The OPIl Gene of Saccharomyces cereuisiae, a Negative Regulator of

THE JOURNAL OF BIOLOGICAL CHEMISTRY (c) 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 2, Issue of January 15, , pp. 863-872.1991 Printed in U.S.A.

The OPIl Gene of Saccharomyces cereuisiae, a Negative Regulator of Phospholipid Biosynthesis, Encodes a Protein Containing Polyglutamine Tracts and a Leucine Zipper*

(Received for publication, July 16, 1990)

Michael J. White$, Jeanne P. Hirschs, and Susan A. Henry$ll From the $Department of Biological Sciences, Carnegie Mellon Uniuersity, Pittsburgh, Pennsylvania 15213 and the §Department of Biological Sciences, Columbia Uniuersity, New York, New York 10027

In Saccharomyces cerevisiae, recessive mutations at the OPIl locus result in constitutively derepressed expression of inositol 1-phosphate synthase, the product of the INOl gene. Many of the other enzymes involved in phospholipid biosynthesis are also expressed at high derepressed levels in opil mutants. Thus, the OPIl gene is believed to encode a negative regulator that is required to repress a whole subset of structural genes encoding for phospholipid biosynthetic enzymes. In this study, the OPIl gene was mapped to chromosome VI11 and cloned. When transformed into an opil mutant, the cloned DNA was capable of complementing the mutant phenotype and re- storing correct regulation to the INOl structural gene. Construction of two opil disruption alleles and subsequent genetic analysis of strains bearing these alleles confirmed that the cloned DNA was homologous to the genomic OPIl locus. Furthermore, the OPIl gene was found to be nonessential to the organism since mutants bearing the null allele were viable and exhibited a phenotype similar to that of previously isolated opil mutants. Similar to other opil mutants, the opil disruption mutants accumulated INOl mRNA constitutively to a level 2-%fold higher than that observed in wild-type cells. The cloned OPIl gene was sequenced, and translation of the open reading frame predicted a protein composed of 404 amino acid residues with a molecular weight of 40,036. The predicted Opil protein contained a well defined heptad repeat of leucine residues that has been observed in other regulatory proteins. In addition, the predicted protein contained polyglutamine residue stretches which have also been reported in yeast genes having regulatory functions. Sequencing of opil mutant alleles, isolated after chem- ical mutagenesis, revealed that several were the result of a chain termination mutation located within the largest polyglutamine residue stretch.

Phospholipid biosynthesis in Saccharomyces cerevisiae is regulated in a coordinated fashion. Inositol 1-phosphate synthase (Culbertson et al., 1976; Donahue and Henry, 1981a; Hirsch and Henry, 1986), cytidine diphosphate-diacylglycerol

* This work was supported by Grant GM 19629 from the National Institutes of Health (to S. A. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact,

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505727.

1 To whom correspondence should be addressed.

synthase (Homann et al., 1985; Klig et al., 1988), phosphatidylserine synthase (Klig et al., 1985; Poole et al., 1986; Bailis et al., 1987; Homann et al., 1987), phosphatidylserine decarboxylase,’ and the phospholipid methyltransferases (Yamash- ita et al., 1982), which convert phosphatidylethanolamine to phosphatidylcholine, are all subject to regulation by inositol and choline (Fig. 1). All of these enzymes show various degrees of repression in cells grown in the presence of inositol, and all display maximal repression when cells are grown in the presence of inositol and choline (Carman and Henry, 1989; White e t al., 1991). However, the enzyme ultimately responsible for the production of phosphatidylinositol, phosphatidylinositol synthase, is not regulated in response to phospholipid precursors (Klig et al., 1985; Fischl et al., 1986).

Several of the phospholipid biosynthetic enzyme activities have been shown to be regulated at the level of transcription of structural genes. The steady-state levels of INOl mRNA (Hirsch and Henry, 1986) and CHOI mRNA (Bailis et al., 1987), encoding for inositol 1-phosphate synthase and phosphatidylserine synthase, respectively, are reduced when wild- type cells are grown in the presence of inositol. The mRNA levels are further reduced when both inositol and choline are present in the growth medium. A similar response has also been observed with the C H 0 2 and OPI3 genes2 encoding the phospholipid methyltransferase (Fig. 1).

Several of the coregulated structural genes have also been shown to be controlled by a common set of regulatory genes. Many mutants with defects in the regulation of phospholipid metabolism were originally isolated on the basis of a defect in inositol 1-phosphate synthase regulation (Culbertson and Henry, 1975; Greenberg et al., 1982a, 1982b). The wild-type products of the regulatory genes IN02 and IN04 are required for expression of INOl (Donahue and Henry, 1981b; Loewy and Henry, 1984). Mutations at either of these two loci result in failure of cells to express inositol 1-phosphate synthase, leading to inositol auxotrophy (Donahue and Henry, 1981b). The in02 and in04 mutants are also unable to derepress the entire set of enzymes that are subject to coordinate control by inositol and choline (Henry et al., 1984; Loewy and Henry, 1984; Bailis et al., 1987).

Mutants with lesions at the OPIl locus were originally isolated by Greenberg et al. (1982a, 1982b) on the basis of an overproduction of inositol phenotype (Opi-). The opil mutants constitutively express derepressed levels of inositol 1- phosphate synthase as well as many of the other coregulated enzymes (Bailis et al., 1987; Homann et al., 1985, 1987; Klig et al., 1985, 1988). The effect of the opil regulatory mutation

E. Lamping, S. D. Koblwein, S. A. Henry, and F. Paultauf (1990) J . Bacteriol., submitted for publication.

* S. Toutenhoofd and T. Gill, personal communication.

863

864 Negative Regulation of Phospholipid Biosynthesis T K e n n e d y Pathway

E MME DME C

v m PA- CDP-DG -PI

uu!!,2 us Membrane

Cytoplasm @ I

G6P- IlP- Inositol !Lp

FIG. 1. Pathways for phospholipid biosynthesis in S. cereuisiae. The phospholipid precursor CDP-DG, which is derived from phosphatidic acid (PA), is the direct source of phosphatidyl moiety for the synthesis of PI and PS. In the presence of PI synthase (PIS), CDP-DG and inositol form PI. Glucose 6-phosphate (G6P) is con- verted to inositol 1-phosphate (IlP) in the presence of inositol 1- phosphate synthase (IIPS). This is dephosphorylated to give free inositol. CDP-DG and serine in the presence of P S synthase (PSS) give rise to PS. Phosphatidylethanolamine (PE) is synthesized via the decarboxylation of PS, and subsequent production of phosphatidylcholine (PC) occurs via three sequential methylations involving the phospholipid N-methyl transferases. This pathway was first described by Lester and colleagues (Waechter and Lester, 1971, 1973; Steiner and Lester, 1972). The Kennedy pathway (Kennedy and Weiss, 1956) is an alternative pathway that can be used to synthesize phosphatidylcholine if the appropriate soluble precursors are supplied to the growth medium. Enzymes catalyzing steps in the phospholipid pathway are abbreviated and underlined. Structural genes encoding for many of the enzymes are encircled. PMME, phosphatidylmono- methylethanolamine; PDME, phosphatidyldimethylethanolamine; CDP-DGS, CDP-DG synthase; PSD, PS decarboxylase; PMT, phospholipid N-methyltransferase.

is also apparent at the level of mRNA. In opil mutants, IN01 transcript is constitutively overexpressed regardless of the growth condition (Hirsch and Henry, 1986). Thus, the OPIl gene is believed to encode a negative regulatory factor that is required to repress the whole subset of enzymes that are coordinately controlled by inositol and choline.

Results from a deletion analysis of the 5"untranslated region of ZNOl suggest that there are &-acting regulatory sites that act to reduce transcription of this gene under repressing growth condition^.^ In order to begin to understand the mechanism by which the OPIl gene and its product interact with other regulatory genes and the structural genes under their control, a detailed molecular analysis of the OPIl gene has been initiated. In this report, we present the genetic mapping, cloning, and molecular analysis of the OPIl gene.

