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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts

Definitions

• This invention relates to methods for screening and identifying inhibitors of promoters or inhibitors of transcriptional activation by use of a collision construct.
• the collision construct provides for increased expression of reporter gene signal in the presence of an appropriate inhibitor.
• This invention thus, relates to the collision construct as well as methods for making and producing the collision construct, and to vectors, host cells and kits containing the collision construct.
• Structural genes have a transcription regulatory region that contains one or more sequences, referred to herein as response elements, that are capable of binding to certain proteins, referred to herein as binding proteins, that activate or repress transcription or facilitate elongation of a mRNA transcript.
• binding proteins include transcription factors, such as those described in Faisst & Meyer (1992), Nucleic Acids Res. 20. 3-26; THE
• a transcription factor such as an activator
• Transcription activators may also contain another domain that interacts with other transcription factors to initiate transcription or to allow elongation of the RNA transcript.
• a molecule that inhibits binding of an activator to a response element either by competitively binding to the DNA-binding domain of the activator or to the response element, or a molecule that blocks the transcription factor-interacting domain of the activator, would be expected to inhibit transcriptional activation.
• Repressors also mostly proteins, function in a similar manner, except that instead of activating transcription, the repressor binds to the response element, for example, an operator, and blocks transcription.
• Molecules that are capable of competitively binding to repressors activate transcription by removing the repressor from the response element.
• an inhibitor to the molecule that competitively binds to the repressor would be expected also to inhibit transcriptional activation.
• a decrease in biological function when researchers look for an inhibitor of a biological function, they look for a decrease in biological function or a decrease in a reporter signal that reflects inhibition of that function.
• a decrease in signal is difficult to inte ⁇ ret because the decrease may be the result of factors other than the presence of the supposed inhibitor being tested.
• the decrease in signal may be caused by the presence of extraneous matter including toxic chemicals in the media, inappropriate incubation temperature, inappropriate incubation time, poor condition of the cells used in the test, etc.
• a number of time- consuming experiments have to be run with a number of controls.
• an object of the present invention to provide a screening test for inhibitors that is capable of generating an increase in reporter signal in the presence of an inhibitor. It is also an object of the present invention to provide for materials that can be used in such a screening test.
• a construct termed a collision construct, that contains a nucleic acid molecule, comprising a first regulatory sequence that comprises a first promoter, a reporter gene that is under transcriptional control of the first promoter, where the reporter gene is capable of providing a detectable signal upon transcription and translation thereof, and a second regulatory sequence that comprises a second promoter, where the direction of transcription under the first promoter is opposite to the direction of transcription under the second promoter, where regulation of transcription under the second promoter alters the reporter gene signal, and where the first promoter is different from the second promoter.
• the collision construct as above where the second promoter or the second regulatory sequence comprises a first response element that is capable of binding to a first binding protein to form a first binding pair, and the formation of the first binding pair regulates the activity of the second promoter.
• the collision construct as above where the last nucleotide of the stop codon of the reporter gene is separated from the 3' terminus of the second promoter by a distance of about less than about 2050 nucleotides.
• the collision construct as above where one or both of the first promoter and second promoter are derived from a promoter or promoter/enhancer region of a gene selected from the group consisting of: a viral gene, a bacteriophage gene, a prokaryotic gene, and an eukaryotic gene.
• the eukaryotic gene can be a yeast or other fungal gene, an avian gene, an insect gene or a mammalian gene.
• the promoter or promoter/enhancer may be synthetically made, or partly derived and partly synthesized.
• the collision construct as above where one or both of the first response element and the second response element, the latter optionally present in the first regulatory region, are derived from the regulatory sequence of a gene selected from the group consisting of: a viral gene, a bacteriophage gene, a prokaryotic gene, and an eukaryotic gene.
• the eukaryotic gene can be, for example, a yeast or other fungal gene, an insect gene, an avian gene, or a mammalian gene.
• a method of using the collision construct as above for screening or identifying a candidate inhibitor for its ability to inhibit transcription under a target promoter comprising the steps of providing a cell that contains the collision construct, where the second promoter in the construct is the target promoter and the cell is capable of expressing the collision construct to produce a reporter gene signal, determining reporter gene signal in the absence and presence of the candidate inhibitor, respectively, and comparing reporter gene signals obtained.
• An appropriate inhibitor is one that is capable of generating an increased reporter signal in the presence of an inhibitor.
• the second promoter or the second regulatory region in the collision construct comprises a response element that is capable of binding to a binding protein.
• the binding protein can be provided by coexpression in a cell of the collision construct and a vector that comprises a coding sequence for binding protein.
• the binding protein can be provided by a cell that produces it constitutively and the collision construct is then introduced into the cell.
• the binding protein can be added directly to the cell that contains the collision construct.
• a method of making the collision construct by providing and linking together a first regulatory sequence that comprises a first promoter, a reporter gene that is capable of providing a detectable signal upon transcription and translation, a second regulatory sequence that comprises a second promoter, where the reporter gene is placed under regulatory control of the first promoter, the direction of transcription under the first promoter is opposite the direction of transcription of the second promoter, and the first promoter is different from the second promoter.
• a method of production of the collision construct by culturing a host cell that comprises the collision construct, for example, a prokaryotic or eukaryotic host cell, for example, a bacterial or yeast cell.
• a kit that comprises the collision construct as above, or a vector or host cell containing the collision construct, with instructions for use thereof in accordance with the method described above.
• FIG. 1 is a schematic representation of a collision construct, transformed in HeLa cells, containing the CMV promoter and the HIV-1 promoter running in opposite directions and another construct for control in which the HIV-1 promoter was absent.
• the graph depicts Tat-dependent inhibition of CMV promoter activity over a range of Tat levels, in micrograms of Tat expression vector, from about 0 to 2.
• the symbol (•) represents the alkaline phosphatase gene expression in the collision construct.
• the symbol (D) represents alkaline phosphatase gene expression in the control construct showing nonspecific reduction of CMV promoter activity by Tat protein.
• Other abbreviations include AP, representing alkaline phosphatase; and CMV, representing the cytomegalovirus promoter/enhancer.
• FIG. 2 is a schematic representation of five different collision constructs, designated as #1152, #1161, #1225, #1162, and #1163, and the reporter gene signals in percent alkaline phosphatase activity, generated by expression of 1 ⁇ g DNA each, respectively, and determined in the presence of 0.5 ⁇ g of Tat protein expression plasmid (" +Tat") or 0.5 ⁇ g inactive Tat protein expression plasmid ("-Tat”) in HeLa cells.
• FIG. 2 shows that specific reduction of alkaline phosphatase expression in the presence of HIV-1 Tat protein is dependent on a functional TAR sequence in the LTR.
• the collision construct #1152 was used, in the presence of inactive Tat plasmid, reporter gene signal was high.
• FIG. 3 is a schematic representation of five different reporter constructs, designated as #1085, #1166, #1213, #1167, and #1168, and the reporter gene signals, indicated as percent alkaline phosphatase activity, generated by expression of 1 ⁇ g of each, respectively, in the presence of 0.5 ⁇ g Tat protein expression plasmid (" +Tat") or 0.5 ⁇ g inactive Tat protein expression plasmid ("-Tat”) in HeLa cells.
• the alkaline phos ⁇ hata-.e activity measured wilh construct #1085 in the presence of active Tat protein expression plasmid was set to 100% .
• FIG. 3 illustrates that HIV-1 promoter activation by Tat protein requires the presence of the TAR sequence and that TAR may possibly have a silencer function because when a portion of TAR was deleted, reporter gene signal in the absence of Tat increased as compared to that when the complete TAR sequence was present.
