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P VII.21 Effects of weak ELF magnetic fields and nalidixic acid on induction of recA-lacZ fusion in E. coli cells
S52
S-VII: Inducible responses to genotoxic stress
Ip VII.lsl
Uvr+-dependent Induction of the aidBI gene In E,· cherkhia coli and Salmonella typhimurium by anaerobiosis and by acidification
NataSa Peric l , Mirela Ivanfic, Vi~nja Bacun-Druzina, Faculty of FOod Technology and Biotechnology. Pierottijeva 6, 10 000 Zagreb, Croatia Exposure of E. coli to sublethal doses of DNA-methylating agents induces the adaptive response which confers resistance to the killing and mutagenic effects of alkylating agent, by inducing the expression of ada and alkA along with at least two other genes, alkB and aidB. The aidB gene of E. coli is subject of dual regulation by ada-dependent alkylation induction and by anaerobiosis or by acetate at pH 6.5 in an ada-independent fashion. In this study we have examined the induction of aidBI gene in E. coli and S. Iyphimurium (wild type, uvrA, uvrB) by anaerobiosis and by addition of sodium acetate to growth media, at acidic pH ranging 6.0 to 6.8. lnducnon of aidBI gene was monitored by assaying jl-galactosidase activity in extracts obtained from strains containing plasmid with fusion of Mudl tbla, lac) to aidBI. Our results have shown that the treatment with 50 mM sodium acetate at an extracellular pH of 6.0 caused an almost four fold increase jl-galactosidase activity in wild type of E. coli. This induction was observed under semianaerobic condition. However aidBJ gene could not be induced by anaerobiosis and by acidification of media in nucleotide excision repair deficient strains of E. coli and S. Iyphimurium.
protein of undetermined biochemical activity which shares epitopes with the bacterial RecA protein. Kin17 and RecA proteins have different primary structures, nevertheless, they display a 47% homology over a 40-residues region. Interestingly, kin 17 protein interacts preferentially with curved DNA through a single Zn-binding domain and seems to participate in illegitimate recombination. We observed an ubiquiious expression of Ktnl r RNA in mouse tissues and in rodent cell lines. We detected an enhanced expression of Kinl 7 in several tumorigenic rodent cell lines like AtT-20, RIN and F9. We assumed a correlation between Xi,,17 gene expression and cellular growth state. We present here the first evidence that the XinI7 gene is preferentially expressed in rapidly diViding cells since the level of Xin17 RNA was fold higher in mid-S phase of serum-stimulated BALB/c 3T3 fibroblasts than in quiescent cells. This observauon suggest that kin 17 protein is normally required for cell division. UVe-irradiation of quiescent fibroblasts enhances Xin17 gene expression. Our results confirm a previous Observation that PUVA-treatment increases Xi" I 7 RNA levels (J. SIMMONS, personal communication). We postulate that XinI7 gene is involved in a late process of cellular recovery after UV irradiation.
s-
Keyword(s): Proliferation; kinl7 protein; curved DNA
Ip VII,211
Effects or weak ELF magnellc fields and Dalldlxlc acid on Induction of recA-lacZ ruslon In E. coli cells
Keyword(s): aidB gene; ada-independent induction; NER
Igor Belyaev', Maria Naslund, Mats Hanns-Ringdahl. Deportment of Radiobiology, Stockholm University, Stockholm, S-106 91, Sweden
Ip VII.191
It is widely accepted, that magnetic fields of extremely low frequency (ELF), under intensities of environmental and residential exposure, don't result in mutagenic effects. However, there are some literature data about possible influence of ELF on inducible responses to genotoxic stress. The effects of ELF (21 microT) on inducible recA-lacZ fusion in E. coli GE499 were studied using the b-galactosidase assay. Within a frequency range of 7- I I Hz, ELF affected cells as measured by cloning efficiency and 1;ly the method of anomalous viscosity time dependencies (AVTD). The AVTD technique provided possibility to analyse the changes in the genome confonnational state of exposed cells. Both effects depended on frequency. Exposure at 8.7 Hz increased the AVTD peaks (15%), cloning efficiency (15%), cell growth (5%) but did not induce the cecA-lacZ fusion. The ELF effect on the genome conformational state was almost blocked by the nalidixic acid at 1-3 mgfml. At these concentrations we did not observe changes in cell survival, but induction of b-galactosidase was significant. Exposure to ELF in combination with nahdixic acid resulted in increase of cell growth and induction of b-galactosidase, These ELF effects were relatively weak (35%), but statisticaljy significant (p < 0.01). The data suppose, that ELF may affect a response of cells to genotoxic stress.
