Proc. Natl. Acad. Sci. USAVol. 81, pp. 7161-7165, November 1984Genetics

Enhancer-dependent expression of human Kc immunoglobulin genesintroduced into mouse pre-B lymphocytes by electroporation

(DNA-mediated transfection/tissue-specific gene expression/B-lymphocyte development/gpt selection)

HUNTINGTON POTTER, LAWRENCE WEIR, AND PHILIP LEDERDepartment of Genetics, Harvard Medical School, Boston, MA 02115

Contributed by Philip Leder, July 16, 1984

ABSTRACT We have developed a general method for in-troducing cloned genes into mammalian cells that affords sub-stantial benefits over current technology. It is simple, rapid,and applicable to many (perhaps all) cell types, including thosethat are refractory to traditional transfection procedures. Themethod involves exposure of a suspension of cells and clonedDNA to a high-voltage electric discharge. In a model applica-tion of this transfection procedure, we have studied the expres-sion of cloned human and mouse Ig K genes stably introducedinto mouse pre-B cells and fibroblasts. We find that there is aB-cell-specific enhancer-activator region in the J-C intron ofthe human Kc gene that is necessary for efficient transcriptionof the cloned gene in mouse pre-B lymphocytes. This suggeststhat both the DNA element and the proteins required for itsregulatory activity have been highly conserved in evolutionand that these elements operate at the pre-B-cell stage of im-munocyte development, a stage that precedes productive Kgene rearrangement.

Studies on the control of gene expression in eukaryotes relyheavily on the ability to induce the integration and stableexpression of cloned genes in mammalian cells. The tradi-tional method of gene transfer, by uptake of calcium phos-phate/DNA co-precipitates (1), works well with fibroblastsbut has proved difficult to apply to other differentiated celltypes such as lymphocytes (but see refs. 2 and 3). Therefore,we have explored a different approach to gene transfer-electroporation (4, 5)-that works for fibroblasts and haveshown that, unlike the calcium phosphate procedure, this ap-proach can be modified to allow the efficient introduction ofDNA into a broad spectrum of cell types.The electroporation method we have developed is the ba-

sis for the experiments described in this paper in which thetissue-specific expression of Ig K genes introduced into Blymphocytes was studied, using fibroblasts as a control. Wehave found that there is a region in the J-C intervening se-quence of the human K gene that is necessary for efficienttranscription in B lymphocytes and appears analogous to theenhancer found in the mouse K gene (6, 7). Further, unlikeviral enhancer sequences, the stimulation of transcription bythe human K sequence is tissue-specific in that it occurs in Blymphocytes but not fibroblasts. Our results also indicatethat both the K enhancer DNA element and the regulatoryproteins that presumably interact with this site have beenhighly conserved in evolution, since the complete human Kgene can be properly transcribed in mouse lymphocytes.Furthermore, the specific factor(s) necessary for transcrib-ing an Ig gene is apparently already present in pre-B cellsthat are not expressing their own K gene.

EXPERIMENTAL METHODSTransfection by Electroporation. DNA was transfected

into various mouse and human cells by applying a high-volt-

age pulse to a suspension of the cells and DNA as follows.Actively growing cells at about 106/ml of medium were cen-trifuged, suspended in ice-cold phosphate-buffered saline(without added MgCl2 or CaCl2), and centrifuged again. Thepellet was resuspended in cold phosphate-buffered saline ata concentration of 1-2 x 107 cells per ml. Plasmid vectorDNA (usually linearized by restriction enzyme digestion, seebelow) was added to the cell suspension to 20 ,ug/ml (al-though concentrations as low as 1 Izg/ml are effective). TheDNA and cells were allowed to sit for -5 min at 00C in one ofthe chambers shown in Fig. 1 and then an electric pulse wasdelivered.The electric pulse was usually applied to the electrodes of

