J. Mol. Biol. (1968) 85, 71-82

Mutations Creating a New Initiation Point for Expression of the Histidine Operon in Salmonella typhimurium

I~IXRIE L. ST. P~ERR~t Biology Department, The John8 HolM:ins University,

Baltimore, Maryland 21218, U.S.A.

(Received 19 January 1968, and in revised form 26 April 1968)

An operator normally controls expression of the histidine operon in SaZmonella typhimurium. Deletion of this operator and a portion of the adjacent (7 gene in mutant hi~(7203 results in loss of expression of the entire operon.

Mutant (7203 does not revert to prototrophy, either spontaneously or following mutagen treatment. However, secondary mut~mts can be selected for tJaeir ability to grow on histidinol, i.e. for restored expression of the intact D gene. Of 145 secondary mutants isolated, at least 91 contain point mutations mapping in the (7 gene. Seventy-nine of these secondary mutants have been found to be identical; another twelve mutants are probably also the same. Each of the 79 point muta- tions maps in region VI of G and appears to be aUelle with the type point mutant , 1306.

The secondary point mutations, in the (7203 background, restore about 50% of the D enzymic activity found in wild-type repressed cells. Histidine repression control, absent in (7203 secondary mutants, is restored when (7203 is transduced to (7203 +. The mutations, in the (7203 + background, elicit a feedback hyper- sensitive G enzyme; this in turn leads to a cold sensitive phenotype. At 37°C the mutants grow like wild-type bacteria; at 20°C they require histidine or histidinol for growtah.

The experimental evidence indicates that the site of the secondary mutation is critical for expression of the operon in (7203. This site may, by base pair transition, become an initiator for transcription or for translation of messenger RNA.

1. I n t r o d u c t i o n

Deletions encompassing the operator end of the histidine operon in Salmonella typhimurium differ f rom deletions located in other portions of the operon in t ha t they result in loss of expression of the remaining intact genes. I n m u t a n t hisG203, for example, only one gene is damaged (Fig. 1), ye t none of the other genes functions normally; enyzmic activities corresponding to these genes cannot be detected in cell extracts (Ames, H a r t m a n & Jacob, 1963). Low-level function can be detected only in complementat ion tests using abort ive transduction (Ames & Har tman , 1962) or in F ' heterogenotes (G. Fink, personal communication).

Mutant G203 does not rever t to growth on minimal medium either spontaneously or as a result of mutagen t rea tment . But, (7203 can mu ta t e to growth on histidinol, the immediate precursor to histidine (Fig. 1). Secondary mutan t s are thus selected for their abil i ty to grow on histidinol, i.e. for expression of the D gene.

t Present address: Bi61ogy Department, Princeton University, Princeton, N.J. 08540, U.S.A. 71

72 M. L. ST. P I E R R E

0 0 I D I c lo,Jo ,-I A I I ' E PR-AMP l PR-ATP pyro- j Gene | p~st~lase enzyme rehydrogenaselTrar~mlnase~Ea~eitrc~e~mer~CF:~sel I~rolase Iphospbohyd~

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FIG. I. Genes and enzymes of the histidine operon. The horizontal line represents a portion of the chromosome of S. typhimurium. The far left end

is the operater end. The extent of deletion hi~Gg03 is represented below the horizontal line. In the biochemical sequence, the following abbreviations are used: P, phosphate; R, ribose; R', ribulose; F, formimino; AIC, amino-imadazole-carboxamide; IG, imidazole glycerol; IA, imada- zole acetol; and HP, histidinol phosphate. This is a modified version of a similar Figure from Loper, Grabnar, Stahl, Hartman & Hartman (1964).

Two types of secondary mutants were described by Ames et al. (1963). These mu- tants produce all enzymes of the histidine biosynthetic pathway with the exception of G (PR-ATP pyrophosphorylase), the gene for which is partially deleted. However, the histidine genes are not under histidine repression control, as are the genes of wild- type bacteria. One type of secondary mutant , designated G203(Pi), has a duplication of the histidine region normally present in G203. The other type of mutan t has an extension of the original G203 deletion.

This paper describes a third class of secondary mutations---point muta t ions - - which enable expression of the undamaged genes in G203.

