DNAVolume 8, Number 6, 1989Mary Ann Liebert, Inc., PublishersPp. 447-455

LABORATORY METHODS

Replica Filter Screening Technique to Detect TransfectedCells Expressing ß2-Adrenergic Receptor

CURTIS A. MACHIDA,* JAMES BUNZOW,ERIC HANNEMAN, DAVID GRANDY, and OLIVIER CIVELLI

ABSTRACT

We have utilized a replica transfer technique to develop a novel screening assay for the identification oftransfectants expressing /32-adrenergic receptors (/32-AR). The hamster 02-AR gene flanked by either its nat-ural promoter or the zinc-inducible mouse metallothionein (MMT) promoter was cotransfected with plas-mids conferring neomycin resistance (pRSVneo) into /32-AR-deficient mouse L cells. Transfectant colonieswere grown on polyester nylon filters and screened by filter binding with [U5I]iodohydroxybenzylpindolol toidentify colonies expressing ß2- AR. Individual colonies were isolated and examined to determine ß2-AR genedosage, mRNA expression, and receptor densities and affinities. Analysis of cell lines expressing ri2-AR indi-cates that this method can identify transfectants containing only a single 02-AR gene copy and expressing as

few as 4,000 02-receptors per cell. This method may be useful as a tool for the molecular cloning of neuro-

transmitter receptor genes and for the measurement of transfection efficiencies and expression of receptorgenes in cells.

INTRODUCTION

An important class of neurotransmitter receptors are

coupled to different effector systems via specific GTPbinding proteins (G proteins) and share sequence similari-ties, suggesting the existence of a large multigene family(Dohlman et al, 1987). These neurotransmitter receptorsinclude the adrenergic receptors (Dixon et al, 1986; Yar-den et al, 1986; Frielle et al., 1987; Kobilka et al,1987a,d; Allen et al., 1988), the muscarinic acetylcholinereceptors (Kubo et al, 1986a,b; Peralta et al., 1987; Bon-ner et al, 1987), the serotonin receptors (Kobilka et al,1987b; Lubbert et al, 1987; Julius et al, 1988; Pritchett etal., 1988), the dopamine D2 receptor (Bunzow et al., 1988),and the substance K receptor (Masu et al., 1987). Severalstructural features of these neurotransmitter receptors ap-pear to be conserved; all display hydropathicity profilesconsistent with seven transmembrane-spanning domains andall retain functionally important amino acid consensus se-

quences necessary for phosphorylation and glycosylation.Structure-function analysis of neurotransmitter recep-

tors and studies examining the regulation of receptor geneexpression often rely on the successful transfection and ex-

pression of receptor genes in eukaryotic cells. These studiescan be expedited by the development of a rapid techniqueto detect transfectants expressing neurotransmitter recep-tors. In addition, since many genes for G-protein-coupledreceptors lack introns, such a technique can be used toclone new neurotransmitter receptor genes.

The 02-adrenergic receptor (j32-AR) is the best character-ized G-protein-coupled receptor. Therefore, we chose the/32-AR system as a model in the development of a receptorscreening assay to identify eukaryotic transfectants whichexpress specific neurotransmitter receptor genes. This as-

say permits rapid evaluation of neurotransmitter receptorgene expression in small numbers of transfectants withinlarger populations of nonexpressors and, when used inconjunction with cDNA expression libraries, can poten-

Vollum Institute for Advanced Biomédical Research, Oregon Health Sciences University, Portland, OR 97201. Present address: Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, OR 97006.

447

448 MACHIDA ET AL.

tially be used as a tool for cloning of neurotransmitter re-

ceptor genes. In addition, this assay may have potentialapplication in (i) the measurement of transfection efficien-cies of receptor-encoding plasmids, (ii) the evaluation ofpromoter strengths, (iii) the determination of ligand selec-tivities for uncharacterized receptors, and (iv) the identifi-cation of mutant receptors with abnormal ligand selectivi-ties. We report the use of a novel neurotransmitter recep-tor screening assay to examine /32-AR gene expression inindividual transfectants and demonstrate the utility of theassay in the measurement of transfection efficiencies andin the evaluation of promoter strength.

