Vectors for constructing kan gene fusions: direct selection of mutations affecting IS10 gene expression

Gene. 90 (1990) 135-140 Elsevier

135

GENE 03553

V e c t o r s f o r c o n s t r u c t i n g k a n g e n e fusions: d i r e c t selection o f m u t a t i o n s affecting I S I O gene expression (Recombinant DNA; transposase; transcriptional terminators; translation; antisense RNA; kanamycin resistance)

Jacqueline K. Sussman', Cynthia Masada-Pepe', Elizabeth L. Simons" and Robert W. Simons "b a Department of Microbiology and b Molecular Biology Institute, University of California, Los Angeles. CA 90024 (U.S.A.) Received by G. Wilcox: 6 November 1989 Accepted: 28 February 1990

SUMMARY

We describe several vectors for constructing translational fusions to the kan gene of TnS. Fusions are constructed in vitro using multi-copy vectors containing unique cloning sites situated between upstream transcriptional terminators and a downstream kan gene lacking transcriptional and translational start signals. Multi-copy fusions can be converted to single-copy chromosomal fusions by in vivo recombination with specific phage ~ vectors and vice versa. We find that/can fusions are often more suitable than lacZ fusions for the direct selection of mutations that increase fusion expression. These vectors were developed for isolating mutations that increase ISIO transposase expression; we describe strategies used to isolate such mutations, which map to IS10 or the Escherichia coil himA, hireD(hip), dam or infC genes.

INTRODUCTION

Genetic fusions are widely used for the analysis of gene expression and regulation in prokaryotes (Silbavy and Beckwith, 1985). For transcriptional fusions, a promoter of interest can be fused to a variety of reporter genes. For

Correspondence to: Dr. R.W. Simons, Department of Microbiology,5304 Life Sciences, University of California, Los Angeles, CA 90024 (U.S.A.) Tel. (213)825-8890; Fax (213)206-5231. Abbreviations: Ap, ampicillin; pGal, p-galactosidase; bla, /~.lactamase-encoding gene ofpBR322; bp, base pair(s); EOP, efficiencyof plating; IS, insertion sequence;/can, Km resistance-encoding gene of TnS; kb, kilobase(s) or 1000 bp; Km, kanamyein; LB, Luria-Bertani broth; MCI, multicopy inhibition; mci, MCI-defective; nt, nucleotide(s); lacZopUVS, lac operon operator and promoter carrying the UV5 mutation; or/, pBR322 plasmid origin of replication; p, promoter; R, resistant; s sensitive; Ti, transcriptional terminator from E. coli rrnB operon; Ti4, four tandem copies of 71; wt, wild type; Tn, transposon; XGal, 5-bromo-4-chloro-3-indolyl-p-D-galaetopyranoside; '(prime), gene truncation at the indicated side; *, altered specificity. 0378-1119/90/$03.50© 1990Elsevier SciencePublishers B.V.(BiomedicalDivision)

protein (translational) fusions, where the N-terminal portion of a gene of interest is fused in-frame to a reporter gene, the E. coli lacZ gene is most often used, primarily because its product,/~Gal, is relatively insensitive to an additional protein sequence at its N terminus. We have used lacZ protein fusions extensively in our studies on regulation of ISIO transposase (tnp) expression (Simons and Kleckner, 1983; Case et al., 1988; 1989; 1990). However, for two reasons we sought to exploit translational fusions to an alternative reporter gene. First, direct selection for increased tnp'-'lacZ expression is often difficult or impossible because the fusion is already phenotypically Lac +. Second, we envisioned mutant isolation strategies involving two different tnp protein fusions in the same cell: one to 'lacZ and the second to another reporter gene. The aim ofthis study was to devise improved multi-copy vectors for constructing protein fusions to the kan gene of Tn$. Such fusions would be sensitive enough to detect very low levels of gene expression, but also able to distinguish high levels of expression, and be easily converted to single copy and vice versa.

