Proc. Natl. Acad. Sci. USAVol. 81, pp. 4722-4726, August 1984Biochemistry

Identification of 17fi-estradiol as the estrogenic substance inSaccharomyces cerevisiae

(yeast/steroid hormone/estrogen receptor)

DAVID FELDMAN*, LASZLO G. TOKtSt, PETER A. STATHIS*, STEVEN C. MILLER*, WALTER KuRzt,AND DENNIS HARVEYt*Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305; and Institute of Organic Chemistry, Syntex Research,Palo Alto, CA 94304

Communicated by Isidore S. Edelman, April 20, 1984

ABSTRACT Saccharomyces cerevisiae possesses a high-af-finity estrogen binding protein and an endogenous ligand thatdisplaces [3H]estradiol from both the yeast binding proteinand mammalian estrogen receptors. Semipurified prepara-tions of this ligand have been shown to exhibit estrogenic activ-ity in mammalian systems. We now describe the purificationprocedure and ultimate identification of the estrogenic sub-stance in extracts of S. cerevisiae as 17,3-estradiol. Organicsolvent extracts of commercially obtained dried yeast were se-quentially chromatographed on silica gel columns and thensubjected to a series of reversed phase HPLC fractionations.Active ligand was monitored by [3H1estradiol displacement ina rat uterine cytosol assay. After seven chromatography steps,the purified and highly active ligand exhibited a single peakwith retention times identical to those of 17.&estradiol on bothHPLC and GC. The yeast material was identified as 17(-es-tradiol by its UV absorbance and mass spectrometric fragmen-tation pattern. In addition, radioimmunoassay confirmed thepresence of approximately the same mass of 17.&estradiol(w8OO ng/1.5 kg of yeast) as estimated both by a competitivebinding assay with estrogen receptor and by mass spectrome-try. Extraneous contamination by estradiol was excluded byrepeat experiments with different batches of starting materialand demonstration of estradiol by RIA in conditioned mediumand cell pellets of laboratory-grown S. cerevisiae whereas non-conditioned medium did not possess the steroid. We concludethat 17j3-estradiol is a yeast product.

The origin of hormones during evolutionary development isunclear. That unicellular organisms communicate by mes-sage molecules is well documented (1-4). Roth et al. (5, 6)have provided evidence that vertebrate peptide hormonesare present in unicellular organisms. Less is known aboutsteroid hormones. Achyla, a water mold, employs the ster-oid hormone-like substances antheridiol and oogoniols asmodulators of sexual division (1, 3, 7, 8). The grain moldFusarium produces zearalenones that, although not steroi-dal, exhibit estrogenic activity via vertebrate estrogen recep-tors (9, 10). The existence of binding proteins with high affin-ity for vertebrate steroid hormones has been demonstratedfor several unicellular fungi including Candida albicans (11-13), Saccharomyces cerevisiae (14, 15), and Paracoccidi-oides brasiliensis (16). Powell et al. (17) reported on thepresence of estradiol and progesterone binding proteins inCoccidioides immitis. It has been hypothesized that thesemacromolecules represent receptors for yeast hormones(technically pheromones) (11-16), and this hypothesis is sup-ported by the finding that lipid extracts of C. albicans pos-sess a substance that can displace [3H]dexamethasone frommammalian glucocorticoid receptor sites and [3H]corticoste-

rone from the corticosteroid binding protein found in the cy-tosol of Candida (11). We proposed that the active substancein the extract represents the endogenous ligand for the Can-dida corticosteroid binding protein. Similarly, lipid extractsof S. cerevisiae exhibit the ability to displace [ H]estradiolfrom both mammalian estrogen receptor preparations andthe estrogen binding protein in S. cerevisiae cytosol (14). Wetherefore felt that S. cerevisiae possessed an endogeflous li-gand for the yeast estrogen binding protein. Importantly, ithas recently been shown that partially purified extracts ofthe S. cerevisiae endogenous ligand exhibit estrogenic activi-ty in mammalian systems (18). This was demonstrated by theinduction of progesterone receptors in cultured MCF-7 hu-man mammary cancer cells, an action that is reversiblyblocked by tamoxifen, an antagonist active at the estrogenreceptor level. The extracts cause both uterine growth andinduction of progesterone receptors when administered invivo to ovariectomized mice (18).

