Gene, 5 (1979) 207--231 207 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

CLONING AND ENDONUCLEASE RESTRICTION ANALYSIS OF argF AND OF THE CONTROL REGION OF THE argECBH BIPOLAR OPERON

IN Escherichiu coil (Arginine regulon; divergent transcription; duplication of ancestral genes; IS insertion sequences; readthrough from pBR322 promoter; recombinant DNA) MARJOLAINE CRABEEL, DANIEL CHARLIER, RAYMOND CUNIN and NICOLAS GLANSDORFF Dienst ErfeUjkheidsleer en Mikrobiologie, Vrije Universiteit Brussel, p. ~ COO VI, Opzoekingsinstituut, Emile Orysonluan, 1, B-1070 Brussels (Belgium) (Received September 14th, 1978) (Accepted November 17th, 1978)

SUMMARY

A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322. The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI. Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, E¢oRI, HaeIII, HincII, HindIII, HpaI and II, PstI and 8alI. Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region. The argF control elements are located in a region accessible to further dissection by BamHI, EeoRI, PstI and HindIII. Carriers of the argF phsmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification Of this enzyme. INTRODUCTION

The bipolar argECBH operon from Escherichia coil is divergently transcribed from an internal control region situated between arg£ and argC. The two wings of the operon are not expressed independently; a genetic and biochemAbbreviations: Ap, ampieillin; bp, bue pairs; kb, kilobase; OTCase, L-ornithine carbamoyltransferase (EC 2.1.3.3); Te, tetracycline; •, deletion; the genetic symbols are as in Bachmann

(1972).

208

ical analysis of a number of deletion and regulatory mutants suggests that the promoter of ArgE and argCBH are locatcd o n t h e far side of their cognate structural genes with partially overlapping operators in between (Elseviers et al., 1972; Boyen et aL, 1978). The o ~ n is primarily regulated ~ t h e transcriptional level ( ~ et al., 1971; ROgers e t aL, 1971). There are indications that a minor second site control may operate as well (McLeUan et al., 1970; Cunin et aL, 1975; Krzyzek et al., 1976a, b). Evidence determinant for o ~ understanding of ~ e regulatory elements involved in divergent transcription would be provided b y nucleotide sequencing of the control region from the wild type strain and from the several available control mutants that do not involve extensive DNA rearrangements (Elseviers et al., 1972; Charlier et al., 1978). Cloning and restriction enzyme analysis of this region was therefore undertaken and the results are reported here. Comparison of regulatory regions controlling different ~enes of the arginine regulon would be of prime interest. We report the cloning and restriction enzyme analysis of the argF gene with particular emphasis on the control region. ArgF expression is peculiar in displaying a very high coefficient of repression (about 500) as compared to argE (15 × ) and argCBH (70 X ). There is evidence that argF may exhibit second site control as well (McLellan et al., 1970). Besides, cloning of that gene on a multicopy vector such as pBR822 is of technical interest in another respect; there is evidence that argF is a ¢~uplicate of argI (Glansdorff et aL, 1967; Legrain et al., 1972; Kikuchi et al., 1976; Gigot et al., 1978), and it appears very likely that both of them are ancestrally related to pyrB, the structural gene for the catalytic chain of aspartate carbamoyltransferase (Legrain et al., 1972; Gigot et al., 1977). Strains harbouring the cloned gene produce extremely high amounts of argF ornithine carbamoyl. transferase (more than 10 times the level achieved in genetically derepressed strains); therefore they facilitate its purification considerably. Amino acid sequencing of the three gene products is in current progress. MATERIALS AND METHODS

Strains, phages and plasmids See Table I.

Construction of C600 OTCC600 (F- rK- mK- leu-) was crossed with a threonine~minus derivative of 3000 × 111 (Hfr ~ pro/ac) :/eu ÷ recombinants were selected in presence of proline. Among the proline- (shown to be simultaneously/ae-) 30% were demonstrated to have retained the proximal rK" mK- marker. Absence of restriction is easily detected by spot test with unmodified ~irulent lambda bacteriophage: a rK* strain gives about thousand times less plaques than arK- one. The pro/o~ deletion removes the argF gene. To mutate the r e m ~ i n g a r g / g e n e , cells lysogenized withbacteriophage Mu c ths~were sub-

209

mi~ to two successive penicillin treatments in the presence of ornithine supplemented medium: citrulline was used to grow the surviving cells. A m u t a n t was obtained (argl-I); a Mu n o n i m m u n e thermoresistant derivative was selected; it did n o t revert to Arg ÷ at a rate higher than 10 -1°.

TABLE I LIST OF USED Escherichia coli K-12 DERIVATIVES, BACTERIOPHAGES ~, AND PLASMIDS Strains

Genotype

Origin

P4X P4X~A019-H2 recA

Hfr P4X metB (~ )÷ Hfr P4X metB A arg sup102 argX (reactivated argE) argH2 recA P4XSA019-H2 recA (pMC7) ÷ Hfr P4X metB argC1 (~ )÷ P4XSB167 (pMC30) ÷

See Bachmann, 1972 Eiseviers et al., 1972

P4XSA019-H2-A7 P4XSB167 P4XBB167-A30 P4XSB167-A31

Crabeel et al., 1977 Glansdorff, N., 1965 This article

P4XSB167 (pMC31)+

C6O0 rKmK- OTC* C600 OTCC600 OTC'A8 C600 OTC--A20 C600 OTC--A21 C600 OTC--A23

F-Thi thr ieu lac7 F-Thi rK-mK-, £ prolac, argl- I C600 OTC-(pBR38)* C600 OTC- (pMC20)* CSO00TC- (pMC21)" C600 OTC- (pMC23)*

P4XargB2 P4XA07 P4XA010 P678C3

Hfr P4X metB argB: :IS5 Hfr P4X metB argB: :IS/ Hfr P4X metB Aargsupl02, control region: :IS2 Hfr P4X metB Aargsup102, control region::lS2 P678-P4X hybrid:thr A carB8 thi OeargF

Legrain et al., 1976

3000Xlll

Hfr A pro lac argF

See Bachmann, 1972

c1857 suaq7 susxis6 • b515 A b519 7,199 d ppc-argECBH-bfc ~,13 carrying argEC1 deletion ~,13 carrying argCBl deletion ~,13 carrying argsup 102 deletion 13 carrying argB: :IS5 ~,13 carrying argB: :lSl ~.13 d argsupl02, control region: :IS2 ~.13 d argsupl02, control region::IS2 ~.199 d argF Cts (thermoinducible)

R. Weisberg

P4XargB5

See construction below This article Charlier et ai., 1978 Charlier et al., 1978 Boyen et al., 1978

Phages X199 ~13 ~.13 dargEC1 ~.13 dargCB1 ~,13 darg Sup102 ~,13 argB2 ~,13 argB5 13 A07 13.3,010 ~, dargF5 Mu-1

Mazaitis et al., 1976

I

Charlier et aL, 1978; Boyen et al., 1978 Legrain et al., 1976b A. Toussaint

Plasmids pMC7 (pMBg-R150a)

Ppc- argECBH*

pBR322 pBR38 (pBR322K12-Bam~rgF) pMC20,21,23(pBR322K12-Bam~rgF 1.6 kb) pMC30 (pBR822,HI3-e 1.8 kb; 2X) pMC31 (pBR322.H13-e 1.8 kb)

ApRTe R ApR argF + ApR argF + ApRTetlt argC ÷ ApltTetlt argC÷

Crabeel et ai., 1977 (Note added in proof, and this article) Rodriguez et al., 1977 This article

210

Culture conditions Minimal medium 132 and 869 broth are described in Glansdorff (1965). Ampicillin or tetracycline were used at a final concentration of 25 ~g/ml, unless otherwise specified.

