Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Patent
1993-02-17
1994-04-12
Schwartz, Richard A.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
4351723, 536 235, C07K 1300, C12N 1500, C12N 1512
Patent
active
053026985
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to DNA coding for a protein which specifically binds to the enhancer of .alpha.-fetoprotein gene and promotes transcription of .alpha.-fetroprotein gene. Since the protein is involved in transcription, this DNA can be used for the construction of a highly efficient gene expression system utilizing animal cells.
BACKGROUND OF THE INVENTION
Expression of genetic information in eucaryotic cells is regulated at the levels of transcription into mRNA, mRNA processing, translation to protein and posttranslational processing. The regulation at the transcription level most strongly influences the expression of genetic information. A promoter which controls transcription is found on the 5' side of genes transcribed by RNA polymerase II and sometimes enhancers, which regulate promoter activity were also found. With the recent discovery of nuclear factors that recognize and bind to specific nucleotide sequences in the promoter and enhancers, it has become evident that the activities of promoter and also enhancers are mediated by the binding of these factors.
Of such factors, for example, studies on proteins which interact with TATA box, CAAT box, or GC box for the stimulation of transcription of RNA polymerase have been undertaken. Cloning of cDNA of CAAT-binding and GC-binding factors has already been accomplished.
Furthermore, factors that bind to enhancers have been investigated and cDNA clones of several transcription factors have been successfully isolated: for example, octamer transcription factor-2 (OTF-2), a B-cell lineage specific factor, that binds to immunoglobulin .kappa.-chain gene enhancer [Michael et al.: Nature, 336, 544-551, (1988)] and OTF-1, which recognizes the same nucleotide sequence and was found ubiquitously in many tissues [R. A. Sturm, G. Das and W. Herr: Gene & Development, 2, 1852 (1988)].
In human .alpha.-fetoprotein gene, presence of an enhancer at 3.5 kb on the upstream of transcription start point has been confirmed. A factor that binds to this enhancer has been discovered and named AFP-1 [Sawadaishi et al.: Molecular and Cellular Biology, 8, 5179-5187 (1988)].
However, the structure and physiological properties of AFP-1, and on the gene which codes for AFP-1 has not been elucidated.
OUTLINE OF THE PRESENT INVENTION
The inventors have been investigating a nuclear factor that specifically interacts with a region, characterized by the TTAATAATTA (see ID NO:3) structure that exists in the enhancer of .alpha.-fetoprotein gene, and isolated a cDNA that encodes this factor. They determined the nucleotide sequence of the cDNA and deduced the amino acid sequence of the factor and accomplished the present invention.
Therefore, this invention offers a DNA, expressed by nucleotide sequence, that codes for a protein which binds to the enhancer of human .alpha.-fetoprotein gene.
The nuclear factor which pertains to this invention and which specifically binds to the enhancer of .alpha.-fetroprotein gene, has the amino acid sequence given in FIG. 1, in its primary structure and present in cell nuclei, and can be extracted from .alpha.-fetroprotein-producing cells. Thus, it is possible to isolate the DNA defined by the nucleotide sequence given in FIG. 2, using cloning procedures starting with messenger RNA that is isolated from .alpha.-fetoprotein-producing cells derived from the liver.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the amino acid sequence (see ID NO:2) of the protein, which binds to the .alpha.-fetoprotein gene enhancer, encoded by the DNA (referred to as .lambda.2cDNA) of this invention.
FIG. 2 shows the nucleotide sequence (see ID NO:1) of said DNA.
FIG. 3 describes briefly the procedure for obtaining cDNA starting with poly(A)RNA to introduce it into .lambda.gtll expression vector in the example. The small arrows indicate the location in the DNA (SEQ ID NO:4) and amino acid (SEQ ID NO:5) sequences of the lacZ gene where the cDNA is to be inserted.
FIG. 4 illustrates a restriction enzyme map of the enhancer region located on t
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Wang et al., "A specific .alpha.-Fetoprotein Gene . . . etc.", Biochemical and BioPhysical Research Communications, vol. 143, No. 1, 1987, pp. 110-115.
Watanabe et al., "Cell-specific enhancer activity . . . etc.", The Journal of Biological Chemistry, vol. 262, Issue of Apr. 5, pp. 4812-4818 (1987).
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Higashio Kanji
Morinaga Tomonori
Tamaoki Taiki
Yasuda Hisataka
Schwartz Richard A.
Snow Brand Milk Products Co. Ltd.
Vogel Nancy T.
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