Proc. Natl. Acad. Sci. USAVol. 90, pp. 5489-5493, June 1993Microbiology

Variant-specific surface proteins of Giardia lamblia arezinc-binding proteins

(metal bindlng/protozoa/antigenic variation)

THEODORE E. NASH* AND MICHAEL R. MOWATTLaboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 126,Bethesda, MD 20892

Communicated by Dr. Louis H. Miller, February 11, 1993 (received for review December 31, 1992)

ABSTRACT GiardYa lamblia undergoes surface antigenicvariation. The variant-specific surface proteins (VSPs) are adistinct family of cysteine-rich proteins. Characteristically,cysteine residues occur mostly as CXXC tetrapeptides. Four ofthe reported five VSPs contain a putative metal-binding do-main that resembles other metal-binding motifs; the fifth isclosely related but lacks an essential histidine. Three differentnative VSPs bound Zn2+. Co2+, Cu2+, and Cd2+ inhibitedZn2+ binding. Analysis of recombinant VSP fusion proteinsshowed that the putative binding motif bound Zn2+. Surpris-ingly, peptide fragments from other regions of the VSP containnumerous CXXCX,CXXC motifs that also bound Zn2+. Anal-ysis of deduced amino acid sequences showed well-conservedCXXC spacing in three out of five VSPs, suggesting conserva-tion of structure despite amino acid sequence divergence. Thefunction of VSPs is unknown, but by binding Zn2+ or othermetals in the intestine, VSPs may contribute to Zn2+ malnu-trition or inhibition of metal-dependent intestinal enzymes,which would lead to malabsorption, a well-known consequenceof giardiasis.

Giardia lamblia is a flagellated binucleated protozoan para-site of humans and other mammals. It is one of the mostcommon disease-causing parasitic pathogens of humansworldwide and is responsible for both endemic and epidemicdiarrhea. In the United States and other developed countries,infections are common in day-care centers (1); among trav-elers (2), backpackers (3), homosexuals (4), and institution-alized persons; and wherever fecal contamination occurs. Indeveloping countries infections are frequent, and in someregions children are universally infected by 2 years ofage (5).

G. lamblia undergoes surface antigenic variation (6-8).The surface and flagella of the trophozoite are covered by aprotein coat composed of a single variant-specific surfaceprotein (VSP) (9, 10). VSP expression spontaneouslychanges, and the rate of change is primarily a function of theisolate or lineage of the parasite but also of the VSP (11). TheVSP repertoires of independent isolates can be overlapping,identical, or mutually exclusive (8, 9, 12).VSPs constitute a distinct family of cysteine-rich proteins

that vary in size from 35 kDa to >200 kDa (8). The sequencesof five VSP genes have been published (7, 13-16), andanalysis of the deduced amino acid sequence shows thatcysteine residues make up 11-12% of the protein and occurprimarily as Cys-Xaa-Xaa-Cys motifs. The biological impor-tance of VSPs is unknown; however, the biological charac-teristics of individual VSPs differ as demonstrated by theirvarying susceptibility to the intestinal proteases trypsin anda-chymotrypsin (17).We recently noted that four of the five reported VSPs

contain a zinc-binding motif that closely resembles that found

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in nuclear DNA-binding proteins (8). In contrast to theseintracellular proteins, VSPs are cell-surface proteins, whichare shed into the culture medium in vitro (18). In the presentreport, we further characterize this motif and show thatnative VSPs, as well as recombinant proteins containing thismotif, bind zinc as well as other metals. Furthermore, otherpreviously defined metal-binding motifs appear sporadicallyamong the VSPs and may also bind zinc.

MATERIALS AND METHODSParasite Culture. Giardia were axenically grown, main-

tained, and harvested as described (18, 19). Clones express-ing defined VSPs included GS/H7 (14, 19, 20), WB/A6 (7, 6,16), and WB/1267 (6, 13). These VSPs were recognizedspecifically by monoclonal antibodies G10/4 (21), 6E7 (6, 22),and 5C1 (6, 17), respectively. Polyclonal antibodies to isolateGS, which expresses VSPH7 predominantly, were producedas described (23).

