and host strains: nucleotide sequences of the …Three kinds of improvements have been introduced...

103 Gene, 33 (1985) 103-l 19

Elsevier

GENE 1167

Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

(Recombinant DNA; molecular cloning; polycloning sites; progressive deletions)

Celeste Yanisch-Perron, Jeffrey Vieira and Joachii Messing

Department of Biochemistry, University of Minnesota, St. Paul, MN 55108 (U.S.A.) Tel. (612) 376 1509

(Received July 27th, 1984) (Accepted September 21st, 1984)

SUMMARY

Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichiu coli host strains have been constructed for the E. coli bacteriophage M 13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M 13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) Ml3 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.

INTRODUCTION

Single-stranded DNA isolation has been facilitated by the properties of the single-stranded bacteriophage Ml3 (Messing et al., 1977). Though it is not a naturally transducing system, recombinant DNA techniques have been used to construct a

Abbreviations: AC, activator; Ap, ampicillin; B-broth, Bacto- tryptone broth; Cm, chloramphenicol; A, deletion; DTI, ditho- threitol; EMS, ethylmethane sulfonate; Exo III and VII, exonuclease III and VII; HA, hydroxylamine hydrochloride; IPTG, isopropyl-B-D-thiogalactopyranoside; LB, Luria broth; M13UC, see RESULTS, section c2; moi, multiplicity of infection; pfu, plaque-forming units; PHS, primer hybridization site; R, resistance; RF, replicative form; RT, room temperature; Sm, strepto- mycin; STE, 10 mM NaCI, 10 mM Tris . HCL pH 7.5, 1 mM EDTA; Tc, tetracycline; Xgal, 5-bromo-4-chloro-indolyl-/?-n-ga- lactopyranoside; YT, yeast tryptone; [I, indicates plasmid- carrier state; A, deletion.

0378-l 119/85/$03.30 0 1985 Elsevier Science Publishers

general transducing system where double-stranded DNA can be introduced into the double-stranded RF of the phage. Upon transfection of appropriate host cells, the DNA strand ligated to the ( + ) strand ofthe RF is strand-separated, packaged and secreted without cell lysis as a recombinant single-stranded DNA phage.

Although inserts seven times longer than the wild- type viral genome have been cloned in Ml3 (Messing, 1981), the presence of large inserts can cause deletions. Accelerated growth of phage containing smaller inserts that arise from deletions makes maintaining large-fragment clones difficult (Messing, 1983). Differentialgrowth can be observed in the plaque-size variety that results from infection of Ml3 clones possessing inserts of > 2000 bp.

With the introduction of a universal primer (Hei- decker et al., 1980), Ml3 was used primarily for

104

the subcloning or shotgun cloning of small fragments (for review, see Messing, 1983). Cloning of 10000 bp fragments now appears possible with deletions occurring less frequently than previously predicted. One of the factors interfering with the cloning of large fragments is the restriction of unmodified DNA. JM103, a restrictionless host strain, was developed to circumvent this problem (Messing et al., 1981), but later lost the mutation (Felton, 1983; Baldwin, T., personal communication, C.Y.-P. and J.M., unpubl.), and instead carried a Pl lysogen which also contains a restriction and modification system.

This paper describes the construction and characterization of a number of new strains developed for use with the Ml3 and pUC cloning vectors. Each strain carries a specific set of mutations that help prevent various cloning problems. A complete

TABLE I

E. coli strains and genotypes

E. coli strain

JM83

JMlOl

JM105

JM106

JM107

JM108

JM109

JMllO

DHl

GM48

SLIO

TDl

MC4100

SK1592

71-18

Genotype

ara, A(lac-proAB). rpsL( = strA), @O, lacZAMl5

supE, thi, A(iac-proAB), [F’, traD36, proAB,

IacIqZAMlS]

thi, qrsL, endA, sbcB15, hspR4, A(lac-proAB),

[F’, traD36,proAB. iacZqZAM15]

endA 1, gvA96, thi, hsdR 17, supE44, reiA 1, I - , A(lac-proAB)

ena2 1, gyrA96, thi. h.sdR 17, supE44, reiA 1,1-,

A(lac-proAB), [F’ , traD 36, proAB,

iacIqZAMlS]

recA1, endAl, gvrA96, thi, hsdR17, supE44,

reiA 1, A(lac-proAB)

recA1. endAl, gyrA96, thi. hsdR17, supE44,

relA1, I-, A(iac-proAB), [F’, traD36,

proAB, iacIqZAM15]

rpsL. thr, leu, thi, lacy, galK. galT, ara. tonA. tsx.

dam, dcm, supE44, A(lac-proAB), [F’,

traD36,proAB, lacIqZAMlS]

F -, recA 1, ena2 1, gyA96, thi, hsdR 17, supE44,

reU1,1-

thr. leu. rhi, lacy, galK, galT, ara, tonA, tsx. dam,

dcm, supE44

Hfr H, thi. sup”. A(iac-proAB), galE, A(pgl-bio)

MC4100, recA56, srlC300::TnIO

araD, rpsL, thi, A(iacIPOZ YA)U 169

thi, supE, enaX. sbcB 15, hsdR4

A(lac-proAB). ihi, supE, [F’, proAB.

lacIqZAM 151

listing of M13mp18 and pUC19 sequences and restriction sites is also presented. The M13mp sequence was compiled from published data (Van Wezenbeek et al., 1980; Messing et al., 1977; 1981; Farabaugh, 1978; Dickson et al., 1975; Kalnins et al., 1983; Messing and Vieira, 1982; Norrander et al., 1983). Re-sequencing of pUC was necessary, as a number of mutations had been introduced into the pBR322 region of the pUC vectors. A new method of generating unidirectional deletions from a full-length Ml3 clone of pUC6 by Exo III and VII was used for this purpose. The results have been combined with those published earlier (Ruther, 1980; Vieira and Messing, 1982; Rubin and Sprad- ling, 1983; Stragier, P., personal communication).