Heteroduplex formation and electron microscopy The procedures used were described by Mazaitis et al. (1976).

DNA preparations Pl~smid DNA was recovered after equilibrium centrifugation in cesium chloride-ethidium bromide gradients of cleared lysates prepared as follows: chloramphenicol (250 #g/ml) was added for overnight mcubation to a log phase culture (usually 800 ml) grown on a medium selective for the retention of the plasmids. The cells were h a r v e Y , washed once with Tris 10 mM EDTA I mM pH 8 (TE buffer), and resuspended at 0°C in Tris 50 mM pH 8, 25% sucrose, in 1/100 the volume of the culture. Per ml of resuspension, 0.2 ml of a fresh lysozyme solution (10 mg/ml in Tris 0.25 M) was added. After 10 rain at 0°C, 0.5 ml of 0.2 M EDTA was added and 10 rain later, 1.6 ml of "detergents" (1% w/v Brij, 0.4% deoxycholate, 0.06 M EDTA in 50 mM Tris, pH 8): the mixture was left for another 10 rain at 0°C. The bulk chromosomal DNA was centrifuged 30 rain at 20 000 rev./min in an R30 rotor and the cleared supernatant was layered on an ethidium bromide-CsCl gradient (the latter was run for 35 h at 37 000 rev./min in a Ti§0 rotor). The lower band (CCC DNA: covalently closed circular) was collected under long wavelength UV using an 18~auge needle equipped with a l-ml syringe. EtBr was removed by repeated extrac. tions (5 X ) with an equal volume of H20 saturated n-b/itanol. DNA was dialysed against TE buffer, pH 8, concentrated to about I ml in the dialysing bag against polyethyleneglycol and dialysed hu-ther against TE buffer, pH 8. The DNA was layered on a 7 ml agarose A15M Biorad (100-200 mesh) column washed with 30 ml TE pH 8 and eluted with the same buffer. About 25 fractions of 16 drops were collected and assayed at 260 nm. The material from the first peak was pooled, concentrated as above and dialysed against TE pH 8. The material in the second peak is RNA, remaining butanol and CsCI. This protocol for purifying plasmids is from P. Wensink (personal communication). It gives about 1 #g of CCC DNA per ml of culture. PL~zgeDNA. Phages were prepared as described in Mazaitis et al. (1976). Purification was usually achieved after two to four equilibrium centrifugations in CsCL CsCI was eliminated from phage suspension by dialysing first against Tris 100 raM, EDTA 10 mM pH 8 (10X TE) + NaCI 0.9 M, then 10 X TE + NaCI 0.4 M and finally 10 X TE. The phage suspension was brought to a concentration giving about 15.0 A units at 260 nm (around 600 #g of DNA/ml). Melted Mallinekrodt phenol, equilibrated 3 X at room temperature with equal volumes of 10 × TE was gently mixed during 3 rain (1:1 volumes) with the phage suspension. After centrifugation at room temperature in a table Sorvall, . the upper aqueous phase was removed by slow pipetting and retreated 2 X

211

with phenol as above. The last upper phase was then dialysed extensively against TE at pH 8; the DNA concentration was evaluated by absorption measurements at 260 nm.

Construction of hybrid plasmids The procedure followed has been described in Herschfield et al. (1974).

Restriction enzymes, T4 DNA ligase and HaelIl digests of ~X174 DNA This material was ordered from New England Biolabs and the enzymes were used according to their instructions.

Electrophoresis According to the molecular weights of the DNA fragments, electrophoreses were performed in one of the following gel types: agarose 0.7%, agarose 1.4%, mixed polyacrylamide 2.5%, agarose 0.5%, polyacrylamide 6%. The mixed gels were poured as in Peacock et al. (1968). Acrylamide, N,N'-methylenebisacrylamide and N,N,N',N'-tetramethylethylenediamine were from EastmanKodak, Rochester. Acrylamide was recrystallized as in Shuster (1971). Agarose was f r o m Miles Laboratories.

Running conditions were as in Crabeel et al. (1977).

DNA fragment isolation Fragments to be used for heteroduplex mapping were isolated as described in Crabeel et al. (1977). For restriction analysis, as some enzymes are very sensitive to agarose traces (in our hands, particularly AluI and HaeIII), fragments were extracted from agarose gels by a procedure from R. Echenlaub (Helinski's lab., communicated by W.K. Maas)" gels were dissolved in 1.5 vol. of 5 M Na-perchlorate at 65°C by repeated inversion of the tube. DNA was adsorbed (5 rain) to I ml hydroxylapatite equilibrated with Tris 100 mM, 50 mM NaCI, 0.1 mM EDTA, pH 7. The adsorbed DNA was collected by centrifugation with a table Sorvall and washed twice with 10 ml of 10 mM Na-phosphate, pH 7. DNA was eluted with 3 ml of I M Na-phosphate, pH 7. The first pellet was eluted again with I ml of the same buffer. The pooled supernatants (cleared by centrifugation 5 rain at 10 000 in Sorvall SS-34 rotor) was dialysed against Tris 10 mM EDTA I raM, pH 8 and precipitated with ethanol. Even with fragments purified by this method, it was necessary to use 10- to 50-fold the recommended amount of restriction enzyme to observe restriction.

Assay of L-ornithine carbamoyltransferase (EC 2.1.3.3) See Glansdorff and Sand (1965).

