A conserved sequence in the immunoglobulin J kappa-C kappa intron: possible enhancer element

Several functionally important genetic elements (such as the TATA box, mRNA splice sequences, poly(A) addition signal) were first detected as short segments of unexplained sequence homology within non-coding regions of different genes. A short region of unknown sequence in the intron between the human J kappa and C kappa immunoglobulin coding regions was found to be sufficiently homologous to the corresponding segment of the mouse gene to form stable heteroduplexes. Although no specific function has yet been definitely ascribed to this region (which we call the kappa intron conserved region, or KICR), some functional significance has been inferred from the findings that (1) activation of B lymphocytes induces a DNase hypersensitivity site in this region and (2) deletions including this region reduce expression of kappa genes introduced into lymphoid cells. To delineate the KICR more precisely and to test the generality of the sequence conservation in a third species, we have sequenced this region of the human and mouse genes and have examined the corresponding region of a recently cloned rabbit kappa gene. We find a segment of about 130 base pairs (bp) that shows striking conservation in all three species, demonstrating homology significantly higher than within the C kappa coding region itself. Two short sequences from the J kappa-C kappa intron that were noted by other investigators to be homologous to proposed 'enhancer' sequences both lie within the conserved region.     

Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice

Transfection of genes into tissue culture cell lines has demonstrated that relatively short DNA sequences can allow expression of immunoglobulin, insulin and chymotrypsin genes in their appropriate cell types. A definitive test of cell-specific gene expression, however, requires testing genes in every possible cell type, an experiment performed easily by introducing the gene in question into the germ line of an animal. Transfer of intact genes into mice has demonstrated that a mouse immunoglobulin kappa gene is expressed specifically in B lymphocytes, a rat elastase I gene is expressed specifically in pancreas and a chicken transferrin gene is expressed preferentially in liver. Mouse metallothionein-growth hormone fusion genes introduced into mice are preferentially expressed in the liver, consistent with the expression of endogenous metallothionein genes, but initial experiments with beta-globin genes have not revealed proper regulation. To identify the DNA elements required for pancreas-specific expression of the rat elastase I gene, we joined the 5'-flanking region of this gene to the human growth hormone (hGH) structural gene and introduced the fusion gene into mice. Here we demonstrate that a fusion gene containing only 213 base pairs (bp) of elastase I gene sequence directs expression of hGH in pancreatic acinar cells.     

Recombination between an expressed immunoglobulin heavy-chain gene and a germline variable gene segment in a Ly 1+ B-cell lymphoma

The early stages of murine B-cell differentiation are characterized by a series of immunoglobulin gene rearrangements which are required for the assembly of heavy(H) and light(L)-chain variable regions from germline gene segments. Rearrangement at the heavy-chain locus is initiated first and consists of the joining of a diversity (DH) gene segment to a joining (JH) gene segment. This forms a DJH intermediate to which a variable (VH) gene segment is subsequently added. Light-chain gene rearrangement follows and consists of the joining of a VL gene segment to a JL gene segment: once a productive light-chain gene has been formed the cell initiates synthesis of surface immunoglobulin M (sIgM) receptors (reviewed in ref. 1). These receptors are clonally distributed and may undergo further diversification either by somatic mutation or possibly by continued recombinational events. Such recombinational events have been detected in the Ly 1+ B-cell lymphoma NFS-5, which has been shown to rearrange both lambda and H-chain genes subsequent to the formation of sIgM (mu kappa) molecules. Here we have analysed a rearrangement of the productive allele of NFS-5 and found that it is due to a novel recombination event between VH genes which results in the replacement of most or all of the coding sequence of the initial VHQ52 rearrangement by a germline VH7183 gene. Embedded in the VH coding sequence close to the site of the cross-over is the sequence 5' TACTGTG 3', which is identical to the signal heptamer found 5' of many DH gene segments. This embedded heptamer is conserved in over 70% of known VH genes. We suggest that this heptamer mediates VH gene replacement and may play an important part in the development of the antibody repertoire.     

