外文翻译--Extremely low frequency electric field induced mutations in Escherichia coli K12 strains

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1、Extremely low frequency electric field induced mutations in Escherichia coli K12 strains Zhiqing Song,Yunzhang Liang School of physical science and technology,Inner Mongolia University,Hohhot,010021,P.R.China AbstractEscherichia coli K12 W3110 was treated by extremely low frequency electric field(EL

2、F EFs)10min with the field intensity of 100，200，300，400kV/m.The mutant frequency reached 31.210-6 at field intensity of 100kV/m,which was about 26-fold over the background(1.210-6)and 12-fold over the controls(2.510-6).This result indicated that the ELF EFs could act as an mutagen.The results also s

3、how that base pair(bp)substitutions were the main type of ELF EFs induced mutations.Although all types of base pair substitutions were observed,30%of the bp substitutions were G:C A:T transitions,70%of the bp substitutions were A:TT:A,G:CT:A,A:TC:G,G:CC:G transversions.When the spontaneous and the E

4、LF EFs exposure group were compared,base-pair substitutions,deletions and additions of the ELF EFs exposure group were significantly increased,and the deletion or the addition of a 5-TGGC-3 sequence at hot spot was decreased from 82 to 26%.But the remarkable increase in absolute Mutation Frequency(M

5、F)of the deletion or the addition of a 5-TGGC-3 sequence at hot spot in the ELF EFs exposure group suggested that ELF EFs did induce the mutations of this type.The spectra of our ELF EFs exposure group with increasing G:C to A:T transitions and A:T to T:A transversions indicate that ELF EFs exposure

6、 may induce SOS response.Keywords-Escherichia coli;extremely low frequency electric field(ELF EFs);lacI gene;Mutation spectrum.I.INTRODUCTION(HEADING 1)Possible health effects of exposure to low frequency electric and magnetic fields are receiving increased interest in the scientific literature.Elec

7、tric field can inhibit the biological properties of the cells membrane protein1,can cause chromosomal aberration of mice bone marrow 2 and serum melatonin levels decrease in male rats3.Previous study also demonstrated that ELF EFs exposure could inhibit the proliferation of human liver cancer cells

8、SMMC7721 and mouse Lewis lung cancer cells 4,5.However,many investigations have shown that exposure to extremely low frequency electric fields cause almost no harmful effects to bacterial 6.But we have found that E.coli cells exposure to 50 Hz ELF EFs can cause DNA damage and gene mutation 7,and res

9、earch on mutagenic mechanisms of ELF EFs exposure was not reported so far.With the aims to investigate the mutational mechanisms of ELF EFs exposure,we chose lacI as target gene because of the advantage of the extensive existing lacI data base in mutational spectra 8-11,and compared the mutational s

10、pectrum of the genomic lacI gene induced ELF EFs exposure in wild type E.coli strain W3110 with that of the spontaneous.II.MATERIALS AND METHODS A.Bacterial strain Wild type strain W3110(F-)of E.coli K12,generously donated by Prof Kaj Frank Jensen,was used for mutational research.E.coli DH5,used as

11、lacZ strain for the scavenging of alternative carbon sources from the minimal agar plate several hours in advance,was stored in our laboratory.B.Reagents and media LB medium and minimal A salts were prepared as described by Miller 12,13.VogelBonner salts 9 were modified,containing 0.2 g MgSO47H2O,2.

12、0 g(NH4)H2PO4,10.0 g K2HPO4,2.8 g sodium citrate per liter.Phenyl-d-galactopyranoside(Pgal)plate consisted of modified VogelBonner salts supplemented with 75 mg Pgal/L and 15 g purified agar/L.5-Bromo-4-chloro-3-indolyl-dgalactopyranoside(Xgal)minimal plates consisted of minimal A salts supplemented

13、 with 1mM MgSO4,5ug/mL thiamine,0.2%glucose,1.5%agar and 45 ug/mL Xgal.C.Extremely low frequency electric field facility The ELF EFs facility mainly consists of a multiple needles-to plate electrode system and a high voltage power supply.The upper electrode is multi-needles array that the length of

14、needles is 2cm and the distance among needles is 4cm,the lower electrode is a aluminums plate.The distance between the electrodes is 6cm.The range of voltage can be adjusted from 0 to 70kV.The frequency of electric field is 50Hz.D.Extremely low frequency electric field exposure and screening of muta

15、nts ELF EFs exposure and screening of mutants was carried out as described previously 7.In brief,a single colony of wild type E.coli W3110 were cultured in 5mL LB medium for overnight at 37.after washed twice with 0.2M PBS(PH 7.0),0.1mL mixture of this sample was spread onto the surface of an empty

