Gene, 120 (1992) 135-141
0 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119~92/~05.~
GENE 0673 1
A selective ;1 phage cloning vector with automatic excision of the insert in a plasmid (Spi selection; Caenorhabditis elegans; cDNA library; bacteriophage P 1; cre-lox site-specific recombination; phagemid)
Ichiro N. Maruyama and Sydney Brenner* MRC Molecular Genetics Unit, Hills Road. Cambridge CB2 2QH, UK
Received by J.A. Hoch: 1 April 1992; Re~s~/Accept~:
21 June/22 June 1992; Received at publishers: 9 July 1992
A bacteriophage Acloning vehicle has been constructed for the generation of cDNA libraries. The vector has the following properties. (I) It has a unique BamHI site engineered into the I gam gene. Segments of DNA can be cloned into this site and clones with an insert can be selected by their ability to grow on an Escherichia coli host lysogenic for phage P2 (Spiphenotype). (2) When the recombinant phage infects a Cre-producing E. colr’strain, a site-specific recombination event results in the excision of a plasmid replicon with the cloned insert. (3) Single-stranded DNAs can be recovered by growing helper Ml3 phages on bacteria harboring such plasmids. The vector, ,JMGU2, has been used to construct a nematode (Caenorha~dit~s elegam) cDNA library.
Efficient genomic and cDNA cloning vectors are important tools in molecular genetic research, because highquality, representative libraries are rich sources for the analysis of many genes. Since the demonstration that DNA
Correspondence to: Dr. I. Maruyama at his present address, Department of Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel. (619)554-4015; Fax (619)5546943. * Present address: Department of Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel. (619)554-4015.
Abbreviations; aa, amino acid(s); Ap, ampicillin; bp, base pair(s); cDNA, DNA complementary to RNA; Cre, phage Pl recombinase encoded by cre gene; ds, double strand(ed); gam, phage 1. gene encoding a protein which inhibits E. coli RecBC enzyme activity; Km, kanamycin; kb, kilobase(s) or 1000 bp; IoxP, phage Pl genetic element recognized by Pl recombinase Cre; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PCR, poiymerase chain reaction; R, resistance~resistant; Spi -, insensitive to P2 interference; ss. single strand(ed); Tc, tetracycline; [ J, denotes plasmidcarrier state.
segments can be cloned and propagated in bacteria in plasmid (Cohen et al., 1973; Morrow et al., 1974) or viral (Murray and Murray, 1974; Rambach and Tiollais, 1974; Thomas et al., 1974) vectors, several cloning vehicles derived from bacte~ophage 1 have been developed. Ligated mixtures of insert and 1 vector DNAs can be packaged in vitro with efficiencies approaching 10% and introduced into bacteria by infection. In addition, positive selections exist to facilitate the recovery of cloned DNA inserts in phage Iz. For example, the Spi- phenotype, the ability of phages lacking gam gene function to grow on E. coli hosts lysogenie for P2, has been used widely for the selection of recombinants in vectors such as 11059 (Ku-n et al., 1980), and derivatives such as IEMBL (Frischauf et al., 1983) and A2001 (Kam et al., 1984). However, these vectors have two disadvantages. They are ‘replacement’ vectors; that is, inserts must be cloned as substitutions of a ‘stuffer’ fragment. Furthermore, they accept rather largeinserts of lo-20 kb to bring the phage genome to a packageable size. Phage E.vectors have other favorable properties for library construction. Phages can be stored at 4” C and larger numbers of plaques may be screened more efficiently than
136 bacterial colonies. On the other hand, vectors based on plasmids, and particularly those containing an additional M 13 replication origin (‘phagemid’ vectors), have other advantages. Their smaller size makes it easier to analyze recombin~t clones by restriction mapping and sequencing. Phagemid vectors can produce ssDNAs for sequencing, site-directed mutagenesis, and use as strand-specific hybridization probes. It would be useful to combine all of these properties into one vector. The vector lZZAP has some of these features (Short et al., 1988). It contains a phagemid within a ,X phage, and excision of the phagemid may be achieved after infection with Ml3 helper phages. Although inserts can be detected by screening for the inactivation of lac complementation, there is no direct selection for inserts. There are selective phage I vectors for recombinant clones, AgtlO (Huynh et al,, 1985) and pAZd39 (Murphy and Schimke, 1991), but none permits the excision of a plasmid in vivo. We report the construction and use of a bacteriophage /z cloning vector in which excision of a phagemid is accomplished by the lox/cre site-specific recombination system. Similar vectors have been developed by others (Palazzolo et al., 1990; Elledge et al., 1991), but our construct enables the positive selection for recombinant clones. For this purpose, an engineered A gum gene containing a BumHI site has been utilized. Cloning into this site allows selection on P2 lysogens of E. coli, We have used these vectors to make cDNA libraries with high efficiencies and show how the clones can be analyzed as 2 phages, plasmids, or Ml3 phages.
