MOLECULAR AND CELLULAR BIOLOGY, May 1985, p. 1111-1121 Vol. 5, No. 50270-7306/85/051111-11$02.00/0Copyright C) 1985, American Society for Microbiology

Isolation of Alcohol Oxidase and Two Other Methanol RegulatableGenes from the Yeast Pichia pastoris

STEVEN B. ELLIS, PAUL F. BRUST, PATRICIA J. KOUTZ, ANN F. WATERS, MICHAEL M. HARPOLD, ANDTHOMAS R. GINGERAS*

Salk Institute BiotechnologylIndiustrial Associates, Inc., La Jolla, California 92037

Received 26 November 1984/Accepted 23 January 1985

The oxidation of methanol follows a well-defined pathway and is similar for several methylotrophic yeasts.The use of methanol as the sole carbon source for the growth of Pichia pastoris stimulates the expression of afamily of genes. Three methanol-responsive genes have been isolated; cDNA copies have been made frommRNAs of these genes, and the protein products from in vitro translations have been examined. Theidentification of alcohol oxidase as one of the cloned, methanol-regulated genes has been made by enzymatic,immunological, and sequence analyses. Methanol-regulated expression of each of these three isolated genes canbe demonstrated to occur at the level of transcription. Finally, DNA subfragments of two of the methanol-re-sponsive genomic clones from P. pastoris have been isolated and tentatively identified as containing the controlregions involved in methanol regulation.

Interest in methanol utilization by yeast has been moti-vated for a variety of reasons such as the use of theseorganisms for single cell protein production (8, 40), the studyof catabolite inactivation of methanol regulatable genes (13,43, 47), and the study of the biosynthesis of peroxisomalpackaged enzymes (14, 16, 33, 36, 44, 45). Other interestinvolves the structure and operation of the regulatory re-gions controlling the expression of the genes for methanolutilization.The ability of yeast to utilize methanol as a sole carbon

source for growth has been determined for genera such asHansenula, Candida, Torulopsis, and Pichia (20). The met-abolic pathway for the conversion of methanol to CO2 andH20 appears to be similar for several of these yeasts (2, 24,30). This pathway has been elucidated in Pichia pastoris(Fig. 1) and involves the enzymes alcohol oxidase, catalase,formaldehyde dehydrogenase, S-formyl-glutathione hydro-lase, and formate dehydrogenase (1, 2). The syntheses ofsome of these enzymes have been shown to be tightlyregulated (13, 33, 35), and several of the genes involved inmethanol utilization appear to be controlled at one level by aglucose catabolite repression-derepression mechanism (12,13, 22, 35). The ability of methanol to induce directly theexpression of any of the methanol-metabolizing enzymesremains unclear.To understand better the mechanism involved in the

regulation of expression of the enzymes in the meuhanolmetabolic pathway, we have begun isolating P. pastorisgenes that are responsive to methanol regulation. In thispaper we report the isolation of three genes from P. pastoriswhose expression varies with the introduction of methanolas the sole carbon source in the growth medium. Analysis ofone of these three methanol-responsive genes has revealed itto be alcohol oxidase. Characterization of the cDNA andcorresponding genomic clones of these three methanol-re-sponsive genes has permitted the identification of DNAfragments containing the regulatory regions for each of thesegenes.

* Corresponding author.

MATERIALS AND METHODS

Growth and preparation of yeast cells. All P. pastoris cellsused in these studies were obtained from Phillips PetroleumCo. P. pastoris was grown under carbon-limited conditionsin continuous culture at 30°C with either methanol or ethanolas sole carbon source in IM1 salts minimal medium. IM1minimal medium contains 36 mM KH2PO4, 23 mM(NH4)2SO4, 2 mM MgSO4, 6.7 mM KCI, 0.7 mM CaC12, 0.2,uM CUSO4 - 5H20, 1.25 ,uM KI, 4.5 ,uM MnSO4, 2 ,uMNa2MoO4, 0.75 ,uM H3BO3, 17.5 ,uM ZnSO4, 44.5 ,uM FeCl2,and 1.6 ,uM Biotin. The cells grown on methanol were grownat a cell density of 140 g/liter (dry weight) with a retentiontime of 12 h. The cells grown on ethanol were grown at a celldensity of 90 g/liter with a retention time of 11.5 h. Whenethanol or methanol was used in a fermentor, feed stockscontaining concentrations of 20 and 45% alcohol, respec-tively, were used.Ten grams of fermenter-grown P. pastoris cells was col-

lected by centrifugation and suspended at ca. 108 cells per mlin 0.1 M Tris (pH 8.0) and 1% 2-mercaptoethanol. Thesecells were incubated for 5 to 10 min at 37°C and collected bycentrifugation. The pellet was washed once with 30 ml of Sbuffer (1.5 M sorbitol, 0.04 M NaPO4 [pH 6.6], and resus-pended in 5 ml of S buffer per g of cells. Zymolyase (MilesBiochemicals) was added to the cell suspension at a finalconcentration of 500 ,ug/ml. The cells were incubated at 37°Cfor 20 min and centrifuged to collect the cell pellet. Thispellet was frozen in liquid nitrogen and stored at -70°C forlater use.

Isolation of yeast RNA. Total cell RNA was prepared by amodification of the method of Shields and Blobel (39). Thefrozen pellet was initially pulverized with a mortar andpestle and further disrupted in a Waring blender in thepresence of liquid nitrogen. The pulverized pellet was addedto PK buffer (0.14 M NaCl, 0.05 M Tris [pH 8.4], 1% sodiumdodecyl sulfate [SDS], 0.01 M EDTA) at a concentration of7.5 ml/g of cells. Proteinase K (Boehringer Mannheim) wasadded to a final concentration of 400 ,ug/ml, and the suspen-sion was incubated at room temperature for 10 min. Thismixture was extracted first with an equal volume of phenol-chloroform-isoamyl alcohol (50:48:2) and then with an equal

1111

1112 ELLIS ET AL.

PEROXI SOMES

G-SH

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\ S

- H-C-OH 7' H-C-O HCOOH + C02 + H20SG NAD NADH SG ' NAD NADH

H+ H+S-HMG S-FG

I: Alcohol oxidase M: MethanolU: Catalase F: Formaldehydem: Formaldehyde dehydrogenase S-HMG: S- Hydroxymethylglutathione]Z: S-Formylglutathione hydrolase S-FG: S - Formylglutathione7: Formate dehydrogenase G-SH: Reduced Glutathione

