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Double isotype production by a neoplastic B cell line. II. Allelically excluded production of mu and gamma 1 heavy chains without CH gene rearrangement

Double isotype production by a neoplastic B cell line. II. Allelically excluded production of mu... DOUBLE ISOTYPE PRODUCTION BY A NEOPLASTIC B CELL LINE 11 . Allelically Excluded Production of A and yl Heavy Chains Without Ct, Gene Rearrangement YUNG-WU CHEN,* CHARLOTTE WORD,* VAITHILINGAM DEV,t JONATHAN W. UHR,* ELLEN S. VITETTA,* AND PHILIP W. TUCKER* From *the Department of Microbiology, Southwestern Medical School, University of Texas Center Dallas, Texas Health Science at Dallas, 75235; and *the Genetics Division, Vanderbilt University, Nashville, Tennessee 37232 Ig class switching is a process in which a single clone of B lymphocytes synthesizes IgM antibody, and subsequently synthesizes another isotype other than IgD with the same specificity for antigen . Thus, the same heavy chain variable region gene (V  ) formerly associated with the CA is gene subsequently associated with one of several constant region genes (C,,) 3' to CS . (reviewed in 1) . of reference Expression C genes downstream to Cb (e .g ., C" C') in C-,, transformed cell lines involves deletion of DNA 5' to the expressed C gene (1, 2) . Studies of the structure of Ig genes in myelomas or hybridomas (reviewed in references 3 and 4) have led to the conclusion that the isotype switch is mediated by DNA recombination between tandemly repeated switch (S)' sequences located 5' to each Cn gene, with the exception of Cb production (5, 6) . Cloned cell lines, including BCL, .B l (7), simultaneously express both A and S genes in the same unrearranged DNA context as in A-producing cells, and therefore, must produce these isotypes by alternative RNA processing and/or termination of a 26-kb RNA transcript (7-11) . Mitogen- or antigen-activated cells are reported to express IgM in combination with isotypes other than S (12-18) . Southern blot analyses have raised the issue of whether these normal cells use downstream CH genes, either singularly or in combination, in the same manner as plasmacytomas and hybridomas . Yaoita et al . (17) presented evidence that CA is not deleted in surface IgM+IgE+ (slgM+slgE+) spleen cells from SJA/9 mice infected with parasites . Their finding that all C genes were retained in the germline context suggested that the expression of IgM and IgE is mediated by the splicing of an 180-kb RNA This work is supported by National Institutes of Health grants AI-11851 and CA-31534 . Address correspondence to Dr . P . Tucker, Department of Microbiology, University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd . Dallas, Texas 75235 . CharlotteJ . Word was a recipient of a Damon Runyon-Walter Winchell Cancer Fund postdoctoral fellowship . Her present address is New Department of Cell Biology, University of Mexico Cancer Center, Albuquerque, New Mexico, Abbreviations used in this paper : s, switch region ; sIg, surface Ig ; UT, untranslated region . 562 J . Exp . Men . © The Rockefeller University Press - 0022-1007/86/08/0562/18 $1 .00 Volume 164 August 1986 562-579 CHEN ET AL . 563 of the induction of avid Fc receptors for IgE after infection transcript . Because parasites, there is a strong possibility that the majority of SJA/9 mice with such sIgE+ cells were not synthesizing IgE, but were absorbing of the ^-10% it from medium . This hypothesis is strongly supported by recent the culture data of et . Studies by Perlmutter and Gilbert (18) also support Katona al (19) . the long- transcript model . Using a combination of cell sorting, Southern blotting, and sandwich RNA hybridization, they concluded that sIgG I' splenic cells have long RNA transcripts generated from unrearranged DNA that contain both -y and A sequences . However, as in the case of the report by Yaoita et al ., their interpre- tations depend upon cell homogeneity . For example, they claimed have to successfully sorted 1-3% sIgG1 + cells to 99% purity, yet no data were shown to estimate the level of contaminating sIgM+ cells in their sIgGl' population . The mechanism of double isotype production could give insights into the genetic regulation of Ig synthesis in differentiating B cells . To analyze such mechanisms requires a constitutively expressing clonal analogue . In our accom- panying paper, we described a switch variant derived from the BCL,B, in vitro line in which each cell expresses both surface IgM and surface IgGI and also secretes both isotypes . The IgM and IgGI share the same idiotype and use the same VDJ rearrangement . In this paper, we present evidence, based on the C gene context of several independently isolated IgM/IgGI-producing clones (col- 2 ), A -yl lectively termed BCL, .B that both and heavy chains are transcribed in germline DNA configuration from a single copy of chromosome 12 . Materials and Methods and. The of the .B, Cell Lines Subdoning derivation two BCL, subclones, BCL,2 .62 (IgM producer) and BCL, .2.58 (IgM/IgGI producer), is described in the accompanying is a sister subclone of BCL,2 For DNA paper . BCL,2.54 .58 . analysis, we have employed additional independently derived IgM/IgGI subclones of the parental BCL, .B, line . The subclone BCL, .13.92 was of the 12 BCL, .B2 subclones obtained at a frequency of 1 .35 10 -2 (12/888) by depositing one cell per well using a FACS III . The medium used for subdoning the described in the is same as accompanying paper. The subclone BCL, .6 .1 subclone of BCL, .58, derived in is a .2 the same way . The subclones BCL,.15 .9 and BCL, .15.19 were two of the seven BCL, .B 2 subclones derived similarly at a frequency of 5 x 10' (7/1429), except that no WEHI 274 SN was added to the medium . in As the case of BCL, .2 .58, all subclones except BCL, .6 .1 secrete more IgM than IgGI and preliminary data of surface immunofluorescence indicate all subclones that express sIgM and sIgG . BCL, .6 .1 secretes more IgGI than IgM (Y.-W . Chen et ., unpublished al data) . Southern Hybridization Analysis . Procedures for genomic DNA isolation and blotting were described in the preceding paper . All washings were done in 3x SSC plus % 0 .1 SDS, three times at room temperature, followed by washing in O.lx SSC, 0 .1% SDS at C 42 ° for 1 h, with the exception of the pe- (see probes) probed filter, which was washed at 65°C . Restriction endonucleases were purchased from Boehringer Mannheim Biochemi- cals (Indianapolis, IN), New England Biolabs (Beverly, MA), and Bethesda Research Laboratories (Gaithersburg, MD) and used according to the manufacturer's instructions . Electrophoresis was carried out as described in the accompanying paper (in 1 % agarose at 40 V for 18 h) . Probes. Genomic or cDNA probes were prepared by gel isolation (20) of restriction fragments from previously characterized recombinant clones, except for pyl and pe where the whole plasmids were used . To insure that no repetitive sequences were present in the probes, particularly in fragments derived from introns, DNAs were digested with various 564 EXPRESSION OF IgM AND IgGI WITHOUT GENE REARRANGEMENT enzymes and subjected to Southern analysis with "P-labeled mouse genomic DNA as a probe . Only nonhybridizing fragments were employed for further experiments . Relative positions of the probes are shown in Figs . 2, A-R and 6, S-U . Ali DNAs were labeled by nick translation (21) to a specific activity of ^-10 8 Probe A : A 700-bp cpm/tag . Pvu II/Pst I fragment of pu3741 (cDNA clone) (22) containing 18 by of C  2, the entire C 3 and half of the C4 region of y chain mRNA . Probe B : A mixture of 1 .95-kbp and 1 .25-kbp Hha I/Bam HI fragments from pB2 (genomic clone) (P . Fell, personal commu- nication) containing the Cpl, Cut, CA3, and CA4 exons of Ck . Probe C : A 900-bp Pst I/Pst I fragment of pb54J (cDNA clone) (23) containing C61, 6H, C63 and the secreted carboxyl terminus (Ss) of S chain mRNA . Probe D : A 1 .3-kbp Bam HI/Bam HI fragment of pCpl3 (genomic clone) (24) containing the carboxyl terminal membrane (M) exons SM1 and bM2 of the C5 gene . Probe E : A 1 .2-kbp Eco RI/Eco RI fragment of y3-13 (genomic clone) (25) containing CS-Cy3 intronic sequences . Probe F : A 1 .0-kbp Eco RI/Eco RI fragment of y3-13 (25), located downstream of probe E. Probe G : A 2.0-kbp Hind III/Hind III fragment of pH311 (subcloned from genomic phage 'y3-25) (25) containing 5' flanking region of the Cy3 gene . Probe H : A 1 .0-kbp Xba I/Hind III fragment of pJW7 (genomic clone) (26) containing the 5' flanking region of the Cy3 I : II/Sph I fragment of pDRI (genomic clone) containing gene . Probe A 473-bp Bste (26) region (UT) the Cy3 gene . Probe J : A 1 .4-kbp Xba most of C.3 and 3' untranslated of (26) containing Cy3, segment and 3' I/Hind III fragment of pJW7 (genomic clone) M2 K : 1 .0-kbp Sac I/Hind III fragment of yl-6 (genomic clone) (25) containing UT . Probe A of Cy1 gene) . Probe L : A 1 .7-kbp the flanking region 5' to SyI (switch region Xba I/Eco containing the flanking region Cyl RI fragment of pyl (genomic clone) (27) 5' of the region) . Probe M : pyl (27) ; pBR322 plasmid containing a 6.6-kbp gene (3' to the Syl and its 5' and flanking regions. Probe N : A .1- Eco RI insert of the entire Cy1 gene 3' 2 Xba I/Sac I fragment of pyl (genomic clone) (27) containing the C'yl structural kbp gene . Probe O : A 1 .2-kbp Sac I/Eco RI fragment of pyl (genomic clone) (27) containing flanking region of the C71 gene . Probe P : A 1 .0-kbp Bam HI/Kpn I fragment of the 3' (cDNA clone) (28) containing the entire coding sequence ofy2b chain message . py2b(11)' Probe Q : pE (29) ; pBR322 plasmid containing a 4.4-kbp Bam HI insert of the CE structural flanking regions . Probe R : A 900-bp Bste II/Bste II fragment of gene and its 5' and 3' 8 (cDNA clone) (30) containing Cal, Ca2, and most of Ca3 of a chain mRNA . paU558)' Probe S : A 2.0-kbp Bam HI/Eco RI fragment of PJ 3J 4 (subcloned from genomic clone CH4A-142 .7) (31) containing J.3 J,,4 and -1 .5 kb of 3' flanking sequence . Probe T : A 674-bp Eco RI/Hind III fragment located just 3' to probe S subcloned from genomic clone CH-28.289 .1 (27 ; P . W . Tucker, unpublished data) . Probe U : A 760-bp Hind III/Hind III fragment located just 3' to probe T (subcloned from genomic clone CH- .289 .1) (P . W . Tucker, unpublished data) . Probe V : 1,774-bp Sph I/Bam HI fragment of pDRI (26) containing the entire C, 8 coding region and 3' UT of the secreted terminus . Karyotyping. Cells from the BCL,B, and BCL, .2.58 lines were grown to exponential phase and cells were harvested for karyotypic G-banding analysis (32, 33) . Densitometry . For band quantification, autoradiographs were scanned with a densitom- eter (model GS 300, Hoefer Instruments), and densities were calculated using a Bio-Rad model 33E2A integrator . Results Carry Two of BCL, .B, and BCL, .2.58 Cells Copies a Translocated Chromosome of of in vivo BCL, line in 12 . Two reports (34, 35) the karyotype the cell were to the copy number and constitution of the IgH-bearing conflict with respect 12 (36) . The single J,, rearrangement observed in the Eco RI blots chromosome in vitro lines (Fig . 