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EP0783509A1 - Facteur iia de transcription humain - Google Patents

Facteur iia de transcription humain

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Publication number
EP0783509A1
EP0783509A1 EP94931757A EP94931757A EP0783509A1 EP 0783509 A1 EP0783509 A1 EP 0783509A1 EP 94931757 A EP94931757 A EP 94931757A EP 94931757 A EP94931757 A EP 94931757A EP 0783509 A1 EP0783509 A1 EP 0783509A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
polynucleotide
tfiia
dna
small subunit
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94931757A
Other languages
German (de)
English (en)
Other versions
EP0783509A4 (fr
Inventor
Paul A. Moore
Craig A. Rosen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Human Genome Sciences Inc
Original Assignee
Human Genome Sciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Human Genome Sciences Inc filed Critical Human Genome Sciences Inc
Publication of EP0783509A1 publication Critical patent/EP0783509A1/fr
Publication of EP0783509A4 publication Critical patent/EP0783509A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is the small ( ⁇ ) subunit of human transcription factor IIA, sometimes hereinafter referred to as "small subunit". The invention also relates to inhibiting the action of such polypeptides.
  • RNA polymerase In prokaryote ⁇ , simply mixing purified RNA polymerase, a template carrying a promoter, nucleoside triphosphates, and appropriate buffer and salts is sufficient to obtain specific gene transcription in vitro beginning at the correct sites.
  • Purified RNA polymerase from eukaryotes initiates transcription very poorly and essentially at random. Accordingly, accessory factors are required for accurate initiation of transcription in eukaryotes. Some of these transcription factors are general factors required for initiation at all promoters, while others are gene-specific and are required only for certain promoters.
  • T Transcription Factor IID
  • A adenosine
  • Other general factors are also involved in the assembly of a multicomponent protein complex at the promoter.
  • transcription factors are found to contain two functional domains, one for DNA-binding and one for transcriptional activation. These functions often reside within circumscribed structural domains that retain their function when removed from their natural context.
  • the DNA- binding domains of transcription factors fall into several structural families based on their primary amino acid sequence.
  • regions of the gene flanking the coding region can be ⁇ equenced. Comparisons of these sequences reveal common patterns near the 5' and 3' ends of different genes. These are predicted to be important for proper transcription by RNA polymerase. The most common motif is the TATA sequence around 30 bp from the transcriptional start site. Other conserved sequences have been found roughly 50 to 100 bp upstream of the transcriptional start site.
  • TFIIA Eukaryotic transcriptional activation requires the characterization of several multiprotein complexes, referred as general transcription factors and coactivators 1 .
  • the heteromeric general transcription factor TFIIA binds directly to the TATA binding protein (TBP) 3,4 and has been implicated in the process of transcriptional activation 5*8 .
  • TBP TATA binding protein
  • the y subunit of TFIIA binds weakly to the TATA binding protein, but strongly stabilized the binding of the large subunit of TFIIA ( ⁇ ⁇ ) to TBP.
  • Recombinant human TFIIA is functional for the transcriptional activation mediated by at leastthree distinct activators. Both the ⁇ and subunits are essential for activator dependent stimulation of TFIID by binding to promoter DNA, thus facilitating the first step in pre- initiation complex formation. This demonstrates that TFIIA is an evolutionary conserved general transcription factor important for activator regulated transcription.
  • TFIIA The interaction of TFIIA with the general transcription factor IID (TFIID) has been shown to be rate-limiting step in the transcriptional activation process 5 .
  • TFIIA binds directly to TBP 3,4 , the DNA binding subunit of the multiprotein TFIID complex 12 .
  • TBP associated factors (TAFs) 12"14 which are essential for activated transcription, are also required for an activator-dependent stimulation of the TFIIA-TFIID- promoter complex 6 . While TFIIA has no known function in unregulated basal transcription 15 , it has been postulated that TFIIA plays a role in preventing inhibitors of TFIID from repressing transcription 16 " 19 .
  • TFIIA homolog has been identified in yeast 9 " 10 , and the genes encoding the two subunits are essential for viability 11 .
  • Human TFIIA consists of three polypeptides ( ⁇ , ⁇ , y ) , but the two largest subunits are derived from a single gene which shares homology to the large subunit of yeast TFIIA 7,20 - 21 . Both the human and yeast protein bind to the evolutionary conserved domain of TBP 22 and stimulate transcription reconstituted with TFIID, but not TBP 17,19 .
  • polypeptide of the present invention has been putatively identified as the subunit of TFIIA. This identification has been made as a result of amino acid sequence homology.
  • polypeptide of the present invention is of human origin.