MATERIALS AND METHODS

Bacterial and Yeast Strains-Escherichia coli DH5a [F-, endA1, hsdR17(rk-, mk+), supE44, thi-1, recA1, gyrA96, relAl, A(argF-lac zya)U169,&30d hcZAM15, X-] (Hanahan, 1983) was used for routine bacterial transformations and maintenance of plasmids, while E. coli XL1-Blue [recAl, endA1, gyrA96, thi, hsdR17(rk-, mk+), supE44, relAl, A-, hc- , IF', proAB, ladQ, lacZAl5, TnlO(tetR)J] (Bullock et al., 1987) was used for single-stranded DNA (ssDNA)~ production.

The genotypes and sources of Saccharomyces cereuisiae strains used in this study are presented in Table I.

Growth Media and Genetic Methods-E. coli DHSa cells used to propagate plasmid DNA were grown in LB medium containing 50 pg

J. P. Hirsch, J . M. Lopes, P. A. Chorgo, and S. A. Henry (1990) Nucleic Acids Res., submitted for publication.

The abbreviations used are: ss, single-stranded; X-Gal, 5-bromo- 4-chloro-3-indolyl 8-D-galactoside; kb, kilobase; bp, base pair; ORF, open reading frame; MOPS, 3-(N-morpholino)propanesulfonic acid; CDP-DG, cytidine diphosphate-diacylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine.

ml" ampicillin. Bacterial strain XL1-Blue used for the production of ssDNA is a lac- AG1 derivative with TnlO, lad', and hcAM15 on the F'. Selection for organisms containing the F' was accomplished by addition of 10 pg ml" tetracycline to the above medium. Plates that had been spread with 50 $1 of 100 mM isopropyl-1-thio-0-D- galactopyranoside and 50 p1 of 2% (w/v) X-Gal in dimethylformamide were used to detect colonies with plasmids containing yeast genomic- DNA inserts. All bacterial strains were incubated a t 37 "C.

Media used for growth and sporulation of yeast were as described by Sherman et al. (1978). For routine culture, YEPD medium (1% yeast extract, 2% peptone, 2% glucose) was used. Synthetic complete medium contained (liter-'): glucose, 20 g; vitamin-free yeast nitrogen base (Difco), 6.7 g; biotin, 2 pg; calcium pantothenate, 400 pg; folic acid, 2 pg; niacin, 400 pg; p-aminobenzoic acid, 200 pg; pyridoxine hydrochloride, 400 yg; myo-inositol, 2 mg; lysine, 20 mg; arginine, 20 mg; methionine, 20 mg; threonine, 300 mg; tryptophan, 20 mg; leucine, 60 mg; histidine, 10 mg; adenine, 20 mg; uracil, 40 mg; and agar (for plates only), 20 g. Auxotrophic markers were checked on medium lacking a single component of the complete medium (drop-out medium). Inositol-free medium was identical to synthetic complete medium with the exception that myo-inositol had been omitted. A buffered medium (Ruby et al., 1983) used to score 0-galactosidase activity consisted of all components of synthetic complete medium except vitamin-free yeast nitrogen base, with the addition of 0.1 M KHzP04 (pH 7.0), 15 mM (NHJ2SO1, 0.8 mM MgS04.7H20, 2 p M FeS04.6H,0, 75 mM KOH, and 0.04 mg ml" X-Gal. Where indicated, media were supplemented with 10 p~ or 75 p~ myo-inositol and/or 1 mM choline chloride. In all studies involving S. cereuisiae, cultures were incubated a t 30 "C.

Bacterial and Yeast Transformations-Bacterial strains were transformed with plasmid DNA following the calcium chloride procedure described by Mandel and Higa (1970). Yeast strains were transformed with isolated plasmid DNA using the lithium acetate method described by Ito et al. (1983) and modified by Hirsch et al. (1986). Where indicated, directed transformations and gapped-plasmid transformations were performed by digesting plasmids at specific endonuclease restriction sites.

Assay for Opi- (Querproduction of Inositol) Phenotype-Strains of S. cereuisiae were tested for the Opi- phenotype by a modification of the method first described by Greenberg et al. (1982a, 1982b). Strains were patched onto inositol-free plates and, after 24-h incubation, sprayed with a suspension of a tester strain (AID) in sterile distilled water. The tester strain is a diploid homozygous for an adel marker, which confers a red phenotype, and an inol marker, which confers inositol auxotrophy (Table I). Overproduction and excretion of inositol by strains results in growth of the tester strain as seen by a red halo around the patch.

Strains transformed with genomic DNA in high copy shuttle vectors or centromeric vectors were assayed using inositol-free plates lacking leucine or uracil.

Chromosomal Mapping, Cloning, and Subcloning of the OPIl Gene-The SPOll-mapping technique first described by Klapholtz and Esposito (1982) was chosen to map the OPIl locus. This procedure utilizes the ability of diploid strains homozygous for the spoll- 1 mutation to undergo chromosomal segregation without appreciable recombination during sporulation. While constructing strains required for the SPOII-mapping technique, we discovered that the OPIl gene and the SPOll gene were on the same chromosome and not separable by recombination. A 9.5-kb yeast genomic clone containing the SPOll gene, p(SPO11)1, was obtained from C. Atcheson and R. Esposito and used to transform an opil mutant strain (JHO- 6D). This 9.5-kb fragment had been cloned into the yeast centromeric plasmid YCpl9 (Stinchcomb et al., 1982; Table 11). Two additional clones obtained from C. Atcheson and R. Esposito, that had been used to localize the SPOll gene, were tested for their ability to complement an opil mutation (Fig. 2). Plasmidp(SP011)3 was a 4.0- kb BamHI-Hind111 fragment from p(SPO11)l cloned into YCp50 (Johnston and Davis, 1984), and p(SP011)S was a Sal1 digest of p(SPO11)l religated (Table 11).

The yeast/E. coli shuttle vectors described by Hill et al. (1986) were used to construct additional subclones (Table 11). The BamHI- SalI, SstI-Hind111 and BglII-Hind111 fragments from p(SPO11)3 were cloned into YEp351 to give pJH344, pJH354, and pJH355, respectively. Fragment XhoI-XhoI from pJH354 was cloned, in both orientations, into the SalI site of YEp352 to give plasmids pMWlO and pMW11. The fragments EcoRV-Hind111 and EcoRV-XhoI from pJH354 were cloned into the SmaI-Hind111 and SmaI-Sal1 sites of YEp352 to give plasmids pMW12 and pMW13, respectively. Cloned

Negative Regulation of Phospholipid Biosynthesis 865

TABLE I List of S. cereuisiae strains used in this stud.y

Strain designation Genotype Source

AID DC5

WT1 le~2-3,-112 MATu This study WT2 ade5 leu2-3,-112 MATO This study JHO-6D opil-l ade5 leu2-3,-112 t rpl- l urd-1 MATa J. P. Hirsch

spoll ade2 MATu J. P. Hirsch opil-2 his3-11,-15 leu2-3,-112 MATa P. McGraw opil-3 his3-11,-15 leu2-3,-112 MATa P. McGraw opil-1 lys2 MATu Greenberg et al. (1982a) opil-12 lys2 MATu Greenberg et al. (1982a)

adelladel inollinol MATalu S. A. Henry his3-11,15 leu2-3,-112 MATa J. Broach

W303-1A ade2-l canl-100 his3-11,-15 leu2-3,-112 trpl-l urd-1 MATa R. Rothstein

JH2-3D opil-l h o d MATa J. P. Hirsch JH2-7C NO80 NO99 OP1 OP12 OP-lac2 opil-l ade5 leu2-3,-112 trpl-l URA3 (pJH394, INOl’lacZ) urd-1 MATa This study OP-A1 opil-::LEU2 leu2-3,-112 his3-11,-15 MATa This study OP-A2 DD1 DD2 DD3

opil-A::LEU2leu2-3,-112 his3-11,-15 MATa This study opil-::LEU2/0PIl his3-l1,-15/HIS3 leu2-3,-112/leu2-3,-112 MATa/u This study opil-::LEUZ/opil-l his3-11,-15/HIS3 leu2-3,-112/LEU2 LYS2/lys2 MATa/u This study opil-::LEU2lopil-l2 his3-11,-15/HIS3 leu2-3,-112/LEU2 LYS2/lys2 MATa/u This study

fragments of pMWlO and p M W l l were also cloned into YIp351, using the SstI-PstI sites of YEp352, to give plasmids pMW14 and pMW15, respectively (Table 11). All of these subclones were tested for their ability to complement the opil mutant phenotype (Fig. 2).

