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1.
Curr Opin Drug Discov Devel ; 4(3): 319-24, 2001 May.
Article in English | MEDLINE | ID: mdl-11560065

ABSTRACT

Knowledge management approaches and technologies are beginning to be implemented by the pharmaceutical industry in support of new drug discovery and development processes aimed at greater efficiencies and effectiveness. This trend coincides with moves to reduce paper, coordinate larger teams with more diverse skills that are distributed around the globe, and to comply with regulatory requirements for electronic submissions and the associated maintenance of electronic records. Concurrently, the available technologies have implemented web-based architectures with a greater range of collaborative tools and personalization through portal approaches. However, successful application of knowledge management methods depends on effective cultural change management, as well as proper architectural design to match the organizational and work processes within a company.


Subject(s)
Computational Biology , Drug Design , Pharmacology/trends , Artificial Intelligence
2.
Biochemistry ; 39(30): 8888-94, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10913301

ABSTRACT

Glucagon-like peptide-2 (GLP-2) is a 33 amino acid gastrointestinal hormone that regulates epithelial growth in the intestine. Dipeptidylpeptidase IV cleaves GLP-2 at the position 2 alanine, resulting in the inactivation of peptide activity. To understand the structural basis for GLP-2 action, we studied receptor binding and activation for 56 GLP-2 analogues with either position 2 substitutions or alanine replacements along the length of the peptide. The majority of position 2 substitutions exhibited normal to enhanced GLP-2 receptor (GLP-2R) binding; in contrast, position 2 substitutions were less well tolerated in studies of receptor activation as only Gly, Ile, Pro, alpha-aminobutyric acid, D-Ala, or nor-Val substitutions exhibited enhanced GLP-2R activation. In contrast, alanine replacement at positions 5,6,17, 20, 22, 23, 25, 26, 30, and 31 led to diminished GLP-2R binding. Position 2 substitutions containing Asp, Leu, Lys, Met, Phe, Trp, and Tyr, and Ala substitutions at positions 12 and 21 exhibited normal to enhanced GLP-2R binding but greater than 75% reduction in receptor activation. D-Ala(2), Pro(2) and Gly(2), Ala(16) exhibited significantly lower EC(50)s for receptor activation than the parent peptide (p < 0.01-0.001). Circular dichroism analysis indicated that the enhanced activity of these GLP-2 analogues was independent of the alpha-helical content of the peptide. These results indicate that single amino acid substitutions within GLP-2 can confer structural changes to the ligand-receptor interface, allowing the identification of residues important for GLP-2R binding and receptor activation.


Subject(s)
Peptide Fragments/genetics , Peptide Fragments/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Circular Dichroism , Glucagon-Like Peptide 2 , Glucagon-Like Peptide-1 Receptor , Glucagon-Like Peptides , Humans , Molecular Sequence Data , Rats , Receptors, Glucagon/metabolism , Receptors, Glucagon/physiology , Sequence Homology, Amino Acid , Structure-Activity Relationship
3.
Proc Natl Acad Sci U S A ; 96(4): 1569-73, 1999 Feb 16.
Article in English | MEDLINE | ID: mdl-9990065

ABSTRACT

Glucagon-like peptide 2 (GLP-2) is a 33-aa proglucagon-derived peptide produced by intestinal enteroendocrine cells. GLP-2 stimulates intestinal growth and up-regulates villus height in the small intestine, concomitant with increased crypt cell proliferation and decreased enterocyte apoptosis. Moreover, GLP-2 prevents intestinal hypoplasia resulting from total parenteral nutrition. However, the mechanism underlying these actions has remained unclear. Here we report the cloning and characterization of cDNAs encoding rat and human GLP-2 receptors (GLP-2R), a G protein-coupled receptor superfamily member expressed in the gut and closely related to the glucagon and GLP-1 receptors. The human GLP-2R gene maps to chromosome 17p13.3. Cells expressing the GLP-2R responded to GLP-2, but not GLP-1 or related peptides, with increased cAMP production (EC50 = 0.58 nM) and displayed saturable high-affinity radioligand binding (Kd = 0.57 nM), which could be displaced by synthetic rat GLP-2 (Ki = 0.06 nM). GLP-2 analogs that activated GLP-2R signal transduction in vitro displayed intestinotrophic activity in vivo. These results strongly suggest that GLP-2, like glucagon and GLP-1, exerts its actions through a distinct and specific novel receptor expressed in its principal target tissue, the gastrointestinal tract.