RESULTS AND DISCUSSION
(a) Vector construction Wild-type ;I bacteriophages cannot grow on E. coli strains lysogenic for bacteriophage P2; phages that form plaques on such strains must have a defective gum gene. In order to use this property to select for the cloning of small DNA fragments, the gum gene was modified to generate a new BamHI site in its coding sequence. As shown in Fig. 1, a BumHI site was successfully created by site-directed mutagenesis, which results in the conservative aa change, L~u~~+ Ile. This change does not inactivate gam. When the Sal1 fragment containing the modified gum gene is cloned into the gum - phage, i/2690, the resulting phage AMGU has a Gam’ phenotype. Thus, 12690 grows on host 4359, a P2 iysogen, and fails to grow on RecA- strains, whereas lMGU grows on the RecA - strain, and fails to grow on 4359. The phage IMGU, with a unique BarnHI site in the gum gene, is a useful phage Avector for the selective cloning of small inserts. In order to provide a phage 3, vector with an ability for
Fig. 1. The nt sequences of part of the mutagenized gam gene. For the site-directed mutagenesis of the Agam gene, a 499-bp Son fragment was cloned into an M 13mp8 vector (Vieira and Messing, 1982) at the SalI site. The 499-bp SafI fragment, nt 3274633244, contains the entire coding region of the gam gene (Daniels et al., 1983). To create a BumHI restriction site, the phage sequence was altered using the synthetic oligo 5’GAAAAAGGGATCCCCCAG and an Ml3 universal primer according to published methods (Zoller and Smith, 1984). After site-directed mutagenesis of a 499-bp Sal1 fragment in M13mpS vector, plaques were transferred onto a nylon membrane and hybridized with the “P-labeled mutagenic oiigonucl~tide to identify the positives. These were contirmed by nt sequencing with an Ml3 universal primer (Sanger et al., 1977). Mutations created by the mutagenesis are labeled with open triangles. The wt sequences are also shown on the right.
