Vol. 174, No. 2

Molecular Genetic Analysis of the Escherichia coli phoP LocusEDUARDO A. GROISMAN,l 2* FRED HEFFRON,2t AND FELIX SOLOMON'

Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue,Box 8230, St. Louis, Missouri 63110-1093,1* and Department of Molecular Biology,

Research Institute of Scripps Clinic, La Jolla, California 920372

Received 6 August 1991/Accepted 31 October 1991

We have cloned the Escherichia coli phoP gene, a member of the family of environmentally responsivetwo-component systems, and found its deduced amino acid sequence to be 93% identical to that of theSalmonella typhimurium homolog, which encodes a major virulence regulator necessary for intramacrophagesurvival and resistance to cationic peptides of phagocytic cells. The phoP gene was mapped to kilobase 1202 onthe Kohara map (25-min region) of the E. coli genome (Y. Kohara, K. Akiyama, and K. Isono, Cell50:495-508, 1987) and found to be transcribed in a counterclockwise direction. Both E. coli and S. typhimuriumphoP mutants were more sensitive than their isogenic wild-type strains to the frog-derived antibacterial peptidemagainin 2, suggesting a role for PhoP in the response to various stresses in both enteric species.

Bacteria modulate expression of their genes in response toenvironmental changes. This modulation is frequently con-trolled at the transcriptional level by members of the familyof two-component systems, in which a membrane-boundsensor-transmitter can detect changes in the environment,such as changes in osmolarity or concentrations of essentialnutrients, and mediate the phosphorylation-dephosphoryla-tion of the regulatory-receiver component (30, 31). We andothers have recently identified the PhoP protein of Salmo-nella typhimurium as a transcriptional regulator which con-trols the expression of genes essential for virulence, survivalwithin macrophages (5, 11, 26), the ability to withstand anacid pH (7), and resistance to antimicrobial peptides ofmammals (5), frogs, and insects (14; see reference 12 for areview). Genetic and DNA sequence analyses revealed thatphoP is part of an operon with phoZ (also called phoQ),which encodes a protein which exhibits homology to thesensors-transmitters (11, 26). To date, five PhoP-regulatedloci in S. typhimurium have been identified: phoN, encod-ing a nonspecific acid phosphatase (NSAP) (13, 17), andpsiD (11), pagA, pagB, and pagC, all of unknown function(26).Because most PhoP-regulated loci identified so far are not

essential for virulence of S. typhimurium, and because phoPhomologs have been detected in several bacterial species(11), PhoP may play a central role in the normal physiologyof gram-negative bacteria. To gain more insights into the roleof PhoP in regulation, we began a molecular genetic analysisof the Escherichia coli phoP locus. Here, we present aphenotypic characterization of a phoP mutant of E. coli, andwe also report the cloning, nucleotide sequence, and mapposition of phoP.

MATERIALS AND METHODSBacterial strains and plasmids. Bacterial strains and plas-

mids are described in Table 1.Media. Luria-Bertani (LB) and M63 minimal media have

been described previously (25). Ampicillin was used at 50

* Corresponding author.t Present address: Department of Microbiology and Immunology,

Oregon Health Sciences University, Portland, OR 97201.

pLg/ml, chloramphenicol was used at 25 ,ug/ml, kanamycinwas used at 40 p.g/ml, and tetracycline was used at 10 p.g/ml.

Peptides. Magainin 2, mastoparan, and melittin were pur-chased from Bachem Inc. (Torrance, Calif.), and cecropinP1 was purchased from Peninsula Laboratories, Inc. (Bel-mont, Calif.). Peptides were dissolved in distilled doublydeionized sterile water to a final concentration of 1 mg/mland kept at -20°C.

Bacterial genetic techniques. Transformation of S. typhi-murium with plasmid DNA was carried out as described byMacLachlan and Sanderson (22). Transformation of E. coliJC7623 was performed by electroporation with a Bio-Radapparatus according to the manufacturer's recommenda-tions. Lysates of mini-Mu replicon phage were prepared andused as described previously (8). Phage P1 transductionswere carried out with Plcml c1rlOC as described previously(25).