Zinc Binding to VSPs Bound to Nitrocellulose. SDS/polyacrylamide gel electrophoresis was carried out as before(14) with commercially obtained 3-17% gradient gels. Theprocedures employed in Western blotting have been de-scribed (22). The ability of VSPs or fusion proteins to bindzinc was determined by using a modification of methodsdevised by Schiff et al. (24) and modified by Grossman andLaimonis (25). The concentration of MgCl2 in the bindingbuffer was increased to 5 mM to decrease nonspecific bind-ing. 65ZnC12 (500 ,uCi/2 ,g; 1 Ci = 37 GBq) was purchasedfrom Amersham. Usually strips or blots were incubated at 1AtCi/ml. Later experiments employed 65ZnCl2 at 3.26mCi/mg (DuPont), and in these experiments 5 mM CaCl2 andMgCl2 were added to the binding buffer to decrease nonspe-cific background activity. Molecular size markers containingcarbonic anhydrase (a Zn-binding protein) and other proteinsserved as additional positive and negative controls. Theidentity of VSPs was confirmed using specific anti-VSPmonoclonal antibodies on adjacent strips or duplicate West-ern blots (6, 20, 22).The capacity of other cations to inhibit 65ZnCl2 binding to

VSPH7 was determined by adding appropriate concentra-tions (0.01 mM-10 mM) of ZnCl2, CuSO4, FeSO4, CdC12,CoCl2, and MnCl2 to binding buffer and measuring thedecrease in signal compared to tests performed withoutinhibiting cation (24, 25). The band corresponding to VSPH7was the most dominant Zn-binding protein in H7; its identitywas confirmed by reactivity with monoclonal antibodyG10/4. The signal corresponding to VSPH7 was quantified bylaser densitometry using a SciScan apparatus and software(United States Biochemical).

Construction ofVSPH7 Fusion Proteins. Two sets offusionproteins (Fig. 1) were made by slightly different procedures,

Abbreviations: VSP, variant-specific surface protein; MBP, maltose-binding protein.*To whom reprint requests should be addressed.

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5490 Microbiology: Nash and Mowatt

but both employed the pMAL-c2 vector (New EnglandBioLabs). A series of oligonucleotides was constructed con-taining (from 5' to 3'), a 24-nt spacer and an internal Pvu IIsite followed by an 18-nt VSPH7-specific sense or antisensesequence (Applied Biosystems). These oligomers were usedas primers to amplify selected gene fragments from thecloned VSPH7 cDNA by PCR; the fragments were thencloned into the Xmn I site of the vector. A second series offusion proteins was produced as above with the followingdifferences: Sense oligonucleotide primers contained anEcoRI recognition sequence, and antisense oligonucleotidescontained a HindIII recognition sequence and a stop codonimmediately 3' to it. After digestion with EcoRI and HindlIl,the fragments were cloned into pMAL-c2. All constructswere verified by direct DNA sequence determination acrosscloning junctions. Fusion proteins were expressed and puri-fied by affinity chromatography as suggested by the manu-facturer. Antibody to the maltose-binding protein (MBP) waspurchased from the manufacturer of the vector.PCR was performed using the manufacturer's reagents

(Perkin-Elmer/Cetus) at 94°C for 30 sec, 50°C for 30 sec, and72°C for 3 min for 31 cycles. PCR products were purified withMagic PCR preps DNA purification system (Promega), andMagic Minipreps (Promega) were used to purify other DNAproducts.

RESULTSFour of the five reported VSPs have motifs similar but notidentical to two known metal-binding domains, the LIM (8,26-29) and RING finger motifs (30). The consensus se-quence, as well as the relationship to the LIM and RINGfinger motifs, is shown in Fig. 2, and the amino acid se-quences ofthe VSPs are shown in Fig. 3. The amino-terminalspacing resembles the LIM motifwhile the carboxyl-terminalend resembles the RING finger motif. One VSP, CRP72, hasa motif that closely resembles the consensus sequence butlacks the histidine, and three of the intervening amino acidsequences differ slightly in length from the other VSPs.Significant areas of amino acid similarity (in the regioncontaining the putative metal-binding domain) are present inVSPs CRP170, 1267, and H7; lesser amounts of homologyexist between TSA417 or CRP72 and the other VSPs (Fig. 3).

65Zn bound to the different VSPs expressed by threedifferent cloned trophozoite lines (Fig. 4). The most promi-nent signals in VSP1267 and VSPH7 corresponded to bandsrecognized by VSP-specific monoclonal antibody. CRP170migrated close to the 200-kDa molecular mass marker.A measure of the affinity of other cations for VSPH7 was

obtained by inhibition of 65Zn2+ binding by various cations.Despite some variation between experiments, the most fre-

l I I l11 III0

-8.19.1

- 10.111.2

12.113.1

14.1

quent ranking ofavidity for VSPH7 was Cd2+ > Zn2+ > Co2+> Cu2+ > Fe2+ >> Mn2+ (Table 1). Although the ranking ofthe most avid cations differs from other Zn-binding proteins(24, 31), they agree in general with Zn2+, Cd2+, Co2+, andCu2+ grouped as the most avid cations, Fe2+ as moderatelyavid, and Mn2+ as the least avid.

