Reduced Binding Activity Research Articles

Molecular characterization of the DNA-binding and dimerization domains of the bZIP transcription factor, EmBP-1.

The wheat basic-leucine zipper (bZIP) DNA-binding protein EmBP-1 has been implicated in the mechanisms of abscisic acid (ABA) mediated gene regulation. Sequence and structural homology to the yeast bZIP protein GCN4 has been used to predict the location of the functional domains of EmBP-1. In order to test these predictions, the presumptive DNA-binding and dimerization domains of EmBP-1 were mapped by producing a series of truncated protein fragments and functionally testing them in vitro. Deletion of 5 amino acids of the predicted basic domain resulted in a loss of all DNA-binding activity. A fragment containing all six leucine repeat elements showed strong DNA-binding activity. Sequential deletion of the leucine repeat elements resulted in first an increase in DNA-binding activity (-L6 and -L5) followed by a reduction in binding activity (-L4) and eventually complete elimination of all detectable DNA-binding activity (-L3 and -L2). This demonstrates the importance of an intact leucine zipper domain of at least 4 repeat elements for efficient DNA-binding. The smallest polypeptide that retained DNA-binding activity is a fragment spanning amino acid residues 248-308 (ca. 8.4 kDa) consisting of minimal basic and leucine zipper domains. Dimerization of EmBP-1 was demonstrated by co-translation of fragments of differing molecular weights and identification of a DNA-protein complex with intermediate mobility to that produced by each fragment alone. A unique leucine-proline repeat element found N-terminal to the DNA-binding domain of EmBP-1 does not appear to play a role in DNA-binding or dimerization. These results confirm the locations of the functional domains of EmBP-1 predicted by similarity to GCN4. The high degree of functional conservation of the bZIP proteins spanning organisms from plants to fungi highlights the ancient origin of this class of transcription factors and of the mechanisms of gene regulation in which they participate.

Monoclonal antibody FDC-6 exhibits binding to human plasma fibronectin: A caveat for cervicovaginal oncofetal fibronectin testing?

Monoclonal antibody FDC-6 has been used clinically to measure cervicovaginal fibronectin and to predict patients at risk for preterm labor. Because bleeding is often a complicating factor during pregnancy, the aim of this study was to determine whether FDC-6 reactive fibronectin is present in normal human plasma. Quantitative Western immunoblots and a commercially available enzyme-linked immunosorbent assay were used to measure FDC-6 binding to nonpregnant human and bovine plasma fibronectin. FDC-6 binding to whole sera was also assessed, as was the effect of enzymatic deglycosylation of human plasma fibronectin. Both assays demonstrated measurable FDC-6-reactive fibronectin in nonpregnant human blood, with plasma concentrations ranging from 3 to 12 micrograms/ml (1% to 4% of total plasma fibronectin). FDC-6 had no detectable binding activity to bovine plasma fibronectin. De-O-glycosylated human plasma fibronectin had markedly reduced binding activity for FDC-6. FDC-6 binds specifically to a clinically significant percentage of circulating O-glycosylated human plasma fibronectin isoforms that are not associated with pregnancy. This caveat should be considered when pregnant patients are evaluated for the presence of FDC-6 reactive oncofetal fibronectin in the cervix or vagina.

Identification of Interactive Sites of Proteins and Protein Receptors by Computer-Assisted Searches for Complementary Peptide Sequences

Protein sites important for receptor binding have been identified in several systems by searching for protein/receptor stretches characterized by hydropathic anti-complementarity. A computer-assisted method [SITESEARCH] has been developed to identify protein sites responsible for receptor recognition, once the amino acid sequences of the protein ligand and its receptor are available. The software first determines the hydropathic profiles of the two polypeptide chains under investigation, and then compares profiles of preselected length, determining at the same time the degree of their hydropathic complementarity. The procedure is repeated until all the different segments in the two chains are compared. Fragments characterized by the maximal level of hydropathic complementarity are selected as putative binding sites. This approach has been initially applied to the interleukin-1β (IL-1β)/receptor type I case. SITESEARCH identified residues 88-99 in IL-1β and 151-162 in the receptor as the sequence pair characterized by the maximal level of hydropathic complementarity. These fragments, once produced by chemical synthesis, have displayed specific recognition properties for each other, as detected by solid-phase binding assays. The IL-1β sequence identified corresponds to a highly exposed part of the protein molecule, and substitution of this sequence with another of the same length but with different hydropathic characteristics generated mutants with drastically reduced binding activity to the receptor. Mutations in this sequence did not alter the protein biological activity, thus suggesting the structural integrity of the mutants. Cumulatively, these results validate the SITESEARCH prediction, suggesting that IL-1β sequence 88-99 is involved in at least a portion of the protein/receptor binding site.

