zgbitchina

TheMutagenicEffectofEthylMethanesulfonateonaNon-acid-fastStrainofMycobacteriumphleig.
KON][(~EKandI.
M~LEKDepartmentofBacterialC~neties,InstituteofMicrobiology,CzechoslovakAcademyofSciences,Prague4ReceivedJune5,1968~kl[~TRAET.
Ethylmethanesulfonatewasusedfortheinductionofthreetypesofmutantsinanon-acid-faststrainofMycobacteriumphlei.
Atotalof20auxotrophiemutantswasisolated.
Themutantswereisolatedmostlywhenusingdosesyieldinghighersurvivalofthecellsofthebasicsuspension.
Theauxo-trophicmutantsisolatedrequiredmostlyaminoacids,twomutantsrequiredpurinesandthreemutantsrequiredvitamins.
Bydeterminingthefrequencyofspontaneousreversions,itwasfoundthat9attxo-trophicmutantscouldbeusedforfurthergeneticstudies.
Theseincludedthefollowingphenotypes:isoleucine-,leueine-,lysine-,nicotinicacid-,pyridoxine-andxanthine-.
Sevenscotochromogenicmutantswereisolatedafterethylmethanesulfonatetreatment.
Onewasochre,theremainingsixwereorange.
Sixachromogenicmutantsweredetected.
Spontaneousauxotrophicmutants,scotochromogenicandachromogenicmutantswerenotisolated.
Thetreatmentwith0.
2MethylmethanesulfonateresultedinanincreaseinthefrequencyofSTM-rcsistantmutants,themaximumphenotypicexpressiontakingplaceafter72hourscultivationinaliquidmediumwithoutthedrug.
ThefrequencyofinducedSTM-resistantmutantsvariedwithintherangeof8.
6.
10-5-1.
0.
10-4ascomparedwiththefrequencyofspontaneousmutants5.
8.
10-e--8.
8.
10-4.
Inthispaperwesummarizeresultsobtainedwhenstudyingthemutageniceffectofethylmethanesulfonateonanon-acid-faststrainofMycobacteriumphlei.
Ethylmethanesulfonatebelongstoagroupofalkylatingagentsandisconsideredtobeaveryeffectivemu-tagen.
Itactsspecificallyonthe7positionofguanine,thetreatmentre-sultinginitsconversionto7-ethylguan-inecausingerrorsinbasepairinginDNA(Bautz&:Freese,1960;Brookes&Lawley,1961).
Undercertainconditionsthealkylationofadenineinpositions1~and3~leadingto1or3ethyladeninemayalsobeconsidered(Pal,1962;Krieg,1963).
Themutageniceffectofethylmethanesulfonatewasstudiedbyobservingtheinductionofauxotrophicmutants,streptomycin(STM)resistantmutantsandtheinductionofscoto-chromogenicandachromogenicmutants,thepigmentationchangesbeingprevi-ouslyfoundusefulasageneticmarkerinmutationstudies(Konieek&M~lek,1967).
Theinactivationeffectwasstudiedonlyinordertoobtaindatarequiredforthestudyofthemutageniceffect.
MATERIALSAND~-~fETHODSAprototrophic,photochromogenic,non-acid-faststrainPNofMycobacte-riumphleiisolatedbyHub~eekandMs{1958)asamutantoftheacid-faststrainPAofMycobacteriumphleiwasusedinthestudy.
ThisstrainislistedunderNo.
727inthecatalogueofmycobacteriabyHaudoroy(1966).
ThesuspensionofthebacterialculturewaspreparedbycultivationinaliquidDubosmediumcontainingTwecn80(finalcon-centrationof0.
05%)at37~for48hours.
Theviablecellcountwasde-terminedbysprayingthesuspensiononasolidmediumwithtryptose(BactotryptoseDifco15.
0,NaCl0.
2,KaHPO40.
2,distilledwaterad1000ml,pH7.
2,1969MU~IAGENESISOFMYCOBACTERIUI~195agar20.
0,10mlof50%glucoseaddedaftersterilization).
Theplateswereexaminedafter4daysat37~C.
Amodi-ficationofthemethodusedbyLinde-gren(Lindegrenetal.
,1965)withSac-charomyceswasadoptedfortheethylmethanesulfonatetreatmentofthePNstrain.
Thecellsuspensionwaswashedandresuspendedin0.
2MphosphatebufferpH7.
6containingTween80.