the electroporation chamber (Fig. 1) by an ISCO model 494electrophoresis dc power supply. Except where indicated inTable 1, the voltage was set at 2.0 kV, the current was set ata maximum of 0.9 mA, and the adjustable current and watt-age dials were set at bare minimum. Four kilovolts was ob-tained by arranging two power supplies in series. The outputleads of the power supply(s) were either touched to thechamber electrodes by hand or through a spark-gap typeswitch (not shown). When the contact was made, the cellswere subjected to a voltage pulse over a distance of 0.5 cm.The cells and DNA were then allowed to sit for -10 min at0C before being added to 10 ml of growth medium. Cellswere grown for about 48 hr before transformants were se-lected in medium supplemented with the appropriate drug towhich the plasmid would confer resistance. Selection medi-um contained either G418 at 800 ,ug/ml (from GIBCO; trueG418 concentration, -370 ,ug/ml) or xanthine at 250 ,Ag/ml,

A

J

IIL

B

FIG. 1. Two designs for shocking chambers for delivering a high-voltage pulse to a suspension of cells and DNA. Each consists of aflat-sided, open-topped, plastic cuvette (from Sarstedt, Princeton,NJ) having two metal electrodes placed against the opposite walls(0.5 cm apart). A high-voltage pulse is applied to the electrodes andthus to the cell-DNA suspension. One version of the apparatus in-corporates heavy aluminum foil electrodes (-1 mil thick) bondeddirectly to the plastic walls of the cuvette (A). The chamber may besterilized with EtOH and reused several times. A second version ofthe shocking chamber uses a fixed pair of stainless steel or platinumelectrodes (-10 mil thick) that can be lowered into a sterile dispos-able cuvette (B).

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Table 1. Transfection frequency by electroporation

Power supplysettings, Elec- Temp., Transfectants/

Cells kV/mA trode 0C 105 live cells

Linear vs. supercoiled DNA1. 1881 (In) 1.2/300 Al 20 1.52. 1881 (sc) 1.2/300 Al 20 0.03

Temperature and voltage effects3. M12 1.2/300 Al 20 2.54. M12 1.2/300 Al 0 15.05. M12/col 1.2/300 Al 20 30.06. M12 4.0/0.9 Al 20 20.07. M12 4.0/capacitor Al 20 1.08. M12 1.2/100 Ss 20 0.39. M12 1.2/100 SS 0 5.0

10. M12 1.2/300 SS 0 10.011. M12 2.0/0.9 Ss 0 20.013. M12 4.0/capacitor Ss 0 1.5

Comparison of cell lines and speciesMouse

14. L 4.0/0.9 Al 0 15.015. 1881 4.0/0.9 Al 0 3.016. M12 4.0/0.9 Al 0 25.017. CTLL 4.0/0.9 Al 0 20.0

Human18. LAZ509 4.0/0.9 Al 0 6.019. Ly65 4.0/0.9 Al 0 1.020. BL18 4.0/0.9 Al 0 10.0

The efficiency of gene transfer by electroporation varies with dif-ferent conditions and recipient cells. The power supply settings referto the position of the central selector dial on the ISCO 494 electro-phoresis power supply. In each case, the variable wattage and cur-rent controls were set at barely above zero and the variable voltagecontrol was set at maximum. Mouse L cells are fibroblasts. M12 is aspontaneous mouse B-cell lymphoma (12), 1881 is an Abelson virus-transformed mouse pre-B cell lymphoma (ref. 13 and M. Kuehl, per-sonal communication), CTLL is a mouse cytotoxic T-lymphocyteline (14), LAZ509 is an Epstein-Barr virus-transformed human lym-phoblastoid line, and BL65 and BL18 are human Burkitt lymphomacell lines (15). The vector used in these transfections (pSV7-neogpt*) was constructed to contain both the neo and the Ecogpt*genes described in the legend to Fig. 2 and Experimental Methods.In all experiments except that reported on line 2, the vector DNAwas linearized with Pvu I before introduction into the cells. The ef-fect of transfecting the cells during mitosis, when their nuclear mem-brane is absent, was tested by incubating the cells with colcemide(col) at 0.1 ,Ag/ml for 24 hr prior to transfection (line 5). For theexperiments reported on lines 8 and 14, 20 capacitors (total capaci-tance, 50 nF) were arranged in parallel, charged to 4.0 kV and dis-charged 10 times across the electrodes of the electroporation cham-ber as described. The recovery of live cells from the procedure wasassayed by trypan blue exclusion and ranged from 60 to 90% exceptfor lines 6 and 8, where it was 25%. SS, stainless steel.

hypoxanthine at 15 ,ug/ml, mycophenolic acid at 1 jig/ml(from Lilly).