5 of companion report) was consistent with either a of the single copy of VDJ or multiple copies of the same VDJ . Resolution of this question, imperative for evaluation of the Southern blotting data presented region derived so, the or Nesbitt been gene(s) XV Ca-Cb variant slides Cb IgGI, fragment, 2from have 1two the light Cy BCL, to within the the composite summarizes required breakpoint region of BCL, locus established insure proximal Alleles in due for that t(12 in Fig from translocated The an region, 12 BCL, chain It whether DNA and lines and an 65 Light BCL, Southern C6 number appears Determining 16 12, 12 unusual 12, cluster to spans Only paper derived 3, insuring IgM-secreting subclones modal cells both the aeither analysis Are (data was per aFranke gene although but context germline Bam careful rehybridization chromosomes on chain (16133) random the absence was the Present the these analyzed) We germline found Therefore, we cell isotypes (Fig the chromosome that in translocation blotting not chromosome (37) one HI or IgM/IgGI-producing probes are expression that performed S,u have found of BCL, cytogenetic (39) expression breakpoint (lanes the normal of These two shown) to at cleaves 1their CH indistinguishable or region, (Fig in loss of BCL,B, in no j,,-C, the the the shown carry subclone every two map analysis and repetitive fragment Germline germline Presumably, We that 4-6, rearrangement of definitive heavy 1(Fig two centromere, microscopic chromosome CH was b) of copies within Gene Similarly, of and this of karyotyped 12 38) restriction by the to cell number the that and (X3) 8) of evaluation this Likewise, chromosomes A locus, Shimizu 1is62 chromosome chain Giemsa of a(lane c) chromosome the and The Context same As in karyotyped Configuration in sequences bands all of could 10 blot in one BCL, Cy, (with band One vitro the expected translocation BCL, an yfrom breakpoint this we all expression in subclones 3), Eco serologically 1generating to of level digestions based distal et banding apparently 25 in of genes are had BCL, 16 of 55-63 conceivably of location have BCL, DNAs BCL, Cb the would al RI those BCL, BCL, 25 3' the cells BCL, present (probe both taken appear (25) from 16 region digests on cells fragment IgM/IgGI-producing during iscarried in chromosomes cells (Materials from [t(12 of in allelically on techniques the From translocate on vitro-adapted is employed t(12 aConsiderable line place has BCL, the BCL, identical and defined C) was chromosome 5' germline shown (lane to (12F2), chromosome synthesize in probed ideogram be the BCL, detected only published 24 to has out and be BCL, that BCL these 63 between generated the preparation 7), out and excluded identical, chromosomes two (57-66 which in one and an several idiotype telomeric translocated with extends One for and The rearranged 9in of Fig results, Methods) both proposed extensive copies the t(12 BCL, parental care (lane Cy maps 52 25 and normal respect CFc 16 in carries 16 modal 2other chro- from 10 cells cells and, gave sub- IgM four and is into and and was has the To 2), 19 of of to 5' at to of in CHEN ET AL . below, and Chromosome .2.58, . clones, chromosome .B, . .2 .58 analyzed) mosomes . .2.58 cells .B, . .B, chromosome . both ;16)], .58, the . . .2 carry . . ;16) ;16), probably the . ;16) .B, . identified .2 .58 chromosome . The the . a) . (36, by . genes chromosome not . .B, .B is chromosome V, either .B, .B2. In Strategy .2.58 accompanying and determine if overlapping .B,, .2 .58 independently . Fig . to . . taken . All . As . lane . .4-kbp .5-kbp fragment . .B, the BCL, .62, .2.58 other .B2 .5- the kbp C6 only .5-kbp EXPRESSION OF IgM AND IgGI WITHOUT GENE REARRANGEMENT r C7 cv . bp o .= ci o r~ ~ ci ro ci c n . 'ro C M . n . o bo .- o c n a. v . CL, V .O bA~ GV G'l C . . .b -?:- .. !v v .ro ..O Ca ~ C: , CL E c , c O cD L y L CO "O c O bp.-7 G. v v"'E°-E .roE~ " o L (O U cE o c a - C ch C O,> ai y, "C blJ f,- a c v C, ct 00 v r ^ v CHEN ET AL . 56'7 ~ = R v oro p . ° R .G v v y~~~roE v o c y 0.% .C RO¢bco GO ^'h E v .n ~v(U o N q 0 a 0. O L ~ 3 v ?IN nn N ¢ N ro of vE .SvVG"'O ~ ." >. bo C L iR . C wm zlw N c6 G C H 4: 'O w bOA .~ ~~ 7 ro v "C ¢ '7° N N v'b>scv `v'v . v L .C G O- e 0 J O a 'c" N v X v 'f ro b0 L W H ro v y A E ~ u-CS Y G R .Y H 4. ti N m -° _° lam u~ o v ' L ~ v N ro G L a nn h .,_ a N m R R Y y~ L , ~ .+ V rq " ti O ° v 3 G L Y N v roc:.ocE >. C 0. .O . C Aa 0 -0 . rozma mo ._i E a o ro vV L '0 ` ^ CC (d ft o. O C O a O." us E,7 0. .T b4~i, Y m 0' .n i c0 w G. C m n cC ro 't T. t0 ~ L y G y. m m ~o m E 2 bbc G ..+ v f v .n E u ^ o O E O .~Lro 'G v L ti c . y G C W n h a Y o v R W v m L croc Nc :: O y ti .b n v a R U G " G ice. R 7 o 0. w U 'O 'O b0 bA Gs ~ c0 . v ` a w a F z ., ~ cs m .n c` a .o uroc N~ ; . v .° C R o.~ os -o n 0 m z prn s o 'IV o > o . E^G G.O m 0 O z m R W .V v I C ti v R ._. v L C N O 4J N bbO m j N E O Of o z ° ro m 3^E v, -0 7 v. .d y )ac O .~ E b0= y, L 0 N E G O C' T v O b0 L N O N R N scLVE yY al W a` W a n bc ~O > .'C + Lo O ro °~ O O u G . m N o F ''b L ° X c;aNV oxy y ~=R i~ .LAL ,Gn a ..ro v .t R yG" ~-°' o J ~~ cd R k,~c .=voosE 568 EXPRESSION OF IgM AND IgG1 WITHOUT GENE REARRANGEMENT Analysis FIGURE 3 . of Cp gene context . High molecular weight DNAs were digested with Bam HI (see Figs . 2 and 6 for relative location of sites), and were fractionated and blotted as described in Materials and Methods, then were hybridized with Cy (probe B) . Lane 1, BALB/c liver DNA ; lane 2, BCL, .B1 ; lane 3, BCL, 4, .2 .62 ; lane BCL, .15 .19 ; lane 5, BCL, .15 .9 ; lane 6, BCL, .13 lane 7, BCL, .92 ; .2 .58 ; lane 8, BCL, .2 .54 . kb markers are shown in margin . conjunction with data from Kpn I digestions presented below, we conclude that no rearrangement involving Su, Cu, or Cb alleles contribute to double production j and in BCL I .B 2 . of yl Genes 3' to C), including Cy,, are in Germline Configuration . The lack All C suggested that y, expression in BCLI .B2 of Cg. rearrangement subclones does occur class-switch recombination . To resolve the issue directly, not by prototypic of probings, which overlapped the entire we carried out an exhaustive set CH and C,., well the DNA region (^-100 kbp) between Cb as as downstream to C,, . The complete analysis was performed on Balb/c liver, BCL I .B I , BCL, and .2.62 BCL, and partial analyses were carried out on additional BCLI .B 2 sub- .2 .58, clones . The data are summarized in Fig . 2, with the most crucial blots for the region between and C,. I presented in Fig . 4 . Digestion with Hind III generates C,3 a diagnostic fragment (fragment 15 in Fig . 2) which spans the 1 switch region 1' and generally detects rearrangement of this locus . This is shown in Fig . 4 a by the two rearranged bands (15 and 16 kbp) in the control digestion of an IgG I secreting hybridoma (lane 1) . In contrast, we detect only the germline band of C,, (23 kbp) and the more faintly crosshybridizing germline fragments of C,26, C., and (9 .2, 6 .6, and 6 .4 kbp, respectively) in liver (lane 2), BCLI IgM s C,2, producers (lanes 4 and 5) and BCL, .B 2 (lanes 3, 6-8) . Likewise, with the more upstream probes L, K and J (Fig . 4,b-d), the BCLI and BCL I .B2 patterns are identical to those of the liver . Although no rearrangement occurred between y3 and y,, it still remained y l might possible that expression of be affected by a recombination between b and y3 . For example, a deletion within this region would appreciably reduce the length of a putative J r-Ca-Cy transcription unit . As summarized in Fig . 2, we detected no rearrangements in BCL I .B, and BCLI .B 2 relative to liver within this exception of only 7 .9 kbp of repetitive DNA ^-55 kbp area with the (between fragment 4 and 7) was difficult to confirm . Similarly, in the BCL I .B 2 cells, that downstream genes from Cy 1 to Ca remain in germline context . Taken together, these results strongly argue that DNA rearrangement of genes is not respon- CH sible for expression of y, in BCL, .B 2 cells . Deletion of the Majority of the C Locus Has Occurred on the Nonproductive Chromosome 12 of BCL I.B2 . As noted above, the Cit and Cb genes of both BCL I .B I BCLI .B 2 are unrearranged and are linked on a Barn HI fragment and FIGURE 4 . C, genes and the region between C,s and C,, are unrearranged in BCL, .B2 double producers. (A) Hybridization of a Hind III digest (Fig. 2, fragment 15) to C,, probe M. Lane 1, 9B 12 (IgG I-secreting hybridoma) ; lane 2, BALB/c liver ; lane 3, BCL, .2 .58 ; lane 4, BCL, .B, ; lane 5, BCL, .2 .62 lane ; 6, BCL, .2 .54 ; lane 7, BCL, .13 .92 lane ; 8, BCL, .15 .9. (B) Hybridization of an Xba I digestion (Fig. 2, fragment 14) to C.3-C ., intronic probe L . Lane 1, BCL, .2 .62 ; lane 2, BCL, .B1 ; lane 3, BCL, .2 .58 ; lane 4, BCL, .6 .1 ; lane 5, Balb/c liver DNA . (C) Hybridi- zation of a Sac I digestion (fragment 13, Fig . 2) to C,3-C,, intronic probe K . Lane 1, BCL, .B, ; lane 2, BCL, .2 .58 ; lane 3, BCL, .6 .1 ; lane 4, Balb/c liver DNA . (D) Hybridization of a Kpn I digestion (fragment 12, Fig . 2) to C,s 3' probe J . Lanes are as in B. The additional bands seen in Fig 4C (13 .1 kbp) and 4d (5 .6 kbp) are residual hybridization from the previous probings of the same filters . kb markers are shown for A-D. IgM AND IgGI WITHOUT GENE REARRANGEMENT 570 EXPRESSION OF carry two different alleles of chromosome 12. Southern blots of FIGURE 5 . BCL, .B2 clones QH3- Fig . 