Proc. Nati. Acad. Sci. USAVol. 84, pp. 7928-7932, November 1987Biochemistry

Accurate 5' splice-site selection in mouse K immunoglobulin lightchain premessenger RNAs is not cell-type-specific

(RNA splicing/tissue-specific gene expression/B lymphocyte)

DEAN H. KEDES AND JOAN A. STEITZHoward Hughes Medical Institute, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street,New Haven, CT 06510

Contributed by Joan A. Steitz, August 14, 1987

ABSTRACT In mature mouse B lymphocytes, immuno-globulin K light chain transcripts contain an interveningsequence separating the recombined variable (V) plus joining(J) exon from the distant constant (C) exon. After V-Jrecombination, this intervening sequence can include as manyas three unused but very similar J-region 5' splice sites. Eachofthese sites is potentially functional ifthe gene is appropriatelyrecombined. It is unclear how the splicing machinery distin-guishes among these 5' splice sites, always choosing the mostupstream site. We used synthetic transcripts of K gene se-quences containing J3 and J4 in both the germ-line and therecombined configurations to study the pattern of 5' splice-siteselection in vitro. We rind that both HeLa cell and lymphocytenuclear extracts fail to discriminate between the J3- andJ4-region 5' splice sites. In contrast, after transfection intoHeLa cells, similar K light chain transcripts are splicedcorrectly at the most upstream 5' splice site-that which is usedin K-producing cells. We conclude that accurate 5' splice-siteselection in the mouse K light chain is neither cell-type- norspecies-specific. Potential mechanisms for this controlling stepin gene expression are discussed.

Despite recent advances in understanding the mechanism ofeukaryotic pre-mRNA splicing, the rules governing splice-site selection remain obscure (for reviews see refs. 1 and 2).Specific in vivo splicing patterns often require the splicingapparatus to disregard certain splice sites while choosingothers. For example, one of a number of 5' splice sites can bejoined to a single 3' splice site or, conversely, a single 5' splicesite can be joined to one of several 3' splice sites. In someinstances, a combination of these events occurs, resulting inthe omission or replacement ofan entire exon. Thus, the finalspliced product from a single pre-mRNA can vary signifi-cantly in different cell types or stages of differentiation (3, 4).Accurate splice-site selection seems to require more than theminimum 5', 3', and branch-point consensus sequences in thepre-mRNA. This suggests that correct splicing of transcriptswith multiple exons or multiple 5' or 3' splice sites mayinvolve factors in addition to those so far characterized ascomponents of the splicing apparatus.One example of highly specific splice-site selection is found

in the splicing of the mouse immunoglobulin K light chainpre-mRNA. The K light chain locus in pre-B cells comprisesseveral hundred variable protein-coding (V) regions [eachwith a promoter and short leader (L) exon upstream] sepa-rated by a large distance (as yet undefined) from a 1.5-kilobase-pair (kbp) cluster of four short (36-38 bp), highlyhomologous (-65%) joining (J) regions (see refs. 5 and 6 fora review of immunoglobulin structure and expression). EachJ sequence is followed immediately by a 5' splice site, and thelast J (J4) is separated by -2.5 kbp from a single downstream

3' splice site adjacent to the constant (C) exon. During B-cellmaturation, DNA recombination converts this germ-lineDNA configuration to one in which a singleLV unit isjoinedto the 5' end ofone ofthe J regions. This creates a V+J exon.During pre-mRNA maturation, the short L exon is spliced tothe 5' end of the V region, but since four J regions exist, thesecond intron can include as many as three unused but almostidentical (each has an 8-out-of-9 fit to the consensus se-quence) 5' splice sites. The mature mRNA in K-producing celllines, however, is generated by exclusive use of the 5' splicesite of the J region that is recombined to the V region; thepotentially functional 5' splice sites of J regions locateddownstream are apparently ignored by the splicing apparatus(7).To begin to clarify how the splicing machinery distinguish-

es among these 5' splice sites, we have compared the in vitroand in vivo splicing patterns of K immunoglobulin genetranscripts in the germ-line and the recombined configura-tions. We find that in vitro splicing systems from either alymphoid or nonlymphoid cell type do not discriminatebetween J-region 5' splice sites in K transcripts. This is similarto the finding of Lowery and Van Ness (8) for in vitro HeLanuclear extracts. Yet in vivo, these same transcripts areprocessed at the correct 5' splice site regardless of cell type.