2. M a t e r i a l s a n d M e t h o d s

(a) Med/a

The salts-glucose medium (E) of Vogel & Bonnet (1956) and Difco nutrient broth were the basic media used. E medium was supplemented with 20 pg T.-histidine-HC1/ml., 150 pg ~-histidinol-HC1/ml. 1.25 vol.% (EM medium) or 2.5 vol.% (2EM) liquid Difco nutrient broth as needed. E or EM media normally contained 0.2% (w/v) glucose; high- glucose media (HGE or ttGEM) contained 2.0% glucose. Soft agar had 0.75% (w/v) agar.

(b) Bacter/a and bacter/ophage

All mutants not isolated during this study were in S. typhimurium LT2 and from the stock collection of Philip E. Hartmau (The Johns Hopkins University, Baltimore, Maryland 21218, U.S.A.). SB391, used for determining the presence of nonsense suppressors, was obtained from B. N. Ames and D. Berkowitz. P22 phage lysates were prepared and stored as described by Hartman (1956). Either wild-type P22 or a Mg-hypersensitive mutant of P22 (Roth e$ a/., 1966) was used in transduction tests, performed by spreading l0 s bacteria and 10 ~ phage together on an HGE plate. A clear plaque-forming mutant of P22 (HS, also known as the v2 mutant of Zinder (1958)) was used to test bacteria for P22 sensitivity.

(e) Iaolat~n o] aecondary rnutan~ Mutants used in this study were isolated as histidinol-growing (Hol+) derivatives of

hisO203. Mutant G203 was originally isolated from S. t~himuriu~n LT2 wild-type bacteria following X-ray treatment (Ames et al., 1963). Each mutant was of independent origin.

(d) ~eparation of secondly rnut~or~ fror~ G203

Separation was based on the observation of Zlata Hartman (personal communication) that secondary point mutations led to wrlnlc]ed colony formation on HGE medium (of. Roth & Hat,man, 1966). The use of a Mg-hypersensitive mutant of P22 (N.D. Zinder, personal communication) enabled isolation of P22.sensitive transductional clones (of.

N E W I N I T I A T I O N P O I N T OF AN O P E R O N 73

Ro th et aL, 1966). Transduct ion was performed on H G E medium, and colonies were selected after a max imum of 48 hr incubat ion a t 37°C. Each was re-tested for persistence of the wrinkled phenotype.

(e) OoZd s~ i t i v l t y and hist~di~ i ~ b i t 6 m About l0 s bacteria were spread on E medium and incubated a t 20°C. Wild-type growth

was visible in 2 days; a cold-sensitive muf~ant did no t grow within 4 days. St imulat ion or inhibi t ion of growth by L-histidine and z,.histidinol was tesf~l by an

agar layer method. About l0 s bacteria were added to 2 ml. soft E agar a t 45°C; the rnlwture was poured on the surface of an E plate and allowed to solidify. Two filter paper discs (Cat. no. 740-E, Schleicher & Schuell Co.) were placed on the hardened layer; one disc was saturated with 0.04 ~-L-histidine-HCl, the other with 0-006 M-L-histidinol-HCL Plates were incubated a t 20aC for 2 days or a t 37°C for 1 day.

(f) M ~ a g e ~ and reversior~ For the selection of histidinol.growing derivatives of hisG203, l0 s bacteria were spread

on the surface of an E plate containing L-histidinol. The mutagen was placed off-center on the plates, and plates were incubated a t 37°C for 48 hr.

The reversion of cold-sensitive mu tan t s to cold insensit ivity was tested in the same manner , except tha t the E plates were incubated at 20°C for 4 to 5 days. A positive result was recorded if the density of colonies around the mutagen spot was greater than tha t on the remainder of the plate. Wi th streptomycin, a band of growth outside the inhibit ion zone was recorded as phenotypi¢ suppression.

Compounds used in liquid form were ~-propiolactone (Eastman Organic G~aemicals), diethyl sulfate (Fisher Scientific Co.), -hr-methyl-2V'-nitro-N-nitrosoguanidine (Aldrich Chemical Co.) in saturated solution, and ICR191 (acridine half-mustard obtained from H. Creech, Ins t i tu te of Cancer Research, Philadelphia, Pa.) in a 0.1 mg]ml, solution. ICR191 was handled in subdued light and the plates were incubated in the dark. Com- pounds used in crystal form were 2-,.mlnopurine ni t ra te (Calbiochem) and streptomycin sulfate (Parke, Davis & Co.). The effects of 2-aminopurine and streptomycin were tested on EM plates where background growth was enhanced.

