Conserved Sequence (B Lymphocyte-Specific Gene Regulation/Promoters) TRISTRAM G
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Proc. Nad. Acad. Sci. USA Vol. 81, pp. 2650-2654, May 1984 Biochemistry Structure of the 5' ends of immunoglobulin genes: A novel conserved sequence (B lymphocyte-specific gene regulation/promoters) TRISTRAM G. PARSLOW*t, DEBRA L. BLAIRt, WILLIAM J. MURPHYt, AND DARYL K. GRANNER* Departments of *Internal Medicine and Biochemistry and the tDiabetes and Endocrinology Research Center, Veterans' Hospital, University of Iowa College of Medicine, Iowa City, IA 52240 Communicated by Leonard A. Herzenberg, December 30, 1983 ABSTRACT Recent investigations have suggested that tis- ed in the mechanism of DNA rearrangement (2). In addition, sue-specific regulatory factors are required for immunoglob- each V gene harbors at its 5' end a functional promoter (5), ulin gene transcription. Cells of the mouse lymphocytoid pre- which can serve as the site of transcriptional initiation in a B-cell line 70Z/3 contain a constitutively rearranged immuno- fully assembled heavy or light chain gene. The precise se- globulin K light chain gene; the nucleotide sequence of this quences required for promoter function in these genes have gene exhibits all the known properties of a functionally compe- not yet been elucidated. tent transcription unit. Nevertheless, transcripts derived from We investigated the structure and expression of an immu- this gene are detectable only after exposure of the cells to bac- noglobulin light chain gene in the mouse leukemia cell line terial lipopolysaccharide, implying that accurate DNA rear- 70Z/3. Under ordinary growth conditions, cells of this line rangement is not sufficient to activate expression of the gene. constitutively express cytoplasmic A heavy chains without Comparison of the sequence of the 70Z/3 K light chain gene associated light chain synthesis, a phenotype characteristic with those encoding other immunoglobulin heavy and light of the early stages of B-lymphocyte ontogeny (6-8). Al- chains has revealed that a distinctive promoter region struc- though these cells harbor a single rearranged K light chain ture is characteristic of this multigene family. The sequence A- gene (7), they ordinarily contain neither K light chain protein T-T-T-G-C-A-T lies approximately 70 base pairs upstream nor its corresponding mRNA. When grown in the presence from the site of transcriptional initiation in every light chain of bacterial lipopolysaccharide (LPS), however, 70Z/3 cells gene examined; in heavy chain genes, the corresponding loca- accumulate cytoplasmic K light chain mRNA (8) and begin to tion is occupied by the precise inverse (A-T-G-C-A-A-A-T) of synthesize K light chain protein; after 12 hr of optimal LPS this sequence. Although adjacent regions of DNA have di- treatment, 70-100% express both heavy and light chain de- verged extensively in evolution, these octanucleotide sequences terminants on their surfaces (6). Therefore, the 70Z/3 cell are stringently conserved at this location among diverse immu- line can serve as a model system for the study of several noglobulin genes from at least two mammalian species. The critical events in early lymphocyte differentiation, including proximity of this conserved octanucleotide block to the site of K light chain gene activation and the induction of surface transcriptional initiation suggests that it may serve as a recog- immunoglobulin expression. nition locus for factors regulating immunoglobulin gene In this report, we present the complete V region and 5' expression in a tissue-specific fashion. flanking DNA sequences of the rearranged K light chain gene of 70Z/3 and further characterize the effects of LPS treat- The extraordinary diversity of immunoglobulin molecules is ment on the expression of RNA transcripts derived from this due, in part, to the existence of a large and heterogeneous gene. In addition, we identify a highly conserved octanucleo- family of genes encoding variable (V) region domains of the tide sequence at the 5' ends of diverse immunoglobulin V heavy chain and light chain polypeptides. The formation of a genes and present evidence that a distinctive promoter re- complete immunoglobulin transcription unit requires specif- gion structure is characteristic of this multigene family. ic DNA rearrangements that fuse a single V gene, along with its 5' flanking sequences, to separate genetic elements cod- MATERIALS AND METHODS ing for the remainder of the polypeptide chain. In the case of Cell Culture. 