the release of a phagemid in vivo, we devised a phagemid, pMGU, which expresses the modified gam gene and contains the bacteriophage PI site-specific recombination site, loxP, as shown in Fig. 2. The modified gam gene, with its BamHI site, was removed by digestion with Sal1 and &a1 enzymes from a replicative form of the Ml3 phage clone. The fragment was then cloned into a plasmid vector pKIS S (Pharmacia, Uppsala, Sweden), as a substitution for the XhoI-SmaI fragment of the ~lpt (KmR) gene, to produce plasmid pKG1. Plasmid pUM13 is a derivative of Bluescribe Ml3 + (Stratagene, La Jolla, CA) made by inserting an oligo in frame into the polylinker sequence to give additional XhoI and NsiI restriction sites. This vector was digested with XhoI, and a 1.17-kb PstI fragment of pKG1, which contains part of the KmR gene and the mutagenized gam gene, was cloned into this site with synthetic oligo linkers containing the restriction enzymes PstI, SacI, KpnI, S$rI and XhoI, to provide pKG2. Plasmid pLP1 was constructed from pRH536 (Abremski et al., 1983) by removing the npt gene after digestion with BamHI+HindIII, followed by replacing the gap with a synthetic oligo linker, 5’-AGCTGTCGAC 3’-CAGCTGCTAG. Plasmid pLP1 has a fragment consisting of two directly repeated IoxP sites separated by a unique Sad restriction site. Finally, pMGU was made by ligating the 162-bp pLP1 XhoI-EcoRI fragment to the pKG2 871-bp XhoI-HindI fragment and cloning the resulting product between the
137 EcoRI ~CGATCATATTCAATAACCCTTAAT~TGTATGC~
SalI ~TAGGTCTGAAGAGGAGTTTACGTCGAGCCAAGCTGJXX&GATCCGGAACCCTTM XJIOI TWmTGTATGCTATACGAAC.TTATTAGGTCCmTACCGCATGC -35 GAGCTCGACCTGCAGGGGGGGGGGGG-GCCACG ZULXGXTCAAAATCTCTGATGTD -10 ~CACAAGATAAAAATATATCATCATGAACAAT-CTGTCTGCTTACATAPACAG
120 180 240 300 360
RRATGGATATTAATACTGACTGAGATC~GCACCCCCTTTCCTG mdintdteikqkhsltpfp TTTTCCTAATCAGCCCGGCATTTCGCGGGCGATATTTTCACAGCTATTTCAGGAGTTCAG vflispa frgryfhsyfrss CCATGAACGCTTATTACATTCAGGATCGTCTTGAGGCTCAGAGCTGGGCGCGTCACTACC aMNAYYIQDRLEAQSWARHY Primer 1 BanHI AGCAGCTCGCCCGTGAAGAG PACGACATGG AAAAAGGGATCC QQLAREEKEAELADDMEKGI Primer 2 CCCAGCACCTGTTTGAATCGCTATGCATCGATCATTTGCAACGCCACGGGGCCAGCPQHLFESLCIDHLQRHGASK AATCCATTACCCGTGCGTTTGATGACGATGTTGAGTTTCAGGAGCGCATGGCAG~CACA TRAFDDDVEFQERMAEH K s I TCCGGTACATGGTTGRAACCATTGCTCACCACCAGGTTGA IAHHQ”DIDSE”* I RYMVET
660 720 780
TTTGGTTGATGCGAGTGATTTTGATGACGAGCGT~TGGCTGGCCTGTTG~C~GTCTG Hind111 GAAAGAAATGCATMGU,T
Fig. 3. Sequence was sequenced
of part of pMGU.
sites are underlined sequences
A 1039-bp EcoRI-Hind111
to confirm the construction. and indicated
of the promoter
by their names.
Daniels sequence. Fig. 2. Construction mid vectors,
of a pMGU
vector. The circular
but inserts relevant to the construction
outside the circles. The arrows
on the restriction
ing region and the transcription
are shown construction
as blackened are described
maps denote plasare depicted
by lines the cod-
from recovered rescued
of the gam letters from
et al., 1982) under
2 was used to sequence
These two primers
tion of insert DNAs
aa sequences code (capital
1 was used to sequence plasmids.
loxt’ sites are shown by two sets
in the single-letter
et al., 1983; lower-case Primer
of the Tn903 KmR gene (Oka et al., 1981) are
of a pair of inverted repeats of arrows. gene product
were also used in PCR amplifica-
of the gam gene. The IoxP sites and pMGU.
in section a.
