Nucleotide sequence of the constant region of a chicken mu heavy chain immunoglobulin mRNA.

We have recently reported the sequence of a chicken Ig lambda light chain cDNA clone, isolated from a spleen partial cDNA library (1). In this paper, we describe the characterization of a cDNA clone coding for the chicken constant (C) region of the secreted mu chain. This is the first report on the nucleotide and amino acid sequence of a chicken Ig heavy chain constant region. It contains the 3' untranslated region of the mu mRNA up to the poly(A) tail, and, in comparison with the mouse Cmu sequence, displays the overall domain size and organization of a secreted mu chain, i.e.: a characteristic COOH-terminal region, a Cmu4, a Cmu3, a Cmu2, and part of a Cmu1 domain. The sequence homology between these two species ranges from 45% for the Cmu4 to 18% for the Cmu2. Thus, the Cmu sequence appears much less conserved between chicken and mouse than their respective lambda light chain constant regions (1). These results, together with some distinctive features of the Cmu2 domain, may be of evolutionary relevance and will be further discussed.     

An enhancer at the 3'' end of the mouse immunoglobulin heavy chain locus.

A tissue-specific enhancer (E mu) lies between the joining (JH) and mu constant region (C mu) gene segments of the immunoglobulin heavy chain (IgH) locus. Since mouse endogenous IgH genes are efficiently transcribed in its absence, the normal function of this enhancer remains ill-defined. Recently, another lymphoid-specific enhancer of equal strength has been identified 3' of the rat IgH locus. We have isolated an analogous sequence from mouse and have mapped it 12.5 kb 3' of the 3'-most constant region gene (C alpha-membrane) of the BALB/c mouse locus. The mouse and rat sequences are 82% homologous and share with other enhancers several DNA sequence motifs capable of binding protein. However, in transient transfection assays, the mouse sequence behaves as a weaker enhancer. The role of this distant element in the expression of endogenous IgH genes, both in E mu-deficient, Ig-producing cell lines and during normal B cell development, is discussed.     

Organization and evolution of variable region genes of the human immunoglobulin heavy chain

We have isolated 23 different cosmid clones of the heavy-chain variable region genes (VH) of human immunoglobulin. These clones encompass about 1000 X 10(3) base-pairs of DNA containing 61 VH genes. Characterization of the 23 clones by Southern blot hybridization showed that VH genes belonging to different families were physically linked in many regions. Cluster 71, which was analyzed in detail, comprised seven VH segments arranged in the same orientation with different intervals. This clone contained internal homology regions, each carrying two VH segments of different families. Comparison of the nucleotide sequences of VH segments within each family showed that profiles of accumulation of mutations in framework (FR) and complementarity-determining (CDR) regions were different. CDR had more mutations at amino-acid-substituting positions than at silent positions, whereas FR had the reverse distribution of mutations. Five out of seven VH segments of this cluster were pseudogenes containing various mutations. VH pseudogenes were classified into two distinct groups; one with a few replacement mutations (conserved pseudogenes), and the other with rather extensive mutations (diverged pseudogenes). The possibility that conserved pseudogenes serve as a reservoir of VH segments is discussed.     

Identification of a matrix-associated region 5'' of an immunoglobulin heavy chain variable region gene.

In the accompanying report (C. F. Webb, C. Das, S. Eaton, K. Calame, and P. Tucker, Mol. Cell. Biol. 11:5197-5205, 1991), we characterize B-cell-specific protein-DNA interactions at -500 and -200 bp upstream of the mu immunoglobulin heavy chain promoter whose abundances were increased by interleukin-5 plus antigen. Because of the high A + T/G + C ratio of these sequences and the consistent findings by others that enhancer- and promoterlike regions are often located near matrix-associated regions, we asked whether these sequences might also be involved in binding to the nuclear matrix. Indeed, DNA fragments containing the -500 binding site were bound by nuclear matrix proteins. Furthermore, UV cross-linking studies showed that the DNA binding site for interleukin-5-plus-antigen-inducible proteins could also bind to proteins solubilized from the nuclear matrix. Nuclear matrix-associated sequences have also been demonstrated on either side of the intronic immunoglobulin heavy chain enhancer. Our data suggest a topological model by which interactions among proteins bound to the promoter and distal enhancer sequences might occur.     

Alternative promoter and 5' exon generate a novel Gs alpha mRNA

Several species of mRNA have been shown to encode the alpha subunit of the stimulatory GTP-binding regulatory protein, Gs alpha. The various Gs alpha mRNAs are generated through alternative splicing of a single precursor RNA and through the use of alternative acceptor splice sites. We now report the existence of a Gs alpha mRNA that uses a previously unidentified promoter and leading exon (termed exon 1'). In both the canine and human Gs alpha genes, exon 1' is located 2.5 kilobases 5' of exon 1. Exon 1' does not contribute an in-frame ATG, and thus its mRNA encodes a truncated form of Gs alpha. Initiation of translation is predicted to begin at an AUG in exon 2, as demonstrated both by in vitro translation and COS cell expression studies.     

Organization of variable region segments of the human immunoglobulin heavy chain: duplication of the D5 cluster within the locus and interchromosomal translocation of variable region segments.

We have studied the organization of variable region (V) genes of the human immunoglobulin heavy chain (H) by cosmid cloning. We isolated two independent immunoglobulin D5 clusters (D5-a and D5-b) from cosmid libraries of the human genome. Restriction maps of these two regions showed that downstream 15 kb portions of the 55 kb overlap were different although upstream 40 kb portions were almost identical. Four more D segments, (DM, DXP, DA and DK) were found around the D5 segment in the conserved region of each cluster. Nucleotide sequences of the corresponding D segments from each cluster were almost identical and they encoded potentially functional D regions. Analysis using human-rodent somatic cell hybrids demonstrated that both clusters were located in the immunoglobulin heavy chain (H) locus on chromosome 14, suggesting that the D5-a and D5-b regions evolved by internal duplication within this locus. We also isolated a 60 kb DNA region carrying four VH segments, designated as VH-F region, which was located on chromosome 16. Nucleotide sequences of the four VH segments were determined. Two of them encoded potentially functional VH segments, and the other two were pseudogenes. Some more VH segments were found to be located outside chromosome 14, by Southern blot hybridization of human-rodent hybrid cell DNAs. These results provide further evidence that the human VH locus has undergone recent reorganization.     

A switch region inversion contributes to the aberrant rearrangement of a mu immunoglobulin heavy chain gene in MPC-11 cells.

We describe the unique features of an aberrantly rearranged mu immunoglobulin heavy chain gene isolated from MPC-11 cells (a gamma 2b producing Balb/c plasmacytoma). A novel rearrangement has occurred 1.5 Kb 5' of the MPC-11 mu gene (denoted 18b mu) resulting in the deletion of the majority of the repetitive switch region (S mu) and 5' flanking DNA including the Joining (JH) sequences. The remainder (275 bp) of the S mu repeat has undergone a complete sequence inversion. DNA sequences 5' of the inverted S mu sequence do not resemble Variable (VH), Diversity (D), JH or their conserved flanking sequences. A DNA sequence localized 5' of the inverted S mu sequence, (p18b mu-1.4) detects a small family of homologous sequences in Balb/c DNA. The 18b mu-1.4 like sequences lack homology to S mu, exhibit flanking sequence polymorphisms in 5 out of 6 inbred mouse strains and undergo partial or complete deletion in 5 out of 10 plasmacytomas tested. Two 18b mu-1.4 homologous sequences display a higher copy number in C57Bl/6, AL/N and CAL9 mouse strains.