• Addition of Tat to cells containing this construct, #1166 which does not allow the formation of a TAR stem loop structure, enhanced reporter gene signal by about 2.5-fold. Additional deletion of the promoter region to include the TATA box and Spl binding sites resulted in complete loss of reporter gene signal, in the absence or presence of Tat.
• FIG. 4 is a schematic representation of different collision constructs containing spacer regions of about 21, 94, 153, 406, 556 and 2047 nucleotides, positioned between the 3' end of the AP coding sequence, as defined by the stop codon TAA, or TGA in construct with a spacer of 21 nucleotides, and the end of the TAR sequence at nucleotide +59 in the HIV-1 promoter, with + 1 nucleotide as the start of transcription. The sequence of this junction is shown in FIG. 5.
• FIG. 5 shows an argument map of the DNA sequence of the 3' end of the AP gene and the 5' end of the mutant HIV-1 promoter in the collision construct #1152.
• the stop codon of the AP coding region is indicated.
• Start of transcription in the HIV- 1 promoter at position 263 is indicated with + 1.
• Nucleotide substitution st position 268 substitution of T to C
• position 271 substitution of C to A
• position 344 substitution of A to C
• FIG. 6 indicates that TAR decoys act as specific inhibitors of HIV-1 transcription.
• FIG 6a shows inhibition of HIV-1 transcription in the presence of TAR decoys.
• FIG. 6a a schematic diagram of an HIV-AP reporter gene construct and a plasmid expressing multimerized (8 copies) of transactivation response sequence, also called TAR decoys.
• the arrows indicate the direction of transcription and translation.
• the black boxes represent the TAR sequence.
• a schematic indicates that HeLa cells were transfected with various combinations of HIV-AP (l ⁇ g) reporter and TAR expression plasmids (0.5 ⁇ g) in the presence or absence of 0.5 ⁇ g of Tat expression vector.
• AP activity was determined as described previously and is expressed as fold activation relative to the level obtained with the HIV-AP plasmid in the absence of Tat (represented in lane 1).
• FIG 6b shows increased reporter gene expression in the collision construct by inhibition of HIV-1 promoter activity.
• FIG. 6b is a schematic representation of th collision construct and a plasmid expressing multimerized copies of the TAR sequence.
• HeLa cells were transfected with various combinations of the collision construct (l ⁇ g) and increasing amounts of a TAR expression plasmid (lanes 3 to 5, l ⁇ g and 2 ⁇ g respectively) in the absence or presence of 0.5 ⁇ g Tat expression plasmid.
• the total DNA concentration in each experiment was kept constant by adding a Tat expression vector containing a premature stop codon and a pBJ plasmid expressing an unrelated (leptin) gene.
• Each column represents the mean of at least three independent experiments. Error bars represent standard error from multiple transfections.
• nucleic acid molecule or “nucleic acid sequence” refers to either a DNA sequence, a RNA sequence, or complementary strands thereof that comprise a nucleotide sequence.
• a “regulatory sequence” refers to a nucleic acid sequence encoding one or more elements that are capable of affecting or effecting expression of a gene sequence, including transcription or translation thereof, when the gene sequence is placed in such a position as to subject it to the control thereof.
• a regulatory sequence can be, for example, a minimal promoter sequence, a complete promoter sequence, an enhancer sequence, an upstream activation sequence ("UAS"), an operator sequence, a downstream termination sequence, a polyadenylation sequence, an optimal 5' leader sequence to optimize initiation of translation, and a Shine-Dalgarno sequence.
• the regulatory sequence can contain a combination enhancer/promoter element.
• the regulatory sequence that is appropriate for expression of the present construct differs depending upon the host system in which the construct is to be expressed. Selection of the appropriate regulatory sequences for use herein is within the capability of one skilled in the art.
• a regulatory sequence in prokaryotes, can include one or more of a promoter sequence, a ribosomal binding site, and a transcription termination sequence.
• such a sequence can include one or more of a promoter sequence and/or a transcription termination sequence. If any necessary component of a regulatory sequence that is needed for expression is lacking in the collision construct, such a component can be supplied by a vector into which the collision construct can be inserted for transformation or reintroduction into a host cell.
• Regulatory sequences suitable for use herein may be derived from any source including a prokaryotic source, an eukaryotic source, a virus, a viral vector, a bacteriophage or a linear or circular plasmid.
• a regulatory sequence is the human immunodeficiency virus ("HIV-1") promoter that is located in the U3 and R region of the HIV-1 long terminal repeat (“LTR").
• the regulatory sequence herein can be a synthetic sequence, for example, one made by combining the UAS of one gene with the remainder of a requisite promoter from another gene, such as the GADP/ADH2 hybrid promoter.
• a “minimal promoter” is a naturally occurring promoter that has been weakened so that it is not 100% active.
• a promoter in which all but a TATA box has been deleted such as the minimal fos promoter, as described in Berkowitz et al. (1989) Mol. Cell. Biol. 5:4272-4281.
• a "reporter gene” refers to a nucleic acid molecule that encodes a polypeptide that is capable of providing a detectable signal either on its own upon transcription or translation or by reaction with another one or more reagents. Reporter genes suitable for use herein are conventional in the art, selection of which is within the capability of one skilled in the art.
• reporter genes include that encoding the enzyme chloramphenicol acetyltransferase ("CAT”), the luc gene from the firefly that encodes luciferase, the bacterial lacZ gene from Esche ⁇ chia coli that encodes ⁇ - galactosidase, alkaline phosphatase (“AP”), human growth hormone (“hGH”), the bacterial ⁇ -glucuronidase (“GUS”), and green fluorescent protein (“GFP”), as described in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1994), (Greene Publishing Associates and John Wiley & Sons, New York, N.Y.).
• CAT chloramphenicol acetyltransferase
• AP alkaline phosphatase
• hGH human growth hormone
• GUS bacterial ⁇ -glucuronidase
• GFP green fluorescent protein
• a “response element” refers to a region of a nucleic acid molecule, usually, from a regulatory region of a gene, that is capable of specifically binding to a binding protein, such as an activator molecule, for activation of transcription or for allowing the elongation of a RNA transcript, or a repressor molecule, for inhibition of transcription. Some response elements are known in the art. Selection of a response element that is suitable for use herein is within the capability of one skilled in the art.
• binding protein refers to a protein that is capable of specifically binding to a response element for regulation of transcription. Some binding proteins are known. Selection of a binding protein suitable for use herein is also within the capability of one skilled in the art. A number of DNA binding proteins as well as response elements of the transcription regulatory regions are described in Wingender (1988), Nucleic Acids Res. 16: 1879-1902; Molecular Cell Biology , J. Darnell, H. Lodish & D. Baltimore, (Scientific American Books, New York 1990); and Dhawale & Lane (1993), Nucleic Acids Res. 2i:5537-5546.
• Tat/TAR combination found in viruses such as human immunodeficiency virus-1 ("HIV-1”), human immunodeficiency virus-2 (“HIV-2”), and simian immunodeficiency virus (“SIV”).
• HIV-1 human immunodeficiency virus-1
• HAV-2 human immunodeficiency virus-2
• SIV simian immunodeficiency virus
• Tat is the binding protein referred to herein
• TAR tr ⁇ ns-activating response element
• TAR is the response element referred to herein, as described in Jones & Peterlin (1994), Ann. Rev. Biochem. 65:717-743; and Antoni et al. (1994), Adv. Virus Res. 45:53-145.
• response elements and binding proteins besides TAR and Tat include Rev response element ("RRE"), a NF- ⁇ B binding site, a Spl binding site, and Gal4 and LexA binding sites.