EcoRI and Hind111 sites of pUM13. The nt sequence of the 1039-bp EcoRI-Hind111 fragment of pMGU is shown in Fig. 3. The construction of phage vectors lMGU 1 and rlMGU2 is outlined in Fig. 4. Vector lMGU1 was constructed by ligating pMGU and A2690, after digestion of the two with S&I. The orientation of the plasmid in phage 1 was determined by restriction mapping. In vector IMGUl, phagemid pMGU DNA was flanked by direct repeats of 1oxP sites. Vector IMGU2 is a deletion derivative of lMGU1 made by removing a 3.2-kb SmaI fragment, and has the capacity to accommodate larger inserts. Although we do not know the exact nt length of the IMGU2 vector, restriction fragment analysis gives an estimated size of 41.7 kb. It can therefore accommodate an insert up to 10 kb in size, a capacity large enough for most cDNAs. Stocks of these gam + phages plate with less than lo- 4 efficiency on the P2 lysogen, 4359.
To excise the phagemid, we used a strain in which the phage cannot grow and which expresses the Cre protein, the trans-acting component of the Pl site-specific recombination system (Sternberg and Hoess, 1983; Sauer and Henderson, 1988). E. coli 1046[pCREl] (Cami and Kourilsky, 1978) is a RecA- strain carrying the plasmid pCRE1 which expresses Cre from its own promoter. Plasmid pCRE1 was constructed by cloning a 1.6-kb PvuII fragment from pRH 147, which has the cre gene of bacteriophage Pl under the control of E. coli IucZ promoter, into pBR322 (Bolivar et al., 1977) at a ScaI site. This insertion results in inactivation of the ApR gene on pBR322. On 1046[pCREl], phages with an inactive gum gene cannot grow productively, and are lost during colony growth. Because excised phagemids can replicate in 1046[pCREl], cells carrying the phagemids can be selected as ApR survivors of phage I infection. The phagemid also carries an M 13 replication origin so that ssDNA can be recovered by infection with M 13 helper phages such as M 13K07 (Vieira and Messing, 1987), using well-established techniques (e.g., Dotto and Horiuchi, 1981; Dente et al., 1983).
h 2690 Wind111
Fig. 4. Construction of lMGUl by digestion with XbaI, followed
and IMGU2 vectors. Phage A2690 is a derivative of 22001 (Karn et al., 1984) from which poiylinkcrs were removed by the removal of the 499-bp Sal1 fragment. The arrows indicate the transcription directions, except for the J and N
genes on lMGU1; the left arm up to the J gene and the right arm up to the N gene are omitted. The IoxP sites are shown as blackened boxes; IG denotes intergenic region. The shaded arrows are the gum gene and the shaded box in AMGU2 is the plasmid pMGU. AMP on the pMGU map denotes the ApR gene, Restriction
to the construction
are shown on the map.
(b) cDNA library cons~uction
To test the vectors, we used AMGU2 for the construction of a cDNA library from the mRNA prepared from a mixed population of the nematode C. eleguns (see legend to Fig. 5). From 0.1 fig of cDNA ligated with 1.5 pg of
dMGU2 DNA-S, followed by in vitro packaging, 10’ independent recombinant phages were recovered on Q359 strain. To test the quality of the library, twelve plaques were picked at random and their inserts were analyzed using the PCR with two primers flanking the BumHI site (Fig. 3). All
Fig. 5, Plasmids recovered from phage Izclones. Plasmids were rescued by the methods described in Table I from phage I clones of the nematode eDNA library. Plasmid DNAs were prepared From 1.5.ml overnight dtures of bacteria rescued according to the p&fished method of Biiboirn and Doly (19793, digested with Not1 enzyme, and separated on a 1.0% agarose gel. Not1 restriction sites were provided as part of the adaptor sequences while constructing the library so that the fragments liberated by the enzyme correspond to the sizes of the insert, The largest fragment in all six lanes is the vector pMGU and the other fragments correspond to the inserts whose positions are shown by arrows on the right (from top down) with lengths of 4.1, 3.15, 2.6, 1.6, 0.94,0.72, 0.56 kb. The nematode cDNA library was constructed with LMGUZ vector by the fohowing methods. Ma~ten~ce and culture of the nematode, C. elegntns strain N2 were previously described (Brenner, 1974; Sulstan and Brenner, 1974). From 5g of animals, total RNA was obtained according to standard methods (GliSin et al., 1974; Ultich et al., 1977) After purification of mRNA from the total RNA by an ohgo(cellulose column chromatography, cDNA was synthesized according to the protocol of the supplier (Amersham, UK), using approximately 3.0 fig of the mRNA preparation. A synthetic adaptor (OS gg) with BnmHI cohesive ends, 5’-GATCCGCGGCCGCATAGGCC 3’-GCGCCGGCGTATCCGG, was ligated to 0.3 pg of the cDNA. Residual unligated adaptors were removed by agarose electrophoresis, and the cDNAs with attached adaptors, 0.5-10.0 kb in length, were purified from the agarose. About 0.1 gg of purified cDNA was ligated with 1.5 pg AMGU2 vector DNA cut with BumHI. The Iigation mixture was packaged in vitro (Hohn and Murray, 1977; Sternberg et al., I977), and plated on the seIective 4359 strain.
