Showing papers by "Laboratory of Molecular Biology published in 1993"
TL;DR: The cloning of a receptor for cannabinoids is reported that is not expressed in the brain but rather in macrophages in the marginal zone of spleen, which helps clarify the non-psychoactive effects of cannabinoids.
Abstract: THE major active ingredient of marijuana, Δ9-tetrahydrocannabi-nol (Δ9-THC), has been used as a psychoactive agent for thousands of years. Marijuana, and Δ9-THC, also exert a wide range of other effects including analgesia, anti-inflammation, immunosuppression, anticonvulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting1. The clinical application of cannabinoids has, however, been limited by their psychoactive effects, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of δ9-THC4,5, and more recently from the cloning of a gene encoding a G-protein-coupled receptor for cannabinoids6. This receptor is expressed in the brain but not in the periphery, except for a low level in testes. It has been proposed that the non-psychoactive effects of cannabinoids are either mediated centrally or through direct interaction with other, non-receptor proteins1,7,8. Here we report the cloning of a receptor for cannabinoids that is not expressed in the brain but rather in macrophages in the marginal zone of spleen.
4,782 citations
TL;DR: The structural constraints for the receptors are used to allocate particular helices to the peaks in the recently published projection map of rhodopsin and to propose a tentative three‐dimensional arrangement of the helices in G protein‐coupled receptors.
Abstract: G protein-coupled receptors form a large family of integral membrane proteins whose amino acid sequences have seven hydrophobic segments containing distinctive sequence patterns. Rhodopsin, a member of the family, is known to have transmembrane alpha-helices. The probable arrangement of the seven helices, in all receptors, was deduced from structural information extracted from a detailed analysis of the sequences. Constraints established include: (1) each helix must be positioned next to its neighbours in the sequence; (2) helices I, IV and V must be most exposed to the lipid surrounding the receptor and helix III least exposed. (1) is established from the lengths of the shortest loops. (2) is determined by considering: (i) sites of the most conserved residues; (ii) other sites where variability is restricted; (iii) sites that accommodate polar residues; (iv) sites of differences in sequence between pairs or within groups of closely related receptors. Most sites in the last category should be in unimportant positions and are most useful in determining the position and extent of lipid-facing surface in each helix. The structural constraints for the receptors are used to allocate particular helices to the peaks in the recently published projection map of rhodopsin and to propose a tentative three-dimensional arrangement of the helices in G protein-coupled receptors.
973 citations
TL;DR: An element near the 5' boundary of the chicken beta-globin domain that insulates a reporter gene from the activating effects of a nearby beta- globin locus control region (5'HS2) when assayed in the human erythroid cell line K562 is characterized.
940 citations
TL;DR: The determinants of the OB‐fold are described and a reinterpretation of the controversial structure of gene 5 ssDNA binding protein is suggested, which exhibits some topological and functional similarities with the OB•fold proteins.
Abstract: A novel folding motif has been observed in four different proteins which bind oligonucleotides or oligosaccharides: staphylococcal nuclease, anticodon binding domain of asp-tRNA synthetase and B-subunits of heat-labile enterotoxin and verotoxin-1. The common fold of the four proteins, which we call the OB-fold, has a five-stranded beta-sheet coiled to form a closed beta-barrel. This barrel is capped by an alpha-helix located between the third and fourth strands. The barrel-helix frameworks can be superimposed with r.m.s. deviations of 1.4-2.2 A, but no similarities can be observed in the corresponding alignment of the four sequences. The nucleotide or sugar binding sites, known for three of the four proteins, are located in nearly the same position in each protein: on the side surface of the beta-barrel, where three loops come together. Here we describe the determinants of the OB-fold, based on an analysis of all four structures. These proposed determinants explain how very different sequences adopt the OB-fold. They also suggest a reinterpretation of the controversial structure of gene 5 ssDNA binding protein, which exhibits some topological and functional similarities with the OB-fold proteins.
928 citations
TL;DR: It is demonstrated that native A68 does not bind to microtubules (MTs), yet dephosphorylated A68 regains the ability to bind to MTs, and phosphorylation of Ser396 may destabilize MTs in AD, resulting in the degeneration of affected cells.