• RRE Rev response element
• binding proteins include Tat, Rev, NF- ⁇ B, Spl , Gal 4, and LexA.
• binding pair refers to a pair of molecules, including a DNA/DNA pair, DNA/RNA pair, protein/DNA pair, protein/RNA pair, and a protein/protein pair in which the components of the pair bind specifically to each other with a higher affinity than to a random molecule, such that upon binding, the pair triggers a biological response, such as activation of transcription or where the binding protein is a repressor, suppresses a biological response, that is, transcription.
• binding in reference to interaction between two molecules indicates a higher affinity binding and a lower dissociation constant than non-specific binding, thus, distinguishing specific binding from background binding.
• the collision construct comprises a reporter gene coding sequence that is linked at its 5' end to a first regulatory sequence that comprises a first promoter such that the reporter gene is placed under transcriptional regulatory control of the first promoter.
• the reporter gene is linked at its 3' end to a second regulatory sequence that comprises a second promoter in such a fashion that transcriptional activity of the second promoter interferes with the transcriptional activity of the first promoter. This can be done, for example, by placing the first regulatory sequence and the second regulatory sequence in such a manner that transcription under the second promoter proceeds in a direction opposite to direction of transcription under the first promoter.
• the collision construct herein can be used, for example, for screening or identification of an inhibitor of transcriptional activity.
• a collision construct as described above is made with the promoter to be inhibited (hereafter "the target promoter") as the second promoter.
• the collision construct so made is inserted into a vector for expression, with or without the use of linker elements.
• the recombinant vector is then introduced into a compatible host cell that can effect the expression of the reporter gene.
• a compatible host cell that can effect the expression of the reporter gene.
• the regulatory sequences suitable for use herein can be any regulatory sequence that is compatible for use with the promoters for expression in a desired host cell.
• a regulatory sequence derived from mammalian systems would be desirable.
• the regulatory sequence can be a sequence naturally associated with the promoters selected for use herein, or can be a synthetic sequence, or partly synthetic or partly derived.
• the promoters suitable for use herein can be any promoter, including those that are constitutively active or those that are inducible or regulatable.
• the promoters can be naturally derived or synthetically made. They can be derived from any genes, viral, prokaryotic or eukaryotic.
• the eukaryotic genes can be yeast or other fungal, insect, mammalian or avian genes.
• the target promoter is derived from a virus or a tumor cell. Examples of suitable promoters are described below in the portion relating to expression systems.
• a suitable promoter for use as the first promoter in the present collision construct is one that possesses a transcriptional activity that is about the same strength as that of the second promoter. If the first promoter is comparatively much stronger than the second promoter, inhibition of reporter gene signal by the presence of the second or target promoter may be low, and an enhanced reporter gene signal in the presence of an inhibitor may be difficult to detect.
• an available promoter to be used in the collision construct as a first promoter is too strong, it may be desirable to weaken the promoter by deleting parts thereof to generate a minimal promoter. This can be done by a number of methods including, for example, restriction enzyme digestion.
• An example of a weakened promoter is one in which all but the TATA box is deleted.
• a promoter is considered too strong herein if it drives the expression of the reporter gene to about the same level regardless of the presence or absence of the second promoter that drives transcription in the opposite direction.
• a reporter gene signal expressed by a transformed host cell containing the collision construct can be first established in the absence of any inhibitors.
• a candidate inhibitor can then be introduced or added to the cells and the reporter gene expression can be monitored.
• the transformed cells containing the collision construct can be placed in a panel of microtiter wells and a panel of candidate inhibitors can be added to the cells, one inhibitor to each well.
• a suitable inhibitor is one that generates an enhanced reporter signal in its presence as compared with the signal produced in its absence.
• the target promoter to be used herein includes promoters that are subject to regulation, such as activation, by the binding of a binding protein to a response element in the proximity of the target promoter.
• the response element can be naturally present in the target promoter or can be artificially linked to the target promoter.
• the present collision construct can be used to identify an inhibitor that can inhibit activation of the target promoter by inhibiting binding between the binding protein and the response element. This can be achieved by identifying an inhibitor that competitively binds either to the binding protein or to the response element.
• the reporter gene activity would retum to a level similar to that in the absence of activation by the binding protein.
• the binding protein can be introduced into the cells by addition thereof to the medium containing the cells and gently scraping the cells from the culture dish.
• the binding protein can be provided in the form of a vector containing the coding sequence of the binding protein and regulatory sequences that would allow expression thereof.
• the vector can be introduced into the host cell either at, before, or after introduction of the collision construct into the cell.
• stable cell lines containing a collision construct or the coding sequence of the binding protein can be first established, and the other sequence introduced later.
• a stable cell line containing both the collision construct and the binding protein can be made and thereafter used for screening inhibitors.
• the collision construct or vector containing the collision construct can be introduced into host cells by conventional techniques including electroporation, calcium phosphate treatment, and lipofectamine transfection.
• the target promoter can be a known promoter with a known nucleotide sequence that can be synthetically made or derived from a natural source such as a viral gene, a tumor cell gene or a fungal gene, for example.
• promoters are excised from the natural source and inserted into the collision construct by use of restriction enzymes and/or linkers.
• the sequence of the promoter may not be precisely known, but the general location of the promoter is known, for example, the promoter can be known to reside in a particular restriction fragment.
• the restricted fragment can be used as the second regulatory sequence of the present collision construct.
• mRNA can be isolated from the tumor cell and compared to that obtained from normal cell by a common procedure known as subtractive hybridization. By substractive hybridization it can be determined which mRNA is present in the tumor cell but absent in normal cell. A cDNA molecule can be constmcted based on the mRNA so obtained, and a fragment of the genomic DNA containing promoter activity can be isolated and used as a target promoter in the present collision.
• the collision construct herein can be inserted into a suitable vector for introduction into a host cell for expression and use thereof.
• a person skilled in the art would be able to select such a vector and host cell for such pu ⁇ oses.
• suitable vectors and host cells are described in greater detail below.
• the reporter gene that is suitable for use herein can be any reporter gene that can be expressed in the desired host expression system, as described previously.
• the reporter gene can be ⁇ -galactosidase, among others.
• the response element suitable for use herein can be any response element to which inhibition is desired. Examples of such response elements are as described above.
• the response element herein may be part of the promoter sequence by conventional techniques such as by synthesis of excision of a known sequence by restriction enzyme and linked to the promoter sequence with or without the use of linkers.
• the binding proteins for use herein may be any binding protein as described above. Such binding proteins may be added to the cells containing the collision construct for use in screening inhibitors. In doing so, the cells can be scraped off the culture dish or well and mixed with the added binding protein.
• the binding proteins can be introduced into the cell in the form of a vector containing the coding sequence of the binding protein and allowing the expression of the coding sequence.
• a stable cell line containing the binding protein can be made and used for screening inhibitors.
• a cell that constitutively produces the binding protein constitutively can be used.
• a stable cell line containing the collision construct can be made. This can be done by introduction of the collision construct into a host cell, by conventional techniques such as electroporation, calcium phosphate treatment, and lipofectamine or transformation, and selecting a cell or cell line that stably expresses the collision construct.
• a candidate inhibitor to be tested for its inhibitory activity on a target promoter, or on transcriptional activity can be added to a cell harboring the collision construct in which the target promoter is the second promoter of the construct, and optimally, is desired, providing the cell also with a binding protein. Expression of reporter gene signal is observed and compared in the absence and in the presence of the candidate inhibitor, respectively.
• stably transformed cell lines containing the collision construct and optionally containing a vector containing the coding sequence of a binding protein can be placed in microtiter wells and a panel of inhibitors is added thereto. Reporter gene signals are also observed and compared in the absence and presence of the candidate inhibitors, respectively.