the clones had inserts which ranged from OS to 4.0 kb in length (data not shown). Six of these were analyzed further by preparing plasmid DNAs from the phage 3, clones. Plasmid clones with cDNA inserts were obtained by infecting E. cofi 1046[pCREII with the hMGU2 clones. The average efficiency of recovery is 20% and is indepen-
dent of insert size (Table I). The size of the inserts was determined by NotI enzyme digestion on preparations of plasmid DNAs (Fig. 5). None of the clones analyzed had only an adaptor or multiple cDNAs joined during ligation as the insert. ssDNAs were also prepared with M13K.07 helper phages, following published methods (Vieira and Messing, 1987). The yield of ssDNA is variable and in the range of 10 bg per ml culture. As shown in Fig. 6, the ssDNA from pMGU1 in Table I, which corresponds to No. 1 in Fig. 5, was sequenced with an Ml3 universal primer, using the dideoxy termination method (Sanger et al., 1977). As expected, only one laxP site is found, and the spacer sequence, including the unique Sal1 site, is deleted. This confirms that the excision of the phagemid has occurred through the site-specific recombination between two ktxP sites on the phage vector. Subsequently about 10’ plaques of this cDNA library were probed with a genomic clone of the unc-13 gene of C. &guns (Maruyama and Brenner, 1991), and seven independent cDNA clones were isolated and confirmed to carry ttnc-I3 DNA by sequence analysis. In addition, five putative homologues of the tine-13 gene were detected and are currently being analyzed by DNA sequencing. Other cDNAs, including mup-2 and unc-53 (T. Bogaert, personal communications, also have been isolated from this library. Moreover, human liver and placenta cDNA libraries have been successfully constructed with the aMGU2 vector (K. Hadfield, personal communication). The JMGU2 vector enables the automatic excision of TABLE I Frequency of recovery of plasmids from phage A clones * Clones
pMGU I pMGU2 pMGU3 pMGU4 pMGU5 pMGU6
Insert sizeb (kb)
Frequency’ (% i: SEM)
2.60 0.94 3.15 0.72 1.60 0.56
14.9 * 10.6 26.6% I1.I 24.7 f 10.0 23.3 + 9.8 19.7 * 4.5 21.1 k6.7
B Phage particles isolated from a single plaque in 0.1 ml of A-dil buffer were added to 0.1 ml of an overnight culture of 1046[pCREl] grown in CY medium containing 15 fig Tcfml and 0.2% maltose. After incubation at 37°C for 20 min, 1.X ml of 2 x YT broth was added and the incubation continued for a further 1.5 h to ahow expression of Apn gene carried by the pMGU vector. Colonies carrying ptasmids were recovered by plating 0.2 ml of the samples with 3.0 ml H top agar on 2 x YT agar plates containing 75 pg Ap/ml. For 2 x YT media see Sambrook et al. (1989). b For insert size, see Fig. 5. c The frequency of the plasmid recoveries is expressed with the ratio of the number of colonies formed on the plate to the number of phageparticle input. Data were the mean 2 SEM of three independent experiments.