(g) Feeding and nonsens~ suppressars The abil i ty of a m u t a n t to feed a histidine auxotroph was f~sted by stre-.lc;ng a sus-

pension of the m u t a n t on the surface of a plate covered by a soft agar layer containing 10 s cells of a histidine auxotrophic strain. After 24 hr incubat ion at 37°C, halos of growth were visible around streaks of feeding mutan t s (of. Sheppard, 1964).

Tests for amber and ochre suppressors entailed replica plat ing of colonies of the strain to be tested onto the surface of a minimal medium plate contahfing lactose as carbon source and seeded with a strain carrying an F' /ac episome with an amber muta t ion (of. Whitfield, Mart in & Ames, 1966). Plates were scored for amber suppressors after 2 days and for ochre suppressors after 4 to 6 days incubat ion a t 87°C.

(h) E n z y ~ assays For crude cell extracts, logarithmically growing cultures a t l0 g eells]ml, were harvested

by centrifugation and washed once with 0.85% (w]v) NaC1. Pellets were resuspended in 2 ml. of 0.01 ~x-Tris-l:fC1 buffer a t pHT-5 and sonicated for 1-5 rain with an MSE sonic disintegrator tuned to 1.5 4. The superna tant fluid resulting from a 30-mln centrifugation a t 37,000 g in a Sorvall centrifuge (model RC2) was used in enzyme assays. All steps were performed at 4°C. Protein content of the crude extract was determined b y the biuret method. Extracts of 25 to 40.ml. cultures usually contained 2 to 7 mg protein/ml.

Histidinol dehydrogenase was assayed by the method of Ames st aL, (1963); P R - A T P pyrophosphorylase by the method of Voll, Appella & Martin (1967).

The concentration of L-histidlne required for 50% inhibit ion of PR-ATP pyrophos- phorylase act ivi ty was determined by adding appropriate solutions of x.-histidine--HC1, in G assay buffer (Voll etal., 1967), to the assay mixture. The degree of in_hibition observed was the same whether the crude extract was pre-incubated with histidine for 10 to 20 mln in the absence of substrate, or whether the histidine was added to the complete incubat ion mixture just prior to assay.

74 M . L . ST . P I E R R E

3. Resul ts

(a) IsoZa~ior~ of 8eco~ry rautc~n~s from hisG203

Secondary mutants of (7203 were selected for the ability to grow on L-histidinol. Spontaneously, G203 gave Hol + colonies at a frequency of about 10-7. This frequency was doubled in the presence of 2-aminopurine but unaffected by the frameshift mutagen ICR191 (cf. Ames & Whitfield, 1966). The use of fl-propioIactone increased the number of Hol ÷ colonies about 25-fold; diethyl sulfate and nitrosoguauidine resulted in more than 50-fold increases. G203 did not grow on histidinol in the presence of streptomycin.

The classes of secondary mutants, according to mutagen used, are listed in Table 1. The 1306-type mutants constitute the largest class, and are the secondary point mutants described in this report. Each secondary mutant is of independent origin.

TABLE 1

Origin of hisG203 Ho~ + ~er~r~ary m~ants

Class N1K DES 2-AP Sport PL UV NG ICR191 Total

1306 203-type 16 23 18 14 13 1 6 0 91 (Pi) 12 2 0 2 0 1 1 0 18 Extended deletions 4 1 0 2 0 2 1 0 10 P22-resistant 0 5 0 4 0 0 7 0 16 Unidentified 2 2 0 0 2 1 3 0 10 Totals 34 33 18 22 15 5 18 0 145

Abbreviations" NM, nitrogen mustard; DES, diethyl sulfate; 2-AP, 2-aminopurine; Spon, spontaneous; PL, flopropiolactone; UV, ultraviolet light~ NG, nitrosoguanidine; Histidinol°growing (HoI +) derivatives of h~G203 are separated into classes by the following tests: sensitivity to P22 phage; segregation of his~idine-requiring eoloniesm(Pi) mutants (of. Ames e~ aL, 1963); ability to give wrinkled recombinants in a cross with h~Gt337~1306 203 class. Of the unidentified secondary mutants, three gave rise to feedback resistant mutants in crosses with wild type, four produce wrlnt: |ed colonies even on 0"2% glucose medium, two had impractically low transduction fre- quencies, and one gave only wild-type recombinants although i t showed no significant extension of the G203 deletion. The single (Pi) type and extended deletion type presumably induced by NG may actually represent spontaneous mutants.

