EDLUND ANDERS (SE)
JOHANSSON THORE (SE)
LEONARDSSON GOERAN (SE)
EKSTRAND JONAS (SE)
EDLUND ANDERS (SE)
JOHANSSON THORE (SE)
LEONARDSSON GOERAN (SE)
| WO1997046675A1 | 1997-12-11 | |||
| WO1999021890A1 | 1999-05-06 |
HOWARD CHRISTIAN PETERS ET AL.: "Mapping genomic structure and polymorphisms of the human GABABR1 receptorgene: evaluation of its involvement in idiopathic generalized epiliepsy", NEUROGENETICS, vol. 2, 1998, pages 47 - 54, XP002932306
| 1. | A nucleic acid molecule constituting a human GABAB receptor 1 promoter Pla. or a functionally equivalent modified form thereof, or an active fragment thereof. |
| 2. | A nucleic acid molecule according to claim I selected from : (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 1; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing. under stringent conditions, to a nucleotide sequence complementary to the polypeptide coding region of a DNA molecule as defined in (a). |
| 3. | A nucleic acid molecule constituting a human GABAB receptor 1 promoter Plb, or a functionally equivalent modified form thereof, or an active fragment thereof. |
| 4. | A nucleic acid molecule according to claim 1 selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 2; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the polypeptide coding region of a DNA molecule as defined in (a). |
| 5. | A nucleic acid molecule comprising a nucleic acid molecule according to claim 1, in combination with a nucleic acid molecule according to claim 3. |
| 6. | A nucleic acid molecule comprising a nucleic acid molecule according to claim 2, in combination with a nucleic acid molecule according to claim 4. |
| 7. | A vector transformed with a nucleic acid molecule according to any one of claims 1 to 6. |
| 8. | A cultured cell host harboring a vector according to claim 7. |
| 9. | An expression system comprising a nucleic acid molecule constituting a human GABAB receptor 1 promoter Pla, or a functionally equivalent modified form thereof, or an active fragment thereof. |
| 10. | An expression system according to claim 9, comprising a nucleic acid molecule selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 1; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the polypeptide coding region of a DNA molecule as defined in (a). |
| 11. | An expression system comprising a nucleic acid molecule constituting a human GABAB receptor 1 promoter Plb, or a functionally equivalent modified form thereof, or an active fragment thereof. |
| 12. | An expression system according to claim 11, comprising a nucleic acid molecule selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 2; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the polypeptide coding region of a DNA molecule as defined in (a). |
| 13. | An expression system comprising a nucleic acid molecule according to claim 1, in combination with a nucleic acid molecule according to claim 3. |
| 14. | An expression system comprising a nucleic acid molecule according to claim 2, in combination with a nucleic acid molecule according to claim 4. |
| 15. | An expression system according to any one of claims 9 to 14, which, in addition, comprises a reporter gene. |
| 16. | An expression system according to claim 15, wherein the reporter gene is selected from: (a) the firefly luciferase gene, (b) the bacterial amphenicol acetyl transferase (CAT) gene, (c) the pgalactosidase (pGAL) gene, and (d) the green fluorescent (GFP) gene. |
| 17. | An expression system according to claim 15 or claim 16, wherein the promoter and the reporter gene are positioned so that the expression system of the reporter gene is regulated by the GABAB receptor 1 promoter. |
| 18. | An expression system according to any one of claims 9 to 17, wherein the said nucleic acid molecule is transformed in a vector. |
| 19. | An expression system according to claim 18, wherein said vector comprises an origin of replication and/or a dominant selection marker. |
| 20. | A host cell transfected with an expression system according to any one of claims 9 to 19. |
| 21. | A method of assay for GABAB receptor promoter activity, comprising the use of a host cell according to claim 20. |
| 22. | A method for screening compounds which are modulators of GABAB receptor 1 transcription, comprising the steps of: (a) transfecting a cell host with a suitable expression system comprising a nucleic acid molecule constituting a human GABAB receptor 1 promoter Pla, and/or a human GABAB receptor 1 promoter Plb, or functionally equivalent modified forms. or active fragments thereof coupled to a reporter gene; (b) contacting a test compound with the cell : and (c) determining whether the test compound modulates the level of expression of the reporter gene. |
| 23. | A method according to claim 22, wherein the said expression system is an expression system according to any one of claims 9 to 19. |
| 24. | A method according to claim 22, wherein the said the reporter gene is selected from: (a) the firefly luciferase gene, (b) the bacterial amphenicol acetyl transferase (CAT) gene, (c) the pgalactosidase (pGAL) gene, and (d) the green fluorescent (GFP) gene. |
| 25. | The method according to claim 22, wherein the host cell endogenously expresses GABAB receptor 1. |
| 26. | The method according to claim 22, wherein the host cell is further transfected with a suitable expression system comprising a nucleic acid molecule encoding one or more specific transcription factors. |
| 27. | The method according to claim 26, where the transcription factor is selected from the group: CREB1, CREB2, CREM1, ATF1, ATF2, ATF3, ATF4, Spl, Sp2, Sp3, Sp4, AP1, and AP2. |
| 28. | A transgenic nonhuman animal whose genome comprises an expression system comprising nucleic acid molecules constituting GABAB receptor promoters Pla and/or Plb, or functionally equivalent modified forms thereof, or active fragments thereof, coupled to a reporter gene. |
| 29. | A transgenic nonhuman animal whose genome comprises an expression system according to any one of claims 9 to 19. |
| 30. | A method for the screening of compounds which are modulators of GABAB receptor 1 transcription, comprising the use of a transgenic nonhuman animal according to claim 28 or claim 29. |
| 31. | A method for the screening of compounds which are modulators of GABAB receptor 1 transcription, comprising the use of tissues or cells isolated from a transgenic non human animal according to claim 28 or claim 29. |
BACKGROUND G. 4BAB receptor I GABA (y-aminobutyric acid) is an endogenous neurotransmitter in the central and peripheral nervous systems. Receptors for GABA have traditionally been divided into GABAA and GABAB receptor subtypes. GABAB receptors (for a review see Kerr, D. I. B. and Ong, J. (1995) Pharmac. Ther. vol. 67, pp. 187-246) belong to the superfamily of G- protein coupled receptors. GABAB receptor agonists are described as being of use in the treatment of CNS disorders, such as muscle relaxation in spinal spasticity, cardiovascular disorders, asthma, gut motility disorders such as irritable bowel syndrome and as prokinetic and anti-tussive agents. GABAB receptor agonists have also been disclosed as useful in the treatment of emesis (WO 96/11680) and reflux disease (WO 98/11885).