Two non-contiguous regions contribute to nidogen binding to a single EGF-like motif of the laminin gamma 1 chain.

High affinity binding of nidogen to laminin is mediated by an EGF-like repeat gamma 1III4 of the mouse laminin gamma 1 chain and has now been restricted to two short noncontiguous regions of its 56 residue sequence by use of synthetic peptides and recombinant mutants. Disulfide loop a,b of the repeat and a modified loop a,c could completely inhibit binding, with a 5000-fold or 300-fold reduced affinity respectively. Synthetic loops c and d lacked inhibitory activity. Some binding contribution of Tyr819 in loop c was, however, shown by mutation and side chain modification. Together with studies of loop chimeras, this indicated a distinct cooperativity between the two binding sites. The major binding site of loop a was localized to the heptapeptide NIDPNAV (position 798-804). A change of Asp800 to Asn or Ala803 to Val caused a strong reduction in binding activity, while only small effects were observed for the changes Pro801 to Gln and Ile799 to Val. The latter replacement corresponds to the single substitution found in the same region of the Drosophila laminin gamma 1 chain. However, the changes Asn802 to Ser or Val804 to Ser, both known to exist in the laminin gamma 2 chain, were deleterious mutations. This demonstrated conservation of binding structures in laminins of distantly related species, but not between homologous chains of laminin isoforms.

Effect of the Order of Antibody Variable Regions on the Expression of the Single-Chain HyHEL10 Fv Fragment in Escherichia coli and the Thermodynamic Analysis of Its Antigen-Binding Properties

In order to physically stabilize the Fv fragment of anti-lysozyme monoclonal antibody, HyHEL10, the variable domains were linked covalently with a flexible linker. A marked difference in the level of expression in E. coli was observed between VH-linker-VL (scFvHL) and VL-linker-VH (scFvLH). The highly expressed scFvLH was purified by a single step of affinity chromatography from the culture supernatant with a typical yield of 3-5 mg per liter of culture. This HyHEL10 scFvLH showed reduced binding activity toward its antigen, HEL, in comparison with Fv. Thermodynamic study showed that this reduced activity was due to entropic loss upon binding to its antigen, although this interaction between scFvLH and its antigen was enthalpically favorable.

A partial structural repeat forms the heterodimer self-association site of all beta-spectrins

The self-polymerization of alpha beta-spectrin heterodimers to form tetramers and higher oligomers is central to its role as a membrane stabilizer and organizer. Mutations near the amino terminus of alpha I sigma 1-spectrin or the COOH terminus of beta I sigma 1-spectrin often lead to profound impairment heterodimer polymerization and to hemolytic disease of varying severity. Previous studies using an 80-kDa univalent fragment of alpha I sigma 1-spectrin have established that the amino-terminal segment of alpha I sigma 1-spectrin mediates the association of the alpha subunit with either intact heterodimers or with isolated beta-spectrin (beta I sigma 1). However, the nature of the self-association site in beta-spectrin has remained unclear. In the present study, native beta-spectrin and recombinant beta-spectrin peptides representing COOH-terminal portions of two alternative transcripts of the gene on chromosome 2 (beta I sigma 1 or "erythrocyte" spectrin and beta I sigma 2 or "muscle" spectrin), and one transcript of the gene on chromosome 14 (beta II sigma 1 or "beta G-fodrin") have been examined for their ability to bind either intact alpha beta-spectrin or the alpha I-spectrin 80-kDa univalent fragment. Deletion of the nonhomologous beta-spectrin sequence downstream of repeat 17 (spectrin domain III) had no discernible effect on binding. Truncations proximal to codon 2085 of beta I sigma 1-spectrin demonstrated a precipitous loss of activity, accounted for by a loss of both binding affinity and capacity. Further truncations to repeat 16 (codon 1979) restored binding activity to levels approximating that of the intact molecule. Repeat 16/17 and 17/16 chimeras displayed reduced binding activity. Collectively, these data indicate that the beta-subunit self-association site is highly sensitive to conformation, involves widespread interactions within the 17th repeat unit, is largely independent of sequences in domain III, and can be recreated by the deletion of all residues distal to the COOH end (codon 1979) of the 16th and presumably other spectrin sequence repeat units. All beta-spectrins appear to use this binding motif, regardless of the nature of the nonhomologous sequence in domain III.