(b) MaT ~ocation

Earlier observations (Zlata Hartman, personal communication) showed that 1306 203 produced two colony types on HGE medium when infected with P22 phage grown on wild-type bacteria. The minority class of wrln~led colonies increased in frequency when h~G337 was used as donor, indicating that these colonies represented the isolated secondary point mutation and that this mutation mapped in or near the G gene. The wriukled phenotype was therefore used to identify and map the separated point mutants. Since the separated point mutants could grow on minimal medium, they were always used as donors in genetic tests.

Of the 91 secondary point mutations, 79 were mapped in region VI of gene G (Fig. 2), since they gave wild-type (smooth) recombinants in crosses with hlsG203, higG1303 and h~sG1304, but gave only donor type (wrinKled) recombinants in crosses with hisG1300, h~(~1301 and h~sG1302. An average of 1000 recombinants was examined for each cross. The 12 additional secondary mutations were mapped in G, but not tested further.

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N E W I N I T I A T I O N P O I N T O F A N O P E R O N 75

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FIe. 2. Map of the G gone of the histidine oporon. Representation of the left-hand portion of Fig. 1. The numbers above the major horizontal

line represent point mutat ions; the Roman numerals below the line designate the regions of the gone, as delineated by the deletion mutants indicated by horizontal lines. Vertical brackets represent aUelio mutations; horizontal brackets indicate tha t the mutants listed above map within the region indicated but have not been more precisely mapped. This genetic map includes revisions by Maxine Levinthal and Gerald Fink (personal comm~mications), and supersedes all earlier maps of the G gone.

(e) Al~lism amo~j separc~e~ point m~tan~

The separated secondary point mutants were tested for allelism with 1306 203 + (Table 2). In one set of transduction tests, 1306 203 was the recipient for each mutant; in reciprocal tests, strains conta,lning G203 were recipients for 1306 203 +.

In these tests, the majority (73) of the 78 mutants tested yielded no wild-type (smooth) recombinants among more than 6000 colonies analyzed for each mutant. The few wild-type colonies arising in other crosses could be explained by the presence

TABLE 2

ANeli.m Zests for separa2~ 9oint m~ants

No. of Reeombinants mutants tested 1306 203 recipient 1306 203 + donor "selBng"

73 0/2000 to 0/6000 0/2200 to 0/5000 not tested 2 2/4000 0/~500 4/6500 3 11/2600 0/3200 25/6000

Transduetion tests were performed between 203+-type donors a~d 1306 203 and between 1306 208 + and 203-type recipients. The last eolumu ("selfing") represents transduetions ~i thhomologous donor and recipient, e.g. 1322 203 + donor and 1322 203 recipient. Each fraction represents the number of smooth (wild type) reeombinants observed among the number of wrinkled (donor type) r ecombi~n t s ; where single fractions are listed, the fraction refers to each of the group of mutants tested; when a range is listed, the numbers refer to the least and most colonies screened for the group of mutants. A total of a t least 6000 colonies was ~screened for any one mutan t (sum of the first two eolnm~s).

76 ~ . L. ST. P I E R R ~

of revertants in the mutant culture upon which phage were prepared. Therecombina. tion frequency between any of the 1306.type mutants tested and 1306 itself is thus less than 0.02%. For comparison, h i8~60 (Fig. 2) gave 8"0~/o wild-type recombinants, hisGl100 0.2% and hie0499 4.6%, when each was tested with the 1306 g03 recipient.

Although the reversion frequencies (about 10 -6 ) of the point mutants limit the sensitivity of tests to determine ff recombination occurs between them, their similarity in a~ characteristics studied (see later sections) indicates that they represent inde- pendent occurrence of the same mutational event. Since the 1306-type mutation could be caused by 2.amlnopurine treatment (Table 1), the mutation is a transition.