• a method for screening inhibitors such as, for example, inhibitors to promoters and transcriptional activators.
• Promoters that can be used for screening inhibitors and used in the collision constmct can be any desired promoters including, for example, promoters from vimses and cancer cells, bacteria and fungi.
• Transcriptional activators that can be inhibited can be any desired transcriptional activator including, for example, Tat, Rev, NFKB and Spl .
• the region of the promoter that can be inhibited herein can be any region that binds transcription factors including, for example, TAR, RRE (Rev response element), NFKB binding site and Spl binding site.
• An embodiment of the present invention can be tailored to screen in vivo in cells a random library of ribozymes for those ribozymes which act as inhibitors of transcription.
• Ribozymes may act by catalytically interrupting transcription by targeting an RNA molecule of a transcription factor that interacts with the promoter or by targeting the mRNA of a reporter gene.
• the use of the invention for screening ribozyme libraries is not limited to any theory of ribozyme function.
• ribozymes catalytically disable an RNA molecule.
• ribozymes which inhibit the second promoter in the collision constmct can be selected from random synthetically derived ribozyme libraries by enhanced reporter gene signal, indicating that a ribozyme is acting to disable the second promoter, or the mRNA for a transcription factor that interacts with that promoter.
• the subunits of a collision constmct including a first regulatory sequence, a reporter gene, optionally, a response element or elements, and a second regulatory sequence, can all be obtained from known sources using conventional techniques of restriction enzyme digestion to remove these elements from such sources.
• these subunits can be made synthetically by chemical synthesis or semi-synthetically by isolating parts thereof from known sources and either combining them or by combining them and making any missing parts synthetically.
• these subunits can be linked together, for example, by use of known linker sequences, so as to place the reporter gene under regulatory control of the first regulatory sequence, with the direction of transcription going in one direction, 5' to 3' and the second regulatory sequence under regulatory control of the response element, with the direction of transcription by the second regulatory sequence mnning in a direction, 5' to 3', but opposite that of the first regulatory sequence. Placement of the response element and the second regulatory sequence is such that activation of transcription of the second regulatory sequence reduces the reporter gene signal upon transcription and translation thereof, presumably as a result of collision between the two transcription units. A mechanism by which the second regulatory sequence generates an anti-sense message that blocks translation of the reporter gene cannot be ruled out.
• the spacing between the reporter gene and the response element can be varied to attain the desired level of inhibition of reporter gene activity.
• the spacing between the 3' end of the reporter gene and the + 1 nucleotide of the promoter of the second regulatory sequence is less than 2200 nucleotides.
• this spacing is less than 1000 nucleotides; more preferably, it is less than 800 nucleotides.
• the spacing is between about 600 nucleotides and about 20 nucleotides. In particular, spacings of about 21 , 94, 153, 406, and 556 base pairs are preferred.
• the target or second promoter of the collision constmct can be optimally placed at a distance of up to 1500 base pairs from the 3' terminus of the first promoter.
• a reporter gene is selected that comprises a sequence that is shorter than or the same as this optimal distance.
• the first response element can also be linked to the second regulatory sequence using linker sequences or the combined first response element and second regulatory sequence can be removed from a known source, again by restriction enzyme digestion.
• the response element will usually be placed at the 5' terminus of the second regulatory region, in accordance with the nature of most promoters which would comprise the second regulatory regions of this invention.
• the response element will be most appropriately placed at the 3' terminus.
• this response element is placed at its natural position in juxtaposition to the promoter being used.
• the response element, TAR is situated 3' to the + 1 nucleotide of the promoter.
• the response element will also be most appropriately positioned at the 3' terminus of the second regulatory region. See for example a discussion of the characteristics of the promoters of such vimses in Cullen, Cell (1993) 73:417-420.
• the response element herein can also be multimerized to produce a more dramatic effect.
• An example of a response element that has been multimerized is the [tet-op]? which is an operator responsive to tetracycline induction.
• the collision constmct can be introduced into an appropriate host cell for expressions thereof, including prokaryotic system such as bacterial, or eukaryotic system, such as yeast, insect cell system, or mammalian system, such as those described below.
• prokaryotic system such as bacterial, or eukaryotic system, such as yeast, insect cell system, or mammalian system, such as those described below.
• the binding protein may also be expressed in the expression systems described below.
• Control elements for use in bacteria include promoters, optionally containing operator sequences, and ribosome binding sites.
• Useful promoters include sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the ⁇ -lactamase (bid) promoter system, bacteriophage ⁇ PL, and T7.
• synthetic promoters can be used, such as the tac promoter.
• ⁇ -lactamase and lactose promoter systems are described in Chang et al., Nature (1978) 275: 615, and Goeddel et al., Nature (1979) 281: 544; the alkaline phosphatase, tryptophan (t ⁇ ) promoter system are described in Goeddel et al. , Nucleic Acids Res. (1980) 8: 4057 and EP 36,776 and hybrid promoters such as the tac promoter is described in U.S. Patent No. 4,551,433 and de Boer et al., Proc. Natl. Acad. Sci. USA (1983) 80: 21-25.
• promoters useful for expression of eukaryotic proteins are also suitable.
• a person skilled in the art would be able to operably ligate such promoters to the coding sequences of interest, for example, as described in Siebenlist et al., Cell (1980) 20: 269, using linkers or adaptors to supply any required restriction sites.
• Promoters for use in bacterial systems also generally will contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the target polypeptide.
• SD Shine-Dalgarno
• the signal sequence can be substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat stable enterotoxin II leaders.
• a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat stable enterotoxin II leaders.
• the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria.
• the foregoing systems are particularly compatible with Escherichia coli.
• numerous other systems for use in bacterial hosts including Gram-negative or Gram-positive organisms such as Bacillus spp. , Streptococcus spp. , Streptomyces spp. , Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, or Serratia marcescans, among others.
• Methods for introducing exogenous DNA into these hosts typically include the use of CaCl2 or other agents, such as divalent cations and DMSO.
• DNA can also be introduced into bacterial cells by electroporation, nuclear injection, or protoplast fusion as described generally in Sambrook et al.
• the host cell should secrete minimal amounts of proteolytic enzymes.
• in vitro methods of cloning e.g. , PCR or other nucleic acid polymerase reactions, are suitable.
• Prokaryotic cells used in this invention are cultured in suitable media, as described generally in Sambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, 2d edition (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).
• Expression and transformation vectors have been developed for transformation into many yeasts.
• expression vectors have been developed for, among others, the following yeasts: Saccharomyces cerevisiae ,as described in Hinnen et al., Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Ito et al, J. Bacteriol. (1983) 755: 163; Candida albicans as described in Kurtz et al., Mol. Cell. Biol. (1986) 6: 142; Candida maltosa, as described in Kunze et al, J. Basic Microbiol.
• Trichoderma reesia as described in EP 0 244 234, and filamentous fungi such as, e.g, Neurospora, Penicillium, Tolypocladium, as described in WO 91/00357.
• Control sequences for yeast vectors are known and include promoters regions from genes such as alcohol dehydrogenase (ADH), as described in EP 0 284 044, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate- dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3- phosphoglycerate mutase, and pyruvate kinase (PyK), as described in EP 0 329 203.
• the yeast PH05 gene, encoding acid phosphatase also provides useful promoter sequences, as described in Myanohara et al., Proc.
• promoter sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase, as described in Hitzeman et al., J. Biol. Chem. (1980) 255:2073, or other glycolytic enzymes, such as pyruvate decarboxylase, triosephosphate isomerase, and phosphoglucose isomerase, as described in Hess et al, J. Adv. Enzyme Reg. (1968) 7: 149 and Holland et al. Biochemistry (1978; 77:4900.