6 for positions of sites) were hybridized in A to probe S Kpn I-digested DNAs (see BCL, .62 ; lane B Lane 1, BALB/c liver; lane 2, BCL, .B1 ; lane 3, .2 JH 4) and in B to probe (Cu) .58 ; lane 8, BCL, .2 .54 . lane 5, BCL, .15.9 ; lane 6, BCL,A3 .92 ; lane 7, BCL, .2 4, BCL, .15.19; -y ju l- (lanes 1-3, genes are not linked in A only (lanes 4-6, BCL, .2 .62) and (C) and C,, JH I were hybridized to producing subclones. DNAs were digested with BgI and BCL,2 .58) JH S and V (lanes 4), (probe V) (lanes 3 and 6), and a mixture of probes (probe S) (lanes 1 and C,s gene lanes 2, 3, 5, and 6, the 17 kbp and 7.5 kbp fragments were C,2n 2 and 5) . In Fig . 5c, W. The 6.2 kbp fragment is the Bgl I fragment containing crosshybridizations with probe located on the H' allele (see Fig. 6) . kb markers are shown . VDJ sequences chromosome . But a single, lines contain two copies of the t(12;16) (Fig . 3). Both RI region rearrangement is observed when Eco apparently identical variable probed with a region BCL I .B I and BCL I .2.62 DNA are JH blots of BCL I .2 .58, accompanying paper) . In addition, a single fragment (probe S and Fig . 6 of Hind III, Bam HI and Bgl I -containing band was obtained in Xba I, VDJ H2 and subclones probed with (accompanying paper digests of all the BCL I .B 2 JH BCL I .B 2 allele possessed an not shown) . These data suggested that each data a rearrangement rearranged VDJ . However, Kpn I digests revealed identically with respect to BCL I .B I . As shown in Fig . 5a, of one of the two BCL I .B2 alleles I fragment found in BCL I .B I (lane the rearranged 17 .8-kbp Kpn in addition to in BCL I .58 (lane 3), we detected a 12 .5-kbp fragment .2 2) and BCL I .2 .62 (lane present in the other BCL I .B 2 subclones 7) . The identically rearranged band is although independently cloned, suggesting that all BCL I .B 2 lines, (lanes 4-6, 8), was parental variant . When a similar Kpn I blot were derived from a common hybridizing band, corresponding to the with Cu (Fig . 5 b), only a single probed CHEN ET AL . 571 from the unrearranged allele of BCL1 .B 1 , was detected in 17 .8-kbp fragment rearrangement more precisely, we performed similar Kpn BCL, .B 2 . To map the JH- U) that extend directly from the Eco RI site in the I blots with probes (T and . only the 17 .8-kbp fragment hybridized (data not shown) . Cp intron In all cases, a single Eco RI band in J3J,,4 blots, the rearrangement had Since there is only occur just 3' (<50 bp) to the Eco RI site in the intron Otherwise, the to J,,-Cp BCL, .B 2 . .5-kbp band would have been detected by probe T or U in that the .5-kbp Hybridization of the Kpn I digests to probe M had shown 12 there are a number fragment did not contain C,., (Fig . 2, fragment 18) . However, there is slight Kpn I sites between C,. 3 and C,, in germline DNA (25), and a of 4 . Therefore, possibility of fragment comigration in the analysis of Fig . we that generates a digested BCL, .B, and BCL 1 .B 2 DNAs with Bgl I, an enzyme . fragment 20a), fragment that spans the entire C,3-S,, germline region (Fig 2, of the Eco RI cuts at a single site between and C, -480 by downstream and JH unpublished results) . As seen site of the rearrangement junction (P . W. Tucker, with (probe S) and C, 3 in Fig . 5c, hybridization either separately or jointly JH . These data (probe V) gave different sized bands (30 and 32 kbp, respectively) of IgM/IgGI producing prove thatJ and C,., are not linked on either allele the variants . yl Allelically Excluded. The Simultaneous Expression of u and in BCL I.B is analyses of the C genes, suggest that the observed above results, taken with the BCL, .B 2 is unusual . It deletes, at a minimum, rearrangement on one allele of by direct inference of the Ckt-C6 linkage (Fig . part of J-Cy intron, all Cy and, patterns (Figs . and 4) and the analysis of Fig . 3), Cb . However, the germline 2 this rearrangement is not accomplished by recombi- 5 c show unequivocally that sequences . Therefore, the 3' side of the rearrange- nation into downstream C to be unrelated to Ig CH DNA, yet it is not large ment in BCL I .B appears cytogenetically . enough to be detected map of two IgH alleles in BCL 1 .B 2 is shown in Fig. 6 . The A proposed the downstream C genes on the productive chromosome (i .e ., model predicts that H+ allele) exist in single copy in BCL I .B but in double copy in BCL1 .B1, on the or liver . On the other hand, J genes should exist in double copy in BCL, .2 .62, parental, variant, and germline DNA. To test this hypothesis, we hybridized Eco digests (Fig . 7a) of the appropriate DNAs to a mixture of (probe S) and RI J,, (probe V), and Hind III digests (Fig . 7b) to a mixture of (probe S) and C,, J,, C,-3 (probe M) . The hybridization intensities were quantified by densitometry and Fig . b) the ratio ofJN/C, 3 (4 kbp:18 kbp, Fig . 7 a) andJ (2.55 kbp:23 kbp, 7 /C,1 ratios in BCL, .B,, were determined . As shown in Table I, the /C,, 3 and J,,/C,1 in the IgM/IgGI-producing variants, BCL, .2.62 are approximately twice those conclude allelic exclusion is operative in BCL, .2.58 and BCL, .13.92 . We that and chains from a single rearranged VDJ. BCL I .B in generating both 2 u 'y, Discussion Two conclusions can drawn from the results of our studies : (a) IgGI major be expressed in the BCL I .B clones in the absence of DNA rearrangement of the is constant region genes ; and (b) the heavy chains of both IgM and IgGI are 572 EXPRESSION OF IgM AND IgGI WITHOUT GENE REARRANGEMENT L~ .b0 .~ F" i.. ° x `n-- 4- ,L s . ~- C .O ro "O + yam'. v > ro a v ._ .- u w ~ CHEN ET AL. 57 3 FIGURE 7 . Quantitative analysis of J , and C,, gene context . (A) Southern blots of Eco C,3, and C,3 RI digests were hybridized separately or with a mixture of J" probes . (B) Hybridization and C,, . Lane 1, BALB/c liver ; 2, BCL, of Hind III digests toj. lane .2 .62 ; lane 3, BCL, .B1 ; 4, .13.92 lane 5, BCL, .2 .58 . Densitometry of the appropriate bands is lane BCL, ; summarized I . are for and in Table kb markers shown A B. TABLE I Allelic Exclusion Is Operative in BCL, .B 2 Double Producers Cells IgH /C,,* JH/C,3 * J BCL, .2 .58 A'y1 1 .25 0.80 BCL, .13 .92 wf1 1 .48 0.77 BCL, .2 .62 A 0.74 0.44 BCL, .Bl A 0.70 0.49 BALB/c liver - 0.71 - * Densitometry was performed on the 4 kbp and 18 kbp bands of Fig. 7a and the 2.55 kbp and 23 kbp bands of Fig. 7b . Ratios represent the average of two JH/C, and three JH/C,, determinations . expressed from a single rearranged VDJ segment on the same chromosome 12 . Therefore, we conclude that simultaneous production of IgM/IgGI in BCLI .B2, as in the case of IgM and IgD in BCL I .B l , is controlled exclusively at the RNA level . Our findings provide the first example of allelically excluded, double isotype expression in a neoplastic B cell clone . A similar conclusion was drawn to explain dual u and ti2b production in a derivative of a 1A-producing Abelson murine leukemia-transformed cell line (40) . Contrary to the initial report, it is now clear (41, 42) that -y2b synthesis in this line is accompanied by deletion of sequences and C,,26 , and thus cannot between be explained by differential RNA process- JH Our support the . ing . data also observations of Yaoita et al (17) and Perlmutter and Gilbert (18), who used purified populations of normal lymphocytes. The caveats associated with these studies (19) are overcome here since we have shown that virtually all cells in BCLI .2.58 synthesize, express, and secrete IgM and 574 EXPRESSION OF IgM AND IgGI WITHOUT GENE REARRANGEMENT IgGI . Furthermore, the clonal BCL, .B 2 lines have provided the opportunity for a more extensive analysis of the DNA context . In addition to establishing germline configuration of sequences anticipated to undergo rearrangement during switch recombination (e .g ., S,, C, and we have also shown that the S,,), remainder of the C locus is unrearranged on the productive allele . This observation argues against the hypothesis that CH genes might be translocated, via nonclassical switch sequences and deletion of Cb , to a position downstream to C,, possibly replacing b with another isotype (7, 11) . Although we have provided strong evidence for use of a single VDJ, it resides on a chromosome 12 that has undergone an unusual translocation, distal to the Ig locus, with chromosome 16 . At the microscopic level, the t(12 ;16) appears to be identically duplicated in both the BCL, .B and BCL, .B 2 cell lines . However, our blotting data define the nonproductive t(12 ;16) allele (H - in Fig . 6) by virtue loss Ig-related sequences 3' to the Eco RI within of its of site the JH-CA intron . These findings have important implications in considering the both derivation the in vivo BCL, leukemia, its subsequent in vitro adaptation, and the eventual of for allelic exclusion in BCLI .B 2 . The in vivo BCL I mechanism leukemia cells appear to have a stable karyotype, with the exception of chromosome 12 . Two early reports (34, 35) are in agreement with our data (Y.-W . Chen and G. V. Dev, unpublished results), with respect to the modal chromosome number (35 chromosomes in reference 34 and our data, and 36 chromosomes in reference 35) . However, Schroeder et al . (34) reported a single normal chromosome 12 and a translocated chromosome 12 to an unidentified recipient in early passages of the line . One year later, Voss et al . (35) found no evidence for normal copies ofchromosome 12, but identified three translocated alleles . We observed a single copy of the same t(12 ;16) in the in vivo line (Y .-W . Chen and G. V. Dev, unpublished results) that is duplicated in the in vitro BCL, .B, and BCL, .B 2 lines, both of which have a modal chromosome number of --60 . We suggest that the precursor to the original leukemic cell carried a normal chromosome 12 as nonproductive and retained it during early passages, at which time the productive underwent translocation(s) . Adaptation to culture chromosome 12 cell resulted of of the karyotype, including ;16), and may in a duplication most t(12 have more subtle event (e .g ., somatic which required a second mutation), was unde- DNA tectable at the cytogenetic or rearrangement level, to effect allelic exclusion in BCL, .B, . Alternatively, both of the t(12 ;16) alleles remain active in BCL, . If the former is true, then the third event, the rearrangement 3' to VDJ on the H- allele of BCL, .B 2 , is not required for allelic exclusion, but may instead reflect some trans-acting regulatory requirement for switching the productive allele ,u/-y from ,u/b to 1 . The significance of this DNA rearrangement is underscored by the fact that all independent BCL, .B 2 variants maintain it . Aside from its unusual karyotype, do the BCI., .B2 variants have a counterpart amongst normal B cells? Based on its surface Ig phenotype, its low level of constitutive IgM secretion (reviewed in reference 43), and its heavy chain gene transcription profile (44), the parental BCL, .B, line appears to represent an immature B cell . It has been employed as a model for numerous functional further cultured studies by virtue of its ability to undergo differentiation when CHEN ET AL . 