In the remainder of this paper, the designation 1306 will be used to represent all 91 mutants of the 1306-type. The actual number of mutants tested for each charac- teristic is designated in the appropriate sections.

(d) Propertle8 of 1306 203 +

The 1306 mutation, as isolated in the 0208 background, restores 50% of the wild- type repressed level of histidinol dehydrogenase activity. This level is constitutive i.e. unaffected by the presence or absence of histidine or histidinol, known to cause de-repression of histidine biosynthetic enzymes in histidine auxotrophs (Ames & Garry, 1959).

The separated point mutant, namely, 1306 203 +, had de-repressed enzyme levels, as indicated by the wrinkled phenotype (cf. Roth etag., 1966). This mutant had high levels of D and G enzymic activities when grown in E medium, and wild.type activity levels after growth in E containing 20 b~g histidine/ml. This shows that normal histi. dine repression control was restored by transduction of Gg03 to G203 +, and that the secondary point mutations result in a defective G enzyme ~yrophosphorylase). Each mutant tested showed about tenfold depression of both I) and G enzymic activities when grown in minimal medium at 37°C.

The 1306 203 + mutants are cold-sensitive (as), growing on minimal medium at 37°C, but requiring histidine or histidinol for growth at 20°G. With several Escherichla co~i c8 mutants, O'Donovan & Ingraham (1965) were able to correlate as with feedback hypersensitivity of P R-ATP pyrophosphorylase. The pyrophosphorylase of 1806 203 + was found to be approximately ten times more sensitive to histidine inhibition than is the enzyme from wild-type bacteria. Growth of this mutant was also inhibited at 37°C by histidine, but not by histidinol. The inhibition was seen on minimM plates as a clear zone surrounding a filter paper disc saturated with histidine (St. Pierre, 1967).

No nonsense suppressors were detected among the 79 secondary point mutants separated. Nor were any such suppressors found among more than 1000 additional Hol + derivatives of 0203.

Mutants of the 1306 208 + type were tested for their ability to revert to cold in- sensitivity upon treatment with a variety of mutagens. Spontaneously the reversion frequency was 10 -6, i.e. 100 revertants for every 106 cells plated. The reversion frequency was increased 25- to 50-fold with nitrosoguanidine, diethyl sulfate and fl-propiolactone. No additional reversion was seen with ICR191. There was a weak but positive reaction with 2-AP (two- to threefold increase over background) and phenotypie suppression with streptomycin.

No nonsense suppressors were found among more than 5000 revertants tested. Cold-insensitive revertant colonies were tested for morphology on HGE plates

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78 M. L . ST . P I E R R E

TABLE 4

Phenotypic characteristics of 1306 203 + revertants

Generat ion D enzyme act iv i ty G enzyme act ivi ty

R e v e r t a n t t ime Morphology Feeder Minimal Hist idine Minimal Inhibi t ion (ml.)

FR1 54(171)t s(s) no 1.1 (1.6) 0.7 NT 2.1 X 10 - s PR3 51(200) s(s) yes 0.8 (0.8) 0.7 NT N T FBA 57(167) s(s) no 1"2 (0"8) 1"0 N T N T P R 6 62(222) sw(w) no 5-4 (16.0) 1.3 no detectable ac t iv i ty PR7 50(182) s(s) yes 1.5 (1.6) 1.0 no detectable ac t iv i ty PR8 56(154) s(s) yes 0.8 (1.2) 1.0 NT NT

LT2 wild 54(179) s(s) no 1-0 (1.2) 1.0 1.0 6.1 × 10 -s 1306 203 + 54(612) w( - - ) no 8-8 (NT) 1.2 8.5 5.6 × 10 -e

Generat ion t ime was determi,~ed by following optical densi ty wi th a K le t t eolorimeter: Abbre- v ia t ions: D enzyme, histidinol dehydrogenase; G enzyme, pyrophosphorylase; s, smooth ; sw, semi-wrlnbled; w, wri,~lded; NT, no t tested.

Numbers for enzyme activities are relat ive specific activit ies based on a value of 1.00 for wild- type cells grown in minimal medium containing 20 ~g hist idine/ml. The column headed "inhibi- t i on" refers to the molar i ty of hist idine required to give 5 0 ~ inhibi t ion of G enzymic act ivi ty. Tests for feeding, inhibi t ion and enzymic activi t ies are described in Materials and Methods.

t The first number or let ter represents the results a t 37°C, the second (in parentheses) the results a t 2O°C.