Construction of Yeast Strains Carrying a n INOl’lacZ Fusion- Mutations in the OPII gene result in constitutive expression of the IN01 gene. In order to obtain a plate phenotype based on the p- galactosidase assay, strains carrying a section of the IN01 promoter fused to the lac2 reporter gene of E. coli were constructed. The integrating plasmid ~ J H 3 3 4 ~ (Table 11) was used to transform yeast strains. This plasmid had been constructed from the hcZ-fusion vector YIp357R (Myers et al., 1986) and contained a 1.0-kb fragment of 5’ IN01 DNA fused in frame with lacZ. In previous studies, this fusion was shown to be fully regulated in response to inositol and choline and to be expressed constitutively in an opil mutant back- g r ~ u n d . ~ Plasmid DNA that had been linearized at the URA3 selectable marker with StuI was used to transform Ura- strains W303-1A and JHO-6D.

The opil-1 strain OP-lac2 (Table I), carrying a single copy of the INOl’lacZ fusion, was also transformed with plasmid pMW14 or pMW15. Each of these were directed to the OPIl locus by linearizing the plasmid at the BglII site. All transformants were tested for p- galactosidase activity using X-Gal plates with and without phospholipid precursors, inositol and choline.

DNA end RNA Isolation-Plasmid DNA was isolated from transformed bacterial strains either by the boiling miniprep method, originally described by Holmes and Quigley (1981), or the CsCI-EtBr gradient procedure used by Clewell and Helinski (1969). Plasmids used for the preparation of sequencing templates were constructed by inserting agarose-gel purified OPIl restriction fragments, from plasmids pMWlO and pMW11, into the vector pGEMTM-5Zf(+) (Pro- mega, Madison, WI; Fig. 6). For induction of ssDNA, E. coli XL1- Blue cells containing pGEM-5Zf(+) recombinants were infected with the helper phage M13K07 (Promega). ssDNA exported from bacterial cells as encapsidated virus-like particles was purified by simple pre- cipitation and extraction procedures.

Isolation of yeast-genomic DNA for Southern blot analysis was performed using the method described by Hoffman and Winston (1987). Total yeast RNA from organisms grown in the presence and absence of phospholipid precursors was isolated using the glass-bead disruption and hot phenol extraction procedure of Elion and Warner ( 1984).

Construction of opil Disruption Alleles-Plasmids containing dis- rupted OPIl fragments were constructed using either pMW10/ pMWll or pJH354 (Table 11). Plasmid pMWlO or p M W l l was linearized at the BglII site, which was internal to the cloned DNA, and a 3.0-kb BglII-BglII LEU2 gene fragment from YEpl3 was inserted in either orientation. This gave rise to plasmids pMW16, pMW17, pMW18, and pMW19 (Table 11). Plasmid pJH354 was digested with the restriction endonuclease XhoI, so as to remove a 2.0-kb fragment from the cloned OPIl gene fragment, and a 2.2-kb SalI-XhoI LEU2 gene fragment from YEpl3 was inserted in either orientation, giving plasmids pMW20 and pMW21 (Table 11). The

OPIl gene fragments containing the selectable LEU2 yeast gene were liberated from plasmids by cutting with SstI-Hind111 (Fig. 3) and used in a one-step disruption transformation of a haploid strain (DC5) wild type for OPIl (Rothstein, 1983).

Southern Blot Analysis-Yeast genomic DNA was digested for 6- 7 h with the appropriate restriction enzymes, as indicated under “Results,” and analyzed by blot hybridization (Southern, 1975). Di- gested DNA was subjected to electrophoresis through a 1% (w/v) agarose gel in 89 mM Tris base, 89 mM boric acid, 2 mM EDTA (pH 8.0) (TBE) and transferred to nitrocellulose (0.45 pm pore size; S&S NC, Schleicher and Schuell) as described by Maniatis et al. (1982). Blots were prehybridized and 32P-labeled nick-translated probes hybridized at 37 “C according to the procedure of Maniatis et al. (1982).

Northern and Slot-blot Analysis-Total yeast RNA was fraction- ated on a 1.2% (w/v) agarose, 3% (v/v) formaldehyde, 20 rnM MOPS (pH 7.4), 1 mM EDTA gel and transferred to nitrocellulose as described by Thomas (1980). Slot blots of total yeast RNA were pre- pared using a Hybrislot slot-blot manifold (Bethesda Research Lab- oratories). Total RNA (1, 2, and 3 pg) was applied directly to nitrocellulose under high salt conditions (3 M NaC1, 0.3 M NanCsH,07. 2H20). Northern blots and slot blots were prehybridized and 32P- labeled ssRNA probes, synthesized using SP6 polymerase (Boehringer Mannheim), hybridized as described for Southern blots with the exception that the incubation temperature was 53 “C. Slot-blot hybridization signals were quantitated, after a visual examination using Kodak X-Omat AR x-ray film (Eastman), by cutting out each slot and counting its radioactivity using a Beckman LS5801 liquid scin- tillation counter (Beckman Instruments).

Sequencing and Sequence Analysis-ssDNA templates were sequenced using the dideoxy chain termination procedure first described by Sanger et al. (1977). The universal M13 primer was annealed to ssDNA templates and DNA sequencing performed using a Sequenase kit (U. S. Biochemical Corp.). The radionucleotide [a-35S]ATP (Amersham Corp.) was used in all labeling reactions. Routinely, 4- 8% (w/v) polyacrylamide, 7 M urea wedge-gels (0.2-0.8 mm) were used and run on an IBI Standard Sequencer (model STS 45; Inter- national Biotechnologies, Inc.) a t a constant 80 watts. Sequencing gels were not fixed before exposure to x-ray film, but dried directly onto Whatman No. 3MM filter paper as described by Kraft et al. (1988).

The DNA Strider program (version 1.0) and PCIGENE (version 6.01) were used for immediate analysis of the OPIl nucleotide and protein sequences. Both GenBank (version 60.0) and EMBL (version 18.0) libraries on BIONET were searched to find nucleotide sequences similar to that of OPIl. Protein data bases NBRF-PIR (version 20.0) and SWISS-Prot (version 10.0) on BIONET were searched for protein sequences similar to the predicted polypeptide sequence of OPI1.

Mapping opil Mutant Alleles Using Gapped Plasmids-Plasmid- borne copies of several opil mutant alleles were obtained using the gap repair procedure described by Orr-Weaver et el. (1983). This technique utilizes the ability of yeast to repair gaps in cloned genes using information from chromosomal DNA. Thus, when a gapped plasmid is used to transform a strain harboring a mutant allele and

866 Negative Regulation of Phospholipid Biosynthesis

TABLE I1 List of plasmid constructions

Plasmid Subclone Vector Source

p(SPO11)l 9.5 kh from yeast genomic DNA YCpl9 C. Atecheson

p(SP011)3

p(SP011)9 Sal1 digest/religation of p(SP011)l YCpl9 C. Atcheson

and R. Esposito

and R. Esposito

and R. Esposito

4.0-kb BamHI-Hind111 from p(SPO11)l YCp50 C. Atcheson

pJH334 1.0-kb EglII-P~t15' IN01

pJH344 2.8-kh EarnHI-Sal1 from p(SPO11)3 YEp351 This study pJH354 2.8-kh SstI-Hind111 from p(SP011)B YEp351 This study pJH355 pMWlO

1.2-kb EglII-Hind111 from p(SPO11)3 YEp351 2.0-kb XhoI-XhoI from pJH354 YEp352

This study

pMWll 2.0-kb XhoI-XhoI YEp352 This study This study

pMW12 1.3-kb EcoRV-Hind111 from pJH354 YEp352 pMW13

This study

pMW14 0.8-kh EcoRV-Hind111 from pJH354 YEp352 This study 2.0-kb SstI-PstI from pMWlO YIp351

pMW15 This study

2.0-kb SstI-PstI from p M W l l YIp351 pMW16

This study 3.0-kb BglII-BglII LEU2 into pMWlO YEp351

pMW17 This study

pMW18 3.0-kh EglII-EglII LEU2 into pMWll YEp352 This study pMW19 3.0-kb EglII-EglII LEU2 YEp352 This study

pMW2O

pMW21 2.2-kb SalI-Xhol LEU2 YEp351 This study

YIp357R DNA in frame with hcZ

Hirsch et aL3

from pJH354 (opposite orientation)

