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1.
Science ; 276(5319): 1696-9, 1997 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-9180079

RESUMO

Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.


Assuntos
Proteínas de Neoplasias , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas/agonistas , Receptores de Citocinas , Trombopoetina/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Plaquetas/citologia , Divisão Celular , Linhagem Celular , Células Cultivadas , Sequência Consenso , Dimerização , Eritropoetina/farmacologia , Hematopoese/efeitos dos fármacos , Humanos , Megacariócitos/citologia , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/metabolismo , Peptídeos/farmacologia , Contagem de Plaquetas , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Trombopoetina , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Trombopoetina/farmacologia , Transfecção
2.
Mol Cell Biol ; 12(9): 4026-37, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1508200

RESUMO

A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.


Assuntos
Proteínas Fúngicas/genética , Histidina/análogos & derivados , Metiltransferases/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Catálise , Clonagem Molecular , DNA Fúngico , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Histidina/biossíntese , Metilação , Metiltransferases/metabolismo , Dados de Sequência Molecular , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência
3.
J Mol Biol ; 235(1): 111-24, 1994 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-7507167

RESUMO

The str operon of Escherichia coli consists of the genes for ribosomal proteins S12 (rpsL) and S7 (rpsG) and elongation factors G (fusA) and Tu (tufA). Previous studies have shown that S7 is a translational feedback repressor and inhibits the synthesis of itself and of elongation factor G. We have now shown that induction of S7 synthesis from the S7 gene fused to the arabinose promoter on a plasmid also leads to inhibition of the synthesis of S12 from the chromosomal S12 gene, and that this regulation takes place using the same target site as that used for distal gene regulation, i.e. S7 retroregulates S12. We have then demonstrated that S7 synthesis is mostly translationally coupled with the translation of the preceding S12 gene. Using a rpsG'-'lacZ fusion gene as a reporter for S7 synthesis, we found that abolishing S12 translation by a mutational alteration of the AUG start codon of the S12 gene leads to about tenfold reduction of S7 synthesis without significantly affecting its rate of transcription. Deletion of the proximal portion of the S12 gene or a premature termination of S12 translation by an amber mutation at the 26th codon also led to a large reduction of S7 synthesis. Unexpectedly, we have discovered that overproduction of S7 in trans from a plasmid leads to repression of the rpsG'-'lacZ fusion gene when the fusion gene is preceded by the intact S12 gene, but not when the S12 gene carried the above-mentioned mutations that abolish S12 translation. Thus, a novel feature of this regulatory system is that translation of S7 achieved by independent initiation is not inhibited by S7 in vivo, whereas translation of S7 achieved by translational coupling is sensitive to S7 repression. These observations also suggest that the coupled S7 translation is probably achieved by the use of ribosomal subunits employed for translation of the upstream S12 gene.


Assuntos
Escherichia coli/metabolismo , Genes Bacterianos , Óperon , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Arabinose/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli , Cinética , Óperon/efeitos dos fármacos , Fator G para Elongação de Peptídeos , Fator Tu de Elongação de Peptídeos/biossíntese , Fator Tu de Elongação de Peptídeos/genética , Fatores de Alongamento de Peptídeos/biossíntese , Fatores de Alongamento de Peptídeos/genética , Plasmídeos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Bacteriano/biossíntese , RNA Mensageiro/biossíntese , Mapeamento por Restrição , Proteína S9 Ribossômica , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética
4.
J Mol Biol ; 204(2): 309-29, 1988 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-2464692

