JOURNAL OF BACTERIOLOGY, Oct. 1973, p. 33-40Copyright 0 1973 American Society for Microbiology

Vol. 116, No. 1Printed in U.S.A.

Duplication-Translocations of TryptophanOperon Genes in Escherichia coli

ETHEL NOLAND JACKSON' AND CHARLES YANOFSKY

Department of Biological Sciences, Stanford University, Stanford, California 94305

Received for publication 11 April 1973

Mutants of Escherichia coli were selected in which a single mutational eventhad both relieved the polar effect of an early trpE mutation on trpB andsimultaneously released the expression of trpB from tryptophan repression. Thefrequency at which these mutations appeared was roughly equal to the frequencyof point mutations. In each of these mutants, the mutation increased thefunction of trpB and also increased the activity of some, but not all, of the otherfour tryptophan operon genes. Genetic analysis showed that the mutations were

not located within the trp operon since in each case the parental trp operon couldbe recovered from the mutants. Each mutant was shown to carry a duplication ofa trp operon segment translocated to a new position near the trp operon. Polarityis relieved since the trpB duplication-translocation is not in the same operon as

the trpE polar mutation. The duplicated and translocated segments are fused tooperons not regulated by tryptophan, so trpB function is no longer subject totryptophan repression. The properties of the mutants indicate that the length ofthe duplicated segment and the position to which it is translocated differ in eachof the seven mutants studied. The duplications are unstable, but the segregationpattem observed is not consistent with a single crossover model for segregation.That such duplication-translocation events generate a variety of new geneticarrangements at a frequency comparable with point mutations suggests they mayplay an important role in evolution.

Among a selected set of spontaneous mutantsof Escherichia coli in which there is a highconstitutive rate of synthesis of a trp operonenzyme, all the mutants characterized haveduplicated segments of the trp operon trans-located to new positions on the chromosome.The duplication-translocation mutations occurat about the same frequency as point muta-tions. Each of the duplication-translocationmutants studied has different properties and,therefore, represents a unique duplication-translocation event. That these duplication-translocation events occur spontaneously at thisfrequency suggests that the mechanisms whichproduce them are normal features of bacteriallife and probably play an important role inevolution.

MATERIALS AND METHODSBacteria. All bacterial strains used are derivatives

of E. coli K-12 strain W3110 from the collection of C.Yanofsky. The characterization and mapping of thevarious trp point mutations are described elsewhere

IPresent address: Department of Human Genetics, Uni-versity of Michigan Medical School, Ann Arbor, Mich. 48104.

(10). Strain trpE9829am trpE985loc AtonBtrpA229carries two strong, polar chain termination pointmutations in trpE and a deletion which covers tonBand the operator-distal end of trpA. Strain cysB-AtonBtrpAE97 carries a cysB- point mutation and atonBtrp deletion which removes the entire trp operon.

Media. The minimal medium of Vogel and Bonnerwas used (9). For solid media it was supplementedwith 0.2% glucose and 1.5% agar. When other supple-ments were added, they were present in the followingamounts: 10 Ag of indole per ml; 1.5 Mg of indole perml plus 50 Mg of DL-5-methyltryptophan per ml; 20 Mgof L-tryptophan per ml; 30 Mg of L-cysteine per ml; and0.5% acid casein hydrolysate (ACH). Nutrient agarcontains 0.8% nutrient broth powder (Difco), 0.5%NaCl, 2% agar, and 30 Mg of L-cysteine per ml. L brothcontains 1% tryptone (Difco), 0.5% yeast extract, 0.5%NaCl, and 0.1% glucose. Assay medium is liquidminimal supplemented with 0.5% glucose, 50 ,Ag ofL-tryptophan per ml, 100 Ag each of L-tyrosine andL-phenylalanine per ml, and 1 Mg each of p-aminoben-zoic acid and p-hydroxybenzoic acid per ml.