3.0-kh EglII-BglII LEU2 YEp352 This study into pMWlO (opposite orientation)

into pmW11 (opposite orientation)

into XhoI-XhoI A of pJH354

into XhoI-XhoI A of pJH354 (opposite orientation)

2.2-kb SalI-XhoI LEU2 YEp351 This study

pMW22 1.5-kb SpeI-XhoI from pMWlO YEp352 This study pMW23 1.4-kb NaeI-XhoI from pMWl0 YEp352 This study pMW24 1.0-kb DraI-XhoI from pMWlO YEp352 This study

the deletion extends past the chromosomal mutation, the mutated sequence is copied onto the plasmid. Using the integrating plasmid pMW14 (Table II), a series of four gappedplasmids were constructed that had sequences deleted from the OPIl coding region. These were a 141-hp SpeI-NaeI deletion, a 602-bp NueI-BglII deletion, a 315-bp BglII-Sal1 deletion, and a 194-hp SalI-NsiI deletion. Three opil mutant strains, JHO-GD, NOBO, and N099, were transformed with each of the four gapped plasmids and all Leu+ transformants assayed for the Opi- phenotype. Transformants that retained the Opi- phenotype were assumed to have an integrated copy of the repaired gapped plasmid, resulting in two copies of the gene each containing the mutant allele. Genomic DNA was isolated from several Leu+Opi- transformants, as previously described. By digesting 5-10 pg of DNA with any restriction endonuclease that cut once within the cloned DNA and ligating under dilute conditions, plasmids containing the mutant alleles were reisolated. Plasmids containing mutant alleles were amplified using the E. coli strain DH5a and the repaired gaps were suhcloned into vectors for ssDNA sequencing, as previously described.

RESULTS AND DISCUSSION

Chromosomal Mapping, Cloning, and Localization of the OPZl Gene-The OPIl gene was assigned to a chromosomal map position to determine its location with respect to other genes involved in phospholipid biosynthesis. Crosses to strains bearing the spoll mutation (Klapholtz and Esposito, 1982) indicated that the OPZI gene was tightly linked to SPOll on chromosome VIII. When a haploid strain carrying an opil-I allele (JH2-3D) was crossed to a haploid strain containing a spoll-1 allele (JH2-7C) and the diploids sporu- lated, of 46 tetrads examined, 43 contained spores with the parental configuration (i.e. opil SPOlI or OPZl spoll). In these tetrads, the opil and SPOl1 alleles segregated 2+:2-. The other three tetrads had a 3:l segregation pattern for the opil mutation and were probably a result of a gene conversion event at the OPIl locus. Thus, OPIl and SPOll are on the

same chromosome and recombine with a frequency of less than 2%.

Three clones, p(SPO11)1, p(SPO11)3, andp(SPO11)9, that had been used to localize the SPOl1 gene were generously provided by C. Atcheson and R. Esposito (Atcheson et al., 1987). The ability of these clones to complement an opil mutation is shown in Fig. 2. Complementation studies revealed that the OPZl gene was distinct from SPOlI since plasmid p(SPO11)3, which does not complement spoll, complemented opil, and p(SPO11)9, which complements spol l , did not complement opil. The ATRZ (adjacent transcript 1) gene, unlike the SPOZI gene, is not expressed during sporulation and its function is unknown (Atcheson et al., 1987). Further subcloning of p(SPO11)l eliminated the possibility that the ATRI RNA encoded the OPIl gene product (Fig. 2). Approximately half of the ATRl transcription unit was deleted in p(SPO11)3, and yet this clone fully complemented the Opil- phenotype. Furthermore, complementation of the opil lesion was also observed with plasmid pJH354, demon- strating that the leftmost 1.8-kb BamHI-SstI fragment was not required for OPIl function. Plasmid pMW12, which in- cluded about 0.6 kb of sequence upstream of the ATRl initiation site, did not complement the Opil- phenotype. Plasmid pJH344, which does not include any of the ATRl transcription unit, was capable of partial complementation, and the 2.0-kb XhoI-XhoI fragment (pMW10 or pMWll) , which includes only a few hundred bases downstream of the ATRI transcription start site, fully complemented the opil lesion. Plasmids pMWlO and pMWll contain (in opposite orientations) the smallest subclone that complemented opil (Fig. 2). Since either orientation of the smallest subclone was capable of complementation, the fragment most probably contained the entire OPZl coding and promoter sequences. Complementa-


g!!!!+ V C C I V I Cvmplcmmlul~on V I Mtl

I! F E X Bg 7 AgDg li I 1 Y i p , .

SI S r R V S I1 E

t p(SPO1113

I " C ;,,,

t p lsPoI I l9

vc;>,n

I pJH344

i "lb,,*! .i I . pJH354

Yf,,l57

?=I YCp35I

pMWlO I 1 YFpl',.'

VC"35I'

rY YEi>lLi.'

p M W l 4 l S ,,f,3,,,

Pi FIG. 2. Restriction mapping. subcloning, and complemen-

tation of the Opi- phenotype. Plasmids p(SPOIl)l, p(SPOI1)R. and p(SPOl1)9 that have been used to localize the SPOII gene were ohtained from C. Atcheson and R. Esposito. The restriction map represents p(SPO11)l. Isolated DNA that was capable of complementing the Opi- phenotype was further subcloned and tested for complementation capabilities as described under "Materials and Methods." The smallest piece of cloned DNA that complemented the Opi- phenotype was a 2-kh Xhol-Xhol fragment. YCp vectors are yeast centromeric plasmids, YEp vectors are high copy yeast episomal plasmids, and YIP vectors are integrating yeast plasmids. Ability of suhclones to complement opil mutant strains is indicated with a (+), inahility to complement is indicated with a (-), and partial complementation is indicated with a (+/-). Unique restriction sites are as follows: R, RamHI; B f , RglII; E, EcoRI: H, HindIII; HV, IkoRV: S , SalI; Ss, SstI.

TABLE 111 The cloned DNA complements the Opil- phenotype and restores

I N 0 1 's response to phospholipid precursors I T ' corresponds to 75 p M inositol and 1 mM choline, the repressed

growth condition. I-C- indicates absence of supplements, the derepressed growth condition. X-Gal, 5-hromo-4-chloro-:~-indolyl &n- galactopyranoside.

Yeast INOl'loeZ I T ' 1 - C Excretion of genotype Fusion + X-Gal + X-Gal inositol

OPII - White White opil- I - White White + opil-1 +pMW14 - White White - opil-1 +pMW15 - White White - 0 PI I + Light blue Dark hlue - opil- I + Dark blue Dark blue + opil-I + pMW14 + Light blue Dark blue - opil-I + pMW1.5 + Light blue Dark blue -

-

tion studies showed that the OPII gene was in fact distinct from SPOl I as well as the closely located gene, ATRI.

Integration of the Cloned DNA, Disruption of the Chromo- somal OPII Locus, and Genetic Analysis of the opil Insertion Allele-The 2.0-kb OPII fragment contained on plasmids pMWlO and pMWll was also capable of complementing an opil mutation in single copy. Integrating plasmids pMW14 and pMW15 (Fig. 2 and Table III), each containing the 2.0- kb OPII fragment in an opposite orientation, were linearized at a unique BglII site (Fig. 2) and used in a directed transformation of an opil mutant strain JHO-6D. Transformants were selected on the basis of leucine prototrophy and subse- quently assayed for the Opil- phenotype as described under

I FUI

J \

q : ' . ! , :::::--::::: '. . I z I + I

FIG. 3. Construction of opi l disruption alleles. The o p i l - complementing suhclone was definitively shown to contain the 01'11 gene by creating disruption alleles (Rothstein, 198.7) using either (a) an insertion wherehy a I X ( 1 2 selectable marker was inserted into the O P I 1 coding region or ( h ) a deletion wherehy the whole O I ' I I coding region was removed and replaced with a LKL'2 selectable marker. Restriction sites are as follows: Bg, Bg/lI; H, Hindlll: HV, !,'CfJH\': S'. Sall; Ss, M I ; X, Xhol.