Variations on the settings for the power supplies with thetwo types of chambers (Fig. 1) have also been used success-fully as indicated in Table 1. Alternatively, a parallel seriesof capacitors (Sprague catalog no. 75GA-D25) with a total of50 nF was charged to 4.0 kV and used to supply high-voltagepulses to the cells but with less efficient transformation (seeTable 1). When the power supply was used directly, the volt-age pulse represented a capacitance of -23 ,f. For the ex-periments described in Figs. 2-4, the transfections were car-ried out at 00C in aluminum foil electrode shocking chambers(Fig. 1A) with two power supplies in series, each set at 2.0kV, 0.9 mA, or in the stainless steel electrode chambers (Fig.

1B) with one power supply at the same setting. Colcemidwas not used in either case.

Transfecting DNA. Plasmid DNA to be transfected was lin-earized with Pvu I for the experiments described in Table 1and with Sal I for the experiments described in Figs. 2-4.Selection was made possible by the presence in the vectorsof either the Eco gpt* gene or the neo gene (8) (see the legendto Figs. 2 and 3A).

Analysis of Cellular DNA and RNA. Clones of transfectedcells were harvested, solubilized in guanidine isothiocya-nate, and layered on a CsCl cushion (9). After centrifugation,RNA and DNA were isolated from the pellet and the CsClcushion, respectively. DNA (10 ,ug) was digested with Hin-dIII and electrophoresed in native (Tris acetate) agarosegels. RNA (15 ug) was electrophoresed in formaldehyde/agarose gels (10). After transfer to nitrocellulose, the DNAand RNA were hybridized with either a K-specific or a gpt-specific radioactive probe and analyzed by autoradiography(11) (see Fig. 3A for a diagrammatic representation and adescription of the EcoRI CK probe and the BamHI Ecogpt*probe).

RESULTSGene Transfer by Electroporation. Some years ago, Zim-

merman and his colleagues discovered that high-voltageelectric discharges induce cells to fuse via their plasma mem-branes, apparently by creating holes or pores in the cellmembrane (for review, see ref. 4). Neumann et al. (5) thenfound that mouse fibroblasts (L cells) take up and expressexogenous DNA when subjected to electric shock. Howev-er, because L cells are easily made to take up DNA by tradi-tional methods, it was not clear that the procedure could beapplied to any other type of cell. In this paper, we describeour extension and modification of electroporation to the in-troduction of exogenous DNA into a number of cell typesincluding mouse and human B lymphocytes, mouse T lym-phocytes, fibroblast lines, and others. Briefly, a suspensionof cells and plasmid DNA is placed in one of the chambersshown in Fig. 1 and then subjected to a high-voltage shock(2.0-4.0 kV). Following the shock and a 2-day growth peri-od, the cells are selected in medium containing either G418or mycophenolic acid and xanthine. Clones can be scoredand recovered 2-3 weeks later.The results of transfecting DNA into several cell types un-

der different conditions are shown in Table 1. Two methodshave been used to deliver a voltage pulse to the cells andDNA in the shocking chamber. The first was to charge aseries of capacitors (connected in parallel to yield 50 nF)with 4.0 kV and then discharge the stored voltage across theelectrodes of the cuvette. This method was successful (lines7 and 13) but did not give as high a transfection frequency asdischarging the electromotive force stored in the power sup-ply directly across the cuvette electrodes. As shown in Table1, the settings of the power supply(s) were varied to establishthose that yielded the highest level of transfection.Table 1 also indicates several important aspects of the

