GB2390606A

GB2390606A - Expression Plasmid

Info

Publication number: GB2390606A
Application number: GB0214381A
Authority: GB
Inventors: Mohammad Saeid Hejazi
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-06-21
Filing date: 2002-06-21
Publication date: 2004-01-14
Also published as: GB0214381D0

Abstract

Plasmid pHEJI provides an inducible double (i.e. negative and positive) selection marker HSV.tk-Zeo (i.e. HSV.tk-Sh.ble fusagene) as the reporter gene. It also contains an independent selection marker (i.e. neomycin) cassette driven by SV40 promoter to select the transfected cells from untransfected cells. Furthermore, it contains suitable cloning sites upstream and downstream of the GAL4 binding sequence (i.e. upstream activator sequence; UAS) to replace the present DNA elements with other DNA sequences. Accordingly, it can be used to study the activity of other transcription activating proteins. Moreover, pHEJI can be used to identify new and possibly tissue specific DNA sequences to which transcription activating proteins bind due to containing a multiple cloning site and inducible selection marker gene(s) as the reporter gene(s). This plasmid also has an independent expression cassette with cytomegalovirus promoter and a multiple cloning site to clone any cDNA within the plasmid. Plasmid pHEJI also encodes ampicillin resistance gene and contains f1 and pUG replication origin.

Description

t pHEJI Plasmid This invention is a new plasmid, which is used to study

the transcription activating function of various transcription activating chimeric proteins containing GAL4 DNA binding domain, such as GAL4-VP16 chimcric transactivating protein. This invented plasmid also can be used to discover new and tissue specific DNA binding sequences.

Additionally, this plasmid can be used to study the transcription activating function of other transcription activating proteins by replacing the present DNA binding sequence with other corresponding sequences using the cloning sites.

Several plasmids have been developed to study the transcription activating function of the transcription activating proteins, but they suffer from at least one or usually some of the following disadvantages. None of them provides a double (i.e. negative and positive) selection marker (i.e. HSV.tk-Zeo fusagene) as the reporter gene. Therefore, by previously developed plasmids it is impossible to select the cells in which the reporter gene is transcriptionally activated from the cells in which the reporter gene remains transcriptionally inactive with two different cytotoxic drugs. Additionally, most of them do not contain an independent selection marker (e.g. neomycin) to select the transfected cells from untransfected cells. Also, some of them do not contain a suitable cloning site upstream and downstream of the DNA binding sequence to replace the present DNA binding sequence with other DNA binding sequences. Accordingly, these plasmids cannot be used to study the activity of other transcription activating chimeric proteins.

Additionally, those plasmids that lack of multiple cloning site and a selection marker as the reporter gene cannot be used to incorporate olionucleotide library in order to identify new and possibly tissue specific DNA sequences to which transcription activating proteins bind.

An object of the present invention is to provide a plasmid that can be used to study the transcription activation function of GAL4 DNA binding domain containing chimeric

transcription activating proteins, or other transcription activating proteins, as well as to identify novel and/or possibly tissue specific DNA binding sequences. Additionally, this invention also aims to provide a plasmid that contains an independent expression cassette with a multiple cloning site, in order to clone and express gene of interest without destructing the reporter gene's cassette or its application and objection.

Accordingly, the plasmid pHEJI is a 7.1kb plasmid containing HSV.tk-Zeo fusagene inducible cassette. This cassette composed of five repeats of 19 bp GAL4 DNA binding sequence followed by TATA box and HSV.tk-Zeo fusagene. The HSV.tk-Zeo fusagene is adenylated by SV40 polyadenylation sequence in this cassette. HSV.TK-Zeo (i.e. HSV.TK-Sh.ble) is a fusion protein composed of HSV.TK and Zeo (i.e. Sh.ble). HSV.TK moiety of this fusion protein confers susceptibility to prodrug GCV (i.e negative selection) and Zeo (i.e. Sh.ble) induces resistance to zeocin (i.e. positive selection).

Therefore, the cells in which the reporter gene (i.e. HSV.tk-Zeo) is transcriptionally activated via a corresponding transcription activating protein can be negatively selected with GCV (i.e. ganciclovir) or positively selected with zeocin from those cells in which the reporter gene remains transcriptionally inactive. Therefore, this plasmid takes the advantage of HSV.TK-Zeo fusion protein as the reporter gene to double select the target cells, either negatively by GCV or positively by zeocin.

