Mapping the Gene Determining Ornithine Escherichia coli B

Vol. 108, No. 2

JOURNAL OF BACTERIOLOGY, Nov. 1971, p. 645-651 Copyright ( 1971 American Society for Microbiology

Printed in U.S.A.

Mapping the Gene Determining Ornithine Transcarbamylase and Its Operator in Escherichia coli B GEORGE A. JACOBY Massachusetts General Hospital, Boston, Massachusetts 02114

Received for publication 27 July 1971

The

gene

determining ornithine transcarbamylase in Escherichia coli B is argI,

not argF. The argI gene has been located at 84 min on the linkage map, close to the gene for aspartate transcarbamylase, with neighboring markers in clockwise order fdp pyrB argI valS. A mutation in its operator locus, argO0, has been

mapped between argI and valS, so that this gene, unlike the argCBH portion of the argECBH cluster, appears to be oriented in the same direction as most of the other known E. coli operons.

Arginine biosynthesis in Escherichia coli re- nized, E. coli B was thought to have its single quires eight enzymes, whose genetic determinants OTC gene at argF (24). are located in several separate regions of the The identity of the gene for OTC in E. coli B chromosome (11, 22). Despite this scattering, has been reexamined. It will be shown that this expression of each gene is controlled by a re- gene is in reality argI. The argI gene has been pressor protein specified by the argR locus (19, more precisely mapped by conjugation with an 23). Mutations which produce an inactive argR Hfr B and by transduction with neighboring product result in constitutive synthesis of all the markers, one of which is pyrB, the gene for asarginine enzymes. It is reasonable to assume that partate transcarbamylase. By using neighboring each arg gene has an associated operator locus markers, the argI operator has been mapped where the common repressor is recognized (11). with respect to its structural gene. Unlike Mutations at such an operator locus should af- argCBH, argI appears to have a counter-clockfect control of only the contiguous gene or genes. wise orientation on the chromosome. A technique has been devised for selecting MATERIALS AND METHODS mutations which specifically relieve repression of the gene for ornithine transcarbamylase (OTC), Bacterial strains. Previously described strains are one of the unlinked genes, in E. coli B (19). Such listed in Table 1. The genotypes of specific strains demutations are cis-dominant, closely linked to the rived during the course of this work are given in Table OTC structural gene, and do not affect control 2, and their derivation is described below. MG1 is a proline auxotroph of P404SmR shown to of other genes in the pathway. Determining on by proC mutants (such as which side of the OTC gene such operator muta- be proAItsince it atis cross-fed least fivefold fewer pro+ transducx342). gives tions map would be of interest, as the only ele- tants with proA donors (such as x462) than with proB ment of the arg pathway with known orientation, or proC donors (X343 and x342), correcting for comargCBH in the argECBH cluster, constitutes one parable numbers of arg+ transductants. MG2 was deof the few exceptions (5) to the generally rived from MGl in several steps. From a spontaneous counter-clockwise orientation of E. coli operons rifampicin-resistant mutant of MGI, an ara mutant was selected on arabinose tetrazolium indicator plates. (30). On the standard E. coli linkage map, OTC is Because the ara marker did not give satisfactory interrupted mating kinetics, the strain was transduced to identified with argF at 5 min on the chromosome ara+ with L44 (ara+ leu-2) used as donor. MG2 is a (29). However, Glansdorff, Sand, and Verhoef nonlysogenic carrying the linked leu-2 (9) showed that in E. coli K there are two dis- marker. MG3transductant was selected from MG2 as a mutant tinct genes for OTC: argF, which is cotransdu- which fails to grow on acetate, glycerol, or succinate as cible with proA at 7 min, and argI, which is carbon sources and which cannot be transduced to do weakly cotransducible with purA at 81 min. Be- so with phage grown on DF411-6 (fdp-5). Accordingly, fore this unusual dual genetic control was recog- MG3 is defective in fdp (7). MG4 was obtained from 645

646

JACOBY

J. BACTERIOL.

TABLE 1. Bacterial strains Pertinent characteristics

Strain

Escherichia coli K K-10

Source

Hfr Cavalli strA+ Hfr Cavalli proC metB strA + Hfr Cavalli proB metB strA + F- proA leu ade trp lys metE thi lac strA FTS- 14 lac+ / (lac pro)xlui suII+ thi strA + F- argG fdp-5 his leu metB F- leu his pyrB thr F- valS