inserts into pMGU in vivo. Although each of the flanking IoxP sites is 49 bp in length, we have seen no spontaneous deletion mutants, even when the phages are grown on recA + strains such as 4358. Such excisions in the parental vector would result in gam - recombinants; stocks grown in a recA+ host have a background of less than 10e4 gamphage, and can be grown on a recA - strain to ensure retention of the gam gene. The host, 1046[pCREl], does not support productive replication of the phages and the excised pMGU plasmids with their cDNA insert survive as ApR colonies. The efficiency of this process is about 20 %, and we have consistently produced colonies from a single plaque of IMGU2 clones. We chose a recA - host rather than a phage I lysogen to prevent growth of the phage, because we found recovery of excised plasmids to be lower in Ivpo~~nc Prprllm~hl~r th;c ;o hnnmsxn- AL--------
141 culture medium as a filamentous phage after supe~nfection of the bacteria with helper M13. Thus, using this system one can analyze and utilize recombinant clones as a phage iE,plasmid, M13, depending on the purpose of the experiment.
We are grateful to R. Hoess of the DuPont Company for providing the plasmids pRH147 and pRH536. We also thank W. Szybalski and N. Kumar for their critical readings of the manuscript.
REFERENCES Abremski, K., Hoess, R. and Stemberg, N.: Studies on the properties of P 1 site-specific recombination: evidence for topologically unlinked products following recombination. Cell 32 (1983) 1301-1311. Bimboim, H.C. and Daly, J.: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7 (1979) 1513-1523. Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W., Crosa, J.H. and Falkow, S.: Construction and characterization of new cloning vehicles, II. A multipurpose cloning system. Gene 2 (1977) 95-113. Brenner, S.: The genetics of Caenorhabditis elegans. Genetics 77 (1974) 71-94. Cami, B. and Kourilsky, P.: Screening of cloned recombinant DNA in bacteria by in situ colony hybridization. Nucleic Acids Res. 5 (1978) 2381-2390. Cohen, S.N., Chang, A.C.Y., Boyer, H.W. and Helling, R.B.: Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA 70 (1973) 3240-3244. Daniels, D.L., Schroeder, J.L., Szybalski, W., Sanger, F., Coulson, A.R., Hong, G.F., Hill, D.F., Petersen, G.B. and Blattner, F.R.: Appendix II: Complete annotated lambda sequence. In: Hendrix, R.W., Roberts, J.W., Stahl, F.W. and Weisberg, R.A. (Eds.), Lambda II. Cofd Spriug Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 469-676. Dente, L., Cesareni, G. and Cortese, R.: pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 11 (1983) 1645-1655. Dotto, G.P. and Horiuchi, K.: Replication of a plasmid containing two origins of bacteriophage fl. J. Mol. Biol. 153 (1981) 169-176. E&edge, S.J., Mulligan, J.T., Ramer, S.W., Spottswood, M. and Davis, R.W.: A YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherikhia coli mutations. Proc. Natl. Acad. Sci. USA 88 (1991) 1731-1735. Frischauf, A., Lehrach, H., Poustka, A. and Murray, N.: Lambda replacement vectors carrying polylinker sequences. J. Mol. Biol. 170 (1983) 827-842. GliSin, V., Crkvenjakov, R. and Byus, C.: Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13 (1974) 2633-2637. Hohn, B. and Murray, K.: Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proc. Natl. Acad. Sci. USA 74 (1977) 3259-3263. Huynh, T.V., Young, R.A. and Davis, R.W.: Constructing and screening cDNA libraries in lgtl0 and &tll. In: Glover,D.M. (Ed.), DNA Cloning. A Practical Approach, Vol. 1. IRL Press, Oxford, 1985, pp.49-18.