MATERIALS AND METHODSRecombinant DNA. X phage clones of the immunoglobulin

K light chain genes (gifts from S. Lewis and D. Baltimore)containing the variable region VK21-C as well as the J and Cregionshavebeendescribed (9). The plasmidLK440(pHPAPr-1) was made available prior to publication by P. Gunning (10).Standard cloning techniques (11) were employed to generatethe SP6 (12) and pHPAPr-1 constructs.Enzymes, Radiochemicals, and Namalwa Cells. All enzymes

and labeled nucleotides were purchased from New EnglandBiolabs, Boehringer-Mannheim, Promega Biotec, New En-gland Nuclear, and Amersham. Namalwa cells (ATCC CRL1432), a human lymphoblastoid cell line from a Burkittlymphoma, were made available by C. Weissmann.

Splicing Extracts. Nuclear extracts from Namalwa cellswere prepared according to Dignam et al. (13). HeLa nuclearextracts were prepared following a modification of the sameprotocol (14).

In Vitro Splicing. Linearized plasmid templates were tran-scribed with SP6 RNA polymerase (12) and the transcriptswere purified by gel electrophoresis as described (15). Splic-ing assays were performed as previously reported (16).

Transfection of HeLa Cells. Transfections and immunoflu-orescence assays to monitor transfection efficiency werecarried out as described (17). Transfection efficiencies typi-

Abbreviations: C, constant; J, joining; L, leader; V, variable; nt,nucleotide(s).

7928

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Proc. Natl. Acad. Sci. USA 84 (1987) 7929

cally ranged between 30% and 40%. Cytoplasmic RNA wasisolated according to Berk and Sharp (18).Primer Extension and RNase Mapping. The 25-nucleotide

(nt) oligodeoxynucleotide complementary to nucleotides9-33 in the C exon (see Fig. 3) was 5'-end-labeled using[y-32P]ATP and polynucleotide kinase (11). Hybridizationand primer extension were then performed as described (19).RNase Ti mapping was performed according to Melton et al.(12) except that RNase A was omitted.

RESULTSIndiscriminate 5' Splice-Site Selection in HeLa Cell Nuclear

Extracts. To study 5' splice-site selection in the mouse Kimmunoglobulin light chain pre-mRNA, we first constructedSP6 plasmids containing gene segments in either the germ-line or the recombined configuration. We chose to studytranscripts that contained only the J3 and J4 5' splice sites so

J3-J4-C 100 n.t.

H-IHincil HaeIli

J3 J.... ..4 * C

aM 0 2h

b0 2h

that the choice of splice-site selection would be a binary one,thereby simplifying the analysis. We also deleted a largesegment (2275 bp) from the intron (2519 bp) between the J4and C exons to produce shorter substrates, more favorablefor in vitro splicing (see Discussion).When the transcripts generated from plasmids containing

the K gene sequences in the configurations J3-J44-C orVLJ3-J4-C (Fig. 1 Upper) were incubated in a HeLa cellnuclear extract (Fig. 1 a and c), surprisingly, splicing oc-curred at both the J3 and the J4 5' splice sites. For theJ3-J4-C transcript (Fig. la), bands with the expected mo-bilities of all splicing products and intermediates were ob-served: the spliced products (164 nt, for the J3-C splice, and502 nt, for the J4-C splice), the 5' exons (99 nt, for the J3splice, and 437 nt, for the J4 splice), and "lariat" interme-diates and products, with and without the second (C) exon,respectively. For the VLJ3-J4-C transcript (Fig. 1c), thespliced products (410 nt, for the VLJ3-C splice, and 748 nt,

VL-J3-J4-C

Hinclil Haelll

V J3 - C

CM 0 2h

d0 2h M

_-- ---

858- -

-

539- w (764)(502)

363- (437))I

D

858- r

539 -

425--408-

(999)_=)

(748)

(622)

(410)

I- -858

- --539

-425--408

363 -

-363

W- -

(164)

(284)

235 -

(99)