MATERIALS AND METHODS

(a) Strains

The bacterial strains listed in Table I are E. coli

K-12 derivatives. SK1592 was obtained from Sidney Kushner, DHl from Jurgen Brosius, GM48 and HBlOl from Raymond Rodriguez, and SLlO and TDl from James Fuchs. Phages lb2c were from Bruno Gronenbom, and f2 from David Pratt. Strains were tested for relevant markers by standard methods and as described below.

(b) Maintenance of strains

Long-term storage of desired strains was accomplished by mixing 1 ml of a stationary-phase culture with 1 ml of glycerol and freezing at -70’ C. Bacteria were revived by streaking aliquots on appropriate selective media and incubating at 37°C. Short-term working strain stocks were maintained at -20°C. Bacteria were revived by inoculating 10 ml of broth with 0.05 ml of the stock and shaking overnight at 37 ‘C. Alternatively, strains containing the F’ from JMlOl were maintained on glucose minimal plates for 2-4 weeks at 4°C.

(c) Media

Bacterial strains were grown in 2YT or LB broth (Miller, 1972) supplemented with 15 pg/ml Tc,

105

500 pg Sm/ml, or 50 pg/ml Ap when required. Bac- teria were plated on M-9 minimal plates (M-9 mini-

mal medium plus 1.5% agar; Miller, 1972) and supplemented with the following as required (per ml): 1 pg thiamine, 40 pg nalidixic acid, 20.1 pg 5-fluorocytosine, 40 pg amino acids, 12.5 pg Cm, and 5OOpg Sm. Fusaric acid medium (Tcs broth plus 1.5% agar; Maloy and Nunn, 1981), B-broth medium (Miller, 1972) supplemented with 0.3% yeast extract and 1.4% agar, 1XA medium (Miller, 1972), or MacConkey plates were also used. B-broth plates were supplemented with 0.1 mM IPTG and 0.004% Xgal per plate. The Xgal and IPTG were stored as 2% and 0.1 M stocks, respectively.

(d) Reagents

Tc, Sm, Cm, Ap, amino acids, 5-fluorocytosine, nalidixic acid, chlorotetracycline, and IPTG were obtained from Sigma. Agar, MacConkey agar, yeast extract, and Bacto tryptone were obtained from Difco. Boehringer Mannheim supplied the Xgal. Restriction endonucleases were provided by Amersham, Bethesda Research Labs, New England Biolabs and PL Biochemicals and used as recommended by their suppliers.

1983; Cohen et al., 1972) was modified by using

50 mM CaCl, and cell harvest at A55,,nm 0.550-0.720 (0.800-0.890 for JM109). Transforma- tion by M 13 RF DNA was as described by Hanahan (1983) and Cohen et al. (1972) except that the heat- shocked cells and DNA were added to B-broth top agar, IPTG, Xgal, and 0.3 ml of host cells grown to t3IlA 550nm of 0.720-1.200 (JM109 to 1.200-1.8). No antibiotic amplification period was required for RF DNA. Transformation of each strain were repeated live or more times.

(i) RF and plasmid DNA preparations

pUC plasmid DNA was prepared by inoculation

to an &.0nm of 0.050 of 2 x YT medium plus Ap with an overnight culture of the plasmid-carrying host strain. The culture was shaken 8-13 h at 37°C before cell harvest. Cm amplification was not required of the multicopy pUC plasmid. RF DNA was prepared by inoculation of 2 x YT medium to CUlA 550nm of 0.05. At A550nm 0.300-0.420, phage supernatant was added to an moi of 10: 1, and the culture was shaken 8-13 h at 37°C. DNA was extracted by the Birnboim and Doly method (1979) and purified on CsCl gradients as described by Messing (1983).

(e) Transductions

(i) Marker tests

Lysates of P 1Cm 1, clr 100 and P 1 vir were prepared by heat induction of lysogenic cells (Miller, 1972). Transductions were performed as described by Miller (1972).

(f) Matings

Matings using F’ and Hfr strains were conducted as described by Miller (1972).

(g) Curing of transposon

Elimination of the Tnl0 transposon was accomplished via selection on Tcs media (Maloy and Nunn, 1981).

(h) Transformations

Preparation of competent cells for transformation by pUC plasmid and Ml3 RF DNA (Ham&an,

The restriction-minus and modification-plus phenotypes were tested by plating 0.1 ml of overnight cultures resuspended in 1XA buffer plus 0.01 M MgSO, on B-broth plates. 10 ~1 of the appropriate phage ilb2c dilutions (either K-12 modified by two serial propagation cycles through JMlOl or K-12 unmodified by two serial propagation cycles through HB 10 1) were spotted on the plates. Plaques were counted after overnight incubation at 37 ‘C. To confirm the modification-plus phenotype, an HB 10 1 lb2c lysate (K-12 unmodified phage) was propagated in the questioned strain for two serial cycles with dilutions of the resultant phage lysate plated on JMlOl and HBlOl. The HBlOl lysate was used as a control. No difference in titer was correlated with a modification-plus phenotype.

Phage M13mplOa containing amber mutations in genes I and II (Messing and Vieira, 1982) and M13mplOw with no amber mutations (Norrander

106

et al., 1983) were tested for the presence of the suI1 suppressor and the F episome that possesses mutation IacZq and deletion lucZAM15. The presence of the suI1 suppressor in F - strains was confirmed by infection with amber phage T4amN130- N82. Tests confinning the lucZq and lacZAM15 deletions via blue plaque production required media supplemented with Xgal and IPTG as described (Messing et al., 1977).

