Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-11-22
2003-02-11
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S325000, C435S471000, C536S023500
Reexamination Certificate
active
06518041
ABSTRACT:
The invention relates to a new HRE (hormone response element, hormone receptor binding site), which is bonded by an androgen and progesterone receptor, its sequence and its use.
Steroid receptors are transcription factors that have a ligand binding site, a DNA binding site and several transactivation functions. If the ligand binds the steroid, the conformation of the receptor changes. By this change in conformation, the receptor can form a dimer and bind to a specific, two-stranded DNA sequence, the so-called HRE (hormone response element, hormone receptor binding site) and interact with coactivators and other transcription factors. As a consequence, the transcription of the target gene, thus the gene that is regulated by the HRE, is activated. These HREs are usually components of promoter regions of such target genes, but they can also be found in the intron areas or in the 3′-part of the gene. For the HRE of the androgen receptor, a consensus sequence (SEQ ID NO: 4) GG(T/A)ACAnnnTGTTCT has been found (Roche et al. 1992, Mol. Endocrinol. 6, 2229-2235). In this case, n refers to any nucleotide. The half-elements, which are the defined nucleotides before or after the “nnn” sequence, are approximately palindromic. It was shown that the sequence (SEQ ID NO: 5) GGTACAtctTGTTCA, which occurs in the promoter of the highly androgen-regulated CRISP-i-gene, has a high binding affinity to the androgen receptor (Schwidetzky et al. 1997, Biochem. J. 321, 325-332). This sequence is referred to in the following consensus-HRE. It is detected, however, not only by the androgen receptor but also by the glucocorticoid and progesterone receptor. The HRE, however, binds the promoter region of the probasin gene selectively to the androgen receptor and not to the glucocorticoid receptor (F. Claessens et al. 1996, J. Biol. Chem. 271, 19013-19016). No information on the binding of the progesterone receptor is present here. By an in vitro selection method, a new DNA sequence named IDR17, which can bind the androgen receptor, was recently found (Zhou et al. 1997, J. Biol. Chem. 272, 8227-8235). Various tests showed that this sequence has a high selectivity for the androgen receptor in comparison to the glucocorticoid receptor. A comparison with the progesterone receptor was not described. The sequence IDR17 is a sequence that was found in vitro; there is no indication as to whether it also occurs in vivo and has a function there as an HRE.
The knowledge on HREs is helpful in the development of medications for the treatment of hormone-dependent diseases. In the treatment of prostate cancer with antiandrogens, which inhibit the binding of the androgen receptor to the consensus HRE, it is frequently shown that not all androgen-regulated genes are adjusted downward and/or the antiandrogens are no longer active after a certain treatment time. These findings thereupon point to the fact that different HREs must be provided that regulate various genes and are responsible for the hormone-dependency. For successful hormone therapy, it is therefore desirable to know other HREs to which the androgen receptor binds. Specifically active natural hormones, synthetic hormones or antihormones could then be identified. The latter can then turn on or off the genes that are relevant for the diseases. To date, only a few genes that are regulated by androgens and their HREs are known. Examples of this are the probasin gene and the C3(1) gene, which are both induced in rat prostates by androgen (F. Claessens et al. 1989, Biochem. Biophys. Res. Commun. 164, 833-840; P. S. Rennie et al. 1993, Mol. Endocrinol. 7, 23-36). In the rat epididymis, the pem gene is expressed in an androgen-dependent manner (S. Malti et al. 1996, J. Biol. Chem. 271, 17536-17546). The approximate position of two initial transcription sites is described, but the HRE is not known.
It is desirable to have available a new HRE for the androgen receptor. The invention provides a new HRE, which binds the androgen and progesterone receptor and which contains
a. a DNA sequence of the general formula (SEQ ID NO: 6) (N)
x
TCTCATTCTGTTCC, whereby N, independently of one another and in any combination, is A, T, C or G, and x is equal to 0-3,
b. a variation of the sequence that is mentioned under a., or
c. a DNA sequence, which is complementary to the DNA sequence that is mentioned under a. or b.
A variation of the sequence can be, e.g., an allelic variation. An allelic variation is defined as a mutation, i.e., a change in the DNA sequence, which is characterized by natural deletion, addition or substitution of nucleotides. Any of these changes can occur alone or in combination with other changes, once or several times in the DNA sequence. The binding properties of the HRE are changed only insignificantly by these changes. Allelic variants are to have an at least 80%, preferably an 85% or most preferably 90% sequence identity in the sequence that is mentioned under a. The DNS sequence according to the invention is present in vivo as a double strand, i.e., it binds to the DNA sequence that is complementary to it.
In addition, a variation can be a sequence that hybridizes with the sequence according to the invention under stringent conditions. For an HRE with 17 nucleotides, stringent conditions are a hybridization temperature of 35° C., preferably 38° C., most preferably 41° C. in a QuickHyb buffer (stratagenes).
Hybridization conditions can be chosen to select nucleic acids which have a desired amount of nucleotide complementarity with the naturally-occurring nucleotide sequences. A nucleic acid capable of hybridizing to such sequence, preferably, possesses, e.g., about 85%, more preferably, 90%, 92%, and even more preferably, 95%, 97%, or 100% complementarity, between the sequences. The present invention particularly relates to nucleic acid sequence variations which hybridize to the nucleotide sequence set forth in SEQ ID NOS. 1-3 under low or high stringency conditions.
Nucleic acids which hybridize to the mentioned sequences can be selected in various ways. For instance, blots (i.e., matrices containing nucleic acid), chip arrays, and other matrices comprising nucleic acids of interest, can be incubated in a prehybridization solution (6×SSC, 0.5% SDS, 100 &mgr;g/ml denatured salmon sperm DNA, 5×Denhardt's solution, and 50% formamide), at 30° C., overnight, and then hybridized with a detectable oligonucleotides probe, (see below) in a hybridization solution (e.g., 6×SSC, 0.5% SDS, 100 &mgr;g/ml denatured salmon sperm DNA and 50% formamide), at 42° C., overnight in accordance with known procedures. Blots can be washed at high stringency conditions that allow, e.g., for less than 5% bp mismatch (e.g., wash twice in 0.1% SSC and 0.1% SDS for 30 min at 65° C.), i.e., selecting sequences having 95% or greater sequence identity. Other non-limiting examples of high stringency conditions includes a final wash at 65° C. in aqueous buffer containing 30 mM NaCl and 0.5% SDS. Another example of high stringent conditions is hybridization in 7% SDS, 0.5 M NaPO4, pH 7, 1 mM EDTA at 50° C., e.g., overnight, followed by one or more washes with a 1% SDS solution at 42° C.
Whereas high stringency washes can allow for less than 5% mismatch, relaxed or low stringency wash conditions (e.g., wash twice in 0.2% SSC and 0.5% SDS for 30 min at 37° C.) can permit up to 20% mismatch. Another non-limiting example of low stringency conditions includes a final wash at 42° C. in a buffer containing 30 mM NaCl and 0.5% SDS. Washing and hybridization can also be performed as described in Sambrook et al., Molecular Cloning, 1989, Chapter 9.
Hybridization can also be based on a calculation of melting temperature (Tm) of the hybrid formed between the probe and its target, as described in Sambrook et al. Generally, the temperature Tm at which a short oligonucleotide (containing 18 nucleotides or fewer) will melt from its target sequence is given by the following equation: Tm=(number of A's and T's)×2° C.+(number of C's and G's)×4
Kunz Gary
Landsman Robert S.
Millen White Zelano & Branigan P.C.
Schering Aktiengesellschaft
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