WO2001096602A2

WO2001096602A2 - Methods and materials to determine the p53 status of a sample by determining the binding of p53 to a vector

Info

Publication number: WO2001096602A2
Application number: PCT/GB2001/002718
Authority: WO
Inventors: Ai Li Yang; Michael Festing
Original assignee: Medical Research Council
Priority date: 2000-06-16
Filing date: 2001-06-18
Publication date: 2001-12-20
Also published as: WO2001096602A3; AU2001274277A1; GB0014820D0

Abstract

The invention provides: (1) a method of determining the p53 status of a sample, the method comprising providing a sample containing a pGL3 vector and determining whether p53 binds to the pGL3 vector; (2) a method of modifying a pGL3 vector, the method comprising deleting or altering a CCCGGG motif of the pGL3 vector and/or deleting or altering a sequence within a 20 bp sequence 5' or 3' of said CCCGGG motif; (3) a modified pGL3 vector having an alteration or deletion in such a site; and (4) the use of nucleic acid having a sequence incorporating the CCCGGG motif to confer promoter activity.

Description

METHODS AND MATERIALS RELATING TO PLASMID VECTORS

The present invention relates to known plasmid vectors, namely the pGL3 luciferase reporter vectors of Promega Corporation, and variants and uses thereof.

The pGL3 reporter vectors contain a modified firefly luciferase cDNA designated luc+ . Four different pGL3 vectors are commercially available from Promega Corporation (Madison, Wisconsin, USA) , namely pGL3-Basic, pGL3-Enhancer, pGL3-Promoter and pGL3-Control . Except for the inclusion of promoters and enhancers, these four vectors are structurally identical. pGL3-Basic lacks both promoter and enhancer; pGL3-Promoter has an SV40 promoter, but lacks an enhancer; pGL3-Enhancer has an SV40 enhancer, but lacks a promoter; pGL3-Control, by contrast, has both an SV40 promoter and an SV40 enhancer.

All four vectors possess a multiple cloning site, into which putative regulatory sequences may be inserted, to investigate their influence on luciferase expression. For more information on the pGL3 vectors, see Promega Technical Manual, pGL3 Luciferase Reporter Vectors, Instructions for use of products E1741, E1751, E1761 and E1771, Printed in USA 11/94, Part# TM033, which is incorporated herein by reference in its entirety for all purposes. (Copies available free of charge from Promega Corporation and from their website: www. promega . com/tbs/ TMO33/tmO33. html) . The sequences of these four commercially available vectors and details of their structural features are included in Annex 1.

In the present context, pGL3 vectors having the sequences set out in Annex 1 will be referred to as the "defined" pGL3 vectors. Other references to pGL3 vectors may be interpreted more widely, to include for example vectors derived from the defined pGL3 vectors, as explained in more detail below.

Surprisingly, the present inventors have found that under suitable conditions, p53 binds to and causes luciferase expression from the defined pGL3-Basic vector, despite its purported lack of any promoter or enhancer elements. Furthermore, by virtue of their structural similarities, it is proposed that other pGL3 vectors, particularly the other defined pGL3 vectors (more particularly the promoterless defined pGL3-Enhancer vector) , will have this property.

p53 protein is a transcription factor that regulates many genes, including those associated with cell cycle control, apoptosis and DNA damage repair (Lane, 1992; Marx, 1994; Wu and Levine, 1994; Polyak et al., 1997). The p53 protein may also regulate the genes associated with oxidative stress responses (Yang et al., 1999). Mutations in the p53 gene have been found to be associated with many forms of human cancer (Hollstein et al., 1991). The DNA-binding activity of wild-type (wt) p53 is believed be essential for its normal function as a tumour suppressor, as mutant p53 that loses tumour suppressor function fails to bind DNA either non- specifically (Bargonetti et al., 1991) or specifically (Raycroft et al., 1990; Weintraub et al., 1991).

Accordingly, the present invention provides a method of determining the p53. status of a sample, the method comprising providing a sample containing a pGL3 vector and determining whether p53 binds to the pGL3 vector.

The determination may occur by determining whether a reporter gene of the vector is expressed (in which case, the sample will comprise an expression system, preferably a cell or tissue sample) . In the defined pGL3 vectors, the reporter gene is luciferase, though the use of variant pGL3 vectors with other reporter genes is also contemplated.

In particular, therefore, the invention provides a method of determining the p53 status of a. cell, the method comprising providing a cell containing (e.g. transfected with) a pGL3 vector and determining whether luciferase is expressed in the cell.

The invention further provides: the use of a pGL3 vector for determining the p53 status of a sample; and a pGL3 vector, and/or cell transfected with a pGL3 vector, packaged with instructions for use in such a method or use for determining p53 status.

Reporter gene (e.g. luciferase)* expression may be determined using any appropriate assay, for example commercially available luciferase assays.

The cell may be cultured prior to determining reporter gene expression under conditions appropri-ate for protein expression.

The cell may be isolated or many cells (for example a cell culture or tissue sample) may be used in the practice of the invention. Reporter gene expression need not be determined for individual cells; for example reporter gene expression may be determined for a tissue sample as a whole.

p53 binding to a pGL3 vector may also be determined in other ways, e.g. in an electrophoresis mobility shift assay, preferably with controls in the presence and absence of competitor nucleic acids known to bind p53.

p53 protein can exist in at least two functional states: non-activated and activated. Non-activated p53 has 3' to 5' exonuclease activity and activated p53 is a transcriptional activator (Albrechtsen et al 1999) . The methods and uses of the present invention therefore additionally provide assays for activated p53 and/or p53- mediated transcriptional activation.

Preferably the vector is a defined pGL3 vector, more preferably the defined pGL3-Basic vector or the defined pGL3-Enhancer vector. Most preferably it is the defined pGL3-Basic vector. However, it is contemplated that the defined pGL3-Promoter and pGL3-Control vectors could also be used in this way in cells in which their SV40 promoter is non-functional (e.g. senescent cells as identified in Rubelj et al 1997 in which the SV40 promoter has been shown to be poorly expressed; and teratocarcinoma stem cells as identified in Thillet et al 1984 in which the SV40 promoter may not function efficiently because such cells do not support SV40 T antigen expression) . It is also contemplated that other pGL3 vectors (as defined below) could be used in the same way.

Preferably the vector lacks any promoter other than the p53-responsive promoter discovered herein and the SV40 promoter of the pGL3-Promoter and pGL3-Control vectors and/or has not had any such other promoter inserted therein. However, it is contemplated that in less preferred embodiments, the vectors could have other promoters which are non-functional for the reporter gene in the cell under investigation. The method may comprise an additional step of treating the cell prior to and/or contemporaneously with the step of determining p53 binding to the pGL3 vector.

Such a treatment step may also be prior to and/or simultaneous with a previous step of transfecting the cell with a pGL3 vector.

In one embodiment, the cell may be subjected to a test treatment, e.g. to determine whether that test affects the p53 status of the cell, e.g. whether it leads to the presence and/or accumulation of activated p53 in the cell. For example, the cell may be exposed to a substance (e.g. a chemical suspected to be mutagenic or carcinogenic) or particular environmental conditions (e.g. conditions suspected to cause DNA damage, e.g. exposure to ionising radiation) . The detection of activated p53 under such circumstances may be indicative of DNA damage. When practising such methods, the cell used is preferably one known to be capable of expressing wild-type (wt) p53 that becomes activated as a transcription factor in response to DNA damage, such as a BALB/c 3T3 mouse cell or a cell from the human cell line Kl, to avoid false negative results which could arise if the cell is incapable of expressing p53 which can be activated.

When the cell is subject to a treatment (e.g. with a chemical suspected to damage DNA) , a microsomal mixture (e.g. a hepatic microsome preparation (S9 mix) consisting of rat liver microsomes and P450 cofactors) may be added to the cell culture medium containing to facilitate metabolic activation of the chemical, or of any intermediate affected by the treatment. For example, such a microsomal mixture provides cytochrome p450 and other drug metabolising enzymes which may be necessary for metabolical activation of DNA-damaging chemicals. However, like the addition of microsomes in the Ames mutagenicity test (see Chapter 6, Genetic Toxicology, by David J. Brusick in 'Toxicology', Hans Marquardt et al (eds) , 1999) , this will not always be necessary, depending on the enzymes expressed by the cell line from which the cell originates.

In an alternative embodiment, the p53 status of a cell may be determined following a treatment known to cause DNA damage (e.g. exposure to adriamycin) , for example for cell typing to determine whether the cell expresses wild- type p53 and/or functional DNA-PK, which may have implications for its suitability for certain applications.

As explained in detail later, such methods have significant advantages over currently available methods of assaying for presence of activated p53 and/or DNA damage .

"Activated p53" as used herein means p53 having the ability to bind DNA, particularly the abiϋty to bind to the defined pGL3 vectors (preferably to the CCCGGG motif) , preferably the ability also to bind to other (e.g. naturally occurring) p53-responsive promoters, and preferably the ability to act as an activator of transcription in such vectors and/or from such promoters. Non-activated and mutant forms of p53 can lack the ability to bind DNA and/or act as an activator of transcription under the control of promoters which are responsive to activated p53. Accordingly, p53-mediated transcriptional activition may be indicative of the ability of a cell to produce wild-type p53, that is p53 which has not through mutation lost the ability to bind DNA and/or cause p53-responsive transcription under the control of suitable promoters. Also, it is thought that p53 is activated (by DNA-dependent protein kinase, DNA- PK, Woo et al 1998) in response to DNA damage. p53 transcriptional activity may therefore also be indicative of DNA damage, for example when cells are treated with suspected carcinogens, and/or the presence of functional DNA-PK.