FIG. 4. Southern blot analysis of opil disruption alleles. Yeast genomic DNA was digested with the restriction endonucleases SslI and Hind111 (Fig. 3 , a and h) . Digested DNA was suhjected to electrophoresis and transferred to nitrocellulose as described under "Materials and Methods." A "1'-labeled nick-translated Sstl-Hind111 DNA fragment from pMW2O (Fig. 3 h ) was used t o hybridize to the Southern blot.


TAME 1V

27 2X

0 0

FIG. 5. Slot-blot analysis of IN01 mRNA from opil mutant strains. Slot Ihts were prohed with an INOI-specific rihoprohe as described under “Materials and Met hods.” Amounts of IN01 mKNA were normalized to mRNA values ohtained for the rihosomal protein gene 7’(’MI and expressed relative to wild-type derepressed levels. All values are the means of‘ three independent determinations.

I ( ) I ’ l l * ‘* I< v

”+ + - - f“ -

(1.5 h b

FIG. 6. Sequencing strategy. The arrows indicate the direction of ssDNA sequencing. ‘The arrow labeled OI’ll indicates the position of the 01’11 coding region and the direction of transcription. Restric- tion sites are as follows: l jg , flglll: I). DroI: NV, b,’coRV: S, Sa l I : Sn , SnaHI; x, XhoI.

“Materials and Methods.” Southern blot analysis of genomic DNA (data not shown) confirmed single copy integrants. Complementation of the opil lesion by a single integrated copy of the OPII clone eliminated the possibility that a suppressor carried in a high copy plasmid may have been responsible for complementation by suppressing the Opil- phenotype.

Insertion and deletion mutations (Table 11) were first constructed in autonomously replicating plasmids, as described under “Materials and Methods.” Disruption of the cloned DNA was confirmed by transforming opil mutant strains with either plasmid pMW16 (containing an insertion of the L E U 2 gene into the smallest OPII subclone) or pMW20 (containing a deletion of the OPII sequence with an insertion of the LEU2 gene). These plasmids failed to complement an opil mutation. To determine the pbenot-ype of a disruption mutation at the genomic 01’11 locus, cloned DNA fragments

0 26 1 0 0 53/54 0 27 1 0 0 .55/.56

26 0 0 12 14 30

Not relevant 1 0 1 9 I O Not relevant

~~ ~

from pMW16 and pMW20 were used in a one-step disruption transformation of a Leu2- haploid strain wild type for O P I l (Fig. 3, a and h) . Leu+ transformants, in both cases, overpro- duced and excreted inositol giving a similar phenot-ype to existing opil alleles in the Opi- test. Integration of the dis- rupted OPIl gene fragments at the O P I I locus by homologous recombination was confirmed by Southern blot analysis (Fig. 4).

A strain containing the insertion-disruption allele opil- ::LEU2 (strain OP-Al; Table I ) was crossed with two O P I I strains and two strains containing different opil alleles, opil- I (OP1) and opil-12 (OP12). Diploids were first tested for their ability to excrete inositol. Diploid strains heterozygous for the OPII wild-type allele and the opiI-::IJ!U2 allele exhibited an Opi’ phenotype whereas diploids produced by crossing haploid strains carrying the opil-I or opil-12 alleles to strains carrying the opil disruption alleles had an Opi- phenotype. Dissection of tetrads from the OPll/opil-::I.E~M diploid gave the expected 2+:2- segregation of the 0pi’:Opi- phenotype. With the exception of 2 tetrads out of -5.5, Leu’ co-segregated with the Opi- phenotype (Table IV). Tetrads from crosses involving strains containing either an opi l - l or opil-12 allele with the strain carrying an opil-::I,EU2 allele showed a 0+:4- segregation pattern of 0pi’:OpiF. Since the strain carrying the opil-::LEU2 allele still contained a mutant leu2 allele, a two-gene segregation pattern of Leu+:Leu- was observed (Table IV). These results confirm that the inserted DNA was linked to the OPII locus and not to the L E i J 2 locus. Since a one-step gene disruption transformation was success- fully performed in a haploid strain wild type for O P I I , this indicated that OPII is not an essential gene and removal of its function is not lethal to the organism.

The opil Insertion-Disruption All& ICrprPssPs IjprPprc.ssed Levels of INOI mRNA Constitutiuc>ly--In order to confirm tha t t he opil gene disruption strains had a phenot-ype similar to opil mutants previously isolated, levels of INOI mRNA were analyzed in strains carrying the opil-;:Ih’iJ2 allele grown under derepressing and repressing growth conditions (Fig. 5 ) . Slot blots of total RNA were probed with a ”P-labeled INOI-specific riboprobe generated from plasmid pJH320 (Hirsch and Henry, 1986). The amounts of INOI mRNA were normalized to mRNA values obtained for the ribosomal protein gene TCMI (Fried and Warner, 1981) to correct for differences in RNA loadings and expressed relative to wild- type derepressed levels. Strains wild type for the OPII gene regulated the level of INOI mRNA in response to soluble phospholipid precursors (Fig. 5 ) . These data on expression of I N 0 1 in wild-type and opil strains are consistent with the data previously reported by Hirsch and Henry (1986). In the strain carrying the opil-::LECI2 allele, IN01 transcription was constitutively derepressed in a manner similar to that found in the strain containing the opil-1 allele (Fig. 5 ) . Both opil mutant strains displayed a 2-&fold increase in IN01 mRNA as compared to wild-type derepressed levels. Thus the phenotype of mutants carrying the insertion-disruption allele


CTCGAGATM 10

20 30 40 50 60 1 0

80 90 100 110 120 130 TTGMGGTC GAAGAGCCAC MTTGCAGTA CCAGTCTTTC CCCTGGGTCA G A T A C S P E

1 4 0 GTCAGCGAC GAAGAGTACG CATATATTGT TCGACGCTTA CGCAGACATC TCATAGATAG

150 160 110 180 190

C M T G G T A CGTTCGTTTT AGT-GA T G G C A C C T T T M T C T T C A T ATGCAACCGG 200 210 220 230 240 250

260 270 200 290 300 310 ; T W T C G G GCGTTCTTAT TTTITTCTTT TCCACCTCAA TGAGAGGGAT TAATAAGAGG

TTGGTCAAC ATTGATTTCG AGATTCCGTA CTGTACATGC AGTGGCCTGA AAGTTGAGTA

320 330 340 350 360 310 t T T G G A G U A C A G C G A T C TGCACTTAGC $A&AWGCA TATCAGGCCA ULACGTGGCA

PTTTGTTTAC AGTGCTGATT M G C G T G T G TATCAGGACA GTGTTTTTAA CGAAGATACT 390 400 4 1 0 420 . 430 380

~ G T C A T T G ~ T C T GAA AAT can CGT TTA GGA TTA TCA GAG GAA GAG G T ~ GAR GCG

a S e r G l u AS" G l n A r g L e u G l y Leu Ser Glu Glu G l u Val G l u A l a

441 450 459 460 411 486

A l a G l u V a l Leu G l y Val Leu L y s Gln Ser C y s A r g Gln L y s Ser G l n PFo Ser GCT GAA GTA CTT GGG GTG TTG BAA CAA TCA TGC AGA CAG AAG TCG CAG CCT TCA

4 3 5 504 513 522 531 540

G l u A s p V a l Ser G l n A l a hsp l y s W P . 0 A l a Ser G l u Ser Ser T h r T h r Pro

GAG GAC GTC TCA CAA GCT GAC BAA m C C G GCA AGT GAG TCG TCT ACG ACG CCG 5 4 3 550 561 576 5 0 5 594

CTA AAC AT? TTG GAT CGC GTA AGT AAC AX4 RTT ATC AGT AAC GTA GTG ACA TTC Leu As" Ile Leu A s p k g Val Ser Asn L y s Ile Ile Ser A s n V a l Val T h r P h e