Selection of mutants. Tryptophan synthetase 02,specified by trpB, has 3% of the activity of thetryptophan synthetase a582 complex in the conversionof indole to tryptophan. A trpB+ trpA deletion mu-tant or a trpB+ trpA nonsense mutant can grow onindole agar because it can derepress its trp operon to

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synthesize enough tryptophan synthetase 02 to sup-

port growth. However, strain trpE9829am trpE9851octrpB+ AtonBtrpA229 will not grow in indole agarbecause the trpE polar mutations reduce the amountof tryptophan synthetase 0, synthesized. We selectedspontaneous mutants of this parental strain.TrpE9829am trpE985loc trpB+ AtonBtrpA229 was

grown from a small inoculum in L broth, and 5 x 107washed cells were plated on indole agar. About 50indole-growing (Ind+) colonies appeared per 5 x 107cells plated. The colonies able to grow on indole agarwere replicated to indole agar containing 5-methyl-tryptophan, a tryptophan analogue which repressesthe trp operon of wild-type E. coli. About 5% of thecolonies which grew on indole agar also grew on

indole-plus-5-methyltryptophan agar. The colonieswhich grew on indole-plus-5-methyltryptophan agar

were tested for deletions by replicating to

080hptEAtrpD- or trpC- transducing phage as de-scribed before (5). No deletions were detected by thismethod among the mutants growing on indole plus5-methyltryptophan. The Plkc transduction methodhas been described (5).

Test for stability of the genes responsible for theInd+ phenotype. To test for loss of the Ind+ propertyonly, a colony growing on indole agar was picked to 5ml of L broth and grown overnight at 37 C. Theculture was diluted and plated on minimal-plus-tryp-tophan agar. Resulting colonies were replicated tominimal-plus-indole agar. Colonies which grow on

minimal plus tryptophan but do not grow on minimalplus indole are scored Ind-.To test for loss of other nutritional markers con-

comitant with loss of the Ind+ character, a colonygrowing on minimal plus indole agar was picked to Lbroth and grown overnight at 37 C, and appropriatedilutions were plated on nutrient agar. After coloniesdeveloped, these plates were replicated to minimalagar supplemented with tryptophan or indole or

cysteine and tryptophan and to ACH agar plustryptophan and cysteine. A nutritional requirementsupplied by nutrient agar or by ACH plus tryptophanand cysteine, but not by minimal medium with thevarious other supplements, was defined as require-ment x.

Enzyme assays. Cultures were grown for enzymeassay in assay medium in the presence of excess

tryptophan, and extracts were prepared as describedelsewhere (5). The enzyme assays used have beendescribed previously (5).

RESULTS

A single mutation can release trpB expres -

sion from a polar effect and from tryptophanrepression. The duplication-translocation mu-

tations were found by a selection procedurewhich we used to obtain internal deletions inthe tryptophan operon of E. coli (Fig. 1) (for a

detailed description of the selection procedureand the media used, see reference 5). Theparent strain from which the translocation-duplications were selected is trpE9829amtrpE985loc AvtonBtrpA229. We selected for relief