846 citations
822 citations
TL;DR: A projection map of rhodopsin showing the configuration of the helices is presented to provide a framework to interpret data, not only for rhodopin but for other G-protein-coupIed receptors.
Abstract: LIGHT absorption by the visual pigment rhodopsin1,2 triggers, through G-protein coupling, a cascade of events in the outer segment of the rod cell of the vertebrate retina that results in membrane hyperpolarization and nerve excitation3–5. Rhodopsin, which contains 348 amino acids6–8, has seven helices that cross the disk membrane6,9 and its amino terminus is extracellular. A wealth of biochemical data is available for rhodopsin: 11-cis retinal is bound10 to lysine 296 in helix VII; glutamic acid 113 on helix III is the counterion to the protonated Schiff's base11,12; a disulphide bridge, cystine 110–187, connects helix III to the second extracellular loop e2 (refs 13, 14); the carboxy terminus has two palmitoylated cysteines forming a cytoplasmic loop i4 (ref. 15); three intracellular loops i2, i3 and i4 mediate activation of the heterotrimeric G protein transducin16,17; glutamic acid 135 and arginine 136 at the cytoplasmic end of helix III affect binding of transducin18. But to provide a framework to interpret these data, not only for rhodopsin but for other G-protein-coupIed receptors, requires the structure to be determined. Here we present a projection map of rhodopsin showing the configuration of the helices.
757 citations
TL;DR: The crystal structure of the fully specific complex between the DNA-binding domain from the estrogen receptor and DNA reveals how the protein recognizes its own half site sequence rather than that of the related glucocorticoid receptor, which differs by only two base pairs.
741 citations
TL;DR: The structure of the nicotinic acetylcholine receptor was determined by electron microscopy of tubular crystals of Torpedo postsynaptic membranes embedded in amorphous ice as mentioned in this paper.
717 citations
TL;DR: The structure of GH5, the globular domain of the linker histone H5, has been solved to 2.5 Å resolution by multiwavelength anomalous diffraction on crystals of the selenomethionyl protein, thereby providing a possible model for the binding ofGH5 to DNA.
Abstract: The structure of GH5, the globular domain of the linker histone H5, has been solved to 2.5 A resolution by multiwavelength anomalous diffraction on crystals of the selenomethionyl protein. The structure shows a striking similarity to the DNA-binding domain of the catabolite gene activator protein CAP, thereby providing a possible model for the binding of GH5 to DNA.
715 citations
TL;DR: Current evidence suggests that protein kinases or protein phosphatases with a specificity for serine/threonine-proline residues are involved in the abnormal phosphorylation of tau.
TL;DR: It is reported that mitochondria‐derived intermediates are not only cytotoxic but, in addition, function as signal transducers of TNF‐induced gene expression and serve as common mediators of the T NF‐cytotoxic and gene‐regulatory signaling pathways.
Abstract: Tumor necrosis factor (TNF) has cytotoxic and gene-inductive activities on several cell types. Previous studies on L929 fibrosarcoma cells have revealed that the mitochondrial electron transport system plays a key role in inducing TNF cytotoxicity, presumably by the formation of reactive oxygen intermediates (ROI). Here we report that mitochondria-derived intermediates are not only cytotoxic but, in addition, function as signal transducers of TNF-induced gene expression. The activation of NF kappa B, which fulfills an important role in TNF-induced gene transcription, could be blocked by interference with the mitochondrial electron transport system. Furthermore, antimycin A, a mitochondrial inhibitor that increases the generation of ROI, potentiated TNF-triggered NF kappa B activation. The dual role of mitochondria-derived intermediates in cytotoxicity and immediate-early gene induction of TNF was further substantiated by isolating L929 subclones which lacked a functional respiratory chain. This depletion of the mitochondrial oxidative metabolism resulted in resistance towards TNF cytotoxicity, as well as in inhibition of NF kappa B activation and interleukin-6 gene induction by TNF. These findings suggest that mitochondria are the source of second messenger molecules and serve as common mediators of the TNF-cytotoxic and gene-regulatory signaling pathways.
