Free Declaration in Support - District Court of California - California

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EXHIBIT 1

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Gene, 33 (1985) 103-l 19 Elsevier GENE 1167

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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, Universityof Minnesota, St. Paul, MN 55108 (U.S.A.) Tel. (612) 376 1509 (Received July 27th, 1984) (Accepted September 21st, 1984)

SUMMARY

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.
Three

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, Bactotryptone broth; Cm, chloramphenicol; A, deletion; DTI, dithothreitol; 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, streptomycin; STE, 10 mM NaCI, 10 mM Tris . HCL pH 7.5, 1 mM EDTA; Tc, tetracycline; Xgal, 5-bromo-4-chloro-indolyl-/?-n-galactopyranoside; YT, yeast tryptone; [I, indicates plasmidcarrier 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 wildtype 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 (Heidecker et al., 1980), Ml3 was used primarily for

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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 and genotypes Genotype ara, A(lac-proAB). rpsL( = strA), @O,

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 Spradling, 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
proAB.

E. coli strains E. coli strain JM83 JMlOl JM105 JM106 JM107

lacZAMl5 supE, thi, A(iac-proAB), IacIqZAMlS] thi, qrsL, endA, sbcB15, hspR4, A(lac-proAB), [F', traD36,proAB. A(lac-proAB) ena2 1, gyrA96, thi. h.sdR17, supE44, reiA 1,1-, A(lac-proAB), iacIqZAMlS] JM108 JM109 recA1, endAl, recA1. endAl, relA1, JMllO I-, gvrA96, thi, hsdR17, supE44, gyrA96, thi. hsdR17, supE44, A(iac-proAB), [F', traD36, reiA 1, A(lac-proAB) iacZqZAM15] endA 1, gvA96, thi, hsdR 17, supE44, reiA 1, I

[F', traD36, proAB,

-,

[F' ,

traD 36,

proAB,

proAB, iacIqZAM15] rpsL. thr, leu, thi, lacy, galK. galT, ara. tonA. tsx. dam, DHl GM48 SLIO TDl MC4100 SK1592 71-18 dcm, supE44, A(lac-proAB), [F', traD36,proAB, reU1,1thr. 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', lacIqZAM 151 lacIqZAMlS] thi, hsdR 17, supE44,

F -, recA 1, ena2 1, gyA96,

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

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

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). Transformation by M 13 RF DNA was as described by Hanahan (1983) and Cohen et al. (1972) except that the heatshocked 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

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.
(e) Transductions

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 phage CUlA 550nm of 0.05. At A550nm 0.300-0.420, 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).
(i) Marker tests

Lysates of P 1Cm 1, clr 100 and P 1vir 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 101) 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

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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 T4amN130N82. 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 (handheld 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. UVresistant 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

(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-TnlOrecA +, 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 M13mploamber. The final strain was designated JM107.
(m) Construction of JMlO8 and JM109

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.

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, reL41,1-, 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

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grow without leucine or threonine confirmed the desired phenotypes. The d&c-proA@ was established 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(lucproA@ 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 requires 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 operon controls conjugation (Achtman et al., 1971), tra mutations have been isolated on F'luc DNA episomes 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 chromosome 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. Therefore, 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 mutation 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

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Ml3 vector and for obtaining Ml3 recombinants with inserts in the opposite orientation (Carlson and Messing, 1984). Since JMlO5 is r-m+, anyunmoditied DNA cloned din&y into wild-type M 13 vectors and propagated in JM105 is modified but not restricted. The relevant markers have been tested as shown in Table II.

(3) Recombination mutants E. coli K-12 restriction was not the only cause for reduced efficiency in cloning larger DNA fragments (> 2000 bp) into Ml3 ; another source of instability was recombining sequences. Since recA mutations reduce recombination, a host with a reck mutation 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 lysis lysis 8 lysis lysis 0 0 r-m+ JM106 lysis lysis 18 lysis lysis 1 lysis r-m+ JM108 lysis lysis 5 lysis lysis 0 lysis

ti2c propagated in HBlOl (r-m-) (1 x lo9 pfu/ml) 2b2c propagated in JMlOl (r+m+) ( 1 x lo8 pfu/ml) T4amN130-N82 propagated in JMlOl (1 x 10" pfu/ml)

lO-2 lo-4 lo-6 1o-2 lo-4 lo-6 lo-2

40 0 0 lysis 40 0 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 Infection with Ml3mpll amber + Xgal + IPTG + Xgal alone Infection with l2 Test for rTest for mm Growth after UV exposurea Growth on nalidixic acid" a Two bottom lines refer to bacterial growth. JM105 JM107 JM109

blue clear 0 + 0

blue clear 0 + + +

blue clear 0 +

+

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Ml3 vectors was thus constructed. Strain DHl, developed for high transformation efficiencies (Ham&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: DHl CaCl, Hanahan 4.2 x lo6 6.3 x lo6 JM105 8.2 x lo6 3.9 x lo5