  • polynucleotides (DNA or RNA) which encode such polypeptide.
  • a process for producing such polypeptide by recombinant techniques is provided.
  • antagonists to such polypeptides which may be used to inhibit the action of such polypeptides, for example, to inhibit transcription of undesired cells, e.g., malignancies.
  • Figure 1(A) depicts the cDNA sequence and corresponding deduced amino acid sequence of the small subunit of human TFIIA.
  • the small subunit polypeptide shown is the mature form of the polypeptide. Standard one-letter abbreviations for amino acids is used.
  • Figure 1(B) illustrates a comparison of the amino acid composition of the human TFIIA small subunit and the yeast (T0A2) TFIIA small subunit.
  • FIG. 1(C) is a schematic diagram depicting the human TFIIA subunits.
  • TFIIA is encoded by two genes, a ⁇ and 7.
  • the a ⁇ protein is processed post-translationally into two polypeptides ( ⁇ and ⁇ ) , approximately 35 and 19 kDa, respectively.
  • Recombinant a and ⁇ polypeptides were designed with a breakpoint at amino acid residue 251, but this may not exactly correspond to the naturally occurring proteolytic cleavage site.
  • Figure 2 illustrates the functional activity of recombinant human, yeast and heterologous TFIIA.
  • Figure 2(A) illustrates the formation of a D-A complex (resulting from the addition of TFIIA small subunit to TBP) bound to DNA detected by polyacrylamide gel EMSA (electrophoresis mobility shift assay) .
  • EMSA electrosis mobility shift assay
  • Partially purified human TFIIA (hllA, lanes 3,4), combinations of recombinant human subunits ( a ⁇ , a , ⁇ , y , lanes 6-16), recombinant yeast a ⁇ with yeast y ( a ⁇ /yy ,lanes 19,20) are indicated above each lane.
  • FIG. 2(B) illustrates the requirement of TFIIA activity in recon ⁇ titution of transcriptional activation by the Epstein-Barr virus encoded activator, Zta transcriptional activator. Transcription reactions were reconstituted with immunoaffinity purified TFIID, recombinant TFIIB, partially purified RNA polymerase II, TFIIE, TFIIF, and USA with (+) or without (-) Zta. Various TFIIA preparations were added to reactions as indicated above each lane. Arrow at the left indicates the correctly initiated transcript.
  • FIG. 3(A) illustrates the interaction of TFIIA subunits with TBP.
  • 3S S-labelled TFIIA (lanes 1-3), a ⁇ (lanes 4-6), a ⁇ +y (lanes 7-9) or T3 luciferase control (lanes 10-12) proteins were incubated with GST (lanes 2, 5, 8, 11,) or GST-TBP (lanes 3, 6, 9, 12) immobilized on glutathione sepharose beads, as indicated above each lane.
  • Lanes marked input (lanes 1, 4, 7, 10) represent approximately 2.5% of the reaction input.
  • Figure 3(B) illustrates that the interaction of TFIIA subunits reveals a strong homotypic binding of TFIIA 7.
  • TFIIA 7 (lanes 1-4), a ⁇ (lanes 5-8), TBP (lanes 9- 12) or T3 luciferase (lanes 13-16) were incubated with GST (lanes 2, 6, 10, 14), GST- ⁇ 3 (lanes 3, 7, 11, 15), or GST-7 (lanes 4, 8, 12, 16) fixed to gluthathione sepharo ⁇ e beads.
  • Figure 4(A) illustrates that recombinant TFIIA restores the ability of three distinct activation domains to function in TFIIA depleted nuclear extracts.
  • the transcriptional activator proteins Zta (lanes 2, 4, 6, 7, 8, 9), GAL4-AH (lanes 10, 12, 14) or VP16 (lanes 16, 18, 20) were incubated with untreated HeLa cell nuclear extract (lane ⁇ 1, 2, 9, 10, 15, 16) or with TFIIA depleted HeLa cell nuclear extracts (lanes 3-9, 11-14, 17-20) in in vitro transcription reactions.
  • TFIIA ⁇ S+7 50 ng of recombinant TFIIA ⁇ S+7 (lane ⁇ 5, 6, 13, 14, 19, 20), TFIIA a ⁇ (lane 7), or TFIIA 7 (lane 8) was supplemented to depleted extracts.
  • Correctly initiated primer extension products for Zta and GAL4 templates are indicated by the arrows at the left and right, respectively.
  • Figure 4(B) illustrates the requirement for TFIIA in the reconstitution of transcriptional activation by an acidic activator with partially purified general transcription factors. Transcription reactions were essentially the same as those described for Fig. 2(B), except that the GAL-AH activator and the G 5 E1BTCAT template were used.