transfection procedure. (i) Linearized plasmid DNA was atleast 20 times more effective in transfecting cells than wascircular DNA (lines 1 and 2). This result probably reflectsthe increased ease with which linear DNA is integrated intothe cellular genome. (ii) Carrying out the electroporation at0°C was 6- to 16-fold more effective than at 20°C (comparelines 3 and 4 and lines 8 and 9). This result may be partly dueto the increased cell survival, but it is also likely due to theslower closing of the membrane pores at 0°C. (iii) Treatmentof the cells with a sublethal concentration of colcemide for18 hr prior to electroporation increased transfection 3- to 10-fold (compare lines 3 and 5). This experiment was based onthe assumption that, in cells temporarily halted in meta-

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phase, the nuclear membrane would be either absent or morepermeable and thus would pose less of a barrier to exoge-nous DNA. Table 1 also shows the results of transfectingseveral mouse and human cell lines under the same (optimal)conditions.Cloned Mouse K Ig Genes Are Expressed in Mouse Lympho-

cytes but Not in Mouse Fibroblasts. To address the questionof how lymphocytes of the B lineage are uniquely able toexpress Ig genes, we constructed a plasmid containing acomplete MOPC 41 mouse K Ig light chain gene along withthe Ecogpt* selectable marker gene, as described (Fig. 2,legend), and introduced the vector by electroporation into1881 pre-B lymphocytes and into mouse L cells. The 1881cell line is derived from BALB/c bone marrow by transfor-mation with Abelson murine leukemia virus (13). 1881 cellsshow ,u heavy chain production but no K protein or mRNA(13). Southern analysis reveals at least one germ line K allele(P. Thistlethwaite and M. Kuehl, personal communication;see also ref. 30). RNA and DNA from clones of permanentlytransfected cells were analyzed for K-specific and Ecogpt*-specific sequences by blot hybridization (Fig. 2). As shown

MOPC 41Cells: Pre-B 1881 L Fibroblasts cytoma 1881 L

+MOPC41K 0 +MOPC4K 0 +MOPC 41 Kri 2 3wrT 5 6 7 8 9 IO1 7 1I7 1:IW-

Probe: Kappa GPT

FIG. 2. Transfection of a complete mouse K gene into mouse pre-B cells and fibroblasts. A clone (in pBR327) of the complete mouse K

Ig light chain gene from the MOPC41 plasmacytoma (16) was linkedto the Ecogpt* gene. [The Ecogpt* gene was modified by Mitch Reffand Martin Rosenberg (personal communication) by deleting the 5'untranslated bacterial sequences from the original eukaryote-expressable Ecogpt gene of Mulligan and Berg (8).] The mouse K-

Ecogpt* plasmid was linearized with Sal I and transfected intomouse 1881 pre-B cells and L cells. Individual clones of cells wereselected with mycophenolic acid/xanthine/hypoxanthine. RNA wasisolated and analyzed by hybridization with radioactive DNAprobes derived from either the K constant region or the Ecogpt*gene. Lanes: 1-4, RNA from three mycophenolic acid-resistantclones of 1881 cells (plus a control lane with untransfected 1881RNA) hybridized with the mouse K probe; 5-11, RNA from six re-sistant clones of L cells (plus a positive MOPC41 control) hybridizedwith the mouse K probe; 12 and 13, RNA from a representative re-sistant 1881 clone and an L-cell clone hybridized with the Ecogpt*probe. The positions of ribosomal RNA (2000 and 5100 bases) areindicated.

(lanes 1-3), the K Ig gene introduced into mouse B lympho-cytes was transcribed into a mRNA of the same size as theauthentic K transcript from the MOPC41 myeloma cells fromwhich this K gene was derived (lane 11). Similarly, theEcogpt* gene was expressed in these cells (lane 12), as ex-pected for cells resistant to mycophenolic acid. In contrast,when transfected mouse L cells were similarly analyzed, lit-tle or no K-specific mRNA could be detected (lanes 5-10)although the Ecogpt* transcript was expressed in abundance(lane 13).