FIG. 1. In vitro splicing of germ-line and recombined K immunoglobulin light chain SP6 transcripts. (Upper) Schematic diagrams of theJ3-J4-C (Left) and VLJ3-J4-C (Right) constructs are drawn to scale (bar indicates 100 nt). Arrow indicates the start site of SP6 transcription.Open boxed regions are protein-coding regions. Broken lines indicate the splicing of each of the 5' splice sites to the single 3' splice site. TheHincII-Hae III 2275-nt deletion in the second intron of each construct is indicated by a labeled triangle. (Lower) Autoradiographs of 4%polyacrylamide/8 M urea gels show the RNA products from 0-hr and 2-hr splicing reactions of uniformly 32P-labeled transcripts from the J3-J4-Cconstruct (a and b) or the VLJ3-J4-C construct (c and d) in HeLa nuclear extracts (a and c) or in Namalwa nuclear extracts (b and d). LanesM show DNA markers with sizes in nt as indicated. A schematic representation of the various RNA species corresponding to each band is alsoshown. Sizes in nt are indicated in parentheses; Note that in this experiment, the lariats corresponding to the J4 splice did not accumulate tothe same extent as those from the J3 splice with both transcripts; however, the difference was not so pronounced in other experiments.

235-

1 66-

-235

Biochemistry: Kedes and Steitz

7930 Biochemistry: Kedes and Steitz

for the J4-C splice), as well as the 5' exon intermediates (284nt, for the VLJ3 splice, and 622 nt, for the J4 splice), wereclearly apparent. In this particular experiment, bands repre-senting the lariats from the J4 splice migrated in the region ofthe precursor and were therefore obscured (Fig. ic). How-ever, other gels with different polyacrylamide concentrationsseparated these lariat bands from the precursor RNA (datanot shown).The identities of all splicing intermediates and products

were determined not oirly by their migration in denaturingpolyacrylamide gels relative to DNA markers (Fig. 1) but alsoby RNase Ti mapping techniques. SP6 transcripts comple-mentary to the precursor RNAs were hybridized to either theeluted RNA from gel-purified bands or the total RNA fromsplicing reactions. Subsequent digestion with RNase Tiyielded products consistent with the designations indicated inFig. 1 (data not shown, but see Fig. 3 Right). In addition, eachband identified as a lariat could be debranched upon incu-bation with a cytoplasmic S-100 fraction (20) (data notshown).Lymphocyte Nuclear Extracts Fail to Exhibit Tissug-Specific

5' Splice-Site Selection. We reasoned that HeLa cell nuclearextracts might lack lymphocyte-specific factors required forspecific splicing of immunoglobulin transcripts. Therefore,we examined the splicing patterns of the J3-J4-C andVLJ3-J4-C transcripts in extracts prepared from a variety oflymphocyte lines. Of these, extracts from Namalwa cells, ahuman pre-B-cell line, were most active in in vitro splicing,but all gave comparable 5' splice-site use.Namalwa cell extracts exhibited no preferential utilization

of the 5' splice site closest to the V region (Fig. 1 b and d).Instead, with either transcript, the first step of the splicingreaction occurred with approximately equal frequency at theJ3 and the J4 5' splice sites, whereas the second step (exonligation and lariat removal) occurred with higher efficiency atthe downstream J4 site. The J4 final product bands at 502 nt(Fig. lb) and 748 nt (Fig. Id) are clearly identified (despite thehigh backgrounds in these regions), whereas the J3 finalproduct bands at 164 nt (Fig. lb) and 410 nt (Fig. Id) are notdetected. This result is the opposite of what might beexpected if the extract contained lymphocyte-specific factorsessential for the 5' splice-site selection observed in K lightchain-producing cells. [Note that the appearance of thereleased lariat from the J3 splice of J3-J44-C diagrammed inFig. lb was not reproducibly delayed compared to that of theVLJ3-J4-C substrate (Fig. Id)]. As in the HeLa nuclearextract experiments, all RNA intermediates and productsresulting from the Namalwa splicing reactions were analyzedby RNase Ti mapping. They proved to be indistinguishablefrom the corresponding species found after incubation in theHeLa extract.