Prior to the present study, it has been shown that a consensus sequence, consisting of. two copies of the double-stranded 10 bp motif 5 ' -PuPuPuC (A/T) (T/A) GPyPyPy- 3' separated by 0-13 residues, such as the GGACATGCCCGGGCATGTCC sequence called p53CON, has affinity for human wild-type (wt) p53 in vitro (El-Deiry et al 1992; Funk et al 1992, Friedman et al 1993) . ("Pu" denotes purine, i.e. a G or A residue, and "Py" denotes pyrimidine, i.e. a C or T/U residue) . The mouse MCK gene and the human p21^WAF1 gene contain p53-responsive elements that have homology to the above consensus sequence. However, these are located far upstream, at -3.1 kb of the MCK gene (Weintraub et al., 1991) and at both -1.3 kb and -2.5 kb of the human p21^mF1 gene (Resnick-Silverman et al., 1998) .

The present study has found that a CCCGGG sequence, which is present as an Sma I restriction site in the multiple cloning site of the defined pGL3 vectors, has affinity on its own for human wt p53 in vitro and that binding to this sequence can lead to luciferase expression in the defined pGL3-Basic vector in the absence of other promoter sequences. It is proposed that this sequence represents a consensus sequence for p53-responsive promoters. It is located at residues 26-31 in the sequence listings given in Annex 1 for each defined pGL3 vector.

Accordingly, in a further aspect, the present invention provides a method of modifying a pGL3 vector, the method comprising deleting or altering a CCCGGG motif located 5' of the reporter gene (e.g. luciferase gene) of the pGL3 vector.

Moreover, by analogy with previous studies into p53 binding, it is thought that the regions of the defined pGL3 vectors which flank the CCCGGG motif may also be involved in p53 binding and hence p53-responsiveness . Accordingly, the present invention also provides a method of modifying a pGL3 vector, the method comprising deleting or altering a sequence within a 20 bp sequence 5' or 3' of a CCCGGG motif located 5' of the reporter gene of the pGL3 vector. Preferably the deletion or alteration is within 15 bp of the CCCGGG motif, more preferably within 10 bp, most preferably within 7bp (since 7bp flanking regions have been implicated previously) , and may be within 5 or 3 bp.

Preferably the CCCGGG motif is located within 500 bp of the start codon of the reporter gene, more preferably within 250 bp, still more preferably within 150 bp or 125 bp, even more preferably within 100 bp. It may even be located within 75 bp.

Having adapted a pGL3 vector according to the previous aspect, in a further aspect, the present invention provides a method of producing modified pGL3 vectors, the method comprising replicating (e.g. in cell culture) the vector so adapted, or a descendent thereof. Further, the present invention provides modified pGL3 vectors having alterations or deletions in the sites mentioned above, preferably in the CCCGGG motif; and cells containing such vectors.

The modified pGL3 vectors may accordingly have reduced p53-responsiveness compared to the unmodified vectors. p53-responsiveness may for example be determined by assaying for reporter gene (e.g. luciferase) activity in BALB/c 3T3 mouse cells transfected with the vector and exposed to adriamycin, e.g. according to experimental procedures described herein.

As used herein, "alteration" includes the insertion, addition, substitution, or transposition of one or more nucleic acids.

Preferably the pGL3 vector is the defined pGL3-Basic or pGL3-Enhancer vector, although the defined pGL3-Promoter and pGP3-Control vectors have also been found to possess the CCCGGG sequence, so may also be so adapted.

Previous studies which implicated various- sequences containing the CCCGGG motif in p53 binding (El-Deiry et al 1992; Funk et al 1992; Shiio et al 1993) either did not determine that these sequences were associated with increased p53-responsive expression, or merely showed such increase when the sequence was upstream of another promoter sequence (e.g. a basal promoter sequence, see e.g. Funk et al 1992 and Shiio et al 1993) . Indeed, Shiio et al 1993 disclosed, in contrast to the present finding, that the motif GCCCGGGC (the "GC₃" motif) cannot on its own act as a positive p53-responsive element. In the present study, it has been found that the level of luciferase reporter protein in mouse BALB/c 3T3 and human Kl cells following transfection of pGL3-basic vector DNA was dependent on the amount of activated p53 present in the cells. Without being bound by any particular theory, we suggest that the failure of Shiio et al to identify p53-responsive promoter activity in the GC₃ element may have been caused by an insufficient quantity of activated p53 in the cells used in their study.

The sequences GAACATGTCCCAACATGTTG and

GAAGAAGACTGGGCATGTCT (from human p21 gene promoter; Resnick-Silverman et al (1998), CCTGCCTGGACTTGCCTGG (PG repeat; Kern et al 1992) have also been shown to be able to function as p53-responsive elements in vitro, but lack the CCCGGG motif.

Moreover, Funk et al did not disclose the CCCGGG motif to be critical for p53-binding. They reported that neither a mutation in which the central CCCGGG block was preserved while the flanking sequence was changed

(competitor A; Fig. 3 lanes 11 and 12) nor a mutation containing an inverted version of this block in which the flanking sequence were maintained (competitor B; Figure 3, lanes 13 and 14) could compete effectively for p53- binding in vitro, suggesting the entire p53CON sequence was required for p53 binding. Again without being bound by any particular theory, we suggest that the EMSA using ³²P is inadequate for detecting p53-binding affinity of the CCCGGG element in p53CON because the strong ³²P signal could make the observation of a small band shift effect difficult.

Accordingly, in a still further aspect, the present invention provides the use of nucleic acid having a sequence incorporating the CCCGGG motif to confer promoter activity.

The CCCGGG motif may be used to confer promoter activity in the absence of other promoter sequences.

Preferably the use comprises bringing the sequence incorporating the CCCGGG motif into operative association with and 5' of nucleic acid encoding a polypeptide of interest (e.g. a reporter gene). Preferably the CCCGGG motif is within 500 bp of the start codon of the polypeptide of interest, more preferably within 250 bp, still more preferably within 150 bp or 125 bp, even more preferably within 100 bp. It may even be located within 75 bp.

The CCCGGG motif alone may be used, or it may be used together with a flanking sequence or sequences.

Flanking sequences may be 5 ' and/or 3 ' of the CCCGGG motif and may for example be: one or both flanking region (s) with which the CCCGGG motif is associated in the defined pGL3-Basic vector; one or both flanking regions with which the CCCGGG motif is found in sequences previously identified as important in p53 binding (e.g. in the studies cited herein) ; or one or both flanking regions with which the CCCGGG motif is found in other p53-responsive promoters identified herein. Preferred 5' and 3 ' flanking regions are all or part of the sequences shown in the following table (all sequences written 5' to 3') .

Preferred combinations of 5' and 3' flanking regions are those in horizontal alignment in the table.

Preferably the or at least one (more preferably each) flanking region is at least 3 bp, more preferably at least 5 bp, most preferably at least 6 bp in length, although it may be at least 7 bp, 9 bp or 11 bp in length. A previous study has shown that the binding of p53 to a (unrelated) 19 bp sequence is sufficient to activate transcription of a CAT reporter gene (Kern et al 1992) .

Preferably the or at least one (more preferably each) flanking region is at most 30 bp, 20 bp, 10 bp or 7 bp in length.

The nucleic acid encoding a polypeptide of interest may for example be a plasmid or other vector (preferably a vector which otherwise lacks a known promoter sequence) containing a coding sequence of interest. Suitable vectors comprising nucleic acid for introduction into cells (e.g. mammalian cells) can be chosen or constructed by the skilled person, containing appropriate other regulatory sequences, including terminator fragments, enhancer sequences, marker genes and other sequences as appropriate. Vectors may for example be plasmids or viral, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs,* metagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al . Eds, John Wiley & Sons 1992. The disclosures of Sambrook et al and Ausubel et al are incorporated herein by reference.

In a further aspect, the present invention provides a method of determining whether a promoter sequence, whose p53 responsiveness is unknown, is likely to be responsive to p53, the method comprising determining whether the promoter contains a CCCGGG motif. Further the present invention provides: the use of a promoter thus identified as being p53-responsive to confer p53-responsiveness on a nucleic acid (e.g. a vector) encoding a polypeptide of interest; the use of a nucleic acid including a promoter thus identified in assays for p53 transcriptional activity; and a nucleic acid including a promoter thus identified packaged for use in such an assay.

pGL Vectors

It is clear that the vectors as sold by Promega could be altered in various ways, and still be appropriate for the practice of the present invention. Accordingly, references herein to pGL3 vectors are not intended to refer solely to vectors having the precise nucleic acid sequences set out in the Promega technical literature (the "defined" pGL3 vectors) , though these are preferred pGL3 vectors. Rather, vectors varying from the defined vectors may also be regarded as a pGL3 vector for the purposes of the present invention, provided that they contain a CCCGGG sequence 5 ' of a reporter gene, preferably within 500, 250, 150, 125, 100 or 75 bp of the start codon of the reporter gene. Thus, ■for example, the defined pGL3 vectors may be subjected to alteration, e.g. by one or more of addition, deletion, substitution and insertion, of one or more nucleic acid residues, and will still be considered to be pGL3 vectors.

The reporter gene is•- preferably a luciferase gene

(preferably the luc+ gene of the defined pGL3 vectors) , though it could be substituted for another reporter gene without departing from the present invention. Similarly, one could use origins of replication derived from different organisms than filamentous phage and E. coli, or (where present) different enhancers from the SV40 enhancer, or a different multiple cloning site from those used in the defined pGL3 vectors (or no multiple cloning site) .

The CCCGGG sequence is preferably flanked by the same or similar 5' and/or (preferably and) 3' sequences as in the defined pGL3 vectors, though variation may be possible without affecting the p53-responsiveness of the reporter gene. In this context, "similar" means that the flanking sequence has at least 50%, preferably 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity (as defined below) with the sequence flanking the CCCGGG motif in the defined pGL3-Basic vector, over a contiguous stretch of at least 4, preferably 5, 7, 8, 10, 12, 15, 20, 25, 30, 40 or 50 nucleotides.

