Search

The introduction of cloned genes into eukaryotic cells has become a major technique in the study of gene expression. Many experiments have demonstrated transcription of cloned genes after transfection into heterologous cell systems--cells in which the genes are not normally active. More recently, several investigators have obtained expression of specialized genes after transfection into cells of the corresponding specialized type, notably the beta-globin gene in erythroleukaemic cells and immunoglobulin genes in myeloma cells. These results allow the study of gene expression during development by comparing transcription of a gene transfected into homologous and heterologous cells. We have shown that a rearranged kappa immunoglobulin gene, cloned from a mouse myeloma, is transcribed transiently at a high level when reintroduced into mouse myeloma cells. We show here, in an internally controlled manner, that the same immunoglobulin gene is not detectably transcribed when transfected into mouse 3T3 or L cells.

Recent studies in which cloned immunoglobulin genes were introduced into cultured cells have produced two significant findings. First, the genes are expressed after transfection into lymphoid cells but not non-lymphoid cells. Second, transcription of an immunoglobulin gene requires, in addition to the promoter region, an enhancer element located downstream of the transcription start site. These findings raise the question of whether it is the promoter or the enhancer region that is responsible for the observed cell-type specificity. It has, in fact, been shown that immunoglobulin enhancers function only in lymphoid cells. We show here that the promoter for an immunoglobulin kappa light-chain gene also is strongly specific for lymphoid cells. Our result reemphasizes the importance of promoters relative to enhancers in determining which cells express which genes.

Enhancers are DNA sequences that stimulate transcription from eukaryotic promoters. This stimulatory effect can be exerted over large distances and from a position either 5' or 3' of a promoter. Enhancers have been found in the genomes of many viruses, and in some cellular genes such as those encoding immunoglobulin heavy chain and kappa light chain. An important feature of both viral and cellular enhancers is the ability of each enhancer to stimulate transcription from many promoters other than the one with which it is found associated. However, the question of whether cellular enhancers stimulate their 'own' promoter more efficiently than other promoters has apparently not been investigated. We show here that the kappa light-chain enhancer stimulates a kappa promoter about 20-fold more than it stimulates either the simian virus 40 (SV40) early promoter or a metallothionein (MT) promoter, two promoters that are very sensitive to other enhancers. Similarly, the heavy-chain enhancer stimulates a heavy-chain promoter much more than it stimulates the SV40 and MT promoters. This synergism between immunoglobulin enhancers and promoters might be due to the action of a protein that binds specifically to each of the regulatory elements.

It has recently been shown that the promoter of a kappa immunoglobulin gene is activated for transcription by a downstream sequence element. Here we mapped the activating element to a resolution of about 20 base pairs by constructing a series of deletions in the cloned kappa gene. After transfection of each deleted gene into myeloma cells, a transient expression assay was used to measure the level of transcription from the kappa promoter. We found that the activating element extends through about 200 base pairs and encompasses a region of sequence that is conserved between mouse and human genes. As successively deeper deletions were made into the conserved region from either the 5' or 3' side, the activating ability was lost gradually rather than abruptly. Although several short segments in this region are homologous to sequences in viral enhancers, they did not seem to play a dominant role in the activating effect. We also found that the activating element remained functional when reversed in orientation or when moved upstream of the kappa gene.

To examine the DNA sequences involved in regulation of kappa immunoglobulin gene expression, an assay was developed whereby transcription of a cloned, rearranged kappa gene could be detected following its transfection into antibody-secreting mouse myeloma cells. In a transient expression assay, RNA transcripts initiating 20 to 25 bp before the AUG translation initiator of the kappa gene were readily detected. The constant region (C kappa) and part of the J kappa-C kappa intron was then deleted from the rearranged gene and a downstream SV40 polyadenylylation signal was provided. No transcription was detected from the deleted gene upon transfection into myeloma cells, suggesting that after rearrangement the kappa variable region promoter is activated by sequences more than 2.6 kb downstream of J kappa. The rearranged kappa gene was also transfected into a nonlymphoid line of monkey cells and RNA transcripts were detected. Transcription in these cells, however, was at a relatively lower level than in myeloma cells, did not depend on the presence of downstream sequences, and initiated from a site about 10 bp downstream of the myeloma site. These experiments define a sequence downstream of the promoter that is necessary for accurate transcription of the kappa chain gene.

We show in this report that the transcription induced by interleukin-2 or pokeweed mitogens of the kappa MOPC 41 immunoglobulin light-chain gene transfected into primary human or murine B lymphocytes initiates from a previously unobserved start site about 26 base pairs upstream of the start site used in myeloma cell lines.