1983) and for examining the expression of mutant proteins in E. coli. Transformation efficiencies of all strains have been tested by the transformation protocols 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 Ausubel, 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

JM107 5.3 x lo6 1.4 x 10'

JM109 1.0 x 106 1.2 x 10'

Method

Transformants per pg Ml3 RF DNA" employing E. coli recipient strain: 71-18 JM105 3.9 x 10s 1.2 x lo5 JM107 2.6 x lo5 2.0 x 106 JM109 3.4 x 105 1.9 x 106

CaCl, Hanahan

7.3 x 10s 3.0 x lo5

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 2160 2190 ~TGKGCTTacTGGwa3GTP6CLTTcAGlwhc 2250 2260 2270 C,WGGCCMTCGTCTGICTGCCTCCTOTC 2360 2330 2340 2350 GGGTGGTGGCTCTG,V~GGTGGCGGTTCTGN~GGTGGCGGCTCT~ 2440 2410 2420 2430 PITTTTGATTFlTGCIMWOT GGCAMaCGCTAIITWVICCTATGfl~ 2490 2510 2540 2%0 GCT~~~CTTGIITTCTGTCGCT~TG~TTPICB 2570 T~TOOTMTffiTGCTACT00T~TTTTOCTODCTCTLLTTCCC~T_TC~TC~T-~T~T~TT~TT 2650 TIPIT~T~TTTCCGTC~TaTTTICCTTCCCTCCCTCCCT~TC~TT~TGTC~~TTTTGTCTTT-~T~T~ 2730 2750 2740 2760 2770 2780 2790 2GOO CC~TIITG~~TTTTCTATTG~TTGT~C~~~T-CTT~TTCCGTGGTGTCTTT-TTTCTTTT~T~TGTT~CTT 2810 2830 2820 2840 2930 2853 2G40 2940 2870 2880 2960 2950 MhMGGGCTTCGGT~ So40 TLTGTLTGT~TTTTCTIICGTTTGCT~~C~T~TGCGT~T~~~~TCTT~TC~T~C~GTTCTTTT~T~TTC~TT 2920 2890 2900 2910 LTT~TTGCGTTTCCTCGGTTTCCTTCT~T~~TTOTTCOTT 2660 2670 2660 2690 2700 2710 2720 2580 2590 2bcm 2610 2620 2630 2b40 2500 2520 2530 245'3 2480 2460 2470 TGCCBIITGA&3ACGCGCThChGTCTGM 2G&3 2370 2400 2380 2390 GGTTCCGGTGGTGGCTCTGGTTCCBeTe 2280 2290 2300 2310 GGCGGCTCTGGTBOTGGTTCTGGT-TCTW 2x0

2240 2200 2210 2220 2230 TGCGCTTTCC&TTCTGGCTTTRATGMGATccATTCGTTTGTGMT6T

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5000 3010 So20 5030 2970 2980 2990 T~GCT~TTGCT~TTTCCITTGTTTCTTGCTCTT~TT~TT-TT~CTC~TTCTTGT~GTT~TCTCTCT~T~TT3080 3090 3100 3110 3050 3060 3070 OCTC~TT~CTCTG~TTTGTTC~~~GGTGTTC~~~TT~TTCTCCCGTCT~T~~TTCCCTGTTTTT~TGTT~TTCT 3140 3130 3140 3150 3170 3180 CTCTGTCI~-TGCTPITTTTCCITTTTTG-TT~--~~TCGTTTCTT~TTTG~TT~~T~T~~T~T-T 3210 3220 3230 GTTT~TTTTGT~CTGGCM~TT~GGCTCTGGM,AG&C 3190

3120

3200 3240 3250 3280 5260 3270 GCTCGTT,WCGTTGGTAAG,3TTChGGATA,WhTTGT$,GCTGG 3330 3340 3340 3350 OOblYQiTTCGCTA MKGCCTCGCGTTC 3400 3480 3410 3490 3420 3430 3440