The defined pGL3 vectors were themselves derived from a previous set of luciferase reporter vectors, namely the pGL2 vectors of Promega, which differ from defined pGL3 vectors in that e.g. the CCCGGG sequence is in a different position (residues 1-6) in the multiple cloning site and in that a different luciferase gene is used. (For more information on the pGL2 vectors^ see Promega Technical Manual, pGL2 Luciferase Reporter Vectors,

Instructions for use of products E1611, E1621, E1631 and E1641, Printed in USA, Revised 10/96, Part# TM003, which is incorporated herein by reference in its entirety for all purposes. Copies are available free of charge from Promega Corporation and from their website: www. promega. com/tbs/TMO03/tmO03. html) ) . Since the pGL2 vectors have the- CCCGGG site identified herein as important for p53 binding and responsiveness, it is proposed that they too may display p53-responsiveness . Vectors having the sequences set out in the Promega technical literature for the pGL2 vectors (the "defined" pGL2 vectors) , preferably the defined pGL2-Basic and pGL2-Enhancer vectors, are therefore also regarded for the purpose of the present invention as pGL3 vectors, as are vectors derived from the defined pGL2 vectors.

The pGL3 vectors used in the practice of the invention are preferably identical to or in fact derived (by nucleic acid deletion or alteration) from the defined pGL2 or pGL3 vectors. This may be indicated by the pGL3 vector containing a contiguous sequence of at least 12, more preferably at least 14, more preferably at least 16, 18 or 20 nucleotides in length which is identical to a contiguous sequence of nucleotides in the backbone of a defined pGL2 or defined pGL3 vector. In relation to the defined pGL3 vectors, the "backbone" means any portion of the vector which does not form part of any of the following structural elements, namely the promoter (where present), the enhancer (s) (where present), the SV40 late poly (A) signal, the luciferase gene, the upstream poly (A) signal, the multiple cloning site, the RV primer3 binding site, the RV primer4 binding site, the GL primer2 binding site, the beta-lactamase gene (Amp^r) , the f1 origin and the ColEl-derived plasmid replication origin. The location of these structural elements in the defined pGL3 vectors is set out in Annex 1. The same definition applies mutatis mutandis to the structural elements set out in the technical literature for the defined pGL2 vectors .

Preferably the pGL3 vectors are at least 70% identical, more preferably 80% identical, more preferably 85%, 90%, 95%, 98% or 99% identical with one of the defined pGL3 vectors over a stretch of at least 40 contiguous nucleotides within a stretch of about 60 contiguous nucleotides immediately 5' of the start codon of the reporter gene.

It will be evident that for detection of p53 binding to the pGL3 vector in an EMSA assay, the presence of a functional reporter gene is not essential. In the context of such detection, therefore, vectors otherwise as described above, but lacking a functional reporter gene, are also considered to be pGL3 vectors.

p53

References herein to p53 are npt to be interpreted as limited to the known, naturally-occurring protein sequence as published in Lamb and Crawford 1986, but also to functional variants or mimetics thereof, which have the property of binding -to and permitting transcription from known p53-responsive promoters (particularly those present in the defined pGL3 vectors and identified herein) . However, the present teaching relates particularly to naturally occurring forms of p53.

For example, the method of the first aspect of the invention may be used to assay for mimetics of p53, e.g. by providing a pGL3 vector in a cell in circumstances in which the presence of activated p53 would not normally be expected (e.g. in a p53-deficient cell), exposing the cell to, or causing it to express, a candidate p53 mimetic and determining whether p53 activity results. A positive result (e.g. luciferase expression) indicates presence of a p53 mimetic.

Moreover, the method can be used to assay for antagonists of p53, e.g. by providing a pGL3 vector in a cell in circumstances in which p53 activity would be expected (e.g. in a BALB/c 3T3 mouse cell exposed to adriamycin) , exposing the cell to, or causing it to express, a candidate p53 antagonist and determining whether p53 activity results. A negative result (e.g. absence of luciferase expression) indicates presence of a p53 antagonist.

Similarly, the present invention also provides a method of identifying a candidate substance (e.g. molecule) as being a potential mimetic or antagonist of p53, the method comprising determining whether the candidate substance is able to bind to nucleic acid comprising or consisting of the CCCGGG motif, e.g. by EMSA or using labelled nucleic acid. Flanking sequences may be present and are preferably as defined in the above aspect relating to the use of the CCCGGG motif to confer promoter activity. Preferred nucleic acids are the pGL3 vectors .

Nucleic acid sequence identity

Percent (%) nucleic acid sequence identity means the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in a reference sequence (e.g. a sequence from a defined pGL2 or pGL3 vector) , after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity, as determined by the BLASTN module of WU-BLAST- 2 [Altschul et al . , Methods in Enzymology, 266:460-480 (1996); http: //blast.wustl/edu/blast/README. html] , set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively. The % nucleic amino acid sequence identity is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region, multiplied by 100. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored) .

Although the present invention mainly relates to the p53 status of cells, it will be appreciated that it may also be applied to expression systems which do not necessarily consist only of intact cells. For example, cell-free coupled transcription and translation systems are known (e.g. the TNT Coupled Reticulocyte Lysate System of Promega, Inc) and it is contemplated that the skilled person could use such a system in the practice of the present invention. Modification of the system to include a p53-responsive transcription complex may be necessary.

It is further contemplated that expression systems based on cell extracts or cell lysates may be used, with or without intact nuclei, for example mammalian cell extracts or cell lysates.

Accordingly the present invention also provides in further aspects analogous methods and materials to those indicated above, employing expression systems which do not necessarily comprise intact cells. Thus, for example, the present invention also provides a method of determining the p53 status in an expression system, the method comprising providing an expression system containing a pGL3 vector and determining whether luciferase is expressed in the expression system. The invention further provides the use of a pGL3 vector in such a method; and provides a pGL3 vector, and/or expression system containing a pGL3 vector, packaged with instructions for use in such a method. Thus, in the foregoing, the term "cell" may be interchanged with "expression system" to include other expression systems. However, cells are the preferred expression systems, mammalian cells being particularly preferred.

For the better understanding of the invention, and to provide more detail on preferred features of the invention, the underlying work will now be described in detail, with reference to the accompanying figures, in which:

Fig. 1 shows the effect of adriamycin on the expression of the luc+ gene in cells transfected with the pGL3-Basic vector. The pGL3-Basic vector and pSV-β-galactosidase vector used as an internal control were co-transfected into BALB/c 3T3 cells, Kl cells and NCI-H358 cells. Following the transfection, cells were incubated for 24 hours in the medium containing 0.9 % NaCl (basal) or adriamycin from 6-14 μg/ml for 3T3 cells and Kl cells and 4-12 μg/ml for NCI-H358 cells. The luciferase activity was then determined and normalised against β- galactosidase activity from the co-transfected pSV-β- galactosidase vector. Data are the average of three independent experiments.

Fig. 2 shows the ability of human wt p53 to recognize the CCCGGG sequence in the cloning region of the pGL3-Basic vector. The pGL3-Basic vector, a supercoiled DNA (Sc; lane 1) , was incubated with Kl nuclear extract (lane 2) followed by further incubation with p53CON (lanes 3 & 4), mutp53CON (lanes 5 & 6) , MNSXB (lanes 7 & 8) and MNSrXB (lanes 9 & 10) at the concentrations indicated. p53CON is a defined p53-binding consensus sequence; mutp53CON is a mutant form of p53CON in which the CCCGGG motif is inverted while the flanking sequences are maintained; MNSXB is a partial cloning sequence of the pGL3-Basic vector consisting of five restriction sites, Mlu I, N e I, Sma I, Xho I and Bgl II; MΝSrXB is a mutant form of MΝSXB in which the CCCGGG motif is inverted while the flanking sequences are maintained. The sequence structures of the competitors are listed below, with the substitutions in the DΝA competitor mutp53COΝ and MNSrXB underlined.

Competitor Name Sequence structure

A p53CON 5 '-GGACATGCCCGGGCATGTCC-3' B mutp53C0N 5 ' -GACATGGGGCCCCATGTC-3 '

C MNSXB 5 ' -ACGCGTGCTAGCCCGGGCTCGAGATCT-3 ^»

D MNSrXB 5 ' -ACGCGTGCTAGGGGCCCCTCGAGATCT-3 '

Summary

The palindrome CCCGGG, a sequence recognised and cut by the restriction enzyme Sma I, in the cloning region of the pGL3-Basic vector can promote transcription of the luc+ gene in a p53-dependent manner. We demonstrate, by electrophoretic mobility shift assay (EMSA) , that human wild type p53 is able to bind to the CCCGGG sequence in vi tro . This suggests that p53 may regulate the luc+ gene in cells transfected with pGL3-Basic vector through direct interaction with the CCCGGG sequence. Thus, the pGL3-Basic vector could be used as an indicator of p53 activity, to determine the p53 status of cell lines and to identify DNA damaging agents that initiate the activation of p53. We suggest that the CCCGGG sequence may be one of the consensus sequences recognized by p53 in vivo and may be used to identify genes whose expression may be controlled by p53.

Experimental Procedures

Cell culture

The mouse BALB/c 3T3 clone A31 cell line (ATCC) was grown in Dulbecco's Modified Eagle's Medium (Gibco-BRL), supplemented with 10% foetal bovine serum (FBS) (Gibco- BRL) and 1% L-glutmax (Gibco-BRL) . The human cell line Kl (ECACC) was grown in DMEM: Ham's F12:MCDB 104 (2:1:1) medium (Gibco-BRL) supplemented with 10% FBS (Gibco-BRL) and the human cell line NCI-H358 (ECACC) was grown in RPMI 1640 medium (Gibco-BRL) , supplemented with 10% FBS (Gibco-BRL) and 1% L-glutamax (Gibco-BRL) . All the cell lines were grown in 5% C0₂ at 37°C.