212

REBULT8

(I) Endonucleolytic dissection of the argECBH bipolar opemn Previous studies have shown that the intact argECBH cluster is liberated on a 17.5 kb fxagment by £coRI (Devine et al., 1977; Crabeel et al., 1977) and on a 7.5 kb fxagment by BamHI (Moran et al., 1977). The HindIII enzyme cuts at two sites within the opemn liberating a 1.8 kb piece, H13~ (Crabeel et aL, 1977; Devine et aL, 1977). The left end of H13-e was shown by us (Crabeel et aL, 1977) to be situated about 0.6 kb to the left of the point which the argECl deletion reaches in argC, thus about 0.4 kb outside the 0.2 kb long overlap between supl02 and argBCl (Mazaitis et al., 1976) (Fig. 1). H13~ should thus catty most or all of the control region at the left end. Further dissection of this region and closer mapping of the control sites are detailed below. In the next section we report the cloning of H13~ on plasmid pBR322. This vector has provided us wi~h a DNA source allowing straightforward purification of fxagments onginsting in the control region. (a) Cloning of the HI3-e fragment. H13-e carries intact the argC gene (Crabeel et al., 1977). Incorporation of H13-e into the DNA of plasmid pBR322 was therefore selected for by tzansducing P4XSB167 (argCI) to Axg° with a ligated mixture of HindIII digests of DNAs f~om pBR322 and ~darg No. 13. The technique followed was from Herschtield et al. (1974). All Arg* colonies obtained were found to be resistant to ampiciUip, thus carders of pBR322. Ten transformants were purified and tested for their resistance toward tetracycline. Studies by Roddguez et al. (1977) strongly suggest that the unique HindIII site of pBR322 lies within the promoter of the gene responsible for resistance to tetracycline. Therefore hybrid plasmids are expected to confer significant resistance to this antibiotic only it the inserted fragment bears a properly oriented promoter and no strong terminator. Of the ten transtonnants four were found to be sensitive to 5 pg/ml of tetracycline on minimal medium, and six resistant to concentrations up to 25 pg/ml (carders of the normal pBR322 usually resist concentxations of 75 to 100 pg/mJ). Moreover this resistance was lowered by the presence of arginine in the medium. This

I control I AargECI ~]eg, o~J I ¢.-...L I ~

L .

H13e--l.8

AargCB ] "

i° 1

Fig. 1. Restriction with HindIH liberated from argECBH DNA on a 1.8 kb fragment, H13-e, delineated with respect to several ~ginine cluster deletions. Distances are in kilobemes.

213

arginine-sensitive tetracycline resistance was taken as a good indication for insertion of the sole H13-e fragment at the HindIII site of pBR322 since: (1) H13-e bears 2 arg promoters, one for each orientation; (2) transcription from either one is repressible by arginine; (3) H13-e bears no transcription terminator. Plasmid DNA was isolated from two transformants, and respectively named pMC30 and 31. In both cases, gel electrophoresis of HindIII digests revealed two bands: one 4.3 kb long (pBR322 DNA) and one 1.85 kb long (H13-e fragment). In the case of pMC31 an extra HindIII fragment of 130 bp was detected; it probably corresponds to a small piece of ~,DNA present to the right of the 0.5 kb fragment bearing gene CI (Robinson and Landy, 1977). Examination in the electron microscope revealed the DNAs of pMC30 and pMC31 to be 12 kb and 6 kb long respectively. This and restriction studies have shown that pMC30 carries a dimer of the plasmid present in pMC31 minus the 130 bp fragment. The latter was shown to be inserted between H13-e and the Tc r gene of pBR322. Considering the above-mentioned, arginine-mediated, reduction of the resistance to Tc, this fragment should bear no terminator of gene expression.

(2) Mapping of the ends of 1-113-e with respect to IS elements inserted into argECBH. The results presented under (a) and (b) are summarized in Fig. 2. (a) Right hand extremity. Two argB mutants have been found to harbour polar insertions (Cha~lier et al., 1978). ArgB2 carries an IS5 and argB5 an ISl element; with respect to argH they are in orientation I and II, respectively

(ibid.). H13-e encompasses the argB5 site; indeed HindIII digests of ~, 13argB5 contain no H13-e fragment but a new band corresponding to the sum of H13-e (1.8 kb) and ISl (0.8 kb) (Fig. 2, line a); Schmidt et al. (1976) and Ohtsubo et al. (1976) have shown that HindIII does not cleave IS1. The IS5 element present in argB2 has been shown to obliterate the HindIII cut, delineat':,lg H13-e on the right hand site (Charlier et al., 1978 and Fig. 2, line b). An ~coRI cut was found in the right half of IS5 in argB2, confirming the origin£, report by Blattner et al. (1974) on the presence of an EcoRI site in IS5: when ~, 13argB2 is digested with EcoRI, two new fragments, respectively 10.6 and 4 kb long replace the 13.5 kb long fragment recovered from ~ 13 (Fig. 2, lines c and d). EM studies have shown that IS5 in argB2 lies about 80 bp to the right of ISl in argB5 (Charlier et al., 1978). Since the secondary promoter present near the argBH boun~;ary is located between argB5 and argB2 (ibid.) it must be very close to the right hand extremity of H13-e. (b) Left hand extremity. The terminus of H13-e on the argE side was determined relatively to IS~ elements inserted within the control region between argE and argC. In mutant P4XA07, IS2 is in orientation II with respect to argE, 120 bp to the left of the argBC-promoter E deletion present in mutant sup102; IS2 is very close to Pcsx and delineates the control region on the left side (Charlier et al., 1978). In mutant P4XA010 IS2 is in orientation I with

214 ! kb

a) .__

, ),13B5 1

r -

ISI-II(o)

I

b}~

Hindlll ,o

,.,, 13.5

ISS-IIo)

,

c)~ :o.s

4,

..M3 } EcoRl ..... A'I3B2

';

d)~

ISS-I{',,) 8.t

e}L

L 65up102

r~

7.2

1.1

7.S

f) L_

IS2-1II..) ~t-~.

g) I kb

h)_

i ) ...

,

L _ . M3A010

IS2-I(-.) L

!

oc 1.1

" £Cl

Sup 102 ,.gs

L

IS2 -I (,,-) .L 0.8 ~r

J ) ....

7.2.

,

IS2-11(*)

~2

JI~ ... )t13 L-.

M3A010

~

M3A07

Hpal

,.4s

F i g . 2. Restriction enzyme analysis of a series of physically characterized arg£CBH trans. dueing 7, phage derivatives bearing IS sequences in the arginine cluster. This allows precise mapping of restriction sites relative to genetic markers having well defined effects on arginine genes expression and regulation. Distanees are in kilobases.

respect to argE, between the previous one and sup102, about 70 bp from the latter (Charlier et aL, 1978). HindHI cleaves IS2 at a well determined site (Schmidt et al., 1976). When HindIII digests of Xdarg13 carrying either one of these IS2 associated with the sup102 deletion are compared with a digest of ~13sup102, the H13 Supl02c fragment (H13-e + H18-c- Asupl02; Crabeel et al., 1977; Fig. 7B) is absent; in both cases two new fragments appear (Fig. 2, lines e,f,g). The lighter one of each pair is expected to carry a short terminal fragment from the left part of H13-e plus a piece of IS2; they measure 1.12 kb and 0.8 kb in the case of IS2 from P4XA07 and P4XA010, respectively. Knowing where HindHI cuts in IS2 (Schmidt et al., 1976) and how this element is oriented in either mutant (Charlier et al., 1978), the data show that the left hand extremity of H 1 3 ~ ~ b e t w e e n 3 3 0 ~ d 390 ~ p a i m to the left o f the sup102 deletion, this compares well with previous estimates of about 0.4 kb (Fig.i 1).