57 5 all LPS (45), anti-Ig (46), or T cell-derived lymphokines (47) . The fact that with .B2 variants isolated for IgG secretion secrete both IgM and IgGI, as well BCL, express them on their surface, may reflect their derivation from a single as precursor . It may also denote a basic difference between these cells and the nonsecreting memory cell populations analyzed by others (17, 18) at the molec- differences in mode of expression are ular level . Regardless of whether these transformed the BCL, .B 2 phenotype real or are a consequence of the state, and the membrane form of mRNA shows that high expression of the secreted from genes in germline configuration . can be generated C mechanisms, the only plausible explanation for the Finally, with regard to and yl mRNA are expressed with a common VDJ gene by present data is that y RNA processing . This could be accomplished by a discontinuous alternative in which the RNA polymerase translocates from the template to mechanism discrete segments of DNA, as proposed for trypanosome variable transcribe antigen (48) and certain viral genes (49, 50) . Alternatively, there could be a continuous transcription mechanism (i .e ., long transcript model), such as that apparently used by the drosophila bithorax locus (51), which has been invoked (17, 18) to account for their results in normal B cells . In the latter by others 1 chains could then be translated from mRNA derived by scenario, the y transcript that includes sequences from both Cp and Cy 1 processing a primary derived either from an identical transcript of -120 genes . The t mRNA may be terminates in the intervening sequence between CU and CS . kb or from one that studies in BCL, .B, cells (44) and other IgM/IgD Based on nuclear transcription favor the latter alternative . This would require double producers (52, 53), we transcriptional termination, perhaps at two points . differential regulation of would in First, the majority of the RNA polymerases unload 3' to Cu, since yl RNA BCL, .B 2 cells, A RNA abundance is greater than abundance (accom- small polym- panying report and data not shown) . Second, a proportion of the erases would read through the 1 termination region and exit the template in BCL, .B 2) . A in downstream of the CH gene to be expressed (Cyl choice the second polymerase unloading event may dictate (and simplify) not only the subsequent RNA splicing decision, but may also bear on the acquisition of isotype commitment ; i.e ., once a cell expresses an isotype other than IgM and IgD, it is committed and restricted to secretion of that isotype upon stimulation (54) . then clonal example of this model is the 1 .29 B cell lymphoma, which The best undergoes spontaneous and/or inducible switch recombination from IgM to IgA The Ca gene is preferentially open, as defined by hypomethylation, in the (55) . cells that are committed to switch to this isotype (56) . Similarly, IgM-synthesizing BCL1 to BCL, .B2 , Cyl appears to be exclusively selected . in the transition of transcription through, and equally important, termination beyond a C Perhaps .B 2) critical in inducing an active chromatin structure for gene (C,, in BCL, is C gene . The resulting prediction is that subsequent expression of that same receiving appropriate signal, would undergo exclusive BCL, .B 2 cells, on the C,,, . This cell line, therefore, provides us with the switch recombination to the mechanisms of double isotype production and its opportunity of testing consequences . 576 EXPRESSION OF IgM AND IgGI WITHOUT GENE REARRANGEMENT Summary In our accompanying paper, described variant (BCL we a switch I .2 .58) that of IgM . IgM IgGI expresses membrane and secreted forms and IgGI Both and share same and use VDJ rearrangement Here, a the idiotype the same . detailed blot analysis of the entire constant region of the Ig heavy chain (Ig Southern C  ) parental (BCL I .B I ) and variants (BCL I .B2 ) DNA showed detectable locus of no rearrangement . Similar analysis of the JH-C/ region led to the conclusion that two heavy chain alleles present in the IgM/IgGI-producing variants carried the same VDJ rearrangement but differed in their 3' flanking regions . One chro- mosome 12 did not carry any Ig C F, genes, whereas, the other chromosome 12 carried one copy of C genes . In BCL I .B I , however, each of the chromosome 12 alleles carried a full copy of C  genes . Karyotypic analysis confirmed the presence of two translocated t(12 ;16) chromosomes in both BCL I .2 .58 and BCL I .B I cells, with a break 5' to the V  locus at the distal region (12F2) of chromosome 12, and at the proximal region below the centromere (16113) of chromosome 16 . We conclude that double production of IgM and IgGI in BCL I .B 2 is accom- plished by transcription of the corresponding C  genes in germline configuration 12 . using a single VDJ on the same chromosome We thank Ms . M.-M . Liu, Ms . C . Das, Mr . J . Rybak and Ms . C . Glover-Humphries for excellent technical assistance and Ms . M . Gardner for secretarial assistance . We thank Dr . P . Howard-Peebles for assistance with the cytogenetics and Dr . D . Yuan for assistance with desitometry . We are grateful to Dr . T . Honjo for providing phages bearing Cy intronic sequences . February in revised May Receivedfor publication 27 1986 and form 6 1986. in of sandwich hybridization and nuclear run-on transcription Note added proof . Results consistent with the long transcript model for dual II. and yl expression in BCL I .B 2 . are References 1 . Marcu, K . B . 1982 Immunoglobulin heavy-chain constant-region genes. Cell . 29:719 . Honjo, T ., and T . Kataoka . 1978 . Organization of immunoglobulin heavy chain 2 . and allelic deletion model . Proc. Natl. Acad . Sci . USA. 75:2140 . genes Marcu, K ., and M . Cooper . 1982 . New views of the immunoglobulin heavy chain 3 . switch . Nature (Loud.) . 298 :327 . 4 . Shimizu, A ., and T . Honjo . 1984 . Immunoglobulin class switching . Cell. 36 :801 . 5 . Richards, J . E ., A . C . Gilliam, A . Shen, P . W . Tucker, and F . R . Blattner . 1983 . Unusua l sequences in the murine immunoglobulin u-6 heavy-chain region . Nature (Lond. ) . 306:483 . 6 . Gilliam, A . C ., A . Shen, J . E . Richards, F . R . Blattner, J . F . Mushinski, and P . W . . generates class switch from to C6 in Tucker 1984 . Illegitimate recombination a CA Proc Natl . Acad . Sci . USA. 81 :4164 . an IgD-secreting plasmacytoma . . M . R ., . Liu, N . Newell, R . B . Ward, P . W . Tucker, S . Strober, and F . 7 . Knapp, C .-P . Simultaneous expression of immunoglobulin A and 6 heavy chains by Blattner 1982 . B-cell lymphoma : a single copy of the V  gene is shared by two adjacent C a cloned . Proc . Natl . Acad. Sci. USA . 79:2996 . genes CHEN ET AL . 577 F . 8 . Tucker, P . W ., C .-P . Liu, J . F . Mushinski, and R . Blattner . 1980 . Mouse immu- noglobulin D : mRNA and genomic DNA sequences . 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Stimulatio n of human B lymphocytes IgM IgG Functional evidence for expression B by antibodies to and : the of IgG on lymphocyte surface membranes . Clin Immunol. Immunopathol . 15:301 . E . F . Assisi, and L . . . 14 . Zan-Bar, L, S . Vitetta, S . Strober 1978 Th e relationship between surface immunoglobulin isotype and function of murine B lymphocytes . III . Expres- sion of a single predominant isotype on primed and unprimed cells . J. Exp . Med . 147:1374 . 15 . Kearney, J . F ., M . D . Cooper, and A . R . Lawton . 1976 . B cell differentiation induced by lipopolysaccharide . IV . Development of immunoglobulin class restriction in pre- cursors of IgG-synthesizing. J. Immunol. 117 :1567 . 16 . Calvert, J . E ., M . F . Kim, W . E . Gathings, and M . D . Cooper . 1983 . Differentiation lineage cells from livers of neonatal mice : generation of immunoglobulin isotype of B Immunol . 131 :1693 diversity in vitro . J. . T . 17 . Yaoita, Y ., Y . Kumagai, K . Okumura, and Honjo . 1982. Expression of lymphocyte require surface IgE does not switch recombination . Nature (Lond .) . 297 :697 . P ., 1984 . 18 . Perlmutter, A . and W . Gilbert . Antibodies of the secondary response can be expressed without switch recombination in normal B cells . Proc. Natl. Acad. Sci . USA . 81 :7189 . 19 . Katona, I . M ., J . F . Urban, and F . D . Finkelman . 1985 . B cells that simultaneously express surface IgM and IgE in Nippostrongylus brasiliensis-infected SJA/9 mice do not provide evidence for isotype switching without gene deletion . Proc. Natl . Acad . Sci . USA . 82:511 . 20 . Maxam, A . M ., andW . Gilbert . 1980 . Sequencing end-labeled DNA with base-specific chemical cleavages . Methods Enzymol . 65:499 . . Rigby, P . W . J ., M . Dieckmann, C . Rhodes, and P . Berg . 1977 . Labeling deoxyribo- nucleic acid high specific activity in vitro by nick translation with DNA to Polymerase I .J. Mol. Biol. 113 :237 . B ., R . Perry . . r 22 . Marcu, K . U . Schibler, and P . 1979 Nuclea transcripts of mouse heavy chain immunoglobulin genes contain only the expressed class of c-region sequences . DC) . Science (Wash . 204 :1087 . . . 23 . Mushinski, J . F ., F . R . Blattner, J D Owens, F . D . Finkelman, S . W . Kessler, L . Fitzmaurice, M . Potter, and P . W . Tucker . 1980 . Mouse immunoglobulin D : Con- struction and characterization of a cloned S chain cDNA . Proc . Natl. Acad . Sci . USA . 77:7405 . 24 . Liu, C .-P ., P . W. Tucker, J . F . Mushinski, and F . R . Blattner . 1980 . Mapping of the heavy chain genes for mouse IgM and IgD . Science (Wash . DC). 209:1348 . 25 . Shimizu, A ., N . Takahashi, Y . Yaoita, and T. Honjo . 