X342 X343 X462

ECO DF411-6 DF634 NP29

R. Curtiss R. Curtiss R. Curtiss J. Beckwith (3) D. Fraenkel (7) D. Fraenkel (7) F. Neidhardt (31)

E. coli B

B40SmR P404SmR B4S-7 H-

argI40 argRB strA40 argI40 argR B strA40 lacZy,4

L44 BB14-1A

argI40argRII strA+ ara+ leu-2 argO, argI+ aroE argRB strA40

L. Gorini (II) P. Strigini L. Gorini L. Breckenridge (4) G. Jacoby (19)

argI47argRBstrA40

TABLE 2. Bacterial strains Strain

argl

argo,

proA

leu

fdp

pyrB

valS

lacZ

rif

strA

argR

MGI MG2 MG3 MG4 MG5 MG6 MG8 MG9

40 40 40 40 + 16

+ + +

-

+

+ + +

+ -

ts +

+

+

+

+

+

+

+

+

+ +

Y14 +

r

+

+ +

ts

MGIOO

+ +

s

+

+ 2 +

+

+

+ +

+

MG1O

40 40 40 40 40 40 40 40 40 40

RB RB RBB R

+ 1

+ +

Y14 Y 14 Y 14 Y 14 + Y 14 Y14

s r r

-

+ -

+ + +

+

+ 2 2 2 + 2

+

MG3 as a uracil-requiring mutant deficient in aspartate transcarbamylase by assay. MG4 therefore carries, in addition, a mutation in pyrB. MG5 is a nonlysogenic argI+ transductant of B4OSmR with NP29 as donor, which carries the valS temperature-sensitive mutation linked to argI+. MG6 is a nonlysogenic pyrB+ transductant of MG4, with an argI mutant of BB14-IA as donor. MG6 carries the argO) and argI 16 mutations present in the donor linked to pyrB+. MG8 is a spontaneous revertant of P404SmR to argI+. MG9 is an Hfr B derived from MG8 by integration of a temperature-sensitive FTslac+ into the chromosomal lac locus (3). Strain ECO was mated with MG8, selecting on minimal lactose streptomycin plates at 30 C Lac+ conjugatants carrying the FTS.l,4Iac+ episome. From such a strain, MG9 was selected on minimal lactose plates at 42 C, a temperature at which the FTslac+ episome fails to replicate unless integrated into the host chromosome. MG9 transfers its chromosome in a counter-clockwise direction. Since it is resistant to streptomycin, rifampicin was used for counterselection. MGIO is a nonlysogenic argI+ transductant of MG4 with MG5 as donor, which carries valS from the donor and retains pyrB of the recipient. MG 100 is an E. coli B derivative which is aroE his but prototrophic for

+

+

r

s r s

s

RBB

R RB

RB

RB R15

Mating type

F F FF FFFHfr FF-

other nutritional markers. It was used as a donor in transduction experiments. Media. Selection plates contained minimal medium A (6), supplemental growth factors as required, 2% agar, and 0.5% glucose. Where indicated, 0.5% acetate, arabinose, glycerol, lactate, or succinate replaced glucose as the carbon source. Supplements were added at the following concentrations (gg/ml): L-arginine, 100; L-leucine, 80; L-proline, 50; and uracil, 20. L plates contained 1% tryptone, 0.5% yeast extract, 0.5% glucose, and 2% agar. Rifampicin-resistant mutants were selected on L plates containing 25 jsg of rifampicin per ml. Rifampicin was the gift of J. G. Fernandez of Ciba Pharmaceutical Co. Arabinose-negative mutants were selected on arabinose tetrazolium indicator plates (15). Suppressibility by streptomycin was tested on minimal plates containing 500 gg of streptomycin per ml in place of the required growth factor. For phage propagation and assay, the media of Lennox were used (20). Mating and transduction were performed in L broth. For ornithine and aspartate transcarbamylase assays, strains were grown aerobically overnight at 37 C in medium A supplemented with growth factors as required and 0.1 % glucose. For semiquantitative OTC assays, medium A was supplemented with arginine-free