Determination of a Monoclonal Antibody Binding Activity Using ImmunoDetection

A method for measuring the binding of chimeric IgG BR-96 to the Lewis Y antigen using the ImmunoDetection technology has been developed. The procedure is rapid (2 min), highly reproducible (<5% CV), and has excellent correlation with the Lewis Y and anti-idiotypic enzyme immunoassays. Samples that were degraded by heat or by repeated freeze-thaw cycles showed reduced binding activity. There was minimal cross-reactivity with other proteins typically found in hybridoma media or with another chimeric IgG directed against a different antigen (L6). The fact that this assay can be performed using conventional HPLC equipment makes it especially attractive because it can be completely automated using equipment readily available.

A cAMP-regulated RNA-binding protein that interacts with phosphoenolpyruvate carboxykinase (GTP) mRNA.

Cyclic-AMP stabilizes phosphoenolpyruvate carboxykinase (GTP) (PEPCK) mRNA against degradation. To investigate the mechanism of this effect, RNA mobility shift assays were used to determine the interaction of cellular proteins with specific domains from the mRNA. We report here the identification of a protein with an affinity for sequences of PEPCK mRNA with a predicted stem-loop structure. RNA-protein complex formation was significantly reduced if the double-stranded RNA probe was preheated to 90 degrees C. The RNA-binding protein did not bind to the hairpin structure of poly(rI)-poly (rC), indicating some degree of sequence specificity and that the RNA-binding protein is not the interferon-induced double-stranded RNA-activated protein kinase. The binding activity was contained in the cytosolic fraction (100,000 x g) of rat hepatoma FTO-2B cells and was significantly enhanced by high concentrations of KCl. Chromatography on an anion exchanger separated the binding activity from a factor which, upon reconstitution, inhibited the interaction with the RNA probe. Incubation of cells with cAMP resulted in a 3-4-fold decrease in the activity of the RNA-binding protein. An inhibition in complex formation was observed with extracts as early as 60 min after exposure of cells to cAMP. Liver extracts from rats starved for 72 h also had reduced binding activity compared to extracts from fed animals. Cellular extracts treated with alkaline phosphatase exhibited an elevated level of complex formation. An analysis by SDS-polyacrylamide gel electrophoresis of the RNA-protein complex after ultraviolet light cross-linking demonstrated that the RNA-binding protein had a molecular mass of approximately 100 kDa. On the basis of these results, we suggest that liver cells contain a protein whose interaction with PEPCK mRNA is regulated by cAMP-dependent phosphorylation and which may be responsible for the cAMP-mediated control of PEPCK mRNA half-life.

Homocysteine, a thrombogenic agent, suppresses anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells.

Previous studies showed that homocysteine, a thrombo-atherogenic and atherogenic agent, inhibits an endothelial thrombomodulin-protein C anticoagulant pathway. We examined whether homocysteine might affect another endothelial anticoagulant mechanism; i.e., heparin-like glycosaminoglycan-antithrombin III interactions. Incubations of porcine aortic endothelial cell cultures with homocysteine reduced the amount of antithrombin III bound to the cell surface in a dose- and time-dependent fashion. The inhibitory effect was observed at a homocysteine concentration as low as 0.1 mM, and the maximal suppression occurred at 1 mM of homocysteine after 24 h. In contrast with a marked reduction in the maximal antithrombin III binding capacity (approximately 30% of control), the radioactivity of [35S]sulfate incorporated into heparan sulfate on the cell surface was minimally (< 15%) reduced. The cells remained viable after homocysteine treatment. Although neither net negative charge nor proportion in total glycosaminoglycans of cell surface heparan sulfate was altered by homocysteine treatment, a substantial reduction in antithrombin III binding capacity of heparan sulfate isolated from homocysteine-treated endothelial cells was found using both affinity chromatography and dot blot assay techniques. The antithrombin III binding activity of endothelial cells decreased after preincubation with 1 mM homocysteine, cysteine, or 2-mercaptoethanol; no reduction in binding activity was observed after preincubation with the same concentration of methionine, alanine, or valine. This sulfhydryl effect may be caused by generation of hydrogen peroxide, as incubation of catalase, but not superoxide dismutase, with homocysteine-treated endothelial cells prevented this reduction, whereas copper augmented the inhibitory effects of the metabolite. Thus, our data suggest that the inhibited expression of anticoagulant heparan sulfate may contribute to the thrombogenic property resulting from the homocysteine-induced endothelial cell perturbation, mediated by generation of hydrogen peroxide through alteration of the redox potential.