of the polar effect of the trpE amber and ochremutations on synthesis of tryptophan synthe-tase fh (the trpB polypeptide) and then ex-amined the strains obtained for constitutivesynthesis of tryptophan synthetase 32. Trypto-phan synthetase 32 has 3% of the activity of thetryptophan synthetase a2df2 complex in conver-sion of indole to tryptophan. This residualindole-to-tryptophan activity of the trpB poly-peptide is sufficient for a trpB+ tonBtrpA dele-tion mutant to grow on indole agar, because onthis medium the cells derepress and high levelsof tryptophan synthetase f32 are produced. How-ever, a strain such as trpE9829am trpE985locAtonBtrpA229, which contains strong polar mu-tations in the trp operon operator-proximal totrpB, plus a tonBtrpA deletion mutation, areunable to grow on indole agar because the polareffect on trpB expression reduces the amount oftryptophan synthetase fl2 synthesized. Howevera fourfold or larger increase in the amount oftryptophan synthetase f32 has been found to besufficient to support growth on indole agar (5).We selected indole growers (Ind+ character)from the trpE polar trpA deletion parent asdescribed in Materials and Methods. This pro-cedure selects mutations which relieve the polareffect of the trpE chain termination mutationson trpB expression. The indole-growers werethen replicated to indole agar containing 50 sgof DL-5-methyltryptophan per ml. In wild-typeE. coli, expression of the trp operon is repressedin the presence of this amount of 5-methyltryp-tophan. Thus growth of the selected indolegrowers on indole-plus-5-methyltryptophan me-dium indicates constitutive synthesis of trypto-phan synthetase fl. We found that mutantswhich grow on indole plus 5-methyltryptophan(abbreviated IndMTR, for indole growth, resist-ant to 5-methyltryptophan) appeared spontane-ously at the rate of about three per 5 x 107 cellsplated. It is not possible to calculate an exactfrequency of these mutational events since thenumber of parental cells probably increased onthe selection plate. However, it is likely thatIndMTR mutants represent between 10-7 and10-8 of the total population.Some trp operon enzyme levels are elevated

and constitutive in the mutants. Eighteen ofthese IndMTR mutants were grown in the pres-ence of excess tryptophan, and their tryptophansynthetase f32 levels were assayed. The trpoperon enzyme specific activities shown inTable 1 for seven indole-growers which areresistant to 5-methyltroptophan are typical ofthe values seen in the set of 18 mutants assayed.All seven mutant strains have higher InGPsynthetase (InGPSase) and tryptophan synthe-

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Nucleotide °pairs

1000 2000 3000 4000 5000 6000

CYS8ftrpO1 Irpi /rpD t/pC IrpO IrpA fonB

thoribosylanfhronilate

t, isomerase-

Anthronilote Phosphobosyl Indole glycerolsynthetose onthronilate phosphOte

tronsferose synthetose

FIG. 1. The tryptophan operon of E. coli. The length of the structural genes is drawn to scale. Theposition and size of genes cysB, trpO, and tonB are not to scale.

tase (TSase) 2 specific activities than theparent strain. Only one mutant, IndMTRE,makes significantly more phosphoribosyl an-

thranilate transferase (PRATase) than thepolar parent strain. All the IndMTR mutantstrains except IndMTRAs are semiconstitutive,that is, they make more InGPSase and TSase f2when grown in excess tryptophan than thecontrol strain W31 10trpA96 which carries a

trpA point mutation. That the synthesis ofInGPSase and TSase levels in these mutants iscoordinate is shown by the ratio of these activi-ties. This ratio for each mutant strain falls inthe range 5 to 10, approximately equal to theratio in the control W310trpA96 which synthe-sizes equimolar amounts of InGPSase andTSase ,8 (7). Only one mutant, IndMTRE,synthesizes PRATase nearly coordinately withthe increased TSase 2 activity, since only thismutant yields a ratio of TSase 2 specificactivity to PRATase activity equal to the con-

trol W31 10trpA96 ratio. (A constitutive low-effi-ciency promoter site located near the middle ofthe trp operon contributes 70% of the InGPSaseand TSase f2 synthesized in wild-type E. coligrown in excess tryptophan, as in the experi-ment of Table 1 (4, 6). Thus, PRATase levels inrepressed wild-type cultures are only semicoor-dinate with InGPSase and TSase 2 levels (6).In the trpEam trpEoc AtonBtrpA229 parent(Table 1), the polar effect generated by the trpEmutations reduces the PRATase level muchmore than the InGPSase and TSase 02 levelssince the polarity does not affect the internalpromoter. Thus in this parent strain PRATasesynthesis is not coordinate with InGPSase andTSase f32 synthesis.The manner in which these mutants were

isolated insures that they represent independ-ent mutational events. That they also representnonidentical mutational events is suggested bythe observation that all mutants exceptIndMTRC and D have significantly differentlevels of the trp operon enzymes.