647

MAPPING OTC IN E. COLI B

VOL. 108, 1971

medium (26). Genetic crosses. Transductions were performed with a strain of P1 by using the procedures of Lennox (20). Control cultures contained L broth without phage or phage grown on the recipient. Lysogeny was tested in transductants by spotting a loopful of a chloroformed overnight culture grown in L broth containing 5 mm Ca2+ on a lawn of indicator Shigella sonnei. For strains containing the valS temperature-sensitive mutation, P1 was propagated at 30 C. Conjugations were done in L broth and interrupted by vibration (21). Counterselection of the male was with streptomycin at 500 gg/ml or, in the case of MG9, with rifampicin at 25 ug/ml. Scoring and selection of mutants. Auxotrophs were obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis (1) and penicillin selection (14). Lac+ and Ara+ recombinants were selected with these compounds as sole carbon sources. Fdp+ recombinants were selected with glycerol as sole carbon source. The valS+ transductants were selected at 42 C. Enzyme assays. OTC was measured quantitatively by using toluenized extracts as described by Gorini (10). Up to 100 samples per day could be processed by using a semi-quantitative assay originated by L. Gorini and modified slightly. In this method, which was used to distinguish argoOl+ and argO)I strains, 0.1 ml of an overnight culture in AF medium was added to 0.4 ml of 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.8). Toluene (0.01 ml) was added, and the mixture was shaken at 37 C for 30 min. The toluenized extract was incubated for 10 min at 37 C in the same tube with 0.5 ml of a mixture containing 45 Mmoles of L-ornithine, 23 Mmoles of carbamyl phosphate, 5 Amoles of MgCl2, and 100 Amoles of Tris (pH 7.8). The reaction was stopped by adding 5 ml of 0.2 M HCI; the mixture was boiled for 10 min and cooled to room temperature. To the same tube was added 2 ml of a 3:

suggests that the OTC defect in MGI is located at a site unlinked to proA and that arg+pro+

cotransductants of MGI from K-10 should still retain the original OTC defect. This prediction can be tested, because the OTC mutation in MGI is suppressible by streptomycin in a strain carrying an appropriate strA allele (13) and derepressed for the arginine pathway (18). Strain B4S-7 11- fulfills these latter requirements but carries a non-suppressible OTC mutation, so that its phenotype is Arg-. With the arg+pro+ transductant of MGI described above as donor, and with B4S-7 11- as recipient, slow-growing transductants, as well as larger argF+ colonies, appeared on selection plates containing streptomycin. The small colonies had the arginine-CSD phenotype: they require either arginine or streptomycin for growth. The donor then must have carried two copies of the OTC gene, argF+, from K-10 and the streptomycin-suppressible OTC mutation from MGI. These experiments show that the OTC gene of strain B is distinct from the argF gene of strain K. Mapping the OTC gene by conjugation. This same conclusion is evident from interrupted mating experiments, provided that an Hfr B strain is employed. If a K Hfr, K-10, is mated with MGI, the result is as in Fig. 1. Recombinants for arg+ pro+ lac+ appear together after a delay related to the restriction operating in K x B crosses (2). If the possibility that two OTC genes exist in K-10 were not recognized, it would

1 (v/v) mixture of 85% H3PO4 and 95 to 98% H2SO,

and 0.2 ml of 3.75% diacetyl monoxime. After thorough mixing, the tubes were placed in a boiling-water bath protected from light for 30 min. When the tubes had cooled to room temperature, the amount of citrulline formed was determined from the optical density at 470 nm. The specific activity of OTC was calculated from the micromoles of citrulline formed per hour, divided by the optical density of the culture at the time of harvest, measured at 490 nm. Determination of OTC control by this simplified method agreed well with the lengthier quantitative procedure, against which it was frequently checked. Aspartate transcarbamylase was assayed by the method of Gerhart and Pardee (8).

K 10 30

x

MG 1

tr

14 org+2I-

+

pro+

a

1k~~~~~~~~~ag,

RESULTS

In E. coli K, one of the OTC genes, argF, is linked to proA (9). MGl is an E. coli B derivative lacking OTC and carrying a proA mutation. With this strain as recipient and K-10 as donor, arg+pro+ cotransduction can be observed at the expected frequency of 25%. However, with several B strains as donors, no cotransduction of arg+ and pro+ could be observed. This result

10

20

30

40

50

60

MINUTES FIG. 1. Mating K-10 x MG]. K-10 and MG] at 108 cells/ml were mixed at a ratio of 1:4, diluted 1:10 at IS min, and sampled periodically for the indicated

recombinants.

