104 DNA molecules per transformant.
Interferon (IFN) regulatory factor-1 (IRF-1) is a transcription factor that has been historically... more Interferon (IFN) regulatory factor-1 (IRF-1) is a transcription factor that has been historically associated with type I IFN activation and antioncogenic properties. We studied IRF-1 expression and DNA-binding capacity in nontransformed and transformed mouse fibroblasts. A 43-kDa nuclear IRF-1 protein was expressed biphasically during the cell cycle in primary mouse embryo fibroblasts, nontransformed NIH 3T3 cells, and ras revertants. IRF-1 expression became constitutive in ras-transformed NIH 3T3 cells and in cells transformed by oncogenes ets, fes, fos, her-2/neu, met, mos, raf, or trk, suggesting that deregulated IRF-1 expression may be associated with loss of growth control. Lysyl oxidase (LO), a ras suppressor that is downregulated in ras transformants, is an IRF-1 target gene, but it is not stimulated by abundant IRF-1 present in transformants, while another IRF-1 target gene (iNOS) is transcribed. IRF-1 from either normal or ras-transformed cells bound to IRF elements in the IFN-beta and LO promoters. IRF-1 in transformants can, therefore, bind to but not transactivate the LO promoter, and the presence of IRF-1 is not sufficient to suppress ras transformation. LO expression may effect the regulated expression of IRF-1: a ras revertant, which was generated by stable transfection of LO cDNA, regained the normal biphasic IRF-1 pattern. A mainly cytoplasmic, constitutively expressed 46-kDa protein with immunologic identity to the 43-kDa nuclear IRF-1 was also present in normal and transformed cells, but as it did not bind to the IRF elements, its function is unclear.
... M. FRIEDMAN, ANNIE YEH, PABLO GUTMAN, SARA CONTENTE, and KAYLENE KENYON ABSTRACT We have prev... more ... M. FRIEDMAN, ANNIE YEH, PABLO GUTMAN, SARA CONTENTE, and KAYLENE KENYON ABSTRACT We have previously shown that prolonged interferon-/} (IFN-/3) treatment of RS485 cells (NIH3T3 cells trans¬ formed with multiple copies of an LTR-cHa-ras oncogene ...
Interferon (IFN) regulatory factor-1 (IRF-1) deregulation in ras-transformed mouse fibroblasts (R... more Interferon (IFN) regulatory factor-1 (IRF-1) deregulation in ras-transformed mouse fibroblasts (RS485) was studied. Treatment with the proteasome inhibitor MG132 did not alter the constitutive IRF-1 protein levels in RS485 but significantly increased them in nontransformed NIH 3T3 cells at 4 h after serum stimulation of synchronized cultures. Because IRF-1 protein levels in NIH 3T3 are minimal at 4 h after serum starvation, the cyclic expression of IRF-1 in NIH 3T3 appears to be partially due to proteasome activity; however, proteasome activity in RS485 did not appear to be defective. In NIH 3T3 and RS485 cells treated with cycloheximide, there were similar rapid drops in IRF-1 protein levels, and the addition of MG132 along with cycloheximide prevented protein loss in both cell lines. Northern blot analyses of synchronized cultures showed that the IRF-1 message closely mirrored the protein expression pattern in both NIH 3T3 and RS485 cells. In synchronized cells treated with the transcription inhibitor actinomycin D, IRF-1 mRNA half-life was only marginally longer in ras-transformed fibroblasts than in the nontransformed cells, and this difference would contribute minimally to protein overexpression. These findings indicate that IRF-1 deregulation in RS485 cells occurs primarily at the transcriptional level.
Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into ... more Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into Bacillus subtilis. Four of these plasmids (pUBllO, pCM194, pSA2100, and pSA0501) have been selected for further study. These plasmids replicate as multicopy autonomous replicons in both Rec+ and Rec-B. subtilis strains. They may be transduced between B. subtilis strains or transformed at a frequency of 104 to 105 transformants per,g of DNA. The molecular weights of
Interferon regulatory factor (IRF)-1 expression was surveyed in nontransformed and oncogene-trans... more Interferon regulatory factor (IRF)-1 expression was surveyed in nontransformed and oncogene-transformed mouse fibroblasts, using Western immunoblot with an IRF-1-specific antiserum, to examine possible differences resulting from cellular transformation. Ten additional proteins that reacted with the IRF-1 antibody and that underwent specific competition by peptide antigen were observed in extracts of both nontransformed and oncogene-transformed cell lines. Cross-reacting proteins were also observed in mouse macrophage extracts. Protein was captured from fibroblast nuclear extracts, using oligonucleotides representing IRF-binding sequences linked to magnetic beads. Captured proteins were eluted and analyzed by immunoblot with anti-IRF-1. Along with 43-kDa IRF-1, 4 of the 7 nuclearly located cross-reacting proteins (97, 90, 66, and 33 kDa) were found to complex with the IRF binding element. These proteins, with an epitope in common with the IRF-1 C-terminal region and IRF element DNA sequence-binding capability, may represent new members of the IRF family.
Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into ... more Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into Bacillus subtilis. Four of these plasmids (pUBllO, pCM194, pSA2100, and pSA0501) have been selected for further study. These plasmids replicate as multicopy autonomous replicons in both Rec+ and Rec-B. subtilis strains. They may be transduced between B. subtilis strains or transformed at a frequency of 104 to 105 transformants per,g of DNA. The molecular weights of these plasmids were estimated, and restriction endonuclease cleavage site maps are presented. Evidence is given that pSA2100, an in vivo recombinant of pSA0501 and pCM194 (S. Iordanescu, J. Bacteriol. 124:597-601, 1975), arose by a fusion of the latter plasmids, possibly by insertion of one element into another as a translocatable element. Genetic information from three other S. aureus plasmids (pK545, pSH2, and pUB101) has also been introduced into B. subtilis, although no covalently closed circular plasmid DNA was recovered.
ABSTRACT The transformation of Bacillus subtilis competent cultures by plasmid DNA is a recE inde... more ABSTRACT The transformation of Bacillus subtilis competent cultures by plasmid DNA is a recE independent first order process which requires multimeric CCC DNA. A model of plasmid transformation is presented which accounts for these properties. Transformation of recipients carrying a homologous resident plasmid by linear plasmid DNA, on the other hand, car. occur by a process which is recE dependent and which requires that homologous sequences flank the selectea donor marker. Evidence is presented that transformation by linear plasmid occurs by recombination and is a valid model for the study of bacterial transformation. The knowledge, obtained in this study is used to develop a system for the cloning of foreign DNA in B. subtilis.
Lysyl oxidase is an extracellular enzyme involved in connective tissue maturation that also acts ... more Lysyl oxidase is an extracellular enzyme involved in connective tissue maturation that also acts as a phenotypic suppressor of the ras oncogene. To understand how this suppressor is controlled, gene transcription was studied and the promoter was characterized. Nuclear runoff transcription assays indicated that the markedly reduced amounts of lysyl oxidase message detected after ras transformation resulted from inhibition of lysyl oxidase transcription. Interferon-mediated phenotypic reversion of ras transformed cells, in which the ras oncogene continued to be expressed, was accompanied by the restoration of lysyl oxidase transcription. Reporter gene assay of a transfected mouse lysyl oxidase promoter indicated that it was active in the transformed background, despite the silencing of the endogenous lysyl oxidase promoter. The detection of comparable amounts of mRNA for transcription factors IRF-1 and IRF-2 in normal and ras-transformed cell lines suggests that the differential trans...
Interferons were first described in 1957, but it was not until 34 years after their discovery tha... more Interferons were first described in 1957, but it was not until 34 years after their discovery that sufficient quantities of it were available for treatment of hepatitis C virus (HCV) infections, Clinicians now have an excellent understanding of the basis for the effectiveness of interferon alpha (IFN-) in the therapy of this disease. Treatment with IFN- is more efficient when it complemented by the antiviral ribavirin and the IFN- is conjugated with polyethylene glycol to form peginterferon. In the near future treatment of HCV with IFN- may involve new anti-HCV agents that are currently under development.