Suppression of S08-lnducinll Ictlvlty of Trp-P·l by
Meso dlhydrogualaretlc acid from Machilas thunbergli In Salmonell4 typhimurlum TAlS3S/pSKI002 /lmu test Mitsuo Miyazawa l , Kayo Oshiro", Yoshiharu Okuno", Hiroyuki Kasahara'; Hideo Shirnamura', Sei-ichi Nakamura2, Hiromu Kameoka': I Department ofApplied Chemistry, FacultyofScienceand Engineering, Kinki University, Kowakae, Higashiosaka-shi, Osaka 577, Japan; 2 Osaka PrefecturalInstitute
ofPublic Health, Nakamichi-I, Higashinari-ku, Osaka 537, Japan A methanol extract from Machi/us thunbergii showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TAI535/pSKlOO2 against the mutagen(3-amino-l,4-dimethyl5H-pyrido[4,3-b]indole(Trp-p-I», which requires liver metabolizing enzymes. The methanol extract from M. thunbergii was re-extracted with chloroform, butanol, and water, respectively. A suppressive compound in the chloroform extract fraction was isolated by Si02 column chromatography and identified as meso-dihydroguaiaretic acid by GC-MS, IH and 13CNMR spectroscopy. Meso-dihydroguaiaretic acid exhibited an inhibition of the SOS-inducing activity of Trp-P-I in the umu test, Gene expression was suppressed 62% at less than 0.18 umol/ml., and the IDso value was 0.08 !tmol/mL. Keyword(s): Machi/us thunbergii; meso-dihydriguaiaretic acid; SOS response; umu test; Salmonellatyphimurium TAI535/pSKlOO2
Ip VII.201
UV-lrradlatlon Induces the expression of a mouse gene encodlnll a nuclear DNA-blndlnll protein akin to E. coil RetA protein
P. Kannouche, A. Tissier, G. Pinon-Lataillade, D.S.F. Biard, Ph. Mauffrey, M. Mezzma", A. Sarasin'; Jaime F, Angulo. Laboratoire de Genetique de
10 Radiosensibi/ite, DRR, DSV,Commlssaritua /'Energie Atomique, BP n"
6; 92265 Fontenay-aux-Roses, France; I UPR 42, IF-I CNRS. 7, rue Guy Moquet, BP n" 8. 94801 Villejuij. France UV-irradiation induces in mammalian cells the expression of a set of genes known as the «UV-response» which may be reminiscent of the bacterial SOS system. The multifunctional protein RecA controls the expression of the SOS genes. We report here the expression profile of a mouse gene conserved among mammals, called XinI7, coding a DNA-binding
Keyword(s): Genotoxic stress; Cell survival; Genome conformation
Ip VII.221
isfA Mutallon Inhibits UV-Induced GC .... AT and AT .... GC transitions In recA+ Itr.lns Ind GC ...... TA and AT ...... TA transverslons In rec.4TJO Itralns
Anna Bebenek', Irena Pietrzykowska. Jnstitute physics PAS, WaJ:v/lMl Poland
0/ Biochemistry and
Bio-
The aim of this study was to investigate the influence of the isfA mutation on the specificity of UV-induced mutations in recA+ strains, as well as in strains constitutively expressing RecA coprotease ativity, recA7JO and ~CA 7JOle:L4(Def) strains. We have previously shown that the IsfA mutation inhibits SOS - induced phenomena, including SOS mutagenesis, most probably by inhibiting RecA coprotease activity. UV- light induces mutations that are predominantly base substitutions (60-75%) followed by smaller contributions of single base frameshifts and deletions. Among base substitutions the largest group consists of the transitions GC .... AT and AT ...... GC. On tho other hand, in ",cAUl and recA7JO strains constitutively expressing tho SOS functions, AT ...... TA and GC ...... TA and GC ...... CO transversions are mainly observed. To analyze the effect of the isfA mutation
S-VII: Inducible responses to genotoxic stress
Ip VII.lsl
Uvr+-dependent Induction of the aidBI gene In E,· cherkhia coli and Salmonella typhimurium by anaerobiosis and by acidification
