Identification of a Mutant Locus by Noncomplementation of a Transposon Insertion Library in Saccharomyces cerevisiae

Corresponding author: Marian Carlson, Columbia University, 701 W. 168th St., HSC922, New York, NY 10032., mbc1{at}columbia.edu (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT Strategy Identification of a... Genetic analysis of the... Recovery of sequence from... LITERATURE CITED

We describe a new approach for identifying the gene corresponding to a mutation in Saccharomyces cerevisiae. A library of mTn-lacZ/LEU2 insertions is tested for failure to complement the mutation, and the noncomplementing insertion is used to obtain sequence. This approach offers an alternative to cloning by complementation with a plasmid library.

A major advantage of the yeast Saccharomyces cerevisiae as a genetic system is that once a mutation is isolated, the mutant locus can be readily identified by cloning the cognate gene. Sometimes, however, a gene proves difficult to clone by conventional methods of complementation with a plasmid library. A gene may be underrepresented in a library or toxic in bacteria, gene dosage effects may cause unexpected phenotypes, or suppressors may be more easily recovered than the gene of interest. We have repeatedly failed to clone the GSF1 gene by complementation; gsf1-1 partially relieves glucose repression of SUC2 and GAL10 and affects glucose induction of the hexose transporter gene HXT1 (SHERWOOD and CARLSON 1997 ). Here we describe a new approach for identifying a mutant locus by noncomplementation with a genomic transposon insertion library. We apply this approach to identify the gsf1-1 locus.

	Strategy

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Fig 1 outlines our strategy. We used a library of mTn-lacZ/LEU2 insertion plasmids (BURNS et al. 1994 ) to generate a collection of haploid mutant strains containing insertions marked with LEU2. The mutant strains were mated to a gsf1-1 strain carrying a glucose-repressible SUC2::HIS3 reporter (TU and CARLSON 1994 ). The gsf1-1 mutation confers a His⁺ phenotype during growth on high glucose, and diploids carrying an insertion mutation that fails to complement gsf1-1 should be His⁺. Such diploids were selected, and genetic tests identified a mutant strain carrying a single insertion mutation that is linked to gsf1. The rescue plasmid pRSQ2-URA3 (website at http://ygac.med.yale.edu) was integrated into the transposon and excised so as to recover DNA sequence flanking the insertion. Sequence analysis then identified the mutant locus.

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Strategy for identification of a mutant locus by noncomplementation of an insertion library. See text for explanation. Transformants were selected on synthetic complete (SC) medium-leu+2% glucose. Mating was carried out on rich medium. Diploids were selected by replica plating to SC-his-leu-trp+5% glucose.

	Identification of a noncomplementing insertion mutation

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We transformed strain MCY4054 (Table 1) with the mTn-lacZ/LEU2 plasmid library cut with NotI and recovered 73,000 Leu⁺ transformants. A strain lacking the 2µ circle plasmid was used so that most transformants would contain insertions into chromosomal loci (BURNS et al. 1994 ).

To identify insertions that fail to complement gsf1-1, the mutant Leu⁺ transformants were mated to strain PS3851-3A (gsf1-1) containing the SUC2::HIS3 reporter, a TRP1-marked centromeric plasmid. His⁺ diploids were selected by replica plating onto selective medium (SC-his-leu-trp) containing 5% glucose, and 52 His⁺ diploids were recovered. We expected that in some cases the His⁺ phenotype resulted from homozygosis of the gsf1-1 allele during selective growth of the diploids. Haploid transformants that had yielded His⁺ diploids were therefore recovered and remated to the gsf1-1 strain to identify transformants that gave rise to His⁺ diploids at high frequency. The diploid derived from one such transformant expressed invertase activity during growth in 4% glucose, indicating that the His⁺ phenotype reflects constitutive expression of the SUC2 promoter.

	Genetic analysis of the His+ diploid

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To determine whether this diploid carries a single insertion mutation that is linked to gsf1, the diploid was subjected to tetrad analysis. Leucine auxotrophy segregated 2:2 in 15 tetrads, confirming the presence of an insertion at a single locus. In 13 tetrads, all segregants expressed invertase activity and/or exhibited a His⁺ phenotype during growth in glucose, indicating that the insertion is linked to gsf1. However, two tetrads yielded one Leu^- segregant that was His^- and glucose-repressed for SUC2. Subsequent identification of the locus as MTH1 (see below) revealed the presence of a recombination hot spot nearby (GERTON et al. 2000 ), suggesting that the gsf1-1 allele was converted to wild type by recombination.

In addition, a Leu⁺ segregant (MCY4059) was crossed to wild type, and the diploid showed glucose-repressed invertase activity, indicating that the insertion mutation is recessive.