Subject(s)
GTP-Binding Proteins/metabolism , Peptides/physiology , Receptors, Glucagon/physiology , Amino Acid Sequence , Animals , Brain/metabolism , COS Cells , Cloning, Molecular , Cyclic AMP/metabolism , Gene Library , Glucagon-Like Peptide 1 , Glucagon-Like Peptide 2 , Glucagon-Like Peptide-1 Receptor , Humans , Intestinal Mucosa/metabolism , Kinetics , Molecular Sequence Data , Organ Specificity , Peptide Fragments/pharmacology , Peptides/pharmacology , Rats , Receptors, Glucagon/chemistry , Receptors, Glucagon/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Structure-Activity Relationship , Transfection
4.
Nat Biotechnol ; 15(7): 673-7, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9219272

ABSTRACT

Species-specific differences in the enzymatic inactivation of peptides is an important consideration in the evaluation of therapeutic efficacy. We demonstrate that glucagon-like peptide 2 (GLP-2), shown to be highly intestinotrophic in mice, promotes an increase in intestinal villus height but has no trophic effect on small bowel weight in rats. The reduced intestinotrophic activity of GLP-2 in rats is attributable to inactivation by the enzyme dipeptidyl peptidase IV (DPP-IV). GLP-2(1-33) was degraded to GLP-2(3-33) following incubation with human placental DPP-IV or rat serum but not by serum from DPP-IV-deficient rats. Administration of rat GLP-2 to DPP-IV-deficient rats was associated with markedly increased bioactivity of rat GLP-2 resulting in a significant increase in small bowel weight. A synthetic GLP-2 analog, r[Gly2]GLP-2, with an alanine to glycine substitution at position 2, was resistant to cleavage by both DPP-IV and rat serum in vitro. Treatment of wild-type rats with r[Gly2]GLP-2 produced a statistically significant increase in small bowel mass. DPP-IV-mediated inactivation of GLP-2 is a critical determinant of the growth factor-like properties of GLP-2.


Subject(s)
Dipeptidyl Peptidase 4/metabolism , Glucagon/pharmacology , Peptides/pharmacology , Animals , Biotechnology , Glucagon/antagonists & inhibitors , Glucagon/metabolism , Glucagon-Like Peptide 2 , Glucagon-Like Peptides , Humans , In Vitro Techniques , Intestine, Small/drug effects , Intestine, Small/growth & development , Mice , Peptides/antagonists & inhibitors , Peptides/metabolism , Protein Engineering , Rats , Rats, Inbred F344 , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology
5.
J Virol ; 70(5): 2825-31, 1996 May.
Article in English | MEDLINE | ID: mdl-8627756

ABSTRACT

An oligocationic peptide compound (ALX40-4C) was developed for consideration in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. This compound was designed to mimic the basic domain of the HIV-1 transactivation protein, Tat, and will competitively inhibit Tat binding to its specific RNA hairpin target (TAR [transactivation region]), found at the 5' end of all HIV-1 transcripts. Blocking Tat-TAR interactions can abrogate HIV-1 replication. ALX40-4C was shown to inhibit replication of HIV-1NL4-3 in a range of cell types, including primary cells and transformed cell lines, by as much as 10(4)-fold. In some experiments, virus rescue was not possible even after removal of ALX40-4C from the cultures. Strain-dependent resistance has been demonstrated for all antiretroviral agents tested; therefore, we tested for variable sensitivity to ALX40-4C. The cloned primary strains, HIV-JR-CSF and HIV-JR-FL, were less sensitive to ALX40-4C inhibition. Unexpectedly, determinants for efficient ALX40-4C inhibition were mapped by using recombinant virus strains to the V3 region of gpl20 and were shown to act at early events in viral replication, which include viral entry. If entry and reverse transcription are bypassed by transfection, a more modest, virus strain-independent inhibition is shown; this inhibition is likely due to blocking of Tat-TAR interaction. Thus, the highly basic oligocationic Tat inhibitor ALX40-4C appears to interfere with initial virus-target cell interactions which involve HIV-1 gp120 V3 determinants, most efficiently for T-cell line-adapted strains.