• Inducible yeast promoters having the additional advantage of transcription controlled by growth conditions, include those from the list above and others including the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
• suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EP 0 073 657.
• Yeast enhancers also are advantageously used with yeast promoters.
• synthetic promoters which do not occur in nature also function as yeast promoters.
• upstream activating sequences (UAS) of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.
• hybrid promoters include the ⁇ DH regulatory sequence linked to the GAP transcription activation region, as described in U.S. Patent Nos. 4,876,197 and 4,880,734.
• Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, or PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK, as described in EP 0 164 556.
• a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.
• yeast expression vectors Other control elements which may be included in the yeast expression vectors are terminators, for example, from GAPDH and from the enolase gene, as described in Holland et al, J. Biol. Chem. (1981) 256: 1385, and leader sequences which encode signal sequences for secretion.
• DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene as described in EP 0 012 873 and JP 62,096,086 and the ⁇ -factor gene, as described in U.S. Patent Nos. 4,588,684, 4,546,083 and 4,870,008 and EP 0 324 274 and WO 89/02463.
• leaders of non-yeast origin such as an interferon leader, also provide for secretion in yeast, as described in EP 0 060 057.
• Methods of introducing exogenous DNA into yeast hosts are well known in the art, and typically include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformations into yeast can be carried out according to the method described in Van Solingen et al, J. Bact. (1977) 750:946 and Hsiao et al., Proc. Natl. Acad. Sci. USA (1979) 76:3829. However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or protoplast fusion may also be used as described generally in Sambrook et al., cited above.
• the native target polypeptide signal sequence may be substituted by the yeast invertase, ⁇ -factor, or acid phosphatase leaders.
• the origin of replication from the 2 ⁇ plasmid origin is suitable for yeast.
• a suitable selection gene for use in yeast is the t l gene present in the yeast plasmid described in Kingsman et al, Gene (1979) 7: 141 or Tschemper et al., Gene (1980) 70:157.
• the trp ⁇ gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan.
• Leu2-def ⁇ cient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
• a sequence encoding a yeast protein can be linked to a coding sequence of the desired polypeptide to produce a fusion protein that can be cleaved intracellularly by the yeast cells upon expression.
• a yeast leader sequence is the yeast ubiquitin gene.
• Baculovirus expression vectors are recombinant insect vimses in which the coding sequence for a foreign gene to be expressed is inserted behind a baculovirus promoter in place of a viral gene, e.g. , polyhedrin, as described in Smith and Summers, U.S. Pat. No. , 4,745,051.
• An expression constmct herein includes a DNA vector useful as an intermediate for the infection or transformation of an insect cell system, the vector generally containing DNA coding for a baculovirus transcriptional promoter, optionally but preferably, followed downstream by an insect signal DNA sequence capable of directing secretion of a desired protein, and a site for insertion of the foreign gene encoding the foreign protein, the signal DNA sequence and the foreign gene being placed under the transcriptional control of a baculovirus promoter, the foreign gene herein being the coding sequence of the desired polypeptide.
• the promoter for use herein can be a baculovirus transcriptional promoter region derived from any of the over 500 baculovimses generally infecting insects, such as, for example, the Orders Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example, but not limited to the viral DNAs of Autographo califomica MNPV, Bombyx mori NPV, rrichoplusia ni MNPV, Rachlplusia ou MNPV or Galleria mellonella MNPV, Aedes aegypti, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.
• the baculovims transcriptional promoter can be, for example, a baculovims immediate-early gene IEI or IEN promoter; an immediate- early gene in combination with a baculovims delayed-early gene promoter region selected from the group consisting of a 39K and a Hindlll fragment containing a delayed-early gene; or a baculovims late gene promoter.
• the immediate-early or delayed-early promoters can be enhanced with transcriptional enhancer elements.
• Particularly suitable for use herein is the strong polyhedrin promoter of the baculovims, which directs a high level of expression of a DNA insert, as described in Friesen et al.
• the plasmid for use herein usually also contains the polyhedrin polyadenylation signal, as described in Miller et al., Ann. Rev. Microbiol. (1988) 42: 177 and a procaryotic ampicillin-resistance (amp) gene and an origin of replication for selection and propagation in E. coli.
• DNA encoding suitable signal sequences can also be included and is generally derived from genes for secreted insect or baculovims proteins, such as the baculovims polyhedrin gene, as described in Carbonell et al., Gene (1988) 75:409, as well as mammalian signal sequences such as those derived from genes encoding human ⁇ -interferon as described in Maeda et al, Nature (1985) 575:592-594; human gastrin-releasing peptide, as described in Lebacq-Verheyden et al., Mol. Cell. Biol. (1988) 5:3129; human IL-2, as described in Smith et al, Proc. Natl. Acad. Sci.
• baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (cate ⁇ illar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fmitfly), and
• Bombyx mori host cells have been identified and can be used herein. See, for example, the description in Luckow et al., Bio/Technology(l9S8) 6:47-55, Miller et al, in GENETIC ENGINEERING (Setlow, J.K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al, Nature, (1985) 575:592-594.
• a variety of such viral strains are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV.
• Such vimses may be used as the vims for transfection of host cells such as Spodoptera frugiperda cells.
• baculovims genes in addition to the polyhedrin promoter may be employed to advantage in a baculovims expression system. These include immediate- early (alpha), delayed-early (beta), late (gamma), or very late (delta), according to the phase of the viral infection during which they are expressed. The expression of these genes occurs sequentially, probably as the result of a "cascade" mechanism of transcriptional regulation. Thus, the immediate-early genes are expressed immediately after infection, in the absence of other viral functions, and one or more of the resulting gene products induces transcription of the delayed-early genes. Some delayed-early gene products, in turn, induce transcription of late genes, and finally, the very late genes are expressed under the control of previously expressed gene products from one or more of the earlier classes.
• IEI a preferred immediate-early gene of Autographo californica nuclear polyhedrosis vims (AcMNPV).
• AcMNPV Autographo californica nuclear polyhedrosis vims
• IEI is expressed in the absence of other viral functions and encodes a product that stimulates the transcription of several genes of the delayed-early class, including the preferred 39K gene, as described in Guarino and Summers, J. Virol. (1986) 57:563-571 and J. Virol. (1987) 67:2091-2099 as well as late genes, as described in Guarino and Summers, Virol. (1988) 762:444-451.
• Immediate-early genes as described above can be used in combination with a baculovims gene promoter region of the delayed-early category. Unlike the immediate-early genes, such delayed-early genes require the presence of other viral genes or gene products such as those of the immediate-early genes.
• the combination of immediate-early genes can be made with any of several delayed-early gene promoter regions such as 39K or one of the delayed-early gene promoters found on the Hindlll fragment of the baculovims genome. In the present instance, the 39K promoter region can be linked to the foreign gene to be expressed such that expression can be further controlled by the presence of IEI, as described in L. A.
• enhancement of the expression of heterologous genes can be realized by the presence of an enhancer sequence in direct cis linkage with the delayed-early gene promoter region.
• enhancer sequences are characterized by their enhancement of delayed-early gene expression in situations where the immediate-early gene or its product is limited.
• the hr5 enhancer sequence can be linked directly, in cis, to the delayed-early gene promoter region, 39K, thereby enhancing the expression of the cloned heterologous DNA as described in Guarino and Summers (1986a), (1986b), and Guarino et al. (1986).