136 EcoRIc

tBuHl

Nbel

pRS1292 Et'dl

pRS1272 8~HI, I~CC

pRS1327

Nhel

BMHI

'aT-

tEt'oltl

s

---

#hal

G G G ~ C 8AT CCG6CCJ U W ~

pP,$1341

B-HI ~"~'~ . , ~

I

[

'/(an ---> 2 3

'/ran ---:,. 3 TGGMT 6AA

£c~I Nhel I~'M"T-~ SAT¢CGGCCAA,G f " " ~ " ~ . . . j

bin

I '

I

Sail

I

8gill 0.9

s.s/o

pRS1292A pRS1327A

lacA &sd41

I Ba~l

1.2 kb

Ikwology cassette A

I

(from pRSISES)

Sell

pRS1292B ~ ' ~ pRS1327B

[

I

Sill

i.d, Honto]ogy cassette S (from pRS1322)

1 Sail

Fig. 1. Multi-copy '/can vectors. Structures and partial restriction maps are shown. Plasmids pRS1292 and pRS1327 contain four unique cloning sites upstream from '/can(the second and third codons of native/canare indicated), pRS1272and pRS1541 are pRS 1292 and pRS 1327 with laeZopUV$ (large arrow). Other derivatives contain 'homologycassettes' for transferring fusions to phage vectors (see Fig, 2). pRS 1292 was constructed from pKM 109-9 (Reiss et ai., 1984),which contains the/cangene with its first eodon replaced by a BamHl site (and unique downstream Sinai and Sail sites): we converted the Smal site to Bglll (pKMI09-9 S/B) and combined the 1.2-kbBamHl-$alI 'kan fragment with the 4.5-kb Sall-BamHI backbone from pRS415. pR$1327 is pKMI09-9 S/B with the BamHI site converted to EcoR! and the 1.2-kbEcoRI-Sall 'kan fragment combined with the 4.5-kb SalI-EcoRI plasmidbackbone from pRS$28.The A and B derivativeswere constructed by inserting'homologycassettes' A or B (FrompRS 1326or pRS 1322)between the Bglll and Sail sites, pR$13Z2contains the BamHl.$all la¢ fragment from pMCI633 (M. Cnsadaban, pers. commun.) + the SalloBamHl backbone of pRS591, with subsequent replacement of sequence between EcoRl in lacZ and a downstream Sail site with a SalI linker, pRS1326 was constructed by replacinglac sequence betweenthe BamHl and the distal Clal site in pRS415 with a BamH[ site. pRS4tS, pRS528 and pRS591 have been described (Simons et al., 1987). Plasmid sequences, which can be inferred from published sources, and details of constructions will be provided with all requests for vectors.

TABLE I Multi-copy ApR plasmids for constructing 'kan protein fusions Plasmida

Cloning sites a

Homology cassette b

Plasmid size (kb)

Max. insert for transfer to ,1.(kb) c

pRS 1292 pRS 1327 pRS 1 2 7 2 pRS 1 3 4 1 pRS 1292A pRS 1292B pRS 1327A pRS 1327B

RI-Sm-Bm-Nh Bm-Sm-RI-Nh Rl-lacZopUVS.Bm-Nh Bm-lacZopUVS-RI.Nh RI-Sm.Bm.Nh RI-Sm-Bm-Nh Bm-Sm-Rl-Nh Bm-Sm-RI-Nh

none none none none A B A B

7.1 7.~. 7.2 7.2 9.9 11.0 9.9 11.0

na na na na 7.1 2.9 7.1 2.9

a See Fig. 1. RI, EcoRl; Sm, Sinai; Bin, BamHl; Nh, Nkel. b See Figs. 1, 2 and sections a, b. Values are estimates ofthe maximumsize of Fragrnentsthat can be cloned in the plasmid ~'~ors and subsequentlycrossed to 2RS45 or ~RS74 (Simons et al., 1987). ha, not applicable.

137

EXPERIMENTAL AND DISCUSSION

(a) Description

gm.;egteA

of multi-copy plasmid vectors for

constructing 'kan fusions The vectors developed are modifications of a kan-based vector (pgM109-9) previously described by Reiss et al. (1984). The structure ofthe vectors is shown in Fig. 1 and their essential features arc summarized in Table L Plasmids pRS1292 and pRS1327 contain the following elements (clockwise): or/and bin of pBR322 (Sutcliffe, 1979) for selecting Ap R transformants; T I 4 to block upstream transcription (Brosius et al., 1981; Simons et al., 1987); unique cloning sites (EcoRI, Sinai, BamHI and NheI) for constructing fusions; and kan lacking transcriptional and translational starts ('/can). pRS 1272 and pRS 1341 have the EcoRI-BamHI fragments in pRS1292 and pRS1327 replaced by lacZopUVS. Such plasmids titrate the lac represser in a lac + host, thereby conferring blue colony color on LB + XGal plates. This simplifies identification of :onstructs in which iacZopUV5 has been replaced by an insert of interest. The other vectors shown in Fig. 1 are derivatives in which segments of the lac operon are inserted downstream from '/can for use in deriving single-copy fusions (see section b).