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3320 3290 5300 3310 GTGC~&WTC)GCACICTP~TCTTG6,TTTAWGCTTC-CTCCCWTC 3370 3380 3390 TTIKiPlllT~CGG~TCIIKiCCTTCT~TaTCTOITT 3490 34LO 3470 GGCTTGCTTGTTCTCGOTG~TGC~T~TTTMTO

3500

SGIO 3590 3650 3640 3670

w20 3600

3560 3570 3530 3540 3550 3580 TTGGTTTCT~CATGCTCGT~TT~T~~T~~~TTTTTCTTGTTC~~TT~TCT~TTGTT~T~~ 3L10 3LZO 3640 3630 GTTCTOCCITT~GCTGIICTGTTGTTT~TTGTCOTTTCT

36Go 3760 3750

3690 3700 3710 3720 37x) 3740 CTTIITTCICTGGCTC~)CICIIITOCCTCT~CT~~TT~~TGTT-GTTGTT~~T~T~TTCT-TT-CCT~C 3770 3780 3790 3800 3810 3820 TGTTOIKICGTTGGCTTT~T~CT~T-~TTTGT~T-C~T~T~T~T~~-TTTTTCT~T~TT~T~TT 3850 3GtKl 3870 CCGGTGTTTCITTCTT~TTT~CGCCTT~TTIITCAC 5880 3890 3900

Filed 01/25/2008

3830 3910

3840

3920

3930 39.93 3940 3950 3970 TTICICT-T~TCITTTGM~~~TTTTCTCOCOTTCTTTGTCTT-TT-TT~~T-~TTT~T~T~TT~ 4010 4020 4040 4030 4050 4060 T.TCI~CCCCWlfCT~PIOCCOOIKiGTT~--T~TCTCTC~-CT~T~TTTT~T-TTC~T~TT-TCT~~ 4120 4090 4100 4110 eGcGTCTTMTCTmGcT?lTcGcT~TGTTTTc~GATTcT 4130 4140 PvWX&MMTTMTTMT-OMTTTB

3990

4000

4070 4160 4150

Page 9 of 18

4200 4210 4170 4180 4190 4240 4220 4230 GGTT~TTC~CTCAC~T~T~TTGFlTTT~TGTRCTGTTTccl3TT&AARMGG T,WTTCWTGkUTTGTTWATGT,V,TT6, 4280 4290 4256 42LO 4270 4soo CITTTTGTTTTCTTG~TGTTTGTTTCCITCTTCTTCTTTT~TC-T~TT~~~T-T~TTC~TCT~~~TTTT 4310 4320

Case 3:07-cv-02845-WHA

Fig. 1A. The nucleotide has been compiled earlier as reference message last digit. NOTE press, [Nature contain ADDED we learned an altered experiments a IN PROOFS: strand. point. (Larson and

sequence

of M13mp18. in RESULTS,

The sequence section b, and Using the programs 1983), the sequence described has been form using the original HincII site

as described Messing, This strand

Document 101-2

entered into an Apple II computer. printed out in the single-strand Numbers

also correspond

represents

the ( + ) or to bases aligned with the

Fig. 1 was modified in proofs because from a publication 311 (1984) 279-2801 to characterize 6125 of the Ml3 wild-type methionine-to-isoleucine (codon 40). The altered wild-type disruption sequence gene II product. sequence change gene II protein

after our paper went into by Dotto that the M13mp a G to T substitution in the and phage They used marker Zinder vectors rescue at position (6967 in Fig. lA), leading to gene II is expressed levels in M 13mp infected cells, but compensates of domain B of the M 13 ori region. The presented has been changed accordingly. protein at normal for the M 13

Filed 01/25/2008 Page 10 of 18

Enrym PO& PO,. Enzyme Site PC... ________________________-_______________---------____________--------_____ PO,. PO..

Sitm F""WI
( 691, (

Pm.