Ethylmethanesulfonate(CHaOSO2CaHs,m.
w.
124,b.
p.
86~at11mmHg)wasaddedtoafinalconcentrationof0.
2M.
ThereactionmixtureatafinalpHof6.
6wasincubatedinthePatoSkaflaskinawaterbathat37~C.
Samplesweretakenatintervalsandtheeffectofethylmethanesulfonateinterruptedbyaddingthe"stopsolution"(6%sodiumpersulphatecontainingTween80,l0minatroomtemperature).
Afterthein-activationthesampleswereconcentratedwithaphysiologicalsalinecontainingTween80totheinitialvolume,diluted,sprayedontothesolidmediumwithtryptoseandincubated5daysat37~C.
Thesamplesweretakenat30and60minintervalsfor6hoursintheexperimentscarriedouttoobtainvaluesfortheconstructionoftherespectivesurvivalcurve.
Thetreatedsuspensionswereinocu-latedonasolidmediumwithtryptoseandthesamemediumcontainingSTM(StreptomycinJenapharm,Jena,Ger-many,]0~zg/ml)wheninducingauxo-trophic,scotochromogenicandachromo-gemcmutants.
Auxotrophicmutantswereisolatedbyreplica-platingofcolon-iesfromthecompletemediumontheDavisminimalmediumandincubationfor3daysat37~(Lederberg&Leder-berg,1952).
NutritionalrequirementsoftheobtainedauxotrophicmutantsweredeterminedbytheauxanographicmethodonthesolidDavisminimalmedium(Lederberg,1950).
Aminoacids,purines,pyrimidines,nucleosides,nucleotidesandvitaminsweretested.
Stabilityoftheclassifiedauxotrophicstrainswasstudiedbydeterminingthefrequencyofspon-taneousreversions.
Washedsuspensionofa48hoursbacterialcultureoftheauxotrophicstrainswasinoculatedonasolidmediumwithtryptoseforthedeterminationoftheviablecellcountandsolidminimalmediumforthede-terminationofspontaneousreversions.
Whenisolatingauxotrophicmutantsa0.
05Mconcentrationofethylmethane-sulfonatewasusedforlong-termtreat-mentinadditionto0.
2Mofthedrug(NeS~sek,1966).
Scotochromogenicmu-tantswereisolatedafter5daysinthedarknessat37~Aehromogenicmu-tantswereisolatedafter3dayexposuretodaylight.
STM-resistantmutantswereobtainedbyincubationoftheethylmethane-sulfonate-treatedbacterialsuspensioninaliquidmediumfor96hoursat37~inordertoallowforthephenotypicexpressionoftheinducedmutants.
Sam-plesweretakenat24hoursintervalsandinoculatedonasolidmediumwithtryp-roseandonthesamemediumwithSTMinordertodeterminetimeofthemaxi-mumoccurrenceofthemutants.
ThefrequencyofspontaneousSTM-resistantmutantswasdeterminedbysprayingthenon-treatedsuspensionontheres-pectivemedia.
RESULTSTheinactivationofcellsofMycobac-teriumphleiPNbybothconcentrationsofethylmethanesulfonateisveryslow;2hourstreatmentwith0.
2MethylmethanesulfonateatpH6.
6resultedinabout80%survival,theinactivationthenbecamefasterreachingvaluesbelow0.
1%(Fig.
1)after5hourstreatment.
Thesurvivalcurve(Fig.
2)wasofabiphasictypeaftertreatmentwith0.
05Mconcentrationofethylmethane-sulfonateusedinordertoinduceauxo-trophicmutantsbymeansoflongtermmutagcnictreatment,theinactivatingeffectofthelatterconcentrationbeingverylow.
AslowdecreaseofthesurvivalI0EI.
O~o0.
O!
T,rne(re,n}969IooSoFig.
1.
SurvivalcurveofMyeobacteriurapldeiPNaftertreatmentwith0.
2Methylmethanesulfonate.
.
curveoccurredonlyafter3hourstreat-ment,50%andlessthan10%cellssurviving6hoursand18hourstreat-ment,respectively.
M'U'TAGENESISOFMYCOBACTERIUM196InductionofauxotrophicmutantsAtotalof17auxotrophicmutants(Table1)wereisolatedafterthetreat-mentofthePNstrainofMycobacteriumphleiwith0.
2~rethylmethanesulfonate.
Themutantswereisolatedmostlyafterthetreatmentwithdosesyieldingahighersurvival.