The cloning of the cDNA encoding the rat GABAB receptors splice isoforms GABABR1 a and GABAgRIb is disclosed by Kaupmann et al. (1997) Nature. vol. 386,239-246. The mature rat GABAgRIb differed from GABAgRIa in that the N-terminal 147 residues were replaced by 18 different residues. It was presumed that the rat GABABR I a and-b receptor variants are derived from the same gene by alternative splicing.
The cloning of the cDNA encoding the human GABAB receptor GABABR I b is disclosed in WO 97/46675.
The cloning of the human GABAB receptor 1 gene and the elucidation of the exon-intron organization is in part or fully disclosed in PCT/SE98/01947. in ENIBL HS971 M'I (GenBankAL031983). EMBLAJ010170toAJ010I91, in Peters. HC et al.. Neurogenetics 2 ; 47-54 (1998) and in Goei. VL et al. Biological Psychiatry. 44 : 659-66 (1998). The human GABAB receptor 1 gene consists of 23 exons, spanning over a distance of 30 kb (Figure 1). The elucidation of the gene organization revealed that the human GABABRIa and GABABRIb are splice variants encoded by a single gene. The GABAsRIa and GABABRIb isoforms are differentially expressed. at least in the rat (Kaupmann et al.
(1997) Nature, vol. 386,239-246). The physiological consequences of multiple GABAB receptor 1 splice isoforms has not yet been determined, but their existence constitute an opportunity for the development of specific pharmaceutical agents.
GABAB receptor 2 Based on its homology with the mammalian GABABRI cDNA, a second member of the GABAB receptor family was identified (Jones, KA et al., Nature 396 ; 674-679 (1998), White, JA et al., Nature 396; 679-682 (1998), Kaupmann, K et al., Nature 396; 683-687 (1998), WO 99/20751). The corresponding protein, GABABR2, forms heteromers with GABABR I a and R I b, resulting in cell surface expression of a functional GABA B receptor (Kuner, R et al. Science 283,74-77 (1999)). At least in recombinant expression systems, GABABRI and R2 coexpression is necessary for the formation of a functional GABAB receptor. Jones et al. (Nature 396; 674-679 (1998)) disclosed that a GABABRI: GABABR2 stochiometry of 1: 1 is an optimal ratio for successful cell surface expression of a ligand binding and functional GABAB receptor. Thus, modulating GABABRI expression could alter the stochiometry between GABABR 1 and other interacting proteins and be a means to regulate signaling through GABAB receptors and thereby interfere with various physiological processes.
Transcriptional regulation Gene regulation is mediated by specific DNA elements in the promoter that directs binding of transcription factors, which thereby mediate transcription of the gene. Eukaryotic transcription factors can be divided in two main groups i) basal transcription factors that
interact with promoter sequences proximal to the start of transcription, thereby initiating transcription upon recruitment of RNA polymerase II and ii) transcription factors that bind to specific distal promoter elements. thereby modulating the transcription upon contact with the basal transcription machinery. The DNA sequence that directs the start of transcription in most eukaryotic genes is the TATA-box. which is often located approximately 30 base pairs upstream from the start of transcription. However, the TATA- box is not a prerequisite for initiation of transcription as there are many promoters, including the GABAB Rl promoters described in this study, that lack a TATA-box. A fundamental physiological process in the eukaryotic organism is that cells can communicate with their environment and respond to extracellular stimuli through signaling molecules, such as hormones and growth factors. The final event for such signaling is the binding of transcription factors to specific distal promoter elements leading to for example up-regulated or tissue specific gene expression. Because of their regulatory role, signaling molecules are putative targets for screening of therapeutic agents. The presence of two distinct and differentially regulated promoters within the human GABAB receptor 1 gene, disclosed in this patent application, makes it possible to screen for therapeutic agents selectively regulating expression of GABAB receptor la and lb-type splice isoforms.
Indications Compounds which are modulators of GABAB receptor 1 transcription are potentially useful in the treatment of disorders which are related to neurally-controlled physiological responses regulated by GABAB receptors, e. g. CNS disorders such as muscle relaxation in spinal spasticity, Alzheimer's disease and other dementias, psychiatric and neurological disorders such as depression, anxiety and epilepsy, cardiovascular disorders, asthma, gut motility disorders such as irritable bowel syndrome, emesis and reflux disease.
In some humans, the lower esophageal sphincter (LES) is prone to relaxing more frequently than in other humans. As a consequence, fluid from the stomach can pass into the esophagus since the mechanical barrier is temporarily lost at such times, an event hereinafter referred to as"reflux".
Gastro-esophageal reflux disease (GERD) is the most prevalent upper gastrointestinal tract disease. Current therapy has aimed at reducing gastric acid secretion. or by reducing esophageal acid exposure by enhancing esophageal clearance, lower esophageal sphincter tone and gastric emptying. The major mechanism behind reflux has been considered to depend on a hypotonic lower esophageal sphincter. However, recent research (e. g.
Holloway & Dent (1990) Gastroenterol. Clin. N. Amer. 19,517-535) has shown that most reflux episodes occur during transient lower esophageal sphincter relaxations (TLESR), i. e. relaxations not triggered by swallows. It has also been shown that gastric acid secretion usually is normal in patients with GERD. Consequently, there is a need for compounds which reduce the incidence of TLESR and thereby prevent reflux.
DESCRIPTION OF THE INVENTION This invention relates to nucleic acid molecules constituting GABAB receptor 1 promoters and fragments of said promoters. By GABAB receptor 1 promoters is understood the nucleic acids sequences upstream of the ATG translation initiation codon of GABAB receptor la of the GABAB receptor 1 gene, designated Pla, and the nucleic acids sequences upstream of the ATG translation initiation codon of GABAB recpetor lb of the GABAB receptor 1 gene, designated Plb, as illustrated in Figure 1.