  • Figure 4(C) shows that recombinant TFIIA promotes an activator and TAF dependent TFIID promoter complex.
  • Mg agarose gel EMSA of DNA binding reactions with immunopurified TFIID (lanes 3-12), Zta (even lane ⁇ , and 13), and a 250 bp probe derived from the Z7E4TCAT promoter.
  • Zta (20 ng), recombinant TFIIA (50 ng), and 0.1 footprinting unit of TFIID were incubated with approximately 1 fmole of radiolabelled promoter DNA for 15 minute ⁇ at room temperature.
  • Sequencing inaccuracies are a common problem when attempting to determine polynucleotide sequences. Accordingly, the sequence of Figure 1A is based on several sequencing runs and the sequencing accuracy is considered to be at least 97%.
  • nucleic acid which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1A or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Depo ⁇ it No. 75809 on June 10, 1994.
  • the polynucleotide of this invention was discovered in a T-cell library. It is structurally related to the small subunit of yeast (T0A2). It contains an open reading frame encoding a protein of 109 amino acid residues. The protein exhibits the highest degree of homology to yeast TOA2 with 40% identity and 50% similarity over the entire amino acid stretch.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1A or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1A or the deposited cDNA.
  • the polynucleotide which encode ⁇ for the mature polypeptide of Figure 1A or for the mature polypeptide encoded by the deposited cDNA includes only the coding sequence for the mature polypeptide since the polypeptide is a nuclear protein which is not excreted to the outside of the cell.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1A or the polypeptide encoded by the cDNA of the deposited clone.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1A or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variant ⁇ of ⁇ uch polynucleotides which variant ⁇ encode for a fragment, derivative or analog of the polypeptide of Figure 1A or the polypeptide encoded by the cDNA of the deposited clone.
  • nucleotide variant ⁇ include deletion variant ⁇ , ⁇ ub ⁇ titution variants and addition or insertion variants.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1A or of the coding sequence of the deposited clone.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which doe ⁇ not ⁇ ub ⁇ tantially alter the function of the encoded polypeptide.
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the ⁇ equence ⁇ .
  • the pre ⁇ ent invention particularly relate ⁇ to polynucleotide ⁇ which hybridize under ⁇ tringent condition ⁇ to the hereinabove-described polynucleotides .
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain sub ⁇ tantially the ⁇ ame biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1A or the depo ⁇ ited cDNA.
  • the present invention further relates to the small subunit of TFIIA polypeptide which has the deduced amino acid sequence of Figure 1A or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivative ⁇ of ⁇ uch polypeptide.
  • fragment when referring to the polypeptide of Figure 1A or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figure 1A or that encoded by the depo ⁇ ited cDNA may be (i) one in which one or more of the amino acid re ⁇ idues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid re ⁇ idue ⁇ include ⁇ a ⁇ ubstituent group, or (iii) one in which the mature polypeptide i ⁇ fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, which are employed for purification of the mature polypeptide.
  • Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptide ⁇ could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the present invention also relate ⁇ to vector ⁇ which include polynucleotides of the present invention, host cells which are genetically engineered with vector ⁇ of the invention and the production of polypeptide ⁇ of the invention by recombinant technique ⁇ .
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vector ⁇ of thi ⁇ invention which may be, for example, a cloning vector or an expre ⁇ ion vector.
  • the vector may be, for example, in the form of a pla ⁇ mid, a viral particle, a phage, etc.
  • the engineered ho ⁇ t cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformant ⁇ or amplifying the TFIIA gene ⁇ .
  • the culture condition ⁇ , ⁇ uch a ⁇ temperature, pH and the like, are those previously used with the host cell selected for expres ⁇ ion, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing polypeptide ⁇ by recombinant technique ⁇ .
  • the polynucleotide may be included in any one of a variety of expression vector ⁇ for expre ⁇ sing a polypeptide.
  • vector ⁇ include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vector ⁇ derived from combinations of plasmids and phage DNA, viral DNA such a ⁇ vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used a ⁇ long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease ⁇ ite( ⁇ ) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expres ⁇ ion vector is operatively linked to an appropriate expression control sequence( ⁇ ) (promoter) to direct RNA ⁇ ynthe ⁇ is.
  • ⁇ promoter
  • ⁇ uch promoter ⁇ there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P L promoter and other promoter ⁇ known to control expre ⁇ sion of genes in prokaryotic or eukaryotic cells or their viru ⁇ e ⁇ .
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more ⁇ electable marker gene ⁇ to provide a phenotypic trait for selection of transformed host cells such a ⁇ dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin re ⁇ i ⁇ tance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate ho ⁇ t to permit the host to express the protein.
