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MUTAGENESIS by ACRIDINE YELLOW in BACZLLUS Subtzlzsl

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MUTAGENESIS by ACRIDINE YELLOW in BACZLLUS Subtzlzsl MUTAGENESIS BY ACRIDINE YELLOW IN BACZLLUS SUBTZLZSl CHARLES R. STEWART2 Department of Genetics and Kennedy Laboratories for Molecular Medicine, Stanfora!University Medical School, Palo Alto, California Received October 13. 1967 ROFLAVIN, acridine yellow, and other amino and methyl derivatives of 'acridine are effective mutagens for bacteriophage (DENIARs1953; ORGELand BRENNER1961), causing the addition or deletion of nucleotides and a resulting shift in the reading frame for translation of the genetic message (CRICKet al. 1961; TERZAGHIet al. 1966). For bacteria, however, successful mutagenesis with acridine compounds is more difficult, and has been achieved only by combining the acridine treatment with irradiation by visible light (WEBBand KUBITSCHEK 1963), by addition of alkylating side chains to the acridine molecules (AMESand WHITFIELD1966), or by such high acridine concentrations as to cause rapid killing of the bacterial population (WITKIN1947; ZAMPIERIand GREENBERG 1965).This paper describes successful mutagenesis in Bacillus subtilis by acridine yellow, in the dark, at concentrations that cause no loss of viability. LERMAN(1963) suggested that acridine induced mutagenesis may be the result of unequal crossing over that occurs because the intercalated acridines cause imprecise pairing of the recombining DNA molecules. This suggestion is con- sistent with the observations that mutagenesis by 5-aminoacridine in yeast occurs only during meiosis (RfAGNI, VONBORSTEL and SORA1964) and that, in T4, a high proportion of prloflavin induced mutants first appear in heterozygotes (DRAKE1964). Howevler, this interpretation is called into question by the facts that increasing the recombination frequency does not increase the mutation fre- quency (DRAKE1964)., that inhibition of recombination does not decrease the mutation frequency (LEERMAN,quoted by STREISINGERet al. 1966), that the heterozygotes associated with proflavin induced mutants are more characteristic of terminally redundant than of internal heterozygotes ( STREISINGERet al. 1966), and that the expected ]production of complementary mutants has not been ob- served (DRAKE1964). If recombination were an integral part of the mechanism of acridine yellow mutagenesis, one would expect that such mutagenesis at a particular locus would be frequently associated with recombination for linked markers. Evidence has been presented that certain types of spontaneous mutagenesis occur in conjunction This nark vas supported by United States Public Health Service Research Training grant GM-295, by National Insti- tute of Alleig>-and Infectious Diseases Research grant AI-5160, and by National Science Foundation grant G-6411. ' Present address: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York iUS(r1. ' Abbrei-iations: met, methionine; try, tryptophan; his, histidine; ilu, isoleucine-valine; gly, glycine; arg, arginine; mr. tyi-omie. thr. threonine; leu, leucine; AY, acridine yellow. Genetics 59: 73-31 May 1968. 24 CHARLES R. STEWART with recombination of linked markers (MAGNI1963; STRIGINI1965; YOSHIKAWA 1966). This paper presents evidence that maximum levels of acridine yellow induced mutagenesis can be obtained under conditions where transformation does not take place, and further, that when acridine yellow induced mutagenesis occurs during transformation, there is no observed association between muta- genesis at a particular locus and transformation for markers linked to that locus. MATERIALS AND METHODS Bacillus subtilis strains: The mutant whose reversion by acridine yellow is most thoroughly studied here is met-63, which is carried in the following strains: SB-54 (met-6 try-2) SB-754 (met-6 his-2), SB-868 (met-6 ilu-6 try-2), and SB-871 (met-6 ilv-5). The met-6 marlier is linked to both ilu markers (presumably it is in the linkage group described by BARAT,ANAGNOSTOPOUL~S and SCHNEIDER1965). Each of the ilu markers gives about 50% cotransfer with met-6 in trans- formation by the DNAs used in these experiments. All other mutants and strains are identified in Table 3. 900 800 700 600 i I I I 500 i I I / 400 I I I I 3oa 20c TIME (MINUTES) FIGURE1.-Mutagenesis by acridine yellow in the dark in non-competent culture. An over- night culture of SB-868 (met-6 ilu-6 try-2) was diluted 1:lOO into C-2 and shaken for seven hours at 30". At this point (time 0) 1 ml was transferred to each of a series of tubes, 10 pg/ml acridine yellow added to half of the tubes and the incubation continued at 30". The cultures were plated at varying times after the addition of AY. 0-0 Met-6+ revertant frequency x 10' with AY. 0-1 Met-6+ revertan,t frequency x 107 without AY. .