RESUMO

The spc ribosomal protein operon of Escherichia coli is feedback-regulated by ribosomal protein S8, a translational repressor. We have analyzed the region of the spc mRNA that is responsible for this regulation. First, we have established that the S8 target site on the mRNA is near the translation start site of the third gene encoding ribosomal protein L5 in the operon. This was done by constructing hybrid plasmids carrying spc operon ribosomal protein genes under lac transcriptional control, as well as their deletion derivatives, and carrying out both in vivo and in vitro protein synthesis experiments. Next, the secondary structure of this region was studied by analyzing 5' end-labeled RNA synthesized from the phage SP6 promoter using structure-specific nucleases. A secondary structure model consistent with the results was deduced with the aid of a computer prediction of RNA folding. In addition, we cloned and sequenced the corresponding region from Salmonella typhimurium, Proteus vulgaris and Serratia marcescens and found five "compensating" substitutions that support some of the deduced helical structures of mRNA. None of the base changes was inconsistent with the deduced secondary structure model. Finally, site-directed mutagenesis experiments have identified bases important for regulation, including two base-paired sites representing each of two helical regions. This has led to the conclusion that some specific nucleotide residues located between these two helical regions are directly involved in S8 recognition, and that the function of the two helical regions is to maintain the proper orientation of these nucleotide residues. Comparison of the structure of the S8 target site on the spc mRNA with the known S8 binding site on rRNA has revealed a striking similarity in both primary and secondary structures. In particular, primary sequences of rRNA conserved among distantly related bacterial species in this region is found to be identical with the sequences at the corresponding positions in mRNA. These results suggest that the same structural features of the S8 repressor protein are involved in the interaction with both 16 S rRNA and the mRNA target site.


Assuntos
Genes Bacterianos , Óperon , Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Sequência de Bases , Deleção Cromossômica , Enterobacteriaceae , Escherichia coli , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Plasmídeos , RNA Bacteriano/genética
5.
Chem Biol ; 6(11): 835-44, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10574783

RESUMO

BACKGROUND: Cell-based reporter assays, which rely on a reporter gene under the control of a regulated promoter, are widely used to screen chemical libraries for novel receptor ligands. Here, we describe a reporter system that is based on ligand-induced DNA recombination to express the reporter gene. This system converts a transient activation of a signal transduction pathway into an amplified, constitutive and heritable expression of the reporter gene. RESULTS: We constructed gene fusions of Cre recombinase and mammalian promoters regulated by calcium, nuclear receptors or cyclic AMP. Reporter systems, comprising a Cre gene fusion and a loxP/reporter gene, were used to study the kinetics and dose responses to compounds that activate or inhibit the corresponding signal transduction pathway. We compared these reporters with conventional reporter systems in which the reporter gene is under the direct control of the responsive promoter. Reporter gene expression of the Cre reporters was greater than that of conventional reporters and could be measured more than a week after adding the stimulus. For all pathways studied here, the dose responses of the Cre reporters are nearly identical to those of conventional reporter systems. CONCLUSIONS: We have shown that Cre recombinase can be regulated by a variety of signal transduction pathways. It should therefore be possible to use receptor ligands to induce phenotypic conversion of mammalian cells for use in a variety of applications. One such application is high-throughput screening, and we developed loxP/luciferase reporter genes that provide an amplified and sustained luminescent response.


Assuntos
Integrases/genética , Proteínas Nucleares , Transdução de Sinais , Proteínas Virais , Animais , Células CHO , Divisão Celular , Cricetinae , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Humanos , Integrases/biossíntese , Células Jurkat , Cinética , Luciferases/genética , Mamíferos , Vírus do Tumor Mamário do Camundongo/genética , Fatores de Transcrição NFATC , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção
6.
Gene ; 132(1): 149-54, 1993 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-8406038

RESUMO

A gene involved in diphthamide biosynthesis, DPH2, was cloned from Saccharomyces cerevisiae by complementation of a diphthamide mutant. DPH2 exists as a single-copy gene in the yeast genome and is located on the left arm of chromosome XI. Sequence analysis of the DPH2 locus predicts that the DPH2 gene product is a 534-amino acid (aa) protein, with a calculated M(r) of 59,772. This conclusion was supported by Northern blot analysis of the DPH2 transcript and gel analysis of the DPH2 protein overproduced in Escherichia coli. Gene disruption studies indicate that the DPH2 gene is not essential for viability of yeast. The role of DPH2 in diphthamide biosynthesis is discussed.