NataSa Peric l , Mirela Ivanfic, Vi~nja Bacun-Druzina, Faculty of FOod Technology and Biotechnology. Pierottijeva 6, 10 000 Zagreb, Croatia Exposure of E. coli to sublethal doses of DNA-methylating agents induces the adaptive response which confers resistance to the killing and mutagenic effects of alkylating agent, by inducing the expression of ada and alkA along with at least two other genes, alkB and aidB. The aidB gene of E. coli is subject of dual regulation by ada-dependent alkylation induction and by anaerobiosis or by acetate at pH 6.5 in an ada-independent fashion. In this study we have examined the induction of aidBI gene in E. coli and S. Iyphimurium (wild type, uvrA, uvrB) by anaerobiosis and by addition of sodium acetate to growth media, at acidic pH ranging 6.0 to 6.8. lnducnon of aidBI gene was monitored by assaying jl-galactosidase activity in extracts obtained from strains containing plasmid with fusion of Mudl tbla, lac) to aidBI. Our results have shown that the treatment with 50 mM sodium acetate at an extracellular pH of 6.0 caused an almost four fold increase jl-galactosidase activity in wild type of E. coli. This induction was observed under semianaerobic condition. However aidBJ gene could not be induced by anaerobiosis and by acidification of media in nucleotide excision repair deficient strains of E. coli and S. Iyphimurium.
protein of undetermined biochemical activity which shares epitopes with the bacterial RecA protein. Kin17 and RecA proteins have different primary structures, nevertheless, they display a 47% homology over a 40-residues region. Interestingly, kin 17 protein interacts preferentially with curved DNA through a single Zn-binding domain and seems to participate in illegitimate recombination. We observed an ubiquiious expression of Ktnl r RNA in mouse tissues and in rodent cell lines. We detected an enhanced expression of Kinl 7 in several tumorigenic rodent cell lines like AtT-20, RIN and F9. We assumed a correlation between Xi,,17 gene expression and cellular growth state. We present here the first evidence that the XinI7 gene is preferentially expressed in rapidly diViding cells since the level of Xin17 RNA was fold higher in mid-S phase of serum-stimulated BALB/c 3T3 fibroblasts than in quiescent cells. This observauon suggest that kin 17 protein is normally required for cell division. UVe-irradiation of quiescent fibroblasts enhances Xin17 gene expression. Our results confirm a previous Observation that PUVA-treatment increases Xi" I 7 RNA levels (J. SIMMONS, personal communication). We postulate that XinI7 gene is involved in a late process of cellular recovery after UV irradiation.
s-
Keyword(s): Proliferation; kinl7 protein; curved DNA
Ip VII,211
Effects or weak ELF magnellc fields and Dalldlxlc acid on Induction of recA-lacZ ruslon In E. coli cells
Keyword(s): aidB gene; ada-independent induction; NER
Igor Belyaev', Maria Naslund, Mats Hanns-Ringdahl. Deportment of Radiobiology, Stockholm University, Stockholm, S-106 91, Sweden
Ip VII.191
It is widely accepted, that magnetic fields of extremely low frequency (ELF), under intensities of environmental and residential exposure, don't result in mutagenic effects. However, there are some literature data about possible influence of ELF on inducible responses to genotoxic stress. The effects of ELF (21 microT) on inducible recA-lacZ fusion in E. coli GE499 were studied using the b-galactosidase assay. Within a frequency range of 7- I I Hz, ELF affected cells as measured by cloning efficiency and 1;ly the method of anomalous viscosity time dependencies (AVTD). The AVTD technique provided possibility to analyse the changes in the genome confonnational state of exposed cells. Both effects depended on frequency. Exposure at 8.7 Hz increased the AVTD peaks (15%), cloning efficiency (15%), cell growth (5%) but did not induce the cecA-lacZ fusion. The ELF effect on the genome conformational state was almost blocked by the nalidixic acid at 1-3 mgfml. At these concentrations we did not observe changes in cell survival, but induction of b-galactosidase was significant. Exposure to ELF in combination with nahdixic acid resulted in increase of cell growth and induction of b-galactosidase, These ELF effects were relatively weak (35%), but statisticaljy significant (p < 0.01). The data suppose, that ELF may affect a response of cells to genotoxic stress.
