Transient and Stable Expression of the FireflyLuciferase Gene in Plant Cells and Transgenic Plants

DAVID W. OW, KEITH V. WOOD, MARLENE DELUCA,JEFFREY R. DE WET,* DONALD R. HELINSKI, STEPHEN H. HOWELLt

The luciferase gene from the firefly, Photinus pyralis, was used as a reporter of geneexpression by light production in transfected plant cells and transgenic plants. Acomplementary DNA clone of the firefly luciferase gene under the control of a plantvirus promoter (cauliflower mosaic virus 35S RNA promoter) was introduced intoplant protoplast cells (Daucus carota) by electroporation and into plants (Nicotianatabacum) by use of the Arobacterum tumefaciens tumor-inducing plasmid. Extractsfrom electroporated celLs (;4 bours after the introduction of DNA) and fromtransgenic plants produce light when mixed with the substrates luciferin and adenosinetriphosphate. Light produced by the action of luciferase was also detected in undis-rupted leaves or cells in culture from transgenic plants incubated in luciferin and inwhole transgenic plants "watered" with luciferin. Although light was detected in mostorgans in intact, transgenic plants (leaves, stems, and roots), the pattern of lumines-cence appeared to reflect both the organ-specific distribution of luciferase and thepathway for uptake of luciferin through the vasculature of the plant.

T HE FIREFLY LUCIFERASE GENE,which encodes an enzyme that cata-lyzes the light-producing, adenosine

triphosphate (ATP)-dependent oxidationof luciferin (1), is a powerful reporter genefor assessing gene expression in eukaryoticorganisms. We report that light emittedfrom firefly luciferase was detected in plantsand plant cells in culture transformed with aluciferase complementary DNA (cDNA)construct driven by a plant virus promoter.The firefly luciferase gene has been ex-pressed in bacteria (2) but is ideal for use asa reporter gene in eukaryotic organisms (3),

because unlike the bacterial lux gene system(4), only one protein is needed for lightproduction if the substrates ATP, 02, andluciferin are available (1).The introduction of DNA into plants for

testing the expression of gene constructs hasbeen made possible by the development ofnew methods for transforming plants andplant cells. Several procedures, includingelectroporation, have been developed to in-troduce naked DNA into plant cells fortesting the transient expression ofgene con-structs (5). The production of transgenicplants, transformed with the tumor-induc-

ing (Ti) plasmid of Agrobacterium tumefa-ciens (6), has permitted investigations intothe regulation of organ-specific gene expres-sion in plants, the development of pathogenor herbicide resistance, and the control ofgene expression linked to plant physiologi-cal processes including responses to light(7).To test the activity of the firefly luciferase

gene in plant cells in a transient expressionassay, we used electroporation (5) to intro-duce a luciferase cDNA construct into pro-toplasts of cultured carrot cells (8). Thebasic plasmid pDO432 (Fig. 1) contains an"intronless" luciferase gene created by deWet et al. (3) from a partial cDNA clonelinked at an Xba I site (129 bp downstreamfrom the start of translation) to the 5' end ofthe firefly gene. The fusion was createdbecause a full-length cDNA covering all ofthe translated region was not obtained in theinitial cDNA cloning effort (2). The lucifer-ase gene was spliced upstream, via a Bam HIsite, to the cauliflower mosaic virus (CaMV)35S RNA promoter, making the expectedstart of transcription 81 bp upstream fromthe predicted start of translation of theluciferase gene (Fig. 1). The luciferase genewas spliced on its downstream side, via aBam HI site, to a segment at the 3' end ofthe nopaline synthase (nos) gene (9).The plasmid pDO432 was introduced

into carrot protoplasts by electroporation,

D. W. Ow, J. R. de Wet, D. R. Helinski, S. H. Howell,Department of Biology, University of California, SanDiego, La Jolla, CA 92093.K. V. Wood and M. DeLuca, Department of Chemistry,Univcrsity of California, San Diego, La Jolla, CA 92093.

*Present address: Stanford University, Stanford, Califor-nia 94305.tTo whom all correspondence should be addressed.