Bactericidal assays. Log-phase cells grown in LB brothwere diluted to 5 x 104 to 1 x 105 CFU/ml in LB broth.Diluted material (50 pl) was placed in 96-well microtiterdishes and added to different amounts of peptide (50 ,ul)diluted in phosphate-buffered saline (PBS) to the final con-centration indicated in Fig. 3. Cells and peptides wereincubated fdr 1 h at 37°C with shaking, and then a portion ofeach sample was diluted in PBS and plated on LB agar platesto assess bacterial viability. Data are presented as percentsurvival relative to the original inoculum. Controls containedPB3S in place of peptide.DNA biochemistry. Restriction endonucleases and phage

T4 DNA ligase were purchased from Bethesda ResearchLaboratories, Inc.; Boehringer Mannheim Biochemicals;and New England BioLabs, Inc., and used according to thesuppliers' specifications. Large-scale isolation of plasmidDNA was carried out by the procedure of Kupersztoch andHelinski (19). Small-scale preparation of plasmid DNA wasdone as described by Holmes and Quigley (15). Dot blothybridization was performed as follows: approximately 250ng of purified plasmid DNA was denatured, transferred to anylon membrane (Nytran; Schleicher & Schuell), and hy-bridized under stringent conditions to a 32P-labeled probecorresponding to the 514-bp EcoRV fragment internal to theS. typhimurium phoP gene (11). Plaque hybridization ofrecombinant phages harboring DNA segments of the 25-minregion of the E. coli genome to a 32P-labeled pEG5424 probe

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TABLE 1. Strains and plasmids used in the study

Strain or Description or Source orplasmid genotypea reference

E. coliFS1000 MC1061 with phoP::kan This workFS1002 MC4100 with phoP::kan This workJC7623 thr-l ara-14 leuB6 A(gpt- 21

proA)62 lacYl sbcC201 tsx-33 supE44 galK2 lambda-rac sbcBIS hisG4 rfbDlrecB21 recC22 rpsL31kdgK51 xyl-S mtl-l argE3thi-i

MC1061 F- araDi39 A(ara-leu)7697 3A(lac)X74 galU galK hsdR2(rK mK ) supE44 thi-irelAl?

MC4100 F- araDJ39 A(lac)U169 LaboratoryrpsL150 relAl thiflbBS301 collectiondeoCi ptsF25 rbsR

MH2923 F+ Mu cts62 hPl-1 araD 4

S. typhimuriumEG5082 galE496 metA22 metESS This work

rpsLi20 xyl404 (Fels2)-Hi-b nml H2-enx (ilv?)hsdL6 hsdSA29phoP7953::TniO Mu hPl

TA2328 purB12 phoP22 17

PlasmidspEG5005 pBCO::Mud5OO5 9pEG5086 pBCO::Mud5086 9pEG5420 Mud5005::phoP+ This workpEG5423 Mud5005::phoA+ proC+ This work

phoBR+pEG5424 Mud5005: :phoP+ This workpEG5425 Mud5OO5::phoA+ proC+ This work

phoBR+pEG5426 Mud5005: :phoP+ This workpEG5484 Apr phoP+ reppMB, This workpEG5513 Apr Kanr phoP::kan repPMBI This workpUC4-K pUC derivative harboring kan Pharmacia

of Tn903a E. coli gene designations are as summarized by Bachmann (1); S.

typhimurium gene designations are as summarized by Sanderson and Roth(27). Apr and Kanr, ampicillin and kanamycin resistance, respectively.

was performed as suggested by Schleicher & Schuell. Otherprotocols were as described by Maniatis et al. (23).DNA sequencing. Double-stranded plasmid DNA sequenc-

ing was carried out on both strands by the dideoxy chaintermination method with the Sequenase kit (United StatesBiochemicals) with 35S-labeled dATP (28). A first sequencerun was carried out with an oligonucleotide primer corre-sponding to the first 20 bases of the S. typhimurium phoPcoding region (11) and pEG5484 as a template. Syntheticoligonucleotides were then used to complete the sequence.Plasmids pEG5420 and pEG5426 were used as templates todetermine the sequence corresponding to nucleotides 1 to160, shown in Fig. 2 (see below). The nucleotide sequencereported in this article is identical to that reported byKasahara et al. in the accompanying article (15a).

Nucleotide sequence accession number. The nucleotidesequence data reported in this paper have been submitted toGenBank and assigned the accession number M81433.