• the polyhedrin gene is classified as a very late gene. Therefore, transcription from the polyhedrin promoter requires the previous expression of an unknown, but probably large number of other viral and cellular gene products. Because of this delayed expression of the polyhedrin promoter, state-of-the-art BEVs, such as the exemplary BEV system described by Smith and Summers in, for example, U.S. Pat. No. , 4,745,051 will express foreign genes only as a result of gene expression from the rest of the viral genome, and only after the viral infection is well underway. This represents a limitation to the use of existing BEVs. The ability of the host cell to process newly synthesized proteins decreases as the baculovims infection progresses.
• gene expression from the polyhedrin promoter occurs at a time when the host cell's ability to process newly synthesized proteins is potentially diminished for certain proteins.
• the expression of secretory giycoproteins in BEV systems is complicated due to incomplete secretion of the cloned gene product, thereby trapping the cloned gene product within the cell in an incompletely processed form.
• an insect signal sequence can be used to express a foreign protein that can be cleaved to produce a mature protein
• the present invention is preferably practiced with a mammalian signal sequence.
• An exemplary insect signal sequence suitable herein is the sequence encoding for a Lepidopteran adipokinetic hormone (AKH) peptide.
• the AKH family consists of short blocked neuropeptides that regulate energy substrate mobilization and metabolism in insects.
• a DNA sequence coding for a Lepidopteran Manduca sexta AKH signal peptide can be used.
• Other insect AKH signal peptides, such as those from the Orthoptera Schistocerca gregaria locus can also be employed to advantage.
• Another exemplary insect signal sequence is the sequence coding for Drosophila cuticle proteins such as CPI, CP2, CP3 or CP4.
• the desired DNA sequence can be inse ⁇ ed into the transfer vector, using known techniques.
• An insect cell host can be cotransformed with the transfer vector containing the inse ⁇ ed desired DNA together with the genomic DNA of wild type baculovims, usually by cotransfection.
• the vector and viral genome are allowed to recombine resulting in a recombinant vims that can be easily identified and purified.
• the packaged recombinant vims can be used to infect insect host cells to express the desired polypeptide.
• Typical promoters for mammalian cell expression include the SV40 early promoter, the CMV promoter, the mouse mammary tumor vims LTR promoter, the adenovims major late promoter (Ad MLP), and the he ⁇ es simplex vims promoter, among others.
• Other non-viral promoters such as a promoter derived from the murine metallothionein gene, will also find use in mammalian constmcts.
• Mammalian expression may be either constitutive or regulated (inducible), depending on the promoter Typically, transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon.
• transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al. (1989), cited previously.
• Introns, containing splice donor and acceptor sites, may also be designed into the constmcts ofthe present invention.
• Enhancer elements can also be used herein to increase expression levels ofthe mammalian constmcts. Examples include the SV40 early gene enhancer, as described in Dijke a et al. , EMBO J.
• a leader sequence can also be present which includes a sequence encoding a signal peptide, to provide for the secretion of the foreign protein in mammalian cells.
• processing sites encoded between the leader fragment and the gene of interest such that the leader sequence can be cleaved either in vivo or in vitro.
• the adenovims tripa ⁇ ite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
• expression vectors that provide for the transient expression in mammalian cells of DNA encoding the target polypeptide.
• transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector.
• Transient expression systems comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired biological or physiological propenies.
• transient expression systems are pa ⁇ icularly useful for pu ⁇ oses of identifying analogs and variants of the target polypeptide that have target polypeptide-like activity.
• the mammalian expression vectors can be used to transform any of several mammalian cells.
• Methods for introduction of heterologous polynucleotides into mammalian cells are known in the an and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
• dextran-mediated transfection calcium phosphate precipitation
• polybrene mediated transfection protoplast fusion
• electroporation electroporation
• encapsulation of the polynucleotide(s) in liposomes and direct microinjection of the DNA into nuclei.
• General aspects of mammalian cell host system transformations have been described by Axel in U.S. Patent No. 4,399,216.
• Mammalian cell lines available as hosts for expression are also known and include many immo ⁇ alized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g. , Hep G2), human embryonic kidney cells, baby hamster kidney cells, mouse se ⁇ oli cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, as well as others.
• the mammalian host cells used to produce the target polypeptide of this invention may be cultured in a variety of media.
• any of these media may be supplemented as necessary with hormones and/or other growth factors such as insulin, transferrin, or epidermal growth factor, salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as GentamycinTM M dmg), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the an.
• the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled a ⁇ isan.
• the collision constmct can be introduced into host cells by conventional techniques including lipofectamine. DEAE-dextran, electroporation, and calcium phosphate, and as described above.
• a stable cell line that contains the collision constmct can be made and selected.
• the collision constmct is electroporated together with a selectable marker gene, for example neomycin. G418 resistant colonies are assayed for the existence and functionality of the collision constmct.
• the cell line can be prokaryotic or eukaryotic in origin.
• the cell line is eukaryotic, more preferably, mammalian.
• the cell line can be derived from HeLa cells, T-cells, B-cells and 293 cells.
• the stable cell line containing the collision constmct can also be cotransfected with a plasmid containing a binding protein.
• the coding sequence for the binding protein can be inse ⁇ ed into an expression plasmid, such as pCG, a pEVRF derivative, described in Giese et al., Genes & Development (1995) 9:995-1008.
• pEVRF is described in Matthias et al., Nucleic Acids Res. (1989) 77:6418.
• pCG has a modified polylinker, and directs expression in mammalian cells from the human cytomegalovirus promoter/enhancer region.
• the coding sequence for the binding protein can also be inse ⁇ ed into the expression plasmid pCDNA (Clontech, Palo Alto, CA).
• the DNA constmct encoding the binding protein can be made by standard methods of recombinant DNA technology as described in Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) and Ausubel et al, cited previously.
• the collision constmct can be transfected into a cell line that constitutively expresses a binding protein, such as, for example, HeLa cells, T-cells, B-cells or 293 cells that have been stably transfected with a vector that directs expression of the binding protein.
• a binding protein such as, for example, HeLa cells, T-cells, B-cells or 293 cells that have been stably transfected with a vector that directs expression of the binding protein.
• the host cell carrying the collision constmct for use in screening can be exposed to a binding protein that is added into the medium containing the transfected cells.
• a Tat protein expression vector is added to a cell line carrying the collision constmct. The amount of the expression vector can be varied depending upon the extent of inhibition of repo ⁇ er gene activity desired.
• the repo ⁇ er gene activity is determined in the absence of the binding protein, in the presence of the binding protein, and in the presence of candidate inhibitors being screened.
• a candidate that increases repo ⁇ er gene activity in the presence of a binding protein that activates transcription for example, can be selected and further tested as an inhibitor to transcriptional activation.
• kits can be made that contain the present collision constmct for screening for inhibitors of transcriptional activation.
• kits can include vectors or host cells containing one or more of the present collision constmcts in suitable containers, along with the reagents and materials required for the conduct of the assay or descriptions of those remaining reagents necessary, as well as a suitable set of assay instmctions.
• Other materials or reagents can include, for example, diluents, buffers, host cells and other reagents, appropriate containers such as tubes, plates, etc. , and may be included in the kit, or described in the instmctions.
• Example 1 Constmction of the Collision Constmct with CMV and HIV-1 Promoters and an Alkaline Phosphatase Repo ⁇ er Gene: Constmct #1152
• the collision constmct containing the human cytomegalovims ("hCMV") promoter, the gene for the secreted form of the human placental heat-stable alkaline phosphatase (“AP”) and the promoter of the human immunodeficiency virus-1 (“HIV-1”) was generated from precursor constmcts as described below.
• a nucleotide sequence comprising the hCMV promoter and a region derived from the he ⁇ es simplex thymidine kinase, tk, gene for the optimal initiation of translation (hereafter "the tk upstream region"), was isolated from plasmid pCG.