Fig. 1. The structure ofthe CaMV 35S RNA promoter-luciferase-nos 3'endconstruct in pUC19 (pDO432). The 35S RNA promoter (35S p) wasobtained by creating a Bam HI site with oligonucleotide-directed mutagene-sis of pCaMV10 (from CaMV isolate CM1841) (14) at the start of 35SRNA transcription (15) (position 7431 as underlined, GGACAC-4GGATCC). The 35S RNA promoter (1586 bases) extends from this newlycreated Bam HI site upstream from the Hind III site at position 5849 on themap of CaMV-CM1841 (14). The luciferase gene was obtained as a HindIII-Bam HI fragment (1886 bases) from pJD201 (3). The upstream HindIII site was filled in and converted to a Bam HI site by the addition of BamHI linkers. The luciferase gene in pJD201 is an intronless gene created by afusion between a partial cDNA clone (Luc23) and the 5' end of thecorresponding firefly luciferase gene, joined in the coding region at an Xba Isite (3). The presumed start of translation is 81 bases downstream from thenewly created Bam HI site, the point offusion ofthe luciferase gene and the35S RNA promoter. A 1028-bp fragment containing polyadenylation sitesfrom the 3' end of the A. tumefaciens nos gene (9) was inserted downstreamfrom the luciferase gene. The 3' end of the luciferase cDNA, in particular,the 139 bp Ssp I-Bam HI fragment, carries a consensus polyadenylationsignal (AATAAA) 28 bases upstream from its 3' end. Plasmid constructswere tested for activity in a transient expression assay in which 10 ,ug ofplasmid DNA and 500 ,ug of carrier calf thymus DNA were introduced byclectroporation (5) into protoplasts prepared from carrot cells growing insuspension (8). Extracts were prepared 24 hours after electroporation byrepeated cycles offreezing and thawing of0.5 x 107 to 1 x 107 carrot cells in

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luciferase extraction buffer, 100 mM potassium phosphate buffer (pH 7.5),and 1 mM dithiothreitol, followed by centrifugation for 5 minutes in amicrocentrifuge (Eppendorf) at 4°C. For luciferase assays, 1/10 volume ofthe supematant fluid was diluted into 400 ,u1 of assay buffer [14 mMglycylglycine buffer (pH 7.8), 14 mM MgCI2, and 6 mM ATP], to which100 ,ul of 1 mM luciferin was added by injection. The peak intensity of theresulting light flash was measured in a luminometer (LKB, model 1250),and the data from a single representative experiment are expressed in lightunits relative to that produced by pDO432.

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and extracts were assayed for activity, usual-ly 24 hours later. Extracts produced a lightflash detected in a luminometer in the pres-ence of the substrates luciferin and ATP. Inthe presence of excess substrates, light pro-duction, measured as peak height, was pro-portional to the amount of plant extractcontaining luciferase activity. No lightabove machine background [<5 light units(LU)] could be detected in the absence ofluciferin, but low levels of light were pro-duced with excess luciferin in the absence ofadded ATP, presumably as a result of ATPin the plant cell extracts. With pDO432,1000 to 3000 LU per 106 cells or 60 to 90LU per microgram of protein were obtainedin transient expression assays correspond-ing, on average, to the activity of300 to 900molecules of luciferase per cell (3).The expression of the luciferase gene in

transient expression assays depended on cer-tain critical features of the DNA construct.For example, transcription was directed bythe 35S RNA promoter, and not by apromoter within the 5' untranslated seg-ment of the luciferase gene. This was indi-cated by the absence of luciferase expressionfrom pDO435 in which the luciferase genewas inverted with respect to the promoter(Fig. 1). Removal of the downstream plantpolyadenylation site (the nos 3' segment), asin pDO446, reduced luciferase activity to66% of full activity. However, when the 3'end of the firefly luciferase gene, whichcontains the polyadenylation signal AA-TAAA that is a consensus sequence for bothanimal and certain plant genes (10), wasdeleted by removing the Ssp I-Bam HIDNA segment, as in pDO445, luciferaseactivity in extracts dropped to 8% of fullactivity. The explanation for this loss inactivity is not clear, but may indicate thatluciferase expression in pDO432 is moredependent on the proximal polyadenylationsignal in the luciferase gene than on a distalsignal in plant DNA.To obtain stable transformants, we intro-