Phage t2 tested for the presence of the traD36

mutation on the F episome. Unlike M13, the traD gene product is required for phage t2 propagation (Achtman et al., 1971). Phage Q’s inability to infect an F’ strain conformed presence of the NIH-recommended traD36 mutation on the F’.

The recA mutation was tested by streaking cells on M9 minimal plates and irradiating with a wavelength of 254 nm for 90 s at a distance of 22.5 cm (hand- held UV lamp model UVGL-25 from UVP, Inc. of San Gabriel, CA). As a control, half the agar plate was masked with a paper card during UV exposure. Cells were then incubated overnight at 37°C. UV- resistant growth indicated absence of the recA mutation.

The dam and dcm mutations in GM48 and JMl 10

were confirmed by propagating pUC plasmids or M 13 phage in these strains for at least two overnight culture cycles, isolating the DNA as described above, and cleaving the DNA with either Mb01 or EcoRII. Mb01 cleavage correlated with the dam mutation and EcoRII cleavage with the dcm mutation. Sau3A cleavage served as a control.

(k) Construction of JM105

Spontaneous SmR mutants of strains SK1592 were selected for on minimal media plates containing high concentrations (500 pg/ml) of Sm. A chromosomal A(lac-proAB) deletion was introduced into SKl5921psL by crossing with SLlO and selected for by growth in the presence of Sm, 5-fluorocytosine, and proline. The F’ episome, carrying mutations ‘traD36, proAB, and lacZqZAM15, was transferred to SK1592+@- A(lac-proAB) by mating with JMlOl. The resulting strain was called JM105 and tested for markers as described above.

(I) Construction of JM106 and JM107

RecA + , TcR derivatives of DHl were obtained by transducing DHl with PlCmlclr-100 propagated in TDl. The desired transductants, DHl-TnlO- recA + , were selected by growth on Tc and nalidixic acid, resistance to UV irradiation, and screened for Cm sensitivity.

The A(lac-proAB) chromosomal deletion was introduced into DHl-TnlO-recA + by mating with SLlO and selected for by growth on minimal media plates containing nalidixic acid, 5-fluorocytosine, and proline. Positive progeny were further tested for resistance to UV irradiation and production of white colonies on MacConkey plates or B-broth plates plus Xgal and IPTG.

The recA+, A(lac-proAB), gyrA96 progeny were tested for retention of the Hsd - and Su + (suppressor-plus) phenotypes. Correct progeny were then cured of the TnlO transposon by selection on fusaric acid medium. This strain, recA+, A(lac-proAB),

endA 1, gyrA 96, thi, hsdR 17, supE44, rel4 1, was called JM106. JM106(F-) was mated with JMlOl(F’), and the F’ transfer confirmed by blue plaque production when cells were infected with M13mp- loamber. The final strain was designated JM107.

(m) Construction of JMlO8 and JM109

The RecA - phenotype was introduced into JM106 by transducing with Plvir propagated on JC10240 (recA-, srlC: :TnlO). Progeny were selected for by growth on Tc, proline, nalidixic acid, and 5-fluorocytosine, and screened for inability to grow following exposure to UV light. Further tests

affirmed the Hsd - and Su + phenotypes and demonstrated white colonies on MacConkey plates and B-broth plates plus Xgal and IPTG.

Positive progeny were cured of the TnlO as before and named JM108. JM108 was mated with JMlOl; progeny were tested for the presence of the F’ . The end product (recA 1, endA 1, gyrA96, thi, hsdR 17, supE44, reL4 1,1-, A(lac-proAB), [F’, traD36,proAB. lacZqZAM151 was called JM109.

(n) Construction of JMllO

Spontaneous SmR in GM48 was selected for by plating on minimal plates plus Sm. Inability to

107

grow without leucine or threonine confirmed the

desired phenotypes. The d&c-proA@ was estab- lished in GM48rpsL by mating with SLlO and selecting for on minimal plates plus proline, leucine, 5-fluorocytosine and Sm.

Correct genotype confirmation was by screening for white colony production on B-broth plates plus Xgal and IPTG, and by lysis with T4amN130-N82. JMllO was the result of mating GM48rpsL-d(luc- proA@ with JMlOl. Infection by M13mplOamber affirmed the F’ presence.

(0) Generation of clones for sequencing

M13UC RF DNA (2 pg) was digested with 10 units each of SstI and BumHI for 1.5 h at 37°C. Next, 0.12 vol. of 80 mM EDTA, 0.4 vol. of 5 M NH, * acetate and 2 ~01s. of isopropanol were added; after vortexing and RT incubation for 15 min, the DNA was pelleted by centrifugation at 9000 x g for 5 min. The pellet was carefully washed with cold 70% ethanol-water and dried under vacuum. It was resuspended in 40 ~1 Exo III buffer (50 mM Tris * HCl pH 8, 5 mM MgCl,, 1 mM DTI’), 8 units of Exo III were added, and it was incubated at 37°C for 20 min, with 2 ~1 aliquots removed at 1-min intervals to a tube on ice containing

2 ~1 of 10 x Exo VII buffer (500 mM K * phosphate, pH 7, 80 mM EDTA, 10 mM DTT). Two tubes, each containing the pooled aliquots from a 10-n+ time period, were thus generated. Then 0.1 unit of Exo VII was added to each, and the tubes were incubated at 37 ‘C for 45 min, and then at 70’ C for 15 min. Next, 1.5 ~1 0.2 M MgCl,, 0,5 units large fragment DNA polymerase, and 1~1 of an 8-mM solution of dATP, dCTP, dGTP, TIP were added and incubated at RT for 30 min. To a 5-~1 (200 ng of DNA) aliquot were added 5 ~110 x ligation buffer (250 mM Tris . HCl pH 7.5,lOO mM MgCl,, 25 mM hexamine cobalt chloride, and 5 mM spermidine), 5 ~1 10 mM ATP, 2.5 ~1 0.1 M DlT, and 2 units DNA ligase and the volume was adjusted to 50 ~1 with H,O. After a 3-h incubation at RT, the DNA was precipitated as described above. The pellet was resuspended in 40 ~1 STE (10 mM NaCl, 10 mM Tris. HCl, pH 7.5, and 1 mM EDTA) and 10 ~1 were used to transform JM105.