Models for the genetic constitution ofIndMTR mutants. We had expected that any ofthe following types of mutations might result inhigh TSase $2 production in the presence of5-methyltryptophan: (i) a mutation in the trpoperon promoter greatly increasing its effi-ciency; (ii) introduction of a new high-efficiencypromoter into the trp operon, such as the one

described by Morse and Yanofsky (8); (iii) a

mutation in the trp operon internal promoterlocated near the trpC end of trpD (4); (iv) fusionof trp operon genes by deletion to anotheroperon not regulated by tryptophan (6); (v)fusion of trp operon genes by translocation toanother operon not regulated by tryptophan.Inspection of Table 1 shows that models (i) and(iv) do not fit the data. A mutation increasingthe efficiency of the trp operon promoter shouldincrease the amount of all trp operon enzymes

measured, but in all mutants except IndMTREPRATase activity is not increased in parallelwith the increases in InGPSase and TSaseactivities. Therefore model (i) can be eliminatedfor all mutants except IndMTRE.

Likewise, the predictions of model (iv) do notcoincide with the properties of the IndMTRmutants shown in Table 1. If the increased rateof synthesis of InGPSase and TSase is due tofusion by deletion to another operon, PRATaseactivity should either be coordinately increasedor completely absent due to deletion of trpD,but all mutants except IndMTRE have the samelow level of PRATase seen in the parent. It waspossible to show directly that trpE and trpDsequences are still present in all the mutantstrains in Table 1. Each of the mutants was

tested for ability to recombine with five trpEpoint mutations distributed throughout thetrpE gene and with the most operator-proximaltrpD mutation known. The trp- point muta-tions were introduced by P1 transduction as

described previously (5). All the mutants inTable 1 gave trp+ recombinants with all thetrp- point mutants tested. Thus none of the

Tryptophtnsynthetase

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TABLE 1. Specific activities of tryptophan biosynthetic enzymes in IndMTR mutant strains grown in thepresence of excess tryptophana

Sp act" Ratio of sp act

Strain PRATase InGPase TSase#2 TSasef2/ TSaseO32/(trpD) (trpC) (trpB) PRATase InGPase

trpE9829am trpE9851oc 0.01 0.084 0.61 61 7.3AtonBtrpA229 (parent)

W3110A96 0.12 0.354 1.62 13.5 4.6trpE9829am trpE9851oc 0.02 0.187 1.85 93 9.9AtonBtrpA229 IndMTR A2

trpE9829am trpE9851oc 0.05 0.582 5.5 110 9.6AtonBtrpA229 IndMT' B

trpE9829am trpE9851oc 0.03 0.474 3.6 120 7.6AtonBtrpA229 IndMTR C

trpE9829am trpE9851oc 0.01 0.564 3.4 340 6.1AtonBtrpA229 IndMTR D

trpE9829am trpE9851oc 0.91 2.10 15.9 17.5 4.6AtonBtrpA229 IndMT5 E

trpE9829am trpE9851oc 0.05 1.09 8.4 168 7.8AtonBtrpA229 indMTR 4

trpE9829am trpE9851oc 0.01 2.64 20.7 2070 7.9AtonBtrpA229 IndMTR 8 I_I_I_II

aCultures were grown with vigorous aeration in assay medium containing excess tryptophan, harvestedduring logarithmic growth, sonically treated, and assayed. Assays of PRATase levels less than 0.05 U/mg arevariable under the assay conditions employed, and therefore differences in this range are not significant. Theenzyme levels in the W3110 A96 control in the experiment presented are slightly higher than usually observed inthis strain.

b Specific activity is units of enzyme activity per milligram of protein.