Fifteen fusion proteins were produced, which includedpeptide fragments encompassing the entire protein except theputative amino-terminal signal peptide of VSPH7. Bacterialcultures not induced with isopropyl ,-D-thiogalactopyrano-side (IPTG) failed to produce a detectable fusion protein, andnone reacted with MBP antiserum or bound 65Zn2+. Incontrast, induced cultures produced fusion proteins thatreacted with MBP antiserum (data not shown), bound Zn2+(Fig. 5), and were specifically recognized by a rabbit poly-clonal antiserum that exhibits VSPH7 reactivity (Fig. 6).MBP alone failed to bind Zn2+ and did not react with the GSantiserum (Figs. 5 and 6).Although these results show that fusion proteins containing

the VSP metal-binding domain, the VSP metal-binding do-main alone, or portions of this domain (1-5, 7, 8.1, 9.1, 10.1,11.2) bind Zn, other metal-binding domains are also present.Constructs 6, 13.1, and 14.1 also bind Zn, although theyexpress fragments that do not contain any or part ofthe motif.Several fusion proteins (6, 7, and 8-1) were purified andanalyzed for Zn binding, but none showed quantitative dif-ferences in ability to bind Zn2+. Therefore, although multiplebinding sites apparently occur within VSPH7, we wereunable to clearly demonstrate differential Zn-binding capac-ity.VSPs, including H7, contain numerous CXXC motifs, and

it is known that CXXCX,CXXC, when n = 13, 19, or 20,binds Zn (32). VSPs were analyzed according to the place-ment of CXXC motifs and the numbers (n) of interveningamino acids (1-30). Fig. 7 shows that the spacing of thetetrapeptides in VSP1267, VSPH7, and CRP170 is remark-ably conserved when these proteins are aligned from thecarboxyl terminus. The spacing in two other VSPs is lessconserved. The number of intervening amino acids (from 1 to30) ranges from 22 to 39 in the various VSPs and depends tosome extent on the size of the protein. n values of 13, 19, and20, which are motifs found in other metal-binding proteins,are present in CRP170, TSA417, and CRP72, but not inVSPH7. However, n values of 12, 14, 18, and 21 (33) arefound in VSPH7 and in other VSPs, and these motifs are mostlikely capable of binding Zn.

Cation analysis by graphite furnace atomic absorption ofGS/H7 trophozoites revealed concentrations ofZn (992 AM)and Cu (63 uM) and undetectable amounts of Mn, Co, andCd.

l l 111111557 A A

-1

34

56

7

FIG. 1. Delineation of the peptide fragments of VSPH7 expressed in various constructs. The location of CXXC tetrapeptides in VSPH7 isindicated by vertical lines, and the extent of the LIM/RING motif is indicated by the overlying horizontal line, which corresponds to aminoacids 255-364. The VSPH7 component ofeach fusion protein is indicated by the horizontal lines below, and the number ofthe construct is shownbeside each line.

n a 0 0 n I I I n a a I I .. . .

Proc. Natl. Acad Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 5491

VSPH7VSP1 267TSA417CRPl 70

CRP72

VSPCONSENSLUS(LIM)

VSPCONSENSUS(RING FINGER)

UM

RING FINGER

CX2CX11CX3CX1o CX2CHX2CX2CX8CX2CXl6CX3CX7CX4CX9CX2CXIOCX2CXI1CX3CX14CX2CHX2CXgC4CX2CX16CX2CX7CX4CXCX2CXIoCX2CX5 CX2CX10CX2CHX2CX2CX7CX2CX16CX3CX8CX2CX9CX2CXI1CX2CX11CX3CX14CX2CHX2CX2CX8CX2CX15CX3CX7CX4CX9CX2CX1o

CX2CX8CX2CX14CX2CGX2CX2CX7CX2CX14CX2CX9CX2CX9CX2CX14

CX2CXS 1CX243CX10,14CX2CHX2CX2CX7.8CX2CX15.16CX2-3CX7-8CX2-4CX9CX2C

CX2CX>1 ICX2-3CX10.I4CX2CHX2CX2CX7,.CX2CX1 5l6CX2-.CX74CX2-4CXgCX2C

CX2CX17.19HX2CX2CX2CX7.11 CX8C

CX2CX9-27CXHX2CX2CX6*17CX2C

FIG. 2. Comparisons ofthe LIM and RING finger motifs in VSPs. Spacing of histidine and cysteine residues in four VSPs (VSPH7, VSP1267,TSA417, and CRP170) that contain the LIM/RING finger metal-binding domains and one VSP (CRP72), which has a glycine residue (in boldfacetype) instead of a histidine residue, is shown. The consensus LIM/RING finger motif in VSPs and the similarity to both the LIM and RINGfinger motifs are given for comparison; the similarity to VSPs is indicated by boldface type. The motifs begin at amino acids 1532, 286, 255,400, and 312 for CRP170, VSP1267, VSPH7, TSA417, and CRP72, respectively.