,O _ O vw ngo C. " C.C.>O m v v v v ven en e1 In v ne

a vL CI Q Cla

pEGS420-pEGS424-pEG5426

1200phoP

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pEG5423pEG5425

400 proc(8.9)

in.

..A a. . .i_. . _

7F9 (239)SF5

20E6 240)ISAS (237)

E<C2 1235)

A u 0O,

FIG. 1. Characterization of E. ccli plasmid clones conferring aPho' phenotype to phoP S. typhimurium. Dot blot hybridization ofPhoP' plasmids to a Salmonella phoP-specific probe is shown at thetop of the figure. Plasmids were hybridized under stringent condi-tions to a 3P-labeled probe corresponding to the 514-bp EcoRVfragment internal to the S. typhimurium phoP gene as described inMaterials and Methods. Controls included plasmid pEG5086, amini-Mu replicon plasmid vector, and pEG5381, harboring the S.typhimurium phoPa gene. Localization ofPhoE plasmids in therestriction map of E. coli K-12 is shown in the middle of the figure.DNA present in the plasmid clones is indicated above the restrictionmap of Kohara et al. (18); restriction sites (lanes, from top tobottom) are BamHl, HindIll, EcoRI, EcoRV, Bgll, Kpnl, Pstl, andPvuil. 1200 and 400 correspond to kilobases on the Kohara map; 8.9refers to position in minutes. Plaque hybridization of recombinantphages harboring DNA segments of the 25-mm region of the E. coligenome to an 2P-labeled, BamHl-linearized pEG5424 probe is shownat the bottom; a positive signal was detected with phage 7F9 but notwith phages 15A8, E4C2, and 20E6.

RESULTS AND DISCUSSION

Cloning of phoP. We used the mini-Mu replicon in vivocloning procedure to prepare a library from E. coli MH2923/pEG5005 into the phoP S. typhimurium strain EG5082 asdescribed previously (8-10). Kanamycin-resistant (encodedby the mini-Mu Mud5005) transductants were selected inM63 minimal glucose media containing the appropriate nu-tritional supplements and kanamycin sulfate and screenedfor complementation of one of the phenotypes of phoP S.typhimurium, production of NSAP, by the procedure of Kieret al. (16). Three deep red and two pale red Pho+ cloneswere identified (against a background of Pho- white colo-nies), and their plasmid DNAs were extracted and digestedwith HindIII, PstI, BamHI, EcoRI, EcoRV, PvuII, BglI, andKpnI (Fig. 1). The three red clones (pEG5420, pEG5424, andpEG5426) had restriction fragments in common, as did thetwo pale clones (pEG5423 and pEG5425). The former clonesharbored the E. coli phoP gene, since they also hybridized toa 514-bp EcoRV fragment internal to the S. typhimuriumphoP gene (Fig. 1). The latter clones, which did not hybrid-ize to the phoP probe, harbored a different region of the E.coli genome which included the phoA gene encoding alkalinephosphatase (data not shown).

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488 GROISMAN ET AL.

TATCAGTGCCGGATGGCGATGCTGTCCGGCCTGCTTATTAAGATTATCCGCTTTTTATTTTTTCACTTTACCTCCCCTCCCCGCTGGTTT

91

181

ATTTAATGTTTAQCCCCATAACCACATAATCGCGTTACACTATTTTAATAATTAAGACAGGOA9.AATAAAAATGCGCGTACTGGTTGTTM R V L V V

M

GAAGACAATGCGTTGTT'ACGTCACCACCTTAAAGTTCAGATTCAGGATGCTGGTCATCAGGTCGATGACGCAGAAGATGCCAAAGAAGCCE D N A L L R H H L K V Q I Q D A G H QV D D A E D A K E A

L S A R

271 GATTATTATCTCAATGAACATATACCGGATATTGCGATTGTCGATCTCGGATTGCCAGACGAGGACGGTCTGTCACTGATTCGCCGCTGGD Y Y L N E H I P D I A I V D L G L P D E D G L S L I R R W

L

361 CGTAGCAACGATGTTTCACTGCCGATTCTGGTATTAACCGCCCGTGAAAGCTGGCAGGACAAAGTCGAAGTATTAAGTGCCGGTGCTGATR S N D V S L P I L V L T A R E S W Q D K V E V L S A G A D