215

(3) H p a / a n d HinclI cleavage sites. (a) Localization of the Hpa/cleavage sites with respect to arg deletions. Comparing HpaI digests of Xdarg13 and ~,199 (parent phage) brings to light 2 bands from X13 (~ and/~) which further analysis shows to contain portions of the argECBH cluster; ~ is 1.05 kb long, 1.55. In digests of X13argEC1, ~ disappears and ~ remains while the reverse is true with ~13argCB1. When ~13sup102 is digested, neither a nor/3 are present but a new band arises, 7, which is 0.78 kb long. The conclusions are summarized in Fig. 3, line a: an HpaI site separating a from/3 is located between the argEC1 and argCB1 deletions in a region which is deleted in mutant sup102; the length of 7 (0.78 kb) corresponds reasonably well to the sum of a and # minus the segment deleted in sup102 (1.05 + 1.55 - 1.8 = 0.80). (b) Localization of Hpa/cleavage sites with respect to 182 insertions in the control region. When DNA from X13A07 and A010, which carry an IS2 associated with the sup102 deletion, is digested with HpaI, the 7 band (see previous section) is replaced by a pair of new bands: 0.80 and 1.45 kb in the case of X13A07, 1.95 and 0.32 in the case of X13A010. IS2 is thus cleaved by HpaI. Knowing the length of this element, its different orientation in either mutant and the distance between the two IS2, the site of HpaI cleavage in the insertion can be placed 0.15 kb from the fight hand extremity when it is in orientation I (Fig. 2, lines h and i). Moreover, as the position of either IS2 is known with respect to the sup102 deletion, the ends of the two fragments replacing -f can be mapped using the same reference; these localizations also correspond to the termini of 7 or of (~ + ~) (Fig. 3, line b ) .

I

• H13e ACBI

__AECl I ,

Jr,.

ASuplO'2

i-' [ ~

O)

1.55

1.05

H 13e:: IS2-11'/H13e

~ 038

...o r~ 226 " 28

c)

1.0

el) e)

J I

0.79 •

7[

Tr

i| ~27~o37_ o~

fig/, fig3

Fig. 3. Location of HpaI (o), HindIII (o) and HincII (o,e) restriction sites relative to each other and to deletions or insertions in the argECBHbipolar operon. Distances are in kilobases. Lines c, d and e summarize results obtained with purified fragments. Fig. 4. Heteroduplexes obtained between a purified fragment isolated after HindHI restriction of ~.13A010 (the fragment consists of the left part of H13-e plus part of IS2). Distances are in kilobases.

216

The HpaI cut which we localize 0.15 kb from the right-hand extremity of IS2-I corresponds well with the HincH site shown to be present in the same region, 0.17 kb from the end (Schmidt et al., 1976). The unique DNA sequence recognized by HpaI (GTTAC) is one of the four sequences recognizable by HincII (GTPyPuAC). (c) H i n d l cleavage'sites. The HincII enzyme recognizes more cleavage sites per unit length of DNA than the 6 base pairs specific endonucleases. Therefore, HincH was used to digest relatively short DNA pieces: either purified restriction fragments or the DNA from plasmid pMC30 and 31, the vectors of cloned H13~. (d) Dissection o f 1t13.e by Hincll and Hpa/. Three fragments appear after digestion of purified H13-e DNA by HincII: a "large" (1.05 kb), a "medium" (0.47 kb) and a "small" one (0.32 kb). The estimates are the average values of at least 10 independent determinations either on agarose or on polyacrylamide gels. HpaI, which recognizes one of the 4 possible HincH sites, gives two fragments respectively 1.05 kb and 0.75 kb long, indicating already that the HincII "medium" and "small" fragments are next to each other. From the position of the HindIII cuts liberating H13-e and of the HpaI cut separating a from $3it can concluded that the "large" HincII fragment contains the righthand part of H13-e. Partial digestions of H13-e by HincII gives a 1.5 kb fragment. The order of the three HincII fragments should thus be: small (0.32) -- medium (0.48) -large (1.05) (Fig. 3, lines c and d). This was confirmed by further restriction analysis reported below in (f) and further on in (6). HD analysis of hybrid molecules formed between fragments purified after digestion of ),13A010 with HindIII and H13-e (Fig. 4) confirm that the left end of H13-e is about 230 bp to the left of the IS2 insertion. The HincII cut between the small (0.32 kb) and the medium (0.48 kb) fragments is thus of special interest, as it is situated about 90 bp to the right of IS2, thus inside the control region. (e) Dissection o f the Hpa/fragments with HinclI. HincII cleaves a into three pieces respectively 0.48, 0.37 and 0.27 kb long. From the overlap between a and H13-e (see Fig. 3) we would expect to recover in this digest the "medium" HincII fragment (0.37 kb) and another one longer than the "small" size HincII piece: the above 0.50 kb long fragment. As the size of the latter and that of the "small" HincII piece do not differ significantly, it was necessary to determine the relative order of the HincII and HindIII sites involved in their delineation. This could be achieved unambiguously through HincII dissection of pMC30 and 31. (f) Dissection o f pMC30 and 31 with HinclI. In pBR322, the plasmid used to construct pMC30 and 31, two HincII sites are known (Rodriguez et al., 1977). Therefore, if we insert H13-e in pBR322 and digest the new plasmid with HincII we expect 4 fragments if HI3-e contains 2 Hinc sites: this is indeed what we obtain (Fig. 5 and accompanying gel electrophoresis pattern). A fifth fragment should have been observed if a third HincII site had been

217

HincI]

-3100

Hincl]

1100

pBR322

137; 87:, Hincll ~ H i n c l l

60C

Hincll

1800

- 3100

ab c d Hincl] Fig. 5. Eiectrophoresis of H i n d I digests of pBR322 (column b) and pMC30 or 31 (columns c and d). pMC31 is a pBR322 hybrid plasmid bearing the H13~ fragment inserted into the unique HindlII site of pBR322. Molecular weight markers are ~X174 DNA, restri~ed by HaeIII (column a): lengths, in base pairs, of some reference bands are given (Banger et ai., 1977). Distances on the plasmid DNA are in base pairs.

present close to the end of H13-e and within it. The data thus place the HincII site discussed at the end of the previous section, outside of H13-e, to the left of it as pictured in Fig. 3 (line e). They also confirm the order of the three HincII subfragments of H13-e: 66 small , 9 -- 66 medium , , -- 66 large 9 , . (g) Other HinclI and Hpa/sites in the argECBH region. DNA from the previously described ColEl-arg plasmid pMC7, derived from pMB9 (Crabeel et al., 1977, note added in proof) has been restricted in single and in mixed digests with HindIII, HincII and HpaI (Fig. 6). The 9.5 kb EcoRI cloned fragment (containing about 1.12 kb of ~,DNA) displays some other sites than the ones described above. To the right of the sup102 deletion (clockwise on the chromosomal map as in Fig. 1, and to the left of it in Fig. 6), two more HincII sites are revealed; the first one (also recognized by HpaI) is situated 0.74 kb from the end of HpaI {J fragment, the second one lies 2.35 kb further. The HincII/HpaI site is still situated in argH, while the HincII site lies outside the argECBH cluster since it is known from the analysis of a tandem duplication of argH (Charlier et al., in preparation) that the size of that gene does not exceed 2.0 kb.

