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WO1993004166A1 - Proteine de liaison d'adn de region purique - Google Patents

Proteine de liaison d'adn de region purique Download PDF

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Publication number
WO1993004166A1
WO1993004166A1 PCT/US1992/006748 US9206748W WO9304166A1 WO 1993004166 A1 WO1993004166 A1 WO 1993004166A1 US 9206748 W US9206748 W US 9206748W WO 9304166 A1 WO9304166 A1 WO 9304166A1
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dna
gabpα
gabpbl
gabp
protein
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PCT/US1992/006748
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English (en)
Inventor
Steven L. Mcknight
Catherine C. Thompson
Karen L. Lamarco
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Carnegie Institution Of Washington
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Priority to JP5504411A priority Critical patent/JPH07500728A/ja
Priority to EP92918552A priority patent/EP0598839A1/fr
Publication of WO1993004166A1 publication Critical patent/WO1993004166A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates, in general, to a DNA binding protein and to a DNA sequence encoding same.
  • the invention relates to a GA binding protein and to DNA segments encoding the subunits thereof.
  • HSV1 immediate early (IE) genes are induced at the outset of the lytic infection by a virion associated protein termed VP16 (Post et al, Cell 24, 555 (1981)). At least two classes of cis-regulatory elements qualify HSV IE genes for induction by VP16. The most essential VP16 ⁇ is-response element is characterized by the nonanucleotide sequence 5*-TAATGARAT-3'
  • VP16 binds tightly to this DNA sequence in a complex with the cellular transcription factor Octl (Preston et al. Cell 52, 425 (1988); O'Hare et al, ibid. p. 435 (1988) ; and Gerster et al, Proc. Natl. Acad. Sci. U.S.A. 85, 6347 (1988)).
  • a second cis-regulatory element required for VP16-mediated induction of HSV IE genes consists of three imperfect repeats of the purine-rich hexanucleotide 5*-CGGAAR-3' (Triezenberg et al, Genes Dev. 2, 730 (1988) and Spector et al, ibid.
  • Applicants have isolated cDNA clones encoding both subunits of GABP and have revealed that one (GABP ⁇ ) is related to the Ets transforming protein, while the other (GABPB) contains a series of 33-amino acid repeats related in sequence to a variety of proteins including Notch of Drosophila melano ⁇ aster.
  • Linl2 and Glpl of Caenorhabditis eleqans and SW14 and SW16 of Saccharormyces cerevisiae ( harton et al, Cell 43, 567 (1985); Greenwald, i id 43, 583 (1985); Yochem et al, Nature 335, 547 (1988); Yochem et al.
  • the present invention relates to a DNA segment encoding GABP ⁇ :, GABPB1 or GABPB2, or portion thereof.
  • the present invention relates to a construct comprising at least one of the above-described segments and to a host cell transformed therewith.
  • Figure l Amino acid sequences of tryptic peptides derived from GA binding proteins.
  • GABP (20 ⁇ g) was purified to homogeneity (inset) as described (LaMarco et al. Genes Dev. 3, 1372 (1989)) except that boiled salmon sperm DNA (20 ⁇ g/ml) was included as a non-specific competitor in the DNA affinity chromatographic step.
  • Approximately 500 picomoles of protein was lyophilized, reduced, acetylated, and subjected to cleavage by trypsin (Boehringer
  • amino acid sequences derived from peaks 1-13 were: 1, SLFDQGVIEK; 2, 7AWALEGY; 3, DEIS?VGDEGEFK; 4, ELESLNQEDFFQR; 5, LQESLDAHEIELQDIQL?P?R; 6, DQISIVGDEGEFK; 7, MAELV; 8, YVQASQLQQMNEIVTIDQP; 9, TPLHMWASEGHA; 10, GEILWS; 11, LIEIEIDGTEK; 12, ILMANGAPFTTD; 13, TGNNGQIQL?QFLLEL?TDR.
  • FIG. 1 Nucleotide and deduced amino acid sequences of cDNAs encoding GABP subunits.
  • A Sequence of GABP ⁇ .
  • B Sequences of GABPBl and &2.
  • An unamplified cDNA library prepared from mouse adipocyte mRNA was screened with a mixture of degenerate oligonucleotides derived from the amino acids sequences of peptides 3, 4, 5, and 8 (Fig. 1) labeled with 32 P using polynucleotide kinase.