Subject(s)
Antiviral Agents/pharmacology , HIV Envelope Protein gp120/drug effects , HIV-1/drug effects , Oligopeptides/pharmacology , Virus Replication/drug effects , Acquired Immunodeficiency Syndrome/drug therapy , Cloning, Molecular , Drug Resistance, Microbial , Gene Products, tat/antagonists & inhibitors , Gene Products, tat/metabolism , HIV Envelope Protein gp120/metabolism , HIV Long Terminal Repeat , HIV-1/isolation & purification , HIV-1/physiology , Humans , Kinetics , Microbial Sensitivity Tests , Proviruses/drug effects , Proviruses/physiology , Restriction Mapping , Transcriptional Activation/drug effects , tat Gene Products, Human Immunodeficiency Virus
6.
Nucl Med Commun ; 17(2): 132-9, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8778637

ABSTRACT

Two 99Tcm-labelled analogues of the chemotactic peptide ForMLF were evaluated as potential agents for imaging inflammation and infection, in the hope that they would be simple to use and would give diagnostically useful images shortly after injection. The peptides differed in the chelation site for 99Tcm and the presence of a hydrophilic spacer. The sequences of RP050 and RP056 were ForNleLFNleYK(G)G-C(Acm)-GPic and ForNleLFNleYKK(DG)GC(Acm)SPic respectively, where Pic is picolinic acid. In in vitro tests of binding to the ForMLF receptor on polymorphonuclear neutrophils and potency for release of myeloperoxidase, RP056 was similar in potency to ForMLF, whereas RP050 was 10 times more potent. When administered in 5-nmol doses to rats, RP050 produced less extensive neutropenia than ForMLF, whereas RP056 produced very little neutropenia. Following labelling by ligand exchange from tartrate or glucoheptonate at 100 degrees C and purification using a C-18 solid-phase extraction cartridge, 4-MBq doses were administered to rats bearing infectious (Escherichia coli) or sterile (zymosan) inflammation sites in the thigh. The inflammation-to-normal muscle ratios at 30 min after injection were 3.9 +/- 0.4 for RP050 and 4.7 +/- 0.3 for RP056 (mean +/- S.E.M., n = 4), and the ratios were maintained for up to 3 h. These peptides are promising agents for imaging inflammation and infection.


Subject(s)
Chemotactic Factors , Inflammation/diagnostic imaging , Oligopeptides , Technetium , Amino Acid Sequence , Animals , Chelating Agents/adverse effects , Chelating Agents/chemistry , Chemotactic Factors/adverse effects , Chemotactic Factors/chemistry , In Vitro Techniques , Male , Molecular Sequence Data , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/chemistry , Neutropenia/etiology , Neutropenia/prevention & control , Oligopeptides/adverse effects , Oligopeptides/chemistry , Radionuclide Imaging , Rats , Rats, Sprague-Dawley , Receptors, Formyl Peptide , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Technetium/adverse effects
7.
Drugs Exp Clin Res ; 21(1): 1-6, 1995.
Article in English | MEDLINE | ID: mdl-7796707