(b) Transferring prophages

multi-copy

fusions

to

single-copy

It is often desirable to transfer 'kan fusions from multi-copy plasmids to the E. coli chromosome. This usually results in lower fusion expression, which may improve the selection of mutants, and permits the use of plasmids that are incompatible with the 'kan plasmids. Without regard to their level of Km R, fusions constructed with the multi-copy 'kan vectors can be transferred to specific ~. phages by genetic recombination in rive, and the recombinant phages integrated as single prophages in the host chromosome (Fig. 2). Transfer is accomplished by homologous recombination at sequences flanking the fusion construct (bla upstream and lac downstream); lac homology is provided by a 'cassette' (Fig. 1; Table I). Two different cassettes (A and B; Fig. 1) were designed, with different recombination strategies in mind (Fig. 2). When plasmids with cassette A (Fig. 2A) are crossed with ZRS74 (Fig. 2B), the desired recombinant phage (Fig. 2C) is isolated from white plaques. When plasmids with cassette B (Fig. 2D) are crossed with ZRS45 (Fig. 2E), the desired recombinant phage (Fig. 2F) is isolated from blue plaques. The choice of strategy depends on the desired phenotype of the resulting recombinant phage: blue plaque-forming phages are easier to isolate but complicate the use of other lac-based elements. In either case, the recombinant phage will he Ap s, permitting subsequent introduction of Ap R elements. Single-copy fusions can be easily recovered by

A

!

°ku fusion vtth cassette A (uhSte. Apt)

/

X

B

i

Plmge ~$74 (btue, ~ )

I

C bcml~.: ~.w, (uhtte, ApS)

Cassette 8 D

•kancassette fus|on II with

I:"

PhaSe~ss4s

F

' e (blue, c m ~ ' ApS) uent''

~

J

t-

~

IL-..~I 'kue Yuslue ~

h~.,

(whtte, Aps)

,,..,

|~cZ"

,--.

,_7

~--

--"

I

pSSl~

~

'kon fusion

~

Fig. 2. Transferring fusions from plasmid to phage and vice versa Strategies for plasmid.to.phage (A-F) phage-to-plasmid (F-G) transfers are shown (see also Fig. ! and section b). Boxes, genes or parts thereof; thin lines, plasmid backbone DNA; double lines, phage DNA; heavy lines, putative recombination arms; bin, the proximal half of bin (bin is oriented right-to-lelt in all elements); lacZ', deletion of terminal ~ 50 bp of lacZ ; 'lacZ, distal one-third of lacZ ; ' Y, distal half o f lacg; °A, distal 90% oftrpA (which contains an uncharacterized promoter; Simons et al., 1987). pRS308, ~1RS45 and ~RS74 (originallyconstructed for transferring 'lacZ-based fusions) and all procedural details are described by Simons et al. (1987).

recombination with plasmid pRS308 (Simons et al., 1987; Fig. 2G). Detailed procedures for plasmid/phage transfers have been described (Simons et al., 1987). (©) Properties of protein fusions to the 'kan gene E coli is naturally resistant to ~ 1 pg Km/ml, and none of the '/can vectors increases Km x beyond 2 pg/ml (Table II). However, when an insert containing a gene of interest is cloned into one ofthese vectors such that a protein fusion is formed, increased Km R is usually detected, even with poorly expressed genes. The properties are illustrated by fusions to the IS10 tnp gene (Table II; Fig. 3). A tnp'-'lacZ fusion (pRS556) expresses only 40 units of/~Gal, which is insufficient for a Lac + phenotype. Nevertheless, the corresponding tnp'-'kan fusion (pRS967) confers 6 #g/mi Km R. Mutations (mci30 and/or HHI04) that increase the activity of the tnp promoter, piN, have coordinate effects on tnp'-'kan and tnp'-lacZ expression (Table II, lines 3-6). Transfer of '/can fusions to single copy decreases Km R ~, 20-fold, as expected from the ~ 20-fold decrease in copy number (cf., pRS1088 and ~.pRS414). Thus, Km R can be