MATERIALS AND METHODSCell lines and tissues

The Syrian hamster melanoma RPMI 1846 (CCL 49)and DDT,MF2 (CRL 1701) cell lines were obtained fromthe American Type Culture Collection (Rockville, MD).The mouse L-cell line was obtained from the Vollum Insti-tute Cell Culture Facility (Portland, OR). All cell cultureswere grown in Dulbecco's modified Eagle's medium(DMEM) (GIBCO) supplemented with 10% fetal calf se-rum and were maintained at 37°C. Syrian hamster lung(untrimmed) was obtained from Pel-Freeze Biologicals(Rogers, AK).

Cloning of the hamster ß2-AR geneThe hamster /32-AR gene was cloned from a hamster

lung X gtlO genomic DnA library [constructed with size-fractionated (5-7 kb) Eco Rl-digested DNA]. Two oligo-nucleotide probes (30-mer, TCTGCTTTCAATCCCCTC-ATCTACTGTCGG; 40-mer, CTATCTTCTGGAGCTG-CCTTTTGGCCACCTGGAAGACCCT; sequence ob-tained from Dixon et al., 1986) were used to screen 500,000independent clones; two genomic clones that hybridizedwith both probes were isolated. Both clones containedidentically sized inserts of approximately 5.2 kb. One ofthe inserts was subcloned into plasmid vector pUC 19; therecombinant (pUC /3-AR) subsequently was analyzed byrestriction endonuclease mapping and partial nucleotidesequence analyses. The results of our analyses are consis-tent with the cloning of the hamster /32-AR gene.

A 1,597-bp Nae l-Xba I restriction fragment containingall the hamster /32-AR coding sequence (see Dixon et al,1986; Nae I at position 128, Xba I at position 1,725) wascloned into the eukaryotic expression vector pZEM 3(Uhler and McKnight, 1987). The restriction fragment was

enzymatically flush-ended to allow ligation of syntheticBam HI linkers, prior to insertion into pZEM 3. The re-

combinant with the receptor-coding sequence in the cor-rect orientation (pZEM /3-AR) was determined by restric-tion endonuclease mapping and verified by partial nucleo-tide sequence analysis.

Plasmid DNA preparation and transfectionsPlasmid DNA was prepared by equilibrium centrifuga-

tion in CsCl-ethidium bromide gradients (Ausubel et al,1987). Transfection of DNA into mouse L cells was donewith the calcium phosphate precipitation method of Gor-man et al. (1983). Plasmid DNA (pRSVneo) conferringneomycin resistance (Southern and Berg, 1982) and pUC/3-AR or pZEM /3-AR were mixed as a calcium phosphateprecipitate and then applied to 5 x 105 L cells in a 100-mmculture dish. The mass of pRSVneo applied to culturesvaried according to the purpose of a specific experiment; 1fig was applied to achieve high colony densities (e.g., Fig.1C) and 0.1-0.2 fig was applied to achieve low colony den-sities (e.g., Table 1). The transfection efficiency ofpRSVneo in L cells (using 1 fig of pRSVneo as a standard)is approximately 1 x 10"3. Drug selection of stable trans-fectants occurred 48 hr later with the addition of neomycinanalog G-418 (Geneticin, GIBCO) to the medium (finalconcentration, 300 /¿g/ml) and maintained for approxi-mately 2 weeks.