F': FormateFIG. 1. Pathway for the oxidation of methanol by P. pastoris.

volume of chloroform-isoamyl alcohol (48:2). Nucleic acidswere precipitated in the presence of 0.25 M NaCl and 2volumes of ethanol, and the pellet was suspended in ETSbuffer (10 mM EDTA, 10 mM Tris [pH 7.4], 0.2% SDS). Thissolution was re-extracted with phenol-chloroform-isoamylalcohol and chloroform-isoamyl alcohol and was ethanolprecipitated. The nucleic acids were redissolved in 10 mMTris (pH 7.4) and 10 mM EDTA. The RNA present in thissolution was enriched either by pelleting through a 4-ml CsClcushion (1 g of CsCl per ml, 10 mM Tris [pH 7.4], 1 mMEDTA) or by precipitation with 2 M LiCl at 4 to 80Covernight and collection by centrifugation. The polyadenyl-ated [poly(A)+] RNA was selected from the solution byaffinity chromatography on oligodeoxythymidylate-cellulosecolumns (Collaborative Research, Inc.) (4). The total andpoly(A)+ RNA were stored under ethanol at -20°C.

Construction of cDNA library. cDNA clones were synthe-sized by a method similar to that reported by Harpold et al.(19). Poly(A)+ RNA (10 ,ug) was suspended in 7 ,u1 of water,brought to a final concentration of 2.7 mM CH3HgOH, andincubated at room temperature for 5 min (31). The firststrand of cDNA was synthesized in 50 ,ul of a solutioncontaining 50 mM Tris (pH 8.3), 10 mM MgCl2, 30 mM2-mercaptoethanol, 70 mM KCI, 500 p.M each of dATP,dGTP, and TTP, 200 p.M dCTP, 25 p.g of oligodeoxythym-idylate per ml (Collaborative Research), 60 p.g of acti-nomycin D per ml, 25 U of RNasin (Promega), 25 p.Ci of[a-32P]dCTP (32.5 pmol), and 120 U of reverse transcriptase(Life Sciences Inc.) at 42°C for 15 min. This reaction mixwas incubated at 37°C for an additional 15 min. The comple-mentary strand to the cDNA was synthesized in a volume of50 p.l containing 50 mM KPO4 (pH 7.4), 5 mM MgC92, 1 mM2-mercaptoethanol, 250 p.M each of dATP, dGTP, and TTP,125 p.M dCTP, 25 p.Ci of [a-32P]dCTP, and 8 U of Klenowfragment DNA Poll (New England BioLabs) at 37°C for 1 h.To ensure the completion of the second strand synthesis, thecDNA was incubated at 42°C for an additional 15 min in asolution of 50 mM Tris (pH 8.3), 10 mM MgCl2, 30 mM2-mercaptoethanol, 70 mM KCI, 500 p.M dXTP, and 150 Uof reverse transcriptase. The cDNA transcripts were di-gested with 1 to 100 U of S1 nuclease (Sigma Chemical Co.)

in a reaction volume of 50 containing 280 mM NaCl, 20mM sodium acetate (pH 4.5), and 4.5 mM ZnSO4 at roomtemperature for 30 min. The cDNA clones were tailed withdeoxycytidine in a reaction volume of 50 containing 10 p.lof 5x terminal transferase buffer (Bethesda Research Labo-ratories), 10 pmol of [a-32P]dCTP, 2.0 p.M dCTP, and 21 U ofterminal transferase (Ratliff Biochem) at 37°C for 30 min.The tailed cDNAs were annealed to deoxyguanidine-tailedpBR322 DNA opened at the PstI site in a volume of 180containing 10 mM Tris (pH 7.4), 0.1 M NaCl, 0.001 MEDTA, 150 ng of double-stranded cDNA, and 900 ng of d(G)pBR322 DNA at 65°C for 3 min and at 42°C for 2 h, afterwhich they were slowly cooled to room temperature. ThecDNA library was used to transform LE392 Escherichia colicells and plated on LB-tetracycline (15 p.g/ml) plates. Rep-lica filters were prepared, and the plasmids were amplified(17) with 200 p.g of chloramphenicol per ml, followed by lysisand baking at 80°C (27).

Northern hybridizations. Poly(A) + mRNA (2 p.g) washeated at 65°C for 5 min in 10 mM NaPO4 (pH 7.4), 50%formamide, 2.2 M formaldehyde, and 0.5 mM EDTA. Thissolution was cooled to room temperature, and an appropri-ate amount of Sx sample buffer (0.5% SDS, 0.025% bromo-phenol blue, 25% glycerol, 25 mM EDTA) was added. Thesamples were loaded on a 1.5% agarose gel prepared in 1.1M formaldehyde-10 mM NaPO4 (pH 7.4) and electropho-resed in the same buffer. The gel was stained with acridineorange (33 p.g/ml) in 10 mM NaPO4 (pH 7.4), destained in thesame buffer, and soaked in 1Ox SSC (lx SSC is 0.15 MNaCl plus 0.015 M sodium citrate), and the RNA transferredto nitrocellulose (5). The sizes of the mRNA transcriptsobserved from these hybridizations were estimated by com-parison with the 25S (3,392 bases) and 17S (1,789 bases)RNAs of Saccharomyces cerevisiae.

Colony hybridization. The cDNA library was denatured,neutralized, and fixed to 150-mm nitrocellulose filters whichwere then prehybridized at 42°C for 5 h in a solutioncontaining 5x SSC (pH 7.0), 5x Denhardt solution, 50%deionized formamide, 0.2% SDS, and 200 p.g of sheared anddenatured herring sperm DNA per ml. The prehybridizationsolution was replaced with hybridization solution (same as

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MOL. CELL. BIOL.

YEAST METABOLISM OF METHANOL 1113

prehybridization except with 1x Denhardt solution). Either32P-labeled poly(A)+ mRNA or 32P-labeled single-strandedcDNA (106 cpm/ml) was hybridized to the filters for 17 h at42°C. After hybridization, the filters were washed briefly in2x SSC at 22°C, followed by two 10-min washes at 65°C in0.lx SSC.