  • bacterial cells such as E. coli. Streptomyces. Salmonella typhimuriu : fungal cells, such as yeast; insect cells such as Drosophila and Sf9 animal cells such a ⁇ CHO, COS or Bowe ⁇ melanoma; plant cells, etc.
  • yeast fungal cells
  • insect cells such as Drosophila and Sf9 animal cells
  • a ⁇ CHO, COS or Bowe ⁇ melanoma plant cells, etc.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plas id or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequence ⁇ , including, for example, a promoter, operably linked to the ⁇ equence.
  • regulatory sequence ⁇ including, for example, a promoter, operably linked to the ⁇ equence.
  • Large number ⁇ of ⁇ uitable vector ⁇ and promoters are known to those of skill in the art, and are commercially available.
  • the following vector ⁇ are provided by way of example.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLNEO, PSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other pla ⁇ mid or vector may be used as long as they are replicable and viable in the ho ⁇ t.
  • Promoter regions can be ⁇ elected from any de ⁇ ired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are PKK232-8 and PCM7.
  • Particular named bacterial promoter ⁇ include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviru ⁇ , and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the ho ⁇ t cell can be a prokaryotic cell, such a ⁇ a bacterial cell.
  • Introduction of the con ⁇ truct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L. , Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide ⁇ ynthe ⁇ izer ⁇ .
  • Mature protein ⁇ can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA construct ⁇ of the pre ⁇ ent invention. Appropriate cloning and expression vector ⁇ for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al.. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancer ⁇ .
  • recombinant expre ⁇ ion vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin re ⁇ i ⁇ tance gene of E. coli and S. cerevi ⁇ iae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such a ⁇ 3-pho ⁇ phoglycerate kinase (PGK), ⁇ -factor, acid phosphatase, or heat shock proteins, among others.
  • the heterologous structural sequence is a ⁇ sembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with ⁇ uitable tran ⁇ lation initiation and termination ⁇ ignal ⁇ in operable reading pha ⁇ e with a functional promoter.
  • the vector will compri ⁇ e one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various specie ⁇ within the genera Pseudomonas, Streptomyce ⁇ , and Staphylococcu ⁇ , although others may also be employed as a matter of choice.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • cloning vector pBR322 ATCC 37017
  • Such commercial vector ⁇ include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expres ⁇ ed.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, di ⁇ rupted by phy ⁇ ical or chemical mean ⁇ , and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be di ⁇ rupted by any convenient method, including freeze-thaw cycling, sonication, mechanical di ⁇ ruption, or u ⁇ e of cell lysing agent ⁇ , ⁇ uch method ⁇ are well know to tho ⁇ e skilled in the art.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibrobla ⁇ t ⁇ , de ⁇ cribed by Gluzman, Cell, 23:175 (1981), and other cell line ⁇ capable of expre ⁇ ing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell line ⁇ .
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the TFIIA small subunit polypeptide ⁇ can be recovered and purified from recombinant cell culture ⁇ by method ⁇ including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic ho ⁇ t (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
  • a prokaryotic or eukaryotic ho ⁇ t for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • TFIIA activity was first assayed for the stabilization of TBP binding to a TATA box containing oligonucleotide probe in polyacrylamide gel EMSA (Fig. 2A) .
  • yeast TBP does not form a ⁇ table complex with a TATA box containing oligonucleotide (lane 2) .
  • Addition of partially purified human TFIIA (hllA) to TBP re ⁇ ulted in the formation of a stable complex (D-A) (lane 4).
  • Recombinant a ⁇ +y also produced a stable D-A complex (lane 6) .
  • TBP failed to form the stable D-A complex when the a ⁇ or 7 subunit was added individually (lanes 8 and 10).
  • the a ⁇ protein was expressed as two independent subunits (Fig. 1C).
  • the a+y had no effect (lane 12), while ⁇ +y had a small effect (lane 14) on TBP binding.
  • the combination of ⁇ , ⁇ and 7 subunits ( a ⁇ +y ) re ⁇ ulted in strong stimulation of TBP binding, while electrophoretic mobility was very similar to the native human TFIIA protein (lane 16).
  • the yeast 7 subunit could also form a stable D-A complex when mixed with the human a ⁇ subunit, demonstrating the interaction between the a ⁇ and 7 subunits is evolutionarily conserved (lane 20).
  • the electrophoretic mobility of the D-A complex was influenced by the different 0/87 forms of TFIIA, ⁇ uggesting that TFIIA is retained in the bound complexes.