(QTCMC'

( 24'

6263,

GCNGC' ( ( 1,:: ( b29, ( 587, ( 73) ( 3b97, ( bl21, 2267 2520 5499 5922 7243 b3bO ( ( ( ( 24, bll, 141 129, ( 8'
So38

acCIcI hh.IK MUI ( ( ( (

(6QmcC

(a6cT'

( 6000, ( 39, ( 2b' ( 534, ( 313' ( 1330, ( 95'

( 104'
3;; 204' b4, 4s)

( ( 180, ( 27,

63 333 1516 3611 Sb29 bl17 6282 bbbb 6976
FOkIA FokIB h.IIPI "..I IS HI,IIC Ha.1 ID HarIII (WATB, (CATCC, (QeCQCC, (NCSCC' (WCBCT' (MCBCT' (WCC'

( ( ( ( ( ( ( ( (

140' blxl, 144b' 484' 331, 9S, 93, b3, 201,

203 933 29b2 4093 S9bO 6212 b37S b729 7177

2311 3131 5513 LOS1

Case 3:07-cv-02845-WHA

AVaIP

(ccCQ66'

(CTCQQ6'

( 2s) ( 111, ( 220, c 6246,

b2b3 bOO0 39 229 1407 3275 5425 bOs3 b217 b48b 6949 b24b

Mf3hcc'

( 5824, ( S913,

(
454,

6130
t+QaIA m&c6c,

(

334,

b4b4

2284 2sSb 4870 sS3s b3S4 sS4b 239 bm0 3558 6445 2709 1395 SOS0 5413 bO36 bS12 526 323b ( 329' ( 849' ( 158' ( 311' ( 257' ( 169' ( 1637, ( 3450, ( 107s, 2244 5238 5724 6293 bbal 21b3 ( ( ( ( ( ( 3W' 1Ob' 214, 102) 341) 315,

2553 5344 5938 bS9S 7022 2478

=: SO79 b2Jl Sb76 b242 LOO0 1248 ( 5226,

6476

CI".IC Pl".IIh 6.1 I BamHI Sh-lIFl BailIll BUIIC BUIID B.nIIB B."IID BbVIcI

62%

CT-' UJSATCC' m6CAcc, U36TACC, ea3wCc, (OOTSCC, (SMCTC' (SWCTC' (BCMC' Sb42 931 bsS4 13bb

BbVIB

(OCTDC'

( (
4bO4, 73,

5535 b427 3131

(

516'

bOS1

[email protected] HgiaIpI H9iaIB HgiaIC Hha1

(OCOTC, h3TBcpIc' (CABCTC) (GTOCTC, WCQC,

(

1154,

4870
b4430 b934

( 5079, ( b2Sl) ( s67L' ( 6242, ( 6000' ( 1248, ( 623.' ( Jb42, ( 931, ( 303, < Is&, ( 1739, ( 6430, < b9s4,
( (
lOS2,

( ( ( ( ( ( ( (

Document 101-2

BP11 ep1rr Cl.1 DdmI

(GCs, (MTCT' (ATCBAT' (CTMB'

( ( (

2S26, 233,

2526
4661 233 141b

( 4355)

( (
HincIIfi Hind111 Hi"+ I (OTCG&C, WASCTT' (OANTC,

46' 15'

( em, ( 367'
109S 1783 1876 2014 2347 4012 4092 4aaO bSOS
7188 472

9bb, 293, 244, 312, ba3, 9, 8, bS, 115,

1010 1469 2710 3407 4994 5511 SSb7 6094 bSb1

( ( ( ( ( ( (

74, 72S, 329, 190, 49S, 22, 434, 331,

1004 2194 3039 3197 5489 x135 bOO1 b42S

( 13' ( 996, ( 39, ( 1.50' ( 672,
7073
"pal

72,

( ( ( ( ( ( ( ( (

272, 63, 241 303' is, 271 28, 3E1, 399,
4149, 4b21

1370 1846 1900 2317 23b2 4039 4120 S2bl b907

( ( ( ( (

Dra1

(TTTAAA,

( 153' ( 189' ( 2162'

(
I

EEORI EcoRIIA EcDRIIB

(SMTTC, (!XA66) KCTOB'

(CCGG)

( 4032, ( (
( 3s3,

5997

FnuDI

I

tceco,

Filed 01/25/2008

771, 889,

( 45' ( 1739, ( 22) ( 503, ( sS46' ( 239, (6000' (5558, ( b44S, ( 2709, ( 1395, ( 2527, ( b9, ( 98, ( 117, ( 526, ( 758, ( 4082' ( 4742' ( b236' t-4, ( 44' ( 92' ( 272' ( 56, ( 714' ( 13, ( 26) ( 28' ( 21, ( bZb.5, ( b281' ( 1X.' ( 21' ( #' ( IbO' ( 234, ( 771' ( bs' ( 253' ( 261' ( 314' ( 829' ( lSt., ( 17b,
(

