Transcriptional regulation of the mu-delta heavy chain locus in normal murine B lymphocytes.

The heavy chain genes for IgM (C mu) and IgD (C delta) are expressed differentially during B cell maturation and activation. We have determined the role that transcription plays in the regulation of these changes by using the method of in vitro nascent RNA chain elongation. In neonatal cells that express much lower densities of IgD than IgM on their surface, transcription of C delta is observed at half the level of C mu. This 3:1 transcriptional ratio of mu to delta is preserved in mature resting cells, which express higher densities of IgD on the surface than IgM. When activated by the mitogen, lipopolysaccharide (LPS), transcription of C mu is preferentially enhanced. However, C delta transcription is not shut off even though the expression of IgD in the stimulated cells is greatly decreased. In all three differentiative stages, polymerase unloading occurs in the vicinity of a large inverted repeat sequence, 5' to C delta and 3' to the mu membrane exons. This suggests that the developmental selection of secreted vs. membrane-bound carboxyl-terminal exons is controlled by RNA cleavage. The data presented here, together with our previous analysis of mRNA and protein synthesis, show that the differential expression of IgM and IgD in normal B lymphocytes is regulated at the transcriptional, translational, and posttranslation levels.

Effect of lipopolysaccharide stimulation on the transcription and translation of messenger RNA for cell surface immunoglobulin M.

Analysis of mu-specific mRNA in the B cell tumor line, BCL1, shows that the cells contain predominantly mRNA for mu chain of membrane-bound immunoglobulin M (IgM) (2.7 kb, mu m mRNA). Stimulation of the cells to Ig secretion by lipopolysaccharide (LPS) results in a 6-12 fold increase in amount of mRNA for the mu chain of secreted IgM (2.4 kb mu s mRNA). The increase in mu s mRNA is accompanied by a 3-4-fold increase in mu m mRNA. The rate of mu chain synthesis of membrane IgM in LPS-stimulated cells is, however, reduced by at least twofold, suggesting that both transcriptional and translational regulatory events are involved in the induction of B lymphocytes to secretion.

Extensive and selective mutation of a rearranged VH5 gene in human B cell chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (CLL) is the malignant, monoclonal equivalent of a human CD5+ B cell. Previous studies have shown that the VH and VL genes rearranged and/or expressed in CLL have few and apparently random mutations. However, in this study, we have found that the rearranged VH251 gene, one of the three-membered VH5 family, has extensive and selective mutations in B-CLL cells. Somatic mutation at the nucleotide level is 6.03% in B-CLLs whereas the somatic mutation levels are much lower in CD5+ and CD5- cord B cells, adult peripheral blood B cells, and Epstein-Barr virus-transformed CD5+ B cell lines (0.45, 0.93, and 1.92%, respectively). Complementary determining region 1 (CDR1) mutation in CLLs is particularly prevalent, and interchanges in CDRs often lead to acquisition of charge. Analysis of somatic mutations and mutations to charged residues demonstrated that the mutations in CLLs are highly selected.

T cell receptor gamma and delta gene junctional sequences in SCID mice: excessive P nucleotide insertion.

The severe combined immunodeficiency (SCID) mutation has been postulated to affect a V(D)J recombinase activity involved in coding joint formation. Analysis of 38 joints from 34 distinct sequences of normally rearranged T cell receptor (TCR) gamma and delta genes from adult, SCID thymocytes reveals coding joints with an increased number of P nucleotides. One-third of P sequences are greater than or equal to 4 nucleotides in length and P elements of up to 15 bases are observed. This suggests that the SCID defect deregulates P nucleotide addition. Consequently, essential V(D)J recombination intermediates may seldom be generated.

COOH terminus of membrane IgM is essential for an antigen-specific induction of some but not all early activation events in mature B cells.

Transfectants of mature B cell lines that bind phosphorylcholine were made in order to understand the role of the COOH terminus of the mu chain of membrane IgM (mIgM) in generation of antigen-specific signals. A chimeric receptor (I-A alpha tail) was constructed by replacing 40 amino acids from the mu COOH terminus with that of major histocompatibility complex class II I-A alpha chain. The effect of wild-type and chimeric tails were studied on representative immediate-early antigen-specific signals. The I-A alpha tail hybrid, but not the wild-type receptor, was defective in antigen-driven Ca2+ mobilization, although it could effectively endocytose ligand-receptor complexes. Signal(s) transduced through the wild-type receptor led to transient induction of selected immediate-early gene messages (Egr-1, c-fos, Jun) above basal levels. However, the signal(s) generated after crosslinking of the I-A alpha tail receptor either showed no effect (c-fos) or actually repressed basal level expression of Egr-1 and Jun. Thus, we have established that receptor-mediated endocytosis can be distinguished from other early events associated with B cell activation, based on their differential dependence upon the structural fidelity of the COOH-terminal sequence of mIgM.

