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Protein Fusions to the URA3 Gene of Yeast

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Protein Fusions to the URA3 Gene of Yeast Copyright 0 1987 by the Genetics Society of America A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast Eric Alani and Nancy Kleckner Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 Manuscript received April 15, 1987 Accepted June 1, 1987 ABSTRACT We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GALI genes and the E. coli lac2 gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5’-phosphate(OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HZS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well. ROTEIN fusions between a gene of interest and KORNBERGand SIMMONS1955; ROSE, GRISAFIand P a gene whose activity can be monitored geneti- BOTSTEIN 1984). (C) OMP decarboxylase is small cally in vivo and assayed biochemically in vitro have (monomer 25 kD) and active as a dimer, and may been used to investigate a wide range of biological therefore be more tractable for some purposes than problems. Fusions bearing some protein of interest at &galactosidase, which is large (monomer 1 16 kD) and the amino terminus and 6-galactosidase at the carboxy functions only as a tetramer (ROSE and BOTSTEIN terminus have been successfully used to characterize 1983; BRODYand WESTHEIMER1979; CONTAXISand regulatory elements, to identify intragenic signal ex- REITHEL1971). (D) The same enzymatic activity is port elements and to distinguish transcriptional from found in Escherichia coli, Drosophila, plants and higher post-transcriptional control (SILHAVYand BECKWITH animal cells, and nucleotide analogs have been used 1985; GUARENTEand PTASHNE1981; ROSE, CASA- in some of these organisms to select against OMP DABAN and BOTSTEIN1984). A number of other genes decarboxylase function (JONES1980). Therefore, the have been identified which retain functional activity possibility exists that the URA3 fusion cassette de- in protein fusions; these include gaLK, cat, npt, phoA, scribed here can be used for genetic analysis in a wide dhfr and uidA (SILHAVYand BECKWITH1985; STUE- range of organisms. BER et al. 1984; GORMAN,MOFFAT and HOWARD 1982; JEFFERSON,BURGESS and HIRSH1986). MATERIALS AND METHODS In this paper we describe protein fusions that con- Bacterial strains: MM294 (F-, endoA, hsdR, supE44, thiA) tain the GALI, HZS4, and lacZ gene products at the was the standard strain used for plasmid amplification and amino terminus and the Saccharomyces cerevisiae URA3 DNA manipulation (GUARENTEet al. 1980). KC-8 (rx1486, gene product, orotidine-5’-phosphate (OMP) decar- M+, K12,leuB600, trpC9830, pyrF::Tn5, hisB463, AlacX74, boxylase at the carboxy terminus. In each case we find strA, galU, galK) was provided by K. STRUHL.KC-8 trans- that OMP decarboxylase is functional and that OMP formed with pNK627 (relevant genotype tet, lacif, N. Kleck- ner laboratory collection) was used to assay the lacZ-URA3 decarboxylase expression is dependent on the regu- gene fusions. lacZ-URA3 gene fusion products were identi- latory elements of the gene to whose product it is tied in NK5830 (F’ lacpro, ladL8, Ara-, Met-, NalA, Rip, fused. The yeast URA3 gene product has a number of srl::TnlO, recA56, argEam, supE, AlacproxlII) (ROBERTSet features that makes it particularly attractive for fusion al. 1985). Yeast strains: DBY745 (MATa, adel-100, leu2-3, leu2- analysis: (A) There are direct genetic selections both 112, ura3-52) was obtained from D. BOTSTEIN.NKY280 for and against URA3 function (BOEKE,LACROUTE (MATa, adel-100, leu2-3, leu2-112, ura3-52, trp1::hisG) and FINK 1984). (B) An extremely sensitive assay for bears a 1.1-kb insertion of Salmonella hisG DNA in the OMP decarboxylase activity exists (LIEBERMAN, TRPl gene of DBY745. The construction of this strain is Genetics 117: 5-12 (September, 1987) 6 E. Alani and N. Kleckner described elsewhere (ALANI,CAO and KLECKNER 1987). by PAULMCDONALD) (RUTHER and MULLER-HILL1983), TD50 (MATa, his4-619, ura3-52) was kindly provided by PRY116 (Gal1,lO upstream regions) (YOCUM et al. 1984), TOMDONAHUE (DONAHUE, FARABAUGH and FINK 1982). YEPl 3 (2p, LEU2) (SHERMAN,FINK and HICKS1982), B115 Media: E. coli were grown in LB broth or on LB agar. E. (URA3, distal HIS4 sequence-gift from T. DONAHUE), coli used for pyrF::Tn 10 complementation experiments were pBR322 (BOLIVARet al. 1977),and pGC-1 (M-13 sequencing grown in M9 salts supplemented with 0.2% glucose and vector) (MYERSand MANIATIS 1985). Further