Curr Genet (1995) 27:309-311 �9 Springer-Verlag 1995

Jacques Daniel

DNA insertion system for complex yeast shuttle vectors

Received: 16 September 1994 /4 October 1994

A b s t r a c t A DNA insertion system, termed marked ho- mologous recombination, was devised for the construction of complex yeast shuttle plasmids. This system, which is efficient, rapid and easy to use, should contribute to our understanding of gene-gene interactions in yeast cells.

Key words Saccharomyces cerivisiae Plasmid construction �9 Homologous recombination E. coli-yeast common markers

It is relatively easy in vitro to insert DNA fragments into E. coti vectors such as the pUC (Yanish-Perron et al. 1985), the pBluescript (Stratagene) or the pGEM-Z (Promega) se- ries of vectors. For this purpose, large polylinker regions are required, as well as a blue/white screening system in- volving [3-galactosidase activity and Xgal. However, when having to construct more complex yeast/E, coli shuttle plasmids which combine together several genes, this ap- proach is more difficult because of the lack of adequate and unique restriction sites, a low construction yield, or problems in transforming E. coli with a very large con- struct. As the demand for complex shuttle plasmids is likely to grow in the future, new techniques for plasmid construc- tion are required.

A novel gene selection method in yeast, called the gene- gene interference (or GGI) method, was recently devel- oped which requires the insertion of specific genes into a large general vector to give a reference plasmid used for subsequent specific GGI selections (Daniel 1993). For per- forming these constructions, it seemed convenient to first insert the specific genes into simple E. coli vectors, using blue/white screening, and then to transfer these genes to the complex yeast shuttle vector by utilizing yeast as the final executant taking advantage of its high propensity for

J. Daniel Centre National de la Recherche Scientifique, Centre de G6n6tique Mol6culaire, Laboratoire associ6 h l'Universit6 Pierre et Marie Curie, F-91198 Gif-snr-Yvette, France

Communicated by K. Wolf

homologous recombination (Ma et al. 1987). Moreover, in order to facilitate the construction of the general recombi- nation-competent vector, as well as the derived plasmid, one could exploit markers recognized both by yeast and E. coli. This simple and efficient molecular construction technique, called marked homologous recombination, is presented here. Though it has essentially been applied to the GGI vector, it could easily be generalized and adapted to other types of yeast shuttle vectors.

E. coli cloning vectors, such as those mentioned above, contain identical regions flanking their specific polylink- ers. These regions correspond, respectively, to the lacOP operator-promoter and to the lacZ'gene used for the inser- tion screening system. Two flanking PvuII restriction sites are located at about 90-95 and 175 base pairs, respectively, from the extremities of the polylinker regions of these vec- tors. The flanking regions thus defined by these PvuII sites can be used as anchorage points for homologous recombi- nation in yeast during the in vivo transfer of a DNA insert from an E. coli vector to an appropriately devised shuttle vector. In order to easily construct such a general recom- bination-competent shuttle vector in E. coli in the first place, and, subsequently, to be able to monitor yeast re- combination events in this vector, markers of yeast origin such as URA3 and LEU2 can be used. Both genes can serve as markers in a yeast carrying the ura3 and leu2 mutations, as well as in E. coli carrying the pyrF and leuB mutations (other markers such as TRP1 might also be used).

The specific application of this method to the GGI vec- tor is described in Fig. 1. The HindIII-HindIII fragment of 1.2 kilobases containing the URA3 marker from YEp24 (Botstein et al. 1979) was inserted into the HindIII site in the potylinker of pUC19 (Yanish-Perron et al. 1985), us- ing the blue/white screening system, to give pUC 19-URA3 (Fig. 1 A). The resulting PvuII-PvuII fragment of 1.5 kilo- bases, including the URA3 marker plus the polylinker flanking regions, was inserted in vitro between the BamHI and SphI sites of the shuttle vector YEp21b-A (Daniel 1993). For this, the recipient E. coli strain transformed by electroporation was MH6 (lacAX74 galU gaIK leuB600 pyrF::Tn5 rpsLr- m+). After selection for growth in LB

310

A- CONSTRUCTION OF RECOMBINATION-COMPETENT YEAST VECTOR (FOR GENERAL USE) :

~JC19 § URA3 r r w k ~

Y~Ib4L B a p) ~111 ~ . ~

B- SPECIFIC GENE INSERTION BY MARKED HOMOLOGOUS RECOMBINATION :

P~JI cut BamHl+Sphl out

r for LEU~ADE§ k ~ prlmePce o~ urlci (yeaJ)