• Plasmid pCG described in Giese et al, Genes and Development (1995) 9:995-1008, is a pEVRF derivative, as described in Matthias et al, cited previously.
• pCG has a modified polylinker, and directs expression in mammalian cells from the human cytomegalovims promoter/enhancer region.
• the hCMV promoter was isolated from pCG by digestion with restriction enzymes EcoRI and Xbal (Boehringer Mannheim, Germany). Restriction digestion for pu ⁇ oses herein was conducted essentially as described in Sambrook et al, cited previously, and Ausubel et al, cited previously, or in accordance with the manufacturer's recommendations. For example, digestions were typically conducted using 2 ⁇ l of 10 x restriction buffer, 0.1 to 4 ⁇ g of DNA in water or T ⁇ buffer, 1-5 U of enzyme per ⁇ g of DNA and water to obtain a total volume of 20 ⁇ l. The components of the digest were incubated at 37°C from about 10 minutes to ovemight, depending on the amount of DNA being digested.
• hCMV promoter sequence isolated from pCG was then ligated into plasmid pTZ19U, purchased from Pharmacia (Piscataway, N.J.), that had been cleaved with the same restriction enzymes, EcoRI and Xbal.
• the resulting plasmid was designated constmct #1080.
• Ligation reactions herein were essentially performed as directed by the manufacturer of the ligase (Boehringer Mannheim, Germany) and along the principles described in Sambrook et al, cited previously, and Ausubel et al, cited previously.
• the coding region of the alkaline phosphatase gene was isolated from plasmid pS ⁇ AP-Basic, purchased from Clontech (Palo Alto, CA) by restriction with Htndlll and Sail and ligated into plasmid Bluescript, purchased from Stratagene (La Jolla, CA) that had been cleaved with the same enzymes.
• the resulting plasmid designated constmct #1067, was cleaved with Clal and Sail, the 5 '-overhangs were filled in by Klenow enzyme, (Boehringer Mannheim, Germany) and the ends were religated. Fill- in reactions described herein were conducted as directed by the manufacturer of the Klenow enzyme (Boehringer Mannheim, Germany).
• the resulting plasmid constmct was designated #1074. This manipulation restored the Sail restriction site.
• the coding region of the alkaline phosphatase gene was isolated from constmct
• the ⁇ IV-1 promoter was isolated from plasmid pHIVSCAT, as described in Selby and Peterlin, Cell (1990) 62:769-776, by treatment with AspllS and Hmdlll. The isolated fragment was ligated into plasmid Bluescript from Stratagene (La Jolla, CA), that had been cleaved with the same enzymes. The resulting plasmid was designated constmct #1075.
• the pa ⁇ icular ⁇ IV-1 promoter in pHIVSCAT contains several point mutations that have been introduced to create restriction enzyme recognition sites. Comparison of the mutant promoter in pHIVSCAT with the wild ⁇ type HIV-1 promoter did not show any significant differences in activity. The sequence of the mutant HIV-1 promoter is listed in FIG. 5.
• the HIV-1 promoter was isolated from constmct #1075 by restriction with EcoRV and then ligated into plasmid pTZ18U from Pharmacia (Piscataway, NJ), that had been cleaved with Smal. The resulting plasmid was designated constmct #1149.
• the HIV-1 promoter was isolated from constmct #1149 by restriction with Sail and AspllS and ligated into constmct #1 1 12 that was cleaved with the same enzymes.
• the resulting plasmid was designated constmct #1152, the collision constmct.
• the direction of transcription from the hCMV and that from the HIV-1 promoter were in opposite directions.
• the distance between the end of the AP coding region, as defined by the stop codon TAA, and the sta ⁇ of transcription in the HIV-1 promoter, as defined by + 1 of the promoter sequence was about 213 nucleotides.
• Plasmid pSV7fd/TAT is herein referred to as the Tat expression plasmid, and its constmction is described below.
• the amount of DNA in each transfection assay was kept constant by adding Tat-inactive plasmid, the constmction of which is also described below. Results are shown in FIG. 1.
• lipofectamine purchased from BRL, Gaithersburg, MD was used in accordance to the manufacturers' instmctions.
• 1 ⁇ g of the collision constmct was used together with either 0.5 ⁇ g of Tat expression plasmid (pSV7fd/TAT) and/or 0.5 ⁇ g of Tat-Inactive plasmid. After approximately 8 hours, the cells were washed and incubated in fresh DME medium, supplemented with 10% fetal calf semm. About 16-20 hours after transfection, aliquots of the supematant were analyzed for alkaline phosphatase activity according to the manufacturers' conditions (Clontech, Palo Alto, CA).
• Plasmid pSV7fd/TAT was obtained from Peterlin at University of California at San Francisco and was constmcted and used as described in Selby and Peterlin, Cell (1990) 62:769-776. Plasmid pSV7fd/TAT contains the coding region for the transcriptional activator Tat from HIV-1. In this plasmid, Tat expression is under control of the SV40 early promoter. Inactive Tat expression plasmid was generated by restriction of plasmid pSV7fd/TAT with Xbal. The DNA ends were filled-in and the plasmid religated.
• This procedure generated a frame shift mutant that resulted in a premature stop codon and no functional Tat protein expression.
• the resulting constmct is referred to as Tat-inactive plasmid, and was used herein to keep the total amount of DNA in each transfection constant.
• FIG. 1 shows a reduction of AP activity that was dependent on the amount of Tat expression plasmid added. Over the range of 0 to 2 ⁇ g of Tat expression plasmid, repo ⁇ er gene activity decreased from about 100% to about 25% , resulting in about 75% inhibition at the highest level tested. Repo ⁇ er activity was about 40% when Tat was present at a level of between about 0.3 to 0.5 ⁇ g of Tat expression vector. At this level, there is almost no nonspecific effect of Tat protein on CMV promoter activity.
• constmct #1152 was introduced into constmct #1152: (1) deletion of a major po ⁇ ion of the TAR sequence, constmct #1161 ; (2) deletion of the entire TAR sequence, constmct #1225; (3) deletion of the entire TAR sequence and the TATA box, constmct #1162; and (4) deletion of the entire TAR sequence, the TATA box, and the three Spl binding sites, constmct #1163.
• Constmct #1152 was restricted with Bglll and BamHI and the ends religated. The resulting plasmid represents constmct #1161. This deletion removes about 36 nucleotides of the TAR sequence.
• TAR sequence Complete removal of the TAR sequence was obtained by deleting nucleotides from position -14 to +59, by PCR using DNA of constmct #1152 as template and primer #613 (5'-GCGAAGCTTTGCAGCTGCTTATATGCAGCA-3') and reverse primer, purchased from New England Biolabs, Beverly, MA.
• the underlined sequence represents nucleotides -35 to -13 of the HIV-1 promoter beginning with Hindlll restriction site; the non-underlined po ⁇ ion includes the Hindlll restriction site that begins after the nucleotides GCG at the 5'-end.
• the resulting DNA fragment was digested with Hindlll and Asp718 and religated into constmct #1152 cleaved with the same enzymes. This manipulation also removed the sta ⁇ of transcription and generated constmct #1225.
• constmct #1162 A third deletion constmct was prepared from constmct #1152 by restriction with Xbal and religation. The resulting plasmid is designated constmct #1162. This deletion removes the complete TAR sequence and the region containing the TATA box sequence.
• constmct #1163 A fou ⁇ h deletion constmct was prepared from constmct #1152 by restriction with Smal and BamHI and the ends religated. The resulting plasmid is designated constmct #1163. This deletion removes the complete TAR sequence, the TATA box sequence and the three Spl binding-site sequences.