In the present context the term"promoter"is meant to include core promoter sequences proximal to the start of transcription and upstream promoter elements which bind constitutively active transcription factors, as well as distal promoter elements which bind specific transcription factors.
Accordingly, the present invention provides a nucleic acid molecule constituting a human GABAB receptor I promoter Pla, or a functionally equivalent modified form thereof, or active fragments thereof. The present invention also provides a nucleic acid molecule constituting a human GABAB receptor 1 promoter Plb, or a functionally equivalent
modified form thereof, or active fragments thereof. By a functionally equivalent modified form is understood nucleic acids modified from the original sequence that can bind transcription factors. By active fragments of the promoters is understood nucleic acid fragments that can bind transcription factors.
In preferred forms of the invention the said nucleic acid molecule is selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 1; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a).
In other preferred forms of the invention the said nucleic acid molecule is selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 2; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a).
In another preferred form of the invention the said nucleic acid molecule may be a nucleic acid molecule constituting a human GABAB receptor 1 promoter PI a, or a functionally equivalent modified form thereof, or active fragments thereof, in combination with a nucleic acid molecule constituting a human GABAB receptor 1 promoter Plb, or a functionally equivalent modified form thereof, or active fragments thereof.
In another preferred form of the invention the said nucleic acid molecule may be a nucleic acid molecule selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 1 ; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a); in combination with a nucleic acid molecule is selected from:
(a) a nucleic acid molecule comprise2 a nucleotide sequence set forth as SEQ ID NO: 2: (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing. under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a).
It should thus be understood that the nucleic acid molecules according to the invention is not to be limited strictly to molecules comprising the sequences set forth as SEQ ID: 1 and 2. Rather the invention encompasses nucleic acid molecules carrying modifications like substitutions, small deletions, insertions or inversions, which nevertheless have substantially the biochemical activity of the GABAB receptor promoters 1 a and/or I b according to the invention. Included in the invention are consequently nucleic acid molecules, the nucleotide sequence of which is at least 95% homologous, preferably at least 96%, 97%, 98% or 99% homologous, with the nucleotide sequence shown as SEQ ID NO: 1 or 2 in the Sequence Listing.
The term"stringent hybridization conditions"is known in the art from standard protocols (e. g. Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994) and could be understood as as stringent or more stringent than those defined by e. g. hybridization to filter-bound DNA in 0.5 M NaHPO4. 7Wo sodium dodecyl sulfate (SDS), 1 mM EDTA at +65°C, and washing in SDS at +68°C.
It will be appreciated that the nucleic acid sequences shown in the Sequence Listing is only an example within a large but definite group of nucleic acid sequences, which will have the GABAB receptor promoter activity.
In yet another aspect, the invention provides a vector transformed with a nucleic acid molecule of the present invention. The said vector can e. g. be a replicable expression vector, which carries a nucleic acid molecule according to the invention. In the present context the term"replicable"means that the vector is able to replicate in a given type of host cell into which is has been introduced. Examples of vectors are viruses such as
bacteriophages. cosmids, plasmids and other recombination vectors. Nucleic acid molecules are inserted into vector genomes by methods well known in the art.
Another embodiment of the present invention is an expression system comprising nucleic acid molecules encoding GABAB receptor promoters Pla and/or Plb, or functionally equivalent modified forms, or active fragments thereof.
In preferred forms of this embodiment of the invention the said nucleic acid molecule is selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 1 and/or SEQ ID NO: 2; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing. under stringent conditions, to a nucleotide sequence complementary to the polypeptide coding region of a DNA molecule as defined in (a).
In another preferred form of this embodiment of the invention the said nucleic acid molecule may be a nucleic acid molecule constituting a human GABAB receptor 1 promoter Pla, or a functionally equivalent modified form thereof, or active fragments thereof, in combination with a nucleic acid molecule constituting a human GABAB receptor 1 promoter Plb, or a functionally equivalent modified form thereof, or active fragments thereof.
In another preferred form of this embodiment of the invention the said nucleic acid molecule may be a nucleic acid molecule selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 1; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing. under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a); in combination with a nucleic acid molecule is selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO : 2 ; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a).
The expression system may. in addition, comprise a reporter gene. the promoter and the reporter gene being positioned so that the expression of the reporter gene is regulated by the GABAB receptor 1 promoters P I a and/or P I b. Suitable expression systems according to the invention are e. g. bacterial or yeast plasmids, wide host range plasmids and vectors derived from combinations of plasmid and phase or virus DNA. Furthermore, an origin of replication and/or a dominant selection marker can be present in the vector according to the invention. Suitable reporter genes that can be used for the construction of expression systems according to the invention are e. g. the firefly luciferase gene, the bacterial chloramphenicol acetyl transferase (CAT) gene, the p-galactosidase (P-GAL) gene, and the green fluorescent protein (GFP).
A further aspect of this embodiment of the invention is a host cell transfected with an expression system comprising nucleic acid molecules constituting GABAB receptor promoters P I a and/or P I b, or functionally equivalent modified forms thereof, or active fragments thereof.
Suitable host cells are cells known to express GABAB receptors or cells known to express transcription factors, which can influence the transcription of GABAB receptors. Host cells transfected with DNA encoding specific transcription factors can preferably be used to study the interaction with defined transcription factors and the GABAB receptor promoter.
Another embodiment of the invention is a method for the assay of GABAB receptor promoter activity said method comprising the use of a host cell transfected with an expression system comprising nucleic acid molecules constituting GABAB receptor promoters P I a and/or P I b, or functionally equivalent modified forms thereof, or active fragments thereof.
A further embodiment of the present invention is a method for the screening of compounds which are modulators of GABAB receptor 1 transcription, said method comprising the use of nucleic acid molecules constituting GABAB receptor Pla and/or Plb promoters.
Accordingly, the present invention provides a method for screening compounds which are modulators of GABAB receptor 1 transcription, comprising the steps of: (a) transfecting a cell host with a suitable expression system comprising a nucleic acid molecule constituting human GABAB receptor 1 promoter P 1 A, and/or a human GABAB receptor 1 promoter P1B or functionally equivalent modified forms, or active fragments thereof coupled to a reporter gene; (b) contacting a test compound with the cell ; and (c) determining whether the test compound modulates the level of expression of the reporter gene.