End-labeling of poly(A)+ mRNA was performed by theaddition of 2 ,ug of poly(A)+ mRNA to a volume of 50 p.1containing 50 mM Tris (pH 9.5); this was heated to 100°C for3 min and rapidly chilled on ice. This RNA solution wasadjusted to 50 mM Tris (pH 9.5), 10 mM MgCl2, 5 mMdithiothreitol, and 50 pmol of [y-32P]ATP in a final volume of200 pu1. Ten units of T4 polynucleotide kinase (BoehringerMannheim) was added, and the reaction mixture was incu-bated at 37°C for 1 h. The kinasing reaction was terminatedby the addition of 10 p.1 of 0.5 M EDTA, and the reactionmixture was extracted with phenol-chloroform-isoamyl alco-hol and chromatographed through Sephadex G-50 to removethe unincorporated label.

Isolation of genomic DNA and clones. Pichia genomic DNAwas isolated by the same method as that used for the RNAisolation. The total nucleic acid was suspended in TE buffer(10 mM Tris [pH 7.4], 1 mM EDTA) and incubated withRNase A at 20 ,ug/ml for 30 min at 37°C. The solution wasbrought to 0.14 M NaCl and treated with proteinase K at 200,ug/ml for 15 min at 22°C. This solution was extracted withphenol-chloroform-isoamyl alcohol and chloroform-isoamylalcohol and was ethanol precipitated. The DNA was sus-pended in TE buffer and centrifuged to clear the superna-tant.

Pichia genomic DNA (15 ,ug) was digested with variousrestriction enzymes (New England BioLabs, Inc.), elec-trophoresed on a 1% agarose gel containing 1 x TAE (40 mMTris, 20 mM acetic acid, 2 mM EDTA [pH 8.3]), and boundto nitrocellulose filters (41). Complementary genomic frag-ments were identified by hybridization to nick-translatedcDNA clones (32). Genomic fragments which hybridized tocDNA clones were sliced from the agarose gel, electroelu-ted, passed through an Elu-tip (Schleicher & Schuell, Inc.),n-butanol extracted, and ethanol precipitated. The fragmentswere ligated to 1 ,ug of pBR322 DNA opened at the appro-priate restriction site (27). This ligation mix was transformedinto LE392 E. coli cells, plated on an appropriate selectionmedium, and prepared for colony hybridization selection.The replica filters of each plate were probed with the32P-labeled cDNA fragments used to identify the originalgenomic fragment.

Large-scale DNA plasmid preparations were isolated andpurified by the method of Clewell and Helinski (7). Min-iplasmid preparations were done by the boiling method ofHolmes and Quigley (21).

Purification of alcohol oxidase. Extracts from P. pastoriscells grown on methanol were provided by Phillips Petro-leum. These extracts were prepared by lysis of yeast cellsfollowed by a clearing spin to remove cell debris. Thecleared lysate was dialyzed against water at 4°C. The crys-talline precipitate resulting from this dialysis was providedto us and used for further purification. Extracts were dia-lyzed against 0.05 M KPO4 (pH 7.5) and applied to a 200-cmSephacryl 200 (Pharmacia Fine Chemicals, Inc.) columnequilibrated with the same buffer. The fractions containingthe peak activity of alcohol oxidase were chromatographed asecond time through an identical column. Fractions of 3.5 mlwere collected at a flow rate of 10 ml/h and assayed foralcohol oxidase activity. The assay for alcohol oxidase isbased on the determination of the levels of hydrogen perox-

ide produced from the metabolism of methanol, utilizinghorseradish peroxidase and a dye precursor, 0-dianisidine,at 25°C (Phillips Chemical Co., Biochemicals TechnicalInformation). Reactions were followed spectrophotometric-ally at 460 nm. A total of 0.1 ,ug of total protein from eachfraction was also assayed by SDS-polyacrylamide gel elec-trophoresis (12%).DNA and protein sequencing and determination of transcrip-

tional initiation site. Determination of DNA sequences wasperformed by the dideoxynucleotide chain elongationmethod, using bacteriophage M13 (37), or by the chemicalmodification method (28). The DNA fragments correspond-ing to the 5' end of the alcohol oxidase gene were insertedinto the M13 mp8 and mp9 vectors or were endlabeled forthe chemical modification method, using restriction enzymesites available in this region (see Fig. 3). The amino acidsequence of alcohol oxidase was performed by Sequemat,Inc., Watertown, Mass., using 2 mg of alcohol oxidase fromP. pastoris.End labeling of DNA fragments for sequencing by the

chemical modification method was done by filling in theHindHI sites with dATP, dGTP, and [a-32P]dCTP. The DNAwas then digested with Sall, and the appropriate fragmentwas isolated for sequencing.For a determination of the location of the start of the

mRNA for alcohol oxidase, a primer extension experimentwas performed by using as primer a synthetic 15-residueoligonucleotide (5' CTTCTCAAGTTGTCG 3') copied fromthe DNA sequence of the 5' end of the alcohol oxidase gene,and as template 10 ,ug of poly(A)+ P. pastoris mRNA. Thisoligonucleotide was synthesized by standard phos-phoamidite chemistry on an Applied Biosystems Synthesizer(model 380A). The primer was extended by using reversetranscriptase reactions similar to those reported by Karan-thanasis (23) modified by the addition of [a-32P]dCTP, 10 ,ugof poly(A)+ mRNA, and 3 ng of oligonucleotide primer. Thereaction was terminated by the addition of 7.5 p.1 of formam-ide dye mix (37). One set of sequencing reactions was donewithout any dideoxynucleotide triphosphate present in themix. All sequencing reactions were electrophoresed on 6 or8% polyacrylamide gradient gels containing 8 M urea (6).mRNA hybridization-selection and in vitro translations.

Positive hybridization-translation experiments were per-formed basically as described by Harpold et al. (18). ClonedDNA (20 ,ug) was linearized by digestion with an appropriaterestriction endonuclease. This DNA was denatured by theaddition of NaOH to a final concentration of 0.3 M, boiledfor 2 min, and incubated at 65°C for 10 min. The DNA wasquickly chilled on ice and neutralized by adjustment to 0.5 MTris-hydrochloride (pH 7.4). An equal volume of 20x SSPE(10) was added immediately before binding of the DNA tonitrocellulose. DNA (10 ,ug) was spotted onto the nitrocel-lulose filters, air dried, and dried under vacuum for 2 h at70°C. Before spotting of the DNA, the nitrocellulose filters(Schleicher & Schuell BA83, 9 mm diameter) were preparedby wetting in water, boiling for 10 min, and rinsing threetimes in lOx SSPE. Before prehybridization, the filters wereplaced in 1 ml of sterile water, heated for 1 min at 100°C,cooled on ice, and rinsed by vortexing in 1 ml of sterilewater, after which 1 ml of prehybridization buffer (contain-ing 200 p.g of rabbit liver tRNA per ml; CollaborativeResearch) was added at 37°C for 2.5 h. Hybridizations forselection of specific mRNAs occurred for 24 h at 42°C. Afterthe hybridization, filters were washed briefly two times in1 x SSPE-0.5% SDS at 22°C, seven times in 1 x SSPE-0.5%SDS at 50°C for 5 min each, three times in 0.1x SSPE at