70Z/3 cells, a gift from R. P. Perry, were the murine K light chain genes, these rearrangements link the grown in suspension culture as described (8) in the presence selected V gene to one of fourjunctional (J) elements located or absence of LPS from Salmonella typhosa (10 ,ug/ml; upstream of the K light chain constant (C) region gene, CK (1, Difco). LPS treatment had no effect on either the doubling 2). Analogous DNA rearrangements are required for assem- time (12 hr) or the total polyadenylylated RNA content of bly of an intact heavy chain gene (3). these cells. Although their nucleotide sequences differ widely, all c Light Chain Gene Isolation and Sequence Determination. functional V genes share certain common structural features DNA was prepared from the nuclei of untreated 70Z/3 cells and are believed to have evolved from a single ancestral se- as described (9) and digested to completion with EcoRI. Di- quence. Each comprises two coding regions (exons) separat- gested DNA (70 jig) was layered over a linear 10-40% su- ed by a short intervening sequence. The first exon encodes a crose gradient and centrifuged for 24 hr at 20°C in an SW 27 hydrophobic signal peptide; the second specifies -95 amino- rotor. Fractions containing restriction fragments greater terminal residues ofthe V domain. Each exon contains splice than 14 kb in length were pooled and concentrated by etha- junctions for RNA processing and specifies a few invariant nol precipitation. The DNA was then ligated into the EcoRI amino acid residues thought to be essential for proper func- arms of the bacteriophage Charon 4A, and a clone library tion of the protein (4). Two short-sequence elements found was prepared by the method of Maniatis et al. (10). This li- near the 3' ends of unrearranged V genes have been implicat- Abbreviations: LPS, bacterial lipopolysaccharide; bp, base pairs; The publication costs of this article were defrayed in part by page charge kb, kilobases or kilobase pairs; V, variable; J, joining; C, constant. payment. This article must therefore be hereby marked "advertisement" tPresent address: Department of Pathology, University ofCalifornia in accordance with 18 U.S.C. §1734 solely to indicate this fact. at San Francisco, San Francisco, CA 94143. 2650 Downloaded by guest on September 26, 2021 Biochemistry: Parslow et aL Proc. NatL Acad. Sci. USA 81 (1984) 2651 brary was screened for chimeric phage harboring sequences homologous to the C, region coding sequence by using the Cytoplasm Nucleus plaque hybridization technique of Benton and Davis (11); a total of 14 K light chain-bearing plaques were identified among the 68,000 plaques tested. Phage containing the rear- mapping of ranged C. allele were identified by restriction - 5.1 purified phage DNA. Portions of the cloned rearranged gene -4.4 were subcloned into the plasmid pBR322 and subjected to * -2.8 nucleotide sequence analysis by the method of Maxam and Gilbert (12). RNA Isolation. For isolation of cytoplasmic RNA, 109 -1.2 washed cells were lysed by gentle stirring for 5 min in 15 ml of ice-cold 10 mM Tris, pH 7.5/4 mM MgCl2/100 mM KCl/0.05% Triton X-100. After centrifugation for 5 min at LPS 3,000 rpm in an HB-4 rotor at 4TC, the supernatant was adjusted to 20 mM EDTA and 2% sodium dodecyl sulfate. FIG. 1. Induction of nuclear and cytoplasm c K light chain-spe- The nuclear pellet was washed in 15 ml of ice-cold 10 mM cific RNA in 70Z/3 cells exposed to bacterial LPS. RNA isolated sodium acetate, pH 5.0/50 mM NaCl and centrifuged as be- from nuclear and cytoplasmic fractions of LPS-treated and untreat- fore. Supernatants were combined with those from the previ- ed cells was subjected to denaturing agarose gel electrophoresis, ous step and then extracted twice against equal volumes of transferred to nitrocellulose membranes, and probed for C, se- phenol/chloroform, 1:1 (vol/vol), and twice against absolute quences. Cytoplasmic RNA samples contained 3 ,g of polyadenyly- The aqueous phases were adjusted to 0.5 M ammoni- lated RNA per lane; polyadenylylated nuclear RNA samples from ether. control and LPS-treated cells contained 64 ,g and 32 Aof RNA per um acetate/10 mM magnesium acetate/1 mM LDTA, and lane, respectively. Sizes of the various transcripts (lOi kb) are indi- RNA was precipitated by addition of 2.5 vol of ethanol. The cated. RNA samples were then incubated at 37°C for 1 hr with pro- teinase K (10 ug/ml) in 10 mM Tris, pH 7.5/0.5% sodium dodecyl sulfate, extracted twice against chloroform, and pre- and, presumably, represents the primary transcript of the cipitated from ethanol as before. gene. The two remaining homologous transcripts (4.4 and 2.8 Nuclear RNA was isolated from the nuclear pellets de- kb) are probably intermediate forms or by-products in pro- scribed above by cesium density centrifugation in the pres- cessing of the primary transcript (17). No such precursor ence ofguanidinium thiocyanate (13). The purified RNA was forms were detectable in the nuclei of cells grown in the ab- enriched for polyadenylylated sequences by two successive sence of LPS; the trace of mature K light chain mRNA found passes over an oligodeoxythymidylate affinity column. in nuclear RNA from untreated cells probably reflects the Hybridization Analysis of Cellular RNA. To detect K light presence of a small minority (<5%) of spontaneously acti- chain-specific transcripts, RNA samples were subjected to vated cells in the untreated population.