H-Ras functions as a signal switch molecule in numerous signaling pathways in the cytoplasm, requ... more H-Ras functions as a signal switch molecule in numerous signaling pathways in the cytoplasm, requiring H-Ras localization to the inner surface of the cytoplasmic membrane, and H-Ras is considered to be a cytoplasmic protein. Immunoblot studies of cells transformed by overexpression of c-H-ras indicated that H-Ras protein was present in both cytoplasmic and nuclear extracts, suggesting a possible correlation of nuclear H-Ras and cellular transformation. Unexpectedly, additional studies revealed that H-Ras protein was also present in the nuclei of nontransformed and primary mouse cells, which do not overexpress H-Ras. Mouse fibroblast NIH 3T3 cells, L cells, and a primary fibroblast line all had H-Ras present in both cytoplasmic and nuclear extracts. Nuclear extracts of cells synchronized by growth without serum displayed an increasing amount of H-Ras and cyclin D1 as cells grew after serum addition. Treatment with farnesyltransferase inhibitor caused loss of H-Ras from the nucleus. Immunofluorescence in situ studies of nuclei from synchronized cultures showed that H-Ras protein appeared in and disappeared from the nuclei as the cells moved through the growth cycle. This cycling occurred in both nontransformed and ras-transformed cells. Flow cytometry measurements on parallel cultures revealed that the time point at which the greatest percentage of cells were in S phase, for each line, corresponded to appearance of a noticeably stronger in situ signal for H-Ras. H-Ras may participate in nuclear signaling pathways associated with replication in addition to its cytoplasmic signaling functions.
A partial complementary DNA was isolated for a gene (rrg) that is normally expressed in mouse NIH... more A partial complementary DNA was isolated for a gene (rrg) that is normally expressed in mouse NIH 3T3 cells, but is down-regulated after cellular transformation by long terminal repeat (LTR)-activated c-H-ras (LTR-c-H-ras). This gene was reexpressed in a nontumorigenic persistent revertant cell line created by prolonged treatment of the transformed cells with mouse interferon alpha/beta. Persistent revertants stably transfected with rrg complementary DNA antisense expression vectors appeared transformed, had decreased amounts of rrg messenger RNA, and were tumorigenic in nude mice. Stable transfection with sense constructs did not alter the normal morphology, message level, or nontumorigenicity of the persistent revertant cell line.
For more than 20 years after the excitement engendered by their discovery in 1957 as antiviral ag... more For more than 20 years after the excitement engendered by their discovery in 1957 as antiviral agents, there were no significant clinical uses of interferons; however, following their cloning they have been employed as effective treatment for several viral, autoimmune, and neoplastic diseases.
The difficulty experienced in the shotgun cloning of chromosomal DNA on plasmid vectors in Bacill... more The difficulty experienced in the shotgun cloning of chromosomal DNA on plasmid vectors in Bacillus subtilis is analyzed and an explanation for this difficulty is offered based on an inherent property of competent cells which imposes a requirement of plasmid multimers in transformation of plasmidfree recipients (Canosi et al,, 1978). A stratagem which uses cloning by recombination between the vector and a resident homologous plasmid is tested and shown to be successful. Several recombinant plasmids are obtained containing Bacillus lichen(formis DNA fragments which complement aromatic amino acid mutants of Bacillus subtilis. The yield of recombinant clones ranges from 6.7 to 210 per gg of chromosomal DNA, depending on the selection and the restriction endonuclease. The various trp clones obtained after cutting chromosomal DNA with BgllI and BclI do not complement trpE and exhibit both orientations with respect to the vector. The location of several restriction endonuclease cleavage sites in the cloned trp fragments is presented, and their relationship to the genetic map of Bacillus licheniformis is described.
Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has... more Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (e) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 103 to 104 DNA molecules per transformant.
Interferon (IFN) regulatory factor-1 (IRF-1) is a transcription factor that has been historically... more Interferon (IFN) regulatory factor-1 (IRF-1) is a transcription factor that has been historically associated with type I IFN activation and antioncogenic properties. We studied IRF-1 expression and DNA-binding capacity in nontransformed and transformed mouse fibroblasts. A 43-kDa nuclear IRF-1 protein was expressed biphasically during the cell cycle in primary mouse embryo fibroblasts, nontransformed NIH 3T3 cells, and ras revertants. IRF-1 expression became constitutive in ras-transformed NIH 3T3 cells and in cells transformed by oncogenes ets, fes, fos, her-2/neu, met, mos, raf, or trk, suggesting that deregulated IRF-1 expression may be associated with loss of growth control. Lysyl oxidase (LO), a ras suppressor that is downregulated in ras transformants, is an IRF-1 target gene, but it is not stimulated by abundant IRF-1 present in transformants, while another IRF-1 target gene (iNOS) is transcribed. IRF-1 from either normal or ras-transformed cells bound to IRF elements in the IFN-beta and LO promoters. IRF-1 in transformants can, therefore, bind to but not transactivate the LO promoter, and the presence of IRF-1 is not sufficient to suppress ras transformation. LO expression may effect the regulated expression of IRF-1: a ras revertant, which was generated by stable transfection of LO cDNA, regained the normal biphasic IRF-1 pattern. A mainly cytoplasmic, constitutively expressed 46-kDa protein with immunologic identity to the 43-kDa nuclear IRF-1 was also present in normal and transformed cells, but as it did not bind to the IRF elements, its function is unclear.