648

J. BACTERIOL.

JACOBY

be natural to conclude from this mating that the OTC defect in B maps near proA and lac. The result of mating with a B Hfr is shown in Fig. 2. MG9, an Hfr derivative of B, was constructed by integration of a temperature-sensitive F'lac into the chromosomal lac locus (3). MG2, the recipient, is like MGI but carries an additional leu marker. In this cross (Fig. 2), the OTC gene of B enters well after proA, in fact distal to leu, in the region of argI, the second OTC gene known in strain K (9). To locate this second OTC gene more precisely, two K strains with markers potentially linked to argI+ were tested as donors in transduction with MG2 as recipient. It was found that, with DF411-6 as donor, 20% of arg+ transductants received the fdp marker from the donor, and, with DF634 as donor, 78% of arg+ transductants received the pyrB mutation of the donor. These are minimal cotransduction frequencies since both donors are K strains and hence argF+ as well as argI+. Nonetheless, these crosses show that fdp and pyrB are closely linked to argI+. To make suitable B recipients, an fdp mutant, MG3, was selected from MG2, and a pyrB mutant, MG4, was obtained from MG3. Figure 3 shows the result of mating the B Hfr MG9 with MG4. The OTC gene of B is seen to enter between leu+ and fdp+, closely linked to pyrB+. It will henceforth be termed argI+. Mapping argI+ by transduction. The order of markers fdp pyrB argI was established by the transduction cross shown in Table 3. The donor

2.

Qi

1.5-

1.0 tj

pro+

eu

py

arg+

fp

z~~~~~~

0. -s

10

20

40 30 M / NlU TES

50

60

FIG. 3. Mating MG9 x MG4. Equal volumes of MG9 at 8 x 107 cells/ml and MG4 at 108 cells/ml were mixed, diluted 1:10 at 5 min, and sampled periodically. TABLE 3. Ordering offdp pyrB and argIa No. of Selected marker

transductants No. carrying unselected markers scored

fdp+

pyrB+ 100 33

pyrB+

200 123

argI+

100 56

argI+ 28

pyrB+ argI+ 28

argI+ 190

fdp+ argI+ 120

pyrB+ 82

fdp+ pyrB+

MG9 x MG2

fdp+ -4.4)

fdp+

l4J

Q3 0)

I0Z 0

Ztj

MINUJTES FIG. 2. Mating MG9 x MG2. Equal volumes of MG9 and MG2 at 108 cells/ml were mixed, diluted 1: 50 at 5 min, and sampled periodically.

56

Donor MGIOO is fdp+ pyrB+ argI+; recipient MG4 is fdp pyrB argl proA leu-2. The fdp+ transductants were selected on glycerol plates supplemented with uracil, arginine, proline, and leucine; pyr+ transductants were selected on glucose plates supplemented with arginine, proline, and leucine; arg+ transductants were selected on glucose plates containing uracil, proline, and leucine. Transductants were scored for unselected markers on similar plates. a

is fdp+ pyrB+ argI+, and the recipient, MG4, is fdp pyrB argI. When fdp+ transductants were selected, 33 % were pyrB+ and 28% argI+, and all argI+ were also pyrB+. When pyrB+ transductants were selected, 61% were fdp+ and 95% were argI+. Selected for pyrB+, not all fdp+ transductants were also argI+, supporting the order fdp pyrB argI. When argI+ transductants were selected, 56% were fdp+ and 82% were