In this communication, we describe experiments that re-sulted in the further purification and finally the identificationof the S. cerevisiae estrogenic substance as 17f3-estradiol.Evidence derived from physical, biochemical, immunologi-cal, and receptor binding approaches are all consistent withthe finding that the S. cerevisiae endogenous ligand is 17,-estradiol.

MATERIALS AND METHODSLigand Purification. Endogenous ligand from intact cells

of S. cerevisiae was extracted with organic solvents and iso-lated by sequential chromatographic steps using both gravityflow and HPLC. Activity was monitored by [3H]estradioldisplacement binding assays in either broken-cell experi-ments using rat uterine and S. cerevisiae a-cell cytosol or bywhole-cell uptake experiments with MCF-7 cells. The purifi-cation scheme is summarized in the flow diagram (Fig. 1).

Extraction Procedure. Batches of dried S. cerevisiae (Sig-ma), 1-2 kg, were extracted with ethanol for 24-48 hr at 60-70°C with stirring. After evaporation of the solvent, the par-tially solid residue was dissolved in ethanol/water, 1:5 (vol/vol) and extracted with ethyl acetate (three 1-liter portions).The residue, after the evaporation of ethyl acetate, was dis-solved in methylene chloride/ethyl acetate/formic acid,95:5:0.05 (vol/vol) and chromatographed on silica gel.

Silica Gel Chromatography. Chromatography was carriedout on a 6-cm (i.d.) column packed with 600 g of silica gel(Merck, Kieselgel 60). The column was eluted stepwise withthe following solvent mixtures: methylene chloride/ethylacetate/methanol, 95:5:0 (3 liters), 75:25:0 (2 liters), 74:25:1(1 liter), 73:25:2 (1 liter), 72:25:3 (1 liter), 70:25:5 (1 liter), and65:25:10 (2 liters) (all vol/vol and containing 0.05% formicacid to avoid streaking of the fatty acids and the active con-stituent). Fractions were assayed for binding activity in[3H]estradiol displacement assays.

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Proc. Natd Acad Sci USA 81 (1984) 4723

Preparative-Scale HPLC. The active fractions after silicagel chromatography were dissolved in 15 ml of acetonitrile/water (60:40) and heated. The precipitate that developed oncooling was removed by filtration through glass wool, andthe soluble portion was chromatographed twice on a What-man Partisil-10 C-8 reversed-phase column (250 x 9.4 mm,i.d.) using 700 psi of (1 psi = 6.9 kPa) pressure. The eluantwas acetonitrile/water at 60:40 and 40:60 for the first andsecond passes, respectively. The chromatograms were moh-itored with a differential refractometer (Waters Associates,model R404) for characteristic marker components. Frac-tions were collected at regular intervals and assayed for[3Hlestradiol displacement activity.

Semi-Preparative- and Analytical-Scale HPLC. These puri-fication steps were carried out by multiple injections on aSpectra Physics (Santa Clara, CA) model 8100 chromato-graph using 250 x 10 and 250 x 4.6 mm (i.#.) Alltech C8 10-,um-particle-size columns (Alltech Aigociates, Los Altos,CA) and a Beckman model 160 UV absorbance detectormonitoring at 214 nm. The Valco valve of the chromatographwas fitted with a syringe port that allowed nearly quantita-tive loading of the samples directly into the injection loop.The mobile phase was acetonitrile/water (40:60). The col-umns were rinsed with 1% acetic acid in acetonitrile betweenruns. The fractions were concentrated under vacuum in arotary evaporator at 40-50'C and then dried in Reacti-Vials(Pierce) at 50'C under a stream of nitrogen that was purifiedthrough a cartridge filled with Drierite (Baker), activatedcharcoal, and a molecular sieve.UV Spectrophotometiy. The UV absorption spectrum of

the purified fraction was measured on a Hewlett-Packardmodel 8540A UV/VIS spectrophotometer using the HPLCmobile phase as the solvent (acetortitrile/water, 40:60).GC/MS. These measurements were carried out on a Finni-