  • the basic SSC protocol was used (Ausubel et al, Current Protocols in Molecular Biology (Wiley & Sons, NY) , 1989) .
  • Hybridization was performed at 48°C for
  • GABPBl and B2 were isolated by screening a day-8.5 mouse embryo cDNA library (Lee, Mol. Endocrinol. 4, 1034 (1990)) with degenerate oligonucleotides corresponding to peptides 9 and 12 (Fig. 1) .
  • Kinased oligonucleotide probes were hybrided in 6X SSC, IX Denhardt's, 0.05% sodium pyrophosphate, and 100 ⁇ g/ml yeast tRNA at 50°C for 14 hours. Washing conditions were 6X SSC, 0.05% sodium pyrophosphate at 55°C. A total of five clones were isolated that hybridized with both oligonucleotide probes.
  • GABPBl and GABPB2 were identical up to nucleotide 1130 except for a three nucleotide insertion (GTA) at position 828 of GABPBl. Sequencing of four other independent isolates of GABPBl were identical to GABPB2 at this site. Peptides identified by amino acid sequencing of purified GABP are underlined in the deduced amino acid sequences. The dashed lines indicate the sequence in GABPBl not found in GABPB2. The sequence for B2 is shown from the point at which it diverges from Bl.
  • RNA was isolated from various rat tissues (Chingwin et al. Biochemistry 18, 5294 (1979)) and mouse L cells (Chomczynski et al. Anal. Biochem. 162, 156 (1987)). lO ⁇ g of poly A+ RNA was separated on a 1% agarose- formaldehyde gel, transferred to Nytran (Schleicher and Schuell) and hybridized with a random-primed probe prepared from GABP ⁇ (A) or GABPBl (B) .
  • FIG. 4 Requirement of GABP ⁇ and GABPBl for sequence specific DNA binding.
  • RNAs were used to program rabbit reticulocyte lysates in the presence of 35 S- methionine under conditions specified by the manufacturer (Promega Biotec) . Unlabeled protein was used for DNA binding experiments. The 35 S- methionine labeled products were separated on a 12.5% SDS-polyacrylamide gel and visualized by fluorography; (-) , anti-sense RNA; (+) , sense strand RNA. Positions of molecular weight markers are indicated in kD. (B) Electrophoretic mobility shift assays with in vitro translated GABP proteins.
  • Proteins were incubated in the presence of a 32 P- labeled DNA fragment from the HSV ICP4 promoter and subjected to electrophoresis on a non-denaturing 5% polyacrylamide gel in .5X TBE (Garner et al, Nucl. Acids Res. 9, 3047 (1981); Fried et al, ibid, p. 6505 (1981)).
  • vitro translated protein were incubated in 25 mM Tris pH 8.0, 10% glycerol, 50 mM CK1, 3 mM MgC12, 0.5mM EDTA, ImM DTT, 50 ⁇ g/ml poly dldC on ice for 10 minutes, then probe was added and incubation continued at room temperature for 10 minutes.
  • the probe was a 180 bp Nco I-Sal I fragment excised from the herpes simplex virus ICP4 promoter. The fragment was labeled by fill-in with the Klenow fragment of DNA polymerase I in the presence of "P-dCTP.
  • Protein:DNA complexes were subjected to electrophoresis on 5% (30:1) polyacrylamide gels in 0.5X TBE. Radioactive DNA and DNA: rotein complexes were visualized by autoradiography. "B” indicates GABP bound DNA, "E” indicates DNA bound by proteins endogenous to reticulocyte lysates.
  • FIG. 5 Schematic diagram of GABP subunits showing regions of amino acid sequence similarity to related proteins.
  • (Top) GABP ⁇ is represented as a rectangle with the NH.-terminus on the left and the COOH-terminus on the right.
  • the region of sequence similarity to Ets-related proteins is shaded (amino acids 316-400) and compared with the sequences of
  • the unique COOH-terminal segment of GABPBl relative to GABPB2 is indicated in black (333-382) .