Ta.BLV. 5

Genetic characteristics of 1306 203 + and its revertants

Rever~ants as donors Rec ip ien~ (FRI , PR3, FR4, PRT, PR7, PR8) 1306 203 + as donor

(i) hisG1302

(2) 1306 203 +

(3) h~-712 his(~1303

(4) h~-712

his{~203

(5) 1306 203

donor type morphology (1000) and feeding abi l i ty (18) donor type morphology (1000) and feeding abili ty (18) no wrinkled (2000)--PR6 not tes ted no wrinkled ($300)--PR6 no t tes ted no cold-sensitive (3000) (FR1, FR4, P R 8 no t tested) no cold-sensitive (3000) (FR1, FR4, P R 8 no t tested) no wild type (2500) (FR1, FR4, P R 8 no t tested)

donor type morphology (1000) no t tes ted no t tes ted no t tes ted 19 wrinkled (103) 700 wrinkled (792) 100 cold sensitive (500)

500 cold sensitive (550)

no wild type (2000)

The listings represent recombinant characterist ics scored in each cross wi th the average n u m b e r of recombinants screened given in parentheses. All t ransduet ion crosses were performed a t 37°0, except for cross 2 done a t 20°C. Crosses 1 and 2 were to determine the pa t t e rn of inher i tance of the rever tan t phenotype. Cross 3 was a n a t t em p t to recover the wrlnlded characterist ic of the original muta t ion , and cross 4 tes ted direct ly for the cold sensi t ivi ty characterist ic. E a c h of these crosses was performed wi th deletions covering opposite ends of the operon, to ellrninate possible effects of orientation. Cross 5 tes ted for recombinat ion between t h e r e v e r s i o n and 1300 203*.

NEW INITIATION POINT OF AN OPERON 79

at 37 and 20°C and for feeding ability, i.e. for evidence of a feedback resistance mutation (cf. Sheppard, 1964). Among 319 spontaneous revertants tested, three types were observed: (1) 246 smooth non-feeders; (2) 41 wrinlded; and (3) 32 feeders. With 2-aminopurine treatment, there were 204 type 1, 3 type 2 and 76 type 3, among 283 colonies tested. Types 1 and 3 are thus transitions; the few type 2 revertants among the 2-aminopurine class presumably represent spontaneous revertants.

(e) Revertant8 of 1306 203 ÷

Several cold-insensitive revertants of 1306 203 ÷, arising spontaneously on HGE plates at 20°C, were selected for more detailed analysis (Table 4). Genetic tests (Table 5) indicated that these revertants were ailelic with the original c~ mutation.

Two revertants (FR1 and FR4, representing type 1), behaved like wild-type bacteria in generation time, morphology, I) and G enzymic activities, in the inability to be inhibited by histidine (at 37°C on minimal plates) and in histidine inhibition of G enzymic activity (Table 4). Three type 3 revertants (PR3, PR7 and PRS) had wild-type generation times, morphology and D enzymic activity; however, they were feeders. In PR7, G enzymic activity was present in intact bacteria (since they grew on minimal medium) but undetectable in cell extracts. The type 2 revertant (PR6) was "pseudowild", i.e. it retained a degree of cold sensitivity, but not enough to~ limit growth at 20°C. D enzymic activity was de-repressed at 37°C and more so at 20°C. G enzymic activity was variably extractable.

In each case, since the reversion was allelic with the original c~ mutation, the re- vertant represented replacement of the original mutation with one leading to a different phenotype.

(f) Reconstruction of Hol + with G203

To demonstrate that the secondary point mutation separated was the same as that resulting in expression of the operon in G203, Hol + recombinants were isolated from a transduetion in which 1306 203 + served as donor for G203. The test was performed on minlmal medium containing histidinol, and recombinant clones were replica-plated to minimal medium. Of 71 recombinants ¢~sted, three grew only when supplied with histidinol, i.e. were like 1306 203 phenotypically. In addition, these recombinants behaved like the original 1306 203 in genetic crosses, i.e. gave no wild-type recombinants with G203 and gave wrinkled recombinants with hisG337 (see section (b)). Two other separated secondary point mutants, when tested in the same manner, gave similar results.