In one aspect of this embodiment of the invention it is provided a method of screening of compounds which are modulators of GABAB receptor 1 transcription, wherein the cell host endogenously expresses GABAB receptor 1.
In another aspect of this embodiment of the invention it is provided a method of screening of compounds which are modulators of GABAB receptor 1 transcription. wherein the said cell host is further transfected with a suitable expression system comprising a nucleic acid molecule encoding one or more specific transcription factors. Preferably, the said transcription factors are selected from the group: CREB-1, CREB-2, CREM-1, ATF-1, ATF-2, ATF-3, ATF-4, Spl, Sp2. Sp3, Sp4, AP-l, andAP-2.
A further embodiment of the invention is a transgenic non-human animal whose genome comprises an expression system comprising nucleic acid molecules constituting GABAB receptor promoters Pla and/or P 1 b, or functionally equivalent modified forms thereof, or active fragments thereof, coupled to a reporter gene.
Such transgenic non-human mammals can be generated by insertion of DNA comprising GABAB receptor promoters by microinjection, retroviral infection or other means well known to those skilled in the art, into appropriate fertilized embryos to produce a
transgenic animal (Hogan B. et al Manipulating the Mouse Embryo. A Laboratory Manual.
Cold Spring Harbor Laboratory (1986)).
Accordingly, the present invention provides a method for the screening of compounds which are modulators of GABAB receptor 1 transcription, comprising the use of a transgenic non-human animal whose genome comprises an expression system comprising nucleic acid molecules constituting GABAB receptor promoters Pla and/or P I b, or functionally equivalent modified forms thereof, or active fragments thereof, coupled to a reporter gene. or tissues or cells isolated from such transgenic animals.
EXAMPLES The following examples are preferred non-limiting examples embodying preferred aspects of the invention.
Example 1. Isolation and identification of human GABAB R1 promoters P I a and P I b Genomic DNA containing the human GABAB receptor gene was isolated from human genomic libraries and genomic DNA. Human genomic libraries were obtained from Clontech (Palo Alto, CA, USA). The libraries were constructed from female leukocyte DNA (catalogue &num HL1111J), cloned into REMBL-3 vector. The average size of inserts are 16 kb and the number of independent clones are 1.7x 106. Human genomic DNA was obtained from Clontech (catalogue # 6550-1). In order to isolate recombinant phases containing exon and intron sequences of the human GABAB receptor gene, 48 individual bacterial plates with a diameter of 150 mm and approximately 4 x 104 individuai plaques per plate, were screened. The methods and solutions used were as described in the Library Protocol Handbook: General Procedures for the Hybridization of Lambda Phage Libraries w/DNA Probes (Clontech) with some modifications as will be apparent from the following.
The experiment was carried out essentially as follows. The numbers are given per plate basis. A sample of the phase library diluted in 0.1 ml sterile lambda diluent was prepared in order to obtain an estimated titer of 40.000 pfu (plaque forming units). A 0.6 ml LB- medium culture of the E. coli host strain K802 (obtained from Clontech) was infected with 40000 pfu recombinant phases for 15 minutes at 37°C. The culture was then mixed with 7 ml top agarose (6.5 g of agarose added per liter LB) and poured onto LB plates. The plates were incubated at 37°C for approximately 7 hours. The plates were then chilled at +4°C.
Plaque hybridization experiments were as follows. Membrane filters. Colony/Plaque Screen (DuPont. Wilmington, DE. USA), were placed onto the top of the plates for 3 minutes. For denaturation of DNA the filters were removed and floated in 0.5 M NaOH on a plastic wrap for 2 minutes, with the plaque side up. This step was repeated once to ensure efficient denaturation. Following neutralization the membrane filters were placed in 1M Tris-HCI pH 7.5, two times 2 minutes and allowed to dry.
To obtain probes for DNA hybridization screening of the membrane filters, a GABAB receptor cDNA clone was digested with SacII and a 479 bp fragment, separated by agarose electrophoresis, excised and transferred to a polypropylene microcentrifuge tube.
Additional probes were obtained by PCR amplification of various regions of the GABAB receptor cDNA. The isolated cDNA fragment was 39P-labeled using Megaprime DNA labeling system (Amersham Pharmacia Biotech, Uppsala, Sweden) by the following procedure. Water was added at a ratio of 3 ml per gram of gel. and placed in a boiling water bath for 7 minutes to melt the gel and denature the DNA. A volume of DNA/agarose solution containing 25 ng of DNA was added to the labeling reaction, according to the supplier's instructions. Labeled nucleotides were removed from DNA labeling reactions using MicroSpin G-50 Columns (Amersham Pharmacia Biotech. Uppsala. Sweden).
The DNA hybridization reaction was performed under stringent conditions according to the method described below. The filter membranes were prehybridized at 65°C for at least 1 hour in a solution composed of 1% SDS. 1M NaCI, and 10% dextran sulfate using a
hybridization oven (Hybaid Ltd, Ashford, UK). Following prehybridization a solution containing denatured herring sperm DNA of a final concentration of 100 ug/ml and the 32p-labeled DNA probe at a concentration <10 ng/ml (for optimal signal to background ratio) was added to the prehybridization solution and the membrane filters were incubated at 65°C for 10-20 hours. Following the removal of the hybridization solution the membrane filters were first washed in a 2xSSC (0.3M NaCl, 0.03M Na-citrate), 1% SDS solution two times for 5 minutes at room temperature. In the next step, the membrane filters were incubated at 60°C two times for 30 minutes each in the same solution. In a third step, the filters were washed two times at room temperature in 0.1xSSC. Finally, the membrane filters were placed on a sheet of filter paper with the DNA face up, and allowed to dry. The dried membrane filters were then exposed to X-ray films and autoradiographed.
Of the approximately 2x 106 individual plaques analyzed, four hybridizing plaques were detected and isolated. These four isolates were designated &num GR 1. &num GR 12, &num GR 13 and #GR41, respectively. After several rescreening experiments, the recombinant phage DNA was purified using Qiagen Lambda Midi Kit (Qiagen GmbH, Germany). The purified DNA was digested with SalI and the fragments representing the inserts were isolated by agarose electrophoresis.