  • the sequence of the four 33 amino acid repeats in GABPBl are shown below; residues that are common to two or more repeats are boxed in black and used to derive the GABPB consensus. Similar criteria were used to derive consensus sequences for the 33 amino acid repeats of cdc 10/SW14,6 (Ares et al, EMBO J. 4, 457 (1985); Andrews et al. Nature, 342, 830 (1989); Breeden et al. Nature 329, 651 (1987)), Notch (Wharton et al. Cell 43, 567 (1985); Greenwald, iJid.
  • FIG. 7 Characterization of the DNA binding site for GABP.
  • A Increasing concentrations of GABP ⁇ , either in the absence (left panel) or presence (right panel) of GABPBl were mixed with a "P-labeled DNA fragment derived from the herpes simplex virus ICP4 enhancer. Free and protein-bound complexes were partially digested with DNase I and subjected to electrophoresis on an 8% polyacrylamide sequencing gel. The positions of three purine-rich repeats within the region of DNA protected from digestion by GABP are indicated by arrows. Lanes l- 6 (left panel) show digestion patterns resulting from GABP ⁇ concentrations starting at 1.5 nM and decreasing in 3-fold increments to 0.005 nM. Lanes 1-6 (right panel) show patterns resulting from addition of the same concentrations of GABP ⁇ that had been supplemented with 0.5 nM of GABPBl.
  • B shows patterns resulting from addition of the same concentrations of GABP ⁇ that had been supplemented with 0.5 nM of G
  • Methylation protection (left panel) and interference (right panel) assays of DNA binding by GABP The same DNA fragment used in (A) was incubated with GABP ⁇ , GABPBl, or an equimolar mixture of the two subunits, and exposed to dimethyl sulfate (DMS) . Partially methylated DNA was recovered, cleaved with piperidine, and run on an 8% polyacrylamide sequencing gel.
  • DMS dimethyl sulfate
  • DNA was partially methylated, incubated with an equimolar mixture of GABP ⁇ and GABPBl, and subjected to electrophoresis on a non-denaturing polyacrylamide gel as described in Fig. 6.
  • Figure 8 Measurements of DNA binding stability of complexes formed by various mixtures of GABP subunits.
  • a M P-labeled oligonucleotide containing a GABP binding site (Fig. 6) was incubated with GABP ⁇ alone, or together with equimolar amounts of either of the two B subunits. After a 10 minute incubation at 24°C, protein:DNA complexes were challenged with a 500-fold excess of unlabeled oligonucleotide.
  • FIG. 9 UV-mediated crosslinking of GABP subunits to DNA. Isolated or mixed GABP subunits were incubated with a M P-labeled oligonucleotide containing a GABP binding site (Chodosh in Current Protocols in Molecular Biology, Vol. II, Ausubel et al eds (Greene Wiley, New York, 1988)) then exposed to ultraviolet light for varying lengths of time.
  • UV crosslinking was performed using an oligonucleotide composed of a GA binding site flanked by 10 bp of non-specific sequence (5' AACCAAGCTTGCGGAACGGAAGCGGAAACCG 3') corresponding to residues located between 280 and 300 bp upstream of the herpes simplex virus gene encoding ICP4.
  • Oligonucleotides were labeled to high specific activity by fill-in reaction with the Klenow fragment of DNA polymerase I in the presence of all four 32 P-labeled dNTPs. DNA binding reactions were performed as described in a 96-well culture dish, followed by exposure to ultraviolet light. Samples were boiled in SDS-sa ple buffer and subjected to electrophoresis on SDS-polyacrylamide gels. Crosslinked protein species were visualized by autoradiography. Samples were denatured by boiling in SDS sample buffer and subjected to electrophoresis on a denaturing 12.5% polyacrylamide gel. Following electrophoresis the gel was dried and exposed to X-ray film. Time of exposure to UV light (minutes) is indicated above each gel lane.
  • FIG. 10 Glutaraldehyde crosslinking of GABPBl and GABPB2 subunits.
  • Bacterially synthesized proteins were incubated in phosphate buffered saline with varying concentrations of glutaraldehyde as indicated below each lane for five minutes at room temperature.
  • Samples were denatured by boiling in SDS sample buffer and subject to electrophoresis on a denaturing 10% polyacrylamide gel. Following electrophoresis the gel was stained with Coomassie brilliant blue. Proteins present in crosslinking reactions are indicated above each lane.