( 6230' ( 5940' ( 1013, < 330, ( 43, ( 57, ( 54) ( 361, C 24' ( 2'
( ( ( b136 196s 6454 347 24bS 3596 4995 9532 bbm( 1% 495, 49b, b3, 27, 1118 3354 3951 5488 bo2B 67% 1393

( ( ( (

3E: 1375' 489' 39,

z b23b S4b4 44 117b 24bb 3095 4311 SSOZ( 5559 6029 b44b b2b3 b2S1 136 511 2496 3417 4071 5119 5437 bO40 6884 314 1923 2551 4017 6247 6.037 1373 2397 2619

( 60' ( 212, ( 346, ( 324' < 45, ( 209, ( 328, ( 221, ( 19' ( bS1' C 4S4' ( 818' < lS9b' ( 21s' ( 123' ( 398' ( 144' ( 6,

216 723 2044 3741 4116 Ss2S( 37bS b2bl 6903 9bS 2377 33b9 Sb13 b4b2 b9bO 1773 2541 2625

( ( ( ( ( ( ( ( ( ( ( (

274' 1287' 413, 9b, 232, 4bl 22, 3b2' 157' 129' IS' 472' 545, IS, b0' 135' 39' 2221'

490 2010 32S7 se37 4340 5374 5787 6b23 7040 lW4 2395 3S41 6158 b480 7020 1908 2SeO 4e4b

Page 11 of 18

F""4HI

(6CNQC)

(

931)

lea1 ( IS, 1972 ( 42, 23s2 ( IS, 3360 ( 652' 4078 ( 14' 4280 C LOO, s9s-J ( 575' 70b0 ( 128' la9 ( 283' 6783 c 290' b230 5940 ( 19b, 1013 ( 952, bS27 ( 127, 43 ( 304, 1175 ( 1290, 3408 ( 190, 4312 ( bS1, 5512 ( 20' bOs0 c 658' 931 c 435'

E"lYnr Bit. PO,. __~_---~~~~~~~___~~______----__------_-------------________-_-____________ 570s 7004 Flsa* ( 241, 5946 (GTRCJ

PO,. PO`.

Hnhl6

(GGTBIIJ

1969
3667 S3E3

WxCCJ 2634 ( 2288, 4922

( 589, < 1025, < 1132, I , ( ( , ( 1164 leea 2132 4189 5483 7164 6236 176a 1904 3466 4379 6243 ( ( < ( ( 27) 65) 201) 1004, 599, ( ( ( , ( 604, 16) 1334, 190, 758,

(GGTACCJ &Bc)TCJ ( ( 332) 153) sac1 Sal I Sl"96I (GWCTCJ (GTCGPIC) (GGNCCJ 1713 6405 < ( 507, 96, 2220 6501

6042

6263
( 190, 5913 ( 24) 5937 196S 6136 ( , (

( ( ( ScrFI ICCNGGJ , Gf."Il BtmIB smar SnaBI WhI TlSI ( ( ( , (GCWCJ (CCCBGGJ (TMGTAJ (GCATGCJ (TCGRJ (GATGCJ ( f ( I , , 910, 571 1, 383, 363, 1696,

2479, 196, 332)

6390 4270 sSa6

Case 3:07-cv-02845-WHA

tln11a

(CCTCJ

3911 4074 5254 6604 373 1923 S997 6247 6637 3Ba( 6545

421 139, 80, PCS) 13) (

6327
1353 6Ssm

( I ( ( ( ( ( ( ( ( ( ( ( < ( ( ( ( ( 5723 6395 1013 5940 6246 MS4 25 4849 3978

-..-

( ( ( ( ( f ( ( ( I 790, 379, 971, 30, XhI XhOI IPI XhoIIB Xh0IID Xmnl These (TCTAMJ (GGATCCJ WGATCC J (PBaTCT J (GMNMNNTTCJ 6246 1267 6275 336 1948 3693 6234 62S7 6251 2219 6934 3s7 (

187) 383, 66, 191 391, 243, 41 269, 604, 149, 69) 2351

Sbo 1036 1296 1344 la96 2262 2676 3320 4306 4920 5415 6255

1126 2527 4664 6264

( f ( (

381, 927) 101e, 618,

1507 34S4 5682 6882

143, 93) 163, 522, 102J 322) ( 6236, ( 6263J ( 5723, ( 458, ( 1013, ( 3975, ( 110, ( 127, ( 25, ( 3496, ( 3978, ( 6246) ( 1267, ( 6275) ( 336) I 441) ( 239, ( SJZJ ( 6257, ( 62SIJ ( 2219, ( 6934) , 357 J 22eaJ 264s