gan-MAT (San Jose, CA) model 311A SS-200 computerizedmass spectrometer interfaced with a Varian model 3700 gaschromatograph by an open split coupling. A 15 m x 0.32 mmDB-1 fused silica column (J&W Scientific, Rancho Cordova,CA) was used at 1.4 kg/cm2 helium pressure, and the oven,injector, and transfer line temperatures were kept at 2100,2500, and 290°C, respectively. A fractidn of the purified Ii-gand and authentic 17a- and 17,l3estradiol samples were si-lylated with N,O-bis(trimethylsilyl)trifluoroacetamide/1%trimethylchlorosilane at 65°C for 40 min. The electron-im-pact (70 eV) mass spectra were measured at 1000 resolution(10% valley). Quantitation of the 17,B-estradiol content offraction G4 was estimated by monitoring the molecular ion(m/z 416.257) of 17,-estradiol disilyl ether. The specificpeak height value obtained for the silylated ligand was com-pared with those of authentic 17,-estradiol samples in the0.1-10 pg range.Receptor Binding Assays. Methods have been described in

detail (14, 15). In brief, rat uterine cytosol was prepared from-240-g ovariectomized rats. Uteri were homogenized inTET buffer (10 mM Tris HCI/1.5 mM EDTA/12 mM mono-thioglycerol, pH 7.8) and cytosol was obtained by centrifu-gation at 204,000 x g for 30 min. Cytosol was incubated with2.6 nM 171-[3H]estradiol (Amersham; 47 Ci/mmol; 1 Ci = 37GBq) for 18 hr at 0°C in the presence of vehicle or variousconcentrations of either unlabeled 17,3estradiol (Steraloids,Wilton, NH) or aliquots of column fractions to be tested forbinding activity. Protein-bound hormone was separated fromfree [3H]estradiol by dextran-coated charcoal (15). Proteinwas measured by the method of Bradford (19). Active ligandwas also tested in the whole-cell assays using MCF-7 cellsand cytosol prepared from a-cell S. cerevisiae (X2180-1B, a-mating type; a gift from Seymour Fogel, University of Cali-fornia, Berkeley, CA) as described (15).

Estradiol RIA. Anti-estradiol-6-bovine serum albumin se-rum 244 was provided by Gordon Niswender (Colorado

State University, Fort Collins, CO). Antibody characteriza-tion, specificity, and assay methodology have been de-scribed in detail (20). Several samples were also tested with adifferent antibody using a commercial estradiol RIA kit (Ra-dioassay Systems Laboratories, Carson, CA).

RESULTSPurification of Endogenous Ligand by Chromatography.

This procedure is summarized in Fig. 1. The organic extractfrom 1.5 kg of dried S. cerevisiae was fractionated on a silicagel column by stepwise elution with methylene thloride/ethyl acetate/methanol. All fractions were tested for dis-placement activity in a rat uterine cytosol assay, and two

S. CEREVISIAE (1.6 kg)

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FIG. 1. Purification scheme. The inserted graphs (A-F) show theactivity profiles of the eluted chromatography fractions as measuredby [3H]estradiol. displacement from rat uterine cytosol. i, Activefractions carried forward to the next step. Dryl weights were notobtained when fractions were -1 mg (D-G). Progressively decreas-ing amounts of ligand (A, 100 pg/ml; B, 20 ptg/mil; C, 10 pg/ml E-G, 1% of fraction volume) were used in the displacement assays,indicating progressive purification of active material.

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4724 Biochemistry: Feldman et aL

zones of activity were noted. Fraction A2 had a high contentof fatty acids, which were found to interfere with the dis-placement assay, so this fraction was not pursued further.The other active fractions (A5-7) were pooled and subjectedto HPLC on a preparative reversed-phase C8 column elutedwith acetonitrile/water (60:40), and activity was again moni-tored by displacement of [3H]estradiol in the rat uterine cy-tosol assay. The active fractions (B2 and 3) were pooled andrechromatographed on the same HPLC system but elutionwas with acetonitrile/water (40:60). The active fractions, by[3H]estradiol displacement activity (C3 and 4), were thenchromatographed in a semi-preparative-scale C8 HPLC sys-tem eluted with acetonitrile/water (40:60). To conserve ac-tive material, the fraction of interest was monitored withbenchmark material rather than by the displacement assay.The benchmark was an active semipurified endogenous li-gand prepared earlier and shown to have estrogenic activityby in vitro binding and in vitro and in vivo functional activity(18). This marker was monitored by a UV detector at 214 nmto indicate the chromatographic area of interest to collectfraction D2. Repeated chromatography of D2 in a similarHPLC system yielded fractions E1-5. Benchmark chroma-tography and binding assay showed predominant activity infraction E4.The mass of active material was now small enough to use