DNA transfection, exposure to DNA-damaging agent and luciferase assay

Two reporter gene vectors, pGL3-Basic vector and the pSV- β-galactosidase vector, were co-transfected into the cells with the Effectene Transfection Reagent (Quiagen) following the manufacturer's protocol. The pSV-β-

Galactosidase vector provides an internal control for monitoring transfection and normalising the level of the luciferase in the cells. Following transfection, the cells were incubated in the culture medium containing 0.9 % NaCl (vehicle control) or adriamycin in 0.9 % NaCI at various concentration. After 24 hours, cells were lysed and the level of the luciferase and β-galatosidase in the lysate were measured using the Luiferase Assay System (Promega) and the β-Galactosidase Enzyme Assay System (Promega) , respectively (Figure 1 ) .

Preparation of nuclear extract

Kl cells were grown to 80% confluency and then treated with adriamycin at 10 μg/ml for 24 hours. After treatment, nuclear extracts were prepared as described (McLure and Lee, 1998), except that cells were scraped off the surface of the tissue culture dish in 1 ml of ice-cold hypotonic buffer and that the 40 μl of nuclear extract (nuclear proteins in hypertonic buffer) were added to 100 μl of hypotonic buffer to reduce the KC1 concentration to 150 mM.

DNA binding analysis

The binding of p53 to pGL3-Basic DNA was analysed by an electrophoretic mobility shift assay (EMSA) . Double- stranded oligonucleotides, p53CON, mutp53CON, MNSXB and MNSrXB (see sequence structures in Figure 2) were used as the specific competitors to compete with pGL3-Basic vector for p53 binding. Each binding reaction contained 5 μl of nuclear extract (~ 5 μg nuclear protein) , 1 μl (0.2 μg) of pGL3-Basic DNA, 1 μl (1 μg) of poly (dl-dC) as a non-specific competitor (Sigma) , 5 μl of either TE buffer or one of the specific competitors, 1.5 μl of glycerol (Sigma), and 1.5 μl of 10 x buffer (buffer J) [1 x buffer J: 50 mM KGl, 10 mM Tris-HCl, 7 mM MgCl₂ and 1 mM DTT, pH 7.6, Promega] in a 10 μl final volume. Reactions were incubated for 10 min on ice, and then 5 μl of TE buffer or specific competitor (8 μg or 12 μg) was added to the reaction followed by a further 10 min incubation ^• on ice. The final reaction products were loaded onto 1% agrose gel (ultrapure agarose; Sigma) containing ethidium bromide at 0.25 μg/ml and electrophoresed in lx TBE buffer at 94 mV/~42 mA for 4-5 hours. The DNA band patterns were then recorded on a Multi-Analyst System (Bio-Rad) .

Results

A p53-regulated luc+ gene in the pGL3-Basic vector

The pGL3-Basic vector is designed ^"for studying putative regulatory sequences. These can be inserted at one of the restriction sites within the cloning region, such as the Sma I site (CCCGGG sequence) , upstream of the luciferase coding gene sequence designated luc+. The construct is transfected into a suitable cell line and the promoter activity of the inserted sequence is mo'nitored through transcriptional activation of the luc+ gene .

Unexpectedly, a high level of luciferase activity was detected in BALB/c 3T3 cells transfected with the pGL3- Basic vector and the observed luciferase Sctivity was further induced by adriamycin, a DNA damaging agent

(Figure 1) . This suggested that the luc+ gene in the pGL3-Basic vector may be regulated by p53 which in many cell lines becomes activated in response to DNA damage (Komarova et al 1997; Woo et al 1998) .

To test this hypothesis, we repeated the transient tranfec^'tion assay in Kl cells expressing wt p53 and the NCI-H358 cells which lack wt p53 (owing to a complete homozygous deletion of the p53 gene) (Bodner et al 1992) In untreated cells, there was minimal luciferase activity in both Kl and NCI-H358 cells transfected with the pGL3-Basic vector. Following exposure to adriamycin, however, the luciferase activity was dramatically increased in Kl cells, whereas little change was detected in NCI-H358 cells (Figure 1) . These results demonstrated that wt p53 is required for the induction of luc+ gene expression.

The p53-binding element in the pGL3-Basic vector

It has been previously shown (see above) that human wt p53 binds in vitro to a palindromic sequence designated p53CON. When this p53CON sequence was upstream both of the CAT reporter gene and a basal promoter, transcription of the CAT gene was induced in a p53-dependent manner (Funk et al 1992) .

Upon investigation of the nucleic acid sequence of the pGL3-Basic vector, in light of the above discovery, it was found that the cloning region of the pGL3-Basic vector contains one CCCGGG sequence, a restriction site cut by the Sma I restriction enzyme, which we propose could be the site which confers p53-responsivity to the pGL-Basic vector.

To determine whether the CCCGGG sequence in the pGL3- Basic vector, without the rest of the p53CON sequence, has affinity for human wt p53, this DNA were incubated with wt p53 contained in the nuclear extract from adriamycin-treated Kl cells and the band shift effect of DNA competitors p53CON, mutp53CON, MNSXB and MNSrXB were analysed by EMSA (Figure 2) . A slow migrating band was observed after incubating the pGL3-Basic vector with the Kl nuclear extract (Figure 2, compare lanes 1 and 2), indicating the formation of DNA/protein complexes. Binding of p53 to the pGL3-Basic vector was confirmed by the observation that the p53CON, at the highest concentration tested (12 μg/reaction) , had significant band shift effect (Figure 2, compare lanes 2 and 4) . At the same concentration tested, the mutp53CON, in which the central CCCGGG sequence of p53CON was inverted, had no detectable band shift effect (Figure 2, compare lanes 2 and 6) , indicating that the CCCGGG sequence is essential for p53CON to compete with the pGL3-Basic DNA for p53-binding. MNSXB, a partial cloning sequence of the pGL3-Basic vector containing the CCCGGG sequence, has showed similar band shift effects to that seen with the p53CON (Figure 2, compare lanes 4 and 8), but the MNSrXB, a mutant form of the MNSXB containing an inverted CCCGGG sequence, could not compete for p53-binding (Figure 2, compare lanes 2 and 10) .

These results clearly show that the CCCGGG sequence in the pGL3-Basic vector is essential and sufficient for p53-binding.

Determination of p53 status of a sample by EMSA

Nuclear extracts prepared from mouse lung and liver tissues were incubated with the pGL3 -Basic vector on ice for 10 min followed by 10 min incubation on ice in the presence or absence of the p53 -binding elements, p53CON or MNSXB. The reaction products were then loaded onto 1 % agarose gel and subjected to EMSA as described above. For all the reactions containing either mouse lung or liver nuclear extract, band shift effects induced by the p53 -binding elements were observed,^' indicating that both lung and liver nuclear extracts contain wtp53. Unlike the conventional EMSA and Western Blot procedures used for detecting DNA-protein interaction and presence of proteins, detecting p53 binding status and presence with the pGL3 -Basic vector does not involve handling radioactive material or the use of p53 antibody. The method is simple, efficient and easy to use.*

Discussion

We have demonstrated that p53 can regulate the luc+ gene contained in the pGL3-Basic vector (Figure 1) , which had previously been assumed to lack any identified eukaryotic promoter, according to the supporting technical literature for this range of vectors. Binding of p53 to the pGL3-Basic vector has been confirmed using the defined p53-binding sequence p53CON as a specific DNA competitor. The DNA competitor MNSXB, a partial sequence from the cloning region of the pGL3-Basic vector, differs from p53CON at its flanking sequence (see sequence structures in Figure 2) . Like p53CON, the MNSXB sequence can effectively compete with pGL3-Basic vector for p53 binding. In fact, the CCCGGG element in both the p53CON and MNSXB competitors were essential for p53 binding as their mutant versions, mutp53CON and MNSrXB (each containing an inverted CCCGGG sequence, see Figure 2) , could not compete effectively with the pGL3-Basic vector for p53 binding. This provides direct evidence that the CCCGGG sequence in the pGL3-Basic vector is a p53-binding site, and that the binding of p53 to the CCCGGG sequence can effectively initiate transcription of the luc+ gene.

The CCCGGG sequence has been found to be present in the promoter regions of a number of p53-regulated genes, such as p21^WAF1 (human/HSU24170 (GenBank accession number: U24170) , where it is present in four copies; mouse/MMU24171 (GenBank: U24171) ; rat/RNU24172 (GenBank: U24172)), fas (human/HSFASXl (GenBank: X82279) ) , bax (human/HSBAXl (GenBank: U17193) ) , ATF3 (human/HS375421 (GenBank: U37542)), human virus type 1 LTR (GenBank:

AF197258), MCK (mouse/MMMCKA (GenBank: M21390)) and bcl-x (mouse/MMBCLXPl (GenBank: U78030)). No particular importance has previously been ascribed to this motif. From this and the above finding, the present inventors propose the CCCGGG sequence as a consensus sequence that in vivo promotes gene transcription in a p53-dependent manner.

The human genome will be fully sequenced soon. A search of the resulting database could indicate which genes have the CCCGGG sequence in their promoter, and which according to the present analysis may be regulated by p^'53.

The pGL3-Basic vector may have other practical uses. Chemicals which damage DNA may be mutagenic and/or carcinogenic, and therefore a hazard to humans and the environment. When the DNA of normal mammalian cells is damaged, p53 becomes activated (Komarova St al., 1997; Woo et al., 1998). The activation of p53 in cultured cells following DNA damage may be monitored either by direct transcriptional activation of the p21^WAF1 gene or by the CAT reporter gene under the control of a p53- responsive promoter element (Woo et al., 1998).