Suppression of S08-lnducinll Ictlvlty of Trp-P·l by
Meso dlhydrogualaretlc acid from Machilas thunbergli In Salmonell4 typhimurlum TAlS3S/pSKI002 /lmu test Mitsuo Miyazawa l , Kayo Oshiro", Yoshiharu Okuno", Hiroyuki Kasahara'; Hideo Shirnamura', Sei-ichi Nakamura2, Hiromu Kameoka': I Department ofApplied Chemistry, FacultyofScienceand Engineering, Kinki University, Kowakae, Higashiosaka-shi, Osaka 577, Japan; 2 Osaka PrefecturalInstitute
ofPublic Health, Nakamichi-I, Higashinari-ku, Osaka 537, Japan A methanol extract from Machi/us thunbergii showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TAI535/pSKlOO2 against the mutagen(3-amino-l,4-dimethyl5H-pyrido[4,3-b]indole(Trp-p-I», which requires liver metabolizing enzymes. The methanol extract from M. thunbergii was re-extracted with chloroform, butanol, and water, respectively. A suppressive compound in the chloroform extract fraction was isolated by Si02 column chromatography and identified as meso-dihydroguaiaretic acid by GC-MS, IH and 13CNMR spectroscopy. Meso-dihydroguaiaretic acid exhibited an inhibition of the SOS-inducing activity of Trp-P-I in the umu test, Gene expression was suppressed 62% at less than 0.18 umol/ml., and the IDso value was 0.08 !tmol/mL. Keyword(s): Machi/us thunbergii; meso-dihydriguaiaretic acid; SOS response; umu test; Salmonellatyphimurium TAI535/pSKlOO2
Ip VII.201
UV-lrradlatlon Induces the expression of a mouse gene encodlnll a nuclear DNA-blndlnll protein akin to E. coil RetA protein
P. Kannouche, A. Tissier, G. Pinon-Lataillade, D.S.F. Biard, Ph. Mauffrey, M. Mezzma", A. Sarasin'; Jaime F, Angulo. Laboratoire de Genetique de
10 Radiosensibi/ite, DRR, DSV,Commlssaritua /'Energie Atomique, BP n"
6; 92265 Fontenay-aux-Roses, France; I UPR 42, IF-I CNRS. 7, rue Guy Moquet, BP n" 8. 94801 Villejuij. France UV-irradiation induces in mammalian cells the expression of a set of genes known as the «UV-response» which may be reminiscent of the bacterial SOS system. The multifunctional protein RecA controls the expression of the SOS genes. We report here the expression profile of a mouse gene conserved among mammals, called XinI7, coding a DNA-binding
Keyword(s): Genotoxic stress; Cell survival; Genome conformation
Ip VII.221
isfA Mutallon Inhibits UV-Induced GC .... AT and AT .... GC transitions In recA+ Itr.lns Ind GC ...... TA and AT ...... TA transverslons In rec.4TJO Itralns
Anna Bebenek', Irena Pietrzykowska. Jnstitute physics PAS, WaJ:v/lMl Poland
0/ Biochemistry and
Bio-
The aim of this study was to investigate the influence of the isfA mutation on the specificity of UV-induced mutations in recA+ strains, as well as in strains constitutively expressing RecA coprotease ativity, recA7JO and ~CA 7JOle:L4(Def) strains. We have previously shown that the IsfA mutation inhibits SOS - induced phenomena, including SOS mutagenesis, most probably by inhibiting RecA coprotease activity. UV- light induces mutations that are predominantly base substitutions (60-75%) followed by smaller contributions of single base frameshifts and deletions. Among base substitutions the largest group consists of the transitions GC .... AT and AT ...... GC. On tho other hand, in ",cAUl and recA7JO strains constitutively expressing tho SOS functions, AT ...... TA and GC ...... TA and GC ...... CO transversions are mainly observed. To analyze the effect of the isfA mutation