Thesurvivalwaslowerthan10~/oonlyinthecaseof3mutants.
Conditionswereusedinvariousexperi-mentsyieldingthesurvivalofcellsofthebasicsuspensionvaryingwithintherangeofseveraltensuptoseveraltenthstohundredthspercent.
Threeauxotrophicpautants(Table2)wereobtainedwhentreatingthecellswiththelowermutagenconcentrationfor4hours(74%survival)and18hours(7.
5%survival).
Outofatotalof20auxotrophiemutantsisolated12re-quiredaminoacids,2mutantsrequiredpurinesand3mutantsrequiredvitamins.
Onemutantwasdoublyauxotrophierequiringinositolandcystineorcysteine(thestraingrewslowlyeveninthepresenceofbothcompounds)andwasisolatedwhenthesurvivalofcellsoftheTable1.
AuxotrophiemutantsofMycobacteriumphleiP-~inducedby0.
2MethylmethanesulfonateNutritionalrequirementNumber%survival*IFrequencyofspon-ItaneousreversionsAlanine-(cysteine-)STMrCystino-(methionine-,cysteine-)Glutamieacid-(aa-ginine-,cystine-)lsoleueine-Leueine-Lysine-Methionine-Xanthine-STMrPurines-Inositol-cystine-(eysteine-)Nicotinica~id-Pyridoxino-Thiamine-Indeterminable110.
111.
316.
5118.
032.
966.
720.
0126.
1118.
0118.
0118.
0110.
1118.
0132.
5150.
124.
8.
10-55.
910-45.
710-e4.
810-72.
510-94.
210-a6.
110-91.
010-71.
910-37.
610-9>10-3**1.
7.
10-73.
8.
10-7>.
lO-a*%survivalofcellsofthebasicsuspensionafterethylmethanesulfonatetreatment;**frequencyofreversionscannotbedeterminedasthestraindoesnotformseparatecoloniesellmi-nimalmedium.
1969MUTAGENESISOFMYCOBACTERIUM197I00.
,,,I0=d1.
0.
o.
ih,~~,,llme(r:~)Fig.
2.
SurvivalcurveofMycobac~eriurnphleiPbTaftertreatmentwith0.
05xrethylmethanosulfonato.
basicsuspensionwas10.
1%.
Require-menstforseveralcompoundswerefoundinthecaseofseveralmutants,however,theadditionofonlyonecompoundissufficient.
Thestrainrequiringcystine(methionine,cysteine)wasverycom-plex,itgrewslowlyonaddinganyoftheaminoacidsinquestion.
Normalgrowthwasobservedaftertheadditionofnicotinicacidorriboflavin;vitaminsalonedidnotsupportgrowth.
TwomutantswerealsoSTM-resistantbeingisolatedontheSTMcontainingmedium.
Morphologyofcoloniesandrodsgrowninliquidmediadidnotchangeaftertreatmentwithethylmethanesulfonate.
Whendeterminingthefrequencyofspontaneousreversionsitwasfoundthatthereversionfrequencyof9auxo-trophicmutantswaslow.
ThereversionfrequencyofthedoublyauxotrophicmutantPNinositol-,cystine-couldnotbedeterminedasthestraininquestiondidnotgrowonaminimalmediumintheformofseparatedsinglecolonies.
ColoniesofspontaneousrevertantsontheminimalmediumwereusuallysmallerthanthoseoftheprototrophicPNstrain.
Theywerebulgy,dullandmostlyround.
Thecoloniesofafewstrainshadir-regularmargins.
Thecoloniesof5strainswereofacauliflower-liketype.
Thistypedisappearsonfurthersubcultures.
Revertantsofthestraincystine-(meth-ionine-,cysteine-)formedbothnormalcoloniesandratherbizzarecoloniestheshapesofwhichwerenotgeneticallyfixed.
Spontaneousauxotrophicmutantswerenotdetected.
InductionofSTM-resistantmutantsThehigherconcentrationofethylmethanesulfonatewasusedforthein-ductionofSTM-resistantmutants.
Itwasfoundthatthenumberofthesemutantsincreasedconsiderably,incuba-tioninaliquidmediumwithoutthedrugafterethylmethanesulfonatetreat-mentbeingessentialforthephenotypicexpression,l~[aximumphenotypicex-pressiontookplaceonlyafter72hoursincubation.
Prolongedincubationbe-yondthisperiodresultedinadecreaseofthenumberofmutants.