Replica copies of cell populations and receptorscreening of transfectants

Polyester nylon cloth (10-/un mesh, Pe Cap fabric) was

obtained from Tetko, Inc. (Elmsford, NY) and preparedfor cell colony overlay as described by Raetz et al (1982).Transfectant colonies were carefully overlaid with 2- to 10-cm-diameter polyester nylon discs, held in place against thecolonies with a monolayer of glass beads (4 mm, AmericanScientific Products). Vertical colony growth into the filtersoccurred within an additional 7 days of growth at 37°C.The transfer of colonies from the dish to the filters was as-sessed by acid fixation and Coomassie blue staining anddetermined to be nearly quantitative. Replica copies oftransfectant populations were removed from the masterdish and placed in individual dishes for either cell colonyrecovery or receptor screening analysis. In the case of discscontaining colonies transfected with pZEM /3-AR, thediscs were first treated with 100 ¡iM zinc sulfate in DMEM(18 hr, 37e C prior to their application in the receptor as-

say. The MMT promoter becomes refractory if zinc stimu-lation is conducted for longer than 24 hr.

/32-AR screening of transfectant populations was con-

ducted by prerinsing the filters with phosphate-bufferedsaline (PBS), followed by incubation with radiolabeled ß2antagonist, [125I]iodohydroxybenzylpindolol (HBP, 2,200Ci/mmole, New England Nuclear, 150 pM in PBS) for 15min at room temperature. Unbound radioactive HBP was

then quickly removed by washing with cold PBS; the filterswere then air-dried and placed under X-ray film (with in-tensifying screens) for a 24-hr exposure at -70°C.

Other methodsWhole-cell receptor binding assays were conducted by

incubating 106 cells in 1 ml of PBS containing 50 pM[125I]HBP and 1% bovine serum albumin (Fraction V,

/32-ADRENERGIC RECEPTOR TRANSFECTANTS 449

Coomassie BlueStaining Autoradiogram

D+°lSr

Coomassie BlueStaining Autoradiogram

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FIG. 1. /32-AR filter screening assays. Replica copies of transfectant populations were screened with [125I]HBP and ana-

lyzed by autoradiography. Filters were subsequently fixed with methanol/acetic acid/water (45:45:10) and stained with1% Coomassie blue (in fixative) for 10 min to see the colonies. A. RPMI 1846 cells transfected withpRSVneo. B. Mouse L cells transfected with pRSVneo. C. Mouse L cells cotransfected with pUC /3-AR/pRSVneo(molar ratio 1:10), two replica filters from the same transfection dish. D. Mouse L cells cotransfected with pUC /3-AR/pRSVneo (molar ratio 5:1). E. Mouse L cells cotransfected with pZEM /3-AR/pRSVneo, two filters from differenttransfection dishes. The arrows point to colonies isolated from the master transfection dishes.

Sigma) for 15 min at room temperature. Displacement of[125I]HBP from receptor sites was conducted by coincuba-tion of cells with 0.6 nM propranolol (Sigma). All assayswere conducted in quadruplicate.

Cell membranes were prepared by hypotonie lysis in 1mM Tris-HCl pH 7.4 as described by Dixon et al. (1987).The membranes were pelleted by centrifugation at 40,000x g for 15 min and resuspended in TME (75 mMTris-HClpH 7.4, 12.5 mM MgCl2, 1.5 mM EDTA) at a concentra-tion of 1 mg/ml. Saturation binding of [,25I]HBP to mem-branes from cell lines was measured using 10-400 pM ra-

diolabeled ligand in an assay volume of 250 ¡A of TME

containing 25 ¡ig of protein. Nonspecific binding of [125I]-HBP in the presence of 10 fiM (-)propranolol was <5<%.Adrenergic agonist [(-)isoproterenol hydrochloride, (-)epi-nephrine bitartrate, and (-)norepinephrine; Sigma)] bind-ing was measured in competition with 30 pM [125I]HBP ata final /32-AR concentration of 5 pM. All membrane assayswere conducted with triplicate samples.