This plasmid also encodes neomycin resistance gene (i.e. neomycin phosphotransferase) driven by SV40 promoter and SV40 polyadenylation signal. The presence of neomycin cassette in this plasmid allows selecting transfected cells with G418. This is in addition to GCV and zeocin selection.

This plasmid also contains an independent expression cassette. This cassette consists of cytomegalovirus promoter (Pcmv) followed by a multiple cloning site (i.e. MCS) and bovine growth hormone polyadenylation sequence (i.e. BGH PolyA). The presence of this cassette allows to clone any cDNA including various transcription activating chimeric proteins encoding sequences within the plasmid. The cloned cDNA will be

driven by Pcmv and be adenylated by BGH poly A. If the expressed transcription activating protein contains GAL4 DNA binding domain, it can activate the transcription of the adjacent reporter gene (i.e. HSV.tk-Zeo) .

The presence of ditterent restriction sites upstream and downstream of DBS (i.e. 5 x i GAL4 DNA binding sequence) allows to replace the present DBS with any other DNA binding sequences. It allows to study the function of other chimeric transcription activating proteins on the transcription of the reporter gene. The presence of these restriction sites upstream and downstream of DBS also allows to incorporate oligonucleotide library in the DBS site in order to discover new and possibly tissue specific DNA binding sequences to which corresponding transcription activating proteins bind. This plasmid also encodes ampicillin resistance gene for selection and maintenance in E.coli. It also contains F1 and pUC replication origin.

The construction and drug resistance induction feature of the invention will now be described with reference to the accompanying drawing in which: FIGURE 1 shows the HSV.tk-Zeo (i.e. HSV.tk-Sh.ble) reporter gene cassette; FIGURE 2 illustrates the construction of pHE!JI plasmid; I; IGURE 3 describes the TK activity of untransfected, pHEJI and pHEJl+pG4VP transfccted HeLa cells; FIGURE 4 describes the Zeo (i.e. Sh.ble) activity of untransfected, pHEJI and pHEJl+pG4VP translected HeLa cells as explained below.

As illustrated in figure 1, the pHEJ1 plasmid composed of HSV.tk-Zeo cassette, an independent expression cassette containing multiple cloning site to clone the gene of interest driven by CMV promoter tor high level expression, neomycin resistance gene for

selection of transfected cells, ampicillin resistance gene for selection of E. coli, pUC origin and fl origin.

As shown in figure 2, the HSV.tk-Zeo reporter gene composed of five repeats of GALA DNA binding sequence followed by TATA box, HSV.tk-Zeo fusion gene and SV40 poly adenylation sequence. The complete sequence of a single box of GAI,4 DNA binding sequence is: TCCGCTCGGAGGACAGTAC.

The HSV.tk-Zeo cassette remains transcriptionally inactive in mammalian cells, unless a transcription activator protein is expressed in the cells. To confirm that the HSV.tk-Zeo cassette cloned within the invented plasmid remains transcriptionally inactive in mammalian cells, HeLa cells were transfectcd with pHEJI plasmid and selected with G418. Then the pilEJI transfected HeLa cells were re-transfected with pG4VP plasmid encoding GAL4-VP16 chimeric transcription activating protein.

To investigate the expression of the HSV.tk-Zeo encoding sequence in these cells the proliferation of the pHEJl and pHEJI+pG4VP cells were analysed in the presence of various concentrations of GCV ranging from 0- 250 /ml using MTT proliferation assay.

In this experiments untransfected HeLa cells were used as negative control. In this experiment 1 x 104 cells/1001 DMEM were seeded in each well of 96-well plates. The next day the cells were treated with a range of different concentrations of GCV from 0-

250 1lg/ml for 6 days. Then the proliferation using MTT method was assayed on day 6 of GCV treatment.