Survivalofcellsofthebasicsuspensionafterthemutagenictreatmentvariedfrom3.
1toTable2.
AuxotrophiemutantsofMycobacteriumphleiPXinducedwith0.
05.
~IethylmethanesulfonateNutritionalrequirementINumber~osurvival*[Frequencyofspon-taneousreversionsICystine-17.
55.
4.
10-sLeucine-17.
51.
1.
10-7Lysine-174.
01.
6.
10-7*~osurvivalofcellsofbasicsuspensionaftertreatmentwith0.
05Methylmethanesulfonate.
198J.
KO~(~EKANDI.
M.
~LEKVol.
14Table3.
FrequencyofSTM-resistantmutantsofMycobav~riumphlsiPNinducedwith0.
2Methylmethane-sulfonateFrequencyo,survival*/oinducedispontaneous[24[48I72I96'*18.
08.
5.
10-s3.
4.
10-59.
0.
10-51.
0.
10-45.
0.
10-53.
16.
0.
l0-61.
0.
10-56.
3.
10-58.
6.
10-s7.
3.
10-5*~survivalofcellsofbasicsuspensionaftertreatmentwithethylmethanesulphonate;**timeofincubationinhoursinaliquidmediumafterethylmethanesulfonatrtreatmentallowingforphcnotypicexpression.
18%indifferentexperiments.
Table3presentsfrequenciesofspontaneousandinducedmutantsin2experiments.
ThefrequencyofspontaneousSTM-resistantmutantsvariedwithintherangeof5.
8.
10-6to8.
8.
10-%ThemaximumincreaseinthefrequencyofinducedSTM-rcsistantmutantsvariedwithintherangeof8.
6.
I0-5to1.
0.
10-4,thein-ducedfrequencyhenceincreasedroughlybyanorderofmagnitudeof1.
1/4ascomparedwiththespontaneousfre-quency.
Coloniesofthemutantswereround,bulgy,dull,theirmarginswereregular,theygrewintoagarandweresmallerthanthecoloniesoftheoriginalPNstrain.
InductionofscotochromogenicandachromogenicmutantsAtotalof7scotochromogenicand6achromogenicmutantswereisolatedaftertreatmentofMycobacleriuraphleiPNwith0.
2~ethylmethanesulfonate.
Scotochromogenicmutantswereinducedbydosesyieldingbothhighandlowsur-vivalofcellsoftheoriginalsuspension(Table4).
Theywereobtainedassec-toredcolonieswiththeexceptionof2mutants.
ThemutantNo.
5wasochre,theremainingmutantswereorange.
ThemutantsgrewwellonallcompletemediaandDavisminimalmedium,onwhichthepigmentationofcolonieswasaspronouncedasonthetryptoseme-dium.
Themutants1and5,whosegrowthwaslessabnndantontheminimalmediumandwhichformedlowerquanti-tiesofpigmentswereexceptionalinthisrespect.
ThemutantNo.
1didnotgrowintoagarascomparedwiththePNstrain.
Morphologyofrodsoftheculturesgrowninliquidmediaandmorphologyofcoloniesofallscoto-chromogenicmutantsdidnotdifferfromthoseofthephotochromogenicPNstrain.
Whenfollowingupthestabilityofthesecolouredmutantsbyobservingpigmen-tationofthecoloniesontryptoseme-diumaftercultivationinthedark,itwasfoundthatonlytheochremutantreversedspontaneouslytotheoriginalTable4.
SeotochromogenicmutantsofMycobac.
teriumphleiPNinducedwith0.
2ethylmethane-sulfonateMu-I%su~-tantrival*IPigmen-tationofcoloniesSTMrl0y/mlSpontaneousreversions**1.
60.
6orange----2.
44.
0orange----3.
18.
0orange----4.
6.
5orange----5.
2.
9ochre--considerable6.
0.
13orange----7.
0.
13orange----*~survivalofcellsofbasicsuspensionaftertreatmentwithethylmethanesulfonate;**spontaneousreversionstooriginaltypeofpig-mentation.
1969MUTAGENESISOF.
71IYCOBACTERIU.
~I199photochromogenictype;theremainingmutantsdidnotreverse.
Achromogeniemutantswereobtainedmostlywhenusingdosesyieldingahighersurvivalofcellsofthebasicsuspension(Table5)andisolatedasminornon-Table5.
AchromogeniemutantsofMycoba~rlumpldeiPNinducedwith0.