TL;DR: The structure of the segment 1 domain of gelsolin, a protein that fragments actin filaments in cells, is reported in complex with actin, providing a basis for understanding the origin of an amyloidosis caused by a gelsolini variant.
Abstract: The structure of the segment 1 domain of gelsolin, a protein that fragments actin filaments in cells, is reported in complex with actin. Segment 1 binds monomer using an apolar patch rimmed by hydrogen bonds in a cleft between actin domains. On the actin filament model it binds tangentially, disrupting only those contacts between adjacent subunits in one helical strand. The segment 1 fold is general for all segments of the gelsolin family because the conserved residues form the core of the structure. It also provides a basis for understanding the origin of an amyloidosis caused by a gelsolin variant.
TL;DR: It is shown that the translocation t(12;16)(q13:p11) in malignant myxoid liposarcoma can be a fusion of the CHOP dominant negative transcription factor gene with a novel gene, FUS, which can result in fusion of.
Abstract: The search for tumour–specific markers is one of the chief goals in cancer biology. We show that the translocation t(12;16)(q13:p11) in malignant myxoid liposarcoma can be a fusion of the CHOP dominant negative transcription factor gene with a novel gene, FUS, which can result in fusion of the FUS glycine–rich protein with the whole CHOP coding region. The data support the concept that protein fusion may commonly occur in solid tumours resulting in tumour–specific markers of potential clinical importance. The data also indicate the importance of transcription disruption in the pathogenesis of solid tumours.
TL;DR: It is shown here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and it is identified as one of the abnormal phosphorylated sites in Alzheimer disease.
Abstract: Tau is a neuronal phosphoprotein whose expression is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult brain, with the fetal isoform corresponding to the shortest of the adult isoforms. Phosphorylation of tau is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer disease, the six adult tau isoforms become abnormally phosphorylated and form the paired helical filament, the major fibrous component of the characteristic neurofibrillary lesions. We show here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and we identify it as one of the abnormal phosphorylation sites in Alzheimer disease. The abnormal phosphorylation of tau at Ser-202 in Alzheimer disease thus recapitulates normal phosphorylation during development.
TL;DR: Electro diffraction studies with two‐dimensional crystals of wild‐type bacteriorhodopsin and the Asp96‐‐>Gly mutant reveal the structure of the M intermediate, which occurs between the proton release and uptake steps of the photocycle.
Abstract: Structural changes are central to the mechanism of light-driven proton transport by bacteriorhodopsin, a seven-helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two-dimensional crystals of wild-type bacteriorhodopsin and the Asp96-->Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.
TL;DR: A model system, involving the lox-Cre site-specific recombination system of bacteriophage PI, to lock together the heavy and light chain genes from two different replicons within an infected bacterium is described.
Abstract: Antibody fragments, comprising paired heavy (VH) and light (VL) chain variable domains, can be displayed on the surface of filamentous bacteriophage, and rare phage (encoding antigen binding activities) selected by binding to antigen (1). The process mimics immune selection and has been used to make human antibody fragments in bacteria, without immunisation, by random combinatorial linkage (2) of diverse repertoires of VH and VL genes from lymphocytes (3, 4). Fragments with a range of binding specificities have been isolated with binding affinities in the range 10 M~'-10 M\" (for reviews see (5, 6)). However larger 'primary' repertoires of phage antibodies should allow higher affinity fragments to be isolated (7, 8). The size of phage antibody repertoires (10) is limited by the efficiency of transformation of E.coli. In principle, larger repertoires could be made by combinatorial infection, for example by transforming E. coli with a repertoire of heavy chains (encoded on plasmids) then infecting with a repertoire of light chains (encoded on phage) (9). Since infection is extremely efficient, and most E.coli cells in an exponential culture can be infected, the combinatorial diversity of Fab fragments displayed on phage could be as large as the number of E.coli in culture (10 per litre). However the heavy and light chain genes would not be packaged together within the same phage particle, and so could not be simultaneously co-selected. Here we describe a model system, involving the lox-Cre site-specific recombination system of bacteriophage PI, to lock together the heavy and light chain genes from two different replicons within an infected bacterium.