S V G S

451 GATTATGTGACTAAACCGTTTCATATTGAAGAGGTGATGGCGCGAATGCAGGCATTAATGCGGCGTAATAGCGGTCTGGCTTCACAGGTCD Y V T K P F H I E E V M A R M Q A L M R R N S G L A S Q V

541 ATTTCGCTCCCCCCGTTTCAGGTTGATCTCTCTCGCCGTGAATTATCTATTAATGACGAAGTGATCAAACTGACCGCGTTCGAATACACCI S L P P F Q V D L S R R E L S Z N D E V I K L T A F E Y T

N I V E

631 ATTATGGAAACGTTGATACGCAATAATGGCAAAGTGGTCAGCAAAGATTCGTTAATGCTCCAACTCTATCCGGATGCGGAGCTGCGGGAAI 1 E T L I R N N G K V V S K D S L M L Q L Y P D A E L R E

721

811

AGCCATACCATTGATGTACTGATGGGACGTCTGCGCAAAAAAATTCAGGCACAATATCCCCAAGAAGTGATTACCACCGTTCGCGGCCAGS H T I D V L M G R L R K K I Q A Q Y P Q E V I T T V R G Q

H D

GGCTATCTGTTCGAATTGCGCTGATGAAAAAATTACTGCGTCTTTTTTTCCCGCTCTCGCTGCGGGTACGTTTTCTGTTGGCAACGGCAGG Y L F E L R * M K K L L R L F F P L S L R V R F L L A T A

N F A H L

FIG. 2. Nucleotide sequence of the region harboring the E. coli phoP gene and deduced amino acid sequence of its product. The putativeribosome binding site is underlined with a wavy line, the Pribnow box is underlined with a double line, and a hexanucleotide repeat isunderlined once. The asterisk indicates the stop codon for phoP. Also shown (following the asterisk) is the sequence corresponding to theN-terminal region ofphoZ. Amino acid residues deduced from the S. typhimurium phoPZ DNA sequences that differ from E. coli PhoP andPhoZ are shown in italics.

Mapping of Pho+ clones. The restriction maps of theclones harboring the phoP homolog were aligned with thepublished physical map of the E. coli K-12 chromosome(Fig. 1) (18). A match to the 25-min region (1,202 kb inKohara map coordinates [18]) was found and verified byplaque hybridization experiments with recombinant lambdaphages of the Kohara collection (18) that span this region(Fig. 1). The purB gene maps to the 25-min region of the E.coli and S. typhimurium chromosomes (1, 27), and plasmidpEG5426 complemented the purine requirement of the purBS. typhimurium strain TA2328, whereas plasmid pEG5424,with the shorter insert, did not. These results showed thatthe phoP gene is located counterclockwise of the purB locus,at approximately the same position at which it is found in S.typhimurium (27). The location of the phoP locus wasconfirmed in genetic experiments using P1 transduction anda phoP::kan strain (constructed as described below). Weestablished phoP::kan to be 28% linked to zce-726::Tn O,46% linked to zcf-117::TnJO, and 39% linked tofadR13::TnJO, which had been previously shown to map to24.25, 25.25, and 25.75 min, respectively, in the E. coli K-12chromosome (29).DNA sequence analysis of phoP. We therefore subcloned a

2.8-kb KpnI-BamHI fragment (including 117 bp from the endof Mud5005) from plasmid pEG5424 into the vector pIBI25to generate plasmid pEG5484. The DNA sequence of 900 bpincluding the phoP gene was obtained as described inMaterials and Methods (Fig. 2). Since phoP is oriented withthe 3' end closer to the KpnI site in plasmid pEG5484, wededuce that it is transcribed in a counterclockwise directionin the E. coli chromosome. The deduced amino acid se-quence of phoP was 93% identical (98% similar) to theSalmonella counterpart, which harbors an extra methionine

