Elsevier

Gene

Volume 252, Issues 1–2, 11 July 2000, Pages 127-135
Gene

Exploring redundancy in the yeast genome: an improved strategy for use of the cre–loxP system

https://doi.org/10.1016/S0378-1119(00)00217-1Get rights and content

Abstract

Gene families having more than three members are a common phenomenon in the Saccharomyces cerevisiae genome. As yeast research enters the post-genome era, the development of existing deletion strategies is crucial for tackling this apparent redundancy, hence a method for performing rapid multiple gene disruptions in this organism has been developed. We constructed three replacement cassettes in which different selectable markers were placed between two loxP loci. Multiple deletions (of members of a gene family) were generated, in one strain, using sequential integration of different replacement markers (kanMX, LYS2, KlURA3 and SpHIS5). Their excision from the genome was performed simultaneously, as the final step, using a new cre recombinase vector, which carries the cycloheximide-resistance gene from Candida maltosa as a selectable marker. Our multiple gene deletion system significantly accelerates and facilitates the functional analysis process and is particularly useful for studying gene families in either laboratory or industrial yeast strains.

Introduction

The complete DNA sequence of all 16 chromosomes of Saccharomyces cerevisiae revealed the presence of about 6000 ORFs, a large portion of which still have completely unknown roles (Goffeau et al., 1996). The next scientific step is to elucidate the function of these novel genes at the biochemical and biological levels. Gene deletion methods and metabolic control analysis (Oliver, 1996), are currently used in the attempt to assign a function to ‘orphan genes’ (Dujon, 1996). The first method allows the replacement of any ORF with an heterologous selectable marker, and the investigation of the resulting phenotype (Wach et al., 1994). The latter aims to exploit such mutants in order to place unknown genes in particular functional domains, starting with global assessments of fitness and metabolic profiles (top-down approach) (Oliver, 1996).

In spite of the fact that S. cerevisiae has a small and compact genome, the sequencing data revealed a high level of apparent genetic redundancy (Mewes et al., 1997, Oliver, 1996, Wolfe and Shields, 1997): blocks of duplicated ORFs (Cluster Homology Regions) (Goffeau et al., 1996) are found both in the telomeric regions and in internal sites of the chromosome arms. For example, the SUC, MAL, MEL and AAD gene families [see Delneri et al. (1999b) and references cited therein] are predominantly associated with chromosomal ends, flocculation genes constitute a new subtelomeric gene set, and there are at least 15 genes belonging to the family of hexose transporters (HXT), spread around the yeast genome (André, 1995). To uncover the real biological role of a gene family of unknown function, all the members of the set need to be disrupted. Within the framework of the EUROFAN project, Node B4 was created to study the biological function of ca. 100 different gene families of three or four members. Moreover, a long-term goal of our laboratory is to construct a ‘minimalist’ yeast (Oliver, 1996) that contains no non-essential genes. In order to carry out these tasks efficiently, it is necessary to devise a method that will allow the creation, in a short time, of multiple mutations within a single strain.

The use of different nutritional markers to delete the members of a family is unsuitable because of the effect that they can have on the final phenotype in different physiological conditions (Baganz et al., 1997). Moreover, the availability of different nutritional markers is limited. On the contrary, the kanr gene was proved to be a ‘safe’ marker to use in functional analysis, exhibiting a negligible effect on specific growth rate. It is, of course, possible to create multiple deletants by classical genetics methods using only kanr marker (Delneri et al., 1999a), but the protocol is laborious when more than three genes need to be deleted in the same strain. Furthermore, meiotic chromosome segregation would be unlikely to redistribute homologous genes located on the same chromosome arm. Thus, for all the above reasons, an efficient marker rescue technique is essential for the study of a gene family with a large number of members.

Different deletion cassettes have been designed for repeated disruptions in the same strain via mitotic recombination events between two homologous regions, like the cassettes containing the removable URA3 gene flanked by direct repeats (250 bp) of Salmonella hisG DNA (Alani et al., 1987). The mitotic recombination frequency between the repeats is, however, very low and so limits the use of this method to markers that can be counter-selected. Furthermore, the length of the homologous sequences causes the preferential integration of subsequent deletion cassettes in the resident ‘scar’, left in the genome from the first marker excision. This last problem was overcome by the construction of a deletion cassette containing an heterologous marker (URA3 gene from Kluyveromyces lactis) flanked by shorter direct repeats (60 bp) fused, via PCR, with sequences from the genome target (Längle-Rouault and Jacobs, 1995). In this case, though, the primers used in the PCR reaction anneal to each other (owing to the presence of direct repeats), rather than the template, causing a very poor product yield. Alternatively, cassettes in which the nutritional markers are flanked by sequences (not found elsewhere in the yeast nuclear genome) that are specific target sites for double-stranded endonucleases, like the FRT (Toh-e, 1995), the I-SceI (Fairhead et al., 1996) and the loxP (Sauer, 1994) were used. Moreover, a deletion cassette loxP–kanMX–loxP, which combines the advantages of the kanr gene with those of the creloxP system, was constructed (Güldener et al., 1996). Nevertheless, these methods are time-consuming because, to allow the pop-out of the marker, an additional transformation step with a plasmid encoding the corresponding site-specific recombinase (cre for loxP, Flp for FRT) or endonuclease (I-SceI) is essential. Moreover, the same plasmid needs to be lost from the strain after the pop-out has occurred.

Recently, a disruption cassette containing the URA3 gene from K. lactis, and the Flp gene flanked by two FRTs sites was developed (Storici et al., 1999): the marker can be eliminated by in vivo site-specific recombination and the selection of the pop-out events can be performed directly onto 5-FOA medium. However, all the marker rescue methods require that the marker genes are excised from their genomic locus in order to proceed with subsequent deletions. It is the marker-recycling step that is rate limiting in the construction of multiply deletant strains. We have developed a procedure in which it is possible to create multiple gene deletions by the sequential use of different replacement markers, but in which a single final step allows for the excision of all markers simultaneously. Using this newly developed method, we have deleted all four members of a gene family whose predicted protein products show significant amino-acid sequence similarity to the dihydroflavonol-4-reductase of Vitae vinifera (the grape vine).

Section snippets

Strains and media

The gene deletions were carried out in the S. cerevisiae background strain BY4742 (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0). Organisms were grown on YPD medium (1% yeast extract, 2% peptone, 2% glucose), or minimal SD medium (0.67% yeast nitrogen base, 2% glucose) supplemented with the auxotrophic requirements, or YPGal medium (1% yeast extract, 2% peptone, 2% galactose). For the selection of the kanr transformants, cells were grown on YPD plates containing 300 μg/ml of geneticin (G418, Gibco BRL).

Plasmid construction

The purpose of this work was to design a strategy for the rapid and efficient deletion of multi-gene families from S. cerevisiae. We constructed a series of pUG6-derived vectors in which different selectable markers are placed in between two loxP sites to permit the ready excision of the markers via the action of cre recombinase. The kanMX marker was replaced with the heterologous nutritional markers KlURA3 and SpHIS5, and with the homologous marker LYS2. Using these vectors, it is possible to

Acknowledgements

We thank Dave Gardner for helpful discussions. This work was supported by a contract within the frame of the EUROFAN Project of the EC (BIO4-CT95-0080) to SGO, and by grants from the BBSRC to SGO and from the Wellcome Trust to SGO and EJL.

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