Loop substitution as a tool to identify active sites of interleukin-1 beta

By computer analysis of the amino acid sequence of human interleukin-1 beta (IL-1 beta) and of the human type I IL-1 receptor (IL-1RI), we have identified two hydropathically complementary peptides (Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989) J. Biol. Chem. 264, 11252-11257) capable of binding to each other. The sequence of the IL-1 beta peptide corresponds to that of residues 88-99 (loop 7 of the crystal structure of mature IL-1 beta) of mature IL-1 beta, one of the exposed and highly charged regions of the molecule. The substitution of this loop with an amino acid sequence of the same length but different hydropathic profile generates a mutant with drastically reduced binding activity to IL-1RI. In contrast, the binding affinity to the type II IL-1R (IL-1RII) is the same as that of wild type IL-1 beta. The results show that 1) loop 7 is part of the binding site of IL-1 beta to IL-1RI, but not to IL-1RII. 2) The structure of the mutant protein is not grossly altered except locally at the position of the substituted loop. 3) The substitution of amino acids by site-directed mutagenesis of the loop 7 region generates mutants with binding affinity constants slightly lower than that of wild type IL-1 beta and not comparable to that of the loop substitution analogue. 4. All mutants analyzed, including the loop substitutions, are biologically active, confirming the structural integrity of the proteins. We propose a binding site in which the cooperation of several low energy bonds extended over a wide area results in a high affinity complex between IL-1 and the type I receptor.

Reduced binding activity of transcription factor NF-κB accounts for MHC class I repression in adenovirus type 12 E1-transformed cells

The early region 1 (E1) of human adenovirus (Ad) type 12 represses the expression of major histocompatibility (MHC) Class I genes in transformed primary rodent cells. In this paper we show that both NF-κB and KBF1 (p50 dimer) binding activity to the H2TF1 element in the Class I promoter is reduced in Ad12-13S-E1A-transformed cells compared to Ad5E1- or Ad12-12S-E1A-transformed cells. Consistently, in Ad 12E1A-13S-transformed cells the H2TF1 element does not contribute to transcriptional activity in transient expression assays, whereas it does contribute in Ad12E1A-12S-transformed cells. Therefore, the most likely explanation is that reduced binding of NF-κB and KBF1 to the H2TF1 element accounts for the down-regulation of MHC Class I expression in Ad12E1- and Ad12E1A-13S-transformed cells.

A single amino acid mutation in CDR3 of the 3-14-9 L chain abolished expression of the IDA 10-defined idiotope and antigen binding.

The ABPC 48 myeloma protein and the 3-14-9 mAb derive their V region genes from the same VH and V kappa gene families. They also share a cross-reactive idiotope defined by the anti-Id mAb IDA 10. Whereas ABPC 48 is specific for bacterial levan, 3-14-9 showed a significant Ag-binding activity to aminophenyl-beta-N-acetylglucosaminide (AZO). In order to define the molecular basis of idiotope expression and Ag-binding activity, we have cloned the genes encoding the 3-14-9 H and L chain V region genes, generated antibodies that carry mutations within the L chain genes, by site-directed mutagenesis, and investigated the effects of those mutations with respect to IDA 10 idiotope expression and binding to AZO. Our findings show that, whereas expression of the IDA 10-defined idiotope requires association of both the H and L chains, a single change (glycine to phenylalanine) at position 91 in the third complementarity-determining region of the L chain abolished both idiotope expression and Ag-binding activity. In addition, a L chain change of alanine to threonine at position 25 allowed idiotope expression to some extent but significantly reduced binding activity to AZO. These data suggest that a single amino acid change can play a crucial role in the functional activity and structural integrity of antibodies.