Preparation and restriction endonuclease digestion ofgenomic DNA, agarose gel electrophoresis, Southerntransfer to nylon membranes (Nytran, Schleicher &Schuell), and hybridization to labeled probes have all beendescribed previously (Ausubel et al., 1987). The 1.3-kb

450 MACHIDA ET AL.

Table 1. Frequency of Receptor Expression WithinTransfectant Populations3

pUC ß-AR/pRSVneo Total Autorad Percentmolar ratio colonies signals positives

Experiment 1

Experiment 2

125

10125

10

2420203047356255

234656

1515

Experiment 1

Experiment 2

125

10125

10

2618193047653250

1210122522392447

815202011172327

pZEM ß-AR/pRSVneo Total Autorad Percentmolar ratio colonies signals positives

4655638347607594

A /3-AR plasmid and pRSVneo were mixed at various molar ratios to form a calcium phos-phate coprecipitate for transfections. Autoradiographic screenings were conducted as de-scribed in Materials and Methods. Coomassie blue staining of colonies is described in the leg-end to Fig. 1.

Hind III fragment containing most of the hamster 02-ARcoding sequence (Dixon et al., 1986) was labeled with[a-32P]dCTP by nick-translation to a specific activity of 5x 108 cpm/fig. RNA was prepared by the guanididium iso-thiocyanate method (Ausubel et al., 1987). Methods forelectrophoresis of RNA in glyoxal gels and transfer tonylon membranes have been previously described (Ausubeletal, 1987).

RESULTSDetection of ß2-AR gene transfectants byreceptor screening assay

/32-AR expression RPMI 1846 and /32-AR-deficient Lcells were transfected with pRSVneo to produce neomycin-resistant colonies that could serve as controls in the recep-tor screening assay. Replica filters containing these cellpopulations were subjected to binding with [125I]iodohy-droxybenzylpindolol (HBP) and analyzed by autoradiogra-phy. Figure 1 shows the autoradiograms of the receptorscreening assays for both the RPMI (Fig. 1A) and L (Fig.IB) transfectant populations. Virtually all the RPMI colo-

nies emit autoradiographic signals; no signals were ob-served for the L-cell transfectants.

When replica filters of L cells cotransfected with pUC ß-AR (/32-AR gene in pUC 19) and pRSVneo were subjectedto the [125I]HBP binding assay, autoradiographic signalswere identified on duplicate filters (Fig. 1C, see arrows).The majority of the neomycin-resistant colonies in this co-transfection (molar ratio of pUC /3-AR/pRSVneo is 1:10)apparently did not take up and/or express pUC /3-AR. Byincreasing the molar ratio of pUC /3-AR/pRSVneo to 5:1,however, the proportion of autoradiographic signals tototal colonies increased (Fig. ID; also see Table 1). Theseobservations suggest that the autoradiographic signalsidentify /32-AR-expressing colonies and that the proportionof receptor-expressing transfectants is moderated by thedose of the transfecting plamid.

Receptor binding assays were also conducted with filterscontaining colonies cotransfected with pZEM /3-AR(/32-AR sequence in eukaryotic expression vector pZEM 3)and pRSVneo (Fig. IE). Colonies identified as positive inthe binding assays examining both pUC /3-AR and pZEM/3-AR transfectant populations were isolated (Fig. 1D,E,see arrows) to verify expression of the transfected receptorgene and to examine more closely the differences in /32-ARgene expression in individual transfectants (see below).

/32-ADRENERGIC RECEPTOR TRANSFECTANTS 451

ß2-AR gene expression in individual pUC ß-ARand pZEM ß-AR transfectants

[125I]HBP binding assays on whole-cell preprations oftransfectant and control (RPMI and L) cells were con-ducted in the presence and absence of the /3-AR antagonistpropranolol to confirm /32-adrenergic expression in thetransfected cell lines. Figure 2 shows perfect correlationbetween receptor expression predicted from the filterscreening assay and the whole-cell binding data of individ-ual transfected clones.