Assuntos
Proteínas Fúngicas/genética , Histidina/análogos & derivados , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , DNA Recombinante , Escherichia coli , Genes Fúngicos , Histidina/biossíntese , Histidina/genética , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA
7.
Curr Opin Drug Discov Devel ; 4(1): 124-34, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11727318

RESUMO

The sequence of the human genome will soon provide researchers with hundreds of new ion channel genes. To create a successful ion channel drug discovery program, it is necessary to quickly develop reliable and robust high-throughput screening (HTS) assays for those ion channels implicated in important diseases. Ion channels are dynamic proteins, and therefore require assays that 'sense' their various functional states. Competition-binding assays, although successfully used for other target classes, often fail to identify ligands that modulate specific ion channel functions. Cell-based functional assays, therefore, are preferred for HTS of ion channel targets. In this review, we evaluate the various cell-based assays available for ion channels, and discuss future directions for assay improvements.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Eletrofisiologia/métodos , Canais Iônicos/fisiologia , Animais , Bioensaio/métodos , Eletrofisiologia/instrumentação , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo
8.
Adv Exp Med Biol ; 419: 45-52, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9193635

RESUMO

Saccharomyces cerevisiae was transformed with expression plasmids carrying the DTA gene under control of the GAL1 promoter; colonies that formed under inducing conditions were selected; and plasmids from these colonies were screened for mutations in DTA that failed to block expression of the protein. Substitutions at three sites were identified, all of which are in the active-site cleft; and each of the substitutions reduced ADP-ribosyltransferase activity by > 10(5). The substitutions include a charge reversal mutation of a catalytically important residue (Glu148Lys) and replacements of either of two glycines (Gly22 and Gly52) with bulky residues. The fact that multiple mutations were identified in these same residues implies that there are relatively few sites at which substitutions ablate ADP-ribosyltransferase activity without blocking expression of the full-length protein. Incorporation of a primary attenuating mutation into the DTA gene allowed S. cerevisiae also to be used to select complementary secondary mutations which altered activity less drastically. Besides elucidating structure-activity relationships, mutations identified by these approaches may be useful in designing new vaccines.


Assuntos
Toxina Diftérica/genética , Ácido Glutâmico , Mutação , Saccharomyces cerevisiae/genética , Sítios de Ligação , Toxina Diftérica/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Estrutura Molecular , Inibidores de Poli(ADP-Ribose) Polimerases , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/isolamento & purificação
11.
J Bacteriol ; 170(10): 4484-92, 1988 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3049533

RESUMO

The spc operon of Escherichia coli encodes 10 ribosomal proteins in the order L14, L24, L5, S14, S8, L6, L18, S5, L30, and L15. This operon is feedback regulated by S8, which binds near the translation start site of L5 and inhibits translation of L5 directly and that of the distal genes indirectly. We constructed plasmids carrying a major portion of the spc operon genes under lac transcriptional control. The plasmids carried a point mutation in the S8 target site which abolished regulation and resulted in overproduction of plasmid-encoded ribosomal proteins upon induction. We showed that alteration of the AUG start codon of L5 to UAG decreased the synthesis rates of plasmid-encoded distal proteins, as well as L5, by approximately 20-fold, with a much smaller (if any) effect on mRNA synthesis rates, indicating coupling of the distal cistrons' translation with the translation of L5. This conclusion was also supported by experiments in which S8 was overproduced in trans. In this case, there was a threefold reduction in the synthesis rates of chromosome-encoded L5 and the distal spc operon proteins, but no decrease in the mRNA synthesis rate. These observations also suggest that transcription from ribosomal protein promoters may be special, perhaps able to overcome transcription termination signals. We also analyzed the state of ribosomal protein mRNA after overproduction of S8 in these experiments and found that repression of ribosomal protein synthesis was accompanied by stimulation of processing (and degradation) of spc operon mRNA. The possible role of mRNA degradation in tightening the regulation is discussed.