DISCUSSIONIn this present report, we show that VSPs of G. lamblia aremetal-binding proteins and contain a distinct motif that hasfeatures common with other metal-binding domains. VSPs ofdifferent cloned trophozoite lines all bound Zn, and recom-binant proteins containing the conserved metal-binding pep-tide in VSPH7 were able to bind Zn. Unexpectedly, otherregions of VSPH7 were also capable of binding Zn, but oncloser inspection of the protein, numerous CXXCX,CXXCof various n values were present. The number of interveningamino acids in VSPH7 is either the same as other proposedZn-binding proteins or closely related; in many cases it differsby 1 amino acid (32, 33). These motifs and others withdifferent n values are probably capable of binding metals.Other metal-binding motifs are scattered among the VSPs andinclude cchc (32) in CRP170, adpribose in VSPH7 (32), andthe iron binding motifs CX6CX2CX5C in CRP170 (34) andCX2CX16CX13C in TSA417 (35).Like other metal-binding proteins, VSPH7 was able to bind

a number of cations. Because of the presence of multiple

CRP1 70VSP1267VSPH7tsa417CRP72

CRP1 70VSP1267VSPH7tsa417CRP72

CRP1 70VSP1 267VSPH7tsa4l7CRP72

binding sites, it is not clear which site or sites are responsiblefor binding. However, the most avid cations generally re-semble those of other metal-binding proteins (24, 25, 31).The surface location of proteins containing similar motifs is

unprecedented (26-28, 30, 36-44). Without exception, suchmotifs are found in intracellular proteins, and most, if not all,proteins except the cysteine-rich intestinal protein, which isbelieved to function as an intracellular Zn epithelium trans-porter (29), are thought to be DNA-binding proteins, controltranscription, repair DNA, or have undefined function.At present, it is not known if native VSPs contain bound

cations or if the binding of Zn in vitro represents onlypotential binding sites. The increased amount of Zn introphozoites supports the notion of Zn binding by the abun-dant VSPs. This requires confirmation by more direct anal-ysis. In prior studies (T.E.N., unpublished observations), thecysteine residues of VSP could not be labeled with thiol-reactive reagents, which suggests that sulflhydryl groups arebound, perhaps by metals, or otherwise unable to react.Although disulfide bond formation is typical within or be-

1 50DKRT ETSGNNLDNVTDI.GVM_ GA

CTKCSSN ?GDAC;VSG.DSAC_FASDE AG

KNGAK PTCS LDG YYNdN GGTV

51 100Y$IPUYS G TAG,

RET E'SN IG AK

X GSVLSGTDN GI IAGSKTA CPT GKALRYGDDCT TG.'TGAGK LTIDGASGND P GFFMK. .GNG PENCVA. CPQGGIDA KESTGPL

101 120

SGFV .PVNGI4C...S TTTE YPQNGC.

KPNRK PAGTSDNYTiY

FIG. 3. Comparison of the amino acid sequences of VSPs in the region of the putative metal-binding motif. Cysteine residues that conformto the motif are boxed, and the histidine residues are indicated in reverse type. Identical amino acids are indicated by dark shading or dark andlight shading when two amino acids are in common. The beginning of the sequences are indicated in the legend to Fig. 2.

Microbiology: Nash and Mowatt

5492 Microbiology: Nash and Mowatt

200

97.4

6 8

4 3

2 9

200.4

97.4

6 8

4 3

2 9

18.4

NO0L >

FIG. 4. Binding of 65Zn to Giardia VSPs. A number of Giardiaproteins bind Zn, but the most predominant band in clones WB/1267,GS/H7, and WB/A6 corresponds to the VSPs. The top, middle, andbottom arrows indicate the migration of CRP170, VSPH7, andVSP1267, respectively, as determined by immunoblot analysis.VSPH7 migrates more slowly in this analysis than in other studies(see Fig. 5). Molecular sizes (in kDa) are given at left.

tween polypeptides found on the surface of eukaryotic cells,this may not be the case with microaerophilic organisms suchas Giardia that are cultured under reducing conditions.Presumably, these conditions are present in vivo as well.