603 612 621 630 633 640

T y r A 9 p G l u I le As" T h r A s n L y g A r g Pro Leu L y s Ser Ile G l y A r g Leu L e u TAC GAT GAA ATA AAC K C AAC M G AGG CCA CTG BAA TCA ATA GGG AGG CTG CTA

651 666 615 604 633 102

A s p A s p A s p A 3 p A s p G l u His A s p A s p T y r Asp Tyr ASn A s p A s p G l u P h e P h e GAC GAT GAC GAT GAC GAG CAT GAT GAT TAC GAC TAC AAC GAC GAT GAG TTC TTC

711 720 129 130 141 156

T h r A s n L y i Arg G l n L y s L e u Ser A r g A l a Ile A l a L Y S G l y L y 9 A s p As" Leu K C AAC AAG AGA CAG AAG CTG TCG CGG GCG ATT GCC AAG GGG AAG GAC AAC TTG

165 174 103 792 001 010

L y a G l u T y r L y 4 Leu A a n M S e r Ile G l u Ser LyS LYS A r g Val T h r C y 3

L 4 A GAG TAC AAG CTG AAC m T C C ATC GAG TCT AAG AAG AGG CTT GTA ACG TGC 019 020 031 046 055 864

Leu His L e u h L y s Leu A l a A s " L y s G l n Lsu Ser A s p L y s Ile Ser C y s h TTG CAT CTT TTA AAG CTG GCC AAT AAG CAG CTT TCC GAT BAA ATC TCG TGT TTA

013 802 091 300 903 910

G l n A s p Leu Val G l u L y s Glu G l n Val His P r o Leu Hi9 L y a Gln asp G l y A 8 n CAG GAC CTT GTT GAA AAG GAG CAG GTG CAT CCT TTG CAC AAG CAR GAT GGA PAT

921 936 9 4 5 354 963 312 A l a A r q T h r T h r T h r G l y ala G l y G l u Asp G l u T h r Ser Ser A s p G l u asp A s p GCT AGG ACG ACC ACT GGA GCT GGC GAG GAC GAG ACA TCG TCA GAC GAA GAC GXC

901 990 999 1000 1011 1026

A s p A s p G l u G l u P h e P h e A s p A l a Ser G l u G l n V a l A a n A l a Ser G l u G l n Ser GAC GAT GAG GAG TTT TTT GAT GCC TCA GAG CAG GTC AAC GCC AGC GAG CAG TCT

1035 1 0 4 4 1053 1062 1071 1000

Ile V a l Val L y r . m G l u V a l V a l G l y T h r Val L y s L y s V a l T y r Ser leu Ile ATT GTG GTG AAA GAG GTG GTC GGC ACA GTC AAG AAA GTC TAC TCG CTG ATA

1089 1030 1107 1116 1125 1134

Ser L y 5 P h e T h r A l a ASn Ser Leu P r o Glu P r o A l a A r q Ser G l n Val A r g G l u TCG M G TTC ACA OCA AAT TCG CTG CCG GAG CCC GCA AGA TCT CAG GTT CGG GAA

1143 1152 1161 1110 1179 1108

AGT CTT CTA AAC TTA CCC ACA AAT TGG TTC GAC AGC GTC CAC AGT hCA TCA CTG Ser Leu Leu A s n Leu P r o T h r A s n T C p P h e Asp Ser Val HIS Ser T h r Ser Leu

1197 1206 1215 1224 1233 1242

PI0 HlS His A l a Sec P h e H i s T y r a l a A s n C y 3 G l u Glu G l n L y s Val Gina CCG CAT CAT GCT TCG TTT CAT TAT GCC M C TGT GAA GAA CAA BAA GTG GAG CAA

I251 1260 1269 1210 1207 1296

/Gin G l n G l n G l n G l n G l n G l n Gln G l n G l n Gln Gln L e n L e u G l n Gln G l n Leu1

CAG CAA CAG C m CAG CAA CAG CAG CAG CAG CAG 0-4 CTT TTG CAG CAG CAA CTC 1305 1314 1323 1332 1341 1350

. f

\Leu G l n G l n G l n G l n G l n l L y s A r g A s n L y 9 A S P G l y Asp A s p Ser A l a Ser Pro

CTG CAA U G CAA CAG 0-4 AX4 AGG AAC AAG GAT GGC GAC GAC TCA GCC TCG CCG 1359 1360 1371 1306 1335 1404

TCC TCC TCC GTA ACT GCG AAT GGG kAA GTA CTC ATT CTC GCC kAA GAA TCC CTG Ser Ser Ser Val Thr A l a As" G l y L y s V a l L e u I l e Leu A l a L y s Glu Ser leu

1413 1422 1431 1440 1443 1458

G l u Met Val Arg A s n V a l Eet G l y Val V a l ASP Ser T h r Leu G l y L y s A l a G l u GAA ATG GTG AGA RAT GTC I\TG GGC GTA GTC GAC TCC ACG TTG GGC M G GCT GAA

1467 1476 1405 1494 1503 1512

GAA TGG GTG AAG CAG BAA CAG GAG GTA PAA GAA ATG ATC AGG GAG CGT TTC TTG G l u T r p V a l L y s G l n L y 9 Gln G l u Val L y s Glu Llst Ile A r g G l u A r g P h e Leu

1521 1530 1533 1540 1557 1566

lGln Gln G l n Gln G l n T y r A r q G l n G l n Gln Gln] LYS A S P G l y A s " T y r Val LyS

CAA CAG CAG CAA CAG TAC AGG CAG CAA CAG CAG AAG GAT GGC AAT TAC GTA AAG 1515 1504 1533 1602 1611 1620

Pro Ser G l n A s p l a " V a l A s p Ser Lys A s p CCC TCT CAG GAC AAC GTG GAT AGC AAG GAC TAA CCGAGAC AGATTGAGGT

1629 1638 1647 1660 1610

1680 1690 1700 1110 1120 1130 CTTTCATGCA TTACCACCAG T U T A T T ATACG-TAmTTA TATAATATCC

1140 ATAATCATAA TCATAATCAT AATCATAATC ATAATCGTGA TATTGTACCA GCCCCGCTTC

1150 1160 1170 1780 1790

TCCCCTTTTG AXCTACCATT ATTATCGGAC CCTCTTTACC TTTGRIITGGC TCAGTAAGGA 1000 1010 1020 1830 1040 1850

1060 1070 1080 1090 CCTTTGCGCG CCGTAAGGGG GTCGGGMTA CATTTCCGGG GTTGATC

FIG. I . Nucleotide sequence and predicted amino acid sequence of the OPIl gene. Possible transcription initiation sites are double underlined, and proposed polyadenylation/termination consensus signal sequences are marked with a broken underline.

at the OPIl locus is identical to the phenotype of previously described opil mutants with regard to overproduction of inositol and INOl expression.

The Cloned D N A Restores Regulation of the INOl Structural Gene in an opil Background-Yeast strains carrying an integrated copy of an INOI'lacZ fusion, as described el~ewhere,~ were analyzed for their response to phospholipid precursors (Table 111). A strain wild type for OPIl expressed @-galactosidase activity under derepressing growth conditions, as seen by a dark blue phenotype on X-Gal plates. Under repressing growth conditions (with inositol and choline) this strain had a light blue phenotype, indicating a basal/repressed level of P-galactosidase activity. The opil mutant carrying the INOl'lacZ fusion (OP- lad ; Table I ) expressed derepressed levels of @-galactosidase under both derepressing and repressing growth conditions. The opil mutant containing an INOl'lacZ fusion, when transformed with either pMW14 or pMW15, resulted in the ability of the INOl promoter to respond to repressing and derepressing growth conditions. All transformants that appeared to have restored INOl regulation were tested for overproduction of inositol (Table 111). Com- plementation of the Opil- phenotype coincided with the restoration of INOl 's response to phospholipid precursors.