  • BN110 is a truncated version of GABPBl missing 110 NH 2 -terminal residues (see Fig. 12B) .
  • FIG. 11 DNA binding and complex formation assays of deleted variants of GABP ⁇ .
  • Top panel shows schematic representation of GABP ⁇ deletion mutants. Individual mutants are designated according to the position of deletion end points with respect to the amino acid sequence of GABP ⁇ . Prefix “N” designates deletions missing residues starting at the NH 2 - terminus of GABP ⁇ , prefix “C” designates deletions missing COOH-terminal residues, numbers indicate the position of the amino acid at which the deletion terminates.
  • the Ets-related segment of GABP ⁇ is highlighted by grey stippling.
  • Bottom panel shows an autoradiographic image of a non-denaturing gel used to separate DNA:protein complexes formed between variants of GABP ⁇ , GABPBl and a M P-labeled oligonucleotide that contained a GABP binding site.
  • Each variant of GABP ⁇ was tested for DNA binding in the absence and presence of GABPBl as indicated above the individual lanes.
  • FIG. 12 Complex formation and UV crosslinking assays of deleted variants of GABPBl.
  • Top panels of (A) and (B) show schematic representations of GABPBl deletion mutants. Individual mutants are designated according to the positions of deletion end points with respect to the amino acid sequence of GABPBl. Prefix “N” designates deletions from the NH 2 - terminus of GABPBl (B) , prefix "C” designates deletions missing COOH-terminal residues. Repeated sequences 33 or 32 amino acids in length that are related to similarly sized repeats in the Notch protein of Drosophila melano ⁇ aster are highlighted by grey stippling.
  • GABPBl and GABPB2 are indicated by black and hatched rectangles at their respective COOH-termini.
  • Deleted formation with GABP ⁇ as shown in the lower left panels of (A) and (B) .
  • Each deletion mutant was also tested in UV crosslinking assays shown in the lower right panels of (A) and (B) . All complex formation and UV crosslinking assays were conducted in the presence of GABP ⁇ and a "P-labeled oligonucleotide containing a GABP binding site.
  • FIG. 13 Model depicting complex formed between GABP and DNA.
  • the sequence of the GABP binding site consists of two hexanucleotide repeats of the sequence 5'-CGGAAR-3' as in lower part of Fig. 13.
  • Oval spheres directly above guanine residues of each hexanucleotide correspond to GABP ⁇ subunits
  • elongated rectangles correspond to 33 amino acid repeats of GABPB subunits.
  • Smaller rectangles shown at top correspond to the region of GABPBl required for formation of stable homodimers.
  • Circular arrows designate flexible regions inferred to occur between the dimer forming region of GABPBl and the 33-amino acid repeats located at its NH 2 -terminus.
  • the present invention relates to a DNA segment encoding all (or a unique portion) of the heteromeric transcriptional regulatory protein termed GA binding protein (GABP) .
  • GABP heteromeric transcriptional regulatory protein
  • the invention further relates to the encoded proteins (or polypeptides) .
  • a "unique portion” as used herein consists of at least five (or six) amino acids or, correspondingly, at least 15 (or 18) nucleotides.
  • the present invention further relates to a recombinant DNA molecule comprising the above DNA segment and to host cells transformed therewith.
  • the present invention relates to a DNA segment that encodes the entire amino acid sequence of GABP ⁇ , GABPBl or GABPB2 given in Figure 2 (the specific DNA segments given in Figure 2 being only examples) , or any unique portion thereof.
  • DNA segments to which the invention relates also include those encoding substantially the same protein subunits as shown in Figure 2 , including, for example, allelic and species variations thereof and functional equivalents of the amino acid sequences of Figure 2.
  • the invention further relates to a DNA segment substantially identical to one of the subunit sequences shown in Figure 2.
  • the invention also relates to nucleotide fragments complementary to such DNA segments. Unique portions of the DNA segment, or complementary fragments, can be used as probes for detecting the presence of respective complementary strands in DNA (or RNA) containing samples.
  • the present invention further relates to GABP, and subunits thereof, substantially free of proteins with which it is normally associated, and more especially, to unique peptide fragments of the subunits of that protein.
  • the GABP protein (or functionally equivalent variations thereof) , or peptide fragments thereof, to which the invention relates, also includes those which are chemically synthesized using known methods.