Assuntos
Escherichia coli/genética , Proteínas Ribossômicas/genética , Northern Blotting , Retroalimentação , Regulação da Expressão Gênica , Genes Bacterianos , Óperon , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteínas Repressoras/fisiologia , Proteínas Ribossômicas/fisiologia , Transcrição Gênica
12.
Yeast ; 9(6): 661-7, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8394042

RESUMO

A 6.8 kbp DNA fragment localized to the left arm of chromosome XI from Saccharomyces cerevisiae was sequenced and analysed (EMBL accession no. X69765). Two genes involved in protein phosphatase activity were identified: YCN2 and an open reading frame encoding a protein that shares 46% amino acid identity with the sds22+ protein from Schizosaccharomyces pombe. A comparison of the genomic YCN2 sequence with the published cDNA sequence suggests the presence of an intron near the 5' end of the gene. Further sequence analysis suggests the presence of three additional genes near YCN2: a mitochondrial acyl-carrier protein, a gene encoding a putative hydrophobic protein, and a new gene coding for a tRNA(Leu) (UAA) isoacceptor located near a delta sequence.


Assuntos
Cromossomos Fúngicos , DNA Fúngico/genética , Genes Fúngicos/genética , Saccharomyces cerevisiae/genética , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos , Sequência de Bases , Calcineurina , Proteínas de Ligação a Calmodulina/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Fosfoproteínas Fosfatases/genética , RNA de Transferência de Leucina/genética
13.
Proc Natl Acad Sci U S A ; 91(19): 9022-6, 1994 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-7522328

RESUMO

We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 10(12) random decapeptides was incubated in an Escherichia coli S30 coupled transcription/translation system. Polysomes were isolated and screened by affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B. The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection. A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA. After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody. Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM. The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems.


Assuntos
Dinorfinas/análogos & derivados , Endorfinas/química , Peptídeos/química , Polirribossomos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Sequência de Bases , Primers do DNA/química , Dinorfinas/química , Epitopos , Escherichia coli , Técnicas In Vitro , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Relação Estrutura-Atividade
14.
Nucleic Acids Res ; 17(18): 7469-86, 1989 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-2508062

RESUMO

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.


Assuntos
Bacillus subtilis/genética , Genes Bacterianos , Óperon , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/genética , Ligação de Hidrogênio , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico
15.
Anal Biochem ; 258(1): 96-102, 1998 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-9527854

RESUMO

Reporter cell lines are often used for high throughput screening of chemical libraries to identify new receptor ligands. Here we show how Cre recombinase can be used in mammalian cells to screen for steroid receptor ligands. A translational fusion of Cre recombinase and the ligand binding domain of the human glucocorticoid receptor was transfected into mammalian cells with a loxP/luciferase reporter gene. The recombinase function of the fusion is dependent on ligand binding to the receptor, and Cre-mediated recombination results in constitutive expression of luciferase from the reporter gene. A stable transfected clone was isolated and used to characterize the kinetics, ligand specificity, and dose response to various receptor ligands. The Cre fusion system, unlike a transcriptional reporter using the mouse mammary tumor virus promoter, can detect binding of the receptor antagonist RU486. We also studied the Cre reporter in a sensitive, miniaturized, assay format using an 864-well plate and show that as few as 560 cells per assay well was sufficient to measure a dose response to ligand.


Assuntos
Genes Reporter , Receptores de Esteroides/agonistas , Receptores de Esteroides/antagonistas & inibidores , Recombinação Genética , Proteínas Virais , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cricetinae , DNA Recombinante/genética , Antagonistas de Hormônios/metabolismo , Humanos , Integrases/genética , Cinética , Ligantes , Luciferases/genética , Camundongos , Mifepristona/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transfecção
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