VOL. 108, 1971

pyrB+. Selected for argI+, all fdp+ transductants were pyrB+, in agreement with the order proposed. Thus, the frequency of cotransduction and the distribution of unselected markers agree with the order fdp pyrB argI, and pyrB and argI, the genes for aspartate transcarbamylase and OTC, respectively, are very closely linked. Tingle and Neidhardt have shown that pyrB and valS are also closely linked in E. coli K (31). MG5 is an E. coli B derivative carrying the temperature-sensitive valS mutation from Neidhardt's strain NP29. With MG5, which is fdp+ pyrB+ argI+ valS, as donor and MG4 as recipient, a transduction cross (Table 4) indicates the order fdp pyrB argI valS. There is 66% cotransduction of fdp+ with argI+ and 71% cotransduction offdp+ with pyrB+. valS is 98% cotransductible with argI+ and 92% cotransducible with pyrB+. There is 97% linkage of argI+ with pyrB+. As expected from the order proposed, when fdp+ argI+ transductants are selected directly, more are valS+ than if argI+ transductants alone are selected. If valS lay betweenfdp+ and argI+, very few valS+ cotransductants would be found, just as very few pyrB transductants are found on selecting for fdp+ argI+. Mapping argO. A method has been described for selecting mutations in the operator locus of the OTC gene in E. coli B (19). The technique relies on the fact that streptomycin suppression of an OTC amber mutation, argI40, is insufficient to allow growth in the absence of arginine unless repression by the argR B regulatory allele is relieved. Mutations at the regulatory gene itself are avoided by making the parent strain diploid for the argRB locus. The original operator mutation argO), as well as those subsequently isolated by the same technique, is partially constitutive for OTC. In addition, whereas arginine stimulates OTC formation in an argR B argO+ strain ( 11), arginine lowers OTC formation in an

argRB argO, mutant toward that of the wild type assay of OTC levels in the presand absence of arginine distinguishes the two control phenotypes. As would be anticipated, the OTC gene and its operator are very closely linked, and mapping the operator proved to be difficult. Preliminary experiments utilizing pyrB+ argI+ argO valS+ donors and argI argOj+ recipients with closely linked markers were inconclusive, because only 4 of over 500 argI+ transductants were still argO1+. In an attempt to enhance recombination near argI+, experiments were next performed with pyrB argI+ argOl+ or argI+ argO+ valS donors and a pyrB+ argI argOI valS+ recipient, selecting for argI+ transductants, but excluding either the closely linked pyrB or valS marker.

(19). Thus, the

ence

649

MAPPING OTC IN E. COLI B TABLE 4. Ordering offdp pyrB argI and valSa No. of

Selected marker

trans-

dduc-

No. carrying unselected markers

tants scored

argl+

400

fdp+ 266

pyrB+ 387

valS 393

valS pyrB+ 398

pyrB+

400

fdp+ 284

argl+ 388

valS 367

valS argl+ 390

fdp+ argl+

200

pyrB+ 199

valS 191

a Donor MG5 is fdp+ pyrB+ argI+ valS; recipient MG4 is fdp pyrB argl valS+ proA leu-2. Transductants were selected at 30 C; arg+ and pyr+ as described in Table 3: fdp+ arg+ on glycerol plates supplemented with uracil, proline, and leucine. valS+ was scored as the ability to grow at 42 C on the appropriately supplemented glucose plate.

At low frequency from each cross argI+ argO+ recombinants were obtained, but a firm conclusion about the location of argO1 could not be made, as the distribution of only one of the two markers linked to argI+ could be followed in each cross. This difficulty was overcome by constructing a donor strain defective in both pyrB and valS. With MGIO (pyrB argI+ argO+ valS) as donor and MG6 (pyrB+ argI argOl) valS+) as recipient, argI+ transductants were again selected in which either pyrB or valS was excluded. They were then screened for receipt of the other closely linked marker. Because the transduction frequency for these specialized classes is low, data from three separate experiments have been combined in Table 5, which also gives the details of the selection. Figure 4 illustrates the cross diagrammatically for the case in which the operator maps on the clockwise side of the structural gene. Two classes of recombinants were analyzed: type A, which have received argI+ valS from the donor but retain pyrB+ of the recipient, and type B, which have received pyrB argI+ of the donor but retain valS+. In only one class are argO)l argI+ transductants found: type B in which a crossover event has occurred between argI and argO5 (Fig. 4). If argO, mapped between pyrB and argI, the opposite result would be predicted. In fact, of 262 type A transductants, none are argOl) argI+. Consequently, the order of markers appears to be pyrB argI argO5 valS, as illustrated.

DISCUSSION In retrospect, the difficulty in locating the gene determining OTC in E. coli B is easy to understand. Since mutants lacking OTC could not be

650 TABLE 5. Ordering ofpjyrB argI argO, and valS

No.

