RESUMEN
Bovine interferon-gamma (IFN-gamma) sequences have been isolated by screening a cDNA library with a human IFN-gamma cDNA probe. The cDNA library was constructed from RNA isolated from concanavalin A-stimulated bovine lymph node cells. The open reading frame predicts that the bovine IFN-gamma precursor is composed of 166 amino acids with a predicted m.w. of 19,393. Alignment of the amino acid sequence with human IFN-gamma indicates that mature bovine IFN-gamma is composed of 143 amino acids with a predicted m.w. of 16,858. It has an amino acid homology of 63% with human IFN-gamma, and 47% with murine IFN-gamma. Biologically active bovine IFN-gamma was synthesized in an Escherichia coli expression system.
Asunto(s)
Clonación Molecular , Interferón gamma/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN/aislamiento & purificación , Escherichia coli/genética , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Ganglios Linfáticos , Hibridación de Ácido Nucleico , ARN Mensajero/análisisRESUMEN
Interleukin 2 (IL-2) cDNA clones have been isolated from both human and murine sources. We report here the isolation of a cDNA clone encoding bovine IL-2. This was accomplished by screening a cDNA library constructed from lectin-stimulated bovine lymph node cells, using a human IL-2 probe. Bovine IL-2 is composed of 155 amino acids and has a predicted molecular weight of 19,555. Alignment of the amino acid sequence with human IL-2 indicates that mature bovine IL-2 is composed of 135 amino acids and has a predicted molecular weight of 15,452. It has an amino acid homology of 65% with human IL-2 and 50% with murine IL-2. Bovine IL-2 is unique among IL-2 homologs in that it has a single N-linked glycosylation site. Biologically active bovine IL-2 was synthesized in an Escherichia coli expression system.
Asunto(s)
Interleucina-2/genética , Proteínas Recombinantes/genética , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , ADN/genética , Escherichia coli/genética , Regulación de la Expresión Génica , Genes , Interleucina-2/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesisRESUMEN
Human granulocyte/macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is essential for the in vitro proliferation and differentiation of precursor cells into mature granulocytes and macrophages. In this report we have used a mouse GM-CSF cDNA clone to isolate human GM-CSF clones from libraries made from HUT-102 messenger RNA and mitogen-stimulated T-lymphocyte messenger RNA. The human cDNA clones contained a single open-reading frame encoding a protein of 144 amino acids with a predicted molecular mass of 16,293 daltons and showed 69% nucleotide homology and 54% amino acid homology to mouse GM-CSF. One of these cDNA clones was shown to direct the synthesis of biologically active GM-CSF using a yeast expression system. The gene for human GM-CSF appears to exist as a single-copy gene.
Asunto(s)
Factores Estimulantes de Colonias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN/genética , ADN Recombinante , Regulación de la Expresión Génica , Genes , Granulocitos , Humanos , Macrófagos , Ratones , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Linfocitos T/fisiologíaRESUMEN
We have cloned the complete genome of an oncogenic primate retrovirus, the San Francisco isolate of gibbon ape leukemia virus, in a lambda phage vector. DNA sequence analysis and restriction endonuclease mapping of the inserted linear provirus demonstrated 9-base pair inverted repeats at its ends, flanking direct terminal repeats 470 base pairs in length. The (-) strong stop region of this DNA showed surprisingly low sequence homology to that of another gibbon ape leukemia virus isolate from an animal with similar disease. Analysis of the clone also revealed the terminal phosphate configuration of the linear provirus. The recombinant phage is suitable for direct use as a hybridization probe to detect homologous retroviral sequences in human cell lines.
Asunto(s)
ADN Viral/genética , Hominidae/microbiología , Hylobates/microbiología , Retroviridae/genética , Animales , Bacteriófago lambda , Secuencia de Bases , Transformación Celular Viral , Clonación Molecular/métodos , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
A positive selection procedure has been devised for isolating mutant strains of Salmonella typhimurium with altered glutamine synthetase activity. Mutants are derived from a histidine auxotroph by selecting for ability to grow on D-histidine as the sole histidine source. We hypothesize that the phenotype may be based on a regulatory increase in the activities of the D-histidine racemizing enzymes, but this has not been established. Spontaneous glutamine-requiring mutants isolated by the above selection procedure have two types of alterations in glutamine synthetase activity. Some have less than 10% of parent activity. Others have significant glutamine synthetase activity, but the enzyme have an altered response to divalent cations. Activity in mutants of the second type mimics that of highly adenylylated wild-type enzyme, which is believed to be in-active in vivo. Glutamine synthetase from one such mutant is more heat labile than wild-type enzyme, indicating that it is structurally altered. Mutations in all strains are probably in the glutamine synthetase structural gene (glnA). They are closely linked on the Salmonella chromosome and lie at about min 125. The mutants have normal glutamate dehydrogenase activity.