The former method is unreliable as the transcription of the p21^NAF1 gene can also be initiated in a p53-independent •manner (Macleod et al., 19^'95) . In addition, the method involves working with RNA and is therefore susceptible to practical problems due to the prevalence of RNases in the environment.

The latter method also needs to use a CAT reporter vector containing a p53-responsive element, but such a vector is not commercially available and the method therefore requires subcloning of these elements onto an appropriate reporter vector. Another disadvantage is that both methods involve handling radioactive material.

Transgenic mice carrying the lacZ reporter gene have been developed for studying p53 activation in vivo and have been used for monitoring p53 activation in several mouse organs after adriamycin treatment (Komarova et al., 1997) . However, this method requires the use of large numbers of laboratory animals and is expensive.

The present study shows that p53 activation in mouse BALB/c 3T3 cells and human Kl cells in response to DNA damage by adriamycin could be effectively monitored by measuring the luciferase activity in cells transfected with the pGL-Basic vector. This suggests that the pGL3- Basic vector could be used as an indicator of p53 ^' ' activity for identifying DNA-damaging agents that initiate the activation of p53. For example, cells transfected with the pGL3-Basic vector could be treated with the test chemical. If this chemical induced DNA damage, p53 would be activated and bind to the CCCGGG sequence upstream of the luc+ gene. This would lead to the production of the luciferase protein which could be detected using the luciferase assay system. It would be relatively easy to assay test chemicals on a large scale.

The pGL3-Basic vector could be used in cell lines that express wt p53, so allowing chemicals to be tested rapidly in a range of different cell lines. Another use of the pGL3-Basic vector is to determine the p53 status of cell lines. The human lung cancer cell line NCI-H358 is known to contain a mutant p53 gene with a homozygous deletion. The p53 messenger RNA in these cells was not detectable by Northern (RNA) blot analysis or by the ribonuclease (RNase) protection assay (Takahashi et al . , 1989). Various mutations that occur outside exons 5-8 of p53 gene (including splicing, nonsense, deletion and missense) have been identified in many other human lung cancer cell lines. These mutations do not result in increased steady-state levels of p53 proteins and therefore cannot be identified by an immunocytochemical (IC) assay (Bodner et al., 1992). Our study shows that luciferase activity in NCI-H358 cells transfected with pGL3-Basic vector did not increase significantly following exposure to adriamycin (Figure 1) . This has confirmed the finding that NCI-H358 cells do not contain wt p53. As many cell lines are used in biomedical research, it would in some cases be useful to know whether they express wt p53. The pGL3-Basic vector can be used as reporter for determining this. For example, the cell line could be transfected with the pGL3-Basic vector, and the luciferase activity determined relative to control cell lines could reflect the presence or absence of wt p53.

References

Albrechtsen et al (1999) Oncogene 18, 7706-7717. Bargonetti et al . (1991) Cell 65, 1083-1091. Bodner et al (1992) Oncogene 7, 734-749. Brusick (1999) Chapter 6 "Genetic Toxicology" in Toxicology, Marquardt et al (eds) , Academic Press (a division of Harcourt, Inc., San Diego, CA, USA) 1999. El-Deiry et al (1992) Nature Genetics 1,45-49. Friedman et al (1993) Proc. Natl. Acad. Sci. USA 90,

3319-3323. Funk et al (1992) Mol . Cell Biol. 12, 2866-2871. Hollstein et al (1991) Science 253, 49-53. Kern et al (1992) Science 256, 827-836. Komarova et al (1997) EMBO J. 16, 1391-1400. Lamb and Crawford (1986) Mol. Cell. Biol. 6, 1379-1385. Lane (1992) Nature 358, 15-16.

Macleod et al (1995) Genes and Develop. 9, 935-944. Marx (1994) Science 266,1321-1322. McLure et al (1998) EMBO J. 17, 3342-3350. Polyak et al (1997) Nature 389,. 300-305. Raycroft et al (1990) Science 249,1049-1051. Resnick-Silverman et al (1998) Genes Dev. 12, 2102-2107. Rubelj et al (1997) Journals of Gerontology Series A - Biological Sciences & Medical Sciences 52(5), B229-B234. Shiio et al 1993 Oncogene 8: 2059-2065. Takahashi et al (1989) Science 246, 491-494. Thillet et al (1984) Experimental Cell Research 151(2), 494-501. Weintraub et al (1991) Proc. Natl. Acad. Sci. USA 88,

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Wu et al (1994) Proc. Natl. Acad. Sci. USA 91, 3602-3606. Yang et al (1999) Pharmacogenetics 9, 183-188.

Annex 1

Structural elements present in the pGL3 vectors and their location in the corresponding sequence listings

luc+ = cDNA encoding the modified firefly luciferase; Amp^r = gene conferring ampicillin resistance in E. coli; fl = origin of replication derived from filamentous phage;

pGL3-Basic Vector

Promoter (none)

Enhancer (none)

SV40 late poly (A) signal 1772-1993

Luciferase gene (luc+) 88-1737 upstream poly (A) signal 4658-4811

Multiple cloning site 1-58

RV primer3 binding site 4760-4779

RV primer4 binding site 2080-2061

GL primer2 binding site 111-89 beta-lactamase gene (Amp^r) 3940-3083 fl origin 4073-4527

ColEl-derived plasmid replication origin 2318

pGL3-Enhancer Vector

Promoter (none)

Enhancer 2005-2249

SV40 late poly (A) signal 1772-1993

Luciferase gene (luc+) 88-1737 upstream poly (A) signal 4904-5057

Multiple cloning site 1-58

RV primer3 binding site 5006-5025

RV primer4 binding site 2326-2307

GL primer2 binding site 111-89 beta-lactamase gene (Amp^r) 4186-3329 fl origin 4319-4773

ColEl-derived plasmid replication origin 2564

pGL3-Promoter Vector

Promoter 49-244

Enhancer (none)

SV40 late poly(A) signal 1964-2185

Luciferase gene (luc+) 280-1929 upstream poly (A) signal 4850-5003

Multiple cloning site 1-41

RV primer3 binding site 4952-4971

RV primer4 binding site 2272-2253

GL primer2 binding site 303-281 beta-lactamase gene (Amp^r) 4132-3275 fl origin 4265-4719

ColEl-derived plasmid replication origin 2510

pGL3-Control Vector

Promoter 49-244

Enhancer 2197-2441

SV40 late poly (A) signal 1964-2185

Luciferase gene (luc+) 280-1929 upstream poly (A) signal 5-096-5249

Multiple cloning site 1-41

RV primer3 binding site 5198-5217

RV primer4 binding site 2518-2499

GL primer2 binding site 303-281 beta-lactamase gene (Amp^r) 4378-3521 fl origin 4511-4965

ColEl-derived plasmid replication origin 2756

In each case, the strand shown is the same as the ssDNA strand produced by this vector and also corresponds to the mRNA synthesized from the luc+ gene. pGL3-Basic Vector Sequence