  • the proteins and peptides of the present invention can be modified, for example, phosphorylated, or unmodified.
  • the present invention also relates to recombinantly produced GABP, or subunits thereof, having the amino acid sequence shown in Figure 2 or functionally equivalent variation thereof.
  • the recombinantly produced protein can be modified, for example phosphorylated, or unmodified.
  • the present invention more particularly, relates to recombinantly produced unique peptide fragments of GABP subunits.
  • the present invention also relates to a recombinant DNA molecule (or construct) and to a host cell transformed therewith.
  • a recombinant DNA molecule comprising a vector and a DNA segment encoding at least one GABP subunit, or a unique portion thereof, can be constructed.
  • Vectors suitable for use in the present invention include plasmid and viral vectors.
  • the vector can be selected so as to be suitable for transforming prokaryotic or eukaryotic cells.
  • the recombinant molecule includes a promoter operably linked to the GABP encoding segment.
  • the recombinant DNA molecule of the invention can be introduced into appropriate host cells by one skilled in the art using method well known in the art.
  • Suitable host cells include prokaryotic cells, such as bacteria, lower eukaryotic cells, such as yeast, and higher eukaryotic cells, such as mammalian cells. These cells can serve as a source of GABP when cultured under appropriate conditions.
  • the amino acid sequences of GABPBl and GABPB2 contain four repeats of a related amino acid sequence located at the NH 2 -termini of both subunits (Fig. 5) .
  • the first two repeats are 32 amino acids in length and the second two contain 33 amino acids.
  • Similar repeats occur in the Notch protein of Drosophila melanoqaster (Wharton et al, Cell 43, 567 (1985); I. Greenwald, ijid. , 583 (1985)), and the Linl2 and Glpl proteins of Caenorhabditis ele ⁇ ans
  • the two subunits of GABP exhibit primary sequence motifs typical of proteins normally found in different cellular compartments. Accordingly, transcriptional regulatory proteins, such as members of the Ets family, might interact with membrane bound proteins that contain the 33-amino acid repeats present in GABPB.
  • the Notch, Glpl and Linl2 proteins might sequester transcription factors at the plasma membrane which could be released in response to appropriate extracellular signaling events.
  • the cytoplasmic segments of these transmembrane proteins might be proteolyzed in response to an extracellular signal, allowing the 33-amino acid repeats to be translocated to the nucleus where they could abet the action of a second subunit. Either scenario would offer a direct pathway of signal transduction.
  • GABP (20 ⁇ g) was purified from rat liver nuclear extracts and cleaved with trypsin. Proteolyzed fragments were separated by high performance liquid chromatography (HPLC) , recovered, and subjected to gas-phase amino acid sequencing (Fig. 1) . Partial sequences were derived from 13 tryptic peptides. Degenerate oligonucleotides capable of encoding four of the thirteen peptide sequences were synthesized and used as hybridization probes to screen an adipocyte cDNA library. Degenerate oligonucleotides were labeled with "P using polynucleotide kinase.
  • SSC basic sodium chloride/sodium citrate
  • the insert of this recombinant was sequenced and found to contain an opening reading frame that encoded a protein of 454 amino acids (Fig. 2A) .
  • the predicted molecular weight of this polypeptide (51.3 kD) corresponded to the size of the GABP ⁇ subunit purified from rat liver nuclei (LaMarco et al, Genes Dev. 3, 1372 (1989); Fig. 1).
  • Inspection of the deduced amino acid sequence revealed segments that corresponded to eight of the 13 peptides isolated by trypsin digestion of intact GABP. On the basis of the latter two observations, this 454 residue polypeptide was tentatively identified as GABP ⁇ .
  • oligonucleotides capable of encoding two of the tryptic peptide sequences not present in GABP ⁇ were synthesized and used as hybridization probes to search for a cDNA clone that encoded GABPB (Lee, Mol. Endocrinol. 4, 1034 (1990) ) .
  • Kinased oligonucleotide probes were hybrided in 6X SSC, IX Denhardt's, 0.05% sodium pyrophosphate, and 100 ⁇ g/ml yeast tRNA at 50°C for 14 hours. Washing conditions were 6X SSC, 0.05% sodium pyrophosphate at 55°C.