seven mutants owe the increase in TSase f2level to deletion of a genetic segment left of trpCand trpB which fuses the trpC and trpB genes toanother operon.An IndMTR mutation is not located within

the parental trp operon. All of the models weconsidered except translocations (v) involvemutational alterations within the parental trpoperon. To reduce the possible explanationsfurther, we performed experiments to testwhether mutations resulting in growth on indoleplus 5-methyltryptophan co-transduce withcysB at the same frequency as the wild-type trpoperon. Plkc transducing lysates were preparedon each IndMTR mutant. All of these mutantstrains carry the cysB+ marker which is closelylinked to trp. Each lysate was then used totransduce a cysB- AtonBtrpAE97 recipient tocysB+ in the presence of tryptophan. The cysB+transductants were then replicated to indoleand to indole plus 5-methyltryptophan to scorethe co-transduction of the IndMTR characterwith cysB+. (The ability of these seven mutantsto grow on indole was never separated from theability to grow on indole plus 5-methyltrypto-phan, and both properties probably result fromthe same mutational event. In later experi-ments we scored only for growth on indole.) The

cysB- AtonBtrpAE97 recipient strain carries acysB point mutation and a deletion of the entiretrp operon. Thus in the transduction experi-ment, all or none of the donor trp operon mustbe incorporated in the recipient. The results areshown in Table 2. As expected, when the donoris the trpEam trpEoc AtonBtrpA229 parent, nocysB+ transductants are Ind+. When a wild-type trp+ strain, W1485, is the donor, the Ind+character co-transduces with cysB+ at the ex-pected high frequency. But when the donor isany IndMTR strain except IndMTRE, the Ind+character co-transduces with cysB+ signifi-cantly less frequently than does the wild-typetrp operon. These frequency differences do notreflect the position of a mutation within the trpoperon since only the whole operon can beincorporated into the trpAE deletion recipient.Therefore, these findings suggest that the muta-tion which enables these strains to grow onindole plus 5-methyltryptophan is not locatedwithin the normal trp operon, although it mustbe nearby on the chromosome since it co-trans-duces with cysB+. These findings suggest thatin all seven mutants (except possiblyIndMTRE), a segment of the trp operon contain-ing intact trpC and trpB has been translocatedto some site on the chromosome near trp and

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fused to some gene or operon not regulated bytryptophan.The cysB+ Ind+ transductants derived from

mutant strain IndMTR8 in the experiment ofTable 2 were examined in more detail forevidence of translocation of trp genes in the

TABLE 2. Position of the Ind+ character with respectto cysB in IndMTR mutants (donors: cysB+ trpEamtrpEoc AtonBtrpA229 IndMTR strains; recipient:

cysB- AtonBtrpAE97)a

cysB + transductantswhich are Ind+

Donor

Fraction Frequency

trpE9829am trpE9851oc 0/569AtonBtrpA229 (parent)

W1485 (wild type) 170/320 53trpE9829am trpE9851oc 1/123 0.8AtonBtrpA229 IndMTR A2

trpE9829am trpE9851oc 2/63 3.2AtonBtrpA229 IndMTR B

trpE9829am trpE985loc 1/109 0.92AtonBtrpA229 IndMTR C

trpE9829am trpE9851oc 1/45 2.2AtonBtrpA229 IndMTR D

trpE9829am trpE9851oc 102/120 85AtonBtrpA229 IndMTR E

trpE9829am trpE985loc 50/216 23AtonBtrpA229 IndMTR 8

a Plkc lysates were prepared on each of the donorstrains listed and used to infect the recipient straincysB- AtonBtrpAE97 in which the entire trp operon isdeleted. The transduction mixture was plated onminimal medium supplemented with tryptophan toselect cysB+ with all trp markers unselected. ThecysB+ transductants were then replicated to minimalmedium plus indole or to minimal medium plusindole and 5-methyltryptophan to score co-transduc-tion of the Ind+ and IndMTR phenotypes with cysB+.The Ind+ and IndMTR classes were identical for allthe IndMT" donors so data is given for the Ind+ classonly. The cross with the trpE9829am trpE985locAtonBA229 parent strain as donor is diagrammedbelow.