1 GGTACCGAGC TCTTACGCGT GCTAGCCCGG GCTCGAGATC TGCGATCTAA

GTAAGCTTGG CATTCCGGTA CTGTTGGTAA AGCCACCATG GAAGACGCCA 101 AAAACATAAA GAAAGGCCCG .GCGCCATTCT ATCCGCTGGA AGATGGAACC

GCTGGAGAGC AACTGCATAA GGCTATGAAG AGATACGCCC TGGTTCCTGG 201 AACAATTGCT TTTACAGATG CACATATCGA GGTGGACATC ACTTACGCTG

AGTACTTCGA AATGTCCGTT CGGTTGGCAG AAGCTATGAA ACGATATGGG 301 CTGAATACAA ATCACAGAAT CGTCGTATGC AGTGAAAACT CTCTTCAATT

CTTTATGCCG GTGTTGGGCG CGTTATTTAT CGGAGTTGCA GTTGCGCCCG 401 CGAACGACAT TTATAATGAA CGTGAATTGC TCAACAGTAT GGGCATTTCG

CAGCCTACCG TGGTGTTCGT TTCCAAAAAG GGGTTGCAAA AAATTTTGAA 501 CGTGCAAAAA AAGCTCCCAA TCATCCAAAA AATTATTATC ATGGATTCTA

AAACGGATTA CCAGGGATTT CAGTCGATGT ACACGTTCGT CACATCTCAT 601 CTACCTCCCG GTTTTAATGA ATACGATTTT GTGCCAGAGT CCTTCGATAG

GGACAAGACA ATTGCACTGA TCATGAACTC CTCTGGATCT ACTGGTCTGC 701 CTAAAGGTGT CGCTCTGCCT CATAGAACTG CCTGCGTGAG ATTCTCGCAT

GCCAGAGATC CTATTTTTGG CAATCAAATC ATTCCGGATA CTGCGATTTT 801 AAGTGTTGTT CCATTCCATC ACGGTTTTGG AATGTTTACT ACACTCGGAT

ATTTGATATG TGGATTTCGA GTCGTCTTAA TGTATAGATT TGAAGAAGAG 901 CTGTTTCTGA GGAGCCTTCA GGATTACAAG ATTCAAAGTG CGCTGCTGGT

GCCAACCCTA TTCTCCTTCT TCGCCAAAAG CACTCTGATT GACAAATACG 1001 ATTTATCTAA TTTACACGAA ATTGCTTCTG GTGGCGCTCC CCTCTCTAAG

GAAGTCGGGG AAGCGGTTGC CAAGAGGTTC CATCTGCCAG GTATCAGGCA 1101 AGGATATGGG CTCACTGAGA CTACATCAGC TATTCTGATT ACACCCGAGG

GGGATGATAA ACCGGGCGCG GTCGGTAAAG TTGTTCCATT T-TTTGAAGCG 1201 AAGGTTGTGG ATCTGGATAC CGGGAAAACG CTGGGCGTTA ATCAAAGAGG

CGAACTGTGT GTGAGAGGTC CTATGATTAT GTCCGGTTAT GTAAACAATC 1301 CGGAAGCGAC CAACGCCTTG ATTGACAAGG ATGGATGGCT ACATTCTGGA

GACATAGCTT ACTGGGACGA AGACGAACAC TTCTTCATCG TTGACCGCCT 1401 GAAGTCTCTG ATTAAGTACA AAGGCTATCA GGTGGCTCCC GCTGAATTGG

AATCCATCTT GCTCCAACAC CCCAACATCT TCGACGCAGG TGTCGCAGGT 1501 CTTCCCGACG ATGACGCCGG TGAACTTCCC GCCGCCGTTG TTGTTTTGGA

GCACGGAAAG ACGATGACGG AAAAAGAGAT CGTGGATTAC GTCGCCAGTC 1601 AAGTAACAAC CGCGAAAAAG TTGCGCGGAG GAGTTGTGTT TGTGGACGAA

GTACCGAAAG GTCTTACCGG AAAACTCGAC GCAAGAAAAA TCAGAGAGAT 1701 CCTCATAAAG GCCAAGAAGG GCGGAAAGAT CGCCGTGTAA TTCTAGAGTC

GGGGCGGCCG GCCGCTTCGA GCAGACATGA TAAGATACAT TGATGAGTTT 1801 GGACAAACCA CAACTAGAAT GCAGTGAAAA AAATGCTTTA TTTGTGAAAT

TTGTGATGCT ATTGCTTTAT TTGTAACCAT TATAAGCTGC AATAAACAAG 1901 TTAACAACAA CAATTGCATT CATTTTATGT TTCAGGTTCA GGGGGAGGTG

TGGGAGGTTT TTTAAAGCAA GTAAAACCTC TACAAATGTG GTAAAATCGA 2001 TAAGGATCCG TCGACCGATG CCCTTGAGAG CCTTCAACCC AGTCAGCTCC

TTCCGGTGGG CGCGGGGCAT GACTATCGTC GCCGCACTTA TGACTGTCTT 2101 CTTTATCATG CAACTCGTAG GACAGGTGCC GGCAGCGCTC TTCCGCTTCC

TCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC 2201 AGCTCACTCA AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG

CAGGAAAGAA CATGTGAGCA AAAGGCCAGC AAAAGGCCAG GAACCGTAAA 2301 AAGGCCGCGT TGCTGGCGTT TTTCCATAGG CT.CCGCCCCC CTGACGAGCA

TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT 2401 AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT

CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG ^' 2501 CGTGGCGCTT TCTCAATGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG

TCGTTCGCTC CAAGCTGGGC TGTGTGCACG AACCCCCCGT TCAGCCCGAC 2601 CGCTGCGCCT TATCCGGTAA CTATCGTCTT GAGTCCAACC CGGTAAGACA

CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT AGCAGAGCGA 2701 GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC

TACACTAGAA GGACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC 2801 CTTCGGAAAA AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG

GTAGCGGTGG TTTTTTTGTT TGCAAGCAGC AGATTACGCG CAGAAAAAAA 2901 GGATCTCAAG AAGATCCTTT GATCTTTTCT ACGGGGTCTG ACGCTCAGTG

GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA TCAAAAAGGA 3001 TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA

AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT T ATCAGTGA 3101 GGCACCTATC TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC

TCCCCGTCGT GTAGATAACT ACGATACGGG AGGGCTTACC ATCTGGCCCC 3201 AGTGCTGCAA TGATACCGCG AGACCCACGC TCACCGGCTC CAGATTTATC

AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT GGTCCTGCAA 3301 CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA

AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTACAGG 3401 CATCGTGGTG TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT

CCCAACGATC AAGGCGAGTT ACATGATCCC CCATGTTGTG CAAAAAAGCG 3501 GTTAGCTCCT TCGGTCCTCC GATCGTTGTC AGAAGTAAGT TGGCCGCAGT

GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT ACTGTCATGC 3601 CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTC

TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAATACG 3701 GGATAATACC GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA

AACGTTCTTC GGGGCGAAAA CTCTCAAGGA TCTTACCGCT GTTGAGATCC

3801 AGTTCGATGT AACCCACTCG TGCACCCAAC TGATCTTCAG CATCTTTTAC

TTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA AATGCCGCAA

3901 AAAAGGGAAT AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTT

TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA

4001 CATATTTGAA TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT

TTCCCCGAAA AGTGCCACCT GACGCGCCCT GTAGCGGCGC ATTAAGCGCG

4101 GCGGGTGTGG TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT

AGCGCCCGCT CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG

4201 GCTTTCCCCG TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT

AGTGCTTTAC GGCACCTCGA CCCCAAAAAA CTTGATTAGG GTGATGGTTC

4301 ACGTAGTGGG CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG

AGTCCACGTT CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC

4401 AACCCTATCT CGGTCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC

GGCCTATTGG TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT

4501 TTAACAAAAT ATTAACGTTT ACAATTTCCC ATTCGCCATT CAGGCTGCGC

AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT TACGCCAGCC

4601 CAAGCTACCA TGATAAGTAA GTAATATTAA GGTACGGGAG GTACTTGGAG

CGGCCGCAAT AAAATATCTT TATTTTCATT ACATCTGTGT GTTGGTTTTT

4701 TGTGTGAATC GATAGTACTA ACATACGCTC TCCATCAAAA CAAAACGAAA

CAAAACAAAC TAGCAAAATA GGCTGTCCCC AGTGCAAGTG CAGGTGCCAG 4801 AACATTTCTC TATCGATA

pGL3-Control Vector Sequence

1 GGTACCGAGC TCTTACGCGT GCTAGCCCGG GCTCGAGATC TGCGATCTGC

ATCTCAATTA GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG 101 CCCCTAACTC CGCCCAGTTC CGCCCATTCT CCGCCCCATC GCTGACTAAT

TTTTTTTATT TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC 201 AGAAGTAGTG AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAAAGCTT

GGCATTCCGG TACTGTTGGT AAAGCCACCA TGGAAGACGC CAAAAACATA 301 AAGAAAGGCC CGGCGCCATT CTATCCGCTG GAAGATGGAA CCGCTGGAGA

GCAACTGCAT AAGGCTATGA AGAGATACGC CCTGGTTCCT GGAACAATTG 401 CTTTTACAGA TG-CACATATC GAGGTGGACA TCACTTACGC TGAGTACTTC

GAAATGTCCG TTCGGTTGGC AGAAGCTATG AAACGATATG GGCTGAATAC 501 AAATCACAGA ATCGTCGTAT GCAGTGAAAA CTCTCTTCAA TTCTTTATGC

CGGTGTTGGG CGCGTTATTT ATCGGAGTTG CAGTTGCGCC CGCGAACGAC 601 ATTTATAATG AACGTGAATT GCTCAACAGT ATGGGCATTT CGCAGCCTAC

CGTGGTGTTC GTTTCCAAAA AGGGGTTGCA AAAAATTTTG AACGTGCAAA 701 AAAAGCTCCC AATCATCCAA AAAATTATTA TCATGGATTC TAAAACGGAT

TACCAGGGAT TTCAGTCGAT GTACACGTTC GTCACATCTC ATCTACCTCC 801 CGGTTTTAAT GAATACGATT TTGTGCCAGA GTCCTTCGAT AGGGACAAGA

CAATTGCACT GATCATGAAC TCCTCTGGAT CTACTGGTCT GCCTAAAGGT 901 GTCGCTCTGC CTCATAGAAC TGCCTGCGTG AGATTCTCGC ATGGCAGAGA

TCCTATTTTT GGCAATCAAA TCATTCCGGA TACTGCGATT TTAAGTGTTG 1001 TTCCATTCCA TCACGGTTTT GGAATGTTTA CTACACTCGG ATATTTGATA

TGTGGATTTC GAGTCGTCTT AATGTATAGA TTTGAAGAAG AGCTGTTTCT 1101 GAGGAGCCTT CAGGATTACA AGATTCAAAG TGCGCTGCTG GTGCCAACCC

TATTCTCCTT CTTCGCCAAA AGCAGTCTGA TTGACAAATA CGATTTATCT 1201 AATTTACACG AAATTGCTTC TGGTGGCGCT CCCCTCTCTA AGGAAGTCGG

GGAAGCGGTT GCCAAGAGGT TCCATCTGCC AGGTATCAGG CAAGGATATG 1301 ^' GGCTCACTGA GACTACATCA GCTATTCTGA TTACACCCGA GGGGGATGAT

AAACCGGGCG CGGTCGGTAA AGTTGTTCCA TTTTTTGAAG CGAAGGTTGT 1401 GGATCTGGAT ACCGGGAAAA CGCTGGGCGT TAATCAAAGA GGCGAACTGT

GTGTGAGAGG TCCTATGATT ATGTCCGGTT ATGTAAACAA TCCGGAAGCG 1501 ACCAACGCCT TGATTGACAA GGATGGATGG CTACATTCTG GAGACATAGC

TTACTGGGAC GAAGACGAAC ACTTCTTCAT CGTTGACCGC CTGAAGTCTC 1601 TGATTAAGTA CAAAGGCTAT CAGGTGGCTC CCGCTGAATT GGAATCCATC

TTGCTCCAAC ACCCCAACAT CTTCGACGCA GGTGTCGCAG GTCTTCCCGA 1701 CGATGACGCC GGTGAACTTC CCGCCGCCGT TGTTGTTTTG GAGCACGGAA

AGACGATGAC GGAAAAAGAG ATCGTGGATT ACGTCGCCAG TCAAGTAACA 1801 ACCGCGAAAA AGTTGCGCGG AGGAGTTGTG TTTGTGGACG AAGTACCGAA

AGGTCTTACC GGAAAACTCG ACGCAAGAAA AATCAGAGAG AJCCTCATAA 1901 AGGCCAAGAA GGGCGGAAAG ATCGCCGTGT AATTCTAGAG TCGGGGCGGC

CGGCCGCTTC GAGCAGACAT GATAAGATAC ATTGATGAGT TTGGACAAAC 2001 CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA ATTTGTGATG