Structure and expression of human germline VH transcripts.

The human VH5 family consists of two functional genes and one pseudogene. We have found a novel 1.2-kb VH5 gene transcript in normal fetal liver and cord blood and in transformed B lineage cells. VH5-positive cDNA clones were isolated from precursor B acute lymphoblastic leukemia, B chronic lymphoblastic leukemia, Epstein-Barr Virus-transformed B cell lines, and cord blood, and were identified as transcripts of unrearranged VH5 genes (germline transcripts). The cDNA clones were derived from both functional and pseudo-VH5 genes. Most germline transcripts appear to initiate at the normal VH promoter and are cleaved and polyadenylated at sites several hundred bases downstream of the VH5 coding region. Correct splicing of the leader intron was observed in all clones. In functional and pseudo-VH5 cDNAs, an open translational reading frame extends from the leader to a termination codon in the nonamer. Only limited polymorphisms were observed in the coding as well as flanking regions of the VH5 transcripts. Functional and pseudo-VH5 transcripts and previously identified murine germline VHJ558 transcripts are discussed.

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

In our accompanying paper, we described a switch variant (BCL1.2.58) that expresses membrane and secreted forms of IgM and IgG1. Both IgM and IgG1 share the same idiotype and use the same VDJ rearrangement. Here, a detailed Southern blot analysis of the entire constant region of the Ig heavy chain (Ig CH) locus of parental (BCL1.B1) and variants (BCL1.B2) DNA showed no detectable rearrangement. Similar analysis of the JH-C mu region led to the conclusion that two heavy chain alleles present in the IgM/IgG1-producing variants carried the same VDJ rearrangement but differed in their 3' flanking regions. One chromosome 12 did not carry any Ig CH genes, whereas, the other chromosome 12 carried one copy of CH genes. In BCL1.B1, however, each of the chromosome 12 alleles carried a full copy of CH genes. Karyotypic analysis confirmed the presence of two translocated t(12;16) chromosomes in both BCL1.2.58 and BCL1.B1 cells, with a break 5' to the VH locus at the distal region (12F2) of chromosome 12, and at the proximal region below the centromere (16B3) of chromosome 16. We conclude that double production of IgM and IgG1 in BCL1.B2 is accomplished by transcription of the corresponding CH genes in germline configuration using a single VDJ on the same chromosome 12.

Double isotype production by a neoplastic B cell line. I. Cellular and biochemical characterization of a variant of BCL1 that expresses and secretes both IgM and IgG1.

We have subcloned the in vitro-adapted murine B cell leukemia, BCL1.B1, to obtain a variant that expresses both IgM and IgG1. By fluorescence analysis, radioiodination, and immunoprecipitation of cell surface Ig, and by RIA of medium from limiting dilution cultures, we have shown that: (a) all the cells express and secrete both isotypes. The heavy chains of both IgG1 and IgM have the apparent molecular weights of membrane mu and gamma 1 chains; (b) both isotypes bear the same idiotype as determined by immunoprecipitation with antiidiotypic antibody, and both use the same VDJ rearrangement as shown by Southern blotting; and (c) the cells express the membrane and secreted forms of mRNA for both mu and gamma 1 but not gamma 2b or gamma 3. Taken together, the data suggest that all the cells are synthesizing, expressing on their surface, and secreting two isotypes that use the same VDJ rearrangement in the DNA and express the same serologically-defined idiotype. The molecular basis responsible for the production of the two isotypes in a single cell is the subject of the accompanying paper.

Differential structure-function requirements of the transmembranal domain of the B cell antigen receptor.

By generating phosphorylcholine (PC)-specific, wild-type (mu), and chimeric (mu-I-A alpha) antigen receptor transfectants of mature B cells, we have shown that the COOH terminus of the mu heavy chain is essential for three major functions: immediate signal transduction (measured as changes in intracellular Ca2+), antigen presentation, and induction of immunoglobulin M secretion. A more detailed analysis of structural requirements of the COOH-terminal domains contributing to these functions was achieved by systematically replacing the spacer, cytoplasmic, and transmembranal domains of the mu-I-A alpha chimeric chain with those of mu. Using this rescue approach, we show that the carboxyl two-thirds of the transmembranal domain (proximal to the cytoplasmic domain) is required for induction of intracellular Ca2+, whereas the complete transmembranal domain is required for the function of antigen presentation but is dispensable for induction of antibody secretion.