TL;DR: MAb 423 labels isolated PHFs prior to Pronase digestion and intracellular granular and neurofibrillary degeneration in Alzheimer's disease tissues, and is described as the minimal protease resistant tau unit of the core PHF.
Abstract: The Alzheimer's disease paired helical filament (PHF), after digestion with Pronase, retains its characteristic morphological features. We term this the protease resistant core PHF. A 12 kDa tau fragment can be released from the core as an essentially pure preparation. Sequence analysis of this fragment revealed six distinct N-termini beginning in the repeat region of tau. The precise C-terminus is unknown, but the fragment is approximately 100 residues long. A monoclonal antibody, mAb 423, which recognizes the core PHF and the 12 kDa tau fragment, does not recognize normal full-length tau. We describe cDNA synthesis and expression of candidate 12 kDa tau analogues which permit the mapping of the mAb 423 epitope. mAb 423 recognizes all and only those analogues which terminate at Glu391, which lies beyond the homology repeat region. Addition or removal of a single residue at the C-terminus abolishes immunoreactivity. Therefore, mAb 423, together with knowledge of the N-terminus, can be used to measure the precise extent of 12 kDa PHF core tau fragment which we term the minimal protease resistant tau unit of the core PHF. This unit is 93-95 residues long, which is equivalent to three repeats, but is 14-16 residues out of phase with respect to the maximum homology organization of the repeat region. mAb 423 labels isolated PHFs prior to Pronase digestion and intracellular granular and neurofibrillary degeneration in Alzheimer's disease tissues. The constraints which determine endogenous truncation at Glu391 appear to be characteristic of an assembled configuration of tau, either within the PHF or its precursor.
TL;DR: It is apparent from this structure that zinc-finger/ DNA recognition is more complex than was originally perceived and may give insight into previous mutagenesis experiments.
Abstract: THE Cys2-His2 zinc-finger is the most widely occurring DNA-binding motif1–3. The first structure of a zinc-finger/DNA complex revealed a fairly simple mechanism for DNA recognition4 suggesting that the zinc-finger might represent a candidate template for designing proteins to recognize DNA5. Residues at three key positions in an α-helical 'reading head' play a dominant role in base-recognition and have been targets for mutagenesis experiments aimed at deriving a recognition code6–8. Here we report the structure of a two zinc-finger DNA-binding domain from the protein Tramtrack complexed with DNA. The amino-terminal zinc-finger and its interaction with DNA illustrate several novel features. These include the use of a serine residue, which is semi-conserved and located outside the three key positions, to make a base contact. Its role in base-recognition correlates with a large, local, protein-induced deformation of the DNA helix at a flexible A-T-A sequence and may give insight into previous mutagenesis experiments9,10. It is apparent from this structure that zinc-finger/ DNA recognition is more complex than was originally perceived.
TL;DR: The restriction of certain Rab proteins to differentiated cell types supports a role for these GTPases in defining the specificity of membrane trafficking.
TL;DR: A minimal set of functional integrase domains are identified that are sufficient for 3′ processing and DNA strand transfer and find that some domains are contributed in trans by separate monomers within the functional complex.
Abstract: HIV-1 integrase protein possesses the 3' processing and DNA strand transfer activities that are required to integrate HIV DNA into a host chromosome. The N-, C-terminal and core domains of integrase are necessary for both activities in vitro. We find that certain pairs of mutant integrase proteins, which are inactive when each protein is assayed alone, can support near wild type levels of activity when both proteins are present together in the reaction mixture. This complementation implies that HIV-1 integrase functions as a multimer and has enabled us to probe the organization of the functional domains within active mixed multimers. We have identified a minimal set of functional integrase domains that are sufficient for 3' processing and DNA strand transfer and find that some domains are contributed in trans by separate monomers within the functional complex.
TL;DR: In this paper, the C-terminal receptor portions (containing transmembrane domains VI and VII) were exchanged between the alpha 2C-adrenergic and the m3 muscarinic receptor.