Recombinant classes

as,sayed

argO0+

argOII

262 76

262 70

0 6

A: pyrB+ argI+ valS B: pyrB argI+ vaIS+

aOg0valS; 16argargO MG6,leu

+ vaIS; MG6, MG10, the donor, ispyrlB argI+ the recipient, is pyrB+ argIl16 argOI) vaiS+ proA leu-2. Type A transductants (pyrB at 30 C on proline, leucine plates (lacking uracil and thus excluding pyrB) and s cored for inability to grow at 42 C (indicating receipt of the temperature-sensitive valS allele). Type B transd uctants (pyrB argI+ valS+) were selected on proline, le ucine, uracil plates at 42 C (to exclude valS) and scc)red for inability to grow without uracil (indicating r4eceipt of pyrB). After punrfication, OTC control was determined by semi-quantitative assay and confirmedI by the quantitative procedure.

DONOR

MG IO

pyr B

+

+

volS

+

arg I

org Ox

+

pyr B

+

+

valS

+

orgl

org Ox

+

All RECIPIENT MG 6 DONOR

J . BACTERIOL.

JACOBY

M G10

RECIPIENT M G 6

FIG. 4. Crossover events type B transductants. For ty

ving, rie.s tor . giving

totypeAand tp

a

and

argO,I recombinant is illusi trated.

selected from strain K-E,2 (now known to contain two genes for OTC) but were available in strain B (which contains only one such gene), Maas and Maas (24) construcited a hybrid F- between nn and aa jLx K uviiYatLIvI denrivative fnr less R qtrqin aiiu all OTC(J JV aLlo>olaiii lVl mapping purposes. This OTC-less hybrid was necessarily deficient in both OTC genes. By chance, matings were done with Hfr K donors which introduce the OTC gene linked to proA as an early marker. Thus, the OTC gene mapped was one present in strain K which happens not to be the homologue of the OTC mutant in the original B strain. An analogous result is evident in Fig. I where, subject to the limitations of a K x B cross, the gene correcting the OTC defect in MGI enters with pro and lac, faithfully representing the location of one OTC gene in the K donor but not the one in the B recipient. Fortunately for mapping purposes, such duplicate genes are rare in E. coli, although, for example, tandem duplications of genes determining several

transfer ribonucleic acid species have been recognized (16, 28). Glansdorff et al. (9) discovered the dual genetic control of OTC in strain K and pointed out that mapping with a B donor would be necessary to determine the location of the single OTC gene in this strain. As shown in Fig. 3, an Hfr B x FB cross places the OTC in B gene at about mi on the linkage The OTC in B 84is map. gene thus argl, not argF. By transduction experiments, the order of markers near argI is fdp pyrB argI valS. The results (Tables 3 and 4) are in agreement with Fraenkel (7), who found 64% cotransduction of fdp and pyrB, and with Tingle and Neidhardt (31), who found 85% cotransduction of valS and pyrB. Both pyrB and valS are very closely linked (about 97%) to argl. The contiguity of pyrB and argI in E. coli is noteworthy, as pyrB codes for aspartate transcarbamylase. OTC and aspartate transcarbamylase are also closely linked in Bacillus subtilis (25) and Proteus mirabilis (27). Each is under separate control in E. coli (12). Both may have arisen by tandem duplication and functional differentiation (17) of a single ancestral transcarbamylase gene. Studies of the homology between pyrB and argI and between argI and argF will be of interest. Most of the known E. coli operons are oriented with the operator locus clockwise to the structural genes (30). Transduction experiments (Table 5) are consistent with a similar orientation for argO, the OTC operator. For operons comprising more than one structural gene, polarity effects provide additional evidence for orientation. Thus, in the argECBH cluster, since certain mutations in argC and argB have polar effects on argH (5), argCBH has a clockwise polarity. Accordingly, among the genes of the arginine pathway, operons with both clockwise and counterclockwise orientation have been demonstrated.

ACKNOWLEDGMENTS I am grateful to the donors listed in Table I for providing bacterial strains. Lorraine Sutton provided excellent technical assistance. This investigation was supported by American Cancer Society grant E499 and by Public Health Service grant AM12133 from the National Institute of Arthritis and Metabolic Diseases.

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VOL. 108, 1971

4. 5.

6. 7. 8.

9. 10. 11.

12. 13. 14. 15.

16.

17.

MAPPING OTC IN E. COLI B

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