Localization of a major nidogen‐binding site to domain III of laminin B2 chain

The large pepsin fragments P1 and P1X, which comprise most of the rod-like domains III of the three short arms of laminin from the mouse Engelbreth-Holm-Swarm tumor, possess full binding activity for nidogen in radioligand assays. Partial reduction (70-80%) of disulfide bonds in P1 did not reduce binding activity and allowed the separation of domain III segments originating from the A, B1 and B2 chains of laminin as demonstrated by sequence analysis. Only the B2 chain segment consisting of seven cysteine-rich repeats with similarity to epidermal growth factor showed substantial nidogen-binding activity. Further degradation of this component to an active 28-kDa fragment was achieved by a second pepsin digestion of partially reduced P1. This indicates that a major binding structure for nidogen is located within three or four cysteine-rich repeats occupying sequence positions 755 to about 920 in the B2 chain. The data also show that fragments P1 and P1X differ by the absence or presence of a large portion, domain IIIb, of the laminin A chain but are indistinguishable in nidogen binding.

The glucocorticoid hormone signal transduction pathway in mice homozygous for chromosomal deletions causing failure of cell type-specific inducible gene expression.

Wild-type newborn mice are characterized by the ability of certain liver-specific genes encoding various enzymes and mapping on different chromosomes to respond to glucocorticoid induction. Newborn mice homozygous for deletions at and around the albino locus on chromosome 7 fail to develop this competence for hormone-inducible gene expression even through they do show normal constitutive expression of the same genes. Studies of the glucocorticoid hormone signal transduction pathway reported here show identical expression of glucocorticoid receptor mRNA and protein in deletion homozygotes and normal littermates. Furthermore, the receptor interacts normally with the 90-kDa heat shock protein hsp90. Elevated glucocorticoid hormone levels in newborn deletion homozygotes, most likely resulting from their stressed condition, provide an explanation for the reduced binding activities of receptors reported previously. The elimination of receptors and hormones as direct targets of the chromosomal deletion effects suggests that the failure of inducible gene expression might reside in defective competence of the affected structural genes to respond to the hormonal stimulus.

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.

Thiopental and ether anaesthesia decrease the prostaglandin receptor density of the rat liver

Male Sprague-Dawley rats were killed either by decapitation or during anaesthesia with thiopental or diethylether by aortectomy. Livers were removed and liver plasma membranes were prepared using standard techniques. Direct binding experiments with 3H-PGE 1 and 3H-iloprost revealed heterogeneity of the binding sites (high and low affinity binding sites), whereas 3H-PGE 2 demonstrated only high affinity binding to the liver. The highest binding capacity for all radioligands was found for livers after decapitation. Livers obtained during anaesthesia showed a significantly (p < 0.05 to p < 0.001) lower binding capacity and binding affinity for 3H-PGE 1, 3H-PGE 2 and 3H-iloprost. The reduction in binding activity was more pronounced in livers obtained during inhalation than thiopental anaesthesia. Specific binding amounted to 82.1 ± 7% for 3H-PGE 2, 75.3 ± 9% for 3H-PGE 1 and 78.9 ± 8% for iloprost in livers obtained after decapitation. In livers obtained during anaesthesia specific prostaglandin binding was significantly (p < 0.01) decreased, again being more pronounced during inhalation than thiopental anaesthesia. These results suggest that some anaesthetics interfere with prostaglandin receptors of the liver.

Mr 46,000 mannose 6-phosphate receptor. The role of histidine and arginine residues for binding of ligand.