VOL. 5, 1985

1114 ELLIS ET AL.

FIG. 2. A 1% agarose gel, showing the cDNA inserts released bydigestion of clones with PstI. pPC8.0 and 8.3 are overlapping cDNAclones covering the 3' and 5' ends, respectively, of the alcoholoxidase gene. pPC6.4 is a 3'-end-specific clone, and pPC6.7 is afull-length copy of the P40 gene. pPC15 is a 3'-end-specific copy ofthe P76 gene.

50°C for 5 min each, and one time in 0.1 x SSPE at 65°C for10 min. The RNA was eluted from the filters by boiling for 1min in 300 ,u1 of water containing 15 ,ug of rabbit liver tRNA.After freezing at -70°C, the filters were removed, and RNAwas precipitated with 2.5 M ammonium acetate and ethanoltwo times and suspended in 100 ,ul of water before beinglyophilized. Translations were performed according to in-structions provided by New England Nuclear Corp. in vitrorabbit reticulocyte lysate translation kits. Protein productswere electrophoresed on 8% polyacrylamide gels containinga 4.5% stacking gel.

Antisera preparations and immunoprecipitation. Antiseraraised in rabbits against an extract from P. pastoris cellscontaining both alcohol oxidase and P76 polypeptides wereprepared by standard protocols. Extracts were dialyzedagainst 1 x phosphate-buffered saline before injections. Overa course of 8 weeks, each rabbit received three injections,each of which consisted of 1 mg of total protein in a volumeof 0.1 ml with 0.2 ml of Freund complete adjuvant. Injec-tions were intradermally delivered to 6 to 10 sites in eachrabbit. At the end of 8 weeks, the rabbits were bled and theirsera were tested against P. pastoris extracts or purifiedalcohol oxidase by the Ouchterlony double diffusion proce-dure.

Affinity-purified rabbit anti-alcohol oxidase and anti-P76protein antibodies were prepared by chromatographing wholeantisera through a CNBr-coupled alcohol oxidase-P76Sepharose 4B column. The column was prepared by stand-ard procedures recommended by Pharmacia, except that thegel was washed with 2 M sodium thiocyanate in 1x phos-phate-buffered saline. The affinity-purified antiserum wasadded to an in vitro translation mix in 1 x phosphate-bufferedsaline-1% Nonidet P-40 (Sigma) and incubated overnight at4°C. The antibody-antigen complex was precipitated withPansorbin (Calbiochem) on ice for 2.5 h. Pansorbin was

prepared by washing in RIPA buffer (1 x phosphate-bufferedsaline, 1% Nonidet P-40, 1% sodium deoxycholate, and0.1% SDS). Pansorbin precipitates were washed four timesin RIPA buffer and dissolved in 1x Laemmli loading buffer(26) at 100°C before electrophoresis.

RESULTSIsolation of three methanol-regulatable genes from P. pas-

toris. A ca. 20,000-member cDNA library was prepared fromP. pastoris cells grown on methanol as the sole carbonsource. The library was screened by hybridization, usingkinase poly(A)+ RNA isolated from P. pastoris grown in thepresence of either methanol or ethanol. After several roundsof this plus-minus screening, three distinct, nonhomologouscDNA clones were identified as being methanol responsive.These clones were designated pPC6.4, 8.0, and 15.0 andwere determined to contain inserts of 470, 750, and 1,100base pairs (bp), respectively (Fig. 2).

Because the average size of inserts in this first cDNAlibrary was relatively short (<1,200 bp), a second cDNAlibrary was prepared by using milder S1 nuclease digestionconditions, and the members of this new library werescreened individually with 32P-labeled cDNA clones pC6.4and 8.0 and 15.0. As a result, larger cDNA clones wereisolated for cDNA clones pPC6.4 and 8.0. pPC6.7 and 8.3were found to contain inserts measuring 1,200 and 2,100nucleotides, respectively (Fig. 2). A cDNA clone possessingan insert larger than the 1,100 nucleotides for pPC15.0 hasnot been observed after the screening of more than 40,000cDNA clones.The isolation of the genomic DNA fragments correspond-

ing to each of these cDNA clones was accomplished bycutting out and electroeluting from agarose gels P. pastorisDNA fragments that hybridized with 32P-labeled pPC15.0,8.0, or 6.4. The eluted genomic DNA fragments were clonedseparately into E. coli and rescreened several times witheach cDNA probe. The relationship of each cDNA clone toits corresponding genomic clone is shown in Fig. 3. pPC15.0is encoded within a 6.0-kb HindIll genomic fragment presentin clone pPG6.0 (Fig. 3A). The 5' end of the gene encoded bypPC15.0 is oriented toward the 1,300-bp HindIII-EcoRIfragment contained in pPG6.0, and the 3' end of the gene isproximal to the PstI sites in pPG6.0. The cDNA clonepPC8.3 is included within the genomic clone pPG4.0 (Fig.3B). pPG4.0 contains an EcoRI-PvuII insert of 4,000 nucle-otides of contiguous genomic DNA. The orientation ofpPC8.3 within pPG4.0 places the 5' end of the gene for thiscDNA clone close to the BamHI site; the 3' end of this geneis located near the PvuII site. The cDNA clone pPC6.7 islocated entirely within a 4,800-nucleotide EcoRI-BamHIgenomic fragment. The 5' end of the gene is positionedcloser to the BamHI end of the genomic clone pPG4.8 (Fig.3C). In all of these genomic clones there is at least 1.2 kb offlanking genomic DNA sequence which is 5' to the genescopied in each of the cDNA clones.

Identification of alcohol oxidase as one of the methanol-regu-latable genes. For a determination of which protein productswere encoded by each of the cDNA clones, poly(A)+ RNAfrom P. pastoris cells grown on methanol was selectivelyhybridized to each of the cDNA clones. The mRNA hybrid-ized to each of the cDNA clones was then translated in vitro,and each of the protein products was resolved by electropho-resis under SDS-denaturing conditions. The results of thesein vitro positive hybridization-translation experiments indi-cated that the cloned pPC15.0, 8.3, and 6.7 encode informa-tion for polypeptides of 76,000, 72,000, and 40,000 daltons,

MOL. CELL. BIOL.

