RESUMO
A mouse metallothionein-I/human growth hormone fusion gene was microinjected into fertilized mouse eggs, the embryos were implanted into pseudopregnant foster mothers, and the offspring analysed. Five of twenty-six mice born after one series of injections contained from one to eight copies of the fusion gene stably integrated into their genomes and had human growth hormone in their serum. When several of these transgenic mice and transgenic offspring were treated with glucocorticoids, serum growth hormone levels were elevated from 1.5- to 6.3-fold. A fourfold induction in fusion gene mRNA in the liver of one of the five mice was also observed after treatment with glucocorticoids. When the fusion gene was transiently transfected into mouse L cells, dexamethasone caused a three- to fourfold induction of fusion gene mRNA and secreted human growth hormone. A deletion analysis of regulatory elements required for inducibility in L cells shows that DNA sequences responsible for the observed inductions are located within the transcribed region of the human growth hormone gene. However, a previously described glucocorticoid receptor binding site in the first intron of the gene is not required for response to the hormone.
Assuntos
Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/genética , Transfecção , Triancinolona/farmacologia , Animais , Clonagem Molecular , Hormônio do Crescimento/sangue , Células L , Metalotioneína/genética , Camundongos , Camundongos Transgênicos , RNA Mensageiro/efeitos dos fármacosRESUMO
A myc-containing recombinant feline leukemia provirus, designated FTT, was molecularly cloned from the cat T-cell lymphoma line F422. Its transforming activity, as well as the nucleotide sequence of the 3' 2.7 kilobases of FTT, including v-myc, was determined. The predicted v-myc protein differs from feline c-myc by three amino acid changes and is truncated by two amino acids at the carboxyl terminus. Comparison with feline leukemia virus (FeLV), feline c-myc, and other FeLV proviruses indicates that recombination junctions involved in the generation of FeLV-onc viruses occur at preferred locations within the virus. They usually follow or occur within the sequence ACCCC at 5' junctions and may result from homologous recombination between sequences of marked purine-pyrimidine strand bias, especially at 3' junctions. Some recombination sites also resemble recombinase recognition sequences utilized in immunoglobulin and T-cell receptor variable-region joining. Transfection of primary rat embryo fibroblasts and subsequent in vivo analysis revealed that morphologic and tumorigenic transformation require cotransfection of FTT with human EJ-ras DNA; neither gene alone is sufficient. FTT v-myc is expressed in these transformed rat cells as a 3.0-kilobase subgenomic RNA; however, in contrast to the depressed level of c-myc expression in v-myc-involved feline tumors, steady-state levels of rat c-myc RNA and protein are apparently unaltered.
Assuntos
Transformação Celular Viral , Vírus da Leucemia Felina/patogenicidade , Proteínas Oncogênicas Virais/genética , Oncogenes , Provírus/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Recombinante , Regulação da Expressão Gênica , Genes Virais , Vírus da Leucemia Felina/genética , Vírus da Leucemia Felina/ultraestrutura , Dados de Sequência Molecular , RNA Viral/genética , Recombinação Genética , Mapeamento por Restrição , Transcrição GênicaRESUMO
The nucleotide sequence of the human growth hormone variant gene, one of the five members of the growth hormone gene family, predicts that it encodes a growth hormone-like protein. As a first step in determining whether this gene is functional in humans, we have expressed a mouse metallothionein I/human growth hormone variant fusion gene in mouse L cells and in transgenic mice. The growth hormone variant protein expressed in transiently transfected L cells is distinct from growth hormone itself with respect to reactivity with anti-growth hormone monoclonal antibodies, behavior during column chromatography, and isoelectric point. Transgenic mice expressing the growth hormone variant protein are 1.4- to 1.9-fold larger than nontransgenic controls, suggesting that the protein has growth-promoting properties.
Assuntos
Fibroblastos/metabolismo , Regulação da Expressão Gênica , Animais , Células Cultivadas , Humanos , Ponto Isoelétrico , Metalotioneína/genética , Camundongos , Camundongos Transgênicos , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismoRESUMO
The human growth hormone (hGH) transient assay system described here is based on the expression of hGH directed by cells transfected with hGH fusion genes. Levels of secreted hGH in the medium, measured by a simple radioimmunoassay, are proportional to both levels of cytoplasmic hGH mRNA and the amount of transfected DNA. The system is extremely sensitive, easy to perform, and is qualitatively different from other transient expression systems in that the medium is assayed and the cells themselves are not destroyed. The hGH transient assay system is appropriate for analyses of regulation of gene expression and was utilized here to investigate the effect of the simian virus 40 enhancer on the herpes simplex virus thymidine kinase promoter and the effect of zinc on the mouse metallothionein-I promoter. The expression of hGH can also be used as an internal control to monitor transfection efficiency along with any other transient expression system. All cell types tested thus far (including AtT-20, CV-1, GC, GH4, JEG, L, and primary pituitary cells) were able to secrete hGH into the medium.