Template preparation, sequencing reaction, gel electrophoresis, and data analysis were as described

(Carlson and Messing, 1984; Messing, 1983; Larson and Messing, 1983).

RESULTSANDDISCUSSION

(a) E. coli hosts

(1) Conjugation mutants The male-specific E. coli bacteriophage Ml3 re-

quires an F episome for infection of host cells. Current NIH guidelines regarding the use of recombinant DNA discourage the use of E. coli strains carrying conjugation proficient plasmids like the F’ episome (Federal Register, 1980). Since the tru ope- ron controls conjugation (Achtman et al., 1971), tra mutations have been isolated on F’luc DNA epi- somes to develop conjugation deficiencies that still allow infection by M13. Ml3 vector utility is also based on proper a-complementation between the phage and host B-galactosidase gene. A host strain containing the d(lac-proA@ deletion on the chromo- some and an F’lac possessing the tra mutation and lacdM15 deletion was constructed and named JMlOl (Messing 1979). JMlOl has a suppressor that permitted growth of M13mp7, 8, 9, 10, and 11 phage that contain amber mutations in genes I and II (Messing et al., 1981; Messing and Vieira, 1982).

(2) Restriction mutants A restrictionless host strain that facilitated cloning

of unmodified DNA was constructed and called JM105.Asdescribedin MATERIALSANDMETHODS,

section k, the d(Zuc-proA@ chromosomal deletion was introduced into SK1592 via an Hfr cross. Although conjugation of F’ with the truD36 mutation was reduced by a factor of 10e5, the leaky mutation allowed conjugation at a reduced rate. There- fore, JMlOl was used as a donor for the F’ traD36AproAB lacPZAM15 episome in mating experiments by using the complementation of proline as selection and drug resistance as counterselection. The resulting strain did not contain the supE muta-

tion of JMlOl, so it permitted growth of wild-type M13mp10, 11, 18, and 19, but not of the amber mutants. This provides selection for transferring inserts from an Ml3 amber phage into a wild-type

108

Ml3 vector and for obtaining Ml3 recombinants (3) Recombination mutants with inserts in the opposite orientation (Carlson and E. coli K-12 restriction was not the only cause for Messing, 1984). Since JMlO5 is r-m+, anyunmodi- reduced efficiency in cloning larger DNA fragments tied DNA cloned din&y into wild-type M 13 vectors (> 2000 bp) into Ml3 ; another source of instability and propagated in JM105 is modified but not was recombining sequences. Since recA mutations restricted. The relevant markers have been tested as reduce recombination, a host with a reck mutation shown in Table II. would be useful. A new recA, r - m + , ~11 host for all

TABLE II

Marker tests (a) Testing for the Hsd - and Su + phenotypes. Lb2c propagated in modifying JMlOl or unmodifying HBlOl for two serial cycles was spotted on lawns of questioned strains and incubated at 37°C overnight. The JMlOl-modified and restricted IZb2c was able to elliciently infect r+ m + strains, while HBlOl unmodified and unrestricted lb2c phage was destroyed in r + m + strains. Amber phage T4amN130-N82 was spotted upon lawns of the strains in question and incubated at 37°C overnight. Only strains possessing the ~11 suppressor gene support growth ofthe amber phage.

Phage Phage dilution Number of plaques on E. coli strain:

r+m+ JMlOl

r-m+ JM105

r-m+ JM106

r-m+ JM108

ti2c propagated in HBlOl (r-m-) (1 x lo9 pfu/ml)

lO-2 40 lysis lysis lysis lo-4 0 lysis lysis lysis lo-6 0 8 18 5

2b2c propagated in JMlOl (r+m+) ( 1 x lo8 pfu/ml)

1o-2 lysis lysis lysis lysis lo-4 40 lysis lysis lysis lo-6 0 0 1 0

T4amN130-N82 propagated in JMlOl (1 x 10” pfu/ml)

lo-2 lysis 0 lysis lysis

(b) Testing for F’, traD36, IacZq, lacZdMl5, and for Su+, Hsd, Ret-, and Gyr phenotypes. Infection of plate lawns by spotted dilutions of amber phage Ml3mpll + XgaI and + / - IPTG demonstrated presence of the suI1 suppressor gene and the need for IPTG induction of the la& gene for proper blue plaque production. Phage f2’s infectious incapability indicated presence of the traD36 mutation in the host strain. Tests for r- and m- were as given above in Table Ha. Plates of freshly streaked cells were subjected to UV irradiation to test for the RecA phenotype. Strain growth on plates containing nalidixic acid affirmed the presence of the gyr mutation.

Test Plaques on E. co/i straina

JMlOl JM105 JM107 JM109

Infection with Ml3mpll amber + Xgal + IPTG + Xgal alone

Infection with l2 Test for r- Test for mm Growth after UV exposurea Growth on nalidixic acid”

blue clear 0 - -

+ 0

blue clear 0 + -

+ +

blue clear 0 +

+

a Two bottom lines refer to bacterial growth.