	Recovery of sequence from the insertion

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We used the rescue plasmid pRSQ2-URA3, cut with BamHI, to transform MCY4059. Three Ura⁺ integrants were crossed to MCY3045 (leu2 ura3), and tetrad analysis showed cosegregation of the Ura⁺ and Leu⁺ phenotypes, confirming that pRSQ2-URA3 integrated into the transposon. In addition, leucine prototrophy was linked to invertase constitutivity. Genomic DNA from the integrants was digested with various restriction enzymes, ligated, and used to transform bacteria. Two integrants did not yield the desired plasmid; the rescue plasmid is able to integrate in a configuration that does not allow recovery of the mutation. A plasmid containing genomic DNA flanking the insertion site was recovered from the third integrant by using EcoRI. Sequence analysis indicated that the insertion occurred at nucleotide 282 of the MTH1 gene. We designate this insertion the mth1::lacZ/LEU2 allele.

Several lines of evidence have implicated MTH1 and its homolog, MSN3 (STD1), in glucose signaling. Increased copy number of MTH1 partially relieves glucose repression of SUC2 (HUBBARD et al. 1994 ). This phenotype accounts for our inability to clone the gene by complementation of gsf1-1 with a plasmid library. The Mth1 protein interacts with the glucose sensors Snf3 and Rgt2 and controls expression of hexose transporter genes, and dominant MTH1 alleles (HTR1, DGT1, BPC1) cause defects in glucose repression and glucose transport (SCHMIDT et al. 1999 ; LAFUENTE et al. 2000 ; SCHULTE et al. 2000 ).

The gsf1-1 and mth1::lacZ/LEU2 alleles are recessive but confer a phenotype distinct from that associated with the null mutation mth11::URA3 (HUBBARD et al. 1994 ), which does not relieve glucose repression of SUC2. It seems likely that gsf1-1 alters function of the gene product but does not confer a mutant phenotype as effectively as the dominant MTH1 alleles. With respect to mth1::lacZ/LEU2, we note the possibility that the C terminus of the protein is expressed.

In summary, we here demonstrate the utility of a new approach for identifying a mutant locus by noncomplementation of an insertion library. We tested a library of mTn-lacZ/LEU2 insertions for noncomplementation of the mutation of interest and used the noncomplementing insertion to sequence the locus. This approach offers a pragmatic alternative to cloning by complementation, and in the example provided here also offers the advantage of allowing a selection rather than a screen. The limitation to this approach is that the library must contain an appropriate insertion that is recessive to the original mutant allele. Essential genes will be identified only if the library contains an insertion that does not result in cell inviability. Although insertions in genes that are toxic in bacteria will be underrepresented, the library should nonetheless contain insertions into plasmids with a partial open reading frame that is nontoxic.

	ACKNOWLEDGMENTS

We thank Michael Snyder for generously providing the insertion library and rescue plasmid. We thank Rodney Rothstein and Lorraine Symington for advice on recombination events. This work was supported by National Institutes of Health grant GM-34095 to M.C.

Manuscript received March 14, 2001; Accepted for publication May 14, 2001.

	LITERATURE CITED

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BURNS, N., B. GRIMWADE, P. B. ROSS-MACDONALD, E.-Y. CHOI, and K. FINBERG et al., 1994  Large-scale analysis of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. Genes Dev. 8:1087-1105[Abstract/Free Full Text].

GERTON, J. L., J. DERISI, R. SHROFF, M. LICHTEN, and P. O. BROWN et al., 2000  Global mapping of meiotic recombination hotspots and coldspots in the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 97:11383-11390[Abstract/Free Full Text].

HUBBARD, E. J. A., R. JIANG, and M. CARLSON, 1994  Dosage-dependent modulation of glucose repression by MSN3 (STD1) in Saccharomyces cerevisiae. Mol. Cell. Biol. 14:1972-1978[Abstract/Free Full Text].

LAFUENTE, M. J., C. GANCEDO, J.-C. JAUNIAUX, and J. M. GANCEDO, 2000  Mth1 receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae. Mol. Microbiol. 35:161-172[Medline].

ROSE, A. B. and J. R. BROACH, 1990  Propagation and expression of cloned genes in yeast: 2-µm circle-based vectors. Methods Enzymol. 185:234-279[Medline].

SCHMIDT, M. C., R. R. MCCARTNEY, X. ZHANG, T. S. TILLMAN, and H. SOLIMEO et al., 1999  Std1 and Mth1 proteins interact with glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae. Mol. Cell. Biol. 19:4561-4571[Abstract/Free Full Text].

SCHULTE, F., R. WIECZORKE, C. P. HOLLENBERG, and E. BOLES, 2000  The HTR1 gene is a dominant negative mutant allele of MTH1 and blocks Snf3- and Rgt2-dependent glucose signaling in yeast. J. Bacteriol. 182:540-542[Abstract/Free Full Text].

SHERWOOD, P. W. and M. CARLSON, 1997  Mutations in GSF1 and GSF2 alter glucose signaling in Saccharomyces cerevisiae. Genetics 147:557-566[Abstract].

TU, J. and M. CARLSON, 1994  The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae. Mol. Cell. Biol. 14:6789-6796[Abstract/Free Full Text].

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