analytical-scale HPLC for final purification. Fraction E4was chromatographed in a HPLC system similar to the onedescribed above but using an analytical column. The eluantwas divided into 12 fractions and binding assay of 1% thevolume of each fraction indicated activity in fractions F6 and7. Some activity was also noted in fractions F1 and F12,which was not pursued. Rechromatography of fractions F6and 7 on the analytical column yielded a symmetrical singlepeak (G4) suggestive of a relatively pure compound (Fig.2A). Binding assay of 1% of each fraction showed extremelypotent material in the G4 fraction with negligible activity inother fractions (Fig. 2B).

Estradiol RIA. Since the binding and functional studies ofsemipurified ligand had previously been indicative of estro-genic activity (18), we were working with the hypothesis thatthe endogenous ligand might be estradiol. Earlier in thework, trace amounts of authentic estradiol were chromato-graphed in the same HPLC system and shown to co-migratein the region of the benchmark material supporting this hy-pothesis. We did not add estradiol to the columns during thepreparation of fractions D2-G4 to avoid possible contamina-tion of our sample. When fractions G1-8 were evaluated ina sensitive estradiol RIA, the assay showed substantialamounts of estradiol immunoreactivity in fraction G4 withnegligible activity in other fractions (Fig. 2C). Estradiolchromatographed with an equivalent retention time as frac-tion G4 in this system. One percent of fraction G4 exhibitedestradiol activity corresponding to -8 ng. Samples of semi-purified ligand were also tested with a different antibodywith comparable results.

Physical Characterization. The UV spectrum of fractionG4 (data not shown) revealed the presence of some contami-nants by intense tail absorption in the 200- to 260-nm regionthat faded away at A400 nm. The spectrum exhibited ashoulder at 221 nm and a low-intensity but clearly definedabsorption maximum at 278 nm that was absent in the adja-cent fractions. By comparison, a dilute solution (0.2 ,g/ml)of 17,3estradiol in the same solvent mixture exhibited a simi-lar absorption maximum at 278 nm.

Strong supporting evidence for the structural assignmentof this ligand as 173-estradiol was provided by GC/MS anal-ysis. The silylation product of a fraction of sample G4 exhib-ited one major component with identical GC retention timeand fragmentation pattern to authentic 17,3-estradiol disilylether (Fig. 3). The observed fragmentation pattern con-

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FIG. 2. Characterization of the active fraction (G4). Fractions F6and 7 were purified by an analytical-scale HPLC procedure to givefractions G1-8. (A) Elution profile was monitored by UV absorptionat 214 nm. The peak in fraction G4 had a retention time of 1034 sec,which is equivalent to authentic 17/3-estradiol in this HPLC system.The peak height was 0.66 absorbance units full scale (AUFS). (B)[3H]Estradiol receptor displacement activity. One percent aliquotsof fractions G1-8 were incubated with 2.6 nM [3H]estradiol in a ratuterine cytosol binding assay. Displacement activity of fraction G4was equivalent to -7 ng of 173-estradiol (or a total activity of -700ng in the full extract) when compared with a standard curve of au-thentic steroid in a competition assay with the same cytosol. (C)Estradiol RIA. Immunoreactivity of 1% aliquots of fractions G1-8was tested in a RIA. Negligible or trad-e activity was detected in allfractions except G4, which exhibited the equivalent of 8 ng per frac-tion or a total activity of '800 ng of 173-estradiol in the full extract.

firmed that the two hydroxyl groups are at the 3 and 17 posi-tions, but it did not differentiate between the two C-17 epi-mers. The 17/3 stereochemical assignment was establishedby the GC retention time of sample G4 because, under theconditions used, an authentic sample of the 17a epimer elut-ed 30 sec faster.

Estradiol Quantitation. The amount of 173-estradiol infraction G4 was estimated by the rat uterine receptor com-petitive binding assay, RIA, and GC/MS. G4 competitivebinding activity was equivalent to -700 ng of 173-estradiol.RIA of fraction G4 indicated -800 ng of 17,B estradiol in thefraction. GC/MS estimates of fraction G4 suggested -600 ngof 17f-estradiol, based on peak-height comparison with 17,&estradiol calibration standards when the mass spectrometerwas tuned to monitor the molecular ion. The yield of -800ng represents the minimum of estradiol present in 1.5 kg ofdried yeast starting material, but the recovery rate has notbeen established.