Murine natural killer cells express functional Fc gamma receptor II encoded by the Fc gamma R alpha gene.

We report evidence that murine NK cells express a functional Fc gamma RII encoded by the Fc gamma RII alpha gene. Several lines of indirect evidence indicate that freshly obtained NK cells from mice of several strains bear a functional Fc gamma RII: (a) anti-Fc gamma RII antibody 2.4G2 detects a small but significant proportion of sIg- cells and a small proportion of the 2.4G2+ cells are included in the Thy-1+ population; (b) sIg- lymphocytes contain 2.4G2+ and Fc gamma R-bearing cells in similar proportions; (c) binding of particulate immune complexes by sIg- lymphocytes is completely inhibited by 2.4G2; (d) 2.4G2+ cells mediate greater than 50% of the spontaneous cytotoxicity in sIg- splenic lymphocytes. Direct evidence for the presence of Fc gamma RII on murine NK cells is provided by the results of two-color immunofluorescence studies performed on splenic lymphocytes from C57BL/6 mice showing coexpression of NK-1.1 and 2.4G2. Studies of in vitro propagated homogeneous NK cell populations confirm that murine NK cells express only Fc gamma RII and that this Fc gamma R is functional, as shown in experiments of inhibition of ADCC by the anti-Fc gamma RII antibody 2.4G2. The results of studies at the molecular level show that an Fc gamma RII alpha transcript identical to that expressed in macrophages is the only molecule encoding Fc gamma RII in murine NK cells.

Molecular analysis of the t(2;14) translocation of childhood chronic lymphocytic leukemia.

Two rare cases of chronic lymphocytic leukemia (CLL) in children have been studied; both are associated with a previously undescribed chromosomal translocation [t(2;14) (p13;q32)]. In one patient the translocation was reciprocal and the breakpoint on chromosome 14 occurred just 5' of the C gamma 2 region on the productive immunoglobulin heavy-chain allele. The breakpoint on chromosome 2 does not involve the K locus but lies within an uncharacterized region that coincides with the position of a constitutive fragile site that occurs within normal lymphocytes. Data on the second patient are consistent with these findings and suggest that these cases represent a rare but distinct subgroup of CLL's with a specific cytogenetic change.

Sequence of the cloned gene for the constant region of murine gamma 2b immunoglobulin heavy chain.

The complete nucleotide sequence of the gamma 2b constant region gene cloned from BALB/c liver DNA is reported. The sequence of approximately 1870 base pairs includes the 5' flanking, 3' untranslated, and 3' flanking regions and three introns. The C gamma 2b coding region is divided by these introns into four segments corresponding to the homology domains and hinge region of the protein. The introns separating the hinge from the CH2 domain and the CH2 from the CH3 domain are small (106 and 119 base pairs). A larger intervening sequence of 314 base pairs separates the CH1 and hinge regions. The stretch of DNA comprising this large intron plus the hinge shows a strong homology with the other CH domains.

Structure of the constant and 3' untranslated regions of the murine gamma 2b heavy chain messenger RNA.

The complete coding sequence for the constant region of the mouse gamma 2b immunoglobulin heavy chain and the 3' untranslated region has been determined. The coding portion of the sequence is 1008 nucleotides long (amino acid residues 114 to 449), and the 3' noncoding region contains 102 nucleotides preceeding the polyadenylate. An extra carboxyl-terminal lysine residue which had not been observed in the gamma 2b or other gamma subclass protein sequences occurs in the nucleotide sequence and is probably processed posttranslationally. A 17-nucleotide sequence occurs with slight variation twice in CH1 and once in CH2 domains in the same relative location but with different translational phase. This sequence may be the site of crossover in a gamma 2b . gamma 2a heavy chain variant, an indication of possible recombinational activity of some kind.

Mouse immunoglobulin D: messenger RNA and genomic DNA sequences.

The molecular structure of a mouse immunoglobulin D from a plasmacytoma tumor and that of the normal mouse gene coding for immunoglobulin D are presented. The DNA sequence results indicate an unusual structure for the tumor delta chain in two respects: (i) Only two constant (C) region domains, termed C delta 1 and C delta 3 by homology considerations, are found; the two domains are separated by an unusual hinge region C delta H that lacks cysteine residues and thus cannot provide the covalent cross-links between heavy chains typically seen in immunoglobulins. The two domains and hinge are all coded on separate exons. (ii) At the carboxyl end of the delta chain there is a stretch of 26 amino acids that is coded from an exon located 2750 to 4600 base pairs downstream from the rest of the gene. Analogy with immunoglobulin M suggests that this distally coded segment C delta DC may have a membrane-binding function; however, it is only moderately hydrophobic. A fifth potential exon (C delta AC), located adjacent to the 3' (carboxyl) end of C delta 3, could code for a stretch of 49 amino acids. The tumor's expression of the delta gene may be aberrant, but the simplest interpretation would be that this tumor expresses one of the several biologically significant forms of the delta chain.