To ascertain the number of receptors expressed by indi-vidual transfectants, membranes were prepared and incu-bated with [125I]HBP. Receptor densities were 300 and1,500 fmoles/mg of protein for the transfected cell linesexpressing highest pUC /3-AR (clone 2) and pZEM ß-AR(clone 5a-2; with zinc stimulation), respectively (Fig. 3A).This is in contrast to a receptor density of approximately50 fmoles/mg of protein observed for the hamster RPMI1846 cell line. Scatchard analysis of [125I]HBP binding topUC /3-AR clone 2 membranes yielded a straight line (notshown), consistent with a single class of saturable bindingsites; the antagonist Kd of 30 pM was consistent with previ-ously published reports (Dixon et al, 1986). Furthermore,the affinity of the transfected receptors (from pUC /3-AR

clone 2) for various adrenergic agonists, as evaluated bycompetition with [125I]HBP (Fig. 3B), is consistent with the/32 subtype (isoproterenol > epinephrine > norepineph-rine).

To analyze more completely the differences in receptorexpression among individual transfectants and to deter-mine the minimum copy number of receptor genes re-

quired for detection in the filter assay Northern and ge-nomic Southern blot analyses were performed. GenomicDNAs from several transfectant cell lines and from ham-ster cell lines and tissues that express the /32-AR were di-gested with Hind III and analyzed by Southern blottingusing the 32P-labeled 1.3-kb Hind III fragment as probe(Fig. 4A). As expected, a 1.3-kb band appears in Hind Ill-digested genomic DNA from the RPMI and DD^MFjhamster cell lines and from hamster lung tissue, but notfrom untransfected mouse L cells or L cells transfectedwith pRSVneo. The 1.3-kb band also appears in pUC/3-AR transfectant clones 1 and 2, consistent with the filter(Fig. ID) and whole-cell binding assays (Fig. 2). The inten-sity of the 1.3-kb Hind III band in hamster lung and RPMIcells appears similar, indicating that only a single /32-ARgene copy is required for detection of colonies in the filterassay. In addition, the 1.3-kb Hind III band from transfec-tant clones 1 and 2 appears more intense than the corre-

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FIG. 2. Whole-cell receptor binding assays. [125I]HBP binding assays on whole-cell preparations of transfectant andcontrol (RPMI 1846 and L) cell lines were conducted in the presence (shaded columns) and absence (clear columns) ofpropranolol. Height of columns corresponds to the mean of four replicate samples; the error bar represents 1 SD. /3-ARclone 2-2 was obtained from a transfection not identified in Fig. 1.

452 MACHIDA ET AL.

5a-2 +2n5a-2BAR 2RPMI5a-45:;

-

hZnL, BAR 3, 7b-5

100 200 300[125I-HBP] (plcomolar)

B

0 (-) NorepinephrintO (-) Epinephrine| (-) Isoproterenol

-log [adrenergic agent]

FIG. 3. A. Saturation binding of [125I]HBP to mem-branes from transfectant and control cell lines. Nonspe-cific binding of [125I]HBP in the presence of 10 fiM pro-panolol was <5%. Membrane sources are identified di-rectly in the figure. B. Adrenergic agonist displacementof [125I]HBP from transfectant cell membranes. Mem-branes were from pUC /3-AR clone 2; adrenergic agonist(isoproterenol, epinephrine, and norepinephrine; see figurefor symbols) binding was measured in competition with 30pM [125I]HBP at a final /32-AR concentration of 5 pM.

sponding bnds observed using hamster cell line or lungDNAs, indicating that the transfectants have acquiredmultiple copies of pUC /3-AR. A 2.2-kb band is also ob-served in clones 1, 2, and 3, and in untransfected L cells;presumably this fragment contains mouse sequencesclosely related to the hamster /32-AR gene.

A 1.9-kb band that hybridizes with the 1.3-kb Hind IIIfragment is observed in genomic DNA prepared fromclones (5a-2 and 5a-4) obtained from the pZEM /3-ARtransfection (Fig. 4A). The size of this fragment is consis-tent with the 1,262-bp Nae l-Hind III sequence containedin the pZEM /3-AR insert plus an additional 600 bp of 5'-flanking vector sequence. These DNAs, as well as DNAfrom the /32-AR-nonexpressing clone 7b-5, also contain the2.2-kb /32-AR-related mouse sequence.