109

Ml3 vectors was thus constructed. Strain DHl, developed for high transformation efficiencies (Ha- m&an, 1983), was selected as the initial strain because it possessed a recA, hsdR 17, supE4.4 genotype and high transformation efficiency. The recA

mutation was transduced to recA+ (Csonka and Clark, 1978) to permit deletion of the d(luc-proAB)

region producing JM106. After introduction of the F’ from JM 101, the resultant strain JM 107 could be used in the same manner as JMlOl for infection by amber or wild-type M 13 or by the pUC plasmids. P 1 transduction of JM106 restored the recA 1 mutation and produced strain JM108. The introduction of JMlOl’s episome into JM108 produced JM109. All four DHl derivative strains, JM106, JM107, JM108, and JM109, have been screened for the correct r-m+ and suI1 phenotype, and JM107 and JM109 have been tested for the dM 15 deletion, the lucZq and truD mutations (Table II).

(4) Applications

These new strains broaden applications of the M13mp and pUC plasmid vector systems. JM106 and JM108 may prove useful as hosts for cosmid libraries, because deletion of the chromosomal lac

DNA can prevent background hybridization. JM109 could be useful as the host for cDNA libraries (Helfman et al., 1983; Heidecker and Messing,

TABLE III

Transformation efficiencies of pUCl8 and Ml3 RF DNAs

Method Transformants per pg pUCl8 DNA” employing E. coli recipient strain:

1983) and for examining the expression of mutant proteins in E. coli. Transformation efficiencies of all strains have been tested by the transformation proto- cols of Cohen et al. (1972) and Hanahan (1983). All strains approximated the efficiencies of standards DHl and 71-18, with JM107 and JM109 proving to be slightly higher (Table III). Higher transformation efficiencies have been reported (Ham&an, 1983) and may be possible for these new strains. This work attempted only to ensure that under defined transformation conditions the new strains gave the same transformation efficiencies as the parental strains.

JM109 has proven useful by virtue of its recA1

mutation. Plasmids form multimers when propagated in recA + strains like JM83 (Bedbrook and Ausu- bel, 1976). JM109 maintains pUC species of a unique size whether monomer or multimer. The recA1 mutation destroys the mechanisms for the recombination and/or replication events that produce the multimers.

Although it is not possible to predict whether large fragments cloned into M 13 and grown in JM109 will experience fewer deletions than when propagated in JMlOl, the following observations have been made. When a 4.5-kb fragment of the maize-controlling element activator (AC) was cloned in Ml3 and propagated in JM107, deletions were found to extend from the AC sequences into M 13 sequences near the

DHl JM105 JM107 JM109

CaCl, 4.2 x lo6 8.2 x lo6 5.3 x lo6 1.0 x 106 Hanahan 6.3 x lo6 3.9 x lo5 1.4 x 10’ 1.2 x 10’

Method Transformants per pg Ml3 RF DNA” employing E. coli recipient strain:

71-18 JM105 JM107 JM109

CaCl, 7.3 x 10s 3.9 x 10s 2.6 x lo5 3.4 x 105 Hanahan 3.0 x lo5 1.2 x lo5 2.0 x 106 1.9 x 106

a Transformation with the plasmid or Ml3 RF DNA into specified host strains, as to compare the traditional CaCl, method (Cohen et al., 1972) with the Hanahan (1983) protocol.

2170

21

60

2190

22

00

2210

22

20

2230

22

40

~TG

KG

CT

Tac

TG

Gw

a3G

TP

6CLT

TcA

Glw

hc

TG

CG

CTT

TCC

&TT

CTG

GC

TTTR

ATG

MG

ATc

cATT

CG

TTTG

TGM

T6T

2250

22

60

2270

22

80

2290

23

00

2310

2x

0 C

,WG

GC

CM

TCG

TCTG

ICTG

CC

TCC

TOTC

G

GC

GG

CTC

TGG

TBO

TGG

TTC

TGG

T-TC

TW

2330

23

40

2350

23

60

2370

23

80

2390

24

00

GG

GT

GG

TG

GC

TC

TG

,V~G

GT

GG

CG

GT

TC

TG

N~G

GT

GG

CG

GC

TC

T~

GG

TTC

CG

GTG

GTG

GC

TCTG

GTT

CC

BeT

e

2410

24

20

2430

24

40

245’