The 16kb insert of isolate &num GR13 was cloned into SalI digested linearized pUC19, resulting in the plasmid pAM364. The insert was analyzed by PCR, restriction mapping and hybridization to 32P-labeled DNA fragments representing various regions of the GABAB receptor cDNA.
The cloned fragment in the plasmid pAM364 was characterized by restriction enzyme mapping, using EcoRI, HindE, PstI, and BamHI. The approximate positions of the exons and the approximate size of the introns were analyzed and determined by PCR-based exon- exon linking and agarose gel electrophoresis.
In order to facilitate nucleotide sequence analysis, 7 restriction sub-fragments derived from pAM364 were isolated and cloned individually into pUC19. The following strategy was
employed : by combining PCR primers located within the pUC 19 sequence either upstream or downstream of the cloning site, with a PCR primer with defined orientation and specific for the GABAB receptor derived subcloned fragment allowed the sequence determination.
The inserts were subjected to nucleotide sequence analysis. The nucleotide sequences for all subclones were determined using a Thermo Sequenase dye terminator cycle sequencing pre-mix kit (Amersham Pharmacia Biotech, Uppsala, Sweden). As primers for sequencing reactions specific oligonucleotides complementary to pUC19 or primers complementary to the human GABAB receptor cDNA were used.
The sequence of the human GABAB receptor gene fragment cloned in the plasmid pAM364 has previously been revealed (see PCT/SE98/01947). This genomic fragment was shown to contain the complete exons 1-11 and the complete introns 1-10 of the human GABAB receptor gene as well as > 3kb sequence upstream of exon 1. The elucidation of the gene organization revealed that the human GABABRla and GABABRIb are splice variants encoded by a single gene.
In order to localize the putative human GABAB receptor promoter, we investigated the genomic sequence for the presence of consensus sequences of known regulatory promoter elements. To our surprise, we found that promoter elements, clustered in two regions: one region upstream of exon 1, and the other region in intron 5, just upstream of exon 6. We concluded that the human GABAB receptor may be regulated by two independent promoters, and not by one single promoter as expected. The first putative promoter, denoted PI a (SEQ ID NO: 1) and described in detail below, may regulate transcription of GABABRIa-type splice variants and the second putative promoter, denoted Plb (SEQ ID NO: 2) and described in detail below, may regulate transcription of GABABRlb-type splice variants.
As indicated in the schematic representation of P I a and Pib (Figure 2), both putative promoters lack a TATA box. However Plb has an initiator (Inr) element in position 4375- 4381 which is located 24-30 bp upstream of the position corresponding to the 5'end of the
lonaest known cDNA isolated with"rapid amplification of cDisA ends (RACE) PCR amplification. The Inr element mav therefore direct the start of transcription from P1b. P1a contains neither a TATA or an Inr element and the transcription from R1a may therefore initiate from different start sites which is often the case in promoters lacking TATA boxes or Inr elements. Both P I a and P I b contain multiple GC rich regions at pos. 3009-3016, 3037-3044 and 3116-3123 in R1a and pos. 4080-4087.4196-4204, 4241-4249 and 4272- 4279 in Rlb, which are potential binding site for the SP1 family of transcription factors.
SP1 binding sites are often found in TATA lacking promoters where they often substantially contribute to transcription. In addition, to the indicated GC sequences in Figure 2 there are also other GC motifs that may function as SP1 binding sites. Pla further contains an activator protein-1 (AP-1) site at position 1497-1503. AP-1 sites are recognized by AP-1 transcription factors which consists of homodimers of members of the Jun family or Fos/Jun heterodimeric complexes. AP-1 complexes also interact, by protein-protein interactions, with members of the steroid receptor family and are therefore also targets for steroid receptor signaling. In addition to the GC motifs Plb also contain an activator protein-2 (AP-2) site at position 3844-3851 and a cAMP responsive element (CRE) at position 4308-4315. Especially the finding of a consensus CRE (TGACGTCA) is interesting as this promoter element is found in many genes regulated by cAMP which are bound and regulated by members of the ATF/CREB gene family. This sequence may therefore be an important target for cAMP mediated signaling via G-protein coupled receptors, including GABAB receptors.
We conclude that transcription of the human GABAB receptor gene may be regulated by two putative promoters, P I a and P I b, that may independently regulate expression of human GABAB receptor la and lb splice isoforms, respectively.
Example 2. Determination of GABAB R 1 promoter P I a and P1 b activitv To experimentally determine if Pla and Plb indeed act as promoters, we fused these regions to a cDNA encoding firefly luciferase to be used as a reporter of promoter activity in transfected cells.
Reporter constructs containing Ria and R1b promoter fragments were generated by PCR using plasmid pAM 364, containing genomic sequence covering the promoter regions, as template. PCR reaction was performed by standard procedure (Perkin Elmer). Briefly. an initial denaturation at 95°C for 4 min was followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, elongation at 72°C for 1 min and finally a 7 min elongation at 72°C. In the sequence for primers used to generate promoter fragments (for details see Table 1. below), a Nhe I and Hind m endonuclease restriction enzyme site was introduced, in 5 and 3 primers respectively, to enable sub-cloning into Nhe I/Hind III digested pGL3-Basic luciferase reporter vector (Promega). Hence, complete promoter- reporter constructs (pAM440, pAM438 and pAM436) contain Ria and R1b promoter fragments (indicated size see table) fused to the luciferase reporter gene. Plasmid DNA were purified using Qiagen tip-100 columns according to suppliers instruction. Correct fragment insertion was verified by DNA sequencing.