DONOR

trpE9829 trpE985locatonBtrpA229

REClIPIENT

_SysB- at.nBtrpAE97

cvsB t-E D £ B A AtonB

I

1 11 It2 3

Diagram 1. The bars represent the tonB deletions.Deletion AtonBtrpA229 extends only a short distanceinto the operator-distal end of trpA. DeletionAtonBtrpAE97 removes the entire trp operon. Thedashed lines indicate points of recombination whichwould yield cysB+ transductants. Recombination atpoints 1 plus 2 does not incorporate the donor trpoperon. Recombination at points 1 plus 3 incorporatesthe entire donor trp operon. Recombination cannot

TABLE 2-Continuedoccur within the trp operon since the recipient carriesa deletion of the whole trp operon. The x's in thedonor trpE gene represent the trpE9829amtrpE985loc strong polar point mutations in the parenttrp operon. In the cross diagrammed, recombinants atpoints 1 plus 3 will be Ind- although they do incorpo-rate trpB, because the trpE polar mutations reducethe expression of trpB. The IndMTR mutations(scored here as Ind+) are present in the parentaltrpEam trpEoc AtonBtrpA229 background. TheIndMTR mutations must be located either within theparental trp operon, or outside of it.

IndMTR8 mutant. All of the cysB+ Ind+ trans-ductants derived from IndMTR8 were alsotrpC+. These cysB+ Ind+ trpC+ transductantswere tested for the presence of a trpE markerwhich is known to be present in the trp operonof the trpEam trpEoc AtonBtrpA229 IndMTR8transduction donor. This trpE marker could notbe rescued from 28% of the cysB+ Ind+ trpC+transductants derived from the IndMTR8 trans-duction donor. This finding indicates that theInd+ and trpC+ characters in the transductantswere not incorporated as part of the parental trpoperon. Since the recipient of the transductionin Table 2 carries a deletion of the entire trpoperon, all or none of the donor trp operon mustbe incorporated into the transductant. Since atrpE marker could not be rescued from 28% ofthe cysB+ Ind+ trpC+ transductants, the trpoperon of IndMTR8 was not incorporated intothese transductants. Therefore the Ind+ andtrpC+ markers were not incorporated at thenormal position. This res-ult shows that in theIndMTR8 mutant the Ind+ and trpC+ markershave been translocated to a new position on thechromosome.An IndMTR mutant carries a duplication of

a trp operon segment translocated to a newposition on the chromosome. Further analysisof this same cross (Table 2) shows that mutantstrain IndMTR8 carries a duplication, as well asa translocation, of the trpC and trpB genes. Inthe same cross of IndMTR8 with the cysB-AtonBtrpAE97 recipient, some cysB+ Ind-transductants were tested for presence of a trpCmarker. A trpC marker was rescued from 18% ofthe cysB+ Ind- transductants. No trpC markerthat is part of the Ind+ translocation will bepresent in these transductants since they areInd- and so have not received the translocation.(Two facts argue that trpC and trpB are trans-located as one unit in IndMTR8. The increasein trpC and trpB polypeptide products is coor-dinate (Table 1). Also, in the cross in Table 2,all cysB+ Ind+ transductants are also trpC+.Thus the Ind+ translocation was never sepa-

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rated from the trpC+ marker.) Thus in 18% ofthe cysB+ Ind- transductants, trpC is presentas part of the original trp operon. Therefore, theIndMTR8 mutant strain which was the trans-duction donor carries two copies of the trpCgene, one within the normal trp operon and oneas part of the IndMTR8 translocation.