Northern blots of RNAs obtained from transfectant andcontrol cell lines and tissue (Fig. 4B) were probed with theHind III fragment. As expected, a 2-kb band appears inRNA preparations from RPMI 1846, DDT,MF2, hamsterlung, and pUC /3-AR clones 1 and 2. The size of this RNA

seerx q _ià

kb kb

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5a-2 5a-4

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FIG. 4. A. Southern blot of genomic DNAs obtainedfrom transfected and control cell lines and tissue. Twentymicrograms of genomic DNA digested with Hind III was

subjected to Southern blot analyses using the 1.3-kb HindIII /3-AR probe. Hybridization was performed in 40%formamide, 5x SSC, at 37°C; the filter was washed in0.2 x SSC at 65CC. The autoradiogram was developedafter a 4-day exposure. Positions of DNA markers andsources of genomic DNAs are indicated. B. Northernblot of RNAs obtained from transfectant and control celllines and tissue. Forty micrograms of total RNA was sub-jected to Northern blot analyses using the 1.3-kb Hind III/3-AR probe. Hybridization was performed in 50% form-amide, 5x SSC, at 42°C; the filter was washed in 0.2 xSSC at 65 °C. The autoradiogram was developed after a 3-day exposure. Positions of RNA markers and sources ofRNAs are indicated.

is consistent with the mRNA size deduced from the ham-ster /32-AR nucleotide sequence (Dixon et al, 1986). Thereduced size of the /3-AR RNAs (1.8 kb) observed in clone5a-2 and 5a-4 cell lines is consistent with the length of themRNA generated from the pZEM /3-AR recombinantDNA. The relative intensities of the /3-AR RNAs observed

&-ADRENERGIC RECEPTOR TRANSFECTANTS 453

in all preparations appear to correlate well with gene copynumber (Fig. 4A) and receptor density (Fig. 3A). As ex-

pected, no bands were observed in RNA preparations frompRSVneo-transfected L cells or in /32-AR-deficient clones 3or 7b-5.

Utility of the assay for measuring transfectionefficiencies and evaluating promoter strength

The in situ receptor screening assay can rapidly measuretransfection efficiencies of receptor-encoding plasmids andcan potentially be used in the evaluation of promoterstrengths. Table 1 shows the percentage of autoradio-graphie signals to total neomycin-resistant colonies for insitu receptor assays of transfectant populations preparedby varying the molar ratios of pUC /3-AR or pZEM /3-ARto pRSVneo. The percentage of receptor-expressing trans-fectants in pUC /3-AR transfections increases linearly withelevating pUC /3-AR/pRSVneo ratios, but reaches only20-27%, even when calcium phosphate precipitates withhigh ratios (10:1) are used. pZEM /3-AR transfections yielda much higher percentage (46%) of receptor-expressingtransfectants even when moderate pZEM /3-AR/pRSVneoratios (1:1) are used and approaches unity at high ratios(10:1). These observations underscore the potential utilityof the filter assay in the measurement of cotransfection ef-ficiencies (e.g., within a single pUC /3-AR/pRSVneo series)and its application in optimizing transfection ratios. Thisassay can also be used to compare promoter strengths(e.g., pUC /3-AR vs. pZEM /3-AR).

DISCUSSION

These data demonstrate the utility of the replica transferand receptor screening assay as as tool for identifyingtransfectants expressing neurotransmitter receptor genes.We show that a single colony expressing the /32-AR can

readily be identified from hundreds of colonies on a singletransfection dish and can be identified in duplicate on rep-licate filters. The predicted receptor expression of severalisolated colonies correlated perfectly with whole-cell bind-ing data. Furthermore, the assay is more rapid than con-ventional binding assays using clonal cell populations andis sensitive, capable of detecting colonies containing only a

single /3-AR gene copy and expressing as few as 4,000 /3-re-ceptors per cell.