Document 101-2

( 1694, ( 3293, ( 318) ( 301, ( 119) < be, ( 143, , ( J: ( 75, ( 404, 1se, ( 351, ( , 74, ( 421, 340, 423, ( 141, ( ( 102, IS, ( f 15) 4, ( ( 799, 947, f 105, f 6SSc 1230 132S ISOS 2019 2672 3051 3702 4771 S346 6020 6933 62S 1833 1878 2334 2368 4819 6393 7190 ( ( ( < < ( ( 747, 1SJ 129) 1SJ 9677) 13, 117, 1372 1848 2007 2349 333S 4834 6510

anzymes
did

not

appcsr.....ArtII

I

"&I tlmt11 NUI

Nat-1
4324) 1080) 47, 499, 698, 99, 55, 187, 232, 18, 211 130, 213) 6247 3802 196 179G 2854 3717 6276 1248 lBO2 2391 S676 6130 6464 ( SW, ( 3043, f 910) ( 23SJ ( 677, < ll6lJ < SW, ( 292, ( 248, ( 32S2J ( 1901 ( 112, , 12, 6037 6845 llD6 2033 3531 5178 beS6 lS40 2050 S643 Se66 6242 6476

CVIaIIB &"*I IB B&XI HlncIIC Not1 Bt.UI

CICCIB AhaIID BrnIIR Bea1 I HincIID NruI TthlllI

#CCIC @aI EWII XC EcDRV HpaI Nsi I XhoI

ACCID Plva1s EC11 HPiaID IllUI sac11 XhoIIC

al.IIA ra".ID Bs,hI HincIIB NC01 SC*1 XIrnIII

Nrl IA NC1 IB Nd.I NIpI

(TGCGCAJ (CCTNRGGJ (GCCGGCJ (BBCBCCJ (CCCGGJ (CCGGGJ (CCITATGJ (MT%,

Filed 01/25/2008

NIaIV

(GGNNCCJ

( ( ( ( ( ( ( ( ( ( ( ( (

P&I PVUI PVUI RsrI < ( bOS2 280 ( ( 322, 741, 93, 107, 6374 ,021

Page 12 of 18

1

(CTGCAGJ (COIITCGJ (C-G, (GTACJ

270, 33, lSO2J 2loEJ 6242) 1381, 4032, 434, 2217) 781) 666, 917, 254, 271 49) 21, 71) 481 6, ( 217, ( 31, ( 391, , 3, ( 263) ( 277, ( 484, ( 339, ( 15, ( 312) , 15) , 683) ( 6121 I 575, ( 6424, ( bSO7J ( SblZJ 16000, ( 6246, ( 1923) ( 2722, f 149, ( 193, I 123, , 87, c ,043, ( ioa1, , 10, ( 3231 ( 121 f 134, c 9, ( 6269J (64WJ ( S9S9J < 173,

5116 5979 lSO2 7030 6242 1381 62S2 6933 2217 781 4936 603 234 Se7 1087 1317 1415 1944 2268 2893 3351 4697 4925 SbBO( 6532 484 1731 la63 2319 2364 4020 5446 7OBS 6424 6507 S612 M)(K) 6246 1923 2722 149 1299 21S6 3618 6221 1061 1SSO 2373 Sbss 6Oao 6251 6269 6404 5959 173

Fig. 1B. Restriction sites of M13mp18. The position (Pos.) and distances (in parentheses) between the neighbouring restriction sites are specified in nucleotides, as compiled and printed out employing the restriction mapping program. (See legend to Fig. 1A).

b

10 20 30 40 TCGCOCGTTTCWT~TOCGGTG~~~CCTCTDTTGTCTGT~CffiAT
50 1670 60 70 GO 140 1720 1730 1740 17sO iSO lb0 lb10 1420 lb30 1640 1630 1460 TATRTOCWTCVIACTTWTCT~C~TTOCCCT~TTC\CITC~T~~CTaTCTC~~TCTGTCT~TTTCGTTC~ 1690 1700 1710 TCCPITLU3TTGCCTGACTCCCCGtCGTLIGCITP.CTT ,770 ,780 1790 1600 lEl0 LICCGC~CICCC~OCTCCICCWCTCC~WTTTATCRDC~TACI~CAOCC~C~~CGaGC~aQ~tWTC lG50 lGL0 1870 1880 1890 t900 CTGCAACTTTFITCCOtCTCC~TCC~GTCT~TTCILITTGTTQCCG~CT~~GTCT~TTCQCC~TTPILQTTTG 1930 1940 ,950 19bO 1970 ,980 CGCCILICGTTGTTDCCaTTQCT~Ca~~TCGTGGTGTCnCGCTCGTCGlTTWTaTWCTTCaTTC~~TCC~TTCCCa 2010 2020 2030 ~CGPITC~CQAQTTRCATGIITCCCCC~TGTTGTGC2040 1G20

