Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1991-09-25
1994-12-06
Ziska, Suzanne E.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
4351723, 4353201, 536 241, C12N 500, C12N 1500, C07H 2100
Patent
active
053710096
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
This invention relates to enhancers and concerns recombinant DNA molecules comprising a novel enhancer, and use of the enhancer in gene expression and gene therapy.
BACKGROUND OF THE INVENTION
Enhancers are DNA sequences which play an important role in the transcription of proteins. A number of different enhancer sequences have been identified from different sources, including enhancers of vital origin, e.g. from the SV40 virus, and of cellular origin. As regards the immunoglobulin (Ig) genes, enhancer elements have been identified in the major introns of both the heavy chain (IgH) (Banerji et al., 1983; Gillies et al., 1983; Neuberger, 1983) and kappa (identified herein as "K") light chain (Picard and Schaffner, 1984; Queen and Stafford, 1984) loci.
The present invention is based on the identification of a novel enhancer in the mouse immunoglobulin K locus, downstream of C.sub.K, in addition to the K-intron enhancer. Functionally equivalent enhancers are to be expected to occur downstrem of C.sub.K of the immunoglobin K locus of other species.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a recombinant DNA molecule comprising an enhancer having at least part or parts of the base sequence occuring downstream of C.sub.K of the immunoglobulin K locus of any species.
The enhancer located in the mouse K locus occurs in the sequence given in FIG. 3A, and the location of the enhancer has been further localised to the sequence given in FIG. 12B.
Thus, in a further aspect the invention provides a recombinant DNA molcule comprising an enhancer having at least part or parts of the following base sequence:
In another aspect the invention provides a recombinant DNA molecule comprising an enhancer having at least part or parts of the base sequence given in FIG. 3A.
The following refers generally to work on the enhancer located in the mouse K locus. It will be apparent to those skilled in the art that functionally equivalent enhancers in other species can be identified by routine investigation.
The novel enhancer is located about 9 kb downstream (3') of C.sub.K, and is known as the K3'-enhancer. Like the K-intron enhancer, the K3'-enhancer is also B cell specific. This novel enhancer is some sevenfold stronger than the K-intron enhancer and the region of the enhancer shows striking sequence homologies to the lymphotropic papovavirus, IgH and K-intron enhancers. The location of the K3'-enhancer between C.sub.K and the RS element means that it is likely to be deleted, at least one allele, in many B cells that express lambda light chains.
The existence of a second enhancer in the mouse K locus explains various previously unexplained observations. For example, locus, transgenes containing all the previously identified K transcription elements were not expressed at high level (Rusconi and Kohler, 1985; Pettersson et al., 1989). Furthermore, the endogenous K gene in the S107 plasmacytoma cell line is well expressed despite the fact that the intron-enhancer is not active in this cell line, which lacks the enhancer binding factor NF-KB (Atchinson and Perry, 1987,1988). The existence of multiple activating sequences in the region of the immunoglobulin loci may also explain why many immunoglobulin transgenes are less transcriptionally active than the endogenous loci; the transgenes may not include the full complement of activating sequences. Indeed, we have found only weak transcriptional activity in transgenic mice that harbour a K transgene that extends only 1.2 kb downstream of the C.sub.K exon.
The molecule of the invention may additionally comprise other elements, and will generally further comprise a promoter and gene or genes coding for a protein or proteins of interest. One or more additional enhancers may also be included.
The novel enhancer may be used in any geometrical configuration of DNA arrangement. The enhancer is preferably closer to a promoter than the natural spacing (in a functionally rearranged K gene the enhancer is about 3 kb downstream
REFERENCES:
Peterson et al. Mol. Cell. Bul 7(12):4194, 1987.
Anderson et al. Science 226:401, 1984.
Stub et al. J. Exp. Med. 164:627, 1986.
Meyer Kerstin B.
Neuberger Michael S.
Medical Research Council
Ziska Suzanne E.
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