The results illustrated in figuec 3, show that the pHEJI transfected and untransfected IleLa cells had a similar proliferation profile, which was quite different from that of the pHEJI+pG4VP cells. lathe pHEJI and untransfected cells appeared to be very resistant to GCV, while the pHEJl+ p(34VP cells were very sensitive to GCV cytotoxicity. For instance, in the presence of 10 g/ml GCV the survival of pHEJI and parental HeLa cells were about or more than 80% and their survival rate did not come less than 40% at the

s highest concentration of GCV (i.e. 250 g/ml). While, the survival of pHEJI+pG4VP cells was only about 3% in the presence of 10 1lg/ml GCV. Furthermore, these results demonstrated that about 0.25 g/ml of GCV was required to kill 50% of pHEJI+pC4VP cells, while this amount for untransfected and pHEJI transfected cells was equal or more than 200 1lg/ml. Comparison of these amounts of GCV indicates that HeWDB+G4VP cells were more than 800-fold more susceptible to GCV than HeLa/DB cells. This experiment confirmed that the HSV.tk-Zeo cassette introduced in HeLa cells using invented plasmid remains transcriptionally silent in the absence of GAL4-VP16 chimeric protein. Moreover, this cassette successfully is transcribed in the presence of GAL4-

VP16 transcription activating protein.

This was further confirmed by the sensitivity of the pHEJI and pHEJI+ pG4VP cells to zeocin toxicity, where the untransfected cells were used as negative control. Similarly, analysis of zeocin resistance, as shown in figure 4, showed that pHEJJ and untransfected HeLa cells were very sensitive to zeocin, while pl-lEJI+pG4VP cells were greatly resistant to zeocin. For instance, in the presence of 1 OO,ug/ml zeocin about 20% of pHEJI transfected and untransfected cells survived, while the survival rate of the pHEJI+pG4VP cells at this concentration of zeocin was more than 95%. As the concentration of zeocin increased, the proliferation of the untransfected and pHEJT transfected HeLa cells continued decreasing so that less than 5% of these cells survived in thc presence of 1000 1lg/ml zeocin. Whilst, the pHEJl+pG4VP cells continued proliferating and their survival rate in the presence of 1000 g/ml zeocin was more than 90%. This finding demonstrates the HSV.tk-Zeo cassette remains transcriptionally inactive in the cells and do not induce zeocin resistance unless GAL4-VP16 is expressed in the cells.

As mentioned earlier the pHEJI plasmid contains an independent expression cassette to clone gene of interest. This cassette contains a multiple cloning site containing NheJ, NotI, EcoRI, HindllI and Aflll unique restriction sites for this purpose.

The Mlul site at 1703 with Ndel site, at 2228, or another MluI site, at 1973 bp, will be used in order to replace the present GAL4 DNA binding sequence and TATA box (DBS+IATA) with other DNA segment. For this purpose BstXI sites at 1730, 2678 and 2704 also can be used. Additionally the combination of any of the MluI, Xbal, BstxT sites with HindlII or AnII sites can be used or this purpose.

This plasmid also allows to replace the present GAL4 DNA binding sequence (DBS) with other transcription activating DNA binding sequences or oligonucleotide library. For instance, XbaI site at 1753 with NotI, l; coRI, HindIII and AflIII sites can be used for this purpose. The complete sequence of the invention is written below.