Hirsch et aL3 have suggested that the OPIl gene product may be a DNA-binding repressor protein that is capable of interacting with specific sites in the promoter of INOl , and possibly other coregulated structural genes (Carman and Henry, 1989), to regulate transcription in response to inositol and choline. Hirsch et aL3 created a series of INOl 5' promoter-deletions fused to the E . coli lacZ reporter gene. When introduced into an opil-1 mutant background, all of these promoter-deletion fusions were constitutively expressed in the absence and presence of inositol and choline. Although several lines of evidence have indicated that the OPIl gene is necessary for the repression of the INOl gene in response to phospholipid precursors, it is not yet certain as to whether the OPIl gene encodes a protein that binds directly to the INOl promoter to control transcription, or, alternatively, whether it regulates other effectors in the pathway.

Nucleotide-Sequence Analysis and Identification of the OPIl Coding Region-The nucleotide sequence of the 2-kb OPIl - complementing fragment was determined using the sequencing strategy shown in Fig. 6. An open reading frame (ORF) of 1212 bp was identified, starting with an ATG codon at nucleotide 439 and terminating with a TAA codon at position 1650 (Fig. 7). Within this ORF there were three other poten- tial translational start sites found, located at nucleotides 565, 829, and 1093 (Fig. 7). Computer analysis identified two other ORFs greater than 200 bp in the opposite strand. However, these did not coincide with the position of the LEU2 gene insertion (Fig. 3a) and were therefore eliminated from further consideration. The cloned OPIl gene was further subcloned using unique restriction sites identified by sequence analysis (Fig. 8). Three 5' deletions of the complementing XhoI-XhoI fragment were cloned into YEp352 to generate plasmids pMW22, pMW23, and pMW24 (Table 11). Plasmids pMW23 and pMW24 failed to complement an opil mutation, presumably because they were unable to encode the complete OPIl protein (Opilp). Plasmid pMW22, however, partially complemented the Opil- phenotype, as seen by a small excretion ring in the Opi- test. This clone contained 10 untranslated nucleotides upstream of the first ATG (Figs. 7 and 8). This

Methionines/ATGs have been bored. The leucine residues of the leucine zipper are underlined and polyglutamine stretches are located within extended boxes. Positions of nonsense codons in isolated opil mutant alleles are marked with an asterisk (see text).


N R Y X Sp 89 S Ni X 0

5 ’ t , , , , ‘ I I 1

ATG 1 A k I ATG ATG

I pMW22

i - / -; pMW23

I I pMW24

1 kb

FIG. 8. 5’ deletions of the cloned OPIZ gene. Unique restriction endonuclease sites identified by sequence analysis were used to successively remove ATGs from the OPIl ORF. Deletions were tested for their ability to complement an opil mutation. Ability to complement is indicated with a (+), inability to complement is indicated with a (-), and partial complementation is indicated with a (+/-).

Ni, NsiI; RV, EcoRV; S, SalI; Sp, SpeI; X , X h I . Unique restriction sites are as follows: Bg, BglII; D, DraI; N , NaeI;

result suggests that the complete OPZl gene product is translated from the sequence contained on pMW22. It is, however, possible that one of the downstream ATGs is used as the start codon, a point that will not be clarified until the OPZl gene product is studied at the protein level. However, the data available does suggest that the ATG at nucleotide 439 is the translation initiation site for Opilp.

Strains bearing the opil mutant alleles, opil-1 (JHO-GD), opil-2 (N080), and opil-3 (N099), were transformed with the gapped plasmids described under “Materials and Meth- ods.” This analysis revealed that the mutations contained in all three strains lie within the same small stretch of DNA. Strains transformed with the gapped plasmids containing SpeI-NaeI, NaeI-BglII, or SalI-NsiI deletions exhibited a complemented, Opi+, phenotype. That is, the gaps in these plasmids did not extend past the chromosomal mutation. When strains were transformed with the BglII-Sal1 gapped plasmid, all retained the Opi- phenotype. Failure of this construct to rescue any of the mutant alleles indicated that, in each case, the mutation must lie between the BglII and the SalI restriction sites of the OPZl locus. Sequencing of EcoRV-Sal1 fragments from reisolated gapped plasmids (Fig. 2) identified nonsense mutations in each of the three alleles, all located within the first polyglutamine stretch of amino acid residues. Alleles opil-3 and opil-2 had a “TAA” codon located at nucleotides 1294 and 1312, respectively, whereas strain opil- 1 had a “TAG” codon a t nucleotide 1315 (Fig. 7).

Due to very low levels of OPZl mRNA, it has not been possible to identify the OPZl transcript definitively or to map the start of transcription. However, several lines of evidence confirmed that the correct coding region had been identified. First, the 5’ deletions of the cloned OPZl gene (Fig. 8) that successively removed sequences from the largest ORF failed to complement an opil mutant allele fully. Second, sequence analysis of three opil mutant alleles revealed three differently located nonsense codons within the ORF. All of these nonsense codons were found within a stretch of DNA sequence that is easily mutated to generate termination codons (CAA- and CAG-rich region encoding polyglutamine residues; Fig. 7). These mutants presumably lack the carboxyl-terminal third of the OPZl gene product, which results in a severe Opi- phenotype. These findings are consistent with the position of the LEU2 gene insertion in the opil-::LEU2 disruption allele. The strain bearing the disruption allele also displays a severe Opi- phenotype and presumably lacks approximately the last third of the translated gene product.

Several elements that are known to be involved in transcription of yeast genes, as well as other eukaryotic genes, were found in untranslated sequences 5’ and 3’ to the OPT1 coding region. In the 5”untranslated region, two “TATA” sequences were found located 205 and 220 nucleotides upstream from the first ATG (Fig. 7). Either one of these may be involved in controlling transcription initiation (Struhl, 1986). In addition to these, several “CAAG” related sequences were also discovered, two of which were in close proximity to a “CT-rich” region (Fig. 7). These sequences are similar to sequences previously described as sites for the start of transcription by yeast RNA polymerase I1 (Burke et al., 1983). In the region 3’ to the ORF, two “AATAA(A)” related sequences, located at nucleotides 1692 and 1707, and a “TAGT” motif, a t nucleotide 1714, were identified (Fig. 7). It has been proposed that these sequences are important for transcription termination and polyadenylation (Zaret and Sherman, 1982; Burke et al., 1983; Humphrey and Proudfoot, 1988). Several other repeated sequences were also found 5‘ and 3’ to the ORF. Most striking were two conserved sequences found in the 3‘-untranslated region. The 7-nucleotide sequence “ATAATAT” was located at nucleotides 1693,1708, and 1722, and “CATAAT” was repeated six times consecutively starting from nucleotide 1730 (Fig. 7). Repeated elements have been shown to be involved in regulation of transcription, either upstream of the coding region (Hinnebusch, 1988) or 3’ to the stop codon (Elion and Warner, 1986; Muller et al., 1988). However, the role of these sequences in OPZl expression remains to be elucidated.

Analysis of the Predicted Opil Protein-Translation of the OPZl ORF predicted a protein (Opilp) composed of 404 amino acids (Fig. 7) with a molecular weight of 40,036. Opilp is a fairly acidic protein having an isoelectric point of 4.77. The codon bias produced for Opilp using the method of Bennetzen and Hall (1982) was 0.089, indicating that the OPZl gene product would probably be a low abundance protein (Ben- netzen and Hall, 1982; Sharp et d . , 1986). This is consistent with the prediction that Opilp is a regulatory protein. Hydro- pathicity analysis (Kyte and Doolittle, 1982) of Opilp indicated no substantial hydrophobic regions, suggesting that there were no membrane-spanning regions. A heptad repeat of leucine residues (a leucine zipper) was identified in the amino acid sequence, starting at nucleotide 853 (Fig. 7).