  • a total of five clones were isolated that hybridized with both oligonucleotide probes.
  • Four of the clones were approximately 2.6 kb and differed only slightly in the length of the 5' untranslated region; these cDNA clones encoded GABPBl.
  • the fifth cDNA clone was approximately 1.4 kb and differed from the other four at its 3' end; this cDNA clone encoded GABPB2.
  • Four additional cDNA clones corresponding to GABPB2 were subsequently identified. Five recombinant bacteriophage were identified according to their capacity to hybridize with both oligonucleotide probes.
  • One of the cDNA clones differed at the 3• end from the other four.
  • the largest cDNA insert of the four (2.6 kb) and the variant (1.4 kb) were sequenced (Fig. 2B) . Both DNA sequences revealed long open reading frames specifying highly similar polypeptides.
  • the open reading frames of both polypeptides contained segments that corresponded to the two tryptic peptides used to design hybridization probes.
  • the predicted molecular weights of the two polypeptides (41.3 and 37 kD) corresponded closely with the size of the GABPB subunit purified from rat liver nuclei (LaMarco et al. Genes Dev. 3, 1372 (1989) ; Fig. 1) .
  • the 41 kD polypeptide was therefore provisionally designated as GABPBl and the 37 kD polypeptide as GABB2.
  • RNA blot assays were used to determine the sizes and tissue distributions of mRNA encoding GABP ⁇ , GABPBl and GABPB2 (Fig. 3) .
  • the cDNA corresponding to GABP ⁇ identified three mRNAs of roughly 5.0, 2.8 and 2.6 kb, which were expressed in a variety of tissues.
  • the GABP ⁇ cDNA which consisted of slightly less than 2.0 kb (Fig. 2A) , represents a partial copy of any of the three mRNAs.
  • Two mRNAs measuring 2.7 and 1.5 kb were identified in northern blots probed with GABPBl cDNA.
  • those encoding GABPBl had a wide tissue distribution.
  • the cDNAs that encoded GABPBl and GABPB2 measured 2.6 and 1.4 kb, respectively (Fig. 2B) , they probably represent nearly full- length copies of the respective mRNAs. Moreover, because the nucleotide sequences of the two cDNAs are identical from their respective 5' termini to the point of abrupt divergence 1.1 kb internal to the mRNA, they likely represent alternatively spliced transcripts derived from the same gene. Consistent with this interpretation is the presence of a potential splice donor site (AG dinucleotide) immediately preceding the point of divergence.
  • reticulocyte lysates were programmed with RNA synthesized from the cDNAs that encode GABPB, GABPBl and GABPB2. Each RNA was translated to form a protein product of the expected size (Fig. 4A) . Individual lysates or mixtures thereof were tested for DNA binding to a fragment from the HSV1 ICP4 promoter that contained three GA repeats. Protein:DNA mixes were subjected to electrophoresis on nondenaturing polyacrylamide gels to separate free DNA from that complexed with protein.
  • Reticulocyte lysate that had not been programmed with exogenous RNA contained protein(s) capable of forming a complex with the oligonucleotide probe that migrated more rapidly than the complex formed by GABP.
  • protein(s) capable of forming a complex with the oligonucleotide probe that migrated more rapidly than the complex formed by GABP.
  • specific protein:DNA complexes were not observed when lysates programmed with GABP ⁇ , GABPBl, or GABPB2 were tested in electrophoretic mobility shift assays.
  • no new DNA binding activity was observed with lysate that had been used to co-translate RNAs encoding GABP ⁇ and GABPB2.
  • cDNA copies of the mRNAs that encode GABP ⁇ and GABPBl were introduced into bacteriophage T7 based vectors that allowed synthesis of the corresponding proteins in Escherichia coli (Studier et al, J. Mol. Biol., 189, 113 (1986)). Polymerase chain reaction was used to introduce a Bam HI site at the 5' end of the open reading frames encoding GABP ⁇ or GABPBl. cDNAs lacking the 3• untranslated region were inserted into a modified pT5 vector, which adds two amino acids (gly-ser) at the NH 2 -terminus of the encoded protein. Each subunit was expressed and purified using conventional chromatographic techniques.