residue at the N terminus (11, 26). Of DNA-binding tran-scriptional regulators sequenced for both S. typhimuriumand E. coli, PhoP is the one, with the exception of AraC,which exhibits the most divergence between these twospecies. Downstream of phoP, we found an open readingframe with a high degree of similarity to the N-terminalsegment of PhoZ, the second component of the PhoP regu-latory system. Interestingly, the conservation between theE. coli and S. typhimurium operons extends to severalpotential regulatory features: (i) the translational couplingstrategy suggested by the overlap between the terminationcodon for phoP and the initiation codon ofphoZ (11, 26), (ii)an identical Pribnow box-like sequence, c-A-T-A-A-T, andabsence of a -35 consensus region, and (iii) the presence ofa hexanucleotide direct repeat (G-T-T-T-A-T, the last Tbeing substituted for a C in the second repeat of the E. colisequence) 11 bp upstream of the Pribnow box. We hadpreviously suggested a role for the hexanucleotide repeat inthe expression of phoP in S. typhimurium (11).Phenotype of a phoP E. coli strain. We constructed a phoP

E. coli strain by first introducing the 1.3-kb BamHI fragmentfrom plasmid pUC4-K (Pharmacia) harboring the kanamycinresistance gene from transposon Tn9O3 into the unique BclIsite of the phoP-containing plasmid pEG5484 (position 585 inFig. 2). The resulting recombinant plasmid, pEG5513, couldno longer complement strain EG5082 for the production ofNSAP as expected of an insertion in the phoP open readingframe. We transferred the plasmid-linked mutation to the E.coli chromosome by transforming the recBC sbcB sbcCstrain JC7623 with uncut pEG5513 plasmid DNA and select-ing for kanamycin resistance. As expected of recBC sbcBsbcC strains, which cannot sustain replication of ColEl-typereplicons (20), most transformants were ampicillin sensitive,

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ESCHERICHIA COLI phoP LOCUS 489

200 Cecropin Pl (3 lwml)100

10

0.1

0:01

.001 MS793s 14028s F-S1002 MC4100phoP

S. typhimwrim

0

phoPE. coli

200 Magainin 2 (50 jngm)100

l0

0.1

0.01

.001 -RI~ -

MS-1933SphoP

S. t)uhimurium

MS1953s 14028s FS1002 MC4100 MS7953s 14028s FS100phoP phoP phoP phoP

S. typhirnmn E. coli S. S)phifawm

FIG. 3. Sensitivity of wild-type and phoP E. coli and S. typhimurium strains to cecropin P1 (Peninsula Laboratories, Inc.), magainin 2(Bachem Inc.), mastoparan (Bachem Inc.), and melittin (Bachem Inc.). Assays were performed as described in Materials and Methods.

and they presumably arose by recombination of the dis-rupted phoP gene in pEG5513 with the chromosomal copy ofphoP. The phoP::kan insertion in strain JC7623 was trans-ferred to strains MC1061 and MC4100 by phage P1-mediatedtransduction. The presence of a disrupted chromosomalphoP gene was confirmed by Southern hybridization analy-sis with phoP and kan probes (data not shown) and geneticlinkage to markers known to map in the region (see above).We tested for phenotypic differences between isogenic

phoP+ and phoP E. coli by investigating the effects ofseveral cationic-peptide antibiotics (magainin 2, cecropin P1,melittin, and mastoparan) which had been shown to have apreferential killing effect upon phoP S. typhimurium (14).The phoP E. coli strain was more susceptible than thewild-type strain to magainin 2, indicative of a phoP require-ment in both enteric organisms for resistance to this peptide.On the other hand, both the wild-type and the phoP E. colistrains were highly and equally susceptible (as susceptible asthe phoP S. typhimurium strain) to melittin, mastoparan, andcecropin P1 (Fig. 3). The phoP+ E. coli strain was asresistant as wild-type S. typhimurium to concentrations ofmagainin 2 up to 40 ,ug/ml; however, at higher concentra-tions of this peptide, S. typhimurium survived better than E.coli. These peptides had no microbicidal activity against thefour strains when tested at the following lower concentra-tions (in micrograms per milliliter): cecropin P1, 1.5; masto-paran, 5; melittin, 5; and magainin 2, 20.