Since the overall splicing efficiency was markedly lessefficient in the Namalwa cell nuclear extracts, we attempteda number ofexperiments using various mixtures ofHeLa andNamalwa nuclear extracts. We found that when the twoextracts were combined in a ratio of 1:1 (based on proteinconcentration), splicing was very efficient (often better thanwith HeLa extract alone) but that use of both 5' splice siteswas observed (data not shown). In addition, the ratio of 5'splice-site selection did not vary either with dilution ofextract or with changes in the method of nuclear extraction.

Correct 5' Splice-Site Selection upon Transfection into HeLaCells. Since in vitro systems regardless of the cell source didnot mimic K-producing lymphocytes in using exclusively the5' splice site of the J region joined to the V region, we askedwhether our splicing substrates were capable of correctsplicing in vivo. We constructed plasmids containing thesame K light chain gene segments inserted downstream fromthe human M-actin promoter and enhancer (10). Fig. 2 shows

LK440-PU WS 1 PL PA

P UT ivs i PL PAA -a -~

V J3 J4 CP UT IVS1 PL PA

V J3 J4 CC UT 1S1 PL PA

J3 J4 C

FIG. 2. K light chain constructs used in HeLa cell transfections.LK440 (pHfiAPr-1 in ref. 9) is the parent plasmid, containing 3 kbpof human 13-actin 5' flanking sequence and promoter (P), 78 bp of 5'untranslated region (UT), 832 bp of the first intervening sequence ofthe 1-actin gene (IVS 1) fused at its 3' splice site to a short polylinker(PL) and followed by a simian virus 40 poly(A)-addition site (PA).The 3' splice site of IVS 1 is inactive in the constructs tested,presumably because no downstream exon sequences are present. Inconstructs A, B, and C, the 307-bp variable region (V), 37-bpjoiningregions (J3 and J4), and the first 51 bp of the constant exon (C) fusedto 71 bp of SP6 sequence at its 3' end are indicated. The trianglebetween J4 and C in constructs A and C represents the deletiondescribed in Fig. 1 legend. Construct B is identical to construct Aexcept that the entire J-C intron (2519 bp) is intact. In construct C,the natural germ-line sequence resides immediately upstream of theJ3 region.

schematic diagrams of these constructs A, B, and C com-pared to the parent vector LK440.HeLa cells were transfected with each of these plasmids.

Two days later, the cytoplasmic RNA was isolated andsubjected to primer extension analyses using a primer com-plementary to the C exon (Fig, 3 Left). With RNA isolatedfrom cells transfected with the parent vector (LK440), nosignal above background was detected. In contrast, analysisof the RNA from cells transfected with clone A revealed asingle band at 456 nt, consistent with the use ofthe J3 5' splicesite alone (Fig. 3 Left, lane A). The 794-nt band predicted forthe J4 splice is notably absent (position of dotted arrow).Analysis of clone B (which has a full-length J-C intron) wasidentical to that for clone A (Fig. 3 Left, compare lanes A andB), showing that the intron deletion does not alter the splicingpattern of the K transcript. No use of the J4 5' splice site couldbe detected. Finally, analysis ofconstruct C, analogous to thein vitro J3-J4-C construct, showed no signal above back-ground (Fig. 3 Left, lane C), possibly reflecting the instabilityof these transcripts in vivo (see Discussion).

Since shorter products are favored in primer-extensionanalyses, we also assayed the RNA from transfected cells forspliced products by the RNase T1 mapping technique, usingtwo uniformly labeled SP6 RNAs (probes P1 and P2) (Fig. 3Right). Whereas the control plasmid LK440 (lanes LK-1 andLK-2) produced no protected bands except for undigested P1and P2 (see Fig. 3 legend), constructs A and B both gaveproducts with lengths predicted by the primer-extensiondata. Lanes A-1 and B-1 each show a band at =214 nt,consistent with the protection of P1 by RNAs spliced at theJ3 5' splice site. Moreover, these two bands shifted to thepredicted length of 268 nt when the RNA from the A and Btransfections was hybridized instead with the longer probe,P2 (see lanes A-2 and B-2). Finally, the protected bands wereeluted from gel slices and subjected to complete RNase T1digestion. The lengths of the resultant T1 oligonucleotidescorresponded to the regions of each probe expected from theJ3 splice (data not shown). Since the probes were uniformlylabeled, the RNase T1 mapping technique should moresensitively detect the longer products resulting from J4splices. Their predicted lengths (approximately 552 nt for P1and 602 nt for P2) are indicated in Fig. 3 Right by the dottedarrows. Even when P1 and P2 were transcribed at a specific