Table 1. Nucleotide sequence of primers used to generate promoter fragments Primer No. Restriction site Promoter sequence Position Sequence 5-3 (AAGCTT)SEQIDNO:13440-3424AAGCTTCTCGGCGCGCGGGCCCG1582Hindl@ (GCTAGC)SEQID1583Nhel 2341-2362GCTAGCCAAGAGCTTCTGGAGCCG1 1584 Nhel (GCTAGC) SEQ ID NO : I 720-741 GCTAGCTGTTACATGCAGAGC AATC 1585 Hindlll (AAGCTT) SEQ ID NO: 2 4439-4421 AAGCTTCCTACGGCCCCCGCG 1586 Nhel (GCT AGC) SEQ ID NO: 2332)-3340GCT AGC GCG CAC TGC AAT GCC CTC
To determine putative promoter activity. the reporter gene constructs were introduced into mammalian cells by transfection. In this study the cell line Nid7/23 (ECACC Ref No: 92090903) was used. ND7/23 is a hybrid cell line originating from a mouse neuroblastoma (N18tg9) fused by PEG to a rat dorsal root ganglion neuron cell line. This cell line was chosen since it express functional GABAB receptors as evidenced by radioligand binding studies. Cells were cultured in a 1: 1 mixture of Dulbecco's modified medium (DMEM) and Ham's FI2 medium supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were grown at 37°C in an atmosphere of 5% CO2. ND7/23 cells (4xx10,) were transfected using the DMRIE-C reagent according to manufacturer's protocol (Gibco). Briefly, cells were seeded in 6 well tissue culture plates (Nunc) the day before transfection. Next day. 2Lg'of of promoter-reporter construct and l) J. g of transfection control construct (pSV-p- Galactosidase, Promega) was mixed with 0.5 ml Optimem media (Gibco). DNA containing media was then mixed with an equal volume (0.5ml) of Optimem containing 4pL1 of DMRE-C reagent and the combined mixture was then incubated for 45 min. After incubation, the transfection mixture was added to the cells, which were washed with Optimem media just prior to addition of transfection mixture. Following 5h incubation at 37°C, an equal volume of 1: 1 mixture of Dulbecco's modifled medium (DMEM) and Ham's F12 medium supplemented with 20% (v/v) fetal bovine serum (FBS) was added to the cells. Cells were incubated for 24h at 37°C with or without cAMP enhancing supplement as indicated in Figure 3. Before cell harvest, cells were washed in PBS (7.6 mM Na2HPO4/NaH2PO4 pH 7.4 and 120 mM NaCl), then cell extracts were prepared by addition of zip reporter lysis buffer (Promega) to cells, followed by transfer of cell suspension to 1.5 ml tubes. Cells were further lysed by one round of freeze-thawing and 15s of vortex. Cell debris were removed by 2 minutes of centrifugation at 12 000 g.
Luciferase activity in cell extracts was measured in a Luciferase Assay System (Promega). where 40p1 of cell extracts was added to a 96-well plate (Maxisorp. Nunc) and mixed with 50p1 of luciferase substrate. Luciferase activity was then measured in a LUMIstar (BMG Lab technologies). As internal control for transfection efficiency,3-Galactosidase (P-Gal.) activity was measured in 96-well plate (Maxisorp. Nunc) using a g3-Galactosidase Enzyme
assay (Promega) according to suppliers protocol. As seen in Figure 3. transient transfection of ND7/23 cells shows that both Pla and Plb has promoter activity as pAM438. pAM436 and pAM440 (Figure 3) result in reporter expression. while the pAM4 ? (vector control) has very low activity. In addition, this experiment demonstrates that especially reporter expression originating from pAM440 (R1b) can be induced by the cAMP activating agent forskolin. Moreover. forskolin induced expression may also be further enhanced in the presence of the phosphodiesterase inhibitor 1-methyl-3- isobutylxanthine (IBMX) although this experiment does not show a significant difference.
In conclusion, this experiment demonstrate that Pla and P I b both have promoter activity and that the degree of activity can be modulated using the cAMP activating agent forskoline.
Example 3. Screening for substances modulating Pla and Plb activity Modulating GABABR1 expression in a controlled way is a means to regulate signaling through GABAB receptors which could be of significant therapeutic value for a variety of conditions. Particularly, the ability to specifically regulate expression of either la-or lb- type GABAB receptor splice isoforms could be of medical value if these isoforms could be attributed to specific conditions.
The experiment presented in Example 2 demonstrates that the use of P 1 a and P I b promoter/reporter constructions can be used to monitor GABAB R 1 expression in screens for therapeutic agents that can modify the expression of GABAB receptor 1 isoforms. A screen for Pla and Plb modulating substances could be performed in ND7/23 cells as described in Example 2. A screen for Pla and P 1 b modulating substances could in addition be performed in any cell type with endogenous expression of GABAB receptor 1 and 2 isoforms. in cells expressing recombinant GABAB receptor 1 and 2 isoforms and in intact cells and in extract or fractions of cells expressing endogenous and recombinant proteins
modulating GABAB receptor function. Such screens could furthermore be done in tissues and in living organisms.
Example 4. Functional analvsis of GABAB-R 1 promoter fragments and modified forms In order to identify functionally active promoter fragments of P I a and P I b. a deletion analysis of DNA fragments containing promoter fragments can be performed in two steps.
It is anticipated that Pla and Plb comprise active fragments that can mediate increased expression by binding of transcription factors that are activators as well as active fragments which can mediate decreased expression by binding of transcription factors that are repressors.
In the first step, promoter fragments mediating expression of a reporter gene when used in reporter constructs can be stepwise deleted or truncated to identify important regions.
Briefly, truncated or deleted promoter fragments are created by PCR using specific primers and the already identified promoter sequences as template. Reporter constructs (as exemplified in this application) comprising the deleted or truncated promoter fragments are then created. These reporter constructs can then be used in transfection experiments, as described above, to identify important regions of the promoters manifested in altered expression from constructs lacking active fragments compared to none-deleted constructs.
In the second step, the exact location and sequence of transcription factor binding sites within active fragments can be determined by PCR technique using specific primers harbouring desired mutations. Such promoter fragments, with specifically mutated promoter regions, can be used in transfection experiments, similar to those described above, to determine the exact sequence of functionally important nuclear factor binding sites within active promoter fragments, manifested in altered expression from constructs with mutated DNA sequence compared to none-mutated control constructs with equal size.
The above mentioned strategy can also be used to identify the specific active promoter fragments which are important for the effect on promoter activity of active substances identified when screening for therapeutic agents regulatina GABAB-R1 expression.