3 24

60

2470

24

80

PITT

TTG

ATT

FlTG

CIM

WO

T G

GC

AM

aCG

CT

AII

TW

VIC

CT

AT

Gfl

~ TG

CC

BII

TGA

&3A

CG

CG

CTh

ChG

TCTG

M

2490

25

00

2510

25

20

2530

25

40

2%0

2G&

3 G

CT

~~~C

TT

GII

TT

CT

GT

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PIC

B

2570

25

80

2590

2b

cm

2610

26

20

2630

2b

40

T~T

OO

TM

Tff

iTG

CT

AC

T00

T~T

TT

TO

CT

OD

CT

CT

LLT

TC

CC

~T_T

C~T

C~T

-~T

~T~T

T~T

T

2650

26

60

2670

26

60

2690

27

00

2710

27

20

TIP

IT~T

~TT

TC

CG

TC

~TaT

TT

ICC

TT

CC

CT

CC

CT

CC

CT

~TC

~TT

~TG

TC

~~T

TT

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TC

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T-~

T~T

~

2730

27

40

2750

27

60

2770

27

80

2790

2G

OO

C

C~T

IIT

G~~

TT

TT

CT

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M 13 origin of replication (Pohhnan, R. and Messing, J., unpubl.). It should be noted that the AC element contains numerous direct and indirect repeats in regions of low and high G + C contents (Pohhnan et al., 1984). When the 4.5kb PstI-BamHI AC fragment is cloned into M 13 and grown in JM 109, stable recombinants have been recovered and no deletions have been detected over numerous generations (not shown).

fs) Methylation

Other E. coli mutations useful for recombinant DNA amplification are DNA methylation deficiencies. For example, Mb01 and BclI can cleave DNA propagated in a dam - E. coli strain while

EcoRII restriction requires the absence of the dcm

product (for review, see Roberts, 1983). If DNA is propagated in E. coli strains lacking the A and C methylases, it is unmodified and can be cleaved by MboI, BclI, and EcoRII. GM48 contains these mutations, but lacks the d(luc-proAB) deletion and the F’ traD36 proAB lacPZAM15 episome required for M13mp and pUC vector use. Strain JMllO was developed to be dam - dcm - by introduction of the A(lac-proAB) deletion and JMlOl episome into GM48. The dam and dcm mutations have been tested as described in MATERIALS AND METHODS,

section j (not shown).

(b) Ml3 phage vectors

Since many specific constructions depend on the knowledge of the vector restriction map, the nucleotide sequences of M13mp18 and pUC19 have been compiled and reprinted here. The wild- type sequence of Ml3 has been determined by Van Wezenbeek et al. (1980). M13’s unique HincII site was used as the sequence reference point. Following this reference, the C in position 3 has been converted to a T to eliminate the HincII site, the G (2220) converted to an A to eliminate the BamHI site, and the C (6917) converted to a T to eliminate the AccI site and introduce the BgfII site (positions are for wild-type M13) (Messing et al., 1981). The luc Hind11 fragment has been inserted into the BsuI site at position 5868 (Messing et al., 1977). The lac sequence has been compiled from the lucl gene (Farabaugh, 1978), the 1acZpo region (Dickson et al., 1975) and the IacZ gene sequences (Kalnins et al., 1983). M 13mp18 and

M13mp19 lack the double amber mutation that M13mplOa and M13mplla had (Messing and Vieira, 1982) but do possess two complementary polylinker regions in the facZ gene (Norrander et al., 1983). The junctions of Iuc DNA and Ml3 DNA were as predicted from the restriction sites used for cloning the lac Hind11 fragment into the BsuI site at position 5868. The junction sequence at the IacZ

gene led to an early ochre termination codon that produced a lac a fragment of 168 amino acid residues ; 18 of them represent the polylinker region. The resulting sequence of M13mp18 is presented in Fig. 1.

(c) The pUC plasmids

(I) Construction The luc Hue11 fragment inserted into pBR322

produced a shorter a peptide than that in Ml3 (Ruther, 1980), yet active. The pBR322 sequence has been modified by removing the EcoRI-PvuII fragment containing the Tc resistance gene via a fill-in reaction and blunt-end ligation. The predicted regen- eration of the EcoRI site failed to occur when sequencing data revealed that the deletion extends across nucleotides 4355-l-2072. Similar findings were reported by Rubin and Spradling (1983) and Stragier, P. (personal communication). Restriction sites were removed from the intermediate plasmid in the following way. EMS mutagenesis resulted in a GC to AT transition in the PstI site at position 3610 (positions are for pBR322) (Vieira and Messing, 1982). Hydroxylamine treatment converted another GC to AT in the HincII site at position 3911. The AccI site was eliminated by BAL31 digestion of nucleotides 2210-2250. The lac sequence was inserted at the Hue11 site at position 2352. The lac

sequence was oriented in the same direction as the bla gene coding for fi-lactamase. The fusion of the 1acZ sequence to the pBR322 DNA at the HaeII site at position 2352 resulted in an a peptide of 107 amino acid residues, 19 encoded by the polylinker region at residue 5. Translation was terminated by the UAG termination codon, which was suppressed in the supE strains. In supE strains the peptide was 15 amino acids longer and terminated by an UGA codon. These small fusion peptides were very unstable in E. coli and detectable only through the highly sensitive complementation test with Xgal. The nucleotide sequence and restriction map for pUC19 are given in Fig. 2.

117

(2) Sequencing with new method

Since the pUC plasmids have been mutagenized with EMS, HA, and treated with BAL31 before introducing the lac DNA into the pBR322 backbone molecule, the pBR322 portion of pUC6 (Vieira and Messing, 1982) had to be resequenced. Earlier sequencing experiments were based on Ml3 shotgun

Cleavage at siteg leaving recessed 3' end

-b restriction -_e enzyme sites

Primer hybridization site

Cleavage at site a leaving protruding 7 end

Klenow -S-NTP

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Cleavage at b

_ 1. r leaving blunt or recessed 3' end _ I.

(2)

(3) Exo VII

-