duced pDO432 into plants via the A. tume-facicns Ti plasmid. The plasmid pDO432was inserted at the Hind III site in the Tiplasmid binary vector Bin-19 (11) and trans-ferred by conjugation into theA. tumefaciensstrain GV3111 bearing the helper plasmidpTiB6S3SE (6). Nicotiana tabacum plants(Wisconsin-38 cultivar) were inoculatedwith the bacterial vector by the leaf diskinoculation method (6), and kanamycin-resistant transformants were selected. Pri-mary transformants were screened for lucif-erase production by grinding a small, frozenleaf disk (diameter, 8 mm) in extractionbuffer (see legend to Fig. 1) to prepare anextract for the luciferase assay. Screening canalso be performed in a semiquantitative

I4 NOVEMBER I986

1 2 3 4 5 6 7

manner by dropping a small leafcutting (-5mm2) in a reaction tube containing sub-strates and reading the light output in aluminometer.

Several primary transformants (transgenicplants B10, B15, B22, and B28) with dem-onstrated luciferase activity were chosen forfurther study. DNA blots indicated the pres-ence of pDO432 in the plant genome,which appeared as a 7-kb band in Hind III-cleaved DNA fragments from transformedplants but not from untransformed plants.The number ofcopies ofpDO432 was quitesimilar in the selected transformants and wasequivalent in reconstructions to one to sev-eral copies per tobacco genome (Fig. 2).The transgenic plants also produced a pro-

2500

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Fig. 2. Blot of genomic DNA from transgenictobacco plants. DNA was extracted from theleaves of primary transformants as indicated,cleaved with Hind III, fractionated on a 1%agarose gel, transferred to a nitrocellulose filter,and hybridized with the nick-translated 1.6-kbHind III-Bam HI fragment from pDO432 con-taining the CaMV 35S promoter (16). Hind IIIcleavage of integrated pDO432 DNA releases alinear 7-kb fragment. Gel migration position ofthe 7-kb fragment is indicated by the arrow on theleft. (Lanes 1 and 2) Copy number standards.Hind III-cut pDO432 DNA in amounts (110and 22 pg) equivalent to ten and two copies pertetraploid tobacco genome; (lanes 3 to 7) 10 jigof Hind III-cut DNA from an untransformedtobacco plant (lane 3) and transformed plantsB10 (lane 4), B15 (lane 5), B22 (lane 6), and B28(lane 7).

tein species of a size expected for fireflyluciferase (12). Protein blots of leaf extractsfrom plant B 15 showed the presence of a62-kD protein band that reacts with antise-rum and migrates with firefly luciferase.The selected transgenic plants (primary

transformants), grown in sterile culture,were examined for the distribution of lucif-erase activity in various plant organs. Wefound that extracts from all organs-leaves,roots and stems-expressed activity to dif-fering extents (Fig. 3). Considerable varia-tion in activity was observed from leaf toleaf, with young, uppermost leaves generallyshowing higher specific activities than older,lower leaves. In general, roots and stems(upper stems were tested) had more lucifer-

* Plant Bl0* Plant BI5O Plant B22O Plant B28

1 2 3 4 5 6 7 8 9 10 11 StemsRootsLeaf number

Fig. 3. Distribution of luciferase activity in the organs of transgenic plants. Plants were grown in glassjars under sterile conditions in a modified Murashige and Skoog agar medium (MS medium) (17) andextracts were prepared when plants reached a height of 12 to 15 cm. Extracts from various plant organswere prepared by grinding a small amount of tissue, frozen in liquid N2 in a 1.5-mi microcentrifugetube, with the extraction buffer described in Fig. 1. The soluble extract obtained after centrifuging for 5minutes at 4°C in a microcentrifuge was assayed for luciferase activity and protein content (18). Leafnumber is the position of the leaf on the plant, starting from the younger leaves at the top of the plantprogressing to the older leaves on the bottom.