The six allelic cold-insensitive revertants were tested in the same series of experi- ments. Among more than 1000 reeombinants tested for each donor with the G203 recipient, no Hol ÷ recombinants were obtained, indicating that the rever~ants cannot restore expression of the opcron in (~203.

4. Discussion

(a) Nature of the G203 mutation

The significance of the G203 deletion lies primarily in the genetic material deleted rather than in base sequences created as a result of the deletion. Although the deletion may result in a frameshift, the frameshfft alone could not explain the absence of histidine biosynthetic activities, since (1) ICR191 did not produce Hol + secondary mutants (cf. Whitfield et al., 1966) and (2) known frameshifts in G grow on histidinol

80 M . L . S T . P I E R R E

although they h~ve as little as 10~ of the D enzymic activity found in wild-type bacteria (G. l~nk~ personal communication).

Creation of nonsense triplets as a result of the deletion cannot be the major defect, since no nonsense suppressors were found among more than a thousand Hol + derivatives of G203 (the possibility of multiple nonsense triplets cannot be excluded by the data, but are considered lmHkely due to the frequency with which Hol + colonies arise).

Also pertinent is the fact that deletions encompassing portions of the histidine operon other than the extreme end of G do not have such drastic effects. The absence of histidine-repression control in 1306 203 supports the conclusion that ~ e major effect of G203 is deletion of the operator for the histidine operon.

Mutation (7203 is presumed to extend greater than five gone-lengths beyond the G gone, outside of the histidine oporon (Benzinger & Hartman, 1962). Thus, in addi- tion to deleting the operator, G203 presumably deletes the "promoter" (Jacob, U]lman & Monod, 1064) for the histidine operon. However, there must be some trans- cription, as well as translation, since enzymic activities for at least part of the operon can be detected in in vivo complementation tests. I t seems reasonable to suspect that the defect in G203 is the absence of an "effective" transcription or translation ini- tiator.

(b) Restoration of operon expression

Operon expression in G203 can be restored in several ways. The (Pi) mutants (Ames etal., 1963) have a duplicated histidine operon (excluding the material en- compassed by G203). In these mutants, restoration of gone expression has been accomplished by removal of a copy of the remaining histidine genes from the damaged genetic region. In one such mutant, sensitive recombination tests indicate that the duplicated gone region contains the distal portion of the G gone, covering sites hisG876 and hisGg99 (Fig. 2) but not hisG1306 (Mark Levinthal, personal communication). At present, the relationship of the duplicated fragment to the chromosome is not known.

A second class of mutants with restored function is the extended deletions. These mutations may Hnl~ the histidine operon to the functional operator of another chromo- somal operon (Ames et al., 1963) or they may shorten the effective mRNA distance to the translation initiator at the beginning of the hext gone, D.

The third class of mutants with restored function are the point mutants described here. These mutations could restore function by creating an initiator or by allowing a pre-existing potential initiator to function.

Table 1 lists a total of ten Hol + derivatives of G203 which were unidentified. Four of these produce wrinkled colonies even on medium with 0-2~/o glucose, and thus could not be tested for 1306 203 characteristics. Two gave such low transduction frequencies that studies with them were impractical. These two groups, and the P22-resistant secondary mutants, neither support nor contradict the suggestion that the 1306 site is the only potential initiator site in G203. However, three secondary mutants, also unidentified, give rise to feedback resistant mutants in crosses with wild type and one, which showed no significant extension of G203, gave onlywild-type recombinants in crosses with hizG337 (see section (b)). The former three (represented by Gl100, Fig. 2) map in region VI of gone G, but are not alielie with 1306 (recombina- tion frequency of 0-2%). The single secondary mutant cannot be mapped. These four

NEW I N I T I A T I O N POINT OF AN OPERON 81

mutants may represent one or more additional initiator sites. The predominance of 1306.type mutants, however, indicates tha t these additional sites, if they do exist, are of minor importance.

(c) Tranacri~tion or translation ?