ABSTRACT

N-alpha-acetyl-nona-D-arginine amide acetate (ALX40-4C) was developed as a competitive inhibitor of the binding of the HIV Tat protein to its RNA target TAR, which is an intracellular interaction dependent on a short, arginine-rich sequence in Tat. ALX40-4C is a simple mimic of that domain, which is stabilised against enzymatic degradation through inclusion of D-amino acids and terminal protection. The drug inhibits HIV-1 in vitro and is currently being assessed in vivo. In the work reported here, potential activities of the compound against other viruses were examined. As expected, there was little or no activity against most viruses examined, except against some herpesviruses: HSV-1, HSV-2 and CMV. Maximal inhibition of HSV-1 in a plaque reduction assay required pre-incubation with the drug. Maximal inhibition of HCMV, which replicates more slowly than HSV-1, requires exposure to the compound within the first few hours of infection. It appears that the drug inhibits an early step in HSV and HCMV infection. Such a mechanism is consistent with that of other cationic, herpesvirus inhibitors.


Subject(s)
Antiviral Agents/pharmacology , Cytomegalovirus/drug effects , Herpesvirus 1, Human/drug effects , Oligopeptides/pharmacology , Herpesvirus 2, Human/drug effects
8.
Protein Expr Purif ; 5(3): 252-8, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7950368

ABSTRACT

In order to rapidly purify human dUTPase, a cDNA fragment that encodes the enzyme was subcloned and expressed using the Escherichia coli plasmid vector pGEX2T. The resulting plasmid expressed high levels of a glutathione S-transferase-dUTPase fusion protein following induction with IPTG. Affinity chromatography was used to purify the fusion protein, and dUTPase was then released from the fusion protein by thrombin treatment. The purified dUTPase has two additional vector-encoded residues at the amino terminus (gly-ser), but they have no apparent effect on the activity of the enzyme since the recombinant dUTPase has catalytic properties similar to those reported for dUTPase purified from human cells (32.3 U/mg, kcat = 25 s-1, Km = 2.6 microM). Enzyme activity was inhibited by 5-mercuri-dUTP and was shown to be sensitive to EDTA. Periodate-oxidized UTP had no effect on the activity of the enzyme, and dTTP caused only slight inhibition. The results of gel filtration experiments are consistent with a homotrimeric subunit composition for dUTPase. The ability to purify human dUTPase from E. coli should allow further characterization of the enzyme and provide material for the screening of potentially useful inhibitors.


Subject(s)
Pyrophosphatases/biosynthesis , Pyrophosphatases/isolation & purification , Recombinant Proteins/biosynthesis , Base Sequence , Chromatography, Gel/methods , Cloning, Molecular/methods , DNA Primers , Electrophoresis, Polyacrylamide Gel/methods , Escherichia coli , Genetic Vectors , Humans , Isoelectric Focusing/methods , Kinetics , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , Plasmids , Polymerase Chain Reaction/methods , Pyrophosphatases/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping
9.
Nucleic Acids Res ; 21(11): 2585-9, 1993 Jun 11.
Article in English | MEDLINE | ID: mdl-8332456

ABSTRACT

We recently developed an approach which allows rapid generation of short, double-stranded oligonucleotides whereby one end of the duplex was joined and stabilized by a synthetic linker of specific design (miniduplexes)(6). Model miniduplexes based on the HIV-1 TAR RNA hairpin were shown to be thermodynamically stable and good substrates for binding by the HIV-1 Tat protein which normally bind to natural TAR (6). In this study, we have extended our studies to the design, synthesis and analysis of the binding properties of covalently closed, double-stranded, cyclic RNA miniduplexes. A strategy using automated chemical synthesis and T4 RNA ligase-catalyzed cyclization was employed to generate cyclic oligoribonucleotides. When both ends of a shortened, wild-type TAR RNA stem (9 bp) were covalently linked through either nucleotidic loops (4-6 nt) or synthetic linkers (derivatized from hexaethylene glycol), the resulting cyclic TAR RNA analogs were good substrates for binding by both Tat-derived peptide or full-length Tat protein. Interestingly, the cyclic TAR analogs failed to show any binding if the synthetic linker was reduced in length (e.g. derivatized from triethylene glycol), although such linkers are acceptable in the hairpin-shaped miniduplexes series (6). This implies that RNA conformational changes are required for Tat binding and that these changes are restricted in certain cyclic variants. Our findings suggest that covalently-closed nucleic acid miniduplexes may be useful both to study nucleic acid-protein interactions as well as to provide a basis for therapeutic intervention as transcription decoys.