... M. FRIEDMAN, ANNIE YEH, PABLO GUTMAN, SARA CONTENTE, and KAYLENE KENYON ABSTRACT We have prev... more ... M. FRIEDMAN, ANNIE YEH, PABLO GUTMAN, SARA CONTENTE, and KAYLENE KENYON ABSTRACT We have previously shown that prolonged interferon-/} (IFN-/3) treatment of RS485 cells (NIH3T3 cells trans¬ formed with multiple copies of an LTR-cHa-ras oncogene ...
Interferon (IFN) regulatory factor-1 (IRF-1) deregulation in ras-transformed mouse fibroblasts (R... more Interferon (IFN) regulatory factor-1 (IRF-1) deregulation in ras-transformed mouse fibroblasts (RS485) was studied. Treatment with the proteasome inhibitor MG132 did not alter the constitutive IRF-1 protein levels in RS485 but significantly increased them in nontransformed NIH 3T3 cells at 4 h after serum stimulation of synchronized cultures. Because IRF-1 protein levels in NIH 3T3 are minimal at 4 h after serum starvation, the cyclic expression of IRF-1 in NIH 3T3 appears to be partially due to proteasome activity; however, proteasome activity in RS485 did not appear to be defective. In NIH 3T3 and RS485 cells treated with cycloheximide, there were similar rapid drops in IRF-1 protein levels, and the addition of MG132 along with cycloheximide prevented protein loss in both cell lines. Northern blot analyses of synchronized cultures showed that the IRF-1 message closely mirrored the protein expression pattern in both NIH 3T3 and RS485 cells. In synchronized cells treated with the transcription inhibitor actinomycin D, IRF-1 mRNA half-life was only marginally longer in ras-transformed fibroblasts than in the nontransformed cells, and this difference would contribute minimally to protein overexpression. These findings indicate that IRF-1 deregulation in RS485 cells occurs primarily at the transcriptional level.
Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into ... more Covalently closed circular DNA from five Staphylococcus aureus plasmids has been introduced into Bacillus subtilis. Four of these plasmids (pUBllO, pCM194, pSA2100, and pSA0501) have been selected for further study. These plasmids replicate as multicopy autonomous replicons in both Rec+ and Rec-B. subtilis strains. They may be transduced between B. subtilis strains or transformed at a frequency of 104 to 105 transformants per,g of DNA. The molecular weights of
Interferon regulatory factor (IRF)-1 expression was surveyed in nontransformed and oncogene-trans... more Interferon regulatory factor (IRF)-1 expression was surveyed in nontransformed and oncogene-transformed mouse fibroblasts, using Western immunoblot with an IRF-1-specific antiserum, to examine possible differences resulting from cellular transformation. Ten additional proteins that reacted with the IRF-1 antibody and that underwent specific competition by peptide antigen were observed in extracts of both nontransformed and oncogene-transformed cell lines. Cross-reacting proteins were also observed in mouse macrophage extracts. Protein was captured from fibroblast nuclear extracts, using oligonucleotides representing IRF-binding sequences linked to magnetic beads. Captured proteins were eluted and analyzed by immunoblot with anti-IRF-1. Along with 43-kDa IRF-1, 4 of the 7 nuclearly located cross-reacting proteins (97, 90, 66, and 33 kDa) were found to complex with the IRF binding element. These proteins, with an epitope in common with the IRF-1 C-terminal region and IRF element DNA sequence-binding capability, may represent new members of the IRF family.
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