1982 . Organization of the constant-region gene family of the mouse immunoglobulin heavy chain . Cell . 28:499 . 578 EXPRESSION OF IgM AND IgG1 WITHOUT GENE REARRANGEMENT . Wels, J . A ., C . J . Word, D . Rimm, G . P . Der-Balan, H . M . Martinez, P . W . Tucker, . . analysis and F . R . Blattner 1984 Structura l of the murine IgG3 constant region gene . EMBO (Eur. Mol . Biol. Organ.) J. 3:2041 . Honjo, T ., M . Obata, Y . Yamawaki-Kataoka, T . Kataoka, T . Kawakami, N . Taka- hashi, and Y . Mano . 1979 . Cloning and complete nucleotide sequence mouse of chain Cell. immunoglobulin yl gene . 18:559 . 28 . Tucker, P. W ., K. B . Marcu, J . L . Slightom, and F . R . Blattner . 1979 . Structure of the constant and 3' untranslated regions of the murine y2b heavy chain messenger RNA . Science (Wash . DC) . 206:1299 . T . Nakai, Kishimoto, I . and 29 . Nishida, Y ., Kataoka, N . Ishicia, S . T. Bottcher, T . Honjo . 1981 . Cloning of mouse immunoglobulin e gene and its location within the heavy chain gene cluster . Proc . Natl. Acad. Sci. USA . 78:1581. P . W ., J . L . Slightom, and F . R . Blattner . 1981 . Mous IgA heavy chain 30 . Tucker, e gene sequence : Implications for evolution of immunoglobulin hinge exons . Proc. Natl. Acad . Sci . USA . 78:7684 . . Newell, N ., J . E . Richards, P . W . Tucker, and F . R . Blattner . 1980 . J genes for heavy chains in mouse Science (Wash. DC) . 209:1128 . immunoglobulin . 32 . Dev, V . G ., and R . Tangravahi. 1982 . Techniques for Chromosomal Analysis . In Techniques in Somatic Cell Genetics . Shay, J ., ed . Plenum, N .Y . p . 493 . . Seabright, M . 1971 . A rapid banding technique for human chromosomes. Lancet . 2:971 . 34 . Schroeder, J ., H . Suomalainen, M . R . Knapp, E . Gronowicz, and S . Strober. 1979 . Karyotypic differentiation in a spontaneous mouse B cell leukemia . Cancer Genet . Cytogenet. 11 :57 . . 1980 . analysis the 35 . Voss, R ., G. Maftzir, and S . Slavin Cytogenetic of spontaneous murine B cell leukemia (BCI  ) . Leuk . Res. 4:325 . . D'Eustachio, P ., D . Pravtcheva, K . Marcu, and F . H . Ruddle. 1980 . Chromosoma l location of the structural gene cluster encoding murine immunoglobulin heavy chains . J. Exp. Med . 151 :1545 . . D'Eustachio, P ., A . L . M . 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Pirrota, F . Schalenghe, Th and T . C . Kaufman . 1983 . e molecular organization of the antennapedia locus of drosophila . Cell. 35:763 . 52 . Mather, E . L ., K. J . Nelson, J . Haimovich, and R . P . Perry . 1984 . Mode of regulation of immunoglobulin j- and 6-chain expression varies during B-lymphocyte maturation . Cell . 36:329 . . . l 53 . Yuan, D ., and P . W . Tucker 1984 Transcriptiona regulation of the IA-6 heavy chain murine B Exp . 160 . locus in normal lymphocytes . J. Med . :564 . . . . . 54 Kincade, P W ., A R . Lawton, D E . Bockman, and M . D Cooper . 1970 . Suppressio n immunoglobulin G a of synthesis as result of antibody-mediated suppression of M synthesis chickens Proc. . immunoglobulin in . Natl Acad. Sci . USA . 69:918 . Sirlin, 55 . Stavnezer, J ., S . and J . Abbot . 1985 . Induction of immunoglobulin isotype switching in cultured 1.29 B lymphoma cells ; Characterization of the accompanying rearrangements of heavy chain genes . J. Exp. Med . 161 :577 . Stavnezerj, J . Abbott, S . Sirlin . 1984 . in 56 . and Immunoglobulin heavy chain switching in cultured 1 .29 murine B lymphoma cells : Commitment to an IgA or IgE switch . Curr. Top. Microbiol. Immunol. 113 :109 . http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Double isotype production by a neoplastic B cell line. II. Allelically excluded production of mu and gamma 1 heavy chains without CH gene rearrangement

The Journal of Experimental Medicine , Volume 164 (2) – Aug 1, 1986

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Abstract

DOUBLE ISOTYPE PRODUCTION BY A NEOPLASTIC B CELL LINE 11 . Allelically Excluded Production of A and yl Heavy Chains Without Ct, Gene Rearrangement YUNG-WU CHEN,* CHARLOTTE WORD,* VAITHILINGAM DEV,t JONATHAN W. UHR,* ELLEN S. VITETTA,* AND PHILIP W. TUCKER* From *the Department of Microbiology, Southwestern Medical School, University of Texas Center Dallas, Texas Health Science at Dallas, 75235; and *the Genetics Division, Vanderbilt University, Nashville, Tennessee 37232 Ig class switching is a process in which a single clone of B lymphocytes synthesizes IgM antibody, and subsequently synthesizes another isotype other than IgD with the same specificity for antigen . Thus, the same heavy chain variable region gene (V  ) formerly associated with the CA is gene subsequently associated with one of several constant region genes (C,,) 3' to CS . (reviewed in 1) . of reference Expression C genes downstream to Cb (e .g ., C" C') in C-,, transformed cell lines involves deletion of DNA 5' to the expressed C gene (1, 2) . Studies of the structure of Ig genes in myelomas or hybridomas (reviewed in references 3 and 4) have led to the conclusion that the isotype switch is mediated by DNA recombination between tandemly repeated switch (S)' sequences located 5' to each Cn gene, with the exception of Cb production (5, 6) . Cloned cell lines, including BCL, .B l (7), simultaneously express both A and S genes in the same unrearranged DNA context as in A-producing cells, and therefore, must produce these isotypes by alternative RNA processing and/or termination of a 26-kb RNA transcript (7-11) . Mitogen- or antigen-activated cells are reported to express IgM in combination with isotypes other than S (12-18) . Southern blot analyses have raised the issue of whether these normal cells use downstream CH genes, either singularly or in combination, in the same manner as plasmacytomas and hybridomas . Yaoita et al . (17) presented evidence that CA is not deleted in surface IgM+IgE+ (slgM+slgE+) spleen cells from SJA/9 mice infected with parasites . Their finding that all C genes were retained in the germline context suggested that the expression of IgM and IgE is mediated by the splicing of an 180-kb RNA This work is supported by National Institutes of Health grants AI-11851 and CA-31534 . Address correspondence to Dr . P . Tucker, Department of Microbiology, University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd . Dallas, Texas 75235 . CharlotteJ . Word was a recipient of a Damon Runyon-Walter Winchell Cancer Fund postdoctoral fellowship . Her present address is New Department of Cell Biology, University of Mexico Cancer Center, Albuquerque, New Mexico, Abbreviations used in this paper : s, switch region ; sIg, surface Ig ; UT, untranslated region . 562 J . Exp . Men . © The Rockefeller University Press - 0022-1007/86/08/0562/18 $1 .00 Volume 164 August 1986 562-579 CHEN ET AL . 563 of the induction of avid Fc receptors for IgE after infection transcript . Because parasites, there is a strong possibility that the majority of SJA/9 mice with such sIgE+ cells were not synthesizing IgE, but were absorbing of the ^-10% it from medium . This hypothesis is strongly supported by recent the culture data of et . Studies by Perlmutter and Gilbert (18) also support Katona al (19) . the long- transcript model . Using a combination of cell sorting, Southern blotting, and sandwich RNA hybridization, they concluded that sIgG I' splenic cells have long RNA transcripts generated from unrearranged DNA that contain both -y and A sequences . However, as in the case of the report by Yaoita et al ., their interpre- tations depend upon cell homogeneity . For example, they claimed have to successfully sorted 1-3% sIgG1 + cells to 99% purity, yet no data were shown to estimate the level of contaminating sIgM+ cells in their sIgGl' population . The mechanism of double isotype production could give insights into the genetic regulation of Ig synthesis in differentiating B cells . To analyze such mechanisms requires a constitutively expressing clonal analogue . In our accom- panying paper, we described a switch variant derived from the BCL,B, in vitro line in which each cell expresses both surface IgM and surface IgGI and also secretes both isotypes . The IgM and IgGI share the same idiotype and use the same VDJ rearrangement . In this paper, we present evidence, based on the C gene context of several independently isolated IgM/IgGI-producing clones (col- 2 ), A -yl lectively termed BCL, .B that both and heavy chains are transcribed in germline DNA configuration from a single copy of chromosome 12 . Materials and Methods and. The of the .B, Cell Lines Subdoning derivation two BCL, subclones, BCL,2 .62 (IgM producer) and BCL, .2.58 (IgM/IgGI producer), is described in the accompanying is a sister subclone of BCL,2 For DNA paper . BCL,2.54 .58 . analysis, we have employed additional independently derived IgM/IgGI subclones of the parental BCL, .B, line . The subclone BCL, .13.92 was of the 12 BCL, .B2 subclones obtained at a frequency of 1 .35 10 -2 (12/888) by depositing one cell per well using a FACS III . The medium used for subdoning the described in the is same as accompanying paper. The subclone BCL, .6 .1 subclone of BCL, .58, derived in is a .2 the same way . The subclones BCL,.15 .9 and BCL, .15.19 were two of the seven BCL, .B 2 subclones derived similarly at a frequency of 5 x 10' (7/1429), except that no WEHI 274 SN was added to the medium . in As the case of BCL, .2 .58, all subclones except BCL, .6 .1 secrete more IgM than IgGI and preliminary data of surface immunofluorescence indicate all subclones that express sIgM and sIgG . BCL, .6 .1 secretes more IgGI than IgM (Y.-W . Chen et ., unpublished al data) . Southern Hybridization Analysis . Procedures for genomic DNA isolation and blotting were described in the preceding paper . All washings were done in 3x SSC plus % 0 .1 SDS, three times at room temperature, followed by washing in O.lx SSC, 0 .1% SDS at C 42 ° for 1 h, with the exception of the pe- (see probes) probed filter, which was washed at 65°C . Restriction endonucleases were purchased from Boehringer Mannheim Biochemi- cals (Indianapolis, IN), New England Biolabs (Beverly, MA), and Bethesda Research Laboratories (Gaithersburg, MD) and used according to the manufacturer's instructions . Electrophoresis was carried out as described in the accompanying paper (in 1 % agarose at 40 V for 18 h) . Probes. Genomic or cDNA probes were prepared by gel isolation (20) of restriction fragments from previously characterized recombinant clones, except for pyl and pe where the whole plasmids were used . To insure that no repetitive sequences were present in the probes, particularly in fragments derived from introns, DNAs were digested with various 564 EXPRESSION OF IgM AND IgGI WITHOUT GENE REARRANGEMENT enzymes and subjected to Southern analysis with "P-labeled mouse genomic DNA as a probe . Only nonhybridizing fragments were employed for further experiments . Relative positions of the probes are shown in Figs . 