We have developed a novel phage lambda replacement cloning vector, lambdapAn. lambdapAn allows one to automatically subclone the insert as a plasmid using the Cre-loxP site. specific recombination system. This eliminates the need to subclone insert fragments and permits the rapid structural analysis of insert DNA. lambdapAn is similar to other phage lambda replacement vectors taking inserts ranging in size from 5 to 19 kb. We have placed the pyrG gene of Aspergillus nidulans on the vector as a nutritional selective marker for transformation. We have developed this vector as part of an overall plan to facilitate the cloning of dominant extragenic suppressor mutations from A. nidulans, but also know that it is a generally useful vector for the purposes of isolating genomic clones without the need to subclone from the phage lambda vector.
The isolation and analysis of genomic DNA sequences has been facilitated by the development of phage lambda replacement vectors that can contain up to 23 kb of contiguous DNA and cosmid vectors that can contain inserts of up to 40kb of DNA. While these cloning vectors have been in general use they each have their limitations. Genomic libraries in phage lambda vectors are easy to screen but present several problems in the analysis of insert DNA and subcloning. Subcloning and analysis each require the preparation of the recombinant lambda clone DNA. While many of these steps have been improved, the subcloning in particular can still require considerable effort (Sambrook et al., 1989; Meese et al., 1990). In contrast, libraries constructed in cosmid vectors require considerable time in screening to isolate pure clones and the inserts are sufficiently large that their analysis can be very demanding (Gibson et al., 1987; Sambrook et al., 1989; Wahl et al., 1987). Even with these limitations both phage lambda and cosmid vectors are used by many laboratories for the purposes of gene cloning and analysis. We describe here the construction of a novel phage lambda replacement vector that does not necessitate the preparation of phage lambda DNA for subcloning and analysis of inserts because the inserts contained in this vector can be converted to plasmids using the Cre-loxP site-specific recombination system (Palazzolo et al., 1990; Elledge et al., 1991). Previously this method for automatic subcloning had been used for CDNA cloning vectors.
To construct lambdapAn we first constructed the plasmid vector pBX (Fig. 1A). To simplify the description of the construction of pBX we will only describe the source and structure of the DNA fragments used in its construction. pBX contains 2064bp of pBR322 from Ndel to EcoRI, an approximately 295bp EcoRI, BamHI fragment containing lox sites flanikng a NotI site from the plasmid pSE 1086(delta)NdeI (a gift from S. Elledge) and a 1565bp XhoI, NdeI fragment containing the pyrG gene from A. nidulans.
The plasmid pBX is too small to clone directly into most phage lambda replacement vectors so we placed an approximately 14kb Xhol fragment of Aspergillus genomic DNA at the XhoI site to act as a stuffer fragment. This plasmid was linearized with NotI and ligated to purified arms of a phage lambda replacement vector related to lambdaEMBL3 that had NotI sites flanking the cloning site. This ligation was transfected into LE392 cells and lambda plaques were screened by hybridization for the presence of the pyrG gene. A positive plaque was picked and amplified to obtain a stock lysate and DNA. We then tested whether this phage could be converted to a plasmid by automatic subcloning in BNN132 cells (Elledge et al., 1991). Individual ampicillin resistant colonies were grown and the plasmid DNA was isolated. In every case examined the expected plasmid was recovered. The structure and partial restriction map of lambdapAn is shown in figure 1B.
The XhoI site is used for cloning of genomic DNA fragments having BamHI compatible ends using the half site fill-in procedure previously described (Zabarovsky and Allikmets, 1986). We have used A. nidulans genomic DNA partially digested with Sau3AI followed by partial filling in with dATP and dGTP in the presence of Pol1k. Insert DNA for the construction of genomic libraries was used in ligations without size fractionation. The cohesive ends of lambda were allowed to anneal and then ligated with T4 DNA ligase. The ligase was heat inactivated for 30 minutes at 65 C and the vector DNA was digested with XhoI, followed by partially filling in with dTTP and dCTP in the presence of Pol1k. The DNAs were extracted twice with phenol and chlorofrom (1:1), recovered by ethanol precipitation from solution in the presence of 0.3 M sodium acetate, air dried and dissolved in water at 0.1 to 1 ug/ul final concentration.
Typical figations contained 1 ug of vector and variable amounts of insert DNA in a final volume of 5 ul of 50 mM Tris-HCL pH 7.5, 10 mM MgCI2 10 mM dithiothreitol, 1 mM ATP, 100 ug/ml BSA and 400 cohesive end units of T4 DNA figase. Ligations were incubated overnight at 4 C. The following day 2 ul of each ligation was packaged into lambda phage particles using in vitro packaging extracts (Stratagene, La Jolla, CA). These particles were titered on LE392 cells and the remainder of the ligation(s) having the highest titers were also packaged. In two separate experiments we have obtained genomic libraries containing 1-2 x I0(6) plaque forming units from a single set of ligations.
One concern of ours early in the development of the vector was that the automatic subcloning would not work with genomic DNA inserts as it had with the previously described cDNA vectors (Elledge et al., 1991). Using the published procedure we have found that the efficiencies of conversion of these genomic DNA containing phage lambda particles was equal to those reported for the cDNA vector. Plasmid DNA isolated from 14 clones selected at random averaged 13.7 kb in size, ranging from 9.7 kb to 19.5 kb. This would give an average insert of 10.3 kb with a range of 6.2 kb to 16 kb in size. In addition, we have idenfified plasmids with inserts of 17 to 18 kb. This indicates that inserts approaching the maximum size can be cloned and are able to undergo automatic subcloning.
We have described lambdapAn, a novel phage replacement cloning vector. While we developed this vector to more efficiently clone genes from A. nidulans, it will be useful in isolating genomic clones from other organisms as well. In addition to the Xhol site for cloning, lambdapAn can also be used to clone DNA fragments having ends compatible with the restriction endonuclease BamHI and still use the automatic subcloning procedure. In this case though, the pyrG gene will be lost and the resulting fragments cannot be used for the direct transformation of A. nidulans.
A similar system for the development of genomic libraries has recently been described that utilizes the site-specific recombination system of the transposon Tn1721 (Altenbuchner, 1993). While the transposon system has properties like those of the Cre- loxP system an advantage of the Cre-loxP system is that it is based on the plasmid pBR322, a well known and widely used cloning vector (Bolivar et al., 1977).
ACKNOWLEDGEMENTS
Support was provided by NIH grant GM41626 to G.S.M. The authors wish to thank Dr.
S. J. Elledge for some of the plasmids and bacterial strains needed for this work.
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Fig. 1. Maps of pBX and lambdapAn. (A) Partial restriction endonuclease map of pBX
showing the organization of DNA fragments used in its construction. (B) Partial
restriction endonulease map for lambdapAn showing the organization of the DNA elements in
the nonrecombinant vector. The 14 kb XhoI stuffer fragment is the hatched region, the
sequences of pBX are clear and the lambda arms are filled.