In sum, although the Ecogpt* gene was transcribed in bothL cells and pre-B cells, Ig gene transcription occurred onlyin the lymphoid cells. This indicates that the cell-type speci-ficity of Ig gene expression is solely dependent on the DNAsequence in the area of the gene and not, for instance, on itschromosome location. Furthermore, it is evident that, al-though the 1881 pre-B cells have not yet undergone a DNArecombination leading to a completed light chain, they arefar enough along the path of B-cell development to be able totranscribe an exogenously added K gene.Cloned Human K Ig Genes Are Expressed in Mouse Lym-

phocytes but Not in Mouse Fibroblasts. We next constructeda plasmid DNA molecule containing a complete rearrangedhuman K Ig gene linked to the Ecogpt* selectable marker asdescribed in Fig. 3A. This plasmid was then transfected into1881 mouse pre-B cells (Fig. 3B) in which it is seen to betranscribed to yield a mRNA of the same size as authentichuman K messenger from BL31 Burkitt lymphoma cells (Fig.3C, right-hand lane). On the other hand, when the same nor-mal human K gene was introduced into mouse L cells, therewas very little K-specific transcription of any kind (Fig. 3D)and the transcripts that were produced were ':300 baseslonger than the authentic human K message. For compari-son, the right-hand lane of Fig. 3E was loaded with 15 ,ug ofRNA from one of the 1881 clones shown in Fig. 3B that re-ceived the normal human K gene.A Region of theJ-C Intron of the K Ig Gene Is Necessary for

Effective Transcription. Because both mouse and human Kgenes were actively transcribed in mouse B cells, but notmouse L cells, it seemed reasonable to ask which region ofthe DNA was responsible for this tissue-specific transcrip-tion. A likely candidate for such a sequence is a region ofDNA in the J-C intron that serves no protein coding func-tion, yet is highly conserved between mouse and human andthus might play a regulatory role in both species (17, 18).Picard and Schaffner (7) have recently found this region tocontain a transcriptional enhancer in the mouse K gene. Witha similar aim, we deleted the relevant portion of the intronfrom the human K gene vector (as described in the legend toFig. 3) and then introduced the altered gene into mouse 1881pre-B cells and L cells as before.The results of subjecting the RNA from transfected clones

of mouse pre-B cells and fibroblasts to hybridization withthe human K constant region probe are shown in Fig. 3.There was a marked decrease in the level of transcription ofK Ig mRNA in the 1881 clones that received the deleted gene(Fig. 3C) compared with those that received the normal gene(Fig. 3B). No further reduction in the low level of aberrant Ktranscripts in L cells resulted from deleting the enhancer se-quence. These results have also been obtained in transient-expression assays using our normal and deleted human Kgenes (unpublished work).To quantitate the level of K-specific mRNA in the individ-

ual clones, the autoradiograms in Fig. 3 and others notshown were analyzed by densitometry. The amount of KmRNA relative to the number of integrated K genes (mea-sured by Southern blot hybridization of HindIII-restrictedDNA) was calculated and is displayed in Fig. 4. Ten individ-ual 1881 clones that received the normal human K gene arecompared with 11 clones that received the "enhancer"-de-

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A

HuKgpt*

pBr322 RI RI VK JsI--- ---- 1I

Sail ClalHuKgpt*/ASst

pBr322 RI RI VK JsI I M._.l

Sal

B

CIa

1881 (Hu K gp t *)