Proc. Natl. Acad. Sci. USA 84 (1987)

Proc. Natl. Acad. Sci. USA 84 (1987) 7931

4 ** (794)

V J3 J4 C

LK A BM 1 2 1 2 1 2 V J3 J4 C

- - -P 2- XN(917)

-P 1 - -- \ (678)-4 (602)

(552)

404 -

622 -

309 -

af - .4- (268)527 -

*4 * (456)

V J3 C

-* w _

404 -

309 -

242 -238 -21 7 -201 -

t - 4- (214)

1 80 -_

160-v

147 -

Mt

FIG. 3. Analysis of K immunoglobulin light chain RNA from transfected HeLa cells. (Left) Primer extension. Autoradiograph of a 4%polyacrylamide/8 M urea gel shows the products of a primer-extension reaction on the cytoplasmic RNA from -5 x 105 cells after transfectionswith constructs LK440, A, B, and C (see Fig. 2). The locations and sizes (nt) of radiolabeled DNA markers are indicated. Diagrams to the leftof the autoradiograph depict the expected structure and length of the primer-extension products corresponding to a J4 (dotted arrow) or J3 (solidarrow) spliced transcript. V, J3, J4, and C are as described in the legend of Fig. 1. Diagrams for construct C and its predicted primer-extendedproducts are not shown (see text). (Right) RNase T1 mapping. Autoradiograph ofa4% polyacrylamide/8 M urea gel shows the uniformly labeledRNA fragments protected from RNase Ti by the K light chain spliced products in HeLa cells transfected with the constructs LK, A, and B.Probes P1 (lanes 1) and P2 (lanes 2) are 32P-labeled complementary RNA probes of lengths 678 and 917 nt, respectively. Lane M shows markers(lengths in nt at left). Horizontal lines in the diagrams of probes P1 and P2 indicate direct complementarity to a segment of the K light chainprecursor, whereas the angled lines indicate noncomplementary SP6 sequence. Dotted arrows show the predicted positions (and lengths) ofprotected P1 (552 nt) and P2 (602 nt) if they had hybridized to J4 spliced products (not seen). Solid arrows indicate bands protected by P1 (214nt) or P2 (268 nt) hybridization to J3 spliced products. (The fainter band migrating just below the 268-nt band is an RNase T1 artifact resultingfrom "breathing" of the hybrid near a guanosine residue 3 nt from the 3' end of the probe.) The small amounts of undigested, full-length probeseen in all the experimental lanes were unchanged with increasing T1 concentrations and disappeared when carrier single-stranded nucleic acidwas added to the reaction.

activity -2 orders of magnitude greater than that used forFig. 3 Right, bands indicative of the J4 splice did not appear(data not shown).

DISCUSSIONUsing the SP6 system to generate transcripts containingmouse immunoglobulin K gene sequences, we found that bothHeLa cell and lymphocyte nuclear extracts fail to discrimi-nate between the J3- and J4-region 5' splice sites. Since thein vitro splicing patterns ofthe transcript with and without thevariable region were identical, we conclude that the juxta-position of V-region sequences is not sufficient to lead to thepreferential use of the J3 5' splice site in vitro. In contrast,HeLa cell transfection experiments examining these same Klight chain gene segments transcribed from the human /3-actinpromoter showed that splicing appeared to occur exclusivelyat the upstream V+J 5' splice site, mimicking the splice-siteselection of K-producing cells. Earlier work demonstratedthat mouse pre-B and B cells, when transfected with a mouseK light chain gene, correctly use the L-exon 5' splice site aswell as the C-exon 3' splice site (21).To facilitate the analysis of in vitro splicing, the V region

in clone VLJ3-J4-C lacked the short L exon (normallyspliced to the 5' end of the V region) and the first 81 nt of the307-nt V region (see Fig. 1). However, an analogous tran-

script containing the full-length V region (though still lackingthe leader exon) also displayed no 5' splice-site selection andinstead gave in vitro results comparable to those shown inFig. 1 a and c (data not shown). For the same technicalreason, much of the second intron (between the J regions andthe C exon) was deleted in the transcripts tested; however,neither the L exon nor the intact second intron is essential foraccurate 5' splice-site selection, as suggested by the in vivoresults discussed below.When HeLa cells were transfected with plasmids contain-