138 TABLE II Properties of '/{an vectors with and without inserts and comparisonwith fusions to the 'lacZ gene ISIO genotype"

! 2 3 4 5 6 7 8 9 10 11

(no insert) (lacZopUV5) R5 mciSO R5 HHI04 R5 mci30 HHI04 HHI04 HHI04 HHI04 (no insert) HHI04 R5

Amount of mp gene (codons)~

mp'-'lmn fusions Elementb

Km~t (pg Km/ml)c

Elementb

/]Gal (units) [Lac+/- ] d

-~ 75 75 75 75 25 75 166

pRS1292 pRS 1272 pRS967 pRSI087 pRSI088 pRSI323 pRSI362 pRS1328 pRS1353 J.RS434 ~.RS414

=1 ~2 6 70 260 >350 170 130 80 1 15

pRS414 pRS476 pRS556 pRSI325 pRS993 pCI291 pRSI301 pRS709 pRSI352 ,tRSI94 ~RS382

<0.01 20 40 565 1560 14525 1450 1235 420 <0.01 20

--

75

mp'-'lacZ fusions

[- ] [- l [+ ] [+] [+ ] [+] [+] [+] [+] [- ] [- ]

" R5 abolishes MCI; mci30 and HHI04 increase piN strength (Case et as., 1988; 1989). b See Figs. ! and 3 and Simons et ad. (1987). ARS434, ARS414, ARS194 and ARS382 were obtained by crossing ARS45 with pRSI292B, pRSI356 (ffi pRSI344 + cassette B), pRS552 and pRS993. c Km~testimated as the #g Km/ml resulting in ~ 50% EOP in E. coil strain DR459 [AlacX74 galOP308 rpsL trpR A(tonB-trpA)905]; in each case, twice the 50% EOP concentration reduced EOP> 100-fold. d /]Gadassayed in DR459 according to Simons et al. (1987); symbols - , +_ and +, no, slow, and normal growth on minimal lactose plates, respectively. c N.terminal codons.

distinguished over a broad range of promoter strengths (360-fold) and Km concentrations (6-350/zg/ml). Reiss et as. (1984) reported that protein sequence fused to the N terminus of 'kan influences the level of Km R. However, we find that the amount of tnp gene included in A ].o.,,eh,

pin

I

I

Sglll

OR

.~

I

I

pOUTA/el

i

-2|0

__ ----

tnp

m mi . , . . ~

lSl

I

Mel

?lcol

336

606

tnp'-'kan and tnp'-'lacZ fusions affects Km R and/~Gal expression equally (Table II, lines 7-9), suggesting that this is not a general problem. (d) Translational control of trip'.' kan fusion expression The tnp gene is controlled at a number of levels including inhibitionof translation by an antisense RNA (RNA-OUT; TABLE Ill Anti-sense RNA control of tnp°.'kan fusion expression Fusion plasmid or phage a

B

Multi.copy ISIO element in trans (genotype) b

Fusion expression [Lac +/- ] c

(AS Multi.copy fusions pRS555 wt pRS556 R5 pRS832 wt pRS967 R5

none none none none

1 40 2 6

(B) Single-copy fusions ~RS382 HHI04 R5 ~RS382 HHI04 R5 ~RS382 HHI04 R5 ~.RS414 HHI04 R5 ,~RS414 HHI04 R5 ~.RS414 HHI04 R5

none pRS449 (wt) pRS450 (RS) none pRS449 (wt) pRS450 (RS)

20 units/]Gal [ - ] 0.5 units/]Gal [ - ] 18 units ~Sal [ - ] 15 #g Km/ml 3 Izg Km/mi 8 #g Km/mi

Prete4n Tuslons to Zr~

trtsmnt

Lensth (bD)

