RESUMO
Macronuclear chromosomes of hypotrichous ciliates are gene-sized molecules carrying the coding sequence flanked by short non-translated regions and bounded by telomeres. We have constructed artificial chromosomes for investigation of transcription in the macronucleus of Euplotes crassus. The neo gene was put under the control of the 5"-non-translated region of the TBP gene of E.crassus. These molecules were introduced into the cell with the help of liposomes. The cells were transformed and survived high concentrations of geneticin. The artificial chromosomes were kept in the macro-nucleus for at least 50 days at a copy number of about 200 per macronucleus. Expression of the gene was shown by reverse transcription of the neo messenger. The transcription start was mapped and found to coincide with that found on the natural macronuclear chromosome encoding TBP in E.crassus.
Assuntos
Núcleo Celular/genética , Cromossomos/genética , Euplotes/genética , Expressão Gênica , Genes Reporter/genética , Transformação Genética/genética , Regiões 5' não Traduzidas/genética , Animais , Sequência de Bases , Proteínas de Ligação a DNA/genética , Euplotes/citologia , Euplotes/efeitos dos fármacos , Euplotes/crescimento & desenvolvimento , Dosagem de Genes , Genes de Protozoários/genética , Gentamicinas/farmacologia , Canamicina Quinase/genética , Lipossomos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Proteína de Ligação a TATA-Box , Telômero/genética , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
The Drosophila don juan gene encodes a basic protein (Don Juan protein), which is solely expressed postmeiotically during spermiogenesis in elongated spermatids and in mature sperm. Transgenic expression of a GFP-tagged Don Juan protein (DJ-GFP) in the male germ line showed an association of the fusion protein with the sperm tail. Detailed examination of DJ-GFP localization revealed novel insights into its distinct temporal and spatial distribution along the sperm tail during the last phase of spermatid maturation. Co-localization of DJ-GFP with actin-labeled cysts demonstrated its emergence in elongated spermatids during individualization. Additionally, the endogenous Don Juan protein was detected with epitope-specific antibodies in finally elongated nuclei of spermatids. After completion of nuclear shaping Don Juan is no longer detectable in the sperm heads with the onset of individualization. Mislocalization of the DJ-GFP protein in flagella of a mutant with defective mitochondrial differentiation provides evidence of mitochondrial association of the fusion protein with flagellar mitochondrial arrays. Ectopically expressed DJ-GFP in premeiotic germ cells as well as salivary gland cells confirmed the capability of the fusion protein to associate with mitochondria. Therefore we suppose that Don Juan is a nuclear-encoded, germ-cell specifically expressed mitochondrial protein, which might be involved in the final steps of mitochondrial differentiation within the flagellum.