Thus, it is clear that mutant IndMTR8 carries(in addition to the parental trp operon) aduplication of the trpC and trpB genes trans-located to a position near trp. That all theIndMTR mutants in Table 1 (except IndMTR4which was not tested) carry the parental trpoperon readily separable from the Ind+ muta-tion is shown by the experiment reported inTable 3. Plkc lysates were grown on each cysB+IndMTR mutant strain and used to transduce a

cysB- trp+ strain to cysB+ in the presence oftryptophan. The transductants were thenscored for ability to grow on indole. The cysB-trp+ parent strain is Ind+ as is each IndMTRdonor. Thus we are looking for the appearanceof Ind- transductants in a cross between twoInd+ parents. CysB+ Ind- transductants werefound at frequencies approaching that seenwhen the donor is the trpEam trpEoc tonBtrpAdeletion parental strain in which the IndMTRmutations were selected. To obtain a cysB+Ind- transductant, cysB+ and the trp operon ofthe donor must be incorporated into the trans-ductant. The trpE polar mutations and thetrpA deletion must both be present for thestrain to be Ind-. Therefore, these cysB+ Ind-transductants have received from the IndMTRdonor strain the parental trp operon carryingtrpE polar and tonBtrpA deletion mutations,but have not received the duplicated, trans-located trpC and trpB genes.

Duplication-translocations are unstable.Duplications of genetic material at nearby posi-tions on the E. coli chromosome have beendescribed by others and found to be unstable(1-3). It is believed that recombination occursbetween the duplicated regions in such a waythat one of the duplicates is lost. We tested eachof the IndMTR strains for stability of the Ind+marker under conditions which did not selectfor the Ind+ character. Isolates of each IndMTRstrain were grown overnight in rich medium,diluted, and plated on medium containing tryp-tophan. Resulting colonies were then replicatedto medium lacking tryptophan but supple-mented with indole. The data displayed inTable 4 show that each IndMTR mutant losesthe ability to grow on indole at a frequency ofabout 10-2. This is additional evidence that thetranslocations conferring ability to grow on

indole are duplications.

TABLE 3. Separation of the parental trp operon fromthe Ind+ character in IndMTR mutants (donors:cysB+ trpE9829am trpE985loc AtonBtrpA229

IndMTR strains; recipient: cysB- trp+)a

cysB+ transductantswhich are Ind

Donor Fraction oftotal trans- Frequencyductants (

trpE9829am trpE985loc 51/208 24AtonBtrpA229 (parent)

trpE9829am trpE9851oc 6/78 8AtonBtrpA229 IndMTR A2

trpE9829am trpE985loc 3/66 5AtonBtrpA229 IndMTR B

trpE9829am trpE9851oc 5/65 8AtonBtrpA229 IndMTR C

trpE9829am trpE9851oc 1/69 1AtonBtrpA229 IndMTR D

trpE9829am trpE985loc 3/91 3AtonBtrpA229 IndMTR E

trpE9829am trpE9851oc 12/66 18AtonBtrpA229 IndMTR 8

aPlkc lysates were prepared on the cysB+ trpEamtrpEoc AtonBtrpA229 IndMTR mutant strains andused to transduce the cysB- trp+ recipient. CysB+transductants were selected on minimal mediumcontaining tryptophan. The cysB+ colonies werescored for ability to grow on indole by replicating tominimal medium containing indole. The cross isdiagrammed below. The donor strain shown is theparent trpEam trpEoc AtonBtrpA229.DONOR

.ysBB trpE9829antrpE985loc Nto.BtBpA229

aBNB trpE D C B A Ato.B

lI iNM I 0

I1

,,.cvs LonBM..,

II %}

Diagram 2. Dotted lines show position of recombi-nation events required to yield a cysB+ Ind- transduc-tant. If the Ind+ mutation were located in the parentaltrp operon, quadruple crossovers would be required toform a cysB+ Ind- transductant.