1530 1540 1550 TTTWTCATG~~TTCITCC~aTCTTCACCTC

1560

1570

1SSO

,590

1600

90 100 110 t20 130 GCCGG~CI~CIPIOECCGTC~G~~GTC~~TGTTGGC~TGTCWGGCT~TT~T~TGC~~TC~Gn 210 220 ES0 -TaCCGCATCLGCGCC 310 320 240

l&E0

170 180 190 200 GC~GATTGTACTG#?GMTQChCCAT~TGCGGTGT~TMXBXC~TGCGT~~

,760

as0 260 270 280 290 300 PlTTCOCCATTCCIGOCTGCOtC\ACfGTtWDFHIWOCDPlTCGGTGCGGGCCTCTTCGCTI\TTCICGCC~TWC~GG 590 400

,850

1840

Case 3:07-cv-02845-WHA

330 340 350 360 370 380 ~TGTGCTOCAAWCGATTCI~GTTGGGTPlLICGCCCIBOGTTTTCCCCIGTCLICG~CGTTGT~CG~~C~GT~~TT 480

1910

1920

410 420 430 440 450 4*0 470 CGI\GCTCGGT~CCCGGGGATCCTCT~G~GTCGLICCTGCAGGCCITBCPlAOCTTGGCGT~TCLITCWCTGTTTCCT s!sO 5LO

1990

2000

490 JO0 510 520 530 540 GtGTGACIPTTGTTPlTCCGCTC~C~~TTCC~C~C~C~T~CG~CGG~~~~T~~~GTGT~~~GCCT~GGT~CT~~TG 640

2030 2060 2070 ZOGO GCGQTTP,QCTCCtTCGGTCCTCCGATCGTTGTC&GhA 2lSO 2160

370 SGO 590 400 610 620 LJO ~GTG~QCTCICICTC~WTTAATTGCOTTGCGTTGCGCtC~tGCCCGCTTTCC~GTCGGG~~CCTGTCGTGCC~GCTGC~TT~~T 710 720

2090 2100 2110 2i20 2130 2140 GT~TTGGCCGCPIDTGTTCITCAttCATODfTCITGDCClOEACTGCPlTMTTCTCTTCICTOTCaT~C~TCCGla~T~ 2170 2180 2190 2200 2210 2220 TTTTCTQTOCICTQGTGCIOTCICTCaaCC~TCaTTCT~TffiTQTaT~WC~~TT~TCTT~CCQGC6TC 2230 2260 2270 2280 2290 AAT~CGGG&kTAP1T`XCGCGCC~C~T~QCAQMCTTTMMQTGCTC~TC~TTG2300

M50 bb0 670 LB0 690 700 GPIPITCGGCC~CGCGCGGPIG(Ki6CGGTTTOCaTCTTCCGCTTCCTCGCTC~CTGCICTCGCTGCDCTCG 770 760 790 800

2230

2240

730 740 750 GTCGTTCWCTGCGGCGLIGCGGTaTCACTCAMCDGG 870 2410 2420 860

7&O

2310 2320 GTfCTTCWGGCQAAAACTCT 2390 2400

Document 101-2

810 620 830 840 BSO EL0 A~GACICATGTG~GC~~~~G5CCffiC~~~~GGCG~GG~~CGT~~~~ffi~CGCGTT~TWCGTTTTTCC~T~GCTCC 940 2490 2500 9GO 960

2330 2340 2350 2340 2370 2380 CPdOQPlTCTTPlCCDCTGTTGAGITCCAOTTCGnTGt~CC~TCGT~nCCC~CT~TCTTC~~TCTTTT~TTTC 2430 2440 2450

090 900 910 920 930 GCCCCCCTGCICGCIGCLlTCaC~lCGPICOCTC~~TC~GGT~Q~~CCC~~TaT~GaTaCC~CG 1040 2570 ,090 2hSO 1200 1100 1110 1120