1 GACGGATCGGGAGATCTCCGAGGGATCCAGACATGATAAGATACATTGAT

51 GAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTG

101 TGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATA

151 AACAAGTTTCGAGGTCGACCCCCCTCGGACCCGTCGGGCCGCGTCGGACC

201 GGCGGTGTTGGTCGGCGTCGGTCAGTCCTGCTCCTCGGCCACGAAGTGCA

251 CGCAGTTGCCGGCCGGGTCGCGCAGGGCGAACTCCCGCCCCCACGGCTGC

301 TCGCCGATCTCGGTCATGGCCGGCCCGGAGGCGTCCCGGAAGTTCGTGGA

351 CACGACCTCCGACCACTCGGCGTACAGCTCGTCCAGGCCGCGCACCCACA

401 CCCAGGCCAGGGTGTTGTCCGGCACCACCTGGTCCTGGACCGCGCTGATG

451 AACAGGGTCACGTCGTCCCGGACCACACCGGCGAAGTCGTCCTCCACGAA

501 GTCCCGGGAGAACCCGAGCCGGTCGGTCCAGAACTCGACCGCTCCGGCGA

551 CGTCGCGCGCGGTGAGCACCGGAACGGCACTGGTCAACTTGGCCATGACG

601 CCATTAGCTCCGCTGATCATCTCTCGGGCAAACGTGCGCGCCAGGTCGCA

651 TATCGTCGGTATGGAGCCGGGGGTGGTGACGTGGGTCTGGACCATCCCGG

701 AGGTAAGTTGCAGCAGGGCGTCCCGGCAGCCGGCGGGCGATTGGTCGTAA

751 TCCAGGATAAAGACGTGCATGGAACGGAGGCGTTTGGCCAAGACGTCCAA

801 GGCCCAGGCAAACACGTTATACAGGTCGCCGTTGGGGGCCAGCAACTCGG

851 GGGCCCGAAACAGGGTAAATAACGTGTCCCCGATATGGGGTCGTGGGCCC

901 GCGTTGCTCTGGGGCTCGGCACCCTGGGGCGGCACGGCCGTCCCCGAAAG

951 CTGTCCCCAGTCCTCCCGCCACGACCCGCCGCACTGCAGATACCGCACCG

1001 TATTGGCAAGTAGCCCGTAAACGCGGCGAATCGCAGCCAGCATAGCCAGG

1051 TCCAGCCGCTCGCCGGGGCGCTGGCGTTTGGCCAGGCGGTCGATGTGTCT

1101 GTCCTCCGGAAGGGCCCCAAGCACGATGTTGGTGCCGGGCAAGGTCGGCG

1151 GGATGAGGGCCACGAACGCCAGCACGGCCTGGGGGGTCATGCTGCCCATA

1201 AGGTACCGCGCGGCCGGGTAGCACAGGAGGGCGGCGATGGGATGGCGGTC

1251 GAAGATGAGGGTGAGGGCCGGGGGCGGGGCATGTGAGCTCCCAGCCTCCC

1301 CCCCGATATGAGGAGCCAGAACGGCGTCGGTCACGGCATAAGGCATGCCC

1351 ATTGTTATCTGGGCGCTTGTCATTACCACCGCCGCGTCCCCGGCCGATAT

1401 CTCACCCTGGTCGAGGCGGTGTTGTGTGGTGTAGATGTTCGCGATTGTCT

1451 CGGAAGCCCCCAGCACCCGCCAGTAAGTCATCGGCTCGGGTACGTAGACG

1501 ATATCGTCGCGCGAACCCAGGGCCACCAGCAGTTGCGTGGTGGTGGTTTT

1551 CCCCATCCCGTGGGGACCGTCTATATAAACCCGCAGTAGCGTGGGCATTT

1601 TCTGCTCCGGGCGGACTTCCGTGGCTTCTTGCTGCCGGCGAGGGCGCAAC

1651 GCCGTACGTCGGTTGCTATGGCCGCGAGAACGCGCAGCCTGGTCGAACGC

1701 AGACGCGTGTTGATGGCCGGGGTACGAGGCCATGGAGCTGGCGCATTATA

1751 TACCCTCTAGAGTCGACGGATCGGAGTACTGTCCTCCGAGCGGAGTACTG

1801 TCCTCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGA

1851 GTACTGTCCTCCGAGCGGATCCCCGGGAATTAGCTTGGCGCGTACTAGTG

1901 ATATCCAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCT

1951 TCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTT

2001 ATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAG

2051 TTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAA

2101 CGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGC

2151 CAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACT

2201 GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTAT

2251 TGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA

2301 CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCT

2351 ATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCG

2401 GTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGA

2451 GTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAAC

2501 TCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTA

2551 TATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTA

2601 TCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTA

2651 AACGGGCCCTCTAGACTCGAGCGGCCGCCACTGTGCTGGATATCTGCAGA

2701 ATTCCACCACACTGGACTAGTGGATCCGAGCTCGGTACCAAGCTTAAGTT

2751 TAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTT

2801 GTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC

2851 TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT

2901 GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGAT

2951 TGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTC

3001 TGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCT

3051 GTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACC

3101 GCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTC

3151 CTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGC

3201 TCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAA

3251 CTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGT

3301 TTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGT

3351 TCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTA

3401 TAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA

3451 ACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTG

3501 TGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATC

3551 TCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGC

3601 AGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCC

3651 CCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTC

3701 CGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC

3751 TCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCT

3801 AGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTG

3851 ATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTG

3901 CACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTG

3951 GGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAG

4001 CGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTG

4051 AATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGG

4101 CGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACT

4151 GGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTT

4201 GCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCA

4251 TACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCA

4301 TCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGAT

4351 CTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCT

4401 CAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATG

4451 CCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATC

4501 GACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGC

4551 TACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCC

4601 TCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTAT

4651 CGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACC

4701 GACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGC

4751 CTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGA

4801 TGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAAC

4851 TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAA

4901 TTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCA

4951 AACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAG

5001 AGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC

5051 GCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCT

5101 GGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTG

5151 CCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGG

5201 CCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCT

5251 CGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCA

5301 GCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC

5351 AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAA

5401 AGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCAT

5451 CACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATA

5501 AAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTC

5551 CGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGC

5601 GTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT

5651 CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC

5701 GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC

5751 GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG

5801 GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT

5851 ACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACC

5901 TTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGG

5951 TAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGAT

6001 CTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAAC

6051 GAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTT

6101 CACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTA

6151 TATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCA

6201 CCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCC

6251 CGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG

6301 CTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCA

6351 ATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTT

6401 ATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTA

6451 GTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATC

6501 GTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCA

6551 ACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTA

6601 GCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTA

6651 TCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATC

6701 CGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAG

6751 AATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGAT

6801 AATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACG

6851 TTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT

6901 CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTC

6951 ACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAA

7001 GGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTC

7051 AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATA

7101 TTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCC

7151 CCGAAAAGTGCCACCTGACGTC

Claims

Plasmid pHEJ1 A plasmid contanmg an inducible HSV.tk-Zco (net HSV.tk-Sh. ble fusagene) double (net negative and positive) selection marker as the reporter gcne(s) preceded by TATA box and multiple GAL4 DNA binding sequences (i.e. upstream activator sequence; UAS) called DBS to study transcription activating function of various chimerc transcription activating proteins containing GAL4 DNA binding domain, rcstncton sites upstream and downstream of DBS (i.e UAS) to replace the present DBS with other DNA elements or otgonucleotide hbrary, an independent expression cassette containing a multiple cloning site In order to clone and express any gene of .. À interest, an mdepcndent neomycin selection marker cassette in order to select À'. the transfected cells from untransfected cells, amplicillin resistance gene for . selection and maintenance in E colt, fl and pUC origin of replication.

.
2. A plasmid as claimed in Clamp, containing inducible HSV.tk-Zco cassette composed of HSV.tk-Zeo double selection marker preceded by TATA box and 5 complete repeats of TCCGCTCGGAGGACAGTAC GAL4 DNA binding sequence (DBS) and followed by SV40 polyadenylation sequence.
3. A plasmid as claimed in Claim 1 and 2, containing inducible HSV.tk-Zeo

cassette whose expression is under the control of GAL4 DNA binding domain containing transcription activating proteins.
4. A plasmidd as clime in Claims 1, 2 and 3, containing an inducible HSV. tk-Zeo double selection marker as the reporter gene(s) that allows to select the cells n1 which the reporter gene(s) is activated from the cells in which the reporter gcne(s) remains silent using GCV and zeocin cytotoxic reagents.

i?
5 A plasmd as claimed in Claim 1, containing Xhal site at 1756, which is used with any of unique Notl, EcoRT, flindlll and AJ7II restriction sites to replace the present DBS with any other DNA elements to study the transcription activating function of other transcription activating proteins.
6. A plasnd as claimed in Claims 1, 2, 3, 4 and 5, containing suitable restriction sites in order to incorporate oligonucleotide library upstream of lISV.tk-Zeo and select the cells in which the reporter gene(s) is activated from the cells m which the reporter gene(s) remains silent In order to identify new DNA fragments to which transcription activating proteins bind.
7. A plasmid as claimed m Claim 1, containing an independent expression cassette composed of cytomegalovirus promoter followed by a multiple '.. cloning site containing Ethel, NotT, EeoRI, ndIII and Af711 unique restriction sales, and bovine growth hormone polyadenylation sequence in order to clone and express and gene of interest.

. Àe À
8. A plasmid as claimed In Claim 1, containing an independent neomycin 2.... selection marker driven by SV40 promoter and adenylated by SV40 A., polyadenylation sequence in order to select transfeeted cells from À untaransfeeted cells.
9. A plasmd substantially as described herein with reference to Figures 14 of the accompanying figures.