Experiments here described do not distinguish whether the new initiation occurs at the level of transcription or of translation. A comparison of this system to those described by others offers little insight into the problem. The 50% restoration of gene expression resulting from the secondary point mutations resembles tha t found for the internal promoter of the t ryp tophan operon (Margolin & Bauerle, 1966), however, the nature of tha t promoter is not known. The secondary point mutations are similar to the "s ta r t " mutat ion in the r l I B cistron (Sarabhai & Brenner, 1967) in tha t a unique site is involved. An elaborate system of frameshifts and nonsense mutations in r I I B enabled Sarabhai & Brenner (1967) to conclude tha t their initiator acts at the translation level. An equally refined system is not available in the G gene of Salmonella; nor has mRNA production been measured in the histi- dine mutants described here.

When more is known about codon suppressibility by streptomycin, i t may be possible to combine streptomycin suppression with the reversion results and dete~- mine the codons involved at the initiator site. The sequence of feedback hyper- sensitive and resistant mutants suggests tha t the codons for cysteine m ay be involved, since Tuli & Moyed (1966) have implicated cysteine as the amino acid critical for feedback inhibition of the G enzyme.

(d) Uniqueness of the 1306 site

Whether the new initiation occurs at the level of translation, transcription, or both, there is a uniqueness to the 1306 site which cannot be at t r ibuted solely to the single base-pair altered; it seems reasonable to conclude tha t adjacent nucleotide pairs, i.e. creation of a sequence, must be important. The predominance of 1306-type mutants among Hol* derivatives strongly suggests tha t an extremely ]imlted number of sites exists which, by transition, can give rise to an initiator. Presumably, deletions other than G203 may have similarly unique sites.

This work was supported, in par~, by Public Health Service Predoctoral Training Grant, by Predoctoral Fellowship 1-F1-GM-33, 864-01 from the National Institute of General Medical Sciences, and by research grant 5 ROI AI 01650 of the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service.

My sincerest thA.nl~ go to Dr Philip E. Hartman for suggesting the research and for his assistance and patience during and after its execution. I also gratefully acknowledge the constructive criticism received from members of Dr Hartman's laboratory, especially Dr Mark Levinthal.

REFERENCES Ames, B. N. & Garry, B. (1959). Prec. Nat. Acad. Sc~., Wash. 45, 1453. Ames, B. N. & Hartman, P. E. (1962). In The Molecular Baaia of 2Vsop/as~, p. 322. Austin:

University of Texas Press. Ames, B. N., Hartman, P. E. & Jacob, F. (1963). J. MoL BioL 7, 23. Ames, B. N. & Whitfield, H., Jr. (1966). Cold Spr. Hath. Sy~v 2. Quant. BioL 31, 22. Benzinger, 1%. & Hartman, P. E. (1962). Virology, 18, 614. Hartman, P. E. (1956). Carnegie Instn. Pub. no. 612, p. 35. ~ Jacob, F., Ullman, A. & Monod, J. (1964). C. R. Acad. Sc~. P a ~ , 258, 3125.

6

82 M . L . ST. P I E R R E

Loper, J . C., Grabnar, M., Stahl, R., Har~man, Z. & Har f~an , P. E. (1964). Broo~ha~r6 ~Fr~. Biol. 17, 15.

Margolin, P. & Bauerle, R. (1966). Gold Spr. HatS. ~ y ~ . Quant. Biol. 31, 311. O'Donovan, G. A. & Ingraham, J. L. (1965). Proc. Nat. Acad. Sci., W~h. 54, 451. Roth, J . R., AntSn, D. N. & Hat ,man, P. E. (1966). J. Mol. BioL 22, 305. Roth, J . R. & Hartman, P. E. (1966). Virology, 27, 297. Sarabhai, A. & Brenner, S. (1967). J. MoL Biol. 27, 145. Sheppard, D. E. (1964). (]e~t~r~, 5¢, 611. St. Pierre, M. L. (1967). Ph.D. dissertation, The Johns Hopkins University. Tuli, V. & Moyed, H. S. (1966). J. Biol. Gh~m. 241, 4564. Vogel, J. & Bonner, D. M. (1956). J. BioL Ghem. 218, 97. Voll, M. J. , Appella, E. & Mar~in, R. G. (1967). J. BioL G~m. 242, 1760. Whitfield, H. J. , Jr., Martin, R. G. & Ames, B. N. (1966). J. MoZ. BioL 21, 335. Zinder~ N. D. (1958). Virology, 5, 291.