ing the same K light chain gene segments inserted down-stream from the human f3-actin promoter, we found that forthe transcript containing the V+J3 and the J4 5' splice sites(see Fig. 2, construct A), only the former was used. Althoughit cannot be completely ruled out that the intron sequencesremaining after a J4 splice might target the RNA for degra-dation, the following observations suggest that these splicedproducts are generated either not at all or only at vanishinglylow frequencies in vivo. (i) When RNase T1 probes withspecific activities -2 orders of magnitude higher than thoseused for Fig. 3 Right were prepared, the only RNAs detectedin addition to the J3 spliced products were the precursors(probably due to nuclear contamination of the cytoplasmicRNA preparation). (ii) Although RNAs containing the J4spliced products could be very unstable after transport to the

Biochemistry: Kedes and Steitz

7932 Biochemistry: Kedes and Steitz

cytoplasm, analysis of nuclear RNA (data not shown) alsofailed to detect these species.

In contrast to the results with constructs A and B (Fig. 2),construct C, which lacks the V region altogether, gave nodetectable signal (Fig. 3 Left). In construct C, the 3' splicesite of the V region, which is normally spliced to the 5'untranslated region (see diagrams in Fig. 3 Left), is missing.Either this splicing event or the presence of the V region itselfmay be essential to ensure the stability of the transcript invivo. We also transfected HeLa cells with a construct inwhich the V region was rearranged to J1, and we detectedonly a single product arising from use of the J1 5' splice site(data not shown). Again, the cell's splicing apparatus appar-ently ignored all theoretically usable 5' splice sites of thedownstream J regions (three in this case). This argues that theresults from the particular transcripts we analyzed (repre-senting the V-J3 recombination event) likely reflect thegeneral mechanism of 5' splice-site selection in all fourpossible K light chain transcripts.

Various models have been proposed to account for thespecific selection of splice sites. The earliest of these werescanning models (22-24) in which components of the"6spliceosome" first bind to one of the two splice sites andthen gather in the adjacent RNA until reaching the firstcomplementary splice site. Subsequently, data arguingagainst such processive mechanisms were reported (25-29).More recent in vitro results indicated that exon sequence andlength, as well as the proximity of 5' and 3' splice sites, alsoplay a role in splice-site selection (30). Likewise, in vivo,exon deletions were observed to affect splicing patterns ofviral transcripts (31). Finally, experiments analyzing the useof cryptic splice sites in the absence of more attractivewild-type sequences (28, 29, 32) suggested that the match ofsplice sites to the consensus sequence can dictate splice-siteselection when alternative patterns are possible.

Since we observed correct 5' splice-site selection in aheterologous system where human HeLa cells weretransfected with rearranged mouse K light chain genes, itseems that 5' splice-site choice in this case is dependent noton cell-type-specific factors but upon the proper interactionof general splicing factors with the sequence of the pre-mRNA itself. Clearly, this 5' splice-site discrimination is lostin vitro. It could be that an essential, common trans-actingfactor(s) is labile, in very low abundance, or lost during thenuclear extraction procedure. On the other hand, attempts toachieve accurate splice-site selection by changing extractconcentration or nuclear extraction procedures did not alterour results. A factor(s) directing correct 5' splice-site selec-tion could recognize the V region (or another exon) sequenceplaced upstream from a J region to direct proper splicing.Alternatively, this factor(s) could respond to a putativeinhibitory sequence that is located upstream from each Jregion and is eliminated upon V-J recombination. Anotherpossibility is that a J hierarchy exists in vivo but is lost invitro. In other words, the splicing apparatus could have adecreasing affinity for the 5' splice sites in the order J1 > J2> J3 > J4. Finally, proper selection may require the cou-pling of transcription with splicing either because the se-lected 5' splice site is the first transcribed [the "first-come,first-served" mechanism (33, 34)] or because correct pack-aging into heterogeneous nuclear ribonucleoproteins mustbe sequential.