Previous workers have favored a simple singlecrossover model to explain segregation of dupli-cated regions (2). Such a model as applied toour duplication-translocation mutants can bediagrammed as in Fig. 2. In this model a singlerecombination event occurs in the duplicatedregion (as shown in Fig. 2). The piece of geneticmaterial between the two duplicate regions isalways lost, but no other genetic materialbeyond the region bounded by the duplicationswould be lost. We performed experiments todetect Ind- segregants from some of theIndMTR strains in a manner which allowed us

to ask whether we could detect the loss of othermarkers when the Ind+ marker was lost. Isolatesof IndMTR mutants were grown overnight in

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rich liquid medium and plated on rich medium.The colonies which grew were replicated toappropriate media to score for loss of Ind+ only,and for loss of cysB+ or of a,bility to synthesizesome other nutrient supplied by rich medium(Table 4). The results show%that some mutants(see IndMTR strains 4, B, nd D) sometimes,but not always, lose markers in addition toInd+. The simple single crossover model forsegregation (Fig. 2) cannot account for these re-

sults.

DISCUSSIONOur results show that each of the seven

IndMTR mutants in Table 1 was generated by a

duplication and translocation event. There is nopolar effect of the trpE mutation on the trans-located trpB gene since it is now part of a new

operon. TrpB expression in these mutants is notrepressed by 5-methyltryptophan because thetranslocated segment is fused to an operon notregulated by tryptophan. Each of the seven

mutants studied represents an independentmutational event. The mutants (except possi-bly IndMTRC and D) differ from one another intrp enzyme levels, in frequency of co-transduc-tion of the Ind+ marker with cysB, and insegregation pattern. Therefore the length andposition of the duplication-translocations inthese strains probably differ. IndMTR mutantsfound in our selection occur spontaneously at a

frequency of about 10-7 to 10-8. Since the seven

mutants we characterized as duplication-trans-location events were randomly chosen from thelarger set of IndMTR mutants selected, duplica-tion-translocation events probably are the mostfrequent mutational change leading to appear-

(a)

1% trp E trp D trp C trp B .trp A %,

trp C trp D trp A ,'.__________ ___ _ _ _ _ _ | r1 1 _ ,,~~~

(b)trpE trpD trp C trp D trpA

trp C trpA \

FIG. 2. Single crossover model for segregation induplication-translocation strains. Symbols; , du-plicated DNA; - -, unduplicated DNA; x, trp- pointmutation; *, deletion; *, single crossover within theduplicated region. (a) Duplicated segment trans-located to trpE side of trp operon; (b) duplication-translocation located at trpA end of trp operon.

TABLE 4. Segregation of markers in IndMTRmutantsa

No. ofStrain colonies Ind only Id - cys Ind - x

tested (%) (%) (%)

W3110 (trp+) 1,511 0 0 0IndMTR4 3,223 0 0.1 0.1IndMTRB 1,209 3 0.1 0IndMTRD 1,025 0.8 0.3 0.3IndMTRE 1,132 2 0 0IndMTR8 3,753 0.5 0 0

a Data obtained as described in Materials andMethods for loss of other markers concomitant withloss of the Ind+ character. The data for each mutantare the sum of three independent tests.

ance of an IndMTR mutant in our selectionprocedure, and their frequency of appearance isroughly comparable with the frequency of pointmutations. It is reasonable to assume that theseduplication-translocations occur equally fre-quently elsewhere in the chromosome of wild-type E. coli. The duplication-translocationevents reported here separate the genes of anoperon and form new operons, yet leave theoriginal operon intact. They generate new ge-netic sequences and change the regulatory sig-nals which govern expression of a gene. Ourresults support the hypothesis which has oftenbeen suggested-that duplication-translocationevents play an important role in evolution bygenerating new arrangements and copies ofgenetic segments without concomitant loss of animportant function. Therefore it is reasonableto expect all organisms to have mechanisms forgenerating such events.

ACKNOWLEDGMENTSWe thank Gifford Noland and Virginia Horn for their

assistance. John Rose also performed an early experimentwhich suggested that a segment of the trp operon wasduplicated in an IndMT" mutant.

This work was supported by National Science Foundationgrant GB 6790, Public Health Service grant GM-09738 fromthe National Institute of General Medical Sciences, and by agrant from the American Heart Association.

E. N. J. is a predoctoral trainee of the Public HealthService. C. Y. is a Career Investigator of the American HeartAssociation.

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