Subject(s)
Gene Products, tat/metabolism , HIV-1/metabolism , Oligoribonucleotides/metabolism , RNA, Double-Stranded/metabolism , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Base Sequence , Molecular Sequence Data , Nucleic Acid Conformation , Oligoribonucleotides/chemical synthesis , RNA, Double-Stranded/chemical synthesis , RNA, Viral/chemical synthesis , tat Gene Products, Human Immunodeficiency Virus
10.
Biochemistry ; 32(7): 1751-8, 1993 Feb 23.
Article in English | MEDLINE | ID: mdl-7679923

ABSTRACT

Double-stranded oligodeoxyribonucleotides or single-stranded oligoribonucleotides with specific secondary structure have been proposed as potential antagonists to target nucleic acid-binding proteins (the sense approach). A major limitation of this strategy is that these derivatives are generally considered to be too large for pharmaceutical applications. We have developed a synthetic linker approach whereby nucleic acid duplexes of a much smaller size (miniduplexes) can be generated directly from a standard oligonucleotide synthesis. In this approach, four synthetic linkers (derivatized respectively from 1,9-nonanediol, triethylene glycol, 1,3-propanediol, and hexaethylene glycol) of different length and hydrophobicity were designed and incorporated into a model RNA molecule based on the TAR stem-loop structure of HIV-1. Their thermal stabilities were evaluated by measuring denaturation profiles (Tm measurements). These linker-derivatized RNA molecules were then assessed for their ability to bind to either a full-length protein (HIV-1 Tat protein) or a short peptide (Tat-derived peptide) through RNA mobility shift assays. Results from this study indicate that such modified miniduplex structures retain full binding activity relative to that of the wild-type sequence (Kd values), while Tm values were increased by 24-31 degrees C compared to an open duplex of the same length. This system provides a new direction in the use of nucleic acid miniduplexes as a novel class of oligonucleotide analogues for both fundamental research and possible therapeutic applications.


Subject(s)
RNA/chemical synthesis , Amino Acid Sequence , Base Sequence , Computer Simulation , Ethylene Glycols/chemistry , Gene Products, tat/metabolism , Glycols , HIV-1/genetics , Hot Temperature , Molecular Sequence Data , Nucleic Acid Denaturation , Peptide Fragments/metabolism , Polyethylene Glycols/chemistry , Propylene Glycols/chemistry , RNA/chemistry , RNA/metabolism , RNA, Viral/chemistry , tat Gene Products, Human Immunodeficiency Virus
11.
Nucleic Acids Res ; 21(1): 151-4, 1993 Jan 11.
Article in English | MEDLINE | ID: mdl-8441609

ABSTRACT

Transactivation of human immunodeficiency virus (HIV) gene expression requires binding of the viral Tat protein to a RNA hairpin-loop structure (TAR) which contains a two or three-nucleotide bulge. Tat binds in the vicinity of the bulge and the two adjacent duplex stems, recognising both specific sequence and structural features of TAR. Binding is mediated by an arginine-rich domain, placing Tat in the family of arginine-rich RNA binding proteins that includes other transactivators, virus capsid proteins and ribosome binding proteins. In order to determine what features of TAR allow Tat to bind efficiently to RNA but not DNA forms, we examined Tat binding to a series of RNA-DNA hybrids. We found that only one specific strand in each duplex stem region needs to be RNA, implying that interaction between Tat and a given stem may be solely or predominantly with one of the two strands. However, the essential strand is not the same one for each stem, suggesting a switch in the bound strand on opposing sides of the bulge.