16、glass Petri dish(90 mm),then dried with a sterile wind for about 10 min.The samples were exposed to ELF EFs 10min with the field intensity of 100，200，300，400kV/m respectively.The temperature of the treat environment was controlled at approximately 4.For mutants selection,100uL 978-1-4244-4713-8/10/$

17、25.00 2010 IEEETABLE I.COMPARISON THE HVPEF EXPOSURE AND SPONTANEOUS LACI MUTATIONS IN E.COLI a The spontaneous mutations was from Murata-Kamiya et al.10 b The TGGC addition is not included.c The TGGC deletion is not included mixture was cultured on minimal plates containing the non-inducing sugar P

18、gal as a sore carbon source at 37 for 3 days.Therefore,only the mutants(lacI and lacOc)can form colonies in 4872 h on the plate.The mutant colonies were scored,and grown on a minimal Xgal plate by overnight incubation at 37.A deep blue colony on the minimal Xgal plate indicates lacI.E.Amplification

19、of the mutant lacI gene and sequence analysis The PCR primers of lacI gene were 5-GACACCATCGAATGGCGC-3(primer 1)and 5-TTCCCAGTCACGACGTTG-3(primer 2)according to Ono et al.9.Purified PCR products were sequenced in TaKaRa(Dalian Corporation)using a Perkin-Elmer Applied Biosystems Model 377 DNA Sequenc

20、er.Mutations were determined on-line(http:/www.ncbi.nih.gov/blast/)by running a blast between the sense strand sequence of the wild type lacI gene(GenBank Accession:NC000913)and those of the mutants.III.RESULTS A.Cell survival and mutation frequency The mutant frequency reached 31.210-6 at field int

21、ensity of 100kV/m,which was about 26-fold over the background(1.210-6)and 12-fold over the controls(2.510-6),and chosen this field intensity to select mutants7.The survival rates did not decrease with increasing field intensity,the curves even show a tendency of first decreased and then increased.B.

22、LacI mutations Fifty-three independent lacI mutants induced by ELF EFs were screened.The types of mutations and their absolute MF 14 are given in Table 1(The spontaneous mutants were from Murata-Kamiya et al.10)According to Murata-Kamiya et al.10,the majority of the spontaneous mutations(82%)involve

23、d the deletion or the addition of a 5-TGGC-3 sequence at position of bases 593604 in the lacI gene,where the TGGC sequence is tandemly repeated three times.The other mutations were base-pair substitutions(10%)and multi-base deletions(6%)and additions(2%).The mutations induced by ELF EFs were+TGGC or

24、 TGGC at the hot spot(26%),base-pair substitutions(57%),one-base deletions(9%),one-base additions(4%),multi-base deletions(4%).Of the base-pair substitutions,transversions were predominant,which accounted for 70%of the total substitutions.All obtained multi-base additions were+TGGC.Interestingly,the

25、 larger deletion(about 283 bases deletion)is found at position 61-343,which was not detected by other researchers before.And the base pair transversions from A:T to T:A was detected in the ELF EFs exposure group,which were not found in the spontaneous group by Murata-Kamiya et al.The distribution an

26、d relative information of all mutations are given in Table 2,included the types of all mutations detected,as well as their mutational sites,amino acid changes,sequences of the contiguous bases.IV.DISCUSSION The mechanism of mutation induction under ELF EFs exposure is not clear.The comparison of mut

27、ational spectra among the spontaneous group and ELF EFs exposure group,as well as those of other ionizing radiation may help us to insight some mutational mechanisms of ELF EFs 8-11,almost all types of mutation were found in the spectra of ELF EFs exposure group(Table 1).That the spectra in our case

28、 were quite different from that of the oxygen free-radical and low energy nitrogen ion induced in the same strain 8-10,The oxygen free-radical and low energy nitrogen ion induced mutations were base-pair substitutions(33%-35%),+TGGC(43%-52%),TGGC(1%-7%).The mutations induced by ELF EFs exposure were

29、 base-pair substitutions(57%),+TGGC(17%),TGGC(9%)and there was a larger deletions,ranging in size from 61 to 343 bp that was not detected in oxygen free-radical and low energy nitrogen ion induced group.These results suggested mutation induced by Mutation HVPEF induced Spontaneousa Cases found Perce

30、ntage(%)Absolute MF(10-6)Cases found Percentage(%)AbsoluteMF(10-6)Base-pair subsitutions 30 57 17.78 5 10 0.15 G:C to A:T 9 17 5.30 1 2 0.03 G:C to T:A 6 11 3.43 3 6 0.09 G:C to C:G 1 2 0.62 1 2 0.03 A:T to C:G 3 6 1.87 0 0 0 A:T to G:C 0 0 0 0 0 0 A:T to T:A 11 21 6.55 0 0 0 One base additions 2 4