ACCROCGtTTCtQQGTGCIQC~A,+,MChGG`MWCAAAhTGCCQC~~G~AT 2JIO 2520 2530

2460 2470 CICIWOCWIChC-TGTTGAAT 2s40 2550

2480

2560

970 980 990 1000 1010 1020 1030 TTTCCCCCTGGPlLIGCTCCCTCGTGCGTCTCCTGTTCCGCICCCTOTTllCCGOCITPlCCTGTCCOCTTTCTCCCTTC

~TCPTACTCTTCCTTTTTCPI~TPITTPlTT~GC~TTTCITCPlOWTTPltfGTCTCATGIIQCWPT~C~TPITTTGACITGT~ 2580 2S90 2600 2610 2620 2630 2640

1030 1060 1070 GGGACIGCGTffiCCICTTTCTCCLCITOCTCACOCT

IOGO

TTT~G~~~T~~PIC\IITCIBBCIOTTCCGCttCICPTTTCCCCMLGTGCCPlCCTGACOTCT~~~~nCCnTT~TTaTC 2&O 2670 2mo 2t.90 2700 27LO 2720

it30 1140 llS0 1160 1170 1180 1190 TGCACBLIClCCCCCCGTTCIlOCCCG~CCGCTGCGCCTTCITCCGGTMCTCITCGTCTTGCIGTCMP1CCCGGTC\PlGPlCPlCDPlC I280

,-,TG~C~TTRACCT6,TA,V,F,~TAGQCGT~TC~C~GQCCCTTTCGTC

1210 ,220 1230 1240 ,250 12k.o 1270 TlnTCGCCaLTQGCnGC~GCC~CTGGTaaC~GGaTTaGCaGaQCG~GGTaTQTnGGCWTGCTaC~GaGTTCTTG~~QTG ,350 ,360 TKCTTCGGAAAAAGAG 1440

Filed 01/25/2008

1340 ,320 ,330 -TGGTCITCTGCGCTCTQCTG&hQCCICiT

1370 1380 1390 1400 1410 1420 ,430 TTGGTPlGCTCTTGPITCCGGCAC\PIC~~PCCICCGCTOOTLIOCOOTOQTTTTTTTGTTTGCCIAGCPlGCP~TTI\CGCGC~GCI 1510 1520

1450 ,460 1470 L4BO 1490 1500 BCICI~PIIIWPITCTC~AG~~TCCTTTGCITCTTTTCT~BGWTCTGCCGCTC~GT~~~~~CTC~GTT~PlT

Page 13 of 18

09
ma. ma.
Pm. ENZYIY SlTE

Case 3:07-cv-02845-WHA

ENIY"E SITE _______________________---------------------------------------------------

Document 101-2

98, 97,

234 I 2324

Filed 01/25/2008

Fig. 2. The nucleotide compiled as described from sequencing earlier (Larson opposite strand, complement in Fig. 1B.

sequence

and restriction and entered and Messing, read from the same strand. is given. The message of pUC19 strand (referred

sites ofpUC19. into an Apple II computer. pUC6 (Fig. 4) was used to make corrections Since all coordinates the single-stranded is obtained

(A) The sequence The information via the programs 1983). Both the blu and the lac a-peptide of the pBR322 DNA with the polarity with the program to as pUC19V). published (B) Restriction sequence that produces

has been derived described gene products are refer to the by Sutcliffe (1979) the reverse site coordinates are as

Page 14 of 18

Case 3:07-cv-02845-WHA
116

Document 101-2

Filed 01/25/2008

Page 15 of 18

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

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 regeneration 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.

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 wildtype 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

Case 3:07-cv-02845-WHA

Document 101-2

Filed 01/25/2008

Page 16 of 18
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

-b restriction -_e enzyme sites Primer hybridization site Cleavage at siteg leaving recessed 3' end

Cleavage at site a leaving protruding 7 end

Klenow -S-NTP

_

I
'
d

_

1.

Cleavage at b leaving blunt or recessed 3' end

r

_

I.

(2)

(3)

Exo VII

-

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0

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Klenow NTP's Ligase

Transformation

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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 regionleft 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 increasingly 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 nomandom sequencing approach based on BAL31 treatment described below (Poncz et al., 1982) and illustrated 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-

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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 exon&ease and ligase treatment, the DNA is transformed into JM105. Plaques are picked from each transformation experiment, 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.

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-FG0284ER13210 and NIH GM31499. J.V. is supported by the NIH training grant No. ST32 GM107094.

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 consecutive 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 opposed 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-

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