CTATTGCTTT ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC 2101 AACAATTGCA TTCATTTTAT GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT

TTTTTAAAGC AAGTAAAACC TCTACAAATG TGGTAAAATC GATAAGGATC 2201 TGAACGATGG AGCGGAGAAT GGGCGGAACT GGGCGGAGTT AGGGGCGGGA

TGGGCGGAGT TAGGGGCGGG ACTATGGTTG CTGACTAATT GAGATGCATG 2301 CTTTGCATAC TTCTGCCTGC TGGGGAGCCT GGGGACTTTC CACACCTGGT

TGCTGACTAA TTGAGATGCA TGCTTTGCAT ACTTCTGCCT GCTGGGGAGC 2401 CTGGGGACTT TCCACACCCT AACTGACACA CATTCCACAG CGGATCCGTC

GACCGATGCC CTTGAGAGCC TTCAACCCAG TCAGCTCCTT CCGGTGGGCG 2501 CGGGGCATGA CTATCGTCGC CGCACTTATG ACTGTCTTCT TTATCATGCA

ACTCGTAGGA CAGGTGCCGG CAGCGCTCTT CCGCTTCCTC GCTCACTGAC 2601 TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG CTCACTCAAA

GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA 2701 TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG

CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG 2801 ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG

CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG 2901 CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC

TCAATGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA 3001 AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA

TCCGGTAACT ' ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC 3101 ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG

GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG 3201 ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT TCGGAAAAAG

AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT 3301 TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA

GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC 3401 ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA

TCCTTTTAAA TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG 3501 TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG CACCTATCTC

AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT 3601 AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG TGCTGCAATG

ATACCGCGAG ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA 3701 GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TTATCCGCCT

CCATCCAGTC TATTAATTGT TGCCGGGAAG CTAGAGTAAG T-iGTTCGCCA 3801 GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTACAGGCA TCGTGGTGTC

ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA 3901 GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC

GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT 4001 GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA TCCGTAAGAT

GCTTTTCTGT GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT 4101 ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAATACGGG ATAATACCGC

GCCACATAGC AGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG 4201 GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG TTCGATGTAA

CCCACTCGTG CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT

4301 TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA AAGGGAATAA

^• GGGCGACACG GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT 4401 TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG

TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG 4501 TGCCACCTGA CGCGCCCTGT AGCGGCGCAT TAAGCGCGGC GGGTGTGGTG

GTTACGCGCA GCGTGACCGC TACACTTGCC AGCGCCCTAG CGCCCGCTCC 4601 TTTCGCTTTC TTCCCTTCCT TTCTCGCCAC GTTCGCCGGC TTTCCCCGTC

AAGCTCTAAA TCGGGGGCTC CCTTTAGGGT TCCGATTTAG TGCTTTACGG 4701 CACCTCGACC CCAAAAAACT TGATTAGGGT GATGGTTCAC GTAGTGGGCC

ATCGCCCTGA TAGACGGTTT TTCGCCCTTT GACGTTGGAG TCCACGTTCT 4801 TTAATAGTGG ACTCTTGTTC CAAACTGGAA CAACACTCAA CCCTATCTCG

GTCTATTCTT TTGATTTATA AGGGATTTTG CCGATTTCGG CCTATTGGTT 4901 AAAAAATGAG CTGATTTAAC AAAAATTTAA CGCGAATTTT AACAAAATAT

TAACGTTTAC AATTTCCCAT TCGCCATTCA GGCTGCGCAA CTGTTGGGAA 5001 GGGCGATCGG TGCGGGCCTC TTCGCTATTA CGCCAGCCCA AGCTACCATG

ATAAGTAAGT AATATTAAGG TACGGGAGGT ACTTGGAGCG GCCGCAATAA 5101 AATATCTTTA TTTTCATTAC ATCTGTGTGT TGGTTTTTTG TGTGAATCGA

TAGTACTAAC ATACGCTCTC CATCAAAACA AAACGAAACA AAACAAACTA 5201 GCAAAATAGG CTGTCCCCAG TGCAAGTGCA GGTGCCAGAA CATTTCTCTA

TCGATA

pGL3Enhancer Vector Sequence

1 GGTACCGAGC TCTTACGCGT GCTAGCCCGG GCTCGAGATC TGCGATCTAA

GTAAGCTTGG CATTCCGGTA CTGTTGGTAA AGCCACCATG GAAGACGCCA 101 AAAACATAAA GAAAGGCCCG GCGCCATTCT ATCCGCTGGA AGATGGAACC

GCCAGAGATC CTATTTTTGG CAATCAAATC ATTCCGGATA CTGCGATTTT 801 AAGTGTTGTT CCATTCCATC ACGGTTTTGG AATGTTTACT ACACTCGGAT ATTTGATATG TGGATTTCGA GTCGTCTTAA TGTATAGATT TGAAGAAGAG

901 CTGTTTCTGA GGAGCCTTCA GGATTACAAG ATTCAAAGTG CGCTGCTGGT

GCCAACCCTA TTCTCCTTCT TCGCCAAAAG CACTCTGATT GACAAATACG

1001 ATTTATCTAA TTTACACGAA ATTGCTTCTG GTGGCGCTCC CCTCTCTAAG

GAAGTCGGGG AAGCGGTTGC CAAGAGGTTC CATCTGCCAG GTATCAGGCA

1101 AGGATATGGG CTCACTGAGA CTACATCAGC TATTCTGATT ACACCCGAGG

GGGATGATAA ACCGGGCGCG GTCGGTAAAG TTGTTCCATT TTTTGAAGCG 1201 AAGGTTGTGG ATCTGGATAC CGGGAAAACG CTGGGCGTTA ATCAAAGAGG

CGAACTGTGT GTGAGAGGTC CTATGATTAT GTCCGGTTAT GTAAACAATC

1301 CGGAAGCGAC CAACGCCTTG ATTGACAAGG ATGGATGGCT ACATTCTGGA

GACATAGCTT ACTGGGACGA AGACGAACAC TTCTTCATCG TTGACCGCCT

1401 GAAGTCTCTG ATTAAGTACA AAGGCTATCA GGTGGCTCCC GCTGAATTGG

AATCCATCTT GCTCCAACAC CCCAACATCT TCGACGCAGG TGTCGCAGGT

1501 CTTCCCGACG ATGACGCCGG TGAACTTCCC GCCGCCGTTG TTGTTTTGGA

GCACGGAAAG ACGATGACGG AAAAAGAGAT CGTGGATTAC GTCGCCAGTC

1601 AAGTAACAAC CGCGAAAAAG TTGCGCGGAG GAGTTGTGTT TGTGGACGAA

GTACCGAAAG GTCTTACCGG AAAACTCGAC GCAAGAAAAA TCAGAGAGAT

1701 CCTCATAAAG GCCAAGAAGG GCGGAAAGAT CGCCGTGTAA TTCTAGAGTC

TTGTGATGCT ATTGCTTTAT TTGTAACCAT TATAAGCTGC AATAAACAAG

1901 TTAACAACAA CAATTGCATT CATTTTATGT TTCAGGTTCA GGGGGAGGTG

TGGGAGGTTT TTTAAAGCAA GTAAAACCTC TACAAATGTG GTAAAATCGA

2001 TAAGGATCTG AACGATGGAG CGGAGAATGG GCGGAACTGG GCGGAGTTAG

GGGCGGGATG GGCGGAGTTA GGGGCGGGAC TATGGTTGCT GACTAATTGA

2101 GATGCATGCT TTGCATACTT CTGCCTGCTG GGGAGCCTGG GGACTTTCCA

CACCTGGTTG CTGACTAATT GAGATGCATG CTTTGCATAC TTCTGCCTGC 2201 TGGGGAGCCT GGGGACTTTC CACACCCTAA CTGACACACA TTCCACAGCG

GATCCGTCGA CCGATGCCCT TGAGAGCCTT CAACCCAGTC AGCTCCTTCC

2301 GGTGGGCGCG GGGCATGACT ATCGTCGCCG CACTTATGAC TGTCTTCTTT

ATCATGCAAC TCGTAGGACA GGTGCCGGCA GCGCTCTTCC GCTTCCTCGC

2401 TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT

CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG

2501 AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG

CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC

2601 AAAAATCGAC GCTCAAGTCA -GAGGTGGCGA AACCCGACAG GACTATAAAG

ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTGCGA ^'

2701 CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT 2801 TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT

GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC 2901 TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA

TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA 3001 CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC

GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG 3101 CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT

CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC 3201 GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT

CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA 3301 TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA

CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC 3401 CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG

CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA 3501 ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT

ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA 3601 GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC

GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA 3701 ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA

GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA 3801 TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC

CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG 3901 AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT

AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG 4001 TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT

CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC 4101 ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA

GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC 4201 AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA

TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC 4301 CCGAAAAGTG CCACCTGACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGG

GTGTGGTGGT TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG 4401 CCCGCTCCTT TCGCTTTCTT CCCTTCCTTT CTCGCCACGT TCGCCGGCTT