Variant (6 ; 15) translocation in a murine plasmacytoma occurs near an immunoglobulin kappa gene but far from the myc oncogene

Chromosome translocations in B-lymphoid tumours are providing intriguing insights and puzzles regarding the role of immunoglobulin genes in the activation of the myc oncogene (reviewed in refs 1, 2). The 15 ; 12 translocations found in most murine plasmacytomas and the analogous 8 ; 14 translocation in human Burkitt's lymphomas involve scissions of murine chromosome 15 (human chromosome 8) near the 5' end of the c-myc gene and subsequent fusion near an immunoglobulin heavy-chain gene. The less well characterized 'variant' translocations found in about 15% of such tumours also involve the myc-bearing chromosome band, but exchange occurs with a chromosome bearing an immunoglobulin light-chain locus--in mice, the kappa-chain locus bearing chromosome 6 (refs 3-5) and, in man, chromosome 2 (or 22), at the same band at which the kappa (or lambda) locus lies (reviewed in ref. 1). The Burkitt variant translocations involve scissions 3' of c-myc; one 8 ; 22 translocation placed the C lambda locus just 3' of c-myc, but usually the chromosome 8 breakpoint is a greater, but unknown, distance away from c-myc, more than 20 kilobases (kb) in one 8 ; 2 translocation involving the C kappa gene. Little is known about the murine 6 ; 15 translocations, although a C kappa gene cloned from one plasmacytoma (PC7183) is linked, via chromosome 12 sequences, to an unidentified region of chromosome 15 (ref. 11). We describe here the chromosome fusion region from plasmacytoma ABPC4, which displays the typical reciprocal 6;15 translocations. We find that the chromosome 6 breakpoint is near C kappa but, unlike those in the heavy-chain locus, not at a position where immunoglobulin genes normally recombine. Moreover, the chromosome 15 sequences involved in the ABPC4 translocation are not derived from the vicinity of c-myc.     

DNase I hypersensitive sites in the chromatin of human mu immunoglobulin heavy-chain genes

An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function.     

The gene encoding the T-cell receptor alpha-chain maps close to the Np-2 locus on mouse chromosome 14

Serological and molecular genetic analyses of T-cell clones have shown that the T-cell antigen receptor apparently comprises two glycosylated, disulphide-linked polypeptide chains (alpha and beta), both of which span the cell membrane. Cloning of the genes encoding the two chains from mouse and human DNA has shown that the alpha- and beta-chains are composed of variable (V) and conserved (C) regions in agreement with peptide mapping data. Gene segments encoding variable and conserved domains of the beta-chain have been identified and undergo rearrangements during T-cell differentiation. The genes encoding the alpha-chain, so far described at the level of complementary DNA clones, also identify DNA rearrangements. Thus, the genes encoding the T-cell receptor show the same structure and dynamic behaviour as immunoglobulin genes, indicating that the two gene families belong to the same supergene family; this evolutionary relationship is supported by the fact that the genes encoding the beta-chain of the T-cell receptor are closely linked to immunoglobulin kappa light-chain genes on chromosome 6 in mouse. In man, however, the beta genes map to chromosome 7 (ref. 14) whereas the kappa-chain genes are located on chromosome 2, indicating that linkage between the two gene families is not needed for proper expression. Here we describe genomic clones encoding the constant portion of the T-cell receptor alpha-chain and map the gene to chromosome 14 in mouse, close to the gene for purine nucleoside phosphorylase (Np-2) which, in man, has been associated with T-cell immunodeficiencies.     

Dispersed human immunoglobulin kappa light-chain genes

The gene segments encoding the constant and variable regions of human immunoglobulin light chains of the kappa type (C kappa, V kappa) have been localized to chromosome 2. The distance between the C kappa and V kappa genes and the number of germline V kappa genes are unknown. As part of our work on the human V kappa locus, we have now mapped two solitary V kappa gene and a cluster of three V kappa genes to chromosomes 1, 15 and 22, respectively. The three genes that have been sequenced are nonprocessed pseudogenes, and the same may be true for the other two genes. This is the first time that V-gene segments have been found outside the C-gene-containing chromosomes. Our finding is relevant to current estimates of the size of the V kappa-gene repertoire. Furthermore, the dispersed gene regions have some unusual characteristics which may help to clarify the mechanism of dispersion.     

Duplications of a mutated simian virus 40 enhancer restore its activity

Enhancers are cis-acting control elements which can stimulate at a distance the activity of a variety of eukaryotic promoters. First identified as a repeated 72 base pair (bp) sequence upstream of the simian virus 40 (SV40) early gene promoter, enhancers have since been shown to be associated with numerous other viral and cellular genes. Although there are no strong homologies between the sequences of different enhancers, a number of short and degenerate consensus sequences have been identified, including the 'core' element GTGGA/TA/TA/TG and stretches of alternating purines and pyrimidines which may have the potential to form left-handed Z DNA. To study the functional significance of two alternating purine and pyrimidine sequences in the SV40 enhancer, we have introduced various combinations of point mutations into a modified SV40 enhancer which contained only one copy of the 72 bp element (W.H., Y.G., A. Nordheim and A. Rich, unpublished results); one of these combinations impaired both the activity of the enhancer and growth of SV40. We describe here the structure of 18 revertants of this mutant and suggest that in each of the 18 revertants, the defects of the original mutant have been overcome by simple tandem duplications in the enhancer region, all of which include the 'core' element.     