218

...

J - ~

~

.,--,

..,,.

~.~ - e

J

I1'

~

.

. , t % ~,

I

o~

p MC 7

, ~

j -J- .i.d.

Fig. 6. £coRl, HindIII, HineII, Sail and HpaI restriction sites in pMC7, a pMB9 hybrid plasmid (heavy line) containing a 9.5 kb long £coRI fragment composed of 1.12 kb x phage material (serrated line) and a piece of chromosomal DNA bearing the whole arg£CBH bipolar operon (thin line). Only the one or the other of the two Hinell sites marked with an ssterisk exists (see text). Distances, in kilobses, are expressed counterclockwise from the EcoRI insertion site. a snd # are the two Hpal fragments covering the control region of the argECBH bipolar operon.

At 0.22 kb to the left of HpaI a fragment (as it is drawn on Figs. 2 and 3, and to the right of it on Fig. 6), thus still in the argE gene, there is a HincII site; it must correspond to the GTCGAC sequence as it is also cut by Sail (see below). Three other HincII sites are present on the arg£ side of that HincII/HpaI sequence in the cloned fragment of pMC7: the first is situated at either 0.47 kb or 1.23 kb from it. The two others delineate a fragment of 0.37 kb which becomes shortened to 0.32 kb after HindIII restriction at the site situated on phage ~DNA in the vicinity of PR (Allet and Bukhari, 1975; Davidson and Szybalski, 1971). Thus the last HincII site is in phage DNA; the other one is probably on chromosomal DNA but outside the arg£CBH cluster since it is k n o ~ from analysis of tandem duplications of argE (Charlier et al., in preparation) t h a t t h e size o f t h a t gene does not exceed 1,9 kb. (4) Sail cleavage sites. (a) Localization with respect to arg deletions. ~Comparing Sail digests of ~dargl3 and of the parent phage ~,199 brings to light

219 three bands which further analysis shows to contain argECBH material: the corresponding fragments measure 7.9, 4 and 0.50 kb. In k13argCB1 (1.4 kb deletion) and ~13supl02 (1.8 kb deletion) only the 7.9 kb band disappears, replaced by a 6.5 kb or a 6.0 kb fragment, respectively. These measurements agree closely with those obtained by subtracting the length of the argCB1 or sup102 deletion from the value of 7.9 kb. In digests of ~13argEC1 all three bands are replaced by a heavier one containing a fragment of about 10.5 kb (thus approximately the sum of all three k 13 fragments minus the 1.4 kb of deletion argEC1). Two SalI sites thus exist in the region lacking in mutant argEC1, but outside the sup lO2-argEC1 overlap. (b) Sal/digestion of H13-e. SalI does not cleave H13-e. This was shown with purified H13-e DNA and with pMC31 DNA. (c) Sal[ sites in the DNA of pMC7. Restriction of pMC7 DNA (carrying the 9.5 kb fragment referred to in section 3g) by SalI, HindIII, HincII or HpaI taken alone or in combination showed that H13-e was present intact in mixed SalI-HindIII digests but that SalI shortens HpaI fragment ~ (1.05 kb) down to 0.78 kb. Thus aSalI site is present between the left-hand termini of H13-e and a. This site corresponds to the HincII site situated immediately to the left of H13-e (see Fig. 3, line e). A 0.5 kb fragment recovered from SalI digests of pMC7, k 13, k 13argEC1 and ~13supl02 DNA disappears in mixed SalI.HpaI digestion; it is replaced by two fragments of 0.22 and 0.26 kb. Thus a further SalI site (also recognized by HincII; see section 3g) delineating the 0.5 kb fragment occurs clockwise of HpaI fragment a (Fig. 6). This site is still in argE (see section 4a and Fig. 6). (5) Pst/cleavage sites in H13.e. PstI has been tested only with purified H13-e DNA and with pMC31 DNA. No cleavage site was found. (6) Dissection of H13-e with restriction enzymes recognizing sequences of 4 nucleotides: Alu/, HaelII, HpaH. HaeIII cuts H13-e in 5 fragments (980, 640, 104, 100 and 48 bp). The 100 and 104 bp fragments are barely resolved when HaeIII digests of pure H13-e DNA are examined. Their existence can be deduced however from the analysis of digests of pMC31 DNA (see further §). In mixed HaeIII.HincII digests, the 980 bp piece disappears; it is replaced by the HincII "small" and "medium" subfragments of 320 and 470 bp respectively; the "large" one is absent while a new one of about 200 bp (980-470+310) appears. The 100 bp fragment is the one furthest to the right in H13-e; indeed, in HaeIII digests of plasmid pMC31 DNA, the 640, 104 and 48 bp fragments are present while the 100 bp piece is replaced by a hybrid one, 360 bp long, which corresponds to the following sum: 100 bp + 130 bp (the small HindIII fragment of ~,DNA present in pMC31, see end of Section 1 (a)) + 130 bp of pBR322 DNA up to the first HaeIII site in this plasmid (following the unpublished nucleotide sequence of pBR322 established by Gregor Sutclfffe). We can thus retain the following scheme (Fig. 7) on which the respective order of the 680, 104 and 48 bp HaeIII fragments remains ambiguous. This uncertainty is resolved below. HpaII delivers 8 bands, respectively 770, 355, 309, 168, 82, 78, 65 and 55

220 H pa F!

Hincll-I Hincg-2 320 i 470 [ -:

,050

1

T oo

Haelll-1

Haelgk

Fig. 7. Results from m ixedHiaclllHaemrestrie*Aon on purified H13,e fragment a n d ~ m on pBR322 and pMC31 D N ~ . D i s t ~ c e s

compated H a e ~ a n d HaeHIIHindlll ~ e t i o n ate in base painL

HpaI-t Hincll.1 Hincl]-2 320 [~ ,.70 [ 1050 770 [ 35613091821?8165155 ]168" Hpal[-1 H pa]]q

Fig. 8. Ruults f~om mi~ed HineHIHpaH restriction on purified H13-e fragment and from compared HlmII and HpaIIIHindIII reatzietion on pBR322 and pMC31 DNAs. Distances

ate in bue pai~

Hpal-1 HincI[-2

Hint]l-1

32o

1=/"

AluI-1

I

sis

,.7o

I

,oso

T 33o l

A[u~2

Alul-3

s o/200/1 3

Fig. 9. R~ults from mized Hinell/AluI and HpaIIAluI restrictions on purified H13-e fragment. Diatances ate in base pairs.

