Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Estrutura Terciária de Proteína , Fator sigma/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Dimerização , Fosforilação , RNA Polimerase Sigma 54 , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Applying a homology search method previously described, we identified a sequence in the extracellular dimerization site of the erythropoietin receptor, distant from the hormone binding site. A peptide identical to that sequence was synthesized. Remarkably, it activated receptor signaling in the absence of erythropoietin. Neither the peptide nor the hormone altered the affinity of the other for the receptor; thus, the peptide does not bind to the hormone binding site. The combined activation of signal transduction by hormone and peptide was strongly synergistic. In mice, the peptide acted like the hormone, protecting against the decrease in hematocrit caused by carboplatin.
Assuntos
Peptídeos/metabolismo , Receptores da Eritropoetina/agonistas , Receptores da Eritropoetina/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Células CHO , Cricetinae , Humanos , Camundongos , Peptídeos/química , Peptídeos/farmacologiaRESUMO
Degenerate PCR probes were used to amplify gene fragments encoding the catalytic domain of sigma54-dependent transcription activators. The procedure should be widely applicable, as it recovered both known and novel gene fragments: 5 from Rhizobium meliloti, 13 from Myxococcus xanthus, and 3 from Bacillus subtilis. No fragments were obtained from Synechococcus sp. strain PCC 7002 or Saccharomyces cerevisiae.
Assuntos
Bacillus subtilis/genética , Cianobactérias/genética , Proteínas de Ligação a DNA , RNA Polimerases Dirigidas por DNA/genética , Myxococcus xanthus/genética , Reação em Cadeia da Polimerase/métodos , Fator sigma/genética , Sinorhizobium meliloti/genética , Transativadores/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano , Genes Bacterianos , Dados de Sequência Molecular , Filogenia , RNA Polimerase Sigma 54 , Saccharomyces cerevisiae/genéticaRESUMO
The glnA gene of the cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002) was isolated by complementing an Escherichia coli strain auxotrophic for glutamine (YMC11) with a PR-6 cosmid library. PR-6 glnA is a single-copy gene that encodes a deduced amino acid sequence that is highly homologous to the deduced glnA amino acid sequences reported for other bacteria. No homology was found between the PR-6 glnA flanking sequences and the ntrB, ntrC, or glnB genes of other bacteria. Northern (RNA) and primer extension analyses of PR-6 RNA revealed one predominant and several minor glnA transcripts of about 1.5 to 1.7 kb. The steady-state amounts of these transcripts increased three- to fivefold when the cells were starved for nitrogen. However, we found that mutant PR-6 cells lacking glnA were still able to use nitrate or ammonium as a sole nitrogen source. Although no RNA homologous to an internal fragment of the glnA gene could be detected in the mutant cells, they retained about 60% of wild-type glutamine biosynthetic activity. The mutant cells were more sensitive than the wild-type cells to methionine sulfoximine, a transition state analog of glutamate, a result that might indicate the presence of an additional glutamine synthetase; however, cell extracts of wild-type PR-6 cells and those lacking glnA were both able to use carbamyl phosphate instead of ammonium as a nitrogen donor for the synthesis of glutamine, a result that indicates the use of carbamyl phosphate synthetase to assimilate ammonium and produce glutamine.