NO. of coootnPn s

~|II-A|ui

401

~tll-Accl

S|6

~ilI-Neoi

826

Number IHO senot~e

tnp'-'k|n pluMds

tnp'-'llcZ pltsmtds

|S

HH|04

pRSIS6|

pRS|S01

26

M; RS mcf30 AS

pRS83~

pRS|S7

1C6

////104 ~ mcf3OMVI04

HlllOS

pRS1087 p~t328 ~ l ~ 8 t 1344 pSSiSSS

pRSSES pRSBS6 pRS]S2S p~70S pUSSS I~J2gl

~04

~$1353

pl~lSS|

Fig. 3. ISIO and tnp'.'iaeZ fusions. (A)The outer ~650bp of ISI0-Right (ISI0) are depicted with a partial restriction map in bp from the outside end ((DE). p i n and pOUT specify RNA-IN and RNA-OUT. (B) Protein fusions to th( tnp gene. All plasmids were constructed by inserting the indicated l~agments into '/can or 'lacZ protein fusion vectors; tnp'-'kan fusions were constructed with pRS 1272 or the closely related pltS735; mp'-'iacZ fusions were constructed with pRS414 or pRS577 (Simons et as., 1987). Plasmids on the same line have isogenic fusion junctions. The R5 mutation abolishes MCI; mci30 and HHI04 mutations increase piN strength ~15- and ~100-fold, respectively (Case et el., 1988; 1989).

ISlO genotype

units/]Gas[-] units/]Gal [ + ] /Jg Km/ml ~g Km/ml

a See Fig. 3 and Table II, footnotes a and b. b pRS449 (wt) and pRS450 (RS) are multicopy plasmids containing the outer 345 bp of ISIO (Case et as, 1989). See Table II, footnotes c and d.

139

Fig. 3A) (Simons and Kleckner, 1983; Case et al., 1989; Ma and Simons, 1990). This control (MCI) is reflected in the inhibition of both tnp'-'lacZ and tnp'-'kan fusion expression. The R5 point mutation severely destabilizes RNA-OUT in vivo and essentially abolishes MCI (Case etal., 1989), increasing both tnp'-'lacZ and tnp'-'kan fusion expression (Table IliA). Table IIIB shows that a multi-copy ISIO plasmid (pRS449) inhibits both single copy tnp'-'lacZ (~RS382) and tnp'-'kan fusions (~RS414), and that R5 (pRS450) alleviates inhibition. These results show that 'kan fusions reflect translational control. (e) Isolation of linked and anlinked mutations that increase tnp'-'kan fusion expression We have used tnp'-'kan protein fusions to select or screen for mutations that increase tnp expression 4-200-fold. In one simple approach, pRS967 (Table II) was mutD-mutagenized (Case et al., 1989) and transformants selected on LB Ap Km (10 pg/ml) media: colonies arose at ~10 -3 and >90~o contained ISIO mutations that increase piN activity. Selection for increased tnp'-'lacZ expression from the corresponding pRS556 plasmid is made difficult by its Lac ± phenotype (Table II).

A

[ ~

ISlOon plasmtd I~

pHS449

M: B liCl)i

~RS414

pRSS3!

~ C

'-'lacg

9 O O

ISJO on

plasMd I~ ectCl

2 6 .,2

Brim1 O.S 22 18

XRS382 m

~'-'k,,n

~__~'-'llc.Z Host Senotype If~ ~; 2

~81

dm ~nfC

45 18

hl~ Itfp

4 4 iS S

]" IgSg3/

l.S S S

Fig.4. Strategiesfor isolatingmutationsthat increasetnp'-'kan fusion expression. See sectione. pRS837 was constructedby combiningthe tnp'-'lacZ and tnp'-'kan fusions from pRS55S and pRSG32 (Fig.3) ArrowsindicateISIOpromoters:thindghtward,wt piN; thickleltward, wt pOUT; thick rightward,HHI04 R5 piN; mcill abolishespOUT (Case et al., 1988).