YEAST METABOLISM OF METHANOL

H2 Pv2H3 H2 RI H2 Xh IRl Pvl H2 H3

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FIG. 3. Restriction enzyme map for the genomic and cDNA clones of methanol-responsive genes (A) P76, (B) alcohol oxidase, and (C)P40. Restriction sites and their abbreviations are: BamHI (B), BgII (B,), CIal (C), EcoRI (RI), EcoRV (E5), HinclI (H,), Hindlll (H3), KpnI(K), PstI (Ps), PvuI (P1, Pvi), PvuIl (Pv,), Sall (S), Stul (St), XbaI (Xb), XhoI (Xh), XmaI (Xm).

respectively (Fig. 4A). These same proteins are visible whentotal poly(A)+ RNA fromn methanol-grown cells is translatedin the same in vitro system. However, only the 40,000-daltonpolypeptide is synthesized from poly(A)+ RNA isolatedfrom cells grown in the presence of ethanol.

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FIG. 4. (A) An autoradiograph detailing the 35S-labeled polypeptides made from mRNA hybridization-selection and in vitro translationsof cDNA clones pPC15.0, pPC8.3, and pPC6.7, as well as total poly(A)t RNA from cells grown on methanol. In vitro translations ofunselected poly(4)+ RNA from cells grown on ethanol shows only P40 product. Lanes labeled total MeOH RNA demonstrate the presenceof the 76-, 72-, qpd 40-kilodalton proteins. Lanes labeled pPC15.0, pPC8.3. and pPC6.7 show the presence of each of these proteinsindividually. The control lane (No DNA) shows the labeled proteins produced by the translation kit without added DNA or RNA. (B) Thesame 8% polyacryl4mide gel as in A, bdt stained with Coomassie blue. Purified alkphol oxidase protein (AO) and molecular weight markersare shown for referi'nnce.

VOL. 5, 1985

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1116 ELLIS ET AL. MOL. CELL. BIOL.

A T G C T T C C A A O A T T C T G G T G G G A A T A C T G C T G A T A G C C T A A C G T T C A T G A T C A AA A T T T A10 20 30 40 50 60

A C T G T T C T A A C C C C T A C T T G A C A G G C A A T A T A T A A A C A G A A G G A A G C T G C C C T G T C T T A A70 80 90 100 110 120

+ * +A C C T T T T T T T T T A T C A T C A T T A T T A G C T T A C T T T C A T A A T T G C G A C T G G T T C C AA T T G A C

130 140 150 160 170 180

A -AG C T T T T G A T T T T A A C G A C T T T T A A C G A C A A C T T G A G A A G A T C A AAA A C A A C T A A T T A

HindlI 190 200 210 220 230 240

MET ALA ILE PRO GLU GLU PHE ASP ILE LEU VAL LEU GLY GLY GLY SER SERT T C GA A A C G A T G G C T A T C C C C GA A G A G T T T G A T A T C C T A G T T C T A G 0 T G G T G G A T C C A G T

250 260 270 280 290 BamHI 300

GLY SER CYS ILE ALA GLY ARG LEU ALA ASN LEU ASP HIS SER LEU LYSGOG A T C C T G T A T T O C C G G A A G A T T G G C A AA C T T G G A C C A C T C C T T G A A A G T

BamHI 310 320 330 340 350 10

FIG. 5. DNA sequence of 350 bases from the 5' end region of the alcohol oxidase gene. The amino acid sequence predicted from thisnucleotide sequence starting at position 250 matches the first 18 amino acids derived from the alcohol oxidase protein. A potential TATA boxregion is denoted by ( ). The 5' ends of the alcohol oxidase mRNA have been determined (Fig. 8) and are denoted as a major species (*)or minor species ( I ) of mRNA transcripts.

resis and was subjected to additional purification by chro-matography through Sephacryl 200 (Fig. 4B). Results fromthis chromatography demonstrated that alcohol oxidase ac-tivity corresponded to that of the purified 72,000-daltonprotein. The size of this protein was identical to that of theprotein product encoded by cDNA clone pPC8.3 (Fig. 4A).For a determination of whether pPC8.3 was in fact the

cDNA clone encoding P. pastoris alcohol oxidase, the

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NH2-terminal amino acid sequence of the 72,000-daltonprotein was derived. The amino acid sequence was deter-mined to be Ala-Ile-Pro-Glu-Glu-Phe-Asp-Ile-Leu-Val-Leu-Gly-Gly-Gly-Ser-Ser-Gly-Ser. In parallel, the nucleotide se-quence of the 5' end of the gene encoded in pPC8.3 andpPG4.0 was determined (Fig. 5). The predicted amino acidsequence derived from the DNA sequences of the clonesagreed perfectly with the first 18 amino acids of the amino

B.z Z0: 0:IK I

0 0oO oi < I

O 0 OH2 2

rl) 0

QL 0-OL ai1

0

a-'2

- - 92 5 K

76 Kd,72 Kd' - 72

-66 2

-45

FIG. 6. (A) Autoradiograph detailing the 35S-labeled polypeptides from in vitro translations of mRNAs hybrid-selected (Fig. 4) that wereprecipitated with anti P76-alcohol oxidase antibodies. P76 was selectively precipitated by the antibody when pPC15.0 was used tohybrid-select mRNA for the in vitro translations. Similarly, alcohol oxidase (72 kilodaltons) was precipitated from translations, using pPC8.3clones as hybrid-selection probe. Both of these proteins were precipitated from translations of total poly(A)+ RNA from cells grown onmethanol. Neither protein was precipitated from translations of total poly(A)+ RNA from cells grown on ethanol. (B) The same 8%polyacrylamide gel as in A, but stained with Coomassie blue. Purified alcohol oxidase protein (AO) and molecular weight markers are shownfor reference.

YEAST METABOLISM OF METHANOL 1117

acid sequence derived from P. pastoris alcohol oxidase (Fig.5).