There are several possible explanations for the observeddifferences in peptide susceptibility between phoP mutantsof E. coli and S. typhimurium. PhoP may regulate differentsets of loci in these two species, perhaps reflecting thesomewhat different niches: these two species occupy innature. This is consistent with the finding that the PhoP-regulated phoN gene is present in S. typhimurium but absentfrom E. coli (13, 33). The observed differences could bebased in the amino acid sequence divergence of PhoP (15 of223 amino acids) and a corresponding divergence of theregulatory regions of the targets of PhoP regulation. Weconsider this unlikely and note that E. coli PhoP, at leastwhen the phoP gene is present in high copy number, canmediate the activation of several Salmonella genes, includ-ing those involved in protamine resistance and production ofNSAP (13). Alternatively, the higher levels of resistance ofwild-type S. typhimurium relative to those of wild-type E.coli K-12 suggest that S. typhimurium harbors a gene(s) notpresent in E. coli which endows it with the ability towithstand the microbicidal effect of these peptides. With theavailable data, we cannot unequivocally establish whethersuch a gene(s) is under the transcriptional control of PhoP.We also examined the abilities of the isogenic pair MC4100

(phoP+) and FS1002 (phoP) to use several compounds as thesole carbon and energy sources. We streaked these strainsside by side onto M63 salts media containing succinate,acetate, glycerol, glucose, L-alanine, and citrate and then

051002uphoP

E. coli

MC4100

E. cdi

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490 GROISMAN ET AL.

compared the sizes of the colonies. Neither strain was ableto grow on citrate, as expected for E. coli, which is missingthe transport genes for citrate. No differences in mediacontaining succinate, acetate, L-alanine, glycerol, or glucosewere observed.What is the role of PhoP in E. coli and other enterobacteria?

The data presented here, as well as those available fromstudies conducted with S. typhimurium (11, 12, 14, 16, 26),indicate an involvement of PhoP in the response to stresssituations such as those which might be present duringstationary phase, when a microorganism may face nutri-tional deprivation, exposure to toxic by-products of metab-olism, or both. For example, in S. typhimurium, the PhoP-regulated phoN is turned on by starvation for a variety ofelements, including phosphorus, carbon, sulfur, and nitro-gen (16), and pag loci are induced by exposure to an acid pH(26). Candidates for PhoP regulation in E. coli include thephosphate starvation-inducible psiG, psiH, psil, psiJ, psiL,and psiO genes, which are also induced by starvation forcarbon and nitrogen and do not seem to be under the controlof PhoB, a PhoP homolog which regulates the expression ofseveral phosphate starvation loci (32). Other candidatesinclude the pex genes, which are involved in the develop-ment of the resistant state during entry to stationary phase(24). The DNA sequence preceding the phoP coding region(Fig. 2) does not resemble a heat shock promoter or themotifs found upstream of genes induced during stationaryphase (24). Moreover, the presence of a potential -10 regionupstream of phoP suggests that it is transcribed by a a70RNA polymerase. The phoP mutant of E. coli described hereshould facilitate evaluation of the role of PhoP in theresponse to diverse environmental stresses.

Cloning with mini-Mu replicon Mud5005. It is interestingthat plasmid pEG5484 (but not pEG5420 or pEG5426) gave aPho+ phenotype although it harbored only 27 bp upstream ofthe phoP coding region and was therefore missing theputative phoP promoter region. This suggests that the vectorMud5005 (9) can promote transcription of genes clonedadjacent to its right end. We believe that transcription isinitiated at the Kan promoter (the Kan fragment in MudS005corresponds to positions 1197 through 2653 from transposonTnS) and reads across 146 nucleotides of polylinker- andMu-derived sequences, which constitute the right end ofMud5005, into adjacent chromosomal DNA. Read-throughtranscription from the Kan promoter had been implicated ina deletion derivative of TnS (2). This previously unrecog-nized property of Mud5005 may underscore its utility for thecloning of DNA sequences not efficiently expressed in E.coli.

ACKNOWLEDGMENTSWe thank Ken Rudd for phage stocks and for sharing unpublished

data, Doug Berg and two anonymous reviewers for comments on themanuscript, and Hideo Shinagawa and coworkers for agreeing toexchange manuscripts before publication.

This work was supported in part by grants from the NSF(DCB-8916403) and the Markey Center at WUMS to E.A.G. andfrom the NIH to F.H. E.A.G. was a fellow of The Jane Coffin ChildsMemorial Fund for Medical Research for the work performed in LaJolla, Calif.

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