I Klenow NTP's Ligase

(4’ 0 0 0 I

Transformation

Fig. 3. Method for generating unidirectional deletions. Details of

the procedure are as follows: (1) Vector is digested with restriction enzymes a and b. Enzyme a leaves a 4-bp 3’ overhang that is resistant to Exo III and protects the PHS site. Enzyme b leaves a recessed 3’ end that is sensitive to Exo III and exposes the insert to digestion. (2) The DNA is treated with Exo III for O-20 min and aliquots removed at 1 min intervals:This generated random insert deletions while leaving’ the PHS intact. (3) Exo VII removed the single-stranded DNA region left by Exo III. (4) The digest was treated with DNA polymerase I to ensure formation of blunt-ended DNA. DNA ligase was added to recircularize the various deletion products, leading to increas- ingly smaller circles with the PHS in the same position.

sequencing of pUC6 (Halling, S., Abbot, A., Kridl, J. and Messing, J., unpubl.). Because the asymmetric Ml3 polylinkers (Vieira and Messing, 1982) could be used to make unidirectional deletions, a different approach of generating pUC6 subclones for sequencing was tested. The polylinker permitted cleavage of the pUC plasmid or the Ml3 RF by two restriction endonucleases, one producing a 3’ protruding end like &I, the other a 5’ protruding end. Since Exo III was double-strand-specific and required a 3’OH end, the PstI end was not accessible to this enzyme. These features simplified the noman- dom sequencing approach based on BAL31 treatment described below (Poncz et al., 1982) and illus- trated in Fig. 3. The method included the following steps: (1 a) pUC6 was linearized with NdeI, and the ends were made flush with the large fragment of DNA polymerase and inserted at the HincII site of M13mp19 to make M13UC. This produced, between the inserted DNA and the PHS, a unique SstI site proximal to the PHS and a unique BamHI site distal to it. (lb) M13UC RF was digested with

SstI and BamHI. SstI leaves a 4-bp 3’ overhang that is resistant to Exo III and protects the PHS from digestion. BamHI leaves a recessed 3’ end that is sensitive to Exo III and exposes the insert to digestion. (2) The DNA was treated with Exo III for O-20 min. Aliquots were removed at I-min intervals. This time course generated random insert deletions while leaving the PHS intact. (3) The single-stranded region of DNA left by Exo III treatment was removed with Exo VII. Since only Exo VII is active in the presence of EDTA, addition of Exo III time-course aliquots to a tube containing Exo VII buffer plus EDTA proves a convenient way to stop the Exo III reaction. (4) To ensure formation of blunt-ended DNA, the digest was treated with DNA polymerase I. DNA ligase was added to recircularize the molecules, which were then used to transform JM105. (5) Phage isolated from transformed cells were used for direct gel electrophoresis (Messing, 1983) to determine clones of appropriate size for sequencing (Fig. 4).

The Exo III, Exo VII, polymerase, and ligase reactions were performed sequentially by adjusting reaction buffers. Alternatively, it was possible to protect the PHS from Exo III digestion via S-NTP incorporation by DNA polymerase at a recessed 3’ end proximal to the PHS left by restriction endo-

118

T PE C A U K yt

Fig. 4. Mapping of the deletion mutants. Since the position of the PHS is unaltered and all deletions occur only at the opposite end, deletion points are mapped by recombinant phage mobility changes indicated by agarose gel electrophoresis. After exo- n&ease and ligase treatment, the DNA is transformed into JM105. Plaques are picked from each transformation experi- ment, grown in small cultures, and supernatant phage used directly for agarose gel electrophoresis as described (Messing, 1983). A picture taken of the agarose gel was used to draw a physical map of the sequenced clones. The first and last lanes (unmarked) contain untreated M13UCl and Ml3mpl9, respectively; the other lanes are labeled alphabetically and represent individual clones. Nine clones from this gel were used to prepare a template for sequencing as described in MATERIALS AND METHODS, section o. The sequence has been entered into an Apple II computer and analyzed using the programs of Larson and Messing (1983). The deletion points are marked in the map by the agarose-gel-derived clone name. The map has been drawn with reference to the N&I sites used to clone pUC6 into the HincII site of Ml3mpl9. The nucleotide numbers in the map are taken from the reverse complement of pUC6, referred to as

pUC6V.

nuclease digestion (Putney et al., 1981; Vieira, J., unpublished results). Cleavage of a site distal to the PHS was then needed to generate an unprotected recessed 3’ end for Exo III treatment. The consecu- tive steps are outlined in Fig. 3.

This approach resembles that described by Poncz et al. (1982), but hastens the construction of recombinant M 13 phage needed for sequencing. As oppos- ed to bidirectional deletions, the creation of unidirectional deletions precludes the need for recloning DNA fragments. The speed by which recombinants can be obtained resembles that of shotgun cloning. Ordering clones on a physical map is simple (Fig. 4) so the redundancies and gaps typical of shotgun sequencing are avoided. Hence, the following se-

quencing approach to larger DNA segments is used. Restriction sites present in the polycloning sites of M13mp18 and M13mp19 are used to clone restriction fragments in both orientations. Fragments need to be inserted such that between the PHS and the insert there exist two unique restriction enzyme sites. The restriction enzyme site proximal to the PHS must produce either a 4 bp 3’ overhang or a recessed 3’ end. The other should leave a blunt or recessed 3’ end next to the insert. Each clone pair representing both orientations is then subjected to Exo III and VII treatment. The optimum insert size for nuclease treatment is 2000-5000 bp. Also, the ExoIII unit activity of different commercial preparations can vary, necessitating the calibration of each enzyme lot. This is accomplished by the electrophoresis of DNA samples taken at two time points from an ExoIII reaction on an agarose gel for size analysis.