2Methylmethanesulfonat~Is=ISpon=o=vival*I0y/m]reversions**1.
74.
5----2.
21.
3--considerable3.
20.
0--rare4.
18.
0§--5.
7.
5----6.
2.
9----*%survivalofcellsofbasicsuspensionaftertreatmentwithethylmethanesulfonato;**spontaneousreversionstooriginalt4~peofpig-mentation.
pigmentingcolonies.
Long-termexposureofthemutantstodaylightandregularsubculturesonsolidmediadidnotchangetheirachromogeniccharacter.
ThemutantsgrewwellonordinarymediausedtocultivatethePNstrain,thegrowthofthemutants2and3onlywasdecreasedontheminimalmedium.
However,itshouldbepointedoutherethatwewerenotdealingwithauxotro-phicmutantsinthiscase.
Themorphologyofcoloniesandrodswasnotchangedinindividualmutants.
OneofthemutantswasalsoSTM-resistantbeingisolatedonthesolidtryptosemediumcontainingSTM.
Spontaneousreversionstophoto-ehromogenywereobservedquitefre-quentlyinonemutantandwereratherrareinanotheronexposuretodaylightforseveraldays.
Spontaneousscotochro-mogenicandachromogenicmutantswerenotdetected.
DIBCUSSIONTheeffectofethylmethanesulfonateontheinductionofthreetypesofmutantsinMycobacteriumphleiPNwasexaminedthusextendingourpreviousstudieswithUV-radiation(Koni6ek&Ms1967,1968).
Ethylmethane-sulfonatewasusedasaneffectivemu-tagenfortheinductionofmetabolicmutantsbyanumbersofauthors.
Linde-grenetal.
(Lindegrenetal.
,1965)ob-tainedconsiderablenumbersofauxo-trophicmutantsinSaccharomyces,manyofwhichrequiredseveralnutrients,andconcludedonthebasicoftheresultsobtainedthatethylmethanesulfonateinducesmanytypesofmutationsre-quiringvariousnitrogenbases,vitaminsandaminoacids.
However,theauthordrewattentionalsotothefactthatmanymutantsweregeneticallyunstableandreversedspontaneouslywithahighfrequency.
ThemutagenwasalsousedwithSchizosaccharomycespombe(Lo-prieno,1966)fortheinductionofadeninerequiringmutantswhichwereeasilydetectedastheyproduceapurplepig-ment.
NeSs(1966)inducedauxo-trophicmutantsinCorynebacteriabylong-termethylmethanesulfonatetreat-mentandfoundthatthefrequencyofinducedmutantsincreasedto15%andeven80~owhentreatingthecellsfor18hourswith0.
05~and0.
1Methylmethanesulfonate,respectively,ascom-paredwiththe0.
5%frequencyobtainedafter30to120mintreatment.
ThesameresultswereobtainedwithBrevibacte-riumaramoniagenes(Honzovsetal.
,1968).
Wewereunabletoobtainacon-siderableincreaseinthenumberofauxotrophicmutantswhenworkingwithourmodelmicroorganisms.
However,perhapsevenlongertreatmentshouldbeused.
NeS~sekalsosuggestsinhispaperthatthespectrumofauxotrophicmutantsinducedwithethylmethane-sulfonateresemblesmorecloselythatobtainedafterUV-treatmentthanthatobtainedwhenusingnitrogenmustard.
Ethylmethanesulfonatewasalsousedfortheinductionofmutantsinbacterio-phages(Loveless,1959;Straus,1961).
Atotaloftwentyauxotrophicmutants200J.
KON~EKANDI.
M/~LEKVOI.
14wereisolatedafterthetreatmentof.
MycobacteriumphleiPNwithbothusedconcentrationsofethylmethanesulfon-ate.
Mostmutantsareaminoacidde-pendent.
AscomparedwithUV-radiation,themutantswereusuallydetectedaftertreatmentwithdosesyieldinghighersurvivalofcellsofthebasicsuspension.
Threemutantsrequiringnicotinicacid,pyridoxineandthiaminewereobtainedcomparedtoUV-treatmentwherenomutantsrequiringvitaminswerede-tected.
Ontheotherhand,manymoremutantsrequiringcomponentsofnucleicacidswereobtainedinthePNstrainasaresultOfUV-irradiation.
Nineauxo-trophicmutantsrepresentingsixdifferentphenotypescouldbeusedinfurthergeneticstudiesduetotheirlowfre-quencyofspontaneousreversions.