  • the ammonium sulfate pellet was resuspended in 25 ml buffer B (25 mM Tris-HCl, pH 8.0, 0.75 mM EDTA, 10% (v/v) glycerol, 1 mM DTT) with 75 mM NaCl and dialyzed against the same buffer.
  • the dialysate was loaded onto a column of Q-Sepharose Fast Flow (Pharmacia) .
  • GABP ⁇ was eluted with a 75-500 mM NaCl gradient in buffer B. Peak fractions were pooled, dialyzed against buffer B and loaded onto a salmon sperm DNA-sepharose column.
  • GABP ⁇ was eluted with a 0-400 mM NaCl gradient. GABP ⁇ was judged by Coomassie Blue staining of SDS polyacrylamide gels to account for greater than 90% of the total protein.
  • GABPBl was solubilized from the particulate fraction of bacterial extracts by sonication in buffer A supplemented with 7 M urea. The urea solubilized fraction was dialyzed against buffer B with 75 mM NaCl and centrifuged at 16,300 x g for one hour. The supernatant was applied to a Q- Sepharose column and eluted with a gradient of 75- 500 mM NaCl. GABPBl was judged to account for greater than 90% of total protein by Coomassie Blue staining of SDS-polyacrylamide gels.
  • the DNA binding properties of the two individual polypeptides and mixtures thereof were first studied by gel retardation using a DNA substrate derived from the enhancer of an immediate early gene of herpes simples virus. Consistent with earlier studies (LaMarco et al, Genes Dev., 3, 1372 (1989)), binding was not observed when DNA was incubated with either of the isolated subunits. When GABP ⁇ and GABPBl were incubated with DNA simultaneously, a DNA:protein complex exhibiting substantially retarded mobility relative to free DNA was observed (Fig. 6, left panel) .
  • Antiserum specific to GABPBl did not affect the mobility of complexes formed between GABP ⁇ DNA, but retarded the complex formed in the presence of both subunits.
  • Polyclonal antisera were generated by rejecting rabbits with purified GABP ⁇ or GABPBl. Antisera were added to gel shift reactions at a dilution of 1:20. Pre- immune sera did not effect the migration of protein:DNA complexes.
  • the HSVl-derived DNA fragment used in binding assays of GABP contains three imperfect repeats of the hexanucleotide sequence 5'-CGGAAR- 3• , which were shown in earlier studies to be protected from DNase I digestion when bound by GABP (Trieenberg et al. Genes Dev.
  • DNase I footprinting assays were performed using bacterially synthesized proteins under conditions that allowed interaction of GABP ⁇ alone. As shown in Fig. 7A, GABP ⁇ was capable of protecting .the repeated hexanucleotide motifs from DNase I digestion when added at a concentration of 0.15 nM. When the GABPBl subunit was added, protection was observed at a 10-fold more dilute concentration of GABP ⁇ (0.015 nM) . In addition, the pattern of nuclease protection was extended slightly beyond the adenine residues of the third repeat.
  • the less retarded of the two bands is interpreted to represent a complex wherein GABP ⁇ is associated with only one of the two hexanucleotide repeats, while the more retarded complex is interpreted to contain GABP ⁇ subunits associated with two hexanucleotide repeats. Binding assays that tested DNA probes containing a single hexanucleotide repeat supported this interpretation. When incubated with GABP ⁇ and assayed in low ionic strength gels, such DNA probes generated only one retarded complex.
  • UV crosslinking was performed using an oligonucleotide composed of a GA binding site flanked by 10 bp of non-specific sequence ((5' AACCAAGCTTGCGGAACGGAAGCGGAAACCG 3') corresponding to residues located between 280 and 300 bp upstream of the herpes simplex virus gene encoding ICP4. Oligonucleotides were labeled to high specific activity by fill-in reaction with the Klenow fragment of DNA polymerase I in the presence of all four 32 P-labeled dNTPs. DNA binding reactions were performed as described in a 96-well culture dish, followed by exposure to ultraviolet light.
  • the Stokes radius was calculated from a plot of (-log K av ) n versus Stokes radius (Ackers, Adv. Prot. Chem. 24, 343 (1970)).
  • GABP ⁇ eluted as a single peak at 15.2 ml; GABPBl at 14.0 ml; GABPB2 at 15.8 ml.
