Assuntos
Hemoglobinas/biossíntese , Cromatografia por Troca Iônica , Escherichia coli/genética , Fermentação , Expressão Gênica , Globinas/genética , Hemoglobinas/genética , Hemoglobinas/isolamento & purificação , Humanos , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Mapeamento por RestriçãoAssuntos
Hemoglobina Falciforme/genética , Hemoglobina Falciforme/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Sequência de Bases , Carboximetilcelulose Sódica , Cromatografia , Cromatografia Líquida de Alta Pressão , DNA Complementar/genética , Escherichia coli/genética , Expressão Gênica , Globinas/genética , Hemoglobina A/genética , Hemoglobina Falciforme/biossíntese , Humanos , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Synthetic genes encoding the human alpha- and beta-globin polypeptides have been expressed from a single operon in Escherichia coli. The alpha- and beta-globin polypeptides associate into soluble tetramers, incorporate heme, and accumulate to greater than 5% of the total cellular protein. Purified recombinant hemoglobin has the correct stoichiometry of alpha- and beta-globin chains and contains a full complement of heme. Each globin chain also contains an additional methionine as an extension to the amino terminus. The recombinant hemoglobin has a C4 reversed-phase HPLC profile essentially identical to that of human hemoglobin A0 and comigrates with hemoglobin A0 on SDS/PAGE. The visible spectrum and oxygen affinity are similar to that of native human hemoglobin A0. The recombinant protein shows a reduction in Bohr and phosphate effects, which may be attributed to the presence of methionine at the amino termini of the alpha and beta chains. We have also expressed the alpha- and beta-globin genes separately and found that the expression of the alpha-globin gene alone results in a marked decrease in the accumulation of alpha-globin in the cell. Separate expression of the beta-globin gene results in high levels of insoluble beta-globin. These observations suggest that the presence of alpha- and beta-globin in the same cell stabilizes alpha-globin and aids the correct folding of beta-globin. This system provides a simple method for expressing large quantities of recombinant hemoglobin and allows facile manipulation of the genes encoding hemoglobin to produce functionally altered forms of this protein.
Assuntos
Escherichia coli/genética , Globinas/genética , Hemoglobinas/genética , Óperon , Sequência de Bases , Clonagem Molecular , Genes Sintéticos , Hemoglobinas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/síntese química , Oxiemoglobinas/metabolismo , Ácido Fítico/farmacologia , Proteínas Recombinantes/metabolismo , Mapeamento por RestriçãoRESUMO
Insulin-like growth factors (IGFs) bind to specific proteins present in extracellular fluids. One of these binding proteins (IGF-BP) was purified from human amniotic fluid and was shown to potentiate the effects of IGF-I in vitro (10). In these studies, a polyclonal antibody to this protein was used to isolate a cDNA clone from a human decidua library. This clone encodes a polypeptide of 25,832 daltons that includes the sequences of 9 tryptic peptides that had been prepared from the purified IGF-BP. The protein has 15 cysteines that are clustered at the amino and carboxy ends of the molecule. The protein has an RGD sequence near its C-terminus, which may account for its ability to attach to cells and to potentiate the biological actions of IGF-I.
Assuntos
Proteínas de Transporte/genética , Clonagem Molecular , Regulação da Expressão Gênica , Sequência de Aminoácidos , Sequência de Bases , Feminino , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Dados de Sequência Molecular , Mapeamento de Peptídeos , PlasmídeosRESUMO
A genomic clone (lambda ScG7) from Saccharomyces cerevisiae encoded a 650-nucleotide poly(A)-containing [poly(A)+] RNA that was about 50 times more abundant in MATa cells that had been exposed to the peptide pheromone alpha-factor than in untreated cells. This RNA was transcribed from a cluster of repetitive sequences: both intact and truncated delta and sigma elements adjacent to a tRNATrp gene. Strand-specific probes indicated that this RNA initiated within an intact sigma element and contained sigma sequences at its 5' end. MATa cells produced two other prominent poly(A)+ RNAs (500 and 5,300 bases) in response to alpha-factor that were homologous to the same strand of sigma but transcribed from other locations in the genome. Induction of the sigma-related transcripts was rapid, was not blocked by inhibition of protein synthesis, required a functional receptor (STE2 gene product), and hence appeared to be a primary response to pheromone. Pulse-labeling confirmed that accumulation of sigma RNA following alpha-factor administration was accounted for by an increase in its rate of transcription. The sigma RNAs also were induced in MAT alpha cells that had been treated with a-factor, but were not present at significant levels in MATa/MAT alpha diploids. In MATa cells transformed with a plasmid in which the lambda ScG7 sigma element was inserted just upstream of a gene coding for the intracellular form of invertase (SUC2) lacking its own promoter, a new poly(A)+ RNA (2.2 kilobases) appeared in response to alpha-factor that hybridized to both sigma and SUC2 probes, and intracellular invertase activity was elevated about 10-fold within 30 min. Primer extension showed that transcription from the hybrid gene initiated exclusively within the sigma sequence (117 nucleotides from the 3' end of the element).
Assuntos
Peptídeos/fisiologia , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Regulação da Expressão Gênica , Glicosídeo Hidrolases/genética , Fator de Acasalamento , RNA Fúngico/genética , Proteínas Recombinantes de Fusão/genética , Transcrição Gênica , beta-FrutofuranosidaseRESUMO
A method for identifying yeast genes whose transcription is differentially regulated was developed. The technique is based on incorporation of the analog 4-thiouridine into nascent RNAs, which allows their purification. The purified RNAs are used to prepare cDNA copies for screening of genomic DNA libraries by hybridization. Using this procedure, several cloned yeast DNA segments were found whose transcription in MATa haploids in vivo is apparently modulated in dramatic fashion within 10-15 min after exposure to the mating pheromone, alpha factor. Subsequent analysis indicated that these sequences fall into three major classes: (i) genes expressed in vegetatively growing cells that are no longer transcribed after alpha-factor administration ("turn-off" genes); (ii) genes whose expression is increased 10- to 20-fold after exposure of the MATa cells to alpha factor ("turn-up" genes); and (iii) genes that are expressed only after alpha-factor treatment ("turn-on" genes). The first class may encode products required for cell cycle progression; the third class may code for products uniquely involved in the mating process.
Assuntos
Clonagem Molecular , Genes Fúngicos , Peptídeos/genética , Feromônios/genética , Saccharomyces cerevisiae/genética , Animais , DNA/análise , Cinética , Fator de Acasalamento , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Under some conditions, T4 DNA replication requires the products of the DNA-delay genes, genes 39, 52, 58, and 60. By using an in vitro complementation assay that stimulates DNA replication in T4 39(-)-infected cell extracts, T4 gene 39 protein has been purified. The purified fraction also contains complementing activities for T4 genes 52 and 60. On sodium dodecyl sulfate/polyacrylamide gel analysis the purified preparation exhibits three protein components: a 51,000-dalton protein corresponding to the product of gene 52, a 64,000-dalton protein corresponding to the product of gene 39, and a 110,000-dalton protein. This purified fraction shows a DNA topoisomerase activity that untwists superhelical DNA in an ATP- and Mg2+-dependent reaction. The analogs adenylyl imidodiphosphate and adenyl [beta, gamma-methylene]diphosphonate cannot be used to replace ATP. The topoisomerase activity is not sensitive to the antibiotics oxolinic acid and novobiocin, known antagonists of Escherichia coli DNA gyrase. The possible relationship among the three polypeptides and their biological activities is discussed.