Mu-tantswithchangedpigmentationwere:ReferencesBautz,E.
,Freese,E.
:Onthemutageniceffectofalkylatingagents.
Prec.
nat.
Acad.
Sci.
US.
46:1585,1960.
Brookes,P.
,Lawley,P.
D.
:Thereactionofmonoanddi-functianalalkylatingagentswithnucleicacid.
Biochem.
J.
80:496,1961.
:Hauduroy,P.
:ListsdegermesdugenreMyeo-bacterium.
Instituted'Hygi6nedeLausanne,1966.
Honzovh,H.
,Javdrkovh,B.
,~koda,J.
,Dyr,J.
:AquantitativeevaluationofthemutageniceffectofethylmethanesulfonateinBrevibacteriumam-moniaqenesandqualitativecharacterofthemutantsobtained.
Fol.
microbiol.
13:125,1968.
Hubh~ek,J.
,M~ek,I.
:EinigebiologiseheundbioehemiseheEigenschaftensdurefestsrundnicht.
sdurefesterS~mmedesMyeobacteriumpM,ei.
Fol.
biol.
4:220,1958.
IKoni6ek,J.
,Mhlek,I.
:Theinductionofpigmenta-tionchangeinanon.
acid-faststrainofMyeo-bacteriumphleibyultravioletradiation.
:Fol.
naierobiol.
12:417,1967.
Koni6ek,J.
,MAlek,I.
:Mutayenlceffectofultra.
violetradiationonthenon-acid.
faststrainofMycobacteriumphlei.
Fol.
microbiol.
13:282,1968.
Krieg,D.
R.
:Ethylmethanesulfonate--inducedre-versionofbacteriophageT4rIImutants.
Genetics48:561,1963.
Lederberg,J.
:Isolationandcharacterizationofusuallystableandmayhencealsobeusedinfurthergeneticwork.
0.
2~ethylmethanesulfonateincreasedthefrequencyofinducedSTM-resistantmutantsbyabout1.
114ofanorderofmagnitude,themaximumphenotypicexpressionbeingreachedafter72hoursincubationinadrug-freeliquidmedium.
Themaximumphenotypicexpressionwasreachedalreadyafter48hoursincubationinthesamemediumwhenusingUV-radiation(KoniSek&M~lek,1968).
Verlyetal.
(1967)usedethylmethanesulfonateandnitrousacidfortheinductionofSTM-resistantmutantsinEscherichiacellK12andalsofoundthatitwasnecessarytoincubatethetreatedsuspensionforseveralhoursinaliquidmediumpriortotheapplicationofSTM.
TheauthorswishtothankMrs.
M.
Fingerovt%forherexcellenttechnicalassistance.
biochemicalmutantsofbacteria.
MethodsinMed.
lores.
3:5,1950.
Lederberg,J.
,Loderberg,E.
M.
:Replicaplatingandindirectselectionofbacterialmutants.
J.
Baeteriol.
63:399,1952.
Lindegron,G.
,Hwang,Y.
L.
,Oshinaa,Y.
,Linde-gren,C.
C.
:GenetlcalmutantsinducedbyethylmethanesulfonateinSaccharomyces.
Can.
J.
Genet.
Cytol.
7:491,1965.
Loprieno,N.
:Differentialr~ponseofSchizosac-charomycespombetoethylmethanesulfonateandmethylmethanesulfonate.
~futationI~s.
3:486,1966.
Loveless,A.
:MutationofT2bacteriophageasaconsequenceofalkylationinvitro:theuniquenessofethylation.
Prec.
roy.
Soc.
LondonB150:497,1959.
Ne6hsek,J.
:Mutageniceffectofethylmethane.
eulfonateinCorynebacterium.
Thephysiologyofgeneandmutationexpression.
ProceedingsofaSymposiumholdinPrague1965,Academia--Prague.
115,1966.
Pal,B.
C.
:Studiesonthealkylationofpurinesandpyrimidines.
Biochemistry1:558,1962.
Straus,B.
S.
:Specificityofthemutagenicactionofthealkylatingagents.
Nature191:730,1961.
Verly,W.
,G.
,Barbason,H.
,Dusart,J.
,Petitpas-Dewandre,A.
:AcomparativestudyoftheactionofethylmethanesulfonateandHN02onthemutationtostreptomycinreai~auceofEoeherlchiaeoliK12.
Bit)china.
biophys.
Acts145:752,1967.