Kam, J., Brenner, S., Bamett, L. and Cesareni, G.: Novel bacteriophage R cloning vector. Proc. Natl. Acad. Sci. USA 77 (1980) 5172-5176. Karn, J., Mattes, H.W.D., Gait, M.J. and Brenner, S.: A new selective phage cloning vector, 12001, with sites for XbuI, BamHI, HindHI, EcoRI, SstI, and XhoI. Gene 32 (1984) 217-224. Maruyama, I.N. and Brenner, S.: A phorbol ester/diacyI~ycerol-binding protein encoded by the unr-13 gene of Caenorhabdjti~ elegans. Proc. Natl. Acad. Sci. USA 88 (1991) 5729-5733. Morrow, J.F., Cohen, S.N., Chang, A.C.Y., Boyer, H.W., Goodman, H.M. and Helling, R.B.: Replication and transcription of eukaryotic DNA in Escherichiu co/i. Proc. Natl. Acad. Sci. USA 71(1974) 17431747. Murphy, A.J.M. and Schimke, R.T.: pZZd39: a new type of cDNA expression vector for low background, high efficiency directional cloning. Nucleic Acids Res. 19 (1991) 3403-3408. Murray, N.E. and Murray, K.: Manipulation of restriction targets in phage 1 to form receptor chromosomes for DNA fragments. Nature 251 (1974) 476-481. Oka, A., Sugisaki, H. and Takanami, M.: Nucleotide sequence of the kanamycin resistance transposon Tn903. J. Mol. Biol. 147 (1981) 217-226. Palazzolo, M.J., Hamilton, B.A., Ding, D., Martin, C.H., Mead, D.A., Mierendorf, R.C., Raghavan, K.V., Meyerowitz, E.M. and Lipshitz, H.D.: Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-ioxP automatic plasmid subcloning. Gene 88 (1990) 25-36. Rambach, A. and Tiollais, P.: Bacteriophage 1 having EcoRI endonuclease sites only in the nonessential region of the genome. Proc. Natl. Acad. Sci. USA 71 (1974) 3927-3930. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molder Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 54635467. Sanger, F., Cot&on, A.R., Hong, G.F. and Hill, D.F.: Nucleotide sequence of bacteriophage ,%DNA. J. Mol. Biol. 162 (1982) 729-773. Sauer, B. and Henderson, N.: The cyclization oflinear DNA in Escherichia coli by site-specific recombination. Gene 70 (1988) 33 l-341, Short, J.M., Femandez, J.M., Serge, J.A. and Huse, W.D.: IZAP: a bacteriophage 1 expression vector with in vivo excision properties. Nucleic Acids Res. 16 (1988) 7583-7600. Stemberg, N. and Hoess, R.: The molecular genetics of bacteriophage Pt. Annu. Rev. Genet. 17 (1983) 123-154. Stemberg, N., Tiemeier, D. and Enquist, L.: In vitro packaging of a lDam vector containing EcoRI DNA fragments of Escherikhia co/i and phage Pl. Gene 1 (1977) 255-280. St&ton, J.E. and Brenner, S.: The DNA of Cae~orhabdirj~ elegans. Genetics 77 (1974) 95-104. Thomas, M., Cameron, J.R. and Davis, R.W.: Viable molecular hybrids of bacteriophage lambda and eukaryotic DNA. Proc. Natl. Acad. Sci. USA 71 (1974) 4579-4583. Ullrich, A., Shine, J., Chirgwin, J., Rutter, W.J. and Goodman, H.M.: Rat insulin genes: Const~ction of plasmids containing the coding sequences. Science 196 (1977)1313-1319. Vieira, J. and Messing, J.: The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19 (1982) 259-268. Vieira, J. and Messing, J.: Production of single-stranded plasmid DNA. Methods Enzymol. 153 (1987) 3-11. Zoller, M.J. and Smith, M.: Oligonucieotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA 3 (1984) 479-488.