bp long. The combined action c~ HpaI and HtncH eliminates the longer 770 bp piece which is replaced by the HincII "small" fragment and a slightly shortened "medium" fragment of 460 bp; the "large" H/ncII piece is absent. The 355, 309 and 168 bp HpaH fragments are not shortened and are thus not contiguous to the 770 bp fragment. Comparing HpaII with HpaII/HindIII digestions of pMC31 and pBR322 allowed identification of those HpaII fragments which are terminal on H13-e: the 770 and 168 bp fragments are respectively located at the left and right hand side extremities of H13-e. The order is thus as on Fig. 8; the u n c e ~ t y regarding the respective order of the remaining fragments is cleared partially below. AluI cuts H13-e into 6 fragments of 580, 515, 330, 200, 113 and 108 bp. Mixed AluI-HincII diges~ contain no typical H/ncII fragment, indicating that AluI cleaves all three "small", "medium" and "large". AluI fragments 515 and 330 bp long disappear in the presence of HincH; they are replaced by four new DNA pieces respectively 210, 310 (Z = 520), 162 and 175 bp long (Z = ,the un-

221

the first (from left to right) HaeIII cut in H13-e is immediately to the right of the third AluI site, the 580 and 330 bp AluI fragments cannot be next neighbours. The 580 bp AluI fragment must be entirely comprised within the only HaeIII digestion product which is bigger: the 640 bp fragment. As the AluI specific 4 bp sequence is comprised within the 6 bp of the HindIII site, it is not possible to identify the fragments bearing the extremities of H13-e by comparing single AluI and double AluI-HindIII digestions of plasmid pMC31. Their identification was derived from labelling experiments designed to isolate fragments for DNA sequencing: if fragments from pMC31 DNA digested with both HindIII and HincII are terminally labelled with s2p and then restricted with AluI, the autoradiography identifies 6 labelled fragments originating from H13-e; four which are expected from double AluI.HincII digestions (210, 310, 162, 175 bp, see above) and two other ones (108 and 200 bp) which are respectively the left and right hand side terminal subfragments from H13-e. The AluI sites being at[ ordered, the HaeIII sites fall in place as well; Fig. 10 summarizes the results of this analysis. The order of some as yet undefined HpaII cuts can now be derived from double digestions of H13-e with HpaII and either HaeIII or AluI. The results also confirm previously established positions for HaeIII and AluI restriction sites. In double HpaII-HaeIII digests the 355 bp HpaII fragment is retained while the 309 bp piece is shortened to 245 bp. This suggests the succession of HpaI fragments depicted on Fig. 11a where only the cluster of the four smallest fragments could not be resolved. As shown on Fig. 11b, one expects double HpaII.AluI digestions to shorten the 355 and 309 bp HpaI fragments down to 320 and 250 bp respectively. This is indeed observed. Hael[[-1,2

~o FoeI

Haell[-3.....4

~o~I

~o

51s .... I 33o 11131

seo

Alul-1

AIuI.2

AluI-3 4

~oo 12oo Atui-5

Fig. 10. Results from mixed HaelII/AluI restriction on purified H13-e fragment and from labelling experiments. Distances are in base pairs. Haell[-1 ~. 980 770

Hae I!1-3._:4

[~o,.i 6~o T,=,~I 309 I 356 HpalI.1 Hpall-5

HpaU.6

N, oo I ~e8 Hpall-7

b

Alu[-1

!=I

Alul-2

515 770

I

Atul-3 .4

Alul-5

330 ["31 sso 1~°° l~l"l~ 3o9 ] 3s6 1168 Hpall.1 Hpalt5

Hpal]-6

Hpall.7

Fig. 11. Results from (a) Mixed HpaIIIHaelII and (b) HpaIIIAluI restrictions on purified H13-e f r ~ e n t . Dktances are in base pairs.

222

[

real

S.7 gb

I

:1

]

i

-

argE

,~. ° ° .

''~

:

iI

L. argC ; '"

, !

IS2-II(-)

Aarg E C I

arg H

arg B

1 15141{~1

152-II,-) •I t ' I

A a r g CB1

, .

I lS~(..,) li

i

A arg Supi02 ,

Hindlll

a

Hinc|l

la)

//

m ~Sal!

/ Hindl m ~Hpal

_"

4.

.-

IB

.lOObp

,"

---

" -2.°.°°

!

4L

J

•

Alul

',

•

Haelll

~

x

Hpall

!

_

/

x x x---

X

-

:7,-- :

lJ

Fig. 12. General restriction map of orgECBH DNA. The HindHI, HineH, SalI and HpaI sites were localized relatively to previously ehazaeterized deletions and insertions carried by ~, d arg phages. HaeHI, AluI and HpaH sites were determined by restriction of purified H13-e DNA fragment and plasmid pMC31 DNA. The relative order of the four smallest HpaII fragment being unknown, the three undetermined targets have been indicated below the line. The most interesting cleavage for nueleotide sequence analysis is at the arrow: either end liberated by that HineH cut can be specifically labelled on two distinct HineII AluI fragments.

obcd

db

Fig. 13. Electrophoretic pattern on 6% polyacrylamide of mixed HineII AluI digests of the DNA from pBR322 (column a) and pMC31 (column b, twice). Column e is a somewhat blurred HindI/AluI digest obtained with the purified H13-e fragment. The arrows both to the left and to the right indicate the two AluI HincII fragments involving the control region of argECBH. H~III digests of ~ 174X DNA are used as molecular weight markers (colunm d): the length of some of the fragments is indicated in base pairs (S~mger et al., 1977).

223 (7) General restriction map. Fragments o f interest for DNA sequencing. All data are summarized in Fig. 12. Regarding DNA sequencing the most interesting cleavage is performed by the HincII enzyme (see arrow). It cuts approximately in the middle of the control region. The regions on both sides of this HincII site can be recovered on two fragments delineated by an AluI and a HincII cleavage site, respectively 210 and 310 bp long. Both can be easily purified in one electrophoretic step on gels made of 6% polyacrylamide, starting with pMC30 or pMC31 DNA (Fig. 13). Work in progress has revealed two other enzymes cleaving in the same area, TaqI and HaeII. (II) Endonucleolytic dissection o f the argF gene Cloning o f the argF gene. pBR322 harbours a unique BamHI site and is thus an appropriate vector for any DNA fragment liberated by this enzyme. DNA from E. coli C600 argF ÷ argI* and from pBR322 were digested with BamHI; the subsequently ligated mixture was used to transform C600 OTC-. Arg÷ transformants displaying high OTCase activity were obtained. Plasmid DNA was purified from one of them (pBR38) and found to have a contour length of about 18 kb. After restriction with BamHI and gel electrophoresis, five fragments were recovered, respectively 8, 4.1, 2, 1.7 and 1.4 kb. To purify further the DNA responsible for OTCase activity a BamHI digest of pBR38 DNA was ligated and used to transform the OTCaseless host again. Ten Arg÷ clones were retained and used for plasmid preparation (pMC20 to 29). After examination of their BamHI digestion products, the DNA of both pMC20 and 23 was shown to be composed of only pBR322 DNA (4.1 kb) plus a 1.66 kb long fragment. Further restriction analysis (see below) demonstrated that pMC20 and 23 differ by the orientation of the latter fragment. The isolated OTCase gene is argF. The DNA of pMC20, pMC23 and of the parent pBR322 plasmid were digested by BamHI, EcoRI, HindIII, P~tI, SalI, BglI and HincII. No cuts were obtained in the BamHI insert with the three last enzymes but well with EcoRI, HindIII and PstI. Following the reasoning developed below, based on the results summarized in Table II (single, double and triple digestions) a restriction map was established for pMC20 and pMC23 (Fig. 14). The pBR322 plasmid bears a single restriction site for each of the enzymes £coRI, HindIII and PstI (Rodriguez et al., 1977). As digestion of pMC20 and pMC23 by those enzymes respectively give 2, 2 and 3 bands, the presence of one EcoRI site, one HindIII site and two PstI sites is indicated in the BamHI fragment. From the length of the fragments obtained, the position of the EcoRI and HindIII sites in the BamHI chromosomal fragment can be determined relative to the known corresponding sites in pBR322 (Rodriguez et al., 1977). On pMC20, the EcoRI site is located very near the left-hand extremity of the BamHI fragment (distance = EcoRI fragment of 0.42 kb minus 0.375 kb EcoRI/BamHI fragment of pBR322).

