Selectable cassettes for simplified construction of yeast gene disruption vectors

Gene, 169 (1996) 111-113 © 1996 Elsevier Science B.V. All rights reserved. 0378-1119/96/$15.00

111

GENE 09507

Selectable cassettes for simplified construction of yeast gene disruption vectors (Recombination; gene targeting; plasmid)

Marie C. Earley and Gray F. Crouse Graduate Program in Genetics and Molecular Biology, and Department of Biology, Emory University, Atlanta, GA 30322, USA Received by J. Marmur: 7 August 1995; Revised/Accepted: 9 October/11 October 1995; Received at publishers: 6 November 1995

SUMMARY

Cassettes based on a hisG-URA3-hisG insert have been modified by the addition of a Kmg-encoding gene and flanking polylinker sites, greatly simplifying construction of gene disruption vectors in Escherichia coli. After gene disruption in yeast, URA3 can then be excised by recombination between the hisG repeats flanking the gene, permitting reuse of the URA3 marker.

INTRODUCTION

One problem that is often encountered when transforming yeast strains is that there are few available markers and once a given marker is used, it cannot be used again. One major need for markers is in gene disruption experiments. The pop-in/pop-out replacement method helped solve the problem of marker availability by permitting the reuse of URA3 (Scherer and Davis, 1979). In this method, a plasmid containing a mutated gene is integrated into the genome using URA3 as a selectable marker. In the second step, one selects for excision of the plasmid using 5-fluoroorotic acid (5-FOA); crossovers that take place on the opposite side of the mutant site from the site of integration result in a replacement of the chromosomal allele with the mutated gene. The resulting strain is once again ura3 and URA3 can therefore be Correspondence to: Dr. G.F. Crouse, Department of Biology, Emory University, Atlanta, GA 30322, USA. Tel. (1-404) 727-4236; Fax (1-404) 727-2880; e-mail: [email protected] Abbreviations: Ap, ampicillin; bp, base pair(s); 5-FOA, 5-fluoroorotic acid; hisG, Salmonella typhimurium gene coding for the ATP phosphoribosyltransferase; kb, kilobase(s) or 1000 bp; Km, kanamycin; nt, nucleotide(s); ori, origin of DNA replication; R, resistance/resistant; URA3, Saccharomyces cerevisiae gene coding for orotidine-5'-phosphate decarboxylase. SSDI 0378-1119(95)00805-5

reused as a marker. The major disadvantage with this method is that not all pop-outs result in replacements, and the resulting pop-out strains must be screened for the desired recombination event. This problem was solved by constructing a plasmid, pNKY51, with a cassette containing URA3 flanked by direct repeats of the Salmonella hisG sequence (Alani et al., 1987). The cassette is released by digestion with BamHI + BglII and used to create a disruption plasmid by replacing the desired segment of the gene with the hisG-URA3-hisG cassette, flanked by intact regions of the gene. This plasmid is cleaved with restriction enzymes that have sites in the flanking DNA and is then transformed into yeast to create the desired mutation by single gene replacement. Transformants with the desired integration event are then plated on 5-FOA plates to select for loss of URA3; this loss generally occurs via a recombination event between the flanking hisG repeats such that URA3 is lost and one hisG repeat is left in the genome. This allows URA3 to be used as a marker again.

EXPERIMENTAL AND DISCUSSION

(a) Construction of new cassettes for gene disruption There are two disadvantages in the use of pNKY51. First, the only enzymes that can be used to remove the

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Fig. 1. Map of pHUKH vectors. To construct the plasmids, the hisG-URA3-hisGcassette was removed from pNKY51 using SalI+BgllI and cloned into pMTL22 (Chambers et al., 1988). The Km R gene from pUC4K (Vieira and Messing, 1982) was removed by digestion with PstI and inserted into the unique Pstl site in URA3 within the hisG-URA3-hisGcassette of pNKY51/pMTL22. The hisG-URA3-hisGIKmRcassette was excised with BamHI+BgllI and the ends were treated with T4 DNA polymerase. For pHUKH1, the cassette was inserted into pGLINK (Crouse et al., 1989) digested with Hincll. PvulI, KpnI, EcoRI and BamHI sites flank the cassette. For pHUKH2, the cassette was inserted into pGLINK (Crouse et al., 1989) digested with KpnI and treated with T4 DNA polymerase; only PvulI sites flank the cassette in this construct. For pHUKH3 and pHUKH4, Km R was removed by PstI digestion of pHUKH1 and pHUKH2 and after treatment with T4 DNA polymerase was inserted into the T4 DNA polymerase-treated NheI site of pHUKH1 and pHUKH2, respectively. The orientation of the cassette was confirmed by restriction analysis in each case. Sequences of each vector were assembled from the known sequences of the various components, and are present in GenBank with the following accession Nos.: pHUKH1, U37023; pHUKH2, U37024; pHUKH3, U37068; pHUKH4, U37069.