We are grateful to Susanna Lewis and David Baltimore for gifts ofclones and to Doug Black, Volker Gerke, David Solnick, KimMowry, Ben Rich, Manny Ares, Ursula Bond, and members of theSteitz lab for helpful advice and discussions. This work was sup-ported by Public Health Service Grant GM26154, and computerresources were provided by Public Health Service BIONET Grant 1U41 RR-01685-03, both from the National Institutes of Health.

1. Padgett, R. A., Grabowski, P. J., Konarska, S. S. & Sharp,P. A. (1986) Annu. Rev. Biochem. 55, 1119-1150.

2. Leff, S. E. & Rosenfeld, M. G. (1986) Annu. Rev. Biochem.55, 1091-1117.

3. Tsurushita, N., Avdalovic, N. M. & Korn, L. J. (1987) Nu-cleic Acids Res. 15, 4603-4615.

4. Stavnezer, J. (1986) Nucleic Acids Res. 14, 6129-6144.5. Honjo, T. & Habu, S. (1985) Annu. Rev. Biochem. 54,

803-830.6. Tonegawa, S. (1983) Nature (London) 302, 575-581.7. Kabat, E. A., Wu, T. T., Bilofsky, H., Reid-Miller, M. &

Perry, H. (1983) Sequences ofProteins ofImmunologic Inter-est (U.S. Dept. of Health and Human Services, Bethesda,MD).

8. Lowery, D. E. & Van Ness, B. G. (1987) Mol. Cell. Biol. 7,1346-1351.

9. Lewis, S., Gifford, A. & Baltimore, D. (1984) Nature (London)308, 425-428.

10. Gunning, P., Leavitt, J., Muscat, G., Ng, S. & Kedes, L.(1987) Proc. Natl. Acad. Sci. USA 84, 4831-4835.

11. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) MolecularCloning: A Laboratory Manual (Cold Spring Harbor Labora-tory, Cold Spring Harbor, NY).

12. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T.,Zinn, K. & Green, M. R. (1984) Nucleic Acids Res. 12,7035-7056.

13. Dignam, J. D., Lebovitz, R. M. & Roeder, R. G. (1983) Nu-cleic Acids Res. 11, 1475-1489.

14. Heintz, N. & Roeder, R. G. (1984) Proc. Natl. Acad. Sci. USA81, 2713-3701.

15. Black, D. L., Chabot, B. & Steitz, J. A. (1985) Cell 42,737-750.

16. Krainer, A. R., Maniatis, T., Ruskin, B. & Green, M. R.(1984) Cell 36, 993-1005.

17. Weber, F., de Villiers, J. & Schaffner, W. (1984) Cell 36,983-992.

18. Berk, A. J. & Sharp, P. A. (1977) Cell 12, 721-732.19. Solnick, D. (1985) Cell 43, 667-676.20. Ruskin, B. & Green, M. R. (1985) Science 229, 135-140.21. Weir, L. & Leder, P. (1986) Nucleic Acids Res. 14, 3957-3970.22. Lang, K. M. & Spritz, R. A. (1983) Science 220, 1351-1355.23. Lewin, B. (1980) Cell 22, 324-326.24. Sharp, P. A. (1981) Cell 23, 643-646.25. Kuhne, T., Wieringa, B., Reiser, J. & Weissmann, C. (1983)

EMBO J. 2, 727-733.26. Solnick, D. (1985) Cell 42, 157-164.27. Konarska, M. M., Padgett, R. A., Steitz, J. A. & Sharp, P. A.

(1985) Cell 42, 165-171.28. Treisman, R., Orkin, S. & Maniatis, T. (1983) Nature (London)

302, 591-596.29. Wieringa, B., Meyer, F., Reiser, J. & Weissmann, C. (1983)

Nature (London) 301, 38-43.30. Reed, R. & Maniatis, T. (1986) Cell 46, 681-690.31. Somasekhar, M. B. & Mertz, J. E. (1985) Nucleic Acids Res.

13, 5591-5609.32. Zhuang, Y. & Weiner, A. (1986) Cell 46, 827-835.33. Aebi, M., Hornig, H., Padgett, R. A., Reiser, J. & Weiss-

mann, C. (1986) Cell 47, 555-565.34. Crenshaw, E. B., III, Russo, A. F., Swanson, L. W. &

Rosenfeld, M. G. (1987) Cell 49, 389-398.

Proc. Natl. Acad. Sci. USA 84 (1987)