Aberrant rearrangement of the kappa light-chain locus involving the heavy-chain locus and chromosome 15 in a mouse plasmacytoma

The creation of a functional antibody gene requires the precise recombination of gene segments initially separated on the chromosome. Frequently errors occur in the process, resulting in the formation of a non-functional gene. The non-functional genes can be generated by incomplete rearrangements, frameshifts, or the use of pseudo V or J joining segments. It is likely that these aberrant rearrangements arise by the same mechanism as is used in generating functional genes, a process which we have suggested may involve unequal sister chromatid exchange. Aberrant rearrangements of immunoglobulin genes occur in normal lymphocytes and play a major part in allelic exclusion. However, it has recently been suggested that aberrant rearrangements involving immunoglobulin and non-immunoglobulin genes may be involved in tumorigenesis. This suggestion has been stimulated by the frequent occurrence of translocations involving chromosomes known to carry immunoglobulin genes in B-cell malignancies. The rearrangement of non-immunoglobulin DNA to the heavy-chain locus has recently been reported. Some aberrant rearrangements of the kappa locus appear to be due to rearrangements to sites that do not include the conventional sequence for V gene segment joining. Here we describe an aberrant kappa rearrangement that has led to the joining of DNA from chromosomes 15, 6 and 12, and so appears to be the result of chromosomal translocations or transpositions. As 15/6 or 15/12 translocations have frequently been found in mouse plasmacytomas (as have analogous translocations in human lymphocyte tumours) this aberrant kappa rearrangement may be unique to the plasmacytoma from which it was isolated.     

The immunoglobulin mu constant region gene is expressed in mouse thymocytes

It has been a matter of controversy whether the functional capacity of T cells to discriminate between antigens is mediated via immunoglobulin, an immunoglobulin-like molecule, or by the product(s) of unrelated genes. The progenitors of immunoglobulin-secreting cells, B cells, express membrane-bound immunoglobulin as the antigen-specific receptor on their surface. For T cells, although products of immunoglobulin heavy chain variable region genes are implicated as receptor components, there has been no compelling immunochemical evidence for participation of either immunoglobulin light chains or heavy chain constant regions (see refs 2-6 for the disparate views). Recently, using cloned immunoglobulin DNA sequences as hybridization probes, we have demonstrated that the immunoglobulin Cmu gene, but not the Cmu gene, is expressed as polyadenylated RNA in some T cell tumour (T lymphoma) cell lines. Individual T lymphoma lines yielded up to three discrete mu RNA species of different sizes (1.9, 2.2 and 3.0 kilobases), each species being different in size from the major mu RNA species present in B lymphoma cells (2.4 and 2.7 kilobases). We show here that cells from the normal mouse thymus contain mu RNA species, indistinguishable in size from those in T lymphoma cells, but contain little if any kappa RNA.     

A proteasome-related gene between the two ABC transporter loci in the class II region of the human MHC

It is now possible to paint a detailed picture of how cytoplasmic proteins are handled by the immune system. They are apparently degraded in the cytoplasm into peptides. These are then transported into the endoplasmic reticulum where they encounter class I major histocompatibility complex (MHC) molecules. Once loaded with peptide, the HLA molecules move through the Golgi apparatus to the cell membrane. Until recently, it had not been established how peptides without signal sequences cross the ER membrane. However, a number of papers have now described a pair of membrane transporter genes of the ABC (ATP-binding cassette) super-family which are attractive candidates for this function. Both transporter genes, which may encode two halves of a heterodimer, are situated in the class II region of the MHC. There is evidence that other putative components of the processing machinery, the LMPs (low molecular mass polypeptides), are also encoded in the MHC. Similarities between the properties of the LMPs and a large intracellular protease complex, called proteasome, have led to the suggestion that LMPs are involved in processing antigens. We have now identified a human gene with sequence homology to proteasome components. Remarkably, this gene maps between the two putative peptide transporter genes.