cassette for cloning are BamHI + BglII and digestion with these enzymes produces two fragments that tend to migrate together under standard gel electrophoresis conditions. Secondly, because there is no general selection in Escherichia coli for transformants containing the cassette,

constructing disruption vectors is relatively difficult. To overcome these problems, we have created four new plasmids containing a modified hisG-URA3-hisG cassette (Fig. 1). These plasmids contain Km R inserted into the cassette so that one can select for Km R during construc-

113 tion of disruption vectors; selection for K m R should also prevent the possible loss of URA3 by recombination in a rec + bacterial host. In p H U K H 1 and p H U K H 2 , Km R is inserted into URA3 and must be removed before transformation into yeast by digesting with PstI and religating the plasmid. The advantage of this arrangement is that the Km R gene contains the only occurrence of sites for XhoI, NruI and PflMI in the cassette, and its removal therefore makes those sites available for cleavage of the flanking DNA if needed. In addition, p H U K H 1 contains the cassette flanked by PvuII, KpnI, EcoRI and BamHI sites, whereas in p H U K H 2 the cassette is flanked by PvuII sites only. In p H U K H 3 and p H U K H 4 , K m R is inserted between URA3 and a hisG repeat and does not have to be removed before transformation into yeast. Sequences of all of the plasmids were assembled from known sequences. Because one can generate a hisG-URA3-hisG cassette with blunt ends by digestion with PvuII and can select for rare ligation products of this cassette into a gene of interest with Km, it is relatively easy to create a disruption in any sequence. Not including the restriction sites flanking the cassette, there are 50 commercially available restriction enzymes that do not cleave within the cassette of p H U K H 1 and p H U K H 2 and 48 commercially available restriction enzymes that do not cleave within the cassette of p H U K H 3 and p H U K H 4 , so it is not likely to be a problem to find restriction enzymes that will cleave the flanking yeast DNA in the disruption vector without cleaving the cassette itself. While this work was in preparation, a modified pNKY51 was reported with an inserted K m R gene; however, there were no added sites at the end of the hisG-URA3-hisG cassette and the Km R gene of that cassette could not be removed (Allen and Elledge, 1994).

(b) Conclusions (I) New cassettes that greatly simplify the construction of gene disruption vectors for yeast have been con-

structed, p H U K H 2 and p H U K H 4 have been used for the generation of disruption plasmids for MSH2, MSH3, SRS2, and RIB5 and in all cases have given the desired gene disruption in a variety of strains. (2) It should be noted that one advantage of having multiple genes within one strain all targeted by the same hisG-URA3-hisG cassette is that the molecular genotype of such strains can be verified by hybridization with a probe made from the hisG region of the cassette, rather than relying upon probes made from each of the specific genes.

ACKNOWLEDGEMENTS

This work was supported in part by Public Health Service grant CA54050 from the National Cancer Institute.

REFERENCES Alani, E., Cao, L. and Kleckner, N.: A method for gene disruption that allows repeated use of U RA3 selection in the construction of multiply disrupted yeast strains. Genetics 116 (1987) 541-545. Allen, J.B. and Elledge, S.J.: A family of vectors that facilitate transposon and insertional mutagenesis of cloned genes in yeast. Yeast 10 (1994) 1267-1272. Chambers, S.P., Prior, S.E., Barstow, D.A. and Minton, N.P.: The pMTL nic- cloning vectors, I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68 (1988) 139 149. Crouse, G.F., New, L. and Stivaletta, L.A.: Gene engineering by selectable intraplasmid recombination: construction of novel dihydrofolate reductase minigenes. Gene 84 (1989) 165-172. Scherer, S. and Davis, R.W.: Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc. Natl. Acad. Sci. USA 76 (1979) 4951-4955. 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.