Homology/Enhancer

*RI ICK RI -gpt BamI, K1_ I777 -

SstI SstlSstI Salm -

CK Probe gpt Probe

CK RI --gpt* Bam,_ I _

I -ISst I Sst

;>.C-

z

z

_

-4til

Sal

Kb

C1881 (Hu K gpi /ASst) BL31

I___-

D E 1881L (Hu K gpt*) L(Hu K gpt*/ASst 1) (HuK)

r~~ -1 --I1F-7

FIG. 3. (A) Construction of the normal and deleted human K Iggene vectors. A complete human K gene was constructed by firstsubcloning (in pBR327) an EcoRI (RI) fragment containing an activerearranged VK-J1 gene, originally cloned into phage X from the hu-man B-cell leukemia cell 6410 (unpublished work). This VK-J1 re-gion was then linked to a J-CK subclone (17) at a unique Xho I sitebetween J1 and J2. The Ecogpt* gene (see legend to Fig. 2) is locat-ed on a 2-kilobase (kb) BamHI fragment that was inserted into a BglII site lying 3' to the complete human K gene. The transcription ofthe gpt gene is toward the K gene. The region of the human J-CKintron that is homologous to the mouse intron and thus might con-tain an enhancer sequence is located on a 2-kb Sst fragment (18) thatwas deleted to yield the plasmid shown on the bottom line. Theseplasmids were linearized with Sal I before transfection. (B-E) RNAfrom individual clones of mouse pre-B cells or mouse L cells trans-fected with the normal and deleted human K gene plasmids was elec-trophoresed and analyzed by hybridization with the CK probe. (Band C) Clones of mouse 1881 cells transfected with the normal anddeleted human K gene, respectively, and analyzed together (the last

i0iNormial 11iMIM1 K

i

I)elcted1 hlllumun K

FIG. 4. Relative amounts (normalized for gene dosage) of KmRNA present in clones of mouse 1881 cells transfected with eitherthe normal or deleted human K Ig gene. Results were obtained byusing the number of integrated copies of the human K gene in eachclone (assayed by Southern analysis of HindIl-digested DNA) tonormalize the amount of K RNA from that clone measured by a den-sitometer tracing.

leted human K gene. On average, K mRNA expression in pre-B cells is depressed by a factor of "15 when the K enhanceris deleted. The same conclusion is reached if one uses thelevel of Ecogpt*-specific mRNA in each clone as a standardby which to judge K mRNA expression. In contrast, a similarquantitative comparison of the data from the mouse L cellsshowed no such difference in transcription from the normalvs. the deleted human K gene. In sum, the region of DNAdeleted from the human K intron must contain DNA neces-sary for efficient transcription in B lymphocytes-a proba-ble enhancer sequence analogous to the simian virus 40 en-hancer but showing marked tissue specificity.

It is perhaps worth noting that the enhancer effect in pre-Bcells would be even more dramatic but for the presence of anoccasional clone expressing unusually large amounts of K-specific messenger from a deleted gene (see, for example,the 6th and 11th clones that received the deleted K gene inFig. 4). We might suppose that, in these clones, a transfectedhuman K gene has integrated near a mouse cellular sequencethat can serve as a substitute enhancer. The large distance(up to several thousand base pairs) over which the normal Kenhancer sequence can act lends credence to such an expla-nation. These clones may also be analogous to the naturalsubclones of 1881 cells that express u heavy chain RNAfrom an allele that lacks the heavy chain enhancer because ofdeletion (31). If the two such clones in Fig. 4 are droppedfrom our analysis, the enhancer effect ratio increases to-40. It is also possible that the background level of tran-scription from the enhancer-less K gene may have beenraised by the presence of the simian virus 40 enhancer asso-ciated with the gpt gene.

DISCUSSIONElectroporation: A General Method for Gene Transfer. The

previously available methods for permanently introducingexogenous DNA into mammalian cells have been by proto-plast fusion (19) or by calcium phosphate co-precipitation

lane of C shows the normal human K transcript from the Burkittlymphoma cell line BL31) (15). (D and E) Clones of mouse L cellstransfected with the normal and deleted human K gene, respectively,and analyzed together (the last lane ofE contains RNA from a repre-sentative 1881 clone containing the normal human K gene for com-parison). Comparison of this lane with those in B shows that D and Ehave been overexposed to reveal the low level of K-specific tran-scripts in the fibroblast cells. The horizontal lines to the left indicatethe positions of 18S (2000 bases) and 28S (5100 bases) ribosomalRNA.