Control Experiments. Several approaches were used to ex-clude the possibility that the estradiol detected in S. cerevisi-ae was a contaminant of the yeast rather than a yeast prod-

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Proc. NatL Acad. Sci. USA 81 (1984) 4725

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FIG. 3. Mass spectrum of fraction G4: high mass range of the background-subtracted mass spectrum (70-eV electron impact) of the silyla-tion product of fraction G4. The sample injected onto the GC column represents about 0.5% of the total mass of fraction G4. An identicalspectrum was obtained with authentic silylation product of 17f3-estradiol. TMS, trimethylsilyl.

uct. Different batches of dried yeast (Sigma) starting materi-al yielded similar results to those described above. S.cerevisae a-mating type cells (X2180-1B) grown in our labo-ratory (18) exhibited estradiol by RIA in both semipurifiedextracts of the cell pellet and the conditioned medium. Al-though extraction was not quantitative and values variedfrom experiment to experiment, estradiol was consistentlydetectable; 0.2-1 ng of estradiol in the cells and 0.2-0.8 ng inthe conditioned medium per g (dry weight) of growing yeastcells. We failed to detect estradiol by RIA in extracts of non-conditioned medium. Finally, extracts of a different yeast,C. albicans, did not exhibit detectable estradiol by RIAwhen analyzed in a similar fashion.

DISCUSSIONThe data presented in this paper indicate that 17/3-estradiol isthe endogenous ligand for the estrogen-binding protein of S.cerevisiae. The proof of structure includes demonstrationthat the substance (i) exhibits identical chromatographic be-havior with 17,B-estradiol on both HPLC and GC, (ii) dis-plays UV absorbance and mass spectral fragmentation pat-terns identical to estradiol, and (iii) shows 17,3-estradiol im-munoreactivity in two RIAs. In addition, as previouslyshown, the ligand competes for estrogen receptor bindingsites and induces estrogenic bioresponses in mammalian sys-tems (18). Importantly, estimates of 17,8-estradiol content byreceptor binding, antibody, and GC/MS are all in the same

range, supporting the argument that the activity detected ineach system is predominantly due to 17,8-estradiol. We wishto emphasize that other estrogenic substances may well bepresent in other fractions of the extract and that our prepara-tion of fraction G4 was not quantitative. We therefore can-

not yet estimate the amount of estradiol produced by theyeast.The purification and identification of 17,festradiol was

carried out with commercially purchased dried yeast. How-ever, in subsequent experiments with S. cerevisiae a-matingtype cells grown in our laboratory, RIA ofHPLC fractions inthe region of authentic estradiol showed nanogram amountsof estradiol activity per gram dry weight of yeast in both thecell pellet and the conditioned growth medium. Noncondi-

tioned growth medium failed to show detectable estradiol.We believe these experiments exclude the possibility that es-

tradiol is a contaminant and indicate that it is a true productof the yeast.The finding of estradiol in this unicellular organism has

significance in several spheres. First, the presence of estra-diol in this simple yeast indicates that the enzymatic systemnecessary for steroid synthesis must exist very early in evo-

lution. Although sterol synthesis is well documented in yeastand hormone-like steroids have been identified (7, 8), to ourknowledge true vertebrate hormonal steroid production hasnot previously been reported.Second, the presence of hormonal levels of this fungal

product, in addition to the previously described estrogenbinding protein (14, 15), adds support to our hypothesis thatS. cerevisiae possesses a hormone-receptor system analo-gous to those present in vertebrates. The system is apparent-ly highly conserved since the ligand, 17.&estradiol, is thesame in yeast and humans. However, many differences havebeen identified between the mammalian receptor and the es-

trogen binding protein in S. cerevisiae (15), suggesting possi-ble further specialization of the receptor protein in higherorganisms. Although the hormone appears the same, we donot have data indicating the nature of the functional responsemediated by this system in yeast.The third area of significance involves the possible use of

yeast as a model system to gain better understanding of themechanism of steroid hormone action. Although we have notproven that estradiol acts as a fungal "hormone," the pres-ence of both a receptor-like protein and a hormonal endoge-nous ligand makes this more feasible. If our hypothesisproves to be correct, then these well-understood simple or-

ganisms, which are easily studied by genetic analysis andamenable to recombinant DNA technology, should allowmore accessible probing of the molecular processes involvedin gene regulation by hormones.The fourth area of significance of the current findings is

that they further support the hypothesis of bidirectional hor-monal interactions between fungus and higher organisms(11-16). It has been shown that fungal extracts, presumablycontaining yeast hormones, bind to mammalian receptors