  • the various TFIIA complexes were analyzed for their ability to support transcriptional activation in reactions reconstituted with partially purified general transcription factors, the coactivator USA 25 , and the Epstein-Barr virus encoded activator, Zta 6 .
  • the 7 subunit was essential for transcriptional activation, and the a and ⁇ subunit could be ⁇ upplemented as either a single a ⁇ polypeptide, or as two distinct polypeptides ( a+ ⁇ ) (Fig. 2B) .
  • the formation of the D-A complex in EMSA correlated with the ability of TFIIA to support transcriptional activation by Zta.
  • 3+7 proteins were tested for their ability to interact with glutathione-S-transferase (GST) or GST-TBP fusion protein ⁇ immobilized on glutathione agaro ⁇ e (Fig. 3A) .
  • GST-TBP fusion protein ⁇ immobilized on glutathione agaro ⁇ e (Fig. 3A) .
  • the combination of the a ⁇ +y subunits markedly increased the binding to GST-TBP (lane 9) .
  • the ability of radiolabelled TBP to bind to GST-7 and GST- ⁇ 3 was also examined (Fig. 3B).
  • TBP bound to GST- ⁇ 3 protein but failed to interact with the GST-7 protein (Fig. 3B, lanes 11 and 12).
  • the discrepancy of the binding of 7 to TBP may be a partial result of the steric hindrance of GST fused to the amino terminus of 7.
  • both a ⁇ and 7 are capable of making direct contact with TBP, the heterodimer clearly binds with higher affinity.
  • the interaction of 7 with the a ⁇ polypeptides was also examined by the GST-fusion protein binding assay. Radiolabelled 7, a ⁇ , or T3 control were incubated with GST-7, GST-a ⁇ , or GST alone (Fig. 3B). As expected, the a ⁇ subunit bound to GST-7 and the 7 subunit bound to GST- ⁇ /3.
  • the 7 subunit also bound strongly to GST-7, while a ⁇ did not bind GST- ⁇ 3, suggesting that a homotypic association of the 7 subunit contributes to the oligomerization state of TFIIA.
  • the T3 control protein did not interact with any of the GST proteins tested.
  • TFIIA depleted HeLa cell nuclear extracts were prepared by serial passage over nickel agarose, which bind ⁇ specifically to the a ⁇ subunit of TFIIA. 7,2 ° Addition of Zta to the depleted extract failed to produce significant transcription levels (Fig. 3A, lane 4).
  • TFIIA a ⁇ +y subunits restored activation of the depleted extracts to levels observed in the undepleted extract (compare lane ⁇ 2 and 6) .
  • the a ⁇ or 7 subunit alone failed to restore Zta activation in these depleted extracts indicating that both subunits were equally depleted by the nickel agarose.
  • GAL4-AH did not function in the TFIIA depleted extracts.
  • Addition of recombinant a ⁇ +y subunits restored activator dependent transcription for all three distinct activators.
  • GAL4-AH was also shown to require TFIIA in reconstituted transcription assays (Fig.
  • the TFIIA small ⁇ ubunit polypeptide may be u ⁇ ed to prevent inhibitors of TFIID from repressing transcription, this i ⁇ useful where a particular gene product is desired and is not being produced at the desired levels due to the inhibition of the TFIID complex.
  • the TFIIA small subunit polypeptide may be used to regulate transcription globally or in a gene- specific manner, to obtain desired concentrations of particular proteins.
  • the TFIIA small subunit may be repressed to prevent transcription and in the case where a protein is desired, for example growth hormone, the TFIIA small ⁇ ubunit may enhance transcription and the production of the gene product.
  • the polypeptide of the present invention is also useful for identifying other molecules which have similar biological activity.
  • Labeled oligonucleotide ⁇ having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • This invention provide ⁇ a method of ⁇ creening compound ⁇ to identify those which enhance (agonist ⁇ ) or block (antagoni ⁇ t ⁇ ) interaction of the small subunit of TFIIA with TFIID.
  • An agonist is a compound which increases the natural biological function of the small subunit of TFIIA, while antagonists eliminate such functions.
  • a ⁇ an example, purified RNA polymerase, a template carrying a promoter, nucleo ⁇ ide triphosphate ⁇ and appropriate buffer and ⁇ alt ⁇ may be mixed with TFIIA and TFIID in the pre ⁇ ence of the compound under condition ⁇ where transcription would normally take place. The ability of the compound to enhance or block the binding of TFIIA to the template DNA could then be determined by measuring the level of transcription product.