2, A-R and 6, S-U . Ali DNAs were labeled by nick translation (21) to a specific activity of ^-10 8 Probe A : A 700-bp cpm/tag . Pvu II/Pst I fragment of pu3741 (cDNA clone) (22) containing 18 by of C  2, the entire C 3 and half of the C4 region of y chain mRNA . Probe B : A mixture of 1 .95-kbp and 1 .25-kbp Hha I/Bam HI fragments from pB2 (genomic clone) (P . Fell, personal commu- nication) containing the Cpl, Cut, CA3, and CA4 exons of Ck . Probe C : A 900-bp Pst I/Pst I fragment of pb54J (cDNA clone) (23) containing C61, 6H, C63 and the secreted carboxyl terminus (Ss) of S chain mRNA . Probe D : A 1 .3-kbp Bam HI/Bam HI fragment of pCpl3 (genomic clone) (24) containing the carboxyl terminal membrane (M) exons SM1 and bM2 of the C5 gene . Probe E : A 1 .2-kbp Eco RI/Eco RI fragment of y3-13 (genomic clone) (25) containing CS-Cy3 intronic sequences . Probe F : A 1 .0-kbp Eco RI/Eco RI fragment of y3-13 (25), located downstream of probe E. Probe G : A 2.0-kbp Hind III/Hind III fragment of pH311 (subcloned from genomic phage 'y3-25) (25) containing 5' flanking region of the Cy3 gene . Probe H : A 1 .0-kbp Xba I/Hind III fragment of pJW7 (genomic clone) (26) containing the 5' flanking region of the Cy3 I : II/Sph I fragment of pDRI (genomic clone) containing gene . Probe A 473-bp Bste (26) region (UT) the Cy3 gene . Probe J : A 1 .4-kbp Xba most of C.3 and 3' untranslated of (26) containing Cy3, segment and 3' I/Hind III fragment of pJW7 (genomic clone) M2 K : 1 .0-kbp Sac I/Hind III fragment of yl-6 (genomic clone) (25) containing UT . Probe A of Cy1 gene) . Probe L : A 1 .7-kbp the flanking region 5' to SyI (switch region Xba I/Eco containing the flanking region Cyl RI fragment of pyl (genomic clone) (27) 5' of the region) . Probe M : pyl (27) ; pBR322 plasmid containing a 6.6-kbp gene (3' to the Syl and its 5' and flanking regions. Probe N : A .1- Eco RI insert of the entire Cy1 gene 3' 2 Xba I/Sac I fragment of pyl (genomic clone) (27) containing the C'yl structural kbp gene . Probe O : A 1 .2-kbp Sac I/Eco RI fragment of pyl (genomic clone) (27) containing flanking region of the C71 gene . Probe P : A 1 .0-kbp Bam HI/Kpn I fragment of the 3' (cDNA clone) (28) containing the entire coding sequence ofy2b chain message . py2b(11)' Probe Q : pE (29) ; pBR322 plasmid containing a 4.4-kbp Bam HI insert of the CE structural flanking regions . Probe R : A 900-bp Bste II/Bste II fragment of gene and its 5' and 3' 8 (cDNA clone) (30) containing Cal, Ca2, and most of Ca3 of a chain mRNA . paU558)' Probe S : A 2.0-kbp Bam HI/Eco RI fragment of PJ 3J 4 (subcloned from genomic clone CH4A-142 .7) (31) containing J.3 J,,4 and -1 .5 kb of 3' flanking sequence . Probe T : A 674-bp Eco RI/Hind III fragment located just 3' to probe S subcloned from genomic clone CH-28.289 .1 (27 ; P . W . Tucker, unpublished data) . Probe U : A 760-bp Hind III/Hind III fragment located just 3' to probe T (subcloned from genomic clone CH- .289 .1) (P . W . Tucker, unpublished data) . Probe V : 1,774-bp Sph I/Bam HI fragment of pDRI (26) containing the entire C, 8 coding region and 3' UT of the secreted terminus . Karyotyping. Cells from the BCL,B, and BCL, .2.58 lines were grown to exponential phase and cells were harvested for karyotypic G-banding analysis (32, 33) . Densitometry . For band quantification, autoradiographs were scanned with a densitom- eter (model GS 300, Hoefer Instruments), and densities were calculated using a Bio-Rad model 33E2A integrator . Results Carry Two of BCL, .B, and BCL, .2.58 Cells Copies a Translocated Chromosome of of in vivo BCL, line in 12 . Two reports (34, 35) the karyotype the cell were to the copy number and constitution of the IgH-bearing conflict with respect 12 (36) . The single J,, rearrangement observed in the Eco RI blots chromosome in vitro lines (Fig . 5 of companion report) was consistent with either a of the single copy of VDJ or multiple copies of the same VDJ . Resolution of this question, imperative for evaluation of the Southern blotting data presented region derived so, the or Nesbitt been gene(s) XV Ca-Cb variant slides Cb IgGI, fragment, 2from have 1two the light Cy BCL, to within the the composite summarizes required breakpoint region of BCL, locus established insure proximal Alleles in due for that t(12 in Fig from translocated The an region, 12 BCL, chain It whether DNA and lines and an 65 Light BCL, Southern C6 number appears Determining 16 12, 12 unusual 12, cluster to spans Only paper derived 3, insuring IgM-secreting subclones modal cells both the aeither analysis Are (data was per aFranke gene although but context germline Bam careful rehybridization chromosomes on chain (16133) random the absence was the Present the these analyzed) We germline found Therefore, we cell isotypes (Fig the chromosome that in translocation blotting not chromosome (37) one HI or IgM/IgGI-producing probes are expression that performed S,u have found of BCL, cytogenetic (39) expression breakpoint (lanes the normal of These two shown) to at cleaves 1their CH indistinguishable or region, (Fig in loss of BCL,B, in no j,,-C, the the the shown carry subclone every two map analysis and repetitive fragment Germline germline Presumably, We that 4-6, rearrangement of definitive heavy 1(Fig two centromere, microscopic chromosome CH was b) of copies within Gene Similarly, of and this of karyotyped 12 38) restriction by the to cell number the that and (X3) 8) of evaluation this Likewise, chromosomes A locus, Shimizu 1is62 chromosome chain Giemsa of a(lane c) chromosome the and The Context same As in karyotyped Configuration in sequences bands all of could 10 blot in one BCL, Cy, (with band One vitro the expected translocation BCL, an yfrom breakpoint this we all expression in subclones 3), Eco serologically 1generating to of level digestions based distal et banding apparently 25 in of genes are had BCL, 16 of 55-63 conceivably of location have BCL, DNAs BCL, Cb the would al RI those BCL, BCL, 25 3' the cells BCL, present (probe both taken appear (25) from 16 region digests on cells fragment IgM/IgGI-producing during iscarried in chromosomes cells (Materials from [t(12 of in allelically on techniques the From translocate on vitro-adapted is employed t(12 aConsiderable line place has BCL, the BCL, identical and defined C) was chromosome 5' germline shown (lane to (12F2), chromosome synthesize in probed ideogram be the BCL, detected only published 24 to has out and be BCL, that BCL these 63 between generated the preparation 7), out and excluded identical, chromosomes two (57-66 which in one and an several idiotype telomeric translocated with extends One for and The rearranged 9in of Fig results, Methods) both proposed extensive copies the t(12 BCL, parental care (lane Cy maps 52 25 and normal respect CFc 16 in carries 16 modal 2other chro- from 10 cells cells and, gave sub- IgM four and is into and and was has the To 2), 19 of of to 5' at to of in CHEN ET AL . below, and Chromosome .2.58, . clones, chromosome .B, . .2 .58 analyzed) mosomes . .2.58 cells .B, . .B, chromosome . both ;16)], .58, the . . .2 carry . . ;16) ;16), probably the . ;16) .B, . identified .2 .58 chromosome . The the . a) . (36, by . genes chromosome not . .B, .B is chromosome V, either .B, .B2. In Strategy .2.58 accompanying and determine if overlapping .B,, .2 .58 independently . Fig . to . . taken . All . As . lane . .4-kbp .5-kbp fragment . .B, the BCL, .62, .2.58 other .B2 .5- the kbp C6 only .5-kbp EXPRESSION OF IgM AND IgGI WITHOUT GENE REARRANGEMENT r C7 cv . bp o .= ci o r~ ~ ci ro ci c n . 'ro C M . n . o bo .- o c n a. v . CL, V .O bA~ GV G'l C . . .b -?:- .. !v v .ro ..O Ca ~ C: , CL E c , c O cD L y L CO "O c O bp.-7 G. v v"'E°-E .roE~ " o L (O U cE o c a - C ch C O,> ai y, "C blJ f,- a c v C, ct 00 v r ^ v CHEN ET AL . 56'7 ~ = R v oro p . ° R .G v v y~~~roE v o c y 0.% .C RO¢bco GO ^'h E v .n ~v(U o N q 0 a 0. O L ~ 3 v ?IN nn N ¢ N ro of vE .SvVG"'O ~ ." >. bo C L iR . C wm zlw N c6 G C H 4: 'O w bOA .~ ~~ 7 ro v "C ¢ '7° N N v'b>scv `v'v . v L .C G O- e 0 J O a 'c" N v X v 'f ro b0 L W H ro v y A E ~ u-CS Y G R .Y H 4. ti N m -° _° lam u~ o v ' L ~ v N ro G L a nn h .,_ a N m R R Y y~ L , ~ .+ V rq " ti O ° v 3 G L Y N v roc:.ocE >. C 0. .O . C Aa 0 -0 . rozma mo ._i E a o ro vV L '0 ` ^ CC (d ft o. O C O a O." us E,7 0. .T b4~i, Y m 0' .n i c0 w G. C m n cC ro 't T. t0 ~ L y G y. m m ~o m E 2 bbc G ..+ v f v .n E u ^ o O E O .~Lro 'G v L ti c . y G C W n h a Y o v R W v m L croc Nc :: O y ti .b n v a R U G " G ice. R 7 o 0. w U 'O 'O b0 bA Gs ~ c0 . v ` a w a F z ., ~ cs m .n c` a .o uroc N~ ; . v .° C R o.~ os -o n 0 m z prn s o 'IV o > o . E^G G.O m 0 O z m R W .V v I C ti v R ._. v L C N O 4J N bbO m j N E O Of o z ° ro m 3^E v, -0 7 v. .d y )ac O .~ E b0= y, L 0 N E G O C' T v O b0 L N O N R N scLVE yY al W a` W a n bc ~O > .'C + Lo O ro °~ O O u G . m N o F ''b L ° X c;aNV oxy y ~=R i~ .LAL ,Gn a ..ro v .t R yG" ~-°' o J ~~ cd R k,~c .=voosE 568 EXPRESSION OF IgM AND IgG1 WITHOUT GENE REARRANGEMENT Analysis FIGURE 3 . of Cp gene context . High molecular weight DNAs were digested with Bam HI (see Figs . 2 and 6 for relative location of sites), and were fractionated and blotted as described in Materials and Methods, then were hybridized with Cy (probe B) . Lane 1, BALB/c liver DNA ; lane 2, BCL, .B1 ; lane 3, BCL, 4, .2 .62 ; lane BCL, .15 .19 ; lane 5, BCL, .15 .9 ; lane 6, BCL, .13 lane 7, BCL, .92 ; .2 .58 ; lane 8, BCL, .2 .54 . kb markers are shown in margin . conjunction with data from Kpn I digestions presented below, we conclude that no rearrangement involving Su, Cu, or Cb alleles contribute to double production j and in BCL I .B 2 . of yl Genes 3' to C), including Cy,, are in Germline Configuration . The lack All C suggested that y, expression in BCLI .B2 of Cg. rearrangement subclones does occur class-switch recombination . To resolve the issue directly, not by prototypic of probings, which overlapped the entire we carried out an exhaustive set CH and C,., well the DNA region (^-100 kbp) between Cb as as downstream to C,, . The complete analysis was performed on Balb/c liver, BCL I .B I , BCL, and .2.62 BCL, and partial analyses were carried out on additional BCLI .B 2 sub- .2 .58, clones . The data are summarized in Fig . 