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and that mammalian hormones bind to steroid binding pro-teins in fungi. In the context of a fungal pathogen, it wasspeculated that the host hormonal environment, acting viathe fungal binding protein, could either support or inhibitgrowth and infectivity of the fungus (11, 16). Recent datawith P. brasiliensis show that indeed this fungal-host inter-action is possible and that, importantly, estrogens inhibit themycelial to yeast form conversion in P. brasiliensis. We be-lieve that circulating host estrogenic hormones, binding tofungal estrogen "receptors" and inhibiting conversion to theinvasive yeast form, is the molecular mechanism explainingthe extremely high preponderance ofP. brasiliensis infectionin men as compared with women (16). The possibility of fun-gal hormones interacting with the host receptors was alsoraised for consideration, especially for C. albicans, whichproduce a factor that binds to mammalian corticosteroid re-ceptors (11). The recently published (18) and current find-ings with S. cerevisiae raise the possibility of fungal hor-mones affecting mammalian receptors, even if the yeast isnot a pathogen. Because S. cerevisiae is extensively used inthe fermentation and baking industries and dried yeast is in-gested directly as a dietary supplement, we postulate thatyeast-produced estradiol may enter the human food supplyand exert estrogenic activity. However, since the levels wehave detected thus far are quite low, the importance ofyeast-produced estradiol as an environmental source of es-trogen contamination remains to be determined.

We thank Dr. Robin Clark of Syntex Research for his helpful com-ments on the separation procedures and Ms. Diane K. Cho, also ofSyntex Research, for the GC/MS analysis of various semipurifiedfractions and of the purified ligand. The work was supported in partby Grant GM 28825 from the National Institutes of Health.

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3. Timberlake, W. E. (1976) Dev. Biol. 51, 202-214.4. Sandor, T. & Mehdi, A. Z. (1979) in Hormones and Evolution,

ed. Barrington, E. J. W. (Academic, New York), Vol. 1, pp.1-72.

5. Roth, J., LeRoith, D., Shiloach, J., Rosenzweig, J. L., Les-niak, M. A. & Havrankova, J. (1982) N. Engl. J. Med. 306,523-527.

6. Roth, J., LeRoith, D., Shiloach, J. & Rubinovitz, C. (1983)Clin. Res. 31, 354-363.

7. Arsenault, G. P., Biemann, K., Barksdale, A. W. & McMor-ris, T. C. (1968) J. Am. Chem. Soc. 90, 5635-5636.

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10. Katzenellenbogen, B. S., Katzenellenbogen, J. A. & Morde-cai, D. (1979) Endocrinology 105, 33-40.

11. Loose, D. S., Schurman, D. & Feldman, D. (1981) Nature(London) 293, 477-479.

12. Loose, D. S. & Feldman, D. (1982) J. Biol. Chem. 257, 4925-4930.

13. Loose, D. S., Stevens, D. A., Schurman, D. J. & Feldman, D.(1983) J. Gen. Microbiol. 129, 2379-2385.

14. Feldman, D., Do, Y., Burshell, A., Stathis, P. & Loose, D.(1982) Science 218, 297-298.

15. Burshell, A., Stathis, P. A., Do, Y., Miller, S. C. & Feldman,D. (1984) J. Biol. Chem. 259, 3450-3456.

16. Loose, D. S., Stover, E. P., Restrepo, A., Stevens, D. A. &Feldman, D. (1983) Proc. NatI. Acad. Sci. USA 80, 7659-7663.

17. Powell, B. L., Drutz, D. J., Huppert, M. & Sun, S. H. (1983)Infect. Immun. 40, 478-485.

18. Feldman, D., Stathis, P. A., Hirst, M. A., Stover, E. P., Do,Y. S. & Kurz, W. (1984) Science 224, 1109-1111.

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