• FIG. 2 shows the results of measuring the specific reduction of alkaline phosphatase expression in the presence of 0.5 ⁇ g HIV-1 Tat protein expression plasmid in the deletion constmcts #1161 , #1225, #1162 and #1163, as compared with constmct #1152.
• activity of the AP gene was set to 100% .
• activity of the AP gene was reduced to 36% +_ 7%.
• expression of AP was reduced by about 64% .
• expression of the AP gene in the absence of Tat was about 55% .
• Constmct #1225 containing a deletion in the TAR sequence, produced about 150% AP gene activity in the absence of Tat, and about 140% AP gene activity in the presence of Tat.
• Constmct #1162 containing a deletion in the TAR sequence and in the TATA box, produced about 150% AP gene expression in the absence of Tat protein, and about 153% AP gene expression in the presence of Tat.
• Constmction of TAT-Inactive Plasmid Other constmcts were made to test the effect of deletion of po ⁇ ions of a promoter region on transcriptional activation, using the AP gene as the repo ⁇ er gene.
• Constmct #1085 was made using the mutant HIV-1 promoter as described in Example 3, as an Asp718/ Hindlll DNA fragment isolated from pHIVSTAT and ligated into plasmid pSEAP-Basic (Clontech, Palo Alto, CA) that was cleaved with the same enzymes. This operation linked the HIV- 1 promoter to the AP gene.
• constmct #1085 contains the entire HIV-1 promoter.
• Constmct #1166 lacks about 36 nucleotides of the TAR sequence.
• Constmct #1213 lacks all of the TAR sequence and nucleotides comprising the original sta ⁇ of transcription.
• Constmct #1167 lacks the TAR sequence and the TATA box.
• Constmct #1168 lacks the TAR sequence, the TATA box, and the three Spl binding sites. HeLa cells were transiently transfected with the latter constmcts as described before and AP gene expression was observed as described before.
• FIG. 3 shows that in the absence of Tat, the full-length HIV-1 promoter in constmct #1085, containing the TAR sequence, was unable to induce significant expression of the AP gene, resulting in about 2% AP gene activity.
• constmct #1085 activated AP gene expression.
• the AP gene activity produced by constmct #1085 was set to 100%.
• Deletion constmct #1166 that lacked most of the TAR sequence showed only about 2% AP activity in the presence of Tat protein.
• the tmncated HIV-1 promoter produced about 12% AP activity.
• Deletion constmcts #1213, #1162 and #1163 showed no basal and also no Tat-inducible promoter activity.
• Constmcts to Study the Effect of Spacing on the Function of the Collision Constmct Constmcts having varying distances or spacer regions between the first and second promoters or between the second promoter and repo ⁇ er gene were made to study the effect of spacing on the function of the collision constmct.
• the various collision constmcts made contained spacer regions of 21 nucleotides (constmct # 1190), 94 nucleotides (constmct #1181), 153 nucleotides (constmct #1187), 406 nucleotides (constmct #1188), 556 nucleotides (constmct #1189) and 2047 nucleotides (constmct #1159), positioned between the 3' end of the AP coding sequence, as defined by the stop codon, and the end of the TAR sequence at +59 nucleotide in the HIV-1 promoter, with + 1 nucleotide as the sta ⁇ of transcription.
• Constmct #1190 was made by restriction digest of constmct #1152 with Hpal and Hindlll and religation. This manipulation changed the stop codon from TAA to TGA.
• Constmct #1181 was made by restriction digest of constmct #1152 with Sail and Hindlll and religation.
• Constmcts #1187, #1188 and #1189, respectively, were made by inse ⁇ ion of pans of the PEBP2 ⁇ coding region, as described in Ogawa et al. (1993) Proc. Natl Acad. Sci.
• Constmct #1159 was made by insertion of the luciferase gene isolated from plasmid pT3/T7-Luc (Clontech, Palo Alto, CA) as a Sall/Asp718 fragment into constmct #1152 cleaved with the same enzymes. Results are shown in FIG. 4.
• FIG. 4 shows that
• AP activity in constmcts with spacing from about 21 nucleotides to about 556 nucleotides is between 40% and 46%, and thereafter, as the spacing increased to about 2047 nucleotides, the alkaline phosphatase activity increased, indicating a certain space requirement for collision.
• Transient transfections were done using 1 ⁇ g of collision constmct DNA and 0.5 ⁇ g of either active or inactive Tat expression plasmid.
• Example 6 HeLa Cells Stably-Transfected with Tat Protein Expression Plasmid HeLa cells were transfected with 10 ⁇ g of either active Tat expression plasmid or with Tat-inactive plasmid together with 1 ⁇ g of plasmid pSVNeo (purchased from Clontech, Palo Alto, CA ) for selection by electroporation using a BioRad Gene Pulser (Purchased from BioRad, Hercules, CA). Electroporation was conducted in accordance with the manufacturer's instmctions.
• the conditions for electroporation are 1000 uF and 300 volts at room temperature in a final volume of 500 ⁇ l medium containing 10% fetal calf semm, and 50 ⁇ g/ml each of penicillin and streptomycin.
• Stable Tat expression colonies were identified by resistance to 400 ⁇ g/ml gentamicin (purchased from GIBCO BRL) after about 10 to 14 days. Stable colonies were picked, amplified, and analyzed for Tat protein expression by transfection with constmct #1085, the plasmid with an HIV-1 promoter linked to the AP reporter gene.
• Positive Tat-expressing cell lines were identified by measuring alkaline phosphatase activity, according to directions described by the manufacturer of pSVNeo (Clontech, Palo Alto, CA).
• the following assay is designed to screen for inhibitors of the Tat protein, the Tat/TAR interaction or any other HIV-1 promoter target.
• the inhibition sought is identified by significant inhibition of long terminal repeat (LTR) activity.
• LTR long terminal repeat
• Cell lines are stably transfected as described in Example 6 above with the collision constmct #1152 and the Tat plasmid. Those stably transfected cell lines that produce a steady- state alkaline phosphatase activity of about ⁇ 30% to 40% compared to control HeLa cells stably transfected with only the collision constmct are selected for screening.
• inhibitors are introduced into the culture medium and, after about 16 to 20 hours, the supematant is analyzed for alkaline phosphatase activity, as described in Example 6 Using a 96-wel! assay plate, for example, 12 different inhibitors, at 8 different concentrations, are tested simultaneously. Those inhibitors that produce an increase in alkaline phosphatase activity are further characterized by transient transfection experiments. Transient transfections are conducted as described in Example 2. For example, HeLa cells are transiently transfected with constmct #1152 and a controlled amount of Tat plasmid in the presence of an inhibitor. The inhibitor is then tested for dose responsiveness to Tat by titrating the inhibitor or the Tat expression plasmid. Separate transient transfection assays are repeated for each inhibitor selected by the screening process described above.
• Example 7 Constmcts Including TAR Decoys
• TAR decoys have proved sufficient at inhibiting HIV-1 promoter activity by squelching Tat-mediated transactivation, as described in Sullenger et al, Cell 63: 601-608 (1990) and Graham and Maio, Proc. Nat'l Acad Sci USA 87: 5817-5821 (1991).
• the plasmid pBJ-TAR8 was constmcted by inse ⁇ ion of multimerized TAR sequences isolated from pHIVSCAT with Xbal and Hindlll restriction enzymes (nucleotides -40 to +59) downstream of the Sra promoter in pBJ, as described in Takebe et al Mol. Cell. Biol. 8: 466-472 (1988).
• the leptin gene as described in Giese et al Embo J. 12:4667-4676 (1993) with a similar size compared to the TAR sequences was inse ⁇ ed into the pBJ vector.

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