  • the a ⁇ ay may be a cell-ba ⁇ ed a ⁇ ay wherein a TFIIA-inducible promoter drives the expression of a marker gene. TFIIA and the compound to be screened would then be added to mea ⁇ ure the level of production of the marker gene. Additionally, thi ⁇ cell-based as ⁇ ay could be u ⁇ ed in tandem with the binding assay to determine if the effects on transcription are specific to TFIIA agonism or antagonism.
  • Potential antagonists include an antibody, or in some cases, an oligonucleotide, which binds to the small subunit of TFIIA.
  • a potential antagoni ⁇ t may be a closely related protein which binds to the TBP protein of the TFIID complex but do not initiate transcription.
  • An example of such a closely related protein is a negative dominant mutant, wherein one the two subunits of TFIIA are mutated and do not retain function. The negative dominant mutant, however, still recognizes substrate but doe ⁇ —not initiate transcription.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucl.
  • the antisen ⁇ e RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the TFIIA small ⁇ ubunit (anti ⁇ en ⁇ e - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expres ⁇ ion, CRC Press, Boca Raton, FL (1988)).
  • the oligonucleotides de ⁇ cribed above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the small subunit of TFIIA.
  • Potential antagonists include a small molecule which binds to and occupies the active site of the polypeptide such that TFIIA is unable to activate TFIID and initiate transcription.
  • small molecules include but are not limited to small peptides or peptide-like molecules.
  • the antagonists may be employed to inhibit the transcription of undesired polypeptides.
  • the antagonists mentioned above may be used to prevent transcription of that polypeptide.
  • An example of this is the transcription and differentiation of cancerous cells.
  • the antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
  • compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provide ⁇ a pharmaceutical pack or kit compri ⁇ ing one or more container ⁇ filled with one or more of the ingredient ⁇ of the pharmaceutical compositions of the invention.
  • Associated with such container(s) can be a notice in the form pre ⁇ cribed by a governmental agency regulating the manufacture, u ⁇ e or sale of pharmaceuticals or biological product ⁇ , which notice reflect ⁇ approval by the agency of manufacture, use or sale for human administration.
  • the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.
  • the pharmaceutical composition ⁇ may be administered in a convenient manner such as by the topical, intravenou ⁇ , intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes.
  • the pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at lea ⁇ t about 10 ⁇ g/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage i ⁇ from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
  • TFIIA and agonists and antagonists which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo , which is often referred to as "gene therapy. "
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cell ⁇ then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • cell ⁇ may be engineered by procedure ⁇ known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the pre ⁇ ent invention may be administered to a patient for engineering cell ⁇ in vivo and expres ⁇ ion of the polypeptide in vivo .
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenoviru ⁇ which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • Fragments of the full length TFIIA small subunit gene may be used a ⁇ a hybridization probe for a cDNA library to isolate the full length TFIIA small subunit gene and to isolate other genes which have a high sequence similarity to the gene.
  • Probes of this type can be, for example, 30, 40, 50 75, 90, 100 or 150 bases. Preferably, however, the probes have between 30 and 50 base pairs.
  • the probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exon ⁇ , and introns.
  • the probe may be labelled, for example, by radioactivity to facilitate identification of hypbridization.
  • sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNA ⁇ to chromo ⁇ ome ⁇ according to the present invention is an important first step in correlating those sequences with genes associated with disea ⁇ e.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
  • Computer analy ⁇ i ⁇ of the cDNA i ⁇ used to rapidly ⁇ elect primers that do not ⁇ pan more than one exon in the genomic DNA, thus complicating the amplification process.
  • These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clone ⁇ in an analogou ⁇ manner.
  • Other mapping strategies that can similarly be u ⁇ ed to map to it ⁇ chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with cDNA as short as 500 or 600 bases; however, clone ⁇ larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp i ⁇ good, 4,000 is better, and more than 4,000 i ⁇ probably not necessary to get good results a reasonable percentage of the time.
  • the phy ⁇ ical po ⁇ ition of the ⁇ equence on the chromo ⁇ ome can be correlated with genetic map data.
  • genetic map data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) .
  • linkage analysi ⁇ coinheritance of physically adjacent gene ⁇ .
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative gene ⁇ . (Thi ⁇ assumes 1 megabase mapping resolution and one gene per 20 kb).
  • the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used a ⁇ an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated again ⁇ t the polypeptide ⁇ corre ⁇ ponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodie ⁇ (Cole, et al., 1985, in Monoclonal Antibodie ⁇ and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • “Plas id ⁇ ” are designated by a lower case p preceded and/or followed by capital letter ⁇ and/or number ⁇ .
  • the ⁇ tarting pla ⁇ mids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain ⁇ equence ⁇ in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were u ⁇ ed a ⁇ would be known to the ordinarily skilled artisan.