224 TABLE H RESTRICTION ENZYME ANALYSIS OF argF HYBRID PLASMIDS (VALUES ARE IN

sim0e dilests BamHl EeoRI H/ndHI Pstl

Bsnds~ p M C 2 0 "

....

pMc23

I

4.1

2

1.66

4.1 1.66

1 2 1 2 I 2 3

5.8 0.42 5 0.71 3.1 1.45 1.25

8.75 1.95 4.12 1.61 3.05 1.45 1.32

I 2 3 4 5 1 2 3 4 5 1 2 3 4

3.1 1.16 0.815 0.410 0.290 3.1 1.45 0.79 0.42 b 5 0.385 0.320 c

2 3 4 5 6 7 1 2 4 4 5 6

1.16' 0.785 0.385 0.290 b c ~3.1 d 1.45 0.795 0.385 0.172

Double digests HindlH + h t l

Pstl + E c o R I

HindllI

+

EcoRI

Triple digests HindIII + Pstl + EcoRI

B a m H I + PstI + £ e o R l

7}

b

b -The values for restrictS'on of pMC20 are means o f 2 to § experiments. In those conditionsof gel eleetrophormk, fragments shorter than 100 hase pairs are not o

bedln Rodri~ez et Jd. (1977)

d accuracy.

emmot be determined with

225

pMC20 EcoRl EcoRl Pstl

lHinm

Pstl BamHl

I-

_1

1.66 Kb

--1

i

argF

pMC 23 EcoRl

EcoRl PstlJ'--HI

Pstl

F 1.66 Kb r-

argF.

J

-

Fig. 14. EeoRI, HindIII, BamHI and PstI enzyme restriction maps for two pBR322 hybrid plasmids bearing a BamHI delineated argF chromosomal DNA fragment in opposite orientation. The approximate location of the I kb long DNA fragment sufficient to encode the argF enzyme is indicated; the polarity of transcription is also shown (arrow). The plasmid DNA is drawn opened at the PstI site.

The HindIII site lies 0.37 kb from the same BamHI extremity (HindIII fragment of 0.71 kb minus 0.34 bp of the HindIII/BamHI fragment of pBR322). The position of the two PstI sites in the BamHI fragment can be determined relative to the HindIII site by comparing the HindIII + PstI double digest with the HindIII and PstI single digest (see Table II): summing the lengths of the following couples of fragments from the double digests (0.815 kb + 0.41 kb; 0.41 kb + 0.29 kb; 0.29 kb + 1.16 kb) respectively restore the 1.25 kb PstI fragment, the 0.71 kb HindIII fragment and the 1.45 kb PstI fragment. The relative order of the latter two PstI fragments is thus determined; the overlap between them and the HindIII fragments situates the first chromosomal PstI site at 0.29 kb to ~he left of the HindIII chromosomal site and the second one at 1.16 kb to the right of it. The EcoRI site described above is situated 0.32 kb to the left of the HindIII site; this derives from the existence of a 0.32 kb fragment in the EcoRI/ HindIII double digests and from the following operation: 0.710 kb HindIII fragment + 0,03 kb EcoRI/HindIII fragment of pBR322 -- the EcoRI fragment of 0.42 kb from pMC20. We therefore felt it necessary to establish unam-

226

biguously that the proximal PstI cut was situated only about 30 bp (0.32 kb-0.29 kb) to the right of the EcoRI site. This was done by running in parallel as illustrated in Fig. 15 the following digests of pMC20 DNA: EcoRI, EcoRI + PstI, H/ndIII + EeoRI, HindIII + EcoRI + PstI and HindIII + PstI. Comparison of the bands present in the first two digests (columns a and b) made it clear that the EeoRI fragment of 420 bp was not shortened in the presence of PstI; this should have been the case had the PstI site been located to the left of the EeoRI site. Furthermore in columns d and e of Fig. 15, it can be seen that the HindIH/PstI fragment of 0.41 kb is shortened by EcoRI restriction to a fragment of 0.385 kb while the PstI/HindIII fragment of 0.29 kb (on the BamHI insert; see above) is not modified. In agreement with this result comparison of columns e and f of Fig. 15 shows that the presence of PstI does not modify the mobility of the HindIII/EcoRI fragment of 0.385 kb while it shortens to 0.29 kb the 0.32 kb EcoRIIHindHI chromosomal band.

c

m

abc

de

f

0

1342 1078

872 --. 606 420..-..

310

278) 271

m m

410R

320, 0

Fig. 15. Restriction of pMC20 DNA by EcoRI, PstI and H i n d l I I enzymes taken in various combinations: (a) EcoRI + PstI; (b) EcoRI; (d) H i n d l l I + PstI; (e) H i n d l l I + E ¢ o R I + PstI; (f) H i n d l H + EcoRI. In (e), ~174X DNA digested by HaelII provides reference fragments, the length of these fragments in base pairs, is given in a column drawn to the left of the figure (Sanger et al., 1977). The ~ m e n t s of interest in this experiment designed to establish the relative order of the EeoRI and PstI sites (see text) are shown by arrows and their lengths are indicated in base pairs.