ACKNOWLEDGEMENTS

We thank Kris Kohn, Tammy Krogmann, Mike Zarowitz, and Stephanie Young for help in preparing the manuscript, Drs. J. Felton, T.Baldwin and P. Stragier for making results available to us prior to publication, and Betsy Kren and James Fuchs for helpful discussions. The work was supported by the Department of Energy DE-FG02- 84ER13210 and NIH GM31499. J.V. is supported by the NIH training grant No. ST32 GM107094.

REFERENCES

Achtman, M., Willets, H. and Clark, A.J.: Beginning a genetic analysis of conjugational transfer determined by the F-factor in Escheric~iu coli by isolation and characterization of transfer-deficient mutants. J. Bacterial. 106 (1971) 529-538.

Bedbrook, J.R. and Ausubel, F.M.: Recombination between bacterial plasmids leading to the formation of plasmid multimers. Cell 9 (1976) 707-716.

Birnboim, H.L. and Doly, J.: A rapid alkaline extraction procedure for screening ofrecombinant plasmid DNA. Nucl. Acids Res. 7 (1979) 1513-1523.

Carlson, J. and Messing, J.: Efficiency in cloning and sequencing using the single-stranded bacteriophage M 13. J. Biotechnol. (1984) in press.

119

Cohen, S.N., Chang, A.C.Y. and Hsu, L.: Non-chromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R factor DNA. Proc. Natl. Acad. Sci. USA 69 (1972) 2110-2114.

Csonka, L. and Clark, A.: Construction of an Hfr strain useful for transferring recA mutations between Escherichia coli

strains. J. Bacterial. 143 (1980) 529-530. Dickson, R.C., Abelson, J., Barnes, W.M. and Reznikoff, W.S.:

Genetic regulation: the lac control region. Science 187 (1975) 27-35.

Federal Register Vol. 45, No. 147 (1980) 50525. Felton, J.: Ml3 host strain JM103 contains two restriction sys-

tems. Biotechniques I (1983) 42-43.

Farabaugh, P.J.: Sequence of the lacl gene. Nature 274 (1978) 765-769.

Hanahan, D.: Studies on transformation of Escherichia colt’ with plasmids. J. Mol. Biol. 166 (1983) 557-580.

Helfman, D.M., Feramisco, J.R., Fiddes, J.C., Thomas, G.P. and Highes, S.H.: Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc. Natl. Acad. Sci. USA 80 (1983) 31-35.

Heidecker, G., Messing, J. and Gronenborn, B.: A versatile primer fbr DNA sequencing in the Ml3mp2 cloning system. Gene 10 (1980) 69-73.

Heidecker, G. and Messing, J.: Sequence analysis ofzein cDNAs obtained by an efficient mRNA cloning method. Nucl. Acids Res. 11 (1983) 4891-4906.

Hu, N-T. and Messing, J.: The making of strand-specific Ml3

probes. Gene 17 (1982) 271-277. Kalnins, A., Otto, K., Ruther, U. and Mtiller-Hill, B.: Sequence

of the IacZ gene of Escherichia coli. EMBO J. 2 (1983) 593-597.

Larson, R. and Messing, J.: Apple II computer software for DNA and protein sequence data. DNA 2 (1983) 31-35.

Malay, S. and Nunn, W.: Selection for loss of tetracycline resistance by Escherichia coli. J. Bacterial. 145 (1981) 1110-1112.

Messing, J., Gronenborn, B., Miiller-Hill, B. and Hofschneider, P.H.: Filamentous coliphage Ml3 as a cloning vehicle: insertion of a Hind11 fragment of the lac regulatory region in the Ml3 replicative form in vitro. Proc. Natl. Acad. Sci. USA 75 (1977) 3642-3646.

Messing, J.: A multipurpose cloning system based on the single- stranded DNA bacteriophage M13. Recombinant DNA Technical Bulletin, NIH Publication No. 79-99, Vol. 2, No. 2 (1979) 43-48.

Messing, J.: Ml3mp2 and derivatives: a molecular cloning system for DNA sequencing, strand specific hybridization, and in vitro mutagenesis, in Walton, A.G. (Ed.), Proceedings of

the Third Cleveland Symposium on Macromolecules. El- sevier, Amsterdam, 1981, pp. 143-153.

Messing, J., Crea, R., and Seeburg, P.H.: A system for shotgun DNA sequencing. Nucl. Acids Res. 9 (1981) 309-321.

Messing, J. and Vieira, J.: A new pair of Ml3 vectors for selecting either strand of a double-digest restriction fragment. Gene 19 (1982) 269-276.

Messing, J.: New Ml3 vectors for cloning. Methods Enzymol. 101 (1983) 20-79.

Miller, J.H.: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972.

Norrander, J., Kempe, T. and Messing, J.: Improved M 13 vectors using oligonucleotide-directed mutagenesis. Gene 26 (1983) 101-106.

Pohlman, R., Fedoroff, N. and Messing, J.: The nucleotide sequence of the maize controlling element activator. Cell 37 (1984) 635-643.

Poncz, M., Solowiejczyk, D., Ballantine, M., Schwartz, E. and Surrey, S.: “Non-random” DNA sequence analysis in bacteriophage Ml3 by the dideoxy chain-termination method. Proc. Natl. Acad. Sci. USA 79 (1982) 4298-4302.

Putney, S.D., BenkoJic, S.J. and Schimmel, P.R.: A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo. Proc. Natl. Acad. Sci. USA 78 (1981) 7350-7354.

Roberts, R.: Restriction and modification enzymes and their recognition sequences. Nucl. Acids Res. 12 (1984) r167-204.

Rubin, G.M. and Spradling, A.C.: Vectors for P element- mediated gene transfer in Drosophila. Nucl. Acids Res. 11 (1983) 6341-6348.

Ruther, U.: Construction and properties of a new cloning vehicle, allowing direct screening for recombinant plasmids. Mol. Gen. Genet. 178 (1980) 475-477.

Sanger, F., Nicklen, S., and Coulson, A.R.: DNA sequencing

with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467.

Sutcliffe, J.G.: Complete nucleotide sequence of the Escherichia

coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol. 43 (1979) 77-90.

Van Wezenbeek, P.M.G.F., Hulsebos, T.J.M. and Schoenma- kers, J.G.G.: Nucleotide sequence of filamentous bacteriophage Ml3 DNA genome: comparison with phage fd. Gene 11 (1980) 129-148.

Vieira, J. and Messing, J.: The pUC plasmids, and M13mp7- derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19 (1982) 259-268.

Communicated by A.J. Podhajska.

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