31、1.25 0 0 0 G:C 1 2 0.62 0 0 0 A:T 1 2 0.62 0 0 0 One base deletions 5 9 2.81 0 0 0 G:C 2 4 1.25 0 0 0 A:T 3 6 1.87 0 0 0 Multi-base additionsb 0 0 0 1 2 0.03 Multi-base deletionsc 2 4 1.25 3 6 0.09+TGGC 9 17 5.30 38 71 1.065-TGGC 5 9 2.81 6 11 0.165 Total 53 53 TABLE II.MUTATED SITES,AMINO ACID CHAN

32、GES AND RELATED INFORMATION IN MUTATIONS INDUCED BY ELF EFS(FIRST RESIDUE OF INITIATOR(GTG)IN LACI GENE WAS DETERMINED AS 1)Mutation type HVEF induced position sequence Amino acid change Transitions GC to AT 660 GAC TGG AGT Trp to stop 871 AAA CAG GAT Gln to stop 380 TAT CCG CTG Pro to Leu 559 GCG G

33、GC CCA Gly to Ser 631 ATT CAG CCG Gln to stop 163 CAA CAA CTG Gln to stop 3 AAT GTG AAA Val to Val 178 AAA CAG TCG Gln to stop 313 GTC GAA GCC Glu to Lys Transversions AT to TA 185 TCG TTG CTG Leu to stop 728 ATG CTG GTT Leu to Gln 125 GCG ATG GCG Met to Lys 612 AAA TAT CTC Tyr to stop 139 AAT TAC A

34、TT Tyr to Asn 73 GTG AAC CAG Asn to Tyr 858 CCG TTA ACC Leu to Phe 322 TGT AAA GCG Lys to stop 356 CGC GTC AGT Val to Asp 113 AAAGTG GAA Val to Glu GC to CG 92 GTT TCT GCG Ser to Cys AT to CG 44 GGT GTC TCT Val to Gly 144 TAC ATT CCC Ile to Met 902 AGC GTG GAC Val to Gly GC to TA 763 ATG CGC GCC Arg

35、 to Ser 230 CCG TCG CAA Ser to stop 88 CAC GTT TCT Val to Phe 158 GTG GCA CAA Ala to Glu 649 CGG GAA GGC Glu to stop 983 AAA ACC ACC Thr to Asn One base addition 827 GATACCC GAA frameshift 331 GCG GCGT GTG frameshift One base deletion 675 TCC GGT TTT frameshift 54 TAT CAG ACC frameshift 753 GCG CTG

36、GGC frameshift 107 CGG GAA AAA frameshift 978 AAA AGA AAA frameshift Multi-base deletion 590-591 CTG CGT CTG frameshift 61-343 frameshift TGGC 593-604 frameshift ELF EFs exposure is not the indirect effects such as oxygen free-radical.The mutational spectra occurring in-ray irradiated E.coli lacI ge

37、ne had been reported 11.In ELF EFs exposure group base-pair substitutions occurred at A:T site(47%)and at G:C site(53%),but in-ray irradiation group were at A:T site(24%)and at G:C site(76%).Mutations induced by-ray irradiation were also supposed to be the indirect effect,mainly via the radicals gen

38、erated by-radiation.These results suggested there were some different effects between the ELF EFs exposure and-irradiation.The percentage of+TGGC or TGGC at hot spot in irradiated group was remarkablely decreased from 82 to 26%,but its absolute MF was about 8-fold greater than the background.Previou

39、s studies had demonstrated that abasic sites(AP)might be the main reason of the mutation at the hot spot in lacI gene 15,16.Our data strongly implied that ELF EFs exposure induced+TGGC orTGGC at hot spot in lacI gene in E.coli.The clear difference between the spontaneous and the ELF EFs exposure gro

40、up in this work ascribed to the increase of the frequencies of base-pair substitutions in the ELF EFs exposure group(5.5-fold in percentage,11 4-fold in absolute MF),especially in G:C to A:T pair(8.5-fold in percentage and 176.7-fold in absolute MF).In addition,A:T to T:A transversion and larger del

41、etion(Table 2),which was not found in the spontaneous mutations obtained by Murata-Kamiya et al,was detected in the ELF EFs exposure group of our case.It has been demonstrated that the SOS response has the effect to contribute to the increases of G:C to A:T and A:T to T:A transversions 17,18.The inc

42、reased fraction in the ELF EFs exposure group is similar to these previously reported results,this might allow for the speculative conclusion that the increases of G:C to A:T,A:T to T:A transversions in the ELF EFs exposure group in this work was likewise a consequence of SOS processing.ACKNOWLEDGEM

43、ENTS We would like to thank Professor Liaofu Luo for enlightening discussions.Professor Kaj Frank Jensen who kindly present the E.coli strain W3110.Xiangsheng Zhang and Changjiang Ding gives a proofread to make the language more complete and fluid.This work was funded by the National Science Foundat

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