Additional support that clones pPC8.3 and pPG4.0 encodethe alcohol oxidase gene was obtained by means of an

immunological approach. The extract isolated from P. pas-

toris containing both the 76,000- and 72,000-dalton proteinswas used to obtain antisera specific for these proteins. Whenthe hybrid-selected poly(A)+ RNA from pPC8.3 was trans-lated in vitro, only the 72,000-dalton translation product wasprecipitated by the antisera made against the extracts fromP. pastoris cells (Fig. 6).The 76,000-dalton protein present as a result of in vitro

translations of total poly(A)+ RNA from cells grown onmethanol comigrates during electrophoresis with the proteinproduct encoded by clones pPC15.0 and pPG6.0 (Fig. 4A).In addition, antisera made against the mixture of 76,000- and72,000-dalton proteins was able to immunoprecipitate onlythe in vitro-translated 76,000-dalton protein encoded bymRNA hybrid selected with pPC15.0 (Fig. 6A), furtherindicating that the 76,000-dalton protein isolated from meth-anol-grown cells appears to be encoded by the gene se-quences present in clones pPC15.0 and pPG6.

Effect of methanol on RNA transcription of P76, P40, andalcohol oxidase genes. The influence of methanol on theexpression of each of the cloned genes can be observed inpart by studying the effects on transcription of these genes.

Isolated poly(A)+ RNA from P. pastoris cells grown withethanol or methanol as sole carbon source was used toprepare Northern hybridization filters. Three identical pairsof filters from methanol- and ethanol-grown cells wereprobed separately with 32P-labeled pPC15.0 (P76), 8.0 (AO),and 6.4 (P40). The clones pPC15.0, 8.0, and 6.4 hybridized toRNA molecules from methanol-grown cells of ca. 2,400,2,300, and 1,300 nucleotides respectively (Fig. 7). No hy-bridization was observed with RNA obtained from cellsgrown in the presence of ethanol with pPC15.0 and 8.0 ashybridization probes. However, when RNA isolated fromcells grown with ethanol was probed with pPC6.4, the clonehybridized to a 1,300-nucleotide RNA molecule identical to

PROBESS cDNA-8

C_,

v _ =E< "i

cDNA-15

E C

cDNA 6I T

0

WholePlosmid

FIG. 7. Northern hybridizations, showing poly(A)+ RNA whichhybridized to nick-translated cDNA probes from pPC8.0. pPC15.0,pPC6.4, and (whole plasmid) vector pBR322. Equivalent amounts ofpoly(A)+ RNA from cells grown on methanol, ethanol, and an

alcohol oxidase-negative P. pastoris mutant were loaded into each

lane and bound to the filter.

that seen with methanol-grown cells but at a ca. fivefoldlower level.

In the case of poly(A)+ RNA hybridized to pPC8.0, thereissignificant hybridization to an RNA species smaller than1,000 nucleotides. The identification of this RNA species isunclear. However, there is DNA sequence homology be-tween pPC8.0 and other regions of the P. pastoris genome(see below). Although the size of this smaller RNA is notlarge enough to encode alcohol oxidase (molecular weight72,000), this smaller species may be large enough to encodeanother gene ofP. pastoris which shares partial homology toalcohol oxidase. In vitro translations of mRNA selected byclone pPC8.0 show polypeptides of less than 40 kilodaltons(Fig. 4A). These polypeptides are not precipitated by anti-bodies made against P76 or alcohol oxidase (Fig. 6A).

Poly(A)+ RNA was prepared from a mutant strain of P.pastoris which could not metabolize methanol and wascharacterized as alcohol oxidase deficient (J. Cruze and G.Sperl. unpublished data). This mutant has no mRNA foreither alcohol oxidase or P76. However, the level of P40mRNA appears normal (Fig. 7). Southern hybridization ofthe mutant genome show no gross deletions of either thealcohol oxidase or P76 genes (data not shown).

Organization of alcohol oxidase gene. For a determinationof where RNA transcription for the alcohol oxidase gene isinitiated, the DNA sequence around the 5' end of this genewas determined from the genomic clone pPG4.0 and thecDNA clone pPC8.3. This sequence was then matched to thesequence obtained from the 5' end of the alcohol oxidasemRNA by means of a primer extension experiment. cDNAclone pPC8.3 contains 100 nucleotides of a 5' untranslatedregion of the alcohol oxidase gene. Based upon the sequenceobtained from pPC8.3, a complementary oligonucleotide of15 bases (5' CTTCTCAAGTTGTCG 3') was synthesized andused as a primer to extend along the alcohol oxidase mRNAto reach the 5' end. The sequence from this primer extensionexperiment revealed three different transcriptional initiationpoints for P. pastoris alcohol oxidase mRNA (Fig. 8). Themajor transcript begins at a rAp residue 114 nucleotides fromthe translational initiation codon (Fig. 5). Two minor alter-native transcripts begin also at rAp 117 and 111 nucleotidesupstream from the alcohol oxidase AUG codon. Conse-quently, there can be as much as 117 nucleotides of untran-slated RNA sequence from the start of the mRNA to thestart of the alcohol oxidase coding sequence.The DNA sequence preceding the 5' end of the alcohol

oxidase mRNA transcript was determined from subfrag-ments of the EcoRI-BamHI fragment from pPG4.0 (Fig. 3B).The 55 nucleotides preceding the start of alcohol oxidasemRNA contain structural features resembling a Goldberg-Hogness box (TATAA box). The sequence TATATAAAoccurs at position -50 from the 5' end of the predominanttranscript for alcohol oxidase RNA and 161 nucleotidesupstream from the initiation codon for this protein.

Examination of the DNA sequence flanking the transla-tional initiation codons of several eucaryotic genes suggestssome consensus signals. Kozak (25) indicated that the se-quence most favored by eucaryotic translational machineryresembles the form-3XXAUGG- In addition, Dobson et al. (4)suggested that a U at position +6 should also be included inthis consensus sequence. The DNA sequence around theinitiation codon for alcohol oxidase is ACG 1T +4 `6iniiatoncodnfr lcool xiaseis CGATG GCT Thissequence appears to support the consensus sequence sug-gested by Kozak (25) and Dobson et al. (11).