REPORTS 857

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ase activity (usually more than 2000 LU permicrogram of protein) than leaves. Thisfinding is consistent with the observationsof Fluhr et al. (13), who demonstrated asimilar organ-specific distribution for chlor-amphenicol acetyltransferase (CAT) activity

driven by the CaMV 35S promoter. Manyfactors may contribute to variation in theoverall activity or the distribution of lucifer-ase activity in plant organs, including differ-ences in developmental stage, plant growthconditions, stability ofthe enzyme, and vari-ation in the expression of the gene, possiblydue to position effects of integration of theluciferase gene in the plant genome (13).We also attempted to determine the dis-

tribution of luciferase activity in variousplant organs by introducing luciferin into

Fig. 4. Leaf luminescence recorded on x-ray film(autoluminograph). Small leaf (2.5 by 4.5 cm)from primary N. tabacum transformant B15 illu-minated with ordinary white light (left). The leafwas soaked for 2.5 hours in 1 mM luciferinsolution containing 100 mM sodium citrate (pH5.0) and 20% dimethyl sulfoxide. The same leaf(right) was exposed by contact to x-ray film(Kodak OG).

intact plant tissue. We found that by soakinga young leaf from a transgenic plant in asolution containing 1 mM luciferin, wecould detect emitted light by exposing theleaf to x-ray film (Kodak OG) for 15 to 60minutes (Fig. 4). Light emission was en-hanced by adding dimethyl sulfoxide(DMSO) up to 20% and sodium citrate (pH5) to the luciferin solution. These additionsprobably aided in the uptake of the sub-strate. No background light was detected onx-ray film from leaves of untransformedplants or from leaves of transformed plantsthat were not soaked in luciferin. Finally,luciferin was introduced through roots intowhole plants by watering small, sterile plantswith a luciferin solution. Plant B21 (a pri-mary transformant with high luciferase ac-tivity) was watered for 6 hours with 5 ml ofa 1 mM luciferin solution and exposed bycontact to Kodak Ektachrome 200 film (4by 5 inch sheet) for 24 hours (Fig. 5). Thepattem of light emission appeared to reflectboth the distribution of luciferase activity inthe plant and the movement of luciferinthrough the vasculature of the plant. Lightemission was most intense from roots,stems, and young, upper leaves. However,many factors may contribute to the pattemof light emission from intact plants, includ-ing possible organ differences in the accessi-bility of luciferase to luciferin, compartmen-talization of luciferase, and absorption ofemitted light.

Fig. 5. Luminescence from transgenic tobacco plant B21 bearing the firefly luciferase gene. The plantwas watered with a 1 mM solution of luciferin, the luciferase substrate, and exposed by contact toKodak Ektachrome 200 film.

858

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Fig. 6. Detection of luciferase activity in cellsdistributed in a 96-well microtiter dish (Falcon3072, Becton Dickinson). Portions (200 ,ul) of acell suspension culture derived from transgenictobacco plant B15 (columns A to C) and anuntransformed tobacco (column D) plant weredistributed into microtiter dish wells as indicated.Numbers to the left indicate the number of cellsper well in each row. Luciferin in MS mediumwas added to the wells (column A, 400 pM; B, 80pM; C, 16 pM; and D, 400 pJM) and the platewas exposed by contact with x-ray film (KodakOG) for 1 hour. The edges of the microtiter dishwere milled so that the bottoms of the wells werein direct contact with the film.

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Because it would be useful for cell screen-ing procedures to detect luciferase activity inundisrupted cultured cells, we tried to assayfor the presence of luciferase activity inintact, living cells distributed in a microtiterplate. A cell line (B15-1) was derived fromleaf protoplasts of the transgenic plant B15,and extracts from cells grown in suspensionculture showed moderate levels of luciferaseactivity (210 LU per microgram ofprotein).Various numbers of cells were distributedinto a microtiter dish, incubated in growthmedium in the presence of different concen-trations of luciferin and exposed for 1 hourto x-ray film (Kodak OG). Wells with lucif-erase-containing cells produced strong lightsignals (Fig. 6). After 1 hour of exposure,minimal signals were detected from 375 to750 cells in 80 pM luciferin. Cells survivedthe lower concentrations of luciferin (80,uM or less) in long-term incubations (sever-al weeks), but higher luciferin concentra-tions (400 pM and above) were apparentlytoxic and blocked further growth of thecells. Hence, at lower luciferin concentra-tions, this assay constitutes a simple, nondis-ruptive procedure for screening reasonablylarge numbers of independent cell lines forluciferase activity.The luciferase gene is an important new

