Asunto(s)
Transformación Celular Neoplásica/genética , Cocarcinogénesis , Neoplasias Hepáticas Experimentales/genética , Animales , Carcinógenos , Transformación Celular Neoplásica/inducido químicamente , Dietilnitrosamina , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Femenino , Genes ras , Hormonas Esteroides Gonadales/fisiología , Hormonas Esteroides Gonadales/toxicidad , Hepatectomía/efectos adversos , Neoplasias Hepáticas Experimentales/etiología , Regeneración Hepática , Masculino , Ratones , Ratones Endogámicos , Modelos Biológicos , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/genética , Ratas , Especificidad de la EspecieRESUMEN
We have determined the nucleotide sequences of 10 intragenic human HPRT gene deletion junctions isolated from thioguanine-resistant PSV811 Werner syndrome fibroblasts or from HL60 myeloid leukemia cells. Deletion junctions were located by fine structure blot hybridization mapping and then amplified with flanking oligonucleotide primer pairs for DNA sequence analysis. The junction region sequences from these 10 HPRT mutants contained 13 deletions ranging in size from 57 bp to 19.3 kb. Three DNA inversions of 711, 368, and 20 bp were associated with tandem deletions in two mutants. Each mutant contained the deletion of one or more HPRT exon, thus explaining the thioguanine-resistant cellular phenotype. Deletion junction and donor nucleotide sequence alignments suggest that all of these HPRT gene rearrangements were generated by the nonhomologous recombination of donor DNA duplexes that share little nucleotide sequence identity. This result is surprising, given the potential for homologous recombination between copies of repeated DNA sequences that constitute approximately a third of the human HPRT locus. No difference in deletion structure or complexity was observed between deletions isolated from Werner syndrome or from HL60 mutants. This suggests that the Werner syndrome deletion mutator uses deletion mutagenesis pathway(s) that are similar or identical to those used in other human somatic cells.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Secuencia de Bases , Deleción Cromosómica , Mapeo Cromosómico , ADN/genética , Análisis Mutacional de ADN , Exones , Amplificación de Genes , Humanos , Datos de Secuencia Molecular , Recombinación GenéticaRESUMEN
We have determined the genetic stability of three independent intragenic human HPRT gene duplications and the structure of each duplication at the nucleotide sequence level. Two of the duplications were isolated as spontaneous mutations from the HL60 human myeloid leukemia cell line, while the third was originally identified in a Lesch-Nyhan patient. All three duplications are genetically unstable and have a reversion rate approximately 100-fold higher than the rate of duplication formation. The molecular structures of these duplications are similar, with direct duplication of HPRT exons 2 and 3 and of 6.8 kb (HL60 duplications) or 13.7 kb (Lesch-Nyhan duplication) of surrounding HPRT sequence. Nucleotide sequence analyses of duplication junctions revealed that the HL60-derived duplications were generated by unequal homologous recombination between clusters of Alu repeats contained in HPRT introns 1 and 3, while the Lesch-Nyhan duplication was generated by the nonhomologous insertion of duplicated HPRT DNA into HPRT intron 1. These results suggest that duplication substrates of different lengths can be generated from the human HPRT exon 2-3 region and can undergo either homologous or nonhomologous recombination with the HPRT locus to form gene duplications.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Familia de Multigenes , Secuencia de Bases , ADN/genética , Análisis Mutacional de ADN , Humanos , Síndrome de Lesch-Nyhan/enzimología , Síndrome de Lesch-Nyhan/genética , Modelos Genéticos , Datos de Secuencia Molecular , ARN Mensajero/genética , Células Tumorales Cultivadas/enzimologíaRESUMEN
We have determined the nucleotide sequence of the rat hprt (hypoxanthine phosphoribosyltransferase; EC 2.4.2.8.) mRNA coding region and of adjacent, untranslated 5' and 3' mRNA, and we have designed an oligonucleotide primer pair for efficient PCR amplification of the rat hprt coding region. These sequence data and rat-specific primer pair will aid workers interested in coupling well-developed rat toxicologic and carcinogenicity bioassays with quantitative and molecular analyses of somatic mutation induction in rat cells in vivo and in vitro.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ratas , Ratas EndogámicasRESUMEN
The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector. A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E. coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3. The primary construction, designated pLB5001, is under transcriptional control of the tandem lipoprotein promoter (lppP) lactose promoter-operator (lacPO), and is regulated by the lactose repressor. Upon induction, a polypeptide of MW = 14,600 was produced which had GM-CSF activity in a human bone marrow colony assay. The linker sequence between the ompA signal peptide and the amino terminus of the mature GM-CSF was removed by oligonucleotide-directed site-specific mutagenesis to produce GM-CSF with an authentic amino terminus. The resulting construct, designated pLB5001-4, expressed authentic GM-CSF with a specific activity similar to that observed for the pLB5001 specified GM-CSF. Both versions of GM-CSF were associated with the membrane fraction after osmotic shock, and were purified to homogeneity by DEAE-Sephacel chromatography, followed by reversed-phase HPLC. Amino acid sequencing from the amino terminus of the purified GM-CSF established that the ompA signal peptide was cleaved at its normal processing site in both cases.
Asunto(s)
Factores Estimulantes de Colonias/biosíntesis , Escherichia coli/metabolismo , Vectores Genéticos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas Bacterianas/genética , Ensayo de Unidades Formadoras de Colonias , Factores Estimulantes de Colonias/genética , Factores Estimulantes de Colonias/aislamiento & purificación , ADN/genética , ADN Recombinante , Escherichia coli/genética , Escherichia coli/ultraestructura , Granulocitos , Humanos , Macrófagos , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Caulobacter crescentus accumulated guanosine tetraphosphate in response to nitrogen starvation but not in response to amino acid starvation. Nitrogen starvation also acted specifically to inhibit certain transitions in the C. crescentus life cycle, and guanosine tetraphosphate may act as an intracellular regulator of cell cycle events.