A A _ _ _ _ _ _ _ _ _ _ _ 1 1_ _ w _ v v _ _ w v v _n11

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(1). Because these procedures have been, respectively, cum-bersome and difficult to apply to lymphoid cells (see, forexample, refs. 2 and 3), we sought to develop an easier andmore general method of gene transfer. Transfection by elec-troporation is reproducible, rapid, and applicable to manytypes of cells. In addition to allowing the transfection ofmouse L cells, mouse B and T lymphocytes, human Burkitttumor cells, and human lymphoblastoid cells as reportedhere, the technique has also been used successfully withmouse erythroleukemia cells, rat pituitary cells, human pro-myelocytic leukemia cells, and Escherichia coli bacteria (un-published work). We believe that electroporation offers away to introduce DNA permanently into any cell type. Inaddition, electroporation has an advantage over calciumphosphate in that the number of integrated copies of plasmidDNA transfected by electroporation is low-in the range of1-15-which makes possible detailed analysis of the DNA atthe integration site.

Tissue-Specific Expression of K Ig Genes. One ideal use ofthe above method is in the evaluation of gene or promoter (orboth) activity in living cells. In particular, the B lymphocyteand its hematologic antecedents are a family of cells withinteresting systems of gene control. One step in the pathwayof B-cell differentiation is the specialized DNA recombina-tion that serves to bring one of many variable region genesinto juxtaposition with a constant region gene. Inasmuch asthis DNA rearrangement occurs only in B lymphocytes, itseemed possible that the recombination event alone might besufficient to activate an Ig gene. However, our results andthose of others (6, 20, 21) indicate that the construction of acompleted K gene is, by itself, not sufficient to allow expres-sion in nonlymphoid cells-for instance, in fibroblasts. Sim-ilarly, it has been found that a complete Ig heavy chain genecan be expressed only in lymphoid cells (22, 23). Thus, theremust be factors exclusively present in B cells that are neededfor transcription of Ig genes. Our results also indicate thatthe factors necessary for K expression must be present inpre-B cells that have yet to undergo a correct V-J recombi-nation and are consequently not expressing their own lightchain genes (see also ref. 2).Enhancer Sequences in Ig Genes. Several lines of inquiry,

including DNA heteroduplex comparison of mouse and hu-man genes (17), DNase-hypersensitivity analysis (24, 25),and direct DNA sequencing (18) point to a regulatory role fora region of the J-C intron of the K Ig gene. Inasmuch as viralenhancer sequences are the only regulatory elements knownto exert their influence over the required distance (26, 27), itwas reasonable to ask whether the evolutionarily conservedregion of the K intron was a cellular transcriptional enhancerof the K Ig gene. Our experiments with cloned human andmouse Ig genes have established that a lymphocyte-specifictranscriptional enhancer-like sequence resides in the J-C in-tron of the human K light chain gene, as has been shown forthe mouse K gene by Queen and Baltimore (6), Queen andStafford (28), and Picard and Shaffner (7) using transient-expression assays. Similar tissue-specific enhancer se-quences have been found in the heavy chain Ig locus (22, 23,29). Our findings and those of others that K and heavy chainIg transcription in B cells is much reduced when the en-hancers are deleted, suggests that these sequences are large-ly responsible for the tissue-specific expression of the Iggenes. That is, the ability to recognize a specific enhancer isone aspect of the differentiated phenotype of a lymphoidcell. However, the fact that we find the low level of K tran-scription in fibroblasts to be aberrant (see also ref. 21), while

the transcription in B cells is correct even in the absence ofthe enhancer, implies that some of the tissue-specific recog-nition of this gene is due to DNA elements other than theenhancer-perhaps the K promoter.

We are grateful to Ms. T. Broderick for her expert assistance inthe preparation of this manuscript. This work was supported bygrants from the American Business for Cancer Research Founda-tion, E. I. DuPont de Nemours, Inc. and National Institutes ofHealth Grant GM17088. L.W. is a North Atlantic Treaty Organiza-tion Fellow. H.P. was supported by the Anna Fuller Fund and theLeopold Schepp Foundation.

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