227 In summary the BamHI chromosomal fragments bearing the OTCase gene bear from left to right (polarity of pMC20): an EcoRI site at 50 +- 15 bp followed closely by a first PstI site 22 -+ 8 bp further (those two distances are means of three determinations established by difference using the values given in Table II); next the HindIII site at 0.29 kb from the first PstI site, then a second PstI site at 0.172 kb from the distal BamHI terminus. EcoRI and HindIII digests of phage ~ vectors carrying the E. coli argF and argI OTCase genes have already been described by Kikuchi and Gorini (1976). They found in argF DNA a pair of closely linked HindIII and EcoRI sites (separated by at most 0.5 kb) in a region which was shown to carry the genetic information for OTCase synthesis on the basis of an heteroduplex analysis of ~dargF versus ~dargI phages. This observation has been repeated by Moore and James, 1978, with ~80dargF DNA. Moreover in PstI or BamHI digests of the DNA from ~dargF5 isolated in our laboratory (Legrain et al., 1976b), we have observed the presence of PstI and BamHI fragments respectively 1.45 and 1.66 kb long. The restriction pattern of the argl locus is quite different (Kikuchi and Gorini, 1975). It is thus clear that the present plasmid carries argF. The results of Kikuchi and G~,dni (1975) concerning the orientation of argF with respect to transcriptional polarity, allow us to situate the regulatory elements of that gene in the BamHI fragment described here: they should be located in the region where the EcoRI, PstI and HindIII sites are clustered, thus, as indicated in Fig. 14 at the extreme left of the BamHI insert in pMC20, and at the extreme right in pMC23 (next section). The hybrid plasmida As shown in Table III, OTCase synthesis in pMC20 carrier cells is extremely active (at least 10 times the rate achieved in genetic derepression) and it is but weakly repressible by arginine (24%). Because of the orientation of the fragment in pMC20, the presumptive control region of argF is situated downstream from the Tc promoter (from which resistance to tetracycline is expressed); readthrough synthesis is known to be relatively insensitive to repression exerted at sites distal to a transcription start (Reznikoff et al., 1969; Franklin, 1971). This phenomenon rather than an incomplete argF regulatory region, appears to be the cause of the lowered repressibility. Indeed, (1) a much greater repressibility (69%) is observed with pBR38 carriers (see above), where the same BamI delineated argF DNA plus three unidentified chromosomal fragments are present in the composite plasmid; (2) extensive repressibility (83%) is observed in the case of pMC23, where the argF fragment is oriented opposite to what it is in pMC20 and where OTCase synthesis is also down to the level encoul~tered in pBR38. In pMC23 no readthrough should occur from the Tc promoter. The difference between 83% and the 95% repression characteristic of the wild type (Table III) may be due to an abnormally high ratio between the number of argF copies and the number of repressor molecules.

228 TABLE 111 ENZYMATIC ASSAY OF ORNITHINE CARBAMOYLTRANSFERASE Strains

OTCase activitya after growth on minimal medium

+Apb

% repression

Mean

(%)

÷Apb + ~ e

100 ~glml

c6oo OTC- (pBRSS)

860 1,160

353 265

60 78

69

CS00 OTC- (pMC20)

12,480 5,502 5,411

6,887 3,911 6,790

44 29 0

24

c6oo OTC- (t~C2S)

1,192 745

217 130

83 83

83

18 520 -60

4

a r ~ * ~rl[- a - ' ~ -~ PeTsca (Oear~)d P4X srgF ÷ argI*

380 1.5--2.0

95

aActivity is in/zmoles of eitrulline per hour per mg of protein. bAp. ampic_,,n (25 #g/ml) is added to maintain selective pressure when plasmids are present. CGlanadorff et al. (1967). dLegrainet aL (1976).

From the analysis of argF/argIheteroduplexes performed by Kikuchi and Gorini (1975) it can be concluded that the homology between carriers of the two genes ends at the most 0.4 kb to the left of the EcoRI site in argF. The EcoRI, PstI and HindIII sites are thus favourably located for sequencing the control region of argF. This conclusion is supported by the observation (Cleary et al., unpublished, quoted in Moore and James, 1978) that EcoRI digestion does not abolish the template activity of ~80dargF DNA for cell-free OTCase synthesis, while treatment with HindIII, which cuts about 300 nucleotides further to the right of the EcoRI site, does. DISCUSSION

The physical mapping studies reported in this paper and the availability of new hybrid plasmids bring us to the stage where DNA s~quencing and in vitro studies on well defined fragments have become possible with two loci coding for control elements of the same regulon. The interest of these forthcoming experiments will be enhanced by the availability of control mutants affecting argF (Legrain et al., 1976; mutants of the ancestral argI gene are available also; ibidem; Jacoby and Gorini, 1969) and argECBH(reviewed in Boyen et al., 1978). Parallel analysis of the control region of ¢rgA (T. Eckhardt, pets. comm.)

229

and o f the carAB cluster of genes, involved in the synthesis of an enzyme cumulatively repressed by arginine and pyrimidines (unpublished results of this laboratory) will widen the scope of these studies. The pattern of divergent transcription displayed by the argECBH cluster makes it a particularly relevant subject of investigation regarding problems of structure and origin of regulatory elements. Previous considerations on the structure of the control region, assumed to contain two promoters facing each other over the control region (PcBHO PE ) will now be approached directly thanks to: (1) the possibility of isolating the two AluI-HincII fragments which originate in the middle of the control region and can be recovered easily from digests of the cloned H13-e fragments. The delineation of these fragments with respect to genetic markers (insertions, deletions) with well characterized effects on the regulation of the ArgECBH cluster already pinpoints probable locations for the promoter of argCBH and the operator region (Fig. 12); (2) the possibility of easily transferring the variety of control mutations available on H13-e since cloning of vectors carrying this fragment is readily achieved by selecting for the ArgC÷ phenotype with the appropriate transformable recipient strain; (3) the possibility of examining patterns of RNA polymerase binding and in vitro transcription using subfragments of H13-e: mixed HindIII-HpaI digests of pMC31 DNA give a 0.8 kb fragment containing the whole control region and part of the flanking genes; HincII cuts this fragment between the two facing promoters. The present results also open the possibility of sequencing the region where the secondary promoter is located, in the 80 bp separating the point of insertion of IS1 in mutant argB5 and the right~hand terminus of H13-e. The sequences flanking the IS1 integration site also become accessible to analysis since an extensive restriction map of this element is available (Calos et al., 1978; Grindley, 1978). Regarding the structure o f the argF control region, the recent sequencing in our laboratory (Gigot et al., 1978 and unpublished) of the 55 aminoproximal residues of the argF OTCase will no doubt be useful. The availability of OTCase overproducers like the pMC20 carrier is of obvious interest for the large-scale purification of the enzyme; it may be worth mentioning that OTCase is used beyond academic circles in clinical tests. ACKNOWLEDGEMENTS

This work has been supported by the Belgian F.K.F.O.-F.R.F.C. (Foundation for CollectiveFundamental Research). REFERENCES Allet, B. and Bukhari, A., Analysis of bacteriophage Mu and Lambda-Mu hybrid DNAs by specific endonu¢leases, J. Mol. Biol., 92 (1975) 529--540. Bachmann, B., Pedigrees of some mutant strains of Escherichia ¢oU K-12, Baeteriol. Rev., 86 (1972) 59.5-557.

230

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