tool for studying gene expression in bothplant and animal cells. Recently, de Wet etal. (3) demonstrated the expression of anSV40 promoter-driven luciferase gene inmnammalian cells (monkey CV-1 cells). Forplant genetic engineering purposes, the lu-ciferase gene can be particularly valuable as areporter of gene expression, as a marker ingenetic crosses, and as a probe for a varietyof plant cell functions. The speed and easeby which the luciferase assay can be per-formed permit more rapid screening of largenumbers oftransgenic plants or transformedcell lines. The luciferase gene is a sensitivereporter gene, because the luciferase assayitself can detect the light output from 3 x106 molecules of luciferase (10 LU on aLKB model 1250 luminometer), which is atleast 100 times more sensitive than a stan-dard CAT assay (3). Furthermore, luciferaseactivity can be detected by simple x-ray filmexposure or by more elaborate devices suchas luminometers or image-intensifying videoequipment. Luciferase offers many new pos-sibilities as a reporter gene since detection ofactivity can be noninvasive and nondestruc-tive, and luciferase activity can be assessedperiodically over time, such as through thecourse of plant development. In addition,luciferase can be used as a cell marker toidentify cells and as a tag to follo'w thetargeting of proteins to various destinationswithin and outside of cells. Finally, becauseluciferase activity is influenced by a varietyI4 NOVEMBER I986

of parameters (1), light production fromluciferase-expressing cells potentially couldbe used to monitor such events as ATPproduction and pH changes.

REFERENCES AND NOTES1. M. DeLuca and W. D. McElroy, Methods Enzymol.

57, 3 (1978).2. J. R. de Wet, K. V. Wood, D. R. Helinski, M.

DeLuca, Proc. Natl. Acad. Sci. u.SA. 82, 7870(1985).

3. J. R. de Wet, K. V. Wood, M. DeLuca, D. R.Helinski, S. Subramani, in preparation.

4. J. Engebrecht, M. Simon, M. Silverman, Science227, 1345 (1985).

5. M. Fromrnm, L. P. Taylor, V. Walbot, Proc. Natl.Acad. Sci. U.SA. 82, 5824 (1985).

6. R. B. Horsch et al., Scicnce 227, 1229 (1985).7. R. N. Beachy et al., EMBO J. 4, 3047 (1985); L.

Comai et al., Nature (London) 317, 741 (1985); G.K. Lamppa, G. Morelli, N.-H. Chua,Mol. Cell. Biol.5, 1370 (1985); G. K. Lampa F. Nagy, N.-H.Chua, Nature (London) 316, 750 (1985); F. Nagy,G. Morelli, R. T. Fraley, S. G. Rogers, N.-H. Chua,EMBOJ. 4, 3063 (1985); C. Sengupta-Gopalan, N.A. Reichert, R. F. Barker, T. C. Hall, J. D. Kemp,Proc. Nati. Acad. Sci. U.SA. 82, 3320 (1985); J.Simpson et al., EMBO J. 4, 2723 (1985); R. Fluhrand N.-H. Chua, Proc. Natl. Acad. Sci. U.SA. 83,2358 (1986); P. Powell ct al., Science 232, 738(1986); J. Simpson, M. Van Montagu, L. Herrera-Estrella, ibid. 233, 34 (1986).

8. Daucus carota WOOlC suspension culture line fromZ. Renee Sung.

9. The 3' end fragment of the nos gene was obtainedfrom pNCAT4 provided by J. Schell. The fragmentextends from nucleotide position 1119 to 91 on the

THHE LYMPHOKINE INTERLEUKIN-2

(IL-2) is synthesized and secreted byactivated T cells and plays a critical

role in the proliferative expansion of T-lymphocyte effector cells during the immuneresponse (1). Concomitant with IL-2 pro-duction, two distinct classes of receptorswith radically different affinities for IL-2 are

expressed by activated lymphocytes (2). Theratio of low-affinity sites [with a reported

nos gene sequence from pTiT37, as described by M.Bevan, W. M. Barnes, and M.-D. Chilton [NuclicAcidsRes. 11, 369 (1983)].

