Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-01-20
1998-06-02
Degen, Nancy
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 913, 4353201, 4353723, 536 241, C12P 2100, C12P 1934, C12N 1579, C07H 2104
Patent
active
057598053
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The production of RNA and polypeptides in recombinant systems, more specifically the use of transcription regulatory elements from the CD69 gene in the production of RNA and polypeptides.
BACKGROUND
During the process of T cell activation, the expression of several new cell-surface glycoproteins is induced. These glycoproteins are in turn involved in further aspects of cellular activation. CD69 is among the earliest of these newly synthesized cell-surface activation molecules induced on activated T cells. CD69 expression is seen within 60 minutes of T-cell stimulation, but is absent on resting cells (Hara et al., J. Exp. Med. 164:1988, 1986; Cosulich et al., Proc. Acad. Sci. USA 84:4205, 1987; and Cebrian et al., J. Exp. Med. 168:1621, 1988). CD69 expression is also inducible on thymocytes, B cells, natural killer (NK) cells and neutrophils (Ziegler et al., J. Immunol., in press; Risso et al., Eur. J. Immunol. 19:323, 1989; Lanier et al., J. Exp. Med. 167:1572, 1988; and Gavioli et al., Cell. Immunol. 142:186, 1992). In addition, CD69 expression is constitutive on a subset of CD3.sup.bright thymocytes and platelets (Testi et al., J. Immunol. 141:2557, 1988; and Testi et al., J. Exp. Med. 172:701, 1990). While a physiological ligand for CD69 is not known, CD69 appears to be involved in cellular activation. For example, cross-linking CD69 on T cells in the presence of a second signal such as phorbol ester results in proliferation involving the induction of the interleukin-2 (IL-2) and IL-2 receptor a-chain genes (Cosulich, supra; Cebrian, supra, Nakamura et al., J. Exp. Med. 169:677, 1989; Risso, supra; Testi et al., J. Immunol. 143:1123, 1989; and Testi et al., J. Immunol. 142:1854, 1989). Binding of the CD69 molecule with a specific antibody is capable of activating each of the expressing cell types (Testi, 1988, supra; Testi, 1990, supra; Lanier, supra; Gavioli, supra; and Moretta et al., J. Exp. Med. 174:1393, 1991). CD69 expression on platelets is reported to mediate platelet activation and aggregation (Testi, 1990, supra).
Recently several groups have reported the molecular cloning of a cDNA encoding human CD69 and the mouse homolog (Ziegler et al., Eur. J. Immuno. 23:1643, 1993; and Hamann et al., J. Immunol. 150:4920, 1993). The predicted amino acid sequence of CD69 showed it to be a member of the C-type lectin family, most closely related to two families of NK cell activation molecules, NKR-P1 and Ly-49 (Chambers et al., Glycobiology (1993) 3:9; and Drickamer, J. Biol. Chem. (1988) 263:9557). These two gene families are expressed almost exclusively on NK cells and have been shown to be involved in NK cell function.
The effective use of expression vectors in recipient cells requires that the expression of the coding sequence of interest be regulated by transcriptional regulatory regions. Vectors developed for the expression of recombinant genes have utilized various viral and non-viral regulatory sequences. The ability to control the expression of recombinant genes in the appropriate cell type or in an inducible or constitutive manner is paramount for studies aimed at examining the function or therapeutic value of the recombinant gene. The invention described below presents regulatory sequences which permit both activation regulated and constitutive transcription and expression in T cells and presumably in other cell types where the CD69 gene is expressed.
SUMMARY OF THE INVENTION
The present invention provides isolated murine and human genomic DNA encoding the CD69 gene plus recombinant expression vectors containing CD69 transcription regulatory elements, including promoter, enhancer, and repressor sequences. The nucleotide sequence of the cloned CD69 promoter, enhancer, and repressor regions are provided.
Embodiments of the invention include the following.
An isolated polynucleotide consisting essentially of a CD69 promoter or active fragment thereof.
A recombinant polynucleotide comprised of a CD69 promoter or active fragment thereof.
An isolated polynucleotide consisting es
REFERENCES:
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Cebrian et al., "Triggering of T cell proliferation through aim, an activation inducer molecule expressed on activated human lymphocytes" J. Exp. Med. (1988) 168:1621-1637.
Risso et al., "MLRe molecule is an activation antigen shared by human B, T lymphocytes and T cell precursors" Eur. J. Immunol. (1989) 19:323-328.
Cosulich et al., "Functional characterization of an antigen involved in an early step of T-cell activation" Proc. Natl. Acad. Sci. USA (1987) 84:4205-4209.
Lanier et al., "Interleukin 2 activation of natural killer cells rapidy induces the expression and phosphorylation of the Leu-23 activation antigen" J. Exp. Med. (1988) 167: 1572-1585.
Gavioli et al. "CD69 molecule in human neutrophils: Its expression and role in signal-transducing mechanisms" Cellular Immunol. (1992) 142:186-196.
Testi et al., "Constitutive expression of a phosphorylated activation antigen (Leu 23) by CD3.sup.bright human thymocytes" J. Immunol. (1988) 141:2557-2563.
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Feldhaus Andrew L.
Ziegler Steven F.
Degen Nancy
Schwartzman Robert
Targeted Genetics Corporation and Immunex
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