Subject(s)
Gene Products, tat/metabolism , HIV-1/metabolism , RNA, Viral/metabolism , Amino Acid Sequence , Base Sequence , DNA/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Viral/chemistry , Ribonucleotides/metabolism , tat Gene Products, Human Immunodeficiency Virus
12.
Nucleic Acids Res ; 20(20): 5311-20, 1992 Oct 25.
Article in English | MEDLINE | ID: mdl-1437550

ABSTRACT

A new method is described for the direct construction of randomly mutagenized genes by applying the polymerase chain reaction (PCR) to an oligonucleotide synthesized using doped nucleotide reservoirs. We have demonstrated the utility of this method by generating a library of mutant HIV-1 tat genes. Several arbitrarily selected, inactive tat clones were sequenced to evaluate the extent of the mutagenesis. Moreover, fourteen recombinants encoding varying levels of transcriptional trans-activator activity were isolated by transient transfection of sub-library pools into a HeLa cell line bearing an HIV-LTR-chloramphenicol acetyltransferase (CAT) reporter gene. Sequence data revealed a spectrum of alterations including nucleotide substitutions, insertions, and deletions, suggesting that mutations arose from both the doped DNA synthesis and the subsequent PCR 'rescue' of full-length product. Sequence comparison between inactive and active Tat clones revealed a selection pressure against amino-acid substitutions within the N-terminal domains of Tat, indicating the importance of this region to trans-activation competence. In addition, single and double missense mutations within the basic-rich, TAR RNA-binding domain were seen to be tolerated within active Tat clones.


Subject(s)
Gene Products, rev/genetics , Genes, tat/genetics , HIV Long Terminal Repeat/genetics , HIV-1/genetics , Mutagenesis , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Gene Library , HeLa Cells , Humans , Molecular Sequence Data , Plasmids/genetics , Polymerase Chain Reaction , Recombinant Fusion Proteins/genetics , rev Gene Products, Human Immunodeficiency Virus
13.
J Virol ; 66(5): 3018-25, 1992 May.
Article in English | MEDLINE | ID: mdl-1560535

ABSTRACT

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.


Subject(s)
Acquired Immunodeficiency Syndrome/genetics , HIV-1/genetics , Nucleic Acid Conformation , RNA, Viral/genetics , Regulatory Sequences, Nucleic Acid/genetics , Base Sequence , Binding Sites , Gene Products, tat/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Structure-Activity Relationship , Transcriptional Activation , tat Gene Products, Human Immunodeficiency Virus
14.
J Virol ; 65(10): 5196-202, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1895380

ABSTRACT

The human immunodeficiency virus type 1 Tat protein binds to an RNA stem-loop structure called TAR which is present at the 5' end of all human immunodeficiency virus type 1 transcripts. This binding is centered on a bulge within the stem of TAR and is an essential step in the trans-activation process which results in a dramatic increase in viral gene expression. By analysis of a series of TAR derivatives produced by transcription or direct chemical synthesis, we determined the structural and chemical requirements for Tat binding. Tat binds well to structures which have a bulge of two to at least five unpaired bases bounded on both sides by a double-stranded RNA stem. This apparent flexibility in bulge size is in contrast to an absolute requirement for an unpaired uridine (U) in the 5'-most position of the bulge (+23). Substitution of the U with either natural bases or chemical analogs demonstrated that the imido group at the N-3 position and, possibly, the carbonyl group at the C-4 position of U are critical for Tat binding. Cytosine (C), which differs from U at only these positions, is not an acceptable substitute. Furthermore, methylation at N-3 abolishes binding. While methylation of U at the C-5 position has little effect on binding, fluorination reduces it, possibly because of its effects on relative tautomer stability at the N-3 and C-4 positions. Thus, we have identified key moieties in the U residue that are of importance for the binding of Tat to TAR RNA. We hypothesize that the invariant U is involved in hydrogen bond interactions with either another part of TAR or the TAR-binding domain in Tat.