J genes for heavy chain immunoglobulins of mouse.

A 15,8-kilobase pair fragment of BALB/c mouse liver DNA, cloned in the Charon 4A lambda phage vector system, was shown to contain the mu heavy chain constant region (CHmu) gene for the mouse immunoglobulin M. In addition, this fragment of DNA contains at least two J genes, used to code for the carboxyl terminal portion of heavy chain variable regions. These genes are located in genomic DNA about eight kilobase pairs to the 5' side of the CHmu gene. The complete nucleotide sequence of a 1120-base pair stretch of DNA that includes the two J genes has been determined.

Mapping of heavy chain genes for mouse immunoglobulins M and D.

A single DNA fragment containing both mu and delta immunoglobulin heavy chain genes has been cloned from normal BALB/c mouse liver DNA with a new lambda phage vector Charon 28. The physical distance between the membrane terminal exon of mu and the first domain of delta is 2466 base pairs, with delta on the 3' side of mu. A single transcript could contain a variable region and both mu and delta constant regions. The dual expression of immunoglobulins M and D on spleen B cells may be due to alternate splicing of this transcript.

Human immunoglobulin D: genomic sequence of the delta heavy chain.

The DNA coding for the human immunoglobulin D(IgD) heavy chain (delta, delta) has been sequenced including the membrane and secreted termini. Human delta, like that of the mouse, has a separate exon for the carboxyl terminus of the secreted form. This feature of human and mouse IgD distinguishes it from all other immunoglobulins regardless of species or class. The human gene is different from that of the mouse; it has three, rather than two, constant region domains; and its lengthy hinge is encoded by two exons rather than one. Except for the third constant region, the human and mouse genes are only distantly related.

Somatic mutation in genes for the variable portion of the immunoglobulin heavy chain.

The size of the gene pool potentially encoding antibodies to p-azophenyl arsonate has been examined. A heavy chain-specific full-length complementary DNA clone has been constructed with the use of messenger RNA from a hybridoma that produces antibodies to the arsonate hapten and bears nearly a full complement of the determinants comprising the cross-reactive idiotype (CRI). The sequences of both the complementary DNA clone and the corresponding immunoglobulin heavy chain have been independently determined. A probe for the variable region gene was prepared from the original heavy chain complementary DNA clone and used to analyze, by Southern filter hybridization, genomic DNA from both A/J (CRI positive) and BALB/c (CRI negative) mice. Approximately 20 to 25 restriction fragments containing "germline" variable region gene segments were detected in both strains, and many are shared by both, Since 35 CRI-positive heavy chains have been partially sequenced thus far and 31 are different, the results of the hybridization analysis suggest that somatic mutation events involving the variable region gene segments of the heavy chain play a role in the origin of the amino acid sequence diversity seen in this system.

Cloning human fetal gamma globin and mouse alpha-type globin DNA: preparation and screening of shotgun collections.

Shotgun collections of Charon 3A bacteriophages containing Eco RI fragments of human and mouse DNA were constructed with the use of in vitro packaging. Plaques were screened by hybridization, and globin-specific clones were isolated from both human (Charon 3AHs51.1) and mouse (Charon 3AMm30.5). The fragments cloned were detected in unfractionated genomic DNA by the Southern method of hybridization.

Cloning human fetal gamma globin and mouse alpha-type globin DNA: characterization and partial sequencing.

Two globin-related clones isolated from collections of bacteriophages containing unfractionated Eco RI fragments of human and mouse DNA were characterized. Charon3AHs51.1Hbgamma includes 2.7 kilobase pairs of human DNA containing a large part of a fetal gamma globin chain structural gene; Charon 3AMm30.5 includes 4.7 kilobase pairs of mouse DNA related to alpha globin. The human fetal gamma globin gene has within its coding region two intervening sequences of noncoding DNA, IVS 1 and IVS 2, of approximately 1-0 and 900 base pairs. Sequence IVS 1 is located at the position of one of the two intervening sequences occurring in adult globin genes; IVS 2 is located at the position of the other.