RESUMO
The half-life of the green fluorescent protein (GFP) was determined biochemically in cultured mouse LA-9 cells. The wild-type protein was found to be stable with a half-life of approximately 26 h, but could be destabilized by the addition of putative proteolytic signal sequences derived from proteins with shorter half-lives. A C-terminal fusion of a PEST sequence from the mouse ornithine decarboxylase gene reduced the half-life to 9.8 h, resulting in a GFP variant suitable for the study of dynamic cellular processes. In an N-terminal fusion containing the mouse cyclin B1 destruction box, it was reduced to 5.8 h, with most degradation taking place at metaphase. The combination of both sequences produced a similar GFP half-life of 5.5 h. Thus, the stability of this marker protein can be controlled in predetermined ways by addition of the appropriate proteolytic signals.
Assuntos
Proteínas Luminescentes/metabolismo , Animais , Linhagem Celular , Ciclina B/genética , Ciclina B1 , Citometria de Fluxo , Fluorescência , Proteínas de Fluorescência Verde , Cinética , Camundongos , Ornitina Descarboxilase/genética , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Fase S , TransfecçãoRESUMO
Clines of P-induced hybrid dysgenesis provide a means for monitoring the evolution of transposition repression over space and time. We have studied the molecular and phenotypic profiles of flies taken from a 2900 km cline along the eastern coast of Australia, which had previously been characterized over 10 years ago as having P populations in the north, Q populations at central sites and M' populations in the south. We have found that Q and M' populations of flies have increased their range within the cline at the expense of P lines. Q populations were found to be in the north of the cline and M' populations in the south. Some of the northern Q lines transmit repression through both sexes and type I deletion elements have been isolated from them. We suggest that these elements are responsible for Q type repression. The results support our model that populations made up of Q individuals with strong biparentally transmitted repression form an evolutionarily stable strategy for the repression of hybrid dysgenesis in Drosophila melanogaster.
Assuntos
Evolução Biológica , Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Disgenesia Gonadal , Animais , Sequência de Bases , Primers do DNA , Feminino , FenótipoRESUMO
We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.
Assuntos
Aberrações Cromossômicas , Citometria de Fluxo/métodos , Indicadores e Reagentes , Proteínas Luminescentes , Mitose/genética , Aneuploidia , Animais , Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Soluções Tampão , Cafeína/farmacologia , Fusão Celular , Cromossomos Artificiais de Levedura/genética , Demecolcina/farmacologia , Etoposídeo/farmacologia , Glucose/farmacologia , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Hibridização in Situ Fluorescente/métodos , Mamíferos , Camundongos , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Nocodazol/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Plasmídeos/genética , LevedurasAssuntos
Centrômero , Evolução Molecular , Cromossomo Y , Idoso , Animais , DNA/genética , Humanos , Masculino , Modelos GenéticosRESUMO
Type I repressors control P element transposition and comprise full length elements and elements with small 3' deletions in the final exon. Using a sensitive assay for measuring the strength of repression of P element transposition in somatic and germline tissues, we have isolated and characterized a naturally occurring type I repressor element from a Q population of Drosophila melanogaster. We demonstrate that the almost complete repression of transposition in this population is a mixture of KP elements with intermediate levels of repression, and the strong contribution of a single 2.6 kb P element deletion derivative, which we call SR (Strong Repressor). A deletion in the final intron of SR allows for the constitutive production of a putative 75 kDa repressor protein in germline tissues in addition to the production of the 66 kDa repressor in the soma, which would result in a biparental mode of inheritance of repression. Based on the four observed classes of natural Q populations, we propose a model in which populations containing SR-like elements, capable of producing strong type I repressor constitutively, have a selective advantage over populations which rely either on maternally transmitted P cytotype or on KP-induced weak levels of repression. Such populations may subsequently spread and constitute an evolutionary stable strategy for the repression of hybrid dysgenesis in Drosophila melanogaster.