10. G. Heidecker and J. Messing, Annu. Rev. PlantPhysiol. 37, 439 (1986).

11. M. Bevan, NuckicAcids Res. 12, 8711 (1984).12. K. V. Wood, J. R. de Wet, N. Dewji, M. DeLuca,

Biochem. Biaphys. Res. Comm. 124, 592 (1985).13. R. Fluhr, C. Kuhlemeier, F. Nagy, N.-H. Chua

Science 232, 1106 (1986).14. The plasmid pCaMV10 described by R. C. Gardner

et at [Nucleic Acids Res. 9, 2871 (1981)] wasprovided by R. J. Shepherd.

15. S. N. Covey, G. P. Lomonossoff, R. Hull, NucleicAcids Res. 9, 6735 (1981); H. Guilley, R. K.Dudley, G. Jonard, E. Balazs, K. E, Richards, Cell30, 763 (1982).

16. T. Maniatis, E. F. Fritsch, J. Sambrook, MoklcdarCloning:A LaboratoryManual (Cold Spring HarborLaboratory, Cold Spring Harbor, NY, 19812).

17. T. Murashige and T. Skoog, Pbysiol. Plant 15, 473(1962).

18. M. M. Bradford, Anal. Biochem. 72, 248 (1976).19. We thank M. Fromm for advice on electroporation

procedures, S. C. Lee for assistance in plant tissueculture, Z. Renee Sung for WOO1C cels, J. Schellfor plasmid pNCAT4, R. J. Shepherd for plasmidpCaMV10, M. Bevan for the Ti plasmid binaryvector Bin-19, and S. Rogers for A. tumefaciemGV3111 containing the helper Ti-plasmidspTiT37SE and pTiB6S3SE. Supported by grant 85-CRCR-1-1540 from the U.S. Department of Agri-culture Competitive Research Grants Program (toS.H.H.), grant PCM83-05446 from the NationalScience Foundation (to M.D.), by the University ofCalifornia Biotechnology Research and EducationProgram, and by an NSF postdoctoral fellowship inplant biology (to D.W.O.).22 August 1986; accepted 7 October 1986

dissociation constant (Kd) of 1 to 30 nM] tohigh-affinity sites (reported Kd of 5 to 90pM) is approximately 10:1 in both normal

M. Sharon, R. D. Klausner, W. J. Leonard, Cell Biologyand Metabolism -Branch, National Institute of CMdHealth and Human Development, National Institutes ofHealth, Bethesda, MD 20892.B. R. Cullen and R. Chizzonite, Department ofMolecu-lar Genetics, Hoffmann-La Roche, Inc., Nudey, NJ07110.

REPORTS 859

Novel Interleukin-2 Receptor Subunit Detected by -

Cross-Linking Under High-Affinity Conditions

MICHAEL SHARON, RIcHARD D. KLAUSNER, BRYAN R. CULLEN,RIcHARD CHIZZONITE, WARREN J. LEONARD

Interleukin-2 (IL-2) binds to both high- and low-affinity classes of IL-2 receptors onactivated T lymphocytes. Only the high-affinity receptors are involved in receptor-mediated endocytosis and normaily transduce the mitogenic signals of1L-2; however,the structural features distinguishing the high- and low-affinity receptors are un-known. When 1251-labeled II-2 was chemically cross-linked to activated human Tlymphocytes, two major bands were identified. First, as predicted, a 68- to 72-kilodalton band, consisting ofIL-2 (15.5 kilodaltons) cross-linked to the IL-2 receptor(55 kilodaltons), was observed. Second, an unpredicted 85- to 92-kilodalton moietywas detected. This band was not present when I-2 was cross-linked to transfectedC127 cells, which exclusively express low-affinity receptors. The data presented aremost consistent with the existence ofa 70- to 77-kilodalton glycoprotein subunit (p70)which, upon associating with the 55-kilodalton low-affinity receptor (p55), transformsit into a high-affinity site. It is proposed that p55 and p70 be referred to as the a and ,subunits, respectively, of the high-affinity I-2 receptor.

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