Restriction enzyme mapping of the 3' end of the alcoholoxidase gene in clones pPG4.0, pPC8.0, and 8.3 revealed

VOL. 5, 1985

1118 ELLIS ET AL.

a-

x0

Methanol

Poly A* RNA

T~ zA C G T x

Genomn:c ;'con-e

..

n0

A% 4ot

a~m

x~ ~ ~ w.Xs

.,.WI~~~~~~~~~~~~~~~~~~~~~~~as-

.zu m

-2

9

TTA G

_A

A*Gi

- A- G:

-AAA

AA --n

FIG. 8. Autoradiograph of an 8% polyacrylamide sequencing gelcontaining DNA sequence frotm the 5' ends of the alcohol oxidase(AO) mRNA and genomic regions. The sequence from both tem-plates was generated by using an oligonucleotide primer to extend tothe end of the mRNA template or through this region on the genomicfragment cloned into a single-stranded M13 phage. The centerchannel contains no dideoxynucleotides as chain terminators, andthus 5' ends of the alcohol oxidase mRNA are indicated as runofftranscripts. These terminal nucleotides are indicated in the sequencewith asterisks (*) above the dTp residues to the right of the figure.

that cDNA clone pPC8.3 is missing ca. 250 nucleotidescorresponding to the alcohol oxidase mRNA sequence (Fig.38). The sequences present at the 3' end of the alcoholoxidase mRNA are present in cDNA clone pPC8.0, whichoverlaps pPC8.3 by ca. 800 nucleotides.DNA fragments containing methanol regulatable promot-

ers. The expression of P76 and alcohol oxidase appears to betightly controlled at the level of transcription (Fig. 7).Restriction mapping of pPG4.0 and pPC8.3 indicates that theregulatory region for the alcohol oxidase is potentiallylocated within a 1-kb HindIII-PstI fragment at the 5' end ofthis gene (Fig. 3B).

Similarly, restriction mapping of pPC15.0 and pPG6.0reveals that a 1,300-bp HindIII-EcoRI DNA fragment poten-tially contains a similarly regulatable promoter (Fig. 3A). Atransformation system has been developed for P. pastoris (J.Cregg, manuscript in preparation). Expression vectors con-taining subfragments of both of these putative regulatoryregions have been constructed and used to regulate theexpression of the E. coli lacZ gene in P. pastoris. Results ofthese experiments indicate that both of these putative regu-latory regions control the expression of P-galactosidase intransformed P. pastoris cells (J. Tschopp, manuscript inpreparation).

DISCUSSIONOf interest to us is the regulation of the expression of the

enzymes required for methanol utilization. As the first steptoward this goal, three nonhomologous cDNA clones(pPC15.0, pPC8.3, and pPC6.7) were constructed frommRNA whose expression appeared to be regulated by the

presence of methanol in the growth medium (Fig. 7). Thesethree cDNA clones were used to selectively hybridize ho-mologous mRNA transcripts from metlianol-grown cells. Invitro translations of these RNAs indicated that cDNA clonepPC15.0 encoded a 76,000-dajton protein and pPC8.3 andpPC6.7 encoded 72,000- and 40,000-dalton proteins, respec-tively (Fig. 4A). A search for the genomic DNA containingthe genes encoding P76, P72, and P40 led to the isolation andidentification of three genomic clones. Genomic clonespPG6.0, pPG4.0, and pPG4.8 were shown to be related topPC15.0, pPC8.3, and pPC6.7, respectively, by restrictionenzyme mapping, Southern hybridization, and DNA se-quence analysis. Ofinterest was the observation that pPC15.0and pPC8,.3 each also hybridized to a second set of genomicDNA fragments (data not shown). pPC15.0 hybridized to a4,400-bp genomic fragment that shares only partial restric-tion site homology with pPC15.0 or pPG6.0. A cDNA clonehas been isolated and shown to be derived from this 4.4-kbgenomic region. Restriction mapping of the 4.4-kb genomicand corresponding cDNA clones indicate several differentsites not seen in clones pPG6.0 and pPC15.0. Also, inaddition to hybridizing to pPG4.0, pPC8.3 hybridized to a3,000-bp genomic fragment that is related but not identical topPG4.0. The relationship of these secondary homologousgenomic DNA fragments to genes P76 and P72 is as yetunclear and is currently being investigated.The identity of the P72 gene as alcohol oxidase was made

possible by the isolation of purified alcohol oxidase from P.pastoris (Fig. 4B). lthe NH2-terminal sequence of the puri-fied alcohol oxidase was derived, revealing a sequence of 18amino acids which agreed perfectly with the N-terminalamino acid sequence derived from the DNA present inclones pPC8.3 and pPG4.0 (Fig. 5). In addition, two otherlines of evidence indicated that pPC8.3 and pPG4.0 encodethe gene for alcohol oxidase. First, the in vitro-translatedprotein product obtained by positive hybridization-transla-tion experiments with pPC8.3 was identical in molecularweight to purified alcohol oxidase. Second, antisera pre-pared against a mixture of both the 76,000-dalton and alcoholoxidase proteins from P. pastoris reacted only with the invitrO 72,000-dalton hybrid-selected translation product.The metabolic pathway for the oxidation of methanol

outlined in Fig. 1 seems generally applicable for mostmethanol-utilizing yeasts (7). As indicated in Fig. 1, the firststeps of this pathway are compartmentalized within theperoxisomes. Ultrastructural studies of yeast cells grown onmethahol have shown an enlargement as well as multiplica-tion of yeast peroxisomes to accommodate the accumulationof alcohol oxidase required for the first step in the oxidationof methanol to formaldehyde (46-48). The levels of accumu-lated alcohol oxidase have been found to be as much as 33 to35% of ihe protein content of P. pastoris (9) and of Hans-enula polymorpha (43). Interest in peroxisome biogenesis inmethanol-utilizing yeasts has motivated others to studythese organisms (16, 44). Comparison of the DNA sequencesof pPC8.3 and protein sequences of peroxisomal packagedalcohol oxidase indicates that there is no signal peptide thatis processed during packaging in the yeast peroxisomes.However, the methionine of alcohol oxidase is processedafter translation. It will be interesting to discern whetheralcohol oxidase possesses a nonprocessed signal sequencenecessary for transport into the peroxisomes. Urate oxidase(15) and catalase (15, 34) are examples of enzymes packagedin liver peroxisomes without processing of signal peptides.Of the enzymes involved in the methanol utilization path-

way of P. pastoris (Fig. 1), alcohol oxidase (9), formate

MOL. CELL. BIOL.