2, with the most crucial blots for the region between and C,. I presented in Fig . 4 . Digestion with Hind III generates C,3 a diagnostic fragment (fragment 15 in Fig . 2) which spans the 1 switch region 1' and generally detects rearrangement of this locus . This is shown in Fig . 4 a by the two rearranged bands (15 and 16 kbp) in the control digestion of an IgG I secreting hybridoma (lane 1) . In contrast, we detect only the germline band of C,, (23 kbp) and the more faintly crosshybridizing germline fragments of C,26, C., and (9 .2, 6 .6, and 6 .4 kbp, respectively) in liver (lane 2), BCLI IgM s C,2, producers (lanes 4 and 5) and BCL, .B 2 (lanes 3, 6-8) . Likewise, with the more upstream probes L, K and J (Fig . 4,b-d), the BCLI and BCL I .B2 patterns are identical to those of the liver . Although no rearrangement occurred between y3 and y,, it still remained y l might possible that expression of be affected by a recombination between b and y3 . For example, a deletion within this region would appreciably reduce the length of a putative J r-Ca-Cy transcription unit . As summarized in Fig . 2, we detected no rearrangements in BCL I .B, and BCLI .B 2 relative to liver within this exception of only 7 .9 kbp of repetitive DNA ^-55 kbp area with the (between fragment 4 and 7) was difficult to confirm . Similarly, in the BCL I .B 2 cells, that downstream genes from Cy 1 to Ca remain in germline context . Taken together, these results strongly argue that DNA rearrangement of genes is not respon- CH sible for expression of y, in BCL, .B 2 cells . Deletion of the Majority of the C Locus Has Occurred on the Nonproductive Chromosome 12 of BCL I.B2 . As noted above, the Cit and Cb genes of both BCL I .B I BCLI .B 2 are unrearranged and are linked on a Barn HI fragment and FIGURE 4 . C, genes and the region between C,s and C,, are unrearranged in BCL, .B2 double producers. (A) Hybridization of a Hind III digest (Fig. 2, fragment 15) to C,, probe M. Lane 1, 9B 12 (IgG I-secreting hybridoma) ; lane 2, BALB/c liver ; lane 3, BCL, .2 .58 ; lane 4, BCL, .B, ; lane 5, BCL, .2 .62 lane ; 6, BCL, .2 .54 ; lane 7, BCL, .13 .92 lane ; 8, BCL, .15 .9. (B) Hybridization of an Xba I digestion (Fig. 2, fragment 14) to C.3-C ., intronic probe L . Lane 1, BCL, .2 .62 ; lane 2, BCL, .B1 ; lane 3, BCL, .2 .58 ; lane 4, BCL, .6 .1 ; lane 5, Balb/c liver DNA . (C) Hybridi- zation of a Sac I digestion (fragment 13, Fig . 2) to C,3-C,, intronic probe K . Lane 1, BCL, .B, ; lane 2, BCL, .2 .58 ; lane 3, BCL, .6 .1 ; lane 4, Balb/c liver DNA . (D) Hybridization of a Kpn I digestion (fragment 12, Fig . 2) to C,s 3' probe J . Lanes are as in B. The additional bands seen in Fig 4C (13 .1 kbp) and 4d (5 .6 kbp) are residual hybridization from the previous probings of the same filters . kb markers are shown for A-D. IgM AND IgGI WITHOUT GENE REARRANGEMENT 570 EXPRESSION OF carry two different alleles of chromosome 12. Southern blots of FIGURE 5 . BCL, .B2 clones QH3- Fig . 6 for positions of sites) were hybridized in A to probe S Kpn I-digested DNAs (see BCL, .62 ; lane B Lane 1, BALB/c liver; lane 2, BCL, .B1 ; lane 3, .2 JH 4) and in B to probe (Cu) .58 ; lane 8, BCL, .2 .54 . lane 5, BCL, .15.9 ; lane 6, BCL,A3 .92 ; lane 7, BCL, .2 4, BCL, .15.19; -y ju l- (lanes 1-3, genes are not linked in A only (lanes 4-6, BCL, .2 .62) and (C) and C,, JH I were hybridized to producing subclones. DNAs were digested with BgI and BCL,2 .58) JH S and V (lanes 4), (probe V) (lanes 3 and 6), and a mixture of probes (probe S) (lanes 1 and C,s gene lanes 2, 3, 5, and 6, the 17 kbp and 7.5 kbp fragments were C,2n 2 and 5) . In Fig . 5c, W. The 6.2 kbp fragment is the Bgl I fragment containing crosshybridizations with probe located on the H' allele (see Fig. 6) . kb markers are shown . VDJ sequences chromosome . But a single, lines contain two copies of the t(12;16) (Fig . 3). Both RI region rearrangement is observed when Eco apparently identical variable probed with a region BCL I .B I and BCL I .2.62 DNA are JH blots of BCL I .2 .58, accompanying paper) . In addition, a single fragment (probe S and Fig . 6 of Hind III, Bam HI and Bgl I -containing band was obtained in Xba I, VDJ H2 and subclones probed with (accompanying paper digests of all the BCL I .B 2 JH BCL I .B 2 allele possessed an not shown) . These data suggested that each data a rearrangement rearranged VDJ . However, Kpn I digests revealed identically with respect to BCL I .B I . As shown in Fig . 5a, of one of the two BCL I .B2 alleles I fragment found in BCL I .B I (lane the rearranged 17 .8-kbp Kpn in addition to in BCL I .58 (lane 3), we detected a 12 .5-kbp fragment .2 2) and BCL I .2 .62 (lane present in the other BCL I .B 2 subclones 7) . The identically rearranged band is although independently cloned, suggesting that all BCL I .B 2 lines, (lanes 4-6, 8), was parental variant . When a similar Kpn I blot were derived from a common hybridizing band, corresponding to the with Cu (Fig . 5 b), only a single probed CHEN ET AL . 571 from the unrearranged allele of BCL1 .B 1 , was detected in 17 .8-kbp fragment rearrangement more precisely, we performed similar Kpn BCL, .B 2 . To map the JH- U) that extend directly from the Eco RI site in the I blots with probes (T and . only the 17 .8-kbp fragment hybridized (data not shown) . Cp intron In all cases, a single Eco RI band in J3J,,4 blots, the rearrangement had Since there is only occur just 3' (<50 bp) to the Eco RI site in the intron Otherwise, the to J,,-Cp BCL, .B 2 . .5-kbp band would have been detected by probe T or U in that the .5-kbp Hybridization of the Kpn I digests to probe M had shown 12 there are a number fragment did not contain C,., (Fig . 2, fragment 18) . However, there is slight Kpn I sites between C,. 3 and C,, in germline DNA (25), and a of 4 . Therefore, possibility of fragment comigration in the analysis of Fig . we that generates a digested BCL, .B, and BCL 1 .B 2 DNAs with Bgl I, an enzyme . fragment 20a), fragment that spans the entire C,3-S,, germline region (Fig 2, of the Eco RI cuts at a single site between and C, -480 by downstream and JH unpublished results) . As seen site of the rearrangement junction (P . W. Tucker, with (probe S) and C, 3 in Fig . 5c, hybridization either separately or jointly JH . These data (probe V) gave different sized bands (30 and 32 kbp, respectively) of IgM/IgGI producing prove thatJ and C,., are not linked on either allele the variants . yl Allelically Excluded. The Simultaneous Expression of u and in BCL I.B is analyses of the C genes, suggest that the observed above results, taken with the BCL, .B 2 is unusual . It deletes, at a minimum, rearrangement on one allele of by direct inference of the Ckt-C6 linkage (Fig . part of J-Cy intron, all Cy and, patterns (Figs . and 4) and the analysis of Fig . 3), Cb . However, the germline 2 this rearrangement is not accomplished by recombi- 5 c show unequivocally that sequences . Therefore, the 3' side of the rearrange- nation into downstream C to be unrelated to Ig CH DNA, yet it is not large ment in BCL I .B appears cytogenetically . enough to be detected map of two IgH alleles in BCL 1 .B 2 is shown in Fig. 6 . The A proposed the downstream C genes on the productive chromosome (i .e ., model predicts that H+ allele) exist in single copy in BCL I .B but in double copy in BCL1 .B1, on the or liver . On the other hand, J genes should exist in double copy in BCL, .2 .62, parental, variant, and germline DNA. To test this hypothesis, we hybridized Eco digests (Fig . 7a) of the appropriate DNAs to a mixture of (probe S) and RI J,, (probe V), and Hind III digests (Fig . 7b) to a mixture of (probe S) and C,, J,, C,-3 (probe M) . The hybridization intensities were quantified by densitometry and Fig . b) the ratio ofJN/C, 3 (4 kbp:18 kbp, Fig . 7 a) andJ (2.55 kbp:23 kbp, 7 /C,1 ratios in BCL, .B,, were determined . As shown in Table I, the /C,, 3 and J,,/C,1 in the IgM/IgGI-producing variants, BCL, .2.62 are approximately twice those conclude allelic exclusion is operative in BCL, .2.58 and BCL, .13.92 . We that and chains from a single rearranged VDJ. BCL I .B in generating both 2 u 'y, Discussion Two conclusions can drawn from the results of our studies : (a) IgGI major be expressed in the BCL I .B clones in the absence of DNA rearrangement of the is constant region genes ; and (b) the heavy chains of both IgM and IgGI are 572 EXPRESSION OF IgM AND IgGI WITHOUT GENE REARRANGEMENT L~ .b0 .~ F" i.. ° x `n-- 4- ,L s . ~- C .O ro "O + yam'. v > ro a v ._ .