In a structural analysis of several proteins that are involved in gene regulation, Landschulz et al. (1988) identified a leucine zipper encoded in the yeast transcriptional regulatory gene, GCN4, and the mammalian enhancer binding protein, C/EBP, as well as the proto-oncogene products belonging to the Jun, Fos, and Myc family of nuclear transforming proteins. This structure is thought to form a stable a-helix whereby the leucines, repeated every seventh residue, form a hydrophobic spine down one face of the helix. It is also believed that dimerization of monomers is stabilized by hydrophobic interactions between closely opposed a-helical leucine repeats (Landschulz et al., 1988). The formation of these protein complexes has been postulated to be involved in the juxtapo- sitioning of basic domains, or other DNA-binding domains, that facilitate binding of the protein to DNA (Sassone-Corsi et al., 1988; Landschulz et al., 1988, 1989; Kouzarides and Ziff, 1988, 1989; Turner and Tjian, 1989; Vinson et al., 1989). When represented in the form of a helical structure (Fig. 9), the Opilp leucine zipper region exhibits amphipathicity that is consistent with many of the aforementioned DNA-binding proteins. The leucine residues of the zipper align down one face every second turn of the a-helix (Fig. 9, l a ) , while immediately opposite is a preponderance of amino acid resi-

Negative Regulation of Phospholipid Biosynthesis 87 1

FIG. 9. The Opilp leucine zipper motif represented dia- grammatically as a coiled coil. Here the leucine residues ( l a , l a ‘ ) are shown to align down one face of the structure every second turn of an a-helix. Further stability of the coiled coil may come from interhelical hydrophobic interactions involving hydrophobic residues positioned a t l e , l e ’ . This figure represents the Opilp as a homodlmer. Quite possibly, this protein could interact with another protein encoded by a gene unrelated to OPIl to form a heterodimer (see text).

dues having either charged or uncharged polar side chains (Fig. 9, IC and I f ) . In addition, there are more hydrophobic residues that lie adjacent to the leucine residues at three out of four positions (Fig. 9, l e ) . It is believed that these adjacently positioned hydrophobic residues may add to the stability of hetero- or homodimers in the form of a coiled coil, as dia- grammed in Fig. 9 (Gentz et al., 1989; Landschulz et al., 1989; O’Shea et al., 1989a, 1989b; Ransone et al., 1989; Smeal et al., 1989).

Immediately NHz-terminal to the Opilp leucine zipper is a 30-amino acid residue region, encoded by nucleotides 763-852 (Fig. 7), that contains a net basic charge. The basic domain is believed to be directly involved in protein-DNA binding and, together, the leucine zipper and basic domain have been implicated as having a role in some DNA-binding proteins that regulate transcription (Landschulz et al., 1988, 1989; Brendel and Karlin, 1989; Kouzarides and Ziff, 1989; Vinson et al., 1989). Recently, Vinson et al. (1989) have proposed a “scissors grip” model explaining how leucine zipper proteins may bind to DNA. This is based on the recognition of a basic- residue consensus sequence located within the 30-amino acid region that precedes many of the identified leucine zippers. Their model predicts that the basic domain forms a non- amphipathic a-helix that extends from the leucine zipper region. In a dimeric structure the a-helices of the basic domains would repel each other causing them to bifurcate from the dimerization contact points of the leucine zipper. These NH2-terminal extensions of the zipper’s a-helices would then form two tightly linked DNA-contact surfaces, each matching the two halves of a cis-acting recognition sequence. This model further predicts that there is a point a t which the basic a- helices break or become angulated, possibly only in the presence of DNA, that would allow for continued contact between the basic domains and DNA. This is thought to occur through N-capping the a-helix by way of an asparagine residue that is centrally located within the basic domain of many leucine zipper proteins (Vinson et al., 1989).

While the 30-amino acid residues NHz-terminal to the Opilp leucine zipper do not display the basic amino acid consensus sequence identified by Vinson et al. (1989), Opilp does have some properties that fit the scissors grip model. An analysis of secondary structure using the methods of Garnier et al. (1978) and Gascuel and Golmard (1988) programmed into PC/GENE (version 6.01) predicts that the basic domain of Opilp could form an a-helical structure that extends from the leucine zipper. In addition, there is also an asparagine residue centrally located within the Opilp basic domain (nu-

cleotides 802-804; Fig. 7). This residue could be involved in N-capping the a-helix to enable maximal contact with the INOl promoter, and possibly other promoters, as the scissors grip model proposes (Vinson et al., 1989).

Based on the identification of cis-acting regulatory elements in the 5’ promoter region of IN01: Lopes’ has employed DNA-binding/mobility-shift assays along with oligonucleo- tide competition experiments to assess the possible interac- tion of DNA-binding proteins with the INOl promoter. Using protein extracts from cells wild type for OPIl as well as extracts from strains carrying the opil-1 and opil disruption alleles, an OPIl-dependent protein-DNA complex was identified. When these studies were repeated using extracts from a strain carrying the opil-1 allele transformed with the cloned OPIl gene, the protein-DNA complex reappeared.’ That Opilp contains a well defined leucine zipper and associated basic domain makes it a good candidate for a DNA-binding repressor protein that binds directly to the INOl promoter, and possibly the promoters of other structural genes involved in the phospholipid biosynthetic pathway. Whether Opilp dimerizes with itself in the form of a coiled coil, as depicted in Fig. 9, similar to the yeast transcriptional activator encoded by GCN4 (Kouzarides and Ziff, 1989; O’Shea et al., 1989a), or whether it complexes with another protein to form a heterodimer, similar to the c-jun and c-fos proto-oncogene products (Kouzarides and Ziff, 1989; Ransone et al., 1989), is unknown.

Two polyglutamine residue stretches were also found in the translated OPIl sequence, starting at nucleotides 1294 and 1567 (Fig. 7). One consisted of 21 glutamine residues with two intervening leucine doublets, and a smaller one of 9 glutamine residues was interrupted by a single tyrosine and arginine residue. Searching the nucleotide and protein sequence data bases identified several genes with similarities to OPIl. How- ever, this was largely due to the presence of sequences encoding polyglutamine residues. Although the exact function of these is unknown a t present, it is striking that glutamine-rich regions and polyglutamine tracts are being reported in an increasing number of predicted yeast proteins that have regulatory functions (Schultz and Carlson, 1987; Sengstag and Hinnen, 1987; Trumbly, 1988; Passmore et al., 1988, 1989; Fassler and Winston, 1989; Garrett and Broach, 1989; Jarvis et al., 1989; Pfeifer et al., 1989) as well as regulatory proteins from higher eukaryotic organisms (Miesfeld et al., 1986; Boh- mann et al., 1987; Courey and Tjian, 1988; Brendel and Karlin, 1989; Courey et al., 1989; Mitchell and Tjian, 1989). When Opilp was analyzed using the FLEXPRO program in PC/ GENE (version 6.01) the polyglutamine tracts were predicted to confer flexibility to the protein. This property has been hypothesized to be involved in the orientation of binding domains to specific sites on DNA and/or other DNA-binding proteins (Bohmann et al., 1987; Brendel and Karlin, 1989). The opil nonsense mutations and the opil insertion-disruption mutation all truncate Opilp, removing the polyglutamine tracts. This causes the disappearance of an OPIl-dependent DNA-protein complex involving the INOl promoter,’ possibly implicating a role for this structural motif in DNA binding. This interpretation is further supported by the fact that plasmidpJH344 (Fig. 2), which contains the first long stretch of polyglutamine residues but has the most carboxyl-terminal stretch deleted, partially complemented an opil mutation.

The nucleotide sequence of OPIl has identified a gene product that has properties consistent with its predicted role as a DNA-binding protein. The finding that Opilp possesses structural motifs, such as a leucine zipper and polyglutamine tracts (Fig. 7), that are found in a wide variety of DNA-

s J. Lopes, personal communication.


binding proteins is a particularly significant one. Although the roles of the leucine zipper and polyglutamine tracts in protein-protein and DNA-protein interactions involving Opilp are purely speculative, identification of these structural motifs has provided possible models for further structural/ functional analysis of the OPIl gene and product. Studies that are currently in progress include further sequence analysis of opil mutant alleles, site-directed mutagenesis of the OPIl gene combined with identification and isolation of Opilp, and further analysis of OPIl -dependent DNA-protein interactions with the IN01 promoter. Collectively, these experiments will define a role for the OPIl gene and its product in regulating gene expression and, ultimately, phospholipid biosynthesis.

Acknowledgment-We thank Susan L. Haslett for expert secreta- rial assistance in preparing the manuscript.

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Documents

The OPIl Gene of Saccharomyces cereuisiae, a Negative Regulator of