Similar spacing of CXXC motifs among different VSPssuggests conservation of an important biological function.Except for the placement of cysteine residues, there is littleconservation of the intervening amino acids among all theVSPs, although there is modest conservation among some.These proteins are for the most part antigenically distinct (18).VSPs have previously been shown to differ in susceptibility

to proteases (17). As mentioned above, one VSP, CRP72, hasdifferent CXXC spacing and alterations in the LIM/RINGfinger-like motif. VSPs also differ in the number and type ofCXXCX,CXXC motifs, iron-binding motifs, and other metal-binding domains. These structural differences may result indivergent biological characteristics; for example, VSPs mayconfer enhanced or diminished virulence, broaden host

Table 1. Percent binding of 65Zn to VSPH7 in the presence ofvarious cations compared to binding in the absence ofinhibitory cations

% 65Zn bound at cation concentration0.01 mM 0.1 1.0

mM mMExp. Zn2+ Cu2+ Cd2+ Co2+ Fe2+ Mn2+

1 52 65 66 89 97 412 76 43 73 71 -3 63 84 50 69 76 984 61 90 56 73 895 76 - 60

Although the concentrations of inhibitory cations ranged over 3orders of magnitude for each cation, the results after incubation foronly one concentration are shown. Cd2+, Zn2+, Co2+, and Cu2+ arethe most inhibitory followed by Fe2+ and Mn2+. CaCl2 was studiedonce (Exp. 1) and gave 61% binding at 0.1 mM.

I. X. m 0o 0 0 C) )

0n M n in _)_ 40_.-

a a4.

FIG. 5. Binding of 65Zn to recombinant VSPH7 fusion proteins.The "-". and "+" indicate uninduced and induced cultures. MBPindicates cultures of nonrecombinant pMAL-c2; lane H7 is totalprotein extract from GS/M clone H7. The numbers conform to theconstructs in Fig. 1; the letters indicate specific clones containing theindicated constructs. Molecular sizes (in kDa) are given at left.

range, or confer greater stability in the intestinal environ-ment.The potential metal-binding properties of G. lamblia sug-

gest unusual ways that the parasite may interact with its host.In some infections, the parasite burden is large, and the villiof the small intestine are covered with trophozoites. VSPscould bind Zn and inhibit the function of Zn or metal-requiring intestinal enzymes such as carboxypeptidases or

MBP 7C

9 7.4

68

4 3 .

2 9

2 C

z l_.co

r l:L r-z m X2 (0

z-

rp

FIG. 6. Western blot of induced cultures expressing MBP aloneor construct 7C. Anti-MBP and anti-GS/H7 sera recognize the 7Cfusion protein, which contains a VSPH7 finment. In contrast, MBPalone is only recognized by anti-MBP serum. Molecular sizes (inkDa) are given at left. NR, normal rabbit serum.

w_

Proc. Natl. Acad Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 5493

llLLI5II 11111 11 11 1I I I I I I I II I III I

I1I IIII I I1liiiI 111

LLlIllHl

VSP 267

VSPH7,7 .-. -A . .... . . . . . C P7

CRP1 70

r 11 1I I I1IL 11111 111111 11

.I II I 111111 III 111111111

l111111I I 11111 TSA417

CRP72

FIG. 7. Comparison of the spacing of CXXC tetrapeptides among VSPs. The vertical lines indicate each CXXC tetrapeptide, and thehorizontal line above VSP1267 signifies the LIM/RING finger-like motif in VSPH7. For ease in presentation, CRP170 is shown with only 2 ofits 21 repetitive units, which are in the amino-terminal region. All VSPs are drawn to the same scale.

compete with the host for Zn and contribute to Zn malnu-trition, which is increasingly recognized (45) and occurs inpopulations and areas where Giardia is prevalent. If this werethe case, only the presence of enough trophozoites, but notnecessarily the presence of disease, would be needed tocause Zn malnutrition.The benefit of metal binding of VSPs to Giardia is un-

known. Metal ions could maintain structure by noncovalentbridging or could bind neighboring VSPs. Another function ofmetal binding may be to prevent oxidation, which is detri-mental to Giardia, by free metal cations and oxygen.Note. After submission of this manuscript, a paper was published byZhang et al. (46), which documents 65Zn binding to membraneproteins of Giardia. These are most likely VSPs.

We appreciate the technical support of John T. Conrad and JamesW. Merritt and the editorial assistance of Sheryl Rathke. We alsoacknowledge C. Graham Clark for critical evaluation of the manu-script.

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