  • For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment i ⁇ used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotide ⁇ have no 5' pho ⁇ phate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A ⁇ ynthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligaation refer ⁇ to the process of forming phosphodie ⁇ ter bond ⁇ between two double ⁇ tranded nucleic acid fragment ⁇ (Maniati ⁇ , T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("liga ⁇ e") per 0.5 ⁇ g of approximately equimolar amount ⁇ of the DNA fragment ⁇ to be ligated.
  • liga ⁇ e T4 DNA ligase
  • the DNA sequence encoding for the small TFIIA subunit, ATCC # 75809, is initially amplified using PCR oligonucleotide primers corresponding to the 5' and sequences of the processed TFIIA subunit protein and the vector sequences 3' to the small subunit of TFIIA gene. Additional nucleotides corresponding to the small subunit of TFIIA were added to the 5' and 3' sequences respectively.
  • the 5' oligonucleotide primer has the ⁇ equence 5'GCGGCGGATCCATGGCATATCAGGTATAC3' contain ⁇ a Bam HI restriction enzyme site followed by 18 nucleotides of the small subunit of TFIIA (underlined) coding sequence starting from the presumed terminal amino acid of the processed protein codon .
  • the 3 ' s equence 5'GCGGCAAGCTTATTCTGTAGTATTGG3' contains complementary sequences to HindiII site and is followed by 13 nucleotides of the small subunit of TFIIA (underlined).
  • the restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc.
  • pQE-9 encode ⁇ antibiotic re ⁇ i ⁇ tance (Amp r ), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/0), a ribo ⁇ ome binding site (RBS), a 6-Hi ⁇ tag and re ⁇ triction enzyme ⁇ ite ⁇ .
  • pQE-9 was then digested with Bam HI and Hind III. The amplified sequences were ligated into pQE-9 and were inserted in frame with 6 His re ⁇ idue ⁇ fused to the amino terminus. The ligation mixture was then used to transform E.
  • M15/rep4 contains multiple copies of the plasmid pREP4, which expres ⁇ e ⁇ the lad repres ⁇ or and al ⁇ o confers kanamycin resi ⁇ tance (Kan r ). Tran ⁇ formant ⁇ are identified by their ability to grow on LB plate ⁇ and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysi ⁇ .
  • Clone ⁇ containing the desired constructs were grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ l).
  • the 0/N culture i ⁇ u ⁇ ed to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cells were grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG "Isopropyl-B-D- thiogalacto pyranoside" was then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expres ⁇ ion.
  • TFIIA solubilized TFIIA subunits was purified from this solution by chromatography on a Nickel-Agarose column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)).
  • the small subunit of TFIIA (95% pure) was eluted from the column in 6 molar guanidine HCl pH 5.0 and were allowed to renature by themselves of in stoichiometric combination with the specified protein (see Fig. 2).
  • Gel Mobility Shift a ⁇ ays were used to separate the transcription products as they appear in the Figure.
  • Bacterial extracts of GST or GST fusion proteins were incubated with glutathione sepharo ⁇ e-4B beads (6-9 ⁇ g of GST- fusion protein/20 ⁇ l of beads) with shaking at 4°C. After 2 hours the bead ⁇ were wa ⁇ hed with 50 column volume ⁇ of cold buffer A (20 mM NaH 2 P0 4 (pH 7.0) 150 mM NaCl, 1 mM DTT, 1 mM PMSF). Wa ⁇ hed bead ⁇ (20 ⁇ l) were then incubated with reticulocyte lysate ⁇ containing 2x10 4 cpm of 35 S labelled protein in 300 ⁇ l of protein binding buffer (PBB) for 1 hour at room temperature.
  • PBB protein binding buffer
  • PBB contained 20 mM Hepe ⁇ (pH 7.9), 20% glycerol, 0.5 mM EDTA, 60 mM KC1, 5 mM MgC12, 0.1% NP40, and 5 mM /3-mercaptoethanol.
  • the bead ⁇ were subsequently washed 4 times in PBB and labeled proteins were eluted with 1M KC1.
  • Samples were analyzed on 15% SDS polyacrylamide gels, enhanced with NaSalycilate, and visualized by autoradiography.
  • Example 3 Transcription reactions utilizing TFIIA
  • Transcription reactions contained 100 ng of the Z7E4TCAT 29 or G5E1BTCAT 26 template, approximately 200 ng of activator protein, and 40 ⁇ g of nuclear extract in a 50 ⁇ l final reaction volume.