Abstract: We have tested the hypothesis that guanine-nucleotide-binding-protein-coupled receptors may be able to interact with each other at a molecular level. To address this question, we have initially created two chimeric receptors, alpha 2/m3 and m3/alpha 2, in which the C-terminal receptor portions (containing transmembrane domains VI and VII) were exchanged between the alpha 2C-adrenergic and the m3 muscarinic receptor. Transfection of COS-7 cells with either of the two chimeric constructs alone did not result in any detectable binding activity for the muscarinic ligand N-[3H]methylscopolamine or the adrenergic ligand [3H]rauwolscine. However, cotransfection with alpha 2/m3 and m3/alpha 2 resulted in the appearance of specific binding sites (30-35 fmol/mg of membrane protein) for both radioligands. These sites displayed ligand binding properties similar to those of the two wild-type receptors. Furthermore, COS-7 cells cotransfected with alpha 2/m3 and m3/alpha 2 were able to mediate a pronounced stimulation of phosphatidylinositol hydrolysis upon stimulation with the muscarinic agonist carbachol (Emax approximately 40-50% of wild-type m3). A mutant m3 receptor (containing 16 amino acids of m2 receptor sequence at the N terminus of the third cytoplasmic loop) that was capable of binding muscarinic ligands but was virtually unable to stimulate phosphatidylinositol hydrolysis was also used in various cotransfection experiments. Coexpression of this chimeric receptor with other functionally impaired mutant muscarinic receptors (e.g., with an m3 receptor containing a Pro-->Ala point mutation in transmembrane region VII) resulted in a considerable stimulation of phosphatidylinositol breakdown after carbachol treatment (Emax approximately 40-50% of wild-type m3). Thus, these data suggest that guanine-nucleotide-binding-protein-coupled receptors can interact with each other at a molecular level. One may speculate that the formation of receptor dimers involving the intermolecular exchange of N- and C-terminal receptor domains (containing transmembrane domains I-V and VI and VII, respectively) may underlie this phenomenon.
TL;DR: It is shown that two additional C. elegans HOM-C genes also specify positional identity and that together they function along the anteroposterior axis in the same order as their homologs in other organisms.
TL;DR: It appears that selectivity of degradation by ClpP in vivo is determined by interaction ofclpP with different regulatory ATPase subunits, and Mutations in either clpX or clpP abolish degradation of the highly unstable lambda O protein in vivo.
TL;DR: A model for the events of PC is presented and how the poles of the mitotic spindle mediate the formation of the contractile ring during cytokinesis in C. elegans is suggested.
Abstract: We have examined the cortex of Caenorhabditis elegans eggs during pseudocleavage (PC), a period of the first cell cycle which is important for the generation of asymmetry at first cleavage (Strome, S. 1989. Int. Rev. Cytol. 114: 81-123). We have found that directed, actin dependent, cytoplasmic, and cortical flow occurs during this period coincident with a rearrangement of the cortical actin cytoskeleton (Strome, S. 1986. J. Cell Biol. 103: 2241-2252). The flow velocity (4-7 microns/min) is similar to previously determined particle movements driven by cortical actin flows in motile cells. We show that directed flows occur in one of the daughters of the first division that itself divides asymmetrically, but not in its sister that divides symmetrically. The cortical and cytoplasmic events of PC can be mimicked in other cells during cytokinesis by displacing the mitotic apparatus with the microtubule polymerization inhibitor nocodazole. In all cases, the polarity of the resulting cortical and cytoplasmic flows correlates with the position of the attenuated mitotic spindle formed. These cortical flows are also accompanied by a change in the distribution of the cortical actin network. The polarity of this redistribution is similarly correlated with the location of the attenuated spindle. These observations suggest a mechanism for generating polarized flows of cytoplasmic and cortical material during embryonic cleavages. We present a model for the events of PC and suggest how the poles of the mitotic spindle mediate the formation of the contractile ring during cytokinesis in C. elegans.
TL;DR: Because the stabilizing disulfide bond was introduced in between two framework residues in a position that is conserved in most Fv molecules, this method of linkage between the heavy- and light-chain variable regions should be generally applicable to construct immunotoxins and dsFv molecules using other antibodies.