TCCCCGTCAA GCTCTAAATC GGGGGCTCCC TTTAGGGTTC CGATTTAGTG 4501 CTTTACGGCA CCTCGACCCC AAAAAACTTG ATTAGGGTGA TGGTTCACGT

AGTGGGCCAT CGCCCTGATA GACGGTTTTT CGCCCTTTGA CGTTGGAGTC 4601 CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA ACACTCAACC CTATCTCGGT CTATTCTTTT GATTTATAAG GGATTTTGCC GATTTCGGCC 4701 TATTGGTTAA AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTTTAA

CAAAATATTA ACGTTTACAA TTTCCCATTC GCCATTCAGG CTGCGCAACT 4801 GTTGGGAAGG GCGATCGGTG CGGGCCTCTT CGCTATTACG CCAGCCCAAG

CTACCATGAT AAGTAAGTAA TATTAAGGTA CGGGAGGTAC TTGGAGCGGC 4901 CGCAATAAAA TATCTTTATT TTCATTACAT CTGTGTGTTG GTTTTTTGTG

TGAATCGATA GTACTAACAT ACGCTCTCCA TCAAAACAAA ACGAAACAAA 5001 ACAAACTAGC AAAATAGGCT GTCCCCAGTG CAAGTGCAGG TGCCAGAACA

TTTCTCTATC GATA

pGL3-Promoter Vector Sequence

1 GGTACCGAGC TCTTACGCGT GCTAGCCCGG GCTCGAGATC TGCGATCTGC

GCAACTGCAT AAGGCTATGA AGAGATACGC CCTGGTTCCT GGAACAATTG 401 CTTTTACAGA TGCACATATC GAGGTGGACA TCACTTACGC TGAGTACTTC

CAATTGCACT GATCATGAAC TCCTCTGGAT CTACTGGTCT GCCTAAAGGT 901 GTCGCTCTGC CTCATAGAAC TGCCTGCGTG AGATTCTCGC ATGCCAGAGA

TATTCTCCTT CTTCGCCAAA AGCACTCTGA TTGACAAATA CGATTTATCT 1201 AATTTACACG AAATTGCTTC TGGTGGCGCT CCCCTCTCTA AGGAAGTCGG

GGAAGCGGTT GCCAAGAGGT TCCATCTGCC AGGTATCAGG CAAGGATATG 1301 GGCTCACTGA GACTACATCA GCTATTCTGA TTACACCCGA GGGGGATGAT

AGGTCTTACC GGAAAACTCG ACGCAAGAAA AATCAGAGAG ATCCTCATAA 1901 AGGCCAAGAA GGGCGGAAAG ATCGCCGTGT AATTCTAGAG TCGGGGCGGC

TTTTTAAAGC AAGTAAAACC TCTACAAATG TGGTAAAATC GATAAGGATC 2201 CGTCGACCGA TGCCCTTGAG AGCCTTCAAC CCAGTCAGCT CCTTCCGGTG

GGCGCGGGGC ATGACTATCG TCGCCGCACT TATGACTGTC TTCTTTATCA 2301 TGCAACTCGT AGGACAGGTG CCGGCAGCGC TCTTCCGCTT CCTCGCTCAC

TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT 2401 CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG

AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC 2501 GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA

ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT AJAAAGATAC 2601 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT

GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC 2701 TTTCTCAATG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC

TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC 2801 CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT

CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA 2901 GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG

AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA 3001 AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT

GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA 3101 AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA

ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC 3201 TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA

TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA 3301 TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC

GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC 3401 AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA

ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC 3501 GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC

GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG 3601 TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA

TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC 3701 CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC

TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA 3801 AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA

GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA 3901 CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT

TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT 4001 GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA

GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA 4101 ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA

TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG 4201 AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA

AAAGTGCCAC CTGACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT 4301 GGTGGTTACG CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG

CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC 4401 CGTCAAGCTC TAAATCGGGG GCTCCCTTTA GGGTTCCGAT TTAGTGCTTT

ACGGCACCTC GACCCCAAAA AACTTGATTA GGGTGATGGT T-CACGTAGTG 4501 GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT GGAGTCCACG

TTCTTTAATA GTGGACTCTT GTTCCAAACT GGAACAACAC TCAACCCTAT 4601 CTCGGTCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT TCGGCCTATT

GGTTAAAAAA TGAGCTGATT TAACAAAAAT TTAACGCGAA TTTTAACAAA 4701 ATATTAACGT TTACAATTTC CCATTCGCCA TTCAGGCTGC GCAACTGTTG

GGAAGGGCGA TCGGTGCGGG CCTCTTCGCT ATTACGCCAG CCCAAGCTAC 4801 CATGATAAGT AAGTAATATT AAGGTACGGG AGGTACTTGG AGCGGCCGCA

ATAAAATATC TTTATTTTCA TTACATCTGT GTGTTGGTTT TTTGTGTGAA 4901 TCGATAGTAC TAACATACGC TCTCCATCAA AACAAAACGA AACAAAACAA

ACTAGCAAAA TAGGCTGTCC . CCAGTGCAAG TGCAGGTGCC AGAACATTTC 5001 TCTATCGATA

Claims

1. A method of determining the p53 status of a sample, the method comprising providing a sample containing a pGL3 vector and determining whether p53 binds to the pGL3 vector.

2. A method according to claim 1 wherein the step of determining whether p53 binds to the pGL3 vector is carried out using an electrophoretic mobility shift assay.

3. A method according to claim 2 wherein the assay is carried out in the presence and absence of competitor nucleic acids known to bind p53.

. A method according to claim 1 wherein the sample is an expression system.

5. A method according to claim 4 wherein the step of determining whether p53 binds to the pGL3 vector comprises determining whether a reporter gene is expressed in the expression system.

6. A method according to claim 5 wherein the reporter gene is luciferase.

7. A method according to claim any preceding claim wherein the pGL3 vector has substantially the sequence of any one of the vectors whose- sequences are shown in Annex 1.

8. A method according to claim 7 wherein the pGL3 vector has substantially the sequence of the pGL3-Basic vector or the pGL3-Enhancer vector whose sequence is shown in Annex 1.

9. A method according to any preceding claim wherein the sample is a cell or tissue sample.

10. A method according to claim 9 further comprising the step of treating the cell or tissue sample prior to and/or contemporaneously with the step of determining whether p53 binds to the pGL3 vector.

11. A method according to claim 10 wherein the cell or tissue sample is one known to be capable of expressing wild-type (wt) p53 that becomes activated as a transcription factor in response to DNA damage.

12. A method according to claim 11 wherein the treatment is one known to cause DNA damage.

13. The use of a pGL3 vector for determining the p53 status of a sample.

14. A pGL3 vector, or a cell transfected with a pGL3 vector, packaged with instructions for use in a method according to any one of claims 1 to 12 or for use according to claim 13.

15. A method of modifying a pGL3 vector, the method comprising deleting or altering a CCCGGG motif of the pGL3 vector and/or deleting or altering a sequence within a 20 bp sequence 5' or 3 ' of said CCCGGG motif.

16. A method according to claim 15 wherein the pGL3 vector comprises a reporter gene and the CCCGGG motif is located 5' of the reporter gene.

17. A method of producing modified pGL3 vectors, having modified a pGL3 vector according to the method of claim 15 or claim 16, the method comprising replicating the vector so modified, or a descendent thereof.

18. A modified pGL3 vector having an alteration or deletion in a site specified in claim 15 or claim 16.

19. A cell containing the vector of claim 18.

20. A method, vector or cell according to any one of claims 15 to 19 wherein the pGL3 vector prior to modification has the sequence of any one of the vectors whose sequences are shown in Annex 1.

21. A method, vector or cell according to claim 20 wherein the pGL3 vector prior to modification has the sequence of the pGL3-Basic vector or the pGL3-Enhancer vector whose sequence is shown in Annex 1.

22. A method, vector or cell according to any one of claims 15 to 19 wherein the pGL3 vector prior to modification is a vector derived from any one of the vectors whose sequences are shown in Annex 1.

23. The use of nucleic acid having a sequence incorporating the CCCGGG motif to confer promoter activity.

24. Use according to claim 23 comprising bringing the sequence incorporating the CCCGGG motif into operative association with and 5' of nucleic acid encoding a polypeptide of interest.

25. Use according to claim 16 or claim 17 wherein the CCCGGG motif is used together with a flanking sequence or sequences .

26. A method of determining whether a promoter sequence, whose p53 responsiveness is unknown, is likely to be responsive to p53, the method comprising determining whether the promoter contains a CCCGGG motif.

27. The use of a promoter identified according^' to the method of claim 26 as being p53-responsive, to confer p53- responsiveness on a nucleic acid encoding a polypeptide of interest.

28. The use of a nucleic acid including a promoter identified according to the method of claim 26 as being p53- responsive, in assays for p53 transcriptional activity.

29. A nucleic acid including a promoter identified according to the method of claim 26 as being p53-responsive, packaged for use in an assay for p53 transcriptional activity.

30. A method according to any one of claims 1 to 10 for assaying for mimetics of p53, the method comprising: providing a pGL3 vector in a sample in circumstances in which the presence of activated p53 wcιld not normally be expected, exposing the sample to, or causing it to express, a candidate p53 mimetic, and determining whether apparent p53 binding to the pGL3 vector occurs in the sample, apparent p53 binding indicating the presence of a p53 mimetic.

31. A method according to any one of claims 1 to 10 for assaying for antagonists of p53, the method comprising: providing a pGL3 vector in a sample in circumstances in which p53 activity would be expected, exposing the sample to, or causing it to express, a candidate p53 antagonist, and determining whether apparent p53 binding to the pGL3 vector occurs in the sample, the absence of apparent p53 binding indicating the presence of a p53 antagonist.

32. A method of identifying a candidate substance as being a potential mimetic or antagonist of p53, the method comprising determining whether the candidate substance is able to bind to nucleic acid comprising or consisting of a CCCGGG motif, optionally with flanking sequences.