YEAST METABOLISM OF METHANOL 1119

dehydrogenase (1), and formaldehyde dehydrogenase (2)have previously been purified and characterized. The nativeform of alcohol oxidase from P. pastoris was determined tobe a flavoprotein of 675,000 daltons composed of eightidentical subunits containing 1 mol of FAD per mol ofenzyme. Couderc and Baratti (9) determined the sequence ofthe first 12 amino acids from the NH2-terminal end. Theamino acid sequence derived from our isolated alcoholoxidase and from the DNA sequence of pPC8.3 and pPG4.0(Fig. 5) is in perfect agreement with their sequence. How-ever, they estimate the mass of the alcohol oxidase mono-mer to be 80,000 daltons as compared with the 72,000daltons measured in this study (Fig. 4). This discrepancymay be due to a difference in the range of molecular weightstandards used in the two studies. Couderc and Baratti (9)used standards of 24,000, 43,000, 60,000, and 130,000 daltonsto estimate the size of alcohol oxidase on a 5.6% SDS-acrylamide gel. Our standards included proteins of 21,000,31,000, 45,000, 66,200, and 92,000 daltons on a 6.0% SDS-polyacrylamide gel. With no molecular weight marker be-tween 60,000 and 130,000, an error of ca. 10% seemspossible in the original estimation of 80,000 daltons.The organization of the alcohol oxidase gene appears to be

very similar to that of several genes described for S. cere-visiae. The sequence preceding the coding region of alcoholoxidase consists of an AT-rich (68%) region of 165 nucleo-tides. RNA transcribed from this gene begins at threedistinct rAp residues located at positions -117, -114, and-111 (the rAp of the translation initiation codon AUG isdesignated position +1). The predominant transcript is theone starting at position -114 (Fig. 8). This results in anuntranslated RNA sequence that contains several signals fortranscription and translation that are similar to those foundin S. cerevisiae. Besides conforming well to the sequencessuggested by Kozak (25) and Dobson et al. (11), the se-quence CAAAAAACAA occurring at position -26 (Fig. 5)resembles CA-rich sequences found -10 to -36 basesupstream from the translational initiation codons of severalS. cerevisiae genes (3, 11, 29, 42). At position -161, thesequence TATATAAA occurs. This Goldberg-Hogness-like sequence is located 47 nucleotides from the start oftranscription for alcohol oxidase. The distance from thisTATAA box to the site of transcription initiation resemblesthe distances observed for S. cerevisiae genes (35 to 180nucleotides) (38).

Identification of P76 and P40 is as yet undetermined. Roaand Blobel (33) have reported that in H. polymorpha twomajor polypeptides of molecular weight 78,000 and 42,000appear to be regulated by the presence of methanol in thegrowth medium. Like the 76,000-dalton protein from P.pastoris, the 78,000-dalton polypeptide from H. polymorphacofractionates with alcohol oxidase (33). In addition, theexpression of both the 76,000- and 78,000-dalton proteinsappears to be strongly regulated by the presence of metha-nol. Recently, Roggenkamp et al. (35) have identified twohigh-molecular-weight proteins that result from in vitrotranslations of poly(A)+ RNA isolated from H. polvmorphagrown on methanol as the sole carbon source. These pro-teins comigrated on SDS-polyacrylamide gels with purifiedalcohol oxidase and dihydroxyacetone synthase. The simi-larities in size and electrophoretic mobility to that of P76 andour alcohol oxidase suggest the possibility that P76 isdihydroxyacetone synthase.The properties of the 40,000-dalton protein encoded by

pPC6.7 and pPG4.8 again parallel those of the 42,000-daltonpolypeptide observed in H. polymorpha (33). Results from

the Northern hybridization experiments indicate that mRNAfrom the P40 gene is made constitutively but is amplifiedupon introduction of methanol to the medium (Fig. 7). In H.polymorpha, the synthesis of the 42,000-dalton protein ap-pears very early after the introduction of methanol into themedia and appears to increase with time (33). Thus, it seemsthat a low level of expression of this gene occurs constitu-tively and is not obvious at the protein product level untilmethanol increases the expression. Similarly, in Fig. 4 and 7,it is evident that P40 and its mRNA are present in cellsgrown on either ethanol or methanol. However, the P40mRNA shows an increase of severalfold when methanol isintroduced. It is also interesting to note that the levels of P40protein observed in the reticulocyte lysate in vitro transla-tion experiments (Fig. 4) indicate that translation of the P40mRNA is very efficient, leading to large accumulations ofP40 protein. However, such an accumulation of P40 in cellsgrown with or without methanol is not observed in vivo.Regulation of P42 expression in H. polymorpha and P40 inP. pastoris appears different from that of P76 and alcoholoxidase.The alcohol oxidase mutant ofP. pastoris lacks mRNA for

both alcohol oxidase and P76 genes when grown in thepresence of methanol (Fig. 7). Because this mutant wasgenerated by nitrosoguanidine treatment, a deletion of alarge portion of the P. pastoris genome is unlikely. Rather,this mutant may be a double point mutant affecting bothgenes or a regulatory mutant that affects the expression ofthese two methanol-responsive genes. If this alcohol oxidase-negative strain contains a regulatory mutation, the unaf-fected levels of P40 mRNA (Fig. 7) again indicate a differentmode of control compared with P76 and alcohol oxidasegenes.The regulation of expression of alcohol oxidase, formal-

dehyde dehydrogenase, formate dehydrogenase, and cat-alase has been studied in H. polymorpha and Candidaboidinii (13). The results of this study (13) suggest that in thepresence of ethanol, no alcohol oxidase activity is observed.Our results for P. pastoris agree with these observations atthe level of RNA transcription, and we are now quantifyingthe transcriptional activity of these genes when P. pastoris isgrown in a variety of other carbon sources. The precisemechanism of how the genes for P76, P40, and alcoholoxidase in P. pastoris are regulated is as yet undetermined.By the use of ,B-galactosidase gene fusions controlled by theregulatory regions from the P76 and alcohol oxidase genes, itwill be possible to dissect at a molecular level the types ofregulation that control expression of the genes needed formethanol metabolism.

ACKNOWLEDGMENTS

We thank D. Stroman, J. Cruze, and T. Hopkins of PhillipsPetroleum Company for their assistance and discussions throughoutthis project, and G. Sperl for the gift of alcohol oxidase-negativemutant. We also thank T. Hunter for assistance in the preparation ofantisera, L. Wondrack for purification of alcohol oxidase, L. Blonskifor synthesis of oligonucleotides, and R. Buckholz, J. Cregg, G.Davis, T. Hunter, G. Thill, J. Tschopp, and G. Wahl for their helpfuldiscussions and comments. We thank Susan Konchal for her help inthe preparation of this manuscript.

This research was supported by Phillips Petroleum Company.

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