Fig. 4A shows a strategy for selecting mci- mutants. Expression from ~.RS414 is subject to MCI by pRS449 (Table IIIB). When pRS449 was mmD-mutagenized and transformants of the ~RS414 lysngen selected on LB Km (10pg/ml) plates, colonies arose at ~10 -3 and > 8 5 ~ contained mutations in pRS449 that alleviate MCI. Selection with the corresponding mp'-'/acZ fusion (,~RS832) does not work; it remains Lac- in the absence of MCI (Table IIIB). Fig. 4B shows a strategy for isolating mutants in which the specificity of MCI is altered (mci*). Expression from 3RS382 (HHI04 R5 tnp'-'lacZ) is subject to MCI (Table IIIB), in this case by a wt mp'-'kan fusion plasmid (pRS832) which is itself under MCI (Table IliA). pRS832 was mutD-mutagenized and transformants of the ~RS382 lysogen selected on LB-Ap-XGal plates, screened for increased tnp'-' lacZ expression, and then tested for KmR. Most had increased KmR ( ~ 6 #g/ml) and contained reelmutations in pRS832; a rarer class did not exhibit increased Km a and contained mci* mutations in pRS832 (e.g., mciCl). In other words, the mci* mutants control the tnp'-'kan fusion but not the tnp'-'lacZ fusions. Such altered specificity mutations have been characterized (Kirtle et al., 1989). Finally, Fig. 4C illustrates a selection for E. coh" mutations that increase tnp expression, pRS837 contains tandem wt tnp'-'kan and mp'-'lacZ fusions and confers resistance to ~ 2 # g Km/ml and expresses ~ 1.5 units of pGal. Without mutagenesis, transformants were selected on LB XGal plates containing 4-12 #g Km/ml and then screened for increased tnp'-'lacZ fusion expression (blue). Genetic experiments (not shown) revealed that these isolates contained mutations in E. coil genes: (i) mutations in dam (DNA-adenine methylase) increase transcription from piN (Roberts et al., 1985); (ii) mutations in him,4 or hireD(hip) [subunits of integration host factor (IHF); Friedman, 1988] also increase p i n activity suggesting that IHF represses thepIN promoter (see also Kur et al., 1989); (ill)mutations in infC (translation initiation factor III; Springer et al., 1979) appear to increase translation (J.K.S. and R.W.S., manuscript in preparation). This strategy is efficient and illustrates the usefulness of dual fusions.

ACKNOWLEDGEMENTS

We thank Chaitanya Jain for strains. J.K.S. and C.M.-P. were supported by Predoctoral National Research Service Awards. R.W.S. was supported by a Junior Faculty Research Award from the American Cancer Society. This research was supported by a USPHS grant to R.W.S. (GM35322).

140 REFERENCES

Brosins,J., Dull, TJ., Sleeter, D.D. and NoUer, H.F.: Gene organization and primary structure of a ribosomal RNA operon. J. MoL Biol. 148 (1981) 107-127. Case, C.C., Roels, S.M., Simons,E.L. and Simons, R.W.:Analysisof the promoters and transcripts involvedin ISIO anti-sense RNA control. Gene 72 (1988) 219-236. Case, CC., Roels, S.R., Jensen, P., Lee, J., Kleckner, N. and Simons, R.W.: The unusual stabilityof the ISIO anti-sense RNA is critical for its function and is determined by the structure of its stem domain. EMBO J. 8 (1989) 4297--4305. Case, C.C., Simons, E.L. and Simons, R.W.: The ISIO transposase mRNA is destabilized during antisense RNA control. EMBO J. 9 (1990) 1259-1266. Friedman, D.I.: Integration host factor: a protein for all reasons. Cell 55 (1988) 545-554. Kittle, J.D., Simons, R.W., Lee, J. and Kleckner, N.: ISIO anti-sense pairing initiates by an interaction between the 5' end of the target RNA and a loop in the anti-sense RNA. J. Mol. Biol. 210 (1989) 561-572. Kur, J., Hasan, N. and Szybalski, W.: Physical and biological consequences of interactions between the integration host factor (IHF) and the coliphage lambda late p~ promoter and its mutants. Gene 81 (1989) 1-15.

Ma, C. and Simons, R.W.: The ISIO antisense RNA blocks ribosome binding at the transposase translation initiation site. EMBO J. 9 0990) 1267-1274. Reiss, B., Sprengel, R. and Schaller, H.: Protein fusions with the kanamycin resistance gene from transposon Tn5. EMBO •. 3 (1984) 3317-3322. Roberts, D., Hoopes, B.C., McClure, W.R. and Kleckner, H.: IS10 transposition is regulated by DNA adenine methylation. Cell 43 0985) 117-130. Siihavy, TJ. and Beckwith, J.R.: Uses of lac fusions for the study of biological problems. Microbiol. Rev. 49 (1985) 398-418. Simons, R.W. and Kleckner, N.: Translational control of ISIO transposition. Cell 34 (1983) 683-691. Simons, R.W., Honman, F. and Kleckner, N.: Improved single and multicopylac-based cloning vectors for protein and operon fusions. Gene 53 (1987) 85-96. Springer, M., Graffe, M. and Grunberg-Manago, M." Genetic organization ofthe E. coli chromosomearound the structural gene for initiation factor IlL Mol. Gen. Genet. 169 (1979) 337-343. Sutcliffe, J,G.: Complete nucleotide sequence of the Escherichia coil plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol, 43 (1979) 77-90.