Abstract: B3(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv region of monoclonal antibody B3 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the unstable Fv heterodimer (composed of heavy- and light-chain variable regions) is held together and stabilized by a disulfide bond [termed disulfide-stabilized Fv (dsFV)]. A computer modeled structure of the B3(Fv), made by mutating and energy minimizing the amino acid sequence and structure of McPC603, enabled us to identify positions in conserved framework regions that "hypothetically" could be used for disulfide stabilization without changing the structure or affecting antigen binding. This prediction was evaluated experimentally by constructing a disulfide-linked two-chain dsFv-immunotoxin that was produced in Escherichia coli. The activity and specificity of this immunotoxin was indistinguishable from its single-chain Fv (scFv) counterpart, indicating that, as in B3(scFv), the structure of the binding region is retained in B3(dsFv). Because we introduced the stabilizing disulfide bond in between two framework residues in a position that is conserved in most Fv molecules, this method of linkage between the heavy- and light-chain variable regions should be generally applicable to construct immunotoxins and dsFv molecules using other antibodies. Furthermore, the finding that B3(dsFv) was much more stable at 37 degrees C in human plasma than B3(scFv) indicates that dsFvs are possibly more versatile for therapeutic application than scFvs.
TL;DR: Cloned human ADF is cloned and shown to be identical in sequence to porcine destrin, and expressed in Escherichia coli behaves like native ADF from Porcine brain.
Abstract: ADF (actin depolymerizing factor) is an M(r) 19,000 actin-binding protein present in many vertebrate tissues and particularly abundant in neuronal cells. We have cloned human ADF and here show it to be identical in sequence to porcine destrin. Human ADF expressed in Escherichia coli behaves like native ADF from porcine brain. It binds to G-actin at pH 8 with a 1:1 stoichiometry and Kd approximately 0.2 microM, thereby sequestering monomers and preventing polymerization. It does not cosediment with F-actin at this pH, but severs actin filaments in a calcium-insensitive manner. The severing activity is only about 0.1% efficient. By contrast, at pH values below 7, ADF binds to actin filaments in a highly cooperative manner and at a 1:1 ratio to filament subunits. When the pH is raised to 8.0, the decorated filaments are rapidly severed and depolymerized.
TL;DR: Somatic hypermutation in mice carrying an immunoglobulin kappa transgene exhibits specific base substitution preferences with transitions being favored over transversions and it is proposed that these substitution preferences can be used to discriminate intrinsic from antigen-selected hot spots.
Abstract: We have analyzed somatic hypermutation in mice carrying an immunoglobulin kappa transgene in order to discriminate mutations that reflect the intrinsic specificity of the hypermutation mechanism from those highlighted by antigenic selection. We have immunized animals with three different immunogens. With one immunogen, the antigen-specific B cells express a transgenic kappa chain, which does not form part of the antibody; the transgene is a passenger free to accumulate unselected mutations. With the other two immunogens, the transgenic kappa chain constitutes the light chain of the expressed antibody. A comparison of the transgene mutations obtained under these different circumstances allows us to identify common features that we attribute to the intrinsic specificity of the hypermutation process. In particular, it yields only base substitutions and leads to hot spots occurring in individual positions (e.g., the second base of the Ser-31 codon). The mutations preferentially accumulate around the first complementarity-determining region. The process exhibits specific base substitution preferences with transitions being favored over transversions. We propose that these substitution preferences can be used to discriminate intrinsic from antigen-selected hot spots. We also note that hypermutation distinguishes between the coding and noncoding strands since pyrimidines (particularly thymidines) mutate less frequently than purines.
TL;DR: It is shown that the presumptive tips of both the leg and wing, the distal extreme of the proximodistal axis, are characterized by the close association of cells expressing wg, dpp, and the homeobox gene aristaless (al).
TL;DR: The binding site for tat protein on TAR RNA has been defined in quantitative terms using an extensive series of mutations and the binding of a series of peptides spanning the basic "arginine-rich" sequence of tat was examined using both filter-binding and gel mobility shift assays.