-e Viable count x lO-G/ml with AY. E-= Viable count x lP/ml without AY. MUTAGENESIS BY ACRIDINE YELLOW 25 Reagents: B. subtilis DNA was prepared by the method of MARMUR(1961). Acridine yellow was purchased from K and K Laboratories. Media C-l = SPIZIZEN'Sminimal medium (SPIZIZEN1958) supplemented with 0.5% glucose and amino acid groups I-V at 6.2 x the concentrations recommended by LEDERBERG(l%O) (except 3.1 x for histidine). C-2 = SPIZIZEN'Sminimal medium supplemented with: 0.5% glucose; 0.02% casein hydrolysate (acid hydrolyzed and neutralized to pH 7.0) ; 10 pg/ml each of histidine, phenylalanine, tryptophan, and tyrosine; and 0.2 pg/ml each of p-aminobenzoic acid and p-hydroxybenzoic acid. Transformant and revertant colonies were scored on plates containing 1.5% Difco Bacto-Agar plus 0.5% glucose in SPIZIZEN'Sminimal medium (SPIZIZEN1958). The appropriate amino acid supplements are added to a concentration of 25 pg/ml. Total viable count was scored on plates of Bacto Nutrient Agar. Preparation of competent' cells: A 5 ml solution of Penassay broth (Difco Antibiotic Medium 3) is inoculated from either a slant or a single colony and shaken at 37" for 12-13 hours, centri- fuged, and the pellet resuspended in 5 ml C-l. 0.5 ml is added 'to 5 ml C-l and shaken vigorously at 37" for 4% hours. 0.1 or 0.2 ml aliquots of this culture are then added to 0.9 mI C-2 and incubated, with slow shakin,g, at 30" for 9.0 minutes. At the end of the 90 minute period, DNA and/or acridine yellow is added and the incubation continued in the same medium. Incubation with acridine yellow: During the treatment with acridine yellow. all manipulations are performed in subdued yellow light. and the incubation itself is done in complete darkness. RESULTS Induction of reverse mutations by acridine yellow: Figure 1 shows the time course of mutagenesis during growth at 30" in C-2 in the dark. The revertant frequency increases with time of incubation with AY3 until, at 360 minutes, it is about 33 times the frequency without AY. There are two explanations, alternative to mutagenesis, that could conceivably account for a higher observed revertant frequency with AY than without AY. One is that the AY may be having a selective effect, permitting more rapid growth of revertants than of the bulk of the population. That this is not the case is shown by the reconstruction experiments in Figure 2. met-6- cells were mixed with met-6f revertants, which were present at more than lo3times the usual revertant frequency. Under these conditions, incubation with AY produced no increase in the frequency of revertants, showing that the AY does not confer a selective advantage on the revertants. The other alternative explanation is that competitive auxotrophic suppression (RYAN and LEDERBERG1946; GRIGG1965) may be differentially affecting the plating efficiency. This possibility was tested by a reconstruction experiment in which a pure culture of revertants was plated separately or mixed with a met-6- culture which was plated at the usual cell concentrations. As shown in Table 1, there is no significant auxotrophic suppression. Lack of association of mutagenesis with recombination: The experiment shown in Figure 1 was done with non-competent cells, as shown by the failure of ali- quots, taken from the cultures at various times during incubation. to be trans- formed by DNA from prototrophic B. subtilis. Similar (but not higher) levels of mutagenesis with AY can be achieved with competent cells, although in this case there is usually a lag of about two hours after the addition of AY before the 26 CHARLES R. STEWART 800 700 600 500 400 300 FIGURE2.-Lack of selective effect of acridine yellow. met-6- cells were mixed with a 100 fold lower concentration of met-64- revertants and grown at 30" in C-2 with and without 10 pg/ml AY. Samples were plated at various times after the addition of AY. 0-0 met-6f frequency x 104 with AY. 0--0 met-6f trequency x 104 without AY. 0-0 Viable count x 10-F/ml with AY. E--. Viable count x IP/ml without AY. revertant frequency begins to increase, and the revertant frequency often reaches a maximum after about 4 hours incubation with AY in C-2 at 30". The addition of DNA carrying the same marker makes no difference in the reversion frequency. The latter observation, added to the success of mutagenesis in non-competent TABLE 1 Luck of auxotrophic suppression Number of Mean number of Culture plated colonies per plate colonies per plate SB-754 427,520,516,541 501 met-64- revertant 505,591,684,618 600 SB-754 and met-6f revertant 1113,1192,1257,1492 1264. Cultures of both SB-754 (his-2 met-6) and its met-6f revertant were grown through the usual competence procedure plus the four hour incubation standardly used for the mutagenesis pro- cedure. 0.1 ml aliquots of the SB-754 culture and of a l:lO5 dilution of the met-Cf revertant culture were plated separately and together on plates lacking methionine but containing histidine.
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