Example 5. Activitv of GABAB R I promoter P I a and Pib fragments.
Reporter constructs containing Pla and Plb promoter fragments were generated by PCR as described in Example 2 and fused to the firefly luciferase reporter gene. The generated constructs are visualise in Figure 4.
Putative transcription initiation sites were identified at position 3207 in SEQ ID NO: 1 for Pla and at position 4405 in SEQ ID NO: 2 for Plb. The positions in the promoter region shown in Figure 4 were calculated setting the transcription initiation sites as position +1.
The generated promoter reporter constructs (see Figure 4) were used to transfect ND7/23 cells. As shown in Figure 4, deletion of Pla promoter region in between position-2549 bp and-361 caused an increase in expression, indicating putative repressor regions between position-2549 and-361. Moreover, when 175 bp of the 5'untranslated region were removed (compare the third and fourth Pla constructs from the top) there was no detected difference in promoter activity and constructs with the shorter Pla 5'-untranslated region were therefore used for mutational analysis throughout the rest of the study. Additional deletions of the Pla promoter from position-361 to-46 caused a stepwise decrease of the expression that seem to correlate with removal of promoter regions containing GC elements (I-ILI). This indicate that 361 bp of the Pla promoter can confer optimal expression and that promoter elements essential for R1 a-type expression, including GC elements I-E are located between position-361 and-46. Similar to the Pla promoter, deletion of Plb promoter region from position-3238 bp to-390 caused an increase in expression, indicating putative repressor regions between-1084 and-390. Also, additional deletions of the P I b promoter from position-390 to-88 caused a stepwise decrease of the expression that correlate with removal of promoter region GC elements (N-VII) as well as the consensus CRE. Although there was still some promoter activity left when the Plb
promoter region was deleted down to position-88. the major promoter elements essential for Rlb-type expression seem to be located between position-390 and-88. Comparison of Pla and Plb expression also indicates that. Plb mediated expression was higher (approximately 3-4 fold) than for PI a.
Example 6. Mutational analvsis of Pla and Plb promoter element function As shown in Figure. 4, reporter constructs-361 in P I a (short 5ETTR) and-390 in P I b conferred optimal expression of the respective promoters. In order to determine the importance of the promoter elements that was found within these regions, Pla (-361) and Plb (-390) constructs were used as templates to obtain reporter constructs with promoter regions mutated at specific sites. For the mutated promoter constructs, a 3 bp substitution was introduced by"quick change"site directed mutagenesis.
Site-directed m2xtagenesis : Mutant constructs were prepared with a QuickChange site- directed mutagenesis kit (Strategene, La Jolla, CA) according to the manufacturer's instructions. The mutagenic oligonucleotide primers are listed in Table 2. All constructs were verified by DNA sequencing.
Table 2. Oliaonucleotide primers used in the mutasenesis experiments Primer Primer Sequences 1949 P Rlb Cre Fwd CGCCGCCCGTTTGGTCAGAGCCCCCT 1950 P Rlb Cre Rev AGGGGGCTCTGACCAAACGGGCGGCG 1951 P Rla GCI Fwd CTCTCTTCCCCCCTAACTGCCTTCCC 1952 PRIaGCIRev GGGAAGGCAGTTAGGGGGGAAGAGAG 1953 P Rla GCII Fwd GGCGGTCCAGTTAGGGGCTGGGATCC 1954 PRlaGCIIRev IGGATCCCAGCCCCTAACTGGACCGCC R1aGCIIIFwd#CCTCTCCACCGCCCTAACCACCGCGTCTGTG2051P 2052 P R 1 a GCIII Rev CACAGCGCGGTGGTTAGGGCGGTGGAGAGG 2053 P Rlb GCIVs Fwd CCCCAGCTCCCGCCCTAACCCCCACCCC 2054 P Rlb GCIVs Rev GGGGTGGGGGTTAGGGCGGGAGCTGGGG 2055 P Rlb GCV Fwd CGCTTCCCTCCCCTAACCCTTCCTGCC 2056 P Rlb GCV Rev GGCAGGAAGGGTTAGGGGAGGGAAGCG 2057 P Rlb GCVI Fwd CCCTCCCCTCCCCTAACCTCCGACTGT 2058 P Rlb GCVI Rev ACAGTCGGAGGTTAGGGGAGGGGAGGG 2059 P Rlb GCVII Fwd CTCCGCCCACCCCTAACTCCTGGCAC 2060 P Rlb GCVII Rev GTGCCAGGAGTTAGGGGTGGGCGGAG 2146 P Rlb GCIVd Fwd CCCCAGCTCCCTAACTAACCCCCACCCC 2147 P Rlb GCIVd Rev GGGGTGGGGGTTAGTTAGGGAGCTGGGG
Bases that are labelled bold correspond to designed mutations Obtained promoter constructs were then used to transfect ND7/23 cells. As shown in Figure 5, single point mutations of GC elements I, II and m reduced Pla expression to approximately 65-75% compared to wild-type, while double-mutations (I/II, I/III and HAll, respectively) resulted in a further decrease to approximately 55-60% of wild-type expression levels. When all three GC elements (1-hui) were mutated, the reporter expression was reduced to approximately one third (33%) of wild-type expression. Together, these results suggest that the three GC-elements found in Pla, all contribute to Pla mediated expression in a substantial and additive manner. However, the fact that one third of the Pla promoter activity still remains suggests that there are additional promoter elements within Pla contributing to Pla mediated expression.
In contrast to PI a. single mutations of the four PI b GC elements (IV. V. VI and VII) only caused small reductions of expression to approximately 75-90cl compared to wild-type.
The relatively modest contribution by the four PI b GC elements was also demonstrated by a promoter construct were all four GC elements (IV-VII) had been mutated. This construct retained 63% of Plb expression compared to the wild-type construct. However, when the P1b CRE consensus site was mutated (CRE), a dramatic reduction of Plb mediated expression (approximately 50%) was obtained. Moreover, when the CRE mutation was combined with mutations of each GC element respectively, a further reduction was observed. Most notably, the promoter construct containing a double mutation of the CRE and GC V (CRE/V) promoter elements resulted in a substantial reduction of P1b expression to approximately 26cas. similar to the construct where all five P1b promoter elements were mutated (CRE/IV-VII) where 24% of the expression still remained compared to wild-type. Together these data demonstrates the absolute importance of the consensus CRE site for P1b expression, alone or in combination with the GC elements (IV, V, VI and VII), of which GC element V seems to contribute most. The fact that one fourth of Plb promoter activity still remains suggests that, as for Pla, there are additional promoter elements within P lb that may contribute to P1 b mediated expression.