  • TFIIA depleted nuclear extracts were prepared by dialyzing HeLa cell nuclear extract in buffer D in 20 mM Hepes (pH 7.9), 20% glycerol, 5 mM ⁇ - mercaptoethanol, 1 mM PMSF, containing 500 mM KCl, followed by two sequential incubations with Nickel agarose bead ⁇ (150 ul packed bead ⁇ /1 mg of nuclear extract) for 20 minute ⁇ at 4°C rotating. Depleted extract ⁇ were dialyzed into D buffer containing 100 mM KCl. The recon ⁇ tituted tran ⁇ cription reactions and the Mg agarose EMSA were described previously 6 .
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • a ⁇ p Ly ⁇ Val Ly ⁇ lie Val Ala Cy ⁇ A ⁇ p Gly Ly ⁇ A ⁇ n Thr Gly Ser

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Abstract

L'invention concerne la petite sous-unité (η) du facteur de transcription IIA humain et l'ADN (ARN) codant ce polypeptide et procédé de production d'un tel polypeptide par des techniques de recombinaison. L'invention concerne également des procédés d'utilisation de ce polypeptide pour réguler la transcription globale et spécifique aux gènes. Les antagonistes contre ce polypeptide et leur utilisation pour empêcher la transcription sont également décrits.
EP94931757A 1994-09-20 1994-09-20 Facteur iia de transcription humain Withdrawn EP0783509A4 (fr)

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Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
BERNSTEIN, RICHARD ET AL: "Characterization of the highly conserved TFIIA small subunit from Drosophila melanogaster." JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 269, NO. 39, PP. 24361-24366 ISSN: 0021-9258.,30 September 1994, XP002092352 *
COULOMBE, BENOIT ET AL: "Topological Localization of the Human Transcription Factors IIA, IIB, TATA Box-binding Protein, and RNA Polymerase II-associated Protein 30 on a Class II Promoter." JOURNAL OF BIOLOGICAL CHEMISTRY, (1994) VOL. 269, NO. 31, PP. 19962-19967 ISSN: 0021-9258., XP002092347 *
J. DEJONG ET AL.: "A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA" GENES & DEVELOPMENT, vol. 7, no. 11, November 1993, pages 2220-2234, XP002092348 *
J.A. RANISH ET AL.: "The yeast general transcription factor TFIIA is composed of two polypeptide subunits" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 266, no. 29, 15 October 1991, pages 19320-19327, XP002092354 *
K. YOKOMORI ET AL.: "Drosophila TFIIA-L is processed into two subunits that are associated with the TBP/TAF complex" GENES & DEVELOPMENT, vol. 7, no. 11, November 1993, pages 2235-2245, XP002092355 *
MA, DONGMIN ET AL: "Isolation of a cDNA encoding the largest subunit of TFIIA reveals functions important for activated transcription." GENES & DEVELOPMENT, (1993) VOL. 7, NO. 11, PP. 2246-2257. ISSN: 0890-9369., XP002092345 *
OZER, JOSEF ET AL: "Molecular cloning of the small ( gamma ) subunit of human TFIIA reveals functions critical for activated transcription." GENES & DEVELOPMENT, VOL. 8, NO. 19, PP. 2324-2335. ISSN: 0890-9369., 1 October 1994, XP002092351 *
P. CORTES ET AL.: "Factors involved in specific transcription by mammalian RNA polymerase II: Purification and analysis of transcription Factor IIA and identification of transcription factor IIJ" MOLECULAR AND CELLULAR BIOLOGY, vol. 12, January 1992, pages 413-421, XP002093031 *
RANISH, JEFFREY A. ET AL: "Isolation of two genes that encode subunits of the yeast transcription factor IIA" SCIENCE (WASHINGTON, D. C., 1883-) (1992), 255(5048), 1127-9 CODEN: SCIEAS;ISSN: 0036-8075,28 February 1992, XP002092346 *
S. HAHN ET AL.: "Identification of a yeast protein homologous in function to the mammalian gneral transcription factor, TFIIA" THE EMBO JOURNAL , vol. 8, no. 11, 1989, pages 3379-3382, XP002092353 *
See also references of WO9609311A1 *
SUN, XIAOQING ET AL: "Reconstitution of human TFIIA activity from recombinant polypeptides: A role in TFIID-mediated transcription." GENES & DEVELOPMENT, (1994) VOL. 8, NO. 19, PP. 2336-2348. ISSN: 0890-9369., XP002092349 *
YOKOMORI, KYOKO (1) ET AL: "Drosophila TFIIA directs cooperative DNA binding with TBP and mediates transcriptional activation." GENES & DEVELOPMENT, VOL. 8, NO. 19, PP. 2313-2323. ISSN: 0890-9369., 1 October 1994, XP002092350 *

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