The filter screening assay can be used either to analyzethe expression of a neurotransmitter receptor gene in largetransfectant populations (e.g., cotransfection efficiencies)or directly to identity and isolate specific receptor-express-ing transfectants. We have used this assay to achieve bothobjectives. This assay, when used in conjunction with eu-

karyotic expression libraries, potentially may be used as atool in the molecular cloning of neurotransmitter receptorgenes. Assuming that a typical mammalian genome con-

sists of approximately 3 x 109 bp of DNA and that eachtransfectant incorporates 500-1,000 kb of DNA (Ruddle etal., 1984), a fully represented eukaryotic expression libraryconsists of a theoretical minimum of 6,000 transfectantcolonies. Additional factors such as chromosomal posi-tioning of the transfected DNA may determine the actualminimum number of transfectant colonies in a fully repre-sented genomic DNA expression library. Alternatively,cDNAs under the control of a strong promoter could betransfected into eukaryotic cells to produce expression li-braries; however, this would necessitate screening a muchlarger number of transfectant colonies. Assuming a maxi-mum transfectant density of 500 colonies per dish, a li-brary screening would consist of a minimum of 12 sets offilters. While we have not duplicated the exact transfectionconditions required for a full library screening, we haveperformed transfection experiments that would simulateexpected results. Our experiments show that a single posi-tive signal can be identified in duplicate (Fig. 1C) from sev-eral hundred transfectant colonies on a single transfectiondish. In addition to its potential utilization as a tool for themolecular cloning of neurotransmitter receptor genes, thereplica transfer method has been used to identify somaticcell mutants defective in the expression of low-density lipo-protein receptor (Esko, 1986) and the nicotinic acetylcho-line receptor (Black and Hall, 1985).

The variation in /32-AR expression in individual transfec-tants appears to correlate with gene dosage. The hamster/32-AR gene is a single-copy gene (Kobilka et al., 1987c).Our hybridization probe, which encompasses most of thehamster /32-AR coding sequence, detects a faint band in ge-nomic blots of hamster lung DNA (Fig. 4A). The intensityof the 1.3-kb band obtained from hamster lung DNA issimilar to those obtained from hamster melanoma cell lineDNAs, indicating that the RPMI and DDT,MF2 hamstercell lines also contain only a single copy of the /32-AR gene.The 1.3-kb band observed in genomic DNAs obtainedfrom pUC /3-AR transfectant clones 1 and 2 are ast leastfive times as intense as those observed for the hamsterDNAs. This indicates that either multiple-copy entry of thetransfecting plasmid occurred into the cell or that geneamplification occurred as a pretext or consequence to plas-mid integration (Ruddle et al, 1984). pUC /3-AR clone 2expresses approximately 25,000 /32-ARs per cell (comparedwith RPMI, which expresses 4,000 /32-ARs per cell), consis-tent with the increased /32 receptor copy number.

The replica transfer and receptor screening assay canalso be used to measure transfection efficiencies and toevaluate promoter strengths. Receptor expression amongindividual transfectants within a population appears to ap-proach unity at high pZEM /3-AR/pRSVneo ratios, butplateaus at equivalent ratios in the pUC /3-AR transfec-tants (Table 1). This information is useful in the optimiza-tion of ratios of cotransfecting plasmids and potentiallycan be applied in the rank ordering of promoter strengths.The optimization of ratios and the choice of promoters are

important parameters required for the development of eu-

karyotic expression libraries.

454 MACHIDA ET AL.

ACKNOWLEDGMENTS

We thank Drs. Hubert van Toi, John Salon, and PaulAlbert for their support and helpful discussions through-out this study. This work was supported by a grant fromthe National Institute of Diabetes, Digestive, and KidneyDiseases (DK 37231).

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Address reprint requests to:Dr. Olivier Civelli

Vollum Institute for Advanced Biomédical ResearchOregon Health Sciences University3181 SW Sam Jackson Park Road

Portland, OR 97201

Received for publication November 29, 1988, and in revised formMarch 24, 1989.