  • GABPBl Gel filtration and gradient sedimentation assays indicated that GABPBl might exist as a dimer. This interpretation was tested using glutaraldehyde crosslinking assays. Bacterially expressed GABPBl and GABPB2 were exposed to glutaraldehyde and subjected to electrophoresis on a denaturing polyacrylamide gel. Incubation of GABPBl with glutaraldehyde led to the formation of a second polypeptide band exhibiting an apparent molecular weight roughly double that of the monomeric form of the protein (Fig. 10) . Similar experiments conducted with GABPB2 failed to yield an analogous product.
  • GABP ⁇ should contain at least two functional components, one that facilitates DNA binding and another that allows complex formation with GABPB.
  • the GABPBl polypeptide should contain at least three components, facilitating self- dimerization, heterodimerization with GABP ⁇ , and direct contact with some part of the purine-rich DNA substrate. Recombinant copies of the genes that encoded each subunit were systematically deleted to localize these components. Deletion mutants of GABP ⁇ were generated by polymerase chain reaction and expressed in pT5 as described (Breeden et al. Nature 329, 651 (1987)). Soluble bacterial extracts containing deleted variants of GABP ⁇ were used for binding reactions.
  • NH 2 -terminal deletions of GABPBl were generated by exonuclease III digestion, followed by digestion with SI nuclease and ligation of Bam HI linkers. All deletions were sequenced and subcloned into the appropriate pET3 vector (Rosenberg et al., Gene 56, 125 (1987)) to maintain the proper reading frame. COOH-terminal deletions were generated using 3' deletions of the cDNA inserted in Bluescript (Stratagene) by subcloning
  • GABPBl Although about 70% of GABPBl could be deleted from its COOH-terminus without eliminating interaction with GABP ⁇ and DNA, removal of only a small segment from the NH 2 -terminus resulted in deleterious effects.
  • cDNA clones encoding human GABP alpha, beta3 and beta4 were isolated by screening a human fetal brain cDNA library.
  • cDNAs for human GABP betal and beta2 were isolated from a HeLa cell cDNA library.
  • the probes used to screen for human GABP alpha were the 865 bp Ava 1-Sst 1 and 678 bp Bam Hl- Sst 1 fragments of the mouse GABP alpha cDNA.
  • the probe used to isolate the human beta was an 850 base pair fragment from the 5' end of the mouse GABP beta2 cDNA.
  • 1A human GABPB, Eco RI fragment common to all beta isoforms, in Bluescript KS+, isolated from HeLa cDNA library.
  • A human GABPBl Eco RI fragment of Bluescript KS+, isolated from HeLa cDNA library.
  • 5A human GABPB2, Eco Rl fragment in Bluescript KS+, isolated from HeLa cDNA library.
  • F human GABPB3, Eco Rl-Xho l fragment in Bluescript SK-, isolated from human fetal brain cDNA library.
  • J human GABPB4, Eco Rl- Xho 1 fragment in Bluescript SK-, isolated from human fetal brain cDNA library.
  • G human GABP ⁇ , Eco Rl- Xho 1 fragment in Bluescript SK-, from human fetal brain cDNA library.

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Abstract

L'invention concerne une protéine de liaison GA ainsi que des segments d'ADN codant ses sous-unités.
PCT/US1992/006748 1991-08-16 1992-08-17 Proteine de liaison d'adn de region purique WO1993004166A1 (fr)

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WO2001060408A3 (fr) * 2000-02-17 2002-08-29 Sci Pharmaceuticals Inc Micro-competition et maladie humaine

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WO1990005745A1 (fr) * 1988-11-18 1990-05-31 The General Hospital Corporation Proteine de liaison stimulatrice de transcription reagissant au monophosphate d'adenosine cyclique
WO1991007423A1 (fr) * 1989-11-17 1991-05-30 Arch Development Corporation Proteines de liaison d'adn comprenant un recepteur d'androgene
EP0449170A1 (fr) * 1990-03-30 1991-10-02 BEHRINGWERKE Aktiengesellschaft Protéine d'attachement cellulaire ayant une affinité pour la région régulatrice du papilloma virus humain de type 18

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001060408A3 (fr) * 2000-02-17 2002-08-29 Sci Pharmaceuticals Inc Micro-competition et maladie humaine

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