details are 0.004% each of histidine, tryptophan and leucine. Ampicil- available upon request. lin and tetracycline were supplemented at 100 q/ml and pNKY2009, 2024: pNKY2009 was built from four other 15 pglml, respectively. Yeast were grown in either YPD or plasmids. The backbone, pNKY2003, was made by deleting minimal selective media. Selective media contained 0.7% the URA3 gene from YEP24 with a partial HindIII digest. yeast nitrogen base (Difco), 2% agar and 2% glucose. Ad- A 4.0 kb Sphl fragment encoding HIS4 (isolated from a B54 dition of 0.004% leucine, tryptophan, adenine and uracil plasmid molecule whose SstI site had been converted into was made according to strain requirements. In the galactose an SphI site) was inserted into the SphI site of pNKY2003 induction study, yeast were grown in media containing 2% to form pNKY2005. A 2.5-kb BglII fragment bearing LEU2 of each of the following carbon sources: galactose plus was isolated from YEPl 3 and inserted into the BamHl site sucrose, galactose plus glucose and galactose. 5-Fluoro-or- of pNKY2005 to form pNKY2010. Finally, a 1.5-kb BgllI- otic acid (5-FOA)was purchased from SCM Specialty Chem- BamHI fragment encoding the URA3 gene was isolated from icals, Gainsville, Florida. 5-FOA plates were prepared as a YEP24 derivative containing a BglII linker at the EcoRl described previously (BOEKE,LACROUTE and FINK 1984). site closest to the 5' end of URA3 and inserted into the BglIl Yeast transformation: Yeast lithium acetate transforma- site of pNKY2010 to form pNKY2009. BAL31 deletions tion were performed by standard methods (ITO et al. 1983). Integration of the HIS4-URA3 fusion into NKY280 was were performed at the BglII site of pNKY2009 to form the performed as follows: pNKY54 was digested with SphI and protein fusion vector pNKY2024 (Figure 1). the 4.5-kb fragment bearing the HIS4-URA3 fusion and the B184: B115, a pBR322 derived plasmid bearing the distal TRPl gene was introduced into NKY280 by homologous region of HIS4 was digested with BglII and integrated into recombination between the 5' and 3' ends of the fragment the HIS4C locus of TD50 (his4-619). Chromosomal DNA of and the chromosomal HIS4 locus. Sixteen Trp+ transform- this integrant was made, cleaved with BamHI, ligated and ants were tested for uracil and histidine prototrophy. Eleven transformed into MM294. Plasmids bearing the entire HIS4 of these candidates were His-, suggesting that a homologous locus (12 kb) were identified by restriction mapping. recombination event occurred between the HlS4-URA3, pNKY2028: pNKY2028 was made by replacing the 1.4- TRPZ fragment and the chromosomal HIS4 locus. Ten of kb SphI-XhoI fragment which spans the HIS4 initiation co- these 1 1 His- candidates were Ura+, sensitive to 5-FOA and don in pNKY2024 with the corresponding fragment from yielded similar colony sizes on media with and without B184. uracil. Genomic DNA from Trp+,Ura+, His- transformants pNKY54: pNKY2024 was digested with SmaI and BstE2 was digested with PstI and blotted with a nick translated and a BglIl 8-bp linker was inserted to form pNKY2029. HIS4 probe. NKY280 displayed a IO-kb band while 6.3- and After a BglII linker was inserted into the EcoRI site of YRP7 3.7-kb bands were detected in the strains containing the closest to the 5' end of the TRPl, an 850-bp BamHI-BglII HZS4-URA3 integrant. This result confirmed that the ob- fragment containing an intact TRPl gene was isolated and served phenotype resulted from a single copy integration inserted into the BglIl site of pNKY2029. An Sphl fragment event within the HIS4 locus (blot not shown). from pNKY2029 which encodes the HIS44JRA3 fusion and Protein gels: A 1.0-ml culture of NK5830 transformed the TRPl gene was isolated and inserted into the Sphl site with the 1ac.Z-URA3 protein fusion plasmid (pNKY59) was of pBR322 to form pNKY54. grown to exponential phase, treated with 1 mM isopropyl p- pNKY48: In order to sequence the HIS4-URA3 fusion D-thiogalactopyranoside (IPTG) and grown for 2 hr more. junction, a HindIlI fragment from pNKY2029, which en- Cells were then suspended in 50 pl of STET (8% sucrose, codes 93 amino acids of HIS4 fused to 267 amino acids of 50 mM EDTA, 50 mM Tris, 5% Triton X-100, pH 8.0); and UKAS, was isolated and inserted into the Hind111 site of treated with 90 fig of lysozyme for 5 min on ice. After an pNKY47. The sequencing primer in pNKY48 is 290 bp equal volume of 0.1% SDS loading buffer was added, the from the HIS4-URA3 fusion junction.
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