The chemical modification of histidine and arginine residues results in a loss of binding of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) to a phosphomannan affinity matrix (Stein, M., Meyer, J. E., Hasilik, A., and von Figura, K. (1987) Biol. Chem. Hoppe-Seyler 368, 927-936). Reversal of the modification or presence of mannose 6-phosphate during the modification partially restores or protects the binding activity, indicating that histidine and arginine residues contribute to the mannose 6-phosphate binding site. The 5 histidine and 8 arginine residues within the luminal domain of MPR 46, which contains the ligand binding site, were exchanged by site-directed mutagenesis. Only the conservative replacement of His-131 and Arg-137 by serine and lysine, respectively, results in a loss of binding activity without affecting other properties of the receptor such as the presence of intramolecular disulfide bonds, immunoreactivity, processing of N-linked oligosaccharides, formation of dimers, intracellular distribution, and surface expression. Conservative replacement of other histidine and arginine residues did not affect the binding activity. Nonconservative replacement of several arginine residues reduced binding activity and immunoreactivity, indicating that the loss of a positive charge at these positions alters the folding of MPR 46. We conclude from these results that His-131 and Arg-137 are essential for binding of ligands by MPR 46.

Comparisons of various acidic treatments of bovine serum on insulin-like growth factor-I immunoreactivity and binding activity

Untreated serum exhibited two forms of insulin-like growth factor-I (IGF-I)-binding protein complexes during gel chromatography: one of Mr 150,000 and the other of Mr 40,000-45,000. The majority of the immunoreactive IGF-I was associated with the Mr 150,000 complex. Following acid-ethanol extraction of serum, the binding activity at Mr 150,000 disappeared and a reduced binding activity appeared in the albumin size range. Acid incubation of serum was slightly less effective than acid-ethanol extraction in reducing the binding activity. Acid-ethanol-extracted or acid-incubated serum were parallel to IGF-I standard in the dose-response displacement of iodinated IGF-I. Gel filtration of serum with 1 mol acetic acid/l almost completely separated IGF-I and the binding proteins. Binding-protein fractions from gel filtration interfered with the immunoreactivity of IGF-I with its antibodies, causing a non-parallel displacement curve in the radioimmunoassay (RIA). Serum IGF-I could be isolated as a single peak by high performance C18 reverse-phase liquid chromatography (HPLC). The concentrations of IGF-I measured in bovine sera by RIA were similar between acid gel filtration and HPLC; the concentrations by acid-ethanol extraction and acid incubation being about 30% smaller than those measured with former methods. The lower concentration of IGF-I measured in bovine serum with acid-ethanol extraction or acid incubation appears to be due to interference of IGF-binding proteins not removed by either treatment.

Evidence for a Stem Cell-Specific Repressor of Moloney Murine Leukemia Virus Expression in Embryonal Carcinoma Cells

A negative regulatory element (NRE) spanning the tRNA primer-binding site (PBS) of Moloney murine leukemia virus (M-MuLV) mediates repression of M-MuLV expression specifically in embryonal carcinoma (EC) cells. We precisely defined the element by base-pair mutagenesis to an 18-base-pair segment of the tRNA PBS and showed that the element also restricted expression when moved upstream of the long terminal repeat. A DNA-binding activity specific for the M-MuLV NRE was detected in vitro by using crude EC nuclear extracts in exonuclease III protection assays. Binding was strongly correlated with repression in EC cells. Mutations within the NRE that relieved repression disrupted binding activity. Also, nuclear extracts prepared from permissive, differentiated EC cell cultures showed reduced binding activity for the NRE. These results indicate the presence of a stem cell-specific repressor that extinguishes M-MuLV expression via the NRE at the tRNA PBS.

Evidence for a stem cell-specific repressor of Moloney murine leukemia virus expression in embryonal carcinoma cells.

A negative regulatory element (NRE) spanning the tRNA primer-binding site (PBS) of Moloney murine leukemia virus (M-MuLV) mediates repression of M-MuLV expression specifically in embryonal carcinoma (EC) cells. We precisely defined the element by base-pair mutagenesis to an 18-base-pair segment of the tRNA PBS and showed that the element also restricted expression when moved upstream of the long terminal repeat. A DNA-binding activity specific for the M-MuLV NRE was detected in vitro by using crude EC nuclear extracts in exonuclease III protection assays. Binding was strongly correlated with repression in EC cells. Mutations within the NRE that relieved repression disrupted binding activity. Also, nuclear extracts prepared from permissive, differentiated EC cell cultures showed reduced binding activity for the NRE. These results indicate the presence of a stem cell-specific repressor that extinguishes M-MuLV expression via the NRE at the tRNA PBS.