- u w ~ CHEN ET AL. 57 3 FIGURE 7 . Quantitative analysis of J , and C,, gene context . (A) Southern blots of Eco C,3, and C,3 RI digests were hybridized separately or with a mixture of J" probes . (B) Hybridization and C,, . Lane 1, BALB/c liver ; 2, BCL, of Hind III digests toj. lane .2 .62 ; lane 3, BCL, .B1 ; 4, .13.92 lane 5, BCL, .2 .58 . Densitometry of the appropriate bands is lane BCL, ; summarized I . are for and in Table kb markers shown A B. TABLE I Allelic Exclusion Is Operative in BCL, .B 2 Double Producers Cells IgH /C,,* JH/C,3 * J BCL, .2 .58 A'y1 1 .25 0.80 BCL, .13 .92 wf1 1 .48 0.77 BCL, .2 .62 A 0.74 0.44 BCL, .Bl A 0.70 0.49 BALB/c liver - 0.71 - * Densitometry was performed on the 4 kbp and 18 kbp bands of Fig. 7a and the 2.55 kbp and 23 kbp bands of Fig. 7b . Ratios represent the average of two JH/C, and three JH/C,, determinations . expressed from a single rearranged VDJ segment on the same chromosome 12 . Therefore, we conclude that simultaneous production of IgM/IgGI in BCLI .B2, as in the case of IgM and IgD in BCL I .B l , is controlled exclusively at the RNA level . Our findings provide the first example of allelically excluded, double isotype expression in a neoplastic B cell clone . A similar conclusion was drawn to explain dual u and ti2b production in a derivative of a 1A-producing Abelson murine leukemia-transformed cell line (40) . Contrary to the initial report, it is now clear (41, 42) that -y2b synthesis in this line is accompanied by deletion of sequences and C,,26 , and thus cannot between be explained by differential RNA process- JH Our support the . ing . data also observations of Yaoita et al (17) and Perlmutter and Gilbert (18), who used purified populations of normal lymphocytes. The caveats associated with these studies (19) are overcome here since we have shown that virtually all cells in BCLI .2.58 synthesize, express, and secrete IgM and 574 EXPRESSION OF IgM AND IgGI WITHOUT GENE REARRANGEMENT IgGI . Furthermore, the clonal BCL, .B 2 lines have provided the opportunity for a more extensive analysis of the DNA context . In addition to establishing germline configuration of sequences anticipated to undergo rearrangement during switch recombination (e .g ., S,, C, and we have also shown that the S,,), remainder of the C locus is unrearranged on the productive allele . This observation argues against the hypothesis that CH genes might be translocated, via nonclassical switch sequences and deletion of Cb , to a position downstream to C,, possibly replacing b with another isotype (7, 11) . Although we have provided strong evidence for use of a single VDJ, it resides on a chromosome 12 that has undergone an unusual translocation, distal to the Ig locus, with chromosome 16 . At the microscopic level, the t(12 ;16) appears to be identically duplicated in both the BCL, .B and BCL, .B 2 cell lines . However, our blotting data define the nonproductive t(12 ;16) allele (H - in Fig . 6) by virtue loss Ig-related sequences 3' to the Eco RI within of its of site the JH-CA intron . These findings have important implications in considering the both derivation the in vivo BCL, leukemia, its subsequent in vitro adaptation, and the eventual of for allelic exclusion in BCLI .B 2 . The in vivo BCL I mechanism leukemia cells appear to have a stable karyotype, with the exception of chromosome 12 . Two early reports (34, 35) are in agreement with our data (Y.-W . Chen and G. V. Dev, unpublished results), with respect to the modal chromosome number (35 chromosomes in reference 34 and our data, and 36 chromosomes in reference 35) . However, Schroeder et al . (34) reported a single normal chromosome 12 and a translocated chromosome 12 to an unidentified recipient in early passages of the line . One year later, Voss et al . (35) found no evidence for normal copies ofchromosome 12, but identified three translocated alleles . We observed a single copy of the same t(12 ;16) in the in vivo line (Y .-W . Chen and G. V. Dev, unpublished results) that is duplicated in the in vitro BCL, .B, and BCL, .B 2 lines, both of which have a modal chromosome number of --60 . We suggest that the precursor to the original leukemic cell carried a normal chromosome 12 as nonproductive and retained it during early passages, at which time the productive underwent translocation(s) . Adaptation to culture chromosome 12 cell resulted of of the karyotype, including ;16), and may in a duplication most t(12 have more subtle event (e .g ., somatic which required a second mutation), was unde- DNA tectable at the cytogenetic or rearrangement level, to effect allelic exclusion in BCL, .B, . Alternatively, both of the t(12 ;16) alleles remain active in BCL, . If the former is true, then the third event, the rearrangement 3' to VDJ on the H- allele of BCL, .B 2 , is not required for allelic exclusion, but may instead reflect some trans-acting regulatory requirement for switching the productive allele ,u/-y from ,u/b to 1 . The significance of this DNA rearrangement is underscored by the fact that all independent BCL, .B 2 variants maintain it . Aside from its unusual karyotype, do the BCI., .B2 variants have a counterpart amongst normal B cells? Based on its surface Ig phenotype, its low level of constitutive IgM secretion (reviewed in reference 43), and its heavy chain gene transcription profile (44), the parental BCL, .B, line appears to represent an immature B cell . It has been employed as a model for numerous functional further cultured studies by virtue of its ability to undergo differentiation when CHEN ET AL . 57 5 all LPS (45), anti-Ig (46), or T cell-derived lymphokines (47) . The fact that with .B2 variants isolated for IgG secretion secrete both IgM and IgGI, as well BCL, express them on their surface, may reflect their derivation from a single as precursor . It may also denote a basic difference between these cells and the nonsecreting memory cell populations analyzed by others (17, 18) at the molec- differences in mode of expression are ular level . Regardless of whether these transformed the BCL, .B 2 phenotype real or are a consequence of the state, and the membrane form of mRNA shows that high expression of the secreted from genes in germline configuration . can be generated C mechanisms, the only plausible explanation for the Finally, with regard to and yl mRNA are expressed with a common VDJ gene by present data is that y RNA processing . This could be accomplished by a discontinuous alternative in which the RNA polymerase translocates from the template to mechanism discrete segments of DNA, as proposed for trypanosome variable transcribe antigen (48) and certain viral genes (49, 50) . Alternatively, there could be a continuous transcription mechanism (i .e ., long transcript model), such as that apparently used by the drosophila bithorax locus (51), which has been invoked (17, 18) to account for their results in normal B cells . In the latter by others 1 chains could then be translated from mRNA derived by scenario, the y transcript that includes sequences from both Cp and Cy 1 processing a primary derived either from an identical transcript of -120 genes . The t mRNA may be terminates in the intervening sequence between CU and CS . kb or from one that studies in BCL, .B, cells (44) and other IgM/IgD Based on nuclear transcription favor the latter alternative . This would require double producers (52, 53), we transcriptional termination, perhaps at two points . differential regulation of would in First, the majority of the RNA polymerases unload 3' to Cu, since yl RNA BCL, .B 2 cells, A RNA abundance is greater than abundance (accom- small polym- panying report and data not shown) . Second, a proportion of the erases would read through the 1 termination region and exit the template in BCL, .B 2) . A in downstream of the CH gene to be expressed (Cyl choice the second polymerase unloading event may dictate (and simplify) not only the subsequent RNA splicing decision, but may also bear on the acquisition of isotype commitment ; i.e ., once a cell expresses an isotype other than IgM and IgD, it is committed and restricted to secretion of that isotype upon stimulation (54) . then clonal example of this model is the 1 .29 B cell lymphoma, which The best undergoes spontaneous and/or inducible switch recombination from IgM to IgA The Ca gene is preferentially open, as defined by hypomethylation, in the (55) . cells that are committed to switch to this isotype (56) . Similarly, IgM-synthesizing BCL1 to BCL, .B2 , Cyl appears to be exclusively selected . in the transition of transcription through, and equally important, termination beyond a C Perhaps .B 2) critical in inducing an active chromatin structure for gene (C,, in BCL, is C gene . The resulting prediction is that subsequent expression of that same receiving appropriate signal, would undergo exclusive BCL, .B 2 cells, on the C,,, . This cell line, therefore, provides us with the switch recombination to the mechanisms of double isotype production and its opportunity of testing consequences . 576 EXPRESSION OF IgM AND IgGI WITHOUT GENE REARRANGEMENT Summary In our accompanying paper, described variant (BCL we a switch I .2 .58) that of IgM . IgM IgGI expresses membrane and secreted forms and IgGI Both and share same and use VDJ rearrangement Here, a the idiotype the same . detailed blot analysis of the entire constant region of the Ig heavy chain (Ig Southern C  ) parental (BCL I .B I ) and variants (BCL I .B2 ) DNA showed detectable locus of no rearrangement . Similar analysis of the JH-C/ region led to the conclusion that two heavy chain alleles present in the IgM/IgGI-producing variants carried the same VDJ rearrangement but differed in their 3' flanking regions . One chro- mosome 12 did not carry any Ig C F, genes, whereas, the other chromosome 12 carried one copy of C genes . In BCL I .B I , however, each of the chromosome 12 alleles carried a full copy of C  genes . Karyotypic analysis confirmed the presence of two translocated t(12 ;16) chromosomes in both BCL I .2 .58 and BCL I .B I cells, with a break 5' to the V  locus at the distal region (12F2) of chromosome 12, and at the proximal region below the centromere (16113) of chromosome 16 . We conclude that double production of IgM and IgGI in BCL I .B 2 is accom- plished by transcription of the corresponding C  genes in germline configuration 12 . using a single VDJ on the same chromosome We thank Ms . M.-M . Liu, Ms . C . Das, Mr . J . Rybak and Ms . C . Glover-Humphries for excellent technical assistance and Ms . M . Gardner for secretarial assistance . We thank Dr . P . Howard-Peebles for assistance with the cytogenetics and Dr . D . Yuan for assistance with desitometry . We are grateful to Dr . T . Honjo for providing phages bearing Cy intronic sequences . February in revised May Receivedfor publication 27 1986 and form 6 1986. in of sandwich hybridization and nuclear run-on transcription Note added proof . Results consistent with the long transcript model for dual II. and yl expression in BCL I .B 2 . are References 1 . Marcu, K . B . 1982 Immunoglobulin heavy-chain constant-region genes. Cell . 29:719 . Honjo, T ., and T . Kataoka . 1978 . Organization of immunoglobulin heavy chain 2 . and allelic deletion model . Proc. Natl. Acad . Sci . USA. 75:2140 . genes Marcu, K ., and M . Cooper . 1982 . New views of the immunoglobulin heavy chain 3 . switch . Nature (Loud.) . 298 :327 . 4 . Shimizu, A ., and T . Honjo . 1984 . 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Journal

The Journal of Experimental MedicinePubmed Central

Published: Aug 1, 1986

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