YEp21b-A-PvART1Pv

ART1

$.s.: U R A 3 marke r (-) - , "

\ , , ,

1 Fig. 1 A, B Gene insertion by marked homologous recombination. A construction of the general recombination-competent yeast shutt- le vector. B insertion of a specific gene (here ART1) in the general recombination-competent yeast shuttle vector; see text. For the ge- notype of E. coli strain XL1-Blue (Stratagene) see Sambrook et al. (1989). "s.s." stands for screening system

ampicillin plates, replica plating on GTE supplemented with leucine, with or without uracil, allowed the screening of URA § transformants (Sambrook et al. 1989). All four URA § clones tested among the dozens present (several per cent of transformants) corresponded to the correct inser- tion in both orientations. The vector thus obtained was called YEp21b-A-PvURA3Pv (Fig. 1 A). For inserting a specific gene (here ART1) into this complex shuttle vec- tor, one then proceeds in the following way (Fig. 1 B). The ART1 gene (XhoI-PvuII fragment of about 3 kilobases) was first cloned in vitro into pKS(+) (Stratagene) between the

XhoI and EcoRV sites, using the blue/white screening system, to give pKS(+)-ART1. Then, the yeast strain C90-A (MATa leu2-3,112 his3-A1 trpl ura3-52 ade2::TRP1) (Daniel 1993) was co-transformed (Ito et al. 1983) with (1) pKS(+)-ART1 linearized by cutting outside the PvuII-ART1-PvulI region (for instance with PvuI, or at the extremities of the region with PvulI), and (2) YEp21b- A-PvURA3Pv linearized by cutting twice in the polylinker next to URA3 and on the same side of URA3 (for instance with BamHI and SphI). By selecting for ADE+/LEU § in the presence of uracil (Sherman et al. 1987), one could obtain transformants that resulted from homologous recombina- tion events within the polylinker flanking regions, giving rise to an exchange of URA3 of the GGI vector with ART1, and thus restoring the continuity of this vector by includ- ing ART1 and excluding URA3. In fact, 17 transformants out of 20 tested (the number of transformants obtained was several hundreds) were found to be URA-; three of these,

further analysed after recovery of their plasmid in E. coli, all contained the expected YEp21b-A-PvART1Pv con- struct. Transforming solely with the linear YEp21b-A- PvURA3Pv plasmid gave only several dozen transfor- mants, among which no more than 5% were URA3 . Ad- ditionally, performing the co-transformation as above ex- cept for pKS(+)-ART1 remaining circular, did not result in a significantly larger number of transformants than in the control experiment with only the linear YEp21b-A- PvURA3Pv plasmid. Therefore, co-transformation with linearized plasmids appears to be an essential requirement in this construction method, most likely because in yeast linear DNA is much more efficient than circular DNA for transformation and homologous recombination (Ma et al. 1987).

To recover the plasmid in E. coli, an efficient and sim- ple "single colony" technique has been devised. Starting from a yeast colony (around 1 mm diameter; or equivalent material from a patch), cells are resuspended into a micro- fuge tube containing 200 gl of TSSTE buffer (2% Triton X100; 1% sodium dodecyl sulphate; 1 0 0 m M NaC1; l0 mM Tris-HC1, pH 8.0; 1 mM EDTA); 200 gl of a phe- nol/chloroform mix (I: 1) and 0.2 g of acid-washed glass beads (0.45 ~tm) are then added. The tube is vigorously vortexed from 2 to 5 min (plasmid yield significantly in- creases with increased vortex time). After centrifugation for 5 rain at room temperature, the upper phase is removed and DNA precipitated by the addition of 25 pl of 3 M so- dium acetate (pH 5.2) and 750 gl of ethanol, followed by washing of the precipitate with 70% cold ethanol. After solubilization in 10 gl of distilled water, 5 gl are used for electxoporation of E. coli (Gene Pulser, Bio-Rad) giving generally hundreds of transformants. The method works well with 2-micron- as well as centromeric-derived plas- mids although the recovery is somewhat smaller with the latter plasmid type.

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In conclusion, the molecular construction method in- volving marked homologous recombination has been suc- cessfully used for the insertion of several genes into the reference GGI vector, and has also been extended to other less complex vectors (such as YCp50). Thus, part of the sequences (lacOP and lacZ') and polylinker regions uni- versally used for DNA insertions could also serve for the transfer of these DNA fragments into complex yeast shuttle vectors.

Acknowledgements This research was supported by the Minerva Foundation, Munich, Germany, and by the Groupement de Recherch- es et d'Etudes sur les G6nomes MRE 92 H 0737, France. I thank P. Netter for his kind hospitality.

References

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