Example 7. Identification of factors interacting with the P1b CRE site Electrophoretic mobility shift assays (EMSA) Electrophoretic mobility shift assays (EMSA) can be used to identify nuclear factors that interact with P I a and P I b promoter elements. In an attempt to identify factors interacting with the Plb CRE site, we performed gelshift analysis with super-shift antibodies that recognise members of the CREB/ATF family of transcription factors. The DNA-binding reactions (12 were done as follows; 9-3 fmol 32P-labeled double-stranded oligonucleotide corresponding to the Plb CRE site was mixed with 5 u. crude nuclear extracts from ND7/23 cells and 1 fig poly (dI-dC).'5mM Hepes (pH 7,9), 150 mM KC1,5 mM dithiothreitol and 10% glycerol (Schneider et al 1986, Nucleic Acids Research.
14: 1303-17). In the supershift lane (Figure 6) the ATF-1 p35/CREB-1 p43/CREM-l
reactive antibody (sc-270 from Santa Cruz Biotechnotogy. Santa Cruz. CA). was pre- incubated at room temperature for 20 min before addition of'2P-labeled probe. After incubation at room temperature for 15 min, protein-DNA complexes were resolved on precasted DNA-retard gels (Novex') containing 6% polyacrylamide prepared with 0.5x TBE as gel buffer., Following electrophoresis, gels were dried and visualise by autoradiography.
As shown in Figure 6. addition of a monoclonal CREB/ATF supershift antibody (reactive with members of the ATF/CREB family such as ATF-1 p3O. CREB-1 p43 and CREM-1 of mouse, rat and human origin) into the EMSA reaction mixture results in a distinct shift of the nuclear factor (s) that interacts with the PI b CRE site. while no super-shift was observed when the same antibody was added to other EMSA reactions containing other promoter elements (data not shown). This data suggests that the complex formed with the P I b CRE site contains a member of the CREB/ATF family.
Similar further studies can be performed in order to determine which factor (s) that interact with the various promoter elements in Pla and Plb.
Example 8. Use of recombinant transcription factors in the studv of P I a and P lb promoter activities.
Cells with stably integrated or with transiently transfected P 1 a/P 1 b reporter constructs can be transfected with cDNA encoding specific transcription factors to make reporter cells suitable for investigations ofPIa and P I b activities mediated by said transcription factors.
Reporter cells can alternatively be generated by delivery of transcription factors into similar cells by various means such as e. g. microinjection and lipofection. Reporter cells can be utilise for screening of compounds, which are modulators of GABAB receptor 1 transcription.
BREF DESCRIPTION OF DRAWINGS Figure 1. The human GABAB receptor gene The figure shows the organization of the human GABAB receptor gene. Exons, represented by vertical squares/bars, are numbered (1-23). Translational start and stop sites are indicated by arrows. Location of human GABAB receptor 1 promoters P I a (SEQ ID NO: 1) and Plb (SEQ ID NO: 2) are indicated. The extent of human GABAB receptor genomic sequence cloned in plasmid pAM 364 is indicated by a horizontal bar.
Figure 2. Schematic representation of the Pla and Plb promoters DNA fragments used to generate reporter constructs, corresponding to the positions in the P I a and P I b promoter sequence, are shown below each promoter. Putative promoter elements in each promoter are indicated. Arrows indicate putative positions for start of transcription.
Figure 3. Determination of GABAs receptor 1 promoter Pla and Plb activit ND7/23 cells (4x 105) were transfected with promoter-luciferase constructs as described above. After transfection, cells were cultured in media without supplement (basal) or in the presence of Forskolin (10M) or Forskolin (10M) + 1-methyl-3-isobutylxanthine (0.125mM) for 24h. After incubation, cells were harvested and luciferase activity measured. Luciferase activity, minus background, is shown as arbitrary light units measured in 40 ul of cell extracts. Relative values represent the mean+SEM of two individual experiments.
Figure 4: Deletion analysis of the GBR1 promoters in ND7/23 cells ND7/23 cells (4x 105) were transfected with P I a and P I b promoter-luciferase constructs as outlined above. After transfection, cells were cultured for 24h. harvested and luciferase activity was measured. Luciferase activity, minus background, is shown as arbitrary light units measured in 10 ul of cell extracts. Relative values represent the mean+SEM of duplicate samples from at least three individual experiments.
Figure 5. Effect of P I a and P) b promoter element mutations on promoter activities.
ND7/23 cells (4x 105) were transfected with wild-type and mutated P I a and P I b promoter- luciferase constructs as described above. The point mutations in the promoter constructs are outlined to the left. After transfection, the cells were cultured for 24h. After incubation, cells were harvested and luciferase activity measured. Luciferase activity, minus background, is shown as arbitrary light units measured in 10 ril of cell extracts. Relative values represent the mean+SEM of duplicate samples from at least three individual experiments. Relative expression level of mutated constructs are indicated to the left with the Pla and Plb"wild-type"expression level set as 100%.
Figure 6. Identification of nuclear factors bindina to the Plb consensus CRE site usina CREB/ATF super-shift antibodies.
Nuclear extracts (5 u. g) from ND7/23 cells were incubated with double-stranded 32 p- labeled oligonucleotides containing the Plb consensus CRE site (sense: 5'- CGCCGCCCGTGACGTCAGAGCCCCCT-3'). In lane 1, no antibody was added. In lane 2, a mouse monoclonal antibody (sc-270 Santa Cruz Biotechnology. Santa Cruz, CA) reactive with members of the ATF/CREB family such asATF-1 p35, CREB-1 p43 and
CREM-1 was pre-incubated at room temperature for 20 min before addition of 2P-labeled probe. The specific complex between nuclear factors and the CRE is indicated by a star and the super-shifted complex is indicated by two stars.