Subject(s)
Gene Products, tat/metabolism , HIV-1/genetics , RNA, Viral/metabolism , Transcriptional Activation , Base Composition , Base Sequence , Binding Sites , Chromosome Deletion , Cytosine , Genes, Viral , HIV-1/metabolism , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Oligonucleotide Probes , RNA, Viral/genetics , Transcription, Genetic , Uracil , tat Gene Products, Human Immunodeficiency Virus
15.
Proc Natl Acad Sci U S A ; 88(14): 6234-8, 1991 Jul 15.
Article in English | MEDLINE | ID: mdl-2068104

ABSTRACT

The basic domain of Tat is required for trans-activation of viral gene expression. We have performed scanning peptide studies to demonstrate that only this domain is capable of binding to the TAR RNA stem-loop. Strikingly, the basic domain of the other human immunodeficiency virus trans-acting factor, Rev, but no other region, is also capable of binding to TAR. Peptide derivatives of Tat do not require the highly conserved glutamine residue at position 54 for TAR binding, since it may be substituted or deleted. In addition, the two lysine residues may be replaced by arginines. Analysis of binding and trans-activation demonstrated that homopolymers of arginine can completely substitute for the basic domain. Such homopolymers have high affinity for wild-type TAR RNA and lower affinity for mutant TAR. Homopolymers of six to nine arginines substituting for the basic domain of Tat enable full trans-activation in vivo. Homopolymers of at least seven arginines are required for detectable in vitro complex formation, although approximately 30% trans-activation is achieved with a mutant Tat containing only five arginines.


Subject(s)
Arginine , Gene Products, tat/genetics , HIV Long Terminal Repeat , HIV-1/genetics , RNA, Viral/genetics , Transcriptional Activation , Amino Acid Sequence , Binding Sites , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Gene Products, rev/genetics , Gene Products, rev/metabolism , Gene Products, tat/metabolism , Genes, tat , HeLa Cells/metabolism , Humans , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Transfection , rev Gene Products, Human Immunodeficiency Virus , tat Gene Products, Human Immunodeficiency Virus
16.
Science ; 242(4885): 1548-51, 1988 Dec 16.
Article in English | MEDLINE | ID: mdl-3144042

ABSTRACT

The fidelity of protein biosynthesis in any cell rests on the accuracy of aminoacylation of tRNA. The exquisite specificity of this reaction is critically dependent on the correct recognition of tRNA by aminoacyl-tRNA synthetases. It is shown here that the relative concentrations of a tRNA and its cognate aminoacyl-tRNA synthetase are normally well balanced and crucial for maintenance of accurate aminoacylation. When Escherichia coli Gln-tRNA synthetase is overproduced in vivo, it incorrectly acylates the supF amber suppressor tRNA(Tyr) with Gln. This effect is abolished when the intracellular concentration of the cognate tRNA(Gln2) is also elevate. These data indicate that the presence of aminoacyl-tRNA synthetase and the cognate tRNAs in complexed form, which requires the proper balance of the two macromolecules, is critical in maintaining the fidelity of protein biosynthesis. Thus, limits exist on the relative levels of tRNAs and aminoacyl-tRNA synthetases within a cell.


Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Escherichia coli/genetics , RNA, Transfer, Amino Acid-Specific/metabolism , RNA, Transfer, Gln/metabolism , Amino Acyl-tRNA Synthetases/genetics , Escherichia coli/enzymology , Kinetics , Plasmids , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
17.
Gene ; 60(1): 115-27, 1987.
Article in English | MEDLINE | ID: mdl-3126097

ABSTRACT

We have tested several different synthesis designs and assembly methodologies to develop an improved gene synthesis strategy which enables significantly longer nucleotide sequences to be easily constructed. This strategy, based in part upon our ability to synthesize high-quality extended-length oligodeoxynucleotides (over 100-mer in length), together with the use of chemical 5'-phosphorylation, and simplified low-melting-temperature agarose gel purification methods, combines ease, speed and high overall efficiency. We show that it is now feasible to synthesize routinely even long genes (at least 1-2 kb). To demonstrate this capability we have chemically synthesized and assembled two different versions of the gene encoding the bovine enzyme prochymosin (prorennin). One gene is essentially the natural bovine prochymosin gene sequence. In the second gene the codons have been optimized with regard to the codon bias of highly expressed yeast genes. Each synthetic gene was in excess of 1100 bp, yet they were assembled from only 13 or 14 pairs of complementary oligodeoxynucleotides (oligos), the average lengths of which were 87 and 82 bp, respectively. The 'mutation' rate was low enough to assess that more than 75% of all such oligo pairs (160-170 total nt) were error-free.


Subject(s)
Chymosin/genetics , Enzyme Precursors/genetics , Genes, Synthetic , Animals , Base Sequence , Cattle , Ligation , Molecular Sequence Data , Oligodeoxyribonucleotides/chemical synthesis , Phosphorylation
18.
Nucleic Acids Res ; 13(11): 3905-916, 1985 Jun 11.
Article in English | MEDLINE | ID: mdl-3839304

ABSTRACT

We have sequenced two genomic clones for wheat alpha/beta-gliadin storage protein genes. Comparison with a known sequence reveals close homology between the three and confirms the previously suspected evolutionary relatedness of members of this gliadin family. The coding region can be divided into six domains. Two unusual structures were found within this region: (i) The P-boxes which are composed of 12 codons, six of which are for proline, that are tandemly repeated four or five times; and (ii) Two polyglutamine stretches which consist of 18-22 tandemly repeated glutamine codons in one case, and 7-28 in the second. Analysis of the P-box structures revealed that certain mutations were probably present in the hypothetical ancestral alpha/beta-gliadin gene prior to gene multiplication. None of the genes have introns. All of the genes appear to contain typical eukaryotic promoters and also possess the double polyadenylation signal of plants.


Subject(s)
Genes , Gliadin/genetics , Plant Proteins/genetics , Plants/genetics , Amino Acid Sequence , Base Sequence , Biological Evolution , Cloning, Molecular , DNA/metabolism , Genetic Variation , Mutation , Plants/metabolism , RNA, Messenger/genetics , Triticum/genetics , Triticum/metabolism
19.
Gene ; 36(3): 333-40, 1985.
Article in English | MEDLINE | ID: mdl-3000884

ABSTRACT

We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene.


Subject(s)
Galactosidases/genetics , Genes, Viral , Genes , Saccharomyces/genetics , alpha-Galactosidase/genetics , Amino Acid Sequence , Base Sequence , Enzyme Induction , Plasmids , Saccharomyces/enzymology , alpha-Galactosidase/biosynthesis
20.
Fed Proc ; 43(15): 2972-6, 1984 Dec.
Article in English | MEDLINE | ID: mdl-6389180

ABSTRACT

Escherichia coli glutaminyl-tRNA synthetase (GlnRS) (EC 6.1.1.18) is a monomeric polypeptide of 553 amino acids. Its amino acid sequence and its gene (glnS) sequence are known. A structural gene mutation, glnS7, codes for a mischarging GlnRS, which acylates some noncognate tRNA species (e.g., su+3 tRNATyr) with glutamine. The mutant enzyme was shown to catalyze in vitro the acylation of glutamine to su+3 tRNATyr, but not to wild-type tRNATyr. The mutation responsible produces an amino acid change in the amino-terminal half of the enzyme. Unexpectedly, overproduction of wild-type GlnRS also leads to in vivo mischarging of su+3 tRNATyr. In vitro and in vivo studies have not revealed evidence for an attenuation or autogenous regulation mechanism for GlnRS, but have implicated transcriptional and translational control in the expression of this enzyme.


Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Glutamine/metabolism , Amino Acid Sequence , Amino Acyl-tRNA Synthetases/genetics